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Sample records for npm1 molecular basis

  1. Nucleophosmin (NPM1) Mutations in Acute Myeloid Leukemia: An Ongoing (Cytoplasmic) Tale of Dueling Mutations and Duality of Molecular Genetic Testing Methodologies

    OpenAIRE

    2008-01-01

    This Commentary highlights two articles that focus on molecular techniques to identify mutations in nucleophosmin (NPM1), which is the most frequently mutated gene in cytogenetically normal acute myeloid leukemia (CN-AML)

  2. Mouse models of NPM1-mutated acute myeloid leukemia: biological and clinical implications.

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    Sportoletti, P; Varasano, E; Rossi, R; Mupo, A; Tiacci, E; Vassiliou, G; Martelli, M P; Falini, B

    2015-02-01

    Acute myeloid leukemia (AML) carrying nucleophosmin (NPM1) mutations displays distinct biological and clinical features that led to its inclusion as a provisional disease entity in the 2008 World Health Organization (WHO) classification of myeloid neoplasms. Studies of the molecular mechanisms underlying the pathogenesis of NPM1-mutated AML have benefited greatly from several mouse models of this leukemia developed over the past few years. Immunocompromised mice xenografted with NPM1-mutated AML served as the first valuable tool for defining the biology of the disease in vivo. Subsequently, genetically engineered mouse models of the NPM1 mutation, including transgenic and knock-in alleles, allowed the generation of mice with a constant genotype and a reproducible phenotype. These models have been critical for investigating the nature of the molecular effects of these mutations, defining the function of leukemic stem cells in NPM1-mutated AML, identifying chemoresistant preleukemic hemopoietic stem cells and unraveling the key molecular events that cooperate with NPM1 mutations to induce AML in vivo. Moreover, they can serve as a platform for the discovery and validation of new antileukemic drugs in vivo. Advances derived from the analysis of these mouse models promise to greatly accelerate the development of new molecularly targeted therapies for patients with NPM1-mutated AML.

  3. How does the NPM1 mutant induce leukemia?

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    Paolo Sportoletti

    2011-06-01

    Full Text Available NPM1 is the most frequently mutated gene in AML and the role of the NPM1 mutant in acute myeloid leukemia along with its leukemogenic potential are still under investigation. NPM1 genetic alterations can contribute to leukemogenesis through the direct oncogenic effect of the mutant protein and the concomitant loss of one functional allele. Npm1 loss determines tumor development in the mouse while in human NPM1 maps in a chromosomal region frequently loss in myelodysplastic syndrome (MDS. The NPM1 mutant cytoplasmic delocalization in leukemic blasts alters multiple cellular pathways through either loss or gain of function effects on different protein partners. Here we discuss the most relevant studies on the role of the NPM1 molecule in hematological malignancies and both in vitro and in vivo studies that are trying to elucidate the way by which the NPM1 mutation induces leukemia.

  4. Mutation of NPM1 and FLT3 Genes in Acute Myeloid Leukemia and Their Association with Clinical and Immunophenotypic Features

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    Pradeep Singh Chauhan

    2013-01-01

    Full Text Available Background. Mutations in NPM1 and FLT3 genes represent the most frequent genetic alterations and important diagnostic and prognostic indicators in patients with acute myeloid leukemia (AML. Objective. We investigated the prevalence and clinical characteristics of NPM1 and FLT3 mutations in 161 patients of de novo AML including adults and children. Results. NPM1 mutation was found in 21% and FLT3 mutation in 25% of the AML patients. Thirteen (8% samples were positive for both NPM1 and FLT3/ITD mutations. Adult patients had significantly higher frequency of NPM1 mutation than children (25.8% versus 8.8%; P=0.02. Further, NPM1 mutation was found to be more frequent in patients above 45 years of age (P=0.02. NPM1 mutation was significantly associated with higher platelet count (P=0.05 and absence of hepatosplenomegaly (P=0.01, while FLT3/ITD mutation was associated with higher white blood count (P=0.01. Immunophenotypically, NPM1 mutation was associated with the lack of CD34 (P<0.001 and HLD-DR expression (P<0.001, while FLT3/ITD mutation was positively associated with the expression of CD7 (P=0.04. No correlation was found between NPM1 mutation and fusion gene. Interestingly, FLT3/ITD mutation was found to be inversely associated with AML/ETO fusion gene (P=0.04. Conclusions. The results suggest that distinct clinical and immunophenotypic characteristics of NPM1 and FLT3/ITD mutations present further insight into the molecular mechanism of leukemogenesis.

  5. A new transcriptional variant and small azurophilic granules in an acute promyelocytic leukemia case with NPM1/RARA fusion gene.

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    Kikuma, Tomoe; Nakamachi, Yuji; Noguchi, Yoriko; Okazaki, Yoko; Shimomura, Daisuke; Yakushijin, Kimikazu; Yamamoto, Katsuya; Matsuoka, Hiroshi; Minami, Hironobu; Itoh, Tomoo; Kawano, Seiji

    2015-12-01

    We report here the first case of NPM1/RARA-positive acute promyelocytic leukemia (APL) preceded by myeloid sarcoma (MS) in the vertebra. A 52-year-old man was diagnosed with MS, as the tumor cells were positive for myeloperoxidase and CD68 but negative for CD163. After treatment with steroids and radiation, the size of the tumor was markedly reduced and peripheral blood count was normal. Bone marrow examination showed 89.2% consisted of unclassified promyelocytes characterized by round nuclei and abundant small azurophilic granules but no Auer rods. The results of chromosome analysis showed 46,XY,t(5;17)(q35;q12). Reverse-transcription polymerase chain reaction amplified the NPM1/RARA fusion transcripts derived from a combination of NPM1 exon 4 and RARA exon 5, or of NPM1 exon 1 and RARA exon 5; the latter of these has not been reported previously. Electron microscopic examination of the promyelocyte nuclei showed they were oval with mild nuclear chromatin condensation and small- to medium-sized nucleoli. Hematological and molecular complete remission was attained after induction therapy including all-trans retinoic acid. As MS was also diagnosed in two of the seven other reported cases of APL with NPM1/RARA, MS may occur more frequently in APL with NPM1/RARA than APL with PML/RARA.

  6. Detection of NPM1 exon 12 mutations and FLT3 – internal tandem duplications by high resolution melting analysis in normal karyotype acute myeloid leukemia

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    Carney Dennis A

    2008-07-01

    Full Text Available Abstract Background Molecular characterisation of normal karyotype acute myeloid leukemia (NK-AML allows prognostic stratification and potentially can alter treatment choices and pathways. Approximately 45–60% of patients with NK-AML carry NPM1 gene mutations and are associated with a favourable clinical outcome when FLT3-internal tandem duplications (ITD are absent. High resolution melting (HRM is a novel screening method that enables rapid identification of mutation positive DNA samples. Results We developed HRM assays to detect NPM1 mutations and FLT3-ITD and tested diagnostic samples from 44 NK-AML patients. Eight were NPM1 mutation positive only, 4 were both NPM1 mutation and FLT3-ITD positive and 4 were FLT3-ITD positive only. A novel point mutation Y572C (c.1715A>G in exon 14 of FLT3 was also detected. In the group with de novo NK-AML, 40% (12/29 were NPM1 mutation positive whereas NPM1 mutations were observed in 20% (3/15 of secondary NK-AML cases. Sequencing was performed and demonstrated 100% concordance with the HRM results. Conclusion HRM is a rapid and efficient method of screening NK-AML samples for both novel and known NPM1 and FLT3 mutations. NPM1 mutations can be observed in both primary and secondary NK-AML cases.

  7. Expression and significance of Wnt5a gene and NPM1 gene in chronic myeloid leukemia patients

    Institute of Scientific and Technical Information of China (English)

    Liang-Tuo Wang; Xiang-Lan Zhang; Jian-Yu Situ; Yuan-Ying Huang

    2015-01-01

    Objective:To detect Wnt5a gene expression and NPM1 gene in chronic myeloid leukemia bone marrow cells and to explore its relevance and significance.Methods:Sixty cases of chronic myeloid leukemia patients in our hospital were included in the experimental group, taking up 20 cases in the blastic phase, 20 cases in the accelerated phase, and 20 cases in the chronic phase; and 60 cases with benign hematologic disease were included in the control group. Cultured bone marrow cells were detected by RT-PCR methods to analyze the significance of Wnt5a and NPM1 gene expressions.Results:Wnt5a mRNA in the three sub-groups of the experimental group had low semi-quantitative mean values and positive rates compared with the control group. NPM1 mRNA positive rates and median expression levels were higher, and the difference was statistically significant. In the three subgroups of the experimental group, Wnt5a mRNA semi-quantitative mean values and positive rates in the blastic phase and accelerated phase were low, while NPM1 mRNA positive rates and median expression levels were higher; and the difference was statistically significant. Wnt5a and NPM1 expression levels in patients' white blood cell count were associated with bone marrow blast cells; and the difference was statistically significant. However, this was not associated with age, hemoglobin content and platelet count; and the difference was not statistically significant.Conclusion: There is a certain degree of correlation between Wnt5a and NPM1 genes and the occurrence and development of chronic myeloid leukemia. Wnt5a gene may inhibit its occurrence, while NPM1 gene positivity promotes its progression, which may be applied in the diagnosis and treatment of chronic myeloid leukemia, providing a new way of analyzing prognosis and a theoretical basis for clinical application.

  8. Recurrent mutations, including NPM1c, activate a BRD4-dependent core transcriptional program in acute myeloid leukemia.

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    Dawson, M A; Gudgin, E J; Horton, S J; Giotopoulos, G; Meduri, E; Robson, S; Cannizzaro, E; Osaki, H; Wiese, M; Putwain, S; Fong, C Y; Grove, C; Craig, J; Dittmann, A; Lugo, D; Jeffrey, P; Drewes, G; Lee, K; Bullinger, L; Prinjha, R K; Kouzarides, T; Vassiliou, G S; Huntly, B J P

    2014-02-01

    Recent evidence suggests that inhibition of bromodomain and extra-terminal (BET) epigenetic readers may have clinical utility against acute myeloid leukemia (AML). Here we validate this hypothesis, demonstrating the efficacy of the BET inhibitor I-BET151 across a variety of AML subtypes driven by disparate mutations. We demonstrate that a common 'core' transcriptional program, which is HOX gene independent, is downregulated in AML and underlies sensitivity to I-BET treatment. This program is enriched for genes that contain 'super-enhancers', recently described regulatory elements postulated to control key oncogenic driver genes. Moreover, our program can independently classify AML patients into distinct cytogenetic and molecular subgroups, suggesting that it contains biomarkers of sensitivity and response. We focus AML with mutations of the Nucleophosmin gene (NPM1) and show evidence to suggest that wild-type NPM1 has an inhibitory influence on BRD4 that is relieved upon NPM1c mutation and cytosplasmic dislocation. This leads to the upregulation of the core transcriptional program facilitating leukemia development. This program is abrogated by I-BET therapy and by nuclear restoration of NPM1. Finally, we demonstrate the efficacy of I-BET151 in a unique murine model and in primary patient samples of NPM1c AML. Taken together, our data support the use of BET inhibitors in clinical trials in AML.

  9. Establishment of xenotransplantation model of human CN-AML with FLT3-ITD (mut) /NPM1 (-) in NOD/SCID mice.

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    Shang, Zhen; Wang, Jue; Wang, Di; Xiao, Min; Li, Tong-juan; Wang, Na; Huang, Liang; Zhou, Jian-feng

    2013-06-01

    Patients with FLT3-ITD (mut) /NPM1 (-) cytogenetically normal acute myeloid leukemia (CN-AML), as high-risk molecular group in CN-AML, are associated with a worse prognosis than other CN-AML patients. It is beneficial to generate xenotransplantation model of FLT3-ITD (mut) /NPM1 (-) CN-AML to better understand the pathogenesis and therapeutic strategies of such AML subtype. The purpose of present study was to establish the xenotransplantation model in NOD/SCID mice with FLT3-ITD (mut) /NPM1 (-) CN-AML primary cells. The FLT3-ITD (mut) /NPM1 (-) CN-AML primary cells from 3 of 7 cases were successfully transplanted into NOD/SCID mice, and human CD45 positive cells were detected in the peripheral blood, spleen and bone marrow of mice by using flow cytometry. Infiltration of human leukemia cells in various organs of mice was observed by using immunohistochemistry. Gene analysis confirmed sustained FLT3/ITD mutation without NPM1 mutation in mice. By performing serial transplantation, it was found that characteristics of the leukemia cells in secondary and tertiary generation models remained unchanged. Moreover, in vivo cytarabine administration could extend survival of NOD/SCID mice, which was consistent with clinical observation. In conclusion, we successfully established xenotransplantation model of human FLT3-ITD (mut) /NPM1 (-) CN-AML in NOD/SCID mice. The model was able to present primary disease and suitable to evaluate the curative effects of new drugs or therapy strategies.

  10. Determining the contribution of NPM1 heterozygosity to NPM-ALK-induced lymphomagenesis.

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    Mduff, Fiona K E; Hook, C Elizabeth; Tooze, Reuben M; Huntly, Brian J; Pandolfi, Pier Paolo; Turner, Suzanne D

    2011-09-01

    Heterozygous expression of Nucleophosmin (NPM1) predisposes to hematological malignancies in the mouse and cooperates with Myc in lymphomagenesis. NPM1 is therefore regarded as a haploinsufficient tumor suppressor. Heterozygous loss of NPM1 occurs as a result of the t(2;5), which generates the oncogenic fusion tyrosine kinase, NPM-anaplastic lymphoma kinase (ALK), a molecule underlying the pathogenesis of anaplastic large cell lymphoma (ALCL). Given the aforementioned role of NPM1 as a tumor suppressor, we hypothesized that NPM1 heterozygosity would cooperate with NPM-ALK in lymphomagenesis. In the event, we observed no difference in tumor latency, incidence or phenotype in NPM-ALK-transgenic mice heterozygous for NPM1 relative to transgenic mice expressing both NPM1 alleles. We propose that although the t(2;5) simultaneously reduces NPM1 allelic dosage and creates the NPM-ALK fusion protein, the two events do not cooperate in the pathogenesis of ALCL in our mouse model. These data indicate that a tumor-suppressive role for NPM1 may depend on cellular and/or genetic context.

  11. NPM1 deletion is associated with gross chromosomal rearrangements in leukemia.

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    Roberta La Starza

    Full Text Available BACKGROUND: NPM1 gene at chromosome 5q35 is involved in recurrent translocations in leukemia and lymphoma. It also undergoes mutations in 60% of adult acute myeloid leukemia (AML cases with normal karyotype. The incidence and significance of NPM1 deletion in human leukemia have not been elucidated. METHODOLOGY AND PRINCIPAL FINDINGS: Bone marrow samples from 145 patients with myelodysplastic syndromes (MDS and AML were included in this study. Cytogenetically 43 cases had isolated 5q-, 84 cases had 5q- plus other changes and 18 cases had complex karyotype without 5q deletion. FISH and direct sequencing investigated the NPM1 gene. NPM1 deletion was an uncommon event in the "5q- syndrome" but occurred in over 40% of cases with high risk MDS/AML with complex karyotypes and 5q loss. It originated from large 5q chromosome deletions. Simultaneous exon 12 mutations were never found. NPM1 gene status was related to the pattern of complex cytogenetic aberrations. NPM1 haploinsufficiency was significantly associated with monosomies (p<0.001 and gross chromosomal rearrangements, i.e., markers, rings, and double minutes (p<0.001, while NPM1 disomy was associated with structural changes (p=0.013. Interestingly, in complex karyotypes with 5q- TP53 deletion and/or mutations are not specifically associated with NPM1 deletion. CONCLUSIONS AND SIGNIFICANCE: NPM1/5q35 deletion is a consistent event in MDS/AML with a 5q-/-5 in complex karyotypes. NPM1 deletion and NPM1 exon 12 mutations appear to be mutually exclusive and are associated with two distinct cytogenetic subsets of MDS and AML.

  12. FLT3 and NPM1 mutations in Chinese patients with acute myeloid leukemia and normal cytogenetics.

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    Wang, Lei; Xu, Wei-lai; Meng, Hai-tao; Qian, Wen-bin; Mai, Wen-yuan; Tong, Hong-yan; Mao, Li-ping; Tong, Yin; Qian, Jie-jing; Lou, Yin-jun; Chen, Zhi-mei; Wang, Yun-gui; Jin, Jie

    2010-10-01

    Mutations of fms-like tyrosine kinase 3 (FLT3) and nucleophosmin (NPM1) exon 12 genes are the most common abnormalities in adult acute myeloid leukemia (AML) with normal cytogenetics. To assess the prognostic impact of the two gene mutations in Chinese AML patients, we used multiplex polymerase chain reaction (PCR) and capillary electrophoresis to screen 76 AML patients with normal cytogenetics for mutations in FLT3 internal tandem duplication (FLT3/ITD) and exon 12 of the NPM1 gene. FLT3/ITD mutation was detected in 15 (19.7%) of 76 subjects, and NPM1 mutation in 20 (26.3%) subjects. Seven (9.2%) cases were positive for both FLT3/ITD and NPM1 mutations. Significantly more FLT3/ITD aberration was detected in subjects with French-American-British (FAB) M1 (42.8%). NPM1 mutation was frequently detected in subjects with M5 (47.1%) and infrequently in subjects with M2 (11.1%). FLT3 and NPM1 mutations were significantly associated with a higher white blood cell count in peripheral blood and a lower CD34 antigen expression, but not age, sex, or platelet count. Statistical analysis revealed that the FLT3/ITD-positive group had a lower complete remission (CR) rate (53.3% vs. 83.6%). Survival analysis showed that the FLT3/ITD-positive/NPM1 mutation-negative group had worse overall survival (OS) and relapse-free survival (RFS). The FLT3/ITD-positive/NPM1 mutation-positive group showed a trend towards favorable survival compared with the FLT3/ITD-positive/NPM1 mutation-negative group (P=0.069). Our results indicate that the FLT3/ITD mutation might be a prognostic factor for an unfavorable outcome in Chinese AML subjects with normal cytogenetics, while NPM1 mutation may be a favorable prognostic factor for OS and RFS in the presence of FLT3/ITD.

  13. Molecular basis of alcoholism.

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    Most, Dana; Ferguson, Laura; Harris, R Adron

    2014-01-01

    Acute alcohol intoxication causes cellular changes in the brain that last for hours, while chronic alcohol use induces widespread neuroadaptations in the nervous system that can last a lifetime. Chronic alcohol use and the progression into dependence involve the remodeling of synapses caused by changes in gene expression produced by alcohol. The progression of alcohol use, abuse, and dependence can be divided into stages, which include intoxication, withdrawal, and craving. Each stage is associated with specific changes in gene expression, cellular function, brain circuits, and ultimately behavior. What are the molecular mechanisms underlying the transition from recreational use (acute) to dependence (chronic)? What cellular adaptations result in drug memory retention, leading to the persistence of addictive behaviors, even after prolonged drug abstinence? Research into the neurobiology of alcoholism aims to answer these questions. This chapter will describe the molecular adaptations caused by alcohol use and dependence, and will outline key neurochemical participants in alcoholism at the molecular level, which are also potential targets for therapy.

  14. Targeting Chromatin Regulators Inhibits Leukemogenic Gene Expression in NPM1 Mutant Leukemia.

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    Kühn, Michael W M; Song, Evelyn; Feng, Zhaohui; Sinha, Amit; Chen, Chun-Wei; Deshpande, Aniruddha J; Cusan, Monica; Farnoud, Noushin; Mupo, Annalisa; Grove, Carolyn; Koche, Richard; Bradner, James E; de Stanchina, Elisa; Vassiliou, George S; Hoshii, Takayuki; Armstrong, Scott A

    2016-10-01

    Homeobox (HOX) proteins and the receptor tyrosine kinase FLT3 are frequently highly expressed and mutated in acute myeloid leukemia (AML). Aberrant HOX expression is found in nearly all AMLs that harbor a mutation in the Nucleophosmin (NPM1) gene, and FLT3 is concomitantly mutated in approximately 60% of these cases. Little is known about how mutant NPM1 (NPM1(mut)) cells maintain aberrant gene expression. Here, we demonstrate that the histone modifiers MLL1 and DOT1L control HOX and FLT3 expression and differentiation in NPM1(mut) AML. Using a CRISPR/Cas9 genome editing domain screen, we show NPM1(mut) AML to be exceptionally dependent on the menin binding site in MLL1. Pharmacologic small-molecule inhibition of the menin-MLL1 protein interaction had profound antileukemic activity in human and murine models of NPM1(mut) AML. Combined pharmacologic inhibition of menin-MLL1 and DOT1L resulted in dramatic suppression of HOX and FLT3 expression, induction of differentiation, and superior activity against NPM1(mut) leukemia.

  15. Prognostic Impact of Nucleophosmin 1 (NPM1 Gene Mutations in Egyptian Acute Myeloid Leukemia Patients

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    Magda Zidan

    2013-06-01

    Full Text Available OBJECTIVE: Somatic mutations of the nucleophosmin gene (NPM1, which alter the subcellular localization of the product, are the most frequent mutations in patients with acute myeloid leukemia. The aim of the study was to assess the prevalence and prognostic impact of NPM1 gene mutations in adult AML patients. METHODS: Polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP were used to screen 55 AML patients for mutations of NPM1 gene. RESULTS: NPM1 mutations were found in 12 (21.8% of 55 patients, significantly associated with higher total leukocytie count, marrow blast percentage (p=0.03 and p=0.02, respectively, and M5 subtype (p<0.001. Patients with NPM1 mutations had significantly higher complete remission rates (p=0.003 and a trend to lower rates of mortality, relapse and refractory disease (p=0.28, p=0.45 and p=0.08, respectively. Survival analysis showed significantly longer disease-free survival (mean 18.635±1.229 versus 11.041±1.250 months, p=0.044 and overall survival (mean 19.810±1.624 versus 12.063±1.244 months, p=0.041 in patients with NPM1 mutations compared with those without. Multivariate analyses confirmed NPM1 mutation as a significant independent predictor for disease-free survival (HR=0.066, p=0.001 and overall survival (HR=0.125, p=0.002. CONCLUSION: NPM1 mutation is a prognostic factor for a favorable outcome in Egyptian population. This finding is of major clinical importance since it strongly suggests that NPM1 mutations may allow one to divide the heterogeneous patient group of AML into prognostically different subgroups.

  16. NPM1 mutations in therapy-related acute myeloid leukemia with uncharacteristic features

    DEFF Research Database (Denmark)

    Andersen, Morten Tolstrup; Andersen, Mette Klarskov; Christiansen, D.H.

    2008-01-01

    Frameshift mutations of the nucleophosmin gene (NPM1) were recently reported as a frequently occurring abnormality in patients with de novo acute myeloid leukemia (AML). To evaluate the frequency of NPM1 mutations in patients with therapy-related myelodysplasia (t-MDS) and therapy-related AML (t......-/-7, the most frequent abnormalities of t-MDS/t-AML, were not observed (P=0.002). This raises the question whether some of the cases presenting NPM1 mutations were in fact cases of de novo leukemia. The close association to class I mutations and the inverse association to class II mutations suggest...

  17. Structural polymorphism in the N-terminal oligomerization domain of NPM1.

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    Mitrea, Diana M; Grace, Christy R; Buljan, Marija; Yun, Mi-Kyung; Pytel, Nicholas J; Satumba, John; Nourse, Amanda; Park, Cheon-Gil; Madan Babu, M; White, Stephen W; Kriwacki, Richard W

    2014-03-25

    Nucleophosmin (NPM1) is a multifunctional phospho-protein with critical roles in ribosome biogenesis, tumor suppression, and nucleolar stress response. Here we show that the N-terminal oligomerization domain of NPM1 (Npm-N) exhibits structural polymorphism by populating conformational states ranging from a highly ordered, folded pentamer to a highly disordered monomer. The monomer-pentamer equilibrium is modulated by posttranslational modification and protein binding. Phosphorylation drives the equilibrium in favor of monomeric forms, and this effect can be reversed by Npm-N binding to its interaction partners. We have identified a short, arginine-rich linear motif in NPM1 binding partners that mediates Npm-N oligomerization. We propose that the diverse functional repertoire associated with NPM1 is controlled through a regulated unfolding mechanism signaled through posttranslational modifications and intermolecular interactions.

  18. FLT3 and NPM-1 mutations in a cohort of acute promyelocytic leukemia patients from India

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    Suchitra Swaminathan

    2014-01-01

    Results: Frequency of FLT3/internal tandem duplication and FLT3/tyrosine kinase domain was found to be 25% and 7% respectively. We observed a high frequency of NPM1 mutation (45% in the present population of APL patients.

  19. Down-regulated expression of NPM1 in IMS-M2 cell line by (-)-epigallocatechin-3-gallate

    Institute of Scientific and Technical Information of China (English)

    Hoang Thanh Chi; Bui Thi Kim Ly; Hoang Anh Vu; Yuko Sato; Phu Chi Dung; Phan Thi Xinh

    2014-01-01

    Objective: To investigate the inhibited effect of epigallocatechin-3-gallate (EGCG) on the expression of NPM1 in IMS-M2 cells harboring the NPM1 mutations.Methods:Cell proliferation assay was performed to test the effects of EGCG on cell growth of IMS-M2 cells harboring the NPM1 mutations. Western blot analysis were performed to test the protein expression of NPM1, AKT, those associated with apoptosis. Results: EGCG can down-regulate the expression of NPM1 in IMS-M2 cells harboring the NPM1 mutations. Moreover, EGCG also suppressed the cell proliferation and induced apoptosis in IMS-M2 cells.Conclusions:The results suggested that EGCG could be considered as a reagent for treatment of AML patients with NPM1 mutations.

  20. Phosphorylation of multifunctional nucleolar protein nucleophosmin (NPM1) by aurora kinase B is critical for mitotic progression.

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    Shandilya, Jayasha; Senapati, Parijat; Dhanasekaran, Karthigeyan; Bangalore, Suma S; Kumar, Manoj; Kishore, A Hari; Bhat, Akshay; Kodaganur, Gopinath S; Kundu, Tapas K

    2014-06-27

    The functional association of NPM1 with Aurora kinases is well documented. Surprisingly, although NPM1 is a well characterized phosphoprotein, it is unknown whether it is a substrate of Aurora kinases. We have found that Aurora kinases A and B can phosphorylate NPM1 at a single serine residue, Ser125, in vitro and in vivo. Phosphorylated-S125-NPM1 (pS125-NPM1) localizes to the midbody region during late cytokinesis where it colocalizes with Aurora B. The overexpression of mutant (S125A) NPM1 resulted in the deregulation of centrosome duplication and mitotic defects possibly due to cytokinesis failure. These data suggest that Aurora kinase B-mediated phosphorylation of NPM1 plays a critical role during mitosis, which could have wider implications in oncogenesis.

  1. The frequency of NPM1 mutations in childhood acute myeloid leukemia

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    Karamolegou Kalliopi

    2010-10-01

    Full Text Available Abstract Background Mutations in the nucleophosmin (NPM1 gene have been solely associated with childhood acute myeloid leukemia (AML. We evaluated the frequency of NPM1 mutations in childhood AML, their relation to clinical and cytogenetic features and the presence of common FLT3 and RAS mutations. Results NPM1 mutations were found in 8% of cases. They involved the typical type 'A' mutation and one novel mutation characterized by two individual base pair substitutions, which resulted in 2 amino acid changes (W290 and (S293 in the NPM protein. FLT3/ITD mutations were observed in 12% of the cases and in one NPM1-mutated case bearing also t(8;21 (q22;q22. No common RAS mutations were identified. Conclusions A relatively consistent NPM1 mutation rate was observed, but with variations in types of mutations. The role of different types of NPM1 mutations, either individually or in the presence of other common gene mutations may be essential for childhood AML prognosis.

  2. New Quantitative Method to Identify NPM1 Mutations in Acute Myeloid Leukaemia

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    Sarah Huet

    2013-01-01

    Full Text Available Somatic mutations in the NPM1 gene, which encodes for nucleophosmin, have been reported to be the most frequent genetic abnormalities found in acute myeloid leukaemia (AML. Their identification and quantification remain crucial for the patients’ residual disease monitoring. We investigated a new method that could represent a novel reliable alternative to sequencing for its identification. This method was based on high-resolution melting analysis in order to detect mutated patients and on an allele-specific oligonucleotide real-time quantitative polymerase chain reaction (ASO-RQ-PCR for the identification and quantification of the transcripts carrying NPM1 mutations (NPM1m. Few patients carrying known NPM1m enabled us to set up a table with the different primers’ ΔCT values, identifying a profile for each mutation type. We then analysed a series of 337 AML patients' samples for NPM1 mutational status characterization and confirmed the ASO-RQ-PCR results by direct sequencing. We identified some mutations in 86 samples, and the results were fully correlated in 100% of the 36 sequenced samples. We also detected other rare NPM1m in two samples, that we confirmed by direct sequencing. This highly specific method provides a novel quick, useful, and costless tool, easy to use in routine practice.

  3. 急性髓系白血病患者骨髓涂片FMS样酪氨酸激酶3、NPM1和c-kit基因检测及临床研究%Detection and clinical research of FMS-like tyrosine kinase-3, NPM1 and c-kit genes in bone marrow slides of patients with acute myeloid leukemia

    Institute of Scientific and Technical Information of China (English)

    潘莹; 龚五星; 梁翠微; 杜均祥; 彭东旭; 谢云; 郑礼平; 张楠; 黄思超

    2016-01-01

    Objective To study the FMS-like tyrosine kinase-3 (FLT3) gene, NPM1 gene and c-kit gene mutations in acute myeloid leukemia (AML) by extracting DNA from the storage of bone marrow slides, and to investigate the relationship between the three gene mutations and clinical features in AML. Methods The bone marrow slides of 55 patients diagnosed with AML were enrolled in this study. The PCR, DNA sequencing and molecular cloning were used to detect and analyse the FLT3-ITD, NPM1 and c-kit gene mutations. Patients' remission, progression and survival time were also recorded. Results The DNA was successfully extracted from the bone marrow slides with -20 ℃ frozen storage without Wright stained, chemically fixed, and room temperature storage Wright stained discoloured by phenol ∶ chloroform ∶ isoamyl alcohol method, which can be used in PCR, direct sequencing and molecular cloning sequencing analysis. 10 of the 55 cases (18.2 %) were FLT3-ITD positive, including 9 cases with heterozygous mutations and 1 case with homozygous mutation. FLT3-ITD positive group had lower complete remission (CR) rate, shorter event-free survival (EFS) time and overall survival (OS) time than the negative group (P< 0.05). 9 of the 55 cases (16.4 %) had NPM1 heterozygous gene mutations, all belonging to type A. The EFS rate of the patients with NPM1 mutation was higher in 10 months and the OS rate was higher in 19 months (P< 0.05). 3 of 9 NPM1 mutations patients were FLT3-ITD positive. The CR rates of the four groups after initial remission induction therapy in order were NPM1+FLT3-ITD-, NPM1-FLT3-ITD-, NPM1-FLT3-ITD+, NPM1+FLT3-ITD+(P<0.05). Besides, NPM1-FLT3-ITD+was a risk factor affecting the OS (RR=1.250, P=0.005). 2 of the 55 cases (3.6 %) had c-kit gene mutations, namely mutant D816H and mutant D816V. The c-kit gene mutations were not found in patients with FLT3-ITD and NPM1 mutations. Conclusions The FLT3-ITD mutation is a poor prognosis molecular marker in AML, and NPM1 mutation is

  4. Mutation of the NPM1 gene contributes to the development of donor cell-derived acute myeloid leukemia after unrelated cord blood transplantation for acute lymphoblastic leukemia.

    Science.gov (United States)

    Rodríguez-Macías, Gabriela; Martínez-Laperche, Carolina; Gayoso, Jorge; Noriega, Víctor; Serrano, David; Balsalobre, Pascual; Muñoz-Martínez, Cristina; Díez-Martín, José L; Buño, Ismael

    2013-08-01

    Donor cell leukemia (DCL) is a rare but severe complication after allogeneic stem cell transplantation. Its true incidence is unknown because of a lack of correct recognition and reporting, although improvements in molecular analysis of donor-host chimerism are contributing to a better diagnosis of this complication. The mechanisms of leukemogenesis are unclear, and multiple factors can contribute to the development of DCL. In recent years, cord blood has emerged as an alternative source of hematopoietic progenitor cells, and at least 12 cases of DCL have been reported after unrelated cord blood transplantation. We report a new case of DCL after unrelated cord blood transplantation in a 44-year-old woman diagnosed as having acute lymphoblastic leukemia with t(1;19) that developed acute myeloid leukemia with normal karyotype and nucleophosmin (NPM1) mutation in donor cells. To our knowledge, this is the first report of NPM1 mutation contributing to DCL development.

  5. A knock-in Npm1 mutation in mice results in myeloproliferation and implies a perturbation in hematopoietic microenvironment.

    Directory of Open Access Journals (Sweden)

    Shiu-Huey Chou

    Full Text Available Somatic Nucleophosmin (NPM1 mutation frequently occurs in acute myeloid leukemia (AML, but its role in leukemogenesis remains unclear. This study reports the first "conventional" knock-in mouse model of Npm1 mutation, which was achieved by inserting TCTG after nucleotide c.857 (c.854_857dupTCTG to mimic human mutation without any "humanized" sequence. The resultant mutant peptide differed slightly different from that in humans but exhibited cytoplasmic pulling force. Homozygous (Npm1(c+/c+ mice showed embryonic lethality before day E8.5, wheras heterozygous (Npm1(wt/c+ mice appeared healthy at birth and were fertile. Approximately 36% of Npm1(wt/c+ mice developed myeloproliferative disease (MPD with extramedullary hematopoiesis. Those Npm1(wt/c+ mice that did not develop MPD nevertheless gradually developed monocytosis and showed increased numbers of marrow myeloid precursors. This second group of Npm1(wt/c+ mice also showed compromised cobblestone area formation, suggesting pathology in the hematopoietic niche. Microarray experiments and bioinformatic analysis on mice myeloid precursor cells and 227 human samples revealed the expression of CXCR4/CXCL12-related genes was significantly suppressed in mutant cells from both mice and humans. Thus, our mouse model demonstrated that Npm1 mutation can result in MPD, but is insufficient for leukemogenesis. Perturbation of hematopoietic niche in mutant hematopoietic stem cells (implied by underrepresentation of CXCR4/CXCL12-related genes may be important in the pathogenesis of NPM1 mutations.

  6. NPM1 Gene Type A Mutation in Bulgarian Adults with Acute Myeloid Leukemia: A Single-Institution Study

    Directory of Open Access Journals (Sweden)

    Gueorgui Balatzenko

    2014-03-01

    Full Text Available OBJECTIVE: Mutations of the nucleophosmin (NPM1 gene are considered as the most frequent acute myeloid leukemia (AML- associated genetic lesion, reported with various incidences in different studies, and type A (NPM1-A is the most frequent type. However, since most series in the literature report on the features of all patients regardless of the type of mutation, NPM1-A(+ cases have not been well characterized yet. Therefore, we evaluated the prevalence of NPM1-A in Bulgarian AML patients and searched for an association with clinical and laboratory features. METHODS: One hundred and four adults (51 men, 53 women were included in the study. NPM1-A status was determined using allele-specific reverse-transcription polymerase chain reaction with co-amplification of NPM1-A and β-actin and real-time quantitative TaqMan-based polymerase chain reaction. Patients received conventional induction chemotherapy and were followed for 13.2±16.4 months. RESULTS: NPM1-A was detected in 26 (24.8% patients. NPM1-A mutation was detected in all AML categories, including in one patient with RUNX1-RUNX1T1. There were no differences associated with the NPM1-A status with respect to age, sex, hemoglobin, platelet counts, percentage of bone marrow blasts, splenomegaly, complete remission rates, and overall survival. NPM1-A(+ patients, compared to NPM1-A(- patients, were characterized by higher leukocyte counts [(75.4±81.9x109/L vs. (42.5±65.9x109/L; p=0.049], higher frequency of normal karyotype [14/18 (77.8% vs. 26/62 (41.9%; p=0.014], higher frequency of FLT3-ITD [11/26 (42.3% vs. 8/77 (10.4%; p=0.001], and lower incidence of CD34(+ [6/21 (28.8% vs. 28/45 (62.2%; p=0.017]. Within the FLT3-ITD(- group, the median overall survival of NPM1-A(- patients was 14 months, while NPM1-A(+ patients did not reach the median (p=0.10. CONCLUSION: The prevalence of NPM1-A mutation in adult Bulgarian AML patients was similar to that reported in other studies. NPM1-A(+ patients

  7. NPM1 Gene Type A Mutation in Bulgarian Adults with Acute Myeloid Leukemia: A Single-Institution Study

    Science.gov (United States)

    Balatzenko, Gueorgui; Spassov, Branimir; Stoyanov, Nikolay; Ganeva, Penka; Dikov, Tihomit; Konstantinov, Spiro; Hrischev, Vasil; Romanova, Malina; Toshkov, Stavri; Guenova, Margarita

    2014-01-01

    Objective: Mutations of the nucleophosmin (NPM1) gene are considered as the most frequent acute myeloid leukemia (AML)-associated genetic lesion, reported with various incidences in different studies, and type A (NPM1-A) is the most frequent type. However, since most series in the literature report on the features of all patients regardless of the type of mutation, NPM1-A(+) cases have not been well characterized yet. Therefore, we evaluated the prevalence of NPM1-A in Bulgarian AML patients and searched for an association with clinical and laboratory features. Materials and Methods: One hundred and four adults (51 men, 53 women) were included in the study. NPM1-A status was determined using allele-specific reverse-transcription polymerase chain reaction with co-amplification of NPM1-A and β-actin and real-time quantitative TaqMan-based polymerase chain reaction. Patients received conventional induction chemotherapy and were followed for 13.2±16.4 months. Results: NPM1-A was detected in 26 (24.8%) patients. NPM1-A mutation was detected in all AML categories, including in one patient with RUNX1-RUNX1T1. There were no differences associated with the NPM1-A status with respect to age, sex, hemoglobin, platelet counts, percentage of bone marrow blasts, splenomegaly, complete remission rates, and overall survival. NPM1-A(+) patients, compared to NPM1-A(-) patients, were characterized by higher leukocyte counts [(75.4±81.9)x109/L vs. (42.5±65.9)x109/L; p=0.049], higher frequency of normal karyotype [14/18 (77.8%) vs. 26/62 (41.9%); p=0.014], higher frequency of FLT3-ITD [11/26 (42.3%) vs. 8/77 (10.4%); p=0.001], and lower incidence of CD34(+) [6/21 (28.8%) vs. 28/45 (62.2%); p=0.017]. Within the FLT3-ITD(-) group, the median overall survival of NPM1-A(-) patients was 14 months, while NPM1-A(+) patients did not reach the median (p=0.10). Conclusion: The prevalence of NPM1-A mutation in adult Bulgarian AML patients was similar to that reported in other studies. NPM1-A

  8. NPM1基因沉默的HL-60及其耐药细胞株的建立%Establishment of Nucleophosmin Gene Silenced HL-60 and Its Resistant Cell Line

    Institute of Scientific and Technical Information of China (English)

    林敏辉; 胡建达

    2011-01-01

    本研究旨在构建针对HL-60细胞及其耐药细胞株(HL-60/ADR)的NPM1-RNAi的细胞模型,为研究NPM1基因在白血病耐药过程中的作用奠定实验基础.通过针对NPM1基因的shRNA与线性化的pGCSIL-GFP 载体进行连接转化,对获得的重组子(pGCSIL-GFP-NPM1-shRNA)进行PCR和测序鉴定.采用慢病毒载体系统将pHelper 1.0载体、pHelper 2.0载体与pGCSIL-GFP-NPM1-shRNA共转染293T细胞,包装成NPM1 -RNAi-LV,将慢病毒载体感染HL-60及HL-60/ADR细胞.采用实时定量RT-PCR和Western blot法分别从mRNA和蛋白水平验证建立的细胞株的转染效率.结果表明,成功构建了重组真核表达载体pGCSIL-GFP-NPM1-shRNA,并通过慢病毒载体系统包装成NPM-1-RNAi-LV;将NPM1-RNAi-LV转染至HL-60及HL-60/ADR细胞后,从mRNA水平上看,对细胞的NPM1 mRNA表达有显著抑制,达到90%以上(p<0.05);从蛋白水平上看,对细胞的NPM蛋白表达具有显著的抑制效果,表明转染后的HL-60及HL-60/ ADR细胞的NPM1基因特异性沉默.NPM1基因沉默后,HL-60/ADR对阿霉素的耐药性有一定程度的下降.结论:成功构建了NPM1-RNAi的HL-60及HL-60/ADR细胞模型,为进一步研究NPM1基因在白血病耐药过程中的作用建立了良好的实验基础.%This study was aimed to construct model cell line of NPMl-RNAi in HL-60 cells and its resistant line (HL-60/ADR) so as to provide a experimental basis for investigating the potential role of NPM1 gene in leukemia drug resistance. The shRNA targeting to NPM1 was ligated into linear pGCSIL-GFP vector, and transformed into E. Coli DH5a. Positive clone was identified by PCR and DNA sequencing. pHelper 1.0, pHelper 2. 0 and pGCSIL-GFP-NPMl-shRNA were cotransformed into 293T cells by lentivius vector system. NPMl-RNAi-LV was transfected into HL-60 and HL-60/ADR cell lines. The efficiency of NPM-RNAi-LV was detected by using real-time quantitative RT-PCR and Western blot. The results showed that the recombinant eukaryotic

  9. Molecular basis for mitochondrial signaling

    CERN Document Server

    2017-01-01

    This book covers recent advances in the study of structure, function, and regulation of metabolite, protein and ion translocating channels, and transporters in mitochondria. A wide array of cutting-edge methods are covered, ranging from electrophysiology and cell biology to bioinformatics, as well as structural, systems, and computational biology. At last, the molecular identity of two important channels in the mitochondrial inner membrane, the mitochondrial calcium uniporter and the mitochondrial permeability transition pore have been established. After years of work on the physiology and structure of VDAC channels in the mitochondrial outer membrane, there have been multiple discoveries on VDAC permeation and regulation by cytosolic proteins. Recent breakthroughs in structural studies of the mitochondrial cholesterol translocator reveal a set of novel unexpected features and provide essential clues for defining therapeutic strategies. Molecular Basis for Mitochondrial Signaling covers these and many more re...

  10. TET2 gene mutation is unfavorable prognostic factor in cytogenetically normal acute myeloid leukemia patients with NPM1+ and FLT3-ITD - mutations.

    Science.gov (United States)

    Tian, Xiaopeng; Xu, Yang; Yin, Jia; Tian, Hong; Chen, Suning; Wu, Depei; Sun, Aining

    2014-07-01

    Cytogenetically normal acute myeloid leukemia (cn-AML) is a group of heterogeneous diseases. Gene mutations are increasingly used to assess the prognosis of cn-AML patients and guide risk-adapted treatment. In the present study, we analyzed the molecular genetics characteristics of 373 adult cn-AML patients and explored the relationship between TET2 gene mutations or different genetic mutation patterns and prognosis. We found that 16.1 % of patients had TET2 mutations, 31.6 % had FLT3 internal tandem duplications (ITDs), 6.2 % had FLT3 tyrosine kinase domain mutations, 2.4 % had c-KIT mutations, 37.8 % had NPM1 mutations, 11.3 % had WT1 mutations, 5.9 % had RUNX1 mutations, 11.5 % had ASXL1 mutations, 3.8 % had MLL-PTDs, 7.8 % had IDH1 mutations, 7.8 % had NRAS mutations, 12.3 % had IDH2 mutations, 1.6 % had EZH2 mutations, and 14.7 % had DNMT3A mutations, while none had CBL mutations. Gene mutations were detected in 76.94 % (287/373) of all patients. In the NPM1m(+) patients, those with TET2 mutations were associated with a shorter median overall survival (OS) as compared to TET2 wild-type (wt) patients (9.9 vs. 27.0 months, respectively; P = 0.023); Interestingly, the TET2 mutation was identified as an unfavorable prognostic factor and was closely associated with a shorter median OS as compared to TET2-wt (9.5 vs. 32.2 months, respectively; P = 0.013) in the NPM1m(+)/FLT3-ITDm(-) patient group. Thus, identification of TET2 combined with classic NPM1 and FLT3-ITD mutations allowed us to stratify cn-AML into distinct subtypes.

  11. Favorable prognostic impact of NPM1 gene mutations in childhood acute myeloid leukemia, with emphasis on cytogenetically normal AML.

    NARCIS (Netherlands)

    Hollink, I.H.; Zwaan, C.M.; Zimmermann, M.; Arentsen-Peters, T.C.; Pieters, R.; Cloos, J.; Kaspers, G.J.L.; Graaf, S.S.N. de; Harbott, J.; Creutzig, U.; Reinhardt, D.; Heuvel-Eibrink, M.M. van den; Thiede, C.

    2009-01-01

    Nucleophosmin (NPM1) mutations occur frequently in adult cytogenetically normal acute myeloid leukemia (CN-AML) and confer favorable outcome. We investigated the frequency and prognostic significance of NPM1 mutations in childhood AML (n=298), specifically focusing on the CN-AML subgroup (n=100). Mu

  12. Molecular Basis of Cardiac Myxomas

    Directory of Open Access Journals (Sweden)

    Pooja Singhal

    2014-01-01

    Full Text Available Cardiac tumors are rare, and of these, primary cardiac tumors are even rarer. Metastatic cardiac tumors are about 100 times more common than the primary tumors. About 90% of primary cardiac tumors are benign, and of these the most common are cardiac myxomas. Approximately 12% of primary cardiac tumors are completely asymptomatic while others present with one or more signs and symptoms of the classical triad of hemodynamic changes due to intracardiac obstruction, embolism and nonspecific constitutional symptoms. Echocardiography is highly sensitive and specific in detecting cardiac tumors. Other helpful investigations are chest X-rays, magnetic resonance imaging and computerized tomography scan. Surgical excision is the treatment of choice for primary cardiac tumors and is usually associated with a good prognosis. This review article will focus on the general features of benign cardiac tumors with an emphasis on cardiac myxomas and their molecular basis.

  13. Importância da detecção das mutações no gene FLT3 e no gene NPM1 na leucemia mieloide aguda - Classificação da Organização Mundial de Saúde 2008 Importance of detecting FLT3 and NPM1 gene mutations in acute myeloid leukemia -World Health Organization Classification 2008

    Directory of Open Access Journals (Sweden)

    Marley Aparecida Licínio

    2010-01-01

    Full Text Available As leucemias mieloides agudas (LMA constituem um grupo de neoplasias malignas caracterizadas pela proliferação descontrolada de células hematopoéticas, decorrente de mutações que podem ocorrer em diferentes fases da diferenciação de células precursoras mieloides. Em 2008, a Organização Mundial da Saúde (OMS-2008 publicou uma nova classificação para neoplasias do sistema hematopoético e linfoide. De acordo com essa classificação, para um diagnóstico mais preciso e estratificação de prognóstico de pacientes com leucemias mieloides agudas, devem-se pesquisar mutações nos genes FLT3 e NPM1. Sabe-se que a presença de mutações no gene FLT3 é de prognóstico desfavorável e que as mutações no gene NPM1 do tipo A são de prognóstico favorável. Assim, nos países desenvolvidos, a análise das mutações no gene FLT3 e NPM1 tem sido considerada como um fator de prognóstico importante na decisão terapêutica em pacientes com diagnóstico de leucemias mieloides agudas. Considerando essas informações, é de extrema importância a análise das mutações no gene FLT3 (duplicação interna em tandem - DIT - e mutação pontual D835 e no gene NPM1 como marcadores moleculares para o diagnóstico, o prognóstico e a monitoração de doença residual mínima em pacientes com leucemias mieloides agudas.Acute myeloid leukemia (AML is a group of malignancies characterized by uncontrolled proliferation of hematopoietic cells resulting from mutations that occur at different stages in the differentiation of myeloid precursor cells. In 2008, the World Health Organization (WHO-2008 published a new classification for cancers of the hematopoietic and lymphoid system. According to this classification, FLT3 and NPM1 gene mutations should be investigated for a more precise diagnosis and prognostic stratification of AML patients. It is well known that the presence of FLT3 gene mutations is considered an unfavorable prognostic factor and type

  14. The Molecular Basis of Evolution.

    Science.gov (United States)

    Wilson, Allan C.

    1985-01-01

    Discovery that mutations accumulate at steady rates over time in the genes of all lineages of plants and animals has led to new insights into evolution at the molecular and organismal levels. Discusses molecular evolution, examining deoxyribonuclei acid (DNA) sequences, morphological distances, and codon rate of change. (DH)

  15. Incidence and significance of FLT3-ITD and NPM1 mutations in patients with normal karyotype acute myeloid leukaemia.

    LENUS (Irish Health Repository)

    Haslam, K

    2012-02-01

    BACKGROUND: Acute myeloid leukaemia (AML) is a heterogeneous clonal disorder of haematopoietic progenitor cells. Approximately half of all adult AML patients have a normal karyotype (NK-AML) and an intermediate risk prognosis. AIMS: To determine the incidence and prognostic significance of NPM1 and FLT3-ITD mutations in a population of patients with NK-AML. METHODS: FLT3-ITD and NPM1 mutation status was retrospectively sought in presentation samples from 44 NK-AML patients. RESULTS: FLT3-ITD and NPM1 mutations were detected in 45.5 and 54.5% of patients, respectively, allowing stratification according to genotype. CONCLUSIONS: FLT3-ITD and NPM1 mutation status can be defined in NK-AML. Prospective screening for these mutations is advocated in all NK-AML patients, as the genotype is of clinical importance when considering treatment options including stem cell transplantation.

  16. Molecular basis of mechanosensory transduction

    Science.gov (United States)

    Gillespie, Peter G.; Walker, Richard G.

    2001-09-01

    Mechanotransduction - a cell's conversion of a mechanical stimulus into an electrical signal - reveals vital features of an organism's environment. From hair cells and skin mechanoreceptors in vertebrates, to bristle receptors in flies and touch receptors in worms, mechanically sensitive cells are essential in the life of an organism. The scarcity of these cells and the uniqueness of their transduction mechanisms have conspired to slow molecular characterization of the ensembles that carry out mechanotransduction. But recent progress in both invertebrates and vertebrates is beginning to reveal the identities of proteins essential for transduction.

  17. Molecular Basis for Mucolytic Therapy

    Directory of Open Access Journals (Sweden)

    Bonnie Dasgupta

    1995-01-01

    Full Text Available Airway mucus is a complex, viscoelastic gel that has a three-dimensional structure. It is composed of water, mucous glycoproteins, low molecular weight ions, proteins and lipids. The three-dimensional structure of the mucous gel depends on a number of forms of bonding, such as ionic bonds and disulphide bridges. Airway obstruction in cystic fibrosis (CF lung disease is accompanied by the accumulation of thick and viscous secretions resulting from chronic infection and inflammation, promoting recurrent exacerbations. The normal, free-flow ing airway mucus becomes thick and purulent in patients suffering from CF lung disease. Therefore, current approaches to the treatment of CF include strategics for changing the physical properties of pulmonary secretions with the goal of improving airway clearance. Some of the same strategies may be applicable in the larger group of patients with chronic obstructive airway diseases, including bronchiectasis and chronic obstructive pulmonary disease. This paper reviews various approaches to mucolysis based on the molecular nature of crosslinking and bonding in mucin gels. A brief review of the structure and biochemistry of airway mucus is followed by a discussion of the various physical and biochemical approaches to mucolysis. Seven representative mucotropic modalities are presented: N-acetylcysteine; urea; hypertonic saline; recombinant human DNase; gelsolin; oscillation; and surfactants. Each of these mucotropic modalities acts on a different component within the mucous gel. Finally, the possibilities of mucolytic synergism among these various agents are conside red.

  18. The molecular basis of memory.

    Science.gov (United States)

    Marx, Gerard; Gilon, Chaim

    2012-08-15

    We propose a tripartite biochemical mechanism for memory. Three physiologic components are involved, namely, the neuron (individual and circuit), the surrounding neural extracellular matrix, and the various trace metals distributed within the matrix. The binding of a metal cation affects a corresponding nanostructure (shrinking, twisting, expansion) and dielectric sensibility of the chelating node (address) within the matrix lattice, sensed by the neuron. The neural extracellular matrix serves as an electro-elastic lattice, wherein neurons manipulate multiple trace metals (n > 10) to encode, store, and decode coginive information. The proposed mechanism explains brains low energy requirements and high rates of storage capacity described in multiples of Avogadro number (N(A) = 6 × 10(23)). Supportive evidence correlates memory loss to trace metal toxicity or deficiency, or breakdown in the delivery/transport of metals to the matrix, or its degradation. Inherited diseases revolving around dysfunctional trace metal metabolism and memory dysfunction, include Alzheimer's disease (Al, Zn, Fe), Wilson's disease (Cu), thalassemia (Fe), and autism (metallothionein). The tripartite mechanism points to the electro-elastic interactions of neurons with trace metals distributed within the neural extracellular matrix, as the molecular underpinning of "synaptic plasticity" affecting short-term memory, long-term memory, and forgetting.

  19. Somatic mosaic mutations of IDH1 and NPM1 associated with cup-like acute myeloid leukemia in a patient with Maffucci syndrome.

    Science.gov (United States)

    Akiyama, Masaharu; Yamaoka, Masayoshi; Mikami-Terao, Yoko; Ohyama, Wataru; Yokoi, Kentaro; Arakawa, Yasuhiro; Takita, Junko; Suzuki, Hideaki; Yamada, Hisashi

    2015-12-01

    Maffucci syndrome is a nonhereditary congenital disorder characterized by multiple enchondromas and with soft-tissue hemangiomas. Somatic mutations of the isocitrate dehydrogenase (IDH) gene have been detected in enchondroma and hemangioma tissue from patients with Maffucci syndrome. The rate of malignant transformation in Maffucci syndrome is high, with enchondromas transforming into chondrosarcomas and the development of secondary neoplasms, including pancreatic and hepatic adenocarcinoma, mesenchymal ovarian tumors, and brain tumors such as glioma. However, hematopoietic malignancies arising in Maffucci syndrome are rare. We report a 7-year-old girl with Maffucci syndrome in whom acute myeloid leukemia (AML) with cup-like nuclear invagination developed. Both leukemic cells and hemangioma had the same gene mutations: an insertion frameshift c.863_864insTCTG (p.W288 fs) in the nucleophosmin (NPM1) gene and a missense mutation c.392_395GTCG > CTCT (p.G131_R132 > AL) in the IDH1 gene. However, buccal mucosa cells and peripheral blood mononuclear cells harvested after two cycles of chemotherapy showed wild-type genotypes. These results suggest that the multiple somatic mutations of the IDH1 and NPM1 genes in hemangioblasts are related to the development of cup-like AML associated with Maffucci syndrome. However, further studies are needed to identify additional molecular events in AML but not in hemangioma.

  20. NPM1 mutation is a stable marker for minimal residual disease monitoring in acute myeloid leukaemia patients with increased sensitivity compared to WT1 expression*

    DEFF Research Database (Denmark)

    Kristensen, Thomas; Møller, Michael B; Friis, Lone;

    2011-01-01

    Mutation in the NPM1 gene occurs in 60% of acute myeloid leukaemia (AML) patients with normal karyotype. NPM1 mutation is potentially a superior minimal residual disease (MRD) marker compared to WT1 gene overexpression by being specific to the malignant clone, although experimental evidence...... published so far includes very limited numbers of relapsed cases. Also, the stability of the NPM1 mutation has been questioned by reports of the mutation being lost at relapse. In the present study we compared NPM1 mutation and WT1 overexpression as MRD markers in 20 cases of relapsed AML. The 20 patients...... experienced a total of 28 morphological relapses. Karyotypic evolution was detected in 56% of relapses. All relapses were accompanied by high levels of NPM1 mutation, along with high WT1 mRNA levels, thus demonstrating complete stability of both markers during relapse. Detectable NPM1 mutation following...

  1. 急性髓系白血病NPM1基因突变检测的临床意义%Detection of NPM1 gene mutation in acute myeloid leukemia

    Institute of Scientific and Technical Information of China (English)

    马亮; 钟明华; 丰岱荣; 龙红; 沈军; 马一盖; 黄尚志

    2012-01-01

    目的 探讨初诊急性髓细胞白血病(acute myeloid leukemia,AML)患者NPM1基因突变发生率及其与染色体核型和FAB亚型之间的关系,并分析NPM1基因的突变类型.方法 选取2004至2010年中日友好医院血液科99例初诊AML患者.采集患者骨髓标本,采用聚合酶链反应(polymerase chain reaction,PCR)扩增基因组DNA,并采用变性聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis,PAGE)和毛细管电泳两种方法对AML患者NPM1基因突变进行检测.应用G显带方法对其中72例初诊AML患者进行细胞遗传学分析,同时对10例NPM1突变阳性患者进行直接测序分析.NPM1插入突变在各亚型患者中发生率的比较采用x2检验.变性PAGE和毛细管电泳两种方法检测NPM1基因突变发生率的比较采用McNemar检验.结果 毛细管电泳法与变性PAGE法检测AML患者NPM1基因插入突变发生率分别为15% (15/99)和11% (11/99),差异无统计学意义(x2 =2.25,P>0.05).NPM1插入突变在各亚型患者中发生率分别为:急性粒细胞白血病部分分化型(M2)(27%,8/30)、急性单核细胞白血病(M5)(32%,6/19)、红白血病(M6)(13%,1/8),差异无统计学意义(x2=1.06,P>0.05),其余亚型未检测到NPM1插入突变.49例AML异常核型患者的NPM1插入突变发生率为4% (2/49),23例正常核型患者的NPM1插入突变发生率为26% (6/23),差异有统计学意义(x2=5.61,P<0.05).10例NPM1基因插入突变均为A型突变(c.860_863 dupTCTG).突变导致NPM蛋白羧基末端读码框移,末尾7个氨基酸WQWRKSL被11个氨基酸CLAVEEVSLRK所代替.2例患者检测到内含子缺失突变,分别为IVS10-18_-15delCTTT和IVS10-17-15delTTT.结论 NPM1插入突变为AML患者常见基因改变,正常核型患者插入突变发生率高于异常核型患者.在NPM1基因内含子区发现2例缺失突变.%Objective To analyze the frequency of NPM1 mutation in de novo acute myeloid leukemia (AML) patients and the relationship

  2. The Molecular Basis of β-Thalassemia

    Science.gov (United States)

    Thein, Swee Lay

    2013-01-01

    The β-thalassemias are characterized by a quantitative deficiency of β-globin chains underlaid by a striking heterogeneity of molecular defects. Although most of the molecular lesions involve the structural β gene directly, some down-regulate the gene through distal cis effects, and rare trans-acting mutations have also been identified. Most β-thalassemias are inherited in a Mendelian recessive fashion but there is a subgroup of β-thalassemia alleles that behave as dominant negatives. Unraveling the molecular basis of β-thalassemia has provided a paradigm for understanding of much of human genetics. PMID:23637309

  3. The molecular basis of peanut allergy

    Science.gov (United States)

    Peanut allergens can trigger a potent and sometimes dangerous immune response in an increasing number of people. The molecular structures of these allergens form the basis for understanding this response. This review describes the currently known peanut allergen structures, and discusses how modif...

  4. Nucleophosmin1胞浆表达的结肠癌组织中不存在其第12外显子突变%Non-presence of mutations in exon 12 of nucleophosmin1 (NPM1) gene in colorectal carcinomas with NPM1 expression in cytoplasm

    Institute of Scientific and Technical Information of China (English)

    杨宇峰; 张锡迎; 杨梅; 谢树瑞; 张宝庭

    2014-01-01

    Objective To investigate whether there is a presence of mutations in exon 12 of nucleophosmin1 (NPM1) gene in colorectal carcinomas (CRC). Methods We investigated NPM expression by immunohistochemis-try in histologically normal to malignant colorectal tissues and also investigate whether exon 12 of the NPM1 gene is mutated in CRC with NPM expression located in cytoplasm. Results Among the 115 samples of colorectal carcino-mas, 92.1% (106/115) of the tumors showed NPM1-positive expression only in the nucleus, 7.0% (8/115) showed NPM1-positive expression in cytoplasm, and 0.9%(1/115) showed NPM1-negative expression in cytoplasm and nucle-us. NPM expression was found significantly up-regulated in CRC compared to adjacent colorectal tissue, villous ade-noma, tubular adenoma and normal colorectal tissues, respectively (P<0.05 for all). Sequencing of exon 12 of NPM1 gene in CRC was the same as those in normal colorectal tissue. Conclusion NPM expression is found significantly up-regulated in CRC, suggesting that NPM contributes to oncogenesis of CRC. However, NPM1 exon 12 is not mutat-ed in CRC, which suggests that NPM1 play a role in the tumorigenesis of CRC through unclear mechanisms rather than NPM1 mutation.%目的:观察Nucleophosmin1(NPM1)在结肠癌组织中是否存在NPM1基因第12外显子突变。方法免疫组化检测115例结肠癌组织及相应癌旁组织、10例结肠绒毛状腺瘤、10例管状腺瘤和10例正常结肠黏膜组织NPM1表达;检查NPM1胞浆表达的结肠癌组织中是否存在NPM1基因第12外显子突变。结果在115例结肠癌样本中有92.1%(106/115)的病例NPM1表达定位于胞核,7.0%(8/115)的病例存在胞浆NPM1表达,0.9%(1/115)的病例胞核与胞浆NPM1阴性表达;NPM1在结肠癌组织中呈高表达,并且明显高于在相应癌旁组织、绒毛状腺瘤、管状腺瘤和正常结肠黏膜组织的表达(P均<0.05)。结肠癌组织中NPM1第12外显子结构与正常黏膜野生型NPM

  5. Effect of NPM1 Mutations on the Invasion Potential of Leukemic Cells in vitro%NPM1基因突变对急性白血病细胞系体外侵袭能力的影响

    Institute of Scientific and Technical Information of China (English)

    邵会媛; 苗宗玉; 覃凤娴; 陈先春; 谭诗; 高玉洁; 张伶

    2010-01-01

    核仁磷酸蛋白基因(nucleophosmin,NPM1)突变是目前急性髓系白血病发生突变率最高的基因改变,与白血病的发生发展密切相关.为探讨NPM1突变对白血病细胞体外侵袭能力的影响,将载体pEGFPC1-NPM1-mA转染THP-1白血病细胞系,筛选稳定表达NPM A型突变蛋白(NPM1-mA)的白血病细胞株(THP-1-mA).通过transwell迁移实验、Matrigel侵袭实验以及细胞粘附实验来观察THP-1-mA细胞体外浸润转移能力的改变.结果发现,THP-1-mA细胞的体外迁移能力和侵袭能力明显高于亲代THP-1细胞;此外,THP-1-mA细胞对纤维连接蛋白的粘附能力也显著高于THP-1细胞.因此,我们的研究结果提示,NPM1突变可增强白血病细胞的体外侵袭能力,这有利于进一步明确NPMJ突变基因在白血病细胞恶性转化中的调控作用.

  6. A one-mutation mathematical model can explain the age incidence of acute myeloid leukemia with mutated nucleophosmin (NPM1)

    NARCIS (Netherlands)

    A. Liso (Arcangelo); F. Castiglione (Filippo); A. Cappuccio (Antonio); F. Stracci (Fabrizio); R.F. Schlenk (Richard); S. Amadori (Sergio); C. Thiede (Christian); S. Schnittger (Susanne); P.J.M. Valk (Peter); K. Döhner (Konstanze); M.F. Martelli (Massimo F.); M. Schaich (Markus); J. Krauter; A. Ganser (Arnold); N. Bolli (Niccolò); B. Löwenberg (Bob); T. Haferlach (Torsten); G. Ehninger (Gerhard); F. Mandelli (Franco); F. Michor (Franziska); B. Falini

    2008-01-01

    textabstractAcute myeloid leukemia with mutated NPM1 gene and aberrant cytoplasmic expression of nucleophosmin (NPMc+acute myeloid leukemia) shows distinctive biological and clinical features. Experimental evidence of the oncogenic potential of the nucleophosmin mutant is, however, still lacking, an

  7. Study on NPM1 gene mutations in patients with primary myelodysplastic syndromes%原发性骨髓增生异常综合征患者NPM1基因突变的研究

    Institute of Scientific and Technical Information of China (English)

    李璘; 张悦; 马晓瑭; 杨琳; 徐泽锋; 肖志坚

    2010-01-01

    目的 探讨原发性骨髓增生异常综合征(MDS)患者NPM1基因突变情况及其与MDS患者临床特征的关系.方法 对232例原发性MDS患者采用基因组DNA聚合酶链反应(PCR)扩增NPM1基因第12外显子,直接测序检测突变状态,克隆后测序鉴定突变类型.比较NPM1突变患者与野生型患者的临床及实验室特征.结果 232例患者中NPM1突变9例(3.9%),均为A型突变.NPM1突变患者中性粒细胞绝对值(ANC)较低[突变型和野生型患者分别为0.60(0.12~2.91)×109/L和1.02(0~10.23)×109/L,P=0.046],骨髓原始细胞比例较高[突变型和野生型患者分别为0.050(0~0.090)和0.025(0~0.190),P=0.035],干/祖细胞培养后红系爆式集落(BFU-E)数量减低[突变型和野生型患者105个骨髓单个核细胞中集落数分别为0(0~0)和6(0~40),P=0.038],血清维生素(Vit)B12水平较高[突变型和野生型患者分别为936.40(373.80~2400.00)pmol/L和557.85(17.00~3032.10)pmol/L,P=0.045].NPM1突变患者以正常核型为主.结论 NPM1基因突变的MDS患者具有一些独特的临床和实验室特征,此为进一步研究NPM1基因突变与原发性MDS发生及转变为白血病的关系提供了重要线索.%Objective To investigate NPM1 gene mutations in patients with primary myelodysplastic syndromes (MDS) and the clinical characteristics of patients with NPM1 mutants. Methods Genomic DNA Corresponding to exon 12 of NPM1 gene was amplified by polymerase chain reaction(PCR) in 232 patients with primary MDS. Identification of mutants was by direct sequencing and classification of mutation types by sequencing followed by plasmid cloning. Results NPM1 mutants were found in 9 patients(3.9% ) . All the mutants were type A. As compared with those with NPM1 wild type, patients with the mutant were of lower ANC [0.60 (0. 12 - 2.91 ) × 109/L vs 1.02 (0 - 10. 23 ) × 109/L, P = 0.046], higher blast percent in bone marrow [0.050(0 -0. 090) vs 0. 025(0 -0. 190) ,P =0. 035], decreased

  8. Simultaneous detection of mutations and copy number variation of NPM1 in the acute myeloid leukemia using multiplex ligation-dependent probe amplification

    Energy Technology Data Exchange (ETDEWEB)

    Marcinkowska-Swojak, Malgorzata, E-mail: m-marcinkowska@o2.pl [European Center of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan (Poland); Handschuh, Luiza, E-mail: luizahan@ibch.poznan.pl [European Center of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan (Poland); Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Szamarzewskiego 82/84, 60-569 Poznan (Poland); Wojciechowski, Pawel, E-mail: Pawel.Wojciechowski@cs.put.poznan.pl [European Center of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan (Poland); Institute of Computing Science, Poznan University of Technology, Piotrowo 2, 60-965 Poznan (Poland); Goralski, Michal, E-mail: mgoralsk@ibch.poznan.pl [European Center of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan (Poland); Tomaszewski, Kamil, E-mail: kamil.tomaszewsky@gmail.com [European Center of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan (Poland); Kazmierczak, Maciej, E-mail: maciej.kazmierczak@onet.eu [Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Szamarzewskiego 82/84, 60-569 Poznan (Poland); Lewandowski, Krzysztof, E-mail: krzysztof.lewandowski@skpp.edu.pl [Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Szamarzewskiego 82/84, 60-569 Poznan (Poland); Komarnicki, Mieczyslaw, E-mail: mak7@pro.onet.pl [Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Szamarzewskiego 82/84, 60-569 Poznan (Poland); and others

    2016-04-15

    Highlights: • The NPM1 mutations were detected exclusively in AML accounting for 25% of cases. • The NPM1 gene did not reveal any copy number alterations. • The NPM1mut+ assay is a reliable test for the analysis of mutations and CNA in NPM1. - Abstract: The NPM1 gene encodes nucleophosmin, a protein involved in multiple cell functions and carcinogenesis. Mutation of the NPM1 gene, causing delocalization of the protein, is the most frequent genetic lesion in acute myeloid leukemia (AML); it is considered a founder event in AML pathogenesis and serves as a favorable prognostic marker. Moreover, in solid tumors and some leukemia cell lines, overexpression of the NPM1 gene is commonly observed. Therefore, the purpose of this study was to develop a new method for the detection of NPM1 mutations and the simultaneous analysis of copy number alterations (CNAs), which may underlie NPM1 gene expression deregulation. To address both of the issues, we applied a strategy based on multiplex ligation-dependent probe amplification (MLPA). A designed NPM1mut+ assay enables the detection of three of the most frequent NPM1 mutations: A, B and D. The accuracy of the assay was tested using a group of 83 samples from Polish patients with AML and other blood-proliferative disorders. To verify the results, we employed traditional Sanger sequencing and next-generation transcriptome sequencing. With the use of the NPM1mut+ assay, we detected mutations A, D and B in 14, 1 and 0 of the analyzed samples, respectively. All of these mutations were confirmed by complementary sequencing approaches, proving the 100% specificity and sensitivity of the proposed test. The performed sequencing analysis allowed the identification of two additional rare mutations (I and ZE). All of the mutations were identified exclusively in AML cases, accounting for 25% of those cases. We did not observe any CNAs (amplifications) of the NPM1 gene in the studied samples, either with or without the mutation. The

  9. Double CEBPA mutations are prognostically favorable in non-M3 acute myeloid leukemia patients with wild-type NPM1 and FLT3-ITD.

    Science.gov (United States)

    Wen, Xiang-Mei; Lin, Jiang; Yang, Jing; Yao, Dong-Ming; Deng, Zhao-Qun; Tang, Chun-Yan; Xiao, Gao-Fei; Yang, Lei; Ma, Ji-Chun; Hu, Jia-Bo; Qian, Wei; Qian, Jun

    2014-01-01

    This study is aimed to investigate the pattern of CEBPA mutations and its clinical significance in Chinese non-M3 acute myeloid leukemia (AML) patients. The entire coding region of CEBPA gene was amplified by PCR and then sequenced in samples from 233 non-M3 AML patients. Fifty mutations were identified in 37 (15.8%) patients with eleven (4.7%) double mutated CEBPA (dmCEBPA) and twenty-six (11.1%) single mutated CEBPA (smCEBPA). dmCEBPA was exclusively observed in M1 and M2 subtypes of FAB classification (P = 0.008), whereas smCEBPA occurred in almost all subtypes (P = 0.401). Patients with dmCEBPA had significantly younger age and higher WBC counts than those with wtCEBPA (P = 0.016 and 0.043, respectively). Both dmCEBPA and smCEBPA were mainly present in cytogenetically normal patients. Patients with dmCEBPA achieved higher rate of complete (CR) than wtCEBPA patients (88% vs. 51%, P = 0.037), whereas smCEBPA and wtCEBPA groups are similar (47% vs. 51%, P = 0.810). Patients with dmCEBPA had a superior overall survival (OS) compared with patients with wtCEBPA (P = 0.033), whereas patients with smCEBPA had a similar OS as patients with wtCEBPA (P = 0.976). dmCEBPA but not smCEBPA was also associated with favorable outcome in patients with wild-type NPM1 and FLT3-ITD (NPM1(wt)FLT3-ITD(wt) ). Our data confirm that dmCEBPA but not smCEBPA is prognostically favorable in NPM1(wt)FLT3-ITD(wt) AML, and suggest that the entity AML with mutated CEBPA should be definitely designated as AML with dmCEBPA in WHO classification and smCEBPA should be excluded from the favorable risk of molecular abnormalities.

  10. Simultaneous detection of mutations and copy number variation of NPM1 in the acute myeloid leukemia using multiplex ligation-dependent probe amplification.

    Science.gov (United States)

    Marcinkowska-Swojak, Malgorzata; Handschuh, Luiza; Wojciechowski, Pawel; Goralski, Michal; Tomaszewski, Kamil; Kazmierczak, Maciej; Lewandowski, Krzysztof; Komarnicki, Mieczyslaw; Blazewicz, Jacek; Figlerowicz, Marek; Kozlowski, Piotr

    2016-04-01

    The NPM1 gene encodes nucleophosmin, a protein involved in multiple cell functions and carcinogenesis. Mutation of the NPM1 gene, causing delocalization of the protein, is the most frequent genetic lesion in acute myeloid leukemia (AML); it is considered a founder event in AML pathogenesis and serves as a favorable prognostic marker. Moreover, in solid tumors and some leukemia cell lines, overexpression of the NPM1 gene is commonly observed. Therefore, the purpose of this study was to develop a new method for the detection of NPM1 mutations and the simultaneous analysis of copy number alterations (CNAs), which may underlie NPM1 gene expression deregulation. To address both of the issues, we applied a strategy based on multiplex ligation-dependent probe amplification (MLPA). A designed NPM1mut+ assay enables the detection of three of the most frequent NPM1 mutations: A, B and D. The accuracy of the assay was tested using a group of 83 samples from Polish patients with AML and other blood-proliferative disorders. To verify the results, we employed traditional Sanger sequencing and next-generation transcriptome sequencing. With the use of the NPM1mut+ assay, we detected mutations A, D and B in 14, 1 and 0 of the analyzed samples, respectively. All of these mutations were confirmed by complementary sequencing approaches, proving the 100% specificity and sensitivity of the proposed test. The performed sequencing analysis allowed the identification of two additional rare mutations (I and ZE). All of the mutations were identified exclusively in AML cases, accounting for 25% of those cases. We did not observe any CNAs (amplifications) of the NPM1 gene in the studied samples, either with or without the mutation. The presented method is simple, reliable and cost-effective. It can be easily introduced into clinical practice or developed to target both less-frequent mutations in the NPM1 gene and other cancer-related genes.

  11. The Molecular Basis of Human Brain Evolution.

    Science.gov (United States)

    Enard, Wolfgang

    2016-10-24

    Humans are a remarkable species, especially because of the remarkable properties of their brain. Since the split from the chimpanzee lineage, the human brain has increased three-fold in size and has acquired abilities for vocal learning, language and intense cooperation. To better understand the molecular basis of these changes is of great biological and biomedical interest. However, all the about 16 million fixed genetic changes that occurred during human evolution are fully correlated with all molecular, cellular, anatomical and behavioral changes that occurred during this time. Hence, as humans and chimpanzees cannot be crossed or genetically manipulated, no direct evidence for linking particular genetic and molecular changes to human brain evolution can be obtained. Here, I sketch a framework how indirect evidence can be obtained and review findings related to the molecular basis of human cognition, vocal learning and brain size. In particular, I discuss how a comprehensive comparative approach, leveraging cellular systems and genomic technologies, could inform the evolution of our brain in the future. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Molecular basis of chloroplast photorelocation movement.

    Science.gov (United States)

    Kong, Sam-Geun; Wada, Masamitsu

    2016-03-01

    Chloroplast photorelocation movement is an essential physiological response for sessile plant survival and the optimization of photosynthetic ability. Simple but effective experiments on the physiological, cell biological and molecular genetic aspects have been widely used to investigate the signaling components of chloroplast photorelocation movement in Arabidopsis for the past few decades. Although recent knowledge on chloroplast photorelocation movement has led us to a deeper understanding of its physiological and molecular basis, the biochemical roles of the downstream factors remain largely unknown. In this review, we briefly summarize recent advances regarding chloroplast photorelocation movement and propose that a new high-resolution approach is necessary to investigate the molecular mechanism underlying actin-based chloroplast photorelocation movement.

  13. Multilineage dysplasia as assessed by immunophenotype has no impact on clinical-biological features and outcome of NPM1-mutated acute myeloid leukemia.

    Science.gov (United States)

    Mannelli, Francesco; Ponziani, Vanessa; Bonetti, Maria Ida; Bencini, Sara; Cutini, Ilaria; Gianfaldoni, Giacomo; Scappini, Barbara; Pancani, Fabiana; Rondelli, Tommaso; Benelli, Matteo; Caporale, Roberto; Grazia Gelli, Anna Maria; Peruzzi, Benedetta; Longo, Giovanni; Bosi, Alberto

    2015-10-01

    The presence of multilineage dysplasia (MLD) by morphology at diagnosis in acute myeloid leukemia (AML) defines a separate subset in the World Health Organization classification with still-debated prognostic value. A major controversy concerns MLD's role in NPM1-mutated (NPM1⁺) AML, which correlates with good prognosis. We used flow cytometry (FC), an emerging technique for assessing dysplasia, to investigate MLD in NPM1⁺ AML by an immunophenotypic score (IPS), a technique previously adopted in myelodysplastic syndrome. Eighty-five intensively treated NPM1⁺ AML cases were studied. Patients were grouped according to the combination of data in maturing cell compartments. FC-assessed dysplasia showed a significant correlation with morphology-assessed dysplasia, showing the efficacy of this method in highlighting dysplasia in AML. Except for MLD, IPS did not influence any patient- or disease-related characteristics at diagnosis. Furthermore, IPS did not influence complete remission rate, disease-free survival, or overall survival. By investigating NPM1 status on separated cell compartments, we established a correlation between FC-assessed MLD and belonging to AML clone. This study shows that dysplasia evaluated by immunophenotype has no impact on clinical-biological characteristics or on outcome of NPM1⁺ AML. Dysplasia is part of the spectrum of NPM1⁺ AML, and the prognostic stratification of this category of patients should not be based upon it.

  14. Basis set independent calculation of molecular polarizabilities

    Science.gov (United States)

    Talman, James D.

    2012-08-01

    It is shown that e-F, where F is the Hartree-Fock (HF) operator, can be inverted, for molecular systems, in numerical Cartesian coordinates. The method was originally applied to finding corrections to approximate Hartree-Fock orbitals [J. D. Talman, Phys. Rev. APLRAAN1050-294710.1103/PhysRevA.82.052518 82, 052518 (2010)]. The approach is applied to determine basis set independent dipole polarizabilities for the water molecule using the Sternheimer method within the uncoupled HF and coupled perturbed HF approximations.

  15. Molecular Basis for Saccharomyces cerevisiae Biofilm Development

    DEFF Research Database (Denmark)

    Andersen, Kaj Scherz

    of translation of FLO11. In conclusion, I have conducted the first global study of the genetic program for yeast biofilm formation on polystyrene. This work provide several target genes as good basis for further research of biofilm, that I believe can contribute to fields such as cell biology, genetics, system......In this study, I sought to identify genes regulating the global molecular program for development of sessile multicellular communities, also known as biofilm, of the eukaryotic microorganism, Saccharomyces cerevisiae (yeast). Yeast biofilm has a clinical interest, as biofilms can cause chronic......, but only a small subset is previously described as regulators of FLO11. These results reveal that the regulation of biofilm formation and FLO11 is even more complex than what has previously been described. I find that the molecular program for biofilm formation shares many essential components with two...

  16. Molecular basis of ancestral vertebrate electroreception.

    Science.gov (United States)

    Bellono, Nicholas W; Leitch, Duncan B; Julius, David

    2017-03-16

    Elasmobranch fishes, including sharks, rays, and skates, use specialized electrosensory organs called ampullae of Lorenzini to detect extremely small changes in environmental electric fields. Electrosensory cells within these ampullae can discriminate and respond to minute changes in environmental voltage gradients through an unknown mechanism. Here we show that the voltage-gated calcium channel CaV1.3 and the big conductance calcium-activated potassium (BK) channel are preferentially expressed by electrosensory cells in little skate (Leucoraja erinacea) and functionally couple to mediate electrosensory cell membrane voltage oscillations, which are important for the detection of specific, weak electrical signals. Both channels exhibit unique properties compared with their mammalian orthologues that support electrosensory functions: structural adaptations in CaV1.3 mediate a low-voltage threshold for activation, and alterations in BK support specifically tuned voltage oscillations. These findings reveal a molecular basis of electroreception and demonstrate how discrete evolutionary changes in ion channel structure facilitate sensory adaptation.

  17. Molecular Basis of Laminin-Integrin Interactions.

    Science.gov (United States)

    Yamada, Masashi; Sekiguchi, Kiyotoshi

    2015-01-01

    Laminins are composed of three polypeptide chains, designated as α, β, and γ. The C-terminal region of laminin heterotrimers, containing coiled-coil regions, short tails, and laminin globular (LG) domains, is necessary and sufficient for binding to integrins, which are the major laminin receptor class. Laminin recognition by integrins critically requires the α chain LG domains and a glutamic acid residue of the γ chain at the third position from the C-terminus. Furthermore, the C-terminal region of the β chain contains a short amino acid sequence that modulates laminin affinity for integrins. Thus, all three of the laminin chains act cooperatively to facilitate integrin binding. Mammals possess 5 α (α1-5), 3 β (β1-3), and 3 γ (γ1-3) chains, combinations of which give rise to 16 distinct laminin isoforms. Each isoform is expressed in a tissue-specific and developmental stage-specific manner, exerting its functions through binding of integrins. In this review, we detail the current knowledge surrounding the molecular basis and physiological relevance of specific interactions between laminins and integrins, and describe the mechanisms underlying laminin action through integrins.

  18. The molecular basis of lactose intolerance.

    Science.gov (United States)

    Campbell, Anthony K; Waud, Jonathan P; Matthews, Stephanie B

    2009-01-01

    A staggering 4000 million people cannot digest lactose, the sugar in milk, properly. All mammals, apart from white Northern Europeans and few tribes in Africa and Asia, lose most of their lactase, the enzyme that cleaves lactose into galactose and glucose, after weaning. Lactose intolerance causes gut and a range of systemic symptoms, though the threshold to lactose varies considerably between ethnic groups and individuals within a group. The molecular basis of inherited hypolactasia has yet to be identified, though two polymorphisms in the introns of a helicase upstream from the lactase gene correlate closely with hypolactasia, and thus lactose intolerance. The symptoms of lactose intolerance are caused by gases and toxins produced by anaerobic bacteria in the large intestine. Bacterial toxins may play a key role in several other diseases, such as diabetes, rheumatoid arthritis, multiple sclerosis and some cancers. The problem of lactose intolerance has been exacerbated because of the addition of products containing lactose to various foods and drinks without being on the label. Lactose intolerance fits exactly the illness that Charles Darwin suffered from for over 40 years, and yet was never diagnosed. Darwin missed something else--the key to our own evolution--the Rubicon some 300 million years ago that produced lactose and lactase in sufficient amounts to be susceptible to natural selection.

  19. Mutation Analysis of JAK2V617F, FLT3-ITD, NPM1, and DNMT3A in Chinese Patients with Myeloproliferative Neoplasms

    Directory of Open Access Journals (Sweden)

    Min Wang

    2014-01-01

    Full Text Available Since the discovery of JAK2V617F tyrosine kinase-activating mutation, several genes have been found mutated in myeloproliferative neoplasms (MPNs. FLT3-ITD, NPM1, and DNMT3A mutations frequently occurred in AML patients and have been found conferred with myeloproliferative neoplasms in mouse model. Therefore, we sought to search for mutations in JAK2V617F, FLT3-ITD, NPM1, and DNMT3A in 129 cases including 120 classic MPN cases and 9 MDS/MPN cases. JAK2V617F mutation was found in 60% of the 120 classic MPNs. However, none of the patients displayed FLT3-ITD and NPM1 mutations; only 2 patients harbored DNMT3A R882 mutation. Further studies including whole-genome sequence will be conducted to investigate the possible involvement of these genes in MPN.

  20. Dynamic conformations of nucleophosmin (NPM1 at a key monomer-monomer interface affect oligomer stability and interactions with granzyme B.

    Directory of Open Access Journals (Sweden)

    Wei D Duan-Porter

    Full Text Available Nucleophosmin (NPM1 is an abundant, nucleolar tumor antigen with important roles in cell proliferation and putative contributions to oncogenesis. Wild-type NPM1 forms pentameric oligomers through interactions at the amino-terminal core domain. A truncated form of NPM1 found in some hepatocellular carcinoma tissue formed an unusually stable oligomer and showed increased susceptibility to cleavage by granzyme B. Initiation of translation at the seventh methionine generated a protein (M7-NPM that shared all these properties. We used deuterium exchange mass spectrometry (DXMS to perform a detailed structural analysis of wild-type NPM1 and M7-NPM, and found dynamic conformational shifts or local "unfolding" at a specific monomer-monomer interface which included the β-hairpin "latch." We tested the importance of interactions at the β-hairpin "latch" by replacing a conserved tyrosine in the middle of the β-hairpin loop with glutamic acid, generating Y67E-NPM. Y67E-NPM did not form stable oligomers and further, prevented wild-type NPM1 oligomerization in a dominant-negative fashion, supporting the critical role of the β-hairpin "latch" in monomer-monomer interactions. Also, we show preferential cleavage by granzyme B at one of two available aspartates (either D161 or D122 in M7-NPM and Y67E-NPM, whereas wild-type NPM1 was cleaved at both sites. Thus, we observed a correlation between the propensity to form oligomers and granzyme B cleavage site selection in nucleophosmin proteins, suggesting that a small change at an important monomer-monomer interface can affect conformational shifts and impact protein-protein interactions.

  1. The molecular basis of fertilization (Review)

    Science.gov (United States)

    Georgadaki, Katerina; Khoury, Nikolas; Spandidos, Demetrios A.; Zoumpourlis, Vasilis

    2016-01-01

    Fertilization is the fusion of the male and female gamete. The process involves the fusion of an oocyte with a sperm, creating a single diploid cell, the zygote, from which a new individual organism will develop. The elucidation of the molecular mechanisms of fertilization has fascinated researchers for many years. In this review, we focus on this intriguing process at the molecular level. Several molecules have been identified to play a key role in each step of this intriguing process (the sperm attraction from the oocyte, the sperm maturation, the sperm and oocyte fusion and the two gamete pronuclei fusion leading to the zygote). Understanding the molecular mechanisms of the cell-cell interactions will provide a better understanding of the causes of fertility issues due to fertilization defects. PMID:27599669

  2. Cellular and Molecular Basis of Cerebellar Development

    Directory of Open Access Journals (Sweden)

    Salvador eMartinez

    2013-06-01

    Full Text Available Historically, the molecular and cellular mechanisms of cerebellar development were investigated through structural descriptions and studying spontaneous mutations in animal models and humans. Advances in experimental embryology, genetic engineering and neuroimaging techniques render today the possibility to approach the analysis of molecular mechanisms underlying histogenesis and morphogenesis of the cerebellum by experimental designs. Several genes and molecules were identified to be involved in the cerebellar plate regionalization, specification and differentiation of cerebellar neurons, as well as the establishment of cellular migratory routes and the subsequent neuronal connectivity. Indeed, pattern formation of the cerebellum requires the adequate orchestration of both key morphogenetic signals, arising from distinct brain regions, and local expression of specific transcription factors. Thus, the present review wants to revisit and discuss these morphogenetic and molecular mechanisms taking place during cerebellar development in order to understand causal processes regulating cerebellar cytoarchitecture, its highly topographically ordered circuitry and its role in brain function.

  3. The molecular basis of aminoacylase 1 deficiency

    DEFF Research Database (Denmark)

    Sommer, Anke; Christensen, Ernst; Schwenger, Susanne

    2011-01-01

    deficiency have not been characterized so far. This has prompted us to approach expression studies of all mutations known to occur in aminoacylase 1 deficient individuals in a human cell line (HEK293), thus providing the authentic human machinery for posttranslational modifications. Mutations were inserted...... by their intramolecular localization and molecular characteristics. In contrast to aminoacylase 1 variants which showed no detectable aminoacylase 1 activity, aminoacylase 1 proteins with the mutations p.Arg378Trp, p.Arg378Gln and p.Arg393His were also detected in Western blot analysis. Investigations of the molecular...

  4. The molecular basis of eucaryotic transcription.

    Science.gov (United States)

    Kornberg, R D

    2007-12-01

    Thanks to the Nobel Foundation for permission to publish this Lecture. We report here the Nobel Lecture delivered by Professor RD Kornberg describing his research in the understanding of transcription in eucaryotes. The amazing work by Professor Kornberg goes from the discovery of the nucleosome to the structural and functional studies of pol II transcription complexes. His research sheds light on fundamental molecular biology problems such as transcription initiation, fidelity of transcription, RNA release at the end of transcription, and many more. This is a beautiful report on how structural and functional studies can be combined to really understand in an accurate and detailed way how proteins combine in huge molecular complexes to regulate one of the most important cellular processes: gene transcription.

  5. Molecular Basis for Saccharomyces cerevisiae Biofilm Development

    DEFF Research Database (Denmark)

    Andersen, Kaj Scherz

    In this study, I sought to identify genes regulating the global molecular program for development of sessile multicellular communities, also known as biofilm, of the eukaryotic microorganism, Saccharomyces cerevisiae (yeast). Yeast biofilm has a clinical interest, as biofilms can cause chronic...... infections in humans. Biofilm is also interesting from an evolutionary standpoint, as an example of primitive multicellularity. By using a genome-wide screen of yeast deletion mutants, I show that 71 genes are essential for biofilm formation. Two-thirds of these genes are required for transcription of FLO11......, but only a small subset is previously described as regulators of FLO11. These results reveal that the regulation of biofilm formation and FLO11 is even more complex than what has previously been described. I find that the molecular program for biofilm formation shares many essential components with two...

  6. Molecular basis of cleft palates in mice

    Institute of Scientific and Technical Information of China (English)

    Noriko; Funato; Masataka; Nakamura; Hiromi; Yanagisawa

    2015-01-01

    Cleft palate, including complete or incomplete cleft palates, soft palate clefts, and submucosal cleft palates, is the most frequent congenital craniofacial anomaly in humans. Multifactorial conditions, including genetic and environmental factors, induce the formation of cleft palates. The process of palatogenesis is temporospatially regulated by transcription factors, growth factors, extracellular matrix proteins, and membranous molecules; a single ablation of these molecules can result in a cleft palate in vivo. Studies on knockout mice were reviewed in order to identify genetic errors that lead to cleft palates. In this review, we systematically describe these mutant mice and discuss the molecular mechanisms of palatogenesis.

  7. Molecular basis of cardiotoxicity upon cobra envenomation.

    Science.gov (United States)

    Cher, C D N; Armugam, A; Zhu, Y Z; Jeyaseelan, K

    2005-01-01

    Various clinical manifestations leading to death have been documented in most cases of bites caused by venomous snakes. Cobra envenomation is an extremely variable process and known to cause profound neurological abnormalities. The complexity of cobra venom can induce multiple-organ failure, leading to death in case of severe envenomation. Intramuscular administration of Malayan spitting cobra (Naja sputatrix) crude venom at 1 microg/g dose caused death in mice in approximately 3 h. Analysis of gene expression profiles in the heart, brain, kidney, liver and lung revealed 203 genes whose expression was altered by at least 3-fold in response to venom treatment. Of these, 50% were differentially expressed in the heart and included genes involved in inflammation, apoptosis, ion transport and energy metabolism. Electrocardiogram recordings and serum troponin T measurements indicated declining cardiac function and myocardial damage. This not only sheds light on the cardiotoxicity of cobra venom but also reveals the molecular networks affected during envenomation.

  8. Molecular basis of HFE-hemochromatosis

    Directory of Open Access Journals (Sweden)

    Maja eVujic Spasic

    2014-03-01

    Full Text Available Iron-overload disorders owing to genetic misregulation of iron acquisition are referred to as hereditary hemochromatosis (HH. The most prevalent genetic iron overload disorder in Caucasians is caused by mutations in the HFE gene, an atypical MHC class I molecule. Recent studies classified HFE/Hfe-hemochromatosis as a liver disease with the primarily failure in the production of the liver iron hormone hepcidin in hepatocytes. Inadequate hepcidin expression signals for excessive iron absorption from the diet and iron deposition in tissues causing multiple organ damage and failure. This review focuses on the molecular actions of the HFE/Hfe and hepcidin in maintaining systemic iron homeostasis and approaches undertaken so far to combat iron overload in HFE/Hfe-HH. In the light of the recent investigations, novel roles of extra-hepatocytic Hfe are discussed raising a question to the relevance of the multipurpose functions of Hfe for the understanding of HH associated pathologies.

  9. Molecular Basis of Microbial One-Carbon Metabolism

    Energy Technology Data Exchange (ETDEWEB)

    None

    2002-07-12

    The Gordon Research Conference (GRC) on Molecular Basis of Microbial One-Carbon Metabolism was held at Connecticut College, New London, Connecticut. Emphasis was placed on current unpublished research and discussion of the future target areas in this field.

  10. Molecular basis for hyperkalemic periodic paralysis.

    Science.gov (United States)

    Brown, R H

    probability of entering a non-inactivating mode was very low and independent of potassium. On the other hand, for the abnormal channel the probability of entering an inactive mode rises up to 5-fold with hyperkalemic. Four mutations have recently been detected in individuals with cold-sensitive paramyotenia congenital. Two of the cause amino acid substitutions within the III-IV intracytoplasmic loop. It is striking that one substitutes a valine for a glycine. An analysis of the molecular biology of each mutation should illuminate not only the disease phenotype but also biophysical properties of specific sub-regions of this muscle sodium channel.

  11. Social parasitism and the molecular basis of phenotypic evolution

    Directory of Open Access Journals (Sweden)

    Alessandro eCini

    2015-02-01

    Full Text Available Contrasting phenotypes arise from similar genomes through a combination of losses, gains, co-option and modifications of inherited genomic material. Understanding the molecular basis of this phenotypic diversity is a fundamental challenge in modern evolutionary biology. Comparisons of the genes and their expression patterns underlying traits in pairs of closely related species offer an unrivalled opportunity to evaluate the extent to which genomic material is reorganised to produce novel traits. Advances in molecular methods now allow us to dissect the molecular machinery underlying phenotypic diversity in almost any organism, from single-celled organisms to the most complex vertebrates. Here we discuss how comparisons of social parasites and their free-living hosts may provide unique insights into the molecular basis of phenotypic evolution. Social parasites evolve from a social ancestor and are specialised to exploit the socially acquired resources of their closely-related, free-living social host. Molecular comparisons of such species pairs can reveal how genomic material is re-organised in the loss of ancestral traits (i.e. of free-living traits in the parasites and the gain of new ones (i.e. specialist traits required for a parasitic lifestyle. We define hypotheses on the molecular basis of phenotypes in the evolution of social parasitism and discuss their wider application in understanding the molecular basis of phenotypic diversity within the theoretical framework of phenotypic plasticity and shifting reaction norms. Currently there are no data available to test these hypotheses, and so we also provide some proof of concept data for our conceptual model using the paper wasp social parasite-host system (Polistes sulcifer - Polistes dominula. This conceptual framework and first empirical data provide a spring-board for directing future genomic analyses on exploiting social parasites as a route to understanding the evolution of phenotypic

  12. Molecular Basis for Mycophenolic Acid Biosynthesis in Penicillium brevicompactum

    DEFF Research Database (Denmark)

    Regueira, Torsten Ulrik Bak; Kildegaard, Kanchana Rueksomtawin; Hansen, Bjarne Gram

    2011-01-01

    Mycophenolic acid (MPA) is the active ingredient in the increasingly important immunosuppressive pharmaceuticals CellCept (Roche) and Myfortic (Novartis). Despite the long history of MPA, the molecular basis for its biosynthesis has remained enigmatic. Here we report the discovery of a polyketide...

  13. Molecular Basis for Mycophenolic Acid Biosynthesis in Penicillium brevicompactum

    DEFF Research Database (Denmark)

    Regueira, Torsten Ulrik Bak; Kildegaard, Kanchana Rueksomtawin; Hansen, Bjarne Gram;

    2011-01-01

    Mycophenolic acid (MPA) is the active ingredient in the increasingly important immunosuppressive pharmaceuticals CellCept (Roche) and Myfortic (Novartis). Despite the long history of MPA, the molecular basis for its biosynthesis has remained enigmatic. Here we report the discovery of a polyketide...

  14. 干扰核仁磷酸蛋白对人白血病OCI/AML3细胞凋亡的影响及其机制%Effect of nucleophosmin(NPM) 1 knockdown on the apoptosis of OCI/AML3 leukemic cell with mutant NPM1 and its mechanisms

    Institute of Scientific and Technical Information of China (English)

    王娟; 陈莎娜; 全静; 贺金刚; 张帅帅; 鲜敬荣; 张伶

    2014-01-01

    目的:探讨抑制核仁磷酸蛋白(nucleophosmin,NPM)1基因对人白血病细胞凋亡的影响及相关机制.方法:将空载慢病毒pGIPZ和NPM干扰慢病毒shNPM分别感染携带NPM1突变的OCI/AML3白血病细胞株以及对照白血病细胞株HL60.采用qRT-PCR、Western blot和免疫细胞化学法检测白血病细胞的shNPM组以及Mock和pGIPZ组细胞及NPM1A型突变(NPM1-mA)基因和蛋白的表达,流式细胞仪和瑞氏染色法观察OCI/AML3的shNPM组以及Mock和pGIPZ组细胞凋亡情况,qRT-PCR和Western blot检测OCI/AML3的shNPM组以及Mock和pGIPZ组凋亡相关蛋白(Bax/Bcl-2)和p-ERK信号分子表达;流式细胞仪检测丝裂原活化蛋白激酶胞外信号调节激酶信号通路抑制剂(PD98059)处理后OCI/AML3的shNPM组以及Mock和pGIPZ组细胞凋亡率的改变.结果:干扰NPM1明显抑制OCI/AML3细胞株NPM1-mA的mRNA水平(F=20.078;P=0.000,PMock vs shNPM0.001,PpGIPz vs shNPM=0.003,PMock vs pGIPZ=0.333)和蛋白水平,伴有胞质NPM突变蛋白表达明显减弱;干扰NPM1能明显上调OCI/AML3细胞凋亡率(F=25.236,P=0.000,PMock vs shNPM=0.001,PpGIPz vs shNPM=0.001,PMock vs pGIPz=0.729),同时光镜下观察到干扰组细胞出现凋亡小体等凋亡形态学特征.此外,干扰NPM1可上调OCI/AML3细胞中促凋亡分子Bax的蛋白和mRNA表达(F=7.649,P=0.000;PMock vs shNPM=0.015,PpGIPZ vs shNPM=0.015,PMock vs PGIPz=0.984)、下调抗凋亡分子Bcl-2的蛋白和mRNA表达(F=5.190,P=0.000;PMock vs shNPM=0.034,PMock vs shNPM=0.029,PMock vs shNPM=0.909);干扰NPM1能明显下调p-ERK的表达,PD98059抑制剂阻断MAPK通路可促进OCI/AML3细胞凋亡(F=25.643,P=0.007).结论:NPM1突变基因在白血病细胞抗凋亡特性中发挥重要作用,潜在的分子机制可能与ERK信号分子及Bax/Bcl-2表达有关.

  15. NPM1 mutation regulates the proliferation and invasion of THP-1 cell in vitro and its mechanism%NPM1基因突变调控THP-1细胞体外增殖和侵袭及其机制

    Institute of Scientific and Technical Information of China (English)

    苗宗玉; 吴红; 邵会媛; 李倩; 邢艳艳; 张伶; 孙成铭

    2013-01-01

    Objective To explore the effect of NPM 1 mutations on proliferation and invasion of THP-1 cells in vitro and its associated mechanism.Methods The THP-1 cells were allocated to THP-1-mA,nil vector transfection and nil treatment groups,respectively.In group THP-1-mA,the pEGFPC1-NPM1-mA plasmid vector with NPM 1 mutation A (NPM 1-mA) was transfected into THP-1 cells for constructing the THP-1-mA cells,the leukemia cell line,with stable expression of NPM1-mA protein.In nil vector transfection group,the pseudo vector pEGFPC1 was employed to transfect the THP-1 cells.Transfection was not processed in nil treatment group.Reverse transcriptase polymerase chain reaction and immunohistochemistry assay were employed to assay the expression of NPM-mA gene and protein,and the cell growth curve was used to determine the proliferation potential in vitro.Flow cytometry was applied to detect the distribution of cell cycles.The invasiveness of cells in vitro was also assayed.The expression of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) mRNA were assayed by quantitative real-time polymerase chain reaction.Results The THP-l-mA cell strain stably expressing NPM 1-mA protein was established successfully.Compared with nil vector transfection and nil treatment groups,the THP-1-mA cells stably expressing NPM-mA protein were characterized by significantly augmented capacity of proliferation in vitro,reduced percentage of cells in phase G1 yet increased proportion in phase S (both P<0.01).These cells demonstrated,as evidenced by invasion assay,markedly augmented ability of invasion in vitro and,as suggested by fluorescent real-time polymerase chain reaction substantially increased expression of Ang-1 mRNA (both P<0.01).Conclusion NPM 1 mutations may promote THP-1 cell proliferation and invasion in vitro,in which Ang-1 may play a crucial role.%目的 探讨NPM1基因突变对THP-1细胞体外增殖和侵袭的影响及其机制.方法 将THP-1细胞分为THP-1-mA组、空载

  16. Unwinding the molecular basis of interval and circadian timing

    Directory of Open Access Journals (Sweden)

    Patricia V. Agostino

    2011-10-01

    Full Text Available Neural timing mechanisms range from the millisecond to diurnal, and possibly annual, frequencies. Two of the main processes under study are the interval timer (seconds-to-minute range and the circadian clock. The molecular basis of these two mechanisms is the subject of intense research, as well as their possible relationship. This article summarizes data from studies investigating a possible interaction between interval and circadian timing and reviews the molecular basis of both mechanisms, including the discussion of the contribution from studies of genetically modified animal models. While a common neurochemical substrate for timing mechanisms in the brain has been related to dopamine-reward systems, circadian modulation of interval timing suggests an interaction of different frequencies in cerebral temporal processes.

  17. Potential Role of Nucleophosmin(NPM1)Gene Mutations in K562 Leukemia Cell Invasion Phenotype%NPM1突变基因调控K562白血病细胞侵袭表型的相关机制

    Institute of Scientific and Technical Information of China (English)

    陈先春; 覃凤娴; 谭诗; 张慧娟; 邵会媛; 张伶

    2011-01-01

    Objective To investigate the role of CXCR4 and Angs in the invasion phenotype in vitro of leukemic cells with NPM1 mutations, and further study the effect of NPM1 mutations on leukemia infiltration. Methods The pEGFPC1-NPM1-mA plasmid vector was transfected into K562 cells. The cells stably expressing NPM1-mA protein were established, named as K562-mA. The expressions of CXCR4 and Ang-1/2 mRNA were assayed by quantitative Real-Time PCR ( qRT-PCR ). The expression of CXCR4 protein was assayed by Western blot and flow cytometry. Results Compared with control groups, CXCR4 mRNA and protein expression levels were significantly increased in K562-mA group. The expression of Ang-1 mRNA was markedly decreased, whereas Ang-2 mRNA expression was increased in K562-mA group. Conclusion CXCR4 and Ang-1/2 may play important roles in the invasion phenotype of leukemic cells with NPM1 mutations in vitro.%目的 探讨CXCR4、Angs在NPM1突变参与调控的白血病细胞浸润转移中的作用,以期进一步明确NPM1突变在白血病浸润转移中的调控机制.方法 通过基因转染构建稳定表达NPM1突变蛋白的K562白血病细胞株(K562-mA).qRT-PCR检测各组细胞CXCR4、Ang-1/2的mRNA表达水平;Western免疫印迹和流式细胞仪分别检测细胞CXCR4总蛋白和膜蛋白的表达.结果 建立了稳定表达NPM突变基因的K562-mA细胞株.与未处理组和空载体转染组相比,K562-mA细胞CXCR4的mRNA和蛋白表达水平显著增高;Ang-1 mRNA表达水平明显降低、Ang-2 mRNA表达水平明显增高.结论 CXCR4、Ang-1/2可能在NPM1突变调控白血病细胞的浸润转移中发挥重要作用.

  18. Molecular basis and regulation of OTULIN-LUBAC interaction

    DEFF Research Database (Denmark)

    Elliott, Paul R.; Al-Saoudi, Sofie Vincents; Marco-Casanova, Paola

    2014-01-01

    Ub. Now, we show that OTULIN binds via a conserved PUB-interacting motif (PIM) to the PUB domain of the LUBAC component HOIP. Crystal structures and nuclear magnetic resonance experiments reveal the molecular basis for the high-affinity interaction and explain why OTULIN binds the HOIP PUB domain......The linear ubiquitin (Ub) chain assembly complex (LUBAC) generates Met1-linked "linear" Ub chains that regulate the activation of the nuclear factor κB (NFκB) transcription factor and other processes. We recently discovered OTULIN as a deubiquitinase that specifically cleaves Met1-linked poly...

  19. Molecular Basis and Current Treatment for Alcoholic Liver Disease

    Directory of Open Access Journals (Sweden)

    Juan Armendariz-Borunda

    2010-04-01

    Full Text Available Alcohol use disorders and alcohol dependency affect millions of individuals worldwide. The impact of these facts lies in the elevated social and economic costs. Alcoholic liver disease is caused by acute and chronic exposure to ethanol which promotes oxidative stress and inflammatory response. Chronic consumption of ethanol implies liver steatosis, which is the first morphological change in the liver, followed by liver fibrosis and cirrhosis. This review comprises a broad approach of alcohol use disorders, and a more specific assessment of the pathophysiologic molecular basis, and genetics, as well as clinical presentation and current modalities of treatment for alcoholic liver disease.

  20. The Genetic and Molecular Basis of Plant Resistance to Pathogens

    Institute of Scientific and Technical Information of China (English)

    Yan Zhang; Thomas Lubberstedt; Mingliang Xu

    2013-01-01

    Plant pathogens have evolved numerous strategies to obtain nutritive materials from their host,and plants in turn have evolved the preformed physical and chemical barriers as well as sophisticated two-tiered immune system to combat pathogen attacks.Genetically,plant resistance to pathogens can be divided into qualitative and quantitative disease resistance,conditioned by major gene(s) and multiple genes with minor effects,respectively.Qualitative disease resistance has been mostly detected in plant defense against biotrophic pathogens,whereas quantitative disease resistance is involved in defense response to all plant pathogens,from biotrophs,hemibiotrophs to necrotrophs.Plant resistance is achieved through interception of pathogen-derived effectors and elicitation of defense response.In recent years,great progress has been made related to the molecular basis underlying host-pathogen interactions.In this review,we would like to provide an update on genetic and molecular aspects of plant resistance to pathogens.

  1. Molecular Basis for Vitamin A Uptake and Storage in Vertebrates.

    Science.gov (United States)

    Chelstowska, Sylwia; Widjaja-Adhi, Made Airanthi K; Silvaroli, Josie A; Golczak, Marcin

    2016-10-26

    The ability to store and distribute vitamin A inside the body is the main evolutionary adaptation that allows vertebrates to maintain retinoid functions during nutritional deficiencies and to acquire new metabolic pathways enabling light-independent production of 11-cis retinoids. These processes greatly depend on enzymes that esterify vitamin A as well as associated retinoid binding proteins. Although the significance of retinyl esters for vitamin A homeostasis is well established, until recently, the molecular basis for the retinol esterification enzymatic activity was unknown. In this review, we will look at retinoid absorption through the prism of current biochemical and structural studies on vitamin A esterifying enzymes. We describe molecular adaptations that enable retinoid storage and delineate mechanisms in which mutations found in selective proteins might influence vitamin A homeostasis in affected patients.

  2. Molecular Basis for Vitamin A Uptake and Storage in Vertebrates

    Science.gov (United States)

    Chelstowska, Sylwia; Widjaja-Adhi, Made Airanthi K.; Silvaroli, Josie A.; Golczak, Marcin

    2016-01-01

    The ability to store and distribute vitamin A inside the body is the main evolutionary adaptation that allows vertebrates to maintain retinoid functions during nutritional deficiencies and to acquire new metabolic pathways enabling light-independent production of 11-cis retinoids. These processes greatly depend on enzymes that esterify vitamin A as well as associated retinoid binding proteins. Although the significance of retinyl esters for vitamin A homeostasis is well established, until recently, the molecular basis for the retinol esterification enzymatic activity was unknown. In this review, we will look at retinoid absorption through the prism of current biochemical and structural studies on vitamin A esterifying enzymes. We describe molecular adaptations that enable retinoid storage and delineate mechanisms in which mutations found in selective proteins might influence vitamin A homeostasis in affected patients. PMID:27792183

  3. The Molecular Basis of Hereditary Enamel Defects in Humans

    Science.gov (United States)

    Carrion, I.A.; Morris, C.

    2015-01-01

    The formation of human enamel is highly regulated at the molecular level and involves thousands of genes. Requisites for development of this highly mineralized tissue include cell differentiation; production of a unique extracellular matrix; processing of the extracellular matrix; altering of cell function during different stages of enamel formation; cell movement and attachment; regulation of ion and protein movement; and regulation of hydration, pH, and other conditions of the microenvironment, to name just a few. Not surprising, there is a plethora of hereditary conditions with an enamel phenotype. The objective of this review was to identify the hereditary conditions listed on Online Mendelian Inheritance in Man (OMIM) that have an associated enamel phenotype and whether a causative gene has been identified. The OMIM database was searched with the terms amelogenesis, enamel, dental, and tooth, and all results were screened by 2 individuals to determine if an enamel phenotype was identified. Gene and gene product function was reviewed on OMIM and from publications identified in PubMed. The search strategy revealed 91 conditions listed in OMIM as having an enamel phenotype, and of those, 71 have a known molecular etiology or linked genetic loci. The purported protein function of those conditions with a known genetic basis included enzymes, regulatory proteins, extracellular matrix proteins, transcription factors, and transmembrane proteins. The most common enamel phenotype was a deficient amount of enamel, or enamel hypoplasia, with hypomineralization defects being reported less frequently. Knowing these molecular defects allows an initial cataloging of molecular pathways that lead to hereditary enamel defects in humans. This knowledge provides insight into the diverse molecular pathways involved in enamel formation and can be useful when searching for the genetic etiology of hereditary conditions that involve enamel. PMID:25389004

  4. The molecular basis of hereditary enamel defects in humans.

    Science.gov (United States)

    Wright, J T; Carrion, I A; Morris, C

    2015-01-01

    The formation of human enamel is highly regulated at the molecular level and involves thousands of genes. Requisites for development of this highly mineralized tissue include cell differentiation; production of a unique extracellular matrix; processing of the extracellular matrix; altering of cell function during different stages of enamel formation; cell movement and attachment; regulation of ion and protein movement; and regulation of hydration, pH, and other conditions of the microenvironment, to name just a few. Not surprising, there is a plethora of hereditary conditions with an enamel phenotype. The objective of this review was to identify the hereditary conditions listed on Online Mendelian Inheritance in Man (OMIM) that have an associated enamel phenotype and whether a causative gene has been identified. The OMIM database was searched with the terms amelogenesis, enamel, dental, and tooth, and all results were screened by 2 individuals to determine if an enamel phenotype was identified. Gene and gene product function was reviewed on OMIM and from publications identified in PubMed. The search strategy revealed 91 conditions listed in OMIM as having an enamel phenotype, and of those, 71 have a known molecular etiology or linked genetic loci. The purported protein function of those conditions with a known genetic basis included enzymes, regulatory proteins, extracellular matrix proteins, transcription factors, and transmembrane proteins. The most common enamel phenotype was a deficient amount of enamel, or enamel hypoplasia, with hypomineralization defects being reported less frequently. Knowing these molecular defects allows an initial cataloging of molecular pathways that lead to hereditary enamel defects in humans. This knowledge provides insight into the diverse molecular pathways involved in enamel formation and can be useful when searching for the genetic etiology of hereditary conditions that involve enamel.

  5. Molecular basis of infantile reversible cytochrome c oxidase deficiency myopathy.

    Science.gov (United States)

    Horvath, Rita; Kemp, John P; Tuppen, Helen A L; Hudson, Gavin; Oldfors, Anders; Marie, Suely K N; Moslemi, Ali-Reza; Servidei, Serenella; Holme, Elisabeth; Shanske, Sara; Kollberg, Gittan; Jayakar, Parul; Pyle, Angela; Marks, Harold M; Holinski-Feder, Elke; Scavina, Mena; Walter, Maggie C; Coku, Jorida; Günther-Scholz, Andrea; Smith, Paul M; McFarland, Robert; Chrzanowska-Lightowlers, Zofia M A; Lightowlers, Robert N; Hirano, Michio; Lochmüller, Hanns; Taylor, Robert W; Chinnery, Patrick F; Tulinius, Mar; DiMauro, Salvatore

    2009-11-01

    Childhood-onset mitochondrial encephalomyopathies are usually severe, relentlessly progressive conditions that have a fatal outcome. However, a puzzling infantile disorder, long known as 'benign cytochrome c oxidase deficiency myopathy' is an exception because it shows spontaneous recovery if infants survive the first months of life. Current investigations cannot distinguish those with a good prognosis from those with terminal disease, making it very difficult to decide when to continue intensive supportive care. Here we define the principal molecular basis of the disorder by identifying a maternally inherited, homoplasmic m.14674T>C mt-tRNA(Glu) mutation in 17 patients from 12 families. Our results provide functional evidence for the pathogenicity of the mutation and show that tissue-specific mechanisms downstream of tRNA(Glu) may explain the spontaneous recovery. This study provides the rationale for a simple genetic test to identify infants with mitochondrial myopathy and good prognosis.

  6. The genetic and molecular basis of congenital cataract

    Directory of Open Access Journals (Sweden)

    Alessandro Santana

    2011-04-01

    Full Text Available Congenital cataracts are one of the most treatable causes of visual impairment and blindness during infancy, with an estimated prevalence of 1 to 6 cases per 10,000 live births. Approximately fifty percent of all congenital cataract cases may have a genetic cause. All three types of Mendelian inheritance have been reported for cataract; however, autosomal dominant transmission seems to be the most frequent. The transparency and high refractive index of the lens are achieved by the precise architecture of the fiber cells and the homeostasis of the lens proteins in terms of their concentration, stability, and supramolecular organization. Research on hereditary congenital cataract led to the identification of several classes of candidate genes that encode proteins such crystallins, lens specific connexins, aquaporine, cytoskeletal structural proteins, and developmental regulators. The purpose of this study was to review the literature on the recent advances made in understanding the molecular genetic basis of congenital cataracts.

  7. Molecular basis of NDM-1, a new antibiotic resistance determinant.

    Directory of Open Access Journals (Sweden)

    Zhongjie Liang

    Full Text Available The New Delhi Metallo-β-lactamase (NDM-1 was first reported in 2009 in a Swedish patient. A recent study reported that Klebsiella pneumonia NDM-1 positive strain or Escherichia coli NDM-1 positive strain was highly resistant to all antibiotics tested except tigecycline and colistin. These can no longer be relied on to treat infections and therefore, NDM-1 now becomes potentially a major global health threat.In this study, we performed modeling studies to obtain its 3D structure and NDM-1/antibiotics complex. It revealed that the hydrolytic mechanisms are highly conserved. In addition, the detailed analysis indicates that the more flexible and hydrophobic loop1, together with the evolution of more positive-charged loop2 leads to NDM-1 positive strain more potent and extensive in antibiotics resistance compared with other MBLs. Furthermore, through biological experiments, we revealed the molecular basis for antibiotics catalysis of NDM-1 on the enzymatic level. We found that NDM-1 enzyme was highly potent to degrade carbapenem antibiotics, while mostly susceptible to tigecycline, which had the ability to slow down the hydrolysis velocity of meropenem by NDM-1. Meanwhile, the mutagenesis experiments, including D124A, C208A, K211A and K211E, which displayed down-regulation on meropenem catalysis, proved the accuracy of our model.At present, there are no effective antibiotics against NDM-1 positive pathogen. Our study will provide clues to investigate the molecular basis of extended antibiotics resistance of NDM-1 and then accelerate the search for new antibiotics against NDM-1 positive strain in clinical studies.

  8. Molecular basis of NDM-1, a new antibiotic resistance determinant.

    Science.gov (United States)

    Liang, Zhongjie; Li, Lianchun; Wang, Yuanyuan; Chen, Limin; Kong, Xiangqian; Hong, Yao; Lan, Lefu; Zheng, Mingyue; Guang-Yang, Cai; Liu, Hong; Shen, Xu; Luo, Cheng; Li, Keqin Kathy; Chen, Kaixian; Jiang, Hualiang

    2011-01-01

    The New Delhi Metallo-β-lactamase (NDM-1) was first reported in 2009 in a Swedish patient. A recent study reported that Klebsiella pneumonia NDM-1 positive strain or Escherichia coli NDM-1 positive strain was highly resistant to all antibiotics tested except tigecycline and colistin. These can no longer be relied on to treat infections and therefore, NDM-1 now becomes potentially a major global health threat.In this study, we performed modeling studies to obtain its 3D structure and NDM-1/antibiotics complex. It revealed that the hydrolytic mechanisms are highly conserved. In addition, the detailed analysis indicates that the more flexible and hydrophobic loop1, together with the evolution of more positive-charged loop2 leads to NDM-1 positive strain more potent and extensive in antibiotics resistance compared with other MBLs. Furthermore, through biological experiments, we revealed the molecular basis for antibiotics catalysis of NDM-1 on the enzymatic level. We found that NDM-1 enzyme was highly potent to degrade carbapenem antibiotics, while mostly susceptible to tigecycline, which had the ability to slow down the hydrolysis velocity of meropenem by NDM-1. Meanwhile, the mutagenesis experiments, including D124A, C208A, K211A and K211E, which displayed down-regulation on meropenem catalysis, proved the accuracy of our model.At present, there are no effective antibiotics against NDM-1 positive pathogen. Our study will provide clues to investigate the molecular basis of extended antibiotics resistance of NDM-1 and then accelerate the search for new antibiotics against NDM-1 positive strain in clinical studies.

  9. Molecular basis of the anti-inflammatory effects of terpenoids.

    Science.gov (United States)

    de las Heras, B; Hortelano, Sonsoles

    2009-03-01

    Natural products play a significant role in human health in relation to the prevention and treatment of inflammatory conditions. Among them, terpenoids (also referred to as terpenes), are the largest and most widespread class of secondary metabolites. They are found in higher plants, mosses, liverworts, algae and lichens, and also in insects, microbes or marine organisms. Some terpenoids have been used for therapeutic purposes for centuries as antibacterial, anti-inflammatory, antitumoral agents, and in recent decades research activity into the clinical potential of this class of compounds has increased continuously as a source of pharmacologically interesting agents. In the present review, molecular basis of the anti-inflammatory action of diterpenoids is presented with special emphasis on their ability to modulate critical cell signaling pathways involved in the inflammatory response of the body such as nuclear transcription factor-kappaB (NF-kappaB) activation. NF-kappaB plays an important role in the regulation of immune and inflammatory responses. Indeed, deregulated NF-kappaB expression is a characteristic phenomenon in several inflammatory diseases and NF-kappaB has become a major target in drug discovery. Hence, this article also introduces our recently elucidated findings about the potential of labdane diterpenoids as anti-inflammatory agents due to their ability to inhibit NF-kappaB. The future development of this class of compounds as anti-inflammatory drugs requires the introduction of novel molecular targets of therapeutic relevance in addition to biotechnological approaches for the production of these molecules.

  10. Insecticide resistance and its molecular basis in urban insect pests.

    Science.gov (United States)

    Naqqash, Muhammad Nadir; Gökçe, Ayhan; Bakhsh, Allah; Salim, Muhammad

    2016-04-01

    Insecticide resistance is one of the most important evolutionary phenomena for researchers. Overuse of chemicals has induced resistance in insect pests that ultimately has led to the collapse of disease control programs in many countries. The erroneous and inappropriate management of insect vectors has resulted in dissemination of many vector-borne diseases like dengue, malaria, diarrhea, leishmaniasis, and many others. In most cases, the emergence of new diseases and the revival of old ones can be related with ecological changes that have favored rapid growth of vector densities. Understanding molecular mechanisms in resistant strains can assist in the development of management programs to control the development and spread of resistant insect populations. The dominant, recessive, and co-dominant forms of genes encoding resistance can be investigated, and furthermore, resistance development can be addressed either by the release of susceptible strains or timely insecticide rotation. The present review discusses the resistance level in all important insect vectors of human diseases; the molecular basis of evolvement of resistance has also been discussed.

  11. Molecular Basis of Chemokine CXCL5-Glycosaminoglycan Interactions.

    Science.gov (United States)

    Sepuru, Krishna Mohan; Nagarajan, Balaji; Desai, Umesh R; Rajarathnam, Krishna

    2016-09-23

    Chemokines, a large family of highly versatile small soluble proteins, play crucial roles in defining innate and adaptive immune responses by regulating the trafficking of leukocytes, and also play a key role in various aspects of human physiology. Chemokines share the characteristic feature of reversibly existing as monomers and dimers, and their functional response is intimately coupled to interaction with glycosaminoglycans (GAGs). Currently, nothing is known regarding the structural basis or molecular mechanisms underlying CXCL5-GAG interactions. To address this missing knowledge, we characterized the interaction of a panel of heparin oligosaccharides to CXCL5 using solution NMR, isothermal titration calorimetry, and molecular dynamics simulations. NMR studies indicated that the dimer is the high-affinity GAG binding ligand and that lysine residues from the N-loop, 40s turn, β3 strand, and C-terminal helix mediate binding. Isothermal titration calorimetry indicated a stoichiometry of two oligosaccharides per CXCL5 dimer. NMR-based structural models reveal that these residues form a contiguous surface within a monomer and, interestingly, that the GAG-binding domain overlaps with the receptor-binding domain, indicating that a GAG-bound chemokine cannot activate the receptor. Molecular dynamics simulations indicate that the roles of the individual lysines are not equivalent and that helical lysines play a more prominent role in determining binding geometry and affinity. Further, binding interactions and GAG geometry in CXCL5 are novel and distinctly different compared with the related chemokines CXCL1 and CXCL8. We conclude that a finely tuned balance between the GAG-bound dimer and free soluble monomer regulates CXCL5-mediated receptor signaling and function.

  12. A new paradigm for the molecular basis of rubber elasticity

    Science.gov (United States)

    Hanson, David E.; Barber, John L.

    2015-07-01

    The molecular basis for rubber elasticity is arguably the oldest and one of the most important questions in the field of polymer physics. The theoretical investigation of rubber elasticity began in earnest almost a century ago with the development of analytic thermodynamic models, based on simple, highly-symmetric configurations of so-called Gaussian chains, i.e. polymer chains that obey Markov statistics. Numerous theories have been proposed over the past 90 years based on the ansatz that the elastic force for individual network chains arises from the entropy change associated with the distribution of end-to-end distances of a free polymer chain. There are serious conceptual objections to this assumption and others, such as the assumption that all network nodes undergo a simple volume-preserving linear motion and that all of the network chains have the same length. Recently, a new paradigm for elasticity in rubber networks has been proposed that is based on mechanisms that originate at the molecular level. Using conventional statistical mechanics analyses, Quantum Chemistry, and Molecular Dynamics simulations, the fundamental entropic and enthalpic chain extension forces for polyisoprene (natural rubber) have been determined, along with estimates for the basic force constants. Concurrently, the complex morphology of natural rubber networks (the joint probability density distributions that relate the chain end-to-end distance to its contour length) has also been captured in a numerical model (EPnet). When molecular chain forces are merged with the network structure in this model, it is possible to study the mechanical response to tensile and compressive strains of a representative volume element of a polymer network. As strain is imposed on a network, pathways of connected taut chains, that completely span the network along strain axis, emerge. Although these chains represent only a few percent of the total, they account for nearly all of the elastic stress at high

  13. Minimally-Myelosuppressive Asparaginase-Containing Induction Regimen for Treatment of a Jehovah’s Witness with mutant IDH1/NPM1/NRAS Acute Myeloid Leukemia

    Directory of Open Access Journals (Sweden)

    Ashkan Emadi

    2016-03-01

    Full Text Available Treatment of patients with acute myeloid leukemia (AML who do not wish to accept blood product transfusion, including Jehovah’s Witnesses, is extremely challenging. The use of conventional chemotherapy for induction of complete remission (CR results in profound anemia and thrombocytopenia requiring frequent transfusions of blood products, without which such treatment will be life-threatening. Finding a well tolerable, minimally myelosuppressive induction regimen for such patients with AML is a clear example of area of unmet medical need. Here, we report a successful treatment of a 52-year-old Jehovah’s Witness with newly diagnosed AML with peg-asparaginase, vincristine and methylprednisolone. The AML was characterized with normal karyotype, and mutations in isocitrate dehydrogenase 1 (IDH1-Arg132Ser, nucleophosmin 1 (NPM1-Trp289Cysfs*12 and neuroblastoma RAS viral oncogene homolog (NRAS-G1y12Va1. After one 28-day cycle of treatment, the patient achieved complete remission with incomplete count recovery (CRi and after the second cycle, he achieved CR with full blood count recovery. The patient has never received any blood products. Notwithstanding that myeloperoxidase-induced oxidative degradation of vincristine results in its lack of activity as monotherapy in AML, its combination with corticosteroid and asparaginase has resulted in a robust remission in this patient. Diminished steroid clearance by asparaginase activity as well as reduction in serum glutamine level induced by glutaminase enzymatic activity of asparaginase may have contributed to effective killing of the myeloblasts that carry IDH1/NPM1/NRAS mutations. In conclusion, asparaginase-containing regimens, which are approved for treatment of acute lymphoblastic leukemia (ALL but not AML, can be used to treat patients with AML who do not accept blood transfusion.

  14. Molecular basis for mycophenolic acid biosynthesis in Penicillium brevicompactum.

    Science.gov (United States)

    Regueira, Torsten Bak; Kildegaard, Kanchana Rueksomtawin; Hansen, Bjarne Gram; Mortensen, Uffe H; Hertweck, Christian; Nielsen, Jens

    2011-05-01

    Mycophenolic acid (MPA) is the active ingredient in the increasingly important immunosuppressive pharmaceuticals CellCept (Roche) and Myfortic (Novartis). Despite the long history of MPA, the molecular basis for its biosynthesis has remained enigmatic. Here we report the discovery of a polyketide synthase (PKS), MpaC, which we successfully characterized and identified as responsible for MPA production in Penicillium brevicompactum. mpaC resides in what most likely is a 25-kb gene cluster in the genome of Penicillium brevicompactum. The gene cluster was successfully localized by targeting putative resistance genes, in this case an additional copy of the gene encoding IMP dehydrogenase (IMPDH). We report the cloning, sequencing, and the functional characterization of the MPA biosynthesis gene cluster by deletion of the polyketide synthase gene mpaC of P. brevicompactum and bioinformatic analyses. As expected, the gene deletion completely abolished MPA production as well as production of several other metabolites derived from the MPA biosynthesis pathway of P. brevicompactum. Our work sets the stage for engineering the production of MPA and analogues through metabolic engineering.

  15. [Molecular basis of stress-evoked psychiatric disturbances].

    Science.gov (United States)

    Beszczyńska, Beata

    2007-11-20

    Stress, defined as coping with environmental challenges, involves the activation of the neuronal and neurohormonal systems. Central monoaminergic (noradrenergic, dopaminergic, serotonergic) neural networks, limbic structures, the sympathoadrenal system, the hypothalamo-pituitary-adrenal axis, and the immune system are considered the most important stress pathways. Their activation determines stress reactivity and pathological consequences on exposure to situations of distress. Both trauma and long-term stress can cause alterations in the activities of neuroanatomical structures and neural networks within the central nervous system. These neurohormonal changes are associated with post-traumatic stress disorder (PTSD), a disturbance thought to be one of the most serious psychiatric illnesses. PTSD may develop in individuals after exposure to a traumatic event (war, violence, accident) and is manifested by various symptoms, such as re-experiencing, flashbacks, intrusive thoughts, impaired memory of the event, sleep disorders, nightmares, panic attacks, and depression. In this review the neurohormonal changes associated with experiencing stress are presented to highlight the molecular and hormonal basis of PTSD.

  16. Molecular basis of colorectal cancer: Towards an individualized management?

    Directory of Open Access Journals (Sweden)

    J. Perea

    Full Text Available Colorectal cancer (CRC has become a highly relevant condition nowadays. In this respect, advances in the understanding of its molecular basis are key for an adequate management. From the time when the adenoma-carcinoma sequence was formulated as a carcinogenesis model to this day, when, among other things, three major carcinogenic pathways have been identified, the CRC concept has evolved from that of a single disease to the notion that each CRC is a differentiated condition in itself. The suppressor or chromosome instability pathway, the mutator or microsatellite instability pathway, and the methylator or CpG island methylation pathway allow various phenotypes to be identified within CRC. Similarly, the presence of different changes in certain genes confers several behaviors on CRC from both the prognostic and responsive standpoints to specific therapies. However, this apparent complexity does help develop the clinical management of this disease through the identification of novel, more specific therapy targets, and also markers for various behaviors within the condition, which will most likely lead us to an individualized management for these patients.

  17. Molecular basis of colorrectal cancer: towards an individualized management?

    Science.gov (United States)

    Perea, J; Lomas, M; Hidalgo, M

    2011-01-01

    Colorectal cancer (CRC) has become a highly relevant condition nowadays. In this respect, advances in the understanding of its molecular basis are key for an adequate management. From the time when the adenoma-carcinoma sequence was formulated as a carcinogenesis model to this day, when -among other things- three major carcinogenic pathways have been identified, the CRC concept has evolved from that of a single disease to the notion that each CRC is a differentiated condition in itself. The suppressor or chromosome instability pathway, the mutator or microsatellite instability pathway, and the methylator or CpG island methylation pathway allow various phenotypes to be identified within CRC. Similarly, the presence of different changes in certain genes confers several behaviors on CRC from both the prognostic and responsive standpoints to specific therapies. However, this apparent complexity does help develop the clinical management of this disease through the identification of novel, more specific therapy targets, and also markers for various behaviors within the condition, which will most likely lead us to an individualized management for these patients.

  18. Microbial biotransformation of DON: molecular basis for reduced toxicity

    Science.gov (United States)

    Pierron, Alix; Mimoun, Sabria; Murate, Leticia S.; Loiseau, Nicolas; Lippi, Yannick; Bracarense, Ana-Paula F. L.; Schatzmayr, Gerd; He, Jian Wei; Zhou, Ting; Moll, Wulf-Dieter; Oswald, Isabelle P.

    2016-07-01

    Bacteria are able to de-epoxidize or epimerize deoxynivalenol (DON), a mycotoxin, to deepoxy-deoxynivalenol (deepoxy-DON or DOM-1) or 3-epi-deoxynivalenol (3-epi-DON), respectively. Using different approaches, the intestinal toxicity of 3 molecules was compared and the molecular basis for the reduced toxicity investigated. In human intestinal epithelial cells, deepoxy-DON and 3-epi-DON were not cytotoxic, did not change the oxygen consumption or impair the barrier function. In intestinal explants, exposure for 4 hours to 10 μM DON induced intestinal lesions not seen in explants treated with deepoxy-DON and 3-epi-DON. A pan-genomic transcriptomic analysis was performed on intestinal explants. 747 probes, representing 323 genes, were differentially expressed, between DON-treated and control explants. By contrast, no differentially expressed genes were observed between control, deepoxy-DON and 3-epi-DON treated explants. Both DON and its biotransformation products were able to fit into the pockets of the A-site of the ribosome peptidyl transferase center. DON forms three hydrogen bonds with the A site and activates MAPKinases (mitogen-activated protein kinases). By contrast deepoxy-DON and 3-epi-DON only form two hydrogen bonds and do not activate MAPKinases. Our data demonstrate that bacterial de-epoxidation or epimerization of DON altered their interaction with the ribosome, leading to an absence of MAPKinase activation and a reduced toxicity.

  19. Structural and molecular basis of starch viscosity in hexaploid wheat.

    Science.gov (United States)

    Ral, J-P; Cavanagh, C R; Larroque, O; Regina, A; Morell, M K

    2008-06-11

    Wheat starch is considered to have a low paste viscosity relative to other starches. Consequently, wheat starch is not preferred for many applications as compared to other high paste viscosity starches. Increasing the viscosity of wheat starch is expected to increase the functionality of a range of wheat flour-based products in which the texture is an important aspect of consumer acceptance (e.g., pasta, and instant and yellow alkaline noodles). To understand the molecular basis of starch viscosity, we have undertaken a comprehensive structural and rheological analysis of starches from a genetically diverse set of wheat genotypes, which revealed significant variation in starch traits including starch granule protein content, starch-associated lipid content and composition, phosphate content, and the structures of the amylose and amylopectin fractions. Statistical analysis highlighted the association between amylopectin chains of 18-25 glucose residues and starch pasting properties. Principal component analysis also identified an association between monoesterified phosphate and starch pasting properties in wheat despite the low starch-phosphate level in wheat as compared to tuber starches. We also found a strong negative correlation between the phosphate ester content and the starch content in flour. Previously observed associations between internal starch granule fatty acids and the swelling peak time and pasting temperature have been confirmed. This study has highlighted a range of parameters associated with increased starch viscosity that could be used in prebreeding/breeding programs to modify wheat starch pasting properties.

  20. Independent Molecular Basis of Convergent Highland Adaptation in Maize

    Science.gov (United States)

    Takuno, Shohei; Ralph, Peter; Swarts, Kelly; Elshire, Rob J.; Glaubitz, Jeffrey C.; Buckler, Edward S.; Hufford, Matthew B.; Ross-Ibarra, Jeffrey

    2015-01-01

    Convergent evolution is the independent evolution of similar traits in different species or lineages of the same species; this often is a result of adaptation to similar environments, a process referred to as convergent adaptation. We investigate here the molecular basis of convergent adaptation in maize to highland climates in Mesoamerica and South America, using genome-wide SNP data. Taking advantage of archaeological data on the arrival of maize to the highlands, we infer demographic models for both populations, identifying evidence of a strong bottleneck and rapid expansion in South America. We use these models to then identify loci showing an excess of differentiation as a means of identifying putative targets of natural selection and compare our results to expectations from recently developed theory on convergent adaptation. Consistent with predictions across a wide parameter space, we see limited evidence for convergent evolution at the nucleotide level in spite of strong similarities in overall phenotypes. Instead, we show that selection appears to have predominantly acted on standing genetic variation and that introgression from wild teosinte populations appears to have played a role in highland adaptation in Mexican maize. PMID:26078279

  1. Combined mutations of ASXL1, CBL, FLT3, IDH1, IDH2, JAK2, KRAS, NPM1, NRAS, RUNX1, TET2 and WT1 genes in myelodysplastic syndromes and acute myeloid leukemias

    Directory of Open Access Journals (Sweden)

    Vey Norbert

    2010-08-01

    Full Text Available Abstract Background Gene mutation is an important mechanism of myeloid leukemogenesis. However, the number and combination of gene mutated in myeloid malignancies is still a matter of investigation. Methods We searched for mutations in the ASXL1, CBL, FLT3, IDH1, IDH2, JAK2, KRAS, NPM1, NRAS, RUNX1, TET2 and WT1 genes in 65 myelodysplastic syndromes (MDSs and 64 acute myeloid leukemias (AMLs without balanced translocation or complex karyotype. Results Mutations in ASXL1 and CBL were frequent in refractory anemia with excess of blasts. Mutations in TET2 occurred with similar frequency in MDSs and AMLs and associated equally with either ASXL1 or NPM1 mutations. Mutations of RUNX1 were mutually exclusive with TET2 and combined with ASXL1 but not with NPM1. Mutations in FLT3 (mutation and internal tandem duplication, IDH1, IDH2, NPM1 and WT1 occurred primarily in AMLs. Conclusion Only 14% MDSs but half AMLs had at least two mutations in the genes studied. Based on the observed combinations and exclusions we classified the 12 genes into four classes and propose a highly speculative model that at least a mutation in one of each class is necessary for developing AML with simple or normal karyotype.

  2. Wrinkled Peas and White-Eyed Fruit Flies: The Molecular Basis of Two Classical Genetic Traits.

    Science.gov (United States)

    Guilfoile, Patrick

    1997-01-01

    Focuses on bridging the gap between classical and molecular genetics for two traits: wrinkled seeds in garden peas and white eye color in fruit flies. Discusses the molecular details of the underlying basis of these traits. Contains 15 references. (JRH)

  3. Molecular basis of potassium channels in pancreatic duct epithelial cells

    DEFF Research Database (Denmark)

    Hayashi, M.; Novak, Ivana

    2013-01-01

    Potassium channels regulate excitability, epithelial ion transport, proliferation, and apoptosis. In pancreatic ducts, K channels hyperpolarize the membrane potential and provide the driving force for anion secretion. This review focuses on the molecular candidates of functional K channels...

  4. Increased molecular damage and heterogeneity as the basis of aging.

    Science.gov (United States)

    Rattan, Suresh I S

    2008-03-01

    Aging at the molecular level is characterized by the progressive accumulation of molecular damage. The sources of damage act randomly through environmental and metabolically generated free radicals, through spontaneous errors in biochemical reactions, and through nutritional components. However, damage to a macromolecule may depend on its structure, localization and interactions with other macromolecules. Damage to the maintenance and repair pathways comprising homeodynamic machinery leads to age-related failure of homeodynamics, increased molecular heterogeneity, altered cellular functioning, reduced stress tolerance, diseases and ultimate death. Novel approaches for testing and developing effective means of intervention, prevention and modulation of aging involve means to minimize the occurrence and accumulation of molecular damage. Mild stress-induced hormesis by physical, biological and nutritional methods, including hormetins, represents a promising strategy for achieving healthy aging and for preventing age-related diseases.

  5. The molecular basis of somatic hypermutation of immunoglobulin genes.

    Science.gov (United States)

    Storb, U

    1996-04-01

    Somatic hypermutation amplifies the variable region repertoire of immunoglobulin genes. Recent experimental evidence has thrown light on various molecular models of somatic hypermutation. A link between somatic hypermutation and transcription coupled DNA repair is shaping up.

  6. Molecular basis of potassium channels in pancreatic duct epithelial cells

    DEFF Research Database (Denmark)

    Hayashi, M.; Novak, Ivana

    2013-01-01

    Potassium channels regulate excitability, epithelial ion transport, proliferation, and apoptosis. In pancreatic ducts, K channels hyperpolarize the membrane potential and provide the driving force for anion secretion. This review focuses on the molecular candidates of functional K channels in pan...

  7. Molecular basis for the thermostability of Newcastle disease virus

    Science.gov (United States)

    Thermostable Newcastle disease virus (NDV) vaccines have been used widely to protect village chickens against Newcastle disease, due to their decreased dependence on cold chain for transport and storage. However, the genetic basis underlying the NDV thermostability is poorly understood. In this stud...

  8. Molecular basis of fungicide resistance in Cercospora beticola

    Science.gov (United States)

    Cercospora leaf spot (CLS), caused by the fungus Cercospora beticola, is the most important foliar disease of sugarbeet. Control measures include the application of sterol demethylation inhibitor (DMI) and quinone outside inhibitor (QoI) fungicides. Understanding the molecular mechanism of fungici...

  9. Coulomb Sturmians as a basis for molecular calculations

    DEFF Research Database (Denmark)

    Avery, John Scales; Avery, James Emil

    2012-01-01

    Almost all modern quantum chemistry programs use Gaussian basis sets even though Gaussians cannot accurately represent the cusp at atomic nuclei, nor can they represent the slow decay of the wave function at large distances. The reason that Gaussians dominate quantum chemistry today is the great...... of hyperspherical harmonics. For the remaining many-centre integrals, Coulomb Sturmians are shown to have advantages over other ETOs. Pilot calculations are performed on N-electron molecules using the Generalized Sturmian Method....

  10. Defining the molecular genetic basis of idiopathic dilated cardiomyopathy.

    Science.gov (United States)

    Olson, T M; Keating, M T

    1997-02-01

    Dilated cardiomyopathy (DCM) is a significant health care problem. The etiology is idiopathic in approximately half of the patients. Recognition that 20%-25% of idiopathic DCM cases are familial has advanced the hypothesis that single gene defects are important in the disease's pathogenesis. General linkage analyses in rare, large DCM families have determined the chromosome location of five idiopathic DCM genes. Candidate-gene mutational analyses in more typical, small pedigrees represent an alternative strategy for DCM gene identification. Human molecular genetics will play a fundamental role in defining pathogenic mechanisms for DCM with the prospect of new, molecular-based diagnostic and therapeutic approaches. (Trends Cardiovasc Med 1997;7:60-63). © 1997, Elsevier Science Inc.

  11. Molecular basis for gene-specific transactivation by nuclear receptors

    DEFF Research Database (Denmark)

    Jørgensen, Mads Aagaard; Siersbæk, Rasmus; Mandrup, Susanne

    2010-01-01

    most likely be accounted for by mechanisms involving receptor-specific interactions with DNA as well as receptor-specific interactions with protein complexes binding to adjacent and distant DNA sequences. Here, we review key molecular aspects of transactivation by NRs with special emphasis......Nuclear receptors (NRs) are key transcriptional regulators of metazoan physiology and metabolism. Different NRs bind to similar or even identical core response elements; however, they regulate transcription in a highly receptor- and gene-specific manner. These differences in gene activation can...... on the recent advances in the molecular mechanisms responsible for receptor- and gene-specific transcriptional activation. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease....

  12. Molecular basis of telomere dysfunction in human genetic diseases.

    Science.gov (United States)

    Sarek, Grzegorz; Marzec, Paulina; Margalef, Pol; Boulton, Simon J

    2015-11-01

    Mutations in genes encoding proteins required for telomere structure, replication, repair and length maintenance are associated with several debilitating human genetic disorders. These complex telomere biology disorders (TBDs) give rise to critically short telomeres that affect the homeostasis of multiple organs. Furthermore, genome instability is often a hallmark of telomere syndromes, which are associated with increased cancer risk. Here, we summarize the molecular causes and cellular consequences of disease-causing mutations associated with telomere dysfunction.

  13. Lysosomal storage disorders: Molecular basis and laboratory testing

    Directory of Open Access Journals (Sweden)

    Filocamo Mirella

    2011-03-01

    Full Text Available Abstract Lysosomal storage disorders (LSDs are a large group of more than 50 different inherited metabolic diseases which, in the great majority of cases, result from the defective function of specific lysosomal enzymes and, in cases, of non-enzymatic lysosomal proteins or non-lysosomal proteins involved in lysosomal biogenesis. The progressive lysosomal accumulation of undegraded metabolites results in generalised cell and tissue dysfunction, and, therefore, multi-systemic pathology. Storage may begin during early embryonic development, and the clinical presentation for LSDs can vary from an early and severe phenotype to late-onset mild disease. The diagnosis of most LSDs--after accurate clinical/paraclinical evaluation, including the analysis of some urinary metabolites--is based mainly on the detection of a specific enzymatic deficiency. In these cases, molecular genetic testing (MGT can refine the enzymatic diagnosis. Once the genotype of an individual LSD patient has been ascertained, genetic counselling should include prediction of the possible phenotype and the identification of carriers in the family at risk. MGT is essential for the identification of genetic disorders resulting from non-enzymatic lysosomal protein defects and is complementary to biochemical genetic testing (BGT in complex situations, such as in cases of enzymatic pseudodeficiencies. Prenatal diagnosis is performed on the most appropriate samples, which include fresh or cultured chorionic villus sampling or cultured amniotic fluid. The choice of the test--enzymatic and/or molecular--is based on the characteristics of the defect to be investigated. For prenatal MGT, the genotype of the family index case must be known. The availability of both tests, enzymatic and molecular, enormously increases the reliability of the entire prenatal diagnostic procedure. To conclude, BGT and MGT are mostly complementary for post- and prenatal diagnosis of LSDs. Whenever genotype

  14. Computational molecular basis for improved silica surface complexation models

    Energy Technology Data Exchange (ETDEWEB)

    Sahai, Nita; Rosso, Kevin M.

    2006-06-06

    The acidity and reactivity of surface sites on amorphous and crystalline polymorphs of silica and other oxides control their thermodynamic stability and kinetic reactivity towards reactants in surface-controlled processes of environmental, industrial, biomedical and technological relevance. Recent advances in computational methodologies such as CPMD and increasing computer power combined with spectroscopic measurements are now making it possible to link, with an impressive degree of accuracy, the molecular-level description of these processes to phenomenological, surface complexation models The future challenge now lies in linking mesoscale properties at the nanometer scale to phenomenological models that will afford a more intuitive understanding of the systems under consideration.

  15. Molecular basis of development in petaloid monocot flowers

    DEFF Research Database (Denmark)

    Johansen, Bo; Frederiksen, Signe; Skipper, Martin

    2006-01-01

    -class genes and at least two copies of A-class genes: one is expressed in floral meristems, the other in inflorescence meristems. In monocots and non-core eudicots the validity of the ABC model is under discussion. Generally, more than one functional copy is found of at least one of the B-class genes. The A......-class genes apparently are expressed in meristems of both flower and inflorescence. Morphologically petaloid stamens and styles are well known within the petaloid monocots, whereas the phenomenon is rare in core eudicots. A simple model based on the extra copies of B-class genes can explain the molecular...

  16. The molecular basis of hereditary fructose intolerance in Italian children.

    Science.gov (United States)

    Santamaria, R; Scarano, M I; Esposito, G; Chiandetti, L; Izzo, P; Salvatore, F

    1993-10-01

    We investigated the molecular defects of the aldolase B gene in five unrelated patients affected by hereditary fructose intolerance. The techniques used were DNA amplification, direct sequencing and allele-specific oligonucleotide (ASO) hybridization. The most frequent substitutions found in the hereditary fructose intolerance alleles analysed were the A174D and the A149P mutations, which account for 50% and 30% of the alleles, respectively. In two unrelated families, we found a rare mutation, the MD delta 4 previously described only in one British family, which may be an important cause of the disease in Italy.

  17. The molecular basis of ethylene signalling in Arabidopsis

    Science.gov (United States)

    Woeste, K.; Kieber, J. J.; Evans, M. L. (Principal Investigator)

    1998-01-01

    The simple gas ethylene profoundly influences plants at nearly every stage of growth and development. In the past ten years, the use of a genetic approach, based on the triple response phenotype, has been a powerful tool for investigating the molecular events that underlie these effects. Several fundamental elements of the pathway have been described: a receptor with homology to bacterial two-component histidine kinases (ETR1), elements of a MAP kinase cascade (CTR1) and a putative transcription factor (EIN3). Taken together, these elements can be assembled into a simple, linear model for ethylene signalling that accounts for most of the well-characterized ethylene mediated responses.

  18. Molecular basis for optical clearing of collagenous tissues

    Science.gov (United States)

    Hirshburg, Jason M.; Ravikumar, Krishnakumar M.; Hwang, Wonmuk; Yeh, Alvin T.

    2010-09-01

    Molecular interactions of optical clearing agents were investigated using a combination of molecular dynamics (MD) simulations and optical spectroscopy. For a series of sugar alcohols with low to high optical clearing potential, Raman spectroscopy and integrating sphere measurements were used to quantitatively characterize tissue water loss and reduction in light scattering following agent exposures. The rate of tissue water loss was found to correlate with agent optical clearing potential, but equivalent tissue optical clearing was measured in native and fixed tissue in vitro, given long-enough exposure times to the polyol series. MD simulations showed that the rate of tissue optical clearing correlated with the preferential formation of hydrogen bond bridges between agent and collagen. Hydrogen bond bridge formation disrupts the collagen hydration layer and facilitates replacement by a chemical agent to homogenize tissue refractive index. However, the reduction in tissue light scattering did not correlate with the agent index of refraction. Our results suggest that a necessary property of optical clearing agents is hyperosmolarity to tissue, but that the most effective agents with the highest rates of optical clearing are a subset with the highest collagen solubilities.

  19. Severe combined immunodeficiency (SCID): from molecular basis to clinical management.

    Science.gov (United States)

    Sponzilli, Ivonne; Notarangelo, Luigi D

    2011-04-01

    Primary immune deficiency diseases (PID) comprise a genetically heterogeneous group of disorders that affect distinct components of the innate and adaptive immune system, such as neutrophils, macrophages, dendritic cells, complement proteins, natural killer cells, as well as T and B lymphocytes. Severe combined immunodeficiency (SCID) is a group of disorders characterized by increased susceptibility to severe infections and early death. The diagnosis of SCID is supported by the demonstration of low absolute lymphocyte count and T cell lymphopenia (variably associated with numerical defects of B and NK cells). In the last two decades, advances in the characterization of the molecular pathophysiology of SCID, have permitted the development of novel diagnostic assays based on analysis of the expression of the disease-associated proteins and mutation analysis. More recently, pilot newborn screening programs for the identification of infants with SCID have been initiated in the United States. Prompt and aggressive treatment of infections, antimicrobial prophylaxis (in particular against Pneumocystis jiroveci) and regular administration of immunoglobulins are essential to reduce the risk of early death. However, survival ultimately depends on reconstitution of immune function, that is usually achieved by means of hematopoietic cell transplantation (HCT). Gene therapy and enzyme replacement therapy have also been used successfully is selected forms of SCID. Here we review the molecular and cellular pathophysiology and the mainstay of treatment of SCID.

  20. Molecular basis and genetic predisposition to intracranial aneurysm.

    Science.gov (United States)

    Tromp, Gerard; Weinsheimer, Shantel; Ronkainen, Antti; Kuivaniemi, Helena

    2014-12-01

    Intracranial aneurysms, also called cerebral aneurysms, are dilatations in the arteries that supply blood to the brain. Rupture of an intracranial aneurysm leads to a subarachnoid hemorrhage, which is fatal in about 50% of the cases. Intracranial aneurysms can be repaired surgically or endovascularly, or by combining these two treatment modalities. They are relatively common with an estimated prevalence of unruptured aneurysms of 2%-6% in the adult population, and are considered a complex disease with both genetic and environmental risk factors. Known risk factors include smoking, hypertension, increasing age, and positive family history for intracranial aneurysms. Identifying the molecular mechanisms underlying the pathogenesis of intracranial aneurysms is complex. Genome-wide approaches such as DNA linkage and genetic association studies, as well as microarray-based mRNA expression studies, provide unbiased approaches to identify genetic risk factors and dissecting the molecular pathobiology of intracranial aneurysms. The ultimate goal of these studies is to use the information in clinical practice to predict an individual's risk for developing an aneurysm or monitor its growth or rupture risk. Another important goal is to design new therapies based on the information on mechanisms of disease processes to prevent the development or halt the progression of intracranial aneurysms.

  1. Genotoxicity of formaldehyde: Molecular basis of DNA damage and mutation

    Directory of Open Access Journals (Sweden)

    Masanobu eKawanishi

    2014-09-01

    Full Text Available Formaldehyde is commonly used in the chemical industry and is present in the environment, such as vehicle emissions, some building materials, food and tobacco smoke. It also occurs as a natural product in most organisms, the sources of which include a number of metabolic processes. It causes various acute and chronic adverse effects in humans if they inhale its fumes. Among the chronic effects on human health, we summarize data on genotoxicity and carcinogenicity in this review, and we particularly focus on the molecular mechanisms involved in the formaldehyde mutagenesis. Formaldehyde mainly induces N-hydroxymethyl mono-adducts on guanine, adenine and cytosine, and N-methylene crosslinks between adjacent purines in DNA. These crosslinks are types of DNA damage potentially fatal for cell survival if they are not removed by the nucleotide excision repair pathway. In the previous studies, we showed evidence that formaldehyde causes intra-strand crosslinks between purines in DNA using a unique method (Matsuda et al. Nucleic Acids Res. 26, 1769-1774,1998. Using shuttle vector plasmids, we also showed that formaldehyde as well as acetaldehyde induces tandem base substitutions, mainly at 5’-GG and 5’-GA sequences, which would arise from the intra-strand crosslinks. These mutation features are different from those of other aldehydes such as crotonaldehyde, acrolein, glyoxal and methylglyoxal. These findings provide molecular clues to improve our understanding of the genotoxicity and carcinogenicity of formaldehyde.

  2. The molecular basis for the high photosensitivity of rhodopsin.

    Science.gov (United States)

    Liu, Robert S H; Colmenares, Leticia U

    2003-12-09

    Based on structural information derived from the F NMR data of labeled rhodopsins, rhodopsin crystal structure, and excited-state properties of model polyenes, we propose a molecular mechanism that accounts specifically for the causes of the well-known enhanced photoreactivity of rhodopsin (increased rates and quantum yield of isomerization). It involves the key features of close proximity of C-187 to H-12 and chromophore bond lengthening upon light absorption. The resultant "sudden punch" to H-12 triggers dual processes of decay of the Franck-Condon-excited rhodopsin, a productive directed photoisomerization and a nonproductive decay returning to the ground state as two separate molecular pathways [based on real-time fluorescence results of Chosrowjan, H., Mataga, N., Shibata, Y., Tachibanaki, S., Kandori, H., Shichida, Y., Okada, T. & Kouyama, T. (1998) J. Am. Chem. Soc. 120, 9706-9707]. The two processes are controlled by the local protein structure: an empty space provided by the intradiscal loop connecting transmembrane helices 4 and 5 and a protein wall composed of amino acid units in transmembrane 3. Suggestions, involving retinal analogs and rhodopsin mutants, to improve the unusually high photosensitivity of rhodopsin are proposed.

  3. Comparison of biomolecules on the basis of Molecular Interaction Potentials

    Directory of Open Access Journals (Sweden)

    Rodrigo Jordi

    2002-01-01

    Full Text Available Molecular Interaction Potentials (MIP are frequently used for the comparison of series of compounds displaying related biological behaviors. These potentials are interaction energies between the considered compounds and relevant probes. The interaction energies are computed in the nodes of grids defined around the compounds. There is a need of detailed and objective comparative analyses of MIP distributions in the framework of structure-activity studies. On the other hand, MIP-based studies do not have to be restricted to series of small ligands, since such studies present also interesting possibilities for the analysis and comparison of biological macromolecules. Such analyses can benefit from the application of new methods and computational approaches. The new software MIPSim (Molecular Interaction Potentials Similarity analysis has recently been introduced with the purpose of analyzing and comparing MIP distributions of series of biomolecules. This program is transparently integrated with other programs, like GAMESS or GRID, which can be used for the computation of the potentials to be analyzed or compared. MIPSim incorporates several definitions of similarity coefficients, and is capable of combining several similarity measures into a single one. On the other hand, MIPSim can perform automatic explorations of the maximum similarity alignments between pairs of molecules.

  4. Molecular basis of glyphosate resistance: Different approaches through protein engineering

    Science.gov (United States)

    Pollegioni, Loredano; Schonbrunn, Ernst; Siehl, Daniel

    2011-01-01

    Glyphosate (N-phosphonomethyl-glycine) is the most-used herbicide in the world: glyphosate-based formulations exhibit broad-spectrum herbicidal activity with minimal human and environmental toxicity. The extraordinary success of this simple small molecule is mainly due to the high specificity of glyphosate towards the plant enzyme enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway leading to biosynthesis of aromatic amino acids. Starting in 1996, transgenic glyphosate-resistant plants were introduced thus allowing the application of the herbicide to the crop (post-emergence) to remove emerged weeds without crop damage. This review focuses on the evolution of mechanisms of resistance to glyphosate as obtained through natural diversity, the gene shuffling approach to molecular evolution, and a rational, structure-based approach to protein engineering. In addition, we offer rationale for the means by which the modifications made have had their intended effect. PMID:21668647

  5. Molecular basis of Post-surgical Peritoneal adhesions - An Overview

    Directory of Open Access Journals (Sweden)

    M.N. Vaze 1

    Full Text Available Post surgical adhesion development remains a frequent occurrence and is often unrecognized by surgeons. Peritoneal adhesions are the leading cause of pelvic pain, bowel obstruction and infertility. The prevention of adhesion till date is speculative due to lack of understanding of mechanisms involved in adhesion development. Adhesions are proposed to the disorder of wound healing and imbalance between fibrinogenesis and fibrinolysis. The unprecedented advancement in Molecular Biology has led us to identify molecules involved in both wound healing and adhesion development. The role of these molecules in peritoneal biological functions is not well understood. Hypoxia is proposed to be major contributing factor for the development of adhesions. The major mechanisms behind adhesion development are increased fibrinogenesis, reduced fibrinolysis, increased Extra Cellular Matrix deposition, increased cytokine production, increased angiogenesis and reduced apoptosis. Better understanding of these events will make efficient management of adhesions possible. [Veterinary World 2010; 3(12.000: 561-566

  6. Molecular and genetic basis of X-linked immunodeficiency disorders

    Energy Technology Data Exchange (ETDEWEB)

    Puck, J.M. (National Center for Human Genome Research, Bethesda, MD (United States))

    1994-03-01

    Within a short time interval the specific gene defects causing three X-linked human immunodeficiencies, agammaglobulinemia (XLA), hyper-IgM syndrome (HIGM), and severe combined immunodeficiency (XSCID), have been identified. These represent the first human disease phenotypes associated with each of three gene families already recognized to be important in lymphocyte development and signaling: XLA is caused by mutations of a B cell-specific intracellular tyrosine kinase; HIGM, by mutations in the TNF-related CD40 ligand, through which T cells deliver helper signals by direct contact with B cell CD40; and XSCID, by mutations in the [gamma] chain of the lymphocyte receptor for IL-2. Each patient mutation analyzed to date has been unique, representing both a challenge for genetic diagnosis and management and an important resource for dissecting molecular domains and understanding the physiologic function of the gene products.

  7. The physical basis of biochemistry the foundations of molecular biophysics

    CERN Document Server

    Bergethon, Peter R

    1998-01-01

    The objective of this book is to provide a unifying approach to the study of biophysical chemistry for the advanced undergraduate who has had a year of physics, organic chem­ istry, calculus, and biology. This book began as a revised edition of Biophysical Chemistry: Molecules to Membranes, which Elizabeth Simons and I coauthored. That short volume was written in an attempt to provide a concise text for a one-semester course in biophysical chemistry at the graduate level. The experience of teaching biophysical chemistry to bi­ ologically oriented students over the last decade has made it clear that the subject requires a more fundamental text that unifies the many threads of modem science: physics, chem­ istry, biology, mathematics, and statistics. This book represents that effort. This volume is not a treatment of modem biophysical chemistry with its rich history and many contro­ versies, although a book on that topic is also needed. The Physical Basis of Biochemistry is an introduction to the philosophy...

  8. The prevalence and molecular basis of hemoglobinopathies in Cambodia.

    Science.gov (United States)

    Carnley, Benedict P; Prior, John F; Gilbert, Anne; Lim, Erna; Devenish, Robyn; Sing, Heng; Sarin, Eng; Guhadasan, Rathi; Sullivan, Sheena G; Wise, Cheryl A; Bittles, Alan H; Chan, Kaimin; Wong, Man-Sim; Chan, Vivian; Erber, Wendy N

    2006-01-01

    Blood counts, hemoglobin (Hb) high performance liquid chromatography (HPLC), and DNA analyses were performed on 260 children, aged 5 months to 16 years, at Siem Reap to assess the prevalence of thalassemia and other hemoglobinopathies in regional Cambodia. Hemoglobinopathies were present in 134 children (51.5%) with 20 abnormal genotypes identified. alpha-Thalassemia (thal) (35.4%) was the most prevalent disorder and the -alpha3.7 gene deletion was the most common alpha-globin gene abnormality. The - -SEA deletion and nondeletional forms of alpha-thal, Hb Constant Spring [Hb CS, alpha142, Term-->Gln, TAA-->CAA (alpha2)], Hb Paksé [alpha142, Term-->Tyr, TAA-->TAT (alpha2)] and triplicated alpha genes, were also present but at low frequencies. Hb E [beta26(B8)Glu-->Lys, GAG-->AAG] (28.8%) was the most common beta-globin gene abnormality, whilst beta-thal was only detected in two children (0.8% of cases). Although hemoglobinopathies were common, the majority of abnormalities detected (heterozygous -alpha3.7 and Hb E) were not clinically significant. On the basis of these findings, and with the majority of abnormalities being mild, it seems improbable that thalassemia represents a major health burden in this region of Cambodia.

  9. Molecular basis for prey relocation in viperid snakes

    Science.gov (United States)

    2013-01-01

    Background Vertebrate predators use a broad arsenal of behaviors and weaponry for overcoming fractious and potentially dangerous prey. A unique array of predatory strategies occur among snakes, ranging from mechanical modes of constriction and jaw-holding in non-venomous snakes, to a chemical means, venom, for quickly dispatching prey. However, even among venomous snakes, different prey handling strategies are utilized, varying from the strike-and-hold behaviors exhibited by highly toxic elapid snakes to the rapid strike-and-release envenomation seen in viperid snakes. For vipers, this mode of envenomation represents a minimal risk predatory strategy by permitting little contact with or retaliation from prey, but it adds the additional task of relocating envenomated prey which has wandered from the attack site. This task is further confounded by trails of other unstruck conspecific or heterospecific prey. Despite decades of behavioral study, researchers still do not know the molecular mechanism which allows for prey relocation. Results During behavioral discrimination trials (vomeronasal responsiveness) to euthanized mice injected with size-fractionated venom, Crotalus atrox responded significantly to only one protein peak. Assays for enzymes common in rattlesnake venoms, such as exonuclease, L-amino acid oxidase, metalloproteinase, thrombin-like and kallikrein-like serine proteases and phospholipase A2, showed that vomeronasal responsiveness was not dependent on enzymatic activity. Using mass spectrometry and N-terminal sequencing, we identified the proteins responsible for envenomated prey discrimination as the non-enzymatic disintegrins crotatroxin 1 and 2. Our results demonstrate a novel and critical biological role for venom disintegrins far beyond their well-established role in disruption of cell-cell and cell-extracellular matrix interactions. Conclusions These findings reveal the evolutionary significance of free disintegrins in venoms as the molecular

  10. Molecular basis of cystic fibrosis in the Republic of Macedonia.

    Science.gov (United States)

    Petreska, L; Koceva, S; Plaseska, D; Chernick, M; Gordova-Muratovska, A; Fustic, S; Nestorov, R; Efremov, G D

    1998-09-01

    Eighty-three cystic fibrosis (CF) patients and their families, belonging to various ethnic groups living in the Republic of Macedonia were studied for molecular defects in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and for the associated extragenic marker loci XV-2c and KM19. The DNA methodology used included characterization of CFTR mutations in 19 exons (and flanking sequences) of the gene and analysis of distribution of the XV-2c/KM19 haplotypes among normal (N) and CF chromosomes by polymerase chain reaction (PCR) amplification followed by dot blot hybridization, restriction digestion, single-strand conformational polymorphism, constant denaturing gel electrophoresis, denaturing gradient gel electrophoresis, and sequencing. We identified 58.4% (97/166) of the CF chromosomes. Nine different CFTR gene mutations, including three novel ones, were found. Eight known and one new CFTR intragene polymorphisms were also characterized. The haplotype analysis of the XV-2c/TaqI and KM19/PstI polymorphic loci have shown that haplotype C is the most frequently found haplotype among the non-deltaF508 CF chromosomes from Macedonia (36.5%). The results demonstrate the broad heterogeneity of CF origin in this part of the Balkan Peninsula.

  11. Cellular and Molecular Basis for Stress-Induced Depression

    Science.gov (United States)

    Seo, Ji-Seon; Wei, Jing; Qin, Luye; Kim, Yong; Yan, Zhen

    2016-01-01

    Chronic stress plays a crucial role in the development of psychiatric diseases, such as anxiety and depression. Dysfunction of the medial prefrontal cortex (mPFC) has been linked to the cognitive and emotional deficits induced by stress. However, little is known about the molecular and cellular determinants in mPFC for stress-associated mental disorders. Here we show that chronic restraint stress induces the selective loss of p11 (also known as annexin II light chain, S100A10), a multifunctional protein binding to 5-HT receptors, in layer II/III neurons of the prelimbic cortex (PrL), as well as depression-like behaviors, both of which are reversed by selective serotonin reuptake inhibitors (SSRIs) and the tricyclic class of antidepressant (TCA) agents. In layer II/III of the PrL, p11 is highly concentrated in dopamine D2 receptor-expressing (D2+) glutamatergic neurons. Viral expression of p11 in D2+ PrL neurons alleviates the depression-like behaviors exhibited by genetically manipulated mice with D2+ neuron-specific or global deletion of p11. In stressed animals, overexpression of p11 in D2+ PrL neurons rescues depression-like behaviors by restoring glutamatergic transmission. Our results have identified p11 as a key molecule in a specific cell type that regulates stress-induced depression, which provides a framework for the development of new strategies to treat stress-associated mental illnesses. PMID:27457815

  12. Clinical applications of molecular basis for Craniosynostosis. A narrative review.

    Directory of Open Access Journals (Sweden)

    Saúl Ernesto Cifuentes-Mendiola

    2016-04-01

    Full Text Available Cranial sutures are specialized structures composed of the sutural mesenchyme, the overlying scalp, the dura and osteogenic fronts. Each one of these structures express important proteins for osteogenic maturation, membranous ossification of skull bones, and homeostasis of cranial sutures in a differential, spatial and temporal manner. These proteins include fibroblast growth factor (FGF and its receptors (FGFR, the transforming growth factor beta (TGF-β, bone morphogenetic proteins (BMPs, as well as transcription factors TWIST and MSX2, among others. The alteration in the expression of one or more of these proteins causes multiple pathological conditions; one of them is the premature closure of one or more cranial sutures, known as craniosynostosis. This malformation is commonly treated with surgery. However, advances in the fields of molecular and cellular biology have allowed to conduct research on some proteins involved in the development of craniosynostosis. The results of these studies can lead to future preventive therapeutic strategies that may be used as a complement to the surgical treatment of craniosynostosis. Possible strategies include the use of specific drugs that can regulate the expression and activation of FGF signaling pathways, TGF-β or BMPs, to prevent or avoid craniosynostosis or re-synostosis after a surgery.

  13. Fitness, nutrition and the molecular basis of chronic disease.

    Science.gov (United States)

    McAtee, C Patrick

    2013-01-01

    The onset of chronic disease is often the prelude to the subsequent physiological and mental twilight in the aging population of modern society. While rates of obesity, specific types of cancer and cardiovascular disorders seem to be on the rise in this group, many new therapies have addressed diseases that have been largely untreatable in the past. Alzheimer's disease has also recently come to the forefront of ongoing maladies most typically associated with an aging population. Ironically, though, many people seem to be living longer than expected. Recent biochemical, nutritional and genomic approaches have been able to elucidate some of the complex mechanisms, which lead to chronic diseases associated with an aging population such as Alzheimer's, metabolic syndrome, tumor metastasis and cardiovascular disease. These diseases and their sequalae seem to be related in many respects, with the common culprit being the inflammatory environment created by the presence of excess fat - particularly within the vascular network. Although a substantial effort has been focused on the development of new-line therapeutics to address these issues, nutrition and overall fitness and their effects on stalling or potentially reversing the advent of these diseases has not been fully embraced in the research arena. This review discusses the role of the inflammatory environment in the development of chronic diseases in the aging population and also proposes a common pathology. The benefits that improvements and dedication in nutrition and fitness approaches may offer at the molecular level are also discussed.

  14. The molecular basis of galactosemia - Past, present and future.

    Science.gov (United States)

    Timson, David J

    2016-09-10

    Galactosemia, an inborn error of galactose metabolism, was first described in the 1900s by von Ruess. The subsequent 100years has seen considerable progress in understanding the underlying genetics and biochemistry of this condition. Initial studies concentrated on increasing the understanding of the clinical manifestations of the disease. However, Leloir's discovery of the pathway of galactose catabolism in the 1940s and 1950s enabled other scientists, notably Kalckar, to link the disease to a specific enzymatic step in the pathway. Kalckar's work established that defects in galactose 1-phosphate uridylyltransferase (GALT) were responsible for the majority of cases of galactosemia. However, over the next three decades it became clear that there were two other forms of galactosemia: type II resulting from deficiencies in galactokinase (GALK1) and type III where the affected enzyme is UDP-galactose 4'-epimerase (GALE). From the 1970s, molecular biology approaches were applied to galactosemia. The chromosomal locations and DNA sequences of the three genes were determined. These studies enabled modern biochemical studies. Structures of the proteins have been determined and biochemical studies have shown that enzymatic impairment often results from misfolding and consequent protein instability. Cellular and model organism studies have demonstrated that reduced GALT or GALE activity results in increased oxidative stress. Thus, after a century of progress, it is possible to conceive of improved therapies including drugs to manipulate the pathway to reduce potentially toxic intermediates, antioxidants to reduce the oxidative stress of cells or use of "pharmacological chaperones" to stabilise the affected proteins.

  15. Molecular basis of chromatic adaptation in pennate diatom Phaeodactylum tricornutum.

    Science.gov (United States)

    Herbstová, Miroslava; Bína, David; Koník, Peter; Gardian, Zdenko; Vácha, František; Litvín, Radek

    2015-01-01

    The remarkable adaptability of diatoms living in a highly variable environment assures their prominence among marine primary producers. The present study integrates biochemical, biophysical and genomic data to bring new insights into the molecular mechanism of chromatic adaptation of pennate diatoms in model species Phaeodactylum tricornutum, a marine eukaryote alga possessing the capability to shift its absorption up to ~700 nm as a consequence of incident light enhanced in the red component. Presence of these low energy spectral forms of Chl a is manifested by room temperature fluorescence emission maximum at 710 nm (F710). Here we report a successful isolation of the supramolecular protein complex emitting F710 and identify a member of the Fucoxanthin Chlorophyll a/c binding Protein family, Lhcf15, as its key building block. This red-shifted antenna complex of P. tricornutum appears to be functionally connected to photosystem II. Phylogenetic analyses do not support relation of Lhcf15 of P. tricornutum to other known red-shifted antenna proteins thus indicating a case of convergent evolutionary adaptation towards survival in shaded environments. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Neuroprotective strategies and the underlying molecular basis of cerebrovascular stroke.

    Science.gov (United States)

    Karsy, Michael; Brock, Andrea; Guan, Jian; Taussky, Phillip; Kalani, M Yashar S; Park, Min S

    2017-04-01

    Stroke is a leading cause of disability in the US. Although there has been significant progress in the area of medical and surgical thrombolytic technologies, neuroprotective agents to prevent secondary cerebral injury and to minimize disability remain limited. Only limited success has been reported in preclinical and clinical trials evaluating a variety of compounds. In this review, the authors discuss the most up-to-date information regarding the underlying molecular biology of stroke as well as strategies that aim to mitigate this complex signaling cascade. Results of historical research trials involving N-methyl-d-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor antagonists, clomethiazole, antioxidants, citicoline, nitric oxide, and immune regulators have laid the groundwork for current progress. In addition, more recent studies involving therapeutic hypothermia, magnesium, albumin, glyburide, uric acid, and a variety of other treatments have provided more options. The use of neuroprotective agents in combination or with existing thrombolytic treatments may be one of many exciting areas of further development. Although past trials of neuroprotective agents in ischemic stroke have been limited, significant insights into mechanisms of stroke, animal models, and trial design have incrementally improved approaches for future therapies.

  17. Subtype-specific patterns of molecular mutations in acute myeloid leukemia.

    Science.gov (United States)

    Rose, D; Haferlach, T; Schnittger, S; Perglerová, K; Kern, W; Haferlach, C

    2017-01-01

    Acute myeloid leukemia (AML) can be grouped into morphologically or genetically defined subtypes. Today, the AML phenotype-genotype associations, that is, FAB/WHO (French-American-British/World Health Organization) definitions and recurrent molecular mutations, are not fully understood. Therefore, we evaluated the impact of molecular mutations on the AML differentiation stage by molecular profiling of 4373 adult de novo AML patients in 7 cytomorphological subtypes. We investigated mutations in 20 genes, including myeloid transcription factors (CEBPA, RUNX1), tumor suppressors (TP53, WT1), DNA modifiers (DNMT3A, IDH1/2, TET2), chromatin modifiers (ASXL1, MLL), signal transduction genes (FLT3, KRAS, NRAS) and NPM1. The most frequently mutated genes per cytomorphological subtype were RUNX1 in M0 (43%), NPM1 in M1 (42%), DNMT3A in M2 (26%), NPM1 in M4 (57%), M5a (49%) and M5b (70%) and TP53 in M6 (36%). Although some gene mutations were frequent in several cytomorphological subtypes, a series of associations of co-occurring mutations with distinct phenotypes were identified for molecularly defined subcohorts. FLT3, NPM1 and WT1 mutations were associated with an immature phenotype in myeloblastic AML, whereas other combinations involving ASXL1, RUNX1, MLL-PTD, CEBPA or KRAS were more frequent in myeloblastic AML with maturation. Within the NPM1 mutated subcohort, ASXL1 mutations were significantly associated with a monoblastic differentiation and DNMT3A mutations with a monocytic phenotype.

  18. A Demonstration of the Molecular Basis of Sickle-Cell Anemia.

    Science.gov (United States)

    Fox, Marty; Gaynor, John J.

    1996-01-01

    Describes a demonstration that permits the separation of different hemoglobin molecules within two to three hours. Introduces students to the powerful technique of gel electrophoresis and illustrates the molecular basis of sickle-cell anemia. (JRH)

  19. Bases biomoleculares do fotoenvelhecimento Molecular basis of photoaging

    Directory of Open Access Journals (Sweden)

    Suelen Montagner

    2009-07-01

    Full Text Available Com o aumento da expectativa de vida, o estudo do processo de envelhecimento orgânico tem sido estimulado. O envelhecimento da pele, órgão que espelha os sinais do tempo, é processo de deterioração progressiva, tempo-dependente, e pode ser intensificado pela exposição solar, então designado fotoenvelhecimento. O dano das radiações sobre diversas estruturas celulares e cutâneas leva a alterações morfológicas nesses componentes, fruto de modificações biomoleculares. Muitas pesquisas são desenvolvidas com o intuito de combater ou minimizar os efeitos do fotoenvelhecimento, porém a principal estratégia nesse sentido continua sendo a prevenção, só conseguida pelo progressivo desvendar dos mecanismos fisiopatogênicos envolvidos nesse processo.As a result of the increase in life expectancy, the study of the organic process of aging has been stimulated. Skin ageing, which reflects the signs of time, is a time-dependent process of progressive deterioration that can be intensified by sun exposure, which is known as photoaging. The damage of radiation on various cell structures and on the skin results in molecular and morphological changes to these components. Many research studies are performed to try to minimize the effects of photoaging; however, the main strategy to manage it is still prevention, which will only be achieved once we learn about the mechanisms involved in the process.

  20. The cellular and molecular basis of cnidarian neurogenesis.

    Science.gov (United States)

    Rentzsch, Fabian; Layden, Michael; Manuel, Michaël

    2017-01-01

    Neurogenesis initiates during early development and it continues through later developmental stages and in adult animals to enable expansion, remodeling, and homeostasis of the nervous system. The generation of nerve cells has been analyzed in detail in few bilaterian model organisms, leaving open many questions about the evolution of this process. As the sister group to bilaterians, cnidarians occupy an informative phylogenetic position to address the early evolution of cellular and molecular aspects of neurogenesis and to understand common principles of neural development. Here we review studies in several cnidarian model systems that have revealed significant similarities and interesting differences compared to neurogenesis in bilaterian species, and between different cnidarian taxa. Cnidarian neurogenesis is currently best understood in the sea anemone Nematostella vectensis, where it includes epithelial neural progenitor cells that express transcription factors of the soxB and atonal families. Notch signaling regulates the number of these neural progenitor cells, achaete-scute and dmrt genes are required for their further development and Wnt and BMP signaling appear to be involved in the patterning of the nervous system. In contrast to many vertebrates and Drosophila, cnidarians have a high capacity to generate neurons throughout their lifetime and during regeneration. Utilizing this feature of cnidarian biology will likely allow gaining new insights into the similarities and differences of embryonic and regenerative neurogenesis. The use of different cnidarian model systems and their expanding experimental toolkits will thus continue to provide a better understanding of evolutionary and developmental aspects of nervous system formation. WIREs Dev Biol 2017, 6:e257. doi: 10.1002/wdev.257 For further resources related to this article, please visit the WIREs website.

  1. Molecular basis for Duarte and Los Angeles variant galactosemia

    Energy Technology Data Exchange (ETDEWEB)

    Langley, S.D.; Lai, K.; Dembure, P.P. [Emory Univ. School of Medicine, Atlanta, GA (United States)] [and others

    1997-02-01

    Human erythrocytes that are homozygous for the Duarte enzyme variant of galactosemia (D/D) have a characteristic isoform on isoelectric focusing and 50% reduction in galactose-1-phosphate uridyltransferase (GALT) enzyme activity. The Duarte biochemical phenotype has a molecular genotype of N314D/N314D. The characteristic Duarte isoform is also associated with a variant called the {open_quotes}Los Angeles (LA) phenotype,{close_quotes} which has increased GALT enzyme activity. We evaluated GALT enzyme activity and screened the GALT genes of 145 patients with one or more N314D-containing alleles. We found seven with the LA biochemical phenotype, and all had a 1721C{r_arrow}T transition in exon 7 in cis with the N314D missense mutation. The 1721C{r_arrow}T transition is a neutral polymorphism for leucine at amino acid 218 (L218L). In pedigree analyses, this 1721C{r_arrow}T transition segregated with the LA phenotype of increased GALT activity in three different biochemical phenotypes (LA/N, LA/G, and LA/D). To determine the mechanism for increased activity of the LA variant, we compared GALT mRNA, protein abundance, and enzyme thermal stability in lymphoblast cell lines of D and LA phenotypes with comparable genotypes. GALT protein abundance was increased in LA compared to D alleles, but mRNA was similar among all genotypes. We conclude that the codon change N314D in cis with the base-pair transition 1721C{r_arrow}T produces the LA variant of galactosemia and that this nucleotide change increases GALT activity by increasing GALT protein abundance without increasing transcription or decreasing thermal lability. A favorable codon bias for the mutated codon with consequently increased translation rates is postulated as the mechanism. 23 refs., 3 figs., 4 tabs.

  2. Molecular basis of ALS and FTD: implications for translational studies.

    Science.gov (United States)

    Liščić, Rajka M

    2015-12-01

    Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative disorders, related by signs of deteriorating motor and cognitive functions, and short survival. The cause is unknown and no effective treatment currently exists. For ALS, there is only a drug Riluzole and a promising substance arimoclomol. The overlap between ALS and FTD occurs at clinical, genetic, and pathological levels. The majority of ALS cases are sporadic (SALS) and a subset of patients has an inherited form of the disease, familial ALS (FALS), with a common SOD1 mutation, also present in SALS. A few of the mutant genes identified in FALS have also been found in SALS. Recently, hexanucleotide repeat expansions in C9ORF72 gene were found to comprise the largest fraction of ALS- and FTD-causing mutations known to date. TAR DNA-binding protein 43 (TDP-43), encoded by the TARDBP gene, has been identified as the pathological protein of FALS, SALS and, less frequently, FTD. The less frequent TDP-43 pathology in other forms of familial FTD has been linked to a range of mutations in GRN, FUS/TLS, rarely VCP, and other genes. TDP-43 and FUS/TLS have striking structural and functional similarities, most likely implicating altered RNA processing as a major event in ALS pathogenesis. The clinical overlap of the symptoms of FTD and ALS is complemented by overlapping neuropathology, with intracellular inclusions composed of microtubule-associated protein tau, TDP-43 and less frequently FUS, or unknown ubiquitinated proteins. Furthermore, new therapeutic approaches continue to emerge, by targeting SOD1, TDP-43 or GRN proteins. This review addresses new advances that are being made in our understanding of the molecular mechanisms of both diseases, which may eventually translate into new treatment options.

  3. A molecular basis for inositol polyphosphate synthesis in Drosophila melanogaster.

    Science.gov (United States)

    Seeds, Andrew M; Sandquist, Joshua C; Spana, Eric P; York, John D

    2004-11-05

    Metabolism of inositol 1,4,5-trisphosphate (I(1,4,5)P3) results in the production of diverse arrays of inositol polyphosphates (IPs), such as IP4, IP5, IP6) and PP-IP5. Insights into their synthesis in metazoans are reported here through molecular studies in the fruit fly, Drosophila melanogaster. Two I(1,4,5)P3 kinase gene products are implicated in initiating catabolism of these important IP regulators. We find dmIpk2 is a nucleocytoplasmic 6-/3-kinase that converts I(1,4,5)P3 to I(1,3,4,5,6)P5, and harbors 5-kinase activity toward I(1,3,4,6)P4, and dmIP3K is a 3-kinase that converts I(1,4,5)P3 to I(1,3,4,5)P4. To assess their relative roles in the cellular production of IPs we utilized complementation analysis, RNA interference, and overexpression studies. Heterologous expression of dmIpk2, but not dmIP3K, in ipk2 mutant yeast recapitulates phospholipase C-dependent cellular synthesis of IP6. Knockdown of dmIpk2 in Drosophila S2 cells and transgenic flies results in a significant reduction of IP6 levels; whereas depletion of dmIP3K, either alpha or beta isoforms or both, does not decrease IP6 synthesis but instead increases its production, possibly by expanding I(1,4,5)P3 pools. Similarly, knockdown of an I(1,4,5)P3 5-phosphatase results in significant increase in dmIpk2/dmIpk1-dependent IP6 synthesis. IP6 production depends on the I(1,3,4,5,6)P5 2-kinase activity of dmIpk1 and is increased in transgenic flies overexpressing dmIpk2. Our studies reveal that phosphatase and kinase regulation of I(1,4,5)P3 metabolic pools directly impinge on higher IP synthesis, and that the major route of IP6 synthesis depends on the activities of dmIpk2 and dmIpk1, but not dmIP3K, thereby challenging the role of IP3K in the genesis of higher IP messengers.

  4. An in silico study of the molecular basis of B-RAF activation and conformational stability

    DEFF Research Database (Denmark)

    Fratev, Filip Filipov; Jonsdottir, Svava Osk

    2009-01-01

    B-RAF kinase plays an important role both in tumour induction and maintenance in several cancers and it is an attractive new drug target. However, the structural basis of the B-RAF activation is still not well understood. RESULTS: In this study we suggest a novel molecular basis of B-RAF activati...

  5. Molecular basis of essential fructosuria: molecular cloning and mutational analysis of human ketohexokinase (fructokinase).

    Science.gov (United States)

    Bonthron, D T; Brady, N; Donaldson, I A; Steinmann, B

    1994-09-01

    Essential fructosuria is one of the oldest known inborn errors of metabolism. It is a benign condition which is believed to result from deficiency of hepatic fructokinase (ketohexokinase, KHK, E.C.2.7.1.3). This enzyme catalyses the first step of metabolism of dietary fructose, conversion of fructose to fructose-1-phosphate. Despite the early recognition of this disorder, the primary structure of human KHK and the molecular basis of essential fructosuria have not been previously defined. In this report, the isolation and sequencing of full-length cDNA clones encoding human ketohexokinase are described. Alternative mRNA species and alternative KHK isozymes are produced by alternative polyadenylation and splicing of the KHK gene. The KHK proteins show a high level of sequence conservation relative to rat KHK. Direct evidence that mutation of the KHK structural gene is the cause of essential fructosuria was also obtained. In a well-characterized family, in which three of eight siblings have fructosuria, all affected individuals are compound heterozygotes for two mutations Gly40Arg and Ala43Thr. Both mutations result from G-->A transitions, and each alters the same conserved region of the KHK protein. Neither mutation was seen in a sample of 52 unrelated control individuals. An additional conservative amino acid change (Val49IIe) was present on the KHK allele bearing Ala43Thr.

  6. Hsp90-Tau complex reveals molecular basis for specificity in chaperone action

    NARCIS (Netherlands)

    Karagöz, G. Elif; Duarte, Afonso M.S.; Akoury, Elias; Ippel, Hans|info:eu-repo/dai/nl/115343415; Biernat, Jacek; Morán Luengo, Tania|info:eu-repo/dai/nl/369406036; Radli, Martina; Didenko, Tatiana|info:eu-repo/dai/nl/304829455; Nordhues, Bryce A.; Veprintsev, Dmitry B.; Dickey, Chad A.; Mandelkow, Eckhard; Zweckstetter, Markus; Boelens, Rolf|info:eu-repo/dai/nl/070151407; Madl, Tobias; Rüdiger, Stefan G.D.|info:eu-repo/dai/nl/314076662

    2014-01-01

    Protein folding in the cell relies on the orchestrated action of conserved families of molecular chaperones, the Hsp70 and Hsp90 systems. Hsp70 acts early and Hsp90 late in the folding path, yet the molecular basis of this timing is enigmatic, mainly because the substrate specificity of Hsp90 is

  7. The Dirac Equation in the algebraic approximation. VII. A comparison of molecular finite difference and finite basis set calculations using distributed Gaussian basis sets

    NARCIS (Netherlands)

    Quiney, H. M.; Glushkov, V. N.; Wilson, S.; Sabin,; Brandas, E

    2001-01-01

    A comparison is made of the accuracy achieved in finite difference and finite basis set approximations to the Dirac equation for the ground state of the hydrogen molecular ion. The finite basis set calculations are carried out using a distributed basis set of Gaussian functions the exponents and pos

  8. The Dirac Equation in the algebraic approximation. VII. A comparison of molecular finite difference and finite basis set calculations using distributed Gaussian basis sets

    NARCIS (Netherlands)

    Quiney, H. M.; Glushkov, V. N.; Wilson, S.; Sabin,; Brandas, E

    2001-01-01

    A comparison is made of the accuracy achieved in finite difference and finite basis set approximations to the Dirac equation for the ground state of the hydrogen molecular ion. The finite basis set calculations are carried out using a distributed basis set of Gaussian functions the exponents and

  9. Molecular basis of Acute Myelogenous Leukemia As bases moleculares da leucemia mielóide aguda

    Directory of Open Access Journals (Sweden)

    Eduardo M. Rego

    2002-01-01

    Full Text Available Acute Myelogenous Leukemia (AML is frequently associated with recurring chromosomal translocations, which lead to the fusion of two genes encoding transcription factors. As the moieties of these fusion proteins retain part of the functional domains of the wild-type proteins, they may interfere directly or indirectly with the transcriptional regulation of the leukemic cell, conferring survival advantage. The majority of the transcription factors commonly involved in recurring chromosomal translocations may be grouped in one of the following families: core binding factor (CBF, retinoic acid receptor alpha (RARalpha, homeobox (HOX family, and mixed lineage leukemia (MLL. In vivo analysis of the molecular basis of leukemogenesis through the generation of transgenic mouse models revealed that a common theme is the recruitment of transcriptional co-activators and co-repressors by these fusion proteins. However, the expression of the fusion protein is not sufficient to induce full blown leukemia, as evidenced in part by the long latencies required for disease development in the transgenic models of leukemia, and therefore, second mutagenic events may contribute to AML pathogenesis.A leucemia mielóide aguda (LMA está freqüentemente associada a translocações cromossômicas recorrentes. Em muitos casos, os genes presentes nos pontos de quebra cromossômica são conhecidos e, quase todos codificam para fatores de transcrição. O gene híbrido, resultante da justaposição de exons de genes distintos, codifica para proteínas de fusão. Como estas retêm a maior parte dos domínios funcionais das proteínas selvagens, elas interferem direta ou indiretamente com regulação da transcrição gênica, conferindo vantagem à sobrevivência das células leucêmicas. A maioria dos fatores de transcrição afetados pelas translocações cromossômicas associadas a LMA pode ser agrupada numa das seguintes famílias: dos core binding factors (CBF, do receptor

  10. Library of molecular associations: curating the complex molecular basis of liver diseases

    Directory of Open Access Journals (Sweden)

    Maass Thorsten

    2010-03-01

    Full Text Available Abstract Background Systems biology approaches offer novel insights into the development of chronic liver diseases. Current genomic databases supporting systems biology analyses are mostly based on microarray data. Although these data often cover genome wide expression, the validity of single microarray experiments remains questionable. However, for systems biology approaches addressing the interactions of molecular networks comprehensive but also highly validated data are necessary. Results We have therefore generated the first comprehensive database for published molecular associations in human liver diseases. It is based on PubMed published abstracts and aimed to close the gap between genome wide coverage of low validity from microarray data and individual highly validated data from PubMed. After an initial text mining process, the extracted abstracts were all manually validated to confirm content and potential genetic associations and may therefore be highly trusted. All data were stored in a publicly available database, Library of Molecular Associations http://www.medicalgenomics.org/databases/loma/news, currently holding approximately 1260 confirmed molecular associations for chronic liver diseases such as HCC, CCC, liver fibrosis, NASH/fatty liver disease, AIH, PBC, and PSC. We furthermore transformed these data into a powerful resource for molecular liver research by connecting them to multiple biomedical information resources. Conclusion Together, this database is the first available database providing a comprehensive view and analysis options for published molecular associations on multiple liver diseases.

  11. Clinical Distribution and Molecular Basis of Traditional Chinese Medicine ZHENG in Cancer

    Directory of Open Access Journals (Sweden)

    Zhen Chen

    2012-01-01

    Full Text Available In traditional Chinese medicine (TCM clinical practice, ZHENG (also known as syndrome helps to guide design of individualized treatment strategies. In this study, we investigated the clinical use of ZHENG in TCM-treated cancer patients by systematically analyzing data from all relevant reports in the Chinese-language scientific literature. We aimed to determine the clinical ZHENG distributions in six common cancers (lung, liver, gastric, breast, colorectal, and pancreatic with the expectation of uncovering a theoretical basis for TCM ZHENG as a clinical cancer treatment. In addition, we also reviewed the molecular basis underlying Xue-Yu (blood stasis, Shi-Re (dampness-heat, Yin-Xu (Yin deficiency, and Pi-Xu (spleen deficiency ZHENG that are commonly found in cancer patients. The results from our summary study provide insights into the potential utility of TCM ZHENG and may contribute to a better understanding of the molecular basis of TCM ZHENG in cancer.

  12. Drugs meeting the molecular basis of diabetic kidney disease: bridging from molecular mechanism to personalized medicine.

    Science.gov (United States)

    Lambers Heerspink, Hiddo J; Oberbauer, Rainer; Perco, Paul; Heinzel, Andreas; Heinze, Georg; Mayer, Gert; Mayer, Bernd

    2015-08-01

    Diabetic kidney disease (DKD) is a complex, multifactorial disease and is associated with a high risk of renal and cardiovascular morbidity and mortality. Clinical practice guidelines for diabetes recommend essentially identical treatments for all patients without taking into account how the individual responds to the instituted therapy. Yet, individuals vary widely in how they respond to medications and therefore optimal therapy differs between individuals. Understanding the underlying molecular mechanisms of variability in drug response will help tailor optimal therapy. Polymorphisms in genes related to drug pharmacokinetics have been used to explore mechanisms of response variability in DKD, but with limited success. The complex interaction between genetic make-up and environmental factors on the abundance of proteins and metabolites renders pharmacogenomics alone insufficient to fully capture response variability. A complementary approach is to attribute drug response variability to individual variability in underlying molecular mechanisms involved in the progression of disease. The interplay of different processes (e.g. inflammation, fibrosis, angiogenesis, oxidative stress) appears to drive disease progression, but the individual contribution of each process varies. Drugs at the other hand address specific targets and thereby interfere in certain disease-associated processes. At this level, biomarkers may help to gain insight into which specific pathophysiological processes are involved in an individual followed by a rational assessment whether a specific drug's mode of action indeed targets the relevant process at hand. This article describes the conceptual background and data-driven workflow developed by the SysKid consortium aimed at improving characterization of the molecular mechanisms underlying DKD at the interference of the molecular impact of individual drugs in order to tailor optimal therapy to individual patients. © The Author 2015. Published by

  13. Molecular Basis of Triple Negative Breast Cancer and Implications for Therapy

    Directory of Open Access Journals (Sweden)

    Parvin F. Peddi

    2012-01-01

    Full Text Available Triple negative breast cancer is an aggressive form of breast cancer with limited treatment options and is without proven targeted therapy. Understanding the molecular basis of triple negative breast cancer is crucial for effective new drug development. Recent genomewide gene expression and DNA sequencing studies indicate that this cancer type is composed of a molecularly heterogeneous group of diseases that carry multiple somatic mutations and genomic structural changes. These findings have implications for therapeutic target identification and the design of future clinical trials for this aggressive group of breast cancer.

  14. Molecular basis for species-specific sensitivity to "hot" chili peppers.

    Science.gov (United States)

    Jordt, Sven-Eric; Julius, David

    2002-02-08

    Chili peppers produce the pungent vanilloid compound capsaicin, which offers protection from predatory mammals. Birds are indifferent to the pain-producing effects of capsaicin and therefore serve as vectors for seed dispersal. Here, we determine the molecular basis for this species-specific behavioral response by identifying a domain of the rat vanilloid receptor that confers sensitivity to capsaicin to the normally insensitive chicken ortholog. Like its mammalian counterpart, the chicken receptor is activated by heat or protons, consistent with the fact that both mammals and birds detect noxious heat and experience thermal hypersensitivity. Our findings provide a molecular basis for the ecological phenomenon of directed deterence and suggest that the capacity to detect capsaicin-like inflammatory substances is a recent acquisition of mammalian vanilloid receptors.

  15. The genetic and molecular basis of cytoplasmic male sterility and fertility restoration in rice

    Institute of Scientific and Technical Information of China (English)

    GUO JingXin; LIU YaoGuang

    2009-01-01

    Cytoplasmic male sterility (CMS) is a maternally inherited characteristic found in many (>150) plant species. CMS/restoration systems are useful tools for hybrid seed production, and are ideal models for study of the interactions between nuclear and mitochondrial genomes. CMS/restoration systems in rice have been widely used for hybrid seed production, greatly contributing to the food supply. This article reviews the progress of the studies on the genetic and molecular basis of cytoplasmic male sterility and fertility restoration in rice.

  16. Evaluation of Molecular Basis of Cross Reactivity between Rye and Bermuda Grass Pollen Allergens

    OpenAIRE

    2009-01-01

    Background: Allergenic cross reactivity between the members of the Pooids (Lolium perenne, Phleum pratense, and Poa pratensis) and Chloridoids (Cynodon dactylon and Paspalum notatum) is well established. Studies using crude extracts in the past have demonstrated limited cross reactivity between the Pooids and the Chloridoids suggesting separate diagnosis and therapy. However, little is known regarding the molecular basis for the limited cross reactivity observed between the 2 groups of grasse...

  17. Vibrational molecular quantum computing: basis set independence and theoretical realization of the Deutsch-Jozsa algorithm.

    Science.gov (United States)

    Tesch, Carmen M; de Vivie-Riedle, Regina

    2004-12-22

    The phase of quantum gates is one key issue for the implementation of quantum algorithms. In this paper we first investigate the phase evolution of global molecular quantum gates, which are realized by optimally shaped femtosecond laser pulses. The specific laser fields are calculated using the multitarget optimal control algorithm, our modification of the optimal control theory relevant for application in quantum computing. As qubit system we use vibrational modes of polyatomic molecules, here the two IR-active modes of acetylene. Exemplarily, we present our results for a Pi gate, which shows a strong dependence on the phase, leading to a significant decrease in quantum yield. To correct for this unwanted behavior we include pressure on the quantum phase in our multitarget approach. In addition the accuracy of these phase corrected global quantum gates is enhanced. Furthermore we could show that in our molecular approach phase corrected quantum gates and basis set independence are directly linked. Basis set independence is also another property highly required for the performance of quantum algorithms. By realizing the Deutsch-Jozsa algorithm in our two qubit molecular model system, we demonstrate the good performance of our phase corrected and basis set independent quantum gates.

  18. A quantum molecular similarity analysis of changes in molecular electron density caused by basis set flotation and electric field application

    Science.gov (United States)

    Simon, Sílvia; Duran, Miquel

    1997-08-01

    Quantum molecular similarity (QMS) techniques are used to assess the response of the electron density of various small molecules to application of a static, uniform electric field. Likewise, QMS is used to analyze the changes in electron density generated by the process of floating a basis set. The results obtained show an interrelation between the floating process, the optimum geometry, and the presence of an external field. Cases involving the Le Chatelier principle are discussed, and an insight on the changes of bond critical point properties, self-similarity values and density differences is performed.

  19. The Molecular Basis of Evolution and Disease: A Cold War Alliance.

    Science.gov (United States)

    Suárez-Díaz, Edna

    2017-03-28

    This paper extends previous arguments against the assumption that the study of variation at the molecular level was instigated with a view to solving an internal conflict between the balance and classical schools of population genetics. It does so by focusing on the intersection of basic research in protein chemistry and the molecular approach to disease with the enactment of global health campaigns during the Cold War period. The paper connects advances in research on protein structure and function as reflected in Christian Anfinsen´s The molecular basis of evolution, with a political reading of Emilé Zuckerkandl and Linus Pauling's identification of molecular disease and evolution. Beyond atomic fallout, these advances constituted a rationale for the promotion of genetic surveys of human populations in the Third World, in connection with international health programs. Light is shed not only on the experimental roots of the molecular challenge but on the broader geopolitical context where the rising role of biomedicine and public health (particularly the malaria eradication campaigns) had an impact on evolutionary biology.

  20. Clinical relevance of molecular aberrations in paediatric acute myeloid leukaemia at first relapse

    NARCIS (Netherlands)

    Bachas, Costa; Schuurhuis, Gerrit Jan; Reinhardt, Dirk; Creutzig, Ursula; Kwidama, Zinia J.; Zwaan, C. Michel; van den Heuvel-Eibrink, Marry M.; De Bont, Evelina S. J. M.; Elitzur, Sarah; Rizzari, Carmelo; de Haas, Valerie; Zimmermann, Martin; Cloos, Jacqueline; Kaspers, Gertjan J. L.

    2014-01-01

    Outcome for relapsed paediatric acute myeloid leukaemia (AML) remains poor. Strong prognostic factors at first relapse are lacking, which hampers optimization of therapy. We assessed the frequency of molecular aberrations (FLT3, NRAS, KRAS, KIT, WT1 and NPM1 genes) at first relapse in a large set (n

  1. Clinical relevance of molecular aberrations in paediatric acute myeloid leukaemia at first relapse

    NARCIS (Netherlands)

    Bachas, Costa; Schuurhuis, Gerrit Jan; Reinhardt, Dirk; Creutzig, Ursula; Kwidama, Zinia J.; Zwaan, C. Michel; van den Heuvel-Eibrink, Marry M.; De Bont, Evelina S. J. M.; Elitzur, Sarah; Rizzari, Carmelo; de Haas, Valerie; Zimmermann, Martin; Cloos, Jacqueline; Kaspers, Gertjan J. L.

    2014-01-01

    Outcome for relapsed paediatric acute myeloid leukaemia (AML) remains poor. Strong prognostic factors at first relapse are lacking, which hampers optimization of therapy. We assessed the frequency of molecular aberrations (FLT3, NRAS, KRAS, KIT, WT1 and NPM1 genes) at first relapse in a large set (n

  2. The molecular basis for inhibition of sulindac and its metabolites towards human aldose reductase.

    Science.gov (United States)

    Zheng, Xuehua; Zhang, Liping; Zhai, Jing; Chen, Yunyun; Luo, Haibin; Hu, Xiaopeng

    2012-01-02

    Sulindac (SLD) exhibits both the highest inhibitory activity towards human aldose reductase (AR) among popular non-steroidal anti-inflammatory drugs and clear beneficial clinical effects on Type 2 diabetes. However, the molecular basis for these properties is unclear. Here, we report that SLD and its pharmacologically active/inactive metabolites, SLD sulfide and SLD sulfone, are equally effective as un-competitive inhibitors of AR in vitro. Crystallographic analysis reveals that π-π stacking favored by the distinct scaffold of SLDs is pivotal to their high AR inhibitory activities. These results also suggest that SLD sulfone could be a potent lead compound for AR inhibition in vivo.

  3. The molecular basis of convergence in hemoglobin function in high-altitude Andean birds

    DEFF Research Database (Denmark)

    Storz, Jay; Natarajan, Chandrasekhar; Witt, Christopher C.

    2016-01-01

    was correct that adaptive modifications of Hb function are typically attributable to a small number of substitutions at key positions, then the clear prediction is that the same mutations will be preferentially fixed in different species that have independently evolved Hbs with similar functional properties....... For example, in high-altitude ertebrates that have convergently evolved elevated Hb-O2 affinities, Perutz’s hypothesis predicts that parallel amino acid substitutions should be pervasive. We investigated the predictability of genetic adaptation by examining the molecular basis of convergence in hemoglobin (Hb...

  4. The molecular basis of metabolic cycles and their relationship to circadian rhythms.

    Science.gov (United States)

    Mellor, Jane

    2016-12-06

    Metabolic cycles result from the partitioning of oxidative and reductive metabolism into rhythmic phases of gene expression and oscillating post-translational protein modifications. Relatively little is known about how these switches in gene expression are controlled, although recent studies have suggested that transcription itself may play a central role. This review explores the molecular basis of the metabolic and gene-expression oscillations in the yeast Saccharomyces cerevisiae, as well as how they relate to other biological time-keeping mechanisms, such as circadian rhythms.

  5. On the molecular basis of D-bifunctional protein deficiency type III.

    Directory of Open Access Journals (Sweden)

    Maija L Mehtälä

    Full Text Available Molecular basis of D-bifunctional protein (D-BP deficiency was studied with wild type and five disease-causing variants of 3R-hydroxyacyl-CoA dehydrogenase fragment of the human MFE-2 (multifunctional enzyme type 2 protein. Complementation analysis in vivo in yeast and in vitro enzyme kinetic and stability determinants as well as in silico stability and structural fluctuation calculations were correlated with clinical data of known patients. Despite variations not affecting the catalytic residues, enzyme kinetic performance (K(m, V(max and k(cat of the recombinant protein variants were compromised to a varying extent and this can be judged as the direct molecular cause for D-BP deficiency. Protein stability plays an additional role in producing non-functionality of MFE-2 in case structural variations affect cofactor or substrate binding sites. Structure-function considerations of the variant proteins matched well with the available data of the patients.

  6. [Molecular ecological basis of high-yielding formation of rice and its application].

    Science.gov (United States)

    Lin, Wenxiong; Liang, Kangjing; Guo, Yuchun; He, Huaqin; Wang, Jingyuan; Liang, Yiyuan; Chen, Fangyu

    2003-12-01

    This paper introduced the developmental genetics and its molecular ecological basis of high yielding formation of rice in the past decade, and analyzed the advantage and the shortage of comparative physiological approach traditionally used in the research work on crop cultivation. It was emphasized to actively introduce the research contents and its methodology from relative disciplines to deeply understand the scientific issue, and suggested that the key to realize stable and high yielding of rice was to develop a rational cultivation system based on the properties of genetic effects on the traits in different developmental stages by controlling and regulating the traits governed by dominant effect genes and additive effect genes x environment in same direction, which was considered as the main characteristics and the technological innovation of modern crop genetic ecological cultivation science. Finally, the development trend of crop cultivation science shifting to molecular crop cultivation science was predicted and discussed.

  7. Molecular Mechanisms and Genetic Basis of Heavy Metal Tolerance/Hyperaccumulation in Plants

    Institute of Scientific and Technical Information of China (English)

    Xiao-E YANG; Xiao-Fen JIN; Ying FENG; Ejazul ISLAM

    2005-01-01

    Phytoremediation has gained increased attention as a cost-effective method for the remediation of heavy metal-contaminated sites. Because some plants possess a range of potential mechanisms that may be involved in the detoxification of heavy metals, they manage to survive under metal stresses. High tolerance to heavy metal toxicity could rely either on reduced uptake or increased plant internal sequestration,which is manifested by an interaction between a genotype and its environment. The growing application of molecular genetic technologies has led to increased understanding of mechanisms of heavy metal tolerance/accumulation in plants and, subsequently, many transgenic plants with increased heavy metal resistance,as well as increased uptake of heavy metals, have been developed for the purpose of phytoremediation. In the present review, our major objective is to concisely evaluate the progress made so far in understanding the molecular/cellular mechanisms and genetic basis that control the uptake and detoxification of metals by plants.

  8. Molecular basis of Rh blood group system in the Malaysian population.

    Science.gov (United States)

    Musa, Rozi Hanisa; Muhamad, Nor Asiah; Hassan, Afifah; Ayob, Yasmin; Yusoff, Narazah Mohd

    2015-01-01

    Rh molecular studies have been previously mainly conducted in Caucasians and African population. There is a limited data on the molecular basis for Rh genotypes among Asians. This study aims to characterize the Rh genes and frequency of the various RH genotypes among blood donors in National Blood Centre (NBC), Kuala Lumpur. A total of 1014 blood samples were obtained from blood donors from four different ethnic groups (360 Malays, 434 Chinese, 164 Indians and 56 others). Serological and molecular analysis of all 1014 blood samples were performed. An automated deoxyribonucleic acid sequencing analysis was performed. Rh phenotypes and RH genotypes showed heterogeneity and significant association with ethnicities. Discrepancies in allele D, C/c and E/e between phenotypes and genotypes results were observed. Discrepancy results in allele D showed significant association with the ethnic groups of the blood donors in NBC. There were multiple novel mutations (23) and published mutations (5) found in this study. Significant associations between discrepancy results and mutations were found in allele D and C/c. Performing RH molecular analysis in Malaysian population provided the basic database for the distribution of Rh genotypes of donors from major ethnic groups in Malaysia.

  9. The molecular basis for the broad substrate specificity of human sulfotransferase 1A1.

    Directory of Open Access Journals (Sweden)

    Ilana Berger

    Full Text Available Cytosolic sulfotransferases (SULTs are mammalian enzymes that detoxify a wide variety of chemicals through the addition of a sulfate group. Despite extensive research, the molecular basis for the broad specificity of SULTs is still not understood. Here, structural, protein engineering and kinetic approaches were employed to obtain deep understanding of the molecular basis for the broad specificity, catalytic activity and substrate inhibition of SULT1A1. We have determined five new structures of SULT1A1 in complex with different acceptors, and utilized a directed evolution approach to generate SULT1A1 mutants with enhanced thermostability and increased catalytic activity. We found that active site plasticity enables binding of different acceptors and identified dramatic structural changes in the SULT1A1 active site leading to the binding of a second acceptor molecule in a conserved yet non-productive manner. Our combined approach highlights the dominant role of SULT1A1 structural flexibility in controlling the specificity and activity of this enzyme.

  10. An in silico study of the molecular basis of B-RAF activation and conformational stability

    Directory of Open Access Journals (Sweden)

    Jónsdóttir Svava

    2009-07-01

    Full Text Available Abstract Background B-RAF kinase plays an important role both in tumour induction and maintenance in several cancers and it is an attractive new drug target. However, the structural basis of the B-RAF activation is still not well understood. Results In this study we suggest a novel molecular basis of B-RAF activation based on molecular dynamics (MD simulations of B-RAFWT and the B-RAFV600E, B-RAFK601E and B-RAFD594V mutants. A strong hydrogen bond network was identified in B-RAFWT in which the interactions between Lys601 and the well known catalytic residues Lys483, Glu501 and Asp594 play an important role. It was found that several mutations, which directly or indirectly destabilized the interactions between these residues within this network, contributed to the changes in B-RAF activity. Conclusion Our results showed that the above mechanisms lead to the disruption of the electrostatic interactions between the A-loop and the αC-helix in the activating mutants, which presumably contribute to the flipping of the activation segment to an active form. Conversely, in the B-RAFD594V mutant that has impaired kinase activity, and in B-RAFWT these interactions were strong and stabilized the kinase inactive form.

  11. Molecular basis for antagonistic activity of anifrolumab, an anti-interferon-α receptor 1 antibody.

    Science.gov (United States)

    Peng, Li; Oganesyan, Vaheh; Wu, Herren; Dall'Acqua, William F; Damschroder, Melissa M

    2015-01-01

    Anifrolumab (anifrolumab) is an antagonist human monoclonal antibody that targets interferon α receptor 1 (IFNAR1). Anifrolumab has been developed to treat autoimmune diseases and is currently in clinical trials. To decipher the molecular basis of its mechanism of action, we engaged in multiple epitope mapping approaches to determine how it interacts with IFNAR1 and antagonizes the receptor. We identified the epitope of anifrolumab using enzymatic fragmentation, phage-peptide library panning and mutagenesis approaches. Our studies revealed that anifrolumab recognizes the SD3 subdomain of IFNAR1 with the critical residue R(279). Further, we solved the crystal structure of anifrolumab Fab to a resolution of 2.3 Å. Guided by our epitope mapping studies, we then used in silico protein docking of the anifrolumab Fab crystal structure to IFNAR1 and characterized the corresponding mode of binding. We find that anifrolumab sterically inhibits the binding of IFN ligands to IFNAR1, thus blocking the formation of the ternary IFN/IFNAR1/IFNAR2 signaling complex. This report provides the molecular basis for the mechanism of action of anifrolumab and may provide insights toward designing antibody therapies against IFNAR1.

  12. Estudo de alterações moleculares e sua relação com dados clínico-laboratoriais em pacientes adultos com leucemia mieloide aguda

    OpenAIRE

    2013-01-01

    Marcadores moleculares, como mutações nos genes FLT3 e NPM1, são ferramentas úteis para a avaliação prognóstica de pacientes com leucemia mieloide aguda (LMA) e, até o momento, não tinham sido estudadas em pacientes com LMA no Estado de Pernambuco. Dessa forma, esse trabalho teve como objetivo caracterizar pacientes adultos com LMA diagnosticados na Fundação HEMOPE de acordo com achados clínico-laboratoriais e as mutações nos genes FLT3 e NPM1. Foram incluídos 115 pacientes com LMA de novo (1...

  13. Implications of the complete basis set limit in valence bond theory : a case study of molecular hydrogen

    NARCIS (Netherlands)

    Duke, Brian J.; Havenith, Remco W. A.

    2016-01-01

    The use of larger basis sets to approach the complete basis limit, now common in quantum chemistry, is applied for the first time to a range of valence bond functions for the simplest case of molecular hydrogen. Good convergence of the energy is slow due to difficulty in getting a correct cusp near

  14. Prognostic impact of white blood cell count in intermediate risk acute myeloid leukemia : relevance of mutated NPM1 and FLT3-ITD

    NARCIS (Netherlands)

    de Jonge, Hendrik J. M.; Valk, Peter J. M.; de Bont, Eveline S. J. M.; Schuringa, Jan Jacob; Ossenkoppele, Gert; Vellenga, Edo; Huls, Gerwin

    2011-01-01

    Background High white blood cell count at presentation is an unfavorable prognostic factor for treatment outcome in intermediate cytogenetic risk acute myeloid leukemia. Since the impact of white blood cell count on outcome of subgroups defined by the molecular markers NPMc(+) and FLT3-internal tand

  15. Molecular Basis of Clay Mineral Structure and Dynamics in Subsurface Engineering Applications

    Science.gov (United States)

    Cygan, R. T.

    2015-12-01

    Clay minerals and their interfaces play an essential role in many geochemical, environmental, and subsurface engineering applications. Adsorption, dissolution, precipitation, nucleation, and growth mechanisms, in particular, are controlled by the interplay of structure, thermodynamics, kinetics, and transport at clay mineral-water interfaces. Molecular details of these processes are typically beyond the sensitivity of experimental and analytical methods, and therefore require accurate models and simulations. Also, basal surfaces and interlayers of clay minerals provide constrained interfacial environments to facilitate the evaluation of these complex processes. We have developed and used classical molecular and quantum methods to examine the complex behavior of clay mineral-water interfaces and dynamics of interlayer species. Bulk structures, swelling behavior, diffusion, and adsorption processes are evaluated and compared to experimental and spectroscopic findings. Analysis of adsorption mechanisms of radionuclides on clay minerals provides a scientific basis for predicting the suitability of engineered barriers associated with nuclear waste repositories and the fate of contaminants in the environment. Similarly, the injection of supercritical carbon dioxide into geological reservoirs—to mitigate the impact of climate change—is evaluated by molecular models of multi-fluid interactions with clay minerals. Molecular dynamics simulations provide insights into the wettability of different fluids—water, electrolyte solutions, and supercritical carbon dioxide—on clay surfaces, and which ultimately affects capillary fluid flow and the integrity of shale caprocks. This work is supported as part of Center for Frontiers of Subsurface Energy Security, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science and by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Geosciences Research Program

  16. Immunological and molecular basis of nonalcoholic steatohepatitis and nonalcoholic fatty liver disease.

    Science.gov (United States)

    Radwan, Mohamed M; Radwan, Basil M; Nandipati, Kalyana C; Hunter, William J; Agrawal, Devendra K

    2013-08-01

    The prevalence of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) is rising worldwide with the increasing incidence of obesity, Type 2 diabetes mellitus and metabolic syndrome. NASH is currently one of the most common indications of liver transplantation in the United States. The immune system plays a major role in the pathogenesis of NAFLD/NASH. The metabolic changes, associated with obesity and metabolic syndrome, induce immunological responses resulting in NAFLD and further aggravation of the metabolic derangement in a feed-forward loop. Genetic and endocrine factors modulate the immunological and metabolic responses and determine the pathophysiological features of NAFLD. Histologically, NAFLD is a spectrum that ranges from simple hepatic steatosis to severe steatohepatitis, liver cirrhosis and/or hepatocellular carcinoma. Liver cirrhosis and hepatocellular carcinoma are responsible for the morbidity and mortality of the disease. This article is a critical evaluation of our current knowledge of the immunological and molecular basis of the disease.

  17. Molecular basis for selective serotonin reuptake inhibition by the antidepressant agent fluoxetine (prozac)

    DEFF Research Database (Denmark)

    Andersen, Jacob; Stuhr-Hansen, Nicolai; Zachariassen, Linda Grønborg

    2014-01-01

    , and computational methods to decipher the molecular basis for high-affinity recognition in SERT and selectivity over NET for the prototypical antidepressant drug fluoxetine (Prozac; Eli Lilly, Indianapolis, IN). We show that fluoxetine binds within the central substrate site of human SERT, in agreement with recent...... X-ray crystal structures of LeuBAT, an engineered monoamine-like version of the bacterial amino acid transporter LeuT. However, the binding orientation of fluoxetine is reversed in our experimentally supported model compared with the LeuBAT structures, emphasizing the need for careful experimental...... verification when extrapolating findings from crystal structures of bacterial transporters to human relatives. We find that the selectivity of fluoxetine and nisoxetine, a NET selective structural congener of fluoxetine, is controlled by residues in different regions of the transporters, indicating a complex...

  18. Molecular basis for the presence of glycosylated onco-foetal fibronectin in oral carcinomas

    DEFF Research Database (Denmark)

    Wandall, Hans H; Dabelsteen, Sally; Sørensen, Jens Ahm;

    2007-01-01

    Glycosylated onco-foetal fibronectin (GOF) deposited in the stroma of oral squamous cell carcinomas correlates with survival. One of the two polypeptide GalNAc-transferases, GalNAc-T3 or GalNAc-T6, is required for the biosynthesis of GOF by the initiation of a unique O-glycan in the alternative...... spliced IIICS region. Using cell culture experiments, immunohistochemical staining of primary tissue, and RT-PCR of tumour cells isolated by laser capture techniques we have examined the molecular basis for the production of GOF in oral carcinomas. Immuno-histochemical investigation confirmed the stromal...... deposition of GOF in oral carcinomas. However, neither GalNAc-T3 nor GalNAc-T6 could be detected in stromal fibroblasts. In contrast both transferases were present in the oral squamous carcinoma cells, suggesting that GOF is produced by the oral cancer cells and not only the stromal cells. RT-PCR analysis...

  19. Cellular and molecular basis of RV hypertrophy in congenital heart disease.

    Science.gov (United States)

    Iacobazzi, D; Suleiman, M-S; Ghorbel, M; George, S J; Caputo, M; Tulloh, R M

    2016-01-01

    RV hypertrophy (RVH) is one of the triggers of RV failure in congenital heart disease (CHD). Therefore, improving our understanding of the cellular and molecular basis of this pathology will help in developing strategic therapeutic interventions to enhance patient benefit in the future. This review describes the potential mechanisms that underlie the transition from RVH to RV failure. In particular, it addresses structural and functional remodelling that encompass contractile dysfunction, metabolic changes, shifts in gene expression and extracellular matrix remodelling. Both ischaemic stress and reactive oxygen species production are implicated in triggering these changes and will be discussed. Finally, RV remodelling in response to various CHDs as well as the potential role of biomarkers will be addressed.

  20. Molecular basis of early stages of Clostridium difficile infection: germination and colonization.

    Science.gov (United States)

    Sarker, Mahfuzur R; Paredes-Sabja, Daniel

    2012-08-01

    Clostridium difficile infections (CDIs) occur when antibiotic therapy disrupts the gastrointestinal flora, favoring infected C. difficile spores to germinate, outgrow, colonize and produce toxins. During CDI, C. difficile vegetative cells initiate the process of sporulation allowing a fraction of the spores to remain adhered to the intestinal surfaces. These spores, which are unaffected by antibiotic therapy commonly used for CDIs, then germinate, outgrow and recolonize the host's GI tract causing relapse of CDI. Consequently, the germination and colonization processes can be considered as the earliest and most essential steps for the development as well as relapse of CDI. The aim of this review is to provide an overview on the molecular basis involved in C. difficile spore germination and colonization.

  1. Structure of the thermolabile mutant aldolase B, A149P: molecular basis of hereditary fructose intolerance.

    Science.gov (United States)

    Malay, Ali D; Allen, Karen N; Tolan, Dean R

    2005-03-18

    Hereditary fructose intolerance (HFI) is a potentially lethal inborn error in metabolism caused by mutations in the aldolase B gene, which is critical for gluconeogenesis and fructose metabolism. The most common mutation, which accounts for 53% of HFI alleles identified worldwide, results in substitution of Pro for Ala at position 149. Structural and functional investigations of human aldolase B with the A149P substitution (AP-aldolase) have shown that the mutation leads to losses in thermal stability, quaternary structure, and activity. X-ray crystallography is used to reveal the structural basis of these perturbations. Crystals of AP-aldolase are grown at two temperatures (4 degrees C and 18 degrees C), and the structure solved to 3.0 angstroms resolution, using the wild-type structure as the phasing model. The structures reveal that the single residue substitution, A149P, causes molecular disorder around the site of mutation (residues 148-159), which is propagated to three adjacent beta-strand and loop regions (residues 110-129, 189-199, 235-242). Disorder in the 110-129-loop region, which comprises one subunit-subunit interface, provides an explanation for the disrupted quaternary structure and thermal instability. Greater structural perturbation, particularly at a Glu189-Arg148 salt bridge in the active-site architecture, is observed in the structure determined at 18 degrees C, which could explain the temperature-dependent loss in activity. The disorder revealed in these structures is far greater than that predicted by homology modeling and underscores the difficulties in predicting perturbations of protein structure and function by homology modeling alone. The AP-aldolase structure reveals the molecular basis of a hereditary disease and represents one of only a few structures known for mutant proteins at the root of the thousands of other inherited disorders.

  2. Convergent Partially Augmented Basis Sets for Post-Hartree-Fock Calculations of Molecular Properties and Reaction Barrier Heights.

    Science.gov (United States)

    Papajak, Ewa; Truhlar, Donald G

    2011-01-11

    We present sets of convergent, partially augmented basis set levels corresponding to subsets of the augmented "aug-cc-pV(n+d)Z" basis sets of Dunning and co-workers. We show that for many molecular properties a basis set fully augmented with diffuse functions is computationally expensive and almost always unnecessary. On the other hand, unaugmented cc-pV(n+d)Z basis sets are insufficient for many properties that require diffuse functions. Therefore, we propose using intermediate basis sets. We developed an efficient strategy for partial augmentation, and in this article, we test it and validate it. Sequentially deleting diffuse basis functions from the "aug" basis sets yields the "jul", "jun", "may", "apr", etc. basis sets. Tests of these basis sets for Møller-Plesset second-order perturbation theory (MP2) show the advantages of using these partially augmented basis sets and allow us to recommend which basis sets offer the best accuracy for a given number of basis functions for calculations on large systems. Similar truncations in the diffuse space can be performed for the aug-cc-pVxZ, aug-cc-pCVxZ, etc. basis sets.

  3. Molecular Origin of Color Variation in Firefly (Beetle) Bioluminescence: A Chemical Basis for Biological Imaging.

    Science.gov (United States)

    Hirano, Takashi

    2016-01-01

    Firefly shows bioluminescence by "luciferin-luciferase" (L-L) reaction using luciferin, luciferase, ATP and O2. The chemical photon generation by an enzymatic reaction is widely utilized for analytical methods including biological imaging in the life science fields. To expand photondetecting analyses with firefly bioluminescence, it is important for users to understand the chemical basis of the L-L reaction. In particular, the emission color variation of the L-L reaction is one of the distinguishing characteristics for multicolor luciferase assay and in vivo imaging. From the viewpoint of fundamental chemistry, this review explains the recent progress in the studies on the molecular mechanism of emission color variation after showing the outline of the reaction mechanism of the whole L-L reaction. On the basis of the mechanism, the progresses in organic synthesis of luciferin analogs modulating their emission colors are also presented to support further developments of red/near infrared in vivo biological imaging utility of firefly bioluminescence.

  4. [Molecular genetic basis for para-Bombay phenotypes in two cases].

    Science.gov (United States)

    He, Yang-Ming; Xu, Xian-Guo; Zhu, Fa-Ming; Yan, Li-Xing

    2007-06-01

    This study was purposed to investigate the molecular genetics basis for para-Bombay phenotype. The para-Bombay phenotype of two probands was identified by routine serological techniques. The full coding region of alpha (1, 2) fucosyltransferase gene (FUT1 and FUT2) in the probands was amplified by polymerase chain reaction and the amplified fragments were directly sequenced, meanwhile the mutations of FUT1 were also identified by TOPO TA cloning sequence method. The results indicated that two heterozygous mutations were detected by directly sequencing in two probands: AG deletion at position 547 - 552 and C to T mutation at position 658. Two different mutations were confirmed to be true compound heterozygotes with each mutation on a separate homologous chromosome by TOPO TA cloning sequence method. AG deletion at position 547 - 552 caused a reading frame shift and a premature stop codon. C658T mutation resulted in Arg-->Cys at amino acid position 220. It is suggested that the FUT1 mutation of two probands are compound heterozygous mutation with different chromosomes, which are named h1h3 and may be the genetics basis of para-Bombay phenotype.

  5. Molecular basis of differential B-pentamer stability of Shiga toxins 1 and 2.

    Directory of Open Access Journals (Sweden)

    Deborah G Conrady

    Full Text Available Escherichia coli strain O157:H7 is a major cause of food poisoning that can result in severe diarrhea and, in some cases, renal failure. The pathogenesis of E. coli O157:H7 is in large part due to the production of Shiga toxin (Stx, an AB(5 toxin that consists of a ribosomal RNA-cleaving A-subunit surrounded by a pentamer of receptor-binding B subunits. There are two major isoforms, Stx1 and Stx2, which differ dramatically in potency despite having 57% sequence identity. Animal studies and epidemiological studies show Stx2 is associated with more severe disease. Although the molecular basis of this difference is unknown, data suggest it is associated with the B-subunit. Mass spectrometry studies have suggested differential B-pentamer stability between Stx1 and Stx2. We have examined the relative stability of the B-pentamers in solution. Analytical ultracentrifugation using purified B-subunits demonstrates that Stx2B, the more deadly isoform, shows decreased pentamer stability compared to Stx1B (EC(50 = 2.3 µM vs. EC(50 = 0.043 µM for Stx1B. X-ray crystal structures of Stx1B and Stx2B identified a glutamine in Stx2 (versus leucine in Stx1 within the otherwise strongly hydrophobic interface between B-subunits. Interchanging these residues switches the stability phenotype of the B-pentamers of Stx1 and Stx2, as demonstrated by analytical ultracentrifugation and circular dichroism. These studies demonstrate a profound difference in stability of the B-pentamers in Stx1 and Stx2, illustrate the mechanistic basis for this differential stability, and provide novel reagents to test the basis for differential pathogenicity of these toxins.

  6. Molecular Basis of Differential B-Pentamer Stability of Shiga Toxins 1 and 2

    Energy Technology Data Exchange (ETDEWEB)

    Conrady, Deborah G.; Flagler, Michael J.; Friedmann, David R.; Vander Wielen, Bradley D.; Kovall, Rhett A.; Weiss, Alison A.; Herr, Andrew B. (UCIN-MED)

    2012-06-27

    Escherichia coli strain O157:H7 is a major cause of food poisoning that can result in severe diarrhea and, in some cases, renal failure. The pathogenesis of E. coli O157:H7 is in large part due to the production of Shiga toxin (Stx), an AB{sub 5} toxin that consists of a ribosomal RNA-cleaving A-subunit surrounded by a pentamer of receptor-binding B subunits. There are two major isoforms, Stx1 and Stx2, which differ dramatically in potency despite having 57% sequence identity. Animal studies and epidemiological studies show Stx2 is associated with more severe disease. Although the molecular basis of this difference is unknown, data suggest it is associated with the B-subunit. Mass spectrometry studies have suggested differential B-pentamer stability between Stx1 and Stx2. We have examined the relative stability of the B-pentamers in solution. Analytical ultracentrifugation using purified B-subunits demonstrates that Stx2B, the more deadly isoform, shows decreased pentamer stability compared to Stx1B (EC{sub 50} = 2.3 {micro}M vs. EC{sub 50} = 0.043 {micro}M for Stx1B). X-ray crystal structures of Stx1B and Stx2B identified a glutamine in Stx2 (versus leucine in Stx1) within the otherwise strongly hydrophobic interface between B-subunits. Interchanging these residues switches the stability phenotype of the B-pentamers of Stx1 and Stx2, as demonstrated by analytical ultracentrifugation and circular dichroism. These studies demonstrate a profound difference in stability of the B-pentamers in Stx1 and Stx2, illustrate the mechanistic basis for this differential stability, and provide novel reagents to test the basis for differential pathogenicity of these toxins.

  7. "Zwitterionic Proton Sponge" Hydrogen Bonding Investigations on the Basis of Car-Parrinello Molecular Dynamics.

    Science.gov (United States)

    Jezierska, Aneta; Panek, Jarosław J

    2015-06-22

    1,8-Bis(dimethylamino)-4,5-dihydroxynaphthalene has been investigated on the basis of static DFT computations and Car-Parrinello molecular dynamics. The simulations were performed in the gas phase and in the solid state. The studied "zwitterionic proton sponge" possesses two, short intramolecular hydrogen bonds (O-H···O and N-H···N) classified as Low Barrier Hydrogen Bonds (LBHBs); therefore, the system studied is strongly anharmonic. In addition, the compound exists as a "zwitterion" in solution and in the solid state, thus the intramolecular hydrogen bonds belong to the class of charge-assisted interactions. The applied quantum-chemical methods enabled investigations of metric and spectroscopic parameters of the molecule. The time-evolution investigations of the H-bonding showed a strong delocalization of the bridge protons and their high mobility, reflected in the low barriers on the free energy surfaces. Frequent proton transfer phenomena were noticed. The power spectra of atomic velocity were computed to analyze the vibrational features associated with O-H and N-H stretching. A broad absorption was indicated for both hydrogen bridges. For the first time, Car-Parrinello molecular dynamics results are reported for the compound, and they indicate a broad, shallow but not barrierless, potential well for each of the bridge protons.

  8. Molecular basis for bacterial peptidoglycan recognition by LysM domains.

    Science.gov (United States)

    Mesnage, Stéphane; Dellarole, Mariano; Baxter, Nicola J; Rouget, Jean-Baptiste; Dimitrov, Jordan D; Wang, Ning; Fujimoto, Yukari; Hounslow, Andrea M; Lacroix-Desmazes, Sébastien; Fukase, Koichi; Foster, Simon J; Williamson, Michael P

    2014-06-30

    Carbohydrate recognition is essential for growth, cell adhesion and signalling in all living organisms. A highly conserved carbohydrate binding module, LysM, is found in proteins from viruses, bacteria, fungi, plants and mammals. LysM modules recognize polysaccharides containing N-acetylglucosamine (GlcNAc) residues including peptidoglycan, an essential component of the bacterial cell wall. However, the molecular mechanism underpinning LysM-peptidoglycan interactions remains unclear. Here we describe the molecular basis for peptidoglycan recognition by a multimodular LysM domain from AtlA, an autolysin involved in cell division in the opportunistic bacterial pathogen Enterococcus faecalis. We explore the contribution of individual modules to the binding, identify the peptidoglycan motif recognized, determine the structures of free and bound modules and reveal the residues involved in binding. Our results suggest that peptide stems modulate LysM binding to peptidoglycan. Using these results, we reveal how the LysM module recognizes the GlcNAc-X-GlcNAc motif present in polysaccharides across kingdoms.

  9. [Study on the molecular genetics basis for one para-Bombay phenotype].

    Science.gov (United States)

    Hong, Xiao-Zhen; Shao, Xiao-Chun; Xu, Xian-Guo; Hu, Qing-Fa; Wu, Jun-Jie; Zhu, Fa-Ming; Fu, Qi-Hua; Yan, Li-Xing

    2005-12-01

    To investigate the molecular genetics basis for one para-Bombay phenotype, the red blood cell phenotype of the proband was characterized by standard serological techniques. Exon 6 and 7 of ABO gene, the entire coding region of FUT1 gene and FUT2 gene were amplified by polymerase chain reaction from genomic DNA of the proband respectively. The PCR products were purified by agarose gels and directly sequenced. The PCR-SSP and genescan were performed to confirm the mutations detected by sequencing. The results showed that the proband ABO genotype was A(102)A(102). Two heterozygous mutations of FUT1 gene, an A to G transition at position 682 and AG deletion at position 547-552 were detected in the proband. A682G could cause transition of Met-->Val at amino acid position 228, AG deletion at position 547-552 caused a reading frame shift and a premature stop codon. The FUT2 genotype was heterozygous for a functional allele Se(357) and a weakly functional allele Se(357), 385 (T/T homozygous at position 357 and A/T heterozygous at 385 position). It is concluded that the compound heterozygous mutation--a novel A682G missense mutation and a 547-552 del AG is the molecular mechanism of this para-Bombay phenotype.

  10. Molecular basis of human transcobalamin II deficiency in an affected family

    Energy Technology Data Exchange (ETDEWEB)

    Li, N.; Seetharam, S.; Seetharam, B. [Medical College of Wisconsin, Milwaukee, WI (United States)] [and others

    1994-09-01

    Transcobalamin II (TC II) deficiency is an autosomal recessive disease leading to cobalamin (Cbl, Vitamin B{sub 12}) deficiency. Patients with this disorder fail to absorb and transport Cbl across cellular membranes and develop Cbl deficiency, symptoms of which include failure to thrive, megaloblastic anemia, impaired immunodefence and neurological disorders. The molecular basis for this disease is not known. By means of Southern blotting and sequence analysis of TC II, cDNA amplified from fibroblasts of an affected child and his parents, we have identified two mutant TC II alleles. The maternally derived allele had a gross deletion, while the paternally derived allele had a 4-nucleotide ({sup 1023}TCTG) deletion which caused a reading frame shift and generation of a premature termination codon, 146 nucleotides downstream from the deletion. Both these deletions caused markedly reduced levels of TC II mRNA and protein. In addition, these two deletions were unique to this family and were not detected in four other unrelated TC II deficient patients who also exhibited the same (TC II protein/mRNA deficiency) phenotypes. Based on this study we suggest, (1) that the molecular defect in the most common form of human TC II deficiency (lack of immunoprecipitable plasma TC II) is heterogeneous and (2) these mutations cause TC II mRNA and protein deficiency leading to defective plasma transport of Cbl and the development of Cbl deficiency.

  11. The molecular basis of autosomal recessive diseases among the Arabs and Druze in Israel.

    Science.gov (United States)

    Zlotogora, Joël

    2010-11-01

    The Israeli population mainly includes Jews, Muslim and Christian Arabs, and Druze In the last decade, data on genetic diseases present in the population have been systematically collected and are available online in the Israeli national genetic database ( http://www.goldenhelix.org/server/israeli ). In the non-Jewish population, up to 1 July 2010, the database included molecular data on six diseases relatively frequent in the whole population: thalassemia, familial Mediterranean fever (FMF), cystic fibrosis, deafness, phenylketonuria and congenital adrenal hyperplasia, as well as data on 195 autosomal recessive diseases among Muslim Israeli Arabs, 11 among the Christian Arabs and 31 among Druze. A single mutation was characterized in 149 out of the 238 rare disorders for which the molecular basis was known. In many diseases, mutation had never been observed in any other population and was present in one family only suggesting that it occurred as a de novo event. In other diseases, the mutation was present in more than one community or even in other populations such as Bedouins from the Arab peninsula or Christians from Lebanon. In the 89 other disorders, more than one mutation was characterized either in the same gene or in more than one gene. While it is probable that most of these cases represent random events in some cases such as Bardet Biedl among the Bedouins, the reason may be a selective advantage to the heterozygotes.

  12. Peptide dynamics by molecular dynamics simulation and diffusion theory method with improved basis sets.

    Science.gov (United States)

    Hsu, Po Jen; Lai, S K; Rapallo, Arnaldo

    2014-03-14

    Improved basis sets for the study of polymer dynamics by means of the diffusion theory, and tests on a melt of cis-1,4-polyisoprene decamers, and a toluene solution of a 71-mer syndiotactic trans-1,2-polypentadiene were presented recently [R. Gaspari and A. Rapallo, J. Chem. Phys. 128, 244109 (2008)]. The proposed hybrid basis approach (HBA) combined two techniques, the long time sorting procedure and the maximum correlation approximation. The HBA takes advantage of the strength of these two techniques, and its basis sets proved to be very effective and computationally convenient in describing both local and global dynamics in cases of flexible synthetic polymers where the repeating unit is a unique type of monomer. The question then arises if the same efficacy continues when the HBA is applied to polymers of different monomers, variable local stiffness along the chain and with longer persistence length, which have different local and global dynamical properties against the above-mentioned systems. Important examples of this kind of molecular chains are the proteins, so that a fragment of the protein transthyretin is chosen as the system of the present study. This peptide corresponds to a sequence that is structured in β-sheets of the protein and is located on the surface of the channel with thyroxin. The protein transthyretin forms amyloid fibrils in vivo, whereas the peptide fragment has been shown [C. P. Jaroniec, C. E. MacPhee, N. S. Astrof, C. M. Dobson, and R. G. Griffin, Proc. Natl. Acad. Sci. U.S.A. 99, 16748 (2002)] to form amyloid fibrils in vitro in extended β-sheet conformations. For these reasons the latter is given considerable attention in the literature and studied also as an isolated fragment in water solution where both experimental and theoretical efforts have indicated the propensity of the system to form β turns or α helices, but is otherwise predominantly unstructured. Differing from previous computational studies that employed implicit

  13. Molecular Basis of β-Thalassemia Intermedia in Erbil Province of Iraqi Kurdistan.

    Science.gov (United States)

    Shamoon, Rawand P; Al-Allawi, Nasir A S; Cappellini, Maria D; Di Pierro, Elena; Brancaleoni, Valentina; Granata, Francesca

    2015-01-01

    β-Thalassemia intermedia (β-TI) is a clinical term describing a range of clinical phenotypes that are intermediate in severity between the carrier state and β-thalassemia major (β-TM). To characterize the molecular basis of β-TI in Erbil Province, Northern Iraq, 83 unrelated patients were investigated. Detection of β-globin gene mutations was carried out by reverse hybridization assay and direct gene sequencing. All patients were screened for the XmnI polymorphism by direct sequencing of HBG2 ((G)γ promoter gene). Detection of α-globin gene deletions and triplication was carried out using the reverse hybridization assay. Four main molecular patterns were identified in association with the β-TI phenotype, namely: β(+)/β(+) (38.5%), β(+)/β(0) (21.6%), β(0)/β(0) (31.3%), and β(0)/wild type (8.4%). IVS-I-6 (T > C) was the most frequently encountered mutation (55 alleles, 34.6%), followed by IVS-II-1 (G > A) and codon 8 (-AA); furthermore, we report for the first time from Iraq two β(+) mutations, -87 (C > G) and 5' untranslated region (5'UTR) +22 (G > A). The XmnI polymorphism was detected in 47.0% of patients, mainly in association with the β(0)/β(0) genotype. The α-globin gene deletions were encountered in four cases, including one case with (- -(FIL)) double gene deletion, a report that is the first from our country. The α-globin gene triplication was detected in five of the seven heterozygous β-thalassemia (β-thal) patients. Similar to other Mediterranean countries, inheritance of mild β-globin mutations was the main molecular pattern underlying β-TI in our patients followed by the ameliorating effect of the XmnI polymorphism.

  14. Molecular basis for the Kallmann syndrome-linked fibroblast growth factor receptor mutation

    Energy Technology Data Exchange (ETDEWEB)

    Thurman, Ryan D.; Kathir, Karuppanan Muthusamy; Rajalingam, Dakshinamurthy [Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701 (United States); Kumar, Thallapuranam K. Suresh, E-mail: sthalla@uark.edu [Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701 (United States)

    2012-08-31

    Highlights: Black-Right-Pointing-Pointer The structural basis of the Kallmann syndrome is elucidated. Black-Right-Pointing-Pointer Kallmann syndrome mutation (A168S) induces a subtle conformational change(s). Black-Right-Pointing-Pointer Structural interactions mediated by beta-sheet G are most perturbed. Black-Right-Pointing-Pointer Ligand (FGF)-receptor interaction(s) is completely abolished by Kallmann mutation. Black-Right-Pointing-Pointer Kallmann mutation directly affects the FGF signaling process. -- Abstract: Kallmann syndrome (KS) is a developmental disease that expresses in patients as hypogonadotropic hypogonadism and anosmia. KS is commonly associated with mutations in the extracellular D2 domain of the fibroblast growth factor receptor (FGFR). In this study, for the first time, the molecular basis for the FGFR associated KS mutation (A168S) is elucidated using a variety of biophysical experiments, including multidimensional NMR spectroscopy. Secondary and tertiary structural analysis using far UV circular dichroism, fluorescence and limited trypsin digestion assays suggest that the KS mutation induces subtle tertiary structure change in the D2 domain of FGFR. Results of isothermal titration calorimetry experiments show the KS mutation causes a 10-fold decrease in heparin binding affinity and also a complete loss in ligand (FGF-1) binding. {sup 1}H-{sup 15}N chemical perturbation data suggest that complete loss in the ligand (FGF) binding affinity is triggered by a subtle conformational change that disrupts crucial structural interactions in both the heparin and the FGF binding sites in the D2 domain of FGFR. The novel findings reported in this study are expected to provide valuable clues toward a complete understanding of the other genetic diseases linked to mutations in the FGFR.

  15. Molecular and Structural Basis of Inner Core Lipopolysaccharide Alterations in Escherichia coli

    Science.gov (United States)

    Klein, Gracjana; Müller-Loennies, Sven; Lindner, Buko; Kobylak, Natalia; Brade, Helmut; Raina, Satish

    2013-01-01

    It is well established that lipopolysaccharide (LPS) often carries nonstoichiometric substitutions in lipid A and in the inner core. In this work, the molecular basis of inner core alterations and their physiological significance are addressed. A new inner core modification of LPS is described, which arises due to the addition of glucuronic acid on the third heptose with a concomitant loss of phosphate on the second heptose. This was shown by chemical and structural analyses. Furthermore, the gene whose product is responsible for the addition of this sugar was identified in all Escherichia coli core types and in Salmonella and was designated waaH. Its deduced amino acid sequence exhibits homology to glycosyltransferase family 2. The transcription of the waaH gene is positively regulated by the PhoB/R two-component system in a growth phase-dependent manner, which is coordinated with the transcription of the ugd gene explaining the genetic basis of this modification. Glucuronic acid modification was observed in E. coli B, K12, R2, and R4 core types and in Salmonella. We also show that the phosphoethanolamine (P-EtN) addition on heptose I in E. coli K12 requires the product of the ORF yijP, a new gene designated as eptC. Incorporation of P-EtN is also positively regulated by PhoB/R, although it can occur at a basal level without a requirement for any regulatory inducible systems. This P-EtN modification is essential for resistance to a variety of factors, which destabilize the outer membrane like the addition of SDS or challenge to sublethal concentrations of Zn2+. PMID:23372159

  16. Molecular basis for effects of carcinogenic heavy metals on inducible gene expression.

    Science.gov (United States)

    Hamilton, J W; Kaltreider, R C; Bajenova, O V; Ihnat, M A; McCaffrey, J; Turpie, B W; Rowell, E E; Oh, J; Nemeth, M J; Pesce, C A; Lariviere, J P

    1998-08-01

    Certain forms of the heavy metals arsenic and chromium are considered human carcinogens, although they are believed to act through very different mechanisms. Chromium(VI) is believed to act as a classic and mutagenic agent, and DNA/chromatin appears to be the principal target for its effects. In contrast, arsenic(III) is considered nongenotoxic, but is able to target specific cellular proteins, principally through sulfhydryl interactions. We had previously shown that various genotoxic chemical carcinogens, including chromium (VI), preferentially altered expression of several inducible genes but had little or no effect on constitutive gene expression. We were therefore interested in whether these carcinogenic heavy metals might target specific but distinct sites within cells, leading to alterations in gene expression that might contribute to the carcinogenic process. Arsenic(III) and chromium(VI) each significantly altered both basal and hormone-inducible expression of a model inducible gene, phosphoenolpyruvate carboxykinase (PEPCK), at nonovertly toxic doses in the chick embryo in vivo and rat hepatoma H411E cells in culture. We have recently developed two parallel cell culture approaches for examining the molecular basis for these effects. First, we are examining the effects of heavy metals on expression and activation of specific transcription factors known to be involved in regulation of susceptible inducible genes, and have recently observed significant but different effects of arsenic(III) and chromium(VI) on nuclear transcription factor binding. Second, we have developed cell lines with stably integrated PEPCK promoter-luciferase reporter gene constructs to examine effects of heavy metals on promoter function, and have also recently seen profound effects induced by both chromium(VI) and arsenic(III) in this system. These model systems should enable us to be able to identify the critical cis (DNA) and trans (protein) cellular targets of heavy metal exposure

  17. The Burmese python genome reveals the molecular basis for extreme adaptation in snakes.

    Science.gov (United States)

    Castoe, Todd A; de Koning, A P Jason; Hall, Kathryn T; Card, Daren C; Schield, Drew R; Fujita, Matthew K; Ruggiero, Robert P; Degner, Jack F; Daza, Juan M; Gu, Wanjun; Reyes-Velasco, Jacobo; Shaney, Kyle J; Castoe, Jill M; Fox, Samuel E; Poole, Alex W; Polanco, Daniel; Dobry, Jason; Vandewege, Michael W; Li, Qing; Schott, Ryan K; Kapusta, Aurélie; Minx, Patrick; Feschotte, Cédric; Uetz, Peter; Ray, David A; Hoffmann, Federico G; Bogden, Robert; Smith, Eric N; Chang, Belinda S W; Vonk, Freek J; Casewell, Nicholas R; Henkel, Christiaan V; Richardson, Michael K; Mackessy, Stephen P; Bronikowski, Anne M; Bronikowsi, Anne M; Yandell, Mark; Warren, Wesley C; Secor, Stephen M; Pollock, David D

    2013-12-17

    Snakes possess many extreme morphological and physiological adaptations. Identification of the molecular basis of these traits can provide novel understanding for vertebrate biology and medicine. Here, we study snake biology using the genome sequence of the Burmese python (Python molurus bivittatus), a model of extreme physiological and metabolic adaptation. We compare the python and king cobra genomes along with genomic samples from other snakes and perform transcriptome analysis to gain insights into the extreme phenotypes of the python. We discovered rapid and massive transcriptional responses in multiple organ systems that occur on feeding and coordinate major changes in organ size and function. Intriguingly, the homologs of these genes in humans are associated with metabolism, development, and pathology. We also found that many snake metabolic genes have undergone positive selection, which together with the rapid evolution of mitochondrial proteins, provides evidence for extensive adaptive redesign of snake metabolic pathways. Additional evidence for molecular adaptation and gene family expansions and contractions is associated with major physiological and phenotypic adaptations in snakes; genes involved are related to cell cycle, development, lungs, eyes, heart, intestine, and skeletal structure, including GRB2-associated binding protein 1, SSH, WNT16, and bone morphogenetic protein 7. Finally, changes in repetitive DNA content, guanine-cytosine isochore structure, and nucleotide substitution rates indicate major shifts in the structure and evolution of snake genomes compared with other amniotes. Phenotypic and physiological novelty in snakes seems to be driven by system-wide coordination of protein adaptation, gene expression, and changes in the structure of the genome.

  18. The Burmese python genome reveals the molecular basis for extreme adaptation in snakes

    Science.gov (United States)

    Castoe, Todd A.; de Koning, A. P. Jason; Hall, Kathryn T.; Card, Daren C.; Schield, Drew R.; Fujita, Matthew K.; Ruggiero, Robert P.; Degner, Jack F.; Daza, Juan M.; Gu, Wanjun; Reyes-Velasco, Jacobo; Shaney, Kyle J.; Castoe, Jill M.; Fox, Samuel E.; Poole, Alex W.; Polanco, Daniel; Dobry, Jason; Vandewege, Michael W.; Li, Qing; Schott, Ryan K.; Kapusta, Aurélie; Minx, Patrick; Feschotte, Cédric; Uetz, Peter; Ray, David A.; Hoffmann, Federico G.; Bogden, Robert; Smith, Eric N.; Chang, Belinda S. W.; Vonk, Freek J.; Casewell, Nicholas R.; Henkel, Christiaan V.; Richardson, Michael K.; Mackessy, Stephen P.; Bronikowski, Anne M.; Yandell, Mark; Warren, Wesley C.; Secor, Stephen M.; Pollock, David D.

    2013-01-01

    Snakes possess many extreme morphological and physiological adaptations. Identification of the molecular basis of these traits can provide novel understanding for vertebrate biology and medicine. Here, we study snake biology using the genome sequence of the Burmese python (Python molurus bivittatus), a model of extreme physiological and metabolic adaptation. We compare the python and king cobra genomes along with genomic samples from other snakes and perform transcriptome analysis to gain insights into the extreme phenotypes of the python. We discovered rapid and massive transcriptional responses in multiple organ systems that occur on feeding and coordinate major changes in organ size and function. Intriguingly, the homologs of these genes in humans are associated with metabolism, development, and pathology. We also found that many snake metabolic genes have undergone positive selection, which together with the rapid evolution of mitochondrial proteins, provides evidence for extensive adaptive redesign of snake metabolic pathways. Additional evidence for molecular adaptation and gene family expansions and contractions is associated with major physiological and phenotypic adaptations in snakes; genes involved are related to cell cycle, development, lungs, eyes, heart, intestine, and skeletal structure, including GRB2-associated binding protein 1, SSH, WNT16, and bone morphogenetic protein 7. Finally, changes in repetitive DNA content, guanine-cytosine isochore structure, and nucleotide substitution rates indicate major shifts in the structure and evolution of snake genomes compared with other amniotes. Phenotypic and physiological novelty in snakes seems to be driven by system-wide coordination of protein adaptation, gene expression, and changes in the structure of the genome. PMID:24297902

  19. Molecular basis for H blood group deficiency in Bombay (Oh) and para-Bombay individuals.

    Science.gov (United States)

    Kelly, R J; Ernst, L K; Larsen, R D; Bryant, J G; Robinson, J S; Lowe, J B

    1994-06-21

    The penultimate step in the biosynthesis of the human ABO blood group oligosaccharide antigens is catalyzed by alpha-(1,2)-fucosyltransferase(s) (GDP-L-fucose: beta-D-galactoside 2-alpha-L-fucosyltransferase, EC 2.4.1.69), whose expression is determined by the H and Secretor (SE) blood group loci (also known as FUT1 and FUT2, respectively). These enzymes construct Fuc alpha 1-->2Gal beta-linkages, known as H determinants, which are essential precursors to the A and B antigens. Erythrocytes from individuals with the rare Bombay and para-Bombay blood group phenotypes are deficient in H determinants, and thus A and B determinants, as a consequence of apparent homozygosity for null alleles at the H locus. We report a molecular analysis of a human alpha-(1,2)-fucosyltransferase gene, thought to correspond to the H blood group locus, in a Bombay pedigree and a para-Bombay pedigree. We find inactivating point mutations in the coding regions of both alleles of this gene in each H-deficient individual. These results define the molecular basis for H blood group antigen deficiency in Bombay and para-Bombay phenotypes, provide compelling evidence that this gene represents the human H blood group locus, and strongly support a hypothesis that the H and SE loci represent distinct alpha-(1,2)-fucosyltransferase genes. Candidate sequences for the human SE locus are identified by low-stringency Southern blot hybridization analyses, using a probe derived from the H alpha-(1,2)-fucosyltransferase gene.

  20. The molecular basis of beta-thalassemia intermedia in southern China: genotypic heterogeneity and phenotypic diversity

    Directory of Open Access Journals (Sweden)

    Sun Manna

    2010-02-01

    Full Text Available Abstract Background The clinical syndrome of thalassemia intermedia (TI results from the β-globin genotypes in combination with factors to produce fetal haemoglobin (HbF and/or co-inheritance of α-thalassemia. However, very little is currently known of the molecular basis of Chinese TI patients. Methods We systematically analyzed and characterized β-globin genotypes, α-thalassemia determinants, and known primary genetic modifiers linked to the production of HbF and the aggravation of α/β imbalance in 117 Chinese TI patients. Genotype-phenotype correlations were analyzed based on retrospective clinical observations. Results A total of 117 TI patients were divided into two major groups, namely heterozygous β-thalassemia (n = 20 in which 14 were characterized as having a mild TI with the Hb levels of 68-95 g/L except for five co-inherited αααanti-3.7 triplication and one carried a dominant mutation; and β-thalassemia homozygotes or compound heterozygotes for β-thalassemia and other β-globin defects in which the β+-thalassemia mutation was the most common (49/97, hemoglobin E (HbE variants was second (27/97, and deletional hereditary persistence of fetal hemoglobin (HPFH or δβ-thalassemia was third (11/97. Two novel mutations, Term CD+32(A→C and Cap+39(C→T, have been detected. Conclusions Chinese TI patients showed considerable heterogeneity, both phenotypically and genotypically. The clinical outcomes of our TI patients were mostly explained by the genotypes linked to the β- and α-globin gene cluster. However, for a group of 14 patients (13 β0/βN and 1 β+/βN with known heterozygous mutations of β-thalassemia and three with homozygous β-thalassemia (β0/β0, the existence of other causative genetic determinants is remaining to be molecularly defined.

  1. Molecular basis of weak D and DEL in Han population in Anhui Province, China

    Institute of Scientific and Technical Information of China (English)

    CHEN Qing; LI Ming; LI Min; LU Xue-song; L(U) Rong; SUN Jun; LIU Zhong

    2012-01-01

    Background Rh blood group system is the most complex and immunogenetic blood group system.Prevalent RHD alleles varied in different populations.The purpose of this study is to determine the molecular basis of weak D and DEL phenotype in Anhui Chinese Han population.Methods The D antigen was determined with IgM monoclonal anti-D conformed to the guidelines for donor testing in China.Weak D samples were identified by an indirect antiglobulin test.DEL phenotype was determined by adsorption and elution test.All the RHD 10 exons were screened by PCR with sequence-specific priming or sequenced for the first-time donors who typed weak D,DEL or D negative by serologic test.Results Of all the 30 799 blood donors,155 blood samples were found D negative with IgM anti-D; 34 blood samples were found D positive by indirect antiglobulin test or absorption elution test.RHD alleles were identified by nucleotide sequencing.Total 4 RHD alleles were found including two new.One hundred and twenty of 155 (77.4%) of the serologically D negative samples lacked the RHD gene.One D negative was RHD(615de12).Thirty-two of 155 (20.6%)carried RHD(K409K) among them one carrying 1227G>A and 845G>A.Two of 155 (1.3%) was weak D type 15.Conclusions In this study at the molecular level,all DEL phenotype is RHD(K40gK); weak D type 15 is the prevalent weak D allele in Anhui Chinese Han population.Additionally,an improved more efficient method was adopted to amplify all the RHD exons in one PCR program.Our study added to the understanding of molecular mechanisms underlying D antigen expression in Anhui Han population and provided useful information for adopting suitable genotyping strategies in routine use.

  2. Molecular basis for the dissociation dynamics of protein A-immunoglobulin G1 complex.

    Directory of Open Access Journals (Sweden)

    Fu-Feng Liu

    Full Text Available Staphylococcus aureus protein A (SpA is the most popular affinity ligand for immunoglobulin G1 (IgG1. However, the molecular basis for the dissociation dynamics of SpA-IgG1 complex is unclear. Herein, coarse-grained (CG molecular dynamics (MD simulations with the Martini force field were used to study the dissociation dynamics of the complex. The CG-MD simulations were first verified by the agreement in the structural and interactional properties of SpA and human IgG1 (hIgG1 in the association process between the CG-MD and all-atom MD at different NaCl concentrations. Then, the CG-MD simulation studies focused on the molecular insight into the dissociation dynamics of SpA-hIgG1 complex at pH 3.0. It is found that there are four steps in the dissociation process of the complex. First, there is a slight conformational adjustment of helix II in SpA. This is followed by the phenomena that the electrostatic interactions provided by the three hot spots (Glu143, Arg146 and Lys154 of helix II of SpA break up, leading to the dissociation of helix II from the binding site of hIgG1. Subsequently, breakup of the hydrophobic interactions between helix I (Phe132, Tyr133 and His137 in SpA and hIgG1 occurs, resulting in the disengagement of helix I from its binding site of hIgG1. Finally, the non-specific interactions between SpA and hIgG1 decrease slowly till disappearance, leading to the complete dissociation of the SpA-hIgG1 complex. This work has revealed that CG-MD coupled with the Martini force field is an effective method for studying the dissociation dynamics of protein-protein complex.

  3. Molecular Basis of NusG-mediated Regulation of Rho-dependent Transcription Termination in Bacteria.

    Science.gov (United States)

    Valabhoju, Vishalini; Agrawal, Sonia; Sen, Ranjan

    2016-10-21

    The bacterial transcription elongation factor NusG stimulates the Rho-dependent transcription termination through a direct interaction with Rho. The mechanistic basis of NusG dependence of the Rho function is not known. Here, we describe Rho* mutants I168V, R221C/A, and P235H that do not require NusG for their termination function. These Rho* mutants have acquired new properties, which otherwise would have been imparted by NusG. A detailed analyses revealed that they have more stable interactions at the secondary RNA binding sites of Rho, which reduced the lag in initiating its ATPase as well as the translocase activities. These more stable interactions arose from the significant spatial re-orientations of the P, Q, and R structural loops of the Rho central channel. We propose that NusG imparts similar conformational changes in the central channel of Rho, yielding faster isomerization of the open to the closed hexameric states of the latter during its RNA-loading step. This acceleration stabilizes the Rho-RNA interactions at many terminators having suboptimal rut sites, thus making Rho-NusG interactions so essential in vivo Finally, identification of the NusG binding sites on the Rho hexamer led us to conclude that the former exerts its effect allosterically. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Deciphering the molecular basis of ammonium uptake and transport in maritime pine.

    Science.gov (United States)

    Castro-Rodríguez, Vanessa; Assaf-Casals, Iman; Pérez-Tienda, Jacob; Fan, Xiaorong; Avila, Concepción; Miller, Anthony; Cánovas, Francisco M

    2016-08-01

    Ammonium is the predominant form of inorganic nitrogen in the soil of coniferous forests. Despite the ecological and economic importance of conifers, the molecular basis of ammonium uptake and transport in this group of gymnosperms is largely unknown. In this study, we describe the functional characterization of members of the AMT gene family in Pinus pinaster: PpAMT1.1, PpAMT1.2 and PpAMT1.3 (subfamily 1) and PpAMT2.1 and PpAMT2.3 (subfamily 2). Our phylogenetic analysis indicates that in conifers, all members of the AMT1 subfamily evolved from a common ancestor that is evolutionarily related to the ancient PpAMT1.2 gene. Individual AMT genes are developmentally and nutritionally regulated, and their transcripts are specifically distributed in different organs. PpAMT1.3 was predominantly expressed in the roots, particularly during N starvation and mycorrhizal interaction, whereas PpAMT2.3 was preferentially expressed in lateral roots. Immunolocalization studies of roots with varied nitrogen availability revealed that PpAMT1 and PpAMT2 proteins play complementary roles in the uptake of external ammonium. Heterologous expression in yeast and Xenopus oocytes revealed that the AMT genes encode functional transporters with different kinetics and with different capacities for ammonium transport. Our results provide new insights on how nitrogen is acquired and transported in conifers.

  5. Genomic analysis reveals the molecular basis for capsule loss in the group B Streptococcus population.

    Science.gov (United States)

    Rosini, Roberto; Campisi, Edmondo; De Chiara, Matteo; Tettelin, Hervé; Rinaudo, Daniela; Toniolo, Chiara; Metruccio, Matteo; Guidotti, Silvia; Sørensen, Uffe B Skov; Kilian, Mogens; Ramirez, Mario; Janulczyk, Robert; Donati, Claudio; Grandi, Guido; Margarit, Immaculada

    2015-01-01

    The human and bovine bacterial pathogen Streptococcus agalactiae (Group B Streptococcus, GBS) expresses a thick polysaccharide capsule that constitutes a major virulence factor and vaccine target. GBS can be classified into ten distinct serotypes differing in the chemical composition of their capsular polysaccharide. However, non-typeable strains that do not react with anti-capsular sera are frequently isolated from colonized and infected humans and cattle. To gain a comprehensive insight into the molecular basis for the loss of capsule expression in GBS, a collection of well-characterized non-typeable strains was investigated by genome sequencing. Genome based phylogenetic analysis extended to a wide population of sequenced strains confirmed the recently observed high clonality among GBS lineages mainly containing human strains, and revealed a much higher degree of diversity in the bovine population. Remarkably, non-typeable strains were equally distributed in all lineages. A number of distinct mutations in the cps operon were identified that were apparently responsible for inactivation of capsule synthesis. The most frequent genetic alterations were point mutations leading to stop codons in the cps genes, and the main target was found to be cpsE encoding the portal glycosyl transferase of capsule biosynthesis. Complementation of strains carrying missense mutations in cpsE with a wild-type gene restored capsule expression allowing the identification of amino acid residues essential for enzyme activity.

  6. Molecular Basis of Inherited Factor XIII- A Deficiency among Patients from Sistan - Baluchestan

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    Hejazi Shabnam

    2010-03-01

    Full Text Available Background: Factor ХШ, the last zymogene in the clotting cascade, converts the loose fibrin polymer into a firm polymer. In the absence of factor ХШ the abnormal fibrin is soluble in acetic acid, as well as 5M urea. Factor ХШ is composed of 2 catalytic A subunit bounds and 2 B subunits as carriers (A2B2. The gene of A chain is located on chromosome 6. Factor ХШ deficiency is rare; with a prevalence of only 1 in 2 million in the general population. The overwhelming majority of cases are due to mutations in subunit A. The aim of this study was to detect the mutations of subunit A.Materials & Methods: In this study we investigated the molecular basis of inherited factor ХШ deficiency among 10 unrelated patients from Sistan and Balouchestan province in 2006. Mutations were detected by amplifying each exon. Those exons exhibiting the presence of heteroduplex by conformation sensitive gel electrophoresis (CSGE were selected for direct sequencing. Sequencing of mutations was carried out by restriction fragment length polymorphism (RFLP.Results: All patients had homologous subsitiation of TGG to CGG in exon 4 which led to change of arginine to tryptophan.Conclusion: The mutation found in this study was in the core domain of enzyme. It seems that the changs in electric charge and affinity of enzyme to substrate‚as a result decreases the level of factor XIII-A activity.

  7. Leukemia-Associated Mutations in Nucleophosmin Alter Recognition by CRM1: Molecular Basis of Aberrant Transport.

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    Igor Arregi

    Full Text Available Nucleophosmin (NPM is a nucleocytoplasmic shuttling protein, normally enriched in nucleoli, that performs several activities related to cell growth. NPM mutations are characteristic of a subtype of acute myeloid leukemia (AML, where mutant NPM seems to play an oncogenic role. AML-associated NPM mutants exhibit altered subcellular traffic, being aberrantly located in the cytoplasm of leukoblasts. Exacerbated export of AML variants of NPM is mediated by the nuclear export receptor CRM1, and due, in part, to a mutationally acquired novel nuclear export signal (NES. To gain insight on the molecular basis of NPM transport in physiological and pathological conditions, we have evaluated the export efficiency of NPM in cells, and present new data indicating that, in normal conditions, wild type NPM is weakly exported by CRM1. On the other hand, we have found that AML-associated NPM mutants efficiently form complexes with CRM1HA (a mutant CRM1 with higher affinity for NESs, and we have quantitatively analyzed CRM1HA interaction with the NES motifs of these mutants, using fluorescence anisotropy and isothermal titration calorimetry. We have observed that the affinity of CRM1HA for these NESs is similar, which may help to explain the transport properties of the mutants. We also describe NPM recognition by the import machinery. Our combined cellular and biophysical studies shed further light on the determinants of NPM traffic, and how it is dramatically altered by AML-related mutations.

  8. Genomic analysis reveals the molecular basis for capsule loss in the group B Streptococcus population.

    Directory of Open Access Journals (Sweden)

    Roberto Rosini

    Full Text Available The human and bovine bacterial pathogen Streptococcus agalactiae (Group B Streptococcus, GBS expresses a thick polysaccharide capsule that constitutes a major virulence factor and vaccine target. GBS can be classified into ten distinct serotypes differing in the chemical composition of their capsular polysaccharide. However, non-typeable strains that do not react with anti-capsular sera are frequently isolated from colonized and infected humans and cattle. To gain a comprehensive insight into the molecular basis for the loss of capsule expression in GBS, a collection of well-characterized non-typeable strains was investigated by genome sequencing. Genome based phylogenetic analysis extended to a wide population of sequenced strains confirmed the recently observed high clonality among GBS lineages mainly containing human strains, and revealed a much higher degree of diversity in the bovine population. Remarkably, non-typeable strains were equally distributed in all lineages. A number of distinct mutations in the cps operon were identified that were apparently responsible for inactivation of capsule synthesis. The most frequent genetic alterations were point mutations leading to stop codons in the cps genes, and the main target was found to be cpsE encoding the portal glycosyl transferase of capsule biosynthesis. Complementation of strains carrying missense mutations in cpsE with a wild-type gene restored capsule expression allowing the identification of amino acid residues essential for enzyme activity.

  9. 2004 Molecular Basis of Microbial One-Carbon Metabolism Gordon Conference - August 1-6, 2004

    Energy Technology Data Exchange (ETDEWEB)

    Joseph A. Krzycki

    2005-09-15

    The Gordon Research Conference (GRC) on 2004 Molecular Basis of Microbial One-Carbon Metabolism Gordon Conference - August 1-6, 2004 was held at Mount Holyoke College, South Hadley, MA from August 1-6, 2004. The Conference was well-attended with 117 participants (attendees list attached). The attendees represented the spectrum of endeavor in this field coming from academia, industry, and government laboratories, both U.S. and foreign scientists, senior researchers, young investigators, and students. In designing the formal speakers program, emphasis was placed on current unpublished research and discussion of the future target areas in this field. There was a conscious effort to stimulate lively discussion about the key issues in the field today. Time for formal presentations was limited in the interest of group discussions. In order that more scientists could communicate their most recent results, poster presentation time was scheduled. Attached is a copy of the formal schedule and speaker program and the poster program. In addition to these formal interactions, 'free time' was scheduled to allow informal discussions. Such discussions are fostering new collaborations and joint efforts in the field.

  10. New Insights into the Molecular Basis of Kidney Governing Bone Theory

    Institute of Scientific and Technical Information of China (English)

    Dong-feng Zhao; Yong-jian Zhao; Cheng-long Wang; Yan-ping Yang; Yong-jun Wang

    2015-01-01

    Kidney governing bone theory plays an important role in treating bone metabolic disease such as osteoporosis, and many tonifying kidney prescriptions/herbs are widely used in Traditional Chinese Medicine (TCM). However, the exact biological basis of kidney governing bone theory in the context of new advances in biology is still not fully established. In this paper, the content of kidney governing bone theory in biology has been fully demonstrated from different aspects. We first propose that bone and kidney mutually affect each other in pathology and physiology, particularly through homeostasis of calcium, phosphorus and fibroblast growth factor-23(FGF-23). Next, we identify that tonifying kidney prescriptions/herbs exert bone protective effects, thus treating osteoporosis by regulating bone formation and bone resorption. Furthermore, the exact molecular mechanisms of tonifying kidney prescriptions, herbs and their effective components in treating osteoporosis have been systematically reviewed. Finally, we come into the conclusion that kidney regulating bone mineral homeostasis, bone protective effects of tonifying kidney herbs and regulatory effects on bone homeostasis are all the manifestations of kidney governing bone theory. Therefore, the new insights into kidney governing bone theory in biology will promote the development of clinical practices, and drugs discovery in treating osteoporosis.

  11. Molecular Basis for the Unique Deubiquitinating Activity of the NF-κB Inhibitor A20

    Energy Technology Data Exchange (ETDEWEB)

    Lin, S.; Chung, J; Lamothe, B; Rajashankar, K; Lu, M; Lo, Y; Lam, A; Darnay, B; Wu, H

    2008-01-01

    Nuclear factor ?B (NF-?B) activation in tumor necrosis factor, interleukin-1, and Toll-like receptor pathways requires Lys63-linked nondegradative polyubiquitination. A20 is a specific feedback inhibitor of NF-?B activation in these pathways that possesses dual ubiquitin-editing functions. While the N-terminal domain of A20 is a deubiquitinating enzyme (DUB) for Lys63-linked polyubiquitinated signaling mediators such as TRAF6 and RIP, its C-terminal domain is a ubiquitin ligase (E3) for Lys48-linked degradative polyubiquitination of the same substrates. To elucidate the molecular basis for the DUB activity of A20, we determined its crystal structure and performed a series of biochemical and cell biological studies. The structure reveals the potential catalytic mechanism of A20, which may be significantly different from papain-like cysteine proteases. Ubiquitin can be docked onto a conserved A20 surface; this interaction exhibits charge complementarity and no steric clash. Surprisingly, A20 does not have specificity for Lys63-linked polyubiquitin chains. Instead, it effectively removes Lys63-linked polyubiquitin chains from TRAF6 without dissembling the chains themselves. Our studies suggest that A20 does not act as a general DUB but has the specificity for particular polyubiquitinated substrates to assure its fidelity in regulating NF-?B activation in the tumor necrosis factor, interleukin-1, and Toll-like receptor pathways.

  12. The neuronal and molecular basis of quinine-dependent bitter taste signaling in Drosophila larvae

    Directory of Open Access Journals (Sweden)

    Anthi A. Apostolopoulou

    2014-01-01

    Full Text Available Bitter sensing can alert an animal that a specific type of food is potentially harmful for the organism and should not be consumed. However, not all bitter compounds are equally toxic and some bitter tastants may even have a positive valence in certain contexts, such as self-medication. Thus, taste systems in general have likely a higher capacity than just alerting the animal. In this study, we investigate bitter sensing and processing in Drosophila larvae, using quinine, a substance perceived by humans as bitter. We show that the four different behaviors choice, feeding, survival and associative olfactory learning are directly affected by quinine. On the cellular level we show that only 12 gustatory sensory receptor neurons expressing both GR66a and GR33a are required for quinine dependent choice and feeding behavior. Interestingly these neurons are not necessary for quinine dependent survival or associative learning. On the molecular level, only the GR33a receptor but not GR66a is required for quinine dependent choice behavior. Screening for single gustatory sensory receptor neurons that trigger quinine dependent choice behavior revealed that a single GR97a positive neuron located in the peripheral terminal sense organ is necessary and sufficient. Taken together, our study shows for the first time that the elementary chemosensory system of the Drosophila larva can serve as a simple model to understand the neuronal basis of taste information processing on the single cell level with respect to different behavioral outputs.

  13. The molecular basis for stability of heterochromatin-mediated silencing in mammals

    Directory of Open Access Journals (Sweden)

    Hiragami-Hamada Kyoko

    2009-11-01

    Full Text Available Abstract The archetypal epigenetic phenomenon of position effect variegation (PEV in Drosophila occurs when a gene is brought abnormally close to heterochromatin, resulting in stochastic silencing of the affected gene in a proportion of cells that would normally express it. PEV has been instrumental in unraveling epigenetic mechanisms. Using an in vivo mammalian model for PEV we have extensively investigated the molecular basis for heterochromatin-mediated gene silencing. Here we distinguish 'epigenetic effects' from other cellular differences by studying ex vivo cells that are identical, apart from the expression of the variegating gene which is silenced in a proportion of the cells. By separating cells according to transgene expression we show here that silencing appears to be associated with histone H3 lysine 9 trimethylation (H3K9me3, DNA methylation and the localization of the silenced gene to a specific nuclear compartment enriched in these modifications. In contrast, histone H3 acetylation (H3Ac and lysine 4 di or tri methylation (H3K4me2/3 are the predominant modifications associated with expression where we see the gene in a euchromatic compartment. Interestingly, DNA methylation and inaccessibility, rather than H3K9me3, correlated most strongly with resistance to de-repression by cellular activation. These results have important implications for understanding the contribution of specific factors involved in the establishment and maintenance of gene silencing and activation in vivo.

  14. Molecular basis for the inhibition of Drosophila eye development by Antennapedia.

    Science.gov (United States)

    Plaza, S; Prince, F; Jaeger, J; Kloter, U; Flister, S; Benassayag, C; Cribbs, D; Gehring, W J

    2001-02-15

    Hox genes encoding homeodomain transcriptional regulators are known to specify the body plan of multicellular organisms and are able to induce body plan transformations when misexpressed. These findings led to the hypothesis that duplication events and misexpression of Hox genes during evolution have been necessary for generating the observed morphological diversity found in metazoans. It is known that overexpressing Antennapedia (Antp) in the head induces antenna-to-leg as well as head-to-thorax transformation and eye reduction. At present, little is known about the exact molecular mechanism causing these phenotypes. The aim of this study is to understand the basis of inhibition of eye development. We demonstrate that Antp represses the activity of the eye regulatory cascade. By ectopic expression, we show that Antp antagonizes the activity of the eye selector gene eyeless. Using both in vitro and in vivo experiments, we demonstrate that this inhibitory mechanism involves direct protein-protein interactions between the DNA-binding domains of EY and ANTP, resulting in mutual inhibition.

  15. New Insights into the Molecular Basis of Kidney Governing Bone Theory

    Directory of Open Access Journals (Sweden)

    Dong-feng Zhao

    2015-07-01

    Full Text Available Kidney governing bone theory plays an important role in treating bone metabolic disease such as osteoporosis, and many tonifying kidney prescriptions/herbs are widely used in Traditional Chinese Medicine (TCM. However, the exact biological basis of kidney governing bone theory in the context of new advances in biology is still not fully established. In this paper, the content of kidney governing bone theory in biology has been fully demonstrated from different aspects. We first propose that bone and kidney mutually affect each other in pathology and physiology, particularly through homeostasis of calcium, phosphorus and fibroblast growth factor-23(FGF-23. Next, we identify that tonifying kidney prescriptions/herbs exert bone protective effects, thus treating osteoporosis by regulating bone formation and bone resorption. Furthermore, the exact molecular mechanisms of tonifying kidney prescriptions, herbs and their effective components in treating osteoporosis have been systematically reviewed. Finally, we come into the conclusion that kidney regulating bone mineral homeostasis, bone protective effects of tonifying kidney herbs and regulatory effects on bone homeostasis are all the manifestations of kidney governing bone theory. Therefore, the new insights into kidney governing bone theory in biology will promote the development of clinical practices, and drugs discovery in treating osteoporosis.

  16. The Molecular Basis of Memory. Part 3: Tagging with emotive neurotransmitters.

    Directory of Open Access Journals (Sweden)

    Gerard eMarx

    2014-04-01

    Full Text Available Many neurons of all animals that exhibit memory (snails, worms, flies, vertebrae present arborized shapes with many varicosities and boutons. These neurons, release neurotransmitters and contain ionotropic receptors that produce and sense electrical signals (ephaptic transmission. The extended shapes maximize neural contact with the surrounding neutrix (neural extracellular matrix (nECM+ diffusible (neurometals and neurotransmitters as well as with other neurons. We propose a tripartite mechanism of animal memory based on the dynamic interactions of splayed neurons with the neutrix. Their interactions form cognitive units of information (cuinfo, metal-centered complexes within the nECM around the neuron. Emotive content is provided by NTs, which embody molecular links between physiologic (body responses and psychic feelings. We propose that neurotransmitters form mixed complexes with cuinfo used for tagging emotive memory.Thus, NTs provide encoding option not available to a Turing, binary-based, device.The neurons employ combinatorially diverse options, with > 10 NMs and > 90 NTs for encoding (flavoring cuinfo with emotive tags. The neural network efficiently encodes, decodes and consolidates related (entangled sets of cuinfo into a coherent pattern, the basis for emotionally imbued memory, critical for determining a behavioral choice aimed at survival. The tripartite mechanism with tagging of NTs permits of a causal connection between physiology and psychology.

  17. Molecular basis for antifreeze activity difference of two insect antifreeze protein isoforms

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The insect spruce budworm(Choristoneura fumiferana) produces antifreeze protein(AFP) to assist in the protection of the over-wintering larval stage and contains multiple isoforms. Structures for two isoforms,known as CfAFP-501 and CfAFP-337,show that both possess similar left-handed β-helical structure,although thermal hysteresis activity of the longer isoform CfAFP-501 is three times that of CfAFP-337. The markedly enhanced activity of CfAFP-501 is not proportional to,and cannot be simply accounted for,by the increased ice-binding site resulting from the two extra coils in CfAFP-501. In or-der to investigate the molecular basis for the activity difference and gain better understanding of AFPs in general,we have employed several different computational methods to systematically study the structural properties and ice interactions of the AFPs and their deletion models. In the context of intact AFPs,a majority of the coils in CfAFP-501 has better ice interaction and causes stronger ice lattice disruption than CfAFP-337,strongly suggesting a cooperative or synergistic effect among β-helical coils. The synergistic effect would play a critical role and make significant contributions to the anti-freeze activity β-helical antifreeze proteins. This is the first time that synergistic effect and its implica-tion for antifreeze activity are reported for β-helical antifreeze proteins.

  18. Molecular basis for antifreeze activity difference of two insect antifreeze protein isoforms

    Institute of Scientific and Technical Information of China (English)

    ZHOU YanXia; TAN HongWei; YANG ZuoYin; JIA ZongChao; LIU RuoZhuang; CHEN GuangJu

    2007-01-01

    The insect spruce budworm (Choristoneura fumiferana) produces antifreeze protein (AFP) to assist in the protection of the over-wintering larval stage and contains multiple isoforms. Structures for two isoforms, known as CfAFP-501 and CfAFP-337, show that both possess similar left-handed β-helical structure, although thermal hysteresis activity of the longer isoform CfAFP-501 is three times that of CfAFP-337. The markedly enhanced activity of CfAFP-501 is not proportional to, and cannot be simply accounted for, by the increased ice-binding site resulting from the two extra coils in CfAFP-501. In order to investigate the molecular basis for the activity difference and gain better understanding of AFPs in general, we have employed several different computational methods to systematically study the structural properties and ice interactions of the AFPs and their deletion models. In the context of intact AFPs, a majority of the coils in CfAFP-501 has better ice interaction and causes stronger ice lattice disruption than CfAFP-337, strongly suggesting a cooperative or synergistic effect among β-helical coils. The synergistic effect would play a critical role and make significant contributions to the antifreeze activity β-helical antifreeze proteins. This is the first time that synergistic effect and its implication for antifreeze activity are reported for β-helical antifreeze proteins.

  19. Monomer Basis Representation Method For Calculating The Spectra Of Molecular Clusters I. The Method And Qualitative Models

    CERN Document Server

    Ocak, Mahir E

    2012-01-01

    Firstly, a sequential symmetry adaptation procedure is derived for semidirect product groups. Then, this sequential symmetry adaptation procedure is used in the development of new method named Monomer Basis Representation (MBR) for calculating the vibration-rotation-tunneling (VRT) spectra of molecular clusters. The method is based on generation of optimized bases for each monomer in the cluster as a linear combination of some primitive basis functions and then using the sequential symmetry adaptation procedure for generating a small symmetry adapted basis for the solution of the full problem. It is seen that given an optimized basis for each monomer the application of the sequential symmetry adaptation procedure leads to a generalized eigenvalue problem instead of a standard eigenvalue problem if the procedure is used as it is. In this paper, MBR method will be developed as a solution of that problem such that it leads to generation of an orthogonal optimized basis for the cluster being studied regardless of...

  20. Potent graft-versus-leukemia effect after reduced-intensity allogeneic SCT for intermediate-risk AML with FLT3-ITD or wild-type NPM1 and CEBPA without FLT3-ITD.

    Science.gov (United States)

    Labouré, Gaëlle; Dulucq, Stéphanie; Labopin, Myriam; Tabrizi, Reza; Guérin, Estelle; Pigneux, Arnaud; Lafarge, Xavier; Leguay, Thibaut; Bouabdallah, Krimo; Dilhuydy, Marie-Sarah; Duclos, Cédric; Lascaux, Axelle; Marit, Gérald; Mahon, François-Xavier; Boiron, Jean-Michel; Milpied, Noël; Vigouroux, Stéphane

    2012-12-01

    To investigate the role of reduced-intensity allogeneic (RIC-allo) stem cell transplant (SCT) as postremission therapy in adult intermediate-risk patients with acute myelogenous leukemia (AML) with FLT3-ITD or wild-type NPM1 and CEBPA without FLT3-ITD, we conducted a single-center retrospective study between January 2001 and December 2010. Sixty-six patients were included: 37 treated with RIC-alloSCT and 29 with nonallogeneic SCT therapies. Both groups were comparable concerning age, WBC count at diagnosis, gender, karyotype, genotype, and number of courses of chemotherapy to reach complete remission (CR1). Median follow-up after CR1 was 37 months (range, 11-112 months) and 48 months (range, 9-83 months) in the allo and no-allo groups, respectively. In the allo versus no-allo groups, the 3-year cumulative incidence of relapse (CIR) rates were 25% ± 8% versus 61% ± 9%; P = .005. The 3-year nonrelapse mortality (NRM), overall survival (OS), and relapse-free survival (RFS) were 22% ± 7% versus 4% ± 4% (P = .005), 52% ± 9% versus 44% ± 10% (P = .75), and 53% ± 9% versus 35% ± 9% (P = .28), respectively. Multivariate analysis indicated that CIR was reduced by allo (hazard ratio [HR], 0.32; P = .01). A landmark analysis performed at day 185 after CR1 confirmed a lower CIR after allo. RIC-allo reduces the risk of relapse, suggesting a potent graft-versus-leukemia (GVL) effect in these patients at a high risk of relapse.

  1. Molecular basis of reduced pyridoxine 5'-phosphate oxidase catalytic activity in neonatal epileptic encephalopathy disorder.

    Science.gov (United States)

    Musayev, Faik N; Di Salvo, Martino L; Saavedra, Mario A; Contestabile, Roberto; Ghatge, Mohini S; Haynes, Alexina; Schirch, Verne; Safo, Martin K

    2009-11-06

    Mutations in pyridoxine 5'-phosphate oxidase are known to cause neonatal epileptic encephalopathy. This disorder has no cure or effective treatment and is often fatal. Pyridoxine 5'-phosphate oxidase catalyzes the oxidation of pyridoxine 5'-phosphate to pyridoxal 5'-phosphate, the active cofactor form of vitamin B(6) required by more than 140 different catalytic activities, including enzymes involved in amino acid metabolism and biosynthesis of neurotransmitters. Our aim is to elucidate the mechanism by which a homozygous missense mutation (R229W) in the oxidase, linked to neonatal epileptic encephalopathy, leads to reduced oxidase activity. The R229W variant is approximately 850-fold less efficient than the wild-type enzyme due to an approximately 192-fold decrease in pyridoxine 5'-phosphate affinity and an approximately 4.5-fold decrease in catalytic activity. There is also an approximately 50-fold reduction in the affinity of the R229W variant for the FMN cofactor. A 2.5 A crystal structure of the R229W variant shows that the substitution of Arg-229 at the FMN binding site has led to a loss of hydrogen-bond and/or salt-bridge interactions between FMN and Arg-229 and Ser-175. Additionally, the mutation has led to an alteration of the configuration of a beta-strand-loop-beta-strand structure at the active site, resulting in loss of two critical hydrogen-bond interactions involving residues His-227 and Arg-225, which are important for substrate binding and orientation for catalysis. These results provide a molecular basis for the phenotype associated with the R229W mutation, as well as providing a foundation for understanding the pathophysiological consequences of pyridoxine 5'-phosphate oxidase mutations.

  2. Molecular basis of substrate selection by the N-end rule adaptor protein ClpS

    Energy Technology Data Exchange (ETDEWEB)

    Román-Hernández, Giselle; Grant, Robert A.; Sauer, Robert T.; Baker, Tania A.; (HHMI)

    2009-06-19

    The N-end rule is a conserved degradation pathway that relates the stability of a protein to its N-terminal amino acid. Here, we present crystal structures of ClpS, the bacterial N-end rule adaptor, alone and engaged with peptides containing N-terminal phenylalanine, leucine, and tryptophan. These structures, together with a previous structure of ClpS bound to an N-terminal tyrosine, illustrate the molecular basis of recognition of the complete set of primary N-end rule amino acids. In each case, the alpha-amino group and side chain of the N-terminal residue are the major determinants of recognition. The binding pocket for the N-end residue is preformed in the free adaptor, and only small adjustments are needed to accommodate N-end rule residues having substantially different sizes and shapes. M53A ClpS is known to mediate degradation of an expanded repertoire of substrates, including those with N-terminal valine or isoleucine. A structure of Met53A ClpS engaged with an N-end rule tryptophan reveals an essentially wild-type mechanism of recognition, indicating that the Met(53) side chain directly enforces specificity by clashing with and excluding beta-branched side chains. Finally, experimental and structural data suggest mechanisms that make proteins with N-terminal methionine bind very poorly to ClpS, explaining why these high-abundance proteins are not degraded via the N-end rule pathway in the cell.

  3. Dissecting the molecular basis of the contribution of source strength to high fructan accumulation in wheat.

    Science.gov (United States)

    Xue, Gang-Ping; Drenth, Janneke; Glassop, Donna; Kooiker, Maarten; McIntyre, C Lynne

    2013-01-01

    Fructans represent the major component of water soluble carbohydrates (WSCs) in the maturing stem of temperate cereals and are an important temporary carbon reserve for grain filling. To investigate the importance of source carbon availability in fructan accumulation and its molecular basis, we performed comparative analyses of WSC components and the expression profiles of genes involved in major carbohydrate metabolism and photosynthesis in the flag leaves of recombinant inbred lines from wheat cultivars Seri M82 and Babax (SB lines). High sucrose levels in the mature flag leaf (source organ) were found to be positively associated with WSC and fructan concentrations in both the leaf and stem of SB lines in several field trials. Analysis of Affymetrix expression array data revealed that high leaf sucrose lines grown in abiotic-stress-prone environments had high expression levels of a number of genes in the leaf involved in the sucrose synthetic pathway and photosynthesis, such as Calvin cycle genes, antioxidant genes involved in chloroplast H(2)O(2) removal and genes involved in energy dissipation. The expression of the majority of genes involved in fructan and starch synthetic pathways were positively correlated with sucrose levels in the leaves of SB lines. The high level of leaf fructans in high leaf sucrose lines is likely attributed to the elevated expression levels of fructan synthetic enzymes, as the mRNA levels of three fructosyltransferase families were consistently correlated with leaf sucrose levels among SB lines. These data suggest that high source strength is one of the important genetic factors determining high levels of WSC in wheat.

  4. Molecular basis of resistance to macrolides, lincosamides and streptogramins in Staphylococcus saprophyticus clinical isolates.

    Science.gov (United States)

    Le Bouter, Anne; Leclercq, Roland; Cattoir, Vincent

    2011-02-01

    The aim of this study was to evaluate the prevalence of resistance to macrolide-lincosamide-streptogramin (MLS) antibiotics as well as to assess the molecular basis of this resistance amongst 72 Staphylococcus saprophyticus urinary isolates collected from 2005 to 2009 in University Hospital of Caen (France). Of the 72 strains studied, 33 (45.8%) were resistant to at least one MLS antibiotic, including 24 (72.7%) with an M phenotype, 5 (15.2%) with an inducible MLS(B) phenotype, 3 (9.1%) with a combined M+L phenotype and 1 (3.0%) with an L phenotype. All isolates were susceptible to the combination of streptogramins A and B. The resistance genes erm(A), erm(B), erm(C), msr(A) and lnu(A) were detected alone in 0, 0, 5 (15.2%), 24 (72.7%) and 1 (3.0%) of the 33 MLS-resistant isolates, respectively, whereas 2 strains (6.1%) were positive for both msr(A) and lnu(A). All msr(A)-positive isolates exhibited an M phenotype, whereas all five erm(C)-positive and all three lnu(A)-positive strains displayed, respectively, an inducible MLS(B) phenotype and an L phenotype with a positive Hodge test. Plasmid analysis indicated that erm(C) and lnu(A) genes were borne by small-size plasmids (ca. 2.5 kb), whereas larger plasmids (30-90 kb) harboured msr(A). In conclusion, these findings show a high prevalence of MLS resistance in S. saprophyticus, which was mainly associated with the presence of the msr(A) gene. Since S. saprophyticus colonises the gastrointestinal tract, it may constitute an unexpected reservoir for MLS resistance genes, in particular msr(A), amongst coagulase-negative staphylococci.

  5. Molecular models of the Mojave rattlesnake (Crotalus scutulatus scutulatus) venom metalloproteinases reveal a structural basis for differences in hemorrhagic activities.

    Science.gov (United States)

    Dagda, Ruben K; Gasanov, Sardar E; Zhang, Boris; Welch, William; Rael, Eppie D

    2014-03-01

    Rattlesnake venom can differ in composition and in metalloproteinase-associated activities. The molecular basis for this intra-species variation in Crotalus scutulatus scutulatus (Mojave rattlesnake) remains an enigma. To understand the molecular basis for intra-species variation of metalloproteinase-associated activities, we modeled the three-dimensional structures of four metalloproteinases based on the amino acid sequence of four variations of the proteinase domain of the C. s. scutulatus metalloproteinase gene (GP1, GP2, GP3, and GP4). For comparative purposes, we modeled the atrolysin metalloproteinases of C. atrox as well. All molecular models shared the same topology. While the atrolysin metalloproteinase molecular models contained highly conserved substrate binding sites, the Mojave rattlesnake metalloproteinases showed higher structural divergence when superimposed onto each other. The highest structural divergence among the four C. s. scutulatus molecular models was located at the northern cleft wall and the S'1-pocket of the substrate binding site, molecular regions that modulate substrate selectivity. Molecular dynamics and field potential maps for each C. s. scutulatus metalloproteinase model demonstrated that the non-hemorrhagic metalloproteinases (GP2 and GP3) contain highly basic molecular and field potential surfaces while the hemorrhagic metalloproteinases GP1 and atrolysin C showed extensive acidic field potential maps and shallow but less dynamic active site pockets. Hence, differences in the spatial arrangement of the northern cleft wall, the S'1-pocket, and the physico-chemical environment surrounding the catalytic site contribute to differences in metalloproteinase activities in the Mojave rattlesnake. Our results provide a structural basis for variation of metalloproteinase-associated activities in the rattlesnake venom of the Mojave rattlesnake.

  6. 'From past to future' - deciphering the molecular basis of Alzheimer's disease through the pages of the Journal of Neurochemistry.

    Science.gov (United States)

    Cappai, Roberto

    2016-10-01

    The Journal of Neurochemistry has made significant contributions to unraveling the molecular basis for Alzheimer's disease during its 60-year history. To mark its 60th anniversary, this review describes the association between the journal and Alzheimer's disease research - from the early years when Alzheimer's disease was a minor topic in the journal through to the molecular era in the mid-1980s. This coincided with a number of the highly cited Alzheimer's disease studies which described fundamental aspects of the neurochemistry of Alzheimer's disease and encompassed the themes of oxidative stress and post-translational modifications, cholinergic system, tau, purification of Aβ, defining the Aβ toxic species, mechanism of amyloid precursor protein processing, and the development of diagnostics and therapeutics. The Journal of Neurochemistry has made significant contributions toward unraveling the molecular, cellular and pathological basis of Alzheimer's disease through its 60 years. This article is part of the 60th Anniversary special issue.

  7. Medicinal Chemistry and Molecular Modeling: An Integration to Teach Drug Structure-Activity Relationship and the Molecular Basis of Drug Action

    Science.gov (United States)

    Carvalho, Ivone; Borges, Aurea D. L.; Bernardes, Lilian S. C.

    2005-01-01

    The use of computational chemistry and the protein data bank (PDB) to understand and predict the chemical and molecular basis involved in the drug-receptor interactions is discussed. A geometrical and chemical overview of the great structural similarity in the substrate and inhibitor is provided.

  8. Molecular basis of proton uptake in single and double mutants of cytochrome c oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Henry, Rowan M; Caplan, David; Pomes, Regis [Molecular Structure and Function, Hospital for Sick Children, Toronto, ON, M5G 1X8 (Canada); Fadda, Elisa, E-mail: pomes@sickkids.ca [Department of Chemistry, University of Galway (Ireland)

    2011-06-15

    Cytochrome c oxidase, the terminal enzyme of the respiratory chain, utilizes the reduction of dioxygen into water to pump protons across the mitochondrial inner membrane. The principal pathway of proton uptake into the enzyme, the D channel, is a 2.5 nm long channel-like cavity named after a conserved, negatively charged aspartic acid (D) residue thought to help recruiting protons to its entrance (D132 in the first subunit of the S. sphaeroides enzyme). The single-point mutation of D132 to asparagine (N), a neutral residue, abolishes enzyme activity. Conversely, replacing conserved N139, one-third into the D channel, by D, induces a decoupled phenotype, whereby oxygen reduction proceeds but not proton pumping. Intriguingly, the double mutant D132N/N139D, which conserves the charge of the D channel, restores the wild-type phenotype. We use molecular dynamics simulations and electrostatic calculations to examine the structural and physical basis for the coupling of proton pumping and oxygen chemistry in single and double N139D mutants. The potential of mean force for the conformational isomerization of N139 and N139D side chains reveals the presence of three rotamers, one of which faces the channel entrance. This out-facing conformer is metastable in the wild-type and in the N139D single mutant, but predominant in the double mutant thanks to the loss of electrostatic repulsion with the carboxylate group of D132. The effects of mutations and conformational isomerization on the pKa of E286, an essential proton-shuttling residue located at the top of the D channel, are shown to be consistent with the electrostatic control of proton pumping proposed recently (Fadda et al 2008 Biochim. Biophys. Acta 1777 277-84). Taken together, these results suggest that preserving the spatial distribution of charges at the entrance of the D channel is necessary to guarantee both the uptake and the relay of protons to the active site of the enzyme. These findings highlight the interplay

  9. Molecular basis of proton uptake in single and double mutants of cytochrome c oxidase

    Science.gov (United States)

    Henry, Rowan M.; Caplan, David; Fadda, Elisa; Pomès, Régis

    2011-06-01

    Cytochrome c oxidase, the terminal enzyme of the respiratory chain, utilizes the reduction of dioxygen into water to pump protons across the mitochondrial inner membrane. The principal pathway of proton uptake into the enzyme, the D channel, is a 2.5 nm long channel-like cavity named after a conserved, negatively charged aspartic acid (D) residue thought to help recruiting protons to its entrance (D132 in the first subunit of the S. sphaeroides enzyme). The single-point mutation of D132 to asparagine (N), a neutral residue, abolishes enzyme activity. Conversely, replacing conserved N139, one-third into the D channel, by D, induces a decoupled phenotype, whereby oxygen reduction proceeds but not proton pumping. Intriguingly, the double mutant D132N/N139D, which conserves the charge of the D channel, restores the wild-type phenotype. We use molecular dynamics simulations and electrostatic calculations to examine the structural and physical basis for the coupling of proton pumping and oxygen chemistry in single and double N139D mutants. The potential of mean force for the conformational isomerization of N139 and N139D side chains reveals the presence of three rotamers, one of which faces the channel entrance. This out-facing conformer is metastable in the wild-type and in the N139D single mutant, but predominant in the double mutant thanks to the loss of electrostatic repulsion with the carboxylate group of D132. The effects of mutations and conformational isomerization on the pKa of E286, an essential proton-shuttling residue located at the top of the D channel, are shown to be consistent with the electrostatic control of proton pumping proposed recently (Fadda et al 2008 Biochim. Biophys. Acta 1777 277-84). Taken together, these results suggest that preserving the spatial distribution of charges at the entrance of the D channel is necessary to guarantee both the uptake and the relay of protons to the active site of the enzyme. These findings highlight the interplay

  10. Bases moleculares de la cancerización de cavidad oral Molecular basis on oral cavity cancerization

    Directory of Open Access Journals (Sweden)

    M.A. González-Moles

    2008-02-01

    Full Text Available Se presenta una revisión bibliográfica breve sobre los principales aspectos moleculares de interés en la cancerización de cavidad oral. Se hace referencia a los conocimientos más recientes sobre las aberraciones cromosómicas más comunes y las alteraciones de los oncogenes y genes supresores tumorales que están implicados en la carcinogénesis oral. Así mismo, se resume la teoría molecular actual que explica el proceso de cancerización de campo.A review about the main molecular aspects on oral cavity cancerization is presented, with special reference to the common chromosomal aberration, oncogenes and tumour suppressor genes implied in oral carcinogenesis. A summary about molecular theory explaining the field cancerization process is also presented.

  11. Design, Synthesis, and Biological Evaluation of Novel Nonsteroidal Farnesoid X Receptor (FXR) Antagonists: Molecular Basis of FXR Antagonism.

    Science.gov (United States)

    Huang, Huang; Si, Pei; Wang, Lei; Xu, Yong; Xu, Xin; Zhu, Jin; Jiang, Hualiang; Li, Weihua; Chen, Lili; Li, Jian

    2015-07-01

    Farnesoid X receptor (FXR) plays an important role in the regulation of cholesterol, lipid, and glucose metabolism. Recently, several studies on the molecular basis of FXR antagonism have been reported. However, none of these studies employs an FXR antagonist with nonsteroidal scaffold. On the basis of our previously reported FXR antagonist with a trisubstituted isoxazole scaffold, a novel nonsteroidal FXR ligand was designed and used as a lead for structural modification. In total, 39 new trisubstituted isoxazole derivatives were designed and synthesized, which led to pharmacological profiles ranging from agonist to antagonist toward FXR. Notably, compound 5s (4'-[(3-{[3-(2-chlorophenyl)-5-(2-thienyl)isoxazol-4-yl]methoxy}-1H-pyrazol-1-yl)methyl]biphenyl-2-carboxylic acid), containing a thienyl-substituted isoxazole ring, displayed the best antagonistic activity against FXR with good cellular potency (IC50 =12.2 ± 0.2 μM). Eventually, this compound was used as a probe in a molecular dynamics simulation assay. Our results allowed us to propose an essential molecular basis for FXR antagonism, which is consistent with a previously reported antagonistic mechanism; furthermore, E467 on H12 was found to be a hot-spot residue and may be important for the future design of nonsteroidal antagonists of FXR.

  12. Can Coulomb Sturmians Be Used as a Basis for N-Electron Molecular Calculations?

    DEFF Research Database (Denmark)

    Avery, John Scales; Avery, James Emil

    2009-01-01

    A method is proposed for using isoenergetic configurations formed from many-center Coulomb Sturmians as a basis for calculations on N-electron molecules. Such configurations are solutions to an approximate N-electron Schrödinger equation with a weighted potential, and they are thus closely analog...

  13. Understanding the molecular basis for differential binding of integrins to collagen and gelatin.

    Science.gov (United States)

    Zaman, Muhammad H

    2007-01-15

    Integrin-mediated cell adhesion plays a central role in cell migration and signaling. Overexpression of integrins is also associated with cancer invasion and metastasis. Although a number of problems in integrin-matrix interactions have been studied in detail, the molecular specificity, which increases integrin adhesion to native collagen but results in poor integrin-gelatin interaction, is not understood. In this report, we study the role of individual amino acids in integrin-collagen and integrin-gelatin interactions using long-term (>100 ns) molecular simulations. The results, which are force-field independent, show that denatured collagen induces helical conformations in integrin amino acids and significantly reduces the poly-proline II content, which stabilizes the integrin-collagen interactions. Our simulations provide a possible explanation of the molecular specificity in integrin binding and suggest new targets for regulating integrin-mediated invasion and metastasis.

  14. Molecular response functions for the polarizable continuum model physical basis and quantum mechanical formalism

    CERN Document Server

    Cammi, Roberto

    2013-01-01

    This Brief presents the main aspects of the response functions theory (RFT) for molecular solutes described within the framework of the Polarizable Continuum Model (PCM). PCM is a solvation model for a Quantum Mechanical molecular system in which the solvent is represented as a continuum distribution of matter. Particular attention is devoted to the description of the basic features of the PCM model, and to the problems characterizing the study of the response function theory for molecules in solution with respect to the analogous theory on isolated molecules.

  15. 27ps DFT Molecular Dynamics Simulation of a-maltose: A Reduced Basis Set Study.

    Science.gov (United States)

    DFT molecular dynamics simulations are time intensive when carried out on carbohydrates such as alpha-maltose, requiring up to three or more weeks on a fast 16-processor computer to obtain just 5ps of constant energy dynamics. In a recent publication [1] forces for dynamics were generated from B3LY...

  16. C-type lectin interactions with Schistosoma mansoni SEA : Molecular basis and function

    NARCIS (Netherlands)

    Liempt, van P.A.G.

    2007-01-01

    Outline of this thesis The studies described in this thesis have been performed to gain more insight in the recognition of Schistosoma mansoni glycans by C-type lectins and the consequences for dendritic cell mediated immune responses. As a first approach to understand the molecular interactions o

  17. Chemometric Analysis of Some Biologically Active Groups of Drugs on the Basis Chromatographic and Molecular Modeling Data.

    Science.gov (United States)

    Stasiak, Jolanta; Koba, Marcin; Baczek, Tomasz; Bucinski, Adam

    2015-01-01

    In this work, three different groups of drugs such as 12 analgesic drugs, 11 cardiovascular system drugs and 36 "other" compounds, respectively, were analyzed with cluster analysis (CA), principal component analysis (PCA) and factor analysis (FA) methods. All chemometric analysis were based on the chromatographic parameters (logk and logk(w)) determined by means of high-performance liquid chromatography (HPLC) and also by molecular modeling descriptors calculated using various computer programs (HyperChem, Dragon, and the VCCLAB). The clustering of compounds were obtained by CA (using various algorithm as e.g. Ward method or unweighted pair-group method using arithmetic averages as well as Euclidean or Manhattan distance), and allowed to build dendrograms linked drugs with similar physicochemical and pharmacological properties were discussed. Moreover, the analysis performed for analyzed groups of compounds with the use of FA or PCA methods indicated that almost all information reached in input chromatographic parameters as well as in molecular modeling descriptors can be explained by first two factors. Additionally, all analyzed drugs were clustered according to their chemical structure and pharmacological activity. Summarized, the performed classification analysis of studied drugs was focused on similarities and differences in methods being used for chemometric analysis as well as focused abilities to drugs classification (clustering) according to their molecular structures and pharmacological activity performed on the basis of chromatographic experimental and molecular modeling data. Thus, the most important application of statistically important molecular descriptors taken from QSRR models to classification analysis allow detailed biological (pharmacological) classification of analyzed drugs.

  18. Final Report: Molecular Basis for Microbial Adhesion and Geochemical Surface Reactions: A Study Across Scales

    Energy Technology Data Exchange (ETDEWEB)

    Dixon, David Adams [The University of Alabama

    2013-06-27

    Computational chemistry was used to help provide a molecular level description of the interactions of Gram-negative microbial membranes with subsurface materials. The goal is to develop a better understanding of the molecular processes involved in microbial metal binding, microbial attachment to mineral surfaces, and, eventually, oxidation/reduction reactions (electron transfer) that can occur at these surfaces and are mediated by the bacterial exterior surface. The project focused on the interaction of the outer microbial membrane, which is dominated by an exterior lipopolysaccharide (LPS) portion, of Pseudomonas aeruginosa with the mineral goethite and with solvated ions in the environment. This was originally a collaborative project with T.P. Straatsma and B. Lowery of the Pacific Northwest National Laboratory. The University of Alabama effort used electronic structure calculations to predict the molecular behavior of ions in solution and the behavior of the sugars which form a critical part of the LPS. The interactions of the sugars with metal ions are expected to dominate much of the microscopic structure and transport phenomena in the LPS. This work, in combination with the molecular dynamics simulations of Straatsma and the experimental electrochemistry and microscopy measurements of Lowry, both at PNNL, is providing new insights into the detailed molecular behavior of these membranes in geochemical environments. The effort at The University of Alabama has three components: solvation energies and structures of ions in solution, prediction of the acidity of the critical groups in the sugars in the LPS, and binding of metal ions to the sugar anions. An important aspect of the structure of the LPS membrane as well as ion transport in the LPS is the ability of the sugar side groups such as the carboxylic acids and the phosphates to bind positively charged ions. We are studying the acidity of the acidic side groups in order to better understand the ability of

  19. Deciphering the Molecular Basis of Human Cardiovascular Disease through Network Biology

    Science.gov (United States)

    Chan, Stephen Y.; White, Kevin; Loscalzo, Joseph

    2012-01-01

    Purpose of review This review introduces the fundamental concepts of network medicine and explores the feasibility and potential impact of network-based methods on predicting and ameliorating individual manifestations of human cardiovascular disease. Recent findings Complex cardiovascular diseases rarely result from an abnormality in a single molecular effector, but, rather, nearly always are the net result of multiple pathobiological pathways that interact through an interconnected network. In the post-genomic era, a framework has emerged of the potential complexity of the interacting pathways that govern molecular actions in the human cell. As a result, network approaches have been developed to understand more comprehensively those interconnections that influence human disease. “Network medicine” has already led to tangible discoveries of novel disease genes and pathways as well as improved mechanisms for rational drug development. Summary As methodologies evolve, network medicine may better capture the complexity of human pathogenesis and, thus, re-define personalized disease classification and therapies. PMID:22382498

  20. Molecular basis of classic galactosemia from the structure of human galactose 1-phosphate uridylyltransferase

    OpenAIRE

    McCorvie, Thomas J; Kopec, Jolanta; Angel L Pey; Fitzpatrick, Fiona; Patel, Dipali; Chalk, Rod; Shrestha, Leela; Yue, Wyatt W.

    2016-01-01

    Classic galactosemia is a potentially lethal disease caused by the dysfunction of galactose 1-phosphate uridylyltransferase (GALT). Over 300 disease-associated GALT mutations have been reported, with the majority being missense changes, although a better understanding of their underlying molecular effects has been hindered by the lack of structural information for the human enzyme. Here, we present the 1.9 Å resolution crystal structure of human GALT (hGALT) ternary complex, revealing a homod...

  1. The molecular basis of ligand interaction at free fatty acid receptor 4 (FFA4/GPR120)

    OpenAIRE

    Hudson, Brian D.; Shimpukade, Bharat; Milligan, Graeme; Ulven, Trond

    2014-01-01

    The long-chain fatty acid receptor FFA4(previously GPR120) is receiving substantial interest as a novel target for the treatment of metabolic and inflammatory disease. The current study examines for the first time the detailed mode of binding of both a long-chain fatty acid and synthetic agonist ligands at FFA4 by integrating molecular modeling, receptor mutagenesis, and ligand structure-activity relationship approaches in an iterative format. In doing so, residues required for binding of fat...

  2. Molecular Basis of Ion Channels and Receptors Involved in Nerve Excitation, Synaptic Transmission and Muscle Contraction

    Science.gov (United States)

    1993-12-20

    BARNARD, MARGARET SUTHERLAND, SHAHID ZAMAN, XMIT5UtIIKO ,MATSUMOTO. NAUSHABA NAVEEJM, TIMi GREEN~, MARK G. DARLITON, and ALAN N . BATESON ...Paralysis of a Japanese Family. By Kiciii ARAHAI A, JIANZ1iOt.: WANG, W. GREGORY FEFiRo. HIROSHI~ HAYAKAWA, Koicii HONDA. HIDEo SLGI-iA, and ERIC P. HOFI...8217 MARK G. DARLISONJ AND ALAN N. BATESON ’ ’Molecular Neurobiology Unit Division of Basic Medical Sciences Royal Free Hospital Scbool of Medicine London NW3

  3. Mass Spectrometry-based Approaches to Understand the Molecular Basis of Memory

    OpenAIRE

    Arthur Henriques Pontes; Marcelo Valle de Sousa

    2016-01-01

    The central nervous system is responsible for an array of cognitive functions such as memory, learning, language and attention. These processes tend to take place in distinct brain regions; yet, they need to be integrated to give rise to adaptive or meaningful behavior. Since cognitive processes result from underlying cellular and molecular changes, genomics and transcriptomics assays have been applied to human and animal models to understand such events. Nevertheless, genes and RNAs are not ...

  4. Mass Spectrometry-Based Approaches to Understand the Molecular Basis of Memory

    OpenAIRE

    Pontes, Arthur H.; Sousa, Marcelo V

    2016-01-01

    The central nervous system is responsible for an array of cognitive functions such as memory, learning, language, and attention. These processes tend to take place in distinct brain regions; yet, they need to be integrated to give rise to adaptive or meaningful behavior. Since cognitive processes result from underlying cellular and molecular changes, genomics and transcriptomics assays have been applied to human and animal models to understand such events. Nevertheless, genes and RNAs are not...

  5. Modeling and analysis of the molecular basis of pain in sensory neurons.

    Directory of Open Access Journals (Sweden)

    Sang Ok Song

    Full Text Available Intracellular calcium dynamics are critical to cellular functions like pain transmission. Extracellular ATP plays an important role in modulating intracellular calcium levels by interacting with the P2 family of surface receptors. In this study, we developed a mechanistic mathematical model of ATP-induced P2 mediated calcium signaling in archetype sensory neurons. The model architecture, which described 90 species connected by 162 interactions, was formulated by aggregating disparate molecular modules from literature. Unlike previous models, only mass action kinetics were used to describe the rate of molecular interactions. Thus, the majority of the 252 unknown model parameters were either association, dissociation or catalytic rate constants. Model parameters were estimated from nine independent data sets taken from multiple laboratories. The training data consisted of both dynamic and steady-state measurements. However, because of the complexity of the calcium network, we were unable to estimate unique model parameters. Instead, we estimated a family or ensemble of probable parameter sets using a multi-objective thermal ensemble method. Each member of the ensemble met an error criterion and was located along or near the optimal trade-off surface between the individual training data sets. The model quantitatively reproduced experimental measurements from dorsal root ganglion neurons as a function of extracellular ATP forcing. Hypothesized architecture linking phosphoinositide regulation with P2X receptor activity explained the inhibition of P2X-mediated current flow by activated metabotropic P2Y receptors. Sensitivity analysis using individual and the whole system outputs suggested which molecular subsystems were most important following P2 activation. Taken together, modeling and analysis of ATP-induced P2 mediated calcium signaling generated qualitative insight into the critical interactions controlling ATP induced calcium dynamics

  6. The Molecular Basis for Calcium-dependent Regulation of Cardiac Structure and Function

    OpenAIRE

    Shimizu, Hirohito

    2014-01-01

    Calcium homeostasis is essential for regulating a wide spectrum of biological processes. In the heart, Ca2+ plays a key role in excitation-contraction coupling, electrophysiological processes, activation of contractile proteins, energy metabolism, cell death, and transcriptional regulation. Alteration of Ca2+ homeostasis is often associated with cardiac pathology such as contractile dysfunction, arrhythmias and heart failure. In order to discover novel molecular mechanisms by which Ca2+ regul...

  7. Communicating the molecular basis of cancer cell-by-cell: an interview with Tatsushi Igaki

    OpenAIRE

    2015-01-01

    ABSTRACT Tatsushi Igaki is currently based at the Kyoto University Graduate School of Biostudies, where he leads a research group dedicated to using Drosophila genetics to build a picture of the cell-cell communications underlying the establishment and maintenance of multicellular systems. His work has provided insight into the molecular bases of cell competition in the context of development and tumorigenesis, including the landmark discovery that oncogenic cells communicate with normal cell...

  8. Plant Adaptation to Acid Soils: The Molecular Basis for Crop Aluminum Resistance.

    Science.gov (United States)

    Kochian, Leon V; Piñeros, Miguel A; Liu, Jiping; Magalhaes, Jurandir V

    2015-01-01

    Aluminum (Al) toxicity in acid soils is a significant limitation to crop production worldwide, as approximately 50% of the world's potentially arable soil is acidic. Because acid soils are such an important constraint to agriculture, understanding the mechanisms and genes conferring resistance to Al toxicity has been a focus of intense research interest in the decade since the last article on crop acid soil tolerance was published in this journal. An impressive amount of progress has been made during that time that has greatly increased our understanding of the diversity of Al resistance genes and mechanisms, how resistance gene expression is regulated and triggered by Al and Al-induced signals, and how the proteins encoded by these genes function and are regulated. This review examines the state of our understanding of the physiological, genetic, and molecular bases for crop Al tolerance, looking at the novel Al resistance genes and mechanisms that have been identified over the past ten years. Additionally, it examines how the integration of molecular and genetic analyses of crop Al resistance is starting to be exploited for the improvement of crop plants grown on acid soils via both molecular-assisted breeding and biotechnology approaches.

  9. Molecular basis of cooperativity in pH-triggered supramolecular self-assembly

    Science.gov (United States)

    Li, Yang; Zhao, Tian; Wang, Chensu; Lin, Zhiqiang; Huang, Gang; Sumer, Baran D.; Gao, Jinming

    2016-10-01

    Supramolecular self-assembly offers a powerful strategy to produce high-performance, stimuli-responsive nanomaterials. However, lack of molecular understanding of stimulated responses frequently hampers our ability to rationally design nanomaterials with sharp responses. Here we elucidated the molecular pathway of pH-triggered supramolecular self-assembly of a series of ultra-pH sensitive (UPS) block copolymers. Hydrophobic micellization drove divergent proton distribution in either highly protonated unimer or neutral micelle states along the majority of the titration coordinate unlike conventional small molecular or polymeric bases. This all-or-nothing two-state solution is a hallmark of positive cooperativity. Integrated modelling and experimental validation yielded a Hill coefficient of 51 in pH cooperativity for a representative UPS block copolymer, by far the largest reported in the literature. These data suggest hydrophobic micellization and resulting positive cooperativity offer a versatile strategy to convert responsive nanomaterials into binary on/off switchable systems for chemical and biological sensing, as demonstrated in an additional anion sensing model.

  10. Identifying the molecular basis of host-parasite coevolution: merging models and mechanisms.

    Science.gov (United States)

    Dybdahl, Mark F; Jenkins, Christina E; Nuismer, Scott L

    2014-07-01

    Mathematical models of the coevolutionary process have uncovered consequences of host-parasite interactions that go well beyond the traditional realm of the Red Queen, potentially explaining several important evolutionary transitions. However, these models also demonstrate that the specific consequences of coevolution are sensitive to the structure of the infection matrix, which is embedded in models to describe the likelihood of infection in encounters between specific host and parasite genotypes. Traditional cross-infection approaches to estimating infection matrices might be unreliable because evolutionary dynamics and experimental sampling lead to missing genotypes. Consequently, our goal is to identify the likely structure of infection matrices by synthesizing molecular mechanisms of host immune defense and parasite counterdefense with coevolutionary models. This synthesis reveals that the molecular mechanisms of immune reactions, although complex and diverse, conform to two basic models commonly used within coevolutionary theory: matching infection and targeted recognition. Our synthesis also overturns conventional wisdom, revealing that the general models are not taxonomically restricted but are applicable to plants, invertebrates, and vertebrates. Finally, our synthesis identifies several important areas for future research that should improve the explanatory power of coevolutionary models. The most important among these include empirical studies to identify the molecular hotspots of genotypic specificity and theoretical studies examining the consequences of matrices that more accurately represent multistep infection processes and quantitative defenses.

  11. Functional basis of a molecular adaptation: prey-specific toxic effects of venom from Sistrurus rattlesnakes.

    Science.gov (United States)

    Gibbs, H Lisle; Mackessy, Stephen P

    2009-05-01

    Understanding the molecular bases of adaptations requires assessing the functional significance of phenotypic variation at the molecular level. Here we conduct such an assessment for an adaptive trait (snake venom proteins) which shows high levels of interspecific variation at the molecular level. We tested the toxicity of venom from four taxa of Sistrurus rattlesnakes with different diets towards 3 representative prey (mice, lizards and frogs). There were significant differences among prey in their overall susceptibility to Sistrurus venom, with frogs being an order of magnitude more resistant than mice or lizards. However, only in mice was there substantial variation in the toxicity of venom from different Sistrurus taxa, with the variation being roughly correlated with the incidence of mammals in the snake's diet. A comparative analysis using published data of the toxicity of rattlesnake and outgroup (Agkistrodon) venoms to mice confirms that both the gain and loss of toxicity to mammals were major modes of venom evolution in Sistrurus catenatus and Sistrurus miliarius. Our findings identify toxicity to mammals as a major axis along which venom evolution has occurred among Sistrurus rattlesnakes, with little evidence for evolutionary changes in toxicity towards the other prey tested. They also emphasize the need to consider ecological and evolutionary factors other than diet alone as causes of variation in venom toxicity.

  12. Molecular basis of hereditary fructose intolerance: mutations and polymorphisms in the human aldolase B gene.

    Science.gov (United States)

    Tolan, D R

    1995-01-01

    Mutations in the human aldolase B gene that result in hereditary fructose intolerance have been characterized extensively. Although the majority of subjects have been from northern Europe, subjects from other geographical regions and ethnic groups have been identified. At present 21 mutations have been reported; 15 of these are single base substitutions, resulting in nine amino acid replacements, four nonsense codons, and two putative splicing defects. Two large deletions, two four-base deletions, a single-base deletion, and a seven-base deletion/one-base insertion have been found. This last mutation leads to a defect in splicing and it is likely that one of the small deletions does as well. Regions of the enzyme where mutations have been observed recurrently are encoded by exons 5 and 9. Indeed, the three most common mutations are found in these exons. Two of these prevalent HFI mutations arose from a common ancestor and spread throughout the population by genetic drift. This finding was based on linkage to two sequence polymorphisms, which are among very few informative polymorphic markers that have been identified within the aldolase B gene. Because of the prevalence of a few HFI alleles, and the recent advances in molecular methods for identifying and screening for mutation, the diagnosis of HFI by molecular screening methods should become routine. These molecular diagnostic methods will be extremely beneficial for this often difficult to diagnose and sometimes fatal disease.

  13. Review and update on the molecular basis of Leber congenital amaurosis.

    Science.gov (United States)

    Chacon-Camacho, Oscar Francisco; Zenteno, Juan Carlos

    2015-02-16

    Inherited retinal diseases are uncommon pathologies and one of the most harmful causes of childhood and adult blindness. Leber congenital amaurosis (LCA) is the most severe kind of these diseases accounting for approximately 5% of the whole retinal dystrophies and 20% of the children that study on blind schools. Clinical ophthalmologic findings including severe vision loss, nystagmus and ERG abnormalities should be suspected through the first year of life in this group of patients. Phenotypic variability is found when LCA patients have a full ophthalmologic examination. However, a correct diagnosis may be carried out; the determination of ophthalmologic clues as light sensibility, night blindness, fundus pigmentation, among other, join with electroretinographics findings, optical coherence tomography, and new technologies as molecular gene testing may help to reach to a precise diagnosis. Several retinal clinical features in LCA may suggest a genetic or gene particular defect; thus genetic-molecular tools could directly corroborate the clinical diagnosis. Currently, approximately 20 genes have been associated to LCA. In this review, historical perspective, clinical ophthalmological findings, new molecular-genetics technologies, possible phenotype-genotypes correlations, and gene therapy for some LCA genes are described.

  14. Targeted metagenomics unveils the molecular basis for adaptive evolution of enzymes to their environment

    Directory of Open Access Journals (Sweden)

    Hikaru eSuenaga

    2015-09-01

    Full Text Available Microorganisms have a wonderful ability to adapt rapidly to new or altered environmental conditions. Enzymes are the basis of metabolism in all living organisms and therefore enzyme adaptation plays a crucial role in the adaptation of microorganisms. Comparisons of homology and parallel beneficial mutations in an enzyme family provide valuable hints of how an enzyme adapted to an ecological system; consequently, a series of enzyme collections is required to investigate enzyme evolution. Targeted metagenomics is a promising tool for the construction of enzyme pools and for studying the adaptive evolution of enzymes. This perspective article presents a summary of targeted metagenomic approaches useful for this purpose.

  15. Targeted metagenomics unveils the molecular basis for adaptive evolution of enzymes to their environment

    Science.gov (United States)

    Suenaga, Hikaru

    2015-01-01

    Microorganisms have a wonderful ability to adapt rapidly to new or altered environmental conditions. Enzymes are the basis of metabolism in all living organisms and, therefore, enzyme adaptation plays a crucial role in the adaptation of microorganisms. Comparisons of homology and parallel beneficial mutations in an enzyme family provide valuable hints of how an enzyme adapted to an ecological system; consequently, a series of enzyme collections is required to investigate enzyme evolution. Targeted metagenomics is a promising tool for the construction of enzyme pools and for studying the adaptive evolution of enzymes. This perspective article presents a summary of targeted metagenomic approaches useful for this purpose. PMID:26441940

  16. Liquid Water through Density-Functional Molecular Dynamics: Plane-Wave vs Atomic-Orbital Basis Sets

    CERN Document Server

    Miceli, Giacomo; Pasquarello, Alfredo

    2016-01-01

    We determine and compare structural, dynamical, and electronic properties of liquid water at near ambient conditions through density-functional molecular dynamics simulations, when using either plane-wave or atomic-orbital basis sets. In both frameworks, the electronic structure and the atomic forces are self-consistently determined within the same theoretical scheme based on a nonlocal density functional accounting for van der Waals interactions. The overall properties of liquid water achieved within the two frameworks are in excellent agreement with each other. Thus, our study supports that implementations with plane-wave or atomic-orbital basis sets yield equivalent results and can be used indiscriminately in study of liquid water or aqueous solutions.

  17. Molecular Basis of the Binding of YAP Transcriptional Regulator to the ErbB4 Receptor Tyrosine Kinase

    OpenAIRE

    Schuchardt, Brett J.; Bhat, Vikas; Mikles, David C.; McDonald, Caleb B.; Sudol, Marius; Farooq, Amjad

    2014-01-01

    The newly discovered transactivation function of ErbB4 receptor tyrosine kinase is believed to be mediated by virtue of the ability of its proteolytically-cleaved intracellular domain (ICD) to physically associate with YAP2 transcriptional regulator. In an effort to unearth the molecular basis of YAP2-ErbB4 interaction, we have conducted a detailed biophysical analysis of the binding of WW domains of YAP2 to PPXY motifs located within the ICD of ErbB4. Our data show that the WW1 domain of YAP...

  18. Deciphering the molecular basis of multidrug recognition: crystal structures of the Staphylococcus aureus multidrug binding transcription regulator QacR.

    Science.gov (United States)

    Schumacher, Maria A; Brennan, Richard G

    2003-03-01

    Multidrug transporters and their transcriptional regulators are key components of bacterial multidrug resistance (MDR). How these multidrug binding proteins can recognize such chemically disparate compounds represents a fascinating question from a structural standpoint and an important question in future drug development efforts. The Staphylococcus aureus multidrug binding regulator, QacR, is soluble and recognizes an especially wide range of structurally dissimilar compounds, properties making it an ideal model system for deciphering the molecular basis of multidrug recognition. Recent structures of QacR have afforded the first view of any MDR protein bound to multiple drugs, revealing key structural features of multidrug recognition, including a multisite binding pocket.

  19. Insights into the molecular basis of L-form formation and survival in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    William A Glover

    Full Text Available L-forms have been shown to occur among many species of bacteria and are suspected to be involved in persistent infections. Since their discovery in 1935, numerous studies characterizing L-form morphology, growth, and pathogenic potential have been conducted. However, the molecular mechanisms underlying the formation and survival of L-forms remain unknown. Using unstable L-form colonies of Escherichia coli as a model, we performed genome-wide transcriptome analysis and screened a deletion mutant library to study the molecular mechanisms involved in formation and survival of L-forms. Microarray analysis of L-form versus classical colonies revealed many up-regulated genes of unknown function as well as multiple over-expressed stress pathways shared in common with persister cells and biofilms. Mutant screens identified three groups of mutants which displayed varying degrees of defects in L-form colony formation. Group 1 mutants, which showed the strongest defect in L-form colony formation, belonged to pathways involved in cell envelope stress, DNA repair, iron homeostasis, outer membrane biogenesis, and drug efflux/ABC transporters. Four (Group 1 mutants, rcsB, a positive response regulator of colanic acid capsule synthesis, ruvA, a recombinational junction binding protein, fur, a ferric uptake regulator and smpA a small membrane lipoprotein were selected for complementation. Complementation of the mutants using a high-copy overexpression vector failed, while utilization of a low-copy inducible vector successfully restored L-form formation. This work represents the first systematic genetic evaluation of genes and pathways involved in the formation and survival of unstable L-form bacteria. Our findings provide new insights into the molecular mechanisms underlying L-form formation and survival and have implications for understanding the emergence of antibiotic resistance, bacterial persistence and latent infections and designing novel drugs and

  20. Molecular basis for identification of species/isolates of gastrointestinal nematode parasites

    Institute of Scientific and Technical Information of China (English)

    Ahmed M; Singh MN; Bera AK; Bandyopadhyay S; Bhattacharya D

    2011-01-01

    Gastrointestinal(GI)parasitism is the most serious constraint throughout the world in small ruminants which causes significant production loss in animals.GI parasites are major contributor to reduce productivity in terms of meat, milk and wool in animals. Control ofGI parasite is done primarily by anthelmintic treatment where choice and schedule of treatment is done after identification and quantitation of individual parasite. Identification ofGI parasites is done through microscopic method by identifying specific morphological characteristics of egg and larva (L3). Since most of parasite eggs are having similar morphological characteristics, identification up to species level through microscopy is not possible in most of cases. To address this issue, molecular techniques are the viable alternative for identification of species as well as molecular level differences within a species (isolates) of parasites. DifferentDNA based molecular techniquesviz.PCR, AFLP, RAPD, RFLP, PCR-SSCP, real timePCR, DNAmicroarrayetc. have been used for identification and to assess the genetic diversity among parasite population. For identification of species, the characteristic sequence of genomicDNAof different species should differ to allow the delineation of species, but at the same time, no/minor variation within the species should exist. In contrast, for purpose of identifying population variants (strains/isolates), a considerable degree of variation in the sequence should exist within a species. Various target regions, including nuclear ribosomal DNA (rDNA), mitochondrial DNA(mtDNA) or repetitiveDNA elements (microsatellite loci), which show considerable variation in the number of repeats within individuals have been employed to achieve the identification of parasites species or strain.

  1. The molecular basis of variable phenotypic severity among common missense mutations causing Rett syndrome.

    Science.gov (United States)

    Brown, Kyla; Selfridge, Jim; Lagger, Sabine; Connelly, John; De Sousa, Dina; Kerr, Alastair; Webb, Shaun; Guy, Jacky; Merusi, Cara; Koerner, Martha V; Bird, Adrian

    2016-02-01

    Rett syndrome is caused by mutations in the X-linked MECP2 gene, which encodes a chromosomal protein that binds to methylated DNA. Mouse models mirror the human disorder and therefore allow investigation of phenotypes at a molecular level. We describe an Mecp2 allelic series representing the three most common missense Rett syndrome (RTT) mutations, including first reports of Mecp2[R133C] and Mecp2[T158M] knock-in mice, in addition to Mecp2[R306C] mutant mice. Together these three alleles comprise ∼25% of all RTT mutations in humans, but they vary significantly in average severity. This spectrum is mimicked in the mouse models; R133C being least severe, T158M most severe and R306C of intermediate severity. Both R133C and T158M mutations cause compound phenotypes at the molecular level, combining compromised DNA binding with reduced stability, the destabilizing effect of T158M being more severe. Our findings contradict the hypothesis that the R133C mutation exclusively abolishes binding to hydroxymethylated DNA, as interactions with DNA containing methyl-CG, methyl-CA and hydroxymethyl-CA are all reduced in vivo. We find that MeCP2[T158M] is significantly less stable than MeCP2[R133C], which may account for the divergent clinical impact of the mutations. Overall, this allelic series recapitulates human RTT severity, reveals compound molecular aetiologies and provides a valuable resource in the search for personalized therapeutic interventions.

  2. Molecular mechanisms of nematode-nematophagous microbe interactions: basis for biological control of plant-parasitic nematodes.

    Science.gov (United States)

    Li, Juan; Zou, Chenggang; Xu, Jianping; Ji, Xinglai; Niu, Xuemei; Yang, Jinkui; Huang, Xiaowei; Zhang, Ke-Qin

    2015-01-01

    Plant-parasitic nematodes cause significant damage to a broad range of vegetables and agricultural crops throughout the world. As the natural enemies of nematodes, nematophagous microorganisms offer a promising approach to control the nematode pests. Some of these microorganisms produce traps to capture and kill the worms from the outside. Others act as internal parasites to produce toxins and virulence factors to kill the nematodes from within. Understanding the molecular basis of microbe-nematode interactions provides crucial insights for developing effective biological control agents against plant-parasitic nematodes. Here, we review recent advances in our understanding of the interactions between nematodes and nematophagous microorganisms, with a focus on the molecular mechanisms by which nematophagous microorganisms infect nematodes and on the nematode defense against pathogenic attacks. We conclude by discussing several key areas for future research and development, including potential approaches to apply our recent understandings to develop effective biocontrol strategies.

  3. Rich diversity and potency of skin antioxidant peptides revealed a novel molecular basis for high-altitude adaptation of amphibians.

    Science.gov (United States)

    Yang, Xinwang; Wang, Ying; Zhang, Yue; Lee, Wen-Hui; Zhang, Yun

    2016-01-27

    Elucidating the mechanisms of high-altitude adaptation is an important research area in modern biology. To date, however, knowledge has been limited to the genetic mechanisms of adaptation to the lower oxygen and temperature levels prevalent at high altitudes, with adaptation to UV radiation largely neglected. Furthermore, few proteomic or peptidomic analyses of these factors have been performed. In this study, the molecular adaptation of high-altitude Odorrana andersonii and cavernicolous O. wuchuanensis to elevated UV radiation was investigated. Compared with O. wuchuanensis, O. andersonii exhibited greater diversity and free radical scavenging potentiality of skin antioxidant peptides to cope with UV radiation. This implied that O. andersonii evolved a much more complicated and powerful skin antioxidant peptide system to survive high-altitude UV levels. Our results provided valuable peptidomic clues for understanding the novel molecular basis for adaptation to high elevation habitats.

  4. Molecular basis for amino acid sensing by family C G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Bräuner-Osborne, Hans

    2009-01-01

    -alpha;-amino acid receptor G-protein-coupled receptor family C, group 6, subtype A (GPRC6A) and seven orphan receptors. Aside from the orphan receptors, the family C GPCRs are dimeric receptors characterized by a large extracellular Venus flytrap domain which bind the endogenous agonists. Except from the GABA(B1......-2) and T1R2-3 receptor, all receptors are either activated or positively modulated by amino acids. In this review, we outline mutational, biophysical and structural studies which have elucidated the interaction of the amino acids with the Venus flytrap domains, molecular mechanisms of receptor selectivity...

  5. Molecular basis of the potential of mesalazine to prevent colorectal cancer

    Institute of Scientific and Technical Information of China (English)

    Carmine Stolfi; Roberto Pellegrini; Eleonora Franzè; Francesco Pallone; Giovanni Monteleone

    2008-01-01

    Patients with ulcerative colitis (UC) and Crohn's disease (CD) are at increased risk for developing colorectal cancer (CRC), and this is believed to be a result of chronic inflammation. Although conclusive evidence is still missing, both epidemiological and experimental observations suggest that certain drugs used to treat inflammation, such as mesalazine,can reduce the incidence of colitis-associated CRC.Therefore, in recent years, several studies have been conducted to dissect the mechanisms by which mesalazine interferes with CRC cell growth and survival. This review summarizes the current information on the molecular mechanisms that underlie the antineoplastic action of mesalazine.

  6. Molecular structure, IR spectra, and chemical reactivity of cisplatin and transplatin: DFT studies, basis set effect and solvent effect.

    Science.gov (United States)

    Wang, Yang; Liu, Qingzhu; Qiu, Ling; Wang, Tengfei; Yuan, Haoliang; Lin, Jianguo; Luo, Shineng

    2015-01-01

    Three different density functional theory (DFT) methods were employed to study the molecular structures of cis-diamminedichloroplatinum(II) (CDDP) and trans-diamminedichloroplatinum(II) (TDDP). The basis set effect on the structure was also investigated. By comparing the optimized structures with the experimental data, a relatively more accurate method was chosen for further study of the IR spectra and other properties as well as the solvent effect. Nineteen characteristic vibrational bands of the title compounds were assigned and compared with available experimental data. The number of characteristic peaks for the asymmetric stretching and deformation vibrations of N-H can serve as a judgment for the isomer between CDDP and TDDP. Significant solvent effect was observed on the molecular structures and IR spectra. The reduced density gradient analysis was performed to study the intramolecular interactions of CDDP and TDDP, and the nature of changes in the structures caused by the solvent was illustrated. Several descriptors determined from the energies of frontier molecular orbitals (HOMO and LUMO) were applied to describe the chemical reactivity of the title compounds. The molecular electrostatic potential (MESP) surfaces showed that the amino groups were the most favorable sites that nucleophilic reagents tend to attack, and CDDP was easier to be attacked by nucleophilic reagents than TDDP.

  7. Molecular basis of RNA guanine-7 methyltransferase (RNMT) activation by RAM.

    Science.gov (United States)

    Varshney, Dhaval; Petit, Alain-Pierre; Bueren-Calabuig, Juan A; Jansen, Chimed; Fletcher, Dan A; Peggie, Mark; Weidlich, Simone; Scullion, Paul; Pisliakov, Andrei V; Cowling, Victoria H

    2016-12-01

    Maturation and translation of mRNA in eukaryotes requires the addition of the 7-methylguanosine cap. In vertebrates, the cap methyltransferase, RNA guanine-7 methyltransferase (RNMT), has an activating subunit, RNMT-Activating Miniprotein (RAM). Here we report the first crystal structure of the human RNMT in complex with the activation domain of RAM. A relatively unstructured and negatively charged RAM binds to a positively charged surface groove on RNMT, distal to the active site. This results in stabilisation of a RNMT lobe structure which co-evolved with RAM and is required for RAM binding. Structure-guided mutagenesis and molecular dynamics simulations reveal that RAM stabilises the structure and positioning of the RNMT lobe and the adjacent α-helix hinge, resulting in optimal positioning of helix A which contacts substrates in the active site. Using biophysical and biochemical approaches, we observe that RAM increases the recruitment of the methyl donor, AdoMet (S-adenosyl methionine), to RNMT. Thus we report the mechanism by which RAM allosterically activates RNMT, allowing it to function as a molecular rheostat for mRNA cap methylation. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Mass Spectrometry-based Approaches to Understand the Molecular Basis of Memory

    Directory of Open Access Journals (Sweden)

    Arthur Henriques Pontes

    2016-10-01

    Full Text Available The central nervous system is responsible for an array of cognitive functions such as memory, learning, language and attention. These processes tend to take place in distinct brain regions; yet, they need to be integrated to give rise to adaptive or meaningful behavior. Since cognitive processes result from underlying cellular and molecular changes, genomics and transcriptomics assays have been applied to human and animal models to understand such events. Nevertheless, genes and RNAs are not the end products of most biological functions. In order to gain further insights toward the understanding of brain processes, the field of proteomics has been of increasing importance in the past years. Advancements in liquid chromatography-tandem mass spectrometry (LC-MS/MS have enable the identification and quantification of thousand of proteins with high accuracy and sensitivity, fostering a revolution in the neurosciences. Herein, we review the molecular bases of explicit memory in the hippocampus. We outline the principles of mass spectrometry (MS-based proteomics, highlighting the use of this analytical tool to study memory formation. In addition, we discuss MS-based targeted approaches as the future of protein analysis.

  9. Molecular basis of classic galactosemia from the structure of human galactose 1-phosphate uridylyltransferase.

    Science.gov (United States)

    McCorvie, Thomas J; Kopec, Jolanta; Pey, Angel L; Fitzpatrick, Fiona; Patel, Dipali; Chalk, Rod; Shrestha, Leela; Yue, Wyatt W

    2016-06-01

    Classic galactosemia is a potentially lethal disease caused by the dysfunction of galactose 1-phosphate uridylyltransferase (GALT). Over 300 disease-associated GALT mutations have been reported, with the majority being missense changes, although a better understanding of their underlying molecular effects has been hindered by the lack of structural information for the human enzyme. Here, we present the 1.9 Å resolution crystal structure of human GALT (hGALT) ternary complex, revealing a homodimer arrangement that contains a covalent uridylylated intermediate and glucose-1-phosphate in the active site, as well as a structural zinc-binding site, per monomer. hGALT reveals significant structural differences from bacterial GALT homologues in metal ligation and dimer interactions, and therefore is a zbetter model for understanding the molecular consequences of disease mutations. Both uridylylation and zinc binding influence the stability and aggregation tendency of hGALT. This has implications for disease-associated variants where p.Gln188Arg, the most commonly detected, increases the rate of aggregation in the absence of zinc likely due to its reduced ability to form the uridylylated intermediate. As such our structure serves as a template in the future design of pharmacological chaperone therapies and opens new concepts about the roles of metal binding and activity in protein misfolding by disease-associated mutants.

  10. Molecular Basis of Medullary Thyroid Carcinoma: The Role of RET Polymorphisms

    Directory of Open Access Journals (Sweden)

    Ana Luiza Maia

    2011-12-01

    Full Text Available Medullary thyroid carcinoma is a rare malignant tumor originating in parafollicular C cells. It accounts for 5 to 8% of all thyroid cancers. MTC develops in either sporadic (75% or hereditary form (25%. Genetic and molecular studies have demonstrated the involvement of the RET proto-oncogene in hereditary MTC and, less often, in its sporadic form. Although a strong genotype-phenotype correlation has been described, wide clinical heterogeneity is observed among families with the same RET mutation or even in carriers of the same kindred. In recent years, several single nucleotide polymorphisms of the RET gene have been described in the general population as well as in patients with MTC. Some studies have reported associations between the presence of polymorphisms and development or progression of MTC. Nonetheless, other studies failed to demonstrate any effect of the RET variants. Differences in the genetic background of distinct populations or methodological approaches have been suggested as potential reasons for the conflicting results. Here, we review current knowledge concerning the molecular pathogenesis of sporadic and hereditary MTC. In particular, we analyze the role of RET polymorphisms in the clinical presentation and prognosis of MTC based on the current literature.

  11. Identifying the molecular basis of functions in the transcriptome of the social amoeba Dictyostelium discoideum.

    Science.gov (United States)

    Whitney, T J; Gardner, D G; Mott, M L; Brandon, M

    2010-03-09

    The unusual life cycle of Dictyostelium discoideum, in which an extra-cellular stressor such as starvation induces the development of a multicellular fruiting body consisting of stalk cells and spores from a culture of identical amoebae, provides an excellent model for investigating the molecular control of differentiation and the transition from single- to multi-cellular life, a key transition in development. We utilized serial analysis of gene expression (SAGE), a molecular method that is unbiased by dependence on previously identified genes, to obtain a transcriptome from a high-density culture of amoebae, in order to examine the transition to multi-cellular development. The SAGE method provides relative expression levels, which allows us to rank order the expressed genes. We found that a large number of ribosomal proteins were expressed at high levels, while various components of the proteosome were expressed at low levels. The only identifiable transmembrane signaling system components expressed in amoebae are related to quorum sensing, and their expression levels were relatively low. The most highly expressed gene in the amoeba transcriptome, dutA untranslated RNA, is a molecule with unknown function that may serve as an inhibitor of translation. These results suggest that high-density amoebae have not initiated development, and they also suggest a mechanism by which the transition into the development program is controlled.

  12. Mass Spectrometry-based Approaches to Understand the Molecular Basis of Memory

    Science.gov (United States)

    Pontes, Arthur; de Sousa, Marcelo

    2016-10-01

    The central nervous system is responsible for an array of cognitive functions such as memory, learning, language and attention. These processes tend to take place in distinct brain regions; yet, they need to be integrated to give rise to adaptive or meaningful behavior. Since cognitive processes result from underlying cellular and molecular changes, genomics and transcriptomics assays have been applied to human and animal models to understand such events. Nevertheless, genes and RNAs are not the end products of most biological functions. In order to gain further insights toward the understanding of brain processes, the field of proteomics has been of increasing importance in the past years. Advancements in liquid chromatography-tandem mass spectrometry (LC-MS/MS) have enable the identification and quantification of thousand of proteins with high accuracy and sensitivity, fostering a revolution in the neurosciences. Herein, we review the molecular bases of explicit memory in the hippocampus. We outline the principles of mass spectrometry (MS)-based proteomics, highlighting the use of this analytical tool to study memory formation. In addition, we discuss MS-based targeted approaches as the future of protein analysis.

  13. Structure of soybean [beta]-cyanoalanine synthase and the molecular basis for cyanide detoxification in plants

    Energy Technology Data Exchange (ETDEWEB)

    Yi, Hankuil; Juergens, Matthew; Jez, Joseph M. (WU)

    2012-09-07

    Plants produce cyanide (CN{sup -}) during ethylene biosynthesis in the mitochondria and require {beta}-cyanoalanine synthase (CAS) for CN{sup -} detoxification. Recent studies show that CAS is a member of the {beta}-substituted alanine synthase (BSAS) family, which also includes the Cys biosynthesis enzyme O-acetylserine sulfhydrylase (OASS), but how the BSAS evolved distinct metabolic functions is not understood. Here we show that soybean (Glycine max) CAS and OASS form {alpha}-aminoacrylate reaction intermediates from Cys and O-acetylserine, respectively. To understand the molecular evolution of CAS and OASS in the BSAS enzyme family, the crystal structures of Gm-CAS and the Gm-CAS K95A mutant with a linked pyridoxal phosphate (PLP)-Cys molecule in the active site were determined. These structures establish a common fold for the plant BSAS family and reveal a substrate-induced conformational change that encloses the active site for catalysis. Comparison of CAS and OASS identified residues that covary in the PLP binding site. The Gm-OASS T81M, S181M, and T185S mutants altered the ratio of OASS:CAS activity but did not convert substrate preference to that of a CAS. Generation of a triple mutant Gm-OASS successfully switched reaction chemistry to that of a CAS. This study provides new molecular insight into the evolution of diverse enzyme functions across the BSAS family in plants.

  14. Molecular basis of classic galactosemia from the structure of human galactose 1-phosphate uridylyltransferase

    Science.gov (United States)

    McCorvie, Thomas J.; Kopec, Jolanta; Pey, Angel L.; Fitzpatrick, Fiona; Patel, Dipali; Chalk, Rod; Shrestha, Leela; Yue, Wyatt W.

    2016-01-01

    Classic galactosemia is a potentially lethal disease caused by the dysfunction of galactose 1-phosphate uridylyltransferase (GALT). Over 300 disease-associated GALT mutations have been reported, with the majority being missense changes, although a better understanding of their underlying molecular effects has been hindered by the lack of structural information for the human enzyme. Here, we present the 1.9 Å resolution crystal structure of human GALT (hGALT) ternary complex, revealing a homodimer arrangement that contains a covalent uridylylated intermediate and glucose-1-phosphate in the active site, as well as a structural zinc-binding site, per monomer. hGALT reveals significant structural differences from bacterial GALT homologues in metal ligation and dimer interactions, and therefore is a zbetter model for understanding the molecular consequences of disease mutations. Both uridylylation and zinc binding influence the stability and aggregation tendency of hGALT. This has implications for disease-associated variants where p.Gln188Arg, the most commonly detected, increases the rate of aggregation in the absence of zinc likely due to its reduced ability to form the uridylylated intermediate. As such our structure serves as a template in the future design of pharmacological chaperone therapies and opens new concepts about the roles of metal binding and activity in protein misfolding by disease-associated mutants. PMID:27005423

  15. MOLECULAR BASIS OF PERIPHERAL OLFACTORY PLASTICITY IN Rhodnius prolixus, A CHAGAS DISEASE VECTOR

    Directory of Open Access Journals (Sweden)

    Jose Manuel Latorre Estivalis

    2015-07-01

    Full Text Available Olfaction is fundamental for most animals and critical for different aspects of triatomine biology, including host-seeking, reproduction, avoidance of predators, and aggregation in shelters. Ethological and physiological aspects of these olfactory-mediated behaviors are well understood, but their molecular bases are still largely unknown. Here we investigated changes in molecular mechanisms at the peripheral olfactory level in response to different physiological and developmental conditions. For this, the antennal expression levels of the odorant (Orco and ionotropic (IR8a, IR25a and IR76b coreceptor genes were determined in Rhodnius prolixus by means of quantitative real-time PCR (qRT-PCR analysis. Gene expression changes were analyzed to test the effect of feeding and imaginal molt for both sexes. Moreover, we analyzed whether expression of these genes changed during the early life of adult bugs. Under these conditions bugs display distinct behavioral responses to diverse chemical stimuli. A significantly decreased expression was induced by blood feeding on all coreceptor genes. The expression of all genes was significantly increased following the imaginal molt. These results show that olfactory coreceptor genes have their expression altered as a response to physiological or developmental changes. Our study suggests that olfactory coreceptor genes confer adaptability to the peripheral olfactory function, probably underlying the known plasticity of triatomine olfactory-mediated behavior.

  16. Structural basis of Staphylococcus epidermidis biofilm formation: mechanisms and molecular interactions

    Science.gov (United States)

    Büttner, Henning; Mack, Dietrich; Rohde, Holger

    2015-01-01

    Staphylococcus epidermidis is a usually harmless commensal bacterium highly abundant on the human skin. Under defined predisposing conditions, most importantly implantation of a medical device, S. epidermidis, however, can switch from a colonizing to an invasive life style. The emergence of S. epidermidis as an opportunistic pathogen is closely linked to the biofilm forming capability of the species. During the past decades, tremendous advance regarding our understanding of molecular mechanisms contributing to surface colonization has been made, and detailed information is available for several factors active during the primary attachment, accumulative or dispersal phase of biofilm formation. A picture evolved in which distinct factors, though appearing to be redundantly organized, take over specific and exclusive functions during biofilm development. In this review, these mechanisms are described in molecular detail, with a highlight on recent insights into multi-functional S. epidermidis cell surface proteins contributing to surface adherence and intercellular adhesion. The integration of distinct biofilm-promoting factors into regulatory networks is summarized, with an emphasis on mechanism that could allow S. epidermidis to flexibly adapt to changing environmental conditions present during colonizing or invasive life-styles. PMID:25741476

  17. The molecular basis of individual differences in phenylthiocarbamide and propylthiouracil bitterness perception.

    Science.gov (United States)

    Bufe, Bernd; Breslin, Paul A S; Kuhn, Christina; Reed, Danielle R; Tharp, Christopher D; Slack, Jay P; Kim, Un-Kyung; Drayna, Dennis; Meyerhof, Wolfgang

    2005-02-22

    Individual differences in perception are ubiquitous within the chemical senses: taste, smell, and chemical somesthesis . A hypothesis of this fact states that polymorphisms in human sensory receptor genes could alter perception by coding for functionally distinct receptor types . We have previously reported evidence that sequence variants in a presumptive bitter receptor gene (hTAS2R38) correlate with differences in bitterness recognition of phenylthiocarbamide (PTC) . Here, we map individual psychogenomic pathways for bitter taste by testing people with a variety of psychophysical tasks and linking their individual perceptions of the compounds PTC and propylthiouracil (PROP) to the in vitro responses of their TAS2R38 receptor variants. Functional expression studies demonstrate that five different haplotypes from the hTAS2R38 gene code for operatively distinct receptors. The responses of the three haplotypes we also tested in vivo correlate strongly with individuals' psychophysical bitter sensitivities to a family of compounds. These data provide a direct molecular link between heritable variability in bitter taste perception to functional variations of a single G protein coupled receptor that responds to compounds such as PTC and PROP that contain the N-C=S moiety. The molecular mechanisms of perceived bitterness variability have therapeutic implications, such as helping patients to consume beneficial bitter-tasting compounds-for example, pharmaceuticals and selected phytochemicals.

  18. Molecular Basis of Neuroimmune Interaction in an In Vitro Coculture Approach

    Institute of Scientific and Technical Information of China (English)

    Mamoru Nakanishi; Tadahide Furuno

    2008-01-01

    A team of researchers from Nagoya, Tokyo and Hamilton developed a unique technique for studying neuro-immune interaction with confocal laser scanning fluorescence microscopy several years ago. It relies on guiding immune and nerve cell interaction by creating an adhesive environment using an in vitro coculture dish. With their technique, they are able to study details of the mechanism of how nerve cells communicate with immune cells (mast cells and T lymphocytes) and vice versa. They showed that nerve-mast cell communication could occur in the absence of an intermediary transducing cell and that the neuropeptide substance P, operating via NK-1 receptors, was a soluble factor of this communication. In addition, recently, they showed that ATP which was released from activated mast cells mediated the activation of nerve cells. Further, with their technique, Nagoya's group was able to study details of the molecular mechanism of nerve-mast cell interaction. N-cadherin and CADM1 (cell adhesion molecule 1) appeared to mediate attachment and promoted the communication between mast cells and nerves predominantly. It would lead to new therapeutic modalities for diseases based on neuroimmune interaction such as neurogenic inflammation, intestinal bowel diseases, asthma, and autoimmune disorders. Cellular & Molecular Immunology. 2008;5(4):249-259.

  19. Comparison of molecular fingerprint methods on the basis of biological profile data.

    Science.gov (United States)

    Steffen, Andreas; Kogej, Thierry; Tyrchan, Christian; Engkvist, Ola

    2009-02-01

    In this study we evaluated a set of molecular fingerprint methods with respect to their capability to reproduce similarities in the biological activity space. The evaluation presented in this paper is therefore different from many other fingerprint studies, in which the enrichment of active compounds binding to the same target as selected query structures was studied. Conversely, our data set was extracted from the BioPrint database, which contains uniformly derived biological activity profiles of mainly marketed drugs for a range of biological assays relevant for the pharmaceutical industry. We compared calculated molecular fingerprint similarity values between all compound pairs of the data set with the corresponding similarities in the biological activity space and additionally analyzed agreements of generated clusterings. A closer analysis of the compound pairs with a high biological activity similarity revealed that fingerprint methods such as CHEMGPS or TRUST4, which describe global features of a molecule such as physicochemical properties and pharmacophore patterns, might be better suited to describe similarity of biological activity profiles than purely structural fingerprint methods. It is therefore suggested that the usage of these fingerprint methods could increase the probability of finding molecules with a similar biological activity profile but yet a different chemical structure.

  20. Phosgene at the complete basis set limit of CCSDT(Q): Molecular structure and rovibrational analysis

    Science.gov (United States)

    Murphy, Kevin V.; Schaefer, Henry F.; Agarwal, Jay

    2017-09-01

    The ground-state (X∼1A″) equilibrium geometry of phosgene is examined with coupled-cluster theory, using derivatives extrapolated to the complete basis set (CBS) limit of CCSDT(Q). The C-O and C-Cl bond lengths are predicted to be 1.1768 Å and 1.7374 Å, respectively; the Cl-C-Cl bond angle is 124.03° and the O-C-Cl bond angle is 111.93°. Anharmonic frequencies are determined with VPT2, using CCSD(T)/cc-pCVQZ cubic and quartic force-fields and a CCSDT(Q)/CBS quadratic force field: ν1 = 1832.9 ; ν2 = 570.5 ; ν3 = 301.2 ; ν4 = 576.3 ; ν5 = 849.4 ; and ν6 = 438.9 cm-1.

  1. Structural and molecular basis for Ebola virus neutralization by protective human antibodies.

    Science.gov (United States)

    Misasi, John; Gilman, Morgan S A; Kanekiyo, Masaru; Gui, Miao; Cagigi, Alberto; Mulangu, Sabue; Corti, Davide; Ledgerwood, Julie E; Lanzavecchia, Antonio; Cunningham, James; Muyembe-Tamfun, Jean Jacques; Baxa, Ulrich; Graham, Barney S; Xiang, Ye; Sullivan, Nancy J; McLellan, Jason S

    2016-03-18

    Ebola virus causes hemorrhagic fever with a high case fatality rate for which there is no approved therapy. Two human monoclonal antibodies, mAb100 and mAb114, in combination, protect nonhuman primates against all signs of Ebola virus disease, including viremia. Here, we demonstrate that mAb100 recognizes the base of the Ebola virus glycoprotein (GP) trimer, occludes access to the cathepsin-cleavage loop, and prevents the proteolytic cleavage of GP that is required for virus entry. We show that mAb114 interacts with the glycan cap and inner chalice of GP, remains associated after proteolytic removal of the glycan cap, and inhibits binding of cleaved GP to its receptor. These results define the basis of neutralization for two protective antibodies and may facilitate development of therapies and vaccines.

  2. Molecular basis and consequences of a deletion in the amelogenin gene, analyzed by capture PCR

    Energy Technology Data Exchange (ETDEWEB)

    Lagerstroem-Fermer, M.; Pettersson, U.; Landegren, U. (Univ. of Uppsala (Sweden))

    1993-07-01

    A mutation that disrupts the gene for one of the major proteins in tooth enamel has been investigated. The mutation is located in the amelogenin gene and causes X-linked amelogenesis imperfecta, characterized by defective mineralization of tooth enamel. The authors have isolated the breakpoints of a 5-kb deletion in the amelogenin gene on the basis of nucleotide sequence information located upstream of the lesion, using a technique termed capture PCR. The deletion removes five of the seven exons, spanning from the second intron to the last exon. Only the first two codons for the mature protein remain, consistent with the relatively severe phenotype of affected individuals in the present family. The mutation appears to have arisen as an illegitimate recombination event since of 11 nucleotide positions immediately surrounding the two breakpoints, 9 are identical. 17 refs., 3 figs., 1 tab.

  3. Rethinking molecular similarity: comparing compounds on the basis of biological activity.

    Science.gov (United States)

    Petrone, Paula M; Simms, Benjamin; Nigsch, Florian; Lounkine, Eugen; Kutchukian, Peter; Cornett, Allen; Deng, Zhan; Davies, John W; Jenkins, Jeremy L; Glick, Meir

    2012-08-17

    Since the advent of high-throughput screening (HTS), there has been an urgent need for methods that facilitate the interrogation of large-scale chemical biology data to build a mode of action (MoA) hypothesis. This can be done either prior to the HTS by subset design of compounds with known MoA or post HTS by data annotation and mining. To enable this process, we developed a tool that compares compounds solely on the basis of their bioactivity: the chemical biological descriptor "high-throughput screening fingerprint" (HTS-FP). In the current embodiment, data are aggregated from 195 biochemical and cell-based assays developed at Novartis and can be used to identify bioactivity relationships among the in-house collection comprising ~1.5 million compounds. We demonstrate the value of the HTS-FP for virtual screening and in particular scaffold hopping. HTS-FP outperforms state of the art methods in several aspects, retrieving bioactive compounds with remarkable chemical dissimilarity to a probe structure. We also apply HTS-FP for the design of screening subsets in HTS. Using retrospective data, we show that a biodiverse selection of plates performs significantly better than a chemically diverse selection of plates, both in terms of number of hits and diversity of chemotypes retrieved. This is also true in the case of hit expansion predictions using HTS-FP similarity. Sets of compounds clustered with HTS-FP are biologically meaningful, in the sense that these clusters enrich for genes and gene ontology (GO) terms, showing that compounds that are bioactively similar also tend to target proteins that operate together in the cell. HTS-FP are valuable not only because of their predictive power but mainly because they relate compounds solely on the basis of bioactivity, harnessing the accumulated knowledge of a high-throughput screening facility toward the understanding of how compounds interact with the proteome.

  4. The Molecular Basis of Toxins’ Interactions with Intracellular Signaling via Discrete Portals

    Directory of Open Access Journals (Sweden)

    Adi Lahiani

    2017-03-01

    Full Text Available An understanding of the molecular mechanisms by which microbial, plant or animal-secreted toxins exert their action provides the most important element for assessment of human health risks and opens new insights into therapies addressing a plethora of pathologies, ranging from neurological disorders to cancer, using toxinomimetic agents. Recently, molecular and cellular biology dissecting tools have provided a wealth of information on the action of these diverse toxins, yet, an integrated framework to explain their selective toxicity is still lacking. In this review, specific examples of different toxins are emphasized to illustrate the fundamental mechanisms of toxicity at different biochemical, molecular and cellular- levels with particular consideration for the nervous system. The target of primary action has been highlighted and operationally classified into 13 sub-categories. Selected examples of toxins were assigned to each target category, denominated as portal, and the modulation of the different portal’s signaling was featured. The first portal encompasses the plasma membrane lipid domains, which give rise to pores when challenged for example with pardaxin, a fish toxin, or is subject to degradation when enzymes of lipid metabolism such as phospholipases A2 (PLA2 or phospholipase C (PLC act upon it. Several major portals consist of ion channels, pumps, transporters and ligand gated ionotropic receptors which many toxins act on, disturbing the intracellular ion homeostasis. Another group of portals consists of G-protein-coupled and tyrosine kinase receptors that, upon interaction with discrete toxins, alter second messengers towards pathological levels. Lastly, subcellular organelles such as mitochondria, nucleus, protein- and RNA-synthesis machineries, cytoskeletal networks and exocytic vesicles are also portals targeted and deregulated by other diverse group of toxins. A fundamental concept can be drawn from these seemingly different

  5. Current understanding of the molecular basis of chloroquine-resistance in Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Jiang H

    2006-01-01

    Full Text Available Chloroquine (CQ is the most successful antimalarial drug ever discovered. Unfortunately, parasites resistant to the drug eventually emerged after its large scale use and are now widespread. Although great progress in our understanding of the mechanisms of CQ action and CQ resistance (CQR has been achieved over the past two decades, including the identification of the molecules responsible for CQR (e.g., Plasmodium falciparum chloroquine resistant transporter, PfCRT many questions remain unanswered. Here we highlight recent advances in our understanding of the genetics and molecular mechanisms of CQR, with particular emphasis on the role of genes such as pfcrt and pfmdr1 in the resistance to CQ and other drugs. New drug development and applications will undoubtedly benefit from a better understanding of CQR, eventually leading to more effective malaria control measures.

  6. Molecular basis of epithelial Ca2+ and Mg2+ transport: insights from the TRP channel family

    DEFF Research Database (Denmark)

    Dimke, Henrik Anthony; Hoenderop, Joost G J; Bindels, René J M

    2011-01-01

    potential (TRP) family identified several new ion channels acting as gatekeepers of Ca(2+) and Mg(2+) transport in these epithelia, greatly increasing our understanding of the molecular processes that facilitate the movement of these minerals. In the intestine, TRP channels contribute to the saturable...... active transcellular movement of divalent cations from the lumen into the enterocyte. Furthermore, in bone, TRPV channels play important roles by influencing the osteoclastic resorption process, thereby contributing importantly to overall bone mineral content. The divalent cation-permeable TRPV5 and TRPM......6 channels are located in the renal distal convolution, the main site of active transcellular Ca(2+) and Mg(2+) transport. The channels are regulated by a multitude of factors and hormones that contribute importantly to keeping the systemic concentrations of Ca(2+) and Mg(2+) within normal limits...

  7. Membrane dynamics of γ-secretase provides a molecular basis for Aβ binding and processing

    DEFF Research Database (Denmark)

    Somavarapu, Arun Kumar; Kepp, Kasper Planeta

    2017-01-01

    γ-secretase produces β-amyloid (Aβ) within its presenilin (PS1) subunit, mutations in which cause Alzheimer’s disease, and current therapies thus seek to modulate its activity. While the general structure is known from recent electron microscopy studies, direct loop- and membrane-interactions and...... a molecular recipe for effective γ-secretase modulators; we provide the full atomic structures for these states that may play a key role in developing selective γ-secretase modulators for treatment of Alzheimer’s disease.......γ-secretase produces β-amyloid (Aβ) within its presenilin (PS1) subunit, mutations in which cause Alzheimer’s disease, and current therapies thus seek to modulate its activity. While the general structure is known from recent electron microscopy studies, direct loop- and membrane...

  8. Molecular basis of physiological heart growth: fundamental concepts and new players

    Science.gov (United States)

    Maillet, Marjorie; van Berlo, Jop H.; Molkentin, Jeffery D.

    2015-01-01

    The heart hypertrophies in response to developmental signals as well as increased workload. Although adult-onset hypertrophy can ultimately lead to disease, cardiac hypertrophy is not necessarily maladaptive and can even be beneficial. Progress has been made in our understanding of the structural and molecular characteristics of physiological cardiac hypertrophy, as well as of the endocrine effectors and associated signalling pathways that regulate it. Physiological hypertrophy is initiated by finite signals, which include growth hormones (such as thyroid hormone, insulin, insulin-like growth factor 1 and vascular endothelial growth factor) and mechanical forces that converge on a limited number of intracellular signalling pathways (such as PI3K, AKT, AMP-activated protein kinase and mTOR) to affect gene transcription, protein translation and metabolism. Harnessing adaptive signalling mediators to reinvigorate the diseased heart could have important medical ramifications. PMID:23258295

  9. Molecular basis of glyphosate resistance-different approaches through protein engineering.

    Science.gov (United States)

    Pollegioni, Loredano; Schonbrunn, Ernst; Siehl, Daniel

    2011-08-01

    Glyphosate (N-phosphonomethyl-glycine) is the most widely used herbicide in the world: glyphosate-based formulations exhibit broad-spectrum herbicidal activity with minimal human and environmental toxicity. The extraordinary success of this simple, small molecule is mainly attributable to the high specificity of glyphosate for the plant enzyme enolpyruvyl shikimate-3-phosphate synthase in the shikimate pathway, leading to the biosynthesis of aromatic amino acids. Starting in 1996, transgenic glyphosate-resistant plants were introduced, thus allowing application of the herbicide to the crop (post-emergence) to remove emerged weeds without crop damage. This review focuses on mechanisms of resistance to glyphosate as obtained through natural diversity, the gene-shuffling approach to molecular evolution, and a rational, structure-based approach to protein engineering. In addition, we offer a rationale for the means by which the modifications made have had their intended effect.

  10. Prenatal Alcohol Exposure in Rodents As a Promising Model for the Study of ADHD Molecular Basis

    Science.gov (United States)

    Rojas-Mayorquín, Argelia E.; Padilla-Velarde, Edgar; Ortuño-Sahagún, Daniel

    2016-01-01

    A physiological parallelism, or even a causal effect relationship, can be deducted from the analysis of the main characteristics of the “Alcohol Related Neurodevelopmental Disorders” (ARND), derived from prenatal alcohol exposure (PAE), and the behavioral performance in the Attention-deficit/hyperactivity disorder (ADHD). These two clinically distinct disease entities, exhibits many common features. They affect neurological shared pathways, and also related neurotransmitter systems. We briefly review here these parallelisms, with their common and uncommon characteristics, and with an emphasis in the subjacent molecular mechanisms of the behavioral manifestations, that lead us to propose that PAE in rats can be considered as a suitable model for the study of ADHD. PMID:28018163

  11. Molecular and Genetic Basis of Hereditary Connective-Tissue Diseases Accompanied by Frequent Fractures

    Directory of Open Access Journals (Sweden)

    G. T. Yakhyaeva

    2016-01-01

    Full Text Available Frequent bone fractures in infancy require the elimination of a large number (> 100 of genetic disorders. The modern diagnostic method of hereditary diseases characterized by debilitating course is a new generation sequencing. The article presents the results of molecular-genetic study conducted in 18 patients with clinical symptoms of connective tissue disorders. 10 (56% patients had mutations in the genes encoding type I collagen chains, leading to the development of osteogenesis imperfecta, 5 (28% — mutations in IV and V type collagen genes that are responsible for the development of Ehlers-Danlos syndrome. 3 (17% patients had mutations in the gene encoding fibrillin-1 protein, deficiency of which is manifested by Marfan syndrome. However, the correlation between patient's phenotype and discovered mutations in the investigated gene is established not in all cases.

  12. The molecular basis for carcinogenesis in metaplastic columnar-lined esophagus.

    Science.gov (United States)

    Souza, R F; Meltzer, S J

    1997-09-01

    A wide variety of biologic events and mechanisms appear to have roles in the development and progression of Barrett's esophagus-associated neoplastic lesions. Figure 5 is a schematic depiction of these events. This is known as an infernogram (named after Dante's Inferno) (S. Kern, unpublished presentations, 1996). Events at the bottom rings of the inferno are high-frequency mutations; nearer to the top of the inferno are the less common events. The next several years promise many further discoveries of not only high-frequency and low-frequency events, but also their application. Some of the molecular alterations already studied show promise as markers for early cancer detection or prognostication. Eventually, applications of these discoveries should yield new and more effective means of preventing and treating the deadly complications of this troublesome premalignant condition.

  13. Structural basis of syndecan-4 phosphorylation as a molecular switch to regulate signaling

    DEFF Research Database (Denmark)

    Koo, Bon-Kyung; Jung, Young Sang; Shin, Joon

    2005-01-01

    The syndecan transmembrane proteoglycans are involved in the organization of the actin cytoskeleton and have important roles as cell surface receptors during cell-matrix interactions. We have shown that the syndecan-4 cytoplasmic domain (4L) forms oligomeric complexes that bind to and stimulate...... PKCalpha activity in the presence of PtdIns(4,5)P2, emphasizing the importance of multimerization in the regulation of PKCalpha activation. Oligomerization of the cytoplasmic domain of syndecan-4 is regulated either positively by PtdIns(4,5)P2 or negatively by phosphorylation of serine 183. Phosphorylation...... analysis showed that the overall conformation of p-4L is a compact intertwined dimer with an unusually symmetric clamp shape, and its molecular surface is mostly positively charged. The two parallel strands form a cavity in the center of the dimeric twist. An especially marked effect of phosphorylation...

  14. Choice of adjuvant drug therapy on the basis of the molecular classification of breast cancer

    Directory of Open Access Journals (Sweden)

    N. S. Besova

    2012-01-01

    Full Text Available Molecular genetic analysis identified some biological subtypes of breast cancer (BC: luminal A, luminal B, HER2 positive, and basal-like (including triple negative. The surrogate clinical and morphological criteria including the immunohistochemical determination of estrogen and progesterone receptors, the hyperexpression and/or amplification of HER2, Ki-67, or tumor grade (G are used to identify the biological subtypes of BC in clinical practice. The biological subtypes are distinguished by their biological course and susceptibility to various systemic treatments, which requires different therapeutic tactics. The paper presents tactics of adjuvant therapy for BC in relation to its biological subtype according to the recommendations of the 12th St. Gallen International Breast Cancer Conference (2011 and considers the place of taxans.

  15. Craniofacial development: current concepts in the molecular basis of Treacher Collins syndrome.

    Science.gov (United States)

    van Gijn, Daniel Richard; Tucker, Abigail S; Cobourne, Martyn T

    2013-07-01

    The human face and skull are an elegant example of the anatomical sophistication that results from the interplay between the molecular cascades and the tissue interactions that are necessary for the proper development of the craniofacial complex. When it fails to develop normally the consequences can have life-long implications for the biological, psychological, and aesthetic wellbeing of an affected person. Among the many syndromes that affect the region, understanding of the biology that underlies Treacher Collins syndrome has advanced in the last decade, particularly concerning the causative TCOF1 gene that encodes TREACLE protein, a serine/alanine-rich nucleolar phosphoprotein with an essential function during ribosome biogenesis in cranial neural crest cells. Abnormal growth and differentiation of these cells affect much of the craniofacial skeleton.

  16. Prediction of static contact angles on the basis of molecular forces and adsorption data

    Science.gov (United States)

    Diaz, M. Elena; Savage, Michael D.; Cerro, Ramon L.

    2016-08-01

    At a three-phase contact line, a liquid bulk phase is in contact with and coexists with a very thin layer of adsorbed molecules. This adsorbed film in the immediate vicinity of a liquid wedge modifies the balance of forces between the liquid and solid phases such that, when included in the balance of forces, a quantitative relationship emerges between the adsorbed film thickness and the static contact angle. This relationship permits the prediction of static contact angles from molecular forces and equilibrium adsorption data by means of quantities that are physically meaningful and measurable. For n-alkanes on polytetrafluoroethylene, for which there are experimental data available on adsorption and contact angles, our computations show remarkable agreement with the data. The results obtained are an improvement on previously published calculations—particularly for alkanes with a low number of carbon atoms, for which adsorption is significant.

  17. Communicating the molecular basis of cancer cell-by-cell: an interview with Tatsushi Igaki

    Directory of Open Access Journals (Sweden)

    2015-12-01

    Full Text Available Tatsushi Igaki is currently based at the Kyoto University Graduate School of Biostudies, where he leads a research group dedicated to using Drosophila genetics to build a picture of the cell-cell communications underlying the establishment and maintenance of multicellular systems. His work has provided insight into the molecular bases of cell competition in the context of development and tumorigenesis, including the landmark discovery that oncogenic cells communicate with normal cells in the tumor microenvironment to induce tumor progression in a non-autonomous fashion. In this interview, he describes his career path, highlighting the shift in his research focus from the basic principles of apoptosis to clonal evolution in cancer, and also explains why Drosophila provides a powerful model system for studying cancer biology.

  18. Molecular basis for the role of Staphylococcus aureus penicillin binding protein 4 in antimicrobial resistance.

    Science.gov (United States)

    Navratna, Vikas; Nadig, Savitha; Sood, Varun; Prasad, K; Arakere, Gayathri; Gopal, B

    2010-01-01

    Penicillin binding proteins (PBPs) are membrane-associated proteins that catalyze the final step of murein biosynthesis. These proteins function as either transpeptidases or carboxypeptidases and in a few cases demonstrate transglycosylase activity. Both transpeptidase and carboxypeptidase activities of PBPs occur at the D-Ala-D-Ala terminus of a murein precursor containing a disaccharide pentapeptide comprising N-acetylglucosamine and N-acetyl-muramic acid-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala. Beta-lactam antibiotics inhibit these enzymes by competing with the pentapeptide precursor for binding to the active site of the enzyme. Here we describe the crystal structure, biochemical characteristics, and expression profile of PBP4, a low-molecular-mass PBP from Staphylococcus aureus strain COL. The crystal structures of PBP4-antibiotic complexes reported here were determined by molecular replacement, using the atomic coordinates deposited by the New York Structural Genomics Consortium. While the pbp4 gene is not essential for the viability of S. aureus, the knockout phenotype of this gene is characterized by a marked reduction in cross-linked muropeptide and increased vancomycin resistance. Unlike other PBPs, we note that expression of PBP4 was not substantially altered under different experimental conditions, nor did it change across representative hospital- or community-associated strains of S. aureus that were examined. In vitro data on purified recombinant S. aureus PBP4 suggest that it is a beta-lactamase and is not trapped as an acyl intermediate with beta-lactam antibiotics. Put together, the expression analysis and biochemical features of PBP4 provide a framework for understanding the function of this protein in S. aureus and its role in antimicrobial resistance.

  19. Molecular time-course and the metabolic basis of entry into dauer in Caenorhabditis elegans.

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    Pan-Young Jeong

    Full Text Available When Caenorhabditis elegans senses dauer pheromone (daumone, signaling inadequate growth conditions, it enters the dauer state, which is capable of long-term survival. However, the molecular pathway of dauer entry in C. elegans has remained elusive. To systematically monitor changes in gene expression in dauer paths, we used a DNA microarray containing 22,625 gene probes corresponding to 22,150 unique genes from C. elegans. We employed two different paths: direct exposure to daumone (Path 1 and normal growth media plus liquid culture (Path 2. Our data reveal that entry into dauer is accomplished through the multi-step process, which appears to be compartmentalized in time and according to metabolic flux. That is, a time-course of dauer entry in Path 1 shows that dauer larvae formation begins at post-embryonic stage S4 (48 h and is complete at S6 (72 h. Our results also suggest the presence of a unique adaptive metabolic control mechanism that requires both stage-specific expression of specific genes and tight regulation of different modes of fuel metabolite utilization to sustain the energy balance in the context of prolonged survival under adverse growth conditions. It is apparent that worms entering dauer stage may rely heavily on carbohydrate-based energy reserves, whereas dauer larvae utilize fat or glyoxylate cycle-based energy sources. We created a comprehensive web-based dauer metabolic database for C. elegans (www.DauerDB.org that makes it possible to search any gene and compare its relative expression at a specific stage, or evaluate overall patterns of gene expression in both paths. This database can be accessed by the research community and could be widely applicable to other related nematodes as a molecular atlas.

  20. Molecular Basis for the Neutralization of Tumor Necrosis Factor α by Certolizumab Pegol in the Treatment of Inflammatory Autoimmune Diseases

    Science.gov (United States)

    Lee, Jee Un; Shin, Woori; Son, Ji Young; Yoo, Ki-Young; Heo, Yong-Seok

    2017-01-01

    Monoclonal antibodies against TNFα, including infliximab, adalimumab, golimumab, and certolizumab pegol, are widely used for the treatment of the inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. Recently, the crystal structures of TNFα, in complex with the Fab fragments of infliximab and adalimumab, have revealed the molecular mechanisms of these antibody drugs. Here, we report the crystal structure of TNFα in complex with the Fab fragment of certolizumab pegol to clarify the precise antigen-antibody interactions and the structural basis for the neutralization of TNFα by this therapeutic antibody. The structural analysis and the mutagenesis study revealed that the epitope is limited to a single protomer of the TNFα trimer. Additionally, the DE loop and the GH loop of TNFα play critical roles in the interaction with certolizumab, suggesting that this drug exerts its effects by partially occupying the receptor binding site of TNFα. In addition, a conformational change of the DE loop was induced by certolizumab binding, thereby interrupting the TNFα-receptor interaction. A comprehensive comparison of the interactions of TNFα blockers with TNFα revealed the epitope diversity on the surface of TNFα, providing a better understanding of the molecular mechanism of TNFα blockers. The accumulation of these structural studies can provide a basis for the improvement of therapeutic antibodies against TNFα. PMID:28124979

  1. Molecular Basis for the Neutralization of Tumor Necrosis Factor α by Certolizumab Pegol in the Treatment of Inflammatory Autoimmune Diseases.

    Science.gov (United States)

    Lee, Jee Un; Shin, Woori; Son, Ji Young; Yoo, Ki-Young; Heo, Yong-Seok

    2017-01-23

    Monoclonal antibodies against TNFα, including infliximab, adalimumab, golimumab, and certolizumab pegol, are widely used for the treatment of the inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. Recently, the crystal structures of TNFα, in complex with the Fab fragments of infliximab and adalimumab, have revealed the molecular mechanisms of these antibody drugs. Here, we report the crystal structure of TNFα in complex with the Fab fragment of certolizumab pegol to clarify the precise antigen-antibody interactions and the structural basis for the neutralization of TNFα by this therapeutic antibody. The structural analysis and the mutagenesis study revealed that the epitope is limited to a single protomer of the TNFα trimer. Additionally, the DE loop and the GH loop of TNFα play critical roles in the interaction with certolizumab, suggesting that this drug exerts its effects by partially occupying the receptor binding site of TNFα. In addition, a conformational change of the DE loop was induced by certolizumab binding, thereby interrupting the TNFα-receptor interaction. A comprehensive comparison of the interactions of TNFα blockers with TNFα revealed the epitope diversity on the surface of TNFα, providing a better understanding of the molecular mechanism of TNFα blockers. The accumulation of these structural studies can provide a basis for the improvement of therapeutic antibodies against TNFα.

  2. Molecular basis of the binding of YAP transcriptional regulator to the ErbB4 receptor tyrosine kinase.

    Science.gov (United States)

    Schuchardt, Brett J; Bhat, Vikas; Mikles, David C; McDonald, Caleb B; Sudol, Marius; Farooq, Amjad

    2014-06-01

    The newly discovered transactivation function of ErbB4 receptor tyrosine kinase is believed to be mediated by virtue of the ability of its proteolytically-cleaved intracellular domain (ICD) to physically associate with YAP2 transcriptional regulator. In an effort to unearth the molecular basis of YAP2-ErbB4 interaction, we have conducted a detailed biophysical analysis of the binding of WW domains of YAP2 to PPXY motifs located within the ICD of ErbB4. Our data show that the WW1 domain of YAP2 binds to PPXY motifs within the ICD in a differential manner and that this behavior is by and large replicated by the WW2 domain. Remarkably, while both WW domains absolutely require the integrity of the PPXY consensus sequence, non-consensus residues within and flanking this motif do not appear to be critical for binding. In spite of this shared mode of binding, the WW domains of YAP2 display distinct conformational dynamics in complex with PPXY motifs derived from ErbB4. Collectively, our study lends new insights into the molecular basis of a key protein-protein interaction involved in a diverse array of cellular processes. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  3. Molecular Basis for the Neutralization of Tumor Necrosis Factor α by Certolizumab Pegol in the Treatment of Inflammatory Autoimmune Diseases

    Directory of Open Access Journals (Sweden)

    Jee Un Lee

    2017-01-01

    Full Text Available Monoclonal antibodies against TNFα, including infliximab, adalimumab, golimumab, and certolizumab pegol, are widely used for the treatment of the inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. Recently, the crystal structures of TNFα, in complex with the Fab fragments of infliximab and adalimumab, have revealed the molecular mechanisms of these antibody drugs. Here, we report the crystal structure of TNFα in complex with the Fab fragment of certolizumab pegol to clarify the precise antigen-antibody interactions and the structural basis for the neutralization of TNFα by this therapeutic antibody. The structural analysis and the mutagenesis study revealed that the epitope is limited to a single protomer of the TNFα trimer. Additionally, the DE loop and the GH loop of TNFα play critical roles in the interaction with certolizumab, suggesting that this drug exerts its effects by partially occupying the receptor binding site of TNFα. In addition, a conformational change of the DE loop was induced by certolizumab binding, thereby interrupting the TNFα-receptor interaction. A comprehensive comparison of the interactions of TNFα blockers with TNFα revealed the epitope diversity on the surface of TNFα, providing a better understanding of the molecular mechanism of TNFα blockers. The accumulation of these structural studies can provide a basis for the improvement of therapeutic antibodies against TNFα.

  4. Molecular basis for the reproductive division of labour in a lower termite

    Directory of Open Access Journals (Sweden)

    Rehli Michael

    2007-06-01

    Full Text Available Abstract Background Polyphenism, the expression of different phenotypes with the same genetic background, is well known for social insects. The substantial physiological and morphological differences among the castes generally are the result of differential gene expression. In lower termites, workers are developmentally flexible to become neotenic replacement reproductives via a single moult after the death of the founding reproductives. Thus, both castes (neotenics and workers are expected to differ mainly in the expression of genes linked to reproductive division of labour, which constitutes the fundamental basis of insect societies. Results Representational difference analysis of cDNAs was used to study differential gene expression between neotenics and workers in the drywood termite Cryptotermes secundus (Kalotermitidae. We identified and, at least partially cloned five novel genes that were highly expressed in female neotenics. Quantitative real-time PCR analysis of all five genes in different castes (neotenics, founding reproductives, winged sexuals and workers of both sexes confirmed the differential expression patterns. In addition, the relative expression of these genes was determined in three body parts of female neotenics (head, thorax, and abdomen using quantitative real-time PCR. Conclusion The identified genes could be involved in the control and regulation of reproductive division of labour. Interestingly, this study revealed an expression pattern partly similar to social Hymenoptera indicating both common and species-specific regulatory mechanisms in hemimetabolous and holometabolous social insects.

  5. Molecular And Structural Basis of Cytokine Receptor Pleiotropy in the Interleukin-4/13 System

    Energy Technology Data Exchange (ETDEWEB)

    LaPorte, S.L.; Juo, Z.S.; Vaclavikova, J.; Colf, L.A.; Qi, X.; Heller, N.M.; Keegan, A.D.; Garcia, K.C.

    2009-05-20

    Interleukin-4 and Interleukin-13 are cytokines critical to the development of T cell-mediated humoral immune responses, which are associated with allergy and asthma, and exert their actions through three different combinations of shared receptors. Here we present the crystal structures of the complete set of type I (IL-4R{alpha}/{gamma}{sub c}/IL-4) and type II (IL-4R/IL-13R{alpha}1/IL-4, IL-4R{alpha}/IL-13R{alpha}1/IL-13) ternary signaling complexes. The type I complex reveals a structural basis for {gamma}{sub c}'s ability to recognize six different {gamma}{sub c}-cytokines. The two type II complexes utilize an unusual top-mounted Ig-like domain on IL-13R{alpha}1 for a novel mode of cytokine engagement that contributes to a reversal in the IL-4 versus IL-13 ternary complex assembly sequences, which are mediated through substantially different recognition chemistries. We also show that the type II receptor heterodimer signals with different potencies in response to IL-4 versus IL-13 and suggest that the extracellular cytokine-receptor interactions are modulating intracellular membrane-proximal signaling events.

  6. Molecular basis for the mutagenic and lethal effects of ultraviolet irradiation. Research accomplishments (1968 to present)

    Energy Technology Data Exchange (ETDEWEB)

    Grossman, L.

    1978-01-01

    Earlier work on the chemical basis of mutagenesis led to certain chemical generalities sufficient to explain how certain mutagens such as uv light and hydroxylamine functioned in information transfer systems (replicative, transcriptive and translational). When such modifications were applied to biologically active DNA in a controlled manner biological expression was non-stoichiometric because much of the damage was removed from the DNA by repair systems. Our efforts were then directed to these systems which led to: (1) the isolation, purification and characterization of endonucleases responsible for the first and controlling step in DNA repair - referred to as incision in both M. luteus and E. coli. The biological role of these enzymes was inferred in appropriate mutants; (2) the isolation, purification and characterization of exonucleases responsible for the removal or excision of damaged nucleotides in M. luteus and human placental trophoblasts; (3) the repair of uv damaged biologically active transforming and transfecting DNAs by purified endonucleases, exonucleases, DNA polymerase I and polynucleotide ligase from M. luteus and E. coli; (4) the characterization of the dual gene control for incision phenomenon in M. luteus and E. coli; and (5) isolation, purification and characterization of repair enzymes from human placenta (currently in progress).

  7. Natural emulsifiers - Biosurfactants, phospholipids, biopolymers, and colloidal particles: Molecular and physicochemical basis of functional performance.

    Science.gov (United States)

    McClements, David Julian; Gumus, Cansu Ekin

    2016-08-01

    There is increasing consumer pressure for commercial products that are more natural, sustainable, and environmentally friendly, including foods, cosmetics, detergents, and personal care products. Industry has responded by trying to identify natural alternatives to synthetic functional ingredients within these products. The focus of this review article is on the replacement of synthetic surfactants with natural emulsifiers, such as amphiphilic proteins, polysaccharides, biosurfactants, phospholipids, and bioparticles. In particular, the physicochemical basis of emulsion formation and stabilization by natural emulsifiers is discussed, and the benefits and limitations of different natural emulsifiers are compared. Surface-active polysaccharides typically have to be used at relatively high levels to produce small droplets, but the droplets formed are highly resistant to environmental changes. Conversely, surface-active proteins are typically utilized at low levels, but the droplets formed are highly sensitive to changes in pH, ionic strength, and temperature. Certain phospholipids are capable of producing small oil droplets during homogenization, but again the droplets formed are highly sensitive to changes in environmental conditions. Biosurfactants (saponins) can be utilized at low levels to form fine oil droplets that remain stable over a range of environmental conditions. Some nature-derived nanoparticles (e.g., cellulose, chitosan, and starch) are effective at stabilizing emulsions containing relatively large oil droplets. Future research is encouraged to identify, isolate, purify, and characterize new types of natural emulsifier, and to test their efficacy in food, cosmetic, detergent, personal care, and other products.

  8. Molecular basis of positive allosteric modulation of GluN2B NMDA receptors by polyamines.

    Science.gov (United States)

    Mony, Laetitia; Zhu, Shujia; Carvalho, Stéphanie; Paoletti, Pierre

    2011-06-17

    NMDA receptors (NMDARs) form glutamate-gated ion channels that have central roles in neuronal communication and plasticity throughout the brain. Dysfunctions of NMDARs are involved in several central nervous system disorders, including stroke, chronic pain and schizophrenia. One hallmark of NMDARs is that their activity can be allosterically regulated by a variety of extracellular small ligands. While much has been learned recently regarding allosteric inhibition of NMDARs, the structural determinants underlying positive allosteric modulation of these receptors remain poorly defined. Here, we show that polyamines, naturally occurring polycations that selectively enhance NMDARs containing the GluN2B subunit, bind at a dimer interface between GluN1 and GluN2B subunit N-terminal domains (NTDs). Polyamines act by shielding negative charges present on GluN1 and GluN2B NTD lower lobes, allowing their close apposition, an effect that in turn prevents NTD clamshell closure. Our work reveals the mechanistic basis for positive allosteric modulation of NMDARs. It provides the first example of an intersubunit binding site in this class of receptors, a discovery that holds promise for future drug interventions.

  9. Seeded fibrillation as molecular basis of the species barrier in human prion diseases.

    Directory of Open Access Journals (Sweden)

    Lars Luers

    Full Text Available Prion diseases are transmissible spongiform encephalopathies in humans and animals, including scrapie in sheep, bovine spongiform encephalopathy (BSE in cattle, chronic wasting disease (CWD in deer, and Creutzfeldt-Jakob disease (CJD in humans. The hallmark of prion diseases is the conversion of the host-encoded prion protein (PrP(C to its pathological isoform PrP(Sc, which is accompanied by PrP fibrillation. Transmission is not restricted within one species, but can also occur between species. In some cases a species barrier can be observed that results in limited or unsuccessful transmission. The mechanism behind interspecies transmissibility or species barriers is not completely understood. To analyse this process at a molecular level, we previously established an in vitro fibrillation assay, in which recombinant PrP (recPrP as substrate can be specifically seeded by PrP(Sc as seed. Seeding with purified components, with no additional cellular components, is a direct consequence of the "prion-protein-only" hypothesis. We therefore hypothesise, that the species barrier is based on the interaction of PrP(C and PrP(Sc. Whereas in our earlier studies, the interspecies transmission in animal systems was analysed, the focus of this study lies on the transmission from animals to humans. We therefore combined seeds from species cattle, sheep and deer (BSE, scrapie, CWD with human recPrP. Homologous seeding served as a control. Our results are consistent with epidemiology, other in vitro aggregation studies, and bioassays investigating the transmission between humans, cattle, sheep, and deer. In contrast to CJD and BSE seeds, which show a seeding activity we can demonstrate a species barrier for seeds from scrapie and CWD in vitro. We could show that the seeding activity and therewith the molecular interaction of PrP as substrate and PrP(Sc as seed is sufficient to explain the phenomenon of species barriers. Therefore our data supports the hypothesis

  10. High-affinity DNA binding sites for H-NS provide a molecular basis for selective silencing within proteobacterial genomes.

    Science.gov (United States)

    Lang, Benjamin; Blot, Nicolas; Bouffartigues, Emeline; Buckle, Malcolm; Geertz, Marcel; Gualerzi, Claudio O; Mavathur, Ramesh; Muskhelishvili, Georgi; Pon, Cynthia L; Rimsky, Sylvie; Stella, Stefano; Babu, M Madan; Travers, Andrew

    2007-01-01

    The global transcriptional regulator H-NS selectively silences bacterial genes associated with pathogenicity and responses to environmental insults. Although there is ample evidence that H-NS binds preferentially to DNA containing curved regions, we show here that a major basis for this selectivity is the presence of a conserved sequence motif in H-NS target transcriptons. We further show that there is a strong tendency for the H-NS binding sites to be clustered, both within operons and in genes contained in the pathogenicity-associated islands. In accordance with previously published findings, we show that these motifs occur in AT-rich regions of DNA. On the basis of these observations, we propose that H-NS silences extensive regions of the bacterial chromosome by binding first to nucleating high-affinity sites and then spreading along AT-rich DNA. This spreading would be reinforced by the frequent occurrence of the motif in such regions. Our findings suggest that such an organization enables the silencing of extensive regions of the genetic material, thereby providing a coherent framework that unifies studies on the H-NS protein and a concrete molecular basis for the genetic control of H-NS transcriptional silencing.

  11. Molecular basis of the evolution of alternative tyrosine biosynthetic routes in plants

    Energy Technology Data Exchange (ETDEWEB)

    Schenck, Craig A.; Holland, Cynthia K.; Schneider, Matthew R.; Men, Yusen; Lee, Soon Goo; Jez, Joseph M.; Maeda, Hiroshi A.

    2017-06-26

    L-Tyrosine (Tyr) is essential for protein synthesis and is a precursor of numerous specialized metabolites crucial for plant and human health. Tyr can be synthesized via two alternative routes by different key regulatory TyrA family enzymes, prephenate dehydrogenase (PDH, also known as TyrAp) or arogenate dehydrogenase (ADH, also known as TyrAa), representing a unique divergence of primary metabolic pathways. The molecular foundation underlying the evolution of these alternative Tyr pathways is currently unknown. Here we characterized recently diverged plant PDH and ADH enzymes, obtained the X-ray crystal structure of soybean PDH, and identified a single amino acid residue that defines TyrA substrate specificity and regulation. Structures of mutated PDHs co-crystallized with Tyr indicate that substitutions of Asn222 confer ADH activity and Tyr sensitivity. Reciprocal mutagenesis of the corresponding residue in divergent plant ADHs further introduced PDH activity and relaxed Tyr sensitivity, highlighting the critical role of this residue in TyrA substrate specificity that underlies the evolution of alternative Tyr biosynthetic pathways in plants.

  12. MicroRNAs as a molecular basis for mental retardation, Alzheimer's and prion diseases.

    Science.gov (United States)

    Provost, Patrick

    2010-06-18

    MicroRNAs (miRNAs) are small, approximately 21- to 23-nucleotide (nt) non-coding RNA species that act as key regulators of gene expression along a central and well-defined cellular process known as RNA silencing, and involving the recognition and translational control of specific messenger RNA (mRNAs). Generated through the well-orchestrated and sequential processing of miRNA precursor molecules, mature miRNAs are subsequently incorporated into miRNA-containing ribonucleoprotein effector complexes to regulate mRNA translation through the recognition of specific binding sites of imperfect complementarity located mainly in the 3' untranslated region. Predicted to regulate up to 90% of the genes in humans, miRNAs may thus control cellular processes in all cells and tissues of the human body. Likely to play a central role in health and disease, a dysfunctional miRNA-based regulation of gene expression may represent the main etiologic factor underlying diseases affecting major organs, such as the brain. In this review article, the molecular mechanisms underlying the role and function of miRNAs in the regulation of genes involved in neurological and neurodegenerative diseases will be discussed, with a focus on the fragile X syndrome, Alzheimer's disease (AD) and prion disease.

  13. Molecular basis for interaction of let-7 microRNAs with Lin28

    Science.gov (United States)

    Nam, Yunsun; Chen, Casandra; Gregory, Richard I; Chou, James J; Sliz, Piotr

    2011-01-01

    SUMMARY MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression. Among these, members of the let-7 miRNA family control many cell fate determination genes to influence pluripotency, differentiation, and transformation. Lin28 is a specific, post-transcriptional inhibitor of let-7 biogenesis. We report crystal structures of mouse Lin28 in complex with sequences from let-7d, let-7-f1, and let-7g precursors. The two folded domains of Lin28 recognize two distinct regions of the RNA and are sufficient for inhibition of let-7 in vivo. We also show by NMR spectroscopy that the linker connecting the two folded domains is flexible, accommodating Lin28 binding to diverse let-7 family members. Protein-RNA complex formation imposes specific conformations on both components that could affect downstream recognition by other processing factors. Our data provide a molecular explanation for Lin28 specificity and a model for how it regulates let-7. PMID:22078496

  14. Molecular Basis for Interaction of let-7 MicroRNAs with Lin28

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Yunsun; Chen, Casandra; Gregory, Richard I.; Chou, James J.; Sliz, Piotr (Harvard-Med)

    2012-02-06

    MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate gene expression. Among these, members of the let-7 miRNA family control many cell-fate determination genes to influence pluripotency, differentiation, and transformation. Lin28 is a specific, posttranscriptional inhibitor of let-7 biogenesis. We report crystal structures of mouse Lin28 in complex with sequences from let-7d, let-7-f1, and let-7g precursors. The two folded domains of Lin28 recognize two distinct regions of the RNA and are sufficient for inhibition of let-7 in vivo. We also show by NMR spectroscopy that the linker connecting the two folded domains is flexible, accommodating Lin28 binding to diverse let-7 family members. Protein-RNA complex formation imposes specific conformations on both components that could affect downstream recognition by other processing factors. Our data provide a molecular explanation for Lin28 specificity and a model for how it regulates let-7.

  15. Radiation and Heat Transfer in the Atmosphere: A Comprehensive Approach on a Molecular Basis

    Directory of Open Access Journals (Sweden)

    Hermann Harde

    2013-01-01

    Full Text Available We investigate the interaction of infrared active molecules in the atmosphere with their own thermal background radiation as well as with radiation from an external blackbody radiator. We show that the background radiation can be well understood only in terms of the spontaneous emission of the molecules. The radiation and heat transfer processes in the atmosphere are described by rate equations which are solved numerically for typical conditions as found in the troposphere and stratosphere, showing the conversion of heat to radiation and vice versa. Consideration of the interaction processes on a molecular scale allows to develop a comprehensive theoretical concept for the description of the radiation transfer in the atmosphere. A generalized form of the radiation transfer equation is presented, which covers both limiting cases of thin and dense atmospheres and allows a continuous transition from low to high densities, controlled by a density dependent parameter. Simulations of the up- and down-welling radiation and its interaction with the most prominent greenhouse gases water vapour, carbon dioxide, methane, and ozone in the atmosphere are presented. The radiative forcing at doubled CO2 concentration is found to be 30% smaller than the IPCC-value.

  16. Microarray Analysis Reveals the Molecular Basis of Antiarthritic Activity of Huo-Luo-Xiao-Ling Dan

    Directory of Open Access Journals (Sweden)

    Hua Yu

    2013-01-01

    Full Text Available Rheumatoid arthritis (RA is a chronic inflammatory disease of autoimmune origin. Huo-luo-xiao-ling dan (HLXL is an herbal mixture that has been used in traditional Chinese medicine over several decades to treat chronic inflammatory diseases including RA. However, the mechanism of the anti-arthritic action of this herbal remedy is poorly understood at the molecular level. In this study, we determined by microarray analysis the effects of HLXL on the global gene expression profile of the draining lymph node cells (LNC in the rat adjuvant arthritis (AA model of human RA. In LNC restimulated in vitro with the disease-related antigen mycobacterial heat-shock protein 65 (Bhsp65, 84 differentially expressed genes (DEG (64 upregulated and 20 downregulated versus 120 DEG (94 upregulated and 26 downregulated were identified in HLXL-treated versus vehicle (Water-treated rats, respectively, and 62 DEG (45 upregulated and 17 downregulated were shared between the two groups. The most affected pathways in response to HLXL treatment included immune response, inflammation, cellular proliferation and apoptosis, and metabolic processes, many of which are directly relevant to arthritis pathogenesis. These results would advance our understanding of the mechanisms underlying the anti-arthritic activity of HLXL.

  17. Molecular basis of Bombay phenotype in Mashhad, Iran: identification of a novel FUT1 deletion.

    Science.gov (United States)

    Zanjani, D S; Afzal Aghaee, M; Badiei, Z; Mehrasa, R; Roodsarabi, A; Khayyami, M E; Shahabi, M

    2016-07-01

    Bombay phenotype is characterized by the lack of H substance both on red blood cell (RBC) surface and in body secretions. Mutations of fucosyltransferase 1 (FUT1) and fucosyltransferase 2 (FUT2) genes are resulted in this rare phenotype. Five unrelated patients were tested by hemagglutination and adsorption/elution techniques for the presence of ABH antigens. The saliva specimens were analysed by hemagglutination inhibition method. The exons 6 and 7 of ABO gene were sequenced to determine ABO genotype. The coding fragments of FUT1 and FUT2 were amplified and sequenced by specific primers. Serologic investigation confirmed Bombay phenotype in all individuals. FUT1 molecular analysis revealed a novel large deletion. Also two novel homozygous mutations were detected; one was a missense mutation (392T>C, L131P) and the other a three nucleotide deletion (668_670delACT, Y224del). FUT2 sequencing showed one reported null allele (428G>A, W143X) and one homozygous deletion of FUT2. Although FUT2 deletion has been reported, this is the first report of FUT1 deletion. Finding two FUT1 novel alleles in Iranian people is indicative of mutation diversity in this gene. © 2016 International Society of Blood Transfusion.

  18. Mechanistic basis for the recognition of a misfolded protein by the molecular chaperone Hsp90.

    Science.gov (United States)

    Oroz, Javier; Kim, Jin Hae; Chang, Bliss J; Zweckstetter, Markus

    2017-02-20

    The critical toxic species in over 40 human diseases are misfolded proteins. Their interaction with molecular chaperones such as Hsp90, which preferentially interacts with metastable proteins, is essential for the blocking of disease progression. Here we used nuclear magnetic resonance (NMR) spectroscopy to determine the three-dimensional structure of the misfolded cytotoxic monomer of the amyloidogenic human protein transthyretin, which is characterized by the release of the C-terminal β-strand and perturbations of the A-B loop. The misfolded transthyretin monomer, but not the wild-type protein, binds to human Hsp90. In the bound state, the Hsp90 dimer predominantly populates an open conformation, and transthyretin retains its globular structure. The interaction surface for the transthyretin monomer comprises the N-terminal and middle domains of Hsp90 and overlaps with that of the Alzheimer's-disease-related protein tau. Taken together, the data suggest that Hsp90 uses a mechanism for the recognition of aggregation-prone proteins that is largely distinct from those of other Hsp90 clients.

  19. Structural and Molecular Basis for Coordination in a Viral DNA Packaging Motor

    Directory of Open Access Journals (Sweden)

    Huzhang Mao

    2016-03-01

    Full Text Available Ring NTPases are a class of ubiquitous molecular motors involved in basic biological partitioning processes. dsDNA viruses encode ring ATPases that translocate their genomes to near-crystalline densities within pre-assembled viral capsids. Here, X-ray crystallography, cryoEM, and biochemical analyses of the dsDNA packaging motor in bacteriophage phi29 show how individual subunits are arranged in a pentameric ATPase ring and suggest how their activities are coordinated to translocate dsDNA. The resulting pseudo-atomic structure of the motor and accompanying functional analyses show how ATP is bound in the ATPase active site; identify two DNA contacts, including a potential DNA translocating loop; demonstrate that a trans-acting arginine finger is involved in coordinating hydrolysis around the ring; and suggest a functional coupling between the arginine finger and the DNA translocating loop. The ability to visualize the motor in action illuminates how the different motor components interact with each other and with their DNA substrate.

  20. The Molecular Basis of Ligand Interaction at Free Fatty Acid Receptor 4 (FFA4/GPR120)*

    Science.gov (United States)

    Hudson, Brian D.; Shimpukade, Bharat; Milligan, Graeme; Ulven, Trond

    2014-01-01

    The long-chain fatty acid receptor FFA4 (previously GPR120) is receiving substantial interest as a novel target for the treatment of metabolic and inflammatory disease. This study examines for the first time the detailed mode of binding of both long-chain fatty acid and synthetic agonist ligands at FFA4 by integrating molecular modeling, receptor mutagenesis, and ligand structure-activity relationship approaches in an iterative format. In doing so, residues required for binding of fatty acid and synthetic agonists to FFA4 have been identified. This has allowed for the refinement of a well validated model of the mode of ligand-FFA4 interaction that will be invaluable in the identification of novel ligands and the future development of this receptor as a therapeutic target. The model reliably predicted the effects of substituent variations on agonist potency, and it was also able to predict the qualitative effect of binding site mutations in the majority of cases. PMID:24860101

  1. The Molecular Basis of Polyunsaturated Fatty Acid Interactions with the Shaker Voltage-Gated Potassium Channel.

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    Samira Yazdi

    2016-01-01

    Full Text Available Voltage-gated potassium (KV channels are membrane proteins that respond to changes in membrane potential by enabling K+ ion flux across the membrane. Polyunsaturated fatty acids (PUFAs induce channel opening by modulating the voltage-sensitivity, which can provide effective treatment against refractory epilepsy by means of a ketogenic diet. While PUFAs have been reported to influence the gating mechanism by electrostatic interactions to the voltage-sensor domain (VSD, the exact PUFA-protein interactions are still elusive. In this study, we report on the interactions between the Shaker KV channel in open and closed states and a PUFA-enriched lipid bilayer using microsecond molecular dynamics simulations. We determined a putative PUFA binding site in the open state of the channel located at the protein-lipid interface in the vicinity of the extracellular halves of the S3 and S4 helices of the VSD. In particular, the lipophilic PUFA tail covered a wide range of non-specific hydrophobic interactions in the hydrophobic central core of the protein-lipid interface, while the carboxylic head group displayed more specific interactions to polar/charged residues at the extracellular regions of the S3 and S4 helices, encompassing the S3-S4 linker. Moreover, by studying the interactions between saturated fatty acids (SFA and the Shaker KV channel, our study confirmed an increased conformational flexibility in the polyunsaturated carbon tails compared to saturated carbon chains, which may explain the specificity of PUFA action on channel proteins.

  2. Structural and Molecular Basis for Coordination in a Viral DNA Packaging Motor

    Science.gov (United States)

    Reyes-Aldrete, Emilio; Sherman, Michael B.; Woodson, Michael; Atz, Rockney; Grimes, Shelley; Jardine, Paul J.; Morais, Marc C.

    2016-01-01

    SUMMARY Ring NTPases are a class of ubiquitous molecular motors involved in basic biological partitioning processes. dsDNA viruses encode ring ATPases that translocate their genomes to near-crystalline densities within pre-assembled viral capsids. Here, X-ray crystallography, cryoEM, and biochemical analyses of the dsDNA packaging motor in bacteriophage phi29 show how individual subunits are arranged in a pentameric ATPase ring, and suggest how their activities are coordinated to translocate dsDNA. The resulting pseudo-atomic structure of the motor and accompanying functional analyses show how ATP is bound in the ATPase active site; identify two DNA contacts, including a potential DNA translocating loop; demonstrate that a trans-acting arginine finger is involved in coordinating hydrolysis around the ring; and suggest a functional coupling between the arginine finger and the DNA translocating loop. The ability to visualize the motor in action illuminates how the different motor components interact with each other and with their DNA substrate. PMID:26904950

  3. Molecular Architecture of G alpha o and the Structural Basis for RGS16-Mediated Deactivation

    Energy Technology Data Exchange (ETDEWEB)

    Slep,K.; Kercher, M.; Wieland, T.; Chen, C.; Simon, M.; Sigler, P.

    2008-01-01

    Heterotrimeric G proteins relay extracellular cues from heptahelical transmembrane receptors to downstream effector molecules. Composed of an a subunit with intrinsic GTPase activity and a {beta} heterodimer, the trimeric complex dissociates upon receptor-mediated nucleotide exchange on the a subunit, enabling each component to engage downstream effector targets for either activation or inhibition as dictated in a particular pathway. To mitigate excessive effector engagement and concomitant signal transmission, the Ga subunit's intrinsic activation timer (the rate of GTP hydrolysis) is regulated spatially and temporally by a class of GTPase accelerating proteins (GAPs) known as the regulator of G protein signaling (RGS) family. The array of G protein-coupled receptors, Ga subunits, RGS proteins and downstream effectors in mammalian systems is vast. Understanding the molecular determinants of specificity is critical for a comprehensive mapping of the G protein system. Here, we present the 2.9 Angstroms crystal structure of the enigmatic, neuronal G protein Gao in the GTP hydrolytic transition state, complexed with RGS16. Comparison with the 1.89 Angstroms structure of apo-RGS16, also presented here, reveals plasticity upon Gao binding, the determinants for GAP activity, and the structurally unique features of Gao that likely distinguish it physiologically from other members of the larger Gai family, affording insight to receptor, GAP and effector specificity.

  4. Molecular basis of active copper resistance mechanisms in Gram-negative bacteria.

    Science.gov (United States)

    Bondarczuk, Kinga; Piotrowska-Seget, Zofia

    2013-12-01

    Copper is a metallic element that is crucial for cell metabolism; however, in extended concentrations, it is toxic for all living organisms. The dual nature of copper has forced organisms, including bacteria, to keep a tight hold on cellular copper content. This challenge has led to the evolution of complex mechanisms that on one hand enable them to deliver the essential element and on the other to protect cells against its toxicity. Such mechanisms have been found in both eukaryotic and prokaryotic cells. In bacteria a number of different systems such as extra- and intracellular sequestration, enzymatic detoxification, and metal removal from the cell enabling them to survive in the presence of high concentration of copper have been identified. Gram-negative bacteria, due to their additional compartment, need to deal with both cytoplasmic and periplasmic copper. Therefore, these bacteria have evolved intricate and precisely regulated systems which interact with each other. In this review the active mechanisms of copper resistance at their molecular level are discussed.

  5. [Molecular basis of one-way serological reaction between SINV and XJ-160 virus].

    Science.gov (United States)

    Wang, Li-hua; Fu, Shi-hong; Yang, Yi-liang; Zhu, Wu-yang; Tang, Qing; Liang, Guo-dong

    2010-05-01

    The purpose of this study is to elucidate the molecular mechanism of one-way serological reaction between XJ-160 virus and SINV by recombinant viruses which exchanged the glycoprotein genes individually or simultaneously. Three recombinant viruses were obtained based on the whole-length infectious cDNA clone of XJ-160 virus. The infectivity and pathogenesis to BHK-21 cells and animals were studied and the gene which controlled this one-way serological reaction phenomenon was searched by MCPENT. The results showed that the E2 glycoprotein was the main factor which influenced the growth rate, plaque morphology and pathogenicity of BHK-21 cells and suckling mice. The results of MCPENT showed that the E2 glycoprotein of SINV played a major role in this one-way serological reaction phenomenon. Our study identified the SINE2 gene was the determined gene for one way serological reaction between XJ-160 virus and SINV, and this research laid the foundation for further analysis of the genomic structure and function of SINV.

  6. Molecular basis of Kar9-Bim1 complex function during mating and spindle positioning

    Science.gov (United States)

    Manatschal, Cristina; Farcas, Ana-Maria; Degen, Miriam Steiner; Bayer, Mathias; Kumar, Anil; Landgraf, Christiane; Volkmer, Rudolf; Barral, Yves; Steinmetz, Michel O.

    2016-01-01

    The Kar9 pathway promotes nuclear fusion during mating and spindle alignment during metaphase in budding yeast. How Kar9 supports the different outcome of these two divergent processes is an open question. Here, we show that three sites in the C-terminal disordered domain of Kar9 mediate tight Kar9 interaction with the C-terminal dimerization domain of Bim1 (EB1 orthologue). Site1 and Site2 contain SxIP motifs; however, Site3 defines a novel type of EB1-binding site. Whereas Site2 and Site3 mediate Kar9 recruitment to microtubule tips, nuclear movement, and karyogamy, only Site2 functions in spindle positioning during metaphase. Site1 in turn plays an inhibitory role during mating. Additionally, the Kar9-Bim1 complex is involved in microtubule-independent activities during mating. Together, our data reveal how multiple and partially redundant EB1-binding sites provide a microtubule-associated protein with the means to modulate its biochemical properties to promote different molecular processes during cell proliferation and differentiation. PMID:27682587

  7. The molecular genetic basis of age-related macular degeneration: an overview

    Indian Academy of Sciences (India)

    Saritha Katta; Inderjeet Kaur; Subhabrata Chakrabarti

    2009-12-01

    Age-related macular degeneration (AMD) is a complex disorder of the eye and the third leading cause of blindness worldwide. With a multifactorial etiology, AMD results in progressive loss of central vision affecting the macular region of the eye in elderly. While the prevalence is relatively higher in the Caucasian populations, it has gradually become a major public health issue among the non-Caucasian populations (including Indians) as well due to senescence, rapidly changing demographics and life-style factors. Recent genome-wide association studies (GWAS) on large case–control cohorts have helped in mapping genes in the complement cascade that are involved in the regulation of innate immunity with AMD susceptibility. Genes involved with mitochondrial oxidative stress and extracellular matrix regulation also play a role in AMD pathogenesis. Majority of the associations observed in complement (CFH, CFB, C2 and C3) and other (ARMS2 and HTRA1) genes have been replicated in diverse populations worldwide. Gene–gene (CFH with ARMS2 and HTRA1) interactions and correlations with environmental traits (smoking and body mass index) have been established as significant covariates in AMD pathology. In this review, we have provided an overview on the underlying molecular genetic mechanisms in AMD worldwide and highlight the AMD-associated-candidate genes and their potential role in disease pathogenesis.

  8. Scarless integumentary wound healing in the mammalian fetus: molecular basis and therapeutic implications.

    Science.gov (United States)

    Kathju, Sandeep; Gallo, Phillip H; Satish, Latha

    2012-09-01

    Adult mammals respond to injury of their skin/integument by forming scar tissue. Scar is useful in rapidly sealing an injured area, but can also lead to significant morbidity. Mammals in fetal life retain the ability to heal integumentary wounds regeneratively, without scar. The critical molecular mechanisms governing this remarkable phenomenon have been a subject of great interest, in the hopes that these could be dissected and recapitulated in the healing adult wound, with the goal of inducing scarless healing in injured patients. Multiple lines of investigation spanning decades have implicated a number of factors in distinguishing scarless from fibrotic wound healing, including most prominently transforming growth factor-β and interleukin-10, among others. Therapeutic interventions to try to mitigate scarring in adult wounds have been developed out of these studies, and have reached the level of clinical trials in humans, although as yet no FDA-approved treatment exists. More recent expressomic studies have revealed many more genes that are differentially expressed in scarlessly healing fetal wounds compared with adult, and microRNAs have also been identified as participating in the fetal wound healing response. These represent an even greater range of potential therapeutics (or targets for therapy) to translate the promise of scarless fetal wound healing to the injured adult patient. Copyright © 2012 Wiley Periodicals, Inc.

  9. Molecular basis of the dynamic structure of the TIM23 complex in the mitochondrial intermembrane space.

    Science.gov (United States)

    Bajaj, Rakhi; Jaremko, Łukasz; Jaremko, Mariusz; Becker, Stefan; Zweckstetter, Markus

    2014-10-07

    The presequence translocase TIM23 is a highly dynamic complex in which its subunits can adopt multiple conformations and undergo association-dissociation to facilitate import of proteins into mitochondria. Despite the importance of protein-protein interactions in TIM23, little is known about the molecular details of these processes. Using nuclear magnetic resonance spectroscopy, we characterized the dynamic interaction network of the intermembrane space domains of Tim23, Tim21, Tim50, and Tom22 at single-residue level. We show that Tim23(IMS) contains multiple sites to efficiently interact with the intermembrane space domain of Tim21 and to bind to Tim21, Tim50, and Tom22. In addition, we reveal the atomic details of the dynamic Tim23(IMS)-Tim21(IMS) complex. The combined data support a central role of the intermembrane space domain of Tim23 in the formation and regulation of the presequence translocase. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Molecular basis for the binding and modulation of V-ATPase by a bacterial effector protein.

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    Jianhua Zhao

    2017-06-01

    Full Text Available Intracellular pathogenic bacteria evade the immune response by replicating within host cells. Legionella pneumophila, the causative agent of Legionnaires' Disease, makes use of numerous effector proteins to construct a niche supportive of its replication within phagocytic cells. The L. pneumophila effector SidK was identified in a screen for proteins that reduce the activity of the proton pumping vacuolar-type ATPases (V-ATPases when expressed in the yeast Saccharomyces cerevisae. SidK is secreted by L. pneumophila in the early stages of infection and by binding to and inhibiting the V-ATPase, SidK reduces phagosomal acidification and promotes survival of the bacterium inside macrophages. We determined crystal structures of the N-terminal region of SidK at 2.3 Å resolution and used single particle electron cryomicroscopy (cryo-EM to determine structures of V-ATPase:SidK complexes at ~6.8 Å resolution. SidK is a flexible and elongated protein composed of an α-helical region that interacts with subunit A of the V-ATPase and a second region of unknown function that is flexibly-tethered to the first. SidK binds V-ATPase strongly by interacting via two α-helical bundles at its N terminus with subunit A. In vitro activity assays show that SidK does not inhibit the V-ATPase completely, but reduces its activity by ~40%, consistent with the partial V-ATPase deficiency phenotype its expression causes in yeast. The cryo-EM analysis shows that SidK reduces the flexibility of the A-subunit that is in the 'open' conformation. Fluorescence experiments indicate that SidK binding decreases the affinity of V-ATPase for a fluorescent analogue of ATP. Together, these results reveal the structural basis for the fine-tuning of V-ATPase activity by SidK.

  11. Genetics of Migraine: Insights into the Molecular Basis of Migraine Disorders.

    Science.gov (United States)

    Sutherland, Heidi G; Griffiths, Lyn R

    2017-04-01

    Migraine is a complex, debilitating neurovascular disorder, typically characterized by recurring, incapacitating attacks of severe headache often accompanied by nausea and neurological disturbances. It has a strong genetic basis demonstrated by rare migraine disorders caused by mutations in single genes (monogenic), as well as familial clustering of common migraine which is associated with polymorphisms in many genes (polygenic). Hemiplegic migraine is a dominantly inherited, severe form of migraine with associated motor weakness. Family studies have found that mutations in three different ion channels genes, CACNA1A, ATP1A2, and SCN1A can be causal. Functional studies of these mutations has shown that they can result in defective regulation of glutamatergic neurotransmission and the excitatory/inhibitory balance in the brain, which lowers the threshold for cortical spreading depression, a wave of cortical depolarization thought to be involved in headache initiation mechanisms. Other putative genes for monogenic migraine include KCKN18, PRRT2, and CSNK1D, which can also be involved with other disorders. There are a number of primarily vascular disorders caused by mutations in single genes, which are often accompanied by migraine symptoms. Mutations in NOTCH3 causes cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a hereditary cerebrovascular disease that leads to ischemic strokes and dementia, but in which migraine is often present, sometimes long before the onset of other symptoms. Mutations in the TREX1 and COL4A1 also cause vascular disorders, but often feature migraine. With respect to common polygenic migraine, genome-wide association studies have now identified single nucleotide polymorphisms at 38 loci significantly associated with migraine risk. Functions assigned to the genes in proximity to these loci suggest that both neuronal and vascular pathways also contribute to the pathophysiology of common

  12. Molecular basis of P450 OleTJE: an investigation of substrate binding mechanism and major pathways

    Science.gov (United States)

    Du, Juan; Liu, Lin; Guo, Li Zhong; Yao, Xiao Jun; Yang, Jian Ming

    2017-05-01

    Cytochrome P450 OleTJE has attracted much attention for its ability to catalyze the decarboxylation of long chain fatty acids to generate alkenes, which are not only biofuel molecule, but also can be used broadly for making lubricants, polymers and detergents. In this study, the molecular basis of the binding mechanism of P450 OleTJE for arachidic acid, myristic acid, and caprylic acid was investigated by utilizing conventional molecular dynamics simulation and binding free energy calculations. Moreover, random acceleration molecular dynamics (RAMD) simulations were performed to uncover the most probable access/egress channels for different fatty acids. The predicted binding free energy shows an order of arachidic acid acid acid. Key residues interacting with three substrates and residues specifically binding to one of them were identified. The RAMD results suggest the most likely channel for arachidic acid, myristic acid, and caprylic acid are 2e/2b, 2a and 2f/2a, respectively. It is suggested that the reaction is easier to carry out in myristic acid bound system than those in arachidic acid and caprylic acid bound system based on the distance of Hβ atom of substrate relative to P450 OleTJE Compound I states. This study provided novel insight to understand the substrate preference mechanism of P450 OleTJE and valuable information for rational enzyme design for short chain fatty acid decarboxylation.

  13. Molecular Basis of the Extracellular Ligands Mediated Signaling by the Calcium Sensing Receptor

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    Chen Zhang

    2016-09-01

    Full Text Available Ca2+-sensing receptors (CaSRs play a central role in regulating extracellular calcium concentration ([Ca2+]o homeostasis and many (pathophysiological processes in multiple organs. This regulation is orchestrated by a cooperative response to extracellular stimuli such as small changes in Ca2+, Mg2+, amino acids and other ligands. In addition, CaSR is a pleiotropic receptor regulating several intracellular signaling pathways, including calcium mobilization and intracellular calcium oscillation. Nearly 200 mutations and polymorphisms have been found in CaSR in relation to a variety of human disorders associated with abnormal Ca2+ homeostasis. In this review, we summarize efforts directed at identifying binding sites for calcium and amino acids. Both homotropic cooperativity among multiple calcium binding sites and heterotropic cooperativity between calcium and amino acid were revealed using computational modeling, predictions, and site-directed mutagenesis coupled with functional assays. The hinge region of the bilobed Venus flytrap (VFT domain of CaSR plays a pivotal role in coordinating multiple extracellular stimuli, leading to cooperative responses from the receptor. We further highlight the extensive number of disease-associated mutations that have also been shown to affect CaSR’s cooperative action via several types of mechanisms. These results provide insights into the molecular bases of the structure and functional cooperativity of this receptor and other members of family C of the G protein-coupled receptors (cGPCRs in health and disease states, and may assist in the prospective development of novel receptor-based therapeutics.

  14. Behavioural pharmacology and its contribution to the molecular basis of memory consolidation.

    Science.gov (United States)

    Izquierdo, I; McGaugh, J L

    2000-11-01

    Recent findings have significantly advanced our understanding the mechanisms of memory formation. Most of these advances stemmed from behavioural pharmacology research involving, particularly, the localized infusion of drugs with specific molecular actions into specific brain regions. This approach has revealed brain structures involved in different memory types and the main neurotransmitter systems and sequence of metabolic cascades that participate in memory consolidation. Biochemical studies and, in several cases, studies of genetically manipulated animals, in which receptors or enzymes affected by the various drugs were absent or overexpressed, have complemented the pharmacological research. Although most studies have concentrated on the involvement of the hippocampus, many have also investigated the entorhinal cortex, other regions of the cortex, and the amygdala. Behavioural pharmacology has been of crucial importance in establishing the major neurohumoral and hormonal systems involved in the modulation of memory formation. These systems act on specific steps of memory formation in the hippocampus and in the entorhinal, parietal, and cingulate cortex. A specialized system mediated by the basolateral amygdaloid nucleus, and involving several neuromodulatory systems, is activated by emotional arousal and serves to regulate memory formation in other brain regions. The core mechanisms involved in the formation of explicit (declarative) memory are in many respects similar to those of long-term potentiation (LTP), particularly in the hippocampus. However, there are also important differences between memory formation and LTP. Memory formation involves numerous modulatory influences, the co-participation of various brain regions other than the hippocampus, and some properties that are specific to memory and absent in LTP (i.e. flexibility of response). We discuss the implications of these similarities and differences for understanding the neural bases of memory.

  15. Molecular basis of a novel adaptation to hypoxic-hypercapnia in a strictly fossorial mole

    Directory of Open Access Journals (Sweden)

    Bonaventura Joseph

    2010-07-01

    Full Text Available Abstract Background Elevated blood O2 affinity enhances survival at low O2 pressures, and is perhaps the best known and most broadly accepted evolutionary adjustment of terrestrial vertebrates to environmental hypoxia. This phenotype arises by increasing the intrinsic O2 affinity of the hemoglobin (Hb molecule, by decreasing the intracellular concentration of allosteric effectors (e.g., 2,3-diphosphoglycerate; DPG, or by suppressing the sensitivity of Hb to these physiological cofactors. Results Here we report that strictly fossorial eastern moles (Scalopus aquaticus have evolved a low O2 affinity, DPG-insensitive Hb - contrary to expectations for a mammalian species that is adapted to the chronic hypoxia and hypercapnia of subterranean burrow systems. Molecular modelling indicates that this functional shift is principally attributable to a single charge altering amino acid substitution in the β-type δ-globin chain (δ136Gly→Glu of this species that perturbs electrostatic interactions between the dimer subunits via formation of an intra-chain salt-bridge with δ82Lys. However, this replacement also abolishes key binding sites for the red blood cell effectors Cl-, lactate and DPG (the latter of which is virtually absent from the red cells of this species at δ82Lys, thereby markedly reducing competition for carbamate formation (CO2 binding at the δ-chain N-termini. Conclusions We propose this Hb phenotype illustrates a novel mechanism for adaptively elevating the CO2 carrying capacity of eastern mole blood during burst tunnelling activities associated with subterranean habitation.

  16. Structural basis of type 2A von Willebrand disease investigated by molecular dynamics simulations and experiments.

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    Gianluca Interlandi

    Full Text Available The hemostatic function of von Willebrand factor is downregulated by the metalloprotease ADAMTS13, which cleaves at a unique site normally buried in the A2 domain. Exposure of the proteolytic site is induced in the wild-type by shear stress as von Willebrand factor circulates in blood. Mutations in the A2 domain, which increase its susceptibility to cleavage, cause type 2A von Willebrand disease. In this study, molecular dynamics simulations suggest that the A2 domain unfolds under tensile force progressively through a series of steps. The simulation results also indicated that three type 2A mutations in the C-terminal half of the A2 domain, L1657I, I1628T and E1638K, destabilize the native state fold of the protein. Furthermore, all three type 2A mutations lowered in silico the tensile force necessary to undock the C-terminal helix α6 from the rest of the A2 domain, the first event in the unfolding pathway. The mutations F1520A, I1651A and A1661G were also predicted by simulations to destabilize the A2 domain and facilitate exposure of the cleavage site. Recombinant A2 domain proteins were expressed and cleavage assays were performed with the wild-type and single-point mutants. All three type 2A and two of the three predicted mutations exhibited increased rate of cleavage by ADAMTS13. These results confirm that destabilization of the helix α6 in the A2 domain facilitates exposure of the cleavage site and increases the rate of cleavage by ADAMTS13.

  17. Molecular basis of rare aminoglycoside susceptibility and pathogenesis of Burkholderia pseudomallei clinical isolates from Thailand.

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    Lily A Trunck

    Full Text Available BACKGROUND: Burkholderia pseudomallei is intrinsically resistant to aminoglycosides and macrolides, mostly due to AmrAB-OprA efflux pump expression. We investigated the molecular mechanisms of aminoglycoside susceptibility exhibited by Thai strains 708a, 2188a, and 3799a. METHODOLOGY/PRINCIPAL FINDINGS: qRT-PCR revealed absence of amrB transcripts in 708a and greatly reduced levels in 2188a and 3799a. Serial passage on increasing gentamicin concentrations yielded 2188a and 3799a mutants that became simultaneously resistant to other aminoglycosides and macrolides, whereas such mutants could not be obtained with 708a. Transcript analysis showed that the resistance of the 2188a and 3799a mutants was due to upregulation of amrAB-oprA expression by unknown mechanism(s. Use of a PCR walking strategy revealed that the amrAB-oprA operon was missing in 708a and that this loss was associated with deletion of more than 70 kb of genetic material. Rescue of the amrAB-oprB region from a 708a fosmid library and sequencing showed the presence of a large chromosome 1 deletion (131 kb and 141 kb compared to strains K96243 and 1710b, respectively. This deletion not only removed the amrAB-oprA operon, but also the entire gene clusters for malleobactin and cobalamin synthesis. Other genes deleted included the anaerobic arginine deiminase pathway, putative type 1 fimbriae and secreted chitinase. Whole genome sequencing and PCR analysis confirmed absence of these genes from 708a. Despite missing several putative virulence genes, 708a was fully virulent in a murine melioidosis model. CONCLUSIONS/SIGNIFICANCE: Strain 708a may be a natural candidate for genetic manipulation experiments that use Select Agent compliant antibiotics for selection and validates the use of laboratory-constructed Delta(amrAB-oprA mutants in such experiments.

  18. Molecular basis for resistance to ACCase-inhibiting herbicides in Pseudosclerochloa kengiana populations.

    Science.gov (United States)

    Yuan, Guohui; Liu, Weitang; Bi, Yaling; Du, Long; Guo, Wenlei; Wang, Jinxin

    2015-03-01

    Pseudosclerochloa kengiana is a troublesome annual grass weed of wheat fields in the rice-wheat double cropping areas in China. Resistance has evolved in P. kengiana under continuously selective pressure of ACCase-inhibiting herbicides. Whole-plant experiments showed that two suspected resistant populations 12-SD-12 and 12-SD-13 were highly resistant to fenoxaprop-P-ethyl (69.9- and 57.2-fold); moderately resistant to clodinafop-propargyl (5.9- and 4.1-fold) and pinoxaden (4.4- and 3.5-fold); lowly resistant to fluazifop-P-butyl (2.2- and 2.0-fold) and sethoxydim (1.8- and 1.6-fold), but were sensitive to clethodim (1.0- and 0.9-fold) and mesosulfuron-methyl (1.1- and 0.9-fold). Molecular analyses confirmed that a Trp1999 to Ser mutation was present in the resistant populations. Two dCAPS markers were also developed to positively determine the wild type Trp and mutant Ser alleles at ACCase position 1999. All 350 individual plants of 12-SD-12 population analysed were heterozygous mutants. Meanwhile, 318 mutant heterozygotes and 32 wild types were detected from the 12-SD-13 population. In addition, the analysis of plant genotype and phenotype showed that all wild type plants were killed after treatment with any one of the ACCase-inhibiting herbicides used, while individual plants carrying the W1999S mutation survived except when treated with clethodim. To our knowledge, this is the first report of pinoxaden resistance and a Trp-1999-Ser mutation in the plastid ACCase for P. kengiana.

  19. Revised molecular basis of the promiscuous carboxylic acid perhydrolase activity in serine hydrolases.

    Science.gov (United States)

    Yin, DeLu Tyler; Kazlauskas, Romas J

    2012-06-25

    Several serine hydrolases catalyze a promiscuous reaction: perhydrolysis of carboxylic acids to form peroxycarboxylic acids. The working hypothesis is that perhydrolases are more selective than esterases for hydrogen peroxide over water. In this study, we tested this hypothesis, and focused on L29P-PFE (Pseudomonas fluorescens esterase), which catalyzes perhydrolysis of acetic acid 43-fold faster than wild-type PFE. This hypothesis predicts that L29P-PFE should be approximately 43-fold more selective for hydrogen peroxide than wild-type PFE, but experiments show that L29P-PFE is less selective. The ratio of hydrolysis to perhydrolysis of methyl acetate at different concentrations of hydrogen peroxide fit a kinetic model for nucleophile selectivity. L29P-PFE (β(0)=170  M(-1)) is approximately half as selective for hydrogen peroxide over water than wild-type PFE (β(0)=330  M(-1)), which contradicts the working hypothesis. An alternative hypothesis is that carboxylic acid perhydrolases increase perhydrolysis by forming the acyl-enzyme intermediate faster. Consistent with this hypothesis, the rate of acetyl-enzyme formation, measured by (18)O-water exchange into acetic acid, was 25-fold faster with L29P-PFE than with wild-type PFE, which is similar to the 43-fold faster perhydrolysis with L29P-PFE. Molecular modeling of the first tetrahedral intermediate (T(d)1) suggests that a closer carbonyl group found in perhydrolases accepts a hydrogen bond from the leaving group water. This revised understanding can help design more efficient enzymes for perhydrolysis and shows how subtle changes can create new, unnatural functions in enzymes. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Molecular Basis of Regulating High Voltage-Activated Calcium Channels by S-Nitrosylation.

    Science.gov (United States)

    Zhou, Meng-Hua; Bavencoffe, Alexis; Pan, Hui-Lin

    2015-12-18

    Nitric oxide (NO) is involved in a variety of physiological processes, such as vasoregulation and neurotransmission, and has a complex role in the regulation of pain transduction and synaptic transmission. We have shown previously that NO inhibits high voltage-activated Ca(2+) channels in primary sensory neurons and excitatory synaptic transmission in the spinal dorsal horn. However, the molecular mechanism involved in this inhibitory action remains unclear. In this study, we investigated the role of S-nitrosylation in the NO regulation of high voltage-activated Ca(2+) channels. The NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) rapidly reduced N-type currents when Cav2.2 was coexpressed with the Cavβ1 or Cavβ3 subunits in HEK293 cells. In contrast, SNAP only slightly inhibited P/Q-type and L-type currents reconstituted with various Cavβ subunits. SNAP caused a depolarizing shift in voltage-dependent N-type channel activation, but it had no effect on Cav2.2 protein levels on the membrane surface. The inhibitory effect of SNAP on N-type currents was blocked by the sulfhydryl-specific modifying reagent methanethiosulfonate ethylammonium. Furthermore, the consensus motifs of S-nitrosylation were much more abundant in Cav2.2 than in Cav1.2 and Cav2.1. Site-directed mutagenesis studies showed that Cys-805, Cys-930, and Cys-1045 in the II-III intracellular loop, Cys-1835 and Cys-2145 in the C terminus of Cav2.2, and Cys-346 in the Cavβ3 subunit were nitrosylation sites mediating NO sensitivity of N-type channels. Our findings demonstrate that the consensus motifs of S-nitrosylation in cytoplasmically accessible sites are critically involved in post-translational regulation of N-type Ca(2+) channels by NO. S-Nitrosylation mediates the feedback regulation of N-type channels by NO.

  1. Microbes on building materials - Evaluation of DNA extraction protocols as common basis for molecular analysis

    Energy Technology Data Exchange (ETDEWEB)

    Ettenauer, Joerg D., E-mail: joerg.ettenauer@boku.ac.at [VIBT-BOKU, University of Natural Resources and Life Sciences, Department of Biotechnology, Muthgasse 11, A-1190 Vienna (Austria); Pinar, Guadalupe, E-mail: Guadalupe.Pinar@boku.ac.at [VIBT-BOKU, University of Natural Resources and Life Sciences, Department of Biotechnology, Muthgasse 11, A-1190 Vienna (Austria); Lopandic, Ksenija, E-mail: Ksenija.Lopandic@boku.ac.at [VIBT-BOKU, University of Natural Resources and Life Sciences, Department of Biotechnology, Muthgasse 11, A-1190 Vienna (Austria); Spangl, Bernhard, E-mail: Bernhard.Spangl@boku.ac.at [University of Natural Resources and Life Sciences, Department of Landscape, Spatial and Infrastructure Science, Institute of Applied Statistics and Computing (IASC), Gregor Mendel-Str. 33, A-1180 Vienna (Austria); Ellersdorfer, Guenther, E-mail: Guenther.Ellersdorfer@boku.ac.at [VIBT-BOKU, University of Natural Resources and Life Sciences, Department of Biotechnology, Muthgasse 11, A-1190 Vienna (Austria); Voitl, Christian, E-mail: Christian.Voitl@boku.ac.at [VIBT-BOKU, University of Natural Resources and Life Sciences, Department of Biotechnology, Muthgasse 11, A-1190 Vienna (Austria); Sterflinger, Katja, E-mail: Katja.Sterflinger@boku.ac.at [VIBT-BOKU, University of Natural Resources and Life Sciences, Department of Biotechnology, Muthgasse 11, A-1190 Vienna (Austria)

    2012-11-15

    The study of microbial life in building materials is an emerging topic concerning biodeterioration of materials as well as health risks in houses and at working places. Biodegradation and potential health implications associated with microbial growth in our residues claim for more precise methods for quantification and identification. To date, cultivation experiments are commonly used to gain insight into the microbial diversity. Nowadays, molecular techniques for the identification of microorganisms provide efficient methods that can be applied in this field. The efficiency of DNA extraction is decisive in order to perform a reliable and reproducible quantification of the microorganisms by qPCR or to characterize the structure of the microbial community. In this study we tested thirteen DNA extraction methods and evaluated their efficiency for identifying (1) the quantity of DNA, (2) the quality and purity of DNA and (3) the ability of the DNA to be amplified in a PCR reaction using three universal primer sets for the ITS region of fungi as well as one primer pair targeting the 16S rRNA of bacteria with three typical building materials - common plaster, red brick and gypsum cardboard. DNA concentration measurements showed strong variations among the tested methods and materials. Measurement of the DNA yield showed up to three orders of magnitude variation from the same samples, whereas A260/A280 ratios often prognosticated biases in the PCR amplifications. Visualization of the crude DNA extracts and the comparison of DGGE fingerprints showed additional drawbacks of some methods. The FastDNA Spin kit for soil showed to be the best DNA extraction method and could provide positive results for all tests with the three building materials. Therefore, we suggest this method as a gold standard for quantification of indoor fungi and bacteria in building materials. -- Highlights: Black-Right-Pointing-Pointer Up to thirteen extraction methods were evaluated with three

  2. MicroRNome analysis unravels the molecular basis of SARS infection in bronchoalveolar stem cells.

    Directory of Open Access Journals (Sweden)

    Bibekanand Mallick

    Full Text Available Severe acute respiratory syndrome (SARS, caused by the coronavirus SARS-CoV, is an acute infectious disease with significant mortality. A typical clinical feature associated with SARS is pulmonary fibrosis and associated lung failure. In the aftermath of the SARS epidemic, although significant progress towards understanding the underlying molecular mechanism of the infection has been made, a large gap still remains in our knowledge regarding how SARS-CoV interacts with the host cell at the onset of infection. The rapidly changing viral genome adds another variable to this equation. We have focused on a novel concept of microRNA (miRNA-mediated host-virus interactions in bronchoalveolar stem cells (BASCs at the onset of infection by correlating the "BASC-microRNome" with their targets within BASCs and viral genome. This work encompasses miRNA array data analysis, target prediction, and miRNA-mRNA enrichment analysis and develops a complex interaction map among disease-related factors, miRNAs, and BASCs in SARS pathway, which will provide some clues for diagnostic markers to view an overall interplay leading to disease progression. Our observation reveals the BASCs (Sca-1+ CD34+ CD45- Pecam-, a subset of Oct-4+ ACE2+ epithelial colony cells at the broncho-alveolar duct junction, to be the prime target cells of SARS-CoV infection. Upregulated BASC miRNAs-17*, -574-5p, and -214 are co-opted by SARS-CoV to suppress its own replication and evade immune elimination until successful transmission takes place. Viral Nucleocapsid and Spike protein targets seem to co-opt downregulated miR-223 and miR-98 respectively within BASCs to control the various stages of BASC differentiation, activation of inflammatory chemokines, and downregulation of ACE2. All these effectively accounts for a successful viral transmission and replication within BASCs causing continued deterioration of lung tissues and apparent loss of capacity for lung repair. Overall, this

  3. Preliminary molecular basis of Danggui-buxue-tang on Qi deficiency and blood stasis syndrome

    Institute of Scientific and Technical Information of China (English)

    LIU Ya; LI Xiao-hui

    2008-01-01

    Objective To study the molecular biology mechanism of Danggui-buxue-tang on tonifying Qi and controlling blood circulation through modulation the immune functional genes. Methods Rats were randomly divided into control group, model group, and Danggui-buxue-tang group. Effect of Dang-guibuxue-tang on mRNA expressions of IL-1β, TNF-α, HSP-70, NF-kB, p38MAPK and JNK in blood cells of rats with Qi deficiency and blood stasis were measured with real-time fluorescent quantitative-PCR at 5th, 14th and 28th day. And that in artery wall were determined at the 28th day. NF-kB/p65 and p-c-jun protein expressions in rat artery wall were detected by western blotting as well. Results In model group, TNF-α, IL-1β, NF-kB, HSP-70, p38MAPK and JNK mRNA expression in blood cell increased significantly compared with control group. Compared with model group, mRNA expression of IL-1βand JNK decreased significantly; TNF-α decreased at 5th, 14th day;HSP-70, p38MAPK decreased at 14th, 28th day; NF-kB decreased at 28th day. In model group, TNF-α, IL-1β, NF-kB, p38MAPK and JNK mRNA expression in artery increased compared with control group, excluded HSP-70, and that in Danggui-buxue-tang group decreased significantly. In model group, NF-kB/p65 and p-c-jun protein expression in artery increased compared with control group, and that in Danggui-buxue-tang group decreased significantly. Conclusions The effects of Dang-guibuxuetang on Qi deficiency and blood stasis syndrome was brought from the regulating of cytokine network at multi-link and multi-target, NF-kB, p38MAPK and JNK signal transduction pathways included. Through which the immune system and whole body reached to a functional balance status.

  4. Molecular Basis of Inactive B-RAF(WT) and B-RAF(V600E) Ligand Inhibition, Selectivity and Conformational Stability: An in Silico Study

    DEFF Research Database (Denmark)

    Fratev, Filip Filipov; Jonsdottir, Svava Osk; Mihaylova, E.

    2009-01-01

    The B-RAF kinase plays an important role both in tumor induction and maintenance in several cancers. The molecular basis of the inactive B-RAF(WT) and B-RAF(V600E) inhibition and selectivity of a series of inhibitors was examined with a combination of molecular dynamics (MD), free energy MM-PBSA ...

  5. Molecular Basis of Ribotype Variation in the Seventh Pandemic Clone and its O139 Variant of Vibrio cholerae

    Directory of Open Access Journals (Sweden)

    Ruiting Lan

    1998-09-01

    Full Text Available Ribotyping has been widely used to characterise the seventh pandemic clone including South American and O139 variants which appeared in 1991 and 1992 respectively. To reveal the molecular basis of ribotype variation we analysed the rrn operons and their flanking regions. All but one variation detected by BglI, the most discriminatory enzyme, was found to be due to changes within the rrn operons, resulting from recombination between operons. The recombinants are detected because of the presence of a BglI site in the 16S gene in three of the nine rrn operons and/or changes of intergenic spacer types of which four variants were identified. As the frequency of rrn recombination is high, ribotyping becomes a less useful tool for evolutionary studies and long term monitoring of the pathogenic clones of Vibrio cholerae as variation could undergo precise reversion by the same recombination event.

  6. Molecular Basis of Spectral Tuning in the Red- and Green-Sensitive (M/LWS) Pigments in Vertebrates

    Science.gov (United States)

    Yokoyama, Shozo; Yang, Hui; Starmer, William T.

    2008-01-01

    Vertebrate vision is mediated by five groups of visual pigments, each absorbing a specific wavelength of light between ultraviolet and red. Despite extensive mutagenesis analyses, the mechanisms by which contemporary pigments absorb variable wavelengths of light are poorly understood. We show that the molecular basis of the spectral tuning of contemporary visual pigments can be illuminated only by mutagenesis analyses using ancestral pigments. Following this new principle, we derive the “five-sites” rule that explains the absorption spectra of red and green (M/LWS) pigments that range from 510 to 560 nm. Our findings demonstrate that the evolutionary method should be used in elucidating the mechanisms of spectral tuning of four other pigment groups and, for that matter, functional differentiations of any other proteins. PMID:18660543

  7. Identification of the molecular basis of inhibitor selectivity between the human and streptococcal type I methionine aminopeptidases.

    Science.gov (United States)

    Arya, Tarun; Reddi, Ravikumar; Kishor, Chandan; Ganji, Roopa Jones; Bhukya, Supriya; Gumpena, Rajesh; McGowan, Sheena; Drag, Marcin; Addlagatta, Anthony

    2015-03-12

    The methionine aminopeptidase (MetAP) family is responsible for the cleavage of the initiator methionine from newly synthesized proteins. Currently, there are no small molecule inhibitors that show selectivity toward the bacterial MetAPs compared to the human enzyme. In our current study, we have screened 20 α-aminophosphonate derivatives and identified a molecule (compound 15) that selectively inhibits the S. pneumonia MetAP in low micromolar range but not the human enzyme. Further bioinformatics, biochemical, and structural analyses suggested that phenylalanine (F309) in the human enzyme and methionine (M205) in the S. pneumonia MetAP at the analogous position render them with different susceptibilities against the identified inhibitor. X-ray crystal structures of various inhibitors in complex with wild type and F309M enzyme further established the molecular basis for the inhibitor selectivity.

  8. Molecular basis for manganese sequestration by calprotectin and roles in the innate immune response to invading bacterial pathogens.

    Science.gov (United States)

    Damo, Steven M; Kehl-Fie, Thomas E; Sugitani, Norie; Holt, Marilyn E; Rathi, Subodh; Murphy, Wesley J; Zhang, Yaofang; Betz, Christine; Hench, Laura; Fritz, Günter; Skaar, Eric P; Chazin, Walter J

    2013-03-05

    The S100A8/S100A9 heterodimer calprotectin (CP) functions in the host response to pathogens through a mechanism termed "nutritional immunity." CP binds Mn(2+) and Zn(2+) with high affinity and starves bacteria of these essential nutrients. Combining biophysical, structural, and microbiological analysis, we identified the molecular basis of Mn(2+) sequestration. The asymmetry of the CP heterodimer creates a single Mn(2+)-binding site from six histidine residues, which distinguishes CP from all other Mn(2+)-binding proteins. Analysis of CP mutants with altered metal-binding properties revealed that, despite both Mn(2+) and Zn(2+) being essential metals, maximal growth inhibition of multiple bacterial pathogens requires Mn(2+) sequestration. These data establish the importance of Mn(2+) sequestration in defense against infection, explain the broad-spectrum antimicrobial activity of CP relative to other S100 proteins, and clarify the impact of metal depletion on the innate immune response to infection.

  9. Defining the molecular basis for the first potent and selective orthosteric agonists of the FFA2 free fatty acid receptor

    DEFF Research Database (Denmark)

    Hudson, Brian D; Due-Hansen, Maria E; Christiansen, Elisabeth

    2013-01-01

    , species ortholog comparisons and mutagenesis studies were then employed to define the molecular basis of selectivity and function of these ligands. From this, we identified key residues within both extracellular loop 2 (ECL2) and the transmembrane domain (TM) regions of FFA2 critical for ligand function......FFA2 is a G protein-coupled receptor that responds to short chain fatty acids (SCFAs) and has generated interest as a therapeutic target for metabolic and inflammatory conditions. However, definition of its functions has been slowed by a dearth of selective ligands that can distinguish it from...... the closely related FFA3. At present, the only selective ligands described for FFA2 suffer from either poor potency, altered signaling due to allosteric modes of action, or a lack of function at non-human orthologs of the receptor. To address the need for novel selective ligands, we synthesized two compounds...

  10. Defining the Molecular Basis for the First Potent and Selective Orthosteric Agonists of the FFA2 Free Fatty Acid Receptor*

    Science.gov (United States)

    Hudson, Brian D.; Due-Hansen, Maria E.; Christiansen, Elisabeth; Hansen, Anna Mette; Mackenzie, Amanda E.; Murdoch, Hannah; Pandey, Sunil K.; Ward, Richard J.; Marquez, Rudi; Tikhonova, Irina G.; Ulven, Trond; Milligan, Graeme

    2013-01-01

    FFA2 is a G protein-coupled receptor that responds to short chain fatty acids and has generated interest as a therapeutic target for metabolic and inflammatory conditions. However, definition of its functions has been slowed by a dearth of selective ligands that can distinguish it from the closely related FFA3. At present, the only selective ligands described for FFA2 suffer from poor potency, altered signaling due to allosteric modes of action, or a lack of function at non-human orthologs of the receptor. To address the need for novel selective ligands, we synthesized two compounds potentially having FFA2 activity and examined the molecular basis of their function. These compounds were confirmed to be potent and selective orthosteric FFA2 agonists. A combination of ligand structure-activity relationship, pharmacological analysis, homology modeling, species ortholog comparisons, and mutagenesis studies were then employed to define the molecular basis of selectivity and function of these ligands. From this, we identified key residues within both extracellular loop 2 and the transmembrane domain regions of FFA2 critical for ligand function. One of these ligands was active with reasonable potency at rodent orthologs of FFA2 and demonstrated the role of FFA2 in inhibition of lipolysis and glucagon-like peptide-1 secretion in murine-derived 3T3-L1 and STC-1 cell lines, respectively. Together, these findings describe the first potent and selective FFA2 orthosteric agonists and demonstrate key aspects of ligand interaction within the binding site of FFA2 that will be invaluable in future ligand development at this receptor. PMID:23589301

  11. Adult T-cell leukemia: molecular basis for clonal expansion and transformation of HTLV-1-infected T cells.

    Science.gov (United States)

    Watanabe, Toshiki

    2017-03-02

    Adult T-cell leukemia (ATL) is an aggressive T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1) that develops through a multistep carcinogenesis process involving 5 or more genetic events. We provide a comprehensive overview of recently uncovered information on the molecular basis of leukemogenesis in ATL. Broadly, the landscape of genetic abnormalities in ATL that include alterations highly enriched in genes for T-cell receptor-NF-κB signaling such as PLCG1, PRKCB, and CARD11 and gain-of function mutations in CCR4 and CCR7 Conversely, the epigenetic landscape of ATL can be summarized as polycomb repressive complex 2 hyperactivation with genome-wide H3K27 me3 accumulation as the basis of the unique transcriptome of ATL cells. Expression of H3K27 methyltransferase enhancer of zeste 2 was shown to be induced by HTLV-1 Tax and NF-κB. Furthermore, provirus integration site analysis with high-throughput sequencing enabled the analysis of clonal composition and cell number of each clone in vivo, whereas multicolor flow cytometric analysis with CD7 and cell adhesion molecule 1 enabled the identification of HTLV-1-infected CD4(+) T cells in vivo. Sorted immortalized but untransformed cells displayed epigenetic changes closely overlapping those observed in terminally transformed ATL cells, suggesting that epigenetic abnormalities are likely earlier events in leukemogenesis. These new findings broaden the scope of conceptualization of the molecular mechanisms of leukemogenesis, dissecting them into immortalization and clonal progression. These recent findings also open a new direction of drug development for ATL prevention and treatment because epigenetic marks can be reprogrammed. Mechanisms underlying initial immortalization and progressive accumulation of these abnormalities remain to be elucidated. © 2017 by The American Society of Hematology.

  12. Molecular basis of differential selectivity of cyclobutyl-substituted imidazole inhibitors against CDKs: insights for rational drug design.

    Directory of Open Access Journals (Sweden)

    Soumya Lipsa Rath

    Full Text Available Cyclin-dependent kinases (CDKs belong to the CMGC subfamily of protein kinases and play crucial roles in eukaryotic cell division cycle. At least seven different CDKs have been reported to be implicated in the cell cycle regulation in vertebrates. These CDKs are highly homologous and contain a conserved catalytic core. This makes the design of inhibitors specific for a particular CDK difficult. There is, however, growing need for CDK5 specific inhibitors to treat various neurodegenerative diseases. Recently, cis-substituted cyclobutyl-4-aminoimidazole inhibitors have been identified as potent CDK5 inhibitors that gave up to 30-fold selectivity over CDK2. Available IC50 values also indicate a higher potency of this class of inhibitors over commercially available drugs, such as roscovitine. To understand the molecular basis of higher potency and selectivity of these inhibitors, here, we present molecular dynamics simulation results of CDK5/p25 and CDK2/CyclinE complexed with a series of cyclobutyl-substituted imidazole inhibitors and roscovitine. The atomic details of the stereospecificity and selectivity of these inhibitors are obtained from energetics and binding characteristics to the CDK binding pocket. The study not only complements the experimental findings, but also provides a wealth of detailed information that could help the structure-based drug designing processes.

  13. Species-level assessment of the molecular basis of fluoroquinolone resistance among viridans group streptococci causing bacteraemia in cancer patients.

    Science.gov (United States)

    Sahasrabhojane, Pranoti; Galloway-Peña, Jessica; Velazquez, Luis; Saldaña, Miguel; Horstmann, Nicola; Tarrand, Jeffrey; Shelburne, Samuel A

    2014-06-01

    Viridans group streptococci (VGS) are a major cause of bacteraemia in neutropenic cancer patients, particularly those receiving fluoroquinolone prophylaxis. In this study, we sought to understand the molecular basis for fluoroquinolone resistance in VGS causing bacteraemia in cancer patients by assigning 115 VGS bloodstream isolates to specific species using multilocus sequence analysis (MLSA), by sequencing the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC and parE, and by testing strain susceptibility to various fluoroquinolones. Non-susceptibility to one or more fluoroquinolones was observed for 78% of isolates, however only 68.7% of patients were receiving fluoroquinolone prophylaxis. All but one of the determinative QRDR polymorphisms occurred in GyrA or ParC, yet the pattern of determinative QRDR polymorphisms was significantly associated with the fluoroquinolone prophylaxis received. By combining MLSA and QRDR data, multiple patients infected with genetically indistinguishable fluoroquinolone-resistant Streptococcus mitis or Streptococcus oralis strains were discovered. Together these data delineate the molecular mechanisms of fluoroquinolone resistance in VGS isolates causing bacteraemia and suggest possible transmission of fluoroquinolone-resistant S. mitis and S. oralis isolates among cancer patients. Copyright © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

  14. Molecular basis of chronic stress-induced hippocampal lateral asymmetry in rats and impact on learning and memory.

    Science.gov (United States)

    Spasojevic, N; Jovanovic, P; Dronjak, Sladjana

    2013-12-01

    Neurochemical lateralization has been demonstrated in the rat brain suggesting that such lateralization might contribute to behavior. Thus, the aim of the present study was to examine neurochemical asymmetry in the hippocampus, molecular basis of neurochemical lateralization and its impact on spatial learning and memory. Changes in noradrenaline content, tyrosine hydroxylase (TH) were studied in the right and left hippocampus of naive control and chronically isolated rats, by applying TaqMan RT-PCR and Western blot analysis. Hippocampal-based spatial learning and memory were evaluated using the Barnes maze. In control rats an asymmetrical right-left distribution of noradrenaline content and gene expression of catecholamine synthesizing enzyme was found. Chronic psychosocial stress further emphasized asymmetry. Isolation stress reduced noradrenaline content only in the right hippocampus. No changes were observed in gene expression and protein levels of TH in the right hippocampus, whereas expression of catecholamine synthesizing enzyme was elevated in the left hippocampus. Reduced noradrenaline content in the right hippocampus did not cause impairment in spatial learning and memory. Our findings suggest that chronic psychosocial stress reduces noradrenaline stores in the right hippocampus which may be caused by molecular asymmetry, but it does not affect spatial learning and memory.

  15. Comparative transcriptome analysis of the Pacific White Shrimp (Litopenaeus vannamei) muscle reveals the molecular basis of residual feed intake.

    Science.gov (United States)

    Dai, Ping; Luan, Sheng; Lu, Xia; Luo, Kun; Kong, Jie

    2017-09-05

    Feed efficiency is an economically important trait in genetic improvement programs of L. vannamei. Residual feed intake (RFI), an ideal measure of feed efficiency, is the difference between observed feed intake and expected feed requirement predicted from maintenance and production. Exploring the molecular basis of RFI is essential to facilitate the genetic breeding of feed efficiency in L. vannamei. However, few studies have been reported in this aspect. In this study, we sequenced muscle transcriptomes of a high-efficiency group, a low-efficiency group and a control group originating from two families, and compared the gene expression patterns between each extreme group and the control group. A total of 383 differentially expressed genes were identified, most of which were involved in cell proliferation, growth and signaling, glucose homeostasis, energy and nutrients metabolism. Functional enrichment analysis of these genes revealed 13 significantly enriched biological pathways, including signaling pathways such as PI3K-Akt signaling pathway, AMPK signaling pathway and mTOR signaling pathway, as well as some important pathways such as ubiquitin mediated proteolysis, cell cycle, pentose phosphate pathway and glycolysis/gluconeogenesis. These genes and pathways provide initial insight into the molecular mechanisms driving the feed efficiency in L. vannamei.

  16. Novel methods for the molecular discrimination of Fasciola spp. on the basis of nuclear protein-coding genes.

    Science.gov (United States)

    Shoriki, Takuya; Ichikawa-Seki, Madoka; Suganuma, Keisuke; Naito, Ikunori; Hayashi, Kei; Nakao, Minoru; Aita, Junya; Mohanta, Uday Kumar; Inoue, Noboru; Murakami, Kenji; Itagaki, Tadashi

    2016-06-01

    Fasciolosis is an economically important disease of livestock caused by Fasciola hepatica, Fasciola gigantica, and aspermic Fasciola flukes. The aspermic Fasciola flukes have been discriminated morphologically from the two other species by the absence of sperm in their seminal vesicles. To date, the molecular discrimination of F. hepatica and F. gigantica has relied on the nucleotide sequences of the internal transcribed spacer 1 (ITS1) region. However, ITS1 genotypes of aspermic Fasciola flukes cannot be clearly differentiated from those of F. hepatica and F. gigantica. Therefore, more precise and robust methods are required to discriminate Fasciola spp. In this study, we developed PCR restriction fragment length polymorphism and multiplex PCR methods to discriminate F. hepatica, F. gigantica, and aspermic Fasciola flukes on the basis of the nuclear protein-coding genes, phosphoenolpyruvate carboxykinase and DNA polymerase delta, which are single locus genes in most eukaryotes. All aspermic Fasciola flukes used in this study had mixed fragment pattern of F. hepatica and F. gigantica for both of these genes, suggesting that the flukes are descended through hybridization between the two species. These molecular methods will facilitate the identification of F. hepatica, F. gigantica, and aspermic Fasciola flukes, and will also prove useful in etiological studies of fasciolosis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  17. Adaptive local basis set for Kohn-Sham density functional theory in a discontinuous Galerkin framework II: Force, vibration, and molecular dynamics calculations

    CERN Document Server

    Zhang, Gaigong; Hu, Wei; Yang, Chao; Pask, John E

    2015-01-01

    Recently, we have proposed the adaptive local basis set for electronic structure calculations based on Kohn-Sham density functional theory in a pseudopotential framework. The adaptive local basis set is efficient and systematically improvable for total energy calculations. In this paper, we present the calculation of atomic forces, which can be used for a range of applications such as geometry optimization and molecular dynamics simulation. We demonstrate that, under mild assumptions, the computation of atomic forces can scale nearly linearly with the number of atoms in the system using the adaptive local basis set. We quantify the accuracy of the Hellmann-Feynman forces for a range of physical systems, benchmarked against converged planewave calculations, and find that the adaptive local basis set is efficient for both force and energy calculations, requiring at most a few tens of basis functions per atom to attain accuracy required in practice. Since the adaptive local basis set has implicit dependence on a...

  18. Selection of non-dioxin-like PCBs for in vitro testing on the basis of environmental abundance and molecular structure.

    Science.gov (United States)

    Stenberg, Mia; Andersson, Patrik L

    2008-05-01

    The non-dioxin-like polychlorinated biphenyls (NDL-PCBs) constitute the major proportion of PCBs found in food and human tissues. It is important to improve our understanding of the toxicity, environmental and human risks associated with the NDL-PCBs, since their toxicology is incompletely characterized and a human health risk assessment is required. This paper discusses the selection of a training set of 20 tri- to hepta-chlorinated biphenyls, PCBs 19,28,47,51,52,53,74,95,100,101,104,118,122,128,136,138,153,170,180, and 190. Suggested for comprehensive screening using in vitro assays to identify critical mechanisms of toxicological action. The selected PCBs form a balanced basis for developing of quantitative structure-activity relationship (QSAR) models for prediction of physicochemical and toxicological properties of non-tested PCB congeners. Chemical and physical properties, environmental abundance and toxicological activities of the congeners were considered during the selection process. A complementary set of PCBs, a reference set, was selected using D-optimal onion design including PCBs 18,20,28,30,37,40,50,54,60,77,82,99,122,132,153,161,170,188,192, and 193. Congeners of this set are well suited for validation of QSAR models developed using the training set. For visualization of the chemical diversity of environmentally abundant PCBs and congeners of the training and reference sets, principal component analysis (PCA) was used. Statistical molecular design was used to verify the structural representation. As a reference structure for dioxin-like PCBs, PCB 126 was added in the training set. The selected set of NDL-PCBs is proposed for use in toxicological testing programs to provide rational basis for risk assessment of the NDL-PCBs.

  19. Dynamical properties of liquid water from ab initio molecular dynamics performed in the complete basis set limit

    Science.gov (United States)

    Lee, Hee-Seung; Tuckerman, Mark E.

    2007-04-01

    Dynamical properties of liquid water were studied using Car-Parrinello [Phys. Rev. Lett. 55, 2471 (1985)] ab initio molecular dynamics (AIMD) simulations within the Kohn-Sham (KS) density functional theory employing the Becke-Lee-Yang-Parr exchange-correlation functional for the electronic structure. The KS orbitals were expanded in a discrete variable representation basis set, wherein the complete basis set limit can be easily reached and which, therefore, provides complete convergence of ionic forces. In order to minimize possible nonergodic behavior of the simulated water system in a constant energy (NVE) ensemble, a long equilibration run (30ps) preceded a 60ps long production run. The temperature drift during the entire 60ps trajectory was found to be minimal. The diffusion coefficient [0.055Å2/ps] obtained from the present work for 32 D2O molecules is a factor of 4 smaller than the most up to date experimental value, but significantly larger than those of other recent AIMD studies. Adjusting the experimental result so as to match the finite-sized system used in the present study brings the comparison between theory and experiment to within a factor of 3. More importantly, the system is not observed to become "glassy" as has been reported in previous AIMD studies. The computed infrared spectrum is in good agreement with experimental data, especially in the low frequency regime where the translational and librational motions of water are manifested. The long simulation length also made it possible to perform detailed studies of hydrogen bond dynamics. The relaxation dynamics of hydrogen bonds observed in the present AIMD simulation is slower than those of popular force fields, such as the TIP4P potential, but comparable to that of the TIP5P potential.

  20. Structural and molecular basis for resistance to aminoglycoside antibiotics by the adenylyltransferase ANT(2″)-Ia.

    Science.gov (United States)

    Cox, Georgina; Stogios, Peter J; Savchenko, Alexei; Wright, Gerard D

    2015-01-01

    The aminoglycosides are highly effective broad-spectrum antimicrobial agents. However, their efficacy is diminished due to enzyme-mediated covalent modification, which reduces affinity of the drug for the target ribosome. One of the most prevalent aminoglycoside resistance enzymes in Gram-negative pathogens is the adenylyltransferase ANT(2″)-Ia, which confers resistance to gentamicin, tobramycin, and kanamycin. Despite the importance of this enzyme in drug resistance, its structure and molecular mechanism have been elusive. This study describes the structural and mechanistic basis for adenylylation of aminoglycosides by the ANT(2″)-Ia enzyme. ANT(2″)-Ia confers resistance by magnesium-dependent transfer of a nucleoside monophosphate (AMP) to the 2″-hydroxyl of aminoglycoside substrates containing a 2-deoxystreptamine core. The catalyzed reaction follows a direct AMP transfer mechanism from ATP to the substrate antibiotic. Central to catalysis is the coordination of two Mg(2+) ions, positioning of the modifiable substrate ring, and the presence of a catalytic base (Asp86). Comparative structural analysis revealed that ANT(2″)-Ia has a two-domain structure with an N-terminal active-site architecture that is conserved among other antibiotic nucleotidyltransferases, including Lnu(A), LinB, ANT(4')-Ia, ANT(4″)-Ib, and ANT(6)-Ia. There is also similarity between the nucleotidyltransferase fold of ANT(2″)-Ia and DNA polymerase β. This similarity is consistent with evolution from a common ancestor, with the nucleotidyltransferase fold having adapted for activity against chemically distinct molecules. IMPORTANCE  : To successfully manage the threat associated with multidrug-resistant infectious diseases, innovative therapeutic strategies need to be developed. One such approach involves the enhancement or potentiation of existing antibiotics against resistant strains of bacteria. The reduction in clinical usefulness of the aminoglycosides is a particular

  1. Molecular basis for variable expression of merozoite surface antigen gp45 among American isolates of Babesia bigemina.

    Science.gov (United States)

    Fisher, T G; McElwain, T F; Palmer, G H

    2001-06-01

    Immunization with the merozoite surface glycoprotein gp45 induces protection against challenge using the homologous Babesia bigemina strain. However, gp45 B-cell epitopes are highly polymorphic among B. bigemina strains isolated from different geographical locations within North and South America. The molecular basis for this polymorphism was investigated using the JG-29 biological clone of a Mexico strain of B. bigemina and comparison with the Puerto Rico, St. Croix, and Texcoco strains. The molecular size and antibody reactivity of gp45 expressed by the JG-29 clone were identical to those of the parental Mexico strain. gp45 cDNA and the genomic locus encompassing gp45 were cloned and sequenced from JG-29. The locus sequence and Southern blot data were consistent with a single gp45 copy in the JG-29 genome. The JG-29 cDNA expressed the full-length protein recognized by the gp45-specific monoclonal antibody 14/1.3.2. The genomes of the Puerto Rico and St. Croix strains of B. bigemina were shown to lack a closely related gp45-like gene by PCR using multiple primer sets and by Southern blots using both full-length and region-specific gp45 probes. This genomic difference was confirmed using unpassaged isolates from a 1999 disease outbreak in Puerto Rico. In contrast, the Texcoco strain retains a gp45 gene, encoding an open reading frame identical to that of JG-29. However, the Texcoco gp45 gene is not transcribed. These two mechanisms, lack of a closely related gp45-like gene and failure to transcribe gp45, result in generation of antigenic polymorphism among B. bigemina strains, and the latter mechanism is unique compared to prior mechanisms of antigenic polymorphism identified in babesial parasites.

  2. Structural and biochemical studies of a fluoroacetyl-CoA-specific thioesterase reveal a molecular basis for fluorine selectivity.

    Science.gov (United States)

    Weeks, Amy M; Coyle, Scott M; Jinek, Martin; Doudna, Jennifer A; Chang, Michelle C Y

    2010-11-02

    We have initiated a broad-based program aimed at understanding the molecular basis of fluorine specificity in enzymatic systems, and in this context, we report crystallographic and biochemical studies on a fluoroacetyl-coenzyme A (CoA) specific thioesterase (FlK) from Streptomyces cattleya. Our data establish that FlK is competent to protect its host from fluoroacetate toxicity in vivo and demonstrate a 10(6)-fold discrimination between fluoroacetyl-CoA (k(cat)/K(M) = 5 × 10⁷ M⁻¹ s⁻¹) and acetyl-CoA (k(cat)/K(M) = 30 M⁻¹ s⁻¹) based on a single fluorine substitution that originates from differences in both substrate reactivity and binding. We show that Thr 42, Glu 50, and His 76 are key catalytic residues and identify several factors that influence substrate selectivity. We propose that FlK minimizes interaction with the thioester carbonyl, leading to selection against acetyl-CoA binding that can be recovered in part by new C═O interactions in the T42S and T42C mutants. We hypothesize that the loss of these interactions is compensated by the entropic driving force for fluorinated substrate binding in a hydrophobic binding pocket created by a lid structure, containing Val 23, Leu 26, Phe 33, and Phe 36, that is not found in other structurally characterized members of this superfamily. We further suggest that water plays a critical role in fluorine specificity based on biochemical and structural studies focused on the unique Phe 36 "gate" residue, which functions to exclude water from the active site. Taken together, the findings from these studies offer molecular insights into organofluorine recognition and design of fluorine-specific enzymes.

  3. Molecular mechanism and structural basis of gain-of-function of STAT1 caused by pathogenic R274Q mutation.

    Science.gov (United States)

    Fujiki, Ryoji; Hijikata, Atsushi; Shirai, Tsuyoshi; Okada, Satoshi; Kobayashi, Masao; Ohara, Osamu

    2017-04-14

    Gain-of-function (GOF) mutations in the STAT1 gene are critical for the onset of chronic mucocutaneous candidiasis (CMC) disease. However, the molecular basis for the gain of STAT1 function remains largely unclear. Here, we investigated the structural features of STAT1 GOF residues to better understand the impact of these pathogenic mutations. We constructed STAT1 alanine mutants of the α3 helix residues of the coiled-coil domain, which are frequently found in CMC pathogenic mutations, and measured their transcriptional activities. Most of the identified GOF residues were located inside the coiled-coil domain stem structure or at the protein surface of the anti-parallel dimer interface. Unlike those, Arg-274 was adjacent to the DNA-binding domain. In addition, Arg-274 was found to functionally interact with Gln-441 in the DNA-binding domain. Because Gln-441 is located at the anti-parallel dimer contact site, Gln-441 reorientation by Arg-274 mutation probably impedes formation of the dimer. Further, the statistical analysis of RNA-seq data with STAT1-deficient epithelial cells and primary T cells from a CMC patient revealed that the R274Q mutation affected gene expression levels of 66 and 76 non-overlapping RefSeq genes, respectively. Because their transcription levels were only slightly modulated by wild-type STAT1, we concluded that the R274Q mutation increased transcriptional activity but did not change dramatically the repertoire of STAT1 targets. Hence, we provide a novel mechanism of STAT1 GOF triggered by a CMC pathogenic mutation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Molecular basis of phosphatidylinositol 4-phosphate and ARF1 GTPase recognition by the FAPP1 pleckstrin homology (PH) domain.

    Science.gov (United States)

    He, Ju; Scott, Jordan L; Heroux, Annie; Roy, Siddhartha; Lenoir, Marc; Overduin, Michael; Stahelin, Robert V; Kutateladze, Tatiana G

    2011-05-27

    Four-phosphate-adaptor protein 1 (FAPP1) regulates secretory transport from the trans-Golgi network (TGN) to the plasma membrane. FAPP1 is recruited to the Golgi through binding of its pleckstrin homology (PH) domain to phosphatidylinositol 4-phosphate (PtdIns(4)P) and a small GTPase ADP-ribosylation factor 1 (ARF1). Despite the critical role of FAPP1 in membrane trafficking, the molecular basis of its dual function remains unclear. Here, we report a 1.9 Å resolution crystal structure of the FAPP1 PH domain and detail the molecular mechanisms of the PtdIns(4)P and ARF1 recognition. The FAPP1 PH domain folds into a seven-stranded β-barrel capped by an α-helix at one edge, whereas the opposite edge is flanked by three loops and the β4 and β7 strands that form a lipid-binding pocket within the β-barrel. The ARF1-binding site is located on the outer side of the β-barrel as determined by NMR resonance perturbation analysis, mutagenesis, and measurements of binding affinities. The two binding sites have little overlap, allowing FAPP1 PH to associate with both ligands simultaneously and independently. Binding to PtdIns(4)P is enhanced in an acidic environment and is required for membrane penetration and tubulation activity of FAPP1, whereas the GTP-bound conformation of the GTPase is necessary for the interaction with ARF1. Together, these findings provide structural and biochemical insight into the multivalent membrane anchoring by the PH domain that may augment affinity and selectivity of FAPP1 toward the TGN membranes enriched in both PtdIns(4)P and GTP-bound ARF1.

  5. Structural Basis for Cooperative Binding of Azoles to CYP2E1 as Interpreted through Guided Molecular Dynamics Simulations

    Science.gov (United States)

    Levy, Joseph W.; Hartman, Jessica H.; Perry, Martin D.; Miller, Grover P.

    2015-01-01

    CYP2E1 metabolizes a wide array of small, hydrophobic molecules, resulting in their detoxification or activation into carcinogens through Michaelis-Menten as well as cooperative mechanisms. Nevertheless, the molecular determinants for CYP2E1 specificity and metabolic efficiency toward these compounds are still unknown. Herein, we employed computational docking studies coupled to Molecular Dynamics simulations to provide a critical perspective for understanding a structural basis for cooperativity observed for an array of azoles from our previous binding and catalytic studies (Hartman, JH et al (2014) Biochem Pharmacol 87, 523-33). The resulting 28 CYP2E1 complexes in this study revealed a common passageway for azoles that included a hydrophobic steric barrier causing a pause in movement toward the active site. The entrance to the active site acted like a second sieve to restrict access to the inner chamber. Collectively, these interactions impacted the final orientation of azoles reaching the active site and hence could explain differences in their biochemical properties observed in our previous studies, such as the consequences of methylation at position 5 of the azole ring. The association of a second azole demonstrated significant differences in interactions stabilizing the bound complex than observed for the first binding event. Intermolecular interactions occurred between the two azoles as well as CYP2E1 residue side chains and backbone and involved both hydrophobic contacts and hydrogen bonds. The relative importance of these interactions depended on the structure of the respective azoles indicating the absence of specific defining criteria for binding unlike the well-characterized dominant role of hydrophobicity in active site binding. Consequently, the structure activity relationships described here and elsewhere are necessary to more accurately identify factors impacting the observation and significance of cooperativity in CYP2E1 binding and catalysis

  6. Molecular basis of antithrombin deficiency in four Japanese patients with antithrombin gene abnormalities including two novel mutations.

    Science.gov (United States)

    Kyotani, Mayu; Okumura, Kaoru; Takagi, Akira; Murate, Takashi; Yamamoto, Koji; Matsushita, Tadashi; Sugimura, Motoi; Kanayama, Naohiro; Kobayashi, Takao; Saito, Hidehiko; Kojima, Tetsuhito

    2007-08-01

    We analyzed the antithrombin (AT) gene in four unrelated Japanese patients with an AT deficiency, and individually identified four distinct mutations in the heterozygous state. There were two novel mutations, 2417delT leading to a frameshift with a premature termination at amino acid -3 (FS-3Stop) and C2640T resulting in a missense mutation (Ala59Val). Previously reported mutations, T5342C (Ser116Pro) and T72C (Met-32Thr), were also found in the other two patients. To understand the molecular basis responsible for the AT deficiency in these patients, in vitro expression experiments were performed using HEK293 cells transfected with either wild type or respective mutant AT expression vector. We found that -3Stop-AT and -32Thr-AT were not secreted into the culture media, whereas 116Pro-AT and 59Val-AT were secreted normally. We further studied the heparin cofactor activity and the binding to heparin of each recombinant AT molecule. Ser116Pro mutation significantly impaired the binding affinity to heparin resulting in a reduced heparin cofactor activity. In contrast, we found that Ala59Val mutant AT unexpectedly showed a normal affinity to heparin, but severely impaired the heparin cofactor activity. Our findings suggested that FS-3Stop and Met-32Thr mutations are responsible for type I AT deficiency, whereas Ser116Pro and Ala59Val mutations contribute to type II AT deficiency, confirming that there were diverse molecular mechanisms of AT deficiency depend upon discrete AT gene abnormalities as reported previously.

  7. Investigating the Molecular Basis of Retinal Degeneration in a Familial Cohort of Pakistani Decent by Exome Sequencing.

    Directory of Open Access Journals (Sweden)

    Bruno Maranhao

    Full Text Available To define the molecular basis of retinal degeneration in consanguineous Pakistani pedigrees with early onset retinal degeneration.A cohort of 277 individuals representing 26 pedigrees from the Punjab province of Pakistan was analyzed. Exomes were captured with commercial kits and sequenced on an Illumina HiSeq 2500. Candidate variants were identified using standard tools and analyzed using exomeSuite to detect all potentially pathogenic changes in genes implicated in retinal degeneration. Segregation analysis was performed by dideoxy sequencing and novel variants were additionally investigated for their presence in ethnicity-matched controls.We identified a total of nine causal mutations, including six novel variants in RPE65, LCA5, USH2A, CNGB1, FAM161A, CERKL and GUCY2D as the underlying cause of inherited retinal degenerations in 13 of 26 pedigrees. In addition to the causal variants, a total of 200 variants each observed in five or more unrelated pedigrees investigated in this study that were absent from the dbSNP, HapMap, 1000 Genomes, NHLBI ESP6500, and ExAC databases were identified, suggesting that they are common in, and unique to the Pakistani population.We identified causal mutations associated with retinal degeneration in nearly half of the pedigrees investigated in this study through next generation whole exome sequencing. All novel variants detected in this study through exome sequencing have been cataloged providing a reference database of variants common in, and unique to the Pakistani population.

  8. Molecular basis for viral selective replication in cancer cells: activation of CDK2 by adenovirus-induced cyclin E.

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    Pei-Hsin Cheng

    Full Text Available Adenoviruses (Ads with deletion of E1b55K preferentially replicate in cancer cells and have been used in cancer therapies. We have previously shown that Ad E1B55K protein is involved in induction of cyclin E for Ad replication, but this E1B55K function is not required in cancer cells in which deregulation of cyclin E is frequently observed. In this study, we investigated the interaction of cyclin E and CDK2 in Ad-infected cells. Ad infection significantly increased the large form of cyclin E (cyclin EL, promoted cyclin E/CDK2 complex formation and increased CDK2 phosphorylation at the T160 site. Activated CDK2 caused pRb phosphorylation at the S612 site. Repression of CDK2 activity with the chemical inhibitor roscovitine or with specific small interfering RNAs significantly decreased pRb phosphorylation, with concomitant repression of viral replication. Our results suggest that Ad-induced cyclin E activates CDK2 that targets the transcriptional repressor pRb to generate a cellular environment for viral productive replication. This study reveals a new molecular basis for oncolytic replication of E1b-deleted Ads and will aid in the development of new strategies for Ad oncolytic virotherapies.

  9. Study on the molecular basis of glucoamylase overproduc-tion of a mutant strain Aspergillus niger T21

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The molecular basis for increasing of the glucoamylase (GLA) production of an Aspergillus niger mutant T21 was investigated . Northern blot analysis showed that the amount of glaA specific mRNA of A . niger T21 was about 20 times higher than that of its start strain A . niger AS 3.795. The two glaA promoter fusions (PglaA)-uidAs were respectively introduced into A . niger. Analysis of GUS activity of the transformants revealed that the PglaA activity of the strain T21 is about 3 times stronger than that of the strain AS 3.795. It is considered to be one of the reasons for the increase of glaA transcriptional level in the strain T21. However, comparing with the 20 times increase in the amount of glaA mRNA the alteration of trans regulation should be the most important reason for that. The results of deletion analysis of 5′-cis region of A . niger T21 glaA gene indicated that the region from - 408 to - 513 bp upstream of ATG is responsible for the high level expression of glaA.

  10. Molecular basis underlying the S5-dependent reproductive isolation and compatibility of indica/japonica rice hybrids.

    Science.gov (United States)

    Ji, Qing; Zhang, Meijing; Lu, Jufei; Wang, Hongmei; Lin, Bing; Liu, Qiaoquan; Chao, Qing; Zhang, Yan; Liu, Chunxia; Gu, Minghong; Xu, Mingliang

    2012-03-01

    The S5 locus regulates spikelet fertility of indica/japonica hybrid rice (Oryza sativa). There are three alleles at the S5 locus, including an indica allele (S5i), a japonica allele (S5j), and a wide-compatibility allele (S5n). This study analyzed the molecular basis for S5-dependent reproductive isolation and compatibility of indica/japonica rice hybrids. Three S5 alleles were expressed at extremely low levels, and only in the ovary. S5n was more similar to S5i in both RNA and protein expression profiles. The S5 locus was not essential for embryo sac development, although deleterious interactions between S5i and S5j resulted in reduced rates of spikelet fertility. The yeast two-hybrid system was used to test direct interactions between S5-encoded proteins. The results indicated that the S5i- and S5j-encoded eukaryotic aspartyl proteases formed both homodimers and heterodimers, whereas the S5n-encoded aspartyl protease was incapable of dimerization. Site-directed mutagenesis revealed that a single amino acid difference between S5i- and S5j-encoded aspartyl proteases (phenylalanine/leucine at residue 273) was primarily responsible for embryo sac abortion. The S5 locus may have promoted the subspeciation of indica and japonica, but it also enables gene flow between them.

  11. Molecular basis of the interaction between gating modifier spider toxins and the voltage sensor of voltage-gated ion channels

    Science.gov (United States)

    Lau, Carus H. Y.; King, Glenn F.; Mobli, Mehdi

    2016-01-01

    Voltage-sensor domains (VSDs) are modular transmembrane domains of voltage-gated ion channels that respond to changes in membrane potential by undergoing conformational changes that are coupled to gating of the ion-conducting pore. Most spider-venom peptides function as gating modifiers by binding to the VSDs of voltage-gated channels and trapping them in a closed or open state. To understand the molecular basis underlying this mode of action, we used nuclear magnetic resonance to delineate the atomic details of the interaction between the VSD of the voltage-gated potassium channel KvAP and the spider-venom peptide VSTx1. Our data reveal that the toxin interacts with residues in an aqueous cleft formed between the extracellular S1-S2 and S3-S4 loops of the VSD whilst maintaining lipid interactions in the gaps formed between the S1-S4 and S2-S3 helices. The resulting network of interactions increases the energetic barrier to the conformational changes required for channel gating, and we propose that this is the mechanism by which gating modifier toxins inhibit voltage-gated ion channels. PMID:27677715

  12. Molecular basis for positive and negative signaling by the natural killer cell receptor 2B4 (CD244).

    Science.gov (United States)

    Eissmann, Philipp; Beauchamp, Lisa; Wooters, Joe; Tilton, John C; Long, Eric O; Watzl, Carsten

    2005-06-15

    Triggering of 2B4 (CD244) can induce natural killer (NK)-cell activation, costimulation, or even inhibition of NK-cell activity. Here, we investigate the molecular basis for the different signals generated by 2B4. We show that the first immunoreceptor tyrosine-based switch motif (ITSM) within the cytoplasmic tail of 2B4 is sufficient for 2B4-mediated NK-cell activation, whereas the third ITSM can negatively influence 2B4 signaling. We further identify signaling molecules that associate with 2B4. Signaling lymphocyte activation molecule-associated protein (SAP) can bind to all 4 ITSMs of 2B4 in a phosphorylation-dependent manner. The phosphorylated third ITSM can additionally recruit the phosphatases SHP-1, SHP-2, SHIP, and the inhibitory kinase Csk. SAP acts as an inhibitor of interactions between 2B4 and these negative regulatory molecules, explaining how 2B4 inhibits NK-cell activation in the absence of functional SAP, as occurs in cells from patients with X-linked lymphoproliferative syndrome (XLP). Recently, another function for SAP was proposed: SAP can recruit the kinase Fyn to the SLAM (CD150) immune receptor. We now show that Fyn can also associate with phosphorylated 2B4. Finally, we demonstrate that Fyn and Csk can both phosphorylate 2B4, suggesting a possible mechanism of 2B4 phosphorylation.

  13. Molecular basis of ALS- and/or ACCase-inhibitor resistance in shortawn foxtail (Alopecurus aequalis Sobol.).

    Science.gov (United States)

    Xia, Wenwen; Pan, Lang; Li, Jun; Wang, Qiong; Feng, Yujuan; Dong, Liyao

    2015-07-01

    Alopecurus aequalis, a predominant weed species in wheat and oilseed rape fields, can no longer be controlled by mesosulfuron-methyl application after continuous use over several years. Based on dose-response studies, the putative resistant populations, JTJY-1 and JHHZ-1, were found to be resistant to mesosulfuron-methyl, with resistance index values of 5.5 and 14, respectively. Sensitivity assays of the mesosulfuron-methyl-resistant populations to other herbicides revealed that the JTJY-1 population had moderate or high cross resistance to sulfonylureas (SUs) and triazolopyrimidines (TPs), but displayed a low level resistance to imidazolinones (IMIs). JTJY-1 also had high multi-resistance to ACCase inhibitors, but remained susceptible to photosystem II inhibitors. The JHHZ-1 population was resistant to all ALS inhibitors tested, but was sensitive to ACCase inhibitors and photosystem II inhibitors. To clarify the molecular basis of resistance in JTJY-1 and JHHZ-1 population, the ALS and ACCase gene were sequenced. Two ALS mutations (Pro-197-Thr or Trp-574-Leu) were detected in the mesosulfuron-methyl-resistant plants. The ACCase gene analysis revealed that the resistant JTJY-1 population had an Ile-1781-Leu mutation. Furthermore, the presence of two different target site resistance (TSR) mechanisms (ALS and ACCase mutations) existing simultaneously in individual A. aequalis was firstly documented in the presented study.

  14. Structural basis for regulation of stability and activity in glyceraldehyde-3-phosphate dehydrogenases. Differential scanning calorimetry and molecular dynamics.

    Science.gov (United States)

    Makshakova, Olga N; Semenyuk, Pavel I; Kuravsky, Mikhail L; Ermakova, Elena A; Zuev, Yuriy F; Muronetz, Vladimir I

    2015-05-01

    Tissue specific isoforms of human glyceraldehyde-3-phosphate dehydrogenase, somatic (GAPD) and sperm-specific (GAPDS), have been reported to display different levels of both stability and catalytic activity. Here we apply MD simulations to investigate molecular basis of this phenomenon. The protein is a tetramer where each subunit consists of two domains - catalytic and NAD-binding one. We demonstrated key residues responsible for intersubunit and interdomain interactions. Effect of several residues was studied by point mutations. Overall we considered three mutations (Glu96Gln, Glu244Gln and Asp311Asn) disrupting GAPDS-specific salt bridges. Comparison of calculated interaction energies with calorimetric enthalpies confirmed that intersubunit interactions were responsible for enhanced thermostability of GAPDS whereas interdomain interactions had indirect influence on intersubunit contacts. Mutation Asp311Asn was around 10Å far from the active center and corresponded to the closest natural substitution in the isoenzymes. MD simulations revealed that this residue had slight interaction with catalytic residues but influenced the hydrogen bond net and dynamics in active site. These effects can be responsible for a strong influence of this residue on catalytic activity. Overall, our results provide new insight into glyceraldehyde-3-phosphate dehydrogenase structure-function relationships and can be used for the engineering of mutant proteins with modified properties and for development of new inhibitors with indirect influence on the catalytic site. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Molecular basis of the interaction between gating modifier spider toxins and the voltage sensor of voltage-gated ion channels

    Science.gov (United States)

    Lau, Carus H. Y.; King, Glenn F.; Mobli, Mehdi

    2016-09-01

    Voltage-sensor domains (VSDs) are modular transmembrane domains of voltage-gated ion channels that respond to changes in membrane potential by undergoing conformational changes that are coupled to gating of the ion-conducting pore. Most spider-venom peptides function as gating modifiers by binding to the VSDs of voltage-gated channels and trapping them in a closed or open state. To understand the molecular basis underlying this mode of action, we used nuclear magnetic resonance to delineate the atomic details of the interaction between the VSD of the voltage-gated potassium channel KvAP and the spider-venom peptide VSTx1. Our data reveal that the toxin interacts with residues in an aqueous cleft formed between the extracellular S1-S2 and S3-S4 loops of the VSD whilst maintaining lipid interactions in the gaps formed between the S1-S4 and S2-S3 helices. The resulting network of interactions increases the energetic barrier to the conformational changes required for channel gating, and we propose that this is the mechanism by which gating modifier toxins inhibit voltage-gated ion channels.

  16. Molecular basis for the wide range of affinity found in Csr/Rsm protein-RNA recognition.

    Science.gov (United States)

    Duss, Olivier; Michel, Erich; Diarra dit Konté, Nana; Schubert, Mario; Allain, Frédéric H-T

    2014-04-01

    The carbon storage regulator/regulator of secondary metabolism (Csr/Rsm) type of small non-coding RNAs (sRNAs) is widespread throughout bacteria and acts by sequestering the global translation repressor protein CsrA/RsmE from the ribosome binding site of a subset of mRNAs. Although we have previously described the molecular basis of a high affinity RNA target bound to RsmE, it remains unknown how other lower affinity targets are recognized by the same protein. Here, we have determined the nuclear magnetic resonance solution structures of five separate GGA binding motifs of the sRNA RsmZ of Pseudomonas fluorescens in complex with RsmE. The structures explain how the variation of sequence and structural context of the GGA binding motifs modulate the binding affinity for RsmE by five orders of magnitude (∼10 nM to ∼3 mM, Kd). Furthermore, we see that conformational adaptation of protein side-chains and RNA enable recognition of different RNA sequences by the same protein contributing to binding affinity without conferring specificity. Overall, our findings illustrate how the variability in the Csr/Rsm protein-RNA recognition allows a fine-tuning of the competition between mRNAs and sRNAs for the CsrA/RsmE protein.

  17. Comparative proteome analysis of psychrophilic versus mesophilic bacterial species: Insights into the molecular basis of cold adaptation of proteins

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    Reddy Boojala

    2009-01-01

    Full Text Available Abstract Background Cold adapted or psychrophilic organisms grow at low temperatures, where most of other organisms cannot grow. This adaptation requires a vast array of sequence, structural and physiological adjustments. To understand the molecular basis of cold adaptation of proteins, we analyzed proteomes of psychrophilic and mesophilic bacterial species and compared the differences in amino acid composition and substitution patterns to investigate their likely association with growth temperatures. Results In psychrophilic bacteria, serine, aspartic acid, threonine and alanine are overrepresented in the coil regions of secondary structures, whilst glutamic acid and leucine are underrepresented in the helical regions. Compared to mesophiles, psychrophiles comprise a significantly higher proportion of amino acids that contribute to higher protein flexibility in the coil regions of proteins, such as those with tiny/small or neutral side chains. Amino acids with aliphatic, basic, aromatic and hydrophilic side chains are underrepresented in the helical regions of proteins of psychrophiles. The patterns of amino acid substitutions between the orthologous proteins of psychrophiles versus mesophiles are significantly different for several amino acids when compared to their substitutions in orthologous proteins of within the mesophiles or psychrophiles. Conclusion Current results provide quantitative substitution preferences (or avoidance of amino acids that lead to the adaptation of proteins to cold temperatures. These finding would help future efforts in selecting mutations for rational design of proteins with enhanced psychrophilic properties.

  18. Fungicide-driven evolution and molecular basis of multidrug resistance in field populations of the grey mould fungus Botrytis cinerea.

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    Matthias Kretschmer

    2009-12-01

    Full Text Available The grey mould fungus Botrytis cinerea causes losses of commercially important fruits, vegetables and ornamentals worldwide. Fungicide treatments are effective for disease control, but bear the risk of resistance development. The major resistance mechanism in fungi is target protein modification resulting in reduced drug binding. Multiple drug resistance (MDR caused by increased efflux activity is common in human pathogenic microbes, but rarely described for plant pathogens. Annual monitoring for fungicide resistance in field isolates from fungicide-treated vineyards in France and Germany revealed a rapidly increasing appearance of B. cinerea field populations with three distinct MDR phenotypes. All MDR strains showed increased fungicide efflux activity and overexpression of efflux transporter genes. Similar to clinical MDR isolates of Candida yeasts that are due to transcription factor mutations, all MDR1 strains were shown to harbor activating mutations in a transcription factor (Mrr1 that controls the gene encoding ABC transporter AtrB. MDR2 strains had undergone a unique rearrangement in the promoter region of the major facilitator superfamily transporter gene mfsM2, induced by insertion of a retrotransposon-derived sequence. MDR2 strains carrying the same rearranged mfsM2 allele have probably migrated from French to German wine-growing regions. The roles of atrB, mrr1 and mfsM2 were proven by the phenotypes of knock-out and overexpression mutants. As confirmed by sexual crosses, combinations of mrr1 and mfsM2 mutations lead to MDR3 strains with higher broad-spectrum resistance. An MDR3 strain was shown in field experiments to be selected against sensitive strains by fungicide treatments. Our data document for the first time the rising prevalence, spread and molecular basis of MDR populations in a major plant pathogen in agricultural environments. These populations will increase the risk of grey mould rot and hamper the effectiveness of

  19. Fungicide-driven evolution and molecular basis of multidrug resistance in field populations of the grey mould fungus Botrytis cinerea.

    Directory of Open Access Journals (Sweden)

    Matthias Kretschmer

    2009-12-01

    Full Text Available The grey mould fungus Botrytis cinerea causes losses of commercially important fruits, vegetables and ornamentals worldwide. Fungicide treatments are effective for disease control, but bear the risk of resistance development. The major resistance mechanism in fungi is target protein modification resulting in reduced drug binding. Multiple drug resistance (MDR caused by increased efflux activity is common in human pathogenic microbes, but rarely described for plant pathogens. Annual monitoring for fungicide resistance in field isolates from fungicide-treated vineyards in France and Germany revealed a rapidly increasing appearance of B. cinerea field populations with three distinct MDR phenotypes. All MDR strains showed increased fungicide efflux activity and overexpression of efflux transporter genes. Similar to clinical MDR isolates of Candida yeasts that are due to transcription factor mutations, all MDR1 strains were shown to harbor activating mutations in a transcription factor (Mrr1 that controls the gene encoding ABC transporter AtrB. MDR2 strains had undergone a unique rearrangement in the promoter region of the major facilitator superfamily transporter gene mfsM2, induced by insertion of a retrotransposon-derived sequence. MDR2 strains carrying the same rearranged mfsM2 allele have probably migrated from French to German wine-growing regions. The roles of atrB, mrr1 and mfsM2 were proven by the phenotypes of knock-out and overexpression mutants. As confirmed by sexual crosses, combinations of mrr1 and mfsM2 mutations lead to MDR3 strains with higher broad-spectrum resistance. An MDR3 strain was shown in field experiments to be selected against sensitive strains by fungicide treatments. Our data document for the first time the rising prevalence, spread and molecular basis of MDR populations in a major plant pathogen in agricultural environments. These populations will increase the risk of grey mould rot and hamper the effectiveness of

  20. Elevated type I interferon-like activity in a subset of multiple sclerosis patients: molecular basis and clinical relevance

    Directory of Open Access Journals (Sweden)

    Hundeshagen Alexander

    2012-06-01

    Full Text Available Abstract Background A subset of patients with multiple sclerosis (MS shows an increased endogenous IFN-like activity before initiation of IFN-beta treatment. The molecular basis of this phenomenon and its relevance to predict individual therapy outcomes are not yet fully understood. We studied the expression patterns of these patients, the prognostic value of an elevated IFN-like activity, and the gene regulatory effects of exogenously administered IFN-beta. Methods Microarray gene expression profiling was performed for 61 MS patients using peripheral blood mononuclear cells obtained before and after 1 month of IFN-beta therapy. Expression levels of genes involved in pathways either inducing or being activated by IFN-beta were compared between patients with high (MX1high cohort and low (MX1low cohort endogenous IFN-like activity. Patients were followed for 5 years and relapses as well as progression on the expanded disability status scale (EDSS were documented. Results Before the start of therapy, 11 patients presented elevated mRNA levels of IFN-stimulated genes indicative of a relatively high endogenous IFN-like activity (MX1high. In these patients, pathogen receptors (for example, TLR7, RIG-I and IFIH1 and transcription factors were also expressed more strongly, which could be attributed to an overactivity of IFN-stimulated gene factor 3 (ISGF3, a complex formed by STAT1, STAT2 and IFN regulatory factor 9. After 1 month of IFN-beta therapy, the expression of many pathway genes was significantly induced in MX1low patients, but remained unaltered in MX1high patients. During follow-up, relapse rate and changes in EDSS were comparable between both patient groups, with differences seen between different types of IFN-beta drug application. Conclusions Therapeutic IFN-beta induces the transcription of several genes involved in IFN-related pathways. In a subgroup of MS patients, the expression of these genes is already increased before therapy

  1. Molecular basis of IgE-recognition of Lol p 5, a major allergen of rye-grass pollen.

    Science.gov (United States)

    Suphioglu, C; Blaher, B; Rolland, J M; McCluskey, J; Schäppi, G; Kenrick, J; Singh, M B; Knox, R B

    1998-04-01

    Grass pollen, especially of rye-grass (Lolium perenne). represents an important cause of type I allergy. Identification of IgE-binding (allergenic) epitopes of major grass pollen allergens is essential for understanding the molecular basis of interaction between allergens and human IgE antibodies and therefore facilitates the devising of safer and more effective diagnostic and immunotherapy reagents. The aim of this study was to identify the allergenic epitopes of Lol p 5, a major allergen of rye-grass pollen, immunodissect these epitopes further so that the amino acid residues critical for antibody binding can be determined and investigate the conservation and nature of these epitopes within the context of the natural grass pollen allergens. Peptides, 12-13 amino acid residues long and overlapping each other by 4 amino acid residues, based on the entire deduced amino acid sequence of the coding region of Lol p 5, were synthesised and assayed for IgE-binding. Two strong IgE-binding epitopes (Lol p 5 (49-60) and (265-276), referred to as peptides 7 and 34, respectively) were identified. These epitopes were further resolved by truncated peptides and amino acid replacement studies and the amino acid residues critical for IgE-binding determined (Lol p 5 (49-60) residue Lys57 and (265-276) residue Lys275). Sequences of these epitopes were conserved in related allergens and may form the conserved allergenic domains responsible for the cross-reactivity observed between pollen allergens of taxonomically related grasses. Furthermore, due to its strong IgE-reactivity, synthetic peptide Lol p 5 (265-276) was used to affinity-purify specific IgE antibodies which recognised proteins of other clinically important grass pollens. further indicating presence of allergenic cross-reactivity at the level of allergenic epitope. Moreover, Lol p 5 (265 276) demonstrated a strong capacity to inhibit IgE-binding to natural rye-grass pollen proteins highlighting the antibody accessibility

  2. Molecular basis of colorectal cancer: Towards an individualized management? Bases moleculares del cáncer colorrectal: ¿Hacia un manejo individualizado?

    Directory of Open Access Journals (Sweden)

    J. Perea

    2011-01-01

    Full Text Available Colorectal cancer (CRC has become a highly relevant condition nowadays. In this respect, advances in the understanding of its molecular basis are key for an adequate management. From the time when the adenoma-carcinoma sequence was formulated as a carcinogenesis model to this day, when, among other things, three major carcinogenic pathways have been identified, the CRC concept has evolved from that of a single disease to the notion that each CRC is a differentiated condition in itself. The suppressor or chromosome instability pathway, the mutator or microsatellite instability pathway, and the methylator or CpG island methylation pathway allow various phenotypes to be identified within CRC. Similarly, the presence of different changes in certain genes confers several behaviors on CRC from both the prognostic and responsive standpoints to specific therapies. However, this apparent complexity does help develop the clinical management of this disease through the identification of novel, more specific therapy targets, and also markers for various behaviors within the condition, which will most likely lead us to an individualized management for these patients.La importancia que está adquiriendo el cáncer colorrectal (CCR hoy en día es importantísima. En este sentido, los avances en el conocimiento de sus bases moleculares son esenciales para su adecuado manejo. Desde la formulación del modelo de carcinogénesis de la secuencia adenoma-carcinoma hasta hoy, en que, entre otros aspectos, se han identificado tres grandes vías de carcinogénesis, el concepto de CCR ha llegado a transformarse desde el de una enfermedad única a la idea de que cada CCR es una entidad diferenciada respecto al resto de CCR. La vía supresora o de la inestabilidad cromosómica, la vía mutadora o de la inestabilidad de microsatélites, y la vía metiladora o del fenotipo metilador de islas CpG, permiten identificar diferentes fenotipos dentro del CCR. De la misma forma

  3. Basis set convergence of CCSD(T) equilibrium geometries using a large and diverse set of molecular structures.

    Science.gov (United States)

    Spackman, Peter R; Jayatilaka, Dylan; Karton, Amir

    2016-09-14

    We examine the basis set convergence of the CCSD(T) method for obtaining the structures of the 108 neutral first- and second-row species in the W4-11 database (with up to five non-hydrogen atoms). This set includes a total of 181 unique bonds: 75 H-X, 49 X-Y, 43 X=Y, and 14 X≡Y bonds (where X and Y are first- and second-row atoms). As reference values, geometries optimized at the CCSD(T)/aug'-cc-pV(6+d)Z level of theory are used. We consider the basis set convergence of the CCSD(T) method with the correlation consistent basis sets cc-pV(n+d)Z and aug'-cc-pV(n+d)Z (n = D, T, Q, 5) and the Weigend-Ahlrichs def2-n ZVPP basis sets (n = T, Q). For each increase in the highest angular momentum present in the basis set, the root-mean-square deviation (RMSD) over the bond distances is decreased by a factor of ∼4. For example, the following RMSDs are obtained for the cc-pV(n+d)Z basis sets 0.0196 (D), 0.0050 (T), 0.0015 (Q), and 0.0004 (5) Å. Similar results are obtained for the aug'-cc-pV(n+d)Z and def2-n ZVPP basis sets. The double-zeta and triple-zeta quality basis sets systematically and significantly overestimate the bond distances. A simple and cost-effective way to improve the performance of these basis sets is to scale the bond distances by an empirical scaling factor of 0.9865 (cc-pV(D+d)Z) and 0.9969 (cc-pV(T+d)Z). This results in RMSDs of 0.0080 (scaled cc-pV(D+d)Z) and 0.0029 (scaled cc-pV(T+d)Z) Å. The basis set convergence of larger basis sets can be accelerated via standard basis-set extrapolations. In addition, the basis set convergence of explicitly correlated CCSD(T)-F12 calculations is investigated in conjunction with the cc-pVnZ-F12 basis sets (n = D, T). Typically, one "gains" two angular momenta in the explicitly correlated calculations. That is, the CCSD(T)-F12/cc-pVnZ-F12 level of theory shows similar performance to the CCSD(T)/cc-pV(n+2)Z level of theory. In particular, the following RMSDs are obtained for the cc-pVnZ-F12 basis sets 0.0019 (D

  4. Basis set convergence of CCSD(T) equilibrium geometries using a large and diverse set of molecular structures

    Science.gov (United States)

    Spackman, Peter R.; Jayatilaka, Dylan; Karton, Amir

    2016-09-01

    We examine the basis set convergence of the CCSD(T) method for obtaining the structures of the 108 neutral first- and second-row species in the W4-11 database (with up to five non-hydrogen atoms). This set includes a total of 181 unique bonds: 75 H—X, 49 X—Y, 43 X=Y, and 14 X≡Y bonds (where X and Y are first- and second-row atoms). As reference values, geometries optimized at the CCSD(T)/aug'-cc-pV(6+d)Z level of theory are used. We consider the basis set convergence of the CCSD(T) method with the correlation consistent basis sets cc-pV(n+d)Z and aug'-cc-pV(n+d)Z (n = D, T, Q, 5) and the Weigend-Ahlrichs def2-n ZVPP basis sets (n = T, Q). For each increase in the highest angular momentum present in the basis set, the root-mean-square deviation (RMSD) over the bond distances is decreased by a factor of ˜4. For example, the following RMSDs are obtained for the cc-pV(n+d)Z basis sets 0.0196 (D), 0.0050 (T), 0.0015 (Q), and 0.0004 (5) Å. Similar results are obtained for the aug'-cc-pV(n+d)Z and def2-n ZVPP basis sets. The double-zeta and triple-zeta quality basis sets systematically and significantly overestimate the bond distances. A simple and cost-effective way to improve the performance of these basis sets is to scale the bond distances by an empirical scaling factor of 0.9865 (cc-pV(D+d)Z) and 0.9969 (cc-pV(T+d)Z). This results in RMSDs of 0.0080 (scaled cc-pV(D+d)Z) and 0.0029 (scaled cc-pV(T+d)Z) Å. The basis set convergence of larger basis sets can be accelerated via standard basis-set extrapolations. In addition, the basis set convergence of explicitly correlated CCSD(T)-F12 calculations is investigated in conjunction with the cc-pVnZ-F12 basis sets (n = D, T). Typically, one "gains" two angular momenta in the explicitly correlated calculations. That is, the CCSD(T)-F12/cc-pVnZ-F12 level of theory shows similar performance to the CCSD(T)/cc-pV(n+2)Z level of theory. In particular, the following RMSDs are obtained for the cc-pVnZ-F12 basis sets 0

  5. Exercise and Prognosis on the Basis of Clinicopathologic and Molecular Features in Early-Stage Breast Cancer: The LACE and Pathways Studies.

    Science.gov (United States)

    Jones, Lee W; Kwan, Marilyn L; Weltzien, Erin; Chandarlapaty, Sarat; Sternfeld, Barbara; Sweeney, Carol; Bernard, Philip S; Castillo, Adrienne; Habel, Laurel A; Kroenke, Candyce H; Langholz, Bryan M; Queensberry, Charles P; Dang, Chau; Weigelt, Britta; Kushi, Lawrence H; Caan, Bette J

    2016-09-15

    To investigate whether the impact of postdiagnosis exercise on breast cancer outcomes in women diagnosed with early-stage breast cancer differs on the basis of tumor clinicopathologic and molecular features. Using a prospective design, 6,211 patients with early-stage breast cancer from two large population-based cohort studies were studied. Age-adjusted and multivariable Cox regression models were performed to determine the relationship between exercise exposure (total MET-hours/week) and recurrence and breast cancer-related death for: (i) all patients ("unselected" cohort), and on the basis of (ii) classic clinicopathologic features, (iii) clinical subtypes, (iv) PAM50-based molecular intrinsic subtypes, and (v) individual PAM50 target genes. After a median follow-up of 7.2 years, in the unselected cohort (n = 6,211) increasing exercise exposure was not associated with a reduction in the risk of recurrence (adjusted Ptrend = 0.60) or breast cancer-related death (adjusted Ptrend = 0.39). On the basis of clinicopathologic features, an exercise-associated reduction in breast cancer-related death was apparent for tumors breast cancer-related death: adjusted HR, 0.57; 95% CI, 0.37-0.86). The impact of exercise on cancer outcomes appears to differ as a function of pathologic and molecular features in early-stage breast cancer. Cancer Res; 76(18); 5415-22. ©2016 AACR.

  6. Periodic local Møller-Plesset second order perturbation theory method applied to molecular crystals: study of solid NH3 and CO2 using extended basis sets.

    Science.gov (United States)

    Maschio, Lorenzo; Usvyat, Denis; Schütz, Martin; Civalleri, Bartolomeo

    2010-04-07

    We have calculated the equilibrium geometry, formation energy, and bulk modulus of two molecular bulk crystals, NH(3) and CO(2), at the periodic post-Hartree-Fock correlated level. The dependence of the results on the basis set has been analyzed, by employing basis sets up to aug-cc-pVQZ quality. In the calculations, we used the periodic local Møller-Plesset second order perturbation theory (LMP2), implemented in the CRYSCOR program. Multipolar expansion techniques, as well as density fitting, are employed in this code to reduce the number of and to factorize the required electron repulsion integrals; as a consequence of that, the computational cost for the correlation part of the calculations is comparable to that of the Hartree-Fock. Auxiliary calculations performed on molecular dimers are also reported to verify the accuracy of the LMP2 approach and of the basis sets used. Furthermore, the effect of spin-component scaling has been investigated for the two crystals. One intention of the present paper is also to lay out and specify the computational setup, which is generally applicable for accurate CRYSCOR calculations on molecular crystals.

  7. Molecular Basis of the Receptor Interactions of Polysialic Acid (polySia), polySia Mimetics, and Sulfated Polysaccharides

    NARCIS (Netherlands)

    Zhang, Ruiyan; Loers, Gabriele; Schachner, Melitta; Boelens, Rolf; Wienk, Hans; Siebert, Simone; Eckert, Thomas; Kraan, Stefan; Rojas-Macias, Miguel A.; Lütteke, Thomas; Galuska, Sebastian P.; Scheidig, Axel; Petridis, Athanasios K.; Liang, Songping; Billeter, Martin; Schauer, Roland; Steinmeyer, Jürgen; Schröder, Jens Michael; Siebert, Hans Christian

    2016-01-01

    Polysialic acid (polySia) and polySia glycomimetic molecules support nerve cell regeneration, differentiation, and neuronal plasticity. With a combination of biophysical and biochemical methods, as well as data mining and molecular modeling techniques, it is possible to correlate specific ligand-rec

  8. Molecular Basis of the Receptor Interactions of Polysialic Acid (polySia), polySia Mimetics, and Sulfated Polysaccharides

    NARCIS (Netherlands)

    Zhang, Ruiyan; Loers, Gabriele; Schachner, Melitta; Boelens, Rolf; Wienk, Hans; Siebert, Simone; Eckert, Thomas; Kraan, Stefan; Rojas-Macias, Miguel A.; Lütteke, Thomas; Galuska, Sebastian P.; Scheidig, Axel; Petridis, Athanasios K.; Liang, Songping; Billeter, Martin; Schauer, Roland; Steinmeyer, Jürgen; Schröder, Jens Michael; Siebert, Hans Christian

    2016-01-01

    Polysialic acid (polySia) and polySia glycomimetic molecules support nerve cell regeneration, differentiation, and neuronal plasticity. With a combination of biophysical and biochemical methods, as well as data mining and molecular modeling techniques, it is possible to correlate specific ligand-rec

  9. Molecular basis of processing-induced changes in protein structure in relation to intestinal digestion in yellow and green type pea (Pisum sativum L.): A molecular spectroscopic analysis.

    Science.gov (United States)

    Yu, Gloria Qingyu; Warkentin, Tom; Niu, Zhiyuan; Khan, Nazir A; Yu, Peiqiang

    2015-12-01

    The objectives of this study were (1) to quantify the protein inherent molecular structural features of green cotyledon (CDC Striker) and yellow cotyledon (CDC Meadow) pea (Pisum sativum L.) seeds using molecular spectroscopic technique (FT/IR-ATR); (2) measure the denaturation of protein molecular makeup in the two types of pea during dry roasting (120°C for 60 min), autoclaving (120°C for 60 min) or microwaving (for 5 min); and (3) correlate the heat-induced changes in protein molecular makeup to the corresponding changes in protein digestibility determined using modified three-step in vitro procedure. Compared with yellow-type, the green-type peas had higher (Ppeas had lower (Ppea-types. However, across the pea types the correlation was not significant. Principal component and hierarchical cluster analyses on the entire spectral data from the amide region (ca. 1727-1480 cm(-1)) were able to visualize and discriminate the structural difference between pea varieties and processing treatments. This study shows that the molecular spectroscopy can be used as a rapid tool to screen the protein value of raw and heat-treated peas.

  10. New insights into heat induced structural changes of pectin methylesterase on fluorescence spectroscopy and molecular modeling basis

    Science.gov (United States)

    Nistor, Oana Viorela; Stănciuc, Nicoleta; Aprodu, Iuliana; Botez, Elisabeta

    2014-07-01

    Heat-induced structural changes of Aspergillus oryzae pectin methylesterase (PME) were studied by means of fluorescence spectroscopy and molecular modeling, whereas the functional enzyme stability was monitored by inactivation studies. The fluorescence spectroscopy experiments were performed at two pH value (4.5 and 7.0). At both pH values, the phase diagrams were linear, indicating the presence of two molecular species induced by thermal treatment. A red shift of 7 nm was observed at neutral pH by increasing temperature up to 60 °C, followed by a blue shift of 4 nm at 70 °C, suggesting significant conformational rearrangements. The quenching experiments using acrylamide and iodide demonstrate a more flexible conformation of enzyme with increasing temperature, especially at neutral pH. The experimental results were complemented with atomic level observations on PME model behavior after performing molecular dynamics simulations at different temperatures. The inactivation kinetics of PME in buffer solutions was fitted using a first-order kinetics model, resulting in activation energy of 241.4 ± 7.51 kJ mol-1.

  11. Calculation of wave-functions with frozen orbitals in mixed quantum mechanics/molecular mechanics methods. II. Application of the local basis equation.

    Science.gov (United States)

    Ferenczy, György G

    2013-04-05

    The application of the local basis equation (Ferenczy and Adams, J. Chem. Phys. 2009, 130, 134108) in mixed quantum mechanics/molecular mechanics (QM/MM) and quantum mechanics/quantum mechanics (QM/QM) methods is investigated. This equation is suitable to derive local basis nonorthogonal orbitals that minimize the energy of the system and it exhibits good convergence properties in a self-consistent field solution. These features make the equation appropriate to be used in mixed QM/MM and QM/QM methods to optimize orbitals in the field of frozen localized orbitals connecting the subsystems. Calculations performed for several properties in divers systems show that the method is robust with various choices of the frozen orbitals and frontier atom properties. With appropriate basis set assignment, it gives results equivalent with those of a related approach [G. G. Ferenczy previous paper in this issue] using the Huzinaga equation. Thus, the local basis equation can be used in mixed QM/MM methods with small size quantum subsystems to calculate properties in good agreement with reference Hartree-Fock-Roothaan results. It is shown that bond charges are not necessary when the local basis equation is applied, although they are required for the self-consistent field solution of the Huzinaga equation based method. Conversely, the deformation of the wave-function near to the boundary is observed without bond charges and this has a significant effect on deprotonation energies but a less pronounced effect when the total charge of the system is conserved. The local basis equation can also be used to define a two layer quantum system with nonorthogonal localized orbitals surrounding the central delocalized quantum subsystem.

  12. Small Atomic Orbital Basis Set First-Principles Quantum Chemical Methods for Large Molecular and Periodic Systems: A Critical Analysis of Error Sources.

    Science.gov (United States)

    Sure, Rebecca; Brandenburg, Jan Gerit; Grimme, Stefan

    2016-04-01

    In quantum chemical computations the combination of Hartree-Fock or a density functional theory (DFT) approximation with relatively small atomic orbital basis sets of double-zeta quality is still widely used, for example, in the popular B3LYP/6-31G* approach. In this Review, we critically analyze the two main sources of error in such computations, that is, the basis set superposition error on the one hand and the missing London dispersion interactions on the other. We review various strategies to correct those errors and present exemplary calculations on mainly noncovalently bound systems of widely varying size. Energies and geometries of small dimers, large supramolecular complexes, and molecular crystals are covered. We conclude that it is not justified to rely on fortunate error compensation, as the main inconsistencies can be cured by modern correction schemes which clearly outperform the plain mean-field methods.

  13. Molecular basis of Refsum disease: sequence variations in phytanoyl-CoA hydroxylase (PHYH) and the PTS2 receptor (PEX7).

    Science.gov (United States)

    Jansen, Gerbert A; Waterham, Hans R; Wanders, Ronald J A

    2004-03-01

    Refsum disease has long been known to be an inherited disorder of lipid metabolism characterized by the accumulation of phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) caused by an alpha-oxidation deficiency of this branched chain fatty acid in peroxisomes. The mechanism of phytanic acid alpha-oxidation and the enzymes involved had long remained mysterious, but they have been resolved in recent years. This has led to the resolution of the molecular basis of Refsum disease. Interestingly, Refsum disease is genetically heterogeneous; two genes, PHYH (also named PAHX) and PEX7, have been identified to cause Refsum disease, as reviewed in this work.

  14. Four new cases of pediatric thoracic aortic aneurysm (TAA) with review of the molecular genetic basis, utilizing the newly published consensus nomenclature.

    Science.gov (United States)

    Deshpande, Shriprasad; Alazraki, Adina; Khoshnam, Nasim; Kantarovich, Diana; Hoseinzadeh, Pooya; Hebson, Camden; Bhatia, Ajay; Schaffer, Lauren; Maccou, Elana; Shehata, Bahig M

    2017-07-27

    The majority of thoracic aortic aneurysms (TAA) in the pediatric population are due to post repair etiology (iatrogenic). Although rare, underlying inheritable disease and congenital cardiac anomalies represent the most common non-iatrogenic cause of TAA among patients in this age group (1-21 years of age). Herein, we present a case series of 9aortic aneurysms with varying underlying etiology. We discuss the molecular genetic basis of these syndromes in conjunction with the radiological findings and histological description utilizing the newly published consensus criteria article. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. T2D-Db: An integrated platform to study the molecular basis of Type 2 diabetes

    Directory of Open Access Journals (Sweden)

    Sriram S

    2008-07-01

    Full Text Available Abstract Background Type 2 Diabetes Mellitus (T2DM is a non insulin dependent, complex trait disease that develops due to genetic predisposition and environmental factors. The advanced stage in type 2 diabetes mellitus leads to several micro and macro vascular complications like nephropathy, neuropathy, retinopathy, heart related problems etc. Studies performed on the genetics, biochemistry and molecular biology of this disease to understand the pathophysiology of type 2 diabetes mellitus has led to the generation of a surfeit of data on candidate genes and related aspects. The research is highly progressive towards defining the exact etiology of this disease. Results T2D-Db (Type 2 diabetes Database is a comprehensive web resource, which provides integrated and curated information on almost all known molecular components involved in the pathogenesis of type 2 diabetes mellitus in the three widely studied mammals namely human, mouse and rat. Information on candidate genes, SNPs (Single Nucleotide Polymorphism in candidate genes or candidate regions, genome wide association studies (GWA, tissue specific gene expression patterns, EST (Expressed Sequence Tag data, expression information from microarray data, pathways, protein-protein interactions and disease associated risk factors or complications have been structured in this on line resource. Conclusion Information available in T2D-Db provides an integrated platform for the better molecular level understanding of type 2 diabetes mellitus and its pathogenesis. Importantly, the resource facilitates graphical presentation of the gene/genome wide map of SNP markers and protein-protein interaction networks, besides providing the heat map diagram of the selected gene(s in an organism across microarray expression experiments from either single or multiple studies. These features aid to the data interpretation in an integrative way. T2D-Db is to our knowledge the first publicly available resource that

  16. Molecular basis for H3K36me3 recognition by the Tudor domain of PHF1

    OpenAIRE

    Musselman, Catherine A.; Avvakumov, Nikita; Watanabe, Reiko; Abraham, Christopher G.; Lalonde, Marie-Eve; Hong, Zehui; Allen, Christopher; Roy, Siddhartha; Nuñez, James K.; Nickoloff, Jac; Kulesza, Caroline A.; Yasui, Akira; Côté, Jacques; Kutateladze, Tatiana G.

    2012-01-01

    The PHD finger protein 1 (PHF1) is essential in epigenetic regulation and genome maintenance. Here, we demonstrate that the Tudor domain of human PHF1 binds to histone H3 trimethylated at Lys36 (H3K36me3). We report a 1.9 Å resolution crystal structure of the Tudor domain in complex with H3K36me3 and describe the molecular mechanism of H3K36me3 recognition using NMR analysis. Binding of PHF1 to H3K36me3 inhibits the ability of the Polycomb PRC2 complex to methylate H3K27 in vitro and in vivo....

  17. Green light emitting diodes accelerate wound healing: characterization of the effect and its molecular basis in vitro and in vivo.

    Science.gov (United States)

    Fushimi, Tomohiro; Inui, Shigeki; Nakajima, Takeshi; Ogasawara, Masahiro; Hosokawa, Ko; Itami, Satoshi

    2012-01-01

    Because light-emitting diodes (LEDs) are low-coherent, quasimonochromatic, and nonthermal, they are an alternative for low level laser therapy, and have photobiostimulative effects on tissue repair. However, the molecular mechanism(s) are unclear, and potential effects of blue and/or green LEDs on wound healing are still unknown. Here, we investigated the effects of red (638 nm), blue (456 nm), and green (518 nm) LEDs on wound healing. In an in vivo study, wound sizes in the skin of ob/ob mice were significantly decreased on day 7 following exposure to green LEDs, and complete reepithelialization was accelerated by red and green LEDs compared with the control mice. To better understand the molecular mechanism(s) involved, we investigated the effects of LEDs on human fibroblasts in vitro by measuring mRNA and protein levels of cytokines secreted by fibroblasts during the process of wound healing and on the migration of HaCat keratinocytes. The results suggest that some cytokines are significantly increased by exposure to LEDs, especially leptin, IL-8, and VEGF, but only by green LEDs. The migration of HaCat keratinocytes was significantly promoted by red or green LEDs. In conclusion, we demonstrate that green LEDs promote wound healing by inducing migratory and proliferative mediators, which suggests that not only red LEDs but also green LEDs can be a new powerful therapeutic strategy for wound healing. © 2012 by the Wound Healing Society.

  18. Molecular modeling study on the tubulin-binding modes of epothilone derivatives: Insight into the structural basis for epothilones activity.

    Science.gov (United States)

    Jiménez, Verónica A; Alderete, Joel B; Navarrete, Karen R

    2017-06-20

    Molecular dynamics (MD) simulations were employed to study the tubulin-binding modes of 20 epothilone derivatives spanning a wide range of antitumor activity. Trajectory analysis revealed that active ligands shared a common region of association and similar binding poses compared to the high-resolution crystal structure of the tubulin complex with epothilone A, the stathmin-like protein RB3, and tubulin tyrosine ligase (PDB code 4I50). Conformational analysis of epothilones in aqueous solution and tubulin-bound states indicated that the bound conformations of active species can be found to a significant extent within the ensemble of conformers available in aqueous solution. On the other hand, inactive derivatives were unable to adopt bound-like conformations in aqueous solution, thus requiring an extensive conformational pre-organization to accomplish an effective interaction with the tubulin receptor. Additionally, MD results revealed that epothilone binding-induced structuring of the M-loop and local flexibility changes in protein regions involved in interdimeric contacts that are relevant for microtubule stabilization. These results provide novel, valuable structural information to increase understanding about the underlying molecular aspects of epothilones activity and support further work on the search for new active tubulin-binding agents. © 2017 John Wiley & Sons A/S.

  19. Recent advances in the pathophysiology and molecular basis of sepsis-associated organ dysfunction: Novel therapeutic implications and challenges.

    Science.gov (United States)

    Hattori, Yuichi; Hattori, Kohshi; Suzuki, Tokiko; Matsuda, Naoyuki

    2017-09-01

    Sepsis is one of the most common reasons for critically ill patients to be admitted to an intensive care unit and, despite advances in overall medical care, it represents a major clinical problem and remains the leading cause of death in the critically ill patient population. Although sepsis has been defined as a systemic inflammatory syndrome, in which there is an identifiable focus of infection, clinical trials aimed at anti-inflammatory therapeutic approaches have largely failed to identify an effective therapeutic target to improve clinical outcomes in sepsis. Very recently, the third international consensus definitions have been advocated for sepsis and septic shock. Thus, sepsis is now defined as life-threatening organ dysfunction due to a dysregulated host response to infection. A better understanding of the molecular mechanisms involved in the pathogenesis of sepsis and its resultant organ failure has been sought, and the development of therapies targeted at preventing or limiting molecular events associated with the progress of fatal organ failure, hence leading to improvement of outcomes, is urgently needed. This review article provides an overview of possible pathogenic mechanisms underlying the development of multiple organ dysfunction in sepsis and discusses pharmacological agents regarded as promising in treatment of this disorder. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Molecular Basis for DNA Double-Strand Break Annealing and Primer Extension by an NHEJ DNA Polymerase

    Directory of Open Access Journals (Sweden)

    Nigel C. Brissett

    2013-11-01

    Full Text Available Nonhomologous end-joining (NHEJ is one of the major DNA double-strand break (DSB repair pathways. The mechanisms by which breaks are competently brought together and extended during NHEJ is poorly understood. As polymerases extend DNA in a 5′-3′ direction by nucleotide addition to a primer, it is unclear how NHEJ polymerases fill in break termini containing 3′ overhangs that lack a primer strand. Here, we describe, at the molecular level, how prokaryotic NHEJ polymerases configure a primer-template substrate by annealing the 3′ overhanging strands from opposing breaks, forming a gapped intermediate that can be extended in trans. We identify structural elements that facilitate docking of the 3′ ends in the active sites of adjacent polymerases and reveal how the termini act as primers for extension of the annealed break, thus explaining how such DSBs are extended in trans. This study clarifies how polymerases couple break-synapsis to catalysis, providing a molecular mechanism to explain how primer extension is achieved on DNA breaks.

  1. Crystal Structures of Glycosyltransferase UGT78G1 Reveal the Molecular Basis for Glycosylation and Deglycosylation of (Iso)flavonoids

    Energy Technology Data Exchange (ETDEWEB)

    Modolo, Luzia V.; Li, Lenong; Pan, Haiyun; Blount, Jack W.; Dixon, Richard A.; Wang, Xiaoqiang; (SRNF)

    2010-09-21

    The glycosyltransferase UGT78G1 from Medicago truncatula catalyzes the glycosylation of various (iso)flavonoids such as the flavonols kaempferol and myricetin, the isoflavone formononetin, and the anthocyanidins pelargonidin and cyanidin. It also catalyzes a reverse reaction to remove the sugar moiety from glycosides. The structures of UGT78G1 bound with uridine diphosphate or with both uridine diphosphate and myricetin were determined at 2.1 {angstrom} resolution, revealing detailed interactions between the enzyme and substrates/products and suggesting a distinct binding mode for the acceptor/product. Comparative structural analysis and mutagenesis identify glutamate 192 as a key amino acid for the reverse reaction. This information provides a basis for enzyme engineering to manipulate substrate specificity and to design effective biocatalysts with glycosylation and/or deglycosylation activity.

  2. Songbird: a unique animal model for studying the molecular basis of disorders of vocal development and communication.

    Science.gov (United States)

    Mori, Chihiro; Wada, Kazuhiro

    2015-01-01

    Like humans, songbirds are one of the few animal groups that learn vocalization. Vocal learning requires coordination of auditory input and vocal output using auditory feedback to guide one's own vocalizations during a specific developmental stage known as the critical period. Songbirds are good animal models for understand the neural basis of vocal learning, a complex form of imitation, because they have many parallels to humans with regard to the features of vocal behavior and neural circuits dedicated to vocal learning. In this review, we will summarize the behavioral, neural, and genetic traits of birdsong. We will also discuss how studies of birdsong can help us understand how the development of neural circuits for vocal learning and production is driven by sensory input (auditory information) and motor output (vocalization).

  3. Molecular basis for vulnerability to mitochondrial and oxidative stress in a neuroendocrine CRI-G1 cell line.

    Directory of Open Access Journals (Sweden)

    Natasha Chandiramani

    Full Text Available BACKGROUND: Many age-associated disorders (including diabetes, cancer, and neurodegenerative diseases are linked to mitochondrial dysfunction, which leads to impaired cellular bioenergetics and increased oxidative stress. However, it is not known what genetic and molecular pathways underlie differential vulnerability to mitochondrial dysfunction observed among different cell types. METHODOLOGY/PRINCIPAL FINDINGS: Starting with an insulinoma cell line as a model for a neuronal/endocrine cell type, we isolated a novel subclonal line (named CRI-G1-RS that was more susceptible to cell death induced by mitochondrial respiratory chain inhibitors than the parental CRI-G1 line (renamed CRI-G1-RR for clarity. Compared to parental RR cells, RS cells were also more vulnerable to direct oxidative stress, but equally vulnerable to mitochondrial uncoupling and less vulnerable to protein kinase inhibition-induced apoptosis. Thus, differential vulnerability to mitochondrial toxins between these two cell types likely reflects differences in their ability to handle metabolically generated reactive oxygen species rather than differences in ATP production/utilization or in downstream apoptotic machinery. Genome-wide gene expression analysis and follow-up biochemical studies revealed that, in this experimental system, increased vulnerability to mitochondrial and oxidative stress was associated with (1 inhibition of ARE/Nrf2/Keap1 antioxidant pathway; (2 decreased expression of antioxidant and phase I/II conjugation enzymes, most of which are Nrf2 transcriptional targets; (3 increased expression of molecular chaperones, many of which are also considered Nrf2 transcriptional targets; (4 increased expression of β cell-specific genes and transcription factors that specify/maintain β cell fate; and (5 reconstitution of glucose-stimulated insulin secretion. CONCLUSIONS/SIGNIFICANCE: The molecular profile presented here will enable identification of individual genes or

  4. DNA cloning of human liver monoamine oxidase A and B: Molecular basis of differences in enzymatic properties

    Energy Technology Data Exchange (ETDEWEB)

    Back, A.W.J.; Lan, N.C.; Johnson, D.L.; Abell, C.W.; Bembenek, M.E.; Kwan, S.W.; Seeburg, P.H.; Shih, J.C. (Univ. of Heidelberg (West Germany))

    1988-07-01

    The monoamine oxidases play a vital role in the metabolism of biogenic amines in the central nervous system and in peripheral tissues. Using oligonucleotide probes derived from three sequenced peptide fragments, the authors have isolated cDNA clones that encode the A and B forms of monoamine oxidase and have determined the nucleotide sequences of these cDNAs. Comparison of the deduced amino acid sequences shows that the A and B forms have subunit molecular weights of 59,700 and 58,800, respectively, and have 70% sequence identity. Both sequences contain the pentapeptide Ser-Gly-Gly-Cys-Tyr, in which the obligatory cofactor FAD is covalently bound to cysteine. Based on differences in primary amino acid sequences and RNA gel blot analysis of mRNAs, the A and B forms of monoamine oxidase appear to be derived from separate genes.

  5. The yellow fever 17D vaccine virus: molecular basis of viral attenuation and its use as an expression vector

    Directory of Open Access Journals (Sweden)

    Galler R.

    1997-01-01

    Full Text Available The yellow fever (YF virus is the prototype flavivirus. The use of molecular techniques has unraveled the basic mechanisms of viral genome structure and expression. Recent trends in flavivirus research include the use of infectious clone technology with which it is possible to recover virus from cloned cDNA. Using this technique, mutations can be introduced at any point of the viral genome and their resulting effect on virus phenotype can be assessed. This approach has opened new possibilities to study several biological viral features with special emphasis on the issue of virulence/attenuation of the YF virus. The feasibility of using YF virus 17D vaccine strain, for which infectious cDNA is available, as a vector for the expression of heterologous antigens is reviewed

  6. Molecular basis for TANK recognition by TRAF1 revealed by the crystal structure of TRAF1/TANK complex.

    Science.gov (United States)

    Kim, Chang Min; Jeong, Jae-Hee; Son, Young-Jin; Choi, Jun-Hyuk; Kim, Sunghwan; Park, Hyun Ho

    2017-02-02

    Tumor necrosis factor receptor-associated factor 1 (TRAF1) is a multifunctional adaptor protein involved in important processes of cellular signaling, including innate immunity and apoptosis. TRAF family member-associated NF-kappaB activator (TANK) has been identified as a competitive intracellular inhibitor of TRAF2 function. Although TRAF recognition by various receptors has been studied extensively in the field of TRAF-mediated biology, molecular and functional details of TANK recognition and interaction with TRAF1 have not been studied. In this study, we report the crystal structure of the TRAF1/TANK peptide complex. Quantitative interaction experiments showed that TANK peptide interacts with both TRAF1 and TRAF2 with similar affinity in a micromolar range. Our structural study also reveals that TANK binds TRAF1 using a minor minimal consensus motif for TRAF binding, Px(Q/E)xT.

  7. Simulation and modeling of synuclein-based 'protofibril' structures: as a means of understanding the molecular basis of Parkinson's disease

    Science.gov (United States)

    Tsigelny, I. F.; Sharikov, Y.; Miller, M. A.; Masliah, E.

    2008-07-01

    The protein α-synuclein (aS) is a 140 amino acid cytosolic protein of unknown function expressed abundantly in neurons and is associated with both environmentally induced and inherited forms of Parkinson's disease, the neurological disorder caused by progressive death of dopaminergic neurons in the substantia nigra. Here we demonstrate that aS aggregation leads to protofibril formation that accompanies aS binding to membranes. The specific surfaces of aS that bind to membranes are likely developed during the folding maturation process, where the 3D combinations of amino acids that are bound to these surfaces are very specific. Using molecular modeling and simulation technique, we elucidate the folding pathway of aS into protofibril structures and its configurations that bind to membranes. These membrane-binding surfaces can be targeted with pharmaceutical intervention for dissolution.

  8. Structural basis of recognition of pathogen-associated molecular patterns and inhibition of proinflammatory cytokines by camel peptidoglycan recognition protein.

    Science.gov (United States)

    Sharma, Pradeep; Dube, Divya; Singh, Amar; Mishra, Biswajit; Singh, Nagendra; Sinha, Mau; Dey, Sharmistha; Kaur, Punit; Mitra, Dipendra K; Sharma, Sujata; Singh, Tej P

    2011-05-06

    Peptidoglycan recognition proteins (PGRPs) are involved in the recognition of pathogen-associated molecular patterns. The well known pathogen-associated molecular patterns include LPS from Gram-negative bacteria and lipoteichoic acid (LTA) from Gram-positive bacteria. In this work, the crystal structures of two complexes of the short form of camel PGRP (CPGRP-S) with LPS and LTA determined at 1.7- and 2.1-Å resolutions, respectively, are reported. Both compounds were held firmly inside the complex formed with four CPGRP-S molecules designated A, B, C, and D. The binding cleft is located at the interface of molecules C and D, which is extendable to the interface of molecules A and C. The interface of molecules A and B is tightly packed, whereas that of molecules B and D forms a wide channel. The hydrophilic moieties of these compounds occupy a common region, whereas hydrophobic chains interact with distinct regions in the binding site. The binding studies showed that CPGRP-S binds to LPS and LTA with affinities of 1.6 × 10(-9) and 2.4 × 10(-8) M, respectively. The flow cytometric studies showed that both LPS- and LTA-induced expression of the proinflammatory cytokines TNF-α and IL-6 was inhibited by CPGRP-S. The results of animal studies using mouse models indicated that both LPS- and LTA-induced mortality rates decreased drastically when CPGRP-S was administered. The recognition of both LPS and LTA, their high binding affinities for CPGRP-S, the significant decrease in the production of LPS- and LTA-induced TNF-α and IL-6, and the drastic reduction in the mortality rates in mice by CPGRP-S indicate its useful properties as an antibiotic agent.

  9. Molecular Basis of Filtering Carbapenems by Porins from β-Lactam-resistant Clinical Strains of Escherichia coli*

    Science.gov (United States)

    Bajaj, Harsha; Scorciapino, Mariano A.; Moynié, Lucile; Page, Malcolm G. P.; Naismith, James H.; Ceccarelli, Matteo; Winterhalter, Mathias

    2016-01-01

    Integral membrane proteins known as porins are the major pathway by which hydrophilic antibiotics cross the outer membrane of Gram-negative bacteria. Single point mutations in porins can decrease the permeability of an antibiotic, either by reduction of channel size or modification of electrostatics in the channel, and thereby confer clinical resistance. Here, we investigate four mutant OmpC proteins from four different clinical isolates of Escherichia coli obtained sequentially from a single patient during a course of antimicrobial chemotherapy. OmpC porin from the first isolate (OmpC20) undergoes three consecutive and additive substitutions giving rise to OmpC26, OmpC28, and finally OmpC33. The permeability of two zwitterionic carbapenems, imipenem and meropenem, measured using liposome permeation assays and single channel electrophysiology differs significantly between OmpC20 and OmpC33. Molecular dynamic simulations show that the antibiotics must pass through the constriction zone of porins with a specific orientation, where the antibiotic dipole is aligned along the electric field inside the porin. We identify that changes in the vector of the electric field in the mutated porin, OmpC33, create an additional barrier by “trapping” the antibiotic in an unfavorable orientation in the constriction zone that suffers steric hindrance for the reorientation needed for its onward translocation. Identification and understanding the underlying molecular details of such a barrier to translocation will aid in the design of new antibiotics with improved permeation properties in Gram-negative bacteria. PMID:26645688

  10. Structural Basis of Recognition of Pathogen-associated Molecular Patterns and Inhibition of Proinflammatory Cytokines by Camel Peptidoglycan Recognition Protein*

    Science.gov (United States)

    Sharma, Pradeep; Dube, Divya; Singh, Amar; Mishra, Biswajit; Singh, Nagendra; Sinha, Mau; Dey, Sharmistha; Kaur, Punit; Mitra, Dipendra K.; Sharma, Sujata; Singh, Tej P.

    2011-01-01

    Peptidoglycan recognition proteins (PGRPs) are involved in the recognition of pathogen-associated molecular patterns. The well known pathogen-associated molecular patterns include LPS from Gram-negative bacteria and lipoteichoic acid (LTA) from Gram-positive bacteria. In this work, the crystal structures of two complexes of the short form of camel PGRP (CPGRP-S) with LPS and LTA determined at 1.7- and 2.1-Å resolutions, respectively, are reported. Both compounds were held firmly inside the complex formed with four CPGRP-S molecules designated A, B, C, and D. The binding cleft is located at the interface of molecules C and D, which is extendable to the interface of molecules A and C. The interface of molecules A and B is tightly packed, whereas that of molecules B and D forms a wide channel. The hydrophilic moieties of these compounds occupy a common region, whereas hydrophobic chains interact with distinct regions in the binding site. The binding studies showed that CPGRP-S binds to LPS and LTA with affinities of 1.6 × 10−9 and 2.4 × 10−8 m, respectively. The flow cytometric studies showed that both LPS- and LTA-induced expression of the proinflammatory cytokines TNF-α and IL-6 was inhibited by CPGRP-S. The results of animal studies using mouse models indicated that both LPS- and LTA-induced mortality rates decreased drastically when CPGRP-S was administered. The recognition of both LPS and LTA, their high binding affinities for CPGRP-S, the significant decrease in the production of LPS- and LTA-induced TNF-α and IL-6, and the drastic reduction in the mortality rates in mice by CPGRP-S indicate its useful properties as an antibiotic agent. PMID:21454594

  11. The Structure of Interleukin-23 Reveals in the Molecular Basis of P40 Subunit Sharing With Interleukin-12

    Energy Technology Data Exchange (ETDEWEB)

    Lupardus, P.J.; Garcia, K.C.

    2009-05-19

    Interleukin-23 is a recently identified member of the IL-12 family of heterodimeric cytokines that modulate subpopulations of T helper cells, and both IL-12 and IL-23 are attractive targets for therapy of autoimmune diseases. IL-23 is a binary complex of a four-helix bundle cytokine (p19) and a soluble class I cytokine receptor p40. IL-12 and IL-23 share p40 as an {alpha}-receptor subunit, yet show only 15% sequence homology between their four-helix cytokines p19 and p35, respectively, and signal through different combinations of shared receptors. In order to elucidate the structural basis of p40 sharing, we have determined a 2.3{angstrom} crystal structure of IL-23 for comparison to the previously determined structure of IL-12. The docking mode of p19 to p40 is altered compared to p35, deviating by a 'tilt' and 'roll' that results in an altered footprint of p40 on the A and D helices of the respective cytokines. Binding of p19 to p40 is mediated primarily by an Arginine residue on helix D of p19 that forms an extensive charge and hydrogen-bonding network with residues at the base of the pocket on p40. This 'Arginine pocket' is lined with an inner shell of hydrophobic interactions that are ringed by an outer shell of polar interactions. Comparative analysis indicates that the IL-23 and IL-12 complexes 'mimic' the network of interactions constituting the central Arginine pocket despite p19 and p35 having limited sequence homology. The majority of the structural epitopes in the two complexes are composed of unique p19 and p35 pair-wise contacts with common residues on p40. Thus, while the critical hotspot is maintained in the two complexes, the majority of the interfaces are structurally distinct and, therefore, provide a basis for the therapeutic targeting of IL-12 versus IL-23 heterodimer formation despite their use of a common receptor subunit.

  12. Molecular basis of the interaction for an essential subunit PA-PB1 in influenza virus RNA polymerase: insights from molecular dynamics simulation and free energy calculation.

    Science.gov (United States)

    Liu, Huanxiang; Yao, Xiaojun

    2010-02-01

    The emergence of the extremely aggressive influenza recently has highlighted the urgent need for new effective treatments. The influenza RNA-dependent RNA polymerase (RdRp) heterotrimer including PA, PB1 and PB2 has crucial roles in viral RNA replication and transcription. The highly conserved PB1 binding site on PA can be considered as a novel potential drug target site. The interaction between PB1 binding site and PA is crucial to many functions of the virus. In this study, to understand the detailed interaction profile and to characterize the binding hot spots in the interactions of the PA-PB1 complex, an 8 ns molecular dynamics simulation of the subunit PA-PB1 combined with MM-PBSA (molecular mechanics Poisson-Boltzmann surface area), MM-GBSA (molecular mechanics generalized Born surface area) computations and virtual alanine scanning were performed. The results from the free energy decomposition indicate that the intermolecular van der Waals interaction and the nonpolar solvation term provide the driving force for binding process. Through the pair interaction analysis and virtual alanine scanning, we identified the binding hot spots of PA and the basic binding motif of PB1. This information can provide some insights for the structure-based RNA-dependent RNA polymerase inhibitors design. The identified binding motif can be used as the starting point for the rational design of small molecules or peptide mimics. This study will also lead to new opportunities toward the development of new generation therapeutic agents exhibiting specificity and low resistance to influenza virus.

  13. Probing the molecular basis of substrate specificity, stereospecificity, and catalysis in the class II pyruvate aldolase, BphI.

    Science.gov (United States)

    Baker, Perrin; Carere, Jason; Seah, Stephen Y K

    2011-05-03

    BphI, a pyruvate-specific class II aldolase found in the polychlorinated biphenyls (PCBs) degradation pathway, catalyzes the reversible C-C bond cleavage of (4S)-hydroxy-2-oxoacids to form pyruvate and an aldehyde. Mutations were introduced into bphI to probe the contribution of active site residues to substrate recognition and catalysis. In contrast to the wild-type enzyme that has similar specificities for acetaldehyde and propionaldehyde, the L87A variant exhibited a 40-fold preference for propionaldehyde over acetaldehyde. The specificity constant of the L89A variant in the aldol addition reaction using pentaldehyde is increased ∼50-fold, making it more catalytically efficient for pentaldehyde utilization compared to the wild-type utilization of the natural substrate, acetaldehyde. Replacement of Tyr-290 with phenylalanine or serine resulted in a loss of stereochemical control as the variants were able to utilize substrates with both R and S configurations at C4 with similar kinetic parameters. Aldol cleavage and pyruvate α-proton exchange activity were undetectable in the R16A variant, supporting the role of Arg-16 in stabilizing a pyruvate enolate intermediate. The pH dependence of the enzyme is consistent with a single deprotonation by a catalytic base with pK(a) values of approximately 7. In H20A and H20S variants, pH profiles show the dependence of enzyme activity on hydroxide concentration. On the basis of these results, a catalytic mechanism is proposed.

  14. Molecular Dynamic Simulation to Explore the Molecular Basis of Btk-PH Domain Interaction with Ins(1,3,4,5P4

    Directory of Open Access Journals (Sweden)

    Dan Lu

    2013-01-01

    Full Text Available Bruton’s tyrosine kinase contains a pleckstrin homology domain, and it specifically binds inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5P4, which is involved in the maturation of B cells. In this paper, we studied 12 systems including the wild type and 11 mutants, K12R, S14F, K19E, R28C/H, E41K, L11P, F25S, Y40N, and K12R-R28C/H, to investigate any change in the ligand binding site of each mutant. Molecular dynamics simulations combined with the method of molecular mechanics/Poisson-Boltzmann solvent-accessible surface area have been applied to the twelve systems, and reasonable mutant structures and their binding free energies have been obtained as criteria in the final classification. As a result, five structures, K12R, K19E, R28C/H, and E41K mutants, were classified as “functional mutations,” whereas L11P, S14F, F25S, and Y40N were grouped into “folding mutations.” This rigorous study of the binding affinity of each of the mutants and their classification provides some new insights into the biological function of the Btk-PH domain and related mutation-causing diseases.

  15. Mixed-culture transcriptome analysis reveals the molecular basis of mixed-culture growth in Streptococcus thermophilus and Lactobacillus bulgaricus.

    Science.gov (United States)

    Sieuwerts, Sander; Molenaar, Douwe; van Hijum, Sacha A F T; Beerthuyzen, Marke; Stevens, Marc J A; Janssen, Patrick W M; Ingham, Colin J; de Bok, Frank A M; de Vos, Willem M; van Hylckama Vlieg, Johan E T

    2010-12-01

    Many food fermentations are performed using mixed cultures of lactic acid bacteria. Interactions between strains are of key importance for the performance of these fermentations. Yogurt fermentation by Streptococcus thermophilus and Lactobacillus bulgaricus (basonym, Lactobacillus delbrueckii subsp. bulgaricus) is one of the best-described mixed-culture fermentations. These species are believed to stimulate each other's growth by the exchange of metabolites such as folic acid and carbon dioxide. Recently, postgenomic studies revealed that an upregulation of biosynthesis pathways for nucleotides and sulfur-containing amino acids is part of the global physiological response to mixed-culture growth in S. thermophilus, but an in-depth molecular analysis of mixed-culture growth of both strains remains to be established. We report here the application of mixed-culture transcriptome profiling and a systematic analysis of the effect of interaction-related compounds on growth, which allowed us to unravel the molecular responses associated with batch mixed-culture growth in milk of S. thermophilus CNRZ1066 and L. bulgaricus ATCC BAA-365. The results indicate that interactions between these bacteria are primarily related to purine, amino acid, and long-chain fatty acid metabolism. The results support a model in which formic acid, folic acid, and fatty acids are provided by S. thermophilus. Proteolysis by L. bulgaricus supplies both strains with amino acids but is insufficient to meet the biosynthetic demands for sulfur and branched-chain amino acids, as becomes clear from the upregulation of genes associated with these amino acids in mixed culture. Moreover, genes involved in iron uptake in S. thermophilus are affected by mixed-culture growth, and genes coding for exopolysaccharide production were upregulated in both organisms in mixed culture compared to monocultures. The confirmation of previously identified responses in S. thermophilus using a different strain combination

  16. Molecular deciphering of the ABO system as a basis for novel diagnostics and therapeutics in ABO incompatible transplantation.

    Science.gov (United States)

    Holgersson, Jan; Rydberg, Lennart; Breimer, Michael E

    2014-01-01

    In recent years ABO incompatible kidney transplantation (KTx) has become a more or less clinical routine procedure with graft and patient survival similar to those of ABO compatible transplants. Antigen-specific immunoadsorption (IA) for anti-A and anti-B antibody removal constitutes in many centers an important part of the treatment protocol. ABO antibody titration by hemagglutination is guiding the treatment; both if the recipient can be transplanted as well as in cases of suspected rejections if antibody removal should be performed. Despite the overall success of ABO incompatible KTx, there is still room for improvements and an extension of the technology to include other solid organs. Based on an increased understanding of the structural complexity and tissue distribution of ABH antigens and the fine epitope specificity of the ABO antibody repertoire, improved IA matrices and ABO antibody diagnostics should be developed. Furthermore, understanding the molecular mechanisms behind accommodation of ABO incompatible renal allografts could make it possible to induce long-term allograft acceptance also in human leukocyte antigen (HLA) sensitized recipients and, perhaps, also make clinical xenotransplantation possible.

  17. Molecular basis of fatty acid selectivity in the zDHHC family of S-acyltransferases revealed by click chemistry

    Science.gov (United States)

    Greaves, Jennifer; Munro, Kevin R.; Davidson, Stuart C.; Riviere, Matthieu; Wojno, Justyna; Smith, Terry K.; Tomkinson, Nicholas C. O.; Chamberlain, Luke H.

    2017-01-01

    S-acylation is a major posttranslational modification, catalyzed by the zinc finger DHHC domain containing (zDHHC) enzyme family. S-acylated proteins can be modified by different fatty acids; however, very little is known about how zDHHC enzymes contribute to acyl chain heterogeneity. Here, we used fatty acid-azide/alkyne labeling of mammalian cells, showing their transformation into acyl-CoAs and subsequent click chemistry-based detection, to demonstrate that zDHHC enzymes have marked differences in their fatty acid selectivity. This difference in selectivity was apparent even for highly related enzymes, such as zDHHC3 and zDHHC7, which displayed a marked difference in their ability to use C18:0 acyl-CoA as a substrate. Furthermore, we identified isoleucine-182 in transmembrane domain 3 of zDHHC3 as a key determinant in limiting the use of longer chain acyl-CoAs by this enzyme. This study uncovered differences in the fatty acid selectivity profiles of cellular zDHHC enzymes and mapped molecular determinants governing this selectivity. PMID:28167757

  18. Molecular basis for the effects of zinc deficiency on spermatogenesis: An experimental study in the Sprague-dawley rat model

    Directory of Open Access Journals (Sweden)

    Alexander E Omu

    2015-01-01

    Full Text Available Introduction: The objective of this study is to investigate the molecular mechanisms underlying the effects of zinc deficiency on spermatogenesis in the Sprague-Dawley (SD rat. Materials and Methods: Three groups of eight adult male SD rats were maintained for 4 weeks on a normal diet as control, zinc deficient diet and zinc deficient diet with zinc supplementation of 28 mg zinc/kg body weight respectively. Using standard techniques, the following parameters were compared between the three groups of experimental animals at the end of 4 weeks: (a Serum zinc, magnesium (Mg, copper (Cu, selenium (Se and cadmium (Cd, (b serum sex hormones, malondialdehyde (MDA, superoxide dismutase (SOD and glutathione peroxidase (GPX, (c interleukin-4 (IL-4, tumor necrosis factor-alpha (TNF-α, Bcl-2, Bax and caspase-3 expression in the testes, (d assessment of apoptosis of testicular cells using electron microscopy and (e testicular volume and histology using the orchidometer and Johnsen score, respectively. Results: The zinc deficient group showed a reduction of testicular volume, serum concentrations of Zn, Cu, Se, Mg, SOD, GPX, IL-4, Bcl-2 and testosterone (P < 0.05, as well as increased levels of serum Cd, MDA and tissue TNF-α, Bax, caspase-3 and apoptosis of the germ cells (P < 0.05 compared with control and zinc supplementation groups. Conclusion: Zinc deficiency is associated with impaired spermatogenesis because of reduced testosterone production, increased oxidative stress and apoptosis. These findings suggest that zinc has a role in male reproduction.

  19. Exploring the Molecular Basis for Selective Binding of Homoserine Dehydrogenase from Mycobacterium leprae TN toward Inhibitors: A Virtual Screening Study

    Science.gov (United States)

    Zhan, Dongling; Wang, Dongmei; Min, Weihong; Han, Weiwei

    2014-01-01

    Homoserine dehydrogenase (HSD) from Mycobacterium leprae TN is an antifungal target for antifungal properties including efficacy against the human pathogen. The 3D structure of HSD has been firmly established by homology modeling methods. Using the template, homoserine dehydrogenase from Thiobacillus denitrificans (PDB Id 3MTJ), a sequence identity of 40% was found and molecular dynamics simulation was used to optimize a reliable structure. The substrate and co-factor-binding regions in HSD were identified. In order to determine the important residues of the substrate (l-aspartate semialdehyde (l-ASA)) binding, the ASA was docked to the protein; Thr163, Asp198, and Glu192 may be important because they form a hydrogen bond with HSD through AutoDock 4.2 software. After use of a virtual screening technique of HSD, the four top-scoring docking hits all seemed to cation–π ion pair with the key recognition residue Lys107, and Lys207. These ligands therefore seemed to be new chemotypes for HSD. Our results may be helpful for further experimental investigations. PMID:24469317

  20. Exploring the Molecular Basis for Selective Binding of Homoserine Dehydrogenase from Mycobacterium leprae TN toward Inhibitors: A Virtual Screening Study

    Directory of Open Access Journals (Sweden)

    Dongling Zhan

    2014-01-01

    Full Text Available Homoserine dehydrogenase (HSD from Mycobacterium leprae TN is an antifungal target for antifungal properties including efficacy against the human pathogen. The 3D structure of HSD has been firmly established by homology modeling methods. Using the template, homoserine dehydrogenase from Thiobacillus denitrificans (PDB Id 3MTJ, a sequence identity of 40% was found and molecular dynamics simulation was used to optimize a reliable structure. The substrate and co-factor-binding regions in HSD were identified. In order to determine the important residues of the substrate (l-aspartate semialdehyde (l-ASA binding, the ASA was docked to the protein; Thr163, Asp198, and Glu192 may be important because they form a hydrogen bond with HSD through AutoDock 4.2 software. neuraminidaseAfter use of a virtual screening technique of HSD, the four top-scoring docking hits all seemed to cation–π ion pair with the key recognition residue Lys107, and Lys207. These ligands therefore seemed to be new chemotypes for HSD. Our results may be helpful for further experimental investigations.

  1. The Risk of Some Veterinary Antimicrobial Agents on Public Health Associated with Antimicrobial Resistance and their Molecular Basis

    Science.gov (United States)

    Hao, Haihong; Sander, Pascal; Iqbal, Zahid; Wang, Yulian; Cheng, Guyue; Yuan, Zonghui

    2016-01-01

    The risk of antimicrobial agents used in food-producing animals on public health associated with antimicrobial resistance continues to be a current topic of discussion as related to animal and human public health. In the present review, resistance monitoring data, and risk assessment results of some important antimicrobial agents were cited to elucidate the possible association of antimicrobial use in food animals and antimicrobial resistance in humans. From the selected examples, it was apparent from reviewing the published scientific literature that the ban on use of some antimicrobial agents (e.g., avoparcin, fluoroquinolone, tetracyclines) did not change drug resistance patterns and did not mitigate the intended goal of minimizing antimicrobial resistance. The use of some antimicrobial agents (e.g., virginiamycin, macrolides, and cephalosporins) in food animals may have an impact on the antimicrobial resistance in humans, but it was largely depended on the pattern of drug usage in different geographical regions. The epidemiological characteristics of resistant bacteria were closely related to molecular mechanisms involved in the development, fitness, and transmission of antimicrobial resistance. PMID:27803693

  2. The challenges involved in elucidating the molecular basis of sperm-egg recognition in mammals and approaches to overcome them.

    Science.gov (United States)

    Wright, Gavin J; Bianchi, Enrica

    2016-01-01

    Sexual reproduction is used by many different organisms to create a new generation of genetically distinct progeny. Cells originating from separate sexes or mating types segregate their genetic material into haploid gametes which must then recognize and fuse with each other in a process known as fertilization to form a diploid zygote. Despite the central importance of fertilization, we know remarkably little about the molecular mechanisms that are involved in how gametes recognize each other, particularly in mammals, although the proteins that are displayed on their surfaces are almost certainly involved. This paucity of knowledge is largely due to both the unique biological properties of mammalian gametes (sperm and egg) which make them experimentally difficult to manipulate, and the technical challenges of identifying interactions between membrane-embedded cell surface receptor proteins. In this review, we will discuss our current knowledge of animal gamete recognition, highlighting where important contributions to our understanding were made, why particular model systems were helpful, and why progress in mammals has been particularly challenging. We discuss how the development of mammalian in vitro fertilization and targeted gene disruption in mice were important technological advances that triggered progress. We argue that approaches employed to discover novel interactions between cell surface gamete recognition proteins should account for the unusual biochemical properties of membrane proteins and the typically highly transient nature of their interactions. Finally, we describe how these principles were applied to identify Juno as the egg receptor for sperm Izumo1, an interaction that is essential for mammalian fertilization.

  3. Molecular basis of the anti-inflammatory property exhibited by cyclo-pentano phenanthrenol isolated from Lippia nodiflora.

    Science.gov (United States)

    Balakrishnan, Gayathri; Janakarajan, Lakshmi; Balakrishnan, Arun; Lakshmi, Baddireddi Subhadra

    2010-01-01

    The objective of this study was to assess the anti-inflammatory potential of the active molecule isolated from Lippia nodiflora and to understand its molecular dynamics in Vitro inflammation models. Human Peripheral Blood Mononuclear Cells were used as models to study mitogen induced lymphocyte proliferation, cytokine mRNA expression (TNF-α, IL-1β and IL-6) and intracellular protein levels of pro-inflammatory mediators (MAPK and NF-κB). The NO release levels, on treatment with the extract and molecule, were correlated with the underlying iNOS mRNA expression in the murine macrophage cell line RAW 264.7. RT-PCR for COX-2, MMP2 and MMP9 were also performed in the cell line. The rat basophilic leukemia cell line RBL-2H3 was used as an in Vitro model for PLA2 activity. Then, 20 μg/ml of Lippia nodiflora crude methanol extract and 10 μg/ml of the purified CPP were used for subsequent studies based on the IC50 values obtained in the proliferation assay. Results demonstrate that the isolated Cyclo-pentano phenanthrenol inhibits TNF-α, IL-1β and IL-6 expression, NO release via iNOS suppression, prostaglandin biosynthesis via PLA2 and COX-2 inhibition and the activation of intracellular targets, MAPK and NF-κB. We conclude, cyclo-pentano phenanthrenol exerts its anti-inflammatory effect via inhibition of MAPK phosphorylation and NF-κB translocation.

  4. Exploring the molecular basis for selective binding of homoserine dehydrogenase from Mycobacterium leprae TN toward inhibitors: a virtual screening study.

    Science.gov (United States)

    Zhan, Dongling; Wang, Dongmei; Min, Weihong; Han, Weiwei

    2014-01-24

    Homoserine dehydrogenase (HSD) from Mycobacterium leprae TN is an antifungal target for antifungal properties including efficacy against the human pathogen. The 3D structure of HSD has been firmly established by homology modeling methods. Using the template, homoserine dehydrogenase from Thiobacillus denitrificans (PDB Id 3MTJ), a sequence identity of 40% was found and molecular dynamics simulation was used to optimize a reliable structure. The substrate and co-factor-binding regions in HSD were identified. In order to determine the important residues of the substrate (L-aspartate semialdehyde (L-ASA)) binding, the ASA was docked to the protein; Thr163, Asp198, and Glu192 may be important because they form a hydrogen bond with HSD through AutoDock 4.2 software. neuraminidaseAfter use of a virtual screening technique of HSD, the four top-scoring docking hits all seemed to cation-π ion pair with the key recognition residue Lys107, and Lys207. These ligands therefore seemed to be new chemotypes for HSD. Our results may be helpful for further experimental investigations.

  5. Molecular basis of multiple resistance to ACCase- and ALS-inhibiting herbicides in Alopecurus japonicus from China.

    Science.gov (United States)

    Bi, Yaling; Liu, Weitang; Guo, Wenlei; Li, Lingxu; Yuan, Guohui; Du, Long; Wang, Jinxin

    2016-01-01

    Fenoxaprop-P-ethyl-resistant Alopecurus japonicus has become a recurring problem in winter wheat fields in eastern China. Growers have resorted to using mesosulfuron-methyl, an acetolactate synthase (ALS)-inhibiting herbicide, to control this weed. A single A. japonicus population (AH-15) resistant to fenoxaprop-P-ethyl and mesosulfuron-methyl was found in Anhui Province, China. The results of whole-plant dose-response experiments showed that AH-15 has evolved high-level resistance to fenoxaprop-P-ethyl (95.96-fold) and mesosulfuron-methyl (39.87-fold). It was shown via molecular analysis that resistance to both fenoxaprop-P-ethyl and mesosulfuron-methyl was due to an amino acid substitution of Ile1781 to Leu in acetyl-CoA carboxylase (ACCase) and a substitution of Trp 574 to Leu in ALS, respectively. Whole-plant bioassays indicated that the AH-15 population was resistant to the ACCase herbicides clodinafop-propargyl, clethodim, sethoxydim and pinoxaden as well as the ALS herbicides pyroxsulam, flucarbazone-Na and imazethapyr, but susceptible to the ACCase herbicide haloxyfop-R-methyl. This work reports for the first time that A. japonicus has developed resistance to ACCase- and ALS-inhibiting herbicides due to target site mutations in the ACCase and ALS genes.

  6. Understanding the molecular basis of plant growth promotional effect of Pseudomonas fluorescens on rice through protein profiling

    Directory of Open Access Journals (Sweden)

    Thiruvengadam Raguchander

    2009-12-01

    Full Text Available Abstract Background Plant Growth Promoting Rhizobacteria (PGPR, Pseudomonas fluorescens strain KH-1 was found to exhibit plant growth promotional activity in rice under both in-vitro and in-vivo conditions. But the mechanism underlying such promotional activity of P. fluorescens is not yet understood clearly. In this study, efforts were made to elucidate the molecular responses of rice plants to P. fluorescens treatment through protein profiling. Two-dimensional polyacrylamide gel electrophoresis strategy was adopted to identify the PGPR responsive proteins and the differentially expressed proteins were analyzed by mass spectrometry. Results Priming of P. fluorescens, 23 different proteins found to be differentially expressed in rice leaf sheaths and MS analysis revealed the differential expression of some important proteins namely putative p23 co-chaperone, Thioredoxin h- rice, Ribulose-bisphosphate carboxylase large chain precursor, Nucleotide diPhosphate kinase, Proteosome sub unit protein and putative glutathione S-transferase protein. Conclusion Functional analyses of the differential proteins were reported to be directly or indirectly involved in growth promotion in plants. Thus, this study confirms the primary role of PGPR strain KH-1 in rice plant growth promotion.

  7. The molecular basis of ubiquitin-like protein NEDD8 deamidation by the bacterial effector protein Cif.

    Science.gov (United States)

    Crow, Allister; Hughes, Richard K; Taieb, Frédéric; Oswald, Eric; Banfield, Mark J

    2012-07-03

    The cycle inhibiting factors (Cifs) are a family of translocated effector proteins, found in diverse pathogenic bacteria, that interfere with the host cell cycle by catalyzing the deamidation of a specific glutamine residue (Gln40) in NEDD8 and the related protein ubiquitin. This modification prevents recycling of neddylated cullin-RING ligases, leading to stabilization of various cullin-RING ligase targets, and also prevents polyubiquitin chain formation. Here, we report the crystal structures of two Cif/NEDD8 complexes, revealing a conserved molecular interface that defines enzyme/substrate recognition. Mutation of residues forming the interface suggests that shape complementarity, rather than specific individual interactions, is a critical feature for complex formation. We show that Cifs from diverse bacteria bind NEDD8 in vitro and conclude that they will all interact with their substrates in the same way. The "occluding loop" in Cif gates access to Gln40 by forcing a conformational change in the C terminus of NEDD8. We used native PAGE to follow the activity of Cif from the human pathogen Yersinia pseudotuberculosis and selected variants, and the position of Gln40 in the active site has allowed us to propose a catalytic mechanism for these enzymes.

  8. Molecular basis of photoperiodic control of reproductive cycle in a subtropical songbird, the Indian weaver bird (Ploceus philippinus).

    Science.gov (United States)

    Srivastava, Amrita; Trivedi, Neerja; Malik, Shalie; Rani, Sangeeta; Kumar, Vinod

    2015-09-01

    Less is known about genetic basis of photoperiodic regulation of reproductive cycle in subtropical birds. This study measured the expression levels of DIO2, DIO3, GnRH, and GnIH genes in Indian weaver birds subjected to short days (8h light:16h darkness, 8L:16D) and long days (16L:8D) for 48weeks. Whereas small, reproductively inactive testes were maintained under short days, weaver birds underwent testis recrudescence - regression cycle under long days. Relative expression levels of DIO2, DIO3, GnRH and GnIH genes were quantified by the real-time PCR (qPCR) in hypothalamus of birds (n=4) sampled at the beginning of the experiments, and after 10 and 48weeks of short and long day exposures. These sample times represented photosensitive unstimulated (day 0), and under long days the recrudescence (photostimulated, after 10weeks) and regression (photorefractory, after 48weeks) testicular phases. Birds under short days served as controls. The expression pattern of these genes corresponded with testicular phases. High and low GnRH and DIO2 levels were found in birds with large and small testes, respectively. By-and-large the converse was true for GnIH and DIO3 expression levels. Thus, after 10weeks of exposure, there was a significant difference in the mRNA levels between short and long day birds, with small and large testes, respectively. The results also suggest for a possible rapid switching between DIO2 and DIO3 and GnRH and GnIH expressions during testis maturation - regression cycle in Indian weaver birds.

  9. Structure of the Hemoglobin-IsdH Complex Reveals the Molecular Basis of Iron Capture by Staphylococcus aureus*♦

    Science.gov (United States)

    Dickson, Claire F.; Kumar, Kaavya Krishna; Jacques, David A.; Malmirchegini, G. Reza; Spirig, Thomas; Mackay, Joel P.; Clubb, Robert T.; Guss, J. Mitchell; Gell, David A.

    2014-01-01

    Staphylococcus aureus causes life-threatening disease in humans. The S. aureus surface protein iron-regulated surface determinant H (IsdH) binds to mammalian hemoglobin (Hb) and extracts heme as a source of iron, which is an essential nutrient for the bacteria. However, the process of heme transfer from Hb is poorly understood. We have determined the structure of IsdH bound to human Hb by x-ray crystallography at 4.2 Å resolution, revealing the structural basis for heme transfer. One IsdH molecule is bound to each α and β Hb subunit, suggesting that the receptor acquires iron from both chains by a similar mechanism. Remarkably, two near iron transporter (NEAT) domains in IsdH perform very different functions. An N-terminal NEAT domain binds α/β globin through a site distant from the globin heme pocket and, via an intervening structural domain, positions the C-terminal heme-binding NEAT domain perfectly for heme transfer. These data, together with a 2.3 Å resolution crystal structure of the isolated N-terminal domain bound to Hb and small-angle x-ray scattering of free IsdH, reveal how multiple domains of IsdH cooperate to strip heme from Hb. Many bacterial pathogens obtain iron from human hemoglobin using proteins that contain multiple NEAT domains and other domains whose functions are poorly understood. Our results suggest that, rather than acting as isolated units, NEAT domains may be integrated into higher order architectures that employ multiple interaction interfaces to efficiently extract heme from host proteins. PMID:24425866

  10. Structure of the hemoglobin-IsdH complex reveals the molecular basis of iron capture by Staphylococcus aureus.

    Science.gov (United States)

    Dickson, Claire F; Kumar, Kaavya Krishna; Jacques, David A; Malmirchegini, G Reza; Spirig, Thomas; Mackay, Joel P; Clubb, Robert T; Guss, J Mitchell; Gell, David A

    2014-03-01

    Staphylococcus aureus causes life-threatening disease in humans. The S. aureus surface protein iron-regulated surface determinant H (IsdH) binds to mammalian hemoglobin (Hb) and extracts heme as a source of iron, which is an essential nutrient for the bacteria. However, the process of heme transfer from Hb is poorly understood. We have determined the structure of IsdH bound to human Hb by x-ray crystallography at 4.2 Å resolution, revealing the structural basis for heme transfer. One IsdH molecule is bound to each α and β Hb subunit, suggesting that the receptor acquires iron from both chains by a similar mechanism. Remarkably, two near iron transporter (NEAT) domains in IsdH perform very different functions. An N-terminal NEAT domain binds α/β globin through a site distant from the globin heme pocket and, via an intervening structural domain, positions the C-terminal heme-binding NEAT domain perfectly for heme transfer. These data, together with a 2.3 Å resolution crystal structure of the isolated N-terminal domain bound to Hb and small-angle x-ray scattering of free IsdH, reveal how multiple domains of IsdH cooperate to strip heme from Hb. Many bacterial pathogens obtain iron from human hemoglobin using proteins that contain multiple NEAT domains and other domains whose functions are poorly understood. Our results suggest that, rather than acting as isolated units, NEAT domains may be integrated into higher order architectures that employ multiple interaction interfaces to efficiently extract heme from host proteins.

  11. The Hawaiian bobtail squid (Euprymna scolopes): a model to study the molecular basis of eukaryote-prokaryote mutualism and the development and evolution of morphological novelties in cephalopods.

    Science.gov (United States)

    Lee, Patricia N; McFall-Ngai, Margaret J; Callaerts, Patrick; de Couet, H Gert

    2009-11-01

    The Hawaiian bobtail squid, Euprymna scolopes, is a cephalopod whose small size, short lifespan, rapid growth, and year-round availability make it suitable as a model organism. E. scolopes is studied in three principal contexts: (1) as a model of cephalopod development; (2) as a model of animal-bacterial symbioses; and (3) as a system for studying adaptations of tissues that interact with light. E. scolopes embryos can be obtained continually and can be reared in the laboratory over an entire generation. The embryos and protective chorions are optically clear, facilitating in situ developmental observations, and can be manipulated experimentally. Many molecular protocols have been developed for studying E. scolopes development. This species is best known, however, for its symbiosis with the luminous marine bacterium Vibrio fischeri and has been used to study determinants of symbiont specificity, the influence of symbiosis on development of the squid light organ, and the mechanisms by which a stable association is achieved. Both partners can be grown independently under laboratory conditions, a feature that offers the unusual opportunity to manipulate the symbiosis experimentally. Molecular and genetic tools have been developed for V. fischeri, and a large expressed sequence tag (EST) database is available for the host symbiotic tissues. Additionally, comparisons between light organ form and function to those of the eye can be made. Both types of tissue interact with light, but have divergent embryonic development. As such, they offer an opportunity to study the molecular basis for the evolution of morphological novelties.

  12. Bicontinuous Fluid Structure with Low Cohesive Energy: Molecular Basis for Exceptionally Low Interfacial Tension of Complex Coacervate Fluids.

    Science.gov (United States)

    Huang, Kuo-Ying; Yoo, Hee Young; Jho, YongSeok; Han, Songi; Hwang, Dong Soo

    2016-05-24

    An exceptionally low interfacial tension of a dense fluid of concentrated polyelectrolyte complexes, phase-separated from a biphasic fluid known as complex coacervates, represents a unique and highly sought-after materials property that inspires novel applications from superior coating to wet adhesion. Despite extensive studies and broad interest, the molecular and structural bases for the unique properties of complex coacervates are unclear. Here, a microphase-separated complex coacervate fluid generated by mixing a recombinant mussel foot protein-1 (mfp-1) as the polycation and hyaluronic acid (HA) as the polyanion at stoichiometric ratios was macroscopically phase-separated into a dense complex coacervate and a dilute supernatant phase to enable separate characterization of the two fluid phases. Surprisingly, despite up to 4 orders of magnitude differing density of the polyelectrolytes, the diffusivity of water in these two phases was found to be indistinguishable. The presence of unbound, bulk-like, water in the dense fluid can be reconciled with a water population that is only weakly perturbed by the polyelectrolyte interface and network. This hypothesis was experimentally validated by cryo-TEM of the macroscopically phase-separated dense complex coacervate phase that was found to be a bicontinuous and biphasic nanostructured network, in which one of the phases was confirmed by staining techniques to be water and the other polyelectrolyte complexes. We conclude that a weak cohesive energy between water-water and water-polyelectrolytes manifests itself in a bicontinuous network, and is responsible for the exceptionally low interfacial energy of this complex fluid phase with respect to virtually any surface within an aqueous medium.

  13. Molecular Basis of the Anti-Cancer Effects of Genistein Isoflavone in LNCaP Prostate Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Hartmann J

    2011-03-01

    Full Text Available Background: Prostate cancer is the most common form of non-skin cancer within the United States and the second leading cause of cancer deaths. Survival rates for the advanced disease remain relatively low, and conventional treatments may be accompanied by significant side effects. As a result, current research is aimed at alternative or adjuvant treatments that will target components of the signal transduction, cell-cycle and apoptosis pathways, to induce cell death with little or no toxic side effects to the patient. In this study, we investigated the effect of genistein isoflavone, a soy derivative, on expression levels of genes involved in these pathways. The mechanism of genistein-induced cell death was also investigated. The chemosensitivity of the LNCaP prostate cancer cells to genistein was investigated using ATP and MTS assays, and a caspase binding assay was used to determine apoptosis induction. Several molecular targets were determined using cDNA microarray and RT-PCR analysis.Results: The overall data revealed that genistein induces cell death in a time- and dose-dependent manner, and regulates expression levels of several genes involved in carcinogenesis and immunity. Several cell-cycle genes were down-regulated, including the mitotic kinesins, cyclins and cyclin-dependent kinases. Various members of the Bcl-2 family of apoptotic proteins were also affected. The DefB1 and the HLA membrane receptor genes involved in immunogenicity were also up-regulated.Conclusion: The results indicate that genistein inhibits growth of the hormone-dependent prostate cancer cells, LNCaP, via apoptosis induction through regulation of some of the genes involved in carcinogenesis of many tumors, and immunogenicity. This study augments the potential phytotherapeutic and immunotherapeutic significance of genistein isoflavone.

  14. Molecular Basis for the Selective Inhibition of Respiratory Syncytial Virus RNA Polymerase by 2'-Fluoro-4'-Chloromethyl-Cytidine Triphosphate.

    Directory of Open Access Journals (Sweden)

    Jerome Deval

    2015-06-01

    Full Text Available Respiratory syncytial virus (RSV causes severe lower respiratory tract infections, yet no vaccines or effective therapeutics are available. ALS-8176 is a first-in-class nucleoside analog prodrug effective in RSV-infected adult volunteers, and currently under evaluation in hospitalized infants. Here, we report the mechanism of inhibition and selectivity of ALS-8176 and its parent ALS-8112. ALS-8176 inhibited RSV replication in non-human primates, while ALS-8112 inhibited all strains of RSV in vitro and was specific for paramyxoviruses and rhabdoviruses. The antiviral effect of ALS-8112 was mediated by the intracellular formation of its 5'-triphosphate metabolite (ALS-8112-TP inhibiting the viral RNA polymerase. ALS-8112 selected for resistance-associated mutations within the region of the L gene of RSV encoding the RNA polymerase. In biochemical assays, ALS-8112-TP was efficiently recognized by the recombinant RSV polymerase complex, causing chain termination of RNA synthesis. ALS-8112-TP did not inhibit polymerases from host or viruses unrelated to RSV such as hepatitis C virus (HCV, whereas structurally related molecules displayed dual RSV/HCV inhibition. The combination of molecular modeling and enzymatic analysis showed that both the 2'F and the 4'ClCH2 groups contributed to the selectivity of ALS-8112-TP. The lack of antiviral effect of ALS-8112-TP against HCV polymerase was caused by Asn291 that is well-conserved within positive-strand RNA viruses. This represents the first comparative study employing recombinant RSV and HCV polymerases to define the selectivity of clinically relevant nucleotide analogs. Understanding nucleotide selectivity towards distant viral RNA polymerases could not only be used to repurpose existing drugs against new viral infections, but also to design novel molecules.

  15. Molecular basis of the mechanism of thiol oxidation by hydrogen peroxide in aqueous solution: challenging the SN2 paradigm.

    Science.gov (United States)

    Zeida, Ari; Babbush, Ryan; Lebrero, Mariano C González; Trujillo, Madia; Radi, Rafael; Estrin, Darío A

    2012-03-19

    The oxidation of cellular thiol-containing compounds, such as glutathione and protein Cys residues, is considered to play an important role in many biological processes. Among possible oxidants, hydrogen peroxide (H(2)O(2)) is known to be produced in many cell types as a response to a variety of extracellular stimuli and could work as an intracellular messenger. This reaction has been reported to proceed through a S(N)2 mechanism, but despite its importance, the reaction is not completely understood at the atomic level. In this work, we elucidate the reaction mechanism of thiol oxidation by H(2)O(2) for a model methanethiolate system using state of the art hybrid quantum-classical (QM-MM) molecular dynamics simulations. Our results show that the solvent plays a key role in positioning the reactants, that there is a significant charge redistribution in the first stages of the reaction, and that there is a hydrogen transfer process between H(2)O(2) oxygen atoms that occurs after reaching the transition state. These observations challenge the S(N)2 mechanism hypothesis for this reaction. Specifically, our results indicate that the reaction is driven by a tendency of the slightly charged peroxidatic oxygen to become even more negative in the product via an electrophilic attack on the negative sulfur atom. This is inconsistent with the S(N)2 mechanism, which predicts a protonated sulfenic acid and hydroxyl anion as stable intermediates. These intermediates are not found. Instead, the reaction proceeds directly to unprotonated sulfenic acid and water.

  16. The genetics of dementias. Part 1: Molecular basis of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17

    Directory of Open Access Journals (Sweden)

    Anna Kowalska

    2009-06-01

    gene have been identified, mainly in families with autosomal dominant FTDP-17 but also in sporadic families with Pick’s disease and AD. The known DNA changes have been classified according to their molecular effects into at least two groups: mutations that change the biochemical properties of tau protein and mutations that alter the alternative splicing of mRNA and lead very often to the overproduction of the 4R tau isoform. The imbalance in the ratio of the synthesized 3R and 4R tau isoforms stimulates protein aggregation and initiates neurodegeneration, leading to the development of dementia.

  17. Exploring the molecular basis for selective binding of Mycobacterium tuberculosis Asp kinase toward its natural substrates and feedback inhibitors: a docking and molecular dynamics study.

    Science.gov (United States)

    Chaitanya, M; Babajan, B; Anuradha, C M; Naveen, M; Rajasekhar, C; Madhusudana, P; Kumar, Chitta Suresh

    2010-08-01

    Tuberculosis (TB) is still a major public health problem, compounded by the human immunodeficiency virus (HIV)-TB co-infection and recent emergence of multidrug-resistant (MDR) and extensively drug resistant (XDR)-TB. In this context, aspartokinase of mycobacterium tuberculosis has drawn attention for designing novel anti-TB drugs. Asp kinase is an enzyme responsible for the synthesis of 4-phospho-L-aspartate from L-aspartate and involved in the branched biosynthetic pathway leading to the synthesis of amino acids lysine, threonine, methionine and isoleucine. An intermediate of lysine biosynthetic branch, mesodiaminopimelate is also a component of the peptidoglycan which is a component of bacterial cell wall. To interfere with the production of all these amino acids and cell wall, it is possible to inhibit Asp kinase activity. This can be achieved using Asp kinase inhibitors. In order to design novel Asp kinase inhibitors as effective anti-TB drugs, it is necessary to have an understanding of the binding sites of Asp kinase. As no crystal structure of the enzyme has yet been published, we built a homology model of Asp kinase using the crystallized Asp kinase from M. Jannaschii, as template structures (2HMF and 3C1M). After the molecular dynamics refinement, the optimized homology model was assessed as a reliable structure by PROCHECK, ERRAT, WHAT-IF, PROSA2003 and VERIFY-3D. The results of molecular docking studies with natural substrates, products and feedback inhibitors are in agreement with the published data and showed that ACT domain plays an important role in binding to ligands. Based on the docking conformations, pharmacophore model can be developed by probing the common features of ligands. By analyzing the results, ACT domain architecture, certain key residues that are responsible for binding to feedback inhibitors and natural substrates were identified. This would be very helpful in understanding the blockade mechanism of Asp kinase and providing insights

  18. Molecular Mechanism and Energy Basis of Conformational Diversity of Antibody SPE7 Revealed by Molecular Dynamics Simulation and Principal Component Analysis

    Science.gov (United States)

    Chen, Jianzhong; Wang, Jinan; Zhu, Weiliang

    2016-11-01

    More and more researchers are interested in and focused on how a limited repertoire of antibodies can bind and correspondingly protect against an almost limitless diversity of invading antigens. In this work, a series of 200-ns molecular dynamics (MD) simulations followed by principal component (PC) analysis and free energy calculations were performed to probe potential mechanism of conformational diversity of antibody SPE7. The results show that the motion direction of loops H3 and L3 is different relative to each other, implying that a big structural difference exists between these two loops. The calculated energy landscapes suggest that the changes in the backbone angles ψ and φ of H-Y101 and H-Y105 provide significant contributions to the conformational diversity of SPE7. The dihedral angle analyses based on MD trajectories show that the side-chain conformational changes of several key residues H-W33, H-Y105, L-Y34 and L-W93 around binding site of SPE7 play a key role in the conformational diversity of SPE7, which gives a reasonable explanation for potential mechanism of cross-reactivity of single antibody toward multiple antigens.

  19. Fundamento molecular de la esteatosis hepática asociada a la obesidad Molecular basis of obesity-related hepatic steatosis

    Directory of Open Access Journals (Sweden)

    X. Buqué

    2008-09-01

    Full Text Available La enfermedad del hígado graso no alcohólico es una enfermedad inflamatoria hepática de carácter crónico de gran relevancia en la actualidad por su fuerte asociación con enfermedades de incidencia creciente como la obesidad y la diabetes mellitus tipo 2. En este trabajo se recoge buena parte del conocimiento existente sobre los mecanismos moleculares implicados en el establecimiento de la esteatosis hepática, el primer estadio de la enfermedad, y en su progreso a esteatohepatitis. Se ha prestado una atención especial al hígado graso asociado a la obesidad, clínica y experimental. En este caso, se valora la rata fa/fa, un modelo animal de obesidad con rasgos fenotípicos similares a los de la obesidad humana, incluyendo la resistencia a la insulina y la dislipemia. La esteatosis hepática se revela como una situación compleja, eminentemente metabólica, en la que se simultanean procesos metabólicos aparentemente contradictorios, así como estrés oxidativo, estrés de retículo endoplasmático, disfunción mitocondrial y descenso en la expresión de genes de supervivencia. En buena medida, en su base se sitúan señales extrahepáticas, como las producidas en una situación de resistencia periférica a la insulina asociada a un aumento de la masa adiposa y de ácidos grasos libres sistémicos, e internas, causantes de un desajuste de las funciones glucostática y lipidostática del hígado y de una mayor vulnerabilidad a otras agresiones.Non-alcoholic fatty liver disease is a chronic inflammation liver condition that is currently highly relevant because of its strong association with increasingly incident diseases such as obesity and type-2 diabetes mellitus. The primary purpose of this paper is to discuss the best part of current knowledge on the molecular mechanisms involved in hepatic steatosis development, the condition's initial stage, and on progression to steatohepatitis. Special attention has been paid to clinical and

  20. Molecular Properties by Quantum Monte Carlo: An Investigation on the Role of the Wave Function Ansatz and the Basis Set in the Water Molecule.

    Science.gov (United States)

    Zen, Andrea; Luo, Ye; Sorella, Sandro; Guidoni, Leonardo

    2013-10-08

    Quantum Monte Carlo methods are accurate and promising many body techniques for electronic structure calculations which, in the last years, are encountering a growing interest thanks to their favorable scaling with the system size and their efficient parallelization, particularly suited for the modern high performance computing facilities. The ansatz of the wave function and its variational flexibility are crucial points for both the accurate description of molecular properties and the capabilities of the method to tackle large systems. In this paper, we extensively analyze, using different variational ansatzes, several properties of the water molecule, namely, the total energy, the dipole and quadrupole momenta, the ionization and atomization energies, the equilibrium configuration, and the harmonic and fundamental frequencies of vibration. The investigation mainly focuses on variational Monte Carlo calculations, although several lattice regularized diffusion Monte Carlo calculations are also reported. Through a systematic study, we provide a useful guide to the choice of the wave function, the pseudopotential, and the basis set for QMC calculations. We also introduce a new method for the computation of forces with finite variance on open systems and a new strategy for the definition of the atomic orbitals involved in the Jastrow-Antisymmetrised Geminal power wave function, in order to drastically reduce the number of variational parameters. This scheme significantly improves the efficiency of QMC energy minimization in case of large basis sets.

  1. Nucleotide sequence analysis of a DNA region involved in capsular polysaccharide biosynthesis reveals the molecular basis of the nontypeability of two Actinobacillus pleuropneumoniae isolates.

    Science.gov (United States)

    Ito, Hiroya; Ogawa, Torata; Fukamizu, Dai; Morinaga, Yuiko; Kusumoto, Masahiro

    2016-11-01

    The aim of our study was to reveal the molecular basis of the serologic nontypeability of 2 Actinobacillus pleuropneumoniae field isolates. Nine field strains of A. pleuropneumoniae, the causative agent of porcine pleuropneumonia, were isolated from pigs raised on the same farm and sent to our diagnostic laboratory for serotyping. Seven of the 9 strains were identified as serovar 15 strains by immunodiffusion tests. However, 2 strains, designated FH24-2 and FH24-5, could not be serotyped with antiserum prepared against serovars 1-15. Strain FH24-5 showed positive results in 2 serovar 15-specific PCR tests, whereas strain FH24-2 was only positive in 1 of the 2 PCR tests. The nucleotide sequence analysis of gene clusters involved in capsular polysaccharide biosynthesis of the 2 nontypeable strains revealed that both had been rendered nontypeable by the action of ISApl1, a transposable element of A. pleuropneumoniae belonging to the IS30 family. The results showed that ISApl1 of A. pleuropneumoniae can interfere with both the serologic and molecular typing methods, and that nucleotide sequence analysis across the capsular gene clusters is the best means of determining the cause of serologic nontypeability in A. pleuropneumoniae. © 2016 The Author(s).

  2. Elucidating the Molecular Basis and Regulation of Chromium (VI) Reduction by Shewanella oneidensis MR-1 Using Biochemical, Genomic, and Proteomic Approaches

    Energy Technology Data Exchange (ETDEWEB)

    Hettich, Robert L.

    2006-10-30

    Although microbial metal reduction has been investigated intensively from physiological and biochemical perspectives, little is known about the genetic basis and regulatory mechanisms underlying the ability of certain bacteria to transform, detoxify, or immobilize a wide array of heavy metals contaminating DOE-relevant environments. The major goal of this work is to elucidate the molecular components comprising the chromium(VI) response pathway, with an emphasis on components involved in Cr(VI) detoxification and the enzyme complex catalyzing the terminal step in Cr(VI) reduction by Shewanella oneidensis MR-1. We have identified and characterized (in the case of DNA-binding response regulator [SO2426] and a putative azoreductase [SO3585]) the genes and gene products involved in the molecular response of MR-1 to chromium(VI) stress using whole-genome sequence information for MR-1 and recently developed proteomic technology, in particular liquid chromatographymass spectrometry (LC-MS), in conjunction with conventional protein purification and characterization techniques. The proteome datasets were integrated with information from whole-genome expression arrays for S. oneidensis MR-1 (as illustrated in Figure 1). The genes and their encoded products identified in this study are of value in understanding metal reduction and bacterial resistance to metal toxicity and in developing effective metal immobilization strategies.

  3. A novel mutation in KVLQT1 is the molecular basis of inherited long QT syndrome in a near-drowning patient's family.

    Science.gov (United States)

    Ackerman, M J; Schroeder, J J; Berry, R; Schaid, D J; Porter, C J; Michels, V V; Thibodeau, S N

    1998-08-01

    After identifying a 10-year-old boy with inherited long QT syndrome (LQTS) after a near-drowning that required defibrillation from torsades de pointes, evaluation of first degree relatives revealed a four-generation kindred comprising 26 individuals with four additional symptomatic and eight asymptomatic members harboring an abnormally prolonged QTc (defined as > or =0.46 s1/2). We set out to determine the molecular basis of their LQTS. The inherited LQTS represents a collection of genetically distinct ion channelopathies with over 40 mutations in four fundamental cardiac ion channels identified. Molecular studies, including linkage analysis and identification of the disease-associated mutation, were performed on genomic DNA isolated from peripheral blood samples from 29 available family members. Genetic linkage analysis excluded the regions for LQT2, LQT3, and LQT5. However, the chromosome 11p15.5 region (LQT1) showed evidence of linkage with a maximum lod score of 3.36. Examination of the KVLQT1 gene revealed a novel 3-bp deletion resulting in an in-frame deltaF339 (phenylalanine) deletion in the proband. This deltaF339 mutation was confirmed in nine additional family members who shared both an assigned affected phenotype and the disease-associated linked haplotype. Importantly, three asymptomatic family members, with a tentative clinical diagnosis based on their QTc, did not have this mutation and could be reclassified as unaffected. It is noteworthy that the proband's ECG suggested the sodium channel-based LQT3 genotype. These findings show the potential importance of establishing a molecular diagnosis rather than initiating genotype-specific interventions based upon inspection of a patient's ECG.

  4. Molecular Basis of Essential Thrombocytosis

    Science.gov (United States)

    2008-06-01

    Beavis RC, Chait BT. Ma- trix-assisted laser desorption/ionization mass spectrometry of biopolymers . Anal Chem. 1991; 63:1193A-1203A. 30. Hillenkamp F...results from the Car - diovascular Health Study. Thromb Haemost. 1998;79:912-915. 81. Madonna P, de Stefano V, Coppola A, et al. Hy- perhomocysteinemia and

  5. Molecular Basis of Essential Thrombocytosis

    Science.gov (United States)

    2007-06-01

    et al. The Kozak sequence polymorphism of platelet glyco- protein Ib and risk of nonfatal myocardial infarc- tion and nonfatal stroke in young...John Gordon and Nicholas M. Barnes UNIVERSITY OF BIRMINGHAM MEDICAL SCHOOL Mouse T lymphocytes unexpectedly produce the classic neurotransmitter seroto...in immune regulation. In fact, this “ neurotransmitter ” is primarily a product of the periphery, with gut enterochromaffin cells the principal factories

  6. Molecular and structural basis of inner core lipopolysaccharide alterations in Escherichia coli: incorporation of glucuronic acid and phosphoethanolamine in the heptose region.

    Science.gov (United States)

    Klein, Gracjana; Müller-Loennies, Sven; Lindner, Buko; Kobylak, Natalia; Brade, Helmut; Raina, Satish

    2013-03-22

    It is well established that lipopolysaccharide (LPS) often carries nonstoichiometric substitutions in lipid A and in the inner core. In this work, the molecular basis of inner core alterations and their physiological significance are addressed. A new inner core modification of LPS is described, which arises due to the addition of glucuronic acid on the third heptose with a concomitant loss of phosphate on the second heptose. This was shown by chemical and structural analyses. Furthermore, the gene whose product is responsible for the addition of this sugar was identified in all Escherichia coli core types and in Salmonella and was designated waaH. Its deduced amino acid sequence exhibits homology to glycosyltransferase family 2. The transcription of the waaH gene is positively regulated by the PhoB/R two-component system in a growth phase-dependent manner, which is coordinated with the transcription of the ugd gene explaining the genetic basis of this modification. Glucuronic acid modification was observed in E. coli B, K12, R2, and R4 core types and in Salmonella. We also show that the phosphoethanolamine (P-EtN) addition on heptose I in E. coli K12 requires the product of the ORF yijP, a new gene designated as eptC. Incorporation of P-EtN is also positively regulated by PhoB/R, although it can occur at a basal level without a requirement for any regulatory inducible systems. This P-EtN modification is essential for resistance to a variety of factors, which destabilize the outer membrane like the addition of SDS or challenge to sublethal concentrations of Zn(2+).

  7. Perspectives of differentiation therapies of acute myeloid leukemia: the search for the molecular basis of patients’ variable responses to 1,25-dihydroxyvitamin D and vitamin D analogs.

    Directory of Open Access Journals (Sweden)

    Aleksandra eMarchwicka

    2014-05-01

    Full Text Available The concept of differentiation therapy of cancer is approximately 40 years old. Despite many encouraging results obtained in laboratories, both in vitro and in vivo studies, the only really successful clinical application of differentiation therapy was all-trans-retinoic acid (ATRA based therapy of acute promyelocytic leukemia (APL. ATRA, which induces granulocytic differentiation of APL leukemic blasts, has revolutionized the therapy of this disease by converting it from a fatal to a curable one. However, ATRA does not work for other acute myeloid leukemias (AMLs. Since 1,25-dihydroxyvitamin D3 (1,25D is capable of inducing monocytic differentiation of leukemic cells, the idea of treating other AMLs with vitamin D analogs (VDAs was widely accepted. Also some types of solid cancers responded to in vitro applied VDAs, and hence it was postulated that VDAs can be used in many clinical applications. However, early clinical trials in which cancer patients were treated either with 1,25D or with VDAs, did not lead to conclusive results. In order to search for a molecular basis of such unpredictable responses of AML patients towards VDAs, we performed ex vivo experiments using patient’s blast cells. Experiments were also performed using 1,25D-responsive and 1,25D-non responsive cell lines, to study their mechanisms of resistance towards 1,25D-induced differentiation. We found that one of the possible reasons might be due to a very low expression level of vitamin D receptor (VDR mRNA in resistant cells, which can be increased by exposing the cells to ATRA. Our considerations concerning the molecular mechanism behind the low VDR expression and its regulation by ATRA are reported in this paper.

  8. Insights from molecular dynamics simulations: structural basis for the V567D mutation-induced instability of zebrafish alpha-dystroglycan and comparison with the murine model.

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    Davide Pirolli

    Full Text Available A missense amino acid mutation of valine to aspartic acid in 567 position of alpha-dystroglycan (DG, identified in dag1-mutated zebrafish, results in a reduced transcription and a complete absence of the protein. Lacking experimental structural data for zebrafish DG domains, the detailed mechanism for the observed mutation-induced destabilization of the DG complex and membrane damage, remained unclear. With the aim to contribute to a better clarification of the structure-function relationships featuring the DG complex, three-dimensional structural models of wild-type and mutant (V567D C-terminal domain of alpha-DG from zebrafish were constructed by a template-based modelling approach. We then ran extensive molecular dynamics (MD simulations to reveal the structural and dynamic properties of the C-terminal domain and to evaluate the effect of the single mutation on alpha-DG stability. A comparative study has been also carried out on our previously generated model of murine alpha-DG C-terminal domain including the I591D mutation, which is topologically equivalent to the V567D mutation found in zebrafish. Trajectories from MD simulations were analyzed in detail, revealing extensive structural disorder involving multiple beta-strands in the mutated variant of the zebrafish protein whereas local effects have been detected in the murine protein. A biochemical analysis of the murine alpha-DG mutant I591D confirmed a pronounced instability of the protein. Taken together, the computational and biochemical analysis suggest that the V567D/I591D mutation, belonging to the G beta-strand, plays a key role in inducing a destabilization of the alpha-DG C-terminal Ig-like domain that could possibly affect and propagate to the entire DG complex. The structural features herein identified may be of crucial help to understand the molecular basis of primary dystroglycanopathies.

  9. Localized orbital corrections applied to thermochemical errors in density functional theory: The role of basis set and application to molecular reactions.

    Science.gov (United States)

    Goldfeld, Dahlia A; Bochevarov, Arteum D; Friesner, Richard A

    2008-12-07

    This paper is a logical continuation of the 22 parameter, localized orbital correction (LOC) methodology that we developed in previous papers [R. A. Friesner et al., J. Chem. Phys. 125, 124107 (2006); E. H. Knoll and R. A. Friesner, J. Phys. Chem. B 110, 18787 (2006).] This methodology allows one to redress systematic density functional theory (DFT) errors, rooted in DFT's inherent inability to accurately describe nondynamical correlation. Variants of the LOC scheme, in conjunction with B3LYP (denoted as B3LYP-LOC), were previously applied to enthalpies of formation, ionization potentials, and electron affinities and showed impressive reduction in the errors. In this paper, we demonstrate for the first time that the B3LYP-LOC scheme is robust across different basis sets [6-31G( *), 6-311++G(3df,3pd), cc-pVTZ, and aug-cc-pVTZ] and reaction types (atomization reactions and molecular reactions). For example, for a test set of 70 molecular reactions, the LOC scheme reduces their mean unsigned error from 4.7 kcal/mol [obtained with B3LYP/6-311++G(3df,3pd)] to 0.8 kcal/mol. We also verified whether the LOC methodology would be equally successful if applied to the promising M05-2X functional. We conclude that although M05-2X produces better reaction enthalpies than B3LYP, the LOC scheme does not combine nearly as successfully with M05-2X than with B3LYP. A brief analysis of another functional, M06-2X, reveals that it is more accurate than M05-2X but its combination with LOC still cannot compete in accuracy with B3LYP-LOC. Indeed, B3LYP-LOC remains the best method of computing reaction enthalpies.

  10. Structural study of the effects of mutations in proteins to identify the molecular basis of the loss of local structural fluidity leading to the onset of autoimmune diseases.

    Science.gov (United States)

    Ali, Ananya; Ghosh, Semanti; Bagchi, Angshuman

    2017-02-26

    Protein-Protein Interactions (PPIs) are crucial in most of the biological processes and PPI dysfunctions are known to be associated with the onsets of various diseases. One of such diseases is the auto-immune disease. Auto-immune diseases are one among the less studied group of diseases with very high mortality rates. Thus, we tried to correlate the appearances of mutations with their probable biochemical basis of the molecular mechanisms leading to the onset of the disease phenotypes. We compared the effects of the Single Amino Acid Variants (SAVs) in the wild type and mutated proteins to identify any structural deformities that might lead to altered PPIs leading ultimately to disease onset. For this we used Relative Solvent Accessibility (RSA) as a spatial parameter to compare the structural perturbation in mutated and wild type proteins. We observed that the mutations were capable to increase intra-chain PPIs whereas inter-chain PPIs would remain mostly unaltered. This might lead to more intra-molecular friction causing a deleterious alteration of protein's normal function. A Lyapunov exponent analysis, using the altered RSA values due to polymorphic and disease causing mutations, revealed polymorphic mutations have a positive mean value for the Lyapunov exponent while disease causing mutations have a negative mean value. Thus, local spatial stochasticity has been lost due to disease causing mutations, indicating a loss of structural fluidity. The amino acid conversion plot also showed a clear tendency of altered surface patch residue conversion propensity than polymorphic conversions. So far, this is the first report that compares the effects of different kinds of mutations (disease and non-disease causing polymorphic mutations) in the onset of autoimmune diseases.

  11. Enzymatic activation of double-targeted 5'-O-L-valyl-decitabine prodrug by biphenyl hydrolase-like protein and its molecular design basis.

    Science.gov (United States)

    Tao, Wenhui; Zhao, Dongyang; Sun, Mengchi; Li, Meng; Zhang, Xiangyu; He, Zhonggui; Sun, Yinghua; Sun, Jin

    2017-04-01

    A primary focus of this research was to explore the activation process and mechanism of decitabine (5-aza-2'-deoxycytidine, DAC) prodrug. Recently, it has been reported that biphenyl hydrolase-like protein (BPHL) can play an important role in the activation of some amino acid nucleoside prodrugs with a general preference for hydrophobic amino acids and 5'-esters. Therefore, we put forward a bold hypothesis that this novel enzyme may be primarily responsible for the activation process of DAC prodrug as well. 5'-O-L-valyl-decitabine (L-val-DAC) was synthesized before and can be transported across biological membranes by the oligopeptide transporter (PEPT1), granting it much greater utility in vivo. In this report, L-val-DAC was found to be a good substrate of BPHL protein (K m 0.59 mM; k cat/K m 553.69 mM(-1) s(-1)). After intestinal absorption, L-val-DAC was rapidly and almost completely hydrolyzed to DAC and L-valine. The catalysis was mainly mediated by the BPHL hydrolase and resulted in the intestinal first-pass effect of L-val-DAC after oral administration in Sprague-Dawley rats with cannulated jugular and portal veins. The structural insights using computational molecular docking showed that BPHL had a unique binding mode for L-val-DAC. As a fundamental basis, the simulation was employed to explain the catalytic mechanism in molecular level. In conclusion, BPHL was at least one of the primary candidate enzymes for L-val-DAC prodrug activation. This promising double-targeted prodrug approach have more advantages than the traditional targeted designs due to its higher transport and more predictable activation, thereby leading to a favorable property for oral delivery.

  12. Bases moleculares do sistema Rh e suas aplicações em obstetrícia e medicina transfusional The molecular basis of RH system and its applications in obstetrics and transfusion medicine

    Directory of Open Access Journals (Sweden)

    Luciano Marcondes Machado Nardozza

    2010-01-01

    Full Text Available O sistema Rh é o mais polimórfico e imunogênico de todos os sistemas de grupos sanguíneos. Atualmente mais de 49 antígenos foram identificados sendo cinco principais os antígenos D, C, c, E, e. O conhecimento das bases moleculares do sistema Rh desde a sua primeira clonagem há 17 anos possibilitou o entendimento tanto do mecanismo do fenótipo Rh negativo quanto das variantes dos antígenos RHD e RHCE. As deleções, rearranjos gênicos e as inserções são as principais mutações encontradas. Nos caucasianos, o mecanismo principal do fenótipo Rh negativo é a completa deleção do gene RHD, enquanto nos afrodescendentes é a presença do pseudogene RHDψ e do gene híbrido RHD-CE (4-7-D. Os autores analisam a estrutura do complexo Rh nas hemácias, as bases moleculares do Sistema Rh, os mecanismos de negatividade RHD, além da Expressão fraca e parcial de D.The Rh system is the most polymorphic and immunogenic for all blood group systems. Currently more than 49 antigens were identified with five major antigens D, C, c, E, e. Knowledge of the Rh system's molecular basis, since its first cloning 17 years ago, allowed to understand the mechanism of Rh-negative phenotype and the variants of antigens as RHD and RHCE. Deletions, gene rearrangements and insertions are the main mutations. In Caucasians the primary mechanism of Rh-negative phenotype is the complete RHD gene deletion, while in African descendants it is the presence of pseudogene and gene RHDψ hybrid RHD-CE (4-7-D. The authors analyze the structure of the Rh complex in red cells, molecular basis of the Rh system, mechanisms of Negativity RHD and weak and incomplete expression of RHD.

  13. Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA.

    Directory of Open Access Journals (Sweden)

    Alessia Liguori

    2016-04-01

    Full Text Available Neisseria adhesin A (NadA is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator both in vitro and in vivo. NadR binds the nadA promoter and represses gene transcription. In the presence of 4-hydroxyphenylacetate (4-HPA, a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i the molecular basis of 'conformational selection' by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii the presence of a leucine residue essential for dimerization and

  14. Exploring the molecular basis of insecticide resistance in the dengue vector Aedes aegypti: a case study in Martinique Island (French West Indies

    Directory of Open Access Journals (Sweden)

    Yébakima André

    2009-10-01

    Full Text Available Abstract Background The yellow fever mosquito Aedes aegypti is a major vector of dengue and hemorrhagic fevers, causing up to 100 million dengue infections every year. As there is still no medicine and efficient vaccine available, vector control largely based on insecticide treatments remains the only method to reduce dengue virus transmission. Unfortunately, vector control programs are facing operational challenges with mosquitoes becoming resistant to commonly used insecticides. Resistance of Ae. aegypti to chemical insecticides has been reported worldwide and the underlying molecular mechanisms, including the identification of enzymes involved in insecticide detoxification are not completely understood. Results The present paper investigates the molecular basis of insecticide resistance in a population of Ae. aegypti collected in Martinique (French West Indies. Bioassays with insecticides on adults and larvae revealed high levels of resistance to organophosphate and pyrethroid insecticides. Molecular screening for common insecticide target-site mutations showed a high frequency (71% of the sodium channel 'knock down resistance' (kdr mutation. Exposing mosquitoes to detoxification enzymes inhibitors prior to bioassays induced a significant increased susceptibility of mosquitoes to insecticides, revealing the presence of metabolic-based resistance mechanisms. This trend was biochemically confirmed by significant elevated activities of cytochrome P450 monooxygenases, glutathione S-transferases and carboxylesterases at both larval and adult stages. Utilization of the microarray Aedes Detox Chip containing probes for all members of detoxification and other insecticide resistance-related enzymes revealed the significant constitutive over-transcription of multiple detoxification genes at both larval and adult stages. The over-transcription of detoxification genes in the resistant strain was confirmed by using real-time quantitative RT

  15. Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA.

    Science.gov (United States)

    Liguori, Alessia; Malito, Enrico; Lo Surdo, Paola; Fagnocchi, Luca; Cantini, Francesca; Haag, Andreas F; Brier, Sébastien; Pizza, Mariagrazia; Delany, Isabel; Bottomley, Matthew J

    2016-04-01

    Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. NadR binds the nadA promoter and represses gene transcription. In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of 'conformational selection' by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in

  16. Molecular and structural basis of glutathione import in Gram-positive bacteria via GshT and the cystine ABC importer TcyBC of Streptococcus mutans.

    Science.gov (United States)

    Vergauwen, Bjorn; Verstraete, Kenneth; Senadheera, Dilani B; Dansercoer, Ann; Cvitkovitch, Dennis G; Guédon, Eric; Savvides, Savvas N

    2013-07-01

    Glutathione (GSH) protects cells against oxidative injury and maintains a range of vital functions across all branches of life. Despite recent advances in our understanding of the transport mechanisms responsible for maintaining the spatiotemporal homeostasis of GSH and its conjugates in eukaryotes and Gram-negative bacteria, the molecular and structural basis of GSH import into Gram-positive bacteria has remained largely uncharacterized. Here, we employ genetic, biochemical and structural studies to investigate a possible glutathione import axis in Streptococcus mutans, an organism that has hitherto served as a model system. We show that GshT, a type 3 solute binding protein, displays physiologically relevant affinity for GSH and glutathione disulfide (GSSG). The crystal structure of GshT in complex with GSSG reveals a collapsed structure whereby the GS-I-leg of GSSG is accommodated tightly via extensive interactions contributed by the N- and C-terminal lobes of GshT, while the GS-II leg extends to the solvent. This can explain the ligand promiscuity of GshT in terms of binding glutathione analogues with substitutions at the cysteine-sulfur or the glycine-carboxylate. Finally, we show that GshT primes glutathione import via the L-cystine ABC transporter TcyBC, a membrane permease, which had previously exclusively been associated with the transport of L-cystine.

  17. The genetic basis of inherited anomalies of the teeth. Part 1: clinical and molecular aspects of non-syndromic dental disorders.

    Science.gov (United States)

    Bailleul-Forestier, Isabelle; Molla, Muriel; Verloes, Alain; Berdal, Ariane

    2008-01-01

    The genetic control of dental development represents a complex series of events, which can very schematically be divided in two pathways: specification of type, size and position of each dental organ, and specific processes for the formation of enamel and dentin. Several genes linked with early tooth positioning and development, belong to signalling pathways and have morphogenesis regulatory functions in morphogenesis of other organs where they are associated with the signalling pathways. Their mutations often show pleïotropic effects beyond dental morphogenesis resulting in syndromic developmental disorders. Some genes affecting early tooth development (MSX1, AXIN2) are associated with tooth agenesis and systemic features (cleft palate, colorectal cancer). By contrast, genes involved in enamel (AMELX, ENAM, MMP20, and KLK4) and dentin (DSPP) structures are highly specific for tooth. Mutations in these genes have been identified as causes of amelogenesis imperfecta, dentinogenesis imperfecta, dentin dysplasias and anomalies of teeth number (hypo-, oligo and anodontia), which only partially overlap with the classical phenotypic classifications of dental disorders. This review of genetic basis of inherited anomalies describes, in this first paper, the molecular bases and clinical features of inherited non-syndromic teeth disorders. And in a second part, the review focus on genetic syndromes with dental involvement.

  18. Molecular basis and specificity of H2A.Z-H2B recognition and deposition by the histone chaperone YL1.

    Science.gov (United States)

    Latrick, Chrysa M; Marek, Martin; Ouararhni, Khalid; Papin, Christophe; Stoll, Isabelle; Ignatyeva, Maria; Obri, Arnaud; Ennifar, Eric; Dimitrov, Stefan; Romier, Christophe; Hamiche, Ali

    2016-04-01

    H2A.Z, a widely conserved histone variant, is evicted from chromatin by the histone chaperone ANP32E. However, to date, no deposition chaperone for H2A.Z is known in metazoans. Here, we identify YL1 as a specific H2A.Z-deposition chaperone. The 2.7-Å-resolution crystal structure of the human YL1-H2A.Z-H2B complex shows that YL1 binding, similarly to ANP32E binding, triggers an extension of the H2A.Z αC helix. The interaction with YL1 is, however, more extensive and includes both the extended acidic patch and the entire DNA-binding surface of H2A.Z-H2B. Substitution of only four amino acid residues of H2A is sufficient for the formation of an H2A.Z-like interface specifically recognized by YL1. Collectively, our data reveal the molecular basis of H2A.Z-specific recognition by YL1 and shed light on the mechanism of H2A.Z transfer to the nucleosome by the ATP-dependent chromatin-remodeling complexes SRCAP and P400-TIP60.

  19. Prevalence of Prostate Cancer Metastases after Intravenous Inoculation Provides Clues into the Molecular Basis of Dormancy in the Bone Marrow Microenvironment

    Directory of Open Access Journals (Sweden)

    Younghun Jung

    2012-05-01

    Full Text Available Bone is the preferred metastasis site of advanced prostate cancer (PCa. Using an in vivo murine model of human PCa cell metastasis to bone, we noted that the majority of animals that develop skeletal metastasis have either spinal lesions or lesions in the bones of the hindlimb. Much less frequently, lesions develop in the bones of the forelimb. We therefore speculated whether the environment of the forelimb bones is not permissive for the growth of PCa. Consequently, data on tumor prevalence were normalized to account for the number of PCa cells arriving after intravascular injection, marrow cellularity, and number of hematopoietic stem cell niches. None of these factors were able to account for the observed differences in tumor prevalence. An analysis of differential gene and protein levels identified that growth arrest specific-6 (GAS6 levels were significantly greater in the forelimb versus hindlimb bone marrow. When murine RM1 cells were implanted into subcutaneous spaces in immune competent animals, tumor growth in the GAS6-/- animals was greater than in GAS6+/+ wild-type animals. In an osseous environment, the human PC3 cell line grew significantly better in vertebral body transplants (vossicles derived from GAS6-/- animals than in vossicles derived from GAS6+/+ animals. Together, these data suggest that the differences in tumor prevalence after intravascular inoculation are a useful model to study the molecular basis of tumor dormancy. Importantly, these data suggest that therapeutic manipulation of GAS6 levels may prove useful as a therapy for metastatic disease.

  20. Functional and Biochemical Analysis of Glucose-6-Phosphate Dehydrogenase (G6PD Variants: Elucidating the Molecular Basis of G6PD Deficiency

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    Saúl Gómez-Manzo

    2017-05-01

    Full Text Available G6PD deficiency is the most common enzymopathy, leading to alterations in the first step of the pentose phosphate pathway, which interferes with the protection of the erythrocyte against oxidative stress and causes a wide range of clinical symptoms of which hemolysis is one of the most severe. The G6PD deficiency causes several abnormalities that range from asymptomatic individuals to more severe manifestations that can lead to death. Nowadays, only 9.2% of all recognized variants have been related to clinical manifestations. It is important to understand the molecular basis of G6PD deficiency to understand how gene mutations can impact structure, stability, and enzymatic function. In this work, we reviewed and compared the functional and structural data generated through the characterization of 20 G6PD variants using different approaches. These studies showed that severe clinical manifestations of G6PD deficiency were related to mutations that affected the catalytic and structural nicotinamide adenine dinucleotide phosphate (NADPH binding sites, and suggests that the misfolding or instability of the 3D structure of the protein could compromise the half-life of the protein in the erythrocyte and its activity.

  1. Human orosomucoid polymorphism: molecular basis of the three common ORM1 alleles, ORM1*F1, ORM1*F2, and ORM1*S.

    Science.gov (United States)

    Yuasa, I; Umetsu, K; Vogt, U; Nakamura, H; Nanba, E; Tamaki, N; Irizawa, Y

    1997-03-01

    The human orosomucoid (ORM) is controlled by two closely linked loci, ORM1 and ORM2, and two tandem genes, AGP1 and AGP2, encoding the proteins produced by the two loci, have been cloned. In this study the molecular basis of ORM1 polymorphism was investigated. For the detection of mutations the products of the six exons of each gene, amplified by the polymerase chain reaction (PCR), were screened by single-strand conformation polymorphism analysis. Subsequently, the exons with an altered migration pattern were gene-specifically amplified by nested PCR. Sequencing of the gene-specific PCR products showed that the three common ORM1 alleles result from A-->G transitions at the codons for amino acid positions 20 in exon 1 and 156 in exon 5 of the AGP1 gene: ORM1*F1 was characterized by CAG (Gln) and GTG (Val), ORM1*F2, by CAG (Gln) and ATG (Met), and ORM1*S, by CGG (Arg) and GTG (Val). The phylogenesis of the genes encoding these three ORM1 alleles is discussed.

  2. A biophysical study with carbohydrate derivatives explains the molecular basis of monosaccharide selectivity of the Pseudomonas aeruginosa lectin LecB.

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    Roman Sommer

    Full Text Available The rise of resistances against antibiotics in bacteria is a major threat for public health and demands the development of novel antibacterial therapies. Infections with Pseudomonas aeruginosa are a severe problem for hospitalized patients and for patients suffering from cystic fibrosis. These bacteria can form biofilms and thereby increase their resistance towards antibiotics. The bacterial lectin LecB was shown to be necessary for biofilm formation and the inhibition with its carbohydrate ligands resulted in reduced amounts of biofilm. The natural ligands for LecB are glycosides of D-mannose and L-fucose, the latter displaying an unusual strong affinity. Interestingly, although mannosides are much weaker ligands for LecB, they do form an additional hydrogen bond with the protein in the crystal structure. To analyze the individual contributions of the methyl group in fucosides and the hydroxymethyl group in mannosides to the binding, we designed and synthesized derivatives of these saccharides. We report glycomimetic inhibitors that dissect the individual interactions of their saccharide precursors with LecB and give insight into the biophysics of binding by LecB. Furthermore, theoretical calculations supported by experimental thermodynamic data suggest a perturbed hydrogen bonding network for mannose derivatives as molecular basis for the selectivity of LecB for fucosides. Knowledge gained on the mode of interaction of LecB with its ligands at ambient conditions will be useful for future drug design.

  3. Sustained productivity in recombinant Chinese Hamster Ovary (CHO) cell lines: proteome analysis of the molecular basis for a process-related phenotype

    LENUS (Irish Health Repository)

    Meleady, Paula

    2011-07-24

    Abstract Background The ability of mammalian cell lines to sustain cell specific productivity (Qp) over the full duration of bioprocess culture is a highly desirable phenotype, but the molecular basis for sustainable productivity has not been previously investigated in detail. In order to identify proteins that may be associated with a sustained productivity phenotype, we have conducted a proteomic profiling analysis of two matched pairs of monoclonal antibody-producing Chinese hamster ovary (CHO) cell lines that differ in their ability to sustain productivity over a 10 day fed-batch culture. Results Proteomic profiling of inherent differences between the two sets of comparators using 2D-DIGE (Difference Gel Electrophoresis) and LC-MS\\/MS resulted in the identification of 89 distinct differentially expressed proteins. Overlap comparisons between the two sets of cell line pairs identified 12 proteins (AKRIB8, ANXA1, ANXA4, EIF3I, G6PD, HSPA8, HSP90B1, HSPD1, NUDC, PGAM1, RUVBL1 and CNN3) that were differentially expressed in the same direction. Conclusion These proteins may have an important role in sustaining high productivity of recombinant protein over the duration of a fed-batch bioprocess culture. It is possible that many of these proteins could be useful for future approaches to successfully manipulate or engineer CHO cells in order to sustain productivity of recombinant protein.

  4. 癌细胞葡萄糖代谢重编程的分子基础%Molecular basis of glucose metabolic reprogramming in cancer cells

    Institute of Scientific and Technical Information of China (English)

    赵世光

    2014-01-01

    代谢的重编程是癌细胞的基本特征之一,其中葡萄糖代谢方式和途径的改变对癌症的发生和发展至关重要。即使在氧气足够充足的情况下,快速增殖的癌细胞生长所需的能量主要由糖酵解而非氧化磷酸化提供,癌细胞这种特殊的糖代谢现象被称为Warburg效应。这种特有的能量获取方式已在多种癌细胞中得到验证,以癌细胞对葡萄糖高摄取率和利用增加为原理的18F-FDG PET/CT显像已广泛应用于临床的癌症诊断。但癌细胞为何利用有氧酵解获取能量以及有氧酵解进行的分子基础目前尚不明确,本文围绕调控癌细胞糖酵解进程中的直接调控酶、癌基因及致癌代谢小分子进行分析和综述。%Cancer metabolic reprogramming is among the basic characteristics of cancer cells. Changes in glucose metabolism are essential for carcinogenesis and cancer development. Previous study indicated that energy is acquired mainly via glycolysis rather than oxidative phosphorylation in the presence of sufficient oxygen levels to promote the rapid proliferation of cells, such as cancer cells. This phenomenon is called the"Warburg effect."Furthermore, this unique approach of energy production in cancer cells has been validated in various types of cancer cells. On the basis of the characteristics of cancer cells with high glucose uptake and utilization, cli-nicians and medical practitioners extensively apply 18-fludeoxyglucose positron emission tomography in clinical diagnosis. Tumor cells undergo aerobic glycolysis to produce energy, but this metabolic pathway is poorly efficient;the molecular basis of aerobic glycol-ysis and the reason for these cells to undergo this metabolic pathway also remain unclear. In this article, glycolysis-related processes, in-cluding enzyme, oncogene, and oncometabolite regulation, in cancer cells are summarized.

  5. Comprehensive molecular pathology analysis of small bowel adenocarcinoma reveals novel targets with potential for clinical utility.

    Science.gov (United States)

    Alvi, Muhammad A; McArt, Darragh G; Kelly, Paul; Fuchs, Marc-Aurel; Alderdice, Matthew; McCabe, Clare M; Bingham, Victoria; McGready, Claire; Tripathi, Shailesh; Emmert-Streib, Frank; Loughrey, Maurice B; McQuaid, Stephen; Maxwell, Perry; Hamilton, Peter W; Turkington, Richard; James, Jacqueline A; Wilson, Richard H; Salto-Tellez, Manuel

    2015-08-28

    Small bowel accounts for only 0.5% of cancer cases in the US but incidence rates have been rising at 2.4% per year over the past decade. One-third of these are adenocarcinomas but little is known about their molecular pathology and no molecular markers are available for clinical use. Using a retrospective 28 patient matched normal-tumor cohort, next-generation sequencing, gene expression arrays and CpG methylation arrays were used for molecular profiling. Next-generation sequencing identified novel mutations in IDH1, CDH1, KIT, FGFR2, FLT3, NPM1, PTEN, MET, AKT1, RET, NOTCH1 and ERBB4. Array data revealed 17% of CpGs and 5% of RNA transcripts assayed to be differentially methylated and expressed respectively (p clinically exploitable markers.

  6. Exploring the molecular pathogenesis and biomarkers of high risk oral premalignant lesions on the basis of long noncoding RNA expression profiling by serial analysis of gene expression.

    Science.gov (United States)

    Jia, Hongcheng; Wang, Xuan; Sun, Zheng

    2017-04-24

    Oral premalignant lesions (OPLs) have malignant transformation potential, with no reliable markers available. This study aimed to assess molecular events to identify biomarkers that can reflect high-risk lesions as predictive factors to tailor clinical decision for patients on the basis of long noncoding RNAs (lncRNA) expression profiling by serial analysis of gene expression. The GSE31021 and GSE8127 datasets were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) and lncRNAs were identified using the LIMMA package in R language. The genes targeted by lncRNAs were predicted among screened DEGs using Pearson's correlation. Gene ontology function and Kyoto Encyclopedia of Genes and Genomes pathway analyses were carried out for genes targeted by lncRNAs using the Database for Annotation, Visualization, and Integrated Discovery online tool. A total of 674 DEGs and differentially expressed lncRNAs were screened. Thirty-two interactions of 10 lncRNAs and 524 target genes were predicted. The lncRNA NEAT1 was among the top 10 lncRNAs. The coregulated target genes RP4-684O24, RP11-283I3, and RP11-350G8 were significantly enriched in the immune response and mannosyl-oligosaccharide mannosidase activity. The target genes coregulated by LINC00665 and MIR378D2 were significantly enriched in the ubiquitin-dependent protein catabolic process, ubiquitin-protein ligase activity, and neurotrophin signaling. The lncRNA NEAT1 may play an important role in high-risk lesions. The novel lncRNAs and DEGs identified in OPLs may mediate the immune response and neurotrophin signaling and show ubiquitin ligase activity. These results improve our understanding of the molecular pathogenesis of OPLs and identify some potential targets for early diagnosis of high risk OPLs.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to

  7. Molecular basis for nucleotide-binding specificity: role of the exocyclic amino group "N2" in recognition by a guanylyl-ribonuclease.

    Science.gov (United States)

    Schrift, Greta L; Waldron, Travis T; Timmons, Mitchell A; Ramaswamy, S; Kearney, William R; Murphy, Kenneth P

    2006-01-06

    Proteins interact with nucleotides to perform a multitude of functions within cells. These interactions are highly specific; however, the molecular basis for this specificity is not well understood. To identify factors critical for protein-guanine nucleotide recognition the binding of two closely related ligands, guanosine 3'-monophosphate (3'GMP) and inosine 3'-monophosphate (3'IMP), to Ribonuclease Sa (RNase Sa), a small, guanylyl-endoribonuclease from Streptomyces aureofaciens, was compared using isothermal titration calorimetry, NMR, X-ray crystallography and molecular dynamics simulations. This comparison has allowed for the determination of the contribution of the exocyclic amino group "N2" of the guanine base to nucleotide binding specificity. Calorimetric measurements indicate that RNase Sa has a higher affinity for 3'GMP (K=(1.5+/-0.2)x10(5)) over 3'IMP (K=(3.1+/-0.2)x10(4)) emphasizing the importance of N2 as a key determinant of RNase Sa guanine binding specificity. This result was unexpected as the published structural data for RNase Sa in complex with 3'GMP showed only a potential long-range interaction (>3.3A) between N2 and the side-chain of Glu41 of RNase Sa. The observed difference in affinity is largely due to a reduction in the favorable enthalpy change by 10 kJ/mol for 3'IMP binding as compared to 3'GMP that is accompanied by a significant difference in the heat capacity changes observed for binding the two ligands. To aid interpretation of the calorimetric data, the first crystal structure of a small, guanylyl ribonuclease bound to 3'IMP was determined to 2.0 A resolution. This structure has revealed small yet unexpected changes in the ligand conformation and differences in the conformations of the side-chains contacting the sugar and phosphate moieties as compared to the 3'GMP complex. The structural data suggest the less favorable enthalpy change is due to an overall lengthening of the contacts between RNase Sa and 3'IMP as compared to 3'GMP

  8. Molecular Dynamics Simulations for Deciphering the Structural Basis of Recognition of Pre-let-7 miRNAs by LIN28.

    Science.gov (United States)

    Sharma, Chhaya; Mohanty, Debasisa

    2017-02-07

    LIN28 protein inhibits biogenesis of miRNAs belonging to the let-7 family by binding to precursor forms of miRNAs. Overexpression of LIN28 and low levels of let-7 miRNAs are associated with several forms of cancer cells. We have performed multiple explicit solvent molecular dynamics simulations ranging from 200 to 500 ns in length on different isoforms of preE-let-7 in complex with LIN28 and also in isolation to identify structural features and key specificity-determining residues (SDRs) that are important for the inhibitory role of LIN28. Our simulations suggest that a conserved structural feature of the loop regions of preE-let-7 miRNAs is more important for LIN28 recognition than sequence conservation among members of the let-7 family or the presence of the GGAG motif in the 3' region. The loop region consisting of a minimum of five nucleotides helps pre-miRNAs to acquire a conformation ideal for binding to LIN28, but pre-let-7c-2 prefers a conformation with a three-nucleotide loop. Thus, our simulations provide a theoretical rationale for the recent experimental observation of the escape of LIN28-mediated repression by pre-let-7c-2. The essential structural and sequence features highlighted in this study might aid in designing synthetic small molecule inhibitors for modulating LIN28-let-7 interaction in malignant cells. We have also identified crucial SDRs of the LIN28-preE-let-7 complex involving 13 residues of LIN28 and 10 residues of the pre-miRNA. On the basis of the conservation profile of these 13 SDRs, we have identified 10 novel proteins that are not annotated as LIN28 like but are similar in sequence, domain, or fold level to LIN28.

  9. Gene amplification, ABC transporters and cytochrome P450s: unraveling the molecular basis of pyrethroid resistance in the dengue vector, Aedes aegypti.

    Directory of Open Access Journals (Sweden)

    Vassiliki Bariami

    Full Text Available BACKGROUND: Pyrethroid insecticides are widely utilized in dengue control. However, the major vector, Aedes aegypti, is becoming increasingly resistant to these insecticides and this is impacting on the efficacy of control measures. The near complete transcriptome of two pyrethroid resistant populations from the Caribbean was examined to explore the molecular basis of this resistance. PRINCIPAL FINDINGS: Two previously described target site mutations, 1016I and 1534C were detected in pyrethroid resistant populations from Grand Cayman and Cuba. In addition between two and five per cent of the Ae. aegypti transcriptome was differentially expressed in the resistant populations compared to a laboratory susceptible population. Approximately 20 per cent of the genes over-expressed in resistant mosquitoes were up-regulated in both Caribbean populations (107 genes. Genes with putative monooxygenase activity were significantly over represented in the up-regulated subset, including five CYP9 P450 genes. Quantitative PCR was used to confirm the higher transcript levels of multiple cytochrome P450 genes from the CYP9J family and an ATP binding cassette transporter. Over expression of two genes, CYP9J26 and ABCB4, is due, at least in part, to gene amplification. SIGNIFICANCE: These results, and those from other studies, strongly suggest that increases in the amount of the CYP9J cytochrome P450s are an important mechanism of pyrethroid resistance in Ae. aegypti. The genetic redundancy resulting from the expansion of this gene family makes it unlikely that a single gene or mutation responsible for pyrethroid resistance will be identified in this mosquito species. However, the results from this study do pave the way for the development of new pyrethroid synergists and improved resistance diagnostics. The role of copy number polymorphisms in detoxification and transporter genes in providing protection against insecticide exposure requires further investigation.

  10. The Binding Affinity and Molecular Basis of the Structure-Binding Relationship between Urinary Tamm-Horsfall Glycoprotein and Tumor Necrosis Factor-α

    Directory of Open Access Journals (Sweden)

    Chia-Li Yu

    2012-10-01

    Full Text Available In a previous study we noted significant THP binding to TNF-α, but did not explore the molecular basis of the structure-binding relationship. In this study, we used lectin-binding ELISA to assess the carbohydrate compositions of THP, BSA, IgG, TNF-α, and IFN-g. We identified β(1,4-N-acetylglucosamine oligomers (GlcNAc and GlcNAc/branched mannose in BSA, IgG, TNF-α, and THP, but not in IFN-g. These carbohydrate moieties mediated binding with THP. Small amounts of Siaα(2,3Gal/ GalNAc, Sia(2,6Gal/GalNAc, and mannose residues were also present in THP and TNF-α. Binding affinity (Kd between THP and TNF-α by Scatchard plot analysis was 1.4–1.7 × 10−6 M, lower than antigen-antibody or ligand-receptor binding affinities. To elucidate the structure-binding relationship of THP-TNF-α, THP was digested with neuraminidase, β-galactosidase, O-sialoglycoprotein endopeptidase, carboxypeptidase Y, or proteinase K. β-galactosidase increased binding capacity of THP for TNF-α. Monosaccharide inhibition suggested that α-methyl-D-mannoside, GlcNAc, and GalNAc, but not sialic acid, suppress THP-TNF-α binding as detected by ELISA. We conclude that sugar-lectin and sugar-protein interactions between cognate sites in THP and TNF-α mediate their binding.

  11. Molecular basis of calcium-sensitizing and desensitizing mutations of the human cardiac troponin C regulatory domain: a multi-scale simulation study.

    Directory of Open Access Journals (Sweden)

    Peter Michael Kekenes-Huskey

    Full Text Available Troponin C (TnC is implicated in the initiation of myocyte contraction via binding of cytosolic Ca²⁺ and subsequent recognition of the Troponin I switch peptide. Mutations of the cardiac TnC N-terminal regulatory domain have been shown to alter both calcium binding and myofilament force generation. We have performed molecular dynamics simulations of engineered TnC variants that increase or decrease Ca²⁺ sensitivity, in order to understand the structural basis of their impact on TnC function. We will use the distinction for mutants that are associated with increased Ca²⁺ affinity and for those mutants with reduced affinity. Our studies demonstrate that for GOF mutants V44Q and L48Q, the structure of the physiologically-active site II Ca²⁺ binding site in the Ca²⁺-free (apo state closely resembled the Ca²⁺-bound (holo state. In contrast, site II is very labile for LOF mutants E40A and V79Q in the apo form and bears little resemblance with the holo conformation. We hypothesize that these phenomena contribute to the increased association rate, k(on, for the GOF mutants relative to LOF. Furthermore, we observe significant positive and negative positional correlations between helices in the GOF holo mutants that are not found in the LOF mutants. We anticipate these correlations may contribute either directly to Ca²⁺ affinity or indirectly through TnI association. Our observations based on the structure and dynamics of mutant TnC provide rationale for binding trends observed in GOF and LOF mutants and will guide the development of inotropic drugs that target TnC.

  12. Molecular Basis Behind Inability of Mitochondrial Holocytochrome c Synthase to Mature Bacterial Cytochromes: DEFINING A CRITICAL ROLE FOR CYTOCHROME c α HELIX-1.

    Science.gov (United States)

    Babbitt, Shalon E; Hsu, Jennifer; Kranz, Robert G

    2016-08-19

    Mitochondrial holocytochrome c synthase (HCCS) is required for cytochrome c (cyt c) maturation and therefore respiration. HCCS efficiently attaches heme via two thioethers to CXXCH of mitochondrial but not bacterial cyt c even though they are functionally conserved. This inability is due to residues in the bacterial cyt c N terminus, but the molecular basis is unknown. Human cyts c with deletions of single residues in α helix-1, which mimic bacterial cyt c, are poorly matured by human HCCS. Focusing on ΔM13 cyt c, we co-purified this variant with HCCS, demonstrating that HCCS recognizes the bacterial-like cytochrome. Although an HCCS-WT cyt c complex contains two covalent links, HCCS-ΔM13 cyt c contains only one thioether attachment. Using multiple approaches, we show that the single attachment is to the second thiol of C(15)SQC(18)H, indicating that α helix-1 is required for positioning the first cysteine for covalent attachment, whereas the histidine of CXXCH positions the second cysteine. Modeling of the N-terminal structure suggested that the serine residue (of CSQCH) would be anchored where the first cysteine should be in ΔM13 cyt c An engineered cyt c with a CQCH motif in the ΔM13 background is matured at higher levels (2-3-fold), providing further evidence for α helix-1 positioning the first cysteine. Bacterial cyt c biogenesis pathways (Systems I and II) appear to recognize simply the CXXCH motif, not requiring α helix-1. Results here explain mechanistically how HCCS (System III) requires an extended region adjacent to CXXCH for maturation.

  13. Molecular Basis of Gene-Gene Interaction: Cyclic Cross-Regulation of Gene Expression and Post-GWAS Gene-Gene Interaction Involved in Atrial Fibrillation.

    Directory of Open Access Journals (Sweden)

    Yufeng Huang

    2015-08-01

    Full Text Available Atrial fibrillation (AF is the most common cardiac arrhythmia at the clinic. Recent GWAS identified several variants associated with AF, but they account for <10% of heritability. Gene-gene interaction is assumed to account for a significant portion of missing heritability. Among GWAS loci for AF, only three were replicated in the Chinese Han population, including SNP rs2106261 (G/A substitution in ZFHX3, rs2200733 (C/T substitution near PITX2c, and rs3807989 (A/G substitution in CAV1. Thus, we analyzed the interaction among these three AF loci. We demonstrated significant interaction between rs2106261 and rs2200733 in three independent populations and combined population with 2,020 cases/5,315 controls. Compared to non-risk genotype GGCC, two-locus risk genotype AATT showed the highest odds ratio in three independent populations and the combined population (OR=5.36 (95% CI 3.87-7.43, P=8.00×10-24. The OR of 5.36 for AATT was significantly higher than the combined OR of 3.31 for both GGTT and AACC, suggesting a synergistic interaction between rs2106261 and rs2200733. Relative excess risk due to interaction (RERI analysis also revealed significant interaction between rs2106261 and rs2200733 when exposed two copies of risk alleles (RERI=2.87, P<1.00×10-4 or exposed to one additional copy of risk allele (RERI=1.29, P<1.00×10-4. The INTERSNP program identified significant genotypic interaction between rs2106261 and rs2200733 under an additive by additive model (OR=0.85, 95% CI: 0.74-0.97, P=0.02. Mechanistically, PITX2c negatively regulates expression of miR-1, which negatively regulates expression of ZFHX3, resulting in a positive regulation of ZFHX3 by PITX2c; ZFHX3 positively regulates expression of PITX2C, resulting in a cyclic loop of cross-regulation between ZFHX3 and PITX2c. Both ZFHX3 and PITX2c regulate expression of NPPA, TBX5 and NKX2.5. These results suggest that cyclic cross-regulation of gene expression is a molecular basis for gene

  14. Correlation consistent basis sets for molecular core-valence effects with explicitly correlated wave functions: The atoms B-Ne and Al-Ar

    Science.gov (United States)

    Hill, J. Grant; Mazumder, Shivnath; Peterson, Kirk A.

    2010-02-01

    Correlation consistent basis sets have been optimized for accurately describing core-core and core-valence correlation effects with explicitly correlated F12 methods. The new sets, denoted cc-pCVnZ-F12 (n =D, T, Q) and aug-cc-pCF12VnZ (n =D, T, Q, 5), were developed by augmenting the cc-pVnZ-F12 and aug-cc-pVnZ families of basis sets with additional functions whose exponents were optimized based on the difference between all-electron and valence-electron correlation energies. The number of augmented functions added is fewer, in general, than in the standard cc-pCVnZ and cc-pwCVnZ families of basis sets. Optimal values of the geminal Slater exponent for use with these basis sets in MP2-F12 calculations are presented and are also recommended for CCSD-F12b calculations. Auxiliary basis sets for use in the resolution of the identity approximation in explicitly correlated calculations have also been optimized and matched to the new cc-pCVnZ-F12 series of orbital basis sets. The cc-pCVnZ-F12 basis sets, along with the new auxiliary sets, were benchmarked in CCSD(T)-F12b calculations of spectroscopic properties on a series of homo- and heteronuclear first and second row diatomic molecules. Comparing the effects of correlating the outer core electrons in these molecules with those from conventional CCSD(T) at the complete basis set limit, which involved calculations with new cc-pCV6Z basis sets for the second row elements that were also developed in the course of this work, it is observed that the F12 values are reasonably well converged already at just the triple-ζ level.

  15. PCR ribotype prevalence and molecular basis of macrolide-lincosamide-streptogramin B (MLSB) and fluoroquinolone resistance in Irish clinical Clostridium difficile isolates.

    LENUS (Irish Health Repository)

    Solomon, Katie

    2011-09-01

    Antimicrobial use is recognized as a risk factor for Clostridium difficile infection (CDI) and outbreaks. We studied the relationship between PCR ribotype, antimicrobial susceptibility and the genetic basis of resistance in response to exposure to antimicrobial agents.

  16. Molecular Genetic Markers in Acute Myeloid Leukemia

    Directory of Open Access Journals (Sweden)

    Sophia Yohe

    2015-03-01

    Full Text Available Genetics play an increasingly important role in the risk stratification and management of acute myeloid leukemia (AML patients. Traditionally, AML classification and risk stratification relied on cytogenetic studies; however, molecular detection of gene mutations is playing an increasingly important role in classification, risk stratification, and management of AML. Molecular testing does not take the place of cytogenetic testing results, but plays a complementary role to help refine prognosis, especially within specific AML subgroups. With the exception of acute promyelocytic leukemia, AML therapy is not targeted but the intensity of therapy is driven by the prognostic subgroup. Many prognostic scoring systems classify patients into favorable, poor, or intermediate prognostic subgroups based on clinical and genetic features. Current standard of care combines cytogenetic results with targeted testing for mutations in FLT3, NPM1, CEBPA, and KIT to determine the prognostic subgroup. Other gene mutations have also been demonstrated to predict prognosis and may play a role in future risk stratification, although some of these have not been confirmed in multiple studies or established as standard of care. This paper will review the contribution of cytogenetic results to prognosis in AML and then will focus on molecular mutations that have a prognostic or possible therapeutic impact.

  17. 2012 Molecular Basis of Microbial One-Carbon Metabolism Gordon Research Conferences and Gordon Research Seminar, August 4-10,2012

    Energy Technology Data Exchange (ETDEWEB)

    Hanson, Thomas

    2012-08-10

    The 2012 Gordon Conference will present and discuss cutting-edge research