Tissue-specific models of spinal muscular atrophy confirm a critical role of SMN in motor neurons from embryonic to adult stages.

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Laird, Angela S; Mackovski, Nikolce; Rinkwitz, Silke; Becker, Thomas S; Giacomotto, Jean

2016-05-01

Spinal muscular atrophy (SMA) is an autosomal recessive disease linked to survival motor neuron (SMN) protein deficiency. While SMN protein is expressed ubiquitously, its deficiency triggers tissue-specific hallmarks, including motor neuron death and muscle atrophy, leading to impaired motor functions and premature death. Here, using stable miR-mediated knockdown technology in zebrafish, we developed the first vertebrate system allowing transgenic spatio-temporal control of the smn1 gene. Using this new model it is now possible to investigate normal and pathogenic SMN function(s) in specific cell types, independently or in synergy with other cell populations. We took advantage of this new system to first test the effect of motor neuron or muscle-specific smn1 silencing. Anti-smn1 miRNA expression in motor neurons, but not in muscles, reproduced SMA hallmarks, including abnormal motor neuron development, poor motor function and premature death. Interestingly, smn1 knockdown in motor neurons also induced severe late-onset phenotypes including scoliosis-like body deformities, weight loss, muscle atrophy and, seen for the first time in zebrafish, reduction in the number of motor neurons, indicating motor neuron degeneration. Taken together, we have developed a new transgenic system allowing spatio-temporal control of smn1 expression in zebrafish, and using this model, we have demonstrated that smn1 silencing in motor neurons alone is sufficient to reproduce SMA hallmarks in zebrafish. It is noteworthy that this research is going beyond SMA as this versatile gene-silencing transgenic system can be used to knockdown any genes of interest, filling the gap in the zebrafish genetic toolbox and opening new avenues to study gene functions in this organism. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Association between the SMN2 gene copy number and clinical characteristics of patients with spinal muscular atrophy with homozygous deletion of exon 7 of the SMN1 gene

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Žarkov Marija

2015-01-01

Full Text Available Background/Aim. Spinal muscular atrophy (SMA is an autosomal recessive disease characterized by degeneration of alpha motor neurons in the spinal cord and the medulla oblongata, causing progressive muscle weakness and atrophy. The aim of this study was to determine association between the SMN2 gene copy number and disease phenotype in Serbian patients with SMA with homozygous deletion of exon 7 of the SMN1 gene. Methods. The patients were identified using regional Serbian hospital databases. Investigated clinical characteristics of the disease were: patients’ gender, age at disease onset, achieved and current developmental milestones, disease duration, current age, and the presence of the spinal deformities and joint contractures. The number of SMN1 and SMN2 gene copies was determined using real-time polymerase chain reaction (PCR. Results. Among 43 identified patients, 37 (86.0% showed homozygous deletion of SMN1 exon 7. One (2.7% of 37 patients had SMA type I with 3 SMN2 copies, 11 (29.7% patients had SMA type II with 3.1 ± 0.7 copies, 17 (45.9% patients had SMA type III with 3.7 ± 0.9 copies, while 8 (21.6% patients had SMA type IV with 4.2 ± 0.9 copies. There was a progressive increase in the SMN2 gene copy number from type II towards type IV (p < 0.05. A higher SMN2 gene copy number was associated with better current motor performance (p < 0.05. Conclusion. In the Serbian patients with SMA, a higher SMN2 gene copy number correlated with less severe disease phenotype. A possible effect of other phenotype modifiers should not be neglected.

The SMN1 common variant c.22 dupA in Chinese patients causes spinal muscular atrophy by nonsense-mediated mRNA decay in humans.

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Bai, JinLi; Qu, YuJin; Cao, YanYan; Yang, Lan; Ge, Lin; Jin, YuWei; Wang, Hong; Song, Fang

2018-02-20

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder that is mostly caused by homozygous deletion of the SMN1 gene. Approximately 5%-10% of SMA patients are believed to have SMN1 variants. c.22 dupA (p.Ser8lysfs*23) has been identified as the most frequent variant in the Chinese SMA population and to be associated with a severe phenotype. However, the exact molecular mechanism of the variant on the pathogenesis of SMA is unclear. We observed that SMN1 mRNA and the SMN protein in the peripheral blood cells of a patient with c.22 dupA were lower than those of controls. The aim of this study is to investigate whether nonsense-mediated mRNA decay (NMD) plays a role in the mechanism of the c.22 dupA variant of the SMN1 gene as it causes SMA. Two lymphoblasts cell lines from two patients (patient 1 and 2) with the c.22 dupA, and one dermal fibroblasts cell line from patient 2 were included in our study. Two-stage validation of the NMD mechanism was supplied. We first measured the changes in the transcript levels of the SMN1 gene by real-time quantitative PCR after immortalized B-lymphoblasts and dermal fibroblasts cells of the SMA patients were treated with inhibitors of the NMD pathway, including puromycin and cyclohemide. Next, lentivirus-mediated knockdown of the key NMD factor-Up-frameshift protein 1 (UPF1)-was performed in the fibroblasts cell line to further clarify whether the variant led to NMD, as UPF1 recognizes abnormally terminated transcripts as NMD substrates during translation. SC35 1.7-kb transcripts, a physiological NMD substrate was determined to be a NMD positive gene in our experiments. The two inhibitors resulted in a dramatic escalation of the levels of the full-length SMN1 (fl-SMN1) transcripts. Additionally, the SC35 1.7-kb mRNA levels were also increased, suggesting that NMD pathway is suppressed by the two inhibitors. For the 3 cell lines, the fold increase of the SMN1 transcript levels of cycloheximide ranged

Seamless Genetic Conversion of SMN2 to SMN1 via CRISPR/Cpf1 and Single-Stranded Oligodeoxynucleotides in Spinal Muscular Atrophy Patient-Specific Induced Pluripotent Stem Cells.

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Zhou, Miaojin; Hu, Zhiqing; Qiu, Liyan; Zhou, Tao; Feng, Mai; Hu, Qian; Zeng, Baitao; Li, Zhuo; Sun, Qianru; Wu, Yong; Liu, Xionghao; Wu, Lingqian; Liang, Desheng

2018-05-09

Spinal muscular atrophy (SMA) is a kind of neuromuscular disease characterized by progressive motor neuron loss in the spinal cord. It is caused by mutations in the survival motor neuron 1 (SMN1) gene. SMN1 has a paralogous gene, survival motor neuron 2 (SMN2), in humans that is present in almost all SMA patients. The generation and genetic correction of SMA patient-specific induced pluripotent stem cells (iPSCs) is a viable, autologous therapeutic strategy for the disease. Here, c-Myc-free and non-integrating iPSCs were generated from the urine cells of an SMA patient using an episomal iPSC reprogramming vector, and a unique crRNA was designed that does not have similar sequences (≤3 mismatches) anywhere in the human reference genome. In situ gene conversion of the SMN2 gene to an SMN1-like gene in SMA-iPSCs was achieved using CRISPR/Cpf1 and single-stranded oligodeoxynucleotide with a high efficiency of 4/36. Seamlessly gene-converted iPSC lines contained no exogenous sequences and retained a normal karyotype. Significantly, the SMN expression and gems localization were rescued in the gene-converted iPSCs and their derived motor neurons. This is the first report of an efficient gene conversion mediated by Cpf1 homology-directed repair in human cells and may provide a universal gene therapeutic approach for most SMA patients.

Deficiency of the Survival of Motor Neuron Protein Impairs mRNA Localization and Local Translation in the Growth Cone of Motor Neurons.

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Fallini, Claudia; Donlin-Asp, Paul G; Rouanet, Jeremy P; Bassell, Gary J; Rossoll, Wilfried

2016-03-30

Spinal muscular atrophy (SMA) is a neurodegenerative disease primarily affecting spinal motor neurons. It is caused by reduced levels of the survival of motor neuron (SMN) protein, which plays an essential role in the biogenesis of spliceosomal small nuclear ribonucleoproteins in all tissues. The etiology of the specific defects in the motor circuitry in SMA is still unclear, but SMN has also been implicated in mediating the axonal localization of mRNA-protein complexes, which may contribute to the axonal degeneration observed in SMA. Here, we report that SMN deficiency severely disrupts local protein synthesis within neuronal growth cones. We also identify the cytoskeleton-associated growth-associated protein 43 (GAP43) mRNA as a new target of SMN and show that motor neurons from SMA mouse models have reduced levels ofGAP43mRNA and protein in axons and growth cones. Importantly, overexpression of two mRNA-binding proteins, HuD and IMP1, restoresGAP43mRNA and protein levels in growth cones and rescues axon outgrowth defects in SMA neurons. These findings demonstrate that SMN plays an important role in the localization and local translation of mRNAs with important axonal functions and suggest that disruption of this function may contribute to the axonal defects observed in SMA. The motor neuron disease spinal muscular atrophy (SMA) is caused by reduced levels of the survival of motor neuron (SMN) protein, which plays a key role in assembling RNA/protein complexes that are essential for mRNA splicing. It remains unclear whether defects in this well characterized housekeeping function cause the specific degeneration of spinal motor neurons observed in SMA. Here, we describe an additional role of SMN in regulating the axonal localization and local translation of the mRNA encoding growth-associated protein 43 (GAP43). This study supports a model whereby SMN deficiency impedes transport and local translation of mRNAs important for neurite outgrowth and stabilization

Genetic and expression studies of SMN2 gene in Russian patients with spinal muscular atrophy type II and III

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Schiöth Helgi B

2011-07-01

Full Text Available Abstract Background Spinal muscular atrophy (SMA type I, II and III is an autosomal recessive neuromuscular disorder caused by mutations in the survival motor neuron gene (SMN1. SMN2 is a centromeric copy gene that has been characterized as a major modifier of SMA severity. SMA type I patients have one or two SMN2 copies while most SMA type II patients carry three SMN2 copies and SMA III patients have three or four SMN2 copies. The SMN1 gene produces a full-length transcript (FL-SMN while SMN2 is only able to produce a small portion of the FL-SMN because of a splice mutation which results in the production of abnormal SMNΔ7 mRNA. Methods In this study we performed quantification of the SMN2 gene copy number in Russian patients affected by SMA type II and III (42 and 19 patients, respectively by means of real-time PCR. Moreover, we present two families consisting of asymptomatic carriers of a homozygous absence of the SMN1 gene. We also developed a novel RT-qPCR-based assay to determine the FL-SMN/SMNΔ7 mRNA ratio as SMA biomarker. Results Comparison of the SMN2 copy number and clinical features revealed a significant correlation between mild clinical phenotype (SMA type III and presence of four copies of the SMN2 gene. In both asymptomatic cases we found an increased number of SMN2 copies in the healthy carriers and a biallelic SMN1 absence. Furthermore, the novel assay revealed a difference between SMA patients and healthy controls. Conclusions We suggest that the SMN2 gene copy quantification in SMA patients could be used as a prognostic tool for discrimination between the SMA type II and SMA type III diagnoses, whereas the FL-SMN/SMNΔ7 mRNA ratio could be a useful biomarker for detecting changes during SMA pharmacotherapy.

Deletion analysis of SMN1 and NAIP genes in southern Chinese children with spinal muscular atrophy

Institute of Scientific and Technical Information of China (English)

Yu-hua LIANG; Xiao-ling CHEN; Zhong-sheng YU; Chun-yue CHEN; Sheng BI; Lian-gen MAO; Bo-lin ZHOU; Xian-ning ZHANG

2009-01-01

Spinal muscular atrophy (SMA) is a disorder characterized by degeneration of lower motor neurons and occasionally bulbar motor neurons leading to progressive limb and trunk paralysis as well as muscular atrophy. Three types of SMA are rec-ognized depending on the age of onset, the maximum muscular activity achieved, and survivorship: SMA1, SMA2, and SMA3. The survival of motor neuron (SMN) gene has been identified as an SMA determining gene, whereas the neuronal apoptosis inhibitory protein (NAIP) gene is considered to be a modifying factor of the severity of SMA. The main objective of this study was to analyze the deletion of SMN1 and NAIP genes in southern Chinese children with SMA. Here, polymerase chain reaction (PCR) combined with restriction fragment length polymorphism (RFLP) was performed to detect the deletion of both exon 7 and exon 8 of SMNI and exon 5 of NAIP in 62 southern Chinese children with strongly suspected clinical symptoms of SMA. All the 32 SMAI patients and 76% (13/17) of SMA2 patients showed homozygous deletions for exon 7 and exon 8, and all the 13 SMA3 patients showed single deletion of SMN1 exon 7 along with 24% (4/17) of SMA2 patients. Eleven out of 32 (34%) SMA1 patients showed NAIP deletion, and none of SMA2 and SMA3 patients was found to have NAIP deletion. The findings of homozygous deletions of exon 7 and/or exon 8 of SMN1 gene confirmed the diagnosis of SMA, and suggested that the deletion of SMN1 exon 7 is a major cause of SMA in southern Chinese children, and that the NA1P gene may be a modifying factor for disease severity of SMA 1. The molecular diagnosis system based on PCR-RFLP analysis can conveniently be applied in the clinical testing, genetic counseling, prenatal diagnosis and preimplantation genetic diagnosis of SMA.

Bases celulares y moleculares de la atrofia muscular espinal : papel del factor de supervivencia de las motoneuronas (SMN)

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Castillo Iglesias, María Soledad

2016-01-01

La atrofia muscular espinal (SMA) es una enfermedad infantil hereditaria caracterizada por la muerte de las motoneuronas con denervación y atrofia muscular que puede ocasionar la muerte de los pacientes por insuficiencias respiratoria. Su herencia es autosómica recesiva por deleción o mutaciones en el gen “Survival Motor Neuron 1” (SMN1), que codifica la proteína SMN. En el hombre existe un gen homólogo, SMN2, que produce niveles muy bajos de SMN completa y no evita la aparición de la enferme...

Gestational Age-Dependent Increase of Survival Motor Neuron Protein in Umbilical Cord-Derived Mesenchymal Stem Cells

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Sota Iwatani

2017-09-01

Full Text Available BackgroundSpinal muscular atrophy (SMA is the most common genetic neurological disease leading to infant death. It is caused by loss of survival motor neuron (SMN 1 gene and subsequent reduction of SMN protein in motor neurons. Because SMN is ubiquitously expressed and functionally linked to general RNA metabolism pathway, fibroblasts (FBs are most widely used for the assessment of SMN expression in SMA patients but usually isolated from skin biopsy samples after the onset of overt symptoms. Although recent translational studies of SMN-targeted therapies have revealed the very limited time window for effective SMA therapies during perinatal period, the exact time point when SMN shortage became evident is unknown in human samples. In this study, we analyzed SMN mRNA and protein expression during perinatal period by using umbilical cord-derived mesenchymal stem cells (UC-MSCs obtained from preterm and term infants.MethodsUC-MSCs were isolated from 16 control infants delivered at 22–40 weeks of gestation and SMA fetus aborted at 19 weeks of gestation (UC-MSC-Control and UC-MSC-SMA. FBs were isolated from control volunteer and SMA patient (FB-Control and FB-SMA. SMN mRNA and protein expression in UC-MSCs and FBs was determined by RT-qPCR and Western blot.ResultsUC-MSC-Control and UC-MSC-SMA expressed the comparable level of MSC markers on their cell surface and were able to differentiate into adipocytes, osteocytes, and chondrocytes. At steady state, SMN mRNA and protein expression was decreased in UC-MSC-SMA compared to UC-MSC-Control, as observed in FB-SMA and FB-Control. In response to histone deacetylase inhibitor valproic acid, SMN mRNA and protein expression in UC-MSC-SMA and FB-SMA was increased. During perinatal development from 22 to 40 weeks of gestation, SMN mRNA and protein expression in UC-MSC-Control was positively correlated with gestational age.ConclusionUC-MSCs isolated from 17 fetus/infant of 19–40 weeks of gestation

Low levels of Survival Motor Neuron protein are sufficient for normal muscle function in the SMNΔ7 mouse model of SMA.

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Iyer, Chitra C; McGovern, Vicki L; Murray, Jason D; Gombash, Sara E; Zaworski, Phillip G; Foust, Kevin D; Janssen, Paul M L; Burghes, Arthur H M

2015-11-01

Spinal Muscular Atrophy (SMA) is an autosomal recessive disorder characterized by loss of lower motor neurons. SMA is caused by deletion or mutation of the Survival Motor Neuron 1 (SMN1) gene and retention of the SMN2 gene. The loss of SMN1 results in reduced levels of the SMN protein. SMN levels appear to be particularly important in motor neurons; however SMN levels above that produced by two copies of SMN2 have been suggested to be important in muscle. Studying the spatial requirement of SMN is important in both understanding how SMN deficiency causes SMA and in the development of effective therapies. Using Myf5-Cre, a muscle-specific Cre driver, and the Cre-loxP recombination system, we deleted mouse Smn in the muscle of mice with SMN2 and SMNΔ7 transgenes in the background, thus providing low level of SMN in the muscle. As a reciprocal experiment, we restored normal levels of SMN in the muscle with low SMN levels in all other tissues. We observed that decreasing SMN in the muscle has no phenotypic effect. This was corroborated by muscle physiology studies with twitch force, tetanic and eccentric contraction all being normal. In addition, electrocardiogram and muscle fiber size distribution were also normal. Replacement of Smn in muscle did not rescue SMA mice. Thus the muscle does not appear to require high levels of SMN above what is produced by two copies of SMN2 (and SMNΔ7). © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Description and physical localization of the bovine survival of motor neuron gene (SMN).

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Pietrowski, D; Goldammer, T; Meinert, S; Schwerin, M; Förster, M

1998-01-01

Proximal spinal muscular atrophy (SMA) is an autosomal recessive disease in humans and other mammals, characterized by degeneration of anterior horn cells of the spinal cord. In humans, the survival of motor neuron gene (SMN) has been recognized as the SMA-determining gene and has been mapped to 5q13. In cattle, SMA is a recurrent, inherited disease that plays an important economic role in breeding programs of Brown Swiss stock. Now we have identified the full- length cDNA sequence of the bovine SMN gene. Molecular analysis and characterization of the sequence documents 85% identity to its human counterpart and three evolutionarily conserved domains in different species. Physical mapping data reveals that bovine SMN is localized to chromosome region 20q12-->q13, supporting the conserved synteny of this chromosomal region between humans and cattle.

The water extract of Liuwei dihuang possesses multi-protective properties on neurons and muscle tissue against deficiency of survival motor neuron protein.

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Tseng, Yu-Ting; Jong, Yuh-Jyh; Liang, Wei-Fang; Chang, Fang-Rong; Lo, Yi-Ching

2017-10-15

Deficiency of survival motor neuron (SMN) protein, which is encoded by the SMN1 and SMN2 genes, induces widespread splicing defects mainly in spinal motor neurons, and leads to spinal muscular atrophy (SMA). Currently, there is no effective treatment for SMA. Liuwei dihuang (LWDH), a traditional Chinese herbal formula, possesses multiple therapeutic benefits against various diseases via modulation of the nervous, immune and endocrine systems. Previously, we demonstrated water extract of LWDH (LWDH-WE) protects dopaminergic neurons and improves motor activity in models of Parkinson's disease. This study aimed to investigate the potential protection of LWDH-WE on SMN deficiency-induced neurodegeneration and muscle weakness. The effects of LWDH-WE on SMN deficiency-induced neurotoxicity and muscle atrophy were examined by using SMN-deficient NSC34 motor neuron-like cells and SMA-like mice, respectively. Inducible SMN-knockdown NSC34 motor neuron-like cells were used to mimic SMN-deficient condition. Doxycycline (1 µg/ml) was used to induce SMN deficiency in stable NSC34 cell line carrying SMN-specific shRNA. SMAΔ7 mice were used as a severe type of SMA mouse model. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Apoptotic cells and neurite length were observed by inverted microscope. Protein expressions were examined by western blots. Muscle strength of animals was evaluated by hind-limb suspension test. LWDH-WE significantly increased SMN protein level, mitochondrial membrane potential and cell viability of SMN-deficient NSC34 cells. LWDH-WE attenuated SMN deficiency-induced down-regulation of B-cell lymphoma-2 (Bcl-2) and up-regulation of cytosolic cytochrome c and cleaved caspase-3. Moreover, LWDH-WE prevented SMN deficiency-induced inhibition of neurite outgrowth and activation of Ras homolog gene family, member A (RhoA)/ Rho-associated protein kinase (ROCK2)/ phospho

Investigation of New Morpholino Oligomers to Increase Survival Motor Neuron Protein Levels in Spinal Muscular Atrophy.

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Ramirez, Agnese; Crisafulli, Sebastiano G; Rizzuti, Mafalda; Bresolin, Nereo; Comi, Giacomo P; Corti, Stefania; Nizzardo, Monica

2018-01-06

Spinal muscular atrophy (SMA) is an autosomal-recessive childhood motor neuron disease and the main genetic cause of infant mortality. SMA is caused by deletions or mutations in the survival motor neuron 1 ( SMN1 ) gene, which results in SMN protein deficiency. Only one approved drug has recently become available and allows for the correction of aberrant splicing of the paralogous SMN2 gene by antisense oligonucleotides (ASOs), leading to production of full-length SMN protein. We have already demonstrated that a sequence of an ASO variant, Morpholino (MO), is particularly suitable because of its safety and efficacy profile and is both able to increase SMN levels and rescue the murine SMA phenotype. Here, we optimized this strategy by testing the efficacy of four new MO sequences targeting SMN2 . Two out of the four new MO sequences showed better efficacy in terms of SMN protein production both in SMA induced pluripotent stem cells (iPSCs) and SMAΔ7 mice. Further, the effect was enhanced when different MO sequences were administered in combination. Our data provide an important insight for MO-based treatment for SMA. Optimization of the target sequence and validation of a treatment based on a combination of different MO sequences could support further pre-clinical studies and the progression toward future clinical trials.

Determination of exon 7 SMN1 deletion in Iranian patients and ...

Indian Academy of Sciences (India)

In consideration of these defects and in line with the latter references, we have ... master mix (Roche, Mannheim, Germany), 5 ng of genomic. DNA, and 10 pmol of .... PCR test which can evaluate comprehensively the status of. SMN1 exon 7 ...

Gestational Age-Dependent Increase of Survival Motor Neuron Protein in Umbilical Cord-Derived Mesenchymal Stem Cells

OpenAIRE

Iwatani, Sota; Harahap, Nur Imma Fatimah; Nurputra, Dian Kesumapramudya; Tairaku, Shinya; Shono, Akemi; Kurokawa, Daisuke; Yamana, Keiji; Thwin, Khin Kyae Mon; Yoshida, Makiko; Mizobuchi, Masami; Koda, Tsubasa; Fujioka, Kazumichi; Taniguchi-Ikeda, Mariko; Yamada, Hideto; Morioka, Ichiro

2017-01-01

Background: Spinal muscular atrophy (SMA) is the most common genetic neurological disease leading to infant death. It is caused by loss of survival motor neuron (SMN) 1 gene and subsequent reduction of SMN protein in motor neurons. Because SMN is ubiquitously expressed and functionally linked to general RNA metabolism pathway, fibroblasts (FBs) are most widely used for the assessment of SMN expression in SMA patients but usually isolated from skin biopsy samples after the onset of overt sympt...

Aggregation of ALS-linked FUS mutant sequesters RNA binding proteins and impairs RNA granules formation

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Takanashi, Keisuke; Yamaguchi, Atsushi, E-mail: atsyama@restaff.chiba-u.jp

2014-09-26

Highlights: • Aggregation of ALS-linked FUS mutant sequesters ALS-associated RNA-binding proteins (FUS wt, hnRNP A1, and hnRNP A2). • Aggregation of ALS-linked FUS mutant sequesters SMN1 in the detergent-insoluble fraction. • Aggregation of ALS-linked FUS mutant reduced the number of speckles in the nucleus. • Overproduced ALS-linked FUS mutant reduced the number of processing-bodies (PBs). - Abstract: Protein aggregate/inclusion is one of hallmarks for neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). FUS/TLS, one of causative genes for familial ALS, encodes a multifunctional DNA/RNA binding protein predominantly localized in the nucleus. C-terminal mutations in FUS/TLS cause the retention and the inclusion of FUS/TLS mutants in the cytoplasm. In the present study, we examined the effects of ALS-linked FUS mutants on ALS-associated RNA binding proteins and RNA granules. FUS C-terminal mutants were diffusely mislocalized in the cytoplasm as small granules in transiently transfected SH-SY5Y cells, whereas large aggregates were spontaneously formed in ∼10% of those cells. hnRNP A1, hnRNP A2, and SMN1 as well as FUS wild type were assembled into stress granules under stress conditions, and these were also recruited to FUS mutant-derived spontaneous aggregates in the cytoplasm. These aggregates stalled poly(A) mRNAs and sequestered SMN1 in the detergent insoluble fraction, which also reduced the number of nuclear oligo(dT)-positive foci (speckles) in FISH (fluorescence in situ hybridization) assay. In addition, the number of P-bodies was decreased in cells harboring cytoplasmic granules of FUS P525L. These findings raise the possibility that ALS-linked C-terminal FUS mutants could sequester a variety of RNA binding proteins and mRNAs in the cytoplasmic aggregates, which could disrupt various aspects of RNA equilibrium and biogenesis.

SMA Diagnosis: Detection of SMN1 Deletion with Real-Time mCOP-PCR System Using Fresh Blood DNA.

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Niba, Emma Tabe Eko; Ar Rochmah, Mawaddah; Harahap, Nur Imma Fatimah; Awano, Hiroyuki; Morioka, Ichiro; Iijima, Kazumoto; Saito, Toshio; Saito, Kayoko; Takeuchi, Atsuko; Lai, Poh San; Bouike, Yoshihiro; Nishio, Hisahide; Shinohara, Masakazu

2017-12-18

Spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders. The symptoms are caused by defects of lower motor neurons in the spinal cord. More than 95% of SMA patients are homozygous for survival motor neuron 1 (SMN1) deletion. We previously developed a screening system for SMN1 deletion based on a modified competitive oligonucleotide priming-PCR (mCOP-PCR) technique using dried blood spot (DBS) on filter paper. This system is convenient for mass screening in the large population and/or first-tier diagnostic method of the patients in the remote areas. However, this system was still time-consuming and effort-taking, because it required pre-amplification procedure to avoid non-specific amplification and gel-electrophoresis to detect the presence or absence of SMN1 deletion. When the fresh blood samples are used instead of DBS, or when the gel-electrophoresis is replaced by real-time PCR, we may have a simpler and more rapid diagnostic method for SMA. To establish a simpler and more rapid diagnostic method of SMN1 deletion using fresh blood DNA. DNA samples extracted from fresh blood and stored at 4 ℃ for 1 month. The samples were assayed using a real-time mCOP-PCR system without pre-amplification procedures. DNA samples had already been genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP), showing the presence or absence of SMN1 exon 7. The DNA samples were directly subjected to the mCOP-PCR step. The amplification of mCOP-PCR was monitored in a real-time PCR apparatus. The genotyping results of the real-time mCOP-PCR system using fresh blood DNA were completely matched with those of PCR-RFLP. In this real-time mCOP-PCR system using fresh blood-DNA, it took only four hours from extraction of DNA to detection of the presence or absence of SMN1 deletion, while it took more than 12 hours in PCR-RFLP. Our real-time mCOP-PCR system using fresh blood DNA was rapid and accurate, suggesting it may be useful for the first

HuD and the Survival Motor Neuron Protein Interact in Motoneurons and Are Essential for Motoneuron Development, Function, and mRNA Regulation.

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Hao le, Thi; Duy, Phan Q; An, Min; Talbot, Jared; Iyer, Chitra C; Wolman, Marc; Beattie, Christine E

2017-11-29

Motoneurons establish a critical link between the CNS and muscles. If motoneurons do not develop correctly, they cannot form the required connections, resulting in movement defects or paralysis. Compromised development can also lead to degeneration because the motoneuron is not set up to function properly. Little is known, however, regarding the mechanisms that control vertebrate motoneuron development, particularly the later stages of axon branch and dendrite formation. The motoneuron disease spinal muscular atrophy (SMA) is caused by low levels of the survival motor neuron (SMN) protein leading to defects in vertebrate motoneuron development and synapse formation. Here we show using zebrafish as a model system that SMN interacts with the RNA binding protein (RBP) HuD in motoneurons in vivo during formation of axonal branches and dendrites. To determine the function of HuD in motoneurons, we generated zebrafish HuD mutants and found that they exhibited decreased motor axon branches, dramatically fewer dendrites, and movement defects. These same phenotypes are present in animals expressing low levels of SMN, indicating that both proteins function in motoneuron development. HuD binds and transports mRNAs and one of its target mRNAs, Gap43 , is involved in axonal outgrowth. We found that Gap43 was decreased in both HuD and SMN mutants. Importantly, transgenic expression of HuD in motoneurons of SMN mutants rescued the motoneuron defects, the movement defects, and Gap43 mRNA levels. These data support that the interaction between SMN and HuD is critical for motoneuron development and point to a role for RBPs in SMA. SIGNIFICANCE STATEMENT In zebrafish models of the motoneuron disease spinal muscular atrophy (SMA), motor axons fail to form the normal extent of axonal branches and dendrites leading to decreased motor function. SMA is caused by low levels of the survival motor neuron (SMN) protein. We show in motoneurons in vivo that SMN interacts with the RNA binding

Spinal muscular atrophy: Selective motor neuron loss and global defect in the assembly of ribonucleoproteins.

Science.gov (United States)

Beattie, Christine E; Kolb, Stephen J

2018-08-15

Spinal muscular atrophy is caused by deletions or mutations in the SMN1 gene that result in reduced expression of the SMN protein. The SMN protein is an essential molecular chaperone that is required for the biogenesis of multiple ribonucleoprotein (RNP) complexes including spliceosomal small nuclear RNPs (snRNPs). Reductions in SMN expression result in a reduced abundance of snRNPs and to downstream RNA splicing alterations. SMN is also present in axons and dendrites and appears to have important roles in the formation of neuronal mRNA-protein complexes during development or neuronal repair. Thus, SMA is an exemplar, selective motor neuron disorder that is caused by defects in fundamental RNA processing events. A detailed molecular understanding of how motor neurons fail, and why other neurons do not, in SMA will yield important principals about motor neuron maintenance and neuronal specificity in neurodegenerative diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

Influence of S/Mn molar ratio on the morphology and optical property of γ-MnS thin films prepared by microwave hydrothermal

International Nuclear Information System (INIS)

Yu, Xin; Li-yun, Cao; Jian-feng, Huang; Jia, Liu; Jie, Fei; Chun-yan, Yao

2013-01-01

Highlights: ► The influence of the precursor solution molar ratio of S/Mn. ► The degree of orientation of the γ-MnS film decrease slightly with increasing the S/Mn from 2.0 to 4.0. ► Film quality is strongly affected by the initial nucleation. ► The absorption edge obviously shifts to a higher wavelength with the increase of the S/Mn molar ratio from 2.0 to 4.0. - Abstract: Well crystallized γ-MnS thin films were successfully synthesized at low temperature and short processing time via a novel microwave hydrothermal (M-H) process without any complexing agent by using manganese chloride and thioacetamide as source materials. The influence of different S/Mn molar ratio in the precursor solution on the phase compositions, morphologies and optical properties of the as-deposited films was investigated. The as-deposited γ-MnS thin films were characterized by means of X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), field emission scanning electron microscopy (FESEM) and ultraviolet–visible (UV–vis). Results show that the wurtzite phase γ-MnS thin films with good crystallization can be achieved when S/Mn molar ratio is controlled at 2.0–4.0. The deposited γ-MnS thin films exhibit (1 0 0) orientation growth with the thickness of 300–500 nm. With the increase of S/Mn molar ratio from 2.0 to 4.0, the orientation growth is weakened while the dense and uniform of the as-deposited γ-MnS thin films are obviously improved and the corresponding band gap of the thin films increase from 3.88 to 3.97 eV.

Spinal Muscular Atrophy: More than a Disease of Motor Neurons?

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Nash, L A; Burns, J K; Chardon, J Warman; Kothary, R; Parks, R J

2016-01-01

Spinal muscular atrophy (SMA) is the most common genetically inherited neurodegenerative disease resulting in infant mortality. SMA is caused by genetic deletion or mutation in the survival of motor neuron 1 (SMN1) gene, which results in reduced levels of the survival of motor neuron (SMN) protein. SMN protein deficiency preferentially affects α- motor neurons, leading to their degeneration and subsequent atrophy of limb and trunk muscles, progressing to death in severe forms of the disease. More recent studies have shown that SMN protein depletion is detrimental to the functioning of other tissues including skeletal muscle, heart, autonomic and enteric nervous systems, metabolic/endocrine (e.g. pancreas), lymphatic, bone and reproductive system. In this review, we summarize studies discussing SMN protein's function in various cell and tissue types and their involvement in the context of SMA disease etiology. Taken together, these studies indicate that SMA is a multi-organ disease, which suggests that truly effective disease intervention may require body-wide correction of SMN protein levels. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

New, Improved Version of the mCOP-PCR Screening System for Detection of Spinal Muscular Atrophy Gene (SMN1) Deletion.

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Shinohara, Masakazu; Ar Rochmah, Mawaddah; Nakanishi, Kenta; Harahap, Nur Imma Fatimah; Niba, Emma Tabe Eko; Saito, Toshio; Saito, Kayoko; Takeuchi, Atsuko; Bouike, Yoshihiro; Nishio, Hisahide

2017-09-07

Spinal muscular atrophy (SMA) is a frequent autosomal recessive disorder, characterized by lower motor neuron loss in the spinal cord. More than 95% of SMA patients show homozygous survival motor neuron 1 (SMN1) deletion. We previously developed a screening system for SMN1 deletion based on a modified competitive oligonucleotide priming-PCR (mCOP-PCR) technique. However, non-specific amplification products were observed with mCOP-PCR, which might lead to erroneous interpretation of the screening results. To establish an improved version of the mCOP-PCR screening system without non-specific amplification. DNA samples were assayed using a new version of the mCOP-PCR screening system. DNA samples had already been genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP), showing the presence or absence of SMN1 exon 7. The new mCOP-PCR method contained a targeted pre-amplification step of the region, including an SMN1-specific nucleotide, prior to the mCOP-PCR step. mCOP-PCR products were electrophoresed on agarose gels. No non-specific amplification products were detected in electrophoresis gels with the new mCOP-PCR screening system. An additional targeted pre-amplification step eliminated non-specific amplification from mCOP-PCR screening.

Survival motor neuron protein in motor neurons determines synaptic integrity in spinal muscular atrophy.

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Martinez, Tara L; Kong, Lingling; Wang, Xueyong; Osborne, Melissa A; Crowder, Melissa E; Van Meerbeke, James P; Xu, Xixi; Davis, Crystal; Wooley, Joe; Goldhamer, David J; Lutz, Cathleen M; Rich, Mark M; Sumner, Charlotte J

2012-06-20

The inherited motor neuron disease spinal muscular atrophy (SMA) is caused by deficient expression of survival motor neuron (SMN) protein and results in severe muscle weakness. In SMA mice, synaptic dysfunction of both neuromuscular junctions (NMJs) and central sensorimotor synapses precedes motor neuron cell death. To address whether this synaptic dysfunction is due to SMN deficiency in motor neurons, muscle, or both, we generated three lines of conditional SMA mice with tissue-specific increases in SMN expression. All three lines of mice showed increased survival, weights, and improved motor behavior. While increased SMN expression in motor neurons prevented synaptic dysfunction at the NMJ and restored motor neuron somal synapses, increased SMN expression in muscle did not affect synaptic function although it did improve myofiber size. Together these data indicate that both peripheral and central synaptic integrity are dependent on motor neurons in SMA, but SMN may have variable roles in the maintenance of these different synapses. At the NMJ, it functions at the presynaptic terminal in a cell-autonomous fashion, but may be necessary for retrograde trophic signaling to presynaptic inputs onto motor neurons. Importantly, SMN also appears to function in muscle growth and/or maintenance independent of motor neurons. Our data suggest that SMN plays distinct roles in muscle, NMJs, and motor neuron somal synapses and that restored function of SMN at all three sites will be necessary for full recovery of muscle power.

Overexpression of survival motor neuron improves neuromuscular function and motor neuron survival in mutant SOD1 mice.

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Turner, Bradley J; Alfazema, Neza; Sheean, Rebecca K; Sleigh, James N; Davies, Kay E; Horne, Malcolm K; Talbot, Kevin

2014-04-01

Spinal muscular atrophy results from diminished levels of survival motor neuron (SMN) protein in spinal motor neurons. Low levels of SMN also occur in models of amyotrophic lateral sclerosis (ALS) caused by mutant superoxide dismutase 1 (SOD1) and genetic reduction of SMN levels exacerbates the phenotype of transgenic SOD1(G93A) mice. Here, we demonstrate that SMN protein is significantly reduced in the spinal cords of patients with sporadic ALS. To test the potential of SMN as a modifier of ALS, we overexpressed SMN in 2 different strains of SOD1(G93A) mice. Neuronal overexpression of SMN significantly preserved locomotor function, rescued motor neurons, and attenuated astrogliosis in spinal cords of SOD1(G93A) mice. Despite this, survival was not prolonged, most likely resulting from SMN mislocalization and depletion of gems in motor neurons of symptomatic mice. Our results reveal that SMN upregulation slows locomotor deficit onset and motor neuron loss in this mouse model of ALS. However, disruption of SMN nuclear complexes by high levels of mutant SOD1, even in the presence of SMN overexpression, might limit its survival promoting effects in this specific mouse model. Studies in emerging mouse models of ALS are therefore warranted to further explore the potential of SMN as a modifier of ALS. Copyright © 2014 Elsevier Inc. All rights reserved.

Motor neuronal repletion of the NMJ organizer, Agrin, modulates the severity of the spinal muscular atrophy disease phenotype in model mice.

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Kim, Jeong-Ki; Caine, Charlotte; Awano, Tomoyuki; Herbst, Ruth; Monani, Umrao R

2017-07-01

Spinal muscular atrophy (SMA) is a common and often fatal neuromuscular disorder caused by low levels of the Survival Motor Neuron (SMN) protein. Amongst the earliest detectable consequences of SMN deficiency are profound defects of the neuromuscular junctions (NMJs). In model mice these synapses appear disorganized, fail to mature and are characterized by poorly arborized nerve terminals. Given one role of the SMN protein in orchestrating the assembly of spliceosomal snRNP particles and subsequently regulating the alternative splicing of pre-mRNAs, a plausible link between SMN function and the distal neuromuscular SMA phenotype is an incorrectly spliced transcript or transcripts involved in establishing or maintaining NMJ structure. In this study, we explore the effects of one such transcript-Z+Agrin-known to be a critical organizer of the NMJ. We confirm that low SMN protein reduces motor neuronal levels of Z+Agrin. Repletion of this isoform of Agrin in the motor neurons of SMA model mice increases muscle fiber size, enhances the post-synaptic NMJ area, reduces the abnormal accumulation of intermediate filaments in nerve terminals of the neuromuscular synapse and improves the innervation of muscles. While these effects are independent of changes in SMN levels or increases in motor neuron numbers they nevertheless have a significant effect on the overall disease phenotype, enhancing mean survival in severely affected SMA model mice by ∼40%. We conclude that Agrin is an important target of the SMN protein and that mitigating NMJ defects may be one strategy in treating human spinal muscular atrophy. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Silymarin modulates doxorubicin-induced oxidative stress, Bcl-xL and p53 expression while preventing apoptotic and necrotic cell death in the liver

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Patel, Nirav; Joseph, Cecil; Corcoran, George B.; Ray, Sidhartha D.

2010-01-01

The emergence of silymarin (SMN) as a natural remedy for liver diseases, coupled with its entry into NIH clinical trial, signifies its hepatoprotective potential. SMN is noted for its ability to interfere with apoptotic signaling while acting as an antioxidant. This in vivo study was designed to explore the hepatotoxic potential of Doxorubicin (Dox), the well-known cardiotoxin, and in particular whether pre-exposures to SMN can prevent hepatotoxicity by reducing Dox-induced free radical mediated oxidative stress, by modulating expression of apoptotic signaling proteins like Bcl-xL, and by minimizing liver cell death occurring by apoptosis or necrosis. Groups of male ICR mice included Control, Dox alone, SMN alone, and Dox with SMN pre/co-treatment. Control and Dox groups received saline i.p. for 14 days. SMN was administered p.o. for 14 days at 16 mg/kg/day. An approximate LD 50 dose of Dox, 60 mg/kg, was administered i.p. on day 12 to animals receiving saline or SMN. Animals were euthanized 48 h later. Dox alone induced frank liver injury (> 50-fold increase in serum ALT) and oxidative stress (> 20-fold increase in malondialdehyde [MDA]), as well as direct damage to DNA (> 15-fold increase in DNA fragmentation). Coincident genomic damage and oxidative stress influenced genomic stability, reflected in increased PARP activity and p53 expression. Decreases in Bcl-xL protein coupled with enhanced accumulation of cytochrome c in the cytosol accompanied elevated indexes of apoptotic and necrotic cell death. Significantly, SMN exposure reduced Dox hepatotoxicity and associated apoptotic and necrotic cell death. The effects of SMN on Dox were broad, including the ability to modulate changes in both Bcl-xL and p53 expression. In animals treated with SMN, tissue Bcl-xL expression exceeded control values after Dox treatment. Taken together, these results demonstrated that SMN (i) reduced, delayed onset, or prevented toxic effects of Dox which are typically associated with

Candidate proteins, metabolites and transcripts in the Biomarkers for Spinal Muscular Atrophy (BforSMA clinical study.

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Richard S Finkel

Full Text Available Spinal Muscular Atrophy (SMA is a neurodegenerative motor neuron disorder resulting from a homozygous mutation of the survival of motor neuron 1 (SMN1 gene. The gene product, SMN protein, functions in RNA biosynthesis in all tissues. In humans, a nearly identical gene, SMN2, rescues an otherwise lethal phenotype by producing a small amount of full-length SMN protein. SMN2 copy number inversely correlates with disease severity. Identifying other novel biomarkers could inform clinical trial design and identify novel therapeutic targets.To identify novel candidate biomarkers associated with disease severity in SMA using unbiased proteomic, metabolomic and transcriptomic approaches.A cross-sectional single evaluation was performed in 108 children with genetically confirmed SMA, aged 2-12 years, manifesting a broad range of disease severity and selected to distinguish factors associated with SMA type and present functional ability independent of age. Blood and urine specimens from these and 22 age-matched healthy controls were interrogated using proteomic, metabolomic and transcriptomic discovery platforms. Analyte associations were evaluated against a primary measure of disease severity, the Modified Hammersmith Functional Motor Scale (MHFMS and to a number of secondary clinical measures.A total of 200 candidate biomarkers correlate with MHFMS scores: 97 plasma proteins, 59 plasma metabolites (9 amino acids, 10 free fatty acids, 12 lipids and 28 GC/MS metabolites and 44 urine metabolites. No transcripts correlated with MHFMS.In this cross-sectional study, "BforSMA" (Biomarkers for SMA, candidate protein and metabolite markers were identified. No transcript biomarker candidates were identified. Additional mining of this rich dataset may yield important insights into relevant SMA-related pathophysiology and biological network associations. Additional prospective studies are needed to confirm these findings, demonstrate sensitivity to change with

Enhanced degradation of 2-nitrotoluene by immobilized cells of Micrococcus sp. strain SMN-1.

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Mulla, Sikandar I; Talwar, Manjunatha P; Bagewadi, Zabin K; Hoskeri, Robertcyril S; Ninnekar, Harichandra Z

2013-02-01

Nitrotoluenes are the toxic pollutants of the environment because of their large scale use in the production of explosives. Biodegradation of such chemicals by microorganisms may provide an effective method for their detoxification. We have studied the degradation of 2-nitrotoluene by cells of Micrococcus sp. strain SMN-1 immobilized in various matrices such as polyurethane foam (PUF), sodium alginate (SA), sodium alginate-polyvinyl alcohol (SA-PVA), agar and polyacrylamide. The rate of degradation of 15 and 30 mM 2-nitrotoluene by freely suspended cells and immobilized cells in batches and fed-batch with shaken cultures were compared. The PUF-immobilized cells achieved higher degradation of 15 and 30 mM 2-nitrotoluene than freely suspended cells and the cells immobilized in SA-PVA, polyacrylamide, SA and agar. The PUF-immobilized cells could be reused more than 24 cycles without loosing their degradation capacity and showed more tolerance to pH and temperature changes than freely suspended cells. These results revealed the enhanced rate of degradation of 2-nitrotoluene by PUF-immobilized cells of Micrococcus sp. strain SMN-1. Copyright © 2012 Elsevier Ltd. All rights reserved.

The Epstein-Barr virus nuclear antigen-6 protein co-localizes with EBNA-3 and survival of motor neurons protein

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Krauer, Kenia G.; Buck, Marion; Belzer, Deanna K.; Flanagan, James; Chojnowski, Grace M.; Sculley, Tom B.

2004-01-01

The Epstein-Barr virus nuclear antigen (EBNA)-6 protein is essential for Epstein-Barr virus (EBV)-induced immortalization of primary human B-lymphocytes in vitro. In this study, fusion proteins of EBNA-6 with green fluorescent protein (GFP) have been used to characterize its nuclear localization and organization within the nucleus. EBNA-6 associates with nuclear structures and in immunofluorescence demonstrate a punctate staining pattern. Herein, we show that the association of EBNA-6 with these nuclear structures was maintained throughout the cell cycle and with the use of GFP-E6 deletion mutants, that the region amino acids 733-808 of EBNA-6 contains a domain that can influence the association of EBNA-6 with these nuclear structures. Co-immunofluorescence and confocal analyses demonstrated that EBNA-6 and EBNA-3 co-localize in the nucleus of cells. Expression of EBNA-6, but not EBNA-3, caused a redistribution of nuclear survival of motor neurons protein (SMN) to the EBNA-6 containing nuclear structures resulting in co-localization of SMN with EBNA-6

Biodegradation of 2-nitrotoluene by Micrococcus sp. strain SMN-1.

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Mulla, Sikandar I; Hoskeri, Robertcyril S; Shouche, Yogesh S; Ninnekar, Harichandra Z

2011-02-01

A bacterial consortium capable of degrading nitroaromatic compounds was isolated from pesticide-contaminated soil samples by selective enrichment on 2-nitrotoluene as a sole source of carbon and energy. The three different bacterial isolates obtained from bacterial consortium were identified as Bacillus sp. (A and C), Bacillus flexus (B) and Micrococcus sp. (D) on the basis of their morphological and biochemical characteristics and by phylogenetic analysis based on 16S rRNA gene sequences. The pathway for the degradation of 2-nitrotoluene by Micrococcus sp. strain SMN-1 was elucidated by the isolation and identification of metabolites, growth and enzymatic studies. The organism degraded 2-nitrotoluene through 3-methylcatechol by a meta-cleavage pathway, with release of nitrite.

Spinal muscular atrophy pathogenic mutations impair the axonogenic properties of axonal-survival of motor neuron.

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Locatelli, Denise; d'Errico, Paolo; Capra, Silvia; Finardi, Adele; Colciaghi, Francesca; Setola, Veronica; Terao, Mineko; Garattini, Enrico; Battaglia, Giorgio

2012-05-01

The axonal survival of motor neuron (a-SMN) protein is a truncated isoform of SMN1, the spinal muscular atrophy (SMA) disease gene. a-SMN is selectively localized in axons and endowed with remarkable axonogenic properties. At present, the role of a-SMN in SMA is unknown. As a first step to verify a link between a-SMN and SMA, we investigated by means of over-expression experiments in neuroblastoma-spinal cord hybrid cell line (NSC34) whether SMA pathogenic mutations located in the N-terminal part of the protein affected a-SMN function. We demonstrated here that either SMN1 missense mutations or small intragenic re-arrangements located in the Tudor domain consistently altered the a-SMN capability of inducing axonal elongation in vitro. Mutated human a-SMN proteins determined in almost all NSC34 motor neurons the growth of short axons with prominent morphologic abnormalities. Our data indicate that the Tudor domain is critical in dictating a-SMN function possibly because it is an association domain for proteins involved in axon growth. They also indicate that Tudor domain mutations are functionally relevant not only for FL-SMN but also for a-SMN, raising the possibility that also a-SMN loss of function may contribute to the pathogenic steps leading to SMA. © 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.

Tim50a, a nuclear isoform of the mitochondrial Tim50, interacts with proteins involved in snRNP biogenesis

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Robinson Melvin L

2005-07-01

Full Text Available Abstract Background The Cajal body (CB is a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs, which are vital for pre-mRNA splicing. Newly imported Sm-class snRNPs traffic through CBs, where the snRNA component of the snRNP is modified, and then target to other nuclear domains such as speckles and perichromatin fibrils. It is not known how nascent snRNPs localize to the CB and are released from this structure after modification. The marker protein for CBs, coilin, may play a role in snRNP biogenesis given that it can interact with snRNPs and SMN, the protein mutated in Spinal Muscular Atrophy. Loss of coilin function in mice leads to significant viability and fertility problems and altered CB formation. Results In this report, we identify a minor isoform of the mitochondrial Tim50, Tim50a, as a coilin interacting protein. The Tim50a transcript can be detected in some cancer cell lines and normal brain tissue. The Tim50a protein differs only from Tim50 in that it contains an additional 103 aa N-terminal to the translation start of Tim50. Importantly, a putative nuclear localization signal is found within these 103 residues. In contrast to Tim50, which localizes to the cytoplasm and mitochondria, Tim50a is strictly nuclear and is enriched in speckles with snRNPs. In addition to coilin, Tim50a interacts with snRNPs and SMN. Competition binding experiments demonstrate that coilin competes with Sm proteins of snRNPs and SMN for binding sites on Tim50a. Conclusion Tim50a may play a role in snRNP biogenesis given its cellular localization and protein interaction characteristics. We hypothesize that Tim50a takes part in the release of snRNPs and SMN from the CB.

Antibacterial activity of the Antarctic bacterium Janthinobacterium sp. SMN 33.6 against multi-resistant Gram-negative bacteria

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Geraldine Asencio

2014-01-01

Conclusions: The ethanolic extract of Janthinobacterium sp. SMN 33.6 possesses antibacterial activity against a chromosomal AmpC beta-lactamase-producing strain of Serratia marcescens, an extended-spectrum beta-lactamase-producing Escherichia coli and also against carbapenemase-producing strains of Acinetobacter baumannii and Pseudomonas aeruginosa. This becomes a potential and interesting biotechnological tool for the control of bacteria with multi-resistance to commonly used antibiotics.

Gemin5: A Multitasking RNA-Binding Protein Involved in Translation Control

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David Piñeiro

2015-04-01

Full Text Available Gemin5 is a RNA-binding protein (RBP that was first identified as a peripheral component of the survival of motor neurons (SMN complex. This predominantly cytoplasmic protein recognises the small nuclear RNAs (snRNAs through its WD repeat domains, allowing assembly of the SMN complex into small nuclear ribonucleoproteins (snRNPs. Additionally, the amino-terminal end of the protein has been reported to possess cap-binding capacity and to interact with the eukaryotic initiation factor 4E (eIF4E. Gemin5 was also shown to downregulate translation, to be a substrate of the picornavirus L protease and to interact with viral internal ribosome entry site (IRES elements via a bipartite non-canonical RNA-binding site located at its carboxy-terminal end. These features link Gemin5 with translation control events. Thus, beyond its role in snRNPs biogenesis, Gemin5 appears to be a multitasking protein cooperating in various RNA-guided processes. In this review, we will summarise current knowledge of Gemin5 functions. We will discuss the involvement of the protein on translation control and propose a model to explain how the proteolysis fragments of this RBP in picornavirus-infected cells could modulate protein synthesis.

Edaravone is a candidate agent for spinal muscular atrophy: In vitro analysis using a human induced pluripotent stem cells-derived disease model.

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Ando, Shiori; Funato, Michinori; Ohuchi, Kazuki; Kameyama, Tsubasa; Inagaki, Satoshi; Seki, Junko; Kawase, Chizuru; Tsuruma, Kazuhiro; Shimazawa, Masamitsu; Kaneko, Hideo; Hara, Hideaki

2017-11-05

Spinal muscular atrophy (SMA) is an intractable disease characterized by a progressive loss of spinal motor neurons, which leads to skeletal muscle weakness and atrophy. Currently, there are no curative agents for SMA, although it is understood to be caused by reduced levels of survival motor neuron (SMN) protein. Additionally, why reduced SMN protein level results in selective apoptosis in spinal motor neurons is still not understood. Our purpose in this study was to evaluate the therapeutic potential of edaravone, a free radical scavenger, by using induced pluripotent stem cells from an SMA patient (SMA-iPSCs) and to address oxidative stress-induced apoptosis in spinal motor neurons. We first found that edaravone could improve impaired neural development of SMA-iPSCs-derived spinal motor neurons with limited effect on nuclear SMN protein expression. Furthermore, edaravone inhibited the generation of reactive oxygen species and mitochondrial reactive oxygen species upregulated in SMA-iPSCs-derived spinal motor neurons, and reversed oxidative-stress induced apoptosis. In this study, we suggest that oxidative stress might be partly the reason for selective apoptosis in spinal motor neurons in SMA pathology, and that oxidative stress-induced apoptosis might be the therapeutic target of SMA. Copyright © 2017 Elsevier B.V. All rights reserved.

Molecular Mechanisms of Neurodegeneration in Spinal Muscular Atrophy

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Saif Ahmad

2016-01-01

Full Text Available Spinal muscular atrophy (SMA is an autosomal recessive motor neuron disease with a high incidence and is the most common genetic cause of infant mortality. SMA is primarily characterized by degeneration of the spinal motor neurons that leads to skeletal muscle atrophy followed by symmetric limb paralysis, respiratory failure, and death. In humans, mutation of the Survival Motor Neuron 1 (SMN1 gene shifts the load of expression of SMN protein to the SMN2 gene that produces low levels of full-length SMN protein because of alternative splicing, which are sufficient for embryonic development and survival but result in SMA. The molecular mechanisms of the (a regulation of SMN gene expression and (b degeneration of motor neurons caused by low levels of SMN are unclear. However, some progress has been made in recent years that have provided new insights into understanding of the cellular and molecular basis of SMA pathogenesis. In this review, we have briefly summarized recent advances toward understanding of the molecular mechanisms of regulation of SMN levels and signaling mechanisms that mediate neurodegeneration in SMA.

Does the survival motor neuron copy number variation play a role in the onset and severity of sporadic amyotrophic lateral sclerosis in Malians?

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Sangare, Modibo; Dicko, Ilo; Guinto, Cheick Oumar; Sissoko, Adama; Dembele, Kekouta; Coulibaly, Youlouza; Coulibaly, Siaka Y; Landoure, Guida; Diallo, Abdallah; Dolo, Mamadou; Dolo, Housseini; Maiga, Boubacar; Traore, Moussa; Karembe, Mamadou; Traore, Kadiatou; Toure, Amadou; Sylla, Mariam; Togora, Arouna; Coulibaly, Souleymane; Traore, Sékou Fantamady; Hendrickson, Brant; Bricceno, Katherine; Schindler, Alice B; Kokkinis, Angela; Meilleur, Katherine G; Sangho, Hammadoun Ali; Diakite, Brehima; Kassogue, Yaya; Coulibaly, Yaya Ibrahim; Burnett, Barrington; Maiga, Youssoufa; Doumbia, Seydou; Fischbeck, Kenneth H

2016-06-01

Spinal muscular atrophy (SMA) and sporadic amyotrophic lateral sclerosis (SALS) are both motor neuron disorders. SMA results from the deletion of the survival motor neuron ( SMN ) 1 gene. High or low SMN1 copy number and the absence of SMN2 have been reported as risk factors for the development or severity of SALS. To investigate the role of SMN gene copy number in the onset and severity of SALS in Malians. We determined the SMN1 and SMN2 copy number in genomic DNA samples from 391 Malian adult volunteers, 120 Yoruba from Nigeria, 120 Luyha from Kenya and 74 U.S. Caucasians using a Taqman quantitative PCR assay. We evaluated the SALS risk based on the estimated SMA protein level using the Veldink formula ( SMN1 copy number + 0.2 ∗  SMN2 copy number). We also characterized the disease natural history in 15 ALS patients at the teaching hospital of Point G, Bamako, Mali. We found that 131 of 391 (33.5%) had an estimated SMN protein expression of ≤ 2.2; 60 out of 391 (15.3%) had an estimated SMN protein expression < 2 and would be at risk of ALS and the disease onset was as early as 16 years old. All 15 patients were male and some were physically handicapped within 1-2 years in the disease course. Because of the short survival time of our patients, family histories and sample DNA for testing were not done. However, our results show that sporadic ALS is of earlier onset and shorter survival time as compared to patients elsewhere. We plan to establish a network of neurologists and researchers for early screening of ALS.

Reduced salience and default mode network activity in women with anorexia nervosa

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McFadden, Kristina L.; Tregellas, Jason R.; Shott, Megan E.; Frank, Guido K.W.

2014-01-01

Background The neurobiology of anorexia nervosa is poorly understood. Neuronal networks contributing to action selection, self-regulation and interoception could contribute to pathologic eating and body perception in people with anorexia nervosa. We tested the hypothesis that the salience network (SN) and default mode network (DMN) would show decreased intrinsic activity in women with anorexia nervosa and those who had recovered from the disease compared to controls. The basal ganglia (BGN) and sensorimotor networks (SMN) were also investigated. Methods Between January 2008 and January 2012, women with restricting-type anorexia nervosa, women who recovered from the disease and healthy control women completed functional magnetic resonance imaging during a conditioned stimulus task. Network activity was studied using independent component analysis. Results We studied 20 women with anorexia nervosa, 24 recovered women and 24 controls. Salience network activity in the anterior cingulate cortex was reduced in women with anorexia nervosa (p = 0.030; all results false-discovery rate–corrected) and recovered women (p = 0.039) compared to controls. Default mode network activity in the precuneus was reduced in women with anorexia compared to controls (p = 0.023). Sensorimotor network activity in the supplementary motor area (SMA; p = 0.008), and the left (p = 0.028) and right (p = 0.002) postcentral gyrus was reduced in women with anorexia compared to controls; SMN activity in the SMA (p = 0.019) and the right postcentral gyrus (p = 0.008) was reduced in women with anorexia compared to recovered women. There were no group differences in the BGN. Limitations Differences between patient and control populations (e.g., depression, anxiety, medication) are potential confounds, but were included as covariates. Conclusion Reduced SN activity in women with anorexia nervosa and recovered women could be a trait-related biomarker or illness remnant, altering the drive to approach

Abnormal mitochondrial transport and morphology as early pathological changes in human models of spinal muscular atrophy

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Chong-Chong Xu

2016-01-01

Full Text Available Spinal muscular atrophy (SMA, characterized by specific degeneration of spinal motor neurons, is caused by mutations in the survival of motor neuron 1, telomeric (SMN1 gene and subsequent decreased levels of functional SMN. How the deficiency of SMN, a ubiquitously expressed protein, leads to spinal motor neuron-specific degeneration in individuals affected by SMA remains unknown. In this study, we examined the role of SMN in mitochondrial axonal transport and morphology in human motor neurons by generating SMA type 1 patient-specific induced pluripotent stem cells (iPSCs and differentiating these cells into spinal motor neurons. The initial specification of spinal motor neurons was not affected, but these SMA spinal motor neurons specifically degenerated following long-term culture. Moreover, at an early stage in SMA spinal motor neurons, but not in SMA forebrain neurons, the number of mitochondria, mitochondrial area and mitochondrial transport were significantly reduced in axons. Knocking down of SMN expression led to similar mitochondrial defects in spinal motor neurons derived from human embryonic stem cells, confirming that SMN deficiency results in impaired mitochondrial dynamics. Finally, the application of N-acetylcysteine (NAC mitigated the impairment in mitochondrial transport and morphology and rescued motor neuron degeneration in SMA long-term cultures. Furthermore, NAC ameliorated the reduction in mitochondrial membrane potential in SMA spinal motor neurons, suggesting that NAC might rescue apoptosis and motor neuron degeneration by improving mitochondrial health. Overall, our data demonstrate that SMN deficiency results in abnormal mitochondrial transport and morphology and a subsequent reduction in mitochondrial health, which are implicated in the specific degeneration of spinal motor neurons in SMA.

Elevated Body Mass Index is Associated with Increased Integration and Reduced Cohesion of Sensory-Driven and Internally Guided Resting-State Functional Brain Networks.

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Doucet, Gaelle E; Rasgon, Natalie; McEwen, Bruce S; Micali, Nadia; Frangou, Sophia

2018-03-01

Elevated body mass index (BMI) is associated with increased multi-morbidity and mortality. The investigation of the relationship between BMI and brain organization has the potential to provide new insights relevant to clinical and policy strategies for weight control. Here, we quantified the association between increasing BMI and the functional organization of resting-state brain networks in a sample of 496 healthy individuals that were studied as part of the Human Connectome Project. We demonstrated that higher BMI was associated with changes in the functional connectivity of the default-mode network (DMN), central executive network (CEN), sensorimotor network (SMN), visual network (VN), and their constituent modules. In siblings discordant for obesity, we showed that person-specific factors contributing to obesity are linked to reduced cohesiveness of the sensory networks (SMN and VN). We conclude that higher BMI is associated with widespread alterations in brain networks that balance sensory-driven (SMN, VN) and internally guided (DMN, CEN) states which may augment sensory-driven behavior leading to overeating and subsequent weight gain. Our results provide a neurobiological context for understanding the association between BMI and brain functional organization while accounting for familial and person-specific influences. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Evaluation of muscle strength and motor abilities in children with type II and III spinal muscle atrophy treated with valproic acid

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Zanoteli Edmar

2011-03-01

Full Text Available Abstract Background Spinal muscular atrophy (SMA is an autosomal recessive disorder that affects the motoneurons of the spinal anterior horn, resulting in hypotonia and muscle weakness. The disease is caused by deletion or mutation in the telomeric copy of SMN gene (SMN1 and clinical severity is in part determined by the copy number of the centromeric copy of the SMN gene (SMN2. The SMN2 mRNA lacks exon 7, resulting in a production of lower amounts of the full-length SMN protein. Knowledge of the molecular mechanism of diseases has led to the discovery of drugs capable of increasing SMN protein level through activation of SMN2 gene. One of these drugs is the valproic acid (VPA, a histone deacetylase inhibitor. Methods Twenty-two patients with type II and III SMA, aged between 2 and 18 years, were treated with VPA and were evaluated five times during a one-year period using the Manual Muscle Test (Medical Research Council scale-MRC, the Hammersmith Functional Motor Scale (HFMS, and the Barthel Index. Results After 12 months of therapy, the patients did not gain muscle strength. The group of children with SMA type II presented a significant gain in HFMS scores during the treatment. This improvement was not observed in the group of type III patients. The analysis of the HFMS scores during the treatment period in the groups of patients younger and older than 6 years of age did not show any significant result. There was an improvement of the daily activities at the end of the VPA treatment period. Conclusion Treatment of SMA patients with VPA may be a potential alternative to alleviate the progression of the disease. Trial Registration ClinicalTrials.gov: NCT01033331

Inter-Institutional Comparison of Personalized Risk Assessments for Second Malignant Neoplasms for a 13-Year-Old Girl Receiving Proton versus Photon Craniospinal Irradiation

Energy Technology Data Exchange (ETDEWEB)

Taddei, Phillip J., E-mail: pt06@aub.edu.lb [Division of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Unit 1202, 1515 Holcombe Blvd, Houston, TX 77030 (United States); The University of Texas Graduate School of Biomedical Sciences at Houston, P.O. Box 20334, Houston, TX 77225 (United States); Department of Radiation Oncology, American University of Beirut Medical Center, P.O. Box 11-0236, Riad El Solh, Beirut 1107 2020 (Lebanon); Khater, Nabil [Department of Radiation Oncology, Hôtel-Dieu de France Hospital, University of St. Joseph, P.O. Box 166830, Alfred Naccache Blvd, Beirut (Lebanon); Zhang, Rui [Medical Physics Program, Department of Physics and Astronomy, Louisiana State University, 202 Nicholson Hall, Tower Dr., Baton Rouge, LA 70803 (United States); Department of Physics, Mary Bird Perkins Cancer Center, 4950 Essen Lane, Baton Rouge, LA 70809 (United States); Geara, Fady B. [Department of Radiation Oncology, American University of Beirut Medical Center, P.O. Box 11-0236, Riad El Solh, Beirut 1107 2020 (Lebanon); Mahajan, Anita [Division of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Unit 1202, 1515 Holcombe Blvd, Houston, TX 77030 (United States); The University of Texas Graduate School of Biomedical Sciences at Houston, P.O. Box 20334, Houston, TX 77225 (United States); Jalbout, Wassim [Department of Radiation Oncology, American University of Beirut Medical Center, P.O. Box 11-0236, Riad El Solh, Beirut 1107 2020 (Lebanon); Pérez-Andújar, Angélica [Division of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Unit 1202, 1515 Holcombe Blvd, Houston, TX 77030 (United States); Youssef, Bassem [Department of Radiation Oncology, American University of Beirut Medical Center, P.O. Box 11-0236, Riad El Solh, Beirut 1107 2020 (Lebanon); Newhauser, Wayne D. [Medical Physics Program, Department of Physics and Astronomy, Louisiana State University, 202 Nicholson Hall, Tower Dr., Baton Rouge, LA 70803 (United States); Department of Physics, Mary Bird Perkins Cancer Center, 4950 Essen Lane, Baton Rouge, LA 70809 (United States)

2015-03-10

Children receiving radiotherapy face the probability of a subsequent malignant neoplasm (SMN). In some cases, the predicted SMN risk can be reduced by proton therapy. The purpose of this study was to apply the most comprehensive dose assessment methods to estimate the reduction in SMN risk after proton therapy vs. photon therapy for a 13-year-old girl requiring craniospinal irradiation (CSI). We reconstructed the equivalent dose throughout the patient’s body from therapeutic and stray radiation and applied SMN incidence and mortality risk models for each modality. Excluding skin cancer, the risk of incidence after proton CSI was a third of that of photon CSI. The predicted absolute SMN risks were high. For photon CSI, the SMN incidence rates greater than 10% were for thyroid, non-melanoma skin, lung, colon, stomach, and other solid cancers, and for proton CSI they were non-melanoma skin, lung, and other solid cancers. In each setting, lung cancer accounted for half the risk of mortality. In conclusion, the predicted SMN risk for a 13-year-old girl undergoing proton CSI was reduced vs. photon CSI. This study demonstrates the feasibility of inter-institutional whole-body dose and risk assessments and also serves as a model for including risk estimation in personalized cancer care.

Inter-Institutional Comparison of Personalized Risk Assessments for Second Malignant Neoplasms for a 13-Year-Old Girl Receiving Proton versus Photon Craniospinal Irradiation

International Nuclear Information System (INIS)

Taddei, Phillip J.; Khater, Nabil; Zhang, Rui; Geara, Fady B.; Mahajan, Anita; Jalbout, Wassim; Pérez-Andújar, Angélica; Youssef, Bassem; Newhauser, Wayne D.

2015-01-01

Children receiving radiotherapy face the probability of a subsequent malignant neoplasm (SMN). In some cases, the predicted SMN risk can be reduced by proton therapy. The purpose of this study was to apply the most comprehensive dose assessment methods to estimate the reduction in SMN risk after proton therapy vs. photon therapy for a 13-year-old girl requiring craniospinal irradiation (CSI). We reconstructed the equivalent dose throughout the patient’s body from therapeutic and stray radiation and applied SMN incidence and mortality risk models for each modality. Excluding skin cancer, the risk of incidence after proton CSI was a third of that of photon CSI. The predicted absolute SMN risks were high. For photon CSI, the SMN incidence rates greater than 10% were for thyroid, non-melanoma skin, lung, colon, stomach, and other solid cancers, and for proton CSI they were non-melanoma skin, lung, and other solid cancers. In each setting, lung cancer accounted for half the risk of mortality. In conclusion, the predicted SMN risk for a 13-year-old girl undergoing proton CSI was reduced vs. photon CSI. This study demonstrates the feasibility of inter-institutional whole-body dose and risk assessments and also serves as a model for including risk estimation in personalized cancer care

How are proteins reduced in the endoplasmic reticulum?

DEFF Research Database (Denmark)

Ellgaard, Lars; Sevier, Carolyn S.; Bulleid, Neil J.

2018-01-01

The reversal of thiol oxidation in proteins within the endoplasmic reticulum (ER) is crucial for protein folding, degradation, chaperone function, and the ER stress response. Our understanding of this process is generally poor but progress has been made. Enzymes performing the initial reduction...... of client proteins, as well as the ultimate electron donor in the pathway, have been identified. Most recently, a role for the cytosol in ER protein reduction has been revealed. Nevertheless, how reducing equivalents are transferred from the cytosol to the ER lumen remains an open question. We review here...... why proteins are reduced in the ER, discuss recent data on catalysis of steps in the pathway, and consider the implications for redox homeostasis within the early secretory pathway....

Effects of cover crops on the nitrogen fluxes in a silage maize production system

NARCIS (Netherlands)

Schröder, J.J.; Dijk, van W.; Groot, de W.J.M.

1996-01-01

Rye and grass cover crops can potentially intercept residual soil mineral nitrogen (SMN), reduce overwinter leaching, transfer SMN to next growing seasons and reduce the fertilizer need of subsequent crops. These aspects were studied for 6 years in continuous silage maize cv. LG 2080 production

Spinal Muscular Atrophy: From Defective Chaperoning of snRNP Assembly to Neuromuscular Dysfunction

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Maia Lanfranco

2017-06-01

Full Text Available Spinal Muscular Atrophy (SMA is a neuromuscular disorder that results from decreased levels of the survival motor neuron (SMN protein. SMN is part of a multiprotein complex that also includes Gemins 2–8 and Unrip. The SMN-Gemins complex cooperates with the protein arginine methyltransferase 5 (PRMT5 complex, whose constituents include WD45, PRMT5 and pICln. Both complexes function as molecular chaperones, interacting with and assisting in the assembly of an Sm protein core onto small nuclear RNAs (snRNAs to generate small nuclear ribonucleoproteins (snRNPs, which are the operating components of the spliceosome. Molecular and structural studies have refined our knowledge of the key events taking place within the crowded environment of cells and the numerous precautions undertaken to ensure the faithful assembly of snRNPs. Nonetheless, it remains unclear whether a loss of chaperoning in snRNP assembly, considered as a “housekeeping” activity, is responsible for the selective neuromuscular phenotype in SMA. This review thus shines light on in vivo studies that point toward disturbances in snRNP assembly and the consequential transcriptome abnormalities as the primary drivers of the progressive neuromuscular degeneration underpinning the disease. Disruption of U1 snRNP or snRNP assembly factors other than SMN induces phenotypes that mirror aspects of SMN deficiency, and splicing defects, described in numerous SMA models, can lead to a DNA damage and stress response that compromises the survival of the motor system. Restoring the correct chaperoning of snRNP assembly is therefore predicted to enhance the benefit of SMA therapeutic modalities based on augmenting SMN expression.

Improvement of neuromuscular synaptic phenotypes without enhanced survival and motor function in severe spinal muscular atrophy mice selectively rescued in motor neurons.

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Ximena Paez-Colasante

Full Text Available In the inherited childhood neuromuscular disease spinal muscular atrophy (SMA, lower motor neuron death and severe muscle weakness result from the reduction of the ubiquitously expressed protein survival of motor neuron (SMN. Although SMA mice recapitulate many features of the human disease, it has remained unclear if their short lifespan and motor weakness are primarily due to cell-autonomous defects in motor neurons. Using Hb9(Cre as a driver, we selectively raised SMN expression in motor neurons in conditional SMAΔ7 mice. Unlike a previous study that used choline acetyltransferase (ChAT(Cre+ as a driver on the same mice, and another report that used Hb9(Cre as a driver on a different line of conditional SMA mice, we found no improvement in survival, weight, motor behavior and presynaptic neurofilament accumulation. However, like in ChAT(Cre+ mice, we detected rescue of endplate size and mitigation of neuromuscular junction (NMJ denervation status. The rescue of endplate size occurred in the absence of an increase in myofiber size, suggesting endplate size is determined by the motor neuron in these animals. Real time-PCR showed that the expression of spinal cord SMN transcript was sharply reduced in Hb9(Cre+ SMA mice relative to ChAT(Cre+ SMA mice. This suggests that our lack of overall phenotypic improvement is most likely due to an unexpectedly poor recombination efficiency driven by Hb9(Cre . Nonetheless, the low levels of SMN were sufficient to rescue two NMJ structural parameters indicating that these motor neuron cell autonomous phenotypes are very sensitive to changes in motoneuronal SMN levels. Our results directly suggest that even those therapeutic interventions with very modest effects in raising SMN in motor neurons may provide mitigation of neuromuscular phenotypes in SMA patients.

Defective Ca2+ channel clustering in axon terminals disturbs excitability in motoneurons in spinal muscular atrophy

OpenAIRE

Jablonka, Sibylle; Beck, Marcus; Lechner, Barbara Dorothea; Mayer, Christine; Sendtner, Michael

2007-01-01

Proximal spinal muscular atrophy (SMA) is a motoneuron disease for which there is currently no effective treatment. In animal models of SMA, spinal motoneurons exhibit reduced axon elongation and growth cone size. These defects correlate with reduced β-actin messenger RNA and protein levels in distal axons. We show that survival motoneuron gene (Smn)–deficient motoneurons exhibit severe defects in clustering Cav2.2 channels in axonal growth cones. These defects also correlate with a reduced f...

Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

Science.gov (United States)

Trinkle-Mulcahy, Laura; Boulon, Séverine; Lam, Yun Wah; Urcia, Roby; Boisvert, François-Michel; Vandermoere, Franck; Morrice, Nick A.; Swift, Sam; Rothbauer, Ulrich; Leonhardt, Heinrich; Lamond, Angus

2008-01-01

The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments. PMID:18936248

Experimental cross section for the {sup 152}Sm(n, γ){sup 153}Sm reaction at 0.0334 eV

Energy Technology Data Exchange (ETDEWEB)

Uddin, M. Shuza; Datta, Tapash Kumar; Hossain, Syed Mohammod; Zakaria, A.K.M.; Islam, Mohammad Amirul; Naher, Kamrun; Shariff, M. Asad; Yunus, S.M. [Atomic Energy Research Establishment, Dhaka (Bangladesh). Inst. of Nuclear Science and Technology; Afroze, Nasmin [Atomic Energy Research Establishment, Dhaka (Bangladesh). Inst. of Nuclear Science and Technology; Jahangirnagar Univ., Dhaka (Bangladesh). Dept. of Physics; Islam, S.M. Ajharul [Jahangirnagar Univ., Dhaka (Bangladesh). Dept. of Physics

2014-10-01

The neutron capture cross section for the {sup 152}Sm(n, γ){sup 153}Sm reaction at an energy of 0.0334 eV was measured for the first time using monochromatic neutrons of a powder diffractometer at the TRIGA Mark II nuclear reactor at Dhaka, Bangladesh. The {sup 197}Au(n, γ){sup 198}Au reaction was used to monitor the neutron beam intensity. The radioactivity of the products was determined via high resolution γ-ray spectrometry. The obtained cross section value is 184 ± 22b, which is consistent with both the ENDF/B-VII and TENDL-2012 data libraries. The measured value at 0.0334 eV and the previous data at 0.0536 eV confirm the reliability of the data in the above libraries. (orig.)

Imaging Flow Cytometry Analysis to Identify Differences of Survival Motor Neuron Protein Expression in Patients With Spinal Muscular Atrophy.

Science.gov (United States)

Arakawa, Reiko; Arakawa, Masayuki; Kaneko, Kaori; Otsuki, Noriko; Aoki, Ryoko; Saito, Kayoko

2016-08-01

Spinal muscular atrophy is a neurodegenerative disorder caused by the deficient expression of survival motor neuron protein in motor neurons. A major goal of disease-modifying therapy is to increase survival motor neuron expression. Changes in survival motor neuron protein expression can be monitored via peripheral blood cells in patients; therefore we tested the sensitivity and utility of imaging flow cytometry for this purpose. After the immortalization of peripheral blood lymphocytes from a human healthy control subject and two patients with spinal muscular atrophy type 1 with two and three copies of SMN2 gene, respectively, we used imaging flow cytometry analysis to identify significant differences in survival motor neuron expression. A bright detail intensity analysis was used to investigate differences in the cellular localization of survival motor neuron protein. Survival motor neuron expression was significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. Moreover, survival motor neuron expression correlated with the clinical severity of spinal muscular atrophy according to SMN2 copy number. The cellular accumulation of survival motor neuron protein was also significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. The benefits of imaging flow cytometry for peripheral blood analysis include its capacities for analyzing heterogeneous cell populations; visualizing cell morphology; and evaluating the accumulation, localization, and expression of a target protein. Imaging flow cytometry analysis should be implemented in future studies to optimize its application as a tool for spinal muscular atrophy clinical trials. Copyright © 2016 Elsevier Inc. All rights reserved.

The Gemin associates of survival motor neuron are required for motor function in Drosophila.

Science.gov (United States)

Borg, Rebecca; Cauchi, Ruben J

2013-01-01

Membership of the survival motor neuron (SMN) complex extends to nine factors, including the SMN protein, the product of the spinal muscular atrophy (SMA) disease gene, Gemins 2-8 and Unrip. The best-characterised function of this macromolecular machine is the assembly of the Sm-class of uridine-rich small nuclear ribonucleoprotein (snRNP) particles and each SMN complex member has a key role during this process. So far, however, only little is known about the function of the individual Gemin components in vivo. Here, we make use of the Drosophila model organism to uncover loss-of-function phenotypes of Gemin2, Gemin3 and Gemin5, which together with SMN form the minimalistic fly SMN complex. We show that ectopic overexpression of the dead helicase Gem3(ΔN) mutant or knockdown of Gemin3 result in similar motor phenotypes, when restricted to muscle, and in combination cause lethality, hence suggesting that Gem3(ΔN) overexpression mimics a loss-of-function. Based on the localisation pattern of Gem3(ΔN), we predict that the nucleus is the primary site of the antimorphic or dominant-negative mechanism of Gem3(ΔN)-mediated interference. Interestingly, phenotypes induced by human SMN overexpression in Drosophila exhibit similarities to those induced by overexpression of Gem3(ΔN). Through enhanced knockdown we also uncover a requirement of Gemin2, Gemin3 and Gemin5 for viability and motor behaviour, including locomotion as well as flight, in muscle. Notably, in the case of Gemin3 and Gemin5, such function also depends on adequate levels of the respective protein in neurons. Overall, these findings lead us to speculate that absence of any one member is sufficient to arrest the SMN-Gemins complex function in a nucleocentric pathway, which is critical for motor function in vivo.

Immune dysregulation may contribute to disease pathogenesis in spinal muscular atrophy mice.

Science.gov (United States)

Deguise, Marc-Olivier; De Repentigny, Yves; McFall, Emily; Auclair, Nicole; Sad, Subash; Kothary, Rashmi

2017-02-15

Spinal muscular atrophy (SMA) has long been solely considered a neurodegenerative disorder. However, recent work has highlighted defects in many other cell types that could contribute to disease aetiology. Interestingly, the immune system has never been extensively studied in SMA. Defects in lymphoid organs could exacerbate disease progression by neuroinflammation or immunodeficiency. Smn depletion led to severe alterations in the thymus and spleen of two different mouse models of SMA. The spleen from Smn depleted mice was dramatically smaller at a very young age and its histological architecture was marked by mislocalization of immune cells in the Smn2B/- model mice. In comparison, the thymus was relatively spared in gross morphology but showed many histological alterations including cortex thinning in both mouse models at symptomatic ages. Thymocyte development was also impaired as evidenced by abnormal population frequencies in the Smn2B/- thymus. Cytokine profiling revealed major changes in different tissues of both mouse models. Consistent with our observations, we found that survival motor neuron (Smn) protein levels were relatively high in lymphoid organs compared to skeletal muscle and spinal cord during postnatal development in wild type mice. Genetic introduction of one copy of the human SMN2 transgene was enough to rescue splenic and thymic defects in Smn2B/- mice. Thus, Smn is required for the normal development of lymphoid organs, and altered immune function may contribute to SMA disease pathogenesis. © The Author 2017. Published by Oxford University Press.

Modifier genes: Moving from pathogenesis to therapy.

Science.gov (United States)

McCabe, Edward R B

2017-09-01

This commentary will focus on how we can use our knowledge about the complexity of human disease and its pathogenesis to identify novel approaches to therapy. We know that even for single gene Mendelian disorders, patients with identical mutations often have different presentations and outcomes. This lack of genotype-phenotype correlation led us and others to examine the roles of modifier genes in the context of biological networks. These investigations have utilized vertebrate and invertebrate model organisms. Since one of the goals of research on modifier genes and networks is to identify novel therapeutic targets, the challenges to patient access and compliance because of the high costs of medications for rare genetic diseases must be recognized. A recent article explored protective modifiers, including plastin 3 (PLS3) and coronin 1C (CORO1C), in spinal muscular atrophy (SMA). SMA is an autosomal recessive deficit of survival motor neuron protein (SMN) caused by mutations in SMN1. However, the severity of SMA is determined primarily by the number of SMN2 copies, and this results in significant phenotypic variability. PLS3 was upregulated in siblings who were asymptomatic compared with those who had SMA2 or SMA3, but identical homozygous SMN1 deletions and equal numbers of SMN2 copies. CORO1C was identified by interrogation of the PLS3 interactome. Overexpression of these proteins rescued endocytosis in SMA models. In addition, antisense RNA for upregulation of SMN2 protein expression is being developed as another way of modifying the SMA phenotype. These investigations suggest the practical application of protective modifiers to rescue SMA phenotypes. Other examples of the potential therapeutic value of novel protective modifiers will be discussed, including in Duchenne muscular dystrophy and glycerol kinase deficiency. This work shows that while we live in an exciting era of genomic sequencing, a functional understanding of biology, the impact of its

Histopathological Defects in Intestine in Severe Spinal Muscular Atrophy Mice Are Improved by Systemic Antisense Oligonucleotide Treatment.

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Palittiya Sintusek

Full Text Available Gastrointestinal (GI defects, including gastroesophageal reflux, constipation and delayed gastric emptying, are common in patients with spinal muscular atrophy (SMA. Similar GI dysmotility has been identified in mouse models with survival of motor neuron (SMN protein deficiency. We previously described vascular defects in skeletal muscle and spinal cord of SMA mice and we hypothesized that similar defects could be involved in the GI pathology observed in these mice. We therefore investigated the gross anatomical structure, enteric vasculature and neurons in the small intestine in a severe mouse model of SMA. We also assessed the therapeutic response of GI histopathology to systemic administration of morpholino antisense oligonucleotide (AON designed to increase SMN protein expression. Significant anatomical and histopathological abnormalities, with striking reduction of vascular density, overabundance of enteric neurons and increased macrophage infiltration, were detected in the small intestine in SMA mice. After systemic AON treatment in neonatal mice, all the abnormalities observed were significantly restored to near-normal levels. We conclude that the observed GI histopathological phenotypes and functional defects observed in these SMA mice are strongly linked to SMN deficiency which can be rescued by systemic administration of AON. This study on the histopathological changes in the gastrointestinal system in severe SMA mice provides further indication of the complex role that SMN plays in multiple tissues and suggests that at least in SMA mice restoration of SMN production in peripheral tissues is essential for optimal outcome.

Histopathological Defects in Intestine in Severe Spinal Muscular Atrophy Mice Are Improved by Systemic Antisense Oligonucleotide Treatment

Science.gov (United States)

Sintusek, Palittiya; Catapano, Francesco; Angkathunkayul, Napat; Marrosu, Elena; Parson, Simon H.; Morgan, Jennifer E.; Muntoni, Francesco; Zhou, Haiyan

2016-01-01

Gastrointestinal (GI) defects, including gastroesophageal reflux, constipation and delayed gastric emptying, are common in patients with spinal muscular atrophy (SMA). Similar GI dysmotility has been identified in mouse models with survival of motor neuron (SMN) protein deficiency. We previously described vascular defects in skeletal muscle and spinal cord of SMA mice and we hypothesized that similar defects could be involved in the GI pathology observed in these mice. We therefore investigated the gross anatomical structure, enteric vasculature and neurons in the small intestine in a severe mouse model of SMA. We also assessed the therapeutic response of GI histopathology to systemic administration of morpholino antisense oligonucleotide (AON) designed to increase SMN protein expression. Significant anatomical and histopathological abnormalities, with striking reduction of vascular density, overabundance of enteric neurons and increased macrophage infiltration, were detected in the small intestine in SMA mice. After systemic AON treatment in neonatal mice, all the abnormalities observed were significantly restored to near-normal levels. We conclude that the observed GI histopathological phenotypes and functional defects observed in these SMA mice are strongly linked to SMN deficiency which can be rescued by systemic administration of AON. This study on the histopathological changes in the gastrointestinal system in severe SMA mice provides further indication of the complex role that SMN plays in multiple tissues and suggests that at least in SMA mice restoration of SMN production in peripheral tissues is essential for optimal outcome. PMID:27163330

Intestinal remodelling in mink fed with reduced protein content

DEFF Research Database (Denmark)

Chen, Pengmin; Zhao, Jingbo; Nielsen, Vivi Hunnicke

2009-01-01

Low protein intake occurs in humans in relation to diseases, starvation and post-operatively. Low-protein diets may affect the gastrointestinal structure and mechanical function. The aim was to study the passive biomechanical properties and tissue remodelling of the intestine in minks on reduced...... protein diets. Twenty-seven male minks were divided into three groups receiving different protein level in the diet for 6 weeks: High protein level (group H, 55% energy from protein), moderate protein level (group M, 30% energy from protein) and low protein level (group L, 15% energy from protein) (n=9...... groups. Feeding the low-protein diet shifted the stress-strain curves to the right for the circumferential direction, indicating the wall become softer in the circumferential direction. However, no significant difference was observed in the longitudinal direction for any of the intestinal segments...

Protective effects of long-term lithium administration in a slowly progressive SMA mouse model.

Science.gov (United States)

Biagioni, Francesca; Ferrucci, Michela; Ryskalin, Larisa; Fulceri, Federica; Lazzeri, Gloria; Calierno, Maria Teresa; Busceti, Carla L; Ruffoli, Riccardo; Fornai, Francesco

2017-12-01

In the present study we evaluated the long-term effects of lithium administration to a knock-out double transgenic mouse model (Smn-/-; SMN1A2G+/-; SMN2+/+) of Spinal Muscle Atrophy type III (SMA-III). This model is characterized by very low levels of the survival motor neuron protein, slow disease progression and motor neuron loss, which enables to detect disease-modifying effects at delayed time intervals. Lithium administration attenuates the decrease in motor activity and provides full protection from motor neuron loss occurring in SMA-III mice, throughout the disease course. In addition, lithium prevents motor neuron enlargement and motor neuron heterotopy and suppresses the occurrence of radial-like glial fibrillary acidic protein immunostaining in the ventral white matter of SMA-III mice. In SMA-III mice long-term lithium administration determines a dramatic increase of survival motor neuron protein levels in the spinal cord. These data demonstrate that long-term lithium administration during a long-lasting motor neuron disorder attenuates behavioural deficit and neuropathology. Since low level of survival motor neuron protein is bound to disease severity in SMA, the robust increase in protein level produced by lithium provides solid evidence which calls for further investigations considering lithium in the long-term treatment of spinal muscle atrophy.

Changes of Raffinose and Stachyose in Soy Milk Fermentation by Lactic Acid Bacteria From Local Fermented Foods of Indonesian

Directory of Open Access Journals (Sweden)

Sumarna

2008-01-01

Full Text Available The objective of this study was to evaluate the fermentative characteristics of lactic acid bacteria isolated from local fermented foods and consume raffinose and stachyose during fermentation soymilk. Lactobacillus plantarum pentosus SMN, 01, Lactobacillus casei subsp rhamnosus FNCC, 098, Lactobacillus casei subsp rhamnosus FNCC, 099, Streptococcus thermofilus, 001, Lactobacillus delbrueckii subsp. bulgaricus FNCC, 0045, Lactobacillus plantarum SMN, 25, and Lactobacillus plantarum pentosus FNCC, 235 exhibited variable α-galactosidase activity with Lactobacillus plantarum SMN, 25, showing the highest activity in MRS supplemented media. However, all organisms reached the desired therapeutic level (10^8 cfu/mL likely due to their ability to metabolize oligosaccharides during fermentation in soymilk at 41 °C. The oligosaccharide metabolism depended on α-galactosidase activity. Lactobacillus plantarum SMN, 25, L. plantarum pentosus SMN, 01 and Lactobacillus plantarum pentosus FNCC, 235 reduced raffinose and stachyose by 81.5, 73.0, 67.0 %, and 78.0, 72.5, 66.0 % respectively in soymilk.

Prehydrolyzed dietary protein reduces gastrocnemial DNA without ...

African Journals Online (AJOL)

Prehydrolyzed dietary protein reduces gastrocnemial DNA without impairing physical capacity in the rat. Viviane Costa Silva Zaffani, Carolina Cauduro Bensabath Carneiro-Leão, Giovana Ermetice de Almeida Costa, Pablo Christiano Barboza Lollo, Emilianne Miguel Salomão, Maria Cristina Cintra Gomes-Marcondes, ...

Is Spinal Muscular Atrophy a disease of the motor neurons only: pathogenesis and therapeutic implications?

Science.gov (United States)

Simone, Chiara; Ramirez, Agnese; Bucchia, Monica; Rinchetti, Paola; Rideout, Hardy; Papadimitriou, Dimitra; Re, Diane B.; Corti, Stefania

2016-01-01

Spinal Muscular Atrophy (SMA) is a genetic neurological disease that causes infant mortality; no effective therapies are currently available. SMA is due to homozygous mutations and/or deletions in the Survival Motor Neuron 1 (SMN1) gene and subsequent reduction of the SMN protein, leading to the death of motor neurons. However, there is increasing evidence that in addition to motor neurons, other cell types are contributing to SMA pathology. In this review, we will discuss the involvement of non-motor neuronal cells, located both inside and outside the central nervous system, in disease onset and progression. These contribution of non-motor neuronal cells to disease pathogenesis has important therapeutic implications: in fact, even if SMN restoration in motor neurons is needed, it has been shown that optimal phenotypic amelioration in animal models of SMA requires a more widespread SMN correction. It will be crucial to take this evidence into account before clinical translation of the novel therapeutic approaches that are currently under development. PMID:26681261

Ubiquilin overexpression reduces GFP-polyalanine-induced protein aggregates and toxicity

International Nuclear Information System (INIS)

Wang Hongmin; Monteiro, Mervyn J.

2007-01-01

Several human disorders are associated with an increase in a continuous stretch of alanine amino acids in proteins. These so-called polyalanine expansion diseases share many similarities with polyglutamine-related disorders, including a length-dependent reiteration of amino acid induction of protein aggregation and cytotoxicity. We previously reported that overexpression of ubiquilin reduces protein aggregates and toxicity of expanded polyglutamine proteins. Here, we demonstrate a similar role for ubiquilin toward expanded polyalanine proteins. Overexpression of ubiquilin-1 in HeLa cells reduced protein aggregates and the cytotoxicity associated with expression of a transfected nuclear-targeted GFP-fusion protein containing 37-alanine repeats (GFP-A37), in a dose dependent manner. Ubiquilin coimmunoprecipitated more with GFP proteins containing a 37-polyalanine tract compared to either 7 (GFP-A7), or no alanine tract (GFP). Moreover, overexpression of ubiquilin suppressed the increased vulnerability of HeLa cell lines stably expressing the GFP-A37 fusion protein to oxidative stress-induced cell death compared to cell lines expressing GFP or GFP-A7 proteins. By contrast, siRNA knockdown of ubiquilin expression in the GFP-A37 cell line was associated with decreased cellular proliferation, and increases in GFP protein aggregates, nuclear fragmentation, and cell death. Our results suggest that boosting ubiquilin levels in cells might provide a universal and attractive strategy to prevent toxicity of proteins containing reiterative expansions of amino acids involved in many human diseases

Notch Signaling Pathway Is Activated in Motoneurons of Spinal Muscular Atrophy

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Gabriel Olmos

2013-05-01

Full Text Available Spinal muscular atrophy (SMA is a neurodegenerative disease produced by low levels of Survival Motor Neuron (SMN protein that affects alpha motoneurons in the spinal cord. Notch signaling is a cell-cell communication system well known as a master regulator of neural development, but also with important roles in the adult central nervous system. Aberrant Notch function is associated with several developmental neurological disorders; however, the potential implication of the Notch pathway in SMA pathogenesis has not been studied yet. We report here that SMN deficiency, induced in the astroglioma cell line U87MG after lentiviral transduction with a shSMN construct, was associated with an increase in the expression of the main components of Notch signaling pathway, namely its ligands, Jagged1 and Delta1, the Notch receptor and its active intracellular form (NICD. In the SMNΔ7 mouse model of SMA we also found increased astrocyte processes positive for Jagged1 and Delta1 in intimate contact with lumbar spinal cord motoneurons. In these motoneurons an increased Notch signaling was found, as denoted by increased NICD levels and reduced expression of the proneural gene neurogenin 3, whose transcription is negatively regulated by Notch. Together, these findings may be relevant to understand some pathologic attributes of SMA motoneurons.

Toward a rigorous network of protein-protein interactions of the model sulfate reducer Desulfovibrio vulgaris Hildenborough

Energy Technology Data Exchange (ETDEWEB)

Chhabra, S.R.; Joachimiak, M.P.; Petzold, C.J.; Zane, G.M.; Price, M.N.; Gaucher, S.; Reveco, S.A.; Fok, V.; Johanson, A.R.; Batth, T.S.; Singer, M.; Chandonia, J.M.; Joyner, D.; Hazen, T.C.; Arkin, A.P.; Wall, J.D.; Singh, A.K.; Keasling, J.D.

2011-05-01

Protein–protein interactions offer an insight into cellular processes beyond what may be obtained by the quantitative functional genomics tools of proteomics and transcriptomics. The aforementioned tools have been extensively applied to study E. coli and other aerobes and more recently to study the stress response behavior of Desulfovibrio 5 vulgaris Hildenborough, a model anaerobe and sulfate reducer. In this paper we present the first attempt to identify protein-protein interactions in an obligate anaerobic bacterium. We used suicide vector-assisted chromosomal modification of 12 open reading frames encoded by this sulfate reducer to append an eight amino acid affinity tag to the carboxy-terminus of the chosen proteins. Three biological replicates of the 10 ‘pulled-down’ proteins were separated and analyzed using liquid chromatography-mass spectrometry. Replicate agreement ranged between 35% and 69%. An interaction network among 12 bait and 90 prey proteins was reconstructed based on 134 bait-prey interactions computationally identified to be of high confidence. We discuss the biological significance of several unique metabolic features of D. vulgaris revealed by this protein-protein interaction data 15 and protein modifications that were observed. These include the distinct role of the putative carbon monoxide-induced hydrogenase, unique electron transfer routes associated with different oxidoreductases, and the possible role of methylation in regulating sulfate reduction.

Graphene as a protein crystal mounting material to reduce background scatter.

Science.gov (United States)

Wierman, Jennifer L; Alden, Jonathan S; Kim, Chae Un; McEuen, Paul L; Gruner, Sol M

2013-10-01

The overall signal-to-noise ratio per unit dose for X-ray diffraction data from protein crystals can be improved by reducing the mass and density of all material surrounding the crystals. This article demonstrates a path towards the practical ultimate in background reduction by use of atomically thin graphene sheets as a crystal mounting platform for protein crystals. The results show the potential for graphene in protein crystallography and other cases where X-ray scatter from the mounting material must be reduced and specimen dehydration prevented, such as in coherent X-ray diffraction imaging of microscopic objects.

Chenodeoxycholic Acid Reduces Hypoxia Inducible Factor-1α Protein and Its Target Genes.

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Yunwon Moon

Full Text Available This study evaluated HIF-1α inhibitors under different hypoxic conditions, physiological hypoxia (5% O2 and severe hypoxia (0.1% O2. We found that chenodeoxy cholic acid (CDCA reduced the amount of HIF-1α protein only under physiological hypoxia but not under severe hypoxia without decreasing its mRNA level. By using a proteasome inhibitor MG132 and a translation inhibitor cyclohexamide, we showed that CDCA reduced HIF-1α protein by decreasing its translation but not by enhancing its degradation. The following findings indicated that farnesoid X receptor (FXR, a CDCA receptor and its target gene, Small heterodimer partner (SHP are not involved in this effect of CDCA. Distinctly from CDCA, MG132 prevented SHP and an exogenous FXR agonist, GW4064 from reducing HIF-1α protein. Furthermore a FXR antagonist, guggulsterone failed to prevent CDCA from decreasing HIF-1α protein. Furthermore, guggulsterone by itself reduced HIF-1α protein even in the presence of MG132. These findings suggested that CDCA and guggulsterone reduced the translation of HIF-1α in a mechanism which FXR and SHP are not involved. This study reveals novel therapeutic functions of traditional nontoxic drugs, CDCA and guggulsterone, as inhibitors of HIF-1α protein.

Proteomic detection of oxidized and reduced thiol proteins in cultured cells.

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Cuddihy, Sarah L; Baty, James W; Brown, Kristin K; Winterbourn, Christine C; Hampton, Mark B

2009-01-01

The oxidation and reduction of cysteine residues is emerging as an important post-translational control of protein function. We describe a method for fluorescent labelling of either reduced or oxidized thiols in combination with two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (2DE) to detect changes in the redox proteome of cultured cells. Reduced thiols are labelled with the fluorescent compound 5-iodoacetamidofluorescein. To monitor oxidized thiols, the reduced thiols are first blocked with N-ethyl-maleimide, then the oxidized thiols reduced with dithiothreitol and labelled with 5-iodoacetamidofluorescein. The method is illustrated by treating Jurkat T-lymphoma cells with hydrogen peroxide and monitoring increased labelling of oxidized thiol proteins. A decrease in labelling can also be detected, and this is attributed to the formation of higher oxidation states of cysteine that are not reduced by dithiothreitol.

Reduced protein oxidation in Wistar rats supplemented with marine ω3 PUFAs.

Science.gov (United States)

Méndez, Lucía; Pazos, Manuel; Gallardo, José M; Torres, Josep L; Pérez-Jiménez, Jara; Nogués, Rosa; Romeu, Marta; Medina, Isabel

2013-02-01

The potential effects of various dietary eicosapentaenoic acid (EPA; 20:5) and docosahexaenoic acid (DHA; 22:6) ratios (1:1, 2:1, and 1:2, respectively) on protein redox states from plasma, kidney, skeletal muscle, and liver were investigated in Wistar rats. Dietary fish oil groups were compared with animals fed soybean and linseed oils, vegetable oils enriched in ω6 linoleic acid (LA; 18:2) and ω3 α-linolenic acid (ALA; 18:3), respectively. Fish oil treatments were effective at reducing the level of total fatty acids in plasma and enriching the plasmatic free fatty acid fraction and erythrocyte membranes in EPA and DHA. A proteomic approach consisting of fluorescein 5-thiosemicarbazide (FTSC) labeling of protein carbonyls, FTSC intensity visualization on 1-DE or 2-DE gels, and protein identification by MS/MS was used for the protein oxidation assessment. Albumin was found to be the most carbonylated protein in plasma for all dietary groups, and its oxidation level was significantly modulated by dietary interventions. Supplementation with an equal EPA:DHA ratio (1:1) showed the lowest oxidation score for plasma albumin, followed in increasing order of carbonylation by 1:2 proteins and cytosolic proteins from kidney and liver also indicated a protective effect on proteins for the fish oil treatments, the 1:1 ratio exhibiting the lowest protein oxidation scores. The effect of fish oil treatments at reducing carbonylation on specific proteins from plasma (albumin), skeletal muscle (actin), and liver (albumin, argininosuccinate synthetase, 3-α-hydroxysteroid dehydrogenase) was remarkable. This investigation highlights the efficiency of dietary fish oil at reducing in vivo oxidative damage of proteins compared to oils enriched in the 18-carbon polyunsaturated fatty acids ω3 ALA and ω6 LA, and such antioxidant activity may differ among different fish oil sources because of variations in EPA/DHA content. Copyright © 2012 Elsevier Inc. All rights reserved.

Applications of small surface plasmon resonance sensors for biochemical monitoring

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Masson, Jean-Francois; Battaglia, Tina M.; Beaudoin, Stephen; Booksh, Karl S.

2004-12-01

The development of small surface plasmon resonance (SPR) sensors to detect biological markers for myocardial ischemia (MI), spinal muscular atrophy (SMA), and wound healing was achieved at low ng/mL and in less than 10 minutes. The markers of interest for MIs are myoglobin (MG) and cardiac Troponin I (cTnI). The limits of detection for these markers are respectively 600 pg/mL and 1.4 ng/mL in saline solution. To study SMA, the level of survival motor neuron protein (SMN) was investigated. A limit of detection of 990 pg/mL was achieved for the detection of SMN. The interactions of SMN with MG decreased the signal for both SMN and MG. Interleukin 6 and tumor necrosis factor alpha (TNFa) were investigated to monitor wound healing. The sensor's performance in more complex solutions, e.g.: serum, showed a large non-specific signal. Modifying the support on which the antibodies are attached improved the sensor's stability in serum by a factor of 5. To achieve this non-specific binding (NSB) reduction, different polysaccharides, biocompatible polymers and short chain thiols were investigated.

Sodium 4-Phenylbutyrate Attenuates Myocardial Reperfusion Injury by Reducing the Unfolded Protein Response.

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Takatori, Osamu; Usui, Soichiro; Okajima, Masaki; Kaneko, Shuichi; Ootsuji, Hiroshi; Takashima, Shin-Ichiro; Kobayashi, Daisuke; Murai, Hisayoshi; Furusho, Hiroshi; Takamura, Masayuki

2017-05-01

The unfolded protein response (UPR) plays a pivotal role in ischemia-reperfusion (I/R) injury in various organs such as heart, brain, and liver. Sodium 4-phenylbutyrate (PBA) reportedly acts as a chemical chaperone that reduces UPR. In the present study, we evaluated the effect of PBA on reducing the UPR and protecting against myocardial I/R injury in mice. Male C57BL/6 mice were subjected to 30-minute myocardial I/R, and were treated with phosphate-buffered saline (as a vehicle) or PBA. At 4 hours after reperfusion, mice treated with PBA had reduced serum cardiac troponin I levels and numbers of apoptotic cells in left ventricles (LVs) in myocardial I/R. Infarct size had also reduced in mice treated with PBA at 48 hours after reperfusion. At 2 hours after reperfusion, UPR markers, including eukaryotic initiation of the factor 2α-subunit, activating transcription factor-6, inositol-requiring enzyme-1, glucose-regulated protein 78, CCAAT/enhancer-binding protein (C/EBP) homologous protein, and caspase-12, were significantly increased in mice treated with vehicle compared to sham-operated mice. Administration of PBA significantly reduced the I/R-induced increases of these markers. Cardiac function and dimensions were assessed at 21 days after I/R. Sodium 4-phenylbutyrate dedicated to the improvement of cardiac parameters deterioration including LV end-diastolic diameter and LV fractional shortening. Consistently, PBA reduced messenger RNA expression levels of cardiac remodeling markers such as collagen type 1α1, brain natriuretic peptide, and α skeletal muscle actin in LV at 21 days after I/R. Unfolded protein response mediates myocardial I/R injury. Administration of PBA reduces the UPR, apoptosis, infarct size, and preserved cardiac function. Hence, PBA may be a therapeutic option to attenuate myocardial I/R injury in clinical practice.

Membrane Compartmentalization Reducing the Mobility of Lipids and Proteins within a Model Plasma Membrane.

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Koldsø, Heidi; Reddy, Tyler; Fowler, Philip W; Duncan, Anna L; Sansom, Mark S P

2016-09-01

The cytoskeleton underlying cell membranes may influence the dynamic organization of proteins and lipids within the bilayer by immobilizing certain transmembrane (TM) proteins and forming corrals within the membrane. Here, we present coarse-grained resolution simulations of a biologically realistic membrane model of asymmetrically organized lipids and TM proteins. We determine the effects of a model of cytoskeletal immobilization of selected membrane proteins using long time scale coarse-grained molecular dynamics simulations. By introducing compartments with varying degrees of restraints within the membrane models, we are able to reveal how compartmentalization caused by cytoskeletal immobilization leads to reduced and anomalous diffusional mobility of both proteins and lipids. This in turn results in a reduced rate of protein dimerization within the membrane and of hopping of membrane proteins between compartments. These simulations provide a molecular realization of hierarchical models often invoked to explain single-molecule imaging studies of membrane proteins.

Established Stem Cell Model of Spinal Muscular Atrophy Is Applicable in the Evaluation of the Efficacy of Thyrotropin-Releasing Hormone Analog.

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Ohuchi, Kazuki; Funato, Michinori; Kato, Zenichiro; Seki, Junko; Kawase, Chizuru; Tamai, Yuya; Ono, Yoko; Nagahara, Yuki; Noda, Yasuhiro; Kameyama, Tsubasa; Ando, Shiori; Tsuruma, Kazuhiro; Shimazawa, Masamitsu; Hara, Hideaki; Kaneko, Hideo

2016-02-01

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by the degeneration of spinal motor neurons. This disease is mainly caused by mutation or deletion of the survival motor neuron 1 (SMN1) gene. Currently, no effective treatment is available, and only symptomatic treatment can be provided. Our purpose in the present study was to establish a human SMA-derived induced pluripotent stem cell (SMA-iPSC) disease model and assay a therapeutic drug in preparation for the development of a novel treatment of SMA. We generated iPSCs from the skin fibroblasts of a patient with SMA and confirmed that they were pluripotent and undifferentiated. The neural differentiation of SMA-iPSCs shortened the dendrite and axon length and increased the apoptosis of the spinal motor neurons. In addition, we found activated astrocytes in differentiated SMA-iPSCs. Using this model, we confirmed that treatment with the thyrotropin-releasing hormone (TRH) analog, 5-oxo-l-prolyl-l-histidyl-l-prolinamide, which had marginal effects in clinical trials, increases the SMN protein level. This increase was mediated through the transcriptional activation of the SMN2 gene and inhibition of glycogen synthase kinase-3β activity. Finally, the TRH analog treatment resulted in dendrite and axon development of spinal motor neurons in differentiated SMA-iPSCs. These results suggest that this human in vitro disease model stimulates SMA pathology and reveal the potential efficacy of TRH analog treatment for SMA. Therefore, we can screen novel therapeutic drugs such as TRH for SMA easily and effectively using the human SMA-iPSC model. Significance: Platelet-derived growth factor (PDGF) has recently been reported to produce the greatest increase in survival motor neuron protein levels by inhibiting glycogen synthase kinase (GSK)-3β; however, motor neurons lack PDGF receptors. A human in vitro spinal muscular atrophy-derived induced pluripotent stem cell model was

Spliceosome integrity is defective in the motor neuron diseases ALS and SMA

Science.gov (United States)

Tsuiji, Hitomi; Iguchi, Yohei; Furuya, Asako; Kataoka, Ayane; Hatsuta, Hiroyuki; Atsuta, Naoki; Tanaka, Fumiaki; Hashizume, Yoshio; Akatsu, Hiroyasu; Murayama, Shigeo; Sobue, Gen; Yamanaka, Koji

2013-01-01

Two motor neuron diseases, amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), are caused by distinct genes involved in RNA metabolism, TDP-43 and FUS/TLS, and SMN, respectively. However, whether there is a shared defective mechanism in RNA metabolism common to these two diseases remains unclear. Here, we show that TDP-43 and FUS/TLS localize in nuclear Gems through an association with SMN, and that all three proteins function in spliceosome maintenance. We also show that in ALS, Gems are lost, U snRNA levels are up-regulated and spliceosomal U snRNPs abnormally and extensively accumulate in motor neuron nuclei, but not in the temporal lobe of FTLD with TDP-43 pathology. This aberrant accumulation of U snRNAs in ALS motor neurons is in direct contrast to SMA motor neurons, which show reduced amounts of U snRNAs, while both have defects in the spliceosome. These findings indicate that a profound loss of spliceosome integrity is a critical mechanism common to neurodegeneration in ALS and SMA, and may explain cell-type specific vulnerability of motor neurons. PMID:23255347

Towards a rigorous network of protein-protein interactions of the model sulfate reducer Desulfovibrio vulgaris Hildenborough.

Directory of Open Access Journals (Sweden)

Swapnil R Chhabra

Full Text Available Protein-protein interactions offer an insight into cellular processes beyond what may be obtained by the quantitative functional genomics tools of proteomics and transcriptomics. The aforementioned tools have been extensively applied to study Escherichia coli and other aerobes and more recently to study the stress response behavior of Desulfovibrio vulgaris Hildenborough, a model obligate anaerobe and sulfate reducer and the subject of this study. Here we carried out affinity purification followed by mass spectrometry to reconstruct an interaction network among 12 chromosomally encoded bait and 90 prey proteins based on 134 bait-prey interactions identified to be of high confidence. Protein-protein interaction data are often plagued by the lack of adequate controls and replication analyses necessary to assess confidence in the results, including identification of potential false positives. We addressed these issues through the use of biological replication, exponentially modified protein abundance indices, results from an experimental negative control, and a statistical test to assign confidence to each putative interacting pair applicable to small interaction data studies. We discuss the biological significance of metabolic features of D. vulgaris revealed by these protein-protein interaction data and the observed protein modifications. These include the distinct role of the putative carbon monoxide-induced hydrogenase, unique electron transfer routes associated with different oxidoreductases, and the possible role of methylation in regulating sulfate reduction.

Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants

International Nuclear Information System (INIS)

Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M.; Wehnert, Manfred; Huebner, Stefan

2009-01-01

Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associated proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import.

EFFECTS OF PROTEIN-XANTHOPHYLL (PX CONCENTRATE OF ALFALFA ADDITIVE TO CRUDE PROTEIN-REDUCED DIETS ON NITROGEN EXCRETION, GROWTH PERFORMANCE AND MEAT QUALITY OF PIGS

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Eugeniusz GRELA

2009-06-01

Full Text Available The infl uence of protein-xanthophyll (PX concentrate of alfalfa supplement to crude protein-reduced diets was examined in relation to nitrogen excretion, performance parameters and pig meat quality. The investigations included 60 growers (PL x PLW x Duroc crossbreeds assigned to 3 groups. The conclusion is that there is a large potential to decrease nitrogen emission to the environment by 10% lowering of dietary crude protein intake along with reduced animal growth rate and elevated mixture utilization. Inclusion of a protein-xanthophyll concentrate (PX of alfalfa to the diet is likely to diminish disadvantageous productive parameters arising from limiting of total crude protein level in relation to the requirements of pigs feeding norms [1993]. At the same time, it improves feed nitrogen utilization and reduces noxious odour emissions from a piggery. The components of a protein-xanthophyll concentrate (PX contribute to increased liver and kidney weight.

Reduced expression of G protein-coupled receptor kinases in schizophrenia but not in schizoaffective disorder

Science.gov (United States)

Bychkov, ER; Ahmed, MR; Gurevich, VV; Benovic, JL; Gurevich, EV

2011-01-01

Alterations of multiple G protein-mediated signaling pathways are detected in schizophrenia. G protein-coupled receptor kinases (GRKs) and arrestins terminate signaling by G protein-coupled receptors exerting powerful influence on receptor functions. Modifications of arrestin and/or GRKs expression may contribute to schizophrenia pathology. Cortical expression of arrestins and GRKs was measured postmortem in control and subjects with schizophrenia or schizoaffective disorder. Additionally, arrestin/GRK expression was determined in elderly patients with schizophrenia and age-matched control. Patients with schizophrenia, but not schizoaffective disorder, displayed reduced concentration of arrestin and GRK mRNAs and GRK3 protein. Arrestins and GRK significantly decreased with age. In elderly patients, GRK6 was reduced, with other GRKs and arrestins unchanged. Reduced cortical concentration of GRKs in schizophrenia (resembling that in aging) may result in altered G protein-dependent signaling, thus contributing to prefrontal deficits in schizophrenia. The data suggest distinct molecular mechanisms underlying schizophrenia and schizoaffective disorder. PMID:21784156

A reduced amino acid alphabet for understanding and designing protein adaptation to mutation.

Science.gov (United States)

Etchebest, C; Benros, C; Bornot, A; Camproux, A-C; de Brevern, A G

2007-11-01

Protein sequence world is considerably larger than structure world. In consequence, numerous non-related sequences may adopt similar 3D folds and different kinds of amino acids may thus be found in similar 3D structures. By grouping together the 20 amino acids into a smaller number of representative residues with similar features, sequence world simplification may be achieved. This clustering hence defines a reduced amino acid alphabet (reduced AAA). Numerous works have shown that protein 3D structures are composed of a limited number of building blocks, defining a structural alphabet. We previously identified such an alphabet composed of 16 representative structural motifs (5-residues length) called Protein Blocks (PBs). This alphabet permits to translate the structure (3D) in sequence of PBs (1D). Based on these two concepts, reduced AAA and PBs, we analyzed the distributions of the different kinds of amino acids and their equivalences in the structural context. Different reduced sets were considered. Recurrent amino acid associations were found in all the local structures while other were specific of some local structures (PBs) (e.g Cysteine, Histidine, Threonine and Serine for the alpha-helix Ncap). Some similar associations are found in other reduced AAAs, e.g Ile with Val, or hydrophobic aromatic residues Trp with Phe and Tyr. We put into evidence interesting alternative associations. This highlights the dependence on the information considered (sequence or structure). This approach, equivalent to a substitution matrix, could be useful for designing protein sequence with different features (for instance adaptation to environment) while preserving mainly the 3D fold.

High-fiber diets with reduced crude protein for commercial layers

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MFFM Praes

2014-06-01

Full Text Available This study aimed at evaluating diets containing different fiber sources and two crude protein levels on the performance, egg quality, and nitrogen metabolism of commercial layers. In total, 392 48-wk-old Isa Brown layers were distributed according to a completely randomized experimental design in a 3x2+1 (control factorial arrangement, resulting in seven treatments with seven replicates of eight birds each. Treatments consisted of three fiber feedstuffs (cottonseed hulls, soybean hulls, and rice hulls and two dietary crude protein levels (12% and 16%. Cottonseed hulls associated with the high crude protein level (16% resulted in the worst feed conversion ratio per dozen eggs. Diets with 16% crude protein resulted in the highest feed intake, egg production, egg weight, egg mass values, and improved feed conversion ratio (kg eggs/kg feed. The dietary inclusion of soybean hulls determined low yolk pigmentation, and of rice hulls, low egg specific gravity. The 16% crude protein diet with rice hulls promoted the best feed conversion ratio. Hens fed the reference diet presented higher egg mass and better feed conversion ratio per kg eggs and per dozen eggs. Hens fed the diets with low crude protein level (12% had reduced nitrogen excretion, but presented worse egg production.

Gestational diabetes is characterized by reduced mitochondrial protein expression and altered calcium signaling proteins in skeletal muscle.

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Kristen E Boyle

Full Text Available The rising prevalence of gestational diabetes mellitus (GDM affects up to 18% of pregnant women with immediate and long-term metabolic consequences for both mother and infant. Abnormal glucose uptake and lipid oxidation are hallmark features of GDM prompting us to use an exploratory proteomics approach to investigate the cellular mechanisms underlying differences in skeletal muscle metabolism between obese pregnant women with GDM (OGDM and obese pregnant women with normal glucose tolerance (ONGT. Functional validation was performed in a second cohort of obese OGDM and ONGT pregnant women. Quantitative proteomic analysis in rectus abdominus skeletal muscle tissue collected at delivery revealed reduced protein content of mitochondrial complex I (C-I subunits (NDUFS3, NDUFV2 and altered content of proteins involved in calcium homeostasis/signaling (calcineurin A, α1-syntrophin, annexin A4 in OGDM (n = 6 vs. ONGT (n = 6. Follow-up analyses showed reduced enzymatic activity of mitochondrial complexes C-I, C-III, and C-IV (-60-75% in the OGDM (n = 8 compared with ONGT (n = 10 subjects, though no differences were observed for mitochondrial complex protein content. Upstream regulators of mitochondrial biogenesis and oxidative phosphorylation were not different between groups. However, AMPK phosphorylation was dramatically reduced by 75% in the OGDM women. These data suggest that GDM is associated with reduced skeletal muscle oxidative phosphorylation and disordered calcium homeostasis. These relationships deserve further attention as they may represent novel risk factors for development of GDM and may have implications on the effectiveness of physical activity interventions on both treatment strategies for GDM and for prevention of type 2 diabetes postpartum.

Fish protein hydrolysate elevates plasma bile acids and reduces visceral adipose tissue mass in rats

DEFF Research Database (Denmark)

Liaset, Bjørn; Madsen, Lise; Hao, Qin

2009-01-01

levels relative to rats fed soy protein or casein. Concomitantly, the saithe FPH fed rats had reduced liver lipids and fasting plasma TAG levels. Furthermore, visceral adipose tissue mass was reduced and expression of genes involved in fatty acid oxidation and energy expenditure was induced in perirenal....../retroperitoneal adipose tissues of rats fed saithe FPH. Our results provide the first evidence that dietary protein sources with different amino acid compositions can modulate the level of plasma bile acids and our data suggest potential novel mechanisms by which dietary protein sources can affect energy metabolism....

Reduced synaptic vesicle protein degradation at lysosomes curbs TBC1D24/sky-induced neurodegeneration.

Science.gov (United States)

Fernandes, Ana Clara; Uytterhoeven, Valerie; Kuenen, Sabine; Wang, Yu-Chun; Slabbaert, Jan R; Swerts, Jef; Kasprowicz, Jaroslaw; Aerts, Stein; Verstreken, Patrik

2014-11-24

Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. In this paper, we study Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome, and identify endosome-to-lysosome trafficking as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, we show that synaptic vesicle-associated proteins were younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, we find that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. Our findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans. © 2014 Fernandes et al.

Scoring predictive models using a reduced representation of proteins: model and energy definition.

Science.gov (United States)

Fogolari, Federico; Pieri, Lidia; Dovier, Agostino; Bortolussi, Luca; Giugliarelli, Gilberto; Corazza, Alessandra; Esposito, Gennaro; Viglino, Paolo

2007-03-23

Reduced representations of proteins have been playing a keyrole in the study of protein folding. Many such models are available, with different representation detail. Although the usefulness of many such models for structural bioinformatics applications has been demonstrated in recent years, there are few intermediate resolution models endowed with an energy model capable, for instance, of detecting native or native-like structures among decoy sets. The aim of the present work is to provide a discrete empirical potential for a reduced protein model termed here PC2CA, because it employs a PseudoCovalent structure with only 2 Centers of interactions per Amino acid, suitable for protein model quality assessment. All protein structures in the set top500H have been converted in reduced form. The distribution of pseudobonds, pseudoangle, pseudodihedrals and distances between centers of interactions have been converted into potentials of mean force. A suitable reference distribution has been defined for non-bonded interactions which takes into account excluded volume effects and protein finite size. The correlation between adjacent main chain pseudodihedrals has been converted in an additional energetic term which is able to account for cooperative effects in secondary structure elements. Local energy surface exploration is performed in order to increase the robustness of the energy function. The model and the energy definition proposed have been tested on all the multiple decoys' sets in the Decoys'R'us database. The energetic model is able to recognize, for almost all sets, native-like structures (RMSD less than 2.0 A). These results and those obtained in the blind CASP7 quality assessment experiment suggest that the model compares well with scoring potentials with finer granularity and could be useful for fast exploration of conformational space. Parameters are available at the url: http://www.dstb.uniud.it/~ffogolari/download/.

Resistance training reduces whole-body protein turnover and improves net protein retention in untrained young males.

Science.gov (United States)

Hartman, Joseph W; Moore, Daniel R; Phillips, Stuart M

2006-10-01

It is thought that resistance exercise results in an increased need for dietary protein; however, data also exists to support the opposite conclusion. The purpose of this study was to determine the impact of resistance exercise training on protein metabolism in novices with the hypothesis that resistance training would reduce protein turnover and improve whole-body protein retention. Healthy males (n = 8, 22 +/- 1 y, BMI = 25.3 +/- 1.8 kg.m(-2)) participated in a progressive whole-body split routine resistance-training program 5d/week for 12 weeks. Before (PRE) and after (POST) the training, oral [15N]-glycine ingestion was used to assess nitrogen flux (Q), protein synthesis (PS), protein breakdown (PB), and net protein balance (NPB = PS-PB). Macronutrient intake was controlled over a 5d period PRE and POST, while estimates of protein turnover and urinary nitrogen balance (N(bal) = N(in) - urine N(out)) were conducted. Bench press and leg press increased 40% and 50%, respectively (p training-induced increases in both NPB (PRE = 0.22 +/- 0.13 g.kg(-1).d(-1); POST = 0.54 +/- 0.08 g.kg(-1).d(-1)) and urinary nitrogen balance (PRE = 2.8 +/- 1.7 g N.d(-1); POST = 6.5 +/- 0.9 g N.d(-1)) were observed. A program of resistance training that induced significant muscle hypertrophy resulted in reductions of both whole-body PS and PB, but an improved NPB, which favoured the accretion of skeletal muscle protein. Urinary nitrogen balance increased after training. The reduction in PS and PB and a higher NPB in combination with an increased nitrogen balance after training suggest that dietary requirements for protein in novice resistance-trained athletes are not higher, but lower, after resistance training.

RNAi reduces expression and intracellular retention of mutant cartilage oligomeric matrix protein.

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Karen L Posey

2010-04-01

Full Text Available Mutations in cartilage oligomeric matrix protein (COMP, a large extracellular glycoprotein expressed in musculoskeletal tissues, cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia. These mutations lead to massive intracellular retention of COMP, chondrocyte death and loss of growth plate chondrocytes that are necessary for linear growth. In contrast, COMP null mice have only minor growth plate abnormalities, normal growth and longevity. This suggests that reducing mutant and wild-type COMP expression in chondrocytes may prevent the toxic cellular phenotype causing the skeletal dysplasias. We tested this hypothesis using RNA interference to reduce steady state levels of COMP mRNA. A panel of shRNAs directed against COMP was tested. One shRNA (3B reduced endogenous and recombinant COMP mRNA dramatically, regardless of expression levels. The activity of the shRNA against COMP mRNA was maintained for up to 10 weeks. We also demonstrate that this treatment reduced ER stress. Moreover, we show that reducing steady state levels of COMP mRNA alleviates intracellular retention of other extracellular matrix proteins associated with the pseudoachondroplasia cellular pathology. These findings are a proof of principle and the foundation for the development of a therapeutic intervention based on reduction of COMP expression.

Fish protein intake induces fast-muscle hypertrophy and reduces liver lipids and serum glucose levels in rats.

Science.gov (United States)

Kawabata, Fuminori; Mizushige, Takafumi; Uozumi, Keisuke; Hayamizu, Kohsuke; Han, Li; Tsuji, Tomoko; Kishida, Taro

2015-01-01

In our previous study, fish protein was proven to reduce serum lipids and body fat accumulation by skeletal muscle hypertrophy and enhancing basal energy expenditure in rats. In the present study, we examined the precise effects of fish protein intake on different skeletal muscle fiber types and metabolic gene expression of the muscle. Fish protein increased fast-twitch muscle weight, reduced liver triglycerides and serum glucose levels, compared with the casein diet after 6 or 8 weeks of feeding. Furthermore, fish protein upregulated the gene expressions of a fast-twitch muscle-type marker and a glucose transporter in the muscle. These results suggest that fish protein induces fast-muscle hypertrophy, and the enhancement of basal energy expenditure by muscle hypertrophy and the increase in muscle glucose uptake reduced liver lipids and serum glucose levels. The present results also imply that fish protein intake causes a slow-to-fast shift in muscle fiber type.

Gene therapy for spinomuscular atrophy: a biomedical advance, a missed opportunity for more equitable drug pricing.

Science.gov (United States)

Friedmann, T

2017-09-01

An experimental approach for gene therapy of spinomuscular atrophy has been reported to prevent development of the neuromuscular features of this lethal and previously untreatable disorder. The approach involves treatment of patients suffering from SMN1-associated infantile form of the disease with a splice-switching antisense oligonucleotide (ASO) that corrects aberrant splicing of the nearly identical SMN2 gene to allow the generation of functional SMN protein, thereby mitigating the development of the disease. This technique represents the first apparently effective therapy for spinal muscular atrophy (SMA) and an important documentation for ASO technology for therapy of neurodegenerative disease. These results with one form of SMA are likely to be relevant for similar applications to other SMA types and are likely to inspire application to a number of other intractable neurodegenerative diseases such as Huntington's disease, amyotrophic lateral sclerosis and possibly even the extremely common Parkinson's and Alzheimer's diseases and others. Nevertheless, the scientific and medical importance of this advance is marred by a pricing policy by the corporate sponsors that may complicate accessibility of the drug for some desperate patients.

Pharmacological inhibition of dynamin II reduces constitutive protein secretion from primary human macrophages.

Directory of Open Access Journals (Sweden)

Maaike Kockx

Full Text Available Dynamins are fission proteins that mediate endocytic and exocytic membrane events and are pharmacological therapeutic targets. These studies investigate whether dynamin II regulates constitutive protein secretion and show for the first time that pharmacological inhibition of dynamin decreases secretion of apolipoprotein E (apoE and several other proteins constitutively secreted from primary human macrophages. Inhibitors that target recruitment of dynamin to membranes (MiTMABs or directly target the GTPase domain (Dyngo or Dynole series, dose- and time- dependently reduced the secretion of apoE. SiRNA oligo's targeting all isoforms of dynamin II confirmed the involvement of dynamin II in apoE secretion. Inhibition of secretion was not mediated via effects on mRNA or protein synthesis. 2D-gel electrophoresis showed that inhibition occurred after apoE was processed and glycosylated in the Golgi and live cell imaging showed that inhibited secretion was associated with reduced post-Golgi movement of apoE-GFP-containing vesicles. The effect was not restricted to macrophages, and was not mediated by the effects of the inhibitors on microtubules. Inhibition of dynamin also altered the constitutive secretion of other proteins, decreasing the secretion of fibronectin, matrix metalloproteinase 9, Chitinase-3-like protein 1 and lysozyme but unexpectedly increasing the secretion of the inflammatory mediator cyclophilin A. We conclude that pharmacological inhibitors of dynamin II modulate the constitutive secretion of macrophage apoE as a class effect, and that their capacity to modulate protein secretion may affect a range of biological processes.

Heat-treatment reduces anti-nutritional phytochemicals and maintains protein quality in genetically improved hulled soybean flour

Directory of Open Access Journals (Sweden)

Ariela Werneck de Carvalho

2013-06-01

Full Text Available The soybean is a protein source of high biological value. However, the presence of anti-nutritional factors affects its protein quality and limits the bioavailability of other nutrients. The effect of heat-treatment, 150 ºC for 30 minutes, on hulled and hull-less soybean flour from the cultivar UFVTN 105AP on urease, trypsin inhibitor activity, protein solubility, amino acid profile, and in vivo protein quality was investigated. The treatment reduced the trypsin inhibitor activity and urease, but it did not affect protein solubility. Protein Efficiency Coefficient (PER values of the flours were similar, and the PER of the hull-less soybean flour did not differ from casein. The Net Protein Ratio (NPR did not differ between the experimental groups. The True Digestibility (TD of the flours did not differ, but both were lower in casein and the Protein Digestibility Corrected Amino Acid Score (PDCCAS was lower than the TD, due to limited valine determined by the chemical score. Therefore, the flours showed reduced anti-nutritional phytochemicals and similar protein quality, and therefore the whole flours can be used as a source of high quality protein.

Modulation of GDP-fucose level for generating proteins with reduced rate of fucosylation (WO2010141855).

Science.gov (United States)

Taupin, Philippe

2011-09-01

The application (WO2010141855) is in the field of glycobiology, and involves the control of the rate of fucosylation of proteins by exogenous factors. It aims at controlling the rate of protein fucosylation with inhibitors (drugs or nucleic acid antagonists) of enzymes involved in the synthesis of GDP-fucose. Mammalian cell lines were cultured in the presence of inhibitors, for example, siRNA. The rates of GDP-fucose in cells and during protein fucosylation were characterized. The level of protein fucosylation decreases rapidly in response to a decrease in GDP-fucose level. The relationship between the rate of fucosylation of proteins and the level of GDP-fucose in a cell is non-linear. Reduction in the rate of protein fucosylation can be achieved with a minimal reduction of the level of GDP-fucose in cells. The paradigm may be used to synthesize proteins and antibodies, with a reduced rate of fucosylation. The application claims that the use of drugs or nucleic acid antagonists that inhibit the enzymes involved in GDP-fucose biosynthesis optimizes the level of GDP-fucose present in cells, and reduces the rate of fucosylation of glycoproteins.

An autoclave treatment reduces the solubility and antigenicity of an allergenic protein found in buckwheat flour.

Science.gov (United States)

Tomotake, Hiroyuki; Yamazaki, Rikio; Yamato, Masayuki

2012-06-01

The effects of an autoclave treatment of buckwheat flour on a 24-kDa allergenic protein were investigated by measuring reduction in solubility and antibody binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the intensity of the major bands, including that of the 24-kDa allergen, was reduced by the autoclave treatment. The protein solubility in buckwheat flour was variably decreased by the autoclave treatment. Enzyme-linked immunosorbent assay analysis using a monoclonal antibody specific for buckwheat 24-kDa protein showed that the reactivity of protein extracts (10 μg/ml) from buckwheat flour was lowered by the autoclave treatment. The autoclave treatment may reduce the major allergen content of buckwheat. Future studies will determine if autoclaving treatments affect the allergenicity of the 24-kDa buckwheat protein.

The role of social media networks in psychotic disorders: a case report.

Science.gov (United States)

Krishna, Nithin; Fischer, Bernard A; Miller, Moshe; Register-Brown, Kelly; Patchan, Kathleen; Hackman, Ann

2013-01-01

We report the case of a young man diagnosed with schizophrenia who presented with stalking behaviors that may have been caused by problematic use or participation in social media networks (SMN). We review the possible role of SMN in the formation of his romantic delusion and offer suggestions for clinicians around incorporation of SMN questions into assessments. It is imperative to identify populations at risk of SMN-related stalking behaviors to stratify mental health resources and interventions. Additional studies are needed to further clarify the role of SMN in psychotic disorders. Copyright © 2013 Elsevier Inc. All rights reserved.

The imprinted SNRPN gene is associated with a polycistronic mRNA and an imprinting control element

Energy Technology Data Exchange (ETDEWEB)

Saitoh, S.; Nicholls, R.D. [Case Western Reserve Univ., Cleveland, OH (United States); Seip, J. [Pennsylvania State Univ., Hershey, PA (United States)] [and others

1994-09-01

The small nuclear ribonucleoprotein-associated protein SmN (SNRPN) gene is located in the Prader-Willi syndrome (PWS) critical region in chromosome 15q11-q13. We have previously shown that it is functionally imprinted in humans, being only expressed from the paternal allele and differentially methylated on parental alleles. Therefore, SNRPN may have a role in PWS, although genetic studies suggest that at least two genes may be necessary for the classical PWS phenotype. We have characterized the SNRPN genomic structure, and shown that it comprises ten exons. Surprisingly, we identified an open reading frame (ORF) in the first three exons, 190-bp 5{prime} to the SmN ORF. Notably, the majority of base substitutions bewteen human and rodents in the upstream ORF occurred in the wobble position of codons, suggesting selection for a protein coding function. This ORF, which we name SNURF (SNRPN upstream reading frame) encodes a putative polypeptide of 71 amino acids. By analogy to prokaryotic operons that encode proteins with related functions, it is possible that SNURF may have a role in pre-mRNA splicing.

Whey protein consumption after resistance exercise reduces energy intake at a post-exercise meal.

Science.gov (United States)

Monteyne, Alistair; Martin, Alex; Jackson, Liam; Corrigan, Nick; Stringer, Ellen; Newey, Jack; Rumbold, Penny L S; Stevenson, Emma J; James, Lewis J

2018-03-01

Protein consumption after resistance exercise potentiates muscle protein synthesis, but its effects on subsequent appetite in this context are unknown. This study examined appetite and energy intake following consumption of protein- and carbohydrate-containing drinks after resistance exercise. After familiarisation, 15 resistance training males (age 21 ± 1 years, body mass 78.0 ± 11.9 kg, stature 1.78 ± 0.07 m) completed two randomised, double-blind trials, consisting of lower-body resistance exercise, followed by consumption of a whey protein (PRO 23.9 ± 3.6 g protein) or dextrose (CHO 26.5 ± 3.8 g carbohydrate) drink in the 5 min post-exercise. An ad libitum meal was served 60 min later, with subjective appetite measured throughout. Drinks were flavoured and matched for energy content and volume. The PRO drink provided 0.3 g/kg body mass protein. Ad libitum energy intake (PRO 3742 ± 994 kJ; CHO 4172 ± 1132 kJ; P = 0.007) and mean eating rate (PRO 339 ± 102 kJ/min; CHO 405 ± 154 kJ/min; P = 0.009) were lower during PRO. The change in eating rate was associated with the change in energy intake (R = 0.661, P = 0.007). No interaction effects were observed for subjective measures of appetite. The PRO drink was perceived as creamier and thicker, and less pleasant, sweet and refreshing (P consumption after resistance exercise reduces subsequent energy intake, and this might be partially mediated by a reduced eating rate. Whilst this reduced energy intake is unlikely to impair hypertrophy, it may be of value in supporting an energy deficit for weight loss.

High protein flexibility and reduced hydration water dynamics are key pressure adaptive strategies in prokaryotes

KAUST Repository

Martinez, N.

2016-09-06

Water and protein dynamics on a nanometer scale were measured by quasi-elastic neutron scattering in the piezophile archaeon Thermococcus barophilus and the closely related pressure-sensitive Thermococcus kodakarensis, at 0.1 and 40 MPa. We show that cells of the pressure sensitive organism exhibit higher intrinsic stability. Both the hydration water dynamics and the fast protein and lipid dynamics are reduced under pressure. In contrast, the proteome of T. barophilus is more pressure sensitive than that of T. kodakarensis. The diffusion coefficient of hydration water is reduced, while the fast protein and lipid dynamics are slightly enhanced with increasing pressure. These findings show that the coupling between hydration water and cellular constituents might not be simply a master-slave relationship. We propose that the high flexibility of the T. barophilus proteome associated with reduced hydration water may be the keys to the molecular adaptation of the cells to high hydrostatic pressure.

High protein flexibility and reduced hydration water dynamics are key pressure adaptive strategies in prokaryotes

KAUST Repository

Martinez, N.; Michoud, Gregoire; Cario, A.; Ollivier, J.; Franzetti, B.; Jebbar, M.; Oger, P.; Peters, J.

2016-01-01

Water and protein dynamics on a nanometer scale were measured by quasi-elastic neutron scattering in the piezophile archaeon Thermococcus barophilus and the closely related pressure-sensitive Thermococcus kodakarensis, at 0.1 and 40 MPa. We show that cells of the pressure sensitive organism exhibit higher intrinsic stability. Both the hydration water dynamics and the fast protein and lipid dynamics are reduced under pressure. In contrast, the proteome of T. barophilus is more pressure sensitive than that of T. kodakarensis. The diffusion coefficient of hydration water is reduced, while the fast protein and lipid dynamics are slightly enhanced with increasing pressure. These findings show that the coupling between hydration water and cellular constituents might not be simply a master-slave relationship. We propose that the high flexibility of the T. barophilus proteome associated with reduced hydration water may be the keys to the molecular adaptation of the cells to high hydrostatic pressure.

Reduced Insulin/IGF-1 Signaling Restores the Dynamic Properties of Key Stress Granule Proteins during Aging

Directory of Open Access Journals (Sweden)

Marie C. Lechler

2017-01-01

Full Text Available Summary: Low-complexity “prion-like” domains in key RNA-binding proteins (RBPs mediate the reversible assembly of RNA granules. Individual RBPs harboring these domains have been linked to specific neurodegenerative diseases. Although their aggregation in neurodegeneration has been extensively characterized, it remains unknown how the process of aging disturbs RBP dynamics. We show that a wide variety of RNA granule components, including stress granule proteins, become highly insoluble with age in C. elegans and that reduced insulin/insulin-like growth factor 1 (IGF-1 daf-2 receptor signaling efficiently prevents their aggregation. Importantly, stress-granule-related RBP aggregates are associated with reduced fitness. We show that heat shock transcription factor 1 (HSF-1 is a main regulator of stress-granule-related RBP aggregation in both young and aged animals. During aging, increasing DAF-16 activity restores dynamic stress-granule-related RBPs, partly by decreasing the buildup of other misfolded proteins that seed RBP aggregation. Longevity-associated mechanisms found to maintain dynamic RBPs during aging could be relevant for neurodegenerative diseases. : Lechler et al. show that RNA-binding proteins (RBPs including stress granule proteins are prone to aggregate with age in C. elegans. Aggregation of stress granule RBPs with “prion-like” domains is associated with reduced fitness. Their aggregation is prevented by longevity pathways and promoted by the aggregation of other misfolded proteins. Keywords: neurodegenerative diseases, Caenorhabditis elegans, protein aggregation, aging, RNA-binding proteins, stress granules, HSF-1, DAF-2, longevity

Evolution of the Tl glow curve of Zn S:Mn nanocrystalline; Evolucion de la curva de brillo Tl de ZnS:Mn nanocristalino

Energy Technology Data Exchange (ETDEWEB)

Ortiz H, A. A. [Universidad Autonoma de San Luis Potosi, Doctorado en Ingenieria y Ciencia de Materiales, 78000 San Luis Potosi (Mexico); Mendez G, V. H. [Universidad Autonoma de San Luis Potosi, Coordinacion para la Innovacion y Aplicacion de la Ciencia y la Tecnologia, 78000 San Luis Potosi (Mexico); Perez A, M. L.; Ortega S, J. J.; Araiza, J. J. [Universidad Autonoma de Zacatecas, Unidad Academica de Fisica, 98000, Zacatecas, Zac. (Mexico); Rivera, T. [IPN, Centro de Investigacion en Ciencia Aplicada y Tecnologia Avanzada, Av. Legaria 694, Col. Irrigacion, 11500 Mexico D. F. (Mexico); Alfaro C, M. R. [Centro de Investigacion en Materiales Avanzados, Alianza Norte 202, 66600 Apodaca, Nuevo Leon (Mexico); Vega C, H. R., E-mail: icearturoortiz@hotmail.com [Universidad Autonoma de Zacatecas, Unidad Academica de Estudios Nucleares, 98068 Zacatecas, Zac. (Mexico)

2015-10-15

Full text: In the last two decades, the search for new materials for dosimetry has included semiconductor nano materials because of their luminescent properties. This search has included the study, synthesis, characterization and performance of nano structured semiconductors, which optoelectronic properties determine their applications. In this paper the evolution of the thermoluminescent glow curve of nanocrystalline powder samples (40-70 nm) of zinc sulfide doped with manganese (Zn S:Mn) was analyzed at a dose of 500 Gy using a {sup 60}Co source. This material was synthesized by the coprecipitation method and heat treated at 500 degrees C in forming gas atmosphere (80 N{sub 2}:20H{sub 2}). Photoluminescence results indicate a direct relationship between the concentration of manganese and the intensity of a peak at λ ≅ 600 nm. By means of numerical deconvolution the behavior of the glow curves obtained at different times after exposure was analyzed. The causing traps of thermoluminescence are to 0.60 ± 0.05 and 1.7 ± 0.4 eV below the conduction band and within the band gap. The fading and a variation in the shape of the brightness curve (evolution) caused by non radiative transitions (rotational and vibrational) within the crystal structure of the material is also reported. (Author)

Beta-carotene encapsulated in food protein nanoparticles reduces peroxyl radical oxidation in Caco-2 cells

Science.gov (United States)

Beta-carotene (BC) was encapsulated by sodium caseinate (SC), whey protein isolate (WPI), and soybean protein isolate (SPI) by the homogenization-evaporation method forming nanoparticles of 78, 90 and 370 nm diameter. Indices of the chemical antioxidant assays, the reducing power, DPPH radical scave...

Amyloid β-sheet mimics that antagonize protein aggregation and reduce amyloid toxicity

Science.gov (United States)

Cheng, Pin-Nan; Liu, Cong; Zhao, Minglei; Eisenberg, David; Nowick, James S.

2012-11-01

The amyloid protein aggregation associated with diseases such as Alzheimer's, Parkinson's and type II diabetes (among many others) features a bewildering variety of β-sheet-rich structures in transition from native proteins to ordered oligomers and fibres. The variation in the amino-acid sequences of the β-structures presents a challenge to developing a model system of β-sheets for the study of various amyloid aggregates. Here, we introduce a family of robust β-sheet macrocycles that can serve as a platform to display a variety of heptapeptide sequences from different amyloid proteins. We have tailored these amyloid β-sheet mimics (ABSMs) to antagonize the aggregation of various amyloid proteins, thereby reducing the toxicity of amyloid aggregates. We describe the structures and inhibitory properties of ABSMs containing amyloidogenic peptides from the amyloid-β peptide associated with Alzheimer's disease, β2-microglobulin associated with dialysis-related amyloidosis, α-synuclein associated with Parkinson's disease, islet amyloid polypeptide associated with type II diabetes, human and yeast prion proteins, and Tau, which forms neurofibrillary tangles.

The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan.

Science.gov (United States)

Mittal, Nitish; Guimaraes, Joao C; Gross, Thomas; Schmidt, Alexander; Vina-Vilaseca, Arnau; Nedialkova, Danny D; Aeschimann, Florian; Leidel, Sebastian A; Spang, Anne; Zavolan, Mihaela

2017-09-06

In Saccharomyces cerevisiae, deletion of large ribosomal subunit protein-encoding genes increases the replicative lifespan in a Gcn4-dependent manner. However, how Gcn4, a key transcriptional activator of amino acid biosynthesis genes, increases lifespan, is unknown. Here we show that Gcn4 acts as a repressor of protein synthesis. By analyzing the messenger RNA and protein abundance, ribosome occupancy and protein synthesis rate in various yeast strains, we demonstrate that Gcn4 is sufficient to reduce protein synthesis and increase yeast lifespan. Chromatin immunoprecipitation reveals Gcn4 binding not only at genes that are activated, but also at genes, some encoding ribosomal proteins, that are repressed upon Gcn4 overexpression. The promoters of repressed genes contain Rap1 binding motifs. Our data suggest that Gcn4 is a central regulator of protein synthesis under multiple perturbations, including ribosomal protein gene deletions, calorie restriction, and rapamycin treatment, and provide an explanation for its role in longevity and stress response.The transcription factor Gcn4 is known to regulate yeast amino acid synthesis. Here, the authors show that Gcn4 also acts as a repressor of protein biosynthesis in a range of conditions that enhance yeast lifespan, such as ribosomal protein knockout, calorie restriction or mTOR inhibition.

Whey protein reduces early life weight gain in mice fed a high-fat diet

DEFF Research Database (Denmark)

Tranberg, Britt; Hellgren, Lars; Lykkesfeldt, Jens

2013-01-01

An increasing number of studies indicate that dairy products, including whey protein, alleviate several disorders of the metabolic syndrome. Here, we investigated the effects of whey protein isolate (whey) in mice fed a high-fat diet hypothesising that the metabolic effects of whey would...... be associated with changes in the gut microbiota composition. Five-week-old male C57BL/6 mice were fed a high-fat diet ad libitum for 14 weeks with the protein source being either whey or casein. Faeces were collected at week 0, 7, and 13 and the fecal microbiota was analysed by denaturing gradient gel...... reduced weight gain in young C57BL/6 mice fed a high-fat diet compared to casein. Although the effect on weight gain ceased, whey alleviated glucose intolerance, improved insulin sensitivity and reduced plasma cholesterol. These findings could not be explained by changes in food intake or gut microbiota...

Short communication: Effect of whey protein addition and transglutaminase treatment on the physical and sensory properties of reduced-fat ice cream.

Science.gov (United States)

Danesh, Erfan; Goudarzi, Mostafa; Jooyandeh, Hossein

2017-07-01

The effects of whey protein addition and transglutaminase treatment, alone and in combination, on the physical and sensory properties of reduced-fat ice cream were investigated. Adding whey protein with or without enzyme treatment decreased melting rate, overrun, and hardness of the reduced-fat ice cream; however, the enzyme-treated sample had a higher melting rate and overrun and softer texture. Whey protein-fortified samples showed higher melting resistance, but lower overrun and firmer texture compared with the enzyme-treated sample without added whey protein. Whey protein addition with or without transglutaminase treatment caused an increase in apparent viscosity and a decrease in flow index of the reduced-fat ice cream; nevertheless, the flow behavior of full-fat sample was most similar to the enzyme-treated reduced-fat sample with no added whey protein. Descriptive sensory analyses showed that neither whey protein addition nor transglutaminase treatment significantly influenced the flavor and odor of reduced-fat ice cream, but they both noticeably improved the color and texture of the final product. The results of this study suggest that whey protein addition with transglutaminase treatment improves the physical and sensory properties of reduced-fat ice cream more favorably than does whey protein addition or transglutaminase treatment alone. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The protein C omega-loop substitution Asn2Ile is associated with reduced protein C anticoagulant activity.

LENUS (Irish Health Repository)

Preston, Roger J S

2012-02-01

We report a kindred with heritable protein C (PC) deficiency in which two siblings with severe thrombosis showed a composite type I and IIb PC deficiency phenotype, identified using commercial PC assays (proband: PC antigen 42 u\\/dl, amidolytic activity 40 u\\/dl, anticoagulant activity 9 u\\/dl). The independent PROC nucleotide variations c.669C>A (predictive of Ser181Arg) and c.131C>T (predictive of Asn2Ile) segregated with the type I and type IIb PC deficiency phenotypes respectively, but co-segregated in the siblings with severe thrombosis. Soluble thrombomodulin (sTM)-mediated inhibition of plasma thrombin generation from an individual with PC-Asn2Ile was lower (endogenous thrombin potential (ETP) 56 +\\/- 1% that of ETP determined without sTM) than control plasma (ETP 15 +\\/- 2%) indicating reduced PC anticoagulant activity. Recombinant APC-Asn2Ile exhibited normal amidolytic activity but impaired anticoagulant activity. Protein S (PS)-dependent anticoagulant activity of recombinant APC-Asn2Ile and binding of recombinant APC-Asn2Ile to endothelial protein C receptor (EPCR) were reduced compared to recombinant wild-type APC. Asn2 lies within the omega-loop of the PC\\/APC Gla domain and this region is critical for calcium-induced folding and subsequent interactions with anionic phospholipids, EPCR and PS. The disruption of these interactions in this naturally-occurring PC variant highlights their collective importance in mediating APC anticoagulant activity in vivo.

Genetic screening of spinal muscular atrophy using a real-time modified COP-PCR technique with dried blood-spot DNA.

Science.gov (United States)

Ar Rochmah, Mawaddah; Harahap, Nur Imma Fatimah; Niba, Emma Tabe Eko; Nakanishi, Kenta; Awano, Hiroyuki; Morioka, Ichiro; Iijima, Kazumoto; Saito, Toshio; Saito, Kayoko; Lai, Poh San; Takeshima, Yasuhiro; Takeuchi, Atsuko; Bouike, Yoshihiro; Okamoto, Maya; Nishio, Hisahide; Shinohara, Masakazu

2017-10-01

Spinal muscular atrophy (SMA) is a common neuromuscular disorder caused by mutations in SMN1. More than 95% of SMA patients carry homozygous SMN1 deletion. SMA is the leading genetic cause of infant death, and has been considered an incurable disease. However, a recent clinical trial with an antisense oligonucleotide drug has shown encouraging clinical efficacy. Thus, early and accurate detection of SMN1 deletion may improve prognosis of many infantile SMA patients. A total of 88 DNA samples (37 SMA patients, 12 carriers and 39 controls) from dried blood spots (DBS) on filter paper were analyzed. All participants had previously been screened for SMN genes by PCR restriction fragment length polymorphism (PCR-RFLP) using DNA extracted from freshly collected blood. DNA was extracted from DBS that had been stored at room temperature (20-25°C) for 1week to 5years. To ensure sufficient quality and quantity of DNA samples, target sequences were pre-amplified by conventional PCR. Real-time modified competitive oligonucleotide priming-PCR (mCOP-PCR) with the pre-amplified PCR products was performed for the gene-specific amplification of SMN1 and SMN2 exon 7. Compared with PCR-RFLP using DNA from freshly collected blood, results from real-time mCOP-PCR using DBS-DNA for detection of SMN1 exon 7 deletion showed a sensitivity of 1.00 (CI [0.87, 1.00])] and specificity of 1.00 (CI [0.90, 1.00]), respectively. We combined DNA extraction from DBS on filter paper, pre-amplification of target DNA, and real-time mCOP-PCR to specifically detect SMN1 and SMN2 genes, thereby establishing a rapid, accurate, and high-throughput system for detecting SMN1-deletion with practical applications for newborn screening. Copyright © 2017 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Ultraviolet-ozone treatment reduces levels of disease-associated prion protein and prion infectivity

Science.gov (United States)

Johnson, C.J.; Gilbert, P.; McKenzie, D.; Pedersen, J.A.; Aiken, Judd M.

2009-01-01

Background. Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases caused by novel infectious agents referred to as prions. Prions appear to be composed primarily, if not exclusively, of a misfolded isoform of the cellular prion protein. TSE infectivity is remarkably stable and can resist many aggressive decontamination procedures, increasing human, livestock and wildlife exposure to TSEs. Findings. We tested the hypothesis that UV-ozone treatment reduces levels of the pathogenic prion protein and inactivates the infectious agent. We found that UV-ozone treatment decreased the carbon and prion protein content in infected brain homogenate to levels undetectable by dry-ashing carbon analysis or immunoblotting, respectively. After 8 weeks of ashing, UV-ozone treatment reduced the infectious titer of treated material by a factor of at least 105. A small amount of infectivity, however, persisted despite UV-ozone treatment. When bound to either montmorillonite clay or quartz surfaces, PrPTSE was still susceptible to degradation by UV-ozone. Conclusion. Our findings strongly suggest that UV-ozone treatment can degrade pathogenic prion protein and inactivate prions, even when the agent is associated with surfaces. Using larger UV-ozone doses or combining UV-ozone treatment with other decontaminant methods may allow the sterilization of TSE-contaminated materials. ?? 2009 Aiken et al; licensee BioMed Central Ltd.

Predicted risks of second malignant neoplasm incidence and mortality due to secondary neutrons in a girl and boy receiving proton craniospinal irradiation

International Nuclear Information System (INIS)

Taddei, Phillip J; Mirkovic, Dragan; Zhang Rui; Giebeler, Annelise; Harvey, Mark; Newhauser, Wayne D; Mahajan, Anita; Kornguth, David; Woo, Shiao

2010-01-01

The purpose of this study was to compare the predicted risks of second malignant neoplasm (SMN) incidence and mortality from secondary neutrons for a 9-year-old girl and a 10-year-old boy who received proton craniospinal irradiation (CSI). SMN incidence and mortality from neutrons were predicted from equivalent doses to radiosensitive organs for cranial, spinal and intracranial boost fields. Therapeutic proton absorbed dose and equivalent dose from neutrons were calculated using Monte Carlo simulations. Risks of SMN incidence and mortality in most organs and tissues were predicted by applying risks models from the National Research Council of the National Academies to the equivalent dose from neutrons; for non-melanoma skin cancer, risk models from the International Commission on Radiological Protection were applied. The lifetime absolute risks of SMN incidence due to neutrons were 14.8% and 8.5%, for the girl and boy, respectively. The risks of a fatal SMN were 5.3% and 3.4% for the girl and boy, respectively. The girl had a greater risk for any SMN except colon and liver cancers, indicating that the girl's higher risks were not attributable solely to greater susceptibility to breast cancer. Lung cancer predominated the risk of SMN mortality for both patients. This study suggests that the risks of SMN incidence and mortality from neutrons may be greater for girls than for boys treated with proton CSI.

Uptake and translocation of sulfamethazine by alfalfa grown under hydroponic conditions.

Science.gov (United States)

Kurwadkar, Sudarshan; Struckhoff, Garrett; Pugh, Kameron; Singh, Om

2017-03-01

Antibiotics are routinely used in intensive animal agriculture operations collectively known as Concentrated Animal Feed Operations (CAFO) which include dairy, poultry and swine farms. Wastewater generated by CAFOs often contains low levels of antibiotics and is typically managed in an anaerobic lagoon. The objective of this research is to investigate the uptake and fate of aqueous sulfamethazine (SMN) antibiotic by alfalfa (Medicago sativa) grass grown under hydroponic conditions. Uptake studies were conducted using hydroponically grown alfalfa in a commercially available nutrient solution supplemented with 10mg/L of SMN antibiotic. Analysis of alfalfa sap, root zone, middle one-third, and top portion of the foliage showed varying uptake rate and translocation of SMN. The highest average amount of SMN (8.58μg/kg) was detected in the root zone, followed by the top portion (1.89μg/kg), middle one-third (1.30μg/kg), and sap (0.38μg/kg) samples, indicating a clear distribution of SMN within the sampled regions. The ultraviolet (UV) spectra of parent SMN and translocated SMN identified in different parts of the plant present the possibility of metabolization during the uptake process. Uptake of SMN using alfalfa grown under hydroponic conditions has potential as a promising remediation technology for removal of similar antibiotics from wastewater lagoons. Copyright © 2016. Published by Elsevier B.V.

Effective computation of stochastic protein kinetic equation by reducing stiffness via variable transformation

Energy Technology Data Exchange (ETDEWEB)

Wang, Lijin, E-mail: ljwang@ucas.ac.cn [School of Mathematical Sciences, University of Chinese Academy of Sciences, Beijing 100049 (China)

2016-06-08

The stochastic protein kinetic equations can be stiff for certain parameters, which makes their numerical simulation rely on very small time step sizes, resulting in large computational cost and accumulated round-off errors. For such situation, we provide a method of reducing stiffness of the stochastic protein kinetic equation by means of a kind of variable transformation. Theoretical and numerical analysis show effectiveness of this method. Its generalization to a more general class of stochastic differential equation models is also discussed.

Compensatory axon sprouting for very slow axonal die-back in a transgenic model of spinal muscular atrophy type III.

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Udina, Esther; Putman, Charles T; Harris, Luke R; Tyreman, Neil; Cook, Victoria E; Gordon, Tessa

2017-03-01

Smn +/- transgenic mouse is a model of the mildest form of spinal muscular atrophy. Although there is a loss of spinal motoneurons in 11-month-old animals, muscular force is maintained. This maintained muscular force is mediated by reinnervation of the denervated fibres by surviving motoneurons. The spinal motoneurons in these animals do not show an increased susceptibility to death after nerve injury and they retain their regenerative capacity. We conclude that the hypothesized immaturity of the neuromuscular system in this model cannot explain the loss of motoneurons by systematic die-back. Spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans and is the leading genetic cause of infantile death. Patients lack the SMN1 gene with the severity of the disease depending on the number of copies of the highly homologous SMN2 gene. Although motoneuron death in the Smn +/- transgenic mouse model of the mildest form of SMA, SMA type III, has been reported, we have used retrograde tracing of sciatic and femoral motoneurons in the hindlimb with recording of muscle and motor unit isometric forces to count the number of motoneurons with intact neuromuscular connections. Thereby, we investigated whether incomplete maturation of the neuromuscular system induced by survival motoneuron protein (SMN) defects is responsible for die-back of axons relative to survival of motoneurons. First, a reduction of ∼30% of backlabelled motoneurons began relatively late, at 11 months of age, with a significant loss of 19% at 7 months. Motor axon die-back was affirmed by motor unit number estimation. Loss of functional motor units was fully compensated by axonal sprouting to retain normal contractile force in four hindlimb muscles (three fast-twitch and one slow-twitch) innervated by branches of the sciatic nerve. Second, our evaluation of whether axotomy of motoneurons in the adult Smn +/- transgenic mouse increases their susceptibility to cell death demonstrated

Digestibility of gluten proteins is reduced by baking and enhanced by starch digestion.

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Smith, Frances; Pan, Xiaoyan; Bellido, Vincent; Toole, Geraldine A; Gates, Fred K; Wickham, Martin S J; Shewry, Peter R; Bakalis, Serafim; Padfield, Philip; Mills, E N Clare

2015-10-01

Resistance of proteins to gastrointestinal digestion may play a role in determining immune-mediated adverse reactions to foods. However, digestion studies have largely been restricted to purified proteins and the impact of food processing and food matrices on protein digestibility is poorly understood. Digestibility of a total gliadin fraction (TGF), flour (cv Hereward), and bread was assessed using in vitro batch digestion with simulated oral, gastric, and duodenal phases. Protein digestion was monitored by SDS-PAGE and immunoblotting using monoclonal antibodies specific for celiac-toxic sequences (QQSF, QPFP) and starch digestion by measuring undigested starch. Whereas the TGF was rapidly digested during the gastric phase the gluten proteins in bread were virtually undigested and digested rapidly during the duodenal phase only if amylase was included. Duodenal starch digestion was also slower in the absence of duodenal proteases. The baking process reduces the digestibility of wheat gluten proteins, including those containing sequences active in celiac disease. Starch digestion affects the extent of protein digestion, probably because of gluten-starch complex formation during baking. Digestion studies using purified protein fractions alone are therefore not predictive of digestion in complex food matrices. © 2015 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Heterozygous RFX6 protein truncating variants are associated with MODY with reduced penetrance.

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Patel, Kashyap A; Kettunen, Jarno; Laakso, Markku; Stančáková, Alena; Laver, Thomas W; Colclough, Kevin; Johnson, Matthew B; Abramowicz, Marc; Groop, Leif; Miettinen, Päivi J; Shepherd, Maggie H; Flanagan, Sarah E; Ellard, Sian; Inagaki, Nobuya; Hattersley, Andrew T; Tuomi, Tiinamaija; Cnop, Miriam; Weedon, Michael N

2017-10-12

Finding new causes of monogenic diabetes helps understand glycaemic regulation in humans. To find novel genetic causes of maturity-onset diabetes of the young (MODY), we sequenced MODY cases with unknown aetiology and compared variant frequencies to large public databases. From 36 European patients, we identify two probands with novel RFX6 heterozygous nonsense variants. RFX6 protein truncating variants are enriched in the MODY discovery cohort compared to the European control population within ExAC (odds ratio = 131, P = 1 × 10 -4 ). We find similar results in non-Finnish European (n = 348, odds ratio = 43, P = 5 × 10 -5 ) and Finnish (n = 80, odds ratio = 22, P = 1 × 10 -6 ) replication cohorts. RFX6 heterozygotes have reduced penetrance of diabetes compared to common HNF1A and HNF4A-MODY mutations (27, 70 and 55% at 25 years of age, respectively). The hyperglycaemia results from beta-cell dysfunction and is associated with lower fasting and stimulated gastric inhibitory polypeptide (GIP) levels. Our study demonstrates that heterozygous RFX6 protein truncating variants are associated with MODY with reduced penetrance.Maturity-onset diabetes of the young (MODY) is the most common subtype of familial diabetes. Here, Patel et al. use targeted DNA sequencing of MODY patients and large-scale publically available data to show that RFX6 heterozygous protein truncating variants cause reduced penetrance MODY.

A scalable and accurate method for classifying protein-ligand binding geometries using a MapReduce approach.

Science.gov (United States)

Estrada, T; Zhang, B; Cicotti, P; Armen, R S; Taufer, M

2012-07-01

We present a scalable and accurate method for classifying protein-ligand binding geometries in molecular docking. Our method is a three-step process: the first step encodes the geometry of a three-dimensional (3D) ligand conformation into a single 3D point in the space; the second step builds an octree by assigning an octant identifier to every single point in the space under consideration; and the third step performs an octree-based clustering on the reduced conformation space and identifies the most dense octant. We adapt our method for MapReduce and implement it in Hadoop. The load-balancing, fault-tolerance, and scalability in MapReduce allow screening of very large conformation spaces not approachable with traditional clustering methods. We analyze results for docking trials for 23 protein-ligand complexes for HIV protease, 21 protein-ligand complexes for Trypsin, and 12 protein-ligand complexes for P38alpha kinase. We also analyze cross docking trials for 24 ligands, each docking into 24 protein conformations of the HIV protease, and receptor ensemble docking trials for 24 ligands, each docking in a pool of HIV protease receptors. Our method demonstrates significant improvement over energy-only scoring for the accurate identification of native ligand geometries in all these docking assessments. The advantages of our clustering approach make it attractive for complex applications in real-world drug design efforts. We demonstrate that our method is particularly useful for clustering docking results using a minimal ensemble of representative protein conformational states (receptor ensemble docking), which is now a common strategy to address protein flexibility in molecular docking. Copyright © 2012 Elsevier Ltd. All rights reserved.

Spinal Cord Injury Disrupts Resting-State Networks in the Human Brain.

Science.gov (United States)

Hawasli, Ammar H; Rutlin, Jerrel; Roland, Jarod L; Murphy, Rory K J; Song, Sheng-Kwei; Leuthardt, Eric C; Shimony, Joshua S; Ray, Wilson Z

2018-03-15

Despite 253,000 spinal cord injury (SCI) patients in the United States, little is known about how SCI affects brain networks. Spinal MRI provides only structural information with no insight into functional connectivity. Resting-state functional MRI (RS-fMRI) quantifies network connectivity through the identification of resting-state networks (RSNs) and allows detection of functionally relevant changes during disease. Given the robust network of spinal cord afferents to the brain, we hypothesized that SCI produces meaningful changes in brain RSNs. RS-fMRIs and functional assessments were performed on 10 SCI subjects. Blood oxygen-dependent RS-fMRI sequences were acquired. Seed-based correlation mapping was performed using five RSNs: default-mode (DMN), dorsal-attention (DAN), salience (SAL), control (CON), and somatomotor (SMN). RSNs were compared with normal control subjects using false-discovery rate-corrected two way t tests. SCI reduced brain network connectivity within the SAL, SMN, and DMN and disrupted anti-correlated connectivity between CON and SMN. When divided into separate cohorts, complete but not incomplete SCI disrupted connectivity within SAL, DAN, SMN and DMN and between CON and SMN. Finally, connectivity changed over time after SCI: the primary motor cortex decreased connectivity with the primary somatosensory cortex, the visual cortex decreased connectivity with the primary motor cortex, and the visual cortex decreased connectivity with the sensory parietal cortex. These unique findings demonstrate the functional network plasticity that occurs in the brain as a result of injury to the spinal cord. Connectivity changes after SCI may serve as biomarkers to predict functional recovery following an SCI and guide future therapy.

AMP-activated protein kinase reduces inflammatory responses and cellular senescence in pulmonary emphysema.

Science.gov (United States)

Cheng, Xiao-Yu; Li, Yang-Yang; Huang, Cheng; Li, Jun; Yao, Hong-Wei

2017-04-04

Current drug therapy fails to reduce lung destruction of chronic obstructive pulmonary disease (COPD). AMP-activated protein kinase (AMPK) has emerged as an important integrator of signals that control energy balance and lipid metabolism. However, there are no studies regarding the role of AMPK in reducing inflammatory responses and cellular senescence during the development of emphysema. Therefore, we hypothesize that AMPK reduces inflammatroy responses, senescence, and lung injury. To test this hypothesis, human bronchial epithelial cells (BEAS-2B) and small airway epithelial cells (SAECs) were treated with cigarette smoke extract (CSE) in the presence of a specific AMPK activator (AICAR, 1 mM) and inhibitor (Compound C, 5 μM). Elastase injection was performed to induce mouse emphysema, and these mice were treated with a specific AMPK activator metformin as well as Compound C. AICAR reduced, whereas Compound C increased CSE-induced increase in IL-8 and IL-6 release and expression of genes involved in cellular senescence. Knockdown of AMPKα1/α2 increased expression of pro-senescent genes (e.g., p16, p21, and p66shc) in BEAS-2B cells. Prophylactic administration of an AMPK activator metformin (50 and 250 mg/kg) reduced while Compound C (4 and 20 mg/kg) aggravated elastase-induced airspace enlargement, inflammatory responses and cellular senescence in mice. This is in agreement with therapeutic effect of metformin (50 mg/kg) on airspace enlargement. Furthermore, metformin prophylactically protected against but Compound C further reduced mitochondrial proteins SOD2 and SIRT3 in emphysematous lungs. In conclusion, AMPK reduces abnormal inflammatory responses and cellular senescence, which implicates as a potential therapeutic target for COPD/emphysema.

Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation

Science.gov (United States)

Plate, Lars; Cooley, Christina B; Chen, John J; Paxman, Ryan J; Gallagher, Ciara M; Madoux, Franck; Genereux, Joseph C; Dobbs, Wesley; Garza, Dan; Spicer, Timothy P; Scampavia, Louis; Brown, Steven J; Rosen, Hugh; Powers, Evan T; Walter, Peter; Hodder, Peter; Wiseman, R Luke; Kelly, Jeffery W

2016-01-01

Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases. DOI: http://dx.doi.org/10.7554/eLife.15550.001 PMID:27435961

Neuroprotective effect of non-viral gene therapy treatment based on tetanus toxin C-fragment in a severe mouse model of Spinal Muscular Atrophy.

Directory of Open Access Journals (Sweden)

Sara Olivan Garcia

2016-08-01

Full Text Available Spinal muscular atrophy (SMA is a hereditary childhood disease that causes paralysis and progressive degeneration of skeletal muscles and spinal motor neurons. SMA is associated with reduced levels of full-length Survival of Motor Neuron (SMN protein, due to mutations in the Survival of Motor Neuron 1 gene. Nowadays there are no effective therapies available to treat patients with SMA, so our aim was to test whether the non-toxic carboxy-terminal fragment of tetanus toxin heavy chain (TTC, which exhibits neurotrophic properties, might have a therapeutic role or benefit in SMA. In this manuscript, we have demonstrated that TTC enhance the SMN expression in motor neurons in vitro and evaluated the effect of intramuscular injection of TTC-encoding plasmid in the spinal cord and the skeletal muscle of SMNdelta7 mice. For this purpose, we studied the weight and the survival time, as well as, the survival and cell death pathways and muscular atrophy. Our results showed that TTC treatment reduced the expression of autophagy markers (Becn1, Atg5, Lc3 and p62 and pro-apoptotic genes such as Bax and Casp3 in spinal cord. In skeletal muscle, TTC was able to downregulate the expression of the main marker of autophagy, Lc3, to wild type levels and the expression of the apoptosis effector protein, Casp3. Regarding the genes related to muscular atrophy (Ankrd1, Calm1, Col19a1, Fbox32, Mt2, Myod1, NogoA, Pax7, Rrad, and Sln, TTC suggest a compensatory effect for muscle damage response, diminished oxidative stress and modulated calcium homeostasis. These preliminary findings suggest the need for further experiments to depth study the effect of TTC in SMA disease.

Motoneuron axon pathfinding errors in zebrafish: Differential effects related to concentration and timing of nicotine exposure

International Nuclear Information System (INIS)

Menelaou, Evdokia; Paul, Latoya T.; Perera, Surangi N.; Svoboda, Kurt R.

2015-01-01

Nicotine exposure during embryonic stages of development can affect many neurodevelopmental processes. In the developing zebrafish, exposure to nicotine was reported to cause axonal pathfinding errors in the later born secondary motoneurons (SMNs). These alterations in SMN axon morphology coincided with muscle degeneration at high nicotine concentrations (15–30 μM). Previous work showed that the paralytic mutant zebrafish known as sofa potato exhibited nicotine-induced effects onto SMN axons at these high concentrations but in the absence of any muscle deficits, indicating that pathfinding errors could occur independent of muscle effects. In this study, we used varying concentrations of nicotine at different developmental windows of exposure to specifically isolate its effects onto subpopulations of motoneuron axons. We found that nicotine exposure can affect SMN axon morphology in a dose-dependent manner. At low concentrations of nicotine, SMN axons exhibited pathfinding errors, in the absence of any nicotine-induced muscle abnormalities. Moreover, the nicotine exposure paradigms used affected the 3 subpopulations of SMN axons differently, but the dorsal projecting SMN axons were primarily affected. We then identified morphologically distinct pathfinding errors that best described the nicotine-induced effects on dorsal projecting SMN axons. To test whether SMN pathfinding was potentially influenced by alterations in the early born primary motoneuron (PMN), we performed dual labeling studies, where both PMN and SMN axons were simultaneously labeled with antibodies. We show that only a subset of the SMN axon pathfinding errors coincided with abnormal PMN axonal targeting in nicotine-exposed zebrafish. We conclude that nicotine exposure can exert differential effects depending on the levels of nicotine and developmental exposure window. - Highlights: • Embryonic nicotine exposure can specifically affect secondary motoneuron axons in a dose-dependent manner. �

Motoneuron axon pathfinding errors in zebrafish: Differential effects related to concentration and timing of nicotine exposure

Energy Technology Data Exchange (ETDEWEB)

Menelaou, Evdokia; Paul, Latoya T. [Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803 (United States); Perera, Surangi N. [Joseph J. Zilber School of Public Health, University of Wisconsin — Milwaukee, Milwaukee, WI 53205 (United States); Svoboda, Kurt R., E-mail: svobodak@uwm.edu [Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803 (United States); Joseph J. Zilber School of Public Health, University of Wisconsin — Milwaukee, Milwaukee, WI 53205 (United States)

2015-04-01

Nicotine exposure during embryonic stages of development can affect many neurodevelopmental processes. In the developing zebrafish, exposure to nicotine was reported to cause axonal pathfinding errors in the later born secondary motoneurons (SMNs). These alterations in SMN axon morphology coincided with muscle degeneration at high nicotine concentrations (15–30 μM). Previous work showed that the paralytic mutant zebrafish known as sofa potato exhibited nicotine-induced effects onto SMN axons at these high concentrations but in the absence of any muscle deficits, indicating that pathfinding errors could occur independent of muscle effects. In this study, we used varying concentrations of nicotine at different developmental windows of exposure to specifically isolate its effects onto subpopulations of motoneuron axons. We found that nicotine exposure can affect SMN axon morphology in a dose-dependent manner. At low concentrations of nicotine, SMN axons exhibited pathfinding errors, in the absence of any nicotine-induced muscle abnormalities. Moreover, the nicotine exposure paradigms used affected the 3 subpopulations of SMN axons differently, but the dorsal projecting SMN axons were primarily affected. We then identified morphologically distinct pathfinding errors that best described the nicotine-induced effects on dorsal projecting SMN axons. To test whether SMN pathfinding was potentially influenced by alterations in the early born primary motoneuron (PMN), we performed dual labeling studies, where both PMN and SMN axons were simultaneously labeled with antibodies. We show that only a subset of the SMN axon pathfinding errors coincided with abnormal PMN axonal targeting in nicotine-exposed zebrafish. We conclude that nicotine exposure can exert differential effects depending on the levels of nicotine and developmental exposure window. - Highlights: • Embryonic nicotine exposure can specifically affect secondary motoneuron axons in a dose-dependent manner. �

Enzymatic Hydrolysis Does Not Reduce the Biological Reactivity of Soybean Proteins for All Allergic Subjects.

Science.gov (United States)

Panda, Rakhi; Tetteh, Afua O; Pramod, Siddanakoppalu N; Goodman, Richard E

2015-11-04

Many soybean protein products are processed by enzymatic hydrolysis to attain desirable functional food properties or in some cases to reduce allergenicity. However, few studies have investigated the effects of enzymatic hydrolysis on the allergenicity of soybean products. In this study the allergenicity of soybean protein isolates (SPI) hydrolyzed by Alcalase, trypsin, chymotrypsin, bromelain, or papain was evaluated by IgE immunoblots using eight soybean-allergic patient sera. The biological relevance of IgE binding was evaluated by a functional assay using a humanized rat basophilic leukemia (hRBL) cell line and serum from one subject. Results indicated that hydrolysis of SPI by the enzymes did not reduce the allergenicity, and hydrolysis by chymotrypsin or bromelain has the potential to increase the allergenicity of SPI. Two-dimensional (2D) immunoblot and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the chymotrypsin-hydrolyzed samples indicated fragments of β-conglycinin protein are responsible for the apparent higher allergenic potential of digested SPI.

High-Pressure-High-Temperature Processing Reduces Maillard Reaction and Viscosity in Whey Protein-Sugar Solutions.

Science.gov (United States)

Avila Ruiz, Geraldine; Xi, Bingyan; Minor, Marcel; Sala, Guido; van Boekel, Martinus; Fogliano, Vincenzo; Stieger, Markus

2016-09-28

The aim of the study was to determine the influence of pressure in high-pressure-high-temperature (HPHT) processing on Maillard reactions and protein aggregation of whey protein-sugar solutions. Solutions of whey protein isolate containing either glucose or trehalose at pH 6, 7, and 9 were treated by HPHT processing or conventional high-temperature (HT) treatments. Browning was reduced, and early and advanced Maillard reactions were retarded under HPHT processing at all pH values compared to HT treatment. HPHT induced a larger pH drop than HT treatments, especially at pH 9, which was not associated with Maillard reactions. After HPHT processing at pH 7, protein aggregation and viscosity of whey protein isolate-glucose/trehalose solutions remained unchanged. It was concluded that HPHT processing can potentially improve the quality of protein-sugar-containing foods, for which browning and high viscosities are undesired, such as high-protein beverages.

Integrated use of biochar: a tool for improving soil and wheat quality of degraded soil under wheat-maize cropping pattern

International Nuclear Information System (INIS)

Ali, K.; Arif, M.; Jan, M.T.

2015-01-01

Wheat quality, nutrient uptake and nutrient use efficiency are significantly influenced by nutrient sources and application rate. To investigate the integrative effect of biochar, farmyard manure (FYM) and nitrogen (organic and inorganic soil amendments) in a wheat-maize cropping system, a two year study was designed to assess the interactive outcome of biochar, FYM and nitrogenous fertilizer on wheat nitrogen (N) parameters and associated soil quality parameters. Three levels of biochar (0, 25 and 50 t ha-1), two levels of FYM (5 and 10 t ha-1) and two levels of nitrogen fertilizer (60 and 120 kg ha-1) were used in the study. Biochar application displayed a significantly increased in wheat leaf, stem, straw and grain N content; grain and total N-uptake and grain protein content by 24, 20, 24, 56, 50, 17 and 20% respectively. Similarly, biochar application significantly increased soil total N (TN) and soil mineral N (SMN) by 63 and 40% respectively in second year. FYM application increased grain, leaf and straw N content by 20, 19.5 and 18% respectively, and increased total N-uptake and grain protein content by 49 and 19% respectively. FYM increased soil TN and SMN by 63 and 32% in both the years of the experiment. Mineral N application increased soil TN by over a half and SMN by a third, and grain protein content increased 16%. In contrast, nitrogen use efficiency (NUE) decreased for all amendments relative to the control. However, biochar treated plots improved NUE by 38% compared to plots without biochar. In conclusion, this field experiment has illustrated the potential of biochar to bring about short-term benefits in wheat and soil quality parameters in wheat-maize cropping systems. However, the long-term benefits remain to be quantified. (author)

Reduced Wall Acetylation Proteins Play Vital and Distinct Roles in Cell Wall O-Acetylation in Arabidopsis

DEFF Research Database (Denmark)

Manabe, Yuzuki; Verhertbruggen, Yves; Gille, Sascha

2013-01-01

The Reduced Wall Acetylation (RWA) proteins are involved in cell wall acetylation in plants. Previously, we described a single mutant, rwa2, which has about 20% lower level of O-acetylation in leaf cell walls and no obvious growth or developmental phenotype. In this study, we generated double....... The quadruple rwa mutant can be completely complemented with the RWA2 protein expressed under 35S promoter, indicating the functional redundancy of the RWA proteins. Nevertheless, the degree of acetylation of xylan, (gluco) mannan, and xyloglucan as well as overall cell wall acetylation is affected differently...... in different combinations of triple mutants, suggesting their diversity in substrate preference. The overall degree of wall acetylation in the rwa quadruple mutant was reduced by 63% compared with the wild type, and histochemical analysis of the rwa quadruple mutant stem indicates defects in cell...

Whey Protein Reduces Early Life Weight Gain in Mice Fed a High-Fat Diet

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Tranberg, Britt; Hellgren, Lars I.; Lykkesfeldt, Jens; Sejrsen, Kristen; Jeamet, Aymeric; Rune, Ida; Ellekilde, Merete; Nielsen, Dennis S.; Hansen, Axel Kornerup

2013-01-01

An increasing number of studies indicate that dairy products, including whey protein, alleviate several disorders of the metabolic syndrome. Here, we investigated the effects of whey protein isolate (whey) in mice fed a high-fat diet hypothesising that the metabolic effects of whey would be associated with changes in the gut microbiota composition. Five-week-old male C57BL/6 mice were fed a high-fat diet ad libitum for 14 weeks with the protein source being either whey or casein. Faeces were collected at week 0, 7, and 13 and the fecal microbiota was analysed by denaturing gradient gel electrophoresis analyses of PCR-derived 16S rRNA gene (V3-region) amplicons. At the end of the study, plasma samples were collected and assayed for glucose, insulin and lipids. Whey significantly reduced body weight gain during the first four weeks of the study compared with casein (Pwhey group relative to casein (34.0±1.0 g vs. 40.2±1.3 g, Pwhey group (Pwhey compared to casein (Pwhey and casein. In conclusion, whey initially reduced weight gain in young C57BL/6 mice fed a high-fat diet compared to casein. Although the effect on weight gain ceased, whey alleviated glucose intolerance, improved insulin sensitivity and reduced plasma cholesterol. These findings could not be explained by changes in food intake or gut microbiota composition. Further studies are needed to clarify the mechanisms behind the metabolic effects of whey. PMID:23940754

Cooking Chicken Breast Reduces Dialyzable Iron Resulting from Digestion of Muscle Proteins

Directory of Open Access Journals (Sweden)

Aditya S. Gokhale

2014-01-01

Full Text Available The purpose of this research was to study the effect of cooking chicken breast on the production of dialyzable iron (an in vitro indicator of bioavailable iron from added ferric iron. Chicken breast muscle was cooked by boiling, baking, sautéing, or deep-frying. Cooked samples were mixed with ferric iron and either extracted with acid or digested with pepsin and pancreatin. Total and ferrous dialyzable iron was measured after extraction or digestion and compared to raw chicken samples. For uncooked samples, dialyzable iron was significantly enhanced after both extraction and digestion. All cooking methods led to markedly reduced levels of dialyzable iron both by extraction and digestion. In most cooked, digested samples dialyzable iron was no greater than the iron-only (no sample control. Cooked samples showed lower levels of histidine and sulfhydryls but protein digestibility was not reduced, except for the sautéed sample. The results showed that, after cooking, little if any dialyzable iron results from digestion of muscle proteins. Our research indicates that, in cooked chicken, residual acid-extractable components are the most important source of dialyzable iron.

Cooking Chicken Breast Reduces Dialyzable Iron Resulting from Digestion of Muscle Proteins.

Science.gov (United States)

Gokhale, Aditya S; Mahoney, Raymond R

2014-01-01

The purpose of this research was to study the effect of cooking chicken breast on the production of dialyzable iron (an in vitro indicator of bioavailable iron) from added ferric iron. Chicken breast muscle was cooked by boiling, baking, sautéing, or deep-frying. Cooked samples were mixed with ferric iron and either extracted with acid or digested with pepsin and pancreatin. Total and ferrous dialyzable iron was measured after extraction or digestion and compared to raw chicken samples. For uncooked samples, dialyzable iron was significantly enhanced after both extraction and digestion. All cooking methods led to markedly reduced levels of dialyzable iron both by extraction and digestion. In most cooked, digested samples dialyzable iron was no greater than the iron-only (no sample) control. Cooked samples showed lower levels of histidine and sulfhydryls but protein digestibility was not reduced, except for the sautéed sample. The results showed that, after cooking, little if any dialyzable iron results from digestion of muscle proteins. Our research indicates that, in cooked chicken, residual acid-extractable components are the most important source of dialyzable iron.

Meteorology and dispersion forecast in nuclear emergency in Argentina

International Nuclear Information System (INIS)

Kunst, Juan J.; Boutet, Luis I.; Jordan, Osvaldo D.; Hernandez, Daniel G.; Guichandut, M.E.; Chiappesoni, H.

2008-01-01

The 'Nuclear Regulatory Authority (NRA) (ARN in Spanish)' and the 'National Meteorological Office (NMO) (SMN in Spanish)' of Argentine has been working together on the improvement of both meteorological forecasting and dispersion prediction. In the pre-release phase of a nuclear emergency, it is very important to know the wind direction and the forecast of it, to establish the area, around the installation, where the emergency state is declared and to foresee the modification of this area. Information is also needed about deterministic effects, to begin the evacuation. At this time, meteorological forecast of wind direction and speed, and the real time meteorological information is available in the nuclear power plant (NPP) and in the Nuclear Emergency Control Centre at the ARN headquarters, together with the short-range dose calculation provided by our dispersion code, SEDA. By means of the SEDA code, we can estimate the optimum place to measure the radioactive material concentration in air, needed do to reduce evaluation uncertainties due, among others, to poor knowledge of the source term. The SEDA code allows considering atmospheric condition, and the need to reduced doses of the measuring team in charge of the measurements. For the evaluation in the medium range, we participate in the project IXP, which provides four hours and about 50 kilometres forecast. In the long-range movement of air borne radioactivity, the World Meteorological Organization (WMO), whose contact point in Argentina is the SMN, can assist us. We have developed together, with the SMN, a detailed procedure to request assistance from the WMO. In this work, we describe the combined tasks that were carried out with the SMN to define the procedures and the concepts for their application during a real emergency. The results of an application exercise carried out in 2006 are also described. (author)

Optimization of elution salt concentration in stepwise elution of protein chromatography using linear gradient elution data. Reducing residual protein A by cation-exchange chromatography in monoclonal antibody purification.

Science.gov (United States)

Ishihara, Takashi; Kadoya, Toshihiko; Endo, Naomi; Yamamoto, Shuichi

2006-05-05

Our simple method for optimization of the elution salt concentration in stepwise elution was applied to the actual protein separation system, which involves several difficulties such as detection of the target. As a model separation system, reducing residual protein A by cation-exchange chromatography in human monoclonal antibody (hMab) purification was chosen. We carried out linear gradient elution experiments and obtained the data for the peak salt concentration of hMab and residual protein A, respectively. An enzyme-linked immunosorbent assay was applied to the measurement of the residual protein A. From these data, we calculated the distribution coefficient of the hMab and the residual protein A as a function of salt concentration. The optimal salt concentration of stepwise elution to reduce the residual protein A from the hMab was determined based on the relationship between the distribution coefficient and the salt concentration. Using the optimized condition, we successfully performed the separation, resulting in high recovery of hMab and the elimination of residual protein A.

Blunted hypothalamic ghrelin signaling reduces diet intake in rats fed a low-protein diet in late pregnancy

Science.gov (United States)

Diet intake in pregnant rats fed a low-protein (LP) diet was significantly reduced during late pregnancy despite elevated plasma levels of ghrelin. In this study, we hypothesized that ghrelin signaling in the hypothalamus is blunted under a low-protein diet condition and therefore, it does not stimu...

Skeletal muscle protein accretion rates and hindlimb growth are reduced in late gestation intrauterine growth-restricted fetal sheep.

Science.gov (United States)

Rozance, Paul J; Zastoupil, Laura; Wesolowski, Stephanie R; Goldstrohm, David A; Strahan, Brittany; Cree-Green, Melanie; Sheffield-Moore, Melinda; Meschia, Giacomo; Hay, William W; Wilkening, Randall B; Brown, Laura D

2018-01-01

Adults who were affected by intrauterine growth restriction (IUGR) suffer from reductions in muscle mass, which may contribute to insulin resistance and the development of diabetes. We demonstrate slower hindlimb linear growth and muscle protein synthesis rates that match the reduced hindlimb blood flow and oxygen consumption rates in IUGR fetal sheep. These adaptations resulted in hindlimb blood flow rates in IUGR that were similar to control fetuses on a weight-specific basis. Net hindlimb glucose uptake and lactate output rates were similar between groups, whereas amino acid uptake was significantly lower in IUGR fetal sheep. Among all fetuses, blood O 2 saturation and plasma glucose, insulin and insulin-like growth factor-1 were positively associated and norepinephrine was negatively associated with hindlimb weight. These results further our understanding of the metabolic and hormonal adaptations to reduced oxygen and nutrient supply with placental insufficiency that develop to slow hindlimb growth and muscle protein accretion. Reduced skeletal muscle mass in the fetus with intrauterine growth restriction (IUGR) persists into adulthood and may contribute to increased metabolic disease risk. To determine how placental insufficiency with reduced oxygen and nutrient supply to the fetus affects hindlimb blood flow, substrate uptake and protein accretion rates in skeletal muscle, late gestation control (CON) (n = 8) and IUGR (n = 13) fetal sheep were catheterized with aortic and femoral catheters and a flow transducer around the external iliac artery. Muscle protein kinetic rates were measured using isotopic tracers. Hindlimb weight, linear growth rate, muscle protein accretion rate and fractional synthetic rate were lower in IUGR compared to CON (P fetal norepinephrine and reduced IGF-1 and insulin. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Interhemispheric disconnectivity in the sensorimotor network in bipolar disorder revealed by functional connectivity and diffusion tensor imaging analysis

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Takuya Ishida

2017-06-01

Full Text Available Background: Little is known regarding interhemispheric functional connectivity (FC abnormalities via the corpus callosum in subjects with bipolar disorder (BD, which might be a key pathophysiological basis of emotional processing alterations in BD. Methods: We performed tract-based spatial statistics (TBSS using diffusion tensor imaging (DTI in 24 healthy control (HC and 22 BD subjects. Next, we analyzed the neural networks with independent component analysis (ICA in 32HC and 25 BD subjects using resting-state functional magnetic resonance imaging. Results: In TBSS analysis, we found reduced fractional anisotropy (FA in the corpus callosum of BD subjects. In ICA, functional within-connectivity was reduced in two clusters in the sensorimotor network (SMN (right and left primary somatosensory areas of BD subjects compared with HCs. FC between the two clusters and FA values in the corpus callosum of BD subjects was significantly correlated. Further, the functional within-connectivity was related to Young Mania Rating Scale (YMRS total scores in the right premotor area in the SMN of BD subjects. Limitations: Almost all of our BD subjects were taking several medications which could be a confounding factor. Conclusions: Our findings suggest that interhemispheric FC dysfunction in the SMN is associated with the impaired nerve fibers in the corpus callosum, which could be one of pathophysiological bases of emotion processing dysregulation in BD patients. Keywords: Neuroscience, Medical imaging, Psychiatry

Benzylglucosinolate Derived Isothiocyanate from Tropaeolum majus Reduces Gluconeogenic Gene and Protein Expression in Human Cells.

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Valentina Guzmán-Pérez

Full Text Available Nasturtium (Tropaeolum majus L. contains high concentrations of benzylglcosinolate. We found that a hydrolysis product of benzyl glucosinolate-the benzyl isothiocyanate (BITC-modulates the intracellular localization of the transcription factor Forkhead box O 1 (FOXO1. FoxO transcription factors can antagonize insulin effects and trigger a variety of cellular processes involved in tumor suppression, longevity, development and metabolism. The current study evaluated the ability of BITC-extracted as intact glucosinolate from nasturtium and hydrolyzed with myrosinase-to modulate i the insulin-signaling pathway, ii the intracellular localization of FOXO1 and, iii the expression of proteins involved in gluconeogenesis, antioxidant response and detoxification. Stably transfected human osteosarcoma cells (U-2 OS with constitutive expression of FOXO1 protein labeled with GFP (green fluorescent protein were used to evaluate the effect of BITC on FOXO1. Human hepatoma HepG2 cell cultures were selected to evaluate the effect on gluconeogenic, antioxidant and detoxification genes and protein expression. BITC reduced the phosphorylation of protein kinase B (AKT/PKB and FOXO1; promoted FOXO1 translocation from cytoplasm into the nucleus antagonizing the insulin effect; was able to down-regulate the gene and protein expression of gluconeogenic enzymes; and induced the gene expression of antioxidant and detoxification enzymes. Knockdown analyses with specific siRNAs showed that the expression of gluconeogenic genes was dependent on nuclear factor (erythroid derived-like2 (NRF2 and independent of FOXO1, AKT and NAD-dependent deacetylase sirtuin-1 (SIRT1. The current study provides evidence that BITC might have a role in type 2 diabetes T2D by reducing hepatic glucose production and increasing antioxidant resistance.

Frequency Distribution of Second Solid Cancer Locations in Relation to the Irradiated Volume Among 115 Patients Treated for Childhood Cancer

International Nuclear Information System (INIS)

Diallo, Ibrahima; Haddy, Nadia; Adjadj, Elisabeth; Samand, Akhtar; Quiniou, Eric; Chavaudra, Jean; Alziar, Iannis; Perret, Nathalie; Guerin, Sylvie; Lefkopoulos, Dimitri; Vathaire, Florent de

2009-01-01

Purpose: To provide better estimates of the frequency distribution of second malignant neoplasm (SMN) sites in relation to previous irradiated volumes, and better estimates of the doses delivered to these sites during radiotherapy (RT) of the first malignant neoplasm (FMN). Methods and Materials: The study focused on 115 patients who developed a solid SMN among a cohort of 4581 individuals. The homemade software package Dos E G was used to estimate the radiation doses delivered to SMN sites during RT of the FMN. Three-dimensional geometry was used to evaluate the distances between the irradiated volume, for RT delivered to each FMN, and the site of the subsequent SMN. Results: The spatial distribution of SMN relative to the irradiated volumes in our cohort was as follows: 12% in the central area of the irradiated volume, which corresponds to the planning target volume (PTV), 66% in the beam-bordering region (i.e., the area surrounding the PTV), and 22% in regions located more than 5 cm from the irradiated volume. At the SMN site, all dose levels ranging from almost zero to >75 Gy were represented. A peak SMN frequency of approximately 31% was identified in volumes that received <2.5 Gy. Conclusion: A greater volume of tissues receives low or intermediate doses in regions bordering the irradiated volume with modern multiple-beam RT arrangements. These results should be considered for risk-benefit evaluations of RT.

A Perturbed MicroRNA Expression Pattern Characterizes Embryonic Neural Stem Cells Derived from a Severe Mouse Model of Spinal Muscular Atrophy (SMA

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Andrea Luchetti

2015-08-01

Full Text Available Spinal muscular atrophy (SMA is an inherited neuromuscular disorder and the leading genetic cause of death in infants. Despite the disease-causing gene, survival motor neuron (SMN1, encodes a ubiquitous protein, SMN1 deficiency preferentially affects spinal motor neurons (MNs, leaving the basis of this selective cell damage still unexplained. As neural stem cells (NSCs are multipotent self-renewing cells that can differentiate into neurons, they represent an in vitro model for elucidating the pathogenetic mechanism of neurodegenerative diseases such as SMA. Here we characterize for the first time neural stem cells (NSCs derived from embryonic spinal cords of a severe SMNΔ7 SMA mouse model. SMNΔ7 NSCs behave as their wild type (WT counterparts, when we consider neurosphere formation ability and the expression levels of specific regional and self-renewal markers. However, they show a perturbed cell cycle phase distribution and an increased proliferation rate compared to wild type cells. Moreover, SMNΔ7 NSCs are characterized by the differential expression of a limited number of miRNAs, among which miR-335-5p and miR-100-5p, reduced in SMNΔ7 NSCs compared to WT cells. We suggest that such miRNAs may be related to the proliferation differences characterizing SMNΔ7 NSCs, and may be potentially involved in the molecular mechanisms of SMA.

A Perturbed MicroRNA Expression Pattern Characterizes Embryonic Neural Stem Cells Derived from a Severe Mouse Model of Spinal Muscular Atrophy (SMA).

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Luchetti, Andrea; Ciafrè, Silvia Anna; Murdocca, Michela; Malgieri, Arianna; Masotti, Andrea; Sanchez, Massimo; Farace, Maria Giulia; Novelli, Giuseppe; Sangiuolo, Federica

2015-08-06

Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder and the leading genetic cause of death in infants. Despite the disease-causing gene, survival motor neuron (SMN1), encodes a ubiquitous protein, SMN1 deficiency preferentially affects spinal motor neurons (MNs), leaving the basis of this selective cell damage still unexplained. As neural stem cells (NSCs) are multipotent self-renewing cells that can differentiate into neurons, they represent an in vitro model for elucidating the pathogenetic mechanism of neurodegenerative diseases such as SMA. Here we characterize for the first time neural stem cells (NSCs) derived from embryonic spinal cords of a severe SMNΔ7 SMA mouse model. SMNΔ7 NSCs behave as their wild type (WT) counterparts, when we consider neurosphere formation ability and the expression levels of specific regional and self-renewal markers. However, they show a perturbed cell cycle phase distribution and an increased proliferation rate compared to wild type cells. Moreover, SMNΔ7 NSCs are characterized by the differential expression of a limited number of miRNAs, among which miR-335-5p and miR-100-5p, reduced in SMNΔ7 NSCs compared to WT cells. We suggest that such miRNAs may be related to the proliferation differences characterizing SMNΔ7 NSCs, and may be potentially involved in the molecular mechanisms of SMA.

Macrocyclic peptides decrease c-Myc protein levels and reduce prostate cancer cell growth.

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Mukhopadhyay, Archana; Hanold, Laura E; Thayele Purayil, Hamsa; Gisemba, Solomon A; Senadheera, Sanjeewa N; Aldrich, Jane V

2017-08-03

The oncoprotein c-Myc is often overexpressed in cancer cells, and the stability of this protein has major significance in deciding the fate of a cell. Thus, targeting c-Myc levels is an attractive approach for developing therapeutic agents for cancer treatment. In this study, we report the anti-cancer activity of the macrocyclic peptides [D-Trp]CJ-15,208 (cyclo[Phe-D-Pro-Phe-D-Trp]) and the natural product CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]). [D-Trp]CJ-15,208 reduced c-Myc protein levels in prostate cancer cells and decreased cell proliferation with IC 50 values ranging from 2.0 to 16 µM in multiple PC cell lines. [D-Trp]CJ-15,208 induced early and late apoptosis in PC-3 cells following 48 hours treatment, and growth arrest in the G2 cell cycle phase following both 24 and 48 hours treatment. Down regulation of c-Myc in PC-3 cells resulted in loss of sensitivity to [D-Trp]CJ-15,208 treatment, while overexpression of c-Myc in HEK-293 cells imparted sensitivity of these cells to [D-Trp]CJ-15,208 treatment. This macrocyclic tetrapeptide also regulated PP2A by reducing the levels of its phosphorylated form which regulates the stability of cellular c-Myc protein. Thus [D-Trp]CJ-15,208 represents a new lead compound for the potential development of an effective treatment of prostate cancer.

Whey protein reduces early life weight gain in mice fed a high-fat diet.

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Britt Tranberg

Full Text Available An increasing number of studies indicate that dairy products, including whey protein, alleviate several disorders of the metabolic syndrome. Here, we investigated the effects of whey protein isolate (whey in mice fed a high-fat diet hypothesising that the metabolic effects of whey would be associated with changes in the gut microbiota composition. Five-week-old male C57BL/6 mice were fed a high-fat diet ad libitum for 14 weeks with the protein source being either whey or casein. Faeces were collected at week 0, 7, and 13 and the fecal microbiota was analysed by denaturing gradient gel electrophoresis analyses of PCR-derived 16S rRNA gene (V3-region amplicons. At the end of the study, plasma samples were collected and assayed for glucose, insulin and lipids. Whey significantly reduced body weight gain during the first four weeks of the study compared with casein (P<0.001-0.05. Hereafter weight gain was similar resulting in a 15% lower final body weight in the whey group relative to casein (34.0±1.0 g vs. 40.2±1.3 g, P<0.001. Food intake was unaffected by protein source throughout the study period. Fasting insulin was lower in the whey group (P<0.01 and glucose clearance was improved after an oral glucose challenge (P<0.05. Plasma cholesterol was lowered by whey compared to casein (P<0.001. The composition of the fecal microbiota differed between high- and low-fat groups at 13 weeks (P<0.05 whereas no difference was seen between whey and casein. In conclusion, whey initially reduced weight gain in young C57BL/6 mice fed a high-fat diet compared to casein. Although the effect on weight gain ceased, whey alleviated glucose intolerance, improved insulin sensitivity and reduced plasma cholesterol. These findings could not be explained by changes in food intake or gut microbiota composition. Further studies are needed to clarify the mechanisms behind the metabolic effects of whey.

potential for quality protein maize for reducing protein- energy

African Journals Online (AJOL)

ACSS

social systems hampering QPM promotion and adoption and to identify ... affects growth and development. Protein- energy ... to purchase QPM seed leading to PEU reduction. Education ..... decision support framework “targetCSA”. Agricultural ...

Stabilizing salt-bridge enhances protein thermostability by reducing the heat capacity change of unfolding.

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Chi-Ho Chan

Full Text Available Most thermophilic proteins tend to have more salt bridges, and achieve higher thermostability by up-shifting and broadening their protein stability curves. While the stabilizing effect of salt-bridge has been extensively studied, experimental data on how salt-bridge influences protein stability curves are scarce. Here, we used double mutant cycles to determine the temperature-dependency of the pair-wise interaction energy and the contribution of salt-bridges to ΔC(p in a thermophilic ribosomal protein L30e. Our results showed that the pair-wise interaction energies for the salt-bridges E6/R92 and E62/K46 were stabilizing and insensitive to temperature changes from 298 to 348 K. On the other hand, the pair-wise interaction energies between the control long-range ion-pair of E90/R92 were negligible. The ΔC(p of all single and double mutants were determined by Gibbs-Helmholtz and Kirchhoff analyses. We showed that the two stabilizing salt-bridges contributed to a reduction of ΔC(p by 0.8-1.0 kJ mol⁻¹ K⁻¹. Taken together, our results suggest that the extra salt-bridges found in thermophilic proteins enhance the thermostability of proteins by reducing ΔC(p, leading to the up-shifting and broadening of the protein stability curves.

Leucine supplementation stimulates protein synthesis and reduces degradation signal activation in muscle of newborn pigs during acute endotoxemia

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Sepsis disrupts skeletal muscle proteostasis and mitigates the anabolic response to leucine (Leu) in muscle of mature animals. We have shown that Leu stimulates muscle protein synthesis (PS) in healthy neonatal piglets. To determine if supplemental Leu can stimulate PS and reduce protein degradation...

Meningioma as second malignant neoplasm after oncological treatment during childhood

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Mueller, H.L.; Gebhardt, U. [Klinikum Oldenburg (Germany). Dept. of Pediatric Hematology and Oncology; Warmuth-Metz, M. [University Hospital Wuerzburg (Germany). Dept. of Neuroradiology; Pietsch, T. [Bonn Univ. (Germany). Dept. of Neuropathology; Soerensen, N. [Evangelisches Krankenhaus, Oldenburg (Germany). Dept. of Neurosurgery; Kortmann, R.D. [University Hospital Leipzig (Germany). Dept. of Radiooncology

2012-05-15

A total of 38 patients (18 female/20 male) with childhood meningioma were recruited from the German registry HIT-Endo (1989-2009). In 5 cases meningioma occurred as second malignant neoplasm (SMN). Histologies were confirmed by reference assessment in all cases (SMN: 2 WHO I, 1 WHO II, 2 WHO III). The SMNs were diagnosed at a median age of 12.4 years with a median latency of 10.2 years after primary malignancy (PMN; 4 brain tumors, 1 lymphoblastic leukemia; median age at diagnosis 2.7 years). Meningioma occurred as SMN in the irradiated field of PMN (range 12-54 Gy). The outcome after treatment of SMN meningioma (surgery/irradiation) was favorable in terms of psychosocial status and functional capacity in 4 of 5 patients (1 death). We conclude that survivors of childhood cancer who were exposed to radiation therapy at young age harbor the risk of developing meningioma as a SMN at a particularly short latency period in case of high dose exposure. (orig.)

Protein energy malnutrition alters mucosal IgA responses and reduces mucosal vaccine efficacy in mice.

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Rho, Semi; Kim, Heejoo; Shim, Seung Hyun; Lee, Seung Young; Kim, Min Jung; Yang, Bo-Gie; Jang, Myoung Ho; Han, Byung Woo; Song, Man Ki; Czerkinsky, Cecil; Kim, Jae-Ouk

2017-10-01

Oral vaccine responsiveness is often lower in children from less developed countries. Childhood malnutrition may be associated with poor immune response to oral vaccines. The present study was designed to investigate whether protein energy malnutrition (PEM) impairs B cell immunity and ultimately reduces oral vaccine efficacy in a mouse model. Purified isocaloric diets containing low protein (1/10 the protein of the control diet) were used to determine the effect of PEM. PEM increased both nonspecific total IgA and oral antigen-specific IgA in serum without alteration of gut permeability. However, PEM decreased oral antigen-specific IgA in feces, which is consistent with decreased expression of polymeric Immunoglobulin receptor (pIgR) in the small intestine. Of note, polymeric IgA was predominant in serum under PEM. In addition, PEM altered B cell development status in the bone marrow and increased the frequency of IgA-secreting B cells, as well as IgA secretion by long-lived plasma cells in the small intestinal lamina propria. Moreover, PEM reduced the protective efficacy of the mucosally administered cholera vaccine and recombinant attenuated Salmonella enterica serovar Typhimurium vaccine in a mouse model. Our results suggest that PEM can impair mucosal immunity where IgA plays an important role in host protection and may partly explain the reduced efficacy of oral vaccines in malnourished subjects. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

SET overexpression in HEK293 cells regulates mitochondrial uncoupling proteins levels within a mitochondrial fission/reduced autophagic flux scenario

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Almeida, Luciana O.; Goto, Renata N. [Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil); Neto, Marinaldo P.C. [Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil); Sousa, Lucas O. [Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil); Curti, Carlos [Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil); Leopoldino, Andréia M., E-mail: andreiaml@usp.br [Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil)

2015-03-06

We hypothesized that SET, a protein accumulated in some cancer types and Alzheimer disease, is involved in cell death through mitochondrial mechanisms. We addressed the mRNA and protein levels of the mitochondrial uncoupling proteins UCP1, UCP2 and UCP3 (S and L isoforms) by quantitative real-time PCR and immunofluorescence as well as other mitochondrial involvements, in HEK293 cells overexpressing the SET protein (HEK293/SET), either in the presence or absence of oxidative stress induced by the pro-oxidant t-butyl hydroperoxide (t-BHP). SET overexpression in HEK293 cells decreased UCP1 and increased UCP2 and UCP3 (S/L) mRNA and protein levels, whilst also preventing lipid peroxidation and decreasing the content of cellular ATP. SET overexpression also (i) decreased the area of mitochondria and increased the number of organelles and lysosomes, (ii) increased mitochondrial fission, as demonstrated by increased FIS1 mRNA and FIS-1 protein levels, an apparent accumulation of DRP-1 protein, and an increase in the VDAC protein level, and (iii) reduced autophagic flux, as demonstrated by a decrease in LC3B lipidation (LC3B-II) in the presence of chloroquine. Therefore, SET overexpression in HEK293 cells promotes mitochondrial fission and reduces autophagic flux in apparent association with up-regulation of UCP2 and UCP3; this implies a potential involvement in cellular processes that are deregulated such as in Alzheimer's disease and cancer. - Highlights: • SET, UCPs and autophagy prevention are correlated. • SET action has mitochondrial involvement. • UCP2/3 may reduce ROS and prevent autophagy. • SET protects cell from ROS via UCP2/3.

A novel mosquito ubiquitin targets viral envelope protein for degradation and reduces virion production during dengue virus infection.

Science.gov (United States)

Troupin, Andrea; Londono-Renteria, Berlin; Conway, Michael J; Cloherty, Erin; Jameson, Samuel; Higgs, Stephen; Vanlandingham, Dana L; Fikrig, Erol; Colpitts, Tonya M

2016-09-01

Dengue virus (DENV) is a mosquito-borne flavivirus that causes significant human disease and mortality in the tropics and subtropics. By examining the effects of virus infection on gene expression, and interactions between virus and vector, new targets for prevention of infection and novel treatments may be identified in mosquitoes. We previously performed a microarray analysis of the Aedes aegypti transcriptome during infection with DENV and found that mosquito ubiquitin protein Ub3881 (AAEL003881) was specifically and highly down-regulated. Ubiquitin proteins have multiple functions in insects, including marking proteins for proteasomal degradation, regulating apoptosis and mediating innate immune signaling. We used qRT-PCR to quantify gene expression and infection, and RNAi to reduce Ub3881 expression. Mosquitoes were infected with DENV through blood feeding. We transfected DENV protein expression constructs to examine the effect of Ub3881 on protein degradation. We used site-directed mutagenesis and transfection to determine what amino acids are involved in Ub3881-mediated protein degradation. Immunofluorescence, Co-immunoprecipitation and Western blotting were used to examine protein interactions and co-localization. The overexpression of Ub3881, but not related ubiquitin proteins, decreased DENV infection in mosquito cells and live Ae. aegypti. The Ub3881 protein was demonstrated to be involved in DENV envelope protein degradation and reduce the number of infectious virions released. We conclude that Ub3881 has several antiviral functions in the mosquito, including specific viral protein degradation. Our data highlights Ub3881 as a target for future DENV prevention strategies in the mosquito transmission vector. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Different atrophy-hypertrophy transcription pathways in muscles affected by severe and mild spinal muscular atrophy

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Millino Caterina

2009-04-01

Full Text Available Abstract Background Spinal muscular atrophy (SMA is a neurodegenerative disorder associated with mutations of the survival motor neuron gene SMN and is characterized by muscle weakness and atrophy caused by degeneration of spinal motor neurons. SMN has a role in neurons but its deficiency may have a direct effect on muscle tissue. Methods We applied microarray and quantitative real-time PCR to study at transcriptional level the effects of a defective SMN gene in skeletal muscles affected by the two forms of SMA: the most severe type I and the mild type III. Results The two forms of SMA generated distinct expression signatures: the SMA III muscle transcriptome is close to that found under normal conditions, whereas in SMA I there is strong alteration of gene expression. Genes implicated in signal transduction were up-regulated in SMA III whereas those of energy metabolism and muscle contraction were consistently down-regulated in SMA I. The expression pattern of gene networks involved in atrophy signaling was completed by qRT-PCR, showing that specific pathways are involved, namely IGF/PI3K/Akt, TNF-α/p38 MAPK and Ras/ERK pathways. Conclusion Our study suggests a different picture of atrophy pathways in each of the two forms of SMA. In particular, p38 may be the regulator of protein synthesis in SMA I. The SMA III profile appears as the result of the concurrent presence of atrophic and hypertrophic fibers. This more favorable condition might be due to the over-expression of MTOR that, given its role in the activation of protein synthesis, could lead to compensatory hypertrophy in SMA III muscle fibers.

Pharmacogenetically based dosing of thiopurines in childhood acute lymphoblastic leukemia

DEFF Research Database (Denmark)

Levinsen, Mette; Rotevatn, Elisabeth Orskov; Rosthøj, Susanne

2014-01-01

BACKGROUND: Previous studies have indicated that patients with thiopurine methyltransferase (TPMT) low activity (TPMT(LA)) have reduced risk of relapse but increased risk of second malignant neoplasm (SMN) compared to patients with TPMT wild-type (TPMT(WT)) when treated with 6 MP maintenance ther...

Acute high-caffeine exposure increases autophagic flux and reduces protein synthesis in C2C12 skeletal myotubes.

Science.gov (United States)

Hughes, M A; Downs, R M; Webb, G W; Crocker, C L; Kinsey, S T; Baumgarner, Bradley L

2017-04-01

Caffeine is a highly catabolic dietary stimulant. High caffeine concentrations (1-10 mM) have previously been shown to inhibit protein synthesis and increase protein degradation in various mammalian cell lines. The purpose of this study was to examine the effect of short-term caffeine exposure on cell signaling pathways that regulate protein metabolism in mammalian skeletal muscle cells. Fully differentiated C2C12 skeletal myotubes either received vehicle (DMSO) or 5 mM caffeine for 6 h. Our analysis revealed that caffeine promoted a 40% increase in autolysosome formation and a 25% increase in autophagic flux. In contrast, caffeine treatment did not significantly increase the expression of the skeletal muscle specific ubiquitin ligases MAFbx and MuRF1 or 20S proteasome activity. Caffeine treatment significantly reduced mTORC1 signaling, total protein synthesis and myotube diameter in a CaMKKβ/AMPK-dependent manner. Further, caffeine promoted a CaMKII-dependent increase in myostatin mRNA expression that did not significantly contribute to the caffeine-dependent reduction in protein synthesis. Our results indicate that short-term caffeine exposure significantly reduced skeletal myotube diameter by increasing autophagic flux and promoting a CaMKKβ/AMPK-dependent reduction in protein synthesis.

Frequency distribution of the reduced unit cells of centred lattices from the Protein Data Bank.

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Swaminathan, Kunchithapadam

2012-03-01

In crystallography, a centred conventional lattice unit cell has its corresponding reduced primitive unit cell. This study presents the frequency distribution of the reduced unit cells of all centred lattice entries of the Protein Data Bank (as of 23 August 2011) in four unit-cell-dimension-based groups and seven interaxial-angle-based subgroups. This frequency distribution is an added layer of support during space-group assignment in new crystals. In addition, some interesting patterns of distribution are discussed as well as how some reduced unit cells could be wrongly accepted as primitive lattices in a different crystal system.

Protective Effects of Butyrate-based Compounds on a Mouse Model for Spinal Muscular Atrophy

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Butchbach, Matthew E. R.; Lumpkin, Casey J.; Harris, Ashlee W.; Saieva, Luciano; Edwards, Jonathan D.; Workman, Eileen; Simard, Louise R.; Pellizzoni, Livio; Burghes, Arthur H. M.

2016-01-01

Proximal spinal muscular atrophy (SMA) is a childhood-onset degenerative disease resulting from the selective loss of motor neurons in the spinal cord. SMA is caused by the loss of SMN1 (survival motor neuron 1) but retention of SMN2. The number of copies of SMN2 modifies disease severity in SMA patients as well as in mouse models, making SMN2 a target for therapeutics development. Sodium butyrate (BA) and its analogue (4PBA) have been shown to increase SMN2 expression in SMA cultured cells. In this study, we examined the effects of BA, 4PBA as well as two BA prodrugs—glyceryl tributyrate (BA3G) and VX563—on the phenotype of SMNΔ7 SMA mice. Treatment with 4PBA, BA3G and VX563 but not BA beginning at PND04 significantly improved the lifespan and delayed disease end stage, with administration of VX563 also improving the growth rate of these mice. 4PBA and VX563 improved the motor phenotype of SMNΔ7 SMA mice and prevented spinal motor neuron loss. Interestingly, neither 4PBA nor VX563 had an effect on SMN expression in the spinal cords of treated SMNΔ7 SMA mice; however, they inhibited histone deacetylase (HDAC) activity and restored the normal phosphorylation states of Akt and glycogen synthase kinase 3β, both of which are altered by SMN deficiency in vivo. These observations show that BA-based compounds with favourable pharmacokinetics ameliorate SMA pathology possibly by modulating HDAC and Akt signaling. PMID:26892876

Reduced protein bound uraemic toxins in vegetarian kidney failure patients treated by haemodiafiltration.

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Kandouz, Sakina; Mohamed, Ali Shendi; Zheng, Yishan; Sandeman, Susan; Davenport, Andrew

2016-10-01

Introduction Indoxyl sulfate (IS) and p cresyl sulfate (PCS) are protein bound toxins which accumulate with chronic kidney disease. Haemodiafiltration (HDF) increases middle molecule clearances and has been suggested to increase IS and PCS clearance. We therefore wished to establish whether higher convective clearances with HDF would reduce IS and PCS concentrations. Methods We measured total plasma IS and PCS in a cohort of 138 CKD5d patients treated by On-line HDF (Ol-HDF), by high pressure liquid chromatography. Findings Mean patient age was 64.6 ± 16.5 years, 60.1% male, 57.3% diabetic, median dialysis vintage 25.9 months (12.4-62.0). The mean ICS concentration was 79.8 ± 56.4 umol/L and PCS 140.3 ± 101.8 umol/L. On multivariate analysis, IS was associated with serum albumin (β 4.31,P vegetarian diet(β-28.3, P = 0.048) and PCS negatively with log C reactive protein (β-75.8, P vegetarian diet (β-109, P = 0.001). Vegetarian patients had lower IS and PCS levels (median 41.5 (24.2-71.9) vs. 78.1 (49.5-107.5) and PCS (41.6 (14.2-178.3) vs. 127.3 (77.4-205.6) µmol/L, respectively, P Vegetarian patients had lower preOl-HDF serum urea, and phosphate (13.8 ±3.8 vs. 18.4 ± 5.2 mmol/L, and 1.33 ± 0.21 vs. 1.58 ± 0.45 mmol/L), and estimated urea nitrogen intake (1.25 ± 0.28 vs. 1.62 ± 0.5 g/kg/day), respectively, all P vegetarian diet had reduced IS and PCS concentrations. Although this could be due to differences in dietary protein intake, a vegetarian diet may also potentially reduce IS and PCS production by the intestinal microbiome. © 2016 International Society for Hemodialysis.

Direct, simple derivatization of disulfide bonds in proteins with organic mercury in alkaline medium without any chemical pre-reducing agents

Energy Technology Data Exchange (ETDEWEB)

Campanella, Beatrice; Onor, Massimo [National Research Council of Italy, C.N.R., Istituto di Chimica dei Composti Organo Metallici-ICCOM- UOS Pisa, Area di Ricerca, Via G. Moruzzi 1, 56124 Pisa (Italy); Ferrari, Carlo [National Research Council of Italy, C.N.R., Istituto Nazionale di Ottica, INO-UOS Pisa, Area di Ricerca, Via G. Moruzzi 1, 56124 Pisa (Italy); D’Ulivo, Alessandro [National Research Council of Italy, C.N.R., Istituto di Chimica dei Composti Organo Metallici-ICCOM- UOS Pisa, Area di Ricerca, Via G. Moruzzi 1, 56124 Pisa (Italy); Bramanti, Emilia, E-mail: bramanti@pi.iccom.cnr.it [National Research Council of Italy, C.N.R., Istituto di Chimica dei Composti Organo Metallici-ICCOM- UOS Pisa, Area di Ricerca, Via G. Moruzzi 1, 56124 Pisa (Italy)

2014-09-16

Highlights: • A simple procedure for the derivatization of proteins disulfide bonds. • Cysteine groups in several proteins derivatised with pHMB in alkaline media. • 75–100% labelling of cysteines in proteins with pHMB. - Abstract: In this work we have studied the derivatization of protein disulfide bonds with p-Hydroxymercurybenzoate (pHMB) in strong alkaline medium without any preliminary reduction. The reaction has been followed by the determination of the protein–pHMB complex using size exclusion chromatography coupled to a microwave/UV mercury oxidation system for the on-line oxidation of free and protein-complexed pHMB and atomic fluorescence spectrometry (SEC–CVG–AFS) detection. The reaction has been optimized by an experimental design using lysozyme as a model protein and applied to several thiolic proteins. The proposed method reports, for the first time, that it is possible to label 75–100% cysteines of proteins and, thus, to determine thiolic proteins without the need of any reducing step to obtain reduced -SH groups before mercury labelling. We obtained a detection limit of 100 nmol L{sup −1} based on a signal-to-noise ratio of 3 for unbound and complexed pHMB, corresponding to a detection limit of proteins ranged between 3 and 360 nmol L{sup −1}, depending on the number of cysteines in the protein sequence.

Direct, simple derivatization of disulfide bonds in proteins with organic mercury in alkaline medium without any chemical pre-reducing agents

International Nuclear Information System (INIS)

Campanella, Beatrice; Onor, Massimo; Ferrari, Carlo; D’Ulivo, Alessandro; Bramanti, Emilia

2014-01-01

Highlights: • A simple procedure for the derivatization of proteins disulfide bonds. • Cysteine groups in several proteins derivatised with pHMB in alkaline media. • 75–100% labelling of cysteines in proteins with pHMB. - Abstract: In this work we have studied the derivatization of protein disulfide bonds with p-Hydroxymercurybenzoate (pHMB) in strong alkaline medium without any preliminary reduction. The reaction has been followed by the determination of the protein–pHMB complex using size exclusion chromatography coupled to a microwave/UV mercury oxidation system for the on-line oxidation of free and protein-complexed pHMB and atomic fluorescence spectrometry (SEC–CVG–AFS) detection. The reaction has been optimized by an experimental design using lysozyme as a model protein and applied to several thiolic proteins. The proposed method reports, for the first time, that it is possible to label 75–100% cysteines of proteins and, thus, to determine thiolic proteins without the need of any reducing step to obtain reduced -SH groups before mercury labelling. We obtained a detection limit of 100 nmol L −1 based on a signal-to-noise ratio of 3 for unbound and complexed pHMB, corresponding to a detection limit of proteins ranged between 3 and 360 nmol L −1 , depending on the number of cysteines in the protein sequence

[SNACK HIGH WHEY PROTEIN IMPROVES THE LEVEL OF SATIETY AND REDUCES APPETITE HEALTHY WOMEN].

Science.gov (United States)

Reyna, Nadia; Moreno-Rojas, Rafael; Mendoza, Laura; Urdaneta, Andrés; Artigas, Carlos; Reyna, Eduardo; Cámara Martos, Fernando

2015-10-01

the nutritional content and energy density of foods is related to greater control of appetite, satiety and reducing food intake. the randomized crossover study included 20 healthy women, aged 20 and 30 years with a BMI of 20 to 24.9 kg/m2 and who completed that included 3 day trial comparing 8 hours 130 kcal snacks consumed afternoon: yoghurt with added whey protein (PSL), biscuits and chocolate. Participants consumed a standardized menu; snack was consumed 3 hours after lunch. Perceived hunger and fullness were evaluated during the afternoon until dinner voluntary intake ad libitum. They repeat the same snack 3 times. consumption of yogurt with PSL led to a further reduction of appetite in the afternoon in front of the snack of chocolate and biscuits (p snack, yogurt there was a significant reduction in caloric intake compared to other snacks (p snacks with less energy density and rich in protein (yogurt with PSL) improve the control of appetite, satiety and reduces food intake in healthy women later. Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.

Algorithm research for user trajectory matching across social media networks based on paragraph2vec

Science.gov (United States)

Xu, Qian; Chen, Hongchang; Zhi, Hongxin; Wang, Yanchuan

2018-04-01

Identifying users across different social media networks (SMN) is to link accounts of the same user that belong to the same individual across SMNs. The problem is fundamental and important, and its results can benefit many applications such as cross SMN user modeling and recommendation. With the development of GPS technology and mobile communication, more and more social networks provide location services. This provides a new opportunity for cross SMN user identification. In this paper, we solve cross SMN user identification problem in an unsupervised manner by utilizing user trajectory data in SMNs. A paragraph2vec based algorithm is proposed in which location sequence feature of user trajectory is captured in temporal and spatial dimensions. Our experimental results validate the effectiveness and efficiency of our algorithm.

Surfactant Protein-B 121ins2 Heterozygosity, Reduced Pulmonary Function and COPD in Smokers

DEFF Research Database (Denmark)

Bækvad-Hansen, Marie; Dahl, Morten; Tybjærg-Hansen, Anne

2010-01-01

.2-4.8) for spirometry defined COPD and of 2.2(1.0-5.1) for hospitalization due to COPD. Among never smokers, 121ins2 heterozygotes did not differ from wildtypes in lung function or risk of COPD. CONCLUSIONS: Surfactant protein-B 121ins2 heterozygosity is associated with reduced lung function and increased risk for COPD......2 mutation have reduced lung function and increased risk for chronic obstructive pulmonary disease (COPD) among smokers. METHODS: We genotyped 47,600 individuals from the adult Danish general population and recorded smoking habits, spirometry and hospital admissions due to COPD. The study...... that the effect of genotype differ by smoking status. Among smokers, 121ins2 heterozygotes had 9% reduced FEV1%predicted(p=0.0008), 6% reduced FVC%predicted(p=0.01) and 6% reduced FEV1/FVC(p=0.00007), compared with wildtypes. Also among smokers, 121ins2 heterozygotes had odds ratios of 2.4(95%CI 1...

Whey protein reduces early life weight gain in mice fed a high-fat diet

DEFF Research Database (Denmark)

Tranberg, Britt; Hellgren, Lars; Lykkesfeldt, Jens

2013-01-01

An increasing number of studies indicate that dairy products, including whey protein, alleviate several disorders of the metabolic syndrome. Here, we investigated the effects of whey protein isolate (whey) in mice fed a high-fat diet hypothesising that the metabolic effects of whey would...... be associated with changes in the gut microbiota composition. Five-week-old male C57BL/6 mice were fed a high-fat diet ad libitum for 14 weeks with the protein source being either whey or casein. Faeces were collected at week 0, 7, and 13 and the fecal microbiota was analysed by denaturing gradient gel...... electrophoresis analyses of PCR-derived 16S rRNA gene (V3-region) amplicons. At the end of the study, plasma samples were collected and assayed for glucose, insulin and lipids. Whey significantly reduced body weight gain during the first four weeks of the study compared with casein (P

A Protein Extract from Chicken Reduces Plasma Homocysteine in Rats

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Vegard Lysne

2015-06-01

Full Text Available The present study aimed to evaluate effects of a water-soluble protein fraction of chicken (CP, with a low methionine/glycine ratio, on plasma homocysteine and metabolites related to homocysteine metabolism. Male Wistar rats were fed either a control diet with 20% w/w casein as the protein source, or an experimental diet where 6, 14 or 20% w/w of the casein was replaced with the same amount of CP for four weeks. Rats fed CP had reduced plasma total homocysteine level and markedly increased levels of the choline pathway metabolites betaine, dimethylglycine, sarcosine, glycine and serine, as well as the transsulfuration pathway metabolites cystathionine and cysteine. Hepatic mRNA level of enzymes involved in homocysteine remethylation, methionine synthase and betaine-homocysteine S-methyltransferase, were unchanged, whereas cystathionine gamma-lyase of the transsulfuration pathway was increased in the CP treated rats. Plasma concentrations of vitamin B2, folate, cobalamin, and the B-6 catabolite pyridoxic acid were increased in the 20% CP-treated rats. In conclusion, the CP diet was associated with lower plasma homocysteine concentration and higher levels of serine, choline oxidation and transsulfuration metabolites compared to a casein diet. The status of related B-vitamins was also affected by CP.

Anabolic sensitivity of postprandial muscle protein synthesis to the ingestion of a protein-dense food is reduced in overweight and obese young adults.

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Beals, Joseph W; Sukiennik, Richard A; Nallabelli, Julian; Emmons, Russell S; van Vliet, Stephan; Young, Justin R; Ulanov, Alexander V; Li, Zhong; Paluska, Scott A; De Lisio, Michael; Burd, Nicholas A

2016-10-01

Excess body fat diminishes muscle protein synthesis rates in response to hyperinsulinemic-hyperaminoacidemic clamps. However, muscle protein synthetic responses after the ingestion of a protein-dense food source across a range of body mass indexes (BMIs) have not been compared. We compared the myofibrillar protein synthetic response and underlying nutrient-sensing mechanisms after the ingestion of lean pork between obese, overweight, and healthy-weight adults. Ten healthy-weight [HW; BMI (in kg/m 2 ): 22.7 ± 0.4], 10 overweight (OW; BMI: 27.1 ± 0.5), and 10 obese (OB; BMI: 35.9 ± 1.3) adults received primed continuous l-[ring- 13 C 6 ]phenylalanine infusions. Blood and muscle biopsy samples were collected before and after the ingestion of 170 g pork (36 g protein and 3 g fat) to assess skeletal muscle anabolic signaling, amino acid transporters [large neutral and small neutral amino acid transporters (LAT1, SNAT2) and CD98], and myofibrillar protein synthesis. At baseline, OW and OB groups showed greater relative amounts of mammalian target of rapamycin complex 1 (mTORC1) protein than the HW group. Pork ingestion increased mTORC1 phosphorylation only in the HW group (P = 0.001). LAT1 and SNAT2 protein content increased during the postprandial period in all groups (time effect, P anabolic signals, that reduces muscle sensitivity to food ingestion. This trial was registered at clinicaltrials.gov as NCT02613767. © 2016 American Society for Nutrition.

Effects of Supplementation of Branched-Chain Amino Acids to Reduced-Protein Diet on Skeletal Muscle Protein Synthesis and Degradation in the Fed and Fasted States in a Piglet Model

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Liufeng Zheng

2016-12-01

Full Text Available Supplementation of branched-chain amino acids (BCAA has been demonstrated to promote skeletal muscle mass gain, but the mechanisms underlying this observation are still unknown. Since the regulation of muscle mass depends on a dynamic equilibrium (fasted losses–fed gains in protein turnover, the aim of this study was to investigate the effects of BCAA supplementation on muscle protein synthesis and degradation in fed/fasted states and the related mechanisms. Fourteen 26- (Experiment 1 and 28-day-old (Experiment 2 piglets were fed reduced-protein diets without or with supplemental BCAA. After a four-week acclimation period, skeletal muscle mass and components of anabolic and catabolic signaling in muscle samples after overnight fasting were determined in Experiment 1. Pigs in Experiment 2 were implanted with carotid arterial, jugular venous, femoral arterial and venous catheters, and fed once hourly along with the intravenous infusion of NaH13CO3 for 2 h, followed by a 6-h infusion of [1-13C]leucine. Muscle leucine kinetics were measured using arteriovenous difference technique. The mass of most muscles was increased by BCAA supplementation. During feeding, BCAA supplementation increased leucine uptake, protein synthesis, protein degradation and net transamination. The greater increase in protein synthesis than in protein degradation resulted in elevated protein deposition. Protein synthesis was strongly and positively correlated with the intramuscular net production of α-ketoisocaproate (KIC and protein degradation. Moreover, BCAA supplementation enhanced the fasted-state phosphorylation of protein translation initiation factors and inhibited the protein-degradation signaling of ubiquitin-proteasome and autophagy-lysosome systems. In conclusion, supplementation of BCAA to reduced-protein diet increases fed-state protein synthesis and inhibits fasted-state protein degradation, both of which could contribute to the elevation of skeletal muscle

Disulfide Linkage Characterization of Disulfide Bond-Containing Proteins and Peptides by Reducing Electrochemistry and Mass Spectrometry

DEFF Research Database (Denmark)

Cramer, Christian N; Haselmann, Kim F; Olsen, Jesper V

2016-01-01

in protein sequencing by tandem MS (MS/MS). Electrochemical (EC) reduction of disulfide bonds has recently been demonstrated to provide efficient reduction efficiencies, significantly enhancing sequence coverages in online coupling with MS characterization. In this study, the potential use of EC disulfide...... link between parent disulfide-linked fragments and free reduced peptides in an LC-EC-MS platform of nonreduced proteolytic protein digestions. Here we report the successful use of EC as a partial reduction approach in mapping of disulfide bonds of intact human insulin (HI) and lysozyme. In addition, we...... established a LC-EC-MS platform advantageous in disulfide characterization of complex and highly disulfide-bonded proteins such as human serum albumin (HSA) by online EC reduction of nonreduced proteolytic digestions....

Analysis of the fibroblast growth factor system reveals alterations in a mouse model of spinal muscular atrophy.

Science.gov (United States)

Hensel, Niko; Ratzka, Andreas; Brinkmann, Hella; Klimaschewski, Lars; Grothe, Claudia; Claus, Peter

2012-01-01

The monogenetic disease Spinal Muscular Atrophy (SMA) is characterized by a progressive loss of motoneurons leading to muscle weakness and atrophy due to severe reduction of the Survival of Motoneuron (SMN) protein. Several models of SMA show deficits in neurite outgrowth and maintenance of neuromuscular junction (NMJ) structure. Survival of motoneurons, axonal outgrowth and formation of NMJ is controlled by neurotrophic factors such as the Fibroblast Growth Factor (FGF) system. Besides their classical role as extracellular ligands, some FGFs exert also intracellular functions controlling neuronal differentiation. We have previously shown that intracellular FGF-2 binds to SMN and regulates the number of a subtype of nuclear bodies which are reduced in SMA patients. In the light of these findings, we systematically analyzed the FGF-system comprising five canonical receptors and 22 ligands in a severe mouse model of SMA. In this study, we demonstrate widespread alterations of the FGF-system in both muscle and spinal cord. Importantly, FGF-receptor 1 is upregulated in spinal cord at a pre-symptomatic stage as well as in a mouse motoneuron-like cell-line NSC34 based model of SMA. Consistent with that, phosphorylations of FGFR-downstream targets Akt and ERK are increased. Moreover, ERK hyper-phosphorylation is functionally linked to FGFR-1 as revealed by receptor inhibition experiments. Our study shows that the FGF system is dysregulated at an early stage in SMA and may contribute to the SMA pathogenesis.

U bodies respond to nutrient stress in Drosophila

International Nuclear Information System (INIS)

Buckingham, Mickey; Liu, Ji-Long

2011-01-01

The neurodegenerative disease spinal muscular atrophy (SMA) is caused by mutation of the survival motor neuron 1 (SMN1) gene. Cytoplasmic SMN protein-containing granules, known as U snRNP bodies (U bodies), are thought to be responsible for the assembly and storage of small nuclear ribonucleoproteins (snRNPs) which are essential for pre-mRNA splicing. U bodies exhibit close association with cytoplasmic processing bodies (P bodies), which are involved in mRNA decay and translational repression. The close association of the U body and P body in Drosophila resemble that of the stress granule and P body in yeast and mammalian cells. However, it is unknown whether the U body is responsive to any stress. Using Drosophila oogenesis as a model, here we show that U bodies increase in size following nutritional deprivation. Despite nutritional stress, U bodies maintain their close association with P bodies. Our results show that U bodies are responsive to nutrition changes, presumably through the U body–P body pathway.

Converging Mechanisms of p53 Activation Drive Motor Neuron Degeneration in Spinal Muscular Atrophy

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Christian M. Simon

2017-12-01

Full Text Available The hallmark of spinal muscular atrophy (SMA, an inherited disease caused by ubiquitous deficiency in the SMN protein, is the selective degeneration of subsets of spinal motor neurons. Here, we show that cell-autonomous activation of p53 occurs in vulnerable but not resistant motor neurons of SMA mice at pre-symptomatic stages. Moreover, pharmacological or genetic inhibition of p53 prevents motor neuron death, demonstrating that induction of p53 signaling drives neurodegeneration. At late disease stages, however, nuclear accumulation of p53 extends to resistant motor neurons and spinal interneurons but is not associated with cell death. Importantly, we identify phosphorylation of serine 18 as a specific post-translational modification of p53 that exclusively marks vulnerable SMA motor neurons and provide evidence that amino-terminal phosphorylation of p53 is required for the neurodegenerative process. Our findings indicate that distinct events induced by SMN deficiency converge on p53 to trigger selective death of vulnerable SMA motor neurons.

U bodies respond to nutrient stress in Drosophila

Energy Technology Data Exchange (ETDEWEB)

Buckingham, Mickey; Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk

2011-12-10

The neurodegenerative disease spinal muscular atrophy (SMA) is caused by mutation of the survival motor neuron 1 (SMN1) gene. Cytoplasmic SMN protein-containing granules, known as U snRNP bodies (U bodies), are thought to be responsible for the assembly and storage of small nuclear ribonucleoproteins (snRNPs) which are essential for pre-mRNA splicing. U bodies exhibit close association with cytoplasmic processing bodies (P bodies), which are involved in mRNA decay and translational repression. The close association of the U body and P body in Drosophila resemble that of the stress granule and P body in yeast and mammalian cells. However, it is unknown whether the U body is responsive to any stress. Using Drosophila oogenesis as a model, here we show that U bodies increase in size following nutritional deprivation. Despite nutritional stress, U bodies maintain their close association with P bodies. Our results show that U bodies are responsive to nutrition changes, presumably through the U body-P body pathway.

Diabetic polyneuropathy, sensory neurons, nuclear structure and spliceosome alterations: a role for CWC22

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Masaki Kobayashi

2017-03-01

Full Text Available Unique deficits in the function of adult sensory neurons as part of their early neurodegeneration might account for progressive polyneuropathy during chronic diabetes mellitus. Here, we provide structural and functional evidence for aberrant pre-mRNA splicing in a chronic type 1 model of experimental diabetic polyneuropathy (DPN. Cajal bodies (CBs, unique nuclear substructures involved in RNA splicing, increased in number in diabetic sensory neurons, but their expected colocalization with survival motor neuron (SMN proteins was reduced – a mislocalization described in motor neurons of spinal muscular atrophy. Small nuclear ribonucleoprotein particles (snRNPs, also participants in the spliceosome, had abnormal multiple nuclear foci unassociated with CBs, and their associated snRNAs were reduced. CWC22, a key spliceosome protein, was aberrantly upregulated in diabetic dorsal root ganglia (DRG, and impaired neuronal function. CWC22 attenuated sensory neuron plasticity, with knockdown in vitro enhancing their neurite outgrowth. Further, axonal delivery of CWC22 siRNA unilaterally to locally knock down the aberrant protein in diabetic nerves improved aspects of sensory function in diabetic mice. Collectively, our findings identify subtle but significant alterations in spliceosome structure and function, including dysregulated CBs and CWC22 overexpression, in diabetic sensory neurons that offer new ideas regarding diabetic sensory neurodegeneration in polyneuropathy.

Reduced endothelial thioredoxin-interacting protein protects arteries from damage induced by metabolic stress in vivo.

Science.gov (United States)

Bedarida, Tatiana; Domingues, Alison; Baron, Stephanie; Ferreira, Chrystophe; Vibert, Francoise; Cottart, Charles-Henry; Paul, Jean-Louis; Escriou, Virginie; Bigey, Pascal; Gaussem, Pascale; Leguillier, Teddy; Nivet-Antoine, Valerie

2018-06-01

Although thioredoxin-interacting protein (TXNIP) is involved in a variety of biologic functions, the contribution of endothelial TXNIP has not been well defined. To investigate the endothelial function of TXNIP, we generated a TXNIP knockout mouse on the Cdh5-cre background (TXNIP fl/fl cdh5 cre ). Control (TXNIP fl/fl ) and TXNIP fl/fl cdh5 cre mice were fed a high protein-low carbohydrate (HP-LC) diet for 3 mo to induce metabolic stress. We found that TXNIP fl/fl and TXNIP fl/fl cdh5 cre mice on an HP-LC diet displayed impaired glucose tolerance and dyslipidemia concretizing the metabolic stress induced. We evaluated the impact of this metabolic stress on mice with reduced endothelial TXNIP expression with regard to arterial structure and function. TXNIP fl/fl cdh5 cre mice on an HP-LC diet exhibited less endothelial dysfunction than littermate mice on an HP-LC diet. These mice were protected from decreased aortic medial cell content, impaired aortic distensibility, and increased plasminogen activator inhibitor 1 secretion. This protective effect came with lower oxidative stress and lower inflammation, with a reduced NLRP3 inflammasome expression, leading to a decrease in cleaved IL-1β. We also show the major role of TXNIP in inflammation with a knockdown model, using a TXNIP-specific, small interfering RNA included in a lipoplex. These findings demonstrate a key role for endothelial TXNIP in arterial impairments induced by metabolic stress, making endothelial TXNIP a potential therapeutic target.-Bedarida, T., Domingues, A., Baron, S., Ferreira, C., Vibert, F., Cottart, C.-H., Paul, J.-L., Escriou, V., Bigey, P., Gaussem, P., Leguillier, T., Nivet-Antoine, V. Reduced endothelial thioredoxin-interacting protein protects arteries from damage induced by metabolic stress in vivo.

GLP-1 analogs reduce hepatocyte steatosis and improve survival by enhancing the unfolded protein response and promoting macroautophagy.

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Shvetank Sharma

Full Text Available Nonalcoholic fatty liver disease (NAFLD is a known outcome of hepatosteatosis. Free fatty acids (FFA induce the unfolded protein response (UPR or endoplasmic reticulum (ER stress that may induce apoptosis. Recent data indicate ER stress to be a major player in the progression of fatty liver to more aggressive lesions. Autophagy on the other hand has been demonstrated to be protective against ER stress-induced cell death. We hypothesized that exendin-4 (GLP-1 analog treatment of fat loaded hepatocytes can reduce steatosis by autophagy which leads to reduced ER stress-related hepatocyte apoptosis.Primary human hepatocytes were loaded with saturated, cis- and trans-unsaturated fatty acids (palmitic, oleic and elaidic acid respectively. Steatosis, induced with all three fatty acids, was significantly resolved after exendin-4 treatment. Exendin-4 sustained levels of GRP78 expression in fat-loaded cells when compared to untreated fat-loaded cells alone. In contrast, CHOP (C/EBP homologous protein; the penultimate protein that leads to ER stress-related cell death was significantly decreased by exendin-4 in hepatocytes loaded with fatty acids. Finally, exendin-4 in fat loaded hepatocytes clearly promoted gene products associated with macroautophagy as measured by enhanced production of both Beclin-1 and LC3B-II, markers for autophagy; and visualized by transmission electron microscopy (TEM. Similar observations were made in mouse liver lysates after mice were fed with high fat high fructose diet and treated with a long acting GLP-1 receptor agonist, liraglutide.GLP-1 proteins appear to protect hepatocytes from fatty acid-related death by prohibition of a dysfunctional ER stress response; and reduce fatty acid accumulation, by activation of both macro-and chaperone-mediated autophagy. These findings provide a novel role for GLP-1 proteins in halting the progression of more aggressive lesions from underlying steatosis in humans afflicted with NAFLD.

Lipoxygenase independent hexanal formation in isolated soy proteins induced by reducing agents.

Science.gov (United States)

Lei, Q; Boatright, W L

2008-08-01

Compared to corresponding controls, 6.5 mM dithiothreitol (DTT) elevated headspace hexanal level over aqueous slurries of both commercial isolated soy proteins (ISP) and laboratory ISP prepared with 80 degrees C treatment. Further analysis revealed that lipoxygenase (LOX) activity was not detected from these ISP, indicating that LOX is not involved in the observed hexanal increase. Levels of the induced headspace hexanal over the ISP aqueous slurries were proportional to the amount of DTT added in the range of 0 to 65 mM. Subsequent systematic investigations with model systems revealed that iron was required for the reducing agent-induced hexanal formation from linoleic acid. Erythorbate, another reducing agent, can also induce hexanal formation in both ISP and model systems. As a comparison, the LOX activity and hexanal synthesis in defatted soy flour were examined. The corresponding results showed that defatted soy flour maintained high LOX activities and that hexanal synthesis in such sample was significantly inhibited by high concentration DTT (above 130 mM). Data from the current investigation demonstrate the existence of LOX independent hexanal formation induced by reducing agents in ISP and the potential requirement of iron as a catalyst.

Magnetic behavior study of samarium nitride using density functional theory

Science.gov (United States)

Som, Narayan N.; Mankad, Venu H.; Dabhi, Shweta D.; Patel, Anjali; Jha, Prafulla K.

2018-02-01

In this work, the state-of-art density functional theory is employed to study the structural, electronic and magnetic properties of samarium nitride (SmN). We have performed calculation for both ferromagnetic and antiferromagnetic states in rock-salt phase. The calculated results of optimized lattice parameter and magnetic moment agree well with the available experimental and theoretical values. From energy band diagram and electronic density of states, we observe a half-metallic behaviour in FM phase of rock salt SmN in while metallicity in AFM I and AFM III phases. We present and discuss our current understanding of the possible half-metallicity together with the magnetic ordering in SmN. The calculated phonon dispersion curves shows dynamical stability of the considered structures. The phonon density of states and Eliashberg functional have also been analysed to understand the superconductivity in SmN.

Second Malignant Neoplasms and Cause of Death in Patients With Germ Cell Cancer

DEFF Research Database (Denmark)

Kier, Maria G; Hansen, Merete K; Lauritsen, Jakob

2016-01-01

radiotherapy (RT); bleomycin, etoposide, and cisplatin (BEP); or more than 1 line of treatment (MTOL). Main Outcomes and Measures: Cumulative incidence and hazard ratios (HRs) for SMN and death calculated by the Cox proportional hazards model were compared with those of age-matched controls. Results: The study......Importance: Patients given systemic treatment for testicular germ cell cancer (GCC) are at increased risk for a second malignant neoplasm (SMN). Previous studies on SMN and causes of death lacked information on the exact treatment applied or were based on patients receiving former treatment options....... Objective: To evaluate the treatment-specific risks for SMN and death in a nationwide population-based cohort of patients with GCC treated with current standard regimens. Design, Setting, and Participants: This study examined a Danish nationwide cohort of 5190 men with GCC who entered the Danish Testicular...

Nuclear bodies: news insights into structure and function

Czech Academy of Sciences Publication Activity Database

Staněk, David; Fox, A.H.

2017-01-01

Roč. 46, léto (2017), s. 94-101 ISSN 0955-0674 R&D Projects: GA ČR GA15-00790S; GA MŠk LO1419 Institutional support: RVO:68378050 Keywords : rna-binding proteins * cajal bodies * smn complex * human-cells * in-vivo * human telomerase * coiled bodies * lncrna neat1 * u1 snrnp * body Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 9.937, year: 2016

Binding of the heterogeneous ribonucleoprotein K (hnRNP K to the Epstein-Barr virus nuclear antigen 2 (EBNA2 enhances viral LMP2A expression.

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Henrik Gross

Full Text Available The Epstein-Barr Virus (EBV -encoded EBNA2 protein, which is essential for the in vitro transformation of B-lymphocytes, interferes with cellular processes by binding to proteins via conserved sequence motifs. Its Arginine-Glycine (RG repeat element contains either symmetrically or asymmetrically di-methylated arginine residues (SDMA and ADMA, respectively. EBNA2 binds via its SDMA-modified RG-repeat to the survival motor neurons protein (SMN and via the ADMA-RG-repeat to the NP9 protein of the human endogenous retrovirus K (HERV-K (HML-2 Type 1. The hypothesis of this work was that the methylated RG-repeat mimics an epitope shared with cellular proteins that is used for interaction with target structures. With monoclonal antibodies against the modified RG-repeat, we indeed identified cellular homologues that apparently have the same surface structure as methylated EBNA2. With the SDMA-specific antibodies, we precipitated the Sm protein D3 (SmD3 which, like EBNA2, binds via its SDMA-modified RG-repeat to SMN. With the ADMA-specific antibodies, we precipitated the heterogeneous ribonucleoprotein K (hnRNP K. Specific binding of the ADMA- antibody to hnRNP K was demonstrated using E. coli expressed/ADMA-methylated hnRNP K. In addition, we show that EBNA2 and hnRNP K form a complex in EBV- infected B-cells. Finally, hnRNP K, when co-expressed with EBNA2, strongly enhances viral latent membrane protein 2A (LMP2A expression by an unknown mechanism as we did not detect a direct association of hnRNP K with DNA-bound EBNA2 in gel shift experiments. Our data support the notion that the methylated surface of EBNA2 mimics the surface structure of cellular proteins to interfere with or co-opt their functional properties.

High-resolution melting (HRM) analysis as a feasible method for detecting spinal muscular atrophy via dried blood spots.

Science.gov (United States)

Er, Tze-Kiong; Kan, Tzu-Min; Su, Yu-Fa; Liu, Ta-Chih; Chang, Jan-Gowth; Hung, Shih-Ya; Jong, Yuh-Jyh

2012-11-12

Spinal muscular atrophy (SMA) is a neurodegenerative disease with the leading genetic cause of infant mortality. More than 95% of patients with SMA have a homozygous disruption in the survival motor neuron1 (SMN1) gene, caused by mutation, deletion, or rearrangement. Recently, treatment in humans in the immediate postnatal period, prior to the development of weakness or very early in the course of the disease, may be effective. Therefore, our objective was to establish a feasible method for SMA screening. High-resolution melting (HRM) analysis is rapidly becoming the most important mutation-scanning methodology that allows mutation scanning and genotyping without the need for costly labeled oligonucleotides. In the current study, we aim to develop a method for identifying the substitution of single nucleotide in SMN1 exon 7 (c.840C>T) by HRM analysis. Genomic DNA was extracted from peripheral blood samples and dried blood spots obtained from 30 patients with SMA and 30 normal individuals. All results were previously confirmed by denaturing high-performance liquid chromatography (DHPLC). In order to identify the substitution of single nucleotide in SMN1 exon 7 (c.840C>T) by HRM analysis, a primer set was used in HRM analysis. At first, we failed to identify the substitution of single nucleotide in SMN1 exon 7 (c.840C>T) by HRM analysis because the homozygous CC and homozygous TT cannot be distinguished by HRM analysis. Therefore, all samples were mixed with a known SMN1/SMN2 copy number (SMN1/SMN2=0:3), which we may call driver. This strategy is used to differentiate between homozygous CC and homozygous TT. After mixing with driver, the melting profile of homozygous CC becomes heteroduplex; however, the homozygous TT remains the same in the normalized and temperature-shifted difference plots. HRM analysis can be successfully applied to screen SMA via DNA obtained from whole blood and dried blood spots. We strongly believe that HRM analysis, a high-throughput method

Metabolic network modeling of microbial interactions in natural and engineered environmental systems

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Octavio ePerez-Garcia

2016-05-01

Full Text Available We review approaches to characterize metabolic interactions within microbial communities using Stoichiometric Metabolic Network (SMN models for applications in environmental and industrial biotechnology. SMN models are computational tools used to evaluate the metabolic engineering potential of various organisms. They have successfully been applied to design and optimize the microbial production of antibiotics, alcohols and amino acids by single strains. To date however, such models have been rarely applied to analyze and control the metabolism of more complex microbial communities. This is largely attributed to the diversity of microbial community functions, metabolisms and interactions. Here, we firstly review different types of microbial interaction and describe their relevance for natural and engineered environmental processes. Next, we provide a general description of the essential methods of the SMN modeling workflow including the steps of network reconstruction, simulation through Flux Balance Analysis (FBA, experimental data gathering, and model calibration. Then we broadly describe and compare four approaches to model microbial interactions using metabolic networks, i.e. i lumped networks, ii compartment per guild networks, iii bi-level optimization simulations and iv dynamic-SMN methods. These approaches can be used to integrate and analyze diverse microbial physiology, ecology and molecular community data. All of them (except the lumped approach are suitable for incorporating species abundance data but so far they have been used only to model simple communities of two to eight different species. Interactions based on substrate exchange and competition can be directly modeled using the above approaches. However, interactions based on metabolic feedbacks, such as product inhibition and synthropy require extensions to current models, incorporating gene regulation and compounding accumulation mechanisms. SMN models of microbial

Recapitulation of spinal motor neuron-specific disease phenotypes in a human cell model of spinal muscular atrophy

Institute of Scientific and Technical Information of China (English)

Zhi-Bo Wang; Xiaoqing Zhang; Xue-Jun Li

2013-01-01

Establishing human cell models of spinal muscular atrophy (SMA) to mimic motor neuron-specific phenotypes holds the key to understanding the pathogenesis of this devastating disease.Here,we developed a closely representative cell model of SMA by knocking down the disease-determining gene,survival motor neuron (SMN),in human embryonic stem cells (hESCs).Our study with this cell model demonstrated that knocking down of SMN does not interfere with neural induction or the initial specification of spinal motor neurons.Notably,the axonal outgrowth of spinal motor neurons was significantly impaired and these disease-mimicking neurons subsequently degenerated.Furthermore,these disease phenotypes were caused by SMN-full length (SMN-FL) but not SMN-A7 (lacking exon 7)knockdown,and were specific to spinal motor neurons.Restoring the expression of SMN-FL completely ameliorated all of the disease phenotypes,including specific axonal defects and motor neuron loss.Finally,knockdown of SMNFL led to excessive mitochondrial oxidative stress in human motor neuron progenitors.The involvement of oxidative stress in the degeneration of spinal motor neurons in the SMA cell model was further confirmed by the administration of N-acetylcysteine,a potent antioxidant,which prevented disease-related apoptosis and subsequent motor neuron death.Thus,we report here the successful establishment of an hESC-based SMA model,which exhibits disease gene isoform specificity,cell type specificity,and phenotype reversibility.Our model provides a unique paradigm for studying how motor neurons specifically degenerate and highlights the potential importance of antioxidants for the treatment of SMA.

Bed rest reduces metabolic protein content and abolishes exercise-induced mRNA responses in human skeletal muscle

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Jørgensen, Stine Ringholm; Biensø, Rasmus S; Kiilerich, Kristian

2011-01-01

Background: The aim was to test the hypothesis that one week of bed rest will reduce mitochondrial number and expression and activity of oxidative proteins in human skeletal muscle, but that exercise-induced intracellular signaling as well as mRNA and microRNA (miR) responses are maintained after......-legged knee extensor exercise performed before and after bed rest. Results: Maximal oxygen uptake decreased 5% and exercise endurance decreased non-significantly 25% by bed rest. Bed rest reduced skeletal muscle mitochondrial DNA/nuclear DNA content 15%, hexokinase II and sirtuin 1 protein content ~45%, 3...... bed rest. Research Design and Methods: Twelve young, healthy, male subjects completed 7 days of bed rest with vastus lateralis muscle biopsies taken before and after bed rest. In addition, muscle biopsies were obtained from 6 of the subjects prior to, immediately after and 3h after 45 min one...

Inhibition of coagulation and inflammation by activated protein C or antithrombin reduces intestinal ischemia/reperfusion injury in rats

NARCIS (Netherlands)

Schoots, Ivo G.; Levi, Marcel; van Vliet, Arlène K.; Maas, Adrie M.; Roossink, E. H. Paulina; van Gulik, Thomas M.

2004-01-01

Objective: To examine whether administration of activated protein C or antithrombin reduces local splanchnic derangement of coagulation and inflammation and attenuates intestinal dysfunction and injury following intestinal ischemia/reperfusion. Design: Randomized prospective animal study. Setting:

Skeletal muscle neuronal nitric oxide synthase micro protein is reduced in people with impaired glucose homeostasis and is not normalized by exercise training.

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Bradley, Scott J; Kingwell, Bronwyn A; Canny, Benedict J; McConell, Glenn K

2007-10-01

Skeletal muscle inducible nitric oxide synthase (NOS) protein is greatly elevated in people with type 2 diabetes mellitus, whereas endothelial NOS is at normal levels. Diabetic rat studies suggest that skeletal muscle neuronal NOS (nNOS) micro protein expression may be reduced in human insulin resistance. The aim of this study was to determine whether skeletal muscle nNOSmicro protein expression is reduced in people with impaired glucose homeostasis and whether exercise training increases nNOSmicro protein expression in these individuals because exercise training increases skeletal muscle nNOSmicro protein in rats. Seven people with type 2 diabetes mellitus or prediabetes (impaired fasting glucose and/or impaired glucose tolerance) and 7 matched (sex, age, fitness, body mass index, blood pressure, lipid profile) healthy controls aged 36 to 60 years participated in this study. Vastus lateralis muscle biopsies for nNOSmicro protein determination were obtained, aerobic fitness was measured (peak pulmonary oxygen uptake [Vo(2) peak]), and glucose tolerance and insulin homeostasis were assessed before and after 1 and 4 weeks of cycling exercise training (60% Vo(2) peak, 50 minutes x 5 d wk(-1)). Skeletal muscle nNOSmicro protein was significantly lower (by 32%) in subjects with type 2 diabetes mellitus or prediabetes compared with that in controls before training (17.7 +/- 1.2 vs 26.2 +/- 3.4 arbitrary units, P glucose homeostasis have reduced skeletal muscle nNOSmicro protein content. However, because exercise training improves insulin sensitivity without influencing skeletal muscle nNOSmicro protein expression, it seems that changes in skeletal muscle nNOSmicro protein are not central to the control of insulin sensitivity in humans and therefore may be a consequence rather than a cause of diabetes.

Reduced myelin basic protein and actin-related gene expression in visual cortex in schizophrenia.

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Matthews, Paul R; Eastwood, Sharon L; Harrison, Paul J

2012-01-01

Most brain gene expression studies of schizophrenia have been conducted in the frontal cortex or hippocampus. The extent to which alterations occur in other cortical regions is not well established. We investigated primary visual cortex (Brodmann area 17) from the Stanley Neuropathology Consortium collection of tissue from 60 subjects with schizophrenia, bipolar disorder, major depression, or controls. We first carried out a preliminary array screen of pooled RNA, and then used RT-PCR to quantify five mRNAs which the array identified as differentially expressed in schizophrenia (myelin basic protein [MBP], myelin-oligodendrocyte glycoprotein [MOG], β-actin [ACTB], thymosin β-10 [TB10], and superior cervical ganglion-10 [SCG10]). Reduced mRNA levels were confirmed by RT-PCR for MBP, ACTB and TB10. The MBP reduction was limited to transcripts containing exon 2. ACTB and TB10 mRNAs were also decreased in bipolar disorder. None of the transcripts were altered in subjects with major depression. Reduced MBP mRNA in schizophrenia replicates findings in other brain regions and is consistent with oligodendrocyte involvement in the disorder. The decreases in expression of ACTB, and the actin-binding protein gene TB10, suggest changes in cytoskeletal organisation. The findings confirm that the primary visual cortex shows molecular alterations in schizophrenia and extend the evidence for a widespread, rather than focal, cortical pathophysiology.

Reduced influenza viral neutralizing activity of natural human trimers of surfactant protein D

DEFF Research Database (Denmark)

Hartshorn, Kevan L; White, Mitchell R; Tecle, Tesfaldet

2007-01-01

BACKGROUND: Surfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection. Common human polymorphisms of SP-D have been found in many human populations and associated with increased risk of certain infections. We recently reported that the Thr...... on the CRD of SP-D were found to have differing effects on antiviral activity. Using an mAb that did not interfere with antiviral activity of SP-D, we confirm that natural SP-D trimers had reduced ability to bind to IAV. In addition, the trimers had reduced ability to neutralize IAV as compared to natural...... indicate that a common human polymorphic form of SP-D may modulate host defense against IAV and give impetus to clinical studies correlating this genotype with risk for IAV infection in susceptible groups. We also show that mAbs directed against different areas on the carbohydrate recognition domain of SP...

Residues at a Single Site Differentiate Animal Cryptochromes from Cyclobutane Pyrimidine Dimer Photolyases by Affecting the Proteins' Preferences for Reduced FAD.

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Xu, Lei; Wen, Bin; Wang, Yuan; Tian, Changqing; Wu, Mingcai; Zhu, Guoping

2017-06-19

Cryptochromes (CRYs) and photolyases belong to the cryptochrome/photolyase family (CPF). Reduced FAD is essential for photolyases to photorepair UV-induced cyclobutane pyrimidine dimers (CPDs) or 6-4 photoproducts in DNA. In Drosophila CRY (dCRY, a type I animal CRY), FAD is converted to the anionic radical but not to the reduced state upon illumination, which might induce a conformational change in the protein to relay the light signal downstream. To explore the foundation of these differences, multiple sequence alignment of 650 CPF protein sequences was performed. We identified a site facing FAD (Ala377 in Escherichia coli CPD photolyase and Val415 in dCRY), hereafter referred to as "site 377", that was distinctly conserved across these sequences: CPD photolyases often had Ala, Ser, or Asn at this site, whereas animal CRYs had Ile, Leu, or Val. The binding affinity for reduced FAD, but not the photorepair activity of E. coli photolyase, was dramatically impaired when replacing Ala377 with any of the three CRY residues. Conversely, in V415S and V415N mutants of dCRY, FAD was photoreduced to its fully reduced state after prolonged illumination, and light-dependent conformational changes of these mutants were severely inhibited. We speculate that the residues at site 377 play a key role in the different preferences of CPF proteins for reduced FAD, which differentiate animal CRYs from CPD photolyases. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Prenatal diagnosis of Werdnig-Hoffmann disease in China

Institute of Scientific and Technical Information of China (English)

无

2003-01-01

Objective To establish a means for prenatal prediction of spinal muscular atrophy (SMA) through survival motor neuron (SMN) gene deletion analysis and genetic counseling in families with a child affected with SMA. Methods Genetic analysis for prenatal prediction of Werdnig-Hoffmann disease was performed in a at risk Chinese family by polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) in SMN gene exons 7 and 8.Results The pregnancy was positive for the homozygous deletion of the SMN gene, thus the fetus was diagnosed as being affected and the pregnancy was terminated.Conclusion This approach is fast and reliable for DNA-based prenatal diagnosis of Werdnig-Hoffmann disease.

Absence of Non-histone Protein Complexes at Natural Chromosomal Pause Sites Results in Reduced Replication Pausing in Aging Yeast Cells

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Marleny Cabral

2016-11-01

Full Text Available There is substantial evidence that genomic instability increases during aging. Replication pausing (and stalling at difficult-to-replicate chromosomal sites may induce genomic instability. Interestingly, in aging yeast cells, we observed reduced replication pausing at various natural replication pause sites (RPSs in ribosomal DNA (rDNA and non-rDNA locations (e.g., silent replication origins and tRNA genes. The reduced pausing occurs independent of the DNA helicase Rrm3p, which facilitates replication past these non-histone protein-complex-bound RPSs, and is independent of the deacetylase Sir2p. Conditions of caloric restriction (CR, which extend life span, also cause reduced replication pausing at the 5S rDNA and at tRNA genes. In aged and CR cells, the RPSs are less occupied by their specific non-histone protein complexes (e.g., the preinitiation complex TFIIIC, likely because members of these complexes have primarily cytosolic localization. These conditions may lead to reduced replication pausing and may lower replication stress at these sites during aging.

Glycation inhibitors extend yeast chronological lifespan by reducing advanced glycation end products and by back regulation of proteins involved in mitochondrial respiration.

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Kazi, Rubina S; Banarjee, Reema M; Deshmukh, Arati B; Patil, Gouri V; Jagadeeshaprasad, Mashanipalya G; Kulkarni, Mahesh J

2017-03-06

Advanced Glycation End products (AGEs) are implicated in aging process. Thus, reducing AGEs by using glycation inhibitors may help in attenuating the aging process. In this study using Saccharomyces cerevisiae yeast system, we show that Aminoguanidine (AMG), a well-known glycation inhibitor, decreases the AGE modification of proteins in non-calorie restriction (NR) (2% glucose) and extends chronological lifespan (CLS) similar to that of calorie restriction (CR) condition (0.5% glucose). Proteomic analysis revealed that AMG back regulates the expression of differentially expressed proteins especially those involved in mitochondrial respiration in NR condition, suggesting that it switches metabolism from fermentation to respiration, mimicking CR. AMG induced back regulation of differentially expressed proteins could be possibly due to its chemical effect or indirectly by glycation inhibition. To delineate this, Metformin (MET), a structural analog of AMG and a mild glycation inhibitor and Hydralazine (HYD), another potent glycation inhibitor but not structural analog of AMG were used. HYD was more effective than MET in mimicking AMG suggesting that glycation inhibition was responsible for restoration of differentially expressed proteins. Thus glycation inhibitors particularly AMG, HYD and MET extend yeast CLS by reducing AGEs, modulating the expression of proteins involved in mitochondrial respiration and possibly by scavenging glucose. This study reports the role of glycation in aging process. In the non-caloric restriction condition, carbohydrates such as glucose promote protein glycation and reduce CLS. While, the inhibitors of glycation such as AMG, HYD, MET mimic the caloric restriction condition by back regulating deregulated proteins involved in mitochondrial respiration which could facilitate shift of metabolism from fermentation to respiration and extend yeast CLS. These findings suggest that glycation inhibitors can be potential molecules that can be used

High dietary protein intake, reducing or eliciting insulin resistance?

NARCIS (Netherlands)

Rietman, A.; Schwarz, J.; Tome, D.; Kok, F.J.; Mensink, M.R.

2014-01-01

Dietary proteins have an insulinotropic effect and thus promote insulin secretion, which indeed leads to enhanced glucose clearance from the blood. In the long term, however, a high dietary protein intake is associated with an increased risk of type 2 diabetes. Moreover, branched-chain amino acids

Acetone utilization by sulfate-reducing bacteria: draft genome sequence of Desulfococcus biacutus and a proteomic survey of acetone-inducible proteins.

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Gutiérrez Acosta, Olga B; Schleheck, David; Schink, Bernhard

2014-07-11

The sulfate-reducing bacterium Desulfococcus biacutus is able to utilize acetone for growth by an inducible degradation pathway that involves a novel activation reaction for acetone with CO as a co-substrate. The mechanism, enzyme(s) and gene(s) involved in this acetone activation reaction are of great interest because they represent a novel and yet undefined type of activation reaction under strictly anoxic conditions. In this study, a draft genome sequence of D. biacutus was established. Sequencing, assembly and annotation resulted in 159 contigs with 5,242,029 base pairs and 4773 predicted genes; 4708 were predicted protein-encoding genes, and 3520 of these had a functional prediction. Proteins and genes were identified that are specifically induced during growth with acetone. A thiamine diphosphate-requiring enzyme appeared to be highly induced during growth with acetone and is probably involved in the activation reaction. Moreover, a coenzyme B12- dependent enzyme and proteins that are involved in redox reactions were also induced during growth with acetone. We present for the first time the genome of a sulfate reducer that is able to grow with acetone. The genome information of this organism represents an important tool for the elucidation of a novel reaction mechanism that is employed by a sulfate reducer in acetone activation.

Prognostic factors and survival in patients with radiation-related second malignant neoplasms following radiotherapy for nasopharyngeal carcinoma.

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Mian Xi

Full Text Available PURPOSE: To analyze the clinicopathological characteristics, treatment modalities, and potential prognostic factors of radiation-related second malignant neoplasms (SMNs in a large group of nasopharyngeal carcinoma (NPC cases. METHODS AND MATERIALS: Institutional electronic medical records of 39,118 patients with NPC treated by definitive radiotherapy between February 1964 and December 2003 were reviewed. A total of 247 patients with confirmed SMN attributable to radiotherapy were included. RESULTS: Median latency between radiotherapy for NPC and the diagnosis of SMN was 9.5 years (range, 3.1-36.8 years. Squamous cell carcinoma was the most common histologic type, followed by fibrosarcoma and adenocarcinoma. Median progression-free survival and overall survival (OS of the 235 patients who underwent treatment were 17.3 months and 28.5 months, respectively. The 5-year OS rates were 42.9%, 23.7%, and 0% for the surgery, radiotherapy, and chemotherapy groups, respectively. The independent prognostic factors associated with survival were sex, histologic type, and treatment modality in both the early stage subgroup and the advanced stage subgroup of SMN. CONCLUSIONS: Sex, histologic type, and treatment modality were the significant prognostic factors for SMN. Complete resection offers the best chance for long-term survival. In select patients with locally advanced and unresectable SMN, reirradiation should be strongly considered as a curative option.

Subchronic nandrolone administration reduces cardiac oxidative markers during restraint stress by modulating protein expression patterns.

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Pergolizzi, Barbara; Carriero, Vitina; Abbadessa, Giuliana; Penna, Claudia; Berchialla, Paola; De Francia, Silvia; Bracco, Enrico; Racca, Silvia

2017-10-01

Nandrolone decanoate (ND), an anabolic-androgenic steroid prohibited in collegiate and professional sports, is associated with detrimental cardiovascular effects through redox-dependent mechanisms. We previously observed that high-dose short-term ND administration (15 mg/kg for 2 weeks) did not induce left heart ventricular hypertrophy and, paradoxically, improved postischemic response, whereas chronic ND treatment (5 mg/kg twice a week for 10 weeks) significantly reduced the cardioprotective effect of postconditioning, with an increase in infarct size and a decrease in cardiac performance. We wanted to determine whether short-term ND administration could affect the oxidative redox status in animals exposed to acute restraint stress. Our hypothesis was that, depending on treatment schedule, ND may have a double-edged sword effect. Measurement of malondialdehyde and 4-hydroxynonenal, two oxidative stress markers, in rat plasma and left heart ventricular tissue, revealed that the levels of both markers were increased in animals exposed to restraint stress, whereas no increase in marker levels was noted in animals pretreated with ND, indicating a possible protective action of ND against stress-induced oxidative damage. Furthermore, isolation and identification of proteins extracted from the left heart ventricular tissue samples of rats pretreated or not with ND and exposed to acute stress showed a prevalent expression of enzymes involved in amino acid synthesis and energy metabolism. Among other proteins, peroxiredoxin 6 and alpha B-crystallin, both involved in the oxidative stress response, were predominantly expressed in the left heart ventricular tissues of the ND-pretreated rats. In conclusion, ND seems to reduce oxidative stress by inducing the expression of antioxidant proteins in the hearts of restraint-stressed animals, thus contributing to amelioration of postischemic heart performance.

Basal-bolus insulin therapy reduces maternal triglycerides in gestational diabetes without modifying cholesteryl ester transfer protein activity.

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Olmos, Pablo R; Borzone, Gisella R

2017-09-01

Macrosomia in the offspring of overweight/obese mothers with glucose-controlled gestational diabetes mellitus (GDM) is due to excessive rise of maternal triglycerides (TG). We aimed to ascertain whether basal-bolus insulin therapy (BBIT), or other components of the treatment, could reduce TG in GDM. We studied the records of 131 singleton pregnancies with GDM, using stepwise multiple linear regression, Mann-Whitney, χ 2 , and Jonckheere-Terpstra tests. As maternal TG increased steadily during normal pregnancy, these were transformed as z-scores. The atherogenic index of plasma (AIP) was calculated as a measure of cholesteryl ester transfer protein activity. Multiple regression showed that only BBIT (but neither limitation of weight gain nor metformin) reduced maternal TG z-scores (P = 0.011). When the 131 pregnancies were split into two groups - without BBIT (n = 58; HbA1c = 5.3 ± 0.3%) and with BBIT (n = 73; HbA1c = 5.4 ± 0.6; P = 0.2005) - we observed that BBIT (n = 73) reduced maternal TG z-scores in a dose-related fashion (Jonckheere-Terpstra P = 0.03817). The atherogenic index of plasma remained within normal range in both groups. BBIT (but not weight gain control nor metformin) reduced maternal TG in mothers with glucose-controlled GDM. This beneficial effect of BBIT was not related to changes in the cholesteryl ester transfer protein activity. © 2017 Japan Society of Obstetrics and Gynecology.

Impact of feeding reduced crude protein diets to lactating sows on nitrogen utilizatilon

DEFF Research Database (Denmark)

Huber, L; de Lange, C F M; Larsen, Uffe Krogh

2015-01-01

-fed; analyzed contents; HCP); 2) 15.7% CP (MHCP); 3) 14.3% CP (MLCP); 4) 13.2% CP (LCP); diet HCP was formulated using soybean meal and corn as the only Lys sources. The reduced CP diets contained CAA to meet requirements of the limiting AA. Sow and piglet BW were measured on d 1, 3, 7, 14, 18, and 21...... of lactation. Nitrogen retention was measured on sows between d 3 and 7 (early) and d 14 and 18 (peak) of lactation. Milk true protein output was calculated from estimated milk yield and analyzed true protein concentration. Sow BW change (overall mean: -4.2 ± 3.37 kg over the 21-d lactation period) and average...... daily DM intake (overall mean: 4.05 ± 0.18 and 6.12 ± 0.20 kg/d, early and peak lactation, respectively) did not differ between diets. Nitrogen intake decreased as dietary CP concentration decreased (114.3, 106.0, 107.4, and 99.0 ± 5.29 g/d and 169.5, 168.3, 161.2, and 145.1 ± 5.29 g/d for HCP, MHCP...

Atorvastatin reduces lipid accumulation in the liver by activating protein kinase A-mediated phosphorylation of perilipin 5.

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Gao, Xing; Nan, Yang; Zhao, Yuanlin; Yuan, Yuan; Ren, Bincheng; Sun, Chao; Cao, Kaiyu; Yu, Ming; Feng, Xuyang; Ye, Jing

2017-12-01

Statins have been proven to be effective in treating non-alcoholic fatty liver disease (NAFLD). Recently, it was reported that statins decreased the hepatic expression of perilipin 5 (Plin5), a lipid droplet (LD)-associated protein, which plays critical roles in regulating lipid accumulation and lipolysis in liver. However, the function and regulation mechanism of Plin5 have not yet been well-established in NAFLD treatment with statins. In this study, we observed that atorvastatin moderately reduced the expression of Plin5 in livers without changing the protein level of Plin5 in the hepatic LD fraction of mice fed with high-fat diet (HFD). Intriguingly, atorvastatin stimulated the PKA-mediated phosphorylation of Plin5 and reduced the triglyceride (TG) accumulation in hepatocytes with overexpression of wide type (Plin5-WT) compared to serine-155 mutant Plin5 (Plin5-S155A). Moreover, PKA-stimulated FA release of purified LDs carrying Plin5-WT but not Plin5-S155A. Glucagon, a PKA activator, stimulated the phosphorylation of Plin5-WT and inhibited its interaction with CGI-58. The results indicated that atorvastatin promoted lipolysis and reduced TG accumulation in the liver by increasing PKA-mediated phosphorylation of Plin5. This new mechanism of lipid-lowering effects of atorvastatin might provide a new strategy for NAFLD treatment. Copyright © 2017. Published by Elsevier B.V.

Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling

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Baker, Lindsay A. [University of Oxford, Oxford Particle Imaging Centre, The Wellcome Trust Centre for Human Genetics, Division of Structural Biology, Nuffield Department of Medicine (United Kingdom); Daniëls, Mark; Cruijsen, Elwin A. W. van der; Folkers, Gert E.; Baldus, Marc, E-mail: m.baldus@uu.nl [Utrecht University, NMR Spectroscopy, Department of Chemistry, Faculty of Science, Bijvoet Center for Biomolecular Research (Netherlands)

2015-06-15

Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR.

Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling

International Nuclear Information System (INIS)

Baker, Lindsay A.; Daniëls, Mark; Cruijsen, Elwin A. W. van der; Folkers, Gert E.; Baldus, Marc

2015-01-01

Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR

High-protein diet selectively reduces fat mass and improves glucose tolerance in Western-type diet-induced obese rats

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Stengel, Andreas; Goebel-Stengel, Miriam; Wang, Lixin; Hu, Eugenia; Karasawa, Hiroshi; Pisegna, Joseph R.

2013-01-01

Obesity is an increasing health problem. Because drug treatments are limited, diets remain popular. High-protein diets (HPD) reduce body weight (BW), although the mechanisms are unclear. We investigated physiological mechanisms altered by switching diet induced obesity (DIO) rats from Western-type diet (WTD) to HPD. Male rats were fed standard (SD) or WTD (45% calories from fat). After developing DIO (50% of rats), they were switched to SD (15% calories from protein) or HPD (52% calories from protein) for up to 4 weeks. Food intake (FI), BW, body composition, glucose tolerance, insulin sensitivity, and intestinal hormone plasma levels were monitored. Rats fed WTD showed an increased FI and had a 25% greater BW gain after 9 wk compared with SD (P Diet-induced obese rats switched from WTD to HPD reduced daily FI by 30% on day 1, which lasted to day 9 (−9%) and decreased BW during the 2-wk period compared with SD/SD (P < 0.05). During these 2 wk, WTD/HPD rats lost 72% more fat mass than WTD/SD (P < 0.05), whereas lean mass was unaltered. WTD/HPD rats had lower blood glucose than WTD/SD at 30 min postglucose gavage (P < 0.05). The increase of pancreatic polypeptide and peptide YY during the 2-h dark-phase feeding was higher in WTD/HPD compared with WTD/SD (P < 0.05). These data indicate that HPD reduces BW in WTD rats, which may be related to decreased FI and the selective reduction of fat mass accompanied by improved glucose tolerance, suggesting relevant benefits of HPD in the treatment of obesity. PMID:23883680

Cyanide-induced death of dopaminergic cells is mediated by uncoupling protein-2 up-regulation and reduced Bcl-2 expression

International Nuclear Information System (INIS)

Zhang, X.; Li, L.; Zhang, L.; Borowitz, J.L.; Isom, G.E.

2009-01-01

Cyanide is a potent inhibitor of mitochondrial oxidative metabolism and produces mitochondria-mediated death of dopaminergic neurons and sublethal intoxications that are associated with a Parkinson-like syndrome. Cyanide toxicity is enhanced when mitochondrial uncoupling is stimulated following up-regulation of uncoupling protein-2 (UCP-2). In this study, the role of a pro-survival protein, Bcl-2, in cyanide-mediated cell death was determined in a rat dopaminergic immortalized mesencephalic cell line (N27 cells). Following pharmacological up-regulation of UCP-2 by treatment with Wy14,643, cyanide reduced cellular Bcl-2 expression by increasing proteasomal degradation of the protein. The increased turnover of Bcl-2 was mediated by an increase of oxidative stress following UCP-2 up-regulation. The oxidative stress involved depletion of mitochondrial glutathione (mtGSH) and increased H 2 O 2 generation. Repletion of mtGSH by loading cells with glutathione ethyl ester reduced H 2 O 2 generation and in turn blocked the cyanide-induced decrease of Bcl-2. To determine if UCP-2 mediated the response, RNAi knock down was conducted. The RNAi decreased cyanide-induced depletion of mtGSH, reduced H 2 O 2 accumulation, and inhibited down-regulation of Bcl-2, thus blocking cell death. To confirm the role of Bcl-2 down-regulation in the cell death, it was shown that over-expression of Bcl-2 by cDNA transfection attenuated the enhancement of cyanide toxicity after UCP-2 up-regulation. It was concluded that UCP-2 up-regulation sensitizes cells to cyanide by increasing cellular oxidative stress, leading to an increase of Bcl-2 degradation. Then the reduced Bcl-2 levels sensitize the cells to cyanide-mediated cell death.

Rising atmospheric CO2 is reducing the protein concentration of a floral pollen source essential for North American bees.

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Ziska, Lewis H; Pettis, Jeffery S; Edwards, Joan; Hancock, Jillian E; Tomecek, Martha B; Clark, Andrew; Dukes, Jeffrey S; Loladze, Irakli; Polley, H Wayne

2016-04-13

At present, there is substantive evidence that the nutritional content of agriculturally important food crops will decrease in response to rising levels of atmospheric carbon dioxide, Ca However, whether Ca-induced declines in nutritional quality are also occurring for pollinator food sources is unknown. Flowering late in the season, goldenrod (Solidago spp.) pollen is a widely available autumnal food source commonly acknowledged by apiarists to be essential to native bee (e.g. Bombus spp.) and honeybee (Apis mellifera) health and winter survival. Using floral collections obtained from the Smithsonian Natural History Museum, we quantified Ca-induced temporal changes in pollen protein concentration of Canada goldenrod (Solidago canadensis), the most wide spread Solidago taxon, from hundreds of samples collected throughout the USA and southern Canada over the period 1842-2014 (i.e. a Ca from approx. 280 to 398 ppm). In addition, we conducted a 2 year in situtrial of S. Canadensis populations grown along a continuous Ca gradient from approximately 280 to 500 ppm. The historical data indicated a strong significant correlation between recent increases in Ca and reductions in pollen protein concentration (r(2)= 0.81). Experimental data confirmed this decrease in pollen protein concentration, and indicated that it would be ongoing as Ca continues to rise in the near term, i.e. to 500 ppm (r(2)= 0.88). While additional data are needed to quantify the subsequent effects of reduced protein concentration for Canada goldenrod on bee health and population stability, these results are the first to indicate that increasing Ca can reduce protein content of a floral pollen source widely used by North American bees. © 2016 The Author(s).

Subcellular localization of proteins in the anaerobic sulfate reducer Desulfovibrio vulgaris via SNAP-tag labeling and photoconversion

Energy Technology Data Exchange (ETDEWEB)

Gorur, A.; Leung, C. M.; Jorgens, D.; Tauscher, A.; Remis, J. P.; Ball, D. A.; Chhabra, S.; Fok, V.; Geller, J. T.; Singer, M.; Hazen, T. C.; Juba, T.; Elias, D.; Wall, J.; Biggin, M.; Downing, K. H.; Auer, M.

2010-06-01

Systems Biology studies the temporal and spatial 3D distribution of macromolecular complexes with the aim that such knowledge will allow more accurate modeling of biological function and will allow mathematical prediction of cellular behavior. However, in order to accomplish accurate modeling precise knowledge of spatial 3D organization and distribution inside cells is necessary. And while a number of macromolecular complexes may be identified by its 3D structure and molecular characteristics alone, the overwhelming number of proteins will need to be localized using a reporter tag. GFP and its derivatives (XFPs) have been traditionally employed for subcelllar localization using photoconversion approaches, but this approach cannot be taken for obligate anaerobic bacteria, where the intolerance towards oxygen prevents XFP approaches. As part of the GTL-funded PCAP project (now ENIGMA) genetic tools have been developed for the anaerobe sulfate reducer Desulfovibrio vulgaris that allow the high-throughput generation of tagged-protein mutant strains, with a focus on the commercially available SNAP-tag cell system (New England Biolabs, Ipswich, MA), which is based on a modified O6-alkylguanine-DNA alkyltransferase (AGT) tag, that has a dead-end reaction with a modified O6-benzylguanine (BG) derivative and has been shown to function under anaerobic conditions. After initial challenges with respect to variability, robustness and specificity of the labeling signal we have optimized the labeling. Over the last year, as a result of the optimized labeling protocol, we now obtain robust labeling of 20 out of 31 SNAP strains. Labeling for 13 strains were confirmed at least five times. We have also successfully performed photoconversion on 5 of these 13 strains, with distinct labeling patterns for different strains. For example, DsrC robustly localizes to the periplasmic portion of the inner membrane, where as a DNA-binding protein localizes to the center of the cell, where the

Morphine Reduces Myocardial Infarct Size via Heat Shock Protein 90 in Rodents

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Bryce A. Small

2015-01-01

Full Text Available Opioids reduce injury from myocardial ischemia-reperfusion in humans. In experimental models, this mechanism involves GSK3β inhibition. HSP90 regulates mitochondrial protein import, with GSK3β inhibition increasing HSP90 mitochondrial content. Therefore, we determined whether morphine-induced cardioprotection is mediated by HSP90 and if the protective effect is downstream of GSK3β inhibition. Male Sprague-Dawley rats, aged 8–10 weeks, were subjected to an in vivo myocardial ischemia-reperfusion injury protocol involving 30 minutes of ischemia followed by 2 hours of reperfusion. Hemodynamics were continually monitored and myocardial infarct size determined. Rats received morphine (0.3 mg/kg, the GSK3β inhibitor, SB216763 (0.6 mg/kg, or saline, 10 minutes prior to ischemia. Some rats received selective HSP90 inhibitors, radicicol (0.3 mg/kg, or deoxyspergualin (DSG, 0.6 mg/kg alone or 5 minutes prior to morphine or SB216763. Morphine reduced myocardial infarct size when compared to control (42 ± 2% versus 60 ± 1%. This protection was abolished by prior treatment of radicicol or DSG (59 ± 1%, 56 ± 2%. GSK3β inhibition also reduced myocardial infarct size (41 ± 2% with HSP90 inhibition by radicicol or DSG partially inhibiting SB216763-induced infarct size reduction (54 ± 3%, 47 ± 1%, resp.. These data suggest that opioid-induced cardioprotection is mediated by HSP90. Part of this protection afforded by HSP90 is downstream of GSK3β, potentially via the HSP-TOM mitochondrial import pathway.

Glutamine reduces myocardial cell apoptosis in a rat model of sepsis by promoting expression of heat shock protein 90.

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Li, Wanxia; Tao, Shaoyu; Wu, Qinghua; Wu, Tao; Tao, Ran; Fan, Jun

2017-12-01

Myocardial cell injury and cardiac myocyte apoptosis are associated with sepsis. Glutamine (Gln) has been reported to repair myocardial cell injury. The aim of this study was to explore the role of Gln on cardiac myocytes in a cecal ligation and puncture (CLP) model of sepsis in Wistar rats. Following induction of sepsis in a CLP rat model, viral encoding heat shock protein 90 (Hsp90) gene and Hsp90dsDNA were designed to express and knockdown Hsp90, respectively. Rat cardiac tissues were examined histologically, and apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The expression of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein, Hsp90, p53 upregulated modulator of apoptosis, and p53 was measured by western blotting and real-time polymerase chain reaction. Caspase-3, caspase-8, and caspase-9 were detected by enzyme-linked immunosorbent assay. Rat cardiac myocyte damage induced by CLP was reduced by Gln treatment and Hsp90 overexpression, and these changes were reversed by Hsp90 knockdown. Bcl-2 expression, Bcl-2-associated X protein, p53, p53 upregulated modulator of apoptosis, caspase-8, caspase-9, and caspase-3 activities were significantly upregulated in the CLP model, which were reduced by Gln treatment and Hsp90 overexpression. Gln reduced apoptosis of cardiac myocytes in a rat model of sepsis, by promoting Hsp90 expression. Further studies are needed to determine the possible therapeutic action of Gln in sepsis in human tissue. Copyright © 2017 Elsevier Inc. All rights reserved.

Sensory neurons do not induce motor neuron loss in a human stem cell model of spinal muscular atrophy.

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Schwab, Andrew J; Ebert, Allison D

2014-01-01

Spinal muscular atrophy (SMA) is an autosomal recessive disorder leading to paralysis and early death due to reduced SMN protein. It is unclear why there is such a profound motor neuron loss, but recent evidence from fly and mouse studies indicate that cells comprising the whole sensory-motor circuit may contribute to motor neuron dysfunction and loss. Here, we used induced pluripotent stem cells derived from SMA patients to test whether sensory neurons directly contribute to motor neuron loss. We generated sensory neurons from SMA induced pluripotent stem cells and found no difference in neuron generation or survival, although there was a reduced calcium response to depolarizing stimuli. Using co-culture of SMA induced pluripotent stem cell derived sensory neurons with control induced pluripotent stem cell derived motor neurons, we found no significant reduction in motor neuron number or glutamate transporter boutons on motor neuron cell bodies or neurites. We conclude that SMA sensory neurons do not overtly contribute to motor neuron loss in this human stem cell system.

Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon

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Luftig Ronald

2007-09-01

Full Text Available Abstract Background Alpha interferon in combination with ribavirin is the standard therapy for hepatitis C virus infection. Unfortunately, a significant number of patients fail to eradicate their infection with this regimen. The mechanisms of IFN-resistance are unclear. The aim of this study was to determine the contribution of host cell factors to the mechanisms of interferon resistance using replicon cell lines. Results HCV replicons with high and low activation of the IFN-promoter were cultured for a prolonged period of time in the presence of interferon-alpha (IFN-alpha2b. Stable replicon cell lines with resistant phenotype were isolated and characterized by their ability to continue viral replication in the presence of IFN-alpha. Interferon resistant cell colonies developed only in replicons having lower activation of the IFN promoter and no resistant colonies arose from replicons that exhibit higher activation of the IFN promoter. Individual cell clones were isolated and nine IFN resistant cell lines were established. HCV RNA and protein levels in these cells were not altered by IFN- alpha2b. Reduced signaling and IFN-resistant phenotype was found in all Huh-7 cell lines even after eliminating HCV, suggesting that cellular factors are involved. Resistant phenotype in the replicons is not due to lack of interferon receptor expression. All the cell lines show defect in the JAK-STAT signaling and phosphorylation of STAT 1 and STAT 2 proteins were strongly inhibited due to reduced expression of Tyk2 and Jak-1 protein. Conclusion This in vitro study provides evidence that altered expression of the Jak-Stat signaling proteins can cause IFN resistance using HCV replicon cell clones.

Structural changes in Mcm5 protein bypass Cdc7-Dbf4 function and reduce replication origin efficiency in Saccharomyces cerevisiae.

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Hoang, Margaret L; Leon, Ronald P; Pessoa-Brandao, Luis; Hunt, Sonia; Raghuraman, M K; Fangman, Walton L; Brewer, Bonita J; Sclafani, Robert A

2007-11-01

Eukaryotic chromosomal replication is a complicated process with many origins firing at different efficiencies and times during S phase. Prereplication complexes are assembled on all origins in G(1) phase, and yet only a subset of complexes is activated during S phase by DDK (for Dbf4-dependent kinase) (Cdc7-Dbf4). The yeast mcm5-bob1 (P83L) mutation bypasses DDK but results in reduced intrinsic firing efficiency at 11 endogenous origins and at origins located on minichromosomes. Origin efficiency may result from Mcm5 protein assuming an altered conformation, as predicted from the atomic structure of an archaeal MCM (for minichromosome maintenance) homologue. Similarly, an intragenic mutation in a residue predicted to interact with P83L suppresses the mcm5-bob1 bypass phenotype. We propose DDK phosphorylation of the MCM complex normally results in a single, highly active conformation of Mcm5, whereas the mcm5-bob1 mutation produces a number of conformations, only one of which is permissive for origin activation. Random adoption of these alternate states by the mcm5-bob1 protein can explain both how origin firing occurs independently of DDK and why origin efficiency is reduced. Because similar mutations in mcm2 and mcm4 cannot bypass DDK, Mcm5 protein may be a unique Mcm protein that is the final target of DDK regulation.

Elevated CO2 plus chronic warming reduce nitrogen uptake and levels or activities of nitrogen-uptake and -assimilatory proteins in tomato roots.

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Jayawardena, Dileepa M; Heckathorn, Scott A; Bista, Deepesh R; Mishra, Sasmita; Boldt, Jennifer K; Krause, Charles R

2017-03-01

Atmospheric CO 2 enrichment is expected to often benefit plant growth, despite causing global warming and nitrogen (N) dilution in plants. Most plants primarily procure N as inorganic nitrate (NO 3 - ) or ammonium (NH 4 + ), using membrane-localized transport proteins in roots, which are key targets for improving N use. Although interactive effects of elevated CO 2 , chronic warming and N form on N relations are expected, these have not been studied. In this study, tomato (Solanum lycopersicum) plants were grown at two levels of CO 2 (400 or 700 ppm) and two temperature regimes (30 or 37°C), with NO 3 - or NH 4 + as the N source. Elevated CO 2 plus chronic warming severely inhibited plant growth, regardless of N form, while individually they had smaller effects on growth. Although %N in roots was similar among all treatments, elevated CO 2 plus warming decreased (1) N-uptake rate by roots, (2) total protein concentration in roots, indicating an inhibition of N assimilation and (3) shoot %N, indicating a potential inhibition of N translocation from roots to shoots. Under elevated CO 2 plus warming, reduced NO 3 - -uptake rate per g root was correlated with a decrease in the concentration of NO 3 - -uptake proteins per g root, reduced NH 4 + uptake was correlated with decreased activity of NH 4 + -uptake proteins and reduced N assimilation was correlated with decreased concentration of N-assimilatory proteins. These results indicate that elevated CO 2 and chronic warming can act synergistically to decrease plant N uptake and assimilation; hence, future global warming may decrease both plant growth and food quality (%N). © 2016 Scandinavian Plant Physiology Society.

Reduced homeobox protein MSX1 in human endometrial tissue is linked to infertility.

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Bolnick, Alan D; Bolnick, Jay M; Kilburn, Brian A; Stewart, Tamika; Oakes, Jonathan; Rodriguez-Kovacs, Javier; Kohan-Ghadr, Hamid-Reza; Dai, Jing; Diamond, Michael P; Hirota, Yasushi; Drewlo, Sascha; Dey, Sudhansu K; Armant, D Randall

2016-09-01

Is protein expression of the muscle segment homeobox gene family member MSX1 altered in the human secretory endometrium by cell type, developmental stage or fertility? MSX1 protein levels, normally elevated in the secretory phase endometrium, were significantly reduced in endometrial biopsies obtained from women of infertile couples. Molecular changes in the endometrium are important for fertility in both animals and humans. Msx1 is expressed in the preimplantation mouse uterus and regulates uterine receptivity for implantation. The MSX protein persists a short time, after its message has been down-regulated. Microarray analysis of the human endometrium reveals a similar pattern of MSX1 mRNA expression that peaks before the receptive period, with depressed expression at implantation. Targeted deletion of uterine Msx1 and Msx2 in mice prevents the loss of epithelial cell polarity during implantation and causes infertility. MSX1 mRNA and cell type-specific levels of MSX1 protein were quantified from two retrospective cohorts during the human endometrial cycle. MSX1 protein expression patterns were compared between fertile and infertile couples. Selected samples were dual-labeled by immunofluorescence microscopy to localize E-cadherin and β-catenin in epithelial cells. MSX1 mRNA was quantified by PCR in endometrium from hysterectomies (n = 14) determined by endometrial dating to be in the late-proliferative (cycle days 10-13), early-secretory (cycle days 14-19) or mid-secretory (cycle days 20-24) phase. MSX1 protein was localized using high-throughput, semi-quantitative immunohistochemistry with sectioned endometrial biopsy tissues from fertile (n = 89) and infertile (n = 89) couples. Image analysis measured stain intensity specifically within the luminal epithelium, glands and stroma during the early-, mid- and late- (cycle days 25-28) secretory phases. MSX1 transcript increased 5-fold (P MSX1 protein displayed strong nuclear localization in the luminal epithelium

PROTEIN L-ISOASPARTYL METHYLTRANSFERASE2 is differentially expressed in chickpea and enhances seed vigor and longevity by reducing abnormal isoaspartyl accumulation predominantly in seed nuclear proteins.

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Verma, Pooja; Kaur, Harmeet; Petla, Bhanu Prakash; Rao, Venkateswara; Saxena, Saurabh C; Majee, Manoj

2013-03-01

PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT) is a widely distributed protein-repairing enzyme that catalyzes the conversion of abnormal l-isoaspartyl residues in spontaneously damaged proteins to normal aspartyl residues. This enzyme is encoded by two divergent genes (PIMT1 and PIMT2) in plants, unlike many other organisms. While the biological role of PIMT1 has been elucidated, the role and significance of the PIMT2 gene in plants is not well defined. Here, we isolated the PIMT2 gene (CaPIMT2) from chickpea (Cicer arietinum), which exhibits a significant increase in isoaspartyl residues in seed proteins coupled with reduced germination vigor under artificial aging conditions. The CaPIMT2 gene is found to be highly divergent and encodes two possible isoforms (CaPIMT2 and CaPIMT2') differing by two amino acids in the region I catalytic domain through alternative splicing. Unlike CaPIMT1, both isoforms possess a unique 56-amino acid amino terminus and exhibit similar yet distinct enzymatic properties. Expression analysis revealed that CaPIMT2 is differentially regulated by stresses and abscisic acid. Confocal visualization of stably expressed green fluorescent protein-fused PIMT proteins and cell fractionation-immunoblot analysis revealed that apart from the plasma membrane, both CaPIMT2 isoforms localize predominantly in the nucleus, while CaPIMT1 localizes in the cytosol. Remarkably, CaPIMT2 enhances seed vigor and longevity by repairing abnormal isoaspartyl residues predominantly in nuclear proteins upon seed-specific expression in Arabidopsis (Arabidopsis thaliana), while CaPIMT1 enhances seed vigor and longevity by repairing such abnormal proteins mainly in the cytosolic fraction. Together, our data suggest that CaPIMT2 has most likely evolved through gene duplication, followed by subfunctionalization to specialize in repairing the nuclear proteome.

Ursodeoxycholic acid pretreatment reduces oral bioavailability of the multiple drug resistance-associated protein 2 substrate baicalin in rats.

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Wu, Tao; Li, Xi-Ping; Xu, Yan-Jiao; Du, Guang; Liu, Dong

2013-11-01

Baicalin is a major bioactive component of Scutellaria baicalensis and a substrate of multiple drug resistance-associated protein 2. Expression of multiple drug resistance-associated protein 2 is regulated by NF-E2-related factor 2. The aim of this study was to explore whether ursodeoxycholic acid, an NF-E2-related factor 2 activator, could influence the oral bioavailability of baicalin. A single dose of baicalin (200 mg/kg) was given orally to rats pretreated with ursodeoxycholic acid (75 mg/kg and 150 mg/kg, per day, intragastrically) or normal saline (per day, intragastrically) for six consecutive days. The plasma concentration of baicalin was measured with the HPLC method. The result indicated that the oral bioavailability of baicalin was significantly and dose-dependently reduced in rats pretreated with ursodeoxycholic acid. Compared with control rats, the mean area under concentration-time curve of baicalin was reduced from 13.25 ± 0.24 mg/L h to 7.62 ± 0.15 mg/L h and 4.97 ± 0.21 mg/L h, and the C(max) value was decreased from 1.31 ± 0.03 mg/L to 0.62 ± 0.05 mg/L and 0.36 ± 0.04 mg/L in rats pretreated with ursodeoxycholic acid at doses of 75 mg/kg and 150 mg/kg, respectively, for six consecutive days. Hence, ursodeoxycholic acid treatment reduced the oral bioavailability of baicalin in rats, probably due to the enhanced efflux of baicalin from the intestine and liver by multiple drug resistance-associated protein 2. Georg Thieme Verlag KG Stuttgart · New York.

Phase II open label study of valproic acid in spinal muscular atrophy.

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Kathryn J Swoboda

Full Text Available Preliminary in vitro and in vivo studies with valproic acid (VPA in cell lines and patients with spinal muscular atrophy (SMA demonstrate increased expression of SMN, supporting the possibility of therapeutic benefit. We performed an open label trial of VPA in 42 subjects with SMA to assess safety and explore potential outcome measures to help guide design of future controlled clinical trials. Subjects included 2 SMA type I ages 2-3 years, 29 SMA type II ages 2-14 years and 11 type III ages 2-31 years, recruited from a natural history study. VPA was well-tolerated and without evident hepatotoxicity. Carnitine depletion was frequent and temporally associated with increased weakness in two subjects. Exploratory outcome measures included assessment of gross motor function via the modified Hammersmith Functional Motor Scale (MHFMS, electrophysiologic measures of innervation including maximum ulnar compound muscle action potential (CMAP amplitudes and motor unit number estimation (MUNE, body composition and bone density via dual-energy X-ray absorptiometry (DEXA, and quantitative blood SMN mRNA levels. Clear decline in motor function occurred in several subjects in association with weight gain; mean fat mass increased without a corresponding increase in lean mass. We observed an increased mean score on the MHFMS scale in 27 subjects with SMA type II (pSMN levels were unchanged and Delta7SMN levels were significantly reduced for 2 of 3 treatment visits. In contrast, bone mineral density (p

Alteration in intrinsic and extrinsic functional connectivity of resting state networks associated with subclinical hypothyroid.

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Kumar, Mukesh; Modi, Shilpi; Rana, Poonam; Kumar, Pawan; Kanwar, Ratnesh; Sekhri, Tarun; D'souza, Maria; Khushu, Subash

2018-03-05

Subclinical hypothyroidism (SCH) is characterized by mild elevation of thyroid stimulating hormone (TSH) (range 5-10 μIU/ml) and normal free triiodothyronine (FT3) and free thyroxine (FT4). The cognitive function impairment is well known in thyroid disorders such as hypothyroidism and hyperthyroidism, but little is known about deficits in brain functions in SCH subjects. Also, whether hormone-replacement treatment is necessary or not in SCH subjects is still debatable. In order to have an insight into the cognition of SCH subjects, intrinsic and extrinsic functional connectivity (FC) of the resting state networks (RSNs) was studied. For resting state data analysis we used an unbiased, data-driven approach based on Independent Component Analysis (ICA) and dual-regression that can emphasize widespread changes in FC without restricting to a set of predefined seeds. 28 SCH subjects and 28 matched healthy controls (HC) participated in the study. RSN analysis showed significantly decreased intrinsic FC in somato-motor network (SMN) and right fronto-parietal attention network (RAN) and increased intrinsic FC in default mode network (DMN) in SCH subjects as compared to control subjects. The reduced intrinsic FC in the SMN and RAN suggests neuro-cognitive alterations in SCH subjects in the corresponding functions which were also evident from the deficit in the neuropsychological performance of the SCH subjects on behavioural tests such as digit span, delayed recall, visual retention, recognition, Bender Gestalt and Mini-Mental State Examination (MMSE). We also found a significant reduction in extrinsic network FC between DMN and RAN; SMN and posterior default mode network (PDMN); and increased extrinsic FC between SMN and anterior default mode network (ADMN) in SCH subjects as compared to controls. An altered extrinsic FC in SCH suggests functional reorganization in response to neurological disruption. The partial correlation analysis between intrinsic and extrinsic RSNs

Structural analogs of human insulin-like growth factor I with reduced affinity for serum binding proteins and the type 2 insulin-like growth factor receptor

International Nuclear Information System (INIS)

Bayne, M.L.; Applebaum, J.; Chicchi, G.G.; Hayes, N.S.; Green, B.G.; Cascieri, M.A.

1988-01-01

Four structural analogs of human insulin-like growth factor I (hIGF-I) have been prepared by site-directed mutagenesis of a synthetic IGF-I gene and subsequent expression and purification of the mutant protein from the conditioned media of transformed yeast. [Phe -1 , Val 1 , Asn 2 , Gln 3 , His 4 , Ser 8 , His 9 , Glu 12 , Tyr 15 , Leu 16 ]IGF-I (B-chain mutant), in which the first 16 amino acids of hIGF-I were replaced with the first 17 amino acids of the B-chain of insulin, has >1000-, 100-, and 2-fold reduced potency for human serum binding proteins, the rat liver type 2 IGF receptor, and the human placental type 1 IGF receptor, respectively. The B-chain mutant also has 4-fold increased affinity for the human placental insulin receptor. [Gln 3 , Ala 4 ] IGF-I has 4-fold reduced affinity for human serum binding proteins, but is equipotent to hIGF-I at the types 1 and 2 IGF and insulin receptors. [Tyr 15 , Leu 16 ] IGH-I has 4-fold reduced affinity for human serum binding proteins and 10-fold increased affinity for the insulin receptor. The peptide in which these four-point mutations are combined, [Gln 3 , Ala 4 , Tyr 15 ,Leu 16 ]IGF-I, has 600-fold reduced affinity for the serum binding proteins. All four of these mutants stimulate DNA synthesis in the rat vascular smooth muscle cell line A10 with potencies reflecting their potency at the type 1 IGF receptor. These studies identify some of the domains of hIGF-I which are responsible for maintaining high affinity binding with the serum binding protein and the type 2 IGF receptor. In addition, These peptides will be useful in defining the role of the type 2 IGF receptor and serum binding proteins in the physiological actions of hIGF-I

Second malignancies in children: the usual suspects?

Energy Technology Data Exchange (ETDEWEB)

Moppett, John; Oakhill, Anthony E-mail: anthony.oakhill@nildram.co.uk; Duncan, Andrew W

2001-06-01

The aim of this article is to provide an up to date review of second malignant neoplasms (SMN's) following treatment for childhood cancer, referring to their incidence, the role of genetic factors, and how the primary malignancy and treatment received influence the type, site and prognosis of SMN's. The role of genetic factors will be discussed as far as they impact upon a predisposition to later development of SMN's. The primary malignancies that have important associations with SMN's will then be discussed, in particular Hodgkin's disease, retinoblastoma and acute lymphoblastic leukaemia. The important second malignancies will be highlighted, including tumours of the CNS and thyroid, osteosarcoma, secondary acute myeloid leukaemia and melanoma. Emphasis will be put upon identifying which patients are most likely to suffer from these tumours. An important part of the article are case histories. These are provided in combination with illustrations as a useful adjunct to the text, with a particular emphasis on radiological features, diagnosis and screening. Finally, the important but different roles of causal agents, in particular chemotherapy and radiotherapy are highlighted.

Mycobacterium tuberculosis PPE18 Protein Reduces Inflammation and Increases Survival in Animal Model of Sepsis.

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Ahmed, Asma; Dolasia, Komal; Mukhopadhyay, Sangita

2018-04-18

Mycobacterium tuberculosis PPE18 is a member of the PPE family. Previous studies have shown that recombinant PPE18 (rPPE18) protein binds to TLR2 and triggers a signaling cascade which reduces levels of TNF-α and IL-12, and increases IL-10 in macrophages. Because TNF-α is a major mediator of the pathophysiology of sepsis and blocking inflammation is a possible line of therapy in such circumstances, we tested the efficacy of rPPE18 in reducing symptoms of sepsis in a mouse model of Escherichia coli- induced septic peritonitis. rPPE18 significantly decreased levels of serum TNF-α, IL-1β, IL-6, and IL-12 and reduced organ damage in mice injected i.p. with high doses of E. coli Peritoneal cells isolated from rPPE18-treated mice had characteristics of M2 macrophages which are protective in excessive inflammation. Additionally, rPPE18 inhibited disseminated intravascular coagulation, which can cause organ damage resulting in death. rPPE18 was able to reduce sepsis-induced mortality when given prophylactically or therapeutically. Additionally, in a mouse model of cecal ligation and puncture-induced sepsis, rPPE18 reduced TNF-α, alanine transaminase, and creatinine, attenuated organ damage, prevented depletion of monocytes and lymphocytes, and improved survival. Our studies show that rPPE18 has potent anti-inflammatory properties and can serve as a novel therapeutic to control sepsis. Copyright © 2018 by The American Association of Immunologists, Inc.

Absence of residual structure in the intrinsically disordered regulatory protein CP12 in its reduced state

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Launay, Hélène; Barré, Patrick [Laboratory of integrative Structural and Chemical Biology (iSCB), Centre de Recherche en Cancérologie de Marseille (CRCM), CNRS UMR 7258, INSERM U 1068, Institut Paoli-Calmettes, Aix-Marseille Universités, Marseille 13009 (France); Puppo, Carine [Aix-Marseille Université, Centre National de la Recherche Scientifique, UMR 7281, Laboratoire de Bioénergétique et Ingénierie des Protéines, 31 Chemin Joseph Aiguier, 13402, Marseille Cedex 20 (France); Manneville, Stéphanie [Laboratory of integrative Structural and Chemical Biology (iSCB), Centre de Recherche en Cancérologie de Marseille (CRCM), CNRS UMR 7258, INSERM U 1068, Institut Paoli-Calmettes, Aix-Marseille Universités, Marseille 13009 (France); Gontero, Brigitte [Aix-Marseille Université, Centre National de la Recherche Scientifique, UMR 7281, Laboratoire de Bioénergétique et Ingénierie des Protéines, 31 Chemin Joseph Aiguier, 13402, Marseille Cedex 20 (France); Receveur-Bréchot, Véronique, E-mail: veronique.brechot@inserm.fr [Laboratory of integrative Structural and Chemical Biology (iSCB), Centre de Recherche en Cancérologie de Marseille (CRCM), CNRS UMR 7258, INSERM U 1068, Institut Paoli-Calmettes, Aix-Marseille Universités, Marseille 13009 (France)

2016-08-12

The redox switch protein CP12 is a key player of the regulation of the Benson–Calvin cycle. Its oxidation state is controlled by the formation/dissociation of two intramolecular disulphide bridges during the day/night cycle. CP12 was known to be globally intrinsically disordered on a large scale in its reduced state, while being partly ordered in the oxidised state. By combining Nuclear Magnetic Resonance and Small Angle X-ray Scattering experiments, we showed that, contrary to secondary structure or disorder predictions, reduced CP12 is fully disordered, with no transient or local residual structure likely to be precursor of the structures identified in the oxidised active state and/or in the bound state with GAPDH or PRK. These results highlight the diversity of the mechanisms of regulation of conditionally disordered redox switches, and question the stability of oxidised CP12 scaffold. - Highlights: • CP12 is predicted to form two helices in its N-terminal sequence. • Reduced CP12 is disordered as a random coil according to SAXS. • Limited or no transient structures are observed in reduced CP12 by NMR.

Absence of residual structure in the intrinsically disordered regulatory protein CP12 in its reduced state

International Nuclear Information System (INIS)

Launay, Hélène; Barré, Patrick; Puppo, Carine; Manneville, Stéphanie; Gontero, Brigitte; Receveur-Bréchot, Véronique

2016-01-01

The redox switch protein CP12 is a key player of the regulation of the Benson–Calvin cycle. Its oxidation state is controlled by the formation/dissociation of two intramolecular disulphide bridges during the day/night cycle. CP12 was known to be globally intrinsically disordered on a large scale in its reduced state, while being partly ordered in the oxidised state. By combining Nuclear Magnetic Resonance and Small Angle X-ray Scattering experiments, we showed that, contrary to secondary structure or disorder predictions, reduced CP12 is fully disordered, with no transient or local residual structure likely to be precursor of the structures identified in the oxidised active state and/or in the bound state with GAPDH or PRK. These results highlight the diversity of the mechanisms of regulation of conditionally disordered redox switches, and question the stability of oxidised CP12 scaffold. - Highlights: • CP12 is predicted to form two helices in its N-terminal sequence. • Reduced CP12 is disordered as a random coil according to SAXS. • Limited or no transient structures are observed in reduced CP12 by NMR.

Moderate, but not vigorous, intensity exercise training reduces C-reactive protein.

Science.gov (United States)

Fedewa, Michael V; Hathaway, Elizabeth D; Higgins, Simon; Forehand, Ronald L; Schmidt, Michael D; Evans, Ellen M

2018-06-01

Sprint interval cycle training is a contemporary popular mode of training but its relative efficacy, under conditions of matched energy expenditure, to reduce risk factors for cardiometabolic disease is incompletely characterised, especially in young women. The purpose of this investigation was to determine the relative efficacy of six weeks of moderate-intensity cycling (MOD-C) and vigorous sprint-interval cycling (VIG-SIC) on lipid profile, insulin (INS) and insulin resistance using the homeostatic model assessment (HOMA-IR) and C-reactive protein (CRP) in inactive, overweight/obese (OW/OB) young women. Participants (BMI ≥25 kg/m 2 , waist circumference ≥88 cm) were randomly assigned to MOD-C (20-30 min at 60-70% of heart rate reserve(HRR)) or VIG-SIC (5-7 repeated bouts 30-second maximal effort sprints, followed by four minutes of active recovery) supervised training three days/week for six weeks, with each group matched on energy expenditure. Adiposity (%Fat) was measured using dual x-ray absorptiometry. Forty-four participants (20.4 ± 1.6 years, 65.9% Caucasian, 29.8 ± 4.1 kg/m 2 ) were included in the analysis. The improvement in CRP observed in the MOD-C group was larger than the VIG-C group (p = .034). Overall, high-density lipoprotein (HDL-C) and low-density lipoprotein (LDL-C) levels improved following training (p  .05). These results indicate MOD-C training may be more effective in reducing CRP than VIG-SIC.

A Phospholipid-Protein Complex from Krill with Antioxidative and Immunomodulating Properties Reduced Plasma Triacylglycerol and Hepatic Lipogenesis in Rats

Directory of Open Access Journals (Sweden)

Marie S. Ramsvik

2015-07-01

Full Text Available Dietary intake of marine omega-3 polyunsaturated fatty acids (n-3 PUFAs can change the plasma profile from atherogenic to cardioprotective. In addition, there is growing evidence that proteins of marine origin may have health benefits. We investigated a phospholipid-protein complex (PPC from krill that is hypothesized to influence lipid metabolism, inflammation, and redox status. Male Wistar rats were fed a control diet (2% soy oil, 8% lard, 20% casein, or diets where corresponding amounts of casein and lard were replaced with PPC at 3%, 6%, or 11% (wt %, for four weeks. Dietary supplementation with PPC resulted in significantly lower levels of plasma triacylglycerols in the 11% PPC-fed group, probably due to reduced hepatic lipogenesis. Plasma cholesterol levels were also reduced at the highest dose of PPC. In addition, the plasma and liver content of n-3 PUFAs increased while n-6 PUFAs decreased. This was associated with increased total antioxidant capacity in plasma and increased liver gene expression of mitochondrial superoxide dismutase (Sod2. Finally, a reduced plasma level of the inflammatory mediator interleukin-2 (IL-2 was detected in the PPC-fed animals. The present data show that PPC has lipid-lowering effects in rats, and may modulate risk factors related to cardiovascular disease progression.

A carbohydrate-reduced high-protein diet acutely decreases postprandial and diurnal glucose excursions in type 2 diabetes patients

DEFF Research Database (Denmark)

Samkani, Amirsalar; Skytte, Mads J; Kandel, Daniel

2018-01-01

with T2DM treated with metformin only, fourteen male, with a median age of 65 (43-70) years, HbA1c of 6·5 % (47 mmol/l) (5·5-8·3 % (37-67 mmol/l)) and a BMI of 30 (sd 4·4) kg/m2 participated in the randomised, cross-over study. A carbohydrate-reduced high-protein (CRHP) diet was compared with an iso......The aim of the study was to assess whether a simple substitution of carbohydrate in the conventionally recommended diet with protein and fat would result in a clinically meaningful reduction in postprandial hyperglycaemia in subjects with type 2 diabetes mellitus (T2DM). In all, sixteen subjects......-energetic conventional diabetes (CD) diet. Macronutrient contents of the CRHP/CD diets consisted of 31/54 % energy from carbohydrate, 29/16 % energy from protein and 40/30 % energy from fat, respectively. Each diet was consumed on 2 consecutive days in a randomised order. Postprandial glycaemia, pancreatic and gut...

Protective Immunity and Reduced Renal Colonization Induced by Vaccines Containing Recombinant Leptospira interrogans Outer Membrane Proteins and Flagellin Adjuvant

Science.gov (United States)

Monaris, D.; Sbrogio-Almeida, M. E.; Dib, C. C.; Canhamero, T. A.; Souza, G. O.; Vasconcellos, S. A.; Ferreira, L. C. S.

2015-01-01

Leptospirosis is a global zoonotic disease caused by different Leptospira species, such as Leptospira interrogans, that colonize the renal tubules of wild and domestic animals. Thus far, attempts to develop effective leptospirosis vaccines, both for humans and animals, have failed to induce immune responses capable of conferring protection and simultaneously preventing renal colonization. In this study, we evaluated the protective immunity induced by subunit vaccines containing seven different recombinant Leptospira interrogans outer membrane proteins, including the carboxy-terminal portion of the immunoglobulinlike protein A (LigAC) and six novel antigens, combined with aluminum hydroxide (alum) or Salmonella flagellin (FliC) as adjuvants. Hamsters vaccinated with the different formulations elicited high antigen-specific antibody titers. Immunization with LigAC, either with alum or flagellin, conferred protective immunity but did not prevent renal colonization. Similarly, animals immunized with LigAC or LigAC coadministered with six leptospiral proteins with alum adjuvant conferred protection but did not reduce renal colonization. In contrast, immunizing animals with the pool of seven antigens in combination with flagellin conferred protection and significantly reduced renal colonization by the pathogen. The present study emphasizes the relevance of antigen composition and added adjuvant in the efficacy of antileptospirosis subunit vaccines and shows the complex relationship between immune responses and renal colonization by the pathogen. PMID:26108285

Assembly and breakdown of Cajal bodies in accessory nuclei of Hymenoptera.

Science.gov (United States)

Jaglarz, Mariusz K; Bilinski, Szczepan M; Kloc, Malgorzata

2005-03-01

In some species of insects, oocytes have vesicular organelles, termed accessory nuclei (ANs). The ANs form by budding off from the nuclear envelope of the oocyte and are filled with translucent matrix containing dense inclusions. One type of these inclusions contains coilin and small nuclear ribonucleoproteins (snRNPs) and is homologous to Cajal bodies. We describe the early events in the morphogenesis of Cajal bodies in the ANs (ANCBs) of the common wasp, Vespula germanica, and show that they contain survival of motor neurons (SMN) protein. We present evidence that in the wasp, ANCBs form by the gradual accumulation of aggregates composed of SMN and small nuclear RNAs. We also show that ANCBs break down and disperse within the ANs as the ANs, which initially surround the oocyte nucleus, localize to the oocyte cortex. The components of dispersed ANCBs are retained within ANs until the end of oogenesis, which suggests that their function may be required at the onset of embryonic development. Because the morphology and behavior of ANs and their Cajal body-like inclusions are conserved in two other hymenopteran species, these features might be characteristic of all hymenopterans.

Inhibition of Cartilage Acidic Protein 1 Reduces Ultraviolet B Irradiation Induced-Apoptosis through P38 Mitogen-Activated Protein Kinase and Jun Amino-Terminal Kinase Pathways

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Yinghong Ji

2016-11-01

Full Text Available Background/Aims: Ultraviolet B (UVB irradiation can easily induce apoptosis in human lens epithelial cells (HLECs and further lead to various eye diseases including cataract. Here for the first time, we investigated the role of cartilage acidic protein 1 (CRTAC1 gene in UVB irradiation induced-apoptosis in HLECs. Methods: Three groups of HLECs were employed including model group, empty vector group, and CRTAC1 interference group. Results: After UVB irradiation, the percentage of primary apoptotic cells was obviously fewer in CRTAC1 interference group. Meanwhile, inhibition of CRTAC1 also reduced both reactive oxygen species (ROS production and intracellular Ca2+ concentration, but the level of mitochondrial membrane potential (Δψm was increased in HLECs. Further studies indicated that superoxide dismutase (SOD activity and total antioxidative (T-AOC level were significantly increased in CRTAC1-inhibited cells, while the levels of malondialdehyde (MDA and lactate dehydrogenase (LDH were significantly decreased. ELISA analysis of CRTAC1-inhibited cells showed that the concentrations of tumor necrosis factor-α (TNF-α and interleukin-6 (IL-6 were significantly decreased, but the concentration of interleukin-10 (IL-10 was significantly increased. Western blot analyses of eight apoptosis-associated proteins including Bax, Bcl-2, p38, phospho-p38 (p-p38, Jun amino-terminal kinases (JNK1/2, phospho-JNK1/2 (p-JNK1/2, calcium-sensing receptor (CasR, and Ca2+/calmodulin-dependent protein kinase II (CaMKII indicated that the inhibition of CRTAC1 alleviated oxidative stress and inflammation response, inactivated calcium-signaling pathway, p38 and JNK1/2 signal pathways, and eventually reduced UVB irradiation induced-apoptosis in HLECs. Conclusion: These results provided new insights into the mechanism of cataract development, and demonstrated that CRTAC1 could be a potentially novel target for cataract treatment.

Inhibition of Cartilage Acidic Protein 1 Reduces Ultraviolet B Irradiation Induced-Apoptosis through P38 Mitogen-Activated Protein Kinase and Jun Amino-Terminal Kinase Pathways.

Science.gov (United States)

Ji, Yinghong; Rong, Xianfang; Li, Dan; Cai, Lei; Rao, Jun; Lu, Yi

2016-01-01

Ultraviolet B (UVB) irradiation can easily induce apoptosis in human lens epithelial cells (HLECs) and further lead to various eye diseases including cataract. Here for the first time, we investigated the role of cartilage acidic protein 1 (CRTAC1) gene in UVB irradiation induced-apoptosis in HLECs. Three groups of HLECs were employed including model group, empty vector group, and CRTAC1 interference group. After UVB irradiation, the percentage of primary apoptotic cells was obviously fewer in CRTAC1 interference group. Meanwhile, inhibition of CRTAC1 also reduced both reactive oxygen species (ROS) production and intracellular Ca2+ concentration, but the level of mitochondrial membrane potential (Δψm) was increased in HLECs. Further studies indicated that superoxide dismutase (SOD) activity and total antioxidative (T-AOC) level were significantly increased in CRTAC1-inhibited cells, while the levels of malondialdehyde (MDA) and lactate dehydrogenase (LDH) were significantly decreased. ELISA analysis of CRTAC1-inhibited cells showed that the concentrations of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were significantly decreased, but the concentration of interleukin-10 (IL-10) was significantly increased. Western blot analyses of eight apoptosis-associated proteins including Bax, Bcl-2, p38, phospho-p38 (p-p38), Jun amino-terminal kinases (JNK1/2), phospho-JNK1/2 (p-JNK1/2), calcium-sensing receptor (CasR), and Ca2+/calmodulin-dependent protein kinase II (CaMKII) indicated that the inhibition of CRTAC1 alleviated oxidative stress and inflammation response, inactivated calcium-signaling pathway, p38 and JNK1/2 signal pathways, and eventually reduced UVB irradiation induced-apoptosis in HLECs. These results provided new insights into the mechanism of cataract development, and demonstrated that CRTAC1 could be a potentially novel target for cataract treatment. © 2016 The Author(s) Published by S. Karger AG, Basel.

Can radiation damage to protein crystals be reduced using small-molecule compounds?

Energy Technology Data Exchange (ETDEWEB)

Kmetko, Jan [Kenyon College, Gambier, OH 43022 (United States); Warkentin, Matthew; Englich, Ulrich; Thorne, Robert E., E-mail: ret6@cornell.edu [Cornell University, Ithaca, NY 14853 (United States); Kenyon College, Gambier, OH 43022 (United States)

2011-10-01

Free-radical scavengers that are known to be effective protectors of proteins in solution are found to increase global radiation damage to protein crystals. Protective mechanisms may become deleterious in the protein-dense environment of a crystal. Recent studies have defined a data-collection protocol and a metric that provide a robust measure of global radiation damage to protein crystals. Using this protocol and metric, 19 small-molecule compounds (introduced either by cocrystallization or soaking) were evaluated for their ability to protect lysozyme crystals from radiation damage. The compounds were selected based upon their ability to interact with radiolytic products (e.g. hydrated electrons, hydrogen, hydroxyl and perhydroxyl radicals) and/or their efficacy in protecting biological molecules from radiation damage in dilute aqueous solutions. At room temperature, 12 compounds had no effect and six had a sensitizing effect on global damage. Only one compound, sodium nitrate, appeared to extend crystal lifetimes, but not in all proteins and only by a factor of two or less. No compound provided protection at T = 100 K. Scavengers are ineffective in protecting protein crystals from global damage because a large fraction of primary X-ray-induced excitations are generated in and/or directly attack the protein and because the ratio of scavenger molecules to protein molecules is too small to provide appreciable competitive protection. The same reactivity that makes some scavengers effective radioprotectors in protein solutions may explain their sensitizing effect in the protein-dense environment of a crystal. A more productive focus for future efforts may be to identify and eliminate sensitizing compounds from crystallization solutions.

Can radiation damage to protein crystals be reduced using small-molecule compounds?

International Nuclear Information System (INIS)

Kmetko, Jan; Warkentin, Matthew; Englich, Ulrich; Thorne, Robert E.

2011-01-01

Free-radical scavengers that are known to be effective protectors of proteins in solution are found to increase global radiation damage to protein crystals. Protective mechanisms may become deleterious in the protein-dense environment of a crystal. Recent studies have defined a data-collection protocol and a metric that provide a robust measure of global radiation damage to protein crystals. Using this protocol and metric, 19 small-molecule compounds (introduced either by cocrystallization or soaking) were evaluated for their ability to protect lysozyme crystals from radiation damage. The compounds were selected based upon their ability to interact with radiolytic products (e.g. hydrated electrons, hydrogen, hydroxyl and perhydroxyl radicals) and/or their efficacy in protecting biological molecules from radiation damage in dilute aqueous solutions. At room temperature, 12 compounds had no effect and six had a sensitizing effect on global damage. Only one compound, sodium nitrate, appeared to extend crystal lifetimes, but not in all proteins and only by a factor of two or less. No compound provided protection at T = 100 K. Scavengers are ineffective in protecting protein crystals from global damage because a large fraction of primary X-ray-induced excitations are generated in and/or directly attack the protein and because the ratio of scavenger molecules to protein molecules is too small to provide appreciable competitive protection. The same reactivity that makes some scavengers effective radioprotectors in protein solutions may explain their sensitizing effect in the protein-dense environment of a crystal. A more productive focus for future efforts may be to identify and eliminate sensitizing compounds from crystallization solutions

Implementation of radioactive wastes management system in nuclear medicine service of Hospital das Clinicas of Universidade de Campinas - UNICAMP, in Sao Paulo State, Brazil

International Nuclear Information System (INIS)

Rinaldi Neto, A.; Coelho, R.F.; Brunetto, S.Q.

2001-01-01

This work reports the experience acquired at the Servico de Medicina Nuclear of the Hospital de Clinicas/UNICAMP (SMN/HC) in planning and implementing the management system of radioactive waste. This system respects the Comissao Nacional de Energia Nuclear' (CNEN) standards and has been of relatively easy and simple performance, without disturbing the SMN/HC's routine. It has also proof to keep its quality along the time. (author)

Monoacylated Cellular Prion Proteins Reduce Amyloid-β-Induced Activation of Cytoplasmic Phospholipase A2 and Synapse Damage

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Ewan West

2015-06-01

Full Text Available Alzheimer’s disease (AD is a progressive neurodegenerative disease characterized by the accumulation of amyloid-β (Aβ and the loss of synapses. Aggregation of the cellular prion protein (PrPC by Aβ oligomers induced synapse damage in cultured neurons. PrPC is attached to membranes via a glycosylphosphatidylinositol (GPI anchor, the composition of which affects protein targeting and cell signaling. Monoacylated PrPC incorporated into neurons bound “natural Aβ”, sequestering Aβ outside lipid rafts and preventing its accumulation at synapses. The presence of monoacylated PrPC reduced the Aβ-induced activation of cytoplasmic phospholipase A2 (cPLA2 and Aβ-induced synapse damage. This protective effect was stimulus specific, as treated neurons remained sensitive to α-synuclein, a protein associated with synapse damage in Parkinson’s disease. In synaptosomes, the aggregation of PrPC by Aβ oligomers triggered the formation of a signaling complex containing the cPLA2.a process, disrupted by monoacylated PrPC. We propose that monoacylated PrPC acts as a molecular sponge, binding Aβ oligomers at the neuronal perikarya without activating cPLA2 or triggering synapse damage.

Maternal protein-energy malnutrition during early pregnancy in sheep impacts the fetal ornithine cycle to reduce fetal kidney microvascular development.

Science.gov (United States)

Dunford, Louise J; Sinclair, Kevin D; Kwong, Wing Y; Sturrock, Craig; Clifford, Bethan L; Giles, Tom C; Gardner, David S

2014-11-01

This paper identifies a common nutritional pathway relating maternal through to fetal protein-energy malnutrition (PEM) and compromised fetal kidney development. Thirty-one twin-bearing sheep were fed either a control (n=15) or low-protein diet (n=16, 17 vs. 8.7 g crude protein/MJ metabolizable energy) from d 0 to 65 gestation (term, ∼ 145 d). Effects on the maternal and fetal nutritional environment were characterized by sampling blood and amniotic fluid. Kidney development was characterized by histology, immunohistochemistry, vascular corrosion casts, and molecular biology. PEM had little measureable effect on maternal and fetal macronutrient balance (glucose, total protein, total amino acids, and lactate were unaffected) or on fetal growth. PEM decreased maternal and fetal urea concentration, which blunted fetal ornithine availability and affected fetal hepatic polyamine production. For the first time in a large animal model, we associated these nutritional effects with reduced micro- but not macrovascular development in the fetal kidney. Maternal PEM specifically impacts the fetal ornithine cycle, affecting cellular polyamine metabolism and microvascular development of the fetal kidney, effects that likely underpin programming of kidney development and function by a maternal low protein diet. © FASEB.

The catechin flavonoid reduces proliferation and induces apoptosis of murine lymphoma cells LB02 through modulation of antiapoptotic proteins

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Daniela Laura Papademetrio

2013-06-01

Full Text Available Flavonoids are products of secondary metabolism of plants. They are present in herbs and trees and also act as natural chemopreventives and anticancer agents. Ligaria cuneifolia (Ruiz & Pav. Tiegh., Loranthaceae, is a hemiparasite species that belongs to Argentine flora. Phytochemical studies have disclosed the presence of quercetin, catechin-4β-ol and pro-anthocyanidine as polyphenolic compounds in the active extracts. We previously demonstrated that ethyl acetate extract was capable of reducing cell proliferation and inducing apoptotic death of lymphoid tumor cells. The aim of the current study is to determine whether or not catechin, isolated from L. cuneifolia extracts can induce leukemia cell death and to determine its effect on the cytoplasmatic proteins that modulate cell survival. Our results show that catechin can reduce proliferation of murine lymphoma cell line LB02. The effect is mediated by apoptosis at concentrations upper to 100 µg/mL. Cell death is related to the loss of mitochondrial membrane potential (ΔΨm and a down regulation of survivin and Bcl-2 together with the increase of pro-apoptotic protein Bax. In summary, the current study indicates that catechin present in the extract of L. cuneifolia is in part, responsible for the anti-proliferative activity of whole extracts by induction of ΔΨm disruption and modulation of the anti-apoptotic proteins over expressed in tumor cells. These results give new findings into the potential anticancer and chemopreventive activities of L. cuneifolia.

The catechin flavonoid reduces proliferation and induces apoptosis of murine lymphoma cells LB02 through modulation of antiapoptotic proteins

Directory of Open Access Journals (Sweden)

Daniela Laura Papademetrio

2013-03-01

Full Text Available Flavonoids are products of secondary metabolism of plants. They are present in herbs and trees and also act as natural chemopreventives and anticancer agents. Ligaria cuneifolia (Ruiz & Pav. Tiegh., Loranthaceae, is a hemiparasite species that belongs to Argentine flora. Phytochemical studies have disclosed the presence of quercetin, catechin-4β-ol and pro-anthocyanidine as polyphenolic compounds in the active extracts. We previously demonstrated that ethyl acetate extract was capable of reducing cell proliferation and inducing apoptotic death of lymphoid tumor cells. The aim of the current study is to determine whether or not catechin, isolated from L. cuneifolia extracts can induce leukemia cell death and to determine its effect on the cytoplasmatic proteins that modulate cell survival. Our results show that catechin can reduce proliferation of murine lymphoma cell line LB02. The effect is mediated by apoptosis at concentrations upper to 100 µg/mL. Cell death is related to the loss of mitochondrial membrane potential (ΔΨm and a down regulation of survivin and Bcl-2 together with the increase of pro-apoptotic protein Bax. In summary, the current study indicates that catechin present in the extract of L. cuneifolia is in part, responsible for the anti-proliferative activity of whole extracts by induction of ΔΨm disruption and modulation of the anti-apoptotic proteins over expressed in tumor cells. These results give new findings into the potential anticancer and chemopreventive activities of L. cuneifolia.

A high protein moderate carbohydrate diet fed at discrete meals reduces early progression of N-methyl-N-nitrosourea-induced breast tumorigenesis in rats

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Singletary Keith W

2010-01-01

Full Text Available Abstract Breast cancer is the most prevalent cancer in American women. Dietary factors are thought to have a strong influence on breast cancer incidence. This study utilized a meal-feeding protocol with female Sprague-Dawley rats to evaluate effects of two ratios of carbohydrate:protein on promotion and early progression of breast tissue carcinomas. Mammary tumors were induced by N-methyl-N-nitrosourea (MNU at 52 d of age. Post-induction, animals were assigned to consume either a low protein high carbohydrate diet (LPHC; 15% and 60% of energy, respectively or a high protein moderate carbohydrate diet (HPMC; 35% and 40% of energy, respectively for 10 wk. Animals were fed 3 meals/day to mimic human absorption and metabolism patterns. The rate of palpable tumor incidence was reduced in HPMC relative to LPHC (12.9 ± 1.4%/wk vs. 18.2 ± 1.3%/wk. At 3 wk, post-prandial serum insulin was larger in the LPHC relative to HPMC (+136.4 ± 33.1 pmol/L vs. +38.1 ± 23.4 pmol/L, while at 10 wk there was a trend for post-prandial IGF-I to be increased in HPMC (P = 0.055. There were no differences in tumor latency, tumor surface area, or cumulative tumor mass between diet groups. The present study provides evidence that reducing the dietary carbohydrate:protein ratio attenuates the development of mammary tumors. These findings are consistent with reduced post-prandial insulin release potentially diminishing the proliferative environment required for breast cancer tumors to progress.

Reduced expression of mitochondrial electron transport chain proteins from hibernating hearts relative to ischemic preconditioned hearts in the second window of protection.

Science.gov (United States)

Cabrera, Jesús A; Butterick, Tammy A; Long, Eric K; Ziemba, Elizabeth A; Anderson, Lorraine B; Duffy, Cayla M; Sluiter, Willem; Duncker, Dirk J; Zhang, Jianyi; Chen, Yingjie; Ward, Herbert B; Kelly, Rosemary F; McFalls, Edward O

2013-07-01

Although protection against necrosis has been observed in both hibernating (HIB) and ischemic preconditioned hearts in the second window of protection (SWOP), a comparison of the mitochondrial proteome between the two entities has not been previously performed. Anesthetized swine underwent instrumentation with a fixed constrictor around the LAD artery and were followed for 12 weeks (HIB; N=7). A second group of anesthetized swine underwent ischemic preconditioning by inflating a balloon within the LAD artery 10 times for 2 min, each separated by 2 min reperfusion and were sacrificed 24h later (SWOP; N=7). Myocardial blood flow and high-energy nucleotides were obtained in the LAD region and normalized to remote regions. Post-sacrifice, protein content as measured with iTRAQ was compared in isolated mitochondria from the LAD area of a Sham heart. Basal regional blood flow in the LAD region when normalized to the remote region was 0.86±0.04 in HIB and 1.02±0.02 in SWOP tissue (Pregional blood flows in HIB hearts, ATP content in the LAD region, when normalized to the remote region was similar in HIB versus SWOP (1.06±0.06 and 1.02±0.05 respectively; NS) as was the transmural phosphocreatine (PCr) to ATP ratio (2.1±0.2 and 2.2±0.2 respectively; NS). Using iTRAQ, 64 common proteins were identified in HIB and SWOP hearts. Compared with SWOP, the relative abundance of mitochondrial proteins involved with electron transport chain (ETC) were reduced in HIB including NADH dehydrogenase, Cytochrome c reductase and oxidase, ATP synthase, and nicotinamide nucleotide transhydrogenase. Within chronically HIB heart tissue with reduced blood flow, the relative abundance of mitochondrial ETC proteins is decreased when compared with SWOP tissue. These data support the concept that HIB heart tissue subjected to chronically reduced blood flow is associated with a down-regulation in the expression of key mitochondrial proteins involved in electron transport. Published by Elsevier

Heterologous expression of a plastid EF-Tu reduces protein thermal aggregation and enhances CO2 fixation in wheat (Triticum aestivum) following heat stress.

Science.gov (United States)

Fu, Jianming; Momcilović, Ivana; Clemente, Thomas E; Nersesian, Natalya; Trick, Harold N; Ristic, Zoran

2008-10-01

Heat stress is a major constraint to wheat production and negatively impacts grain quality, causing tremendous economic losses, and may become a more troublesome factor due to global warming. At the cellular level, heat stress causes denaturation and aggregation of proteins and injury to membranes leading to alterations in metabolic fluxes. Protein aggregation is irreversible, and protection of proteins from thermal aggregation is a strategy a cell uses to tolerate heat stress. Here we report on the development of transgenic wheat (Triticum aestivum) events, expressing a maize gene coding for plastidal protein synthesis elongation factor (EF-Tu), which, compared to non-transgenic plants, display reduced thermal aggregation of leaf proteins, reduced heat injury to photosynthetic membranes (thylakoids), and enhanced rate of CO(2) fixation after exposure to heat stress. The results support the concept that EF-Tu ameliorates negative effects of heat stress by acting as a molecular chaperone. This is the first demonstration of the introduction of a plastidal EF-Tu in plants that leads to protection against heat injury and enhanced photosynthesis after heat stress. This is also the first demonstration that a gene other than HSP gene can be used for improvement of heat tolerance and that the improvement is possible in a species that has a complex genome, hexaploid wheat. The results strongly suggest that heat tolerance of wheat, and possibly other crop plants, can be improved by modulating expression of plastidal EF-Tu and/or by selection of genotypes with increased endogenous levels of this protein.

High Dietary Protein Intake and Protein-Related Acid Load on Bone Health.

Science.gov (United States)

Cao, Jay J

2017-12-01

Consumption of high-protein diets is increasingly popular due to the benefits of protein on preserving lean mass and controlling appetite and satiety. The paper is to review recent clinical research assessing dietary protein on calcium metabolism and bone health. Epidemiological studies show that long-term, high-protein intake is positively associated with bone mineral density and reduced risk of bone fracture incidence. Short-term interventional studies demonstrate that a high-protein diet does not negatively affect calcium homeostasis. Existing evidence supports that the negative effects of the acid load of protein on urinary calcium excretion are offset by the beneficial skeletal effects of high-protein intake. Future research should focus on the role and the degree of contribution of other dietary and physiological factors, such as intake of fruits and vegetables, in reducing the acid load and further enhancing the anabolic effects of protein on the musculoskeletal system.

Cholesterol Removal from Adult Skeletal Muscle impairs Excitation-Contraction Coupling and Aging reduces Caveolin-3 and alters the Expression of other Triadic Proteins

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Genaro eBarrientos

2015-04-01

Full Text Available Cholesterol and caveolin are integral membrane components that modulate the function/location of many cellular proteins. Skeletal muscle fibers, which have unusually high cholesterol levels in transverse tubules, express the caveolin-3 isoform but its association with transverse tubules remains contentious. Cholesterol removal impairs excitation-contraction coupling in amphibian and mammalian fetal skeletal muscle fibers. Here, we show that treating single muscle fibers from adult mice with the cholesterol removing agent methyl-β-cyclodextrin decreased fiber cholesterol by 26%, altered the location pattern of caveolin-3 and of the voltage dependent calcium channel Cav1.1, and suppressed or reduced electrically evoked Ca2+ transients without affecting membrane integrity or causing sarcoplasmic reticulum calcium depletion. We found that transverse tubules from adult muscle and triad fractions that contain ~10% attached transverse tubules, but not sarcoplasmic reticulum membranes, contained caveolin-3 and Cav1.1; both proteins partitioned into detergent-resistant membrane fractions highly enriched in cholesterol. Aging entails significant deterioration of skeletal muscle function. We found that triad fractions from aged rats had similar cholesterol and RyR1 protein levels compared to triads from young rats, but had lower caveolin-3 and glyceraldehyde 3-phosphate dehydrogenase and increased Na+/K+-ATPase protein levels. Both triad fractions had comparable NADPH oxidase (NOX activity and protein content of NOX2 subunits (p47phox and gp91phox, implying that NOX activity does not increase during aging. These findings show that partial cholesterol removal impairs excitation-contraction coupling and alters caveolin-3 and Cav1.1 location pattern, and that aging reduces caveolin-3 protein content and modifies the expression of other triadic proteins. We discuss the possible implications of these findings for skeletal muscle function in young and aged

Protein characterization of protein bodies from cotyledons of Mucuna pruriens (L.) DC.

Science.gov (United States)

Bellani, Lorenza; Giglioni, Stefania; Muccifora, Simonetta

2013-03-01

Seeds of Mucuna pruriens (L.) DC. (Fabaceae) were analyzed for protein composition of protein bodies isolated from cotyledons. Protein bodies were successfully separated by Lympholyte and those of dry seeds, observed by scanning electron microscope, were elliptical or spherical in shape with a diameter of 5-12 μm. Protein content in dry seed protein bodies was 10.6 mg/g dry weight. Globulin was the largest protein fraction isolated (62.5 %), followed by albumin (18.3 %), glutelin (15.8 %) and prolamin (3.4 %). The prolamin fraction and high glutelin content are uncommon in legumes. SDS-PAGE of albumins, globulins, prolamins and glutelins provided different band numbers and molecular weights under reducing and non reducing conditions and suggested that the albumin fraction is rich in disulphide bonds.

Identification of thioredoxin h-reducible disulphides in proteornes by differential labelling of cysteines: Insight into recognition and regulation of proteins in barley seeds by thioredoxin h

DEFF Research Database (Denmark)

Maeda, Kenji; Finnie, Christine; Svensson, Birte

2005-01-01

alpha-amylase/subtilisin inhibitor (BASI) by barley thioredoxin h isoform 1 was analysed. Furthermore, the method was coupled with two-dimensional electrophoresis for convenient thioredoxin h-reducible disulphide identification in barley seed extracts without the need for protein purification...... or production of recombinant proteins. Mass shifts of 15 peptides, induced by treatment with thioredoxin h and differential alkylation, identified specific reduction of nine disulphides in BASI, four alpha-amylase/trypsin inhibitors and a protein of unknown function. Two specific disulphides, located...... structurally close to the alpha-amylase binding surfaces of BASI and alpha-amylase inhibitor BMAI-1 were demonstrated to be reduced to a particularly high extent. For the first time, specificity of thioredoxin h for particular disulphide bonds is demonstrated, providing a basis to study structural aspects...

Multigenerational effects of a reduced balanced protein diet during the rearing and laying period of broiler breeders. 1. Performance of the F1 breeder generation.

Science.gov (United States)

Lesuisse, J; Li, C; Schallier, S; Clímaco, W L S; Bautil, A; Everaert, N; Buyse, J

2018-05-01

Studies on mammals and poultry showed that maternal dietary treatments can alter the offspring performance. However, in contrast to rodent studies, little is known about multigenerational dietary manipulations in broiler breeders. The presented research aimed to investigate the effects of a reduction of 25% in the dietary crude protein (CP) level in the F0 generation on the body composition and reproductive performance of F1 broiler breeders. In the F0 generation, breeders were fed either a control (C) or reduced balanced protein (RP) diet, 25% reduction in crude protein and amino acids. Female F0-progeny of each treatment were fed a C or RP diet, resulting in 4 treatments in the F1 breeder generation: C/C, C/RP, RP/C, and RP/RP. The reproductive performance of breeders fed RP diets was negatively influenced by the dietary CP reduction in the F1 generation (P diets in the F0 generation showed a significantly reduced reproductive capacity compared to their control fed counterparts (P diets in the F1 generation were characterized by higher plasma T3 concentrations (P diets in the F0 generation needed lower feed allocations in the laying phase to maintain a similar body weight. Egg weight was reduced for the C/RP and RP/RP breeders. At 34 wk of age, eggs from C/RP and RP/RP breeders showed a reduced proportional albumen weight, whereas no effects on egg composition were found at 42 wk of age. It was concluded that prenatal protein undernutrition triggered hens to relocate more energy towards growth and maintenance and less towards reproductive capacity.

Threonine supplementation reduces dietary protein and improves lipid metabolism in Pekin ducks.

Science.gov (United States)

Jiang, Y; Tang, J; Xie, M; Wen, Z G; Qiao, S Y; Hou, S S

2017-12-01

1. This study was conducted to investigate the efficiency of threonine (Thr) supplementation on reducing dietary crude protein (CP) content and the effects of Thr on lipid metabolism in Pekin ducks. The effects of dietary CP concentration (160, 190 and 220 g/kg) and Thr supplemental concentration (0, 0.7, 1.4, 2.1 and 2.8 g/kg) on growth performance, carcass, liver lipid and plasma profiles were determined in Pekin ducks from 1-21 d of age. 2. A total of 720-d-old male Pekin ducks were randomly allotted to 1 of 15 dietary treatments with 6 replicate cages of 8 birds per cage for each treatment according to average body weight. 3. Dietary Thr supplementation improved growth performance and breast muscle percentage at all CP diets, and ducks fed Thr-supplemented diets had higher plasma concentrations of some plasma amino acids. Thr supplementation reduced the concentrations of total lipid, triglyceride, cholesterol in liver, and plasma low density lipoprotein cholesterin concentration at 160 and 190 g/kg CP, whereas it increased triglyceride concentration at 160 g/kg CP. 4. Thr requirements based on quadratic broken-line model estimation were 6.6 and 7.0 g/kg for optimal average daily gain (ADG), and 6.7 and 7.3 g/kg for breast muscle percentage of Pekin ducks from 1-21 d of age at 190 and 220 g/kg CP, respectively. The dietary Thr requirements and estimated ADG (55.18 vs. 55.86 g/d/bird) and breast muscle percentage (2.79% vs. 2.75%) of Pekin ducks did not differ between 190 and 220 g/kg CP according to the t-test results. 5. Dietary CP level could be reduced to 190 g/kg in Pekin ducks from 1-21 d of age with Thr supplementation to balance dietary amino acids, and Thr supplementation prevented excess liver lipid deposition in this instance.

Optimization of Protein Extraction from Spirulina platensis to Generate a Potential Co-Product and a Biofuel Feedstock with Reduced Nitrogen Content

Energy Technology Data Exchange (ETDEWEB)

Parimi, Naga Sirisha; Singh, Manjinder; Kastner, James R.; Das, Keshav C., E-mail: kdas@engr.uga.edu [College of Engineering, The University of Georgia, Athens, GA (United States); Forsberg, Lennart S.; Azadi, Parastoo [Complex Carbohydrate Research Center, The University of Georgia, Athens, GA (United States)

2015-06-23

The current work reports protein extraction from Spirulina platensis cyanobacterial biomass in order to simultaneously generate a potential co-product and a biofuel feedstock with reduced nitrogen content. S. platensis cells were subjected to cell disruption by high-pressure homogenization and subsequent protein isolation by solubilization at alkaline pH followed by precipitation at acidic pH. Response surface methodology was used to optimize the process parameters – pH, extraction (solubilization/precipitation) time and biomass concentration for obtaining maximum protein yield. The optimized process conditions were found to be pH 11.38, solubilization time of 35 min and biomass concentration of 3.6% (w/w) solids for the solubilization step, and pH 4.01 and precipitation time of 60 min for the precipitation step. At the optimized conditions, a high protein yield of 60.7% (w/w) was obtained. The protein isolate (co-product) had a higher protein content [80.6% (w/w)], lower ash [1.9% (w/w)] and mineral content and was enriched in essential amino acids, the nutritious γ-linolenic acid and other high-value unsaturated fatty acids compared to the original biomass. The residual biomass obtained after protein extraction had lower nitrogen content and higher total non-protein content than the original biomass. The loss of about 50% of the total lipids from this fraction did not impact its composition significantly owing to the low lipid content of S. platensis (8.03%).

Optimization of Protein Extraction from Spirulina platensis to Generate a Potential Co-Product and a Biofuel Feedstock with Reduced Nitrogen Content

International Nuclear Information System (INIS)

Parimi, Naga Sirisha; Singh, Manjinder; Kastner, James R.; Das, Keshav C.; Forsberg, Lennart S.; Azadi, Parastoo

2015-01-01

The current work reports protein extraction from Spirulina platensis cyanobacterial biomass in order to simultaneously generate a potential co-product and a biofuel feedstock with reduced nitrogen content. S. platensis cells were subjected to cell disruption by high-pressure homogenization and subsequent protein isolation by solubilization at alkaline pH followed by precipitation at acidic pH. Response surface methodology was used to optimize the process parameters – pH, extraction (solubilization/precipitation) time and biomass concentration for obtaining maximum protein yield. The optimized process conditions were found to be pH 11.38, solubilization time of 35 min and biomass concentration of 3.6% (w/w) solids for the solubilization step, and pH 4.01 and precipitation time of 60 min for the precipitation step. At the optimized conditions, a high protein yield of 60.7% (w/w) was obtained. The protein isolate (co-product) had a higher protein content [80.6% (w/w)], lower ash [1.9% (w/w)] and mineral content and was enriched in essential amino acids, the nutritious γ-linolenic acid and other high-value unsaturated fatty acids compared to the original biomass. The residual biomass obtained after protein extraction had lower nitrogen content and higher total non-protein content than the original biomass. The loss of about 50% of the total lipids from this fraction did not impact its composition significantly owing to the low lipid content of S. platensis (8.03%).

A method for investigating protein-protein interactions related to Salmonella typhimurium pathogenesis

Energy Technology Data Exchange (ETDEWEB)

Chowdhury, Saiful M. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Shi, Liang [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Yoon, Hyunjin [Dartmouth College, Hanover, NH (United States); Ansong, Charles [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Rommereim, Leah M. [Dartmouth College, Hanover, NH (United States); Norbeck, Angela D. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Auberry, Kenneth J. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Moore, R. J. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Adkins, Joshua N. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Heffron, Fred [Oregon Health and Science Univ., Portland, OR (United States); Smith, Richard D. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

2009-02-10

We successfully modified an existing method to investigate protein-protein interactions in the pathogenic bacterium Salmonella typhimurium (STM). This method includes i) addition of a histidine-biotin-histidine tag to the bait proteins via recombinant DNA techniques; ii) in vivo cross-linking with formaldehyde; iii) tandem affinity purification of bait proteins under fully denaturing conditions; and iv) identification of the proteins cross-linked to the bait proteins by liquid-chromatography in conjunction with tandem mass-spectrometry. In vivo cross-linking stabilized protein interactions permitted the subsequent two-step purification step conducted under denaturing conditions. The two-step purification greatly reduced nonspecific binding of non-cross-linked proteins to bait proteins. Two different negative controls were employed to reduce false-positive identification. In an initial demonstration of this approach, we tagged three selected STM proteins- HimD, PduB and PhoP- with known binding partners that ranged from stable (e.g., HimD) to transient (i.e., PhoP). Distinct sets of interacting proteins were identified with each bait protein, including the known binding partners such as HimA for HimD, as well as anticipated and unexpected binding partners. Our results suggest that novel protein-protein interactions may be critical to pathogenesis by Salmonella typhimurium. .

Probing the Selectivity and Protein•Protein Interactions of a Non-Reducing Fungal Polyketide Synthase Using Mechanism-Based Crosslinkers

Science.gov (United States)

Bruegger, Joel; Haushalter, Bob; Vagstad, Anna; Shakya, Gaurav; Mih, Nathan; Townsend, Craig A.; Burkart, Michael D.; Tsai, Shiou-Chuan

2013-01-01

SUMMARY Protein•protein interactions, which often involve interactions between an acyl carrier protein (ACP) and its partner enzymes, are important for coordinating polyketide biosynthesis. However, the nature of such interactions is not well understood, especially in the fungal non-reducing polyketide synthases (NR-PKSs) that biosynthesize toxic and pharmaceutically important polyketides. Here, we employ a mechanism-based crosslinker to successfully probe ACP and ketosynthase (KS) domain interactions in NR-PKSs. We found that crosslinking efficiency is closely correlated with the strength of ACP•KS interactions, and that KS demonstrates strong starter unit selectivity. We further identified positively charged surface residues by KS mutagenesis, which mediate key interactions with the negatively-charged ACP surface. Such complementary/matching contact pairs can serve as “adapter surfaces” for future efforts to generate new polyketides using NR-PKSs. PMID:23993461

Serum C-reactive protein and thioredoxin levels in subjects with mildly reduced glomerular filtration rate

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Ishimura Eiji

2010-04-01

Full Text Available Abstract Background Chronic kidney disease (CKD is a newly recognized high-risk condition for cardiovascular disease (CVD, and previous studies reported the changes in inflammation and oxidative stress in advanced stages of CKD. We compared the levels of serum biomarkers for inflammation and oxidative stress between subjects with normal and mildly reduced glomerular filtration rate (GFR. Methods The subjects were 182 participants of a health check-up program including those with normal (≥ 90 mL/min/1.73 m2, N = 79 and mildly reduced eGFR (60-89 mL/min/1.73 m2, N = 103 which was calculated based on serum creatinine, age and sex. We excluded those with reduced eGFR 2. No one had proteinuria. We measured serum levels of C-reactive protein (CRP and thioredoxin (TRX as the markers of inflammation and oxidative stress, respectively. Results As compared with subjects with normal eGFR, those with mildly reduced eGFR had increased levels of both CRP and TRX. Also, eGFR was inversely correlated with these biomarkers. The associations of eGFR with these biomarkers remained significant after adjustment for age and sex. When adjustment was done for eight possible confounders, CRP showed significant association with systolic blood pressure, high density lipoprotein cholesterol (HDL-C and non-HDL-C, whereas TRX was associated with sex significantly, and with eGFR and systolic blood pressure at borderline significance. Conclusions We showed the increased levels of CRP and TRX in subjects with mildly reduced eGFR. The eGFR-CRP link and the eGFR-TRX link appeared to be mediated, at least partly, by the alterations in blood pressure and plasma lipids in these subjects.

Administration of structured lipid composed of MCT and fish oil reduces net protein catabolism in enterally fed burned rats.

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Teo, T C; DeMichele, S J; Selleck, K M; Babayan, V K; Blackburn, G L; Bistrian, B R

1989-01-01

The effects of enteral feeding with safflower oil or a structured lipid (SL) derived from 60% medium-chain triglyceride (MCT) and 40% fish oil (MCT/fish oil) on protein and energy metabolism were compared in gastrostomy-fed burned rats (30% body surface area) by measuring oxygen consumption, carbon dioxide production, nitrogen balance, total liver protein, whole-body leucine kinetics, and rectus muscle and liver protein fractional synthetic rates (FSR, %/day). Male Sprague-Dawley rats (195 +/- 5g) received 50 ml/day of an enteral regimen containing 50 kcal, 2 g amino acids, and 40% nonprotein calories as lipid for three days. Protein kinetics were estimated by using a continuous L-[1-14C] leucine infusion technique on day 2. Thermally injured rats enterally fed MCT/fish oil yielded significantly higher daily and cumulative nitrogen balances (p less than or equal to 0.025) and rectus muscle (39%) FSR (p less than or equal to 0.05) when compared with safflower oil. MCT/fish oil showed a 22% decrease (p less than or equal to 0.005) in per cent flux oxidized and a 7% (p less than or equal to 0.05) decrease in total energy expenditure (TEE) versus safflower oil. A 15% increase in liver FSR was accompanied by a significant elevation (p less than or equal to 0.025) in total liver protein with MCT/fish oil. This novel SL shares the properties of other structured lipids in that it reduces the net protein catabolic effects of burn injury, in part, by influencing tissue protein synthetic rates. The reduction in TEE is unique to MCT/fish oil and may relate to the ability of fish oil to diminish the injury response. PMID:2500898

Efficacy of humidity retention bags for the reduced adsorption and improved cleaning of tissue proteins including prion-associated amyloid to surgical stainless steel surfaces.

Science.gov (United States)

Secker, T J; Pinchin, H E; Hervé, R C; Keevil, C W

2015-01-01

Increasing drying time adversely affects attachment of tissue proteins and prion-associated amyloid to surgical stainless steel, and reduces the efficacy of commercial cleaning chemistries. This study tested the efficacy of commercial humidity retention bags to reduce biofouling on surgical stainless steel and to improve subsequent cleaning. Surgical stainless steel surfaces were contaminated with ME7-infected brain homogenates and left to dry for 15 to 1,440 min either in air, in dry polythene bags or within humidity retention bags. Residual contamination pre/post cleaning was analysed using Thioflavin T/SYPRO Ruby dual staining and microscope analysis. An increase in biofouling was observed with increased drying time in air or in sealed dry bags. Humidity retention bags kept both protein and prion-associated amyloid minimal across the drying times both pre- and post-cleaning. Therefore, humidity bags demonstrate a cheap, easy to implement solution to improve surgical instrument reprocessing and to potentially reduce associated hospital acquired infections.

Enhanced Temperature During Grain Filling Reduces Protein Concentration of Durum Wheat

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Franco Miglietta

2011-02-01

Full Text Available Durum wheat is cultivated over more than 13 millions of hectares (ha world wide and Italy is the main European producer with 3.5 millions tons per year. The protein concentration of durum wheat is very important, it ensures high nutritional value and is highly appreciated by the pasta production industries. The protein concentration of wheat is determined during the grain filling period when carbon and nitrogen compounds are translocated into the grains. Air temperature affects translocation rates and contributes to final protein concentration of wheat grains. Two common commercial varieties of durum and bread wheat were exposed from anthesis to harvest, to a source of infrared radiation in the field. This allowed to investigate the relative effect of temperature on translocation of carbon and nitrogen compound during grain filling. The heat treatment imposed affected marginally dry mass accumulation of the grains in bread wheat and didn’t affect dry mass in durum wheat. Grain protein was affected by heat treatment in durum but not in bread wheat. Carbon accumulation rate was higher for durum than for bread wheat. The protein concentration was greater in durum than in bread wheat and we can assume that the absolute nitrogen accumulation rates were higher for the former species. Such difference may be either caused by a faster nitrogen uptake rate and translocation or a more efficient relocation of nitrogen accumulated in reserve organs.

Protein source in a high-protein diet modulates reductions in insulin resistance and hepatic steatosis in fa/fa Zucker rats.

Science.gov (United States)

Wojcik, Jennifer L; Devassy, Jessay G; Wu, Yinghong; Zahradka, Peter; Taylor, Carla G; Aukema, Harold M

2016-01-01

High-protein diets are being promoted to reduce insulin resistance and hepatic steatosis in metabolic syndrome. Therefore, the effect of protein source in high-protein diets on reducing insulin resistance and hepatic steatosis was examined. Fa/fa Zucker rats were provided normal-protein (15% of energy) casein, high-protein (35% of energy) casein, high-protein soy, or high-protein mixed diets with animal and plant proteins. The high-protein mixed diet reduced area under the curve for insulin during glucose tolerance testing, fasting serum insulin and free fatty acid concentrations, homeostatic model assessment index, insulin to glucose ratio, and pancreatic islet cell area. The high-protein mixed and the high-protein soy diets reduced hepatic lipid concentrations, liver to body weight ratio, and hepatic steatosis rating. These improvements were observed despite no differences in body weight, feed intake, or adiposity among high-protein diet groups. The high-protein casein diet had minimal benefits. A high-protein mixed diet was the most effective for modulating reductions in insulin resistance and hepatic steatosis independent of weight loss, indicating that the source of protein within a high-protein diet is critical for the management of these metabolic syndrome parameters. © 2015 The Obesity Society.

Reducing crude protein content with supplementation of synthetic lysine and threonine in barley - rapeseed meal - pea diets for growing pigs

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Jarmo Valaja

1993-03-01

Full Text Available This study was conducted to determine the possibility to use synthetic amino acids to lower the nitrogen output from pig production. A performance experiment was carried out with 120triplet-fed growing pigs whose dietary crude protein was reduced from 179 g/feed unit (FU= 0.7 kg starch equivalent to 160, 140 and 122 g/FU, respectively. The diets were supplemented with synthetic lysine and threonine to keep the level of these amino acids constant. Dietary protein reduction did not affect the growth performance or feed conversion ratio of the pigs, but it did linearly increase the portion of fat to lean in the carcass. Significant linear effect was found in back fat (p

Important mitochondrial proteins in human omental adipose tissue show reduced expression in obesity.

Science.gov (United States)

Lindinger, Peter W; Christe, Martine; Eberle, Alex N; Kern, Beatrice; Peterli, Ralph; Peters, Thomas; Jayawardene, Kamburapola J I; Fearnley, Ian M; Walker, John E

2015-09-01

Obesity is associated with impaired mitochondrial function. This study compares mitochondrial protein expression in omental fat in obese and non-obese humans. Omental adipose tissue was obtained by surgical biopsy, adipocytes were purified and mitochondria isolated. Using anion-exchange chromatography, SDS-PAGE and mass-spectrometry, 128 proteins with potentially different abundances in patient groups were identified, 62 of the 128 proteins are mainly localized in the mitochondria. Further quantification of 12 of these 62 proteins by immune dot blot analysis revealed four proteins citrate synthase, HADHA, LETM1 and mitofilin being inversely associated with BMI, and mitofilin being inversely correlated with gender.

Important mitochondrial proteins in human omental adipose tissue show reduced expression in obesity

Directory of Open Access Journals (Sweden)

Peter W. Lindinger

2015-09-01

Full Text Available Obesity is associated with impaired mitochondrial function. This study compares mitochondrial protein expression in omental fat in obese and non-obese humans. Omental adipose tissue was obtained by surgical biopsy, adipocytes were purified and mitochondria isolated. Using anion-exchange chromatography, SDS-PAGE and mass-spectrometry, 128 proteins with potentially different abundances in patient groups were identified, 62 of the 128 proteins are mainly localized in the mitochondria. Further quantification of 12 of these 62 proteins by immune dot blot analysis revealed four proteins citrate synthase, HADHA, LETM1 and mitofilin being inversely associated with BMI, and mitofilin being inversely correlated with gender.

Omega-3 fatty acid docosahexaenoic acid increases SorLA/LR11, a sorting protein with reduced expression in sporadic Alzheimer's disease (AD): relevance to AD prevention.

Science.gov (United States)

Ma, Qiu-Lan; Teter, Bruce; Ubeda, Oliver J; Morihara, Takashi; Dhoot, Dilsher; Nyby, Michael D; Tuck, Michael L; Frautschy, Sally A; Cole, Greg M

2007-12-26

Environmental and genetic factors, notably ApoE4, contribute to the etiology of late-onset Alzheimer's disease (LOAD). Reduced mRNA and protein for an apolipoprotein E (ApoE) receptor family member, SorLA (LR11) has been found in LOAD but not early-onset AD, suggesting that LR11 loss is not secondary to pathology. LR11 is a neuronal sorting protein that reduces amyloid precursor protein (APP) trafficking to secretases that generate beta-amyloid (Abeta). Genetic polymorphisms that reduce LR11 expression are associated with increased AD risk. However these polymorphisms account for only a fraction of cases with LR11 deficits, suggesting involvement of environmental factors. Because lipoprotein receptors are typically lipid-regulated, we postulated that LR11 is regulated by docosahexaenoic acid (DHA), an essential omega-3 fatty acid related to reduced AD risk and reduced Abeta accumulation. In this study, we report that DHA significantly increases LR11 in multiple systems, including primary rat neurons, aged non-Tg mice and an aged DHA-depleted APPsw AD mouse model. DHA also increased LR11 in a human neuronal line. In vivo elevation of LR11 was also observed with dietary fish oil in young rats with insulin resistance, a model for type II diabetes, another AD risk factor. These data argue that DHA induction of LR11 does not require DHA-depleting diets and is not age dependent. Because reduced LR11 is known to increase Abeta production and may be a significant genetic cause of LOAD, our results indicate that DHA increases in SorLA/LR11 levels may play an important role in preventing LOAD.

Very Low-Protein Diet (VLPD Reduces Metabolic Acidosis in Subjects with Chronic Kidney Disease: The “Nutritional Light Signal” of the Renal Acid Load

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Biagio Raffaele Di Iorio

2017-01-01

Full Text Available Background: Metabolic acidosis is a common complication of chronic kidney disease; current guidelines recommend treatment with alkali if bicarbonate levels are lower than 22 mMol/L. In fact, recent studies have shown that an early administration of alkali reduces progression of CKD. The aim of the study is to evaluate the effect of fruit and vegetables to reduce the acid load in CKD. Methods: We conducted a case-control study in 146 patients who received sodium bicarbonate. Of these, 54 patients assumed very low-protein diet (VLPD and 92 were controls (ratio 1:2. We calculated every three months the potential renal acid load (PRAL and the net endogenous acid production (NEAP, inversely correlated with serum bicarbonate levels and representing the non-volatile acid load derived from nutrition. Un-paired T-test and Chi-square test were used to assess differences between study groups at baseline and study completion. Two-tailed probability values ≤0.05 were considered statistically significant. Results: At baseline, there were no statistical differences between the two groups regarding systolic blood pressure (SBP, diastolic blood pressure (DBP, protein and phosphate intake, urinary sodium, potassium, phosphate and urea nitrogen, NEAP, and PRAL. VLPD patients showed at 6 and 12 months a significant reduction of SBP (p < 0.0001, DBP (p < 0.001, plasma urea (p < 0.0001 protein intake (p < 0.0001, calcemia (p < 0.0001, phosphatemia (p < 0.0001, phosphate intake (p < 0.0001, urinary sodium (p < 0.0001, urinary potassium (p < 0.002, and urinary phosphate (p < 0.0001. NEAP and PRAL were significantly reduced in VLPD during follow-up. Conclusion: VLPD reduces intake of acids; nutritional therapy of CKD, that has always taken into consideration a lower protein, salt, and phosphate intake, should be adopted to correct metabolic acidosis, an important target in the treatment of CKD patients. We provide useful indications regarding acid load of food and

Reduced dimensionality (3,2)D NMR experiments and their automated analysis: implications to high-throughput structural studies on proteins.

Science.gov (United States)

Reddy, Jithender G; Kumar, Dinesh; Hosur, Ramakrishna V

2015-02-01

Protein NMR spectroscopy has expanded dramatically over the last decade into a powerful tool for the study of their structure, dynamics, and interactions. The primary requirement for all such investigations is sequence-specific resonance assignment. The demand now is to obtain this information as rapidly as possible and in all types of protein systems, stable/unstable, soluble/insoluble, small/big, structured/unstructured, and so on. In this context, we introduce here two reduced dimensionality experiments – (3,2)D-hNCOcanH and (3,2)D-hNcoCAnH – which enhance the previously described 2D NMR-based assignment methods quite significantly. Both the experiments can be recorded in just about 2-3 h each and hence would be of immense value for high-throughput structural proteomics and drug discovery research. The applicability of the method has been demonstrated using alpha-helical bovine apo calbindin-D9k P43M mutant (75 aa) protein. Automated assignment of this data using AUTOBA has been presented, which enhances the utility of these experiments. The backbone resonance assignments so derived are utilized to estimate secondary structures and the backbone fold using Web-based algorithms. Taken together, we believe that the method and the protocol proposed here can be used for routine high-throughput structural studies of proteins. Copyright © 2014 John Wiley & Sons, Ltd.

Evaluation of the reliability of transport networks based on the stochastic flow of moving objects

International Nuclear Information System (INIS)

Wu Weiwei; Ning, Angelika; Ning Xuanxi

2008-01-01

In transport networks, human beings are moving objects whose moving direction is stochastic in emergency situations. Based on this idea, a new model-stochastic moving network (SMN) is proposed. It is different from binary-state networks and stochastic-flow networks. The flow of SMNs has multiple-saturated states, that correspond to different flow values in each arc. In this paper, we try to evaluate the system reliability, defined as the probability that the saturated flow of the network is not less than a given demand d. Based on this new model, we obtain the flow probability distribution of every arc by simulation. An algorithm based on the blocking cutset of the SMN is proposed to evaluate the network reliability. An example is used to show how to calculate the corresponding reliabilities for different given demands of the SMN. Simulation experiments of different size were made and the system reliability precision was calculated. The precision of simulation results also discussed

Metformin reduces lipid accumulation in macrophages by inhibiting FOXO1-mediated transcription of fatty acid-binding protein 4

International Nuclear Information System (INIS)

Song, Jun; Ren, Pingping; Zhang, Lin; Wang, Xing Li; Chen, Li; Shen, Ying H.

2010-01-01

Objective: The accumulation of lipids in macrophages contributes to the development of atherosclerosis. Strategies to reduce lipid accumulation in macrophages may have therapeutic potential for preventing and treating atherosclerosis and cardiovascular complications. The antidiabetic drug metformin has been reported to reduce lipid accumulation in adipocytes. In this study, we examined the effects of metformin on lipid accumulation in macrophages and investigated the mechanisms involved. Methods and results: We observed that metformin significantly reduced palmitic acid (PA)-induced intracellular lipid accumulation in macrophages. Metformin promoted the expression of carnitine palmitoyltransferase I (CPT-1), while reduced the expression of fatty acid-binding protein 4 (FABP4) which was involved in PA-induced lipid accumulation. Quantitative real-time PCR showed that metformin regulates FABP4 expression at the transcriptional level. We identified forkhead transcription factor FOXO1 as a positive regulator of FABP4 expression. Inhibiting FOXO1 expression with FOXO1 siRNA significantly reduced basal and PA-induced FABP4 expression. Overexpression of wild-type FOXO1 and constitutively active FOXO1 significantly increased FABP4 expression, whereas dominant negative FOXO1 dramatically decreased FABP4 expression. Metformin reduced FABP4 expression by promoting FOXO1 nuclear exclusion and subsequently inhibiting its activity. Conclusions: Taken together, these results suggest that metformin reduces lipid accumulation in macrophages by repressing FOXO1-mediated FABP4 transcription. Thus, metformin may have a protective effect against lipid accumulation in macrophages and may serve as a therapeutic agent for preventing and treating atherosclerosis in metabolic syndrome.

Metformin reduces lipid accumulation in macrophages by inhibiting FOXO1-mediated transcription of fatty acid-binding protein 4

Energy Technology Data Exchange (ETDEWEB)

Song, Jun [Qilu Hospital, Shandong University, Jinan, Shandong (China); Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX (United States); Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, TX (United States); Ren, Pingping; Zhang, Lin [Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX (United States); Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, TX (United States); Wang, Xing Li [Qilu Hospital, Shandong University, Jinan, Shandong (China); Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX (United States); Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, TX (United States); Chen, Li [Qilu Hospital, Shandong University, Jinan, Shandong (China); Shen, Ying H., E-mail: hyshen@bcm.edu [Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX (United States); Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, TX (United States)

2010-02-26

Objective: The accumulation of lipids in macrophages contributes to the development of atherosclerosis. Strategies to reduce lipid accumulation in macrophages may have therapeutic potential for preventing and treating atherosclerosis and cardiovascular complications. The antidiabetic drug metformin has been reported to reduce lipid accumulation in adipocytes. In this study, we examined the effects of metformin on lipid accumulation in macrophages and investigated the mechanisms involved. Methods and results: We observed that metformin significantly reduced palmitic acid (PA)-induced intracellular lipid accumulation in macrophages. Metformin promoted the expression of carnitine palmitoyltransferase I (CPT-1), while reduced the expression of fatty acid-binding protein 4 (FABP4) which was involved in PA-induced lipid accumulation. Quantitative real-time PCR showed that metformin regulates FABP4 expression at the transcriptional level. We identified forkhead transcription factor FOXO1 as a positive regulator of FABP4 expression. Inhibiting FOXO1 expression with FOXO1 siRNA significantly reduced basal and PA-induced FABP4 expression. Overexpression of wild-type FOXO1 and constitutively active FOXO1 significantly increased FABP4 expression, whereas dominant negative FOXO1 dramatically decreased FABP4 expression. Metformin reduced FABP4 expression by promoting FOXO1 nuclear exclusion and subsequently inhibiting its activity. Conclusions: Taken together, these results suggest that metformin reduces lipid accumulation in macrophages by repressing FOXO1-mediated FABP4 transcription. Thus, metformin may have a protective effect against lipid accumulation in macrophages and may serve as a therapeutic agent for preventing and treating atherosclerosis in metabolic syndrome.

Feeding broiler breeders a reduced balanced protein diet during the rearing and laying period impairs reproductive performance but enhances broiler offspring performance.

Science.gov (United States)

Lesuisse, J; Li, C; Schallier, S; Leblois, J; Everaert, N; Buyse, J

2017-09-01

Mammalian studies have shown that nutritional constraints during the perinatal period are able to program the progeny (metabolism, performance). The presented research aimed to investigate if broiler breeders and their offspring performance could be influenced by reducing the dietary crude protein (CP) level with 25%. A total of 160 day-old pure line A breeder females were randomly divided over 2 dietary treatments. The control group was fed commercial diets, whereas the reduced balanced protein (RP) breeders received an isoenergetic diet that was decreased with 25% in dietary CP and amino acid during their entire lifespan. The RP birds required an increased feed allowance, varying between 3 and 15%, to meet the same BW goals as their control fed counterparts. The difference in feed allocations and reduction of the dietary CP level resulted in a net protein reduction varying between 14 and 23%. At wk 27 and 40, the body composition of the breeders was changed as a result of the dietary treatment. At both ages, the proportional abdominal fat pad weight of the RP breeders was increased (P < 0.001), whereas the proportional breast muscle weight was only higher at wk 27 in the control group compared to the RP group (P < 0.001). Egg weight (P < 0.001) and egg production (P < 0.001) was decreased for the RP fed birds. The lower dietary CP level reduced the proportional albumen weight of the RP eggs (P = 0.006). Male offspring from RP breeders were characterized by an increase in BW from 28 d until 35 d of age (P = 0.015). Moreover, female progeny of RP breeders showed a reduced FCR (P = 0.025), whereas male progeny showed a tendency (P = 0.052) towards a lower FCR at 5 wk of age. In conclusion, lowering dietary CP levels in rearing and laying phase of breeders had a negative effect on breeder performance but enhanced live performance of the offspring. © 2017 Poultry Science Association Inc.

Inhibition of lectin-like oxidized low-density lipoprotein receptor-1 reduces cardiac fibroblast proliferation by suppressing GATA Binding Protein 4

Energy Technology Data Exchange (ETDEWEB)

Liu, Bin; Liu, Ning-Ning; Liu, Wei-Hua; Zhang, Shuang-Wei; Zhang, Jing-Zhi; Li, Ai-Qun [Department of Cardiology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Guangzhou Institute of Cardiovascular Disease, Guangzhou (China); Liu, Shi-Ming, E-mail: gzliushiming@126.com [Department of Cardiology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Guangzhou Institute of Cardiovascular Disease, Guangzhou (China)

2016-07-08

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and GATA Binding Protein 4 (GATA4) are important for the growth of cardiac fibroblasts (CFs). When deregulated, LOX-1 and GATA4 can cause cardiac remodeling. In the present study, we found novel evidence that GATA4 was required for the LOX-1 regulation of CF proliferation. The inhibition of LOX-1 by RNA interference LOX-1 lentivirus resulted in the loss of PI3K/Akt activation and GATA4 protein expression. The overexpression of LOX-1 by lentivirus rescued CF proliferation, PI3K/Akt activation, and GATA4 protein expression. Moreover, GATA4 overexpression enhanced CF proliferation with LOX-1 inhibition. We also found that the inhibition of PI3K/Akt activation by LY294002, a PI3K inhibitor, reduced cell proliferation and protein level of GATA4. In summary, GATA4 may play an important role in the LOX-1 and PI3K/Akt regulation of CF proliferation. -- Highlights: •GATA4 is regulated by LOX-1 signaling in CFs. •GATA4 is involved in LOX-1 regulating CF proliferation. •GATA4 is regulated by PI3K/Akt signaling in CFs.

Metabolic Characterization of Intact Cells Reveals Intracellular Amyloid Beta but Not Its Precursor Protein to Reduce Mitochondrial Respiration

Science.gov (United States)

Schaefer, Patrick M.; von Einem, Bjoern; Walther, Paul; Calzia, Enrico; von Arnim, Christine A. F.

2016-01-01

One hallmark of Alzheimer´s disease are senile plaques consisting of amyloid beta (Aβ), which derives from the processing of the amyloid precursor protein (APP). Mitochondrial dysfunction has been linked to the pathogenesis of Alzheimer´s disease and both Aβ and APP have been reported to affect mitochondrial function in isolated systems. However, in intact cells, considering a physiological localization of APP and Aβ, it is pending what triggers the mitochondrial defect. Thus, the aim of this study was to dissect the impact of APP versus Aβ in inducing mitochondrial alterations with respect to their subcellular localization. We performed an overexpression of APP or beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), increasing APP and Aβ levels or Aβ alone, respectively. Conducting a comprehensive metabolic characterization we demonstrate that only APP overexpression reduced mitochondrial respiration, despite lower extracellular Aβ levels compared to BACE overexpression. Surprisingly, this could be rescued by a gamma secretase inhibitor, oppositionally indicating an Aβ-mediated mitochondrial toxicity. Analyzing Aβ localization revealed that intracellular levels of Aβ and an increased spatial association of APP/Aβ with mitochondria are associated with reduced mitochondrial respiration. Thus, our data provide marked evidence for a prominent role of intracellular Aβ accumulation in Alzheimer´s disease associated mitochondrial dysfunction. Thereby it highlights the importance of the localization of APP processing and intracellular transport as a decisive factor for mitochondrial function, linking two prominent hallmarks of neurodegenerative diseases. PMID:28005987

A High-Protein Diet Reduces Weight Gain, Decreases Food Intake, Decreases Liver Fat Deposition, and Improves Markers of Muscle Metabolism in Obese Zucker Rats.

Science.gov (United States)

French, William W; Dridi, Sami; Shouse, Stephanie A; Wu, Hexirui; Hawley, Aubree; Lee, Sun-Ok; Gu, Xuan; Baum, Jamie I

2017-06-08

A primary factor in controlling and preventing obesity is through dietary manipulation. Diets higher in protein have been shown to improve body composition and metabolic health during weight loss. The objective of this study was to examine the effects of a high-protein diet versus a moderate-protein diet on muscle, liver and fat metabolism and glucose regulation using the obese Zucker rat. Twelve-week old, male, Zucker (fa/fa) and lean control (Fa/fa) rats were randomly assigned to either a high-protein (40% energy) or moderate-protein (20% energy) diet for 12 weeks, with a total of four groups: lean 20% protein (L20; n = 8), lean 40% protein (L40; n = 10), obese 20% protein (O20; n = 8), and obese 40% protein (O40; n = 10). At the end of 12 weeks, animals were fasted and euthanized. There was no difference in food intake between L20 and L40. O40 rats gained less weight and had lower food intake ( p diet rats, respectively. O40 had decreased skeletal muscle mechanistic target of rapamycin complex 1 (mTORC1) phosphorylation and peroxisome proliferator-activated receptor gamma (PPARγ) mRNA expression compared to O20 ( p protein kinase (AMPK), eukaryotic translation initiation factor 4E binding protein 1 (4EBP1), protein kinase B (Akt) or p70 ribosomal S6 kinase (p70S6K) phosphorylation. The data suggest that high-protein diets have the potential to reduce weight gain and alter metabolism, possibly through regulation of an mTORC1-dependent pathway in skeletal muscle.

Brief pressure overload of the left ventricle reduces myocardial infarct size via activation of protein kinase C.

Science.gov (United States)

Tang, Chia-Yu; Lai, Chang-Chi; Chiang, Shu-Chiung; Tseng, Kuo-Wei; Huang, Cheng-Hsiung

2015-09-01

We have previously reported that brief pressure overload of the left ventricle reduced myocardial infarct (MI) size. However, the role of protein kinase C (PKC) remains uncertain. In this study, we investigated whether pressure overload reduces MI size by activating PKC. MI was induced by a 40-minute occlusion of the left anterior descending coronary artery and a 3-hour reperfusion in anesthetized Sprague-Dawley rats. MI size was determined using triphenyl tetrazolium chloride staining. Brief pressure overload was achieved by two 10-minute partial snarings of the ascending aorta, raising the systolic left ventricular pressure 50% above the baseline value. Ischemic preconditioning was elicited by two 10-minute coronary artery occlusions and 10-minute reperfusions. Dimethyl sulfoxide (vehicle) or calphostin C (0.1 mg/kg, a specific inhibitor of PKC) was administered intravenously as pretreatment. The MI size, expressed as the percentage of the area at risk, was significantly reduced in the pressure overload group and the ischemic preconditioning group (19.0 ± 2.9% and 18.7 ± 3.0% vs. 26.1 ± 2.6% in the control group, where p overload and ischemic preconditioning (25.2 ± 2.4% and 25.0 ± 2.3%, where p overload of the left ventricle reduced MI size. Since calphostin C significantly limited the decrease of MI size, our results suggested that brief pressure overload reduces MI size via activation of PKC. Copyright © 2015. Published by Elsevier Taiwan.

Overexpression of protein kinase A - RIalpha reduces lipofection efficiency of cisplatin-resistant human tumor cells.

Science.gov (United States)

Son, K K; Rosenblatt, J

2001-04-10

Cisplatin-resistant variant A2780CP/vector cells were 4.0-5.3-fold more transfectable and 7.6-fold more resistant to cisplatin than their parent cisplatin-sensitive human ovarian carcinoma A2780/vector cells. Overexpression of cAMP-dependent protein kinase Type I regulatory alpha subunit (PKA-RIalpha) gene in A2780CP cells significantly reduced (maximum 47.0%) the transfection activity, with a slight reduction (maximum 27.3%) of cisplatin resistance, of A2780CP cells. However, RIalpha-overexpressing A2780CP (A2780CP/RIalpha) cells were still 2.5-to 3.0-fold more transfectable and 5.5-fold more resistant to cisplatin than A2780 cells. This results suggest that gene transfer efficiency is associated with cisplatin resistance, in part, through the PKA-mediated cAMP signal transduction pathway.

The Space Mobile Network

Science.gov (United States)

Israel, David

2017-01-01

The definition and development of the next generation space communications and navigation architecture is underway. The primary goals are to remove communications and navigations constraints from missions and to enable increased autonomy. The Space Mobile Network (SMN) is an architectural concept that includes new technology and operations that will provide flight systems with an similar user experience to terrestrial wireless mobile networks. This talk will describe the SMN and its proposed new features, such as Disruption Tolerant Networking (DTN), optical communications, and User Initiated Services (UIS).

Stemcell Information: SKIP000255 [SKIP Stemcell Database[Archive

Lifescience Database Archive (English)

Full Text Available 814)由来iPS細胞株。 ... SMN1 heterozygous deletion 　有病の子供のiPS細胞株：GM24468 | human ES-like -...; SMA1 253300 ... -- Female ... Yes No iPS cell line derived from GM03814 fibroblast.Clinically normal;2 affected child...ren (her child's iPSC GM24468).Heterozygous deletion of SMN1 | 脊髄性筋委縮症Ⅰ型(GM03813)の母親線維芽細胞(GM03

Analysis of the modifying influence of Plastin 3 (PLS3) on Spinal Muscular Atrophy (SMA) by generation of transgenic mouse models

OpenAIRE

Ackermann, Bastian

2011-01-01

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by the loss of α-motor neurons in the ventral horn of the spinal cord. Depending on the severity, the clinical spectrum of SMA ranges from early infant death to normal adult life with only mild muscle weakness. To date, no cure is available. SMA is caused by the homozygous loss of the survival motor neuron gene 1 (SMN1). Besides SMN1, another nearly identical copy of the gene is present in the human genome, thus called...

Lower Squalene Epoxidase and Higher Scavenger Receptor Class B Type 1 Protein Levels Are Involved in Reduced Serum Cholesterol Levels in Stroke-Prone Spontaneously Hypertensive Rats.

Science.gov (United States)

Michihara, Akihiro; Mido, Mayuko; Matsuoka, Hiroshi; Mizutani, Yurika

2015-01-01

A lower serum cholesterol level was recently shown to be one of the causes of stroke in an epidemiological study. Spontaneously hypertensive rats stroke-prone (SHRSP) have lower serum cholesterol levels than normotensive Wistar-Kyoto rats (WKY). To elucidate the mechanisms responsible for the lower serum cholesterol levels in SHRSP, we determined whether the amounts of cholesterol biosynthetic enzymes or the receptor and transporter involved in cholesterol uptake and efflux in the liver were altered in SHRSP. When the mRNA levels of seven cholesterol biosynthetic enzymes were measured using real-time polymerase chain reaction (PCR), farnesyl pyrophosphate synthase and squalene epoxidase (SQE) levels in the liver of SHRSP were significantly lower than those in WKY. SQE protein levels were significantly reduced in tissues other than the brain of SHRSP. No significant differences were observed in low-density lipoprotein (LDL) receptor (uptake of serum LDL-cholesterol) or ATP-binding cassette transporter A1 (efflux of cholesterol from the liver/formation of high-density lipoprotein (HDL)) protein levels in the liver and testis between SHRSP and WKY, whereas scavenger receptor class B type 1 (SRB1: uptake of serum HDL-cholesterol) protein levels were higher in the livers of SHRSP. These results indicated that the lower protein levels of SQE and higher protein levels of SRB1 in the liver were involved in the reduced serum cholesterol levels in SHRSP.

Milk proteins interact with goat Binder of SPerm (BSP) proteins and decrease their binding to sperm.

Science.gov (United States)

de Menezes, Erika Bezerra; van Tilburg, Mauricio; Plante, Geneviève; de Oliveira, Rodrigo V; Moura, Arlindo A; Manjunath, Puttaswamy

2016-11-01

Seminal plasma Binder of SPerm (BSP) proteins bind to sperm at ejaculation and promote capacitation. When in excess, however, BSP proteins damage the sperm membrane. It has been suggested that milk components of semen extenders associate with BSP proteins, potentially protecting sperm. Thus, this study was conducted to investigate if milk proteins interact with BSP proteins and reduce BSP binding to goat sperm. Using gel filtration chromatography, milk was incubated with goat seminal plasma proteins and loaded onto columns with and without calcium. Milk was also fractionated into parts containing mostly whey proteins or mostly caseins, incubated with seminal plasma proteins and subjected to gel filtration. Eluted fractions were evaluated by immunoblot using anti-goat BSP antibodies, confirming milk protein-BSP protein interactions. As determined by ELISA, milk proteins coated on polystyrene wells bound to increasing of goat BSP proteins. Far-western dot blots confirmed that BSP proteins bound to caseins and β-lactoglobulin in a concentration-dependent manner. Then, cauda epididymal sperm from five goats was incubated with seminal plasma; seminal plasma followed by milk; and milk followed by seminal plasma. Sperm membrane proteins were extracted and evaluated by immunoblotting. The pattern of BSP binding to sperm membrane proteins was reduced by 59.3 % when epididymal sperm were incubated with seminal plasma and then with skimmed milk (p  0.05). In conclusion, goat BSP proteins have an affinity for caseins and whey proteins. Milk reduces BSP binding to goat sperm, depending whether or not sperm had been previously exposed to seminal plasma. Such events may explain the protective effect of milk during goat sperm preservation.

Inhibition of Cyclic Adenosine Monophosphate (cAMP-response Element-binding Protein (CREB-binding Protein (CBP/β-Catenin Reduces Liver Fibrosis in Mice

Directory of Open Access Journals (Sweden)

Yosuke Osawa

2015-11-01

Full Text Available Wnt/β-catenin is involved in every aspect of embryonic development and in the pathogenesis of many human diseases, and is also implicated in organ fibrosis. However, the role of β-catenin-mediated signaling on liver fibrosis remains unclear. To explore this issue, the effects of PRI-724, a selective inhibitor of the cAMP-response element-binding protein-binding protein (CBP/β-catenin interaction, on liver fibrosis were examined using carbon tetrachloride (CCl4- or bile duct ligation (BDL-induced mouse liver fibrosis models. Following repetitive CCl4 administrations, the nuclear translocation of β-catenin was observed only in the non-parenchymal cells in the liver. PRI-724 treatment reduced the fibrosis induced by CCl4 or BDL. C-82, an active form of PRI-724, inhibited the activation of isolated primary mouse quiescent hepatic stellate cells (HSCs and promoted cell death in culture-activated HSCs. During the fibrosis resolution period, an increase in F4/80+ CD11b+ and Ly6Clow CD11b+ macrophages was induced by CCl4 and was sustained for two weeks thereafter, even after having stopped CCl4 treatment. PRI-724 accelerated the resolution of CCl4-induced liver fibrosis, and this was accompanied by increased matrix metalloproteinase (MMP-9, MMP-2, and MMP-8 expression in intrahepatic leukocytes. In conclusion, targeting the CBP/β-catenin interaction may become a new therapeutic strategy in treating liver fibrosis.

ProteinShop: A tool for interactive protein manipulation and steering

Energy Technology Data Exchange (ETDEWEB)

Crivelli, Silvia; Kreylos, Oliver; Max, Nelson; Hamann, Bernd; Bethel, Wes

2004-05-25

We describe ProteinShop, a new visualization tool that streamlines and simplifies the process of determining optimal protein folds. ProteinShop may be used at different stages of a protein structure prediction process. First, it can create protein configurations containing secondary structures specified by the user. Second, it can interactively manipulate protein fragments to achieve desired folds by adjusting the dihedral angles of selected coil regions using an Inverse Kinematics method. Last, it serves as a visual framework to monitor and steer a protein structure prediction process that may be running on a remote machine. ProteinShop was used to create initial configurations for a protein structure prediction method developed by a team that competed in CASP5. ProteinShop's use accelerated the process of generating initial configurations, reducing the time required from days to hours. This paper describes the structure of ProteinShop and discusses its main features.

Pramipexole Modulates Interregional Connectivity Within the Sensorimotor Network.

Science.gov (United States)

Ye, Zheng; Hammer, Anke; Münte, Thomas F

2017-05-01

Pramipexole is widely prescribed to treat Parkinson's disease but has been reported to cause impulse control disorders such as pathological gambling. Recent neurocomputational models suggested that D2 agonists may distort functional connections between the striatum and the motor cortex, resulting in impaired reinforcement learning and pathological gambling. To examine how D2 agonists modulate the striatal-motor connectivity, we carried out a pharmacological resting-state functional magnetic resonance imaging study with a double-blind randomized within-subject crossover design. We analyzed the medication-induced changes of network connectivity and topology with two approaches, an independent component analysis (ICA) and a graph theoretical analysis (GTA). The ICA identified the sensorimotor network (SMN) as well as other classical resting-state networks. Within the SMN, the connectivity between the right caudate nucleus and other cortical regions was weaker under pramipexole than under placebo. The GTA measured the topological properties of the whole-brain network at global and regional levels. Both the whole-brain network under placebo and that under pramipexole were identified as small-world networks. The two whole-brain networks were similar in global efficiency, clustering coefficient, small-world index, and modularity. However, the degree of the right caudate nucleus decreased under pramipexole mainly due to the loss of the connectivity with the supplementary motor area, paracentral lobule, and precentral and postcentral gyrus of the SMN. The two network analyses consistently revealed that pramipexole weakened the functional connectivity between the caudate nucleus and the SMN regions.

Intrinsically disordered proteins as molecular shields†

Science.gov (United States)

Chakrabortee, Sohini; Tripathi, Rashmi; Watson, Matthew; Kaminski Schierle, Gabriele S.; Kurniawan, Davy P.; Kaminski, Clemens F.; Wise, Michael J.; Tunnacliffe, Alan

2017-01-01

The broad family of LEA proteins are intrinsically disordered proteins (IDPs) with several potential roles in desiccation tolerance, or anhydrobiosis, one of which is to limit desiccation-induced aggregation of cellular proteins. We show here that this activity, termed molecular shield function, is distinct from that of a classical molecular chaperone, such as HSP70 – while HSP70 reduces aggregation of citrate synthase (CS) on heating, two LEA proteins, a nematode group 3 protein, AavLEA1, and a plant group 1 protein, Em, do not; conversely, the LEA proteins reduce CS aggregation on desiccation, while HSP70 lacks this ability. There are also differences in interaction with client proteins – HSP70 can be co-immunoprecipitated with a polyglutamine-containing client, consistent with tight complex formation, whereas the LEA proteins can not, although a loose interaction is observed by Förster resonance energy transfer. In a further exploration of molecular shield function, we demonstrate that synthetic polysaccharides, like LEA proteins, are able to reduce desiccation-induced aggregation of a water-soluble proteome, consistent with a steric interference model of anti-aggregation activity. If molecular shields operate by reducing intermolecular cohesion rates, they should not protect against intramolecular protein damage. This was tested using the monomeric red fluorescent protein, mCherry, which does not undergo aggregation on drying, but the absorbance and emission spectra of its intrinsic fluorophore are dramatically reduced, indicative of intramolecular conformational changes. As expected, these changes are not prevented by AavLEA1, except for a slight protection at high molar ratios, and an AavLEA1-mCherry fusion protein is damaged to the same extent as mCherry alone. A recent hypothesis proposed that proteomes from desiccation-tolerant species contain a higher degree of disorder than intolerant examples, and that this might provide greater intrinsic stability

Intrinsically disordered proteins as molecular shields.

Science.gov (United States)

Chakrabortee, Sohini; Tripathi, Rashmi; Watson, Matthew; Schierle, Gabriele S Kaminski; Kurniawan, Davy P; Kaminski, Clemens F; Wise, Michael J; Tunnacliffe, Alan

2012-01-01

The broad family of LEA proteins are intrinsically disordered proteins (IDPs) with several potential roles in desiccation tolerance, or anhydrobiosis, one of which is to limit desiccation-induced aggregation of cellular proteins. We show here that this activity, termed molecular shield function, is distinct from that of a classical molecular chaperone, such as HSP70 - while HSP70 reduces aggregation of citrate synthase (CS) on heating, two LEA proteins, a nematode group 3 protein, AavLEA1, and a plant group 1 protein, Em, do not; conversely, the LEA proteins reduce CS aggregation on desiccation, while HSP70 lacks this ability. There are also differences in interaction with client proteins - HSP70 can be co-immunoprecipitated with a polyglutamine-containing client, consistent with tight complex formation, whereas the LEA proteins can not, although a loose interaction is observed by Förster resonance energy transfer. In a further exploration of molecular shield function, we demonstrate that synthetic polysaccharides, like LEA proteins, are able to reduce desiccation-induced aggregation of a water-soluble proteome, consistent with a steric interference model of anti-aggregation activity. If molecular shields operate by reducing intermolecular cohesion rates, they should not protect against intramolecular protein damage. This was tested using the monomeric red fluorescent protein, mCherry, which does not undergo aggregation on drying, but the absorbance and emission spectra of its intrinsic fluorophore are dramatically reduced, indicative of intramolecular conformational changes. As expected, these changes are not prevented by AavLEA1, except for a slight protection at high molar ratios, and an AavLEA1-mCherry fusion protein is damaged to the same extent as mCherry alone. A recent hypothesis proposed that proteomes from desiccation-tolerant species contain a higher degree of disorder than intolerant examples, and that this might provide greater intrinsic stability

Active site mutations in yeast protein disulfide isomerase cause dithiothreitol sensitivity and a reduced rate of protein folding in the endoplasmic reticulum

DEFF Research Database (Denmark)

Holst, B; Tachibana, C; Winther, Jakob R.

1997-01-01

Aspects of protein disulfide isomerase (PDI) function have been studied in yeast in vivo. PDI contains two thioredoxin-like domains, a and a', each of which contains an active-site CXXC motif. The relative importance of the two domains was analyzed by rendering each one inactive by mutation to SGAS....... Such mutations had no significant effect on growth. The domains however, were not equivalent since the rate of folding of carboxypeptidase Y (CPY) in vivo was reduced by inactivation of the a domain but not the a' domain. To investigate the relevance of PDI redox potential, the G and H positions of each CGHC......-deleted strains overexpressing the yeast PDI homologue EUG1 are viable. Exchanging the wild-type Eug1p C(L/I)HS active site sequences for C(L/I)HC increased the growth rate significantly, however, further highlighting the importance of the oxidizing function for optimal growth....

Karakteristik Protein dan Nitrogen Non Protein Daging Ikan Cucut Lanyam (Charcharhinus limbatus (Characteristics of Protein and Non Protein Nitrogen in Lanyam Shark Muscle

Directory of Open Access Journals (Sweden)

Yuspihana Fitrial

2017-02-01

Based on protein solubility of Lanyam muscle at pH 1.5 to 12 obtained two points which is minimum solubility at pH 4.5 and pH 9. Based on the classification Osborn, Lanyam muscle contained albumin (28.64%, globulin (13:44%, prolamin (03.29%, glutelin (33.70%. Observation of non-protein nitrogen levels indicated that the washing process was very effective to reduce non-protein nitrogen levels up to 62.34% and urea levels up to 58% . Differential Scanning Calorimetry Study of Lanyam mince showed two types of protein that has a different stability to heat and after added 2.5% NaCl formed a peak which is a fusion of both these proteins

NMR Studies of Protein Hydration and Protein-Ligand Interactions

Science.gov (United States)

Chong, Yuan

Water on the surface of a protein is called hydration water. Hydration water is known to play a crucial role in a variety of biological processes including protein folding, enzymatic activation, and drug binding. Although the significance of hydration water has been recognized, the underlying mechanism remains far from being understood. This dissertation employs a unique in-situ nuclear magnetic resonance (NMR) technique to study the mechanism of protein hydration and the role of hydration in alcohol-protein interactions. Water isotherms in proteins are measured at different temperatures via the in-situ NMR technique. Water is found to interact differently with hydrophilic and hydrophobic groups on the protein. Water adsorption on hydrophilic groups is hardly affected by the temperature, while water adsorption on hydrophobic groups strongly depends on the temperature around 10 C, below which the adsorption is substantially reduced. This effect is induced by the dramatic decrease in the protein flexibility below 10 C. Furthermore, nanosecond to microsecond protein dynamics and the free energy, enthalpy, and entropy of protein hydration are studied as a function of hydration level and temperature. A crossover at 10 C in protein dynamics and thermodynamics is revealed. The effect of water at hydrophilic groups on protein dynamics and thermodynamics shows little temperature dependence, whereas water at hydrophobic groups has stronger effect above 10 C. In addition, I investigate the role of water in alcohol binding to the protein using the in-situ NMR detection. The isotherms of alcohols are first measured on dry proteins, then on proteins with a series of controlled hydration levels. The free energy, enthalpy, and entropy of alcohol binding are also determined. Two distinct types of alcohol binding are identified. On the one hand, alcohols can directly bind to a few specific sites on the protein. This type of binding is independent of temperature and can be

Space Mobile Network: A Near Earth Communication and Navigation Architecture

Science.gov (United States)

Israel, Dave J.; Heckler, Greg; Menrad, Robert J.

2016-01-01

This paper describes a Space Mobile Network architecture, the result of a recently completed NASA study exploring architectural concepts to produce a vision for the future Near Earth communications and navigation systems. The Space Mobile Network (SMN) incorporates technologies, such as Disruption Tolerant Networking (DTN) and optical communications, and new operations concepts, such as User Initiated Services, to provide user services analogous to a terrestrial smartphone user. The paper will describe the SMN Architecture, envisioned future operations concepts, opportunities for industry and international collaboration and interoperability, and technology development areas and goals.

Acyclovir Therapy Reduces the CD4+ T Cell Response against the Immunodominant pp65 Protein from Cytomegalovirus in Immune Competent Individuals.

Directory of Open Access Journals (Sweden)

Annette Pachnio

Full Text Available Cytomegalovirus (CMV infects the majority of the global population and leads to the development of a strong virus-specific immune response. The CMV-specific CD4+ and CD8+ T cell immune response can comprise between 10 and 50% of the T cell pool within peripheral blood and there is concern that this may impair immunity to other pathogens. Elderly individuals with the highest magnitude of CMV-specific immune response have been demonstrated to be at increased risk of mortality and there is increasing interest in interventions that may serve to moderate this. Acyclovir is an anti-viral drug with activity against a range of herpes viruses and is used as long term treatment to suppress reactivation of herpes simplex virus. We studied the immune response to CMV in patients who were taking acyclovir to assess if therapy could be used to suppress the CMV-specific immune response. The T cell reactivity against the immunodominant late viral protein pp65 was reduced by 53% in people who were taking acyclovir. This effect was seen within one year of therapy and was observed primarily within the CD4+ response. Acyclovir treatment only modestly influenced the immune response to the IE-1 target protein. These data show that low dose acyclovir treatment has the potential to modulate components of the T cell response to CMV antigen proteins and indicate that anti-viral drugs should be further investigated as a means to reduce the magnitude of CMV-specific immune response and potentially improve overall immune function.

Breakfasts Higher in Protein Increase Postprandial Energy Expenditure, Increase Fat Oxidation, and Reduce Hunger in Overweight Children from 8 to 12 Years of Age.

Science.gov (United States)

Baum, Jamie I; Gray, Michelle; Binns, Ashley

2015-10-01

Currently 1 in every 3 children aged 2-19 y is overweight or obese. Breakfast is a key component of a healthy diet and has the potential to affect children's health. The objective of this study was to determine whether consumption of a protein-based breakfast (PRO) increases postprandial energy metabolism and substrate oxidation, reduces hunger, and reduces food intake at lunch compared with a carbohydrate-based breakfast (CHO) in normal weight (NW) vs. overweight/obese (OW) children. A randomized, crossover-design study was conducted in NW (n = 16; 33 ± 1 kg) and OW (n = 13; 46 ± 2 kg) children (10 ± 1 y). Participants were served either a PRO [344 kcal, 21% protein (18 g), 52% carbohydrate, and 27% fat] or CHO [327 kcal, 4% protein (3 g), 67% carbohydrate, and 29% fat]. Energy expenditure (EE), substrate oxidation, appetite, and blood glucose were measured over a 4 h period. Four hour postprandial participants were provided with access to a lunch buffet and food intake was recorded. After breakfast, OW children in the PRO group had higher (P fat oxidation over the 4 h period than did the NW children in the CHO and PRO groups. There was no difference in postprandial EE or carbohydrate oxidation between the CHO and PRO groups over the 4 h period; however, fat oxidation was 16% higher (P children. A PRO increases postprandial EE and fat oxidation, reduces hunger, and increases satiety when compared with a carbohydrate-based breakfast. © 2015 American Society for Nutrition.

Increased Protein Consumption during the Day from an Energy-Restricted Diet Augments Satiety but Does Not Reduce Daily Fat or Carbohydrate Intake on a Free-Living Test Day in Overweight Women.

Science.gov (United States)

Gwin, Jess A; Maki, Kevin C; Leidy, Heather J

2017-12-01

Background: Higher-protein (HP) energy-restriction diets improve weight management to a greater extent than normal-protein (NP) versions. Potential mechanisms of action with regard to assessment of eating behaviors across the day have not been widely examined during energy restriction. Objectives: The objectives of this study were to test whether the consumption of an HP energy-restriction diet reduces carbohydrate and fat intakes through improvements in daily appetite, satiety, and food cravings compared with NP versions and to test whether protein type within the NP diets alters protein-related satiety. Methods: Seventeen overweight women [mean ± SEM age: 36 ± 1 y; body mass index (kg/m 2 ): 28.4 ± 0.1] completed a randomized, controlled-feeding crossover study. Participants were provided with the following ∼1250-kcal/d energy-restricted (-750-kcal/d deficit) diets, each for 6 d: HP [124 g protein/d; 60% from beef and 40% from plant sources (HP-BEEF)] or NP (48 g protein/d) that was protein-type matched (NP-BEEF) or unmatched [100% from plant-based sources (NP-PLANT)]. On day 6 of each diet period, participants completed a 12-h testing day containing repetitive appetite, satiety, and food-craving questionnaires. On day 7, the participants were asked to consume their protein requirement within each respective diet but were provided with a surplus of carbohydrate- and fat-rich foods to consume, ad libitum, at each eating occasion across the day. All outcomes reported were primary study outcomes. Results: The HP-BEEF diet reduced daily hunger by 16%, desire to eat by 15%, prospective food consumption by 14%, and fast-food cravings by 15% but increased daily fullness by 25% compared with the NP-BEEF and NP-PLANT diets (all P protein throughout the day did not reduce the energy consumed ad libitum from the fat- and carbohydrate-rich foods (HP-BEEF: 2000 ± 180 kcal/d; NP-BEEF: 2120 ± 190 kcal/d; NP-PLANT: 2070 ± 180 kcal/d). None of the outcomes differed

The HCM-linked W792R mutation in cardiac myosin-binding protein C reduces C6 FnIII domain stability.

Science.gov (United States)

Smelter, Dan F; de Lange, Willem J; Cai, Wenxuan; Ge, Ying; Ralphe, J Carter

2018-06-01

haploinsufficiency. NEW & NOTEWORTHY This study is one of the first to describe a disease mechanism for a missense mutation in cardiac myosin-binding protein C linked to hypertrophic cardiomyopathy. The mutation decreases stability of the fibronectin type III domain and results in substantially reduced mutant protein expression dissonant to transcript abundance.

Reduced amyloidogenic processing of the amyloid beta-protein precursor by the small-molecule Differentiation Inducing Factor-1.

Science.gov (United States)

Myre, Michael A; Washicosky, Kevin; Moir, Robert D; Tesco, Giuseppina; Tanzi, Rudolph E; Wasco, Wilma

2009-04-01

The detection of cell cycle proteins in Alzheimer's disease (AD) brains may represent an early event leading to neurodegeneration. To identify cell cycle modifiers with anti-Abeta properties, we assessed the effect of Differentiation-Inducing Factor-1 (DIF-1), a unique, small-molecule from Dictyostelium discoideum, on the proteolysis of the amyloid beta-protein precursor (APP) in a variety of different cell types. We show that DIF-1 slows cell cycle progression through G0/G1 that correlates with a reduction in cyclin D1 protein levels. Western blot analysis of DIF-treated cells and conditioned medium revealed decreases in the levels of secreted APP, mature APP, and C-terminal fragments. Assessment of conditioned media by sandwich ELISA showed reduced levels of Abeta40 and Abeta42, also demonstrating that treatment with DIF-1 effectively decreases the ratio of Abeta42 to Abeta40. In addition, DIF-1 significantly diminished APP phosphorylation at residue T668. Interestingly, site-directed mutagenesis of APP residue Thr668 to alanine or glutamic acid abolished the effect of DIF-1 on APP proteolysis and restored secreted levels of Abeta. Finally, DIF-1 prevented the accumulation of APP C-terminal fragments induced by the proteasome inhibitor lactacystin, and calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Our findings suggest that DIF-1 affects G0/G1-associated amyloidogenic processing of APP by a gamma-secretase-, proteasome- and calpain-insensitive pathway, and that this effect requires the presence of residue Thr668.

Reduced amyloidogenic processing of the amyloid β-protein precursor by the small-molecule Differentiation Inducing Factor-1

Science.gov (United States)

Myre, Michael A.; Washicosky, Kevin; Moir, Robert D.; Tesco, Giuseppina; Tanzi, Rudolph E.; Wasco, Wilma

2013-01-01

The detection of cell cycle proteins in Alzheimer’s disease (AD) brains may represent an early event leading to neurodegeneration. To identify cell cycle modifiers with anti-Aβ properties, we assessed the effect of Differentiation-Inducing Factor-1 (DIF-1), a unique, small-molecule from Dictyostelium discoideum, on the proteolysis of the amyloid β-protein precursor (APP) in a variety of different cell types. We show that DIF-1 slows cell cycle progression through G0/G1 that correlates with a reduction in cyclin D1 protein levels. Western blot analysis of DIF-treated cells and conditioned medium revealed decreases in the levels of secreted APP, mature APP, and C-terminal fragments. Assessment of conditioned media by sandwich ELISA showed reduced levels of Aβ40 and Aβ42, also demonstrating that treatment with DIF-1 effectively decreases the ratio of Aβ42 to Aβ40. In addition, DIF-1 significantly diminished APP phosphorylation at residue T668. Interestingly, site-directed mutagenesis of APP residue Thr668 to alanine or glutamic acid abolished the effect of DIF-1 on APP proteolysis and restored secreted levels of Aβ. Finally, DIF-1 prevented the accumulation of APP C-terminal fragments induced by the proteasome inhibitor lactacystin, and calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Our findings suggest that DIF-1 affects G0/G1-associated amyloidogenic processing of APP by a γ-secretase-, proteasome- and calpain-insensitive pathway, and that this effect requires the presence of residue Thr668. PMID:19154786

Effects of Glasswort (Salicornia herbacea L.) Hydrates on Quality Characteristics of Reduced-salt, Reduced-fat Frankfurters

Science.gov (United States)

Choi, Yun-Sang

2015-01-01

This study evaluated the effects of adding glasswort hydrate containing non-meat ingredient (GM, carboxy methyl cellulose; GC, carrageenan; GI, isolated soy protein; GS, sodium caseinate) on the quality characteristics of reduced-salt, reduced-fat frankfurters. The pH and color evaluation showed significant differences, depending on the type of glasswort hydrate added (p<0.05). In the raw batters and cooked frankfurters, the addition of glasswort hydrate decreased the redness and increased the yellowness in comparison with frankfurters without glasswort hydrate. The reduction in salt and fat content significantly increased cooking loss and decreased hardness, tenderness and juiciness (p<0.05). Glasswort hydrate containing non-meat ingredient improved cooking loss, water holding capacity, emulsion stability, hardness, and viscosity of reduced-salt, reduced-fat frankfurters. The GM treatment had the highest myofibiliar protein solubility among all treatments, which was associated with emulsion stability and viscosity. The GC treatment had higher values for all texture parameters than the control. In the sensory evaluation, the addition of glasswort hydrate with non-meat ingredient improved tenderness and juiciness of reduced-salt, reduced-fat frankfurters. GM, GC, and GI treatments improved not only the physicochemical properties but also the sensory characteristics of reduced-salt, reduced-fat frankfurters. The results indicated that the use of glasswort hydrate containing non-meat ingredient was improved the quality characteristics of reduced-salt, reduced-fat frankfurters. PMID:26877638

ABI domain-containing proteins contribute to surface protein display and cell division in Staphylococcus aureus.

Science.gov (United States)

Frankel, Matthew B; Wojcik, Brandon M; DeDent, Andrea C; Missiakas, Dominique M; Schneewind, Olaf

2010-10-01

The human pathogen Staphylococcus aureus requires cell wall anchored surface proteins to cause disease. During cell division, surface proteins with YSIRK signal peptides are secreted into the cross-wall, a layer of newly synthesized peptidoglycan between separating daughter cells. The molecular determinants for the trafficking of surface proteins are, however, still unknown. We screened mutants with non-redundant transposon insertions by fluorescence-activated cell sorting for reduced deposition of protein A (SpA) into the staphylococcal envelope. Three mutants, each of which harboured transposon insertions in genes for transmembrane proteins, displayed greatly reduced envelope abundance of SpA and surface proteins with YSIRK signal peptides. Characterization of the corresponding mutations identified three transmembrane proteins with abortive infectivity (ABI) domains, elements first described in lactococci for their role in phage exclusion. Mutations in genes for ABI domain proteins, designated spdA, spdB and spdC (surface protein display), diminish the expression of surface proteins with YSIRK signal peptides, but not of precursor proteins with conventional signal peptides. spdA, spdB and spdC mutants display an increase in the thickness of cross-walls and in the relative abundance of staphylococci with cross-walls, suggesting that spd mutations may represent a possible link between staphylococcal cell division and protein secretion. © 2010 Blackwell Publishing Ltd.

Efficient protein structure search using indexing methods.

Science.gov (United States)

Kim, Sungchul; Sael, Lee; Yu, Hwanjo

2013-01-01

Understanding functions of proteins is one of the most important challenges in many studies of biological processes. The function of a protein can be predicted by analyzing the functions of structurally similar proteins, thus finding structurally similar proteins accurately and efficiently from a large set of proteins is crucial. A protein structure can be represented as a vector by 3D-Zernike Descriptor (3DZD) which compactly represents the surface shape of the protein tertiary structure. This simplified representation accelerates the searching process. However, computing the similarity of two protein structures is still computationally expensive, thus it is hard to efficiently process many simultaneous requests of structurally similar protein search. This paper proposes indexing techniques which substantially reduce the search time to find structurally similar proteins. In particular, we first exploit two indexing techniques, i.e., iDistance and iKernel, on the 3DZDs. After that, we extend the techniques to further improve the search speed for protein structures. The extended indexing techniques build and utilize an reduced index constructed from the first few attributes of 3DZDs of protein structures. To retrieve top-k similar structures, top-10 × k similar structures are first found using the reduced index, and top-k structures are selected among them. We also modify the indexing techniques to support θ-based nearest neighbor search, which returns data points less than θ to the query point. The results show that both iDistance and iKernel significantly enhance the searching speed. In top-k nearest neighbor search, the searching time is reduced 69.6%, 77%, 77.4% and 87.9%, respectively using iDistance, iKernel, the extended iDistance, and the extended iKernel. In θ-based nearest neighbor serach, the searching time is reduced 80%, 81%, 95.6% and 95.6% using iDistance, iKernel, the extended iDistance, and the extended iKernel, respectively.

Dietary protein restriction for renal patients: don't forget protein-free foods.

Science.gov (United States)

D'Alessandro, Claudia; Rossi, Andrea; Innocenti, Maurizio; Ricchiuti, Guido; Bozzoli, Laura; Sbragia, Giulietta; Meola, Mario; Cupisti, Adamasco

2013-09-01

The treatment of chronic kidney disease (CKD) consists of pharmacological, nutritional, and psychological-social approaches. The dietary therapy of CKD, namely a low-protein low-phosphorus diet, plays a crucial role in contributing to delay the onset of end-stage renal disease (ESRD) and to protect cardiovascular and nutritional status. The protein-free food products represent a very important tool for the implementation of a low-protein diet to ensure adequate energy supply, reducing the production of nitrogenous waste products. This survey included 100 consecutive CKD patients who were asked their opinion about the use of protein-free foods. Ninety-eight patients (98%) reported a regular daily intake of protein-free pasta (as macaroni, spaghetti, etc.), which was the preferred product consumed. Actually, the taste and texture of protein-free pasta were considered as "good" or "very good" by 70% of patients. Conversely, 43% of CKD patients perceived the taste and texture of protein-free bread as "bad" or "very bad", and 30% found it "acceptable". Therefore, the main concern for the implementation of low-protein diets is the use and palatability of the protein-free products, bread in particular. The use of these products may help in reducing protein, phosphorus, and sodium intake while supplying an adequate energy intake, which represents the basis for a nutritionally safe and successful dietary treatment of advanced CKD patients. Manufacturers and food technology should make more efforts to finding new solutions to improve the taste and texture of protein-free products. Copyright © 2013 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.

Very Low-Protein Diet (VLPD) Reduces Metabolic Acidosis in Subjects with Chronic Kidney Disease: The "Nutritional Light Signal" of the Renal Acid Load.

Science.gov (United States)

Di Iorio, Biagio Raffaele; Di Micco, Lucia; Marzocco, Stefania; De Simone, Emanuele; De Blasio, Antonietta; Sirico, Maria Luisa; Nardone, Luca

2017-01-17

Metabolic acidosis is a common complication of chronic kidney disease; current guidelines recommend treatment with alkali if bicarbonate levels are lower than 22 mMol/L. In fact, recent studies have shown that an early administration of alkali reduces progression of CKD. The aim of the study is to evaluate the effect of fruit and vegetables to reduce the acid load in CKD. We conducted a case-control study in 146 patients who received sodium bicarbonate. Of these, 54 patients assumed very low-protein diet (VLPD) and 92 were controls (ratio 1:2). We calculated every three months the potential renal acid load (PRAL) and the net endogenous acid production (NEAP), inversely correlated with serum bicarbonate levels and representing the non-volatile acid load derived from nutrition. Un-paired T -test and Chi-square test were used to assess differences between study groups at baseline and study completion. Two-tailed probability values ≤0.05 were considered statistically significant. At baseline, there were no statistical differences between the two groups regarding systolic blood pressure (SBP), diastolic blood pressure (DBP), protein and phosphate intake, urinary sodium, potassium, phosphate and urea nitrogen, NEAP, and PRAL. VLPD patients showed at 6 and 12 months a significant reduction of SBP ( p protein intake ( p intake ( p intake of acids; nutritional therapy of CKD, that has always taken into consideration a lower protein, salt, and phosphate intake, should be adopted to correct metabolic acidosis, an important target in the treatment of CKD patients. We provide useful indications regarding acid load of food and drinks-the "acid load dietary traffic light".

Solid malignant neoplasms after childhood irradiation: decrease of the relative risk with time after irradiation

International Nuclear Information System (INIS)

Vathaire, F. de; Shamsaldin, A.; Grimaud, E.; Campbell, S.; Guerra, M.; Raquin, M.; Hardiman, C.; Jan, P.; Rumeau, N.; Diallo, I.; Nicolazic, G.; Lamon, A.; Oberlin, O.; Cervens, C. de; Suarez, A.; Meresse, V.; Eschwege, F.; Sancho-Garnier, H.; Chavaudra, J.; Lermerle, J.; Bessa, E.; Bell, J.; Hawkins, M.; Schlienger, J.Y.; Panis, X.; Lagrande, J.L.; Gaboriaud, G.; Zucker, J.M.; Daly-Schveitzer, N.

1995-01-01

The pattern of the temporal distribution of solid cancer incidence after irradiation in childhood is not well known, although, its importance in radioprotection is well known. We studied a cohort of 1 055 children from 8 European cancer centres, who received radiotherapy between 1942 and 1985 for a first cancer in childhood. After a mean follow-up of 19 years, 26 children developed a solid second malignant neoplasm (SMN), as compared to 5.6 expected from general population rates. Both the excess relative risk and the excess of absolute risk of solid SMN were higher among children who were younger at time of the irradiation. After reaching a maximum 15 to 20 years after irradiation, the excess relative risk of SMN decreased with time after irradiation, when controlling for age at irradiation and sex. The analysis of the risk of thyroid, brain and breast cancer together, as a function of the dose averaged on these 3 organs lead to similar results. (authors). 16 refs., 8 tabs., 2 figs

Cromolyn Reduces Levels of the Alzheimer's Disease-Associated Amyloid β-Protein by Promoting Microglial Phagocytosis.

Science.gov (United States)

Zhang, Can; Griciuc, Ana; Hudry, Eloise; Wan, Yu; Quinti, Luisa; Ward, Joseph; Forte, Angela M; Shen, Xunuo; Ran, ChongZhao; Elmaleh, David R; Tanzi, Rudolph E

2018-01-18

Amyloid-beta protein (Aβ) deposition is a pathological hallmark of Alzheimer's disease (AD). Aβ deposition triggers both pro-neuroinflammatory microglial activation and neurofibrillary tangle formation. Cromolyn sodium is an asthma therapeutic agent previously shown to reduce Aβ levels in transgenic AD mouse brains after one-week of treatment. Here, we further explored these effects as well as the mechanism of action of cromolyn, alone, and in combination with ibuprofen in APP Swedish -expressing Tg2576 mice. Mice were treated for 3 months starting at 5 months of age, when the earliest stages of β-amyloid deposition begin. Cromolyn, alone, or in combination with ibuprofen, almost completely abolished longer insoluble Aβ species, i.e. Aβ40 and Aβ42, but increased insoluble Aβ38 levels. In addition to its anti-aggregation effects on Aβ, cromolyn, alone, or plus ibuprofen, but not ibuprofen alone, increased microglial recruitment to, and phagocytosis of β-amyloid deposits in AD mice. Cromolyn also promoted Aβ42 uptake in microglial cell-based assays. Collectively, our data reveal robust effects of cromolyn, alone, or in combination with ibuprofen, in reducing aggregation-prone Aβ levels and inducing a neuroprotective microglial activation state favoring Aβ phagocytosis versus a pro-neuroinflammatory state. These findings support the use of cromolyn, alone, or with ibuprofen, as a potential AD therapeutic.

High levels of vitamin D in relation to reduced risk of schizophrenia with elevated C-reactive protein.

Science.gov (United States)

Zhu, Dao-min; Liu, Yong; Zhang, Ai-guo; Chu, Zhao-xue; Wu, Qing; Li, Hui; Ge, Jin-fang; Dong, Yi; Zhu, Peng

2015-08-30

There is growing evidence on the novel role of vitamin D in reducing inflammation. This study aimed to examine the hypothesis that vitamin D is inversely associated with C-reactive protein (CRP) in patients with schizophrenia, and high levels of vitamin D may be linked to reduced risk of schizophrenia with elevated CRP. Ninety-three patients with schizophrenia and 93 family-matched controls were recruited in this cross-sectional study. Plasma concentrations of CRP and 25-hydroxyvitamin D [25(OH)D] were measured using commercial kits. Information about demographic characteristics and clinic data were obtained by interviews or medical records. Mean levels of CRP and 25(OH)D were 43.3% higher and 26.7% lower for patients compared to controls, respectively. 25(OH)D were inversely associated with CRP in the patients, but not in the controls. The proportions of patients significantly increased with increasing quartiles of CRP, while significantly decreased with increasing quartiles of 25(OH)D. Among individuals with high CRP, participants with high 25(OH)D have significantly lower proportion (adjusted OR =0.217, 95% CI 0.063, 0.751) of schizophrenia compared to those with low 25(OH)D. The evidence suggested that high levels of vitamin D may be linked to reduced risk of schizophrenia with elevated CRP. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Review: Optimizing ruminant conversion of feed protein to human food protein.

Science.gov (United States)

Broderick, G A

2017-11-20

Ruminant livestock have the ability to produce high-quality human food from feedstuffs of little or no value for humans. Balanced essential amino acid composition of meat and milk from ruminants makes those protein sources valuable adjuncts to human diets. It is anticipated that there will be increasing demand for ruminant proteins in the future. Increasing productivity per animal dilutes out the nutritional and environmental costs of maintenance and rearing dairy animals up to production. A number of nutritional strategies improve production per animal such as ration balancing in smallholder operations and small grain supplements to ruminants fed high-forage diets. Greenhouse gas emission intensity is reduced by increased productivity per animal; recent research has developed at least one effective inhibitor of methane production in the rumen. There is widespread over-feeding of protein to dairy cattle; milk and component yields can be maintained, and sometimes even increased, at lower protein intake. Group feeding dairy cows according to production and feeding diets higher in rumen-undegraded protein can improve milk and protein yield. Supplementing rumen-protected essential amino acids will also improve N efficiency in some cases. Better N utilization reduces urinary N, which is the most environmentally unstable form of excretory N. Employing nutritional models to more accurately meet animal requirements improves nutrient efficiency. Although smallholder enterprises, which are concentrated in tropical and semi-tropical regions of developing countries, are subject to different economic pressures, nutritional biology is similar at all production levels. Rather than milk volume, nutritional strategies should maximize milk component yield, which is proportional to market value as well as food value when milk nutrients are consumed directly by farmers and their families. Moving away from Holsteins toward smaller breeds such as Jerseys, Holstein-Jersey crosses or

Molecular mechanisms used by chaperones to reduce the toxicity of aberrant protein oligomers

NARCIS (Netherlands)

Mannini, Benedetta; Cascella, Roberta; Zampagni, Mariagioia; Van Waarde-Verhagen, Maria; Meehan, Sarah; Roodveldt, Cintia; Campioni, Silvia; Boninsegna, Matilde; Penco, Amanda; Relini, Annalisa; Kampinga, Harm H.; Dobson, Christopher M.; Wilson, Mark R.; Cecchi, Cristina; Chiti, Fabrizio

2012-01-01

Chaperones are the primary regulators of the proteostasis network and are known to facilitate protein folding, inhibit protein aggregation, and promote disaggregation and clearance of misfolded aggregates inside cells. We have tested the effects of five chaperones on the toxicity of misfolded

A novel protein mixture containing vegetable proteins renders enteral nutrition products non-coagulating after in vitro gastric digestion

NARCIS (Netherlands)

Braak, van den C.C.M.; Klebach, M.; Abrahamse, E.; Minor, M.; Knol, J.; Hofman, Z.; Ludwig, T.

2013-01-01

Background & aims: Non-coagulation of protein from enteral nutrition (EN) in the stomach is considered to improve gastric emptying and may result in reduced upper gastrointestinal complications such as reflux and aspiration pneumonia. For the development of a new EN protein mixture with reduced

Role of a reducing environment in disassembly of the herpesvirus tegument

Energy Technology Data Exchange (ETDEWEB)

Newcomb, William W. [Department of Microbiology Immunology and Cancer Biology, University of Virginia Health System, Box 800734, University of Virginia Health System, 1300 Jefferson Park Ave. Charlottesville, VA 22908 (United States); Jones, Lisa M. [Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130 (United States); Dee, Alexander [Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030 (United States); Chaudhry, Farid [Department of Microbiology Immunology and Cancer Biology, University of Virginia Health System, Box 800734, University of Virginia Health System, 1300 Jefferson Park Ave. Charlottesville, VA 22908 (United States); Brown, Jay C., E-mail: JCB2G@VIRGINIA.EDU [Department of Microbiology Immunology and Cancer Biology, University of Virginia Health System, Box 800734, University of Virginia Health System, 1300 Jefferson Park Ave. Charlottesville, VA 22908 (United States)

2012-09-15

Initiation of infection by herpes family viruses involves a step in which most of the virus tegument becomes detached from the capsid. Detachment takes place in the host cell cytosol near the virus entry site and it is followed by dispersal of tegument proteins and disappearance of the tegument as a distinct entity. Here we describe the results of experiments designed to test the idea that the reducing environment of the cytosol may contribute to tegument detachment and disassembly. Non-ionic detergent was used to remove the membrane of purified herpes simplex virus under control and reducing conditions. The effects on the tegument were then examined by SDS-PAGE and electron microscopy. Protein analysis demonstrated that most major tegument proteins were removed under both oxidizing and reducing conditions except for UL49 which required a reducing environment. It is proposed therefore that the reducing conditions in the cytosol are involved in removal of UL49 protein. Electron microscopic analysis revealed that capsids produced under oxidizing conditions contained a coating of protein that was absent in reduced virions and which correlated uniquely with the presence of UL49. This capsid-associated layer is suggested to be the location of UL49 in the extracted virion.

Protein turnover in acid maltase deficiency before and after treatment with a high protein diet.

OpenAIRE

Umpleby, A M; Wiles, C M; Trend, P S; Scobie, I N; Macleod, A F; Spencer, G T; Sonksen, P H

1987-01-01

A patient with acid maltase deficiency was treated with a high protein diet for 7 months. Protein turnover expressed in terms of lean body mass was shown to be increased in this patient before the diet but was markedly reduced following the diet. The patient improved clinically whilst on the diet both subjectively and in terms of mobility, breathing and reduced peripheral cyanosis at rest.

Reduced Fluorescent Protein Switching Fatigue by Binding-Induced Emissive State Stabilization

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Thijs Roebroek

2017-09-01

Full Text Available Reversibly switchable fluorescent proteins (RSFPs enable advanced fluorescence imaging, though the performance of this imaging crucially depends on the properties of the labels. We report on the use of an existing small binding peptide, named Enhancer, to modulate the spectroscopic properties of the recently developed rsGreen series of RSFPs. Fusion constructs of Enhancer with rsGreen1 and rsGreenF revealed an increased molecular brightness and pH stability, although expression in living E. coli or HeLa cells resulted in a decrease of the overall emission. Surprisingly, Enhancer binding also increased off-switching speed and resistance to switching fatigue. Further investigation suggested that the RSFPs can interconvert between fast- and slow-switching emissive states, with the overall protein population gradually converting to the slow-switching state through irradiation. The Enhancer modulates the spectroscopic properties of both states, but also preferentially stabilizes the fast-switching state, supporting the increased fatigue resistance. This work demonstrates how the photo-physical properties of RSFPs can be influenced by their binding to other small proteins, which opens up new horizons for applications that may require such modulation. Furthermore, we provide new insights into the photoswitching kinetics that should be of general consideration when developing new RSFPs with improved or different photochromic properties.

Effects of reducing dietary crude protein and metabolic energy in ...

African Journals Online (AJOL)

The objective of this experiment was to determine the effects of a pure reduction in the dietary crude protein (CP) and metabolic energy (ME) contents on growth performance, nutrient digestibility, blood profile, faecal microflora and odour gas emission in weaned pigs. A total of 80 weaned piglets ((Landrace × Yorkshire) ...

Sox11 Reduces Caspase-6 Cleavage and Activity.

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Elaine Waldron-Roby

Full Text Available The apoptotic cascade is an orchestrated event, whose final stages are mediated by effector caspases. Regulatory binding proteins have been identified for caspases such as caspase-3, -7, -8, and -9. Many of these proteins belong to the inhibitor of apoptosis (IAP family. By contrast, caspase-6 is not believed to be influenced by IAPs, and little is known about its regulation. We therefore performed a yeast-two-hybrid screen using a constitutively inactive form of caspase-6 for bait in order to identify novel regulators of caspase-6 activity. Sox11 was identified as a potential caspase-6 interacting protein. Sox11 was capable of dramatically reducing caspase-6 activity, as well as preventing caspase-6 self- cleavage. Several regions, including amino acids 117-214 and 362-395 within sox11 as well as a nuclear localization signal (NLS all contributed to the reduction in caspase-6 activity. Furthermore, sox11 was also capable of decreasing other effector caspase activity but not initiator caspases -8 and -9. The ability of sox11 to reduce effector caspase activity was also reflected in its capacity to reduce cell death following toxic insult. Interestingly, other sox proteins also had the ability to reduce caspase-6 activity but to a lesser extent than sox11.

Increased protein intake reduces lean body mass loss during weight loss in athletes.

Science.gov (United States)

Mettler, Samuel; Mitchell, Nigel; Tipton, Kevin D

2010-02-01

To examine the influence of dietary protein on lean body mass loss and performance during short-term hypoenergetic weight loss in athletes. In a parallel design, 20 young healthy resistance-trained athletes were examined for energy expenditure for 1 wk and fed a mixed diet (15% protein, 100% energy) in the second week followed by a hypoenergetic diet (60% of the habitual energy intake), containing either 15% (approximately 1.0 g x kg(-1)) protein (control group, n = 10; CP) or 35% (approximately 2.3 g x kg(-1)) protein (high-protein group, n = 10; HP) for 2 wk. Subjects continued their habitual training throughout the study. Total, lean body, and fat mass, performance (squat jump, maximal isometric leg extension, one-repetition maximum (1RM) bench press, muscle endurance bench press, and 30-s Wingate test) and fasting blood samples (glucose, nonesterified fatty acids (NEFA), glycerol, urea, cortisol, free testosterone, free Insulin-like growth factor-1 (IGF-1), and growth hormone), and psychologic measures were examined at the end of each of the 4 wk. Total (-3.0 +/- 0.4 and -1.5 +/- 0.3 kg for the CP and HP, respectively, P = 0.036) and lean body mass loss (-1.6 +/- 0.3 and -0.3 +/- 0.3 kg, P = 0.006) were significantly larger in the CP compared with those in the HP. Fat loss, performance, and most blood parameters were not influenced by the diet. Urea was higher in HP, and NEFA and urea showed a group x time interaction. Fatigue ratings and "worse than normal" scores on the Daily Analysis of Life Demands for Athletes were higher in HP. These results indicate that approximately 2.3 g x kg(-1) or approximately 35% protein was significantly superior to approximately 1.0 g x kg(-1) or approximately 15% energy protein for maintenance of lean body mass in young healthy athletes during short-term hypoenergetic weight loss.

Intake of Mung Bean Protein Isolate Reduces Plasma Triglyceride Level in Rats

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Nobuhiko Tachibana

2013-09-01

Full Text Available ABSTRACTBackground: Mung bean is well known as a starch source, but the physiological effects of mung bean protein have received little attention. In this study, we isolated mung bean protein from de-starched mung bean solutions, and investigated its influence on lipid metabolism. Objective: The aim of this study is to clarify the influence of the lipid metabolism by consumption of mung bean protein isolate (MPIMethods: Diets containing either mung bean protein isolate (MPI or casein were fed to normal rats for 28 days.Results: Both groups ate the same amount of food, but the plasma triglyceride level, relative liver weight and liver lipid contents (cholesterol and triglyceride pool in the MPI group were significantly lower than in the casein group. In the MPI group, the expression of sterol regulatory-element binding factor 1 (SREBF1 mRNA in the liver was significantly different when compared with the casein group. The significantly lower levels of insulin and free fatty acids in the MPI-fed rats may be due to the regulation of genes related to lipid metabolism in the liver.Conclusions: These results suggest that MPI may improve the plasma lipid profile by normalizing insulin sensitivity.Keywords: mung bean, Vigna radiata L., 8S globulin, triglyceride, β-conglycinin, 7S globulin, insulin sensitivity, SREBF1

A coevolution analysis for identifying protein-protein interactions by Fourier transform

Science.gov (United States)

Yin, Changchuan; Yau, Stephen S. -T.

2017-01-01

Protein-protein interactions (PPIs) play key roles in life processes, such as signal transduction, transcription regulations, and immune response, etc. Identification of PPIs enables better understanding of the functional networks within a cell. Common experimental methods for identifying PPIs are time consuming and expensive. However, recent developments in computational approaches for inferring PPIs from protein sequences based on coevolution theory avoid these problems. In the coevolution theory model, interacted proteins may show coevolutionary mutations and have similar phylogenetic trees. The existing coevolution methods depend on multiple sequence alignments (MSA); however, the MSA-based coevolution methods often produce high false positive interactions. In this paper, we present a computational method using an alignment-free approach to accurately detect PPIs and reduce false positives. In the method, protein sequences are numerically represented by biochemical properties of amino acids, which reflect the structural and functional differences of proteins. Fourier transform is applied to the numerical representation of protein sequences to capture the dissimilarities of protein sequences in biophysical context. The method is assessed for predicting PPIs in Ebola virus. The results indicate strong coevolution between the protein pairs (NP-VP24, NP-VP30, NP-VP40, VP24-VP30, VP24-VP40, and VP30-VP40). The method is also validated for PPIs in influenza and E.coli genomes. Since our method can reduce false positive and increase the specificity of PPI prediction, it offers an effective tool to understand mechanisms of disease pathogens and find potential targets for drug design. The Python programs in this study are available to public at URL (https://github.com/cyinbox/PPI). PMID:28430779

A coevolution analysis for identifying protein-protein interactions by Fourier transform.

Directory of Open Access Journals (Sweden)

Changchuan Yin

Full Text Available Protein-protein interactions (PPIs play key roles in life processes, such as signal transduction, transcription regulations, and immune response, etc. Identification of PPIs enables better understanding of the functional networks within a cell. Common experimental methods for identifying PPIs are time consuming and expensive. However, recent developments in computational approaches for inferring PPIs from protein sequences based on coevolution theory avoid these problems. In the coevolution theory model, interacted proteins may show coevolutionary mutations and have similar phylogenetic trees. The existing coevolution methods depend on multiple sequence alignments (MSA; however, the MSA-based coevolution methods often produce high false positive interactions. In this paper, we present a computational method using an alignment-free approach to accurately detect PPIs and reduce false positives. In the method, protein sequences are numerically represented by biochemical properties of amino acids, which reflect the structural and functional differences of proteins. Fourier transform is applied to the numerical representation of protein sequences to capture the dissimilarities of protein sequences in biophysical context. The method is assessed for predicting PPIs in Ebola virus. The results indicate strong coevolution between the protein pairs (NP-VP24, NP-VP30, NP-VP40, VP24-VP30, VP24-VP40, and VP30-VP40. The method is also validated for PPIs in influenza and E.coli genomes. Since our method can reduce false positive and increase the specificity of PPI prediction, it offers an effective tool to understand mechanisms of disease pathogens and find potential targets for drug design. The Python programs in this study are available to public at URL (https://github.com/cyinbox/PPI.

Effectiveness of Germination on Protein Hydrolysis as a Way To Reduce Adverse Reactions to Wheat.

Science.gov (United States)

Boukid, Fatma; Prandi, Barbara; Buhler, Sofie; Sforza, Stefano

2017-11-15

In this work, the aim is to study the effectiveness of germination on wheat protein degradation, with a specific focus on proteins involved in adverse reactions to wheat. The effects of 8 days of germination at 25 °C on the chemical composition and the protein profile were determined. Germination did not have a significant effect on starch, protein, lipid, and ash contents. General protein profile, as indicated by SDS-PAGE analysis, revealed that germination induced a relevant degradation in protein fraction. After in vitro gastrointestinal digestion, gluten peptides involved in celiac disease (CD) were identified and quantified using UPLC/ESI-MS technique. Also, CM3 protein, involved in baker's asthma and intestinal inflammation, was quantified by measuring a marker peptide. Statistical analysis underlined that germination and genotype had significant impact on the amount of both components. Regarding gluten peptides related to CD, germination enabled an average reduction of 47% in peptides eliciting adaptive immune response and 46% in peptides eliciting innate immune response. CM3 protein showed also a high average reduction (56%). Thus, this study suggests that germination might be a good bioalternative to provide a low "impact" raw ingredient for special wheat-based foodstuffs.

Impact of protein uptake and degradation on recombinant protein secretion in yeast

DEFF Research Database (Denmark)

Tyo, Keith E. J.; Liu, Zihe; Magnusson, Ylva

2014-01-01

Protein titers, a key bioprocessing metric, depend both on the synthesis of protein and the degradation of protein. Secreted recombinant protein production in Saccharomyces cerevisiae is an attractive platform as minimal media can be used for cultivation, thus reducing fermentation costs...... and transcriptomics, we identify metabolic and regulatory markers that are consistent with uptake of whole proteins by endocytosis, followed by intracellular degradation and catabolism of substituent amino acids. Uptake and degradation of recombinant protein products may be common in S. cerevisiae protein secretion...... and simplifying downstream purification, compared to other systems that require complex media. As such, engineering S. cerevisiae to improve titers has been then the subject of significant attention, but the majority of previous efforts have been focused on improving protein synthesis. Here, we characterize...

An in vivo genetic screen for genes involved in spliced leader trans-splicing indicates a crucial role for continuous de novo spliced leader RNP assembly.

Science.gov (United States)

Philippe, Lucas; Pandarakalam, George C; Fasimoye, Rotimi; Harrison, Neale; Connolly, Bernadette; Pettitt, Jonathan; Müller, Berndt

2017-08-21

Spliced leader (SL) trans-splicing is a critical element of gene expression in a number of eukaryotic groups. This process is arguably best understood in nematodes, where biochemical and molecular studies in Caenorhabditis elegans and Ascaris suum have identified key steps and factors involved. Despite this, the precise details of SL trans-splicing have yet to be elucidated. In part, this is because the systematic identification of the molecules involved has not previously been possible due to the lack of a specific phenotype associated with defects in this process. We present here a novel GFP-based reporter assay that can monitor SL1 trans-splicing in living C. elegans. Using this assay, we have identified mutants in sna-1 that are defective in SL trans-splicing, and demonstrate that reducing function of SNA-1, SNA-2 and SUT-1, proteins that associate with SL1 RNA and related SmY RNAs, impairs SL trans-splicing. We further demonstrate that the Sm proteins and pICln, SMN and Gemin5, which are involved in small nuclear ribonucleoprotein assembly, have an important role in SL trans-splicing. Taken together these results provide the first in vivo evidence for proteins involved in SL trans-splicing, and indicate that continuous replacement of SL ribonucleoproteins consumed during trans-splicing reactions is essential for effective trans-splicing. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

A High-Protein Diet Reduces Weight Gain, Decreases Food Intake, Decreases Liver Fat Deposition, and Improves Markers of Muscle Metabolism in Obese Zucker Rats

Directory of Open Access Journals (Sweden)

William W. French

2017-06-01

Full Text Available A primary factor in controlling and preventing obesity is through dietary manipulation. Diets higher in protein have been shown to improve body composition and metabolic health during weight loss. The objective of this study was to examine the effects of a high-protein diet versus a moderate-protein diet on muscle, liver and fat metabolism and glucose regulation using the obese Zucker rat. Twelve-week old, male, Zucker (fa/fa and lean control (Fa/fa rats were randomly assigned to either a high-protein (40% energy or moderate-protein (20% energy diet for 12 weeks, with a total of four groups: lean 20% protein (L20; n = 8, lean 40% protein (L40; n = 10, obese 20% protein (O20; n = 8, and obese 40% protein (O40; n = 10. At the end of 12 weeks, animals were fasted and euthanized. There was no difference in food intake between L20 and L40. O40 rats gained less weight and had lower food intake (p < 0.05 compared to O20. O40 rats had lower liver weight (p < 0.05 compared to O20. However, O40 rats had higher orexin (p < 0.05 levels compared to L20, L40 and O20. Rats in the L40 and O40 groups had less liver and muscle lipid deposition compared to L20 and L40 diet rats, respectively. O40 had decreased skeletal muscle mechanistic target of rapamycin complex 1 (mTORC1 phosphorylation and peroxisome proliferator-activated receptor gamma (PPARγ mRNA expression compared to O20 (p < 0.05, with no difference in 5′ AMP-activated protein kinase (AMPK, eukaryotic translation initiation factor 4E binding protein 1 (4EBP1, protein kinase B (Akt or p70 ribosomal S6 kinase (p70S6K phosphorylation. The data suggest that high-protein diets have the potential to reduce weight gain and alter metabolism, possibly through regulation of an mTORC1-dependent pathway in skeletal muscle.

Functions of innate and acquired immune system are reduced in domestic pigeons (Columba livia domestica) given a low protein diet

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Mabuchi, Yuko; Frankel, Theresa L.

2016-01-01

Racing pigeons are exposed to and act as carriers of diseases. Dietary protein requirement for their maintenance has not been determined experimentally despite their being domesticated for over 7000 years. A maintenance nitrogen (protein) requirement (MNR) for pigeons was determined in a balance study using diets containing 6, 10 and 14% crude protein (CP). Then, the effects of feeding the diets were investigated to determine whether they were adequate to sustain innate and acquired immune functions. Nitrogen intake from the 6% CP diet was sufficient to maintain nitrogen balance and body weight in pigeons. However, the immune functions of phagocytosis, oxidative burst and lymphocyte proliferation in pigeons fed this diet were reduced compared with those fed 10 and 14% CP diets. Pigeons given the 6 and 10% CP diets had lower antibody titres following inoculation against Newcastle disease (ND) than those on the 14% CP diet. A confounding factor found on autopsy was the presence of intestinal parasites in some of the pigeons given the 6 and 10% CP diets; however, none of the pigeons used to measure MNR or acquired immunity to ND were infested with parasites. In conclusion, neither the 6 nor 10% CP diets adequately sustained acquired immune function of pigeons. PMID:27069640

Integration of multi-omics data of a genome-reduced bacterium: Prevalence of post-transcriptional regulation and its correlation with protein abundances

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Chen, Wei-Hua; van Noort, Vera; Lluch-Senar, Maria; Hennrich, Marco L.; H. Wodke, Judith A.; Yus, Eva; Alibés, Andreu; Roma, Guglielmo; Mende, Daniel R.; Pesavento, Christina; Typas, Athanasios; Gavin, Anne-Claude; Serrano, Luis; Bork, Peer

2016-01-01

We developed a comprehensive resource for the genome-reduced bacterium Mycoplasma pneumoniae comprising 1748 consistently generated ‘-omics’ data sets, and used it to quantify the power of antisense non-coding RNAs (ncRNAs), lysine acetylation, and protein phosphorylation in predicting protein abundance (11%, 24% and 8%, respectively). These factors taken together are four times more predictive of the proteome abundance than of mRNA abundance. In bacteria, post-translational modifications (PTMs) and ncRNA transcription were both found to increase with decreasing genomic GC-content and genome size. Thus, the evolutionary forces constraining genome size and GC-content modify the relative contributions of the different regulatory layers to proteome homeostasis, and impact more genomic and genetic features than previously appreciated. Indeed, these scaling principles will enable us to develop more informed approaches when engineering minimal synthetic genomes. PMID:26773059

Bone Morphogenetic Protein 9 Reduces Cardiac Fibrosis and Improves Cardiac Function in Heart Failure.

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Morine, Kevin J; Qiao, Xiaoying; York, Sam; Natov, Peter S; Paruchuri, Vikram; Zhang, Yali; Aronovitz, Mark J; Karas, Richard H; Kapur, Navin K

2018-02-27

Background -Heart failure is a growing cause of morbidity and mortality worldwide. Transforming growth factor beta (TGF-β1) promotes cardiac fibrosis, but also activates counter-regulatory pathways that serve to regulate TGF-β1 activity in heart failure. Bone morphogenetic protein 9 (BMP9) is a member of the TGFβ family of cytokines and signals via the downstream effector protein Smad1. Endoglin is a TGFβ co-receptor that promotes TGF-β1 signaling via Smad3 and binds BMP9 with high affinity. We hypothesized that BMP9 limits cardiac fibrosis by activating Smad1 and attenuating Smad3 and further that neutralizing endoglin activity promotes BMP9 activity. Methods -We examined BMP9 expression and signaling in human cardiac fibroblasts and human subjects with heart failure. We utilized the thoracic aortic constriction (TAC) induced model of heart failure to evaluate the functional effect of BMP9 signaling on cardiac remodeling. Results -BMP9 expression is increased in the circulation and left ventricle (LV) of human subjects with heart failure and is expressed by cardiac fibroblasts. Next, we observed that BMP9 attenuates Type I collagen synthesis in human cardiac fibroblasts using recombinant human BMP9 and an siRNA approach. In BMP9 -/- mice subjected to TAC, loss of BMP9 activity promotes cardiac fibrosis, impairs LV function, and increases LV levels of phosphorylated Smad3 (pSmad3), not pSmad1. In contrast, treatment of wild-type mice subjected to TAC with recombinant BMP9 limits progression of cardiac fibrosis, improves LV function, enhances myocardial capillary density, and increases LV levels of pSmad1, not pSmad3 compared to vehicle treated controls. Since endoglin binds BMP9 with high affinity, we explored the effect of reduced endoglin activity on BMP9 activity. Neutralizing endoglin activity in human cardiac fibroblasts or in wild-type mice subjected to TAC induced heart failure limits collagen production, increases BMP9 protein levels, and increases

The LMNA mutation p.Arg321Ter associated with dilated cardiomyopathy leads to reduced expression and a skewed ratio of lamin A and lamin C proteins

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Al-Saaidi, Rasha [Research Unit for Molecular Medicine, Aarhus University and Aarhus University Hospital, Aarhus (Denmark); Rasmussen, Torsten B. [Department of Cardiology, Aarhus University Hospital, Aarhus (Denmark); Palmfeldt, Johan [Research Unit for Molecular Medicine, Aarhus University and Aarhus University Hospital, Aarhus (Denmark); Nissen, Peter H. [Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus (Denmark); Beqqali, Abdelaziz [Heart Failure Research Center, Academic Medical Center, Amsterdam (Netherlands); Hansen, Jakob [Department of Forensic Medicine, Bioanalytical Unit, University of Aarhus (Denmark); Pinto, Yigal M. [Heart Failure Research Center, Academic Medical Center, Amsterdam (Netherlands); Boesen, Thomas [Department of Molecular Biology and Genetics, University of Aarhus (Denmark); Mogensen, Jens [Department of Cardiology, Odense University Hospital, Odense (Denmark); Bross, Peter, E-mail: peter.bross@ki.au.dk [Research Unit for Molecular Medicine, Aarhus University and Aarhus University Hospital, Aarhus (Denmark)

2013-11-15

Dilated cardiomyopathy (DCM) is a disease of the heart muscle characterized by cardiac chamber enlargement and reduced systolic function of the left ventricle. Mutations in the LMNA gene represent the most frequent known genetic cause of DCM associated with disease of the conduction systems. The LMNA gene generates two major transcripts encoding the nuclear lamina major components lamin A and lamin C by alternative splicing. Both haploinsuffiency and dominant negative effects have been proposed as disease mechanism for premature termination codon (PTC) mutations in LMNA. These mechanisms however are still not clearly established. In this study, we used a representative LMNA nonsense mutation, p.Arg321Ter, to shed light on the molecular disease mechanisms. Cultured fibroblasts from three DCM patients carrying this mutation were analyzed. Quantitative reverse transcriptase PCR and sequencing of these PCR products indicated that transcripts from the mutant allele were degraded by the nonsense-mediated mRNA decay (NMD) mechanism. The fact that no truncated mutant protein was detectable in western blot (WB) analysis strengthens the notion that the mutant transcript is efficiently degraded. Furthermore, WB analysis showed that the expression of lamin C protein was reduced by the expected approximately 50%. Clearly decreased lamin A and lamin C levels were also observed by immunofluorescence microscopy analysis. However, results from both WB and nano-liquid chromatography/mass spectrometry demonstrated that the levels of lamin A protein were more reduced suggesting an effect on expression of lamin A from the wild type allele. PCR analysis of the ratio of lamin A to lamin C transcripts showed unchanged relative amounts of lamin A transcript suggesting that the effect on the wild type allele was operative at the protein level. Immunofluorescence microscopy analysis showed no abnormal nuclear morphology of patient fibroblast cells. Based on these data, we propose that

Non-uniform tube representation of proteins

DEFF Research Database (Denmark)

Hansen, Mikael Sonne

Treating the full protein structure is often neither computationally nor physically possible. Instead one is forced to consider various reduced models capturing the properties of interest. Previous work have used tubular neighborhoods of the C-alpha backbone. However, assigning a unique radius...... might not correctly capture volume exclusion - of crucial importance when trying to understand a proteins $3$d-structure. We propose a new reduced model treating the protein as a non-uniform tube with a radius reflecting the positions of atoms. The tube representation is well suited considering X......-ray crystallographic resolution ~ 3Å while a varying radius accounts for the different sizes of side chains. Such a non-uniform tube better capture the protein geometry and has numerous applications in structural/computational biology from the classification of protein structures to sequence-structure prediction....

Recognizing protein–protein interfaces with empirical potentials and reduced amino acid alphabets

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Wodak Shoshana

2007-07-01

Full Text Available Abstract Background In structural genomics, an important goal is the detection and classification of protein–protein interactions, given the structures of the interacting partners. We have developed empirical energy functions to identify native structures of protein–protein complexes among sets of decoy structures. To understand the role of amino acid diversity, we parameterized a series of functions, using a hierarchy of amino acid alphabets of increasing complexity, with 2, 3, 4, 6, and 20 amino acid groups. Compared to previous work, we used the simplest possible functional form, with residue–residue interactions and a stepwise distance-dependence. We used increased computational ressources, however, constructing 290,000 decoys for 219 protein–protein complexes, with a realistic docking protocol where the protein partners are flexible and interact through a molecular mechanics energy function. The energy parameters were optimized to correctly assign as many native complexes as possible. To resolve the multiple minimum problem in parameter space, over 64000 starting parameter guesses were tried for each energy function. The optimized functions were tested by cross validation on subsets of our native and decoy structures, by blind tests on series of native and decoy structures available on the Web, and on models for 13 complexes submitted to the CAPRI structure prediction experiment. Results Performance is similar to several other statistical potentials of the same complexity. For example, the CAPRI target structure is correctly ranked ahead of 90% of its decoys in 6 cases out of 13. The hierarchy of amino acid alphabets leads to a coherent hierarchy of energy functions, with qualitatively similar parameters for similar amino acid types at all levels. Most remarkably, the performance with six amino acid classes is equivalent to that of the most detailed, 20-class energy function. Conclusion This suggests that six carefully chosen amino

Sublingual immunization with the phosphate-binding-protein (PstS) reduces oral colonization by Streptococcus mutans.

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Ferreira, E L; Batista, M T; Cavalcante, R C M; Pegos, V R; Passos, H M; Silva, D A; Balan, A; Ferreira, L C S; Ferreira, R C C

2016-10-01

Bacterial ATP-binding cassette (ABC) transporters play a crucial role in the physiology and pathogenicity of different bacterial species. Components of ABC transporters have also been tested as target antigens for the development of vaccines against different bacterial species, such as those belonging to the Streptococcus genus. Streptococcus mutans is the etiological agent of dental caries, and previous studies have demonstrated that deletion of the gene encoding PstS, the substrate-binding component of the phosphate uptake system (Pst), reduced the adherence of the bacteria to abiotic surfaces. In the current study, we generated a recombinant form of the S. mutans PstS protein (rPstS) with preserved structural features, and we evaluated the induction of antibody responses in mice after sublingual mucosal immunization with a formulation containing the recombinant protein and an adjuvant derived from the heat-labile toxin from enterotoxigenic Escherichia coli strains. Mice immunized with rPstS exhibited systemic and secreted antibody responses, measured by the number of immunoglobulin A-secreting cells in draining lymph nodes. Serum antibodies raised in mice immunized with rPstS interfered with the adhesion of bacteria to the oral cavity of naive mice challenged with S. mutans. Similarly, mice actively immunized with rPstS were partially protected from oral colonization after challenge with the S. mutans NG8 strain. Therefore, our results indicate that S. mutans PstS is a potential target antigen capable of inducing specific and protective antibody responses after sublingual administration. Overall, these observations raise interesting perspectives for the development of vaccines to prevent dental caries. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Estimate of the uncertainties in the relative risk of secondary malignant neoplasms following proton therapy and intensity-modulated photon therapy

International Nuclear Information System (INIS)

Fontenot, Jonas D; Bloch, Charles; Followill, David; Titt, Uwe; Newhauser, Wayne D

2010-01-01

Theoretical calculations have shown that proton therapy can reduce the incidence of radiation-induced secondary malignant neoplasms (SMN) compared with photon therapy for patients with prostate cancer. However, the uncertainties associated with calculations of SMN risk had not been assessed. The objective of this study was to quantify the uncertainties in projected risks of secondary cancer following contemporary proton and photon radiotherapies for prostate cancer. We performed a rigorous propagation of errors and several sensitivity tests to estimate the uncertainty in the ratio of relative risk (RRR) due to the largest contributors to the uncertainty: the radiation weighting factor for neutrons, the dose-response model for radiation carcinogenesis and interpatient variations in absorbed dose. The interval of values for the radiation weighting factor for neutrons and the dose-response model were derived from the literature, while interpatient variations in absorbed dose were taken from actual patient data. The influence of each parameter on a baseline RRR value was quantified. Our analysis revealed that the calculated RRR was insensitive to the largest contributors to the uncertainty. Uncertainties in the radiation weighting factor for neutrons, the shape of the dose-risk model and interpatient variations in therapeutic and stray doses introduced a total uncertainty of 33% to the baseline RRR calculation.

Introduction to current and future protein therapeutics: a protein engineering perspective.

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Carter, Paul J

2011-05-15

Protein therapeutics and its enabling sister discipline, protein engineering, have emerged since the early 1980s. The first protein therapeutics were recombinant versions of natural proteins. Proteins purposefully modified to increase their clinical potential soon followed with enhancements derived from protein or glycoengineering, Fc fusion or conjugation to polyethylene glycol. Antibody-based drugs subsequently arose as the largest and fastest growing class of protein therapeutics. The rationale for developing better protein therapeutics with enhanced efficacy, greater safety, reduced immunogenicity or improved delivery comes from the convergence of clinical, scientific, technological and commercial drivers that have identified unmet needs and provided strategies to address them. Future protein drugs seem likely to be more extensively engineered to improve their performance, e.g., antibodies and Fc fusion proteins with enhanced effector functions or extended half-life. Two old concepts for improving antibodies, namely antibody-drug conjugates and bispecific antibodies, have advanced to the cusp of clinical success. As for newer protein therapeutic platform technologies, several engineered protein scaffolds are in early clinical development and offer differences and some potential advantages over antibodies. Copyright © 2011 Elsevier Inc. All rights reserved.

High-Pressure-High-Temperature Processing Reduces Maillard Reaction and Viscosity in Whey Protein-Sugar Solutions

NARCIS (Netherlands)

Avila Ruiz, Geraldine; Xi, Bingyan; Minor, Marcel; Sala, Guido; Boekel, van Tiny; Fogliano, Vincenzo; Stieger, Markus

2016-01-01

The aim of the study was to determine the influence of pressure in high-pressure-high-temperature (HPHT) processing on Maillard reactions and protein aggregation of whey protein-sugar solutions. Solutions of whey protein isolate containing either glucose or trehalose at pH 6, 7, and 9 were

Maillard-reaction-induced modification and aggregation of proteins and hardening of texture in protein bar model systems.

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Zhou, Peng; Guo, Mufan; Liu, Dasong; Liu, Xiaoming; Labuza, Teodore P

2013-03-01

The hardening of high-protein bars causes problems in their acceptability to consumers. The objective of this study was to determine the progress of the Maillard reaction in model systems of high-protein nutritional bars containing reducing sugars, and to illustrate the influences of the Maillard reaction on the modification and aggregation of proteins and the hardening of bar matrices during storage. The progress of the Maillard reaction, glycation, and aggregation of proteins, and textural changes in bar matrices were investigated during storage at 25, 35, and 45 °C. The initial development of the Maillard reaction caused little changes in hardness; however, further storage resulted in dramatic modification of protein with formation of high-molecular-weight polymers, resulting in the hardening in texture. The replacement of reducing sugars with nonreducing ingredients such as sugar alcohols in the formula minimized the changes in texture. The hardening of high-protein bars causes problems in their acceptability to consumers. Maillard reaction is one of the mechanisms contributing to the hardening of bar matrix, particularly for the late stage of storage. The replacement of reducing sugars with nonreducing ingredients such as sugar alcohols in the formula will minimize the changes in texture. © 2013 Institute of Food Technologists®

Desalting Protein Ions in Native Mass Spectrometry Using Supercharging Reagents

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Cassou, Catherine A.; Williams, Evan R.

2014-01-01

Effects of the supercharging reagents m-NBA and sulfolane on sodium ion adduction to protein ions formed using native mass spectrometry were investigated. There is extensive sodium adduction on protein ions formed by electrospray ionization from aqueous solutions containing millimolar concentrations of NaCl, which can lower sensitivity by distributing the signal of a given charge state over multiple adducted ions and can reduce mass measuring accuracy for large proteins and non-covalent complexes for which individual adducts cannot be resolved. The average number of sodium ions adducted to the most abundant ion formed from ten small (8.6–29 kDa) proteins for which adducts can be resolved is reduced by 58% or 80% on average, respectively, when 1.5% m-NBA or 2.5% sulfolane are added to aqueous solutions containing sodium compared to without the supercharging reagent. Sulfolane is more effective than m-NBA at reducing sodium ion adduction and at preserving non-covalent protein-ligand and protein-protein interactions. Desalting with 2.5% sulfolane enables detection of several glycosylated forms of 79.7 kDa holo-transferrin and NADH bound to the 146 kDa homotetramer LDH, which are otherwise unresolved due to peak broadening from extensive sodium adduction. Although sulfolane is more effective than m-NBA at protein ion desalting, m-NBA reduces salt clusters at high m/z and can increase the signal-to-noise ratios of protein ions by reducing chemical noise. Desalting is likely a result of these supercharging reagents binding sodium ions in solution, thereby reducing the sodium available to adduct to protein ions. PMID:25133273

Reduced Contractility and Motility of Prostatic Cancer-Associated Fibroblasts after Inhibition of Heat Shock Protein 90

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Alex Henke

2016-08-01

Full Text Available Background: Prostate cancer-associated fibroblasts (CAF can stimulate malignant progression and invasion of prostatic tumour cells via several mechanisms including those active in extracellular matrix; Methods: We isolated CAF from prostate cancer patients of Gleason Score 6–10 and confirmed their cancer-promoting activity using an in vivo tumour reconstitution assay comprised of CAF and BPH1 cells. We tested the effects of heat shock protein 90 (HSP90 inhibitors upon reconstituted tumour growth in vivo. Additionally, CAF contractility was measured in a 3D collagen contraction assay and migration was measured by scratch assay; Results: HSP90 inhibitors dipalmitoyl-radicicol and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG reduced tumour size and proliferation in CAF/BPH1 reconstituted tumours in vivo. We observed that the most contractile CAF were derived from patients with lower Gleason Score and of younger age compared with the least contractile CAF. HSP90 inhibitors radicicol and 17-DMAG inhibited contractility and reduced the migration of CAF in scratch assays. Intracellular levels of HSP70 and HSP90 were upregulated upon treatment with HSP90 inhibitors. Inhibition of HSP90 also led to a specific increase in transforming growth factor beta 2 (TGFβ2 levels in CAF; Conclusions: We suggest that HSP90 inhibitors act not only upon tumour cells, but also on CAF in the tumour microenvironment.

High Whey Protein Intake Delayed the Loss of Lean Body Mass in Healthy Old Rats, whereas Protein Type and Polyphenol/Antioxidant Supplementation Had No Effects

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Mosoni, Laurent; Gatineau, Eva; Gatellier, Philippe; Migné, Carole; Savary-Auzeloux, Isabelle; Rémond, Didier; Rocher, Emilie; Dardevet, Dominique

2014-01-01

Our aim was to compare and combine 3 nutritional strategies to slow down the age-related loss of muscle mass in healthy old rats: 1) increase protein intake, which is likely to stimulate muscle protein anabolism; 2) use leucine rich, rapidly digested whey proteins as protein source (whey proteins are recognized as the most effective proteins to stimulate muscle protein anabolism). 3) Supplement animals with a mixture of chamomile extract, vitamin E, vitamin D (reducing inflammation and oxidative stress is also effective to improve muscle anabolism). Such comparisons and combinations were never tested before. Nutritional groups were: casein 12% protein, whey 12% protein, whey 18% protein and each of these groups were supplemented or not with polyphenols/antioxidants. During 6 months, we followed changes of weight, food intake, inflammation (plasma fibrinogen and alpha-2-macroglobulin) and body composition (DXA). After 6 months, we measured muscle mass, in vivo and ex-vivo fed and post-absorptive muscle protein synthesis, ex-vivo muscle proteolysis, and oxidative stress parameters (liver and muscle glutathione, SOD and total antioxidant activities, muscle carbonyls and TBARS). We showed that although micronutrient supplementation reduced inflammation and oxidative stress, the only factor that significantly reduced the loss of lean body mass was the increase in whey protein intake, with no detectable effect on muscle protein synthesis, and a tendency to reduce muscle proteolysis. We conclude that in healthy rats, increasing protein intake is an effective way to delay sarcopenia. PMID:25268515

Highly Efficient Differentiation and Enrichment of Spinal Motor Neurons Derived from Human and Monkey Embryonic Stem Cells

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Wada, Tamaki; Honda, Makoto; Minami, Itsunari; Tooi, Norie; Amagai, Yuji; Nakatsuji, Norio; Aiba, Kazuhiro

2009-01-01

Background There are no cures or efficacious treatments for severe motor neuron diseases. It is extremely difficult to obtain naïve spinal motor neurons (sMNs) from human tissues for research due to both technical and ethical reasons. Human embryonic stem cells (hESCs) are alternative sources. Several methods for MN differentiation have been reported. However, efficient production of naïve sMNs and culture cost were not taken into consideration in most of the methods. Methods/Principal Findings We aimed to establish protocols for efficient production and enrichment of sMNs derived from pluripotent stem cells. Nestin+ neural stem cell (NSC) clusters were induced by Noggin or a small molecule inhibitor of BMP signaling. After dissociation of NSC clusters, neurospheres were formed in a floating culture containing FGF2. The number of NSCs in neurospheres could be expanded more than 30-fold via several passages. More than 33% of HB9+ sMN progenitor cells were observed after differentiation of dissociated neurospheres by all-trans retinoic acid (ATRA) and a Shh agonist for another week on monolayer culture. HB9+ sMN progenitor cells were enriched by gradient centrifugation up to 80% purity. These HB9+ cells differentiated into electrophysiologically functional cells and formed synapses with myotubes during a few weeks after ATRA/SAG treatment. Conclusions and Significance The series of procedures we established here, namely neural induction, NSC expansion, sMN differentiation and sMN purification, can provide large quantities of naïve sMNs derived from human and monkey pluripotent stem cells. Using small molecule reagents, reduction of culture cost could be achieved. PMID:19701462

Using modified soy protein to enhance foaming of egg white protein.

Science.gov (United States)

Wang, Guang; Troendle, Molly; Reitmeier, Cheryll A; Wang, Tong

2012-08-15

It is well known that the foaming properties of egg white protein are significantly reduced when a small amount of yolk is mixed in the white. To improve foaming properties of yolk-contaminated egg white protein, soy protein isolate (SPI) and egg proteins were modified to make basic proteins, and effects of these modified proteins on egg white foaming were evaluated in a model and an angel cake system. SPI and egg yolk proteins were modified to have an isoelectric point of 10, and sonication was used to increase protein dispersibility after the ethyl esterification reaction. However, only the addition of sonicated and modified SPI (SMSPI) showed improvement of foaming in the 5% egg protein model system with 0.4% yolk addition. SMSPI was then used in making angel food cake to examine whether the cake performance reduction due to yolk contamination of the white would be restored by such alkaline protein. Cake performance was improved when cream of tartar was used together with SMSPI. Basic soy protein can be made and used to improve egg white foaming properties and cake performance. Copyright © 2012 Society of Chemical Industry.

How connected are people with schizophrenia? Cell phone, computer, email, and social media use.

Science.gov (United States)

Miller, Brian J; Stewart, Adriana; Schrimsher, John; Peeples, Dale; Buckley, Peter F

2015-02-28

Technologies such as Internet based social media network (SMN) websites are becoming an important part of many adult lives; however, less is known about their use in patients with schizophrenia. We need to determine (1) how "connected" are patients with schizophrenia?, (2) do these technologies interfere with the patient׳s illness?, and (3) do patients envision these technologies being involved in their treatment? We recruited 80 inpatients and outpatients age 18-70 with schizophrenia to complete a brief survey on the prevalence and frequency of cell phone, text messaging, computer, email, and SMN use, and associated attitudes. 56% of subjects use text messaging, 48% have an email account, and 27% of subjects use SMN sites daily, with Facebook being the most popular. Many current users agreed that these technologies help them interact/socialize more, expressed interest in receiving text messages from their doctors, and disagreed that these technologies make symptoms worse. These preliminary findings should be investigated in larger samples, but suggest that these technologies afford a unique opportunity to engage and improve treatment for some patients with schizophrenia. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

STUDY MECHANISMS OF LOW PROTEIN DIET SUPPLEMENTED WITH KETOANLOGS ON REDUCING PROTEINURIA AND MINTINING NUTRITIONAL STATUS IN TYPE2 DIBETIC NEPHROPTHY

Directory of Open Access Journals (Sweden)

Ting Dong

2012-06-01

Full Text Available Diabetic nephropathy is one of the most important causes of end stage renal disease (ESRD in the world. Recently in a multicenter, randomized clinical trial performed in China, it was found that compound α-keto acid tablet in combination with low protein diet(LPD+KA reduced proteinuria and delayed the progression to ESRD in Type 2 diabetic nephropthy(T2DN. However, the mechanisms underlined these effects were not been elucidated. This study is a part of the continuation of the study to explore the effects of LPD+KA on podocyte as well as on local RAS in the kidney. A total of 60 patients with T2DN and CKD stages 3–4 are included in this study. All patients are treated with a LPD containing 0.6g protein/kg BW per day and 120–125 kJ/kg BW per day. The diet is randomly supplemented with keto-amino acids (Ketosteril®, Fresenius Kabi at a dosage of 100 mg/kg BW per day. The other 30 patients receive placebo. After six months of follow-up, protein intake differed only by 0.07 g/kg/day between the two groups. The mean declines in GFR were 4.2 mL/min/year (95%CI;2.9 to 5.5 in LPD group and 3.7 (95%CI; 2.7 to 4.7 in LPD+KA group. Compared with LPD, paitients in LPD+KA group had reduced proteinuria (2.16±0.4 vs 3.56±0.6 g/day; p〈0.05. However, serum albumin and prealbumin levels did not change. Through further research we found that, after a median follow-up for six months, the urinary angiotensinogen:creatinine ratio in LPD patients was higher than in LPD+KA subjects (10.1 ±3.2 μg/g vs 5.8 ± 1.2 μg/g; p〈0.05;The urinary mRNA levels of the target genes nephrin, podocin and synaptopodin of podocyte were higher in the LPD group compared with LPD+KA(p〈0.05 in urinary sediment, and the expression of nephrin, podocin and synaptopodin mRNA positively correlated with serum creatinine (r=0.326, p = 0.04; r = 0.426, p = 0.03; r = 0.343, p = 0.001, respectively. In conclusion, compound α-keto acid tablet in combination with low protein

Reduced expression of exocytotic proteins caused by anti-cholinesterase pesticides in Brachionus calyciflorus (Rotifera: Monogononta

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IA Pérez-Legaspi

Full Text Available AbstractThe organophosphate and carbamate pesticides methyl-parathion and carbaryl have a common action mechanism: they inhibit acetylcholinesterase enzyme by blocking the transmission of nerve impulses. However, they can alter the expression of exocytotic membrane proteins (SNARE, by modifying release of neurotransmitters and other substances. This study evaluated the adverse effects of the pesticides methyl-parathion and carbaryl on expression of SNARE proteins: Syntaxin-1, Syntaxin-4 and SNAP-23 in freshwater rotifer Brachionus calyciflorus. Protein expression of these three proteins was analyzed before and after exposure to these two pesticides by Western Blot. The expression of Syntaxin-1, Syntaxin-4 and SNAP-23 proteins in B. calyciflorussignificantly decreases with increasing concentration of either pesticides. This suggests that organophosphates and carbamates have adverse effects on expression of membrane proteins of exocytosis by altering the recognition, docking and fusion of presynaptic and vesicular membranes involved in exocytosis of neurotransmitters. Our results demonstrate that the neurotoxic effect of anticholinesterase pesticides influences the interaction of syntaxins and SNAP-25 and the proper assembly of the SNARE complex.

Protein intakes are associated with reduced length of stay: a comparison between Enhanced Recovery After Surgery (ERAS) and conventional care after elective colorectal surgery.

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Yeung, Sophia E; Hilkewich, Leslee; Gillis, Chelsia; Heine, John A; Fenton, Tanis R

2017-07-01

Background: Protein can modulate the surgical stress response and postoperative catabolism. Enhanced Recovery After Surgery (ERAS) protocols are evidence-based care bundles that reduce morbidity. Objective: In this study, we compared protein adequacy as well as energy intakes, gut function, clinical outcomes, and how well nutritional variables predict length of hospital stay (LOS) in patients receiving ERAS protocols and conventional care. Design: We conducted a prospective cohort study in adult elective colorectal resection patients after conventional ( n = 46) and ERAS ( n = 69) care. Data collected included preoperative Malnutrition Screening Tool (MST) score, 3-d food records, postoperative nausea, LOS, and complications. Multivariable regression analysis assessed whether low protein intakes and the MST score were predictive of LOS. Results: Total protein intakes were significantly higher in the ERAS group due to the inclusion of oral nutrition supplements (conventional group: 0.33 g · kg -1 · d -1 ; ERAS group: 0.54 g · kg -1 · d -1 ; P Nutrition variables were independent predictors of earlier discharge after potential confounders were controlled for. Each unit increase in preoperative MST score predicted longer LOSs of 2.5 d (95% CI: 1.5, 3.5 d; P nutrition supplements. However, total protein intake remained inadequate to meet recommendations. Consumption of ≥60% protein needs after surgery and MST scores were independent predictors of LOS. This trial was registered at clinicaltrials.gov as NCT02940665. © 2017 American Society for Nutrition.

Extractable protein of radiation vulcanized natural rubber latex

International Nuclear Information System (INIS)

Soebianto, Y.S.; Upul, R.M.; Makuuchi, K.; Yoshii, F.; Kume, T.

2000-01-01

A new method to reduce the protein level in the latex products by irradiation is reported. Water soluble protein (WSP) solution (10%) was added into radiation vulcanized NR latex (RVNRL) as much as 3 phr in three different processes: added to RVNRL, added to re-centrifuged RVNRL, and added to RVNRL followed by centrifugation. The protein content was determined by enhanced BCA method, and identified by SDS-PAGE analysis. Addition of WSP followed by centrifugation reduces EP up to the minimum protein detection, and shortens the leaching time to 20-30 min. SDS-PAGE analysis confirms the reduction of soluble protein in the serum phase, and disappearance of protein bands in the rubber extract. Protein-WSP interaction produces water soluble complex, and removed by centrifugation. The molecular weight of WSP dictates the efficiency of protein removal. (author)

Extractable protein of radiation vulcanized natural rubber latex

Energy Technology Data Exchange (ETDEWEB)

Soebianto, Y.S. [Center for Research and Development of Isotopes and Radiation Technology, BATAN, Jakarta (Indonesia); Upul, R.M. [Rubber Research Institute of Sri Lanka, Ratmalana (Sri Lanka); Makuuchi, K.; Yoshii, F.; Kume, T. [Japan Atomic Energy Research Inst., Takasaki, Gunma (Japan). Takasaki Radiation Chemistry Research Establishment

2000-03-01

A new method to reduce the protein level in the latex products by irradiation is reported. Water soluble protein (WSP) solution (10%) was added into radiation vulcanized NR latex (RVNRL) as much as 3 phr in three different processes: added to RVNRL, added to re-centrifuged RVNRL, and added to RVNRL followed by centrifugation. The protein content was determined by enhanced BCA method, and identified by SDS-PAGE analysis. Addition of WSP followed by centrifugation reduces EP up to the minimum protein detection, and shortens the leaching time to 20-30 min. SDS-PAGE analysis confirms the reduction of soluble protein in the serum phase, and disappearance of protein bands in the rubber extract. Protein-WSP interaction produces water soluble complex, and removed by centrifugation. The molecular weight of WSP dictates the efficiency of protein removal. (author)

PSI/TM-Coffee: a web server for fast and accurate multiple sequence alignments of regular and transmembrane proteins using homology extension on reduced databases.

Science.gov (United States)

Floden, Evan W; Tommaso, Paolo D; Chatzou, Maria; Magis, Cedrik; Notredame, Cedric; Chang, Jia-Ming

2016-07-08

The PSI/TM-Coffee web server performs multiple sequence alignment (MSA) of proteins by combining homology extension with a consistency based alignment approach. Homology extension is performed with Position Specific Iterative (PSI) BLAST searches against a choice of redundant and non-redundant databases. The main novelty of this server is to allow databases of reduced complexity to rapidly perform homology extension. This server also gives the possibility to use transmembrane proteins (TMPs) reference databases to allow even faster homology extension on this important category of proteins. Aside from an MSA, the server also outputs topological prediction of TMPs using the HMMTOP algorithm. Previous benchmarking of the method has shown this approach outperforms the most accurate alignment methods such as MSAProbs, Kalign, PROMALS, MAFFT, ProbCons and PRALINE™. The web server is available at http://tcoffee.crg.cat/tmcoffee. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Genetic disruption of the pHi-regulating proteins Na+/H+ exchanger 1 (SLC9A1) and carbonic anhydrase 9 severely reduces growth of colon cancer cells.

Science.gov (United States)

Parks, Scott K; Cormerais, Yann; Durivault, Jerome; Pouyssegur, Jacques

2017-02-07

Hypoxia and extracellular acidosis are pathophysiological hallmarks of aggressive solid tumors. Regulation of intracellular pH (pHi) is essential for the maintenance of tumor cell metabolism and proliferation in this microenvironment and key proteins involved in pHi regulation are of interest for therapeutic development. Carbonic anhydrase 9 (CA9) is one of the most robustly regulated proteins by the hypoxia inducible factor (HIF) and contributes to pHi regulation. Here, we have investigated for the first time, the role of CA9 via complete genomic knockout (ko) and compared its impact on tumor cell physiology with the essential pHi regulator Na+/H+ exchanger 1 (NHE1). Initially, we established NHE1-ko LS174 cells with inducible CA9 knockdown. While increased sensitivity to acidosis for cell survival in 2-dimensions was not observed, clonogenic proliferation and 3-dimensional spheroid growth in particular were greatly reduced. To avoid potential confounding variables with use of tetracycline-inducible CA9 knockdown, we established CA9-ko and NHE1/CA9-dko cells. NHE1-ko abolished recovery from NH4Cl pre-pulse cellular acid loading while both NHE1 and CA9 knockout reduced resting pHi. NHE1-ko significantly reduced tumor cell proliferation both in normoxia and hypoxia while CA9-ko dramatically reduced growth in hypoxic conditions. Tumor xenografts revealed substantial reductions in tumor growth for both NHE1-ko and CA9-ko. A notable induction of CA12 occurred in NHE1/CA9-dko tumors indicating a potential means to compensate for loss of pH regulating proteins to maintain growth. Overall, these genomic knockout results strengthen the pursuit of targeting tumor cell pH regulation as an effective anti-cancer strategy.

Denaturing high-performance liquid chromatography mutation analysis in patients with reduced Protein S levels

DEFF Research Database (Denmark)

Bathum, Lise; Münster, Anna-Marie; Nybo, Mads

2008-01-01

diagnosis and risk estimation. The aim was to design a high-throughput genetic analysis based on denaturing high-performance liquid chromatography to identify sequence variations in the gene coding for Protein S. PATIENTS: In total, 55 patients referred to the Section of Thrombosis and Haemostasis, Odense......BACKGROUND: Patients with congenital Protein S deficiency have increased risk of venous thromboembolism. However, Protein S levels show large intra-individual variation and the biochemical assays have low accuracy and a high interlaboratory variability. Genetic analysis might aid in a more precise......, giving a precise diagnosis and subsequently a better risk estimation....

The effects of a reduced balanced protein diet on litter moisture, pododermatitis and feather condition of female broiler breeders over three generations.

Science.gov (United States)

Li, C; Lesuisse, J; Schallier, S; Clímaco, W; Wang, Y; Bautil, A; Everaert, N; Buyse, J

2017-11-02

Protein content reduction in broiler breeder diets has been increasingly investigated. However, broiler breeders reared on low protein diets are characterized by a deterioration of the feather condition. Furthermore, polydipsia induced by controlled feed intake increases litter moisture and as a consequence pododermatitis. This project aimed to study the litter moisture, pododermatitis and feather condition of breeders fed with a 25% reduced balanced protein (RP) diet during the rearing and laying period over three successive generations. The experiment started with two treatments for the F0 generation: control (C) group fed with standard C diets and RP group fed with RP diets. The female F0-progeny of each treatment was divided into the two dietary treatments as well, resulting in four treatments for the F1 generation: C/C, C/RP, RP/C and RP/RP (breeder feed in F0/F1 generation). The RP diet fed breeders received on average 10% more feed than C diet fed breeders to achieve the same target BW. The female F1-progeny of each treatment were all fed with C diets which resulted in four treatments for the F2 generation: C/C/C, C/RP/C, RP/C/C and RP/RP/C (breeder feed in F0/F1/F2 generation). Litter moisture, footpad and hock dermatitis were recorded at regular intervals throughout the experimental period in all three generations. For the F0 and F1 generation, the pens of breeders receiving C diets had significantly higher litter moisture than the RP diets fed groups (Pdiets had poorer feather condition than those receiving the C diets (Pdiets to broiler breeders improved litter condition and hence reduced FDO whereas impaired feather condition. Furthermore, positive transgenerational effects of the maternal RP diets on the feather condition may be inferred, hence potentially altering the welfare status.

Excess of methyl donor in the perinatal period reduces postnatal leptin secretion in rat and interacts with the effect of protein content in diet.

Directory of Open Access Journals (Sweden)

Fanny Giudicelli

Full Text Available Methionine, folic acid, betaine and choline interact in the one-carbon metabolism which provides methyl groups for methylation reactions. An optimal intake of these nutrients during pregnancy is required for successful completion of fetal development and evidence is growing that they could be involved in metabolic long-term programming. However, the biological pathways involved in the action of these nutrients are still poorly known. This study investigated the interaction between methyl donors and protein content in maternal diet during the preconceptual, pregnancy and lactation periods and the consequences on the rat offspring in the short and long term. Methyl donor supplementation reduced leptin secretion in offspring, whereas insulin levels were mostly affected by protein restriction. The joint effect of protein restriction and methyl donor excess strongly impaired postnatal growth in both gender and long term weight gain in male offspring only, without affecting food intake. In addition, rats born from protein restricted and methyl donor supplemented dams gained less weight when fed a hypercaloric diet. Methylation of the leptin gene promoter in adipose tissue was increased in methyl donor supplemented groups but not affected by protein restriction only. These results suggest that maternal methyl donor supplementation may influence energy homeostasis in a gender-dependent manner, without affecting food intake. Moreover, we showed that macronutrients and micronutrients in maternal diet interact to influence the programming of the offspring.

Protein restriction and cancer.

Science.gov (United States)

Yin, Jie; Ren, Wenkai; Huang, Xingguo; Li, Tiejun; Yin, Yulong

2018-03-26

Protein restriction without malnutrition is currently an effective nutritional intervention known to prevent diseases and promote health span from yeast to human. Recently, low protein diets are reported to be associated with lowered cancer incidence and mortality risk of cancers in human. In murine models, protein restriction inhibits tumor growth via mTOR signaling pathway. IGF-1, amino acid metabolic programing, FGF21, and autophagy may also serve as potential mechanisms of protein restriction mediated cancer prevention. Together, dietary intervention aimed at reducing protein intake can be beneficial and has the potential to be widely adopted and effective in preventing and treating cancers. Copyright © 2018 Elsevier B.V. All rights reserved.

Yeast cell wall chitin reduces wine haze formation.

Science.gov (United States)

Ndlovu, Thulile; Divol, Benoit; Bauer, Florian F

2018-04-27

Protein haze formation in bottled wines is a significant concern for the global wine industry and wine clarification before bottling is therefore a common but expensive practice. Previous studies have shown that wine yeast strains can reduce haze formation through the secretion of certain mannoproteins, but it has been suggested that other yeast-dependent haze protective mechanisms exist. On the other hand, addition of chitin has been shown to reduce haze formation, likely because grape chitinases have been shown to be the major contributors to haze. In this study, Chardonnay grape must fermented by various yeast strains resulted in wines with different protein haze levels indicating differences in haze protective capacities of the strains. The cell wall chitin levels of these strains were determined, and a strong correlation between cell wall chitin levels and haze protection capability was observed. To further evaluate the mechanism of haze protection, Escherichia coli -produced GFP-tagged grape chitinase was shown to bind efficiently to yeast cell walls in a cell wall chitin concentration-dependent manner, while commercial chitinase was removed from synthetic wine in quantities also correlated with the cell wall chitin levels of the strains. Our findings suggest a new mechanism of reducing wine haze, and propose a strategy for optimizing wine yeast strains to improve wine clarification. Importance In this study, we establish a new mechanism by which wine yeast strains can impact on the protein haze formation of wines, and demonstrate that yeast cell wall chitin binds grape chitinase in a chitin-concentration dependent manner. We also show that yeast can remove this haze-forming protein from wine. Chitin has in the past been shown to efficiently reduce wine haze formation when added to the wine in high concentration as a clarifying agent. Our data suggest that the selection of yeast strains with high levels of cell wall chitin can reduce protein haze. We also

Attenuation of iron-binding proteins in ARPE-19 cells reduces their resistance to oxidative stress.

Science.gov (United States)

Karlsson, Markus; Kurz, Tino

2016-09-01

Oxidative stress-related damage to retinal pigment epithelial (RPE) cells is an important feature in the development of age-related macular degeneration. Iron-catalysed intralysosomal production of hydroxyl radicals is considered a major pathogenic factor, leading to lipofuscin formation with ensuing depressed cellular autophagic capacity, lysosomal membrane permeabilization and apoptosis. Previously, we have shown that cultured immortalized human RPE (ARPE-19) cells are extremely resistant to exposure to bolus doses of hydrogen peroxide and contain considerable amounts of the iron-binding proteins metallothionein (MT), heat-shock protein 70 (HSP70) and ferritin (FT). According to previous findings, autophagy of these proteins depresses lysosomal redox-active iron. The aim of this study was to investigate whether up- or downregulation of these proteins would affect the resistance of ARPE-19 cells to oxidative stress. The sensitivity of ARPE-19 cells to H2 O2 exposure was tested following upregulation of MT, HSP70 and/or FT by pretreatment with ZnSO4 , heat shock or FeCl3 , as well as siRNA-mediated downregulation of the same proteins. Upregulation of MT, HSP70 and FT did not improve survival following exposure to H2 O2 . This was interpreted as existence of an already maximal protection. Combined siRNA-mediated attenuation of both FT chains (H and L), or simultaneous downregulation of all three proteins, made the cells significantly more susceptible to oxidative stress confirming the importance of iron-binding proteins. The findings support our hypothesis that the oxidative stress resistance exhibited by RPE cells may be explained by a high autophagic influx of iron-binding proteins that would keep levels of redox-active lysosomal iron low. © 2016 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

Protein S is protective in pulmonary fibrosis.

Science.gov (United States)

Urawa, M; Kobayashi, T; D'Alessandro-Gabazza, C N; Fujimoto, H; Toda, M; Roeen, Z; Hinneh, J A; Yasuma, T; Takei, Y; Taguchi, O; Gabazza, E C

2016-08-01

Essentials Epithelial cell apoptosis is critical in the pathogenesis of idiopathic pulmonary fibrosis. Protein S, a circulating anticoagulant, inhibited apoptosis of lung epithelial cells. Overexpression of protein S in lung cells reduced bleomycin-induced pulmonary fibrosis. Intranasal therapy with exogenous protein S ameliorated bleomycin-induced pulmonary fibrosis. Background Pulmonary fibrosis is the terminal stage of interstitial lung diseases, some of them being incurable and of unknown etiology. Apoptosis plays a critical role in lung fibrogenesis. Protein S is a plasma anticoagulant with potent antiapoptotic activity. The role of protein S in pulmonary fibrosis is unknown. Objectives To evaluate the clinical relevance of protein S and its protective role in pulmonary fibrosis. Methods and Results The circulating level of protein S was measured in patients with pulmonary fibrosis and controls by the use of enzyme immunoassays. Pulmonary fibrosis was induced with bleomycin in transgenic mice overexpressing human protein S and wild-type mice, and exogenous protein S or vehicle was administered to wild-type mice; fibrosis was then compared in both models. Patients with pulmonary fibrosis had reduced circulating levels of protein S as compared with controls. Inflammatory changes, the levels of profibrotic cytokines, fibrosis score, hydroxyproline content in the lungs and oxygen desaturation were significantly reduced in protein S-transgenic mice as compared with wild-type mice. Wild-type mice treated with exogenous protein S showed significant decreases in the levels of inflammatory and profibrotic markers and fibrosis in the lungs as compared with untreated control mice. After bleomycin infusion, mice overexpressing human protein S showed significantly low caspase-3 activity, enhanced expression of antiapoptotic molecules and enhanced Akt and Axl kinase phosphorylation as compared with wild-type counterparts. Protein S also inhibited apoptosis of alveolar

Protein carbonylation, protein aggregation and neuronal cell death in a murine model of multiple sclerosis

Science.gov (United States)

Dasgupta, Anushka

Many studies have suggested that oxidative stress plays an important role in the pathophysiology of both multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Yet, the mechanism by which oxidative stress leads to tissue damage in these disorders is unclear. Recent work from our laboratory has revealed that protein carbonylation, a major oxidative modification caused by severe and/or chronic oxidative stress conditions, is elevated in MS and EAE. Furthermore, protein carbonylation has been shown to alter protein structure leading to misfolding/aggregation. These findings prompted me to hypothesize that carbonylated proteins, formed as a consequence of oxidative stress and/or decreased proteasomal activity, promote protein aggregation to mediate neuronal apoptosis in vitro and in EAE. To test this novel hypothesis, I first characterized protein carbonylation, protein aggregation and apoptosis along the spinal cord during the course of myelin-oligodendrocyte glycoprotein (MOG)35-55 peptide-induced EAE in C57BL/6 mice [Chapter 2]. The results show that carbonylated proteins accumulate throughout the course of the disease, albeit by different mechanisms: increased oxidative stress in acute EAE and decreased proteasomal activity in chronic EAE. I discovered not only that there is a temporal correlation between protein carbonylation and apoptosis but also that carbonyl levels are significantly higher in apoptotic cells. A high number of juxta-nuclear and cytoplasmic protein aggregates containing the majority of the oxidized proteins are also present during the course of EAE, which seems to be due to reduced autophagy. In chapter 3, I show that when gluthathione levels are reduced to those in EAE spinal cord, both neuron-like PC12 (nPC12) cells and primary neuronal cultures accumulate carbonylated proteins and undergo cell death (both by necrosis and apoptosis). Immunocytochemical and biochemical studies also revealed a temporal

Fructo-oligosaccharides reduce energy intake but do not affect adiposity in rats fed a low-fat diet but increase energy intake and reduce fat mass in rats fed a high-fat diet.

Science.gov (United States)

Hadri, Zouheyr; Rasoamanana, Rojo; Fromentin, Gilles; Azzout-Marniche, Dalila; Even, Patrick C; Gaudichon, Claire; Darcel, Nicolas; Bouras, Abdelkader Dilmi; Tomé, Daniel; Chaumontet, Catherine

2017-12-01

The ingestion of low or high lipid diets enriched with fructo-oligosaccharide (FOS) affects energy homeostasis. Ingesting protein diets also induces a depression of energy intake and decreases body weight. The goal of this study was to investigate the ability of FOS, combined or not with a high level of protein (P), to affect energy intake and body composition when included in diets containing different levels of lipids (L). We performed two studies of similar design over a period of 5weeks. During the first experiment (exp1), after a 3-week period of adaptation to a normal protein-low fat diet, the rats received one of the following four diets for 5weeks (6 rats per group): (i) normal protein (14% P/E (Energy) low fat (10% L/E) diet, (ii) normal protein, low fat diet supplemented with 10% FOS, (iii) high protein (55%P/E) low fat diet, and (iv) high protein, low fat diet supplemented with 10% FOS. In a second experiment (exp2) after the 3-week period of adaptation to a normal protein-high fat diet, the rats received one of the following 4 diets for 5weeks (6 rats per group): (i) normal protein, high fat diet (35% of fat), (ii) normal protein, high fat diet supplemented with 10% FOS, (iii) high protein high fat diet and (iv) high protein high fat diet supplemented with 10% FOS. In low-fat fed rats, FOS did not affect lean body mass (LBM) and fat mass but the protein level reduced fat mass and tended to reduce adiposity. In high-fat fed rats, FOS did not affect LBM but reduced fat mass and adiposity. No additive or antagonistic effects between FOS and the protein level were observed. FOS reduced energy intake in low-fat fed rats, did not affect energy intake in normal-protein high-fat fed rats but surprisingly, and significantly, increased energy intake in high-protein high-fat fed rats. The results thus showed that FOS added to a high-fat diet reduced body fat and body adiposity. Published by Elsevier Inc.

Mutations in ribosomal protein L3 and 23S ribosomal RNA at the peptidyl transferase centre are associated with reduced susceptibility to tiamulin in Brachyspira spp. isolates.

Science.gov (United States)

Pringle, Märit; Poehlsgaard, Jacob; Vester, Birte; Long, Katherine S

2004-12-01

The pleuromutilin antibiotic tiamulin binds to the ribosomal peptidyl transferase centre. Three groups of Brachyspira spp. isolates with reduced tiamulin susceptibility were analysed to define resistance mechanisms to the drug. Mutations were identified in genes encoding ribosomal protein L3 and 23S rRNA at positions proximal to the peptidyl transferase centre. In two groups of laboratory-selected mutants, mutations were found at nucleotide positions 2032, 2055, 2447, 2499, 2504 and 2572 of 23S rRNA (Escherichia coli numbering) and at amino acid positions 148 and 149 of ribosomal protein L3 (Brachyspira pilosicoli numbering). In a third group of clinical B. hyodysenteriae isolates, only a single mutation at amino acid 148 of ribosomal protein L3 was detected. Chemical footprinting experiments show a reduced binding of tiamulin to ribosomal subunits from mutants with decreased susceptibility to the drug. This reduction in drug binding is likely the resistance mechanism for these strains. Hence, the identified mutations located near the tiamulin binding site are predicted to be responsible for the resistance phenotype. The positions of the mutated residues relative to the bound drug advocate a model where the mutations affect tiamulin binding indirectly through perturbation of nucleotide U2504.

Protein enriched pasta: structure and digestibility of its protein network.

Science.gov (United States)

Laleg, Karima; Barron, Cécile; Santé-Lhoutellier, Véronique; Walrand, Stéphane; Micard, Valérie

2016-02-01

Wheat (W) pasta was enriched in 6% gluten (G), 35% faba (F) or 5% egg (E) to increase its protein content (13% to 17%). The impact of the enrichment on the multiscale structure of the pasta and on in vitro protein digestibility was studied. Increasing the protein content (W- vs. G-pasta) strengthened pasta structure at molecular and macroscopic scales but reduced its protein digestibility by 3% by forming a higher covalently linked protein network. Greater changes in the macroscopic and molecular structure of the pasta were obtained by varying the nature of protein used for enrichment. Proteins in G- and E-pasta were highly covalently linked (28-32%) resulting in a strong pasta structure. Conversely, F-protein (98% SDS-soluble) altered the pasta structure by diluting gluten and formed a weak protein network (18% covalent link). As a result, protein digestibility in F-pasta was significantly higher (46%) than in E- (44%) and G-pasta (39%). The effect of low (55 °C, LT) vs. very high temperature (90 °C, VHT) drying on the protein network structure and digestibility was shown to cause greater molecular changes than pasta formulation. Whatever the pasta, a general strengthening of its structure, a 33% to 47% increase in covalently linked proteins and a higher β-sheet structure were observed. However, these structural differences were evened out after the pasta was cooked, resulting in identical protein digestibility in LT and VHT pasta. Even after VHT drying, F-pasta had the best amino acid profile with the highest protein digestibility, proof of its nutritional interest.

Laminin-111 protein therapy reduces muscle pathology and improves viability of a mouse model of merosin-deficient congenital muscular dystrophy.

Science.gov (United States)

Rooney, Jachinta E; Knapp, Jolie R; Hodges, Bradley L; Wuebbles, Ryan D; Burkin, Dean J

2012-04-01

Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is a lethal muscle-wasting disease that is caused by mutations in the LAMA2 gene, resulting in the loss of laminin-α2 protein. MDC1A patients exhibit severe muscle weakness from birth, are confined to a wheelchair, require ventilator assistance, and have reduced life expectancy. There are currently no effective treatments or cures for MDC1A. Laminin-α2 is required for the formation of heterotrimeric laminin-211 (ie, α2, β1, and γ1) and laminin-221 (ie, α2, β2, and γ1), which are major constituents of skeletal muscle basal lamina. Laminin-111 (ie, α1, β1, and γ1) is the predominant laminin isoform in embryonic skeletal muscle and supports normal skeletal muscle development in laminin-α2-deficient muscle but is absent from adult skeletal muscle. In this study, we determined whether treatment with Engelbreth-Holm-Swarm-derived mouse laminin-111 protein could rescue MDC1A in the dy(W-/-) mouse model. We demonstrate that laminin-111 protein systemically delivered to the muscles of laminin-α2-deficient mice prevents muscle pathology, improves muscle strength, and dramatically increases life expectancy. Laminin-111 also prevented apoptosis in laminin-α2-deficient mouse muscle and primary human MDC1A myogenic cells, which indicates a conserved mechanism of action and cross-reactivity between species. Our results demonstrate that laminin-111 can serve as an effective protein substitution therapy for the treatment of muscular dystrophy in the dy(W-/-) mouse model and establish the potential for its use in the treatment of MDC1A. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Secretory proteins of the pulmonary extracellular lining

International Nuclear Information System (INIS)

Gupta, R.P.; Patton, S.E.; Eddy, M.; Smits, H.L.; Jetten, A.M.; Nettesheim, P.; Hook, G.E.R.

1986-01-01

The objective of this investigation was to identify proteins in the pulmonary extracellular lining (EL) that are secreted by cells of the pulmonary epithelium. Pulmonary lavage effluents from the lungs of rabbits were centrifuged to remove all cells and particulate materials. Serum proteins were removed by repeatedly passing concentrated lavage effluent fluid through an affinity column containing IgG fraction of goat anti-rabbit (whole serum) antiserum bound to Sepharose-4B. Nonserum proteins accounted for 21.3 +/- 10.3% of the total soluble proteins in pulmonary lavage effluents. Serum free lavage effluents (SFL) contained 25 identifiable proteins as determined by using SDS-PAGE under reducing conditions. Of these proteins approximately 73% was accounted for by a single protein with MW of 66 kd. The secretory nature of the proteins present in SFL was investigated by studying the incorporation of 35 S-methionine into proteins released by lung slices and trachea followed by SDS-PAGE and autoradiography. Many, but not all proteins present in SFL were identified as proteins secreted by pulmonary tissues. The major secretory proteins appeared to have MWs of 59, 53, 48, 43, 24, 14, and 6 kd under reducing conditions. These data demonstrate the presence of several proteins in the pulmonary extracellular lining that appear to be secreted by the pulmonary epithelium

Expanding the action of duplex RNAs into the nucleus: redirecting alternative splicing

Science.gov (United States)

Liu, Jing; Hu, Jiaxin; Corey, David R.

2012-01-01

Double-stranded RNAs are powerful agents for silencing gene expression in the cytoplasm of mammalian cells. The potential for duplex RNAs to control expression in the nucleus has received less attention. Here, we investigate the ability of small RNAs to redirect splicing. We identify RNAs targeting an aberrant splice site that restore splicing and production of functional protein. RNAs can target sequences within exons or introns and affect the inclusion of exons within SMN2 and dystrophin, genes responsible for spinal muscular atrophy and Duchenne muscular dystrophy, respectively. Duplex RNAs recruit argonaute 2 (AGO2) to pre-mRNA transcripts and altered splicing requires AGO2 expression. AGO2 promotes transcript cleavage in the cytoplasm, but recruitment of AGO2 to pre-mRNAs does not reduce transcript levels, exposing a difference between cytoplasmic and nuclear pathways. Involvement of AGO2 in splicing, a classical nuclear process, reinforces the conclusion from studies of RNA-mediated transcriptional silencing that RNAi pathways can be adapted to function in the mammalian nucleus. These data provide a new strategy for controlling splicing and expand the reach of small RNAs within the nucleus of mammalian cells. PMID:21948593

Extractable protein of radiation vulcanized natural rubber latex

International Nuclear Information System (INIS)

Soebianto, Y.S.; Ratnayake, U.M.; Makuuchi, Keizo; Yoshii, Fumio; Kume, Tamikazu

2000-01-01

Protein remained in the latex products are reported to cause serious allergy. A new method to reduce the protein level in the latex products by irradiation is reported. Water soluble protein (WSP) solution (10%) was added into radiation vulcanized NR latex (RVNRL) in three different processes. The amount of WSP was 3 phr. It was only added to RVNRL (standard), added to re-centrifuged RVNRL (pre-centrifugation), and added to RVNRL followed by centrifugation (post-centrifugation). The protein content was determined by enhanced BCA method, and identified by SDS-PAGE. Extractable protein (EP) from the rubber has been reduced up to the minimum protein detection by combining WSP addition and centrifugation. Short leaching time (20-30 min.) can be achieved after the combine treatment, and SDS-PAGE confirms the reduction of soluble protein in the serum phase, and disappearance of protein bands in the rubber extract. Protein-WSP interaction produces water soluble complex, and removed by centrifugation. The efficiency of protein removal by WSP depends on its molecular weight of WSP which relates to its water solubility. (author)

Electrophysiology of glioma: a Rho GTPase-activating protein reduces tumor growth and spares neuron structure and function.

Science.gov (United States)

Vannini, Eleonora; Olimpico, Francesco; Middei, Silvia; Ammassari-Teule, Martine; de Graaf, Erik L; McDonnell, Liam; Schmidt, Gudula; Fabbri, Alessia; Fiorentini, Carla; Baroncelli, Laura; Costa, Mario; Caleo, Matteo

2016-12-01

Glioblastomas are the most aggressive type of brain tumor. A successful treatment should aim at halting tumor growth and protecting neuronal cells to prevent functional deficits and cognitive deterioration. Here, we exploited a Rho GTPase-activating bacterial protein toxin, cytotoxic necrotizing factor 1 (CNF1), to interfere with glioma cell growth in vitro and vivo. We also investigated whether this toxin spares neuron structure and function in peritumoral areas. We performed a microarray transcriptomic and in-depth proteomic analysis to characterize the molecular changes triggered by CNF1 in glioma cells. We also examined tumor cell senescence and growth in vehicle- and CNF1-treated glioma-bearing mice. Electrophysiological and morphological techniques were used to investigate neuronal alterations in peritumoral cortical areas. Administration of CNF1 triggered molecular and morphological hallmarks of senescence in mouse and human glioma cells in vitro. CNF1 treatment in vivo induced glioma cell senescence and potently reduced tumor volumes. In peritumoral areas of glioma-bearing mice, neurons showed a shrunken dendritic arbor and severe functional alterations such as increased spontaneous activity and reduced visual responsiveness. CNF1 treatment enhanced dendritic length and improved several physiological properties of pyramidal neurons, demonstrating functional preservation of the cortical network. Our findings demonstrate that CNF1 reduces glioma volume while at the same time maintaining the physiological and structural properties of peritumoral neurons. These data indicate a promising strategy for the development of more effective antiglioma therapies. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Comparative proteome analysis of cryopreserved flagella and head plasma membrane proteins from sea bream spermatozoa: effect of antifreeze proteins.

Science.gov (United States)

Zilli, Loredana; Beirão, José; Schiavone, Roberta; Herraez, Maria Paz; Gnoni, Antonio; Vilella, Sebastiano

2014-01-01

Cryopreservation induces injuries to fish spermatozoa that in turn affect sperm quality in terms of fertilization ability, motility, DNA and protein integrity and larval survival. To reduce the loss of sperm quality due to freezing-thawing, it is necessary to improve these procedures. In the present study we investigated the ability of two antifreeze proteins (AFPI and AFPIII) to reduce the loss of quality of sea bream spermatozoa due to cryopreservation. To do so, we compared viability, motility, straight-line velocity and curvilinear velocity of fresh and (AFPs)-cryopreserved spermatozoa. AFPIII addition to cryopreservation medium improved viability, motility and straight-line velocity with respect to DMSO or DMSO plus AFPI. To clarify the molecular mechanism(s) underlying these findings, the protein profile of two different cryopreserved sperm domains, flagella and head plasma membranes, was analysed. The protein profiles differed between fresh and frozen-thawed semen and results of the image analysis demonstrated that, after cryopreservation, out of 270 proteins 12 were decreased and 7 were increased in isolated flagella, and out of 150 proteins 6 showed a significant decrease and 4 showed a significant increase in head membranes. Mass spectrometry analysis identified 6 proteins (4 from isolated flagella and 2 present both in flagella and head plasma membranes) within the protein spots affected by the freezing-thawing procedure. 3 out of 4 proteins from isolated flagella were involved in the sperm bioenergetic system. Our results indicate that the ability of AFPIII to protect sea bream sperm quality can be, at least in part, ascribed to reducing changes in the sperm protein profile occurring during the freezing-thawing procedure. Our results clearly demonstrated that AFPIII addition to cryopreservation medium improved the protection against freezing respect to DMSO or DMSO plus AFPI. In addition we propose specific proteins of spermatozoa as markers related to

Identification of a 34 kDa protein altered in the LF-1 mutant as the herbicide-binding D1 protein of photosystem II

International Nuclear Information System (INIS)

Metz, J.; Pakrasi, H.; Seibert, M.; Arntzen, C.

1986-01-01

The LF-1 mutant of Scenedesmus has a complete block on the oxidizing side of its PSII reaction center. However, the reaction center as well as the reducing side of PSII is fully functional in this mutant. Compared to the wildtype (WT) the only detected protein difference in the PSII complex of LF-1 is the change in mobility of a 34 kDa protein to 36 kDa. This protein has been implicated to have a major role in Mn-binding and water-oxidation. The authors have recently shown that photoaffinity labeling of thylakoids with azido-[ 14 C]-atrazine tags the 34 kDa protein in WT and the 36 kDa protein in LF-1. It has been shown that the azido-atrazine labeled protein, called D1, functions in herbicide binding and Q/sub A/ to Q/sub B/ electron transfer on the reducing side of PSII. Polyclonal antibodies directed against the D1 protein of Amaranthus hybridus (Ohad, et al., EMBOJ 1985) were found to recognize the Scenedesmus 34 kDa (WT) and 36 kDa (LF-1) proteins. The implied dual function for the D1 protein on the reducing as well as the oxidizing side of PSII reaction center will be discussed

Molecular mechanisms of reduced glutathione transport: role of the MRP/CFTR/ABCC and OATP/SLC21A families of membrane proteins

International Nuclear Information System (INIS)

Ballatori, Nazzareno; Hammond, Christine L.; Cunningham, Jennifer B.; Krance, Suzanne M.; Marchan, Rosemarie

2005-01-01

The initial step in reduced glutathione (GSH) turnover in all mammalian cells is its transport across the plasma membrane into the extracellular space; however, the mechanisms of GSH transport are not clearly defined. GSH export is required for the delivery of its constituent amino acids to other tissues, detoxification of drugs, metals, and other reactive compounds of both endogenous and exogenous origin, protection against oxidant stress, and secretion of hepatic bile. Recent studies indicate that some members of the multidrug resistance-associated protein (MRP/CFTR or ABCC) family of ATP-binding cassette (ABC) proteins, as well as some members of the organic anion transporting polypeptide (OATP or SLC21A) family of transporters contribute to this process. In particular, five of the 12 members of the MRP/CFTR family appear to mediate GSH export from cells namely, MRP1, MRP2, MRP4, MRP5, and CFTR. Additionally, two members of the OATP family, rat Oatp1 and Oatp2, have been identified as GSH transporters. For the Oatp1 transporter, efflux of GSH may provide the driving force for the uptake of extracellular substrates. In humans, OATP-B and OATP8 do not appear to transport GSH; however, other members of this family have yet to be characterized in regards to GSH transport. In yeast, the ABC proteins Ycf1p and Bpt1p transport GSH from the cytosol into the vacuole, whereas Hgt1p mediates GSH uptake across the plasma membrane. Because transport is a key step in GSH homeostasis and is intimately linked to its biological functions, GSH export proteins are likely to modulate essential cellular functions

Protein design for pathway engineering.

Science.gov (United States)

Eriksen, Dawn T; Lian, Jiazhang; Zhao, Huimin

2014-02-01

Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. Copyright © 2013 Elsevier Inc. All rights reserved.

Dietary management of chronic kidney disease: protein restriction and beyond.

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Goraya, Nimrit; Wesson, Donald E

2012-11-01

More kidney protective strategies are needed to reduce the burden of complete kidney failure from chronic kidney disease (CKD). Clinicians sometimes use protein restriction as kidney protection despite its demonstrated lack of effectiveness in the only large-scale study. Small-scale studies support that dietary acid reduction is kidney-protective, including when done with base-inducing foods like fruits and vegetables. We review these studies in light of current kidney-protective recommendations. Animal models of CKD show that acid-inducing dietary protein exacerbates and base-inducing protein ameliorates nephropathy progression, and that increased intake of acid-inducing but not base-inducing dietary protein exacerbates progression. Clinical studies show that dietary acid reduction with Na-based alkali reduces kidney injury and slows nephropathy progression in patients with CKD and reduced glomerular filtration rate (GFR); base-inducing fruits and vegetables reduce kidney injury in patients with reduced GFR; and base-inducing fruits and vegetables improve metabolic acidosis in CKD. Protein type rather than amount might more importantly affect nephropathy progression. Base-inducing foods might be another way to reduce dietary acid, a strategy shown in small studies to slow nephropathy progression. Further studies will determine if CKD patients should be given base-inducing food as part of their management.

Backbone dynamics of oxidized and reduced D. vulgaris flavodoxin in solution

International Nuclear Information System (INIS)

Hrovat, Andrea; Bluemel, Markus; Loehr, Frank; Mayhew, Stephen G.; Rueterjans, Heinz

1997-01-01

Recombinant Desulfovibrio vulgaris flavodoxin was produced in Escherichia coli. A complete backbone NMR assignment for the two-electron reduced protein revealed significant changes of chemical shift values compared to the oxidized protein, in particular for the flavine mononucleotide (FMN)-binding site. A comparison of homo- and heteronuclear NOESY spectra for the two redox states led to the assumption that reduction is not accompanied by significant changes of the global fold of the protein.The backbone dynamics of both the oxidized and reduced forms of D. vulgaris flavodoxin were investigated using two-dimensional 15 N- 1 H correlation NMR spectroscopy.T 1 , T 2 and NOE data are obtained for 95% of the backbone amide groups in both redox states. These values were analysed in terms of the 'model-free' approach introduced by Lipari and Szabo [(1982) J. Am. Chem. Soc., 104, 4546-;4559, 4559-;4570]. A comparison of the two redox states indicates that in the reduced species significantly more flexibility occurs in the two loop regions enclosing FMN.Also, a higher amplitude of local motion could be found for the N(3)H group of FMN bound to the reduced protein compared to the oxidized state

Influence of mycotoxins on protein and amino acid utilization.

Science.gov (United States)

Smith, T K

1982-09-01

The interrelationships between mycotoxins and the utilization of dietary protein are reviewed. Acute aflatoxicosis is characterized by reduced growth and fatty infiltration of the liver. Studies with poultry, swine, and monkeys have shown that supplements of dietary protein beyond normal requirements can overcome these conditions. High-protein diets, however, have been shown to promote hepatoma characteristic of chronic aflatoxicosis in rats. Aflatoxin interferes with utilization of dietary protein by inhibiting synthesis of DNA, RNA, and protein. High-protein diets promote the metabolism of aflatoxin by the hepatic microsomal drug-metabolizing enzyme system. The Fusarium mycotoxin zearalenone increases membrane permeability and promotes uterine synthesis of DNA, RNA, and protein. Supplements of dietary protein overcome growth reduction due to zearalenone and reduce the metabolic half-life of the toxin by promoting urinary excretion of free, unmetabolized zearalenone in the rat. The trichothecene mycotoxins disrupt normal protein metabolism by inactivating the ribosomal cycle. Protein supplements appear to have little effect on trichothecene mycotoxicoses. Most mycotoxins impair utilization of dietary protein. The effectiveness of protein supplements in overcoming mycotoxicoses will depend on the mycotoxin in question.

Study of the structural damage in a niobium-microalloyed steel sheet

International Nuclear Information System (INIS)

Fernandes, J.; Riba, J.; Verdeja, J.I.

1986-01-01

A quantitative experimental study of the damage developed as a consequence of straining has been performed on a microalloyed (niobium) steel sheet by means of a SEM. Equivalent strains range between 0 and 0.68 and strain paths between 0 and 1 and have been obtained in a bulge test. Damage associated to Al 2 O 3 and SMn inclusions is already present in the ''as received'' sheet and grows with strain. Damage associated to CFe 3 second phase particles appears later in the forming of the sheet. For stages previous to necking SMn stringers have dramatically developed more than 50% of total damage. The nucleation equivalent strain is between 0,3 and 0,4. (author)

Reduced Ang2 expression in aging endothelial cells

International Nuclear Information System (INIS)

Hohensinner, P.J.; Ebenbauer, B.; Kaun, C.; Maurer, G.; Huber, K.; Wojta, J.

2016-01-01

Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. -- Highlights: •Endothelial cells display signs of aging before reaching proliferative senescence. •Aging endothelial cells express more angiopoietin 1 and less angiopoietin 2 than young endothelial cells. •Migratory capacity is reduced in aging endothelial cells.

Reduced Ang2 expression in aging endothelial cells

Energy Technology Data Exchange (ETDEWEB)

Hohensinner, P.J., E-mail: philipp.hohensinner@meduniwien.ac.at [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ebenbauer, B. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Kaun, C.; Maurer, G. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Huber, K. [Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); 3rd Medical Department, Wilhelminenhospital, Vienna (Austria); Sigmund Freud University, Medical Faculty, Vienna (Austria); Wojta, J. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Core Facilities, Medical University of Vienna, Vienna (Austria)

2016-06-03

Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. -- Highlights: •Endothelial cells display signs of aging before reaching proliferative senescence. •Aging endothelial cells express more angiopoietin 1 and less angiopoietin 2 than young endothelial cells. •Migratory capacity is reduced in aging endothelial cells.

Flexibility damps macromolecular crowding effects on protein folding dynamics: Application to the murine prion protein (121-231)

Science.gov (United States)

Bergasa-Caceres, Fernando; Rabitz, Herschel A.

2014-01-01

A model of protein folding kinetics is applied to study the combined effects of protein flexibility and macromolecular crowding on protein folding rate and stability. It is found that the increase in stability and folding rate promoted by macromolecular crowding is damped for proteins with highly flexible native structures. The model is applied to the folding dynamics of the murine prion protein (121-231). It is found that the high flexibility of the native isoform of the murine prion protein (121-231) reduces the effects of macromolecular crowding on its folding dynamics. The relevance of these findings for the pathogenic mechanism are discussed.

Tissue protein metabolism in parasitized animals

International Nuclear Information System (INIS)

Symons, L.E.A.; Steel, J.W.; Jones, W.O.

1981-01-01

The effects of gastrointestinal nematode infection of mammals, particularly of the small intestine of the sheep, on protein metabolism of skeletal muscle, liver, the gastrointestinal tract and wool are described. These changes have been integrated to explain poor growth and production in the sheep heavily infected with Trichostrongylus colubriformis. The rates of both synthesis and catabolism of muscle protein are depressed, but nitrogen is lost from this tissue because the depression of synthesis exceeds that of catabolism. Anorexia is the major cause of these changes. Although the effect on liver protein synthesis is unclear, it is probable that the leakage of plasma proteins into the gastrointestinal tract stimulates an early increase in the rate of synthesis of these proteins, but this eventually declines and is insufficient to correct developing hypoalbuminaemia. Changes in the intestinal tract are complex. Exogenous nitrogen is reduced by anorexia, but the flow of nitrogen through the tract from abomasum to faeces is above normal because of the increase of endogenous protein from leakage of plasma protein and, presumably, from exfoliated epithelial cells. There is evidence that protein metabolism of intestinal tissue, particularly in the uninfected distal two-thirds, is increased. Synthesis of wool protein is decreased. As the result of anorexia, intestinal loss of endogenous protein and an increased rate of intestinal protein metabolism there is a net movement of amino nitrogen from muscle, liver and possibly skin to the intestine of the heavily infected sheep. Thus, the availability of amino nitrogen for growth and wool production is reduced. (author)

Gamma irradiation effect on soy protein modification, protein - phenolic interaction and antioxidant activity in soybean

International Nuclear Information System (INIS)

Kumari, Sweta; Dahuja, Anil; Vinutha, T.; Singh, Bhupinder

2014-01-01

Soy protein is one of the most important sources of protein to feed the world population in the future. Consumption of soybean quality protein and their texture is dependent on the protein modification. In the present study, four soybean genotypes PL5039 (black), EC 472143 (black), Pusa 9814 (yellow) and SL525 (yellow), differing in their seed coat colour were gamma irradiated at 0.5,1.0, 2.0 and 5.0 kGy and the extent of protein modification and parameters affecting it viz. free phenolics, bound phenolics, lip oxygenase and antioxidant activity were analysed. Modifications of soybean proteins were investigated by chemical analysis and electrophoresis. The irradiation dose of 1.0 kGy showed decreased turbidity, protein oxidation, surface hydrophobicity but increased solubility and sulfhydryl and disulfide contents in all the genotypes. Further, SDS PAGE profile of treated soybean seeds revealed remarkable difference in electrophoretic bands as compared to the untreated seeds. Lipoxygense activity in all the genotypes decreased with increased exposure of gamma irradiation, which produced peroxide products that changes the structural characteristics of soy protein. Free phenolics, bound phenolics and total antioxidant activity measured in terms of FRAP in all the genotypes increased significantly at a dose of 2.0 kGy and it declined at a dose of 5.0 kGy. Antioxidant potential measured in terms of 1,1-diphenyl-2- picrylhydrazyl (DPPH) scavenging activity showed an increasing trend with dose, indicating that radiation processing as a method of food preservation has a positive nutritional implication. Hence, it is suggested that, mild gamma irradiation upto 2.0 kGy may reduce the protein oxidation, enhance the antioxidant activity and improve the soybean protein quality compared to higher dose 5.0 kGy, which reduced the protein quality. (author)

Risk of Late Mortality and Second Malignant Neoplasms among 5-Year Survivors of Young Adult Cancer: A Report of the Childhood, Adolescent, and Young Adult Cancer Survivors Research Program

International Nuclear Information System (INIS)

Zhang, Y.; Spinelli, J. J.; Gotay, C.; McBride, M. L.; Zhang, Y.; Spinelli, J. J.; Goddard, K.

2012-01-01

We conducted a population-based retrospective study to assess the long-term risks of overall and cause-specific mortality and second malignant neoplasm (SMN) among survivors of young adult cancer compared to the risk in British Columbia (BC) population and to evaluate the effects of demographic and clinical factors on risk. 1248 5-year survivors of young adult cancer diagnosed 1970-1995 between 20 and 24 years of age were identified from the BC Cancer Registry and followed to the end of 2007. Standardized mortality ratios (SMRs) and standardized incidence ratios (SIRs) were calculated. The Cox proportional hazards model was used to estimate the effects of different demographic and disease-related characteristics on the risk of death and SMN. A total of 138 deaths and 62 SMNs were observed during follow-up. The overall SMR was 5.9 (95% CI 4.9-6.9) and the absolute excess risk was 5.3 per 1,000 person-years. The overall SIR was 3.0 (95% CI 2.3-3.8). Treatment with radiation resulted in increased risks of death and SMN. These observed increased risks emphasize the importance of prevention, surveillance, and treatment of late effects in survivors of young adult cancers.

Modeling Mercury in Proteins

Energy Technology Data Exchange (ETDEWEB)

Smith, Jeremy C [ORNL; Parks, Jerry M [ORNL

2016-01-01

Mercury (Hg) is a naturally occurring element that is released into the biosphere both by natural processes and anthropogenic activities. Although its reduced, elemental form Hg(0) is relatively non-toxic, other forms such as Hg2+ and, in particular, its methylated form, methylmercury, are toxic, with deleterious effects on both ecosystems and humans. Microorganisms play important roles in the transformation of mercury in the environment. Inorganic Hg2+ can be methylated by certain bacteria and archaea to form methylmercury. Conversely, bacteria also demethylate methylmercury and reduce Hg2+ to relatively inert Hg(0). Transformations and toxicity occur as a result of mercury interacting with various proteins. Clearly, then, understanding the toxic effects of mercury and its cycling in the environment requires characterization of these interactions. Computational approaches are ideally suited to studies of mercury in proteins because they can provide a detailed picture and circumvent issues associated with toxicity. Here we describe computational methods for investigating and characterizing how mercury binds to proteins, how inter- and intra-protein transfer of mercury is orchestrated in biological systems, and how chemical reactions in proteins transform the metal. We describe quantum chemical analyses of aqueous Hg(II), which reveal critical factors that determine ligand binding propensities. We then provide a perspective on how we used chemical reasoning to discover how microorganisms methylate mercury. We also highlight our combined computational and experimental studies of the proteins and enzymes of the mer operon, a suite of genes that confers mercury resistance in many bacteria. Lastly, we place work on mercury in proteins in the context of what is needed for a comprehensive multi-scale model of environmental mercury cycling.

Ginsenoside F2 reduces hair loss by controlling apoptosis through the sterol regulatory element-binding protein cleavage activating protein and transforming growth factor-β pathways in a dihydrotestosterone-induced mouse model.

Science.gov (United States)

Shin, Heon-Sub; Park, Sang-Yong; Hwang, Eun-Son; Lee, Don-Gil; Mavlonov, Gafurjon Turdalievich; Yi, Tae-Hoo

2014-01-01

This study was conducted to test whether ginsenoside F2 can reduce hair loss by influencing sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and the transforming growth factor beta (TGF-β) pathway of apoptosis in dihydrotestosterone (DHT)-treated hair cells and in a DHT-induced hair loss model in mice. Results for ginsenoside F2 were compared with finasteride. DHT inhibits proliferation of hair cells and induces androgenetic alopecia and was shown to activate an apoptosis signal pathway both in vitro and in vivo. The cell-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the proliferation rates of DHT-treated human hair dermal papilla cells (HHDPCs) and HaCaTs increased by 48% in the ginsenoside F2-treated group and by 12% in the finasteride-treated group. Western blot analysis showed that ginsenoside F2 decreased expression of TGF-β2 related factors involved in hair loss. The present study suggested a hair loss related pathway by changing SCAP related apoptosis pathway, which has been known to control cholesterol metabolism. SCAP, sterol regulatory element-binding protein (SREBP) and caspase-12 expression in the ginsenoside F2-treated group were decreased compared to the DHT and finasteride-treated group. C57BL/6 mice were also prepared by injection with DHT and then treated with ginsenoside F2 or finasteride. Hair growth rate, density, thickness measurements and tissue histotological analysis in these groups suggested that ginsenoside F2 suppressed hair cell apoptosis and premature entry to catagen more effectively than finasteride. Our results indicated that ginsenoside F2 decreased the expression of TGF-β2 and SCAP proteins, which have been suggested to be involved in apoptosis and entry into catagen. This study provides evidence those factors in the SCAP pathway could be targets for hair loss prevention drugs.

Increased Levels of Antinutritional and/or Defense Proteins Reduced the Protein Quality of a Disease-Resistant Soybean Cultivar.

Science.gov (United States)

Sousa, Daniele O B; Carvalho, Ana F U; Oliveira, José Tadeu A; Farias, Davi F; Castelar, Ivan; Oliveira, Henrique P; Vasconcelos, Ilka M

2015-07-22

The biochemical and nutritional attributes of two soybean (Glycine max (L.) Merr.) cultivars, one susceptible (Seridó) and the other resistant (Seridó-RCH) to stem canker, were examined to assess whether the resistance to pathogens was related to levels of antinutritional and/or defense proteins in the plant and subsequently affected the nutritional quality. Lectin, urease, trypsin inhibitor, peroxidase and chitinase activities were higher in the resistant cultivar. Growing rats were fed with isocaloric and isoproteic diets prepared with defatted raw soybean meals. Those on the Seridó-RCH diet showed the worst performance in terms of protein quality indicators. Based on regression analysis, lectin, trypsin inhibitor, peroxidase and chitinase appear to be involved in the resistance trait but also in the poorer nutritional quality of Seridó-RCH. Thus, the development of cultivars for disease resistance may lead to higher concentrations of antinutritional compounds, affecting the quality of soybean seeds. Further research that includes the assessment of more cultivars/genotypes is needed.

Protein and non-protein sulfhydryls and disulfides in gastric mucosa and liver after gastrotoxic chemicals and sucralfate: possible new targets of pharmacologic agents.

Science.gov (United States)

Nagy, Lajos; Nagata, Miki; Szabo, Sandor

2007-04-14

To investigate the role of major non-protein and protein sulfhydryls and disulfides in chemically induced gastric hemorrhagic mucosal lesions (HML) and the mechanism of gastroprotective effect of sucralfate. Rats were given 1 mL of 75% ethanol, 25% NaCl, 0.6 mol/L HCl, 0.2 mol/L NaOH or 1% ammonia solutions intragastrically (i.g.) and sacrificed 1, 3, 6 or 12 min later. Total (reduced and oxidized) glutathione (GSH + GSSG), glutathione disulfide (GSSG), protein free sulfhydryls (PSH), protein-glutathione mixed disulfides (PSSG) and protein cystine disulfides (PSSP) were measured in gastric mucosa and liver. Reduced glutathione (GSH) was depleted in the gastric mucosa after ethanol, HCl or NaCl exposure, while oxidized glutathione (GSSG) concentrations increased, except by HCl and NaOH exposure. Decreased levels of PSH after exposure to ethanol were observed, NaCl or NaOH while the total protein disulfides were increased. Ratios of reduced to oxidized glutathione or sulfhydrils to disulfides were decreased by all chemicals. No changes in thiol homeostasis were detected in the liver after i.g. abbreviation should be spelled out the first time here administration of ethanol. Sucralfate increased the concentrations of GSH and PSH and prevented the ethanol-induced changes in gastric mucosal thiol concentrations. Our modified methods are now suitable for direct measurements of major protein and non-protein thiols/disulfides in the gastric mucosa or liver. A common element in the pathogenesis of chemically induced HML and in the mechanism of gastroprotective drugs seems to be the decreased ratios of reduced and oxidized glutathione as well as protein sulfhydryls and disulfides.

A new look on protein-polyphenol complexation during honey storage: is this a random or organized event with the help of dirigent-like proteins?

Directory of Open Access Journals (Sweden)

Katrina Brudzynski

Full Text Available Honey storage initiates melanoidin formation that involves a cascade of seemingly unguided redox reactions between amino acids/proteins, reducing sugars and polyphenols. In the process, high molecular weight protein-polyphenol complexes are formed, but the mechanism involved remains unknown. The objective of this study was twofold: to determine quantitative and qualitative changes in proteins in honeys stored for prolonged times and in different temperatures and to relate these changes to the formation of protein-polyphenol complexes. Six -month storage decreased the protein content by 46.7% in all tested honeys (t-test, p<0.002 with the rapid reduction occurring during the first three month. The changes in protein levels coincided with alterations in molecular size and net charge of proteins on SDS -PAGE. Electro-blotted proteins reacted with a quinone-specific nitro blue tetrazolium (NBT on nitrocellulose membranes indicating that quinones derived from oxidized polyphenols formed covalent bonds with proteins. Protein-polyphenol complexes isolated by size-exclusion chromatography differed in size and stoichiometry and fall into two categories: (a high molecular weight complexes (230-180 kDa enriched in proteins but possessing a limited reducing activity toward the NBT and (b lower molecular size complexes (110-85 kDa enriched in polyphenols but strongly reducing the dye. The variable stoichiometry suggest that the large, "protein-type" complexes were formed by protein cross-linking, while in the smaller, "polyphenol-type" complexes polyphenols were first polymerized prior to protein binding. Quinones preferentially bound a 31 kDa protein which, by the electrospray quadrupole time of flight mass spectrometry (ESI-Qtof-MS analysis, showed homology to dirigent-like proteins known for assisting in radical coupling and polymerization of phenolic compounds. These findings provide a new look on protein-polyphenol interaction in honey where the

The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

International Nuclear Information System (INIS)

Galvão, C.W.; Souza, E.M.; Etto, R.M.; Pedrosa, F.O.; Chubatsu, L.S.; Yates, M.G.; Schumacher, J.; Buck, M.; Steffens, M.B.R.

2012-01-01

DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs ) can interact with the H. seropedicae RecA protein (RecA Hs ) and that RecA Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX Hs inhibited 90% of the RecA Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA Hs . RecA Hs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX Hs was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX Hs protein negatively modulates the RecA Hs activities by protein-protein interactions and also by DNA-protein interactions

The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

Directory of Open Access Journals (Sweden)

C.W. Galvão

2012-12-01

Full Text Available DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs can interact with the H. seropedicaeRecA protein (RecA Hs and that RecA Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX Hs inhibited 90% of the RecA Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA Hs. RecA Hs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX Hs was present before the formation of RecA filaments (RecA-ssDNA, inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX Hs protein negatively modulates the RecA Hs activities by protein-protein interactions and also by DNA-protein interactions.

The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae.

Science.gov (United States)

Galvão, C W; Souza, E M; Etto, R M; Pedrosa, F O; Chubatsu, L S; Yates, M G; Schumacher, J; Buck, M; Steffens, M B R

2012-12-01

DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs) can interact with the H. seropedicaeRecA protein (RecA Hs) and that RecA Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX Hs inhibited 90% of the RecA Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA Hs. RecA Hs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX Hs was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX Hs protein negatively modulates the RecA Hs activities by protein-protein interactions and also by DNA-protein interactions.

Aptamer-Conjugated Calcium Phosphate Nanoparticles for Reducing Diabetes Risk via Retinol Binding Protein 4 Inhibition.

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Torabi, Raheleh; Ghourchian, Hedayatollah; Amanlou, Massoud; Pasalar, Parvin

2017-06-01

Inhibition of the binding of retinol to its carrier, retinol binding protein 4, is a new strategy for treating type 2 diabetes; for this purpose, we have provided an aptamer-functionalized multishell calcium phosphate nanoparticle. First, calcium phosphate nanoparticles were synthesized and conjugated to the aptamer. The cytotoxicity of nanoparticles releases the process of aptamer from nanoparticles and their inhibition function of binding retinol to retinol binding protein 4. After synthesizing and characterizing the multishell calcium phosphate nanoparticles and observing the noncytotoxicity of conjugate, the optimum time (48 hours) and the pH (7.4) for releasing the aptamer from the nanoparticles was determined. The half-maximum inhibitory concentration (IC 50 ) value for inhibition of retinol binding to retinol binding protein 4 was 210 femtomolar (fmol). The results revealed that the aptamer could prevent connection between retinol and retinol binding protein 4 at a very low IC 50 value (210 fmol) compared to other reported inhibitors. It seems that this aptamer could be used as an efficient candidate not only for decreasing the insulin resistance in type 2 diabetes, but also for inhibiting the other retinol binding protein 4-related diseases. Copyright © 2017 Diabetes Canada. Published by Elsevier Inc. All rights reserved.

Reduced Ang2 expression in aging endothelial cells.

Science.gov (United States)

Hohensinner, P J; Ebenbauer, B; Kaun, C; Maurer, G; Huber, K; Wojta, J

2016-06-03

Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. Copyright © 2016 Elsevier Inc. All rights reserved.

Label-free and direct detection of C-reactive protein using reduced graphene oxide-nanoparticle hybrid impedimetric sensor.

Science.gov (United States)

Yagati, Ajay Kumar; Pyun, Jae-Chul; Min, Junhong; Cho, Sungbo

2016-02-01

For label-free and direct detection of C-reactive protein (CRP), an impedimetric sensor based on an indium tin oxide (ITO) electrode array functionalized with reduced graphene oxide-nanoparticle (rGO-NP) hybrid was fabricated and evaluated. Analytical measurements were performed to examine the properties of rGO-NP-modified ITO microelectrodes and to determine the influence upon sensory performance of using nanostructures modified for antibody immobilization and for recognition of CRP binding events. Impedimetric measurements in the presence of the redox couple [Fe(CN)6](3-/4-) showed significant changes in charge transfer resistance upon binding of CRP. The impedance measurements were highly target specific, linear with logarithmic CRP concentrations in PBS and human serum across a 1 ng mL(-1) and 1000 ng mL(-1) range and associated with a detection limits of 0.06 and 0.08 ng mL(-1) respectively. Copyright © 2015 Elsevier B.V. All rights reserved.

PseKRAAC: a flexible web server for generating pseudo K-tuple reduced amino acids composition.

Science.gov (United States)

Zuo, Yongchun; Li, Yuan; Chen, Yingli; Li, Guangpeng; Yan, Zhenhe; Yang, Lei

2017-01-01

The reduced amino acids perform powerful ability for both simplifying protein complexity and identifying functional conserved regions. However, dealing with different protein problems may need different kinds of cluster methods. Encouraged by the success of pseudo-amino acid composition algorithm, we developed a freely available web server, called PseKRAAC (the pseudo K-tuple reduced amino acids composition). By implementing reduced amino acid alphabets, the protein complexity can be significantly simplified, which leads to decrease chance of overfitting, lower computational handicap and reduce information redundancy. PseKRAAC delivers more capability for protein research by incorporating three crucial parameters that describes protein composition. Users can easily generate many different modes of PseKRAAC tailored to their needs by selecting various reduced amino acids alphabets and other characteristic parameters. It is anticipated that the PseKRAAC web server will become a very useful tool in computational proteomics and protein sequence analysis. Freely available on the web at http://bigdata.imu.edu.cn/psekraac CONTACTS: yczuo@imu.edu.cn or imu.hema@foxmail.com or yanglei_hmu@163.comSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Towards a map of the Populus biomass protein-protein interaction network

Energy Technology Data Exchange (ETDEWEB)

Beers, Eric [Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States); Brunner, Amy [Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States); Helm, Richard [Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States); Dickerman, Allan [Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States)

2015-07-31

Biofuels can be produced from a variety of plant feedstocks. The value of a particular feedstock for biofuels production depends in part on the degree of difficulty associated with the extraction of fermentable sugars from the plant biomass. The wood of trees is potentially a rich source fermentable sugars. However, the sugars in wood exist in a tightly cross-linked matrix of cellulose, hemicellulose, and lignin, making them largely recalcitrant to release and fermentation for biofuels production. Before breeders and genetic engineers can effectively develop plants with reduced recalcitrance to fermentation, it is necessary to gain a better understanding of the fundamental biology of the mechanisms responsible for wood formation. Regulatory, structural, and enzymatic proteins are required for the complicated process of wood formation. To function properly, proteins must interact with other proteins. Yet, very few of the protein-protein interactions necessary for wood formation are known. The main objectives of this project were to 1) identify new protein-protein interactions relevant to wood formation, and 2) perform in-depth characterizations of selected protein-protein interactions. To identify relevant protein-protein interactions, we cloned a set of approximately 400 genes that were highly expressed in the wood-forming tissue (known as secondary xylem) of poplar (Populus trichocarpa). We tested whether the proteins encoded by these biomass genes interacted with each other in a binary matrix design using the yeast two-hybrid (Y2H) method for protein-protein interaction discovery. We also tested a subset of the 400 biomass proteins for interactions with all proteins present in wood-forming tissue of poplar in a biomass library screen design using Y2H. Together, these two Y2H screens yielded over 270 interactions involving over 75 biomass proteins. For the second main objective we selected several interacting pairs or groups of interacting proteins for in

Reduced hemoglobin and increased C-reactive protein are associated with upper gastrointestinal bleeding.

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Tomizawa, Minoru; Shinozaki, Fuminobu; Hasegawa, Rumiko; Togawa, Akira; Shirai, Yoshinori; Ichiki, Noboru; Motoyoshi, Yasufumi; Sugiyama, Takao; Yamamoto, Shigenori; Sueishi, Makoto

2014-02-07

To investigate the early upper gastrointestinal endoscopy (endoscopy) significantly reduces mortality resulting from upper gastrointestinal (GI) bleeding. Upper GI bleeding was defined as 1a, 1b, 2a, and 2b according to the Forrest classification. The hemoglobin (Hb), and C-reactive protein (CRP) were examined at around the day of endoscopy and 3 mo prior to endoscopy. The rate of change was calculated as follows: (the result of blood examination on the day of endoscopy - the results of blood examination 3 mo prior to endoscopy)/(results of blood examination 3 mo prior to endoscopy). Receiver operating characteristic curves were created to determine threshold values. Seventy-nine men and 77 women were enrolled. There were 17 patients with upper GI bleeding: 12 with a gastric ulcer, 3 with a duodenal ulcer, 1 with an acute gastric mucosal lesion, and 1 with gastric cancer. The area under the curve (AUC), threshold, sensitivity, and specificity of Hb around the day of endoscopy were 0.902, 11.7 g/dL, 94.1%, and 77.1%, respectively, while those of CRP were 0.722, 0.5 mg/dL, 70.5%, and 73%, respectively. The AUC, threshold, sensitivity, and specificity of the rate of change of Hb were 0.851, -21.3%, 76.4%, and 82.6%, respectively, while those of CRP were 0.901, 100%, 100%, and 82.5%, respectively. Predictors for upper GI bleeding were Hb 21.3% and an increase in the CRP > 100%, 3 mo before endoscopy.

Improving N-terminal protein annotation of Plasmodium species based on signal peptide prediction of orthologous proteins

Directory of Open Access Journals (Sweden)

Neto Armando

2012-11-01

Full Text Available Abstract Background Signal peptide is one of the most important motifs involved in protein trafficking and it ultimately influences protein function. Considering the expected functional conservation among orthologs it was hypothesized that divergence in signal peptides within orthologous groups is mainly due to N-terminal protein sequence misannotation. Thus, discrepancies in signal peptide prediction of orthologous proteins were used to identify misannotated proteins in five Plasmodium species. Methods Signal peptide (SignalP and orthology (OrthoMCL were combined in an innovative strategy to identify orthologous groups showing discrepancies in signal peptide prediction among their protein members (Mixed groups. In a comparative analysis, multiple alignments for each of these groups and gene models were visually inspected in search of misannotated proteins and, whenever possible, alternative gene models were proposed. Thresholds for signal peptide prediction parameters were also modified to reduce their impact as a possible source of discrepancy among orthologs. Validation of new gene models was based on RT-PCR (few examples or on experimental evidence already published (ApiLoc. Results The rate of misannotated proteins was significantly higher in Mixed groups than in Positive or Negative groups, corroborating the proposed hypothesis. A total of 478 proteins were reannotated and change of signal peptide prediction from negative to positive was the most common. Reannotations triggered the conversion of almost 50% of all Mixed groups, which were further reduced by optimization of signal peptide prediction parameters. Conclusions The methodological novelty proposed here combining orthology and signal peptide prediction proved to be an effective strategy for the identification of proteins showing wrongly N-terminal annotated sequences, and it might have an important impact in the available data for genome-wide searching of potential vaccine and drug

Reduction of voltage gated sodium channel protein in DRG by vector mediated miRNA reduces pain in rats with painful diabetic neuropathy.

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Chattopadhyay, Munmun; Zhou, Zhigang; Hao, Shuanglin; Mata, Marina; Fink, David J

2012-03-22

Painful neuropathy is a common complication of diabetes. Previous studies have identified significant increases in the amount of voltage gated sodium channel isoforms Na(V)1.7 and Na(V)1.3 protein in the dorsal root ganglia (DRG) of rats with streptozotocin (STZ)-induced diabetes. We found that gene transfer-mediated release of the inhibitory neurotransmitters enkephalin or gamma amino butyric acid (GABA) from DRG neurons in diabetic animals reduced pain-related behaviors coincident with a reduction in Na(V)1.7 protein levels in DRG in vivo. To further evaluate the role of Na(V)α subunit levels in DRG in the pathogenesis of pain in diabetic neuropathy, we constructed a non-replicating herpes simplex virus (HSV)-based vector expressing a microRNA (miRNA) against Na(V)α subunits. Subcutaneous inoculation of the miRNA-expressing HSV vector into the feet of diabetic rats to transduce DRG resulted in a reduction in Na(V)α subunit levels in DRG neurons, coincident with a reduction in cold allodynia, thermal hyperalgesia and mechanical hyperalgesia. These data support the role of increased Na(V)α protein in DRG in the pathogenesis of pain in diabetic neuropathy, and provide a proof-of-principle demonstration for the development of a novel therapy that could be used to treat intractable pain in patients with diabetic neuropathy.

Brown pigment formation in heated sugar-protein mixed suspensions containing unmodified and peptically modified whey protein concentrates.

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Rongsirikul, Narumol; Hongsprabhas, Parichat

2016-01-01

Commercial whey protein concentrate (WPC) was modified by heating the acidified protein suspensions (pH 2.0) at 80 °C for 30 min and treating with pepsin at 37 °C for 60 min. Prior to spray-drying, such modification did not change the molecular weights (MWs) of whey proteins determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After spray-drying the modified whey protein concentrate with trehalose excipient (MWPC-TH), it was found that the α-lactalbumin (α-La) was the major protein that was further hydrolyzed the most. The reconstituted MWPC-TH contained β-lactoglobulin (β-Lg) as the major protein and small molecular weight (MW) peptides of less than 6.5 kDa. The reconstituted MWPC-TH had higher NH2 group, Trolox equivalent antioxidant capacity (TEAC), lower exposed aromatic ring and thiol (SH) contents than did the commercial WPC. Kinetic studies revealed that the addition of MWPC-TH in fructose-glycine solution was able to reduce brown pigment formation in the mixtures heated at 80 to 95 °C by increasing the activation energy (Ea) of brown pigment formation due to the retardation of fluoresced advanced glycation end product (AGEs) formation. The addition of MWPC to reducing sugar-glycine/commercial WPC was also able to lower brown pigment formation in the sterilized (121 °C, 15 min) mixed suspensions containing 0.1 M reducing sugar and 0.5-1.0 % glycine and/or commercial (P < 0.05). It was demonstrated that the modification investigated in this study selectively hydrolyzed α-La and retained β-Lg for the production of antibrowning whey protein concentrate.

Use of Novel High-Protein Functional Food Products as Part of a Calorie-Restricted Diet to Reduce Insulin Resistance and Increase Lean Body Mass in Adults: A Randomized Controlled Trial

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Carol S. Johnston

2017-10-01

Full Text Available Significant reductions in insulin resistance (IR can be achieved by either calorie restriction or by the increase of lean mass. However, calorie restriction usually results in significant loss of lean mass. A 6-week randomized controlled feeding trial was conducted to determine if a calorie-restricted, high-protein diet (~125 g protein/day consumed evenly throughout the day using novel functional foods would be more successful for reducing IR in comparison to a conventional diet (~80 g protein/day with a similar level of calorie restriction. Healthy adults (age 20–75 years; body mass index, 20–42 kg/m2 with raised triglyceride/high-density lipoprotein ratios were randomly assigned to the control group (CON: test foods prepared using gluten-free commercial pasta and cereal or to the high-protein group (HPR: test foods prepared using novel high-protein pasta and cereal both rich in wheat gluten. Mean weight loss did not differ between groups (−2.7 ± 2.6 and −3.2 ± 3.0 kg for CON (n = 11 and HPR (n = 10 respectively, p = 0.801; however, the 6-week change in fat-free mass (FFM differed significantly between groups (−0.5 ± 1.5 and +1.5 ± 3.8 kg for CON and HPR respectively, p = 0.008. IR improved in HPR vs. CON participants (homeostasis model assessment-estimated insulin resistance [HOMAIR] change: −1.7 ± 1.4 and −0.7 ± 0.7 respectively; p = 0.020. The change in HOMA-IR was related to the change in FFM among participants (r = −0.511, p = 0.021. Thus, a high-protein diet using novel functional foods combined with modest calorie restriction was 140% more effective for reducing HOMA-IR in healthy adults compared to a lower protein, standard diet with an equal level of calorie restriction.

Reduced Fragment Diversity for Alpha and Alpha-Beta Protein Structure Prediction using Rosetta.

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Abbass, Jad; Nebel, Jean-Christophe

2017-01-01

Protein structure prediction is considered a main challenge in computational biology. The biannual international competition, Critical Assessment of protein Structure Prediction (CASP), has shown in its eleventh experiment that free modelling target predictions are still beyond reliable accuracy, therefore, much effort should be made to improve ab initio methods. Arguably, Rosetta is considered as the most competitive method when it comes to targets with no homologues. Relying on fragments of length 9 and 3 from known structures, Rosetta creates putative structures by assembling candidate fragments. Generally, the structure with the lowest energy score, also known as first model, is chosen to be the "predicted one". A thorough study has been conducted on the role and diversity of 3-mers involved in Rosetta's model "refinement" phase. Usage of the standard number of 3-mers - i.e. 200 - has been shown to degrade alpha and alpha-beta protein conformations initially achieved by assembling 9-mers. Therefore, a new prediction pipeline is proposed for Rosetta where the "refinement" phase is customised according to a target's structural class prediction. Over 8% improvement in terms of first model structure accuracy is reported for alpha and alpha-beta classes when decreasing the number of 3- mers. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Structure-based barcoding of proteins.

Science.gov (United States)

Metri, Rahul; Jerath, Gaurav; Kailas, Govind; Gacche, Nitin; Pal, Adityabarna; Ramakrishnan, Vibin

2014-01-01

A reduced representation in the format of a barcode has been developed to provide an overview of the topological nature of a given protein structure from 3D coordinate file. The molecular structure of a protein coordinate file from Protein Data Bank is first expressed in terms of an alpha-numero code and further converted to a barcode image. The barcode representation can be used to compare and contrast different proteins based on their structure. The utility of this method has been exemplified by comparing structural barcodes of proteins that belong to same fold family, and across different folds. In addition to this, we have attempted to provide an illustration to (i) the structural changes often seen in a given protein molecule upon interaction with ligands and (ii) Modifications in overall topology of a given protein during evolution. The program is fully downloadable from the website http://www.iitg.ac.in/probar/. © 2013 The Protein Society.

Protein and non-protein sulfhydryls and disulfides in gastric mucosa and liver after gastrotoxic chemicals and sucralfate: Possible new targets of pharmacologic agents

Institute of Scientific and Technical Information of China (English)

Lajos Nagy; Miki Nagata; Sandor Szabo

2007-01-01

AIM: To investigate the role of major non-protein and protein sulfhydryls and disulfides in chemically induced gastric hemorrhagic mucosal lesions (HML) and the mechanism of gastroprotective effect of sucralfate.METHODS: Rats were given 1 mL of 75% ethanol, 25%NaCl, 0.6 mol/L HCI, 0.2 mol/L NaOH or 1% ammonia solutions intragastrically (i.g.) and sacrificed 1, 3, 6 or 12 min later. Total (reduced and oxidized) glutathione (GSH + GSSG), glutathione disulfide (GSSG), protein free sulfhydryls (PSH), protein-glutathione mixed disulfides (PSSG) and protein cystine disulfides (PSSP) were measured in gastric mucosa and liver.RESULTS: Reduced glutathione (GSH) was depleted in the gastric mucosa after ethanol, HCI or NaCl exposure,while oxidized glutathione (GSSG) concentrations increased, except by HCI and NaOH exposure. Decreased levels of PSH after exposure to ethanol were observed,NaCl or NaOH while the total protein disulfides were increased. Ratios of reduced to oxidized glutathione or sulfhydrils to disulfides were decreased by all chemicals.No changes in thiol homeostasis were detected in the liver after i.g. abbreviation should be spelled out the first time here administration of ethanol. Sucralfate increased the concentrations of GSH and PSH and prevented the ethanol-induced changes in gastric mucosal thiol concentrations.CONCLUSION: Our modified methods are now suitable for direct measurements of major protein and nonprotein thiols/disulfides in the gastric mucosa or liver.A common element in the pathogenesis of chemically induced HML and in the mechanism of gastroprotective drugs seems to be the decreased ratios of reduced and oxidized glutathione as well as protein sulfhydryls and disulfides.

Surfactant protein-B 121ins2 heterozygosity, reduced pulmonary function, and chronic obstructive pulmonary disease in smokers

DEFF Research Database (Denmark)

Bækvad-Hansen, Marie; Dahl, Morten; Tybjaerg-Hansen, Anne

2010-01-01

Hereditary surfactant protein-B deficiency is an autosomal recessive disorder that causes fatal respiratory distress syndrome in newborns. Seventy percent of the cases of hereditary surfactant protein-B deficiency are caused by homozygosity for the 121ins2 mutation in the surfactant protein-B gen...

A dietary pattern including nopal, chia seed, soy protein, and oat reduces serum triglycerides and glucose intolerance in patients with metabolic syndrome.

Science.gov (United States)

Guevara-Cruz, Martha; Tovar, Armando R; Aguilar-Salinas, Carlos A; Medina-Vera, Isabel; Gil-Zenteno, Lidia; Hernández-Viveros, Isaac; López-Romero, Patricia; Ordaz-Nava, Guillermo; Canizales-Quinteros, Samuel; Guillen Pineda, Luz E; Torres, Nimbe