Corvitin restores metallothionein and glial fibrillary acidic protein levels in rat brain affected by pituitrin-izadrin

OpenAIRE

H. N. Shiyntum; O. O. Dovban; Y. P. Kovalchuk; T. Ya. Yaroshenko2; G. A. Ushakova1

2017-01-01

In this research, we investigated the effect of pituitrin-izadrin induced injury on the levels of metallothionein (MT) and soluble and filament forms of glial fibrillary acidic protein (GFAP) in the hippocampus, cerebellum, thalamus, and the cerebral cortex, and examined the effect of corvitin on the brain under the noted changes. Our results showed oppositely directed changes – a decrease in the level of MT and an increase in GFAP in the rat brain, with a tendency to astrogliosis development...

Intranasal cotinine improves memory, and reduces depressive-like behavior, and GFAP+ cells loss induced by restraint stress in mice.

Science.gov (United States)

Perez-Urrutia, Nelson; Mendoza, Cristhian; Alvarez-Ricartes, Nathalie; Oliveros-Matus, Patricia; Echeverria, Florencia; Grizzell, J Alex; Barreto, George E; Iarkov, Alexandre; Echeverria, Valentina

2017-09-01

Posttraumatic stress disorder (PTSD), chronic psychological stress, and major depressive disorder have been found to be associated with a significant decrease in glial fibrillary acidic protein (GFAP) immunoreactivity in the hippocampus of rodents. Cotinine is an alkaloid that prevents memory impairment, depressive-like behavior and synaptic loss when co-administered during restraint stress, a model of PTSD and stress-induced depression, in mice. Here, we investigated the effects of post-treatment with intranasal cotinine on depressive- and anxiety-like behaviors, visual recognition memory as well as the number and morphology of GFAP+ immunoreactive cells, in the hippocampus and frontal cortex of mice subjected to prolonged restraint stress. The results revealed that in addition to the mood and cognitive impairments, restraint stress induced a significant decrease in the number and arborization of GFAP+ cells in the brain of mice. Intranasal cotinine prevented these stress-derived symptoms and the morphological abnormalities GFAP+ cells in both of these brain regions which are critical to resilience to stress. The significance of these findings for the therapy of PTSD and depression is discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

[Alterations of glial fibrillary acidic protein in rat brain after gamma knife irradiation].

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Ma, Z M; Jiang, B; Ma, J R

2001-08-28

To study glial fibrillary acidic protein (GFAP) immunoreactivity in different time and water content of the rat brain treated with gamma knife radiotherapy and to understand the alteration course of the brain lesion after a single high dose radiosurgical treatment. In the brains of the normal rats were irradiated by gamma knife with 160 Gy-high dose. The irradiated rats were then killed on the 1st day, 7th day, 14th day, and 28th day after radiotherapy, respectively. The positive cells of GFAP in brain tissue were detected by immunostaining; the water content of the brain tissue was measured by microgravimetry. The histological study of the irradiated brain tissue was performed with H.E. and examined under light microscope. The numbers of GFAP-positive astrocytes began to increase on the 1st day after gamma knife irradiation. It was enlarged markedly in the number and size of GFAP-stained astrocytes over the irradiated areas. Up to the 28th day, circumscribed necrosis foci (4 mm in diameter) was seen in the central area of the target. In the brain tissue around the necrosis, GFAP-positive astrocytes significantly increased (P gravity in the irradiated brain tissue the 14th and 28th day after irradiation. The results suggest that GFAP can be used as a marker for the radiation-induced brain injury. The brain edema and disruption of brain-blood barrier can be occurred during the acute stage after irradiation.

Inhalation exposure to white spirit causes region-dependent alterations in the levels of glial fibrillary acidic protein

DEFF Research Database (Denmark)

Lam, Henrik Rye; Ladefoged, Ole; østergaard, G.

2000-01-01

Enhanced expression of glial fibrillary acidic protein (GFAP) is known to be associated with toxicant-induced gliosis, a homotypic response of the central nervous system to neural injury. A variety of neurochemical and neurophysiological effects have been observed in experimental animals exposed ...

Corvitin restores metallothionein and glial fibrillary acidic protein levels in rat brain affected by pituitrin-izadrin

Directory of Open Access Journals (Sweden)

H. N. Shiyntum

2017-06-01

Full Text Available In this research, we investigated the effect of pituitrin-izadrin induced injury on the levels of metallothionein (MT and soluble and filament forms of glial fibrillary acidic protein (GFAP in the hippocampus, cerebellum, thalamus, and the cerebral cortex, and examined the effect of corvitin on the brain under the noted changes. Our results showed oppositely directed changes – a decrease in the level of MT and an increase in GFAP in the rat brain, with a tendency to astrogliosis development, under the influence of systemic deficiencies in myocardial function. The use of corvitin at a dose of 42 mg/kg for five days after a cardiac attack caused by pituitary-izadrin leads to recovery in the balance of the studied proteins.

UCH-L1 and GFAP Serum Levels in Neonates with Hypoxic-Ischemic Encephalopathy: A single center pilot study

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Martha V. Douglas-Escobar

2014-12-01

Full Text Available Objective - We examined two potential biomarkers of brain damage in HIE neonates: glial fibrillary acidic protein (GFAP; a marker of gliosis and ubiquitin C-terminal hydrolase L1 (UCH-L1; a marker of neuronal injury. We hypothesized the biomarkers would be measurable in cord blood of healthy neonates and could serve as a normative reference for brain injury in HIE infants. Further, we hypothesized that serum samples of HIE neonates would have higher levels and would correlate with brain damage on MRI and later developmental outcomes.Study Design - Serum UCH - L1 and GFAP concentrations from HIE neonates(n = 16 were compared with controls(n = 11.Pearson correlation coefficients and a mixed model design examined the relationship between biomarker concentrations of HIE neonates and brain damage(MRI and developmental outcomes(Bayley - III.Result– Both biomarkers were detected in cord blood from control subjects.UCH - L1 concentrations were higher in HIE neonates(p < 0.001 and associated with cortical injury(p < 0.055 and later motor and cognitive developmental outcomes(p < 0.05.The temporal change in GFAP concentrations from birth to 96 hours of age predicted motor developmental outcomes(p < 0.05 and injury to the basal ganglia and white matter.Conclusion– UCH - L1 concentrations correlated with cortical injury and developmental delays and GFAP concentrations correlated with basal ganglia and white matter injury and motor delay in HIE affected patients.Researchers should continue to explore UCH - L1 and GFAP as promising serum biomarkers of brain damage and predictors of neurodevelopmental outcomes in neonates with HIE.

Dampak Hipoksia Sistemik terhadap Malondialdehida, Glial Fibrillary Acidic Protein dan Aktivitas Asetilkolin Esterase Otak Tikus

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Andriani Andriani; Ani Retno Prijanti; Ninik Mudjihartini; Sri Widia A. Jusman

2016-01-01

Hipoksia sistemik menyebabkan berkurangnya oksigen dan energi di otak sehingga memicupenglepasan neurotransmiter asetilkolin, meningkatkan radikal bebas dan glial fibrillary acidic protein (GFAP)yang berfungsi menjaga kekuatan membran. Tujuan penelitian untuk melihat gambaran adaptasi otak padahipoksia sistemik terhadap fungsi asetilkolin esterase, kerusakan membran sel neuron dan astrosit. Penelitiandilakukan di Laboratorium Biokimia & Biologi Molekuler FK Universitas Indonesia, pada ta...

Phenotypic and gene expression modification with normal brain aging in GFAP-positive astrocytes and neural stem cells.

Science.gov (United States)

Bernal, Giovanna M; Peterson, Daniel A

2011-06-01

Astrocytes secrete growth factors that are both neuroprotective and supportive for the local environment. Identified by glial fibrillary acidic protein (GFAP) expression, astrocytes exhibit heterogeneity in morphology and in the expression of phenotypic markers and growth factors throughout different adult brain regions. In adult neurogenic niches, astrocytes secrete vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) within the neurogenic niche and are also a source of special GFAP-positive multipotent neural stem cells (NSCs). Normal aging is accompanied by a decline in CNS function and reduced neurogenesis. We asked whether a decreased availability of astrocyte-derived factors may contribute to the age-related decline in neurogenesis. Determining alterations of astrocytic activity in the aging brain is crucial for understanding CNS homeostasis in aging and for assessing appropriate therapeutic targets for an aging population. We found region-specific alterations in the gene expression of GFAP, VEGF, and FGF-2 and their receptors in the aged brain corresponding to changes in astrocytic reactivity, supporting astrocytic heterogeneity and demonstrating a differential aging effect. We found that GFAP-positive NSCs uniquely coexpress both VEGF and its key mitotic receptor Flk-1 in both young and aged hippocampus, indicating a possible autocrine/paracrine signaling mechanism. VEGF expression is lost once NSCs commit to a neuronal fate, but Flk-1-mediated sensitivity to VEGF signaling is maintained. We propose that age-related astrocytic changes result in reduced VEGF and FGF-2 signaling, which in turn limits NSC and progenitor cell maintenance and contributes to decreased neurogenesis. © 2011 The Authors. Aging Cell © 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.

A Systematic Review of the Usefulness of Glial Fibrillary Acidic Protein for Predicting Acute Intracranial Lesions following Head Trauma

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Teemu M. Luoto

2017-12-01

Full Text Available BackgroundThe extensive use of computed tomography (CT after acute head injury is costly and carries potential iatrogenic risk. This systematic review examined the usefulness of blood-based glial fibrillary acidic protein (GFAP for predicting acute trauma-related CT-positive intracranial lesions following head trauma. The main objective was to summarize the current evidence on blood-based GFAP as a potential screening test for acute CT-positive intracranial lesions following head trauma.MethodsWe screened MEDLINE, EMBASE, PsychInfo, CINAHL, Web of Science, the Cochrane Database, Scopus, Clinical Trials, OpenGrey, ResearchGate, and the reference lists of eligible publications for original contributions published between January 1980 and January 2017. Eligibility criteria included: (i population: human head and brain injuries of all severities and ages; (ii intervention: blood-based GFAP measurement ≤24 h post-injury; and (iii outcome: acute traumatic lesion on non-contrast head CT ≤24 h post-injury. Three authors completed the publication screening, data extraction, and quality assessment of eligible articles.ResultsThe initial search identified 4,706 articles, with 51 eligible for subsequent full-text assessment. Twenty-seven articles were ultimately included. Twenty-four (89% studies reported a positive association between GFAP level and acute trauma-related intracranial lesions on head CT. The area under the receiver operating characteristic curve for GFAP prediction of intracranial pathology ranged from 0.74 to 0.98 indicating good to excellent discrimination. GFAP seemed to discriminate mass lesions and diffuse injury, with mass lesions having significantly higher GFAP levels. There was considerable variability between the measured GFAP averages between studies and assays. No well-designed diagnostic studies with specific GFAP cutoff values predictive of acute traumatic intracranial lesions have been published

Adult-onset Alexander disease, associated with a mutation in an alternative GFAP transcript, may be phenotypically modulated by a non-neutral HDAC6 variant.

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Melchionda, Laura; Fang, Mingyan; Wang, Hairong; Fugnanesi, Valeria; Morbin, Michela; Liu, Xuanzhu; Li, Wenyan; Ceccherini, Isabella; Farina, Laura; Savoiardo, Mario; D'Adamo, Pio; Zhang, Jianguo; Costa, Alfredo; Ravaglia, Sabrina; Ghezzi, Daniele; Zeviani, Massimo

2013-05-01

We studied a family including two half-siblings, sharing the same mother, affected by slowly progressive, adult-onset neurological syndromes. In spite of the diversity of the clinical features, characterized by a mild movement disorder with cognitive impairment in the elder patient, and severe motor-neuron disease (MND) in her half-brother, the brain Magnetic Resonance Imaging (MRI) features were compatible with adult-onset Alexander's disease (AOAD), suggesting different expression of the same, genetically determined, condition. Since mutations in the alpha isoform of glial fibrillary acidic protein, GFAP-α, the only cause so far known of AOAD, were excluded, we applied exome Next Generation Sequencing (NGS) to identify gene variants, which were then functionally validated by molecular characterization of recombinant and patient-derived cells. Exome-NGS revealed a mutation in a previously neglected GFAP isoform, GFAP-ϵ, which disrupts the GFAP-associated filamentous cytoskeletal meshwork of astrocytoma cells. To shed light on the different clinical features in the two patients, we sought for variants in other genes. The male patient had a mutation, absent in his half-sister, in X-linked histone deacetylase 6, a candidate MND susceptibility gene. Exome-NGS is an unbiased approach that not only helps identify new disease genes, but may also contribute to elucidate phenotypic expression.

Astrocytic expression of GFAP and serum levels of IL-1β and TNF-α in rats treated with different pain relievers

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Gisele Ferreira Amaral

Full Text Available ABSTRACT Pro-inflammatory cytokines and glial cells, especially microglial cells, have been implicated in persistent pain sensitization. Less is known about the role of astrocytes in pain regulation. This study aimed to observe the expression of the astrocytic biomarker glial fibrillary acidic protein (GFAP and the serum levels of interleukin 1 beta (IL-1β and tumor necrosis factor alpha (TNF-α after short-term administration of central pain relievers in rats not submitted to noxious stimuli. Male Wistar rats were divided into five groups, receiving for nine days- (1 amitriptyline (Amt-10 mg/kg/day, by gavage; (2 gabapentin (Gb-60 mg/kg/day, by gavage; (3 methadone (Me-4.5 mg/kg/day, intraperitoneal route [IP]; (4 morphine (Mo-10 mg/kg/day, IP; or (5 0.9% saline solution, IP. Brain samples were collected for immunohistochemical study of GFAP expression in the mesencephalon and nucleus accumbens (NAc. The area of GFAP-positive cells was calculated using MetaMorph software and serum levels of IL-1β and TNF-α were measured by enzyme-linked immunosorbent assay. Serum TNF-α levels were decreased in the groups treated with Mo, Me and Gb, but not in the Amt-treated group. IL-1β decreased only in rats treated with Me. The astrocytic expression of GFAP was decreased in the brainstem with all drugs, while it was increased in the NAc with Amt, Me and Mo.

Glial Fibrillary Acidic Protein and Ubiquitin C-Terminal Hydrolase-L1 as Outcome Predictors in Traumatic Brain Injury.

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Takala, Riikka S K; Posti, Jussi P; Runtti, Hilkka; Newcombe, Virginia F; Outtrim, Joanne; Katila, Ari J; Frantzén, Janek; Ala-Seppälä, Henna; Kyllönen, Anna; Maanpää, Henna-Riikka; Tallus, Jussi; Hossain, Md Iftakher; Coles, Jonathan P; Hutchinson, Peter; van Gils, Mark; Menon, David K; Tenovuo, Olli

2016-03-01

Biomarkers ubiquitin C-terminal hydrolase-L1 (UCH-L1) and glial fibrillary acidic protein (GFAP) may help detect brain injury, assess its severity, and improve outcome prediction. This study aimed to evaluate the prognostic value of these biomarkers during the first days after brain injury. Serum UCH-L1 and GFAP were measured in 324 patients with traumatic brain injury (TBI) enrolled in a prospective study. The outcome was assessed using the Glasgow Outcome Scale (GOS) or the extended version, Glasgow Outcome Scale-Extended (GOSE). Patients with full recovery had lower UCH-L1 concentrations on the second day and patients with favorable outcome had lower UCH-L1 concentrations during the first 2 days compared with patients with incomplete recovery and unfavorable outcome. Patients with full recovery and favorable outcome had significantly lower GFAP concentrations in the first 2 days than patients with incomplete recovery or unfavorable outcome. There was a strong negative correlation between outcome and UCH-L1 in the first 3 days and GFAP levels in the first 2 days. On arrival, both UCH-L1 and GFAP distinguished patients with GOS score 1-3 from patients with GOS score 4-5, but not patients with GOSE score 8 from patients with GOSE score 1-7. For UCH-L1 and GFAP to predict unfavorable outcome (GOS score ≤ 3), the area under the receiver operating characteristic curve was 0.727, and 0.723, respectively. Neither UCHL-1 nor GFAP was independently able to predict the outcome when age, worst Glasgow Coma Scale score, pupil reactivity, Injury Severity Score, and Marshall score were added into the multivariate logistic regression model. GFAP and UCH-L1 are significantly associated with outcome, but they do not add predictive power to commonly used prognostic variables in a population of patients with TBI of varying severities. Copyright © 2016 Elsevier Inc. All rights reserved.

Quiescent Oct4+ Neural Stem Cells (NSCs) Repopulate Ablated Glial Fibrillary Acidic Protein+ NSCs in the Adult Mouse Brain.

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Reeve, Rachel L; Yammine, Samantha Z; Morshead, Cindi M; van der Kooy, Derek

2017-09-01

Adult primitive neural stem cells (pNSCs) are a rare population of glial fibrillary acidic protein (GFAP) - Oct4 + cells in the mouse forebrain subependymal zone bordering the lateral ventricles that give rise to clonal neurospheres in leukemia inhibitory factor in vitro. pNSC neurospheres can be passaged to self-renew or give rise to GFAP + NSCs that form neurospheres in epidermal growth factor and fibroblast growth factor 2, which we collectively refer to as definitive NSCs (dNSCs). Label retention experiments using doxycycline-inducible histone-2B (H2B)-green fluorescent protein (GFP) mice and several chase periods of up to 1 year quantified the adult pNSC cell cycle time as 3-5 months. We hypothesized that while pNSCs are not very proliferative at baseline, they may exist as a reserve pool of NSCs in case of injury. To test this function of pNSCs, we obtained conditional Oct4 knockout mice, Oct4 fl/fl ;Sox1 Cre (Oct4 CKO ), which do not yield adult pNSC-derived neurospheres. When we ablated the progeny of pNSCs, namely all GFAP + dNSCs, in these Oct4 CKO mice, we found that dNSCs did not recover as they do in wild-type mice, suggesting that pNSCs are necessary for dNSC repopulation. Returning to the H2B-GFP mice, we observed that the cytosine β-d-arabinofuranoside ablation of proliferating cells including dNSCs-induced quiescent pNSCs to proliferate and significantly dilute their H2B-GFP label. In conclusion, we demonstrate that pNSCs are the most quiescent stem cells in the adult brain reported to date and that their lineage position upstream of GFAP + dNSCs allows them to repopulate a depleted neural lineage. Stem Cells 2017;35:2071-2082. © 2017 AlphaMed Press.

Low-level bisphenol A increases production of glial fibrillary acidic protein in differentiating astrocyte progenitor cells through excessive STAT3 and Smad1 activation

International Nuclear Information System (INIS)

Yamaguchi, Hideaki; Zhu, Jun; Yu, Tao; Sasaki, Kazuo; Umetsu, Hironori; Kidachi, Yumi; Ryoyama, Kazuo

2006-01-01

The effects of bisphenol A (BPA) on the differentiation of serum-free mouse embryo (SFME) cells, the astrocyte progenitor cells in the central nervous system, were examined. SFME cells were exposed to 10 ng/ml leukemia inhibitory factor (LIF) and 10 ng/ml bone morphogenetic protein 2 (BMP2) to increase glial fibrillary acidic protein (GFAP) expression and induce cell differentiation. Various concentrations of BPA (0.1 pg/ml-1 μg/ml) were then added to determine their effects on the cell differentiation. SFME cells were effectively differentiated by LIF and BMP2 in completely serum-free cultures. Cell proliferation following cell differentiation was not significantly affected by low-level BPA. However, GFAP expression was significantly increased in SFME cells in the presence of 1-100 pg/ml BPA. These increases were due to excessive activation of signal transducer and activator of transcription 3 (STAT3) and mothers against decapentaplegic homolog 1 (Smad1) by the low-level BPA

The phosphorylation status and cytoskeletal remodeling of striatal astrocytes treated with quinolinic acid

International Nuclear Information System (INIS)

Pierozan, Paula; Ferreira, Fernanda; Ortiz de Lima, Bárbara; Gonçalves Fernandes, Carolina; Totarelli Monteforte, Priscila; Castro Medaglia, Natalia de; Bincoletto, Claudia; Soubhi Smaili, Soraya; Pessoa-Pureur, Regina

2014-01-01

Quinolinic acid (QUIN) is a glutamate agonist which markedly enhances the vulnerability of neural cells to excitotoxicity. QUIN is produced from the amino acid tryptophan through the kynurenine pathway (KP). Dysregulation of this pathway is associated with neurodegenerative conditions. In this study we treated striatal astrocytes in culture with QUIN and assayed the endogenous phosphorylating system associated with glial fibrillary acidic protein (GFAP) and vimentin as well as cytoskeletal remodeling. After 24 h incubation with 100 µM QUIN, cells were exposed to 32 P-orthophosphate and/or protein kinase A (PKA), protein kinase dependent of Ca 2+ /calmodulin II (PKCaMII) or protein kinase C (PKC) inhibitors, H89 (20 μM), KN93 (10 μM) and staurosporin (10 nM), respectively. Results showed that hyperphosphorylation was abrogated by PKA and PKC inhibitors but not by the PKCaMII inhibitor. The specific antagonists to ionotropic NMDA and non-NMDA (50 µM DL-AP5 and CNQX, respectively) glutamate receptors as well as to metabotropic glutamate receptor (mGLUR; 50 µM MCPG), mGLUR1 (100 µM MPEP) and mGLUR5 (10 µM 4C3HPG) prevented the hyperphosphorylation provoked by QUIN. Also, intra and extracellular Ca 2+ quelators (1 mM EGTA; 10 µM BAPTA-AM, respectively) prevented QUIN-mediated effect, while Ca 2+ influx through voltage-dependent Ca 2+ channel type L (L-VDCC) (blocker: 10 µM verapamil) is not implicated in this effect. Morphological analysis showed dramatically altered actin cytoskeleton with concomitant change of morphology to fusiform and/or flattened cells with retracted cytoplasm and disruption of the GFAP meshwork, supporting misregulation of actin cytoskeleton. Both hyperphosphorylation and cytoskeletal remodeling were reversed 24 h after QUIN removal. Astrocytes are highly plastic cells and the vulnerability of astrocyte cytoskeleton may have important implications for understanding the neurotoxicity of QUIN in neurodegenerative disorders. - Highlights: �

Cytoskeletal proteins in the cerebrospinal fluid as biomarker of multiple sclerosis.

Science.gov (United States)

Madeddu, Roberto; Farace, Cristiano; Tolu, Paola; Solinas, Giuliana; Asara, Yolande; Sotgiu, Maria Alessandra; Delogu, Lucia Gemma; Prados, Jose Carlos; Sotgiu, Stefano; Montella, Andrea

2013-02-01

The axonal cytoskeleton is a finely organized system, essential for maintaining the integrity of the axon. Axonal degeneration is implicated in the pathogenesis of unremitting disability of multiple sclerosis (MS). Purpose of this study is to evaluate levels of cytoskeletal proteins such as neurofilament light protein (NFL), glial fibrillary acidic protein (GFAP), and β-tubulin (β-Tub) isoforms II and III in the cerebrospinal fluid (CSF) of MS patients and their correlation with MS clinical indices. CSF levels of cytoskeletal proteins were determined in 51 patients: 33 with MS and 18 with other neurological diseases (OND). NFL, GFAP and β-Tub II proteins were significantly higher (p 0.05) was found between MS and OND with regard to β-Tub III. Interestingly, levels of β-Tub III and NFL were higher in progressive than in remitting MS forms; on the contrary, higher levels of β-Tub II and GFAP were found in remitting MS forms. However, with the exception of β-Tub III, all proteins tend to decrease their CSF levels concomitantly with the increasing disability (EDSS) score. Overall, our results might indicate β-Tub II as a potential candidate for diagnostic and β-Tub III as a possible prognostic biomarker of MS. Therefore, further analyses are legitimated and desirable.

CSF Neurofilament Proteins Levels are Elevated in Sporadic Creutzfeldt-Jakob Disease

NARCIS (Netherlands)

van Eijk, Jeroen J. J.; van Everbroeck, Bart; Abdo, W. Farid; Kremer, Berry P. H.; Verbeek, Marcel M.

2010-01-01

In this study we investigated the cerebrospinal fluid (CSF) levels of neurofilament light (NFL) and heavy chain (NFHp35), total tau (t-tau), and glial fibrillary acidic protein (GFAP) to detect disease specific profiles in sporadic Creutzfeldt Jakob disease (sCJD) patients and Alzheimer's disease

In Silico Prediction and Validation of Gfap as an miR-3099 Target in Mouse Brain.

Science.gov (United States)

Abidin, Shahidee Zainal; Leong, Jia-Wen; Mahmoudi, Marzieh; Nordin, Norshariza; Abdullah, Syahril; Cheah, Pike-See; Ling, King-Hwa

2017-08-01

MicroRNAs are small non-coding RNAs that play crucial roles in the regulation of gene expression and protein synthesis during brain development. MiR-3099 is highly expressed throughout embryogenesis, especially in the developing central nervous system. Moreover, miR-3099 is also expressed at a higher level in differentiating neurons in vitro, suggesting that it is a potential regulator during neuronal cell development. This study aimed to predict the target genes of miR-3099 via in-silico analysis using four independent prediction algorithms (miRDB, miRanda, TargetScan, and DIANA-micro-T-CDS) with emphasis on target genes related to brain development and function. Based on the analysis, a total of 3,174 miR-3099 target genes were predicted. Those predicted by at least three algorithms (324 genes) were subjected to DAVID bioinformatics analysis to understand their overall functional themes and representation. The analysis revealed that nearly 70% of the target genes were expressed in the nervous system and a significant proportion were associated with transcriptional regulation and protein ubiquitination mechanisms. Comparison of in situ hybridization (ISH) expression patterns of miR-3099 in both published and in-house-generated ISH sections with the ISH sections of target genes from the Allen Brain Atlas identified 7 target genes (Dnmt3a, Gabpa, Gfap, Itga4, Lxn, Smad7, and Tbx18) having expression patterns complementary to miR-3099 in the developing and adult mouse brain samples. Of these, we validated Gfap as a direct downstream target of miR-3099 using the luciferase reporter gene system. In conclusion, we report the successful prediction and validation of Gfap as an miR-3099 target gene using a combination of bioinformatics resources with enrichment of annotations based on functional ontologies and a spatio-temporal expression dataset.

Neural stem cells in the adult ciliary epithelium express GFAP and are regulated by Wnt signaling

International Nuclear Information System (INIS)

Das, Ani V.; Zhao Xing; James, Jackson; Kim, Min; Cowan, Kenneth H.; Ahmad, Iqbal

2006-01-01

The identification of neural stem cells with retinal potential in the ciliary epithelium (CE) of the adult mammals is of considerable interest because of their potential for replacing or rescuing degenerating retinal neurons in disease or injury. The evaluation of such a potential requires characterization of these cells with regard to their phenotypic properties, potential, and regulatory mechanisms. Here, we demonstrate that rat CE stem cells/progenitors in neurosphere culture display astrocytic nature in terms of expressing glial intermediate neurofilament protein, GFAP. The GFAP-expressing CE stem cells/progenitors form neurospheres in proliferating conditions and generate neurons when shifted to differentiating conditions. These cells express components of the canonical Wnt pathway and its activation promotes their proliferation. Furthermore, we demonstrate that the activation of the canonical Wnt pathway influences neuronal differentiation of CE stem cells/progenitors in a context dependent manner. Our observations suggest that CE stem cells/progenitors share phenotypic properties and regulatory mechanism(s) with neural stem cells elsewhere in the adult CNS

Mechanisms and Treatment of OP-Induced Seizures and Neuropathology

Science.gov (United States)

1993-08-18

Millan, M. H., Patel, S., Mello, L. M. and Meldrum , B. S., Focal injection of 2-amino-7- phosphonoheptanoic acid into prepiriform cortex protects against...LIST OF FIGURES Figure 1. Rapid, selective induction of c-fos and glial fibrillary acidic protein (GFAP) in piriform cortex (PC) by a single...specific to astrocytes, glial fibrillary acidic protein (GFAP). We found that there was a robust increase in GFAP staining in layers Il-III of PC that

The phosphorylation status and cytoskeletal remodeling of striatal astrocytes treated with quinolinic acid

Energy Technology Data Exchange (ETDEWEB)

Pierozan, Paula; Ferreira, Fernanda; Ortiz de Lima, Bárbara; Gonçalves Fernandes, Carolina [Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS 90035-003 (Brazil); Totarelli Monteforte, Priscila; Castro Medaglia, Natalia de; Bincoletto, Claudia; Soubhi Smaili, Soraya [Departamento de Farmacologia, Universidade Federal de São Paulo (UNIFESP/EPM), São Paulo, SP (Brazil); Pessoa-Pureur, Regina, E-mail: rpureur@ufrgs.br [Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS 90035-003 (Brazil)

2014-04-01

Quinolinic acid (QUIN) is a glutamate agonist which markedly enhances the vulnerability of neural cells to excitotoxicity. QUIN is produced from the amino acid tryptophan through the kynurenine pathway (KP). Dysregulation of this pathway is associated with neurodegenerative conditions. In this study we treated striatal astrocytes in culture with QUIN and assayed the endogenous phosphorylating system associated with glial fibrillary acidic protein (GFAP) and vimentin as well as cytoskeletal remodeling. After 24 h incubation with 100 µM QUIN, cells were exposed to {sup 32}P-orthophosphate and/or protein kinase A (PKA), protein kinase dependent of Ca{sup 2+}/calmodulin II (PKCaMII) or protein kinase C (PKC) inhibitors, H89 (20 μM), KN93 (10 μM) and staurosporin (10 nM), respectively. Results showed that hyperphosphorylation was abrogated by PKA and PKC inhibitors but not by the PKCaMII inhibitor. The specific antagonists to ionotropic NMDA and non-NMDA (50 µM DL-AP5 and CNQX, respectively) glutamate receptors as well as to metabotropic glutamate receptor (mGLUR; 50 µM MCPG), mGLUR1 (100 µM MPEP) and mGLUR5 (10 µM 4C3HPG) prevented the hyperphosphorylation provoked by QUIN. Also, intra and extracellular Ca{sup 2+} quelators (1 mM EGTA; 10 µM BAPTA-AM, respectively) prevented QUIN-mediated effect, while Ca{sup 2+} influx through voltage-dependent Ca{sup 2+} channel type L (L-VDCC) (blocker: 10 µM verapamil) is not implicated in this effect. Morphological analysis showed dramatically altered actin cytoskeleton with concomitant change of morphology to fusiform and/or flattened cells with retracted cytoplasm and disruption of the GFAP meshwork, supporting misregulation of actin cytoskeleton. Both hyperphosphorylation and cytoskeletal remodeling were reversed 24 h after QUIN removal. Astrocytes are highly plastic cells and the vulnerability of astrocyte cytoskeleton may have important implications for understanding the neurotoxicity of QUIN in neurodegenerative

Astrocytes in development, aging and disease: starring GFAP

NARCIS (Netherlands)

Middeldorp, J.

2010-01-01

We show in this thesis that different subtypes of astrocytes comprise specialized GFAP-IF networks, that change during development, aging and Alzheimer’s disease. The novel functions that have emerged for the IF network suggest these changes can play an important part in the specialized function of

Type III intermediate filaments desmin, glial fibrillary acidic protein (GFAP), vimentin, and peripherin

NARCIS (Netherlands)

Hol, Elly M.; Capetanaki, Yassemi

2017-01-01

Type III intermediate filament (IF) proteins assemble into cytoplasmic homopolymeric and heteropolymeric filaments with other type III and some type IV IFs. These highly dynamic structures form an integral component of the cytoskeleton of muscle, brain, and mesenchymal cells. Here, we review the

Type III Intermediate Filaments Desmin, Glial Fibrillary Acidic Protein (GFAP), Vimentin, and Peripherin

NARCIS (Netherlands)

Hol, Elly M; Capetanaki, Yassemi

2017-01-01

SummaryType III intermediate filament (IF) proteins assemble into cytoplasmic homopolymeric and heteropolymeric filaments with other type III and some type IV IFs. These highly dynamic structures form an integral component of the cytoskeleton of muscle, brain, and mesenchymal cells. Here, we review

The adult spinal cord harbors a population of GFAP-positive progenitors with limited self-renewal potential.

Science.gov (United States)

Fiorelli, Roberto; Cebrian-Silla, Arantxa; Garcia-Verdugo, Jose-Manuel; Raineteau, Olivier

2013-12-01

Adult neural stem cells (aNSCs) of the forebrain are GFAP-expressing cells that are intercalated within ependymal cells of the subventricular zone (SVZ). Cells showing NSCs characteristics in vitro can also be isolated from the periaqueductal region in the adult spinal cord (SC), but contradicting results exist concerning their glial versus ependymal identity. We used an inducible transgenic mouse line (hGFAP-CreERT2) to conditionally label GFAP-expressing cells in the adult SVZ and SC periaqueduct, and directly and systematically compared their self-renewal and multipotential properties in vitro. We demonstrate that a population of GFAP(+) cells that share the morphology and the antigenic properties of SVZ-NSCs mostly reside in the dorsal aspect of the central canal (CC) throughout the spinal cord. These cells are non-proliferative in the intact spinal cord, but incorporate the S-phase marker EdU following spinal cord injury. Multipotent, clonal YFP-expressing neurospheres (i.e., deriving from recombined GFAP-expressing cells) were successfully obtained from both the intact and injured spinal cord. These spheres however showed limited self-renewal properties when compared with SVZ-neurospheres, even after spinal cord injury. Altogether, these results demonstrate that significant differences exist in NSCs lineages between neurogenic and non-neurogenic regions of the adult CNS. Thus, although we confirm that a population of multipotent GFAP(+) cells co-exists alongside with multipotent ependymal cells within the adult SC, we identify these cells as multipotent progenitors showing limited self-renewal properties. Copyright © 2013 Wiley Periodicals, Inc.

The inhibition of subchondral bone lesions significantly reversed the weight-bearing deficit and the overexpression of CGRP in DRG neurons, GFAP and Iba-1 in the spinal dorsal horn in the monosodium iodoacetate induced model of osteoarthritis pain.

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Degang Yu

Full Text Available Chronic pain is the most prominent and disabling symptom of osteoarthritis (OA. Clinical data suggest that subchondral bone lesions contribute to the occurrence of joint pain. The present study investigated the effect of the inhibition of subchondral bone lesions on joint pain.Osteoarthritic pain was induced by an injection of monosodium iodoacetate (MIA into the rat knee joint. Zoledronic acid (ZOL, a third generation of bisphosphonate, was used to inhibit subchondral bone lesions. Joint histomorphology was evaluated using X-ray micro computed tomography scanning and hematoxylin-eosin staining. The activity of osteoclast in subchondral bone was evaluated using tartrate-resistant acid phosphatase staining. Joint pain was evaluated using weight-bearing asymmetry, the expression of calcitonin gene-related peptide (CGRP in the dorsal root ganglion (DRG, and spinal glial activation status using glial fibrillary acidic protein (GFAP and ionized calcium binding adaptor molecule-1 (Iba-1 immunofluorescence. Afferent neurons in the DRGs that innervated the joints were identified using retrograde fluorogold labeling.MIA injections induced significant histomorphological alterations and joint pain. The inhibition of subchondral bone lesions by ZOL significantly reduced the MIA-induced weight-bearing deficit and overexpression of CGRP in DRG neurons, GFAP and Iba-1 in the spinal dorsal horn at 3 and 6 weeks after MIA injection; however, joint swelling and synovial reaction were unaffected.The inhibition of subchondral bone lesions alleviated joint pain. Subchondral bone lesions should be a key target in the management of osteoarthritic joint pain.

Altered astrocytic swelling in the cortex of α-syntrophin-negative GFAP/EGFP mice.

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Miroslava Anderova

Full Text Available Brain edema accompanying ischemic or traumatic brain injuries, originates from a disruption of ionic/neurotransmitter homeostasis that leads to accumulation of K(+ and glutamate in the extracellular space. Their increased uptake, predominantly provided by astrocytes, is associated with water influx via aquaporin-4 (AQP4. As the removal of perivascular AQP4 via the deletion of α-syntrophin was shown to delay edema formation and K(+ clearance, we aimed to elucidate the impact of α-syntrophin knockout on volume changes in individual astrocytes in situ evoked by pathological stimuli using three dimensional confocal morphometry and changes in the extracellular space volume fraction (α in situ and in vivo in the mouse cortex employing the real-time iontophoretic method. RT-qPCR profiling was used to reveal possible differences in the expression of ion channels/transporters that participate in maintaining ionic/neurotransmitter homeostasis. To visualize individual astrocytes in mice lacking α-syntrophin we crossbred GFAP/EGFP mice, in which the astrocytes are labeled by the enhanced green fluorescent protein under the human glial fibrillary acidic protein promoter, with α-syntrophin knockout mice. Three-dimensional confocal morphometry revealed that α-syntrophin deletion results in significantly smaller astrocyte swelling when induced by severe hypoosmotic stress, oxygen glucose deprivation (OGD or 50 mM K(+. As for the mild stimuli, such as mild hypoosmotic or hyperosmotic stress or 10 mM K(+, α-syntrophin deletion had no effect on astrocyte swelling. Similarly, evaluation of relative α changes showed a significantly smaller decrease in α-syntrophin knockout mice only during severe pathological conditions, but not during mild stimuli. In summary, the deletion of α-syntrophin markedly alters astrocyte swelling during severe hypoosmotic stress, OGD or high K(+.

Viral Vector-Based Dissection of Marmoset GFAP Promoter in Mouse and Marmoset Brains.

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Yoichiro Shinohara

Full Text Available Adeno-associated virus (AAV vectors are small in diameter, diffuse easily in the brain, and represent a highly efficient means by which to transfer a transgene to the brain of a large animal. A major demerit of AAV vectors is their limited accommodation capacity for transgenes. Thus, a compact promoter is useful when delivering large transgenes via AAV vectors. In the present study, we aimed to identify the shortest astrocyte-specific GFAP promoter region that could be used for AAV-vector-mediated transgene expression in the marmoset brain. The 2.0-kb promoter region upstream of the GFAP gene was cloned from the marmoset genome, and short promoters (1.6 kb, 1.4 kb, 0.6 kb, 0.3 kb and 0.2 kb were obtained by progressively deleting the original 2.0-kb promoter from the 5' end. The short promoters were screened in the mouse cerebellum in terms of their strength and astrocyte specificity. We found that the 0.3-kb promoter maintained 40% of the strength of the original 2.0-kb promoter, and approximately 90% of its astrocyte specificity. These properties were superior to those of the 1.4-kb, 0.6-kb (20% promoter strength and 0.2-kb (70% astrocyte specificity promoters. Then, we verified whether the 0.3-kb GFAP promoter retained astrocyte specificity in the marmoset cerebral cortex. Injection of viral vectors carrying the 0.3-kb marmoset GFAP promoter specifically transduced astrocytes in both the cerebral cortex and cerebellar cortex of the marmoset. These results suggest that the compact 0.3-kb promoter region serves as an astrocyte-specific promoter in the marmoset brain, which permits us to express a large gene by AAV vectors that have a limited accommodation capacity.

Extended Solution Gate OFET-based Biosensor for Label-free Glial Fibrillary Acidic Protein Detection with Polyethylene Glycol-Containing Bioreceptor Layer.

Science.gov (United States)

Song, Jian; Dailey, Jennifer; Li, Hui; Jang, Hyun-June; Zhang, Pengfei; Wang, Jeff Tza-Huei; Everett, Allen D; Katz, Howard E

2017-05-25

A novel organic field effect transistor (OFET) -based biosensor is described for label-free glial fibrillary acidic protein (GFAP) detection. We report the first use of an extended solution gate structure where the sensing area and the organic semiconductor are separated, and a reference electrode is not needed. Different molecular weight polyethylene glycols (PEGs) are mixed into the bio-receptor layer to help extend the Debye screening length. The drain current change was significantly increased with the help of higher molecular weight PEGs, as they are known to reduce the dielectric constant. We also investigated the sensing performance under different gate voltage (V g ). The sensitivity increased after we decreased V g from -5 V to -2 V, because the lower V g is much closer to the OFET threshold voltage and the influence of attached negatively charged proteins become more apparent. Finally, the selectivity experiments toward different interferents were performed. The stability and selectivity are promising for clinical applications.

Neurobiology of Soman

Science.gov (United States)

1991-06-30

seizures In rats. Neurosc. Let 70. 69-74. Millan, M.H., S. Patel, and B.S. Meldrum (1988). The involvement of excitatory mino acid receptors within...a marker specific to astrocytes, glial fibrillary acidic protein (GFAP). We have used this marker to demonstrate that astrocytes are activated soon...88 I I I I I I IUST OF FIGURES Figure 1. Rapid, selective induction of c-fos and glial fibrillary acidic protein (GFAP) In piriform cortex 3 (PC

The Effect of Silybum marianum on GFAP and Spatial Memory in a Mouse Model of Alzheimer\\'s Disease

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A Hadinia

2010-01-01

Full Text Available Introduction & Objective: Studies have shown that Silybum marianum have high levels of antioxidant polyphenolic substances and have neuro-protective effects on neurodegenerative diseases. Accordingly, this study was conducted to determine the possible effect of Silybum marianum on expression of and spatial memory in a mouse model of Alzheimer's disease. Materials & Methods: This experimental study was conducted at Yasuj University of Medical Sciences in 2009. Thirty adult male Wistar rats were allocated in three groups: sham group, experimental group, and lesion group, each consisting of ten rats. The experimental and lesion groups received Ibotonic acid of the NBM nucleus in stereotaxic apparatus whereas the sham group underwent surgical procedure without any injection. The experimental group received 200mg/kg of Silybum mirianum extract orally, diluted in 1% Arabic gum. Also the sham group received 1% Arabic gum every day for four weeks. The lesion group did not receive anything. The behavioral assessment was measured, after treatment , by using of Y maze test on day 7 and 28 in all groups. The ELISA method was used to measure the GFAP level in Hippocamp at the end of behavioral assessment. The collected data was analyzed by the SPSS software using ANOVA and Repeated Measures of Analysis Variance tests. Results:Improvement of behavioral performance of the experimental animals compared to the lesion and sham groups were increased significantly on day 7 and 28 (P <0.01 & P <0.001 respectively. The ELISA method showed that the level of the GFAP synthesis decreased in the experimental group compared to the lesion and sham groups (P <0.001. Conclusion: The Silybum marianum plant has a protective effect on the nerve tissue in a mouse model of Alzheimer's disease by decreasing of the GFAP synthesis and lead to the improvement of behavioral performance. :

Expression of glial fibrillar acidic protein in the sensorimotor cortex of the cerebral hemispheres in the modeling of transient ischemia against the background of previous sensitization by brain antigen and immunocorrection

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L. M. Yaremenko

2017-12-01

Full Text Available Aim. In order to analyze the dynamics of expression of glial fibrillar acidic protein in the sensorimotor cortex of the large hemispheres in the simulation of transient ischemia against the background of previous sensitization by brain antigen and immunocorrection. Materials and methods. The study is conducted on 185 male mature white rats from Wistar line weighing 260-290 g, in which the damage of the brain was modulated. The brain for study was taken on the 1st, 3rd, 10th, 30th and 90th days after the start of the experiment. The histological, immunohistochemical, morphometric and statistical methods were used. Results. Observations have shown that sensitization by the brain antigen causes neurodegenerative changes in the sensorimotor cortex and a moderate increase in the number of GFAP+-gliocytes, which is gradually increasing. The discirculatory changes that occurred with PO and BCA against the background of previous sensitization practically do not lead to changes in the number of GFAP+-cells. Against the background of sensitization by brain antigen, brain ischemia leads to an increase in the number of gliocytes that are GFAP labeled. In the affected hemisphere, their number reaches a maximum in the end of the acute period of ischemia, after which it decreases. But even in 3 months after transient vascular lesion, there are almost twice as many as in conditionally intact rats. This can be a factor that will significantly affect the function of brain regions after a vascular accident. The increase in the number of GFAP+-gliocytes in the contralateral hemisphere allows us to speak about a certain systemic response of astrocytic glia after ischemic trauma. An early reaction to increase of the number of labeled astrocytes just a day after ischemic attack suggests that some of this type of gliocytes does not expresses GFAP under normal conditions. The action of Imunofan in MEAs results in a less significant decrease in manifestations of

A novel and efficient gene transfer strategy reduces glial reactivity and improves neuronal survival and axonal growth in vitro

OpenAIRE

Desclaux, Mathieu; Teigell, Marisa; Amar, Lahouari; Vogel, Roland; Giménez y Ribotta, Minerva; Privát, Alain M.; Mallet, Jacques

2009-01-01

Background: The lack of axonal regeneration in the central nervous system is attributed among other factors to the formation of a glial scar. This cellular structure is mainly composed of reactive astrocytes that overexpress two intermediate filament proteins, the glial fibrillary acidic protein (GFAP) and vimentin. Indeed, in vitro, astrocytes lacking GFAP or both GFAP and vimentin were shown to be the substrate for increased neuronal plasticity. Moreover, double knockout mice lacking both G...

Green-fluorescent protein+ Astrocytes Attach to beta-Amyloid Plaques in an Alzheimer Mouse Model and GFPare Sensitive for Clasmatodendrosis

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Christian eHumpel

2016-04-01

Full Text Available Alzheimer’s disease (AD is pathologically characterized by beta-amyloid (Aβ plaques and Tau pathology. It is well-established that Aβ plaques are surrounded by reactive astrocytes, highly expressing glial fibrillary acidic protein (GFAP. In order to study the cellular interaction of reactive astrocytes with Aβ plaques, we crossbred mice overexpressing amyloid precursor protein (APP with the Swedish-Dutch-Iowa mutations (APP-SweDI with mice expressing green fluorescent protein (GFP under the GFAP-promotor. Three-dimensional confocal microscopy revealed a tight association and intense sprouting of astrocytic fine branched processes towards Aβ plaques in 12 month old mice. In order to study phagocytosis, 110 µm thick brain slices from 12 month old crossbred mice were cultured overnight, however, we found that the GFP fluorescence faded away, distal processes degenerated and a complete loss of astrocytic morphology was seen (clasmatodendrosis. In summary, our data show that GFP+ reactive astrocytes make intense contact with Aβ plaques but these cells are highly vulnerable for degeneration.

The calcium-modulated proteins, S100A1 and S100B, as potential regulators of the dynamics of type III intermediate filaments

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M. Garbuglia

1999-10-01

Full Text Available The Ca2+-modulated, dimeric proteins of the EF-hand (helix-loop-helix type, S100A1 and S100B, that have been shown to inhibit microtubule (MT protein assembly and to promote MT disassembly, interact with the type III intermediate filament (IF subunits, desmin and glial fibrillary acidic protein (GFAP, with a stoichiometry of 2 mol of IF subunit/mol of S100A1 or S100B dimer and an affinity of 0.5-1.0 µM in the presence of a few micromolar concentrations of Ca2+. Binding of S100A1 and S100B results in inhibition of desmin and GFAP assemblies into IFs and stimulation of the disassembly of preformed desmin and GFAP IFs. S100A1 and S100B interact with a stretch of residues in the N-terminal (head domain of desmin and GFAP, thereby blocking the head-to-tail process of IF elongation. The C-terminal extension of S100A1 (and, likely, S100B represents a critical part of the site that recognizes desmin and GFAP. S100B is localized to IFs within cells, suggesting that it might have a role in remodeling IFs upon elevation of cytosolic Ca2+ concentration by avoiding excess IF assembly and/or promoting IF disassembly in vivo. S100A1, that is not localized to IFs, might also play a role in the regulation of IF dynamics by binding to and sequestering unassembled IF subunits. Together, these observations suggest that S100A1 and S100B may be regarded as Ca2+-dependent regulators of the state of assembly of two important elements of the cytoskeleton, IFs and MTs, and, potentially, of MT- and IF-based activities.

Amino acids and proteins

NARCIS (Netherlands)

van Goudoever, Johannes B.; Vlaardingerbroek, Hester; van den Akker, Chris H.; de Groof, Femke; van der Schoor, Sophie R. D.

2014-01-01

Amino acids and protein are key factors for growth. The neonatal period requires the highest intake in life to meet the demands. Those demands include amino acids for growth, but proteins and amino acids also function as signalling molecules and function as neurotransmitters. Often the nutritional

Archetypal and new families with Alexander disease and novel mutations in GFAP

NARCIS (Netherlands)

Messing, Albee; Li, Rong; Naidu, Sakkubai; Taylor, J. Paul; Silverman, Lital; Flint, Daniel; van der Knaap, Marjo S.; Brenner, Michael

2012-01-01

To describe genetic analyses of the 2 most thoroughly studied, historically seminal multigenerational families with Alexander disease described prior to the identification of GFAP as the related gene, as well as 1 newly discovered family. Clinical histories were obtained and DNA was analyzed from

Assessment of Kerch Bay environmental pollution using neuroglial proteins of ground fish

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H. V. Sukharenko

2014-03-01

Full Text Available The modern ecology situation in waters of the Kerch Strait requires assessment of disturbances in biotopes and monitoring of the degree of impact of industrial pollutants on ecosystem. Deposit of oil products after the 2007 year ships’ accidents might have considerable impact on the water biocenosis area. The investigation of cytoskeleton marker of astrocytes glial fibrillary acidic protein (GFAP in brain of the bullhead (Neogobius fluviatilis, which is the typical representative of the commercial ground fish of the Kerch Strait, has been carried out. The results of comparative analysis of GFAP content in the brain of fish from the Kerch Bay near-shore waters and fish from conditionally clear area of Vorskla river shows the reliable (2.18 times increasing of GFAP in the area of industrial pollution. Rising GFAP content indicates the astrogliosis development as a result of metabolic disturbances which can be induced by higher content of oil products in the near-bottom biotopes of the Kerch Bay. Increase in lipid peroxidation level was observed in the brain of fish from the Kerch Bay. The results provided with regard to violations of the state of astrocyte cytoskeleton and oxidative stress in the brain of bullhead from the Kerch Bay prove the sublethal biology effect of industrial pollutants in hydrobionts from this area. Results of this investigation also indicate the necessity of continuous ecology monitoring and comprehensive study of hydrobiont populations in the industrial regions and ecological disaster zones.

Chronically administered 3-nitropropionic acid produces selective lesions in the striatum and reduces muscle tonus.

Science.gov (United States)

Shimano, Y; Kumazaki, M; Sakurai, T; Hida, H; Fujimoto, I; Fukuda, A; Nishino, H

1995-12-01

Systemically administered 3-nitropropionic acid (3- NPA), irreversible inhibitor of succinate dehydrogenase, produced characteristic bilateral lesions in the striatum (STR) in the rat. Inside the lesion, neutrophils invaded and strong immunoreaction for IgG as well as complement factor C3b/C4b receptor (C3b/C4br) were observed. The core of the lesion lost the immunoreaction for glial fibrillary acidic protein (GFAP) while the marginal area had abundant GFAP-labeled astrocytes around the vessels. Intoxicated rats often became somnolent and were awkward in cooperative movement on a pole climbing test, but they had a quite good memory retention in a passive avoidance learning. Muscle tonus in some of the intoxicated rats became hypotonic with low voltage electromyogram (EMG) activity, especially in lower limbs. In summary, 3-NPA intoxicated rats had selective bilateral lesions in the STR and exhibited disturbances in a cooperative movement owing to the impairment in muscle tonus, thus it would be a useful animal model to deduce the central pathogenesis of Huntington's disease.

Metallothionein prevents neurodegeneration and central nervous system cell death after treatment with gliotoxin 6-aminonicotinamide

DEFF Research Database (Denmark)

Penkowa, Milena; Quintana, Albert; Carrasco, Javier

2004-01-01

Transgenic expression of interleukin-6 (IL-6) in the CNS under the control of the glial fibrillary acidic protein (GFAP) gene promoter (GFAP-IL6 mice) induces significant inflammation and neurodegeneration but also affords neuroprotection against acute traumatic brain injury. This neuroprotection...

Phthalic Acid Chemical Probes Synthesized for Protein-Protein Interaction Analysis

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Chin-Jen Wu

2013-06-01

Full Text Available Plasticizers are additives that are used to increase the flexibility of plastic during manufacturing. However, in injection molding processes, plasticizers cannot be generated with monomers because they can peel off from the plastics into the surrounding environment, water, or food, or become attached to skin. Among the various plasticizers that are used, 1,2-benzenedicarboxylic acid (phthalic acid is a typical precursor to generate phthalates. In addition, phthalic acid is a metabolite of diethylhexyl phthalate (DEHP. According to Gene_Ontology gene/protein database, phthalates can cause genital diseases, cardiotoxicity, hepatotoxicity, nephrotoxicity, etc. In this study, a silanized linker (3-aminopropyl triethoxyslane, APTES was deposited on silicon dioxides (SiO2 particles and phthalate chemical probes were manufactured from phthalic acid and APTES–SiO2. These probes could be used for detecting proteins that targeted phthalic acid and for protein-protein interactions. The phthalic acid chemical probes we produced were incubated with epithelioid cell lysates of normal rat kidney (NRK-52E cells to detect the interactions between phthalic acid and NRK-52E extracted proteins. These chemical probes interacted with a number of chaperones such as protein disulfide-isomerase A6, heat shock proteins, and Serpin H1. Ingenuity Pathways Analysis (IPA software showed that these chemical probes were a practical technique for protein-protein interaction analysis.

CNS development under altered gravity: cerebellar glial and neuronal protein expression in rat neonates exposed to hypergravity

Science.gov (United States)

Nguon, K.; Li, G.-H.; Sajdel-Sulkowska, E. M.

2004-01-01

The future of space exploration depends on a solid understanding of the developmental process under microgravity, specifically in relation to the central nervous system (CNS). We have previously employed a hypergravity paradigm to assess the impact of altered gravity on the developing rat cerebellum [Exp. Biol. Med. 226 (2000) 790]. The present study addresses the molecular mechanisms involved in the cerebellar response to hypergravity. Specifically, the study focuses on the expression of selected glial and neuronal cerebellar proteins in rat neonates exposed to hypergravity (1.5 G) from embryonic day (E)11 to postnatal day (P)6 or P9 (the time of maximal cerebellar changes) comparing them against their expression in rat neonates developing under normal gravity. Proteins were analyzed by quantitative Western blots of cerebellar homogenates; RNA analysis was performed in the same samples using quantitative PCR. Densitometric analysis of Western blots suggested a reduction in glial (glial acidic protein, GFAP) and neuronal (neuronal cell adhesion moiecule, NCAM-L1, synaptophysin) proteins, but the changes in individual cerebellar proteins in hypergravity-exposed neonates appeared both age- and gender-specific. RNA analysis suggested a reduction in GFAP and synaptophysin mRNAs on P6. These data suggest that exposure to hypergravity may interfere with the expression of selected cerebellar proteins. These changes in protein expression may be involved in mediating the effect of hypergravity on the developing rat cerebellum.

GFAP-immunopositive structures in spiny dogfish, Squalus acanthias, and little skate, Raia erinacea, brains: differences have evolutionary implications.

Science.gov (United States)

Kálmán, M; Gould, R M

2001-07-01

GFAP expression patterns were compared between the brains of a spiny dogfish (Squalus acanthias) and a little skate (Raia erinacea). After anesthesia, the animals were perfused with paraformaldehyde. Serial vibratome sections were immunostained against GFAP using the avidin-biotin method. Spiny dogfish brain contained mainly uniformly-distributed, radially arranged ependymoglia. From GFAP distribution, the layered organization in both the telencephalon and the tectum were visible. In the cerebellum, the molecular and granular layers displayed conspicuously different glial structures; in the former a Bergmann glia-like population was found. No true astrocytes (i.e., stellate-shaped cells) were found. Radial glial endfeet lined all meningeal surfaces. Radial fibers also seemed to form endfeet and en passant contacts on the vessels. Plexuses of fine perivascular glial fibers also contributed to the perivascular glia. Compared with spiny dogfish brain, GFAP expression in the little skate brain was confined. Radial glia were limited to a few areas, e.g., segments of the ventricular surface of the telencephalon, and the midline of the diencephalon and mesencephalon. Scarce astrocytes occurred in every brain part, but only the optic chiasm, and the junction of the tegmentum and optic tectum contained large numbers of astrocytes. Astrocytes formed the meningeal glia limitans and the perivascular glia. No GFAP-immunopositive Bergmann glia-like structure was found. Astrocytes seen in the little skate were clearly different from the mammalian and avian ones; they had a different process system - extra large forms were frequently seen, and the meningeal and perivascular cells were spread along the surface instead of forming endfeet by processes. The differences between Squalus and Raia astroglia were much like those found between reptiles versus mammals and birds. It suggests independent and parallel glial evolutionary processes in amniotes and chondrichthyans, seemingly

Hypothalamic GPR40 signaling activated by free long chain fatty acids suppresses CFA-induced inflammatory chronic pain.

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Kazuo Nakamoto

Full Text Available GPR40 has been reported to be activated by long-chain fatty acids, such as docosahexaenoic acid (DHA. However, reports studying functional role of GPR40 in the brain are lacking. The present study focused on the relationship between pain regulation and GPR40, investigating the functional roles of hypothalamic GPR40 during chronic pain caused using a complete Freund's adjuvant (CFA-induced inflammatory chronic pain mouse model. GPR40 protein expression in the hypothalamus was transiently increased at day 7, but not at days 1, 3 and 14, after CFA injection. GPR40 was co-localized with NeuN, a neuron marker, but not with glial fibrillary acidic protein (GFAP, an astrocyte marker. At day 1 after CFA injection, GFAP protein expression was markedly increased in the hypothalamus. These increases were significantly inhibited by the intracerebroventricular injection of flavopiridol (15 nmol, a cyclin-dependent kinase inhibitor, depending on the decreases in both the increment of GPR40 protein expression and the induction of mechanical allodynia and thermal hyperalgesia at day 7 after CFA injection. Furthermore, the level of DHA in the hypothalamus tissue was significantly increased in a flavopiridol reversible manner at day 1, but not at day 7, after CFA injection. The intracerebroventricular injection of DHA (50 µg and GW9508 (1.0 µg, a GPR40-selective agonist, significantly reduced mechanical allodynia and thermal hyperalgesia at day 7, but not at day 1, after CFA injection. These effects were inhibited by intracerebroventricular pretreatment with GW1100 (10 µg, a GPR40 antagonist. The protein expression of GPR40 was colocalized with that of β-endorphin and proopiomelanocortin, and a single intracerebroventricular injection of GW9508 (1.0 µg significantly increased the number of neurons double-stained for c-Fos and proopiomelanocortin in the arcuate nucleus of the hypothalamus. Our findings suggest that hypothalamic GPR40 activated by free long

Hypothalamic GPR40 signaling activated by free long chain fatty acids suppresses CFA-induced inflammatory chronic pain.

Science.gov (United States)

Nakamoto, Kazuo; Nishinaka, Takashi; Sato, Naoya; Mankura, Mitsumasa; Koyama, Yutaka; Kasuya, Fumiyo; Tokuyama, Shogo

2013-01-01

GPR40 has been reported to be activated by long-chain fatty acids, such as docosahexaenoic acid (DHA). However, reports studying functional role of GPR40 in the brain are lacking. The present study focused on the relationship between pain regulation and GPR40, investigating the functional roles of hypothalamic GPR40 during chronic pain caused using a complete Freund's adjuvant (CFA)-induced inflammatory chronic pain mouse model. GPR40 protein expression in the hypothalamus was transiently increased at day 7, but not at days 1, 3 and 14, after CFA injection. GPR40 was co-localized with NeuN, a neuron marker, but not with glial fibrillary acidic protein (GFAP), an astrocyte marker. At day 1 after CFA injection, GFAP protein expression was markedly increased in the hypothalamus. These increases were significantly inhibited by the intracerebroventricular injection of flavopiridol (15 nmol), a cyclin-dependent kinase inhibitor, depending on the decreases in both the increment of GPR40 protein expression and the induction of mechanical allodynia and thermal hyperalgesia at day 7 after CFA injection. Furthermore, the level of DHA in the hypothalamus tissue was significantly increased in a flavopiridol reversible manner at day 1, but not at day 7, after CFA injection. The intracerebroventricular injection of DHA (50 µg) and GW9508 (1.0 µg), a GPR40-selective agonist, significantly reduced mechanical allodynia and thermal hyperalgesia at day 7, but not at day 1, after CFA injection. These effects were inhibited by intracerebroventricular pretreatment with GW1100 (10 µg), a GPR40 antagonist. The protein expression of GPR40 was colocalized with that of β-endorphin and proopiomelanocortin, and a single intracerebroventricular injection of GW9508 (1.0 µg) significantly increased the number of neurons double-stained for c-Fos and proopiomelanocortin in the arcuate nucleus of the hypothalamus. Our findings suggest that hypothalamic GPR40 activated by free long chain fatty

CNS-syndrome. Characterization of rat brain intermediate filaments

International Nuclear Information System (INIS)

Nedzvetskij, V.S.; Busygina, S.G.; Berezin, V.A.; Dvoretskij, A.I.

1990-01-01

A study was made of the effect of ionizing radiation on the content and polypeptide composition of filamentous and soluble glial fibrillary acidic protein (GFAP) in different regions of rat brain. Ionizing radiation was shown to decrease considerably the level of soluble GFAP in cerebral cortex, cerebellum, middle brain and hippocampus. Polypeptide composition of soluble GFAP detected by the immonublot-method was found to be changed considerably in different brain areas of irradiated animals

Correlation of serum GFAP, S100B and NSE contents with posttraumatic oxidative stress response and insulin resistance in patients with traumatic brain injury

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Bing-Feng Tian

2018-07-01

Full Text Available Objective: To study the correlation of serum GFAP, S100B and NSE contents with posttraumatic oxidative stress response and insulin resistance in patients with traumatic brain injury. Methods: A total of 110 patients with traumatic brain injury who were treated in our hospital between January 2015 and December 2016 were collected as the observation group, and 60 healthy subjects who received physical examination in our hospital during the same period were collected as normal control group. Serum GFAP, S100B and NSE levels as well as oxidative stress index and insulin resistance index levels of two groups of subjects were detected, and Pearson test was used to further evaluate the correlation of serum GFAP, S100B and NSE contents with oxidative stress response and insulin resistance in patients with traumatic brain injury. Results: Serum GFAP, S100B and NSE contents of observation group were significantly higher than those of normal control group; serum oxidative stress indexes MDA, MPO and LPO contents were higher than those of normal control group while SOD and TAC contents were lower than those of normal control group; serum insulin resistance indexes GLU, INS and HOMA-IR levels were higher than those of control group. Pearson test showed that serum GFAP, S100B and NSE contents in patients with traumatic brain injury were directly correlated with post-traumatic oxidative stress and insulin resistance. Conclusion: The serum GFAP, S100B and NSE contents increase in patients with traumatic brain injury, and the increase is directly correlated with the oxidative stress and insulin resistance.

An amino acid depleted cell-free protein synthesis system for the incorporation of non-canonical amino acid analogs into proteins.

Science.gov (United States)

Singh-Blom, Amrita; Hughes, Randall A; Ellington, Andrew D

2014-05-20

Residue-specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an efficient amino acid depleted cell-free protein synthesis system that can be used to study residue-specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines a simple methodology and high protein expression titers with a high-efficiency analog substitution into a target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein-wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo methodologies. Copyright © 2014 Elsevier B.V. All rights reserved.

Ethanol stimulates ROS generation by mitochondria through Ca2+ mobilization and increases GFAP content in rat hippocampal astrocytes.

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González, Antonio; Pariente, José A; Salido, Ginés M

2007-10-31

We have employed rat hippocampal astrocytes in culture to investigate the effect of ethanol on reactive oxygen species (ROS) production as well as its effect on [Ca2+]c and GFAP expression. Cells were loaded with the fluorescent probes fura-2 and H2DCFDA for the determination of changes in [Ca2+]c and ROS production respectively, employing spectrofluorimetry. GFAP content was determined by immunocytochemistry and confocal scanning microscopy. Our results show ROS production in response to 50 mM ethanol, that was reduced in Ca2+-free medium (containing 0.5 mM EGTA) and in the presence of the intracellular Ca2+ chelator BAPTA (10 microM). The effect of ethanol on ROS production was significantly reduced in the presence of the alcohol dehydrogenase inhibitor 4-methylpyrazole (1 mM), and the antioxidants resveratrol (100 microM) or catalase (300 U/ml). Preincubation of astrocytes in the presence of 10 microM antimycin plus 10 microM oligomycin to inhibit mitochondria completely blocked ethanol-evoked ROS production. In addition, ethanol led to a sustained increase in [Ca2+]c that reached a constant level over the prestimulation values. Finally, incubation of astrocytes in the presence of ethanol increased the content of GFAP that was significantly reduced in the absence of extracellular Ca2+ and by resveratrol and catalase pretreatment. The data obtained in the present study suggest that astrocytes are able to metabolize ethanol, which induces two effects on intracellular homeostasis: an immediate response (Ca2+ release and ROS generation) and later changes involving GFAP expression. Both effects may underline various signaling pathways which are important for cell proliferation, differentiation and function.

Immunohistochemical study of the sensory formations in the glabrous skin of the rat.

Science.gov (United States)

Vega, J A; Malinovsky, L; del Valle, M E; Hernandez, L C; Dubový, P; Perez-Casas, A

1990-01-01

The presence of some cytoskeletal proteins related to the intermediate filaments glial fibrillary acidic protein -GFAP and vimentin) and S-100 protein has been investigated in sensory formations of the glabrous skin of the rat. A positive reaction both for S-100 protein and vimentin was found in the inner core and related cells of glomerular and simple sensory corpuscles; in contrast, no positive reaction was shown for GFAP. The authors discuss these results on the basis of the glial origin of the inner core and related cells in sensory formations.

Regulatory mechanisms for 3'-end alternative splicing and polyadenylation of the Glial Fibrillary Acidic Protein, GFAP, transcript

DEFF Research Database (Denmark)

Blechingberg, Jenny; Lykke-Andersen, Søren; Jensen, Torben Heick

2007-01-01

(PTB) protein enhanced both exon 7a polyadenylation and exon 7a splicing. Finally, increasing transcription by the VP16 trans-activator did not affect the frequency of use of the exon 7a polyadenylation signal whereas the exon 7a splicing frequency was decreased. Our data suggest a model...

Acylation of cellular proteins with endogenously synthesized fatty acids

International Nuclear Information System (INIS)

Towler, D.; Glaser, L.

1986-01-01

A number of cellular proteins contain covalently bound fatty acids. Previous studies have identified myristic acid and palmitic acid covalently linked to protein, the former usually attached to proteins by an amide linkage and the latter by ester or thio ester linkages. While in a few instances specific proteins have been isolated from cells and their fatty acid composition has been determined, the most frequent approach to the identification of protein-linked fatty acids is to biosynthetically label proteins with fatty acids added to intact cells. This procedure introduces possible bias in that only a selected fraction of proteins may be labeled, and it is not known whether the radioactive fatty acid linked to the protein is identical with that which is attached to the protein when the fatty acid is derived from endogenous sources. We have examined the distribution of protein-bound fatty acid following labeling with [ 3 H]acetate, a general precursor of all fatty acids, using BC 3 H1 cells (a mouse muscle cell line) and A431 cells (a human epidermoid carcinoma). Myristate, palmitate, and stearate account for essentially all of the fatty acids linked to protein following labeling with [ 3 H]acetate, but at least 30% of the protein-bound palmitate in these cells was present in amide linkage. In BC3H1 cells, exogenous palmitate becomes covalently bound to protein such that less than 10% of the fatty acid is present in amide linkage. These data are compatible with multiple protein acylating activities specific for acceptor protein fatty acid chain length and linkage

Manipulating fatty acid biosynthesis in microalgae for biofuel through protein-protein interactions.

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Jillian L Blatti

Full Text Available Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP and thioesterase (TE govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes.

Ebselen increases cytosolic free Ca2+ concentration, stimulates glutamate release and increases GFAP content in rat hippocampal astrocytes

International Nuclear Information System (INIS)

Salazar, Miguel; Pariente, Jose Antonio; Salido, Gines Maria; Gonzalez, Antonio

2008-01-01

We have investigated the effect of the seleno-organic compound and radical scavenger ebselen on rat hippocampal astrocytes in culture. Throughout our study we carried out determinations of [Ca 2+ ] c in fura-2-loaded cells by single cell imaging, glutamate secretion employing an enzymatic-based assay and GFAP expression, which was monitorized by immunocytochemistry and confocal microscopy. Our results show that ebselen (1-20 μM) dose dependently increases [Ca 2+ ] c , stimulates glutamate release and increases GFAP content, a hallmark of astrocyte reactivity. Ebselen did not alter significantly cell viability as assayed by determination of LDH release into the extracellular medium. Ebselen-evoked glutamate release and increase in GFAP content were Ca 2+ -dependent, because incubation of astrocytes in the absence of extracellular Ca 2+ (medium containing 0.5 mM EGTA) and in the presence of the intracellular Ca 2+ chelator BAPTA (10 μM) significantly reduced ebselen-evoked changes in these parameters. The effects of ebselen we have observed may underline various signalling pathways which are important for cell proliferation, differentiation and function. However, aberrations in astroglial physiology could significantly compromise brain function, due to their role as modulators of neuron activity. Therefore, we consider that careful attention should be paid when employing ebselen as a prophylactic agent against brain damage

Comparative analysis of amino acids and amino-acid derivatives in protein crystallization

International Nuclear Information System (INIS)

Ito, Len; Shiraki, Kentaro; Yamaguchi, Hiroshi

2010-01-01

New types of aggregation suppressors, such as amino acids and their derivatives, were focused on as fourth-component additives. Data were obtained that indicated that the additives promote protein crystallization. Optimal conditions for protein crystallization are difficult to determine because proteins tend to aggregate in saturated solutions. This study comprehensively evaluates amino acids and amino-acid derivatives as additives for crystallization. This fourth component of the solution increases the probability of crystallization of hen egg-white lysozyme in various precipitants owing to a decrease in aggregation. These results suggest that the addition of certain types of amino acids and amino-acid derivatives, such as Arg, Lys and esterified and amidated amino acids, is a simple method of improving the success rate of protein crystallization

Amino acid metabolism conflicts with protein diversity

OpenAIRE

Krick, Teresa; Shub, David A.; Verstraete, Nina; Ferreiro, Diego U.; Alonso, Leonardo G.; Shub, Michael; Sanchez, Ignacio E.

2014-01-01

The 20 protein-coding amino acids are found in proteomes with different relative abundances. The most abundant amino acid, leucine, is nearly an order of magnitude more prevalent than the least abundant amino acid, cysteine. Amino acid metabolic costs differ similarly, constraining their incorporation into proteins. On the other hand, a diverse set of protein sequences is necessary to build functional proteomes. Here, we present a simple model for a cost-diversity trade-off postulating that n...

Acid-regulated proteins induced by Streptococcus mutans and other oral bacteria during acid shock.

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Hamilton, I R; Svensäter, G

1998-10-01

Our previous research has demonstrated that with the more aciduric oral bacteria, an acid shock to sub-lethal pH values results in the induction of an acid tolerance response that protects the cells at extremely low pH (pH 3.0-4.0) that kills unadapted control cells maintained at pH 7.5 (Oral Microbiol Immunol 1997: 12: 266-273). In this study, we were interested in comparing the protein profiles of acid-shocked and control cells of nine organisms from three acid-ogenic genera that could be categorized as strong, weak and non-acid responders in an attempt to identify proteins that could be classified as acid-regulated proteins and which may be important in the process of survival at very low pH. For this, log-phase cultures were rapidly acidified from pH 7.5 to 5.5 in the presence of [14C]-amino acids for varying periods up to 2 h, the period previously shown to be required for maximum induction of the acid response. The cells were extracted for total protein and subjected to one-dimensional sodium dodecyl sulfate-polyacrylamide chromatography with comparable control and acid-shocked protein profiles compared by scanning and computer analysis. Of particular interest were the proteins in the acid-shocked cells that showed enhanced labeling (i.e., synthesis) over the control cells, since these were considered acid-regulated proteins of importance in pH homeostasis. Streptococcus mutans LT11 generated the most rapid and complex pattern: a total of 36 acid-regulated proteins showing enhanced synthesis, with 25 appearing within the first 30 min of acid shock. The enhanced synthesis was transient with all proteins, with the exception of two with molecular weights of 50/49 and 33/32 kDa. Within the acid-regulated proteins were proteins having molecular weights comparable to the heat shock proteins and the various subunits of the membrane H+/ATPase. By comparison, the strong responder, Lactobacillus casei 151, showed the enhanced formation of only nine proteins within the

Protein Design Using Unnatural Amino Acids

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Bilgiçer, Basar; Kumar, Krishna

2003-11-01

With the increasing availability of whole organism genome sequences, understanding protein structure and function is of capital importance. Recent developments in the methodology of incorporation of unnatural amino acids into proteins allow the exploration of proteins at a very detailed level. Furthermore, de novo design of novel protein structures and function is feasible with unprecedented sophistication. Using examples from the literature, this article describes the available methods for unnatural amino acid incorporation and highlights some recent applications including the design of hyperstable protein folds.

Protein synthesis in the presence of carbamoyl-amino acids

International Nuclear Information System (INIS)

Kraus, L.M.; Stephens, M.C.

1987-01-01

The role of exogenous carbamoyl-amino acids in protein biosynthesis has been examined in vitro using a mixture of 14 C amino acids to label newly synthesized protein in human reticulocyte rich (8-18%) peripheral blood. Aliquots of the radiolabeled newly synthesized protein were acid precipitated, washed and the radioactivity measured. Control samples which measured the synthetic capacity of the blood were aliquots of the same blood- 14 C amino acid mixture without added carbamoyl-amino acids or cyanate. N-carbamoyl leucine alone or a 3 N-carbamoyl amino acid mixture of leucine, aspartic acid and tyrosine were used to test inhibition of protein synthesis. Also carbamoyl-amino acids were synthesized using cyanate and Pierce hydrolyzate amino acid calibration standards or the mixture of 14 C amino acids. In this system the carbamoylation of endogenous amino acids by cyanate up to 8 μmol/100μl showed a linear decrease in protein synthesis with time which is inversely related to the cyanate concentration. At greater cyanate levels the inhibition of protein synthesis reaches a plateau. When N-carbamoyl-amino acids only are present there is about a 50% decrease in the 14 C protein at 30 minutes as compared to the synthesis of 14 C protein without N-carbamoyl-amino acids. These results indicate that the presence of carbamoyl-amino acids interferes with protein synthesis

A novel and efficient gene transfer strategy reduces glial reactivity and improves neuronal survival and axonal growth in vitro.

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Mathieu Desclaux

Full Text Available BACKGROUND: The lack of axonal regeneration in the central nervous system is attributed among other factors to the formation of a glial scar. This cellular structure is mainly composed of reactive astrocytes that overexpress two intermediate filament proteins, the glial fibrillary acidic protein (GFAP and vimentin. Indeed, in vitro, astrocytes lacking GFAP or both GFAP and vimentin were shown to be the substrate for increased neuronal plasticity. Moreover, double knockout mice lacking both GFAP and vimentin presented lower levels of glial reactivity in vivo, significant axonal regrowth and improved functional recovery in comparison with wild-type mice after spinal cord hemisection. From these results, our objective was to develop a novel therapeutic strategy for axonal regeneration, based on the targeted suppression of astroglial reactivity and scarring by lentiviral-mediated RNA-interference (RNAi. METHODS AND FINDINGS: In this study, we constructed two lentiviral vectors, Lv-shGFAP and Lv-shVIM, which allow efficient and stable RNAi-mediated silencing of endogenous GFAP or vimentin in vitro. In cultured cortical and spinal reactive astrocytes, the use of these vectors resulted in a specific, stable and highly significant decrease in the corresponding protein levels. In a second model -- scratched primary cultured astrocytes -- Lv-shGFAP, alone or associated with Lv-shVIM, decreased astrocytic reactivity and glial scarring. Finally, in a heterotopic coculture model, cortical neurons displayed higher survival rates and increased neurite growth when cultured with astrocytes in which GFAP and vimentin had been invalidated by lentiviral-mediated RNAi. CONCLUSIONS: Lentiviral-mediated knockdown of GFAP and vimentin in astrocytes show that GFAP is a key target for modulating reactive gliosis and monitoring neuron/glia interactions. Thus, manipulation of reactive astrocytes with the Lv-shGFAP vector constitutes a promising therapeutic strategy for

A novel and efficient gene transfer strategy reduces glial reactivity and improves neuronal survival and axonal growth in vitro.

Science.gov (United States)

Desclaux, Mathieu; Teigell, Marisa; Amar, Lahouari; Vogel, Roland; Gimenez Y Ribotta, Minerva; Privat, Alain; Mallet, Jacques

2009-07-14

The lack of axonal regeneration in the central nervous system is attributed among other factors to the formation of a glial scar. This cellular structure is mainly composed of reactive astrocytes that overexpress two intermediate filament proteins, the glial fibrillary acidic protein (GFAP) and vimentin. Indeed, in vitro, astrocytes lacking GFAP or both GFAP and vimentin were shown to be the substrate for increased neuronal plasticity. Moreover, double knockout mice lacking both GFAP and vimentin presented lower levels of glial reactivity in vivo, significant axonal regrowth and improved functional recovery in comparison with wild-type mice after spinal cord hemisection. From these results, our objective was to develop a novel therapeutic strategy for axonal regeneration, based on the targeted suppression of astroglial reactivity and scarring by lentiviral-mediated RNA-interference (RNAi). In this study, we constructed two lentiviral vectors, Lv-shGFAP and Lv-shVIM, which allow efficient and stable RNAi-mediated silencing of endogenous GFAP or vimentin in vitro. In cultured cortical and spinal reactive astrocytes, the use of these vectors resulted in a specific, stable and highly significant decrease in the corresponding protein levels. In a second model -- scratched primary cultured astrocytes -- Lv-shGFAP, alone or associated with Lv-shVIM, decreased astrocytic reactivity and glial scarring. Finally, in a heterotopic coculture model, cortical neurons displayed higher survival rates and increased neurite growth when cultured with astrocytes in which GFAP and vimentin had been invalidated by lentiviral-mediated RNAi. Lentiviral-mediated knockdown of GFAP and vimentin in astrocytes show that GFAP is a key target for modulating reactive gliosis and monitoring neuron/glia interactions. Thus, manipulation of reactive astrocytes with the Lv-shGFAP vector constitutes a promising therapeutic strategy for increasing glial permissiveness and permitting axonal regeneration

Regional Susceptibility to Domoic Acid in Primary Astrocyte Cells Cultured from the Brain Stem and Hippocampus

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Olga M. Pulido

2008-02-01

Full Text Available Domoic acid is a marine biotoxin associated with harmful algal blooms and is the causative agent of amnesic shellfish poisoning in marine animals and humans. It is also an excitatory amino acid analog to glutamate and kainic acid which acts through glutamate receptors eliciting a very rapid and potent neurotoxic response. The hippocampus, among other brain regions, has been identified as a specific target site having high sensitivity to DOM toxicity. Histopathology evidence indicates that in addition to neurons, the astrocytes were also injured. Electron microscopy data reported in this study further supports the light microscopy findings. Furthermore, the effect of DOM was confirmed by culturing primary astrocytes from the hippocampus and the brain stem and subsequently exposing them to domoic acid. The RNA was extracted and used for biomarker analysis. The biomarker analysis was done for the early response genes including c-fos, c-jun, c-myc, Hsp-72; specific marker for the astrocytes- GFAP and the glutamate receptors including GluR 2, NMDAR 1, NMDAR 2A and B. Although, the astrocyte-GFAP and c-fos were not affected, c-jun and GluR 2 were down-regulated. The microarray analysis revealed that the chemokines / cytokines, tyrosine kinases (Trk, and apoptotic genes were altered. The chemokines that were up-regulated included - IL1-a, IL-1B, IL-6, the small inducible cytokine, interferon protein IP-10, CXC chemokine LIX, and IGF binding proteins. The Bax, Bcl-2, Trk A and Trk B were all downregulated. Interestingly, only the hippocampal astrocytes were affected. Our findings suggest that astrocytes may present a possible target for pharmacological interventions for the prevention and treatment of amnesic shellfish poisoning and for other brain pathologies involving excitotoxicity

Differential Effects of High-Protein Diets Derived from Soy and Casein on Blood–Brain Barrier Integrity in Wild-type Mice

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Matthew Snelson

2017-07-01

Full Text Available A number of studies report that a diet high in protein influences cognitive performance, but the results are inconsistent. Studies demonstrated that protein from different food sources has differential effects on cognition. It is increasingly recognized that the integrity of cerebrovascular blood–brain barrier (BBB is pivotal for central nervous system function. However, to date, no studies have reported the effects of high-protein diets on BBB integrity. Therefore, in this study, the effects of diets enriched in casein or soy protein on BBB permeability were investigated. Immunomicroscopy analyses of cerebral parenchymal immunoglobulin G extravasation indicated significant BBB disruption in the cortex of young adult mice maintained on high-casein diet for 12 weeks, while no signs of BBB dysfunction were observed in mice fed with control or high-soy protein diet. Moreover, cortical expression of glial fibrillary acidic protein (GFAP was significantly greater in mice fed the high-casein diet compared to control mice, indicating heightened astrocyte activation, whereas mice maintained on a soy-enriched diet showed no increase of GFAP abundance. Plasma concentrations of homocysteine were markedly greater in mice maintained on a high-casein diet in comparison to control mice. Collectively, these findings suggest that a diet enriched in casein but not soy protein may induce astrocyte activation through exaggerated BBB permeability by increased plasma homocysteine. The outcomes indicate the differential effects of protein sources on BBB and neuroinflammation, which may provide an important implication for dietary guidelines for protein supplementation.

Regulation of intestinal protein metabolism by amino acids.

Science.gov (United States)

Bertrand, Julien; Goichon, Alexis; Déchelotte, Pierre; Coëffier, Moïse

2013-09-01

Gut homeostasis plays a major role in health and may be regulated by quantitative and qualitative food intake. In the intestinal mucosa, an intense renewal of proteins occurs, at approximately 50% per day in humans. In some pathophysiological conditions, protein turnover is altered and may contribute to intestinal or systemic diseases. Amino acids are key effectors of gut protein turnover, both as constituents of proteins and as regulatory molecules limiting intestinal injury and maintaining intestinal functions. Many studies have focused on two amino acids: glutamine, known as the preferential substrate of rapidly dividing cells, and arginine, another conditionally essential amino acid. The effects of glutamine and arginine on protein synthesis appear to be model and condition dependent, as are the involved signaling pathways. The regulation of gut protein degradation by amino acids has been minimally documented until now. This review will examine recent data, helping to better understand how amino acids regulate intestinal protein metabolism, and will explore perspectives for future studies.

Absorption of proteins and amino acids

International Nuclear Information System (INIS)

Jeejeebhoy, K.N.

1976-01-01

Although the absorption of proteins and amino acids is an important issue in nutrition, its measurement is not common because of the methodological difficulties. Complications are attributable in particular to the magnitude of endogenous protein secretion and to the diversity of absorption mechanisms for amino acids either as individual units or as peptides. Methods for studying absorption include balance techniques, tolerance tests, tracer techniques using proteins or amino acids labelled with 131 I, 3 H, or 15 N, intestinal perfusion studies, and others; they must be selected according to the nature of the information sought. Improvements over the current methods would be useful. (author)

Hypochlorous and peracetic acid induced oxidation of dairy proteins.

Science.gov (United States)

Kerkaert, Barbara; Mestdagh, Frédéric; Cucu, Tatiana; Aedo, Philip Roger; Ling, Shen Yan; De Meulenaer, Bruno

2011-02-09

Hypochlorous and peracetic acids, both known disinfectants in the food industry, were compared for their oxidative capacity toward dairy proteins. Whey proteins and caseins were oxidized under well controlled conditions at pH 8 as a function of the sanitizing concentration. Different markers for protein oxidation were monitored. The results established that the protein carbonyl content was a rather unspecific marker for protein oxidation, which did not allow one to differentiate the oxidant used especially at the lower concentrations. Cysteine, tryptophan, and methionine were proven to be the most vulnerable amino acids for degradation upon hypochlorous and peracetic acid treatment, while tyrosine was only prone to degradation in the presence of hypochlorous acid. Hypochlorous acid induced oxidation gave rise to protein aggregation, while during peracetic acid induced oxidation, no high molecular weight aggregates were observed. Protein aggregation upon hypochlorous acid oxidation could primarily be linked to tryptophan and tyrosine degradation.

Interactions between Therapeutic Proteins and Acrylic Acid Leachable.

Science.gov (United States)

Liu, Dengfeng; Nashed-Samuel, Yasser; Bondarenko, Pavel V; Brems, David N; Ren, Da

2012-01-01

Leachables are chemical compounds that migrate from manufacturing equipment, primary containers and closure systems, and packaging components into biopharmaceutical and pharmaceutical products. Acrylic acid (at concentration around 5 μg/mL) was detected as leachable in syringes from one of the potential vendors (X syringes). In order to evaluate the potential impact of acrylic acid on therapeutic proteins, an IgG 2 molecule was filled into a sterilized X syringe and then incubated at 45 °C for 45 days in a pH 5 acetate buffer. We discovered that acrylic acid can interact with proteins at three different sites: (1) the lysine side chain, (2) the N-terminus, and (3) the histidine side chain, by the Michael reaction. In this report, the direct interactions between acrylic acid leachable and a biopharmaceutical product were demonstrated and the reaction mechanism was proposed. Even thought a small amount (from 0.02% to 0.3%) of protein was found to be modified by acrylic acid, the modified protein can potentially be harmful due to the toxicity of acrylic acid. After being modified by acrylic acid, the properties of the therapeutic protein may change due to charge and hydrophobicity variations. Acrylic acid was detected to migrate from syringes (Vendor X) into a therapeutic protein solution (at a concentration around 5 μg/mL). In this study, we discovered that acrylic acid can modify proteins at three different sites: (1) the lysine side chain, 2) the N-terminus, and 3) the histidine side chain, by the Michael reaction. In this report, the direct interactions between acrylic acid leachable and a biopharmaceutical product were demonstrated and the reaction mechanism was proposed.

Tanshinone IIA attenuates the cerebral ischemic injury-induced increase in levels of GFAP and of caspases-3 and -8.

Science.gov (United States)

Zhou, L; Bondy, S C; Jian, L; Wen, P; Yang, F; Luo, H; Li, W; Zhou, Jun

2015-03-12

Tanshinone IIA (TSA) is a lipid soluble agent derived from the root of Salvia miltiorrhiza (Danshen). This plant is a traditional Chinese herb, which has been used widely in China especially for enhancing circulation. However mechanisms underlying its efficacy remain poorly understood. The present study was designed to illuminate events that may underlie the apparently neuroprotective effects of TSA following ischemic insult. Adult Sprague-Dawley rats were subjected to transient focal cerebral ischemia by use of a middle cerebral artery occlusion model. They were then randomly divided into a sham-operated control group, and cerebral ischemia/reperfusion groups receiving a two-hour occlusion. Further subsets of groups received the same durations of occlusion or were sham-operated but then received daily i.p. injections of high or low doses of TSA, for seven or 15days. Hematoxylin and eosin staining revealed lesions in the entorhinal cortex of both rats subject to ischemia and to a lesser extent to those receiving TSA after surgery. Levels of glial fibrillary acidic protein (GFAP), caspase-3 and caspase-8, were quantified by both immunohistochemistry and Western blotting. TSA treatment after middle cerebral artery occlusion, markedly reduced infarct size, and reduced the expression of caspase-3 and caspase-8. These changes were considered protective and were generally proportional to the dose of TSA used. These results suggest that TSA may effect neuroprotection by way of reduction of the extent of cell inflammation and death within affected regions. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Neuroprotection by Combined Administration with Maslinic Acid, a Natural Product from Olea europaea, and MK-801 in the Cerebral Ischemia Model

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Yisong Qian

2016-08-01

Full Text Available Glutamate-mediated excitotoxicity is a major cause of ischemic brain damage. MK-801 confers neuroprotection by attenuating the activation of the N-methyl-d-aspartate (NMDA receptor, but it failed in clinical use due to the short therapeutic window. Here we aim to investigate the effects of maslinic acid, a natural product from Olea europaea, on the therapeutic time window and dose range for the neuroprotection of MK-801. Rats were administered with maslinic acid intracerebroventricularly and cerebral ischemia was induced by middle cerebral artery occlusion (MCAO followed by reperfusion. MK-801 was administered at 1 h, 2 h, 3 h and 4 h after ischemia, respectively. The cerebral infarct volume was determined by 2,3,5-Triphenyltetrazolium chloride (TTC staining, neuronal damage was assessed by Haematoxylin Eosin (H&E staining, and the expression of glial glutamate transporters and glial fibrillary acidic protein (GFAP was evaluated by immunohistochemistry and Western blot post-ischemia. Results showed that the presence of maslinic acid extended the therapeutic time window for MK-801 from 1 h to 3 h. Co-treatment of maslinic acid and MK-801 at a subthreshold dosage obviously induced neuroprotection after ischemia. The combination of these two compounds improved the outcome in ischemic rats. Moreover, maslinic acid treatment promoted the expression of GLT-1 and GFAP post-ischemia. These data suggest that the synergistic effect of maslinic acid on neurological protection might be associated with the improvement of glial function, especially with the increased expression of GLT-1. The combination therapy of maslinic acid and MK-801 may prove to be a potential strategy for treating acute ischemic stroke.

Neuroprotection by Combined Administration with Maslinic Acid, a Natural Product from Olea europaea, and MK-801 in the Cerebral Ischemia Model.

Science.gov (United States)

Qian, Yisong; Tang, Xuzhen; Guan, Teng; Li, Yunman; Sun, Hongbin

2016-08-19

Glutamate-mediated excitotoxicity is a major cause of ischemic brain damage. MK-801 confers neuroprotection by attenuating the activation of the N-methyl-d-aspartate (NMDA) receptor, but it failed in clinical use due to the short therapeutic window. Here we aim to investigate the effects of maslinic acid, a natural product from Olea europaea, on the therapeutic time window and dose range for the neuroprotection of MK-801. Rats were administered with maslinic acid intracerebroventricularly and cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) followed by reperfusion. MK-801 was administered at 1 h, 2 h, 3 h and 4 h after ischemia, respectively. The cerebral infarct volume was determined by 2,3,5-Triphenyltetrazolium chloride (TTC) staining, neuronal damage was assessed by Haematoxylin Eosin (H&E) staining, and the expression of glial glutamate transporters and glial fibrillary acidic protein (GFAP) was evaluated by immunohistochemistry and Western blot post-ischemia. Results showed that the presence of maslinic acid extended the therapeutic time window for MK-801 from 1 h to 3 h. Co-treatment of maslinic acid and MK-801 at a subthreshold dosage obviously induced neuroprotection after ischemia. The combination of these two compounds improved the outcome in ischemic rats. Moreover, maslinic acid treatment promoted the expression of GLT-1 and GFAP post-ischemia. These data suggest that the synergistic effect of maslinic acid on neurological protection might be associated with the improvement of glial function, especially with the increased expression of GLT-1. The combination therapy of maslinic acid and MK-801 may prove to be a potential strategy for treating acute ischemic stroke.

Cytokines: muscle protein and amino acid metabolism

DEFF Research Database (Denmark)

van Hall, Gerrit

2012-01-01

raises TNF-α and IL-6 to moderate levels, has only identified IL-6 as a potent cytokine, decreasing systemic amino acid levels and muscle protein metabolism. The marked decrease in circulatory and muscle amino acid concentrations was observed with a concomitant reduction in both the rates of muscle...... of IL-6 on the regulation of muscle protein metabolism but indirectly via IL-6 reducing amino acid availability. SUMMARY: Recent studies suggest that the best described cytokines TNF-α and IL-6 are unlikely to be the major direct mediators of muscle protein loss in inflammatory diseases. However...

Hemangioblastomas: histogenesis of the stromal cell studied by immunocytochemistry.

Science.gov (United States)

Jurco, S; Nadji, M; Harvey, D G; Parker, J C; Font, R L; Morales, A R

1982-01-01

Twenty-one cases of hemangioblastoma from the cerebellum, spinal cord and retina were studied using the unlabeled antibody peroxidase-antiperoxidase technique with antibodies directed against glial fibrillary acidic protein (GFAP) and factor VIII related antigen (VIIIR:Ag). In 19 of 21 cases studied with anti-GFAP, astrocytes were identified peripherally, and in 13 cases they were found centrally within the tumor. In no instance did stromal cells react positively for GFAP. Sixteen cases with anti-VIIIR:Ag antibody were examined, and in all cases many stromal cells showed positive staining. It is concluded that the stromal cells were of endothelial origin. The occasional stromal cells that other investigators have identified as reacting positively for GFAP may represent stromal cells capable of ingesting extracellular GFAP derived from reactive astrocytes within the tumor, or they may be lipidized astrocytes.

Immunohistochemical study of sensory nerve formations in human glabrous skin.

Science.gov (United States)

Haro, J J; Vega, J A; del Valle, M E; Calzada, B; Zaccheo, D; Malinovsky, L

1991-01-01

The sensory nerve formations (or corpuscles) of normal human glabrous skin from hand and fingers, obtained by punch biopsies, were studied by the streptavidin-biotin method using monoclonal antibodies directed against neurofilament protein (NFP), S-100 protein, glial fibrillary acidic protein (GFAP), cytokeratins, and vimentin. NFP immunoreactivity (IR) was observed in the central axons of most sensory formations, while S-100 protein IR was restricted to non-neuronal cells forming the so-called inner cells core or lamellar cells. Furthermore, vimentin IR was found in the same cells of Meissner's and glomerular corpuscles. None of the sensory nerve formations were stained for GFAP or keratin. The present results suggest that the main nature of the intermediate filaments of the non-neuronal cells of sensory nerve formations from human glabrous skin is represented by vimentin and not by GFAP. Thus, our findings suggest that lamellar and inner core cells of SNF are modified and specialized Schwann cells and not epithelial or perineurial derived cells.

Protein-membrane interaction and fatty acid transfer from intestinal fatty acid-binding protein to membranes. Support for a multistep process.

Science.gov (United States)

Falomir-Lockhart, Lisandro J; Laborde, Lisandro; Kahn, Peter C; Storch, Judith; Córsico, Betina

2006-05-19

Fatty acid transfer from intestinal fatty acid-binding protein (IFABP) to phospholipid membranes occurs during protein-membrane collisions. Electrostatic interactions involving the alpha-helical "portal" region of the protein have been shown to be of great importance. In the present study, the role of specific lysine residues in the alpha-helical region of IFABP was directly examined. A series of point mutants in rat IFABP was engineered in which the lysine positive charges in this domain were eliminated or reversed. Using a fluorescence resonance energy transfer assay, we analyzed the rates and mechanism of fatty acid transfer from wild type and mutant proteins to acceptor membranes. Most of the alpha-helical domain mutants showed slower absolute fatty acid transfer rates to zwitterionic membranes, with substitution of one of the lysines of the alpha2 helix, Lys27, resulting in a particularly dramatic decrease in the fatty acid transfer rate. Sensitivity to negatively charged phospholipid membranes was also reduced, with charge reversal mutants in the alpha2 helix the most affected. The results support the hypothesis that the portal region undergoes a conformational change during protein-membrane interaction, which leads to release of the bound fatty acid to the membrane and that the alpha2 segment is of particular importance in the establishment of charge-charge interactions between IFABP and membranes. Cross-linking experiments with a phospholipid-photoactivable reagent underscored the importance of charge-charge interactions, showing that the physical interaction between wild-type intestinal fatty acid-binding protein and phospholipid membranes is enhanced by electrostatic interactions. Protein-membrane interactions were also found to be enhanced by the presence of ligand, suggesting different collisional complex structures for holo- and apo-IFABP.

In vitro differentiation of bone marrow stromal cells into neurons and glial cells and differential protein expression in a two-compartment bone marrow stromal cell/neuron co-culture system.

Science.gov (United States)

Qi, Xu; Shao, Ming; Peng, Haisheng; Bi, Zhenggang; Su, Zhiqiang; Li, Hulun

2010-07-01

This study was performed to establish a bone marrow stromal cell (BMSC)/neuron two-compartment co-culture model in which differentiation of BMSCs into neurons could occur without direct contact between the two cell types, and to investigate protein expression changes during differentiation of this entirely BMSC-derived population. Cultured BMSCs isolated from Wistar rats were divided into three groups: BMSC culture, BMSC/neuron co-culture and BMSC/neuron two-compartment co-culture. Cells were examined for neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) expression. The electrophysiological behavior of the BMSCs was examined using patch clamping. Proteins that had significantly different expression levels in BMSCs cultured alone and co-cultured with neurons were studied using a protein chip-mass spectroscopy technique. Expression of NSE and GFAP were significantly higher in co-culture cells than in two-compartment co-culture cells, and significantly higher in both co-culture groups than in BMSCs cultured alone. Five proteins showed significant changes in expression during differentiation: TIP39_RAT and CALC_RAT underwent increases, and INSL6_RAT, PNOC_RAT and PCSK1_RAT underwent decreases in expression. We conclude that BMSCs can differentiate into neurons during both contact co-culture with neurons and two-compartment co-culture with neurons. The rate at which BMSCs differentiated into neurons was higher in contact co-culture than in non-contact co-culture.

Amino acid code of protein secondary structure.

Science.gov (United States)

Shestopalov, B V

2003-01-01

The calculation of protein three-dimensional structure from the amino acid sequence is a fundamental problem to be solved. This paper presents principles of the code theory of protein secondary structure, and their consequence--the amino acid code of protein secondary structure. The doublet code model of protein secondary structure, developed earlier by the author (Shestopalov, 1990), is part of this theory. The theory basis are: 1) the name secondary structure is assigned to the conformation, stabilized only by the nearest (intraresidual) and middle-range (at a distance no more than that between residues i and i + 5) interactions; 2) the secondary structure consists of regular (alpha-helical and beta-structural) and irregular (coil) segments; 3) the alpha-helices, beta-strands and coil segments are encoded, respectively, by residue pairs (i, i + 4), (i, i + 2), (i, i = 1), according to the numbers of residues per period, 3.6, 2, 1; 4) all such pairs in the amino acid sequence are codons for elementary structural elements, or structurons; 5) the codons are divided into 21 types depending on their strength, i.e. their encoding capability; 6) overlappings of structurons of one and the same structure generate the longer segments of this structure; 7) overlapping of structurons of different structures is forbidden, and therefore selection of codons is required, the codon selection is hierarchic; 8) the code theory of protein secondary structure generates six variants of the amino acid code of protein secondary structure. There are two possible kinds of model construction based on the theory: the physical one using physical properties of amino acid residues, and the statistical one using results of statistical analysis of a great body of structural data. Some evident consequences of the theory are: a) the theory can be used for calculating the secondary structure from the amino acid sequence as a partial solution of the problem of calculation of protein three

Biocompatibility of silicon-based arrays of electrodes coupled to organotypic hippocampal brain slice cultures

DEFF Research Database (Denmark)

Kristensen, Bjarne Winther; Noraberg, J; Thiébaud, P

2001-01-01

by Nissl staining, Timm sulphide silver-staining, microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP) immunostaining, the slice cultures grown on chips did not differ from conventionally grown slice cultures. Neither were there any signs of astrogliosis or neurodegeneration...

Protein evolution via amino acid and codon elimination

DEFF Research Database (Denmark)

Goltermann, Lise; Larsen, Marie Sofie Yoo; Banerjee, Rajat

2010-01-01

BACKGROUND: Global residue-specific amino acid mutagenesis can provide important biological insight and generate proteins with altered properties, but at the risk of protein misfolding. Further, targeted libraries are usually restricted to a handful of amino acids because there is an exponential...... correlation between the number of residues randomized and the size of the resulting ensemble. Using GFP as the model protein, we present a strategy, termed protein evolution via amino acid and codon elimination, through which simplified, native-like polypeptides encoded by a reduced genetic code were obtained...... simultaneously), while retaining varying levels of activity. Combination of these substitutions to generate a Phe-free variant of GFP abolished fluorescence. Combinatorial re-introduction of five Phe residues, based on the activities of the respective single amino acid replacements, was sufficient to restore GFP...

Incorporation of radioactive amino acids into protein in isolated rat hepatocytes

International Nuclear Information System (INIS)

Seglin, P.O.

1976-01-01

The incorporation of radioactivity from a 14 C-labelled amino acid mixture (algal protein hydrolysate) into protein in isolated rat hepatocytes has been studied. The incorporation rate declined with increasing cell concentration, an effect which could be explained by isotope consumption, partly (and largely) by isotope dilution due to the formation of non-labelled amino acids by the cells. At a high extracellular amino acid concentration, the rate of incorporation into protein became independent of cell concentration because the isotope dilution effect was now quantitatively insignificant. The time course of protein labelling at various cell concentrations correlated better with the intracellular than with the extracellular amino acid specific activity, suggesting that amino acids for protein synthesis were taken from an intracellular pool. With increasing extracellular amino acid concentrations, both the intracellular amino acid concentration, the intracellular radioactivity and the rate of incorporation into protein increased. Protein labelling exhibited a distinct time lag at high amino acid concentrations, presumable reflecting the time-dependent expansion of the intracellular amino acid pool. The gradual increase in the rate of protein labelling could be due either to an increased intracellular specific activity, or to a real stimulation of protein synthesis by amino acids, depending on whether the total intracellular amino acid pool or just the expandable compartment is the precursor pool for protein synthesis

Discrete nuclear structures in actively growing neuroblastoma cells are revealed by antibodies raised against phosphorylated neurofilament proteins

Directory of Open Access Journals (Sweden)

Raabe Timothy D

2003-04-01

Full Text Available Abstract Background Nuclear objects that have in common the property of being recognized by monoclonal antibodies specific for phosphoprotein epitopes and cytoplasmic intermediate filaments (in particular, SMI-31 and RT-97 have been reported in glial and neuronal cells, in situ and in vitro. Since neurofilament and glial filaments are generally considered to be restricted to the cytoplasm, we were interested in exploring the identity of the structures labeled in the nucleus as well as the conditions under which they could be found there. Results Using confocal microscopy and western analysis techniques, we determined 1 the immunolabeled structures are truly within the nucleus; 2 the phosphoepitope labeled by SMI-31 and RT-97 is not specific to neurofilaments (NFs and it can be identified on other intermediate filament proteins (IFs in other cell types; and 3 there is a close relationship between DNA synthesis and the amount of nuclear staining by these antibodies thought to be specific for cytoplasmic proteins. Searches of protein data bases for putative phosphorylation motifs revealed that lamins, NF-H, and GFAP each contain a single tyrosine phosphorylation motif with nearly identical amino acid sequence. Conclusion We therefore suggest that this sequence may be the epitope recognized by SMI-31 and RT-97 mABs, and that the nuclear structures previously reported and shown here are likely phosphorylated lamin intermediate filaments, while the cytoplasmic labeling revealed by the same mABs indicates phosphorylated NFs in neurons or GFAP in glia.

Brain injury-associated biomarkers of TGF-beta1, S100B, GFAP, NF-L, tTG, AbetaPP, and tau were concomitantly enhanced and the UPS was impaired during acute brain injury caused by Toxocara canis in mice

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Ji Dar-Der

2008-06-01

Full Text Available Abstract Background Because the outcomes and sequelae after different types of brain injury (BI are variable and difficult to predict, investigations on whether enhanced expressions of BI-associated biomarkers (BIABs, including transforming growth factor β1 (TGF-β1, S100B, glial fibrillary acidic protein (GFAP, neurofilament light chain (NF-L, tissue transglutaminases (tTGs, β-amyloid precursor proteins (AβPP, and tau are present as well as whether impairment of the ubiquitin-proteasome system (UPS is present have been widely used to help delineate pathophysiological mechanisms in various BIs. Larvae of Toxocara canis can invade the brain and cause BI in humans and mice, leading to cerebral toxocariasis (CT. Because the parasitic burden is light in CT, it may be too cryptic to be detected in humans, making it difficult to clearly understand the pathogenesis of subtle BI in CT. Since the pathogenesis of murine toxocariasis is very similar to that in humans, it appears appropriate to use a murine model to investigate the pathogenesis of CT. Methods BIAB expressions and UPS function in the brains of mice inoculated with a single dose of 250 T. canis embryonated eggs was investigated from 3 days (dpi to 8 weeks post-infection (wpi by Western blotting and RT-PCR. Results Results revealed that at 4 and 8 wpi, T. canis larvae were found to have invaded areas around the choroid plexus but without eliciting leukocyte infiltration in brains of infected mice; nevertheless, astrogliosis, an indicator of BI, with 78.9~142.0-fold increases in GFAP expression was present. Meanwhile, markedly increased levels of other BIAB proteins including TGF-β1, S100B, NF-L, tTG, AβPP, and tau, with increases ranging 2.0~12.0-fold were found, although their corresponding mRNA expressions were not found to be present at 8 wpi. Concomitantly, UPS impairment was evidenced by the overexpression of conjugated ubiquitin and ubiquitin in the brain. Conclusion Further studies

Brain injury-associated biomarkers of TGF-beta1, S100B, GFAP, NF-L, tTG, AbetaPP, and tau were concomitantly enhanced and the UPS was impaired during acute brain injury caused by Toxocara canis in mice.

Science.gov (United States)

Liao, Chien-Wei; Fan, Chia-Kwung; Kao, Ting-Chang; Ji, Dar-Der; Su, Kua-Eyre; Lin, Yun-Ho; Cho, Wen-Long

2008-06-24

Because the outcomes and sequelae after different types of brain injury (BI) are variable and difficult to predict, investigations on whether enhanced expressions of BI-associated biomarkers (BIABs), including transforming growth factor beta1 (TGF-beta1), S100B, glial fibrillary acidic protein (GFAP), neurofilament light chain (NF-L), tissue transglutaminases (tTGs), beta-amyloid precursor proteins (AbetaPP), and tau are present as well as whether impairment of the ubiquitin-proteasome system (UPS) is present have been widely used to help delineate pathophysiological mechanisms in various BIs. Larvae of Toxocara canis can invade the brain and cause BI in humans and mice, leading to cerebral toxocariasis (CT). Because the parasitic burden is light in CT, it may be too cryptic to be detected in humans, making it difficult to clearly understand the pathogenesis of subtle BI in CT. Since the pathogenesis of murine toxocariasis is very similar to that in humans, it appears appropriate to use a murine model to investigate the pathogenesis of CT. BIAB expressions and UPS function in the brains of mice inoculated with a single dose of 250 T. canis embryonated eggs was investigated from 3 days (dpi) to 8 weeks post-infection (wpi) by Western blotting and RT-PCR. Results revealed that at 4 and 8 wpi, T. canis larvae were found to have invaded areas around the choroid plexus but without eliciting leukocyte infiltration in brains of infected mice; nevertheless, astrogliosis, an indicator of BI, with 78.9~142.0-fold increases in GFAP expression was present. Meanwhile, markedly increased levels of other BIAB proteins including TGF-beta1, S100B, NF-L, tTG, AbetaPP, and tau, with increases ranging 2.0~12.0-fold were found, although their corresponding mRNA expressions were not found to be present at 8 wpi. Concomitantly, UPS impairment was evidenced by the overexpression of conjugated ubiquitin and ubiquitin in the brain. Further studies are needed to determine whether there is an

Echinococcus granulosus fatty acid binding proteins subcellular localization.

Science.gov (United States)

Alvite, Gabriela; Esteves, Adriana

2016-05-01

Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.

Absolute quantitation of proteins by Acid hydrolysis combined with amino Acid detection by mass spectrometry

DEFF Research Database (Denmark)

Mirgorodskaya, Olga A; Körner, Roman; Kozmin, Yuri P

2012-01-01

Amino acid analysis is among the most accurate methods for absolute quantification of proteins and peptides. Here, we combine acid hydrolysis with the addition of isotopically labeled standard amino acids and analysis by mass spectrometry for accurate and sensitive protein quantitation...

Marcação imunoistoquímica da expressão astrocitária de proteína glial fibrilar ácida e de vimentina no sistema nervoso central de cães com cinomose Immunohistochemical staining of the astrocytic expression of glial fibrillary acidic protein and vimentin in the central nervous system of dogs with canine distemper

Directory of Open Access Journals (Sweden)

Heloísa Orsini

2007-12-01

immunohistochemical staining of two astrocytic proteins - glial fibrillary acidic protein (GFAP and vimentin (VIM -, comparing samples of cerebellum and brainstem from eight dogs with canine distemper and from two healthy dogs, from different breeds and ages varying from 1 to 4 years old. Histological sections were submitted to the avidin-biotin-peroxidase indirect method of immmunohistochemical staining (ABC and the astrocytic reactivity, observed in light microscopy, was quantified in a computer system for image analysis. It was possible to notice, on most of the sections from sick animals, degenerative lesions that indicate demyelination. The immunostaining for GFAP and VIM was more intense on animals with canine distemper, specially around the ventricules and near degenerated sites. There was no significant difference between the immunostaining (GFAP and VIM of animals with canine distemper with and without inflammatory infiltrate of the cerebellar white matter. The increased immunoreactivity of astrocytes for GFAP and the VIM reexpression in injured areas indicate the astrocytic involvement on nervous tissue response to the demyelinating lesions induced by the canine distemper virus (CDV in the CNS.

Tsc2 gene inactivation causes a more severe epilepsy phenotype than Tsc1 inactivation in a mouse model of tuberous sclerosis complex.

Science.gov (United States)

Zeng, Ling-Hui; Rensing, Nicholas R; Zhang, Bo; Gutmann, David H; Gambello, Michael J; Wong, Michael

2011-02-01

Tuberous Sclerosis Complex (TSC) is an autosomal dominant, multi-system disorder, typically involving severe neurological symptoms, such as epilepsy, cognitive deficits and autism. Two genes, TSC1 and TSC2, encoding the proteins hamartin and tuberin, respectively, have been identified as causing TSC. Although there is a substantial overlap in the clinical phenotype produced by TSC1 and TSC2 mutations, accumulating evidence indicates that TSC2 mutations cause more severe neurological manifestations than TSC1 mutations. In this study, the neurological phenotype of a novel mouse model involving conditional inactivation of the Tsc2 gene in glial-fibrillary acidic protein (GFAP)-positive cells (Tsc2(GFAP1)CKO mice) was characterized and compared with previously generated Tsc1(GFAP1)CKO mice. Similar to Tsc1(GFAP1)CKO mice, Tsc2(GFAP1)CKO mice exhibited epilepsy, premature death, progressive megencephaly, diffuse glial proliferation, dispersion of hippocampal pyramidal cells and decreased astrocyte glutamate transporter expression. However, Tsc2(GFAP1)CKO mice had an earlier onset and higher frequency of seizures, as well as significantly more severe histological abnormalities, compared with Tsc1(GFAP1)CKO mice. The differences between Tsc1(GFAP1)CKO and Tsc2(GFAP1)CKO mice were correlated with higher levels of mammalian target of rapamycin (mTOR) activation in Tsc2(GFAP1)CKO mice and were reversed by the mTOR inhibitor, rapamycin. These findings provide novel evidence in mouse models that Tsc2 mutations intrinsically cause a more severe neurological phenotype than Tsc1 mutations and suggest that the difference in phenotype may be related to the degree to which Tsc1 and Tsc2 inactivation causes abnormal mTOR activation.

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.

Acylation of proteins with myristic acid occurs cotranslationally

International Nuclear Information System (INIS)

Wilcox, C.; Hu, J.S.; Olson, E.N.

1987-01-01

Several proteins of viral and cellular origin are acylated with myristic acid early during their biogenesis. To investigate the possibility that myristylation occurred cotranslationally, the BC 3 H1 muscle cell line, which contains a broad array of myristylated proteins, was pulse-labeled with [ 3 H]myristic acid. Nascent polypeptide chains covalently associated with transfer RNA were isolated subsequently by ion-exchange chromatography. [ 3 H]Myristate was attached to nascent chains through an amide linkage and was identified by thin-layer chromatography after its release from nascent chains by acid methanolysis. Inhibition of cellular protein synthesis with puromycin resulted in cessation of [ 3 H]myristate-labeling of nascent chains, in agreement with the dependence of this modification on protein synthesis in vivo. These data represent a direct demonstration that myristylation of proteins is a cotranslational modification

Amino acid repletion does not decrease muscle protein catabolism during hemodialysis.

Science.gov (United States)

Raj, Dominic S C; Adeniyi, Oladipo; Dominic, Elizabeth A; Boivin, Michel A; McClelland, Sandra; Tzamaloukas, Antonios H; Morgan, Nancy; Gonzales, Lawrence; Wolfe, Robert; Ferrando, Arny

2007-06-01

Intradialytic protein catabolism is attributed to loss of amino acids in the dialysate. We investigated the effect of amino acid infusion during hemodialysis (HD) on muscle protein turnover and amino acid transport kinetics by using stable isotopes of phenylalanine, leucine, and lysine in eight patients with end-stage renal disease (ESRD). Subjects were studied at baseline (pre-HD), 2 h of HD without amino acid infusion (HD-O), and 2 h of HD with amino acid infusion (HD+AA). Amino acid depletion during HD-O augmented the outward transport of amino acids from muscle into the vein. Increased delivery of amino acids to the leg during HD+AA facilitated the transport of amino acids from the artery into the intracellular compartment. Increase in muscle protein breakdown was more than the increase in synthesis during HD-O (46.7 vs. 22.3%, P HD-O compared with pre-HD (-33.7 +/- 1.5 vs. -6.0 +/- 2.3, P acids, the net balance (-16.9 +/- 1.8) did not switch from net release to net uptake. HD+AA induced a proportional increase in muscle protein synthesis and catabolism. Branched chain amino acid catabolism increased significantly from baseline during HD-O and did not decrease during HD+AA. Protein synthesis efficiency, the fraction of amino acid in the intracellular pool that is utilized for muscle protein synthesis decreased from 42.1% pre-HD to 33.7 and 32.6% during HD-O and HD+AA, respectively (P acid repletion during HD increased muscle protein synthesis but did not decrease muscle protein breakdown.

Interaction of milk whey protein with common phenolic acids

Science.gov (United States)

Zhang, Hao; Yu, Dandan; Sun, Jing; Guo, Huiyuan; Ding, Qingbo; Liu, Ruihai; Ren, Fazheng

2014-01-01

Phenolics-rich foods such as fruit juices and coffee are often consumed with milk. In this study, the interactions of α-lactalbumin and β-lactoglobulin with the phenolic acids (chlorogenic acid, caffeic acid, ferulic acid, and coumalic acid) were examined. Fluorescence, CD, and FTIR spectroscopies were used to analyze the binding modes, binding constants, and the effects of complexation on the conformation of whey protein. The results showed that binding constants of each whey protein-phenolic acid interaction ranged from 4 × 105 to 7 × 106 M-n and the number of binding sites n ranged from 1.28 ± 0.13 to 1.54 ± 0.34. Because of these interactions, the conformation of whey protein was altered, with a significant reduction in the amount of α-helix and an increase in the amounts of β-sheet and turn structures.

Reactive Transformation and Increased BDNF Signaling by Hippocampal Astrocytes in Response to MK-801.

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Wenjuan Yu

Full Text Available MK-801, also known as dizocilpine, is a noncompetitive N-methyl-D-aspartic acid (NMDA receptor antagonist that induces schizophrenia-like symptoms. While astrocytes have been implicated in the pathophysiology of psychiatric disorders, including schizophrenia, astrocytic responses to MK-801 and their significance to schizotypic symptoms are unclear. Changes in the expression levels of glial fibrillary acid protein (GFAP, a marker of astrocyte activation in response to a variety of pathogenic stimuli, were examined in the hippocampus of rats treated with the repeated MK-801 injection (0.5 mg/10 ml/kg body weight for 6 days and in primary cultured hippocampal astrocytes incubated with MK-801 (5 or 20 μM for 24 h. Moreover, the expression levels of BDNF and its receptors TrkB and p75 were examined in MK-801-treated astrocyte cultures. MK-801 treatment enhanced GFAP expression in the rat hippocampus and also increased the levels of GFAP protein and mRNA in hippocampal astrocytes in vitro. Treatment of cultured hippocampal astrocytes with MK-801 enhanced protein and mRNA levels of BDNF, TrkB, and p75. Collectively, our results suggest that hippocampal astrocytes may contribute to the pathophysiology of schizophrenia symptoms associated with NMDA receptor hypofunction by reactive transformation and altered BDNF signaling.

Reactive Transformation and Increased BDNF Signaling by Hippocampal Astrocytes in Response to MK-801

Science.gov (United States)

Wang, Yueming; Li, Guanjun; Wang, Lihua; Li, Huafang

2015-01-01

MK-801, also known as dizocilpine, is a noncompetitive N-methyl-D-aspartic acid (NMDA) receptor antagonist that induces schizophrenia-like symptoms. While astrocytes have been implicated in the pathophysiology of psychiatric disorders, including schizophrenia, astrocytic responses to MK-801 and their significance to schizotypic symptoms are unclear. Changes in the expression levels of glial fibrillary acid protein (GFAP), a marker of astrocyte activation in response to a variety of pathogenic stimuli, were examined in the hippocampus of rats treated with the repeated MK-801 injection (0.5 mg/10ml/kg body weight for 6 days) and in primary cultured hippocampal astrocytes incubated with MK-801 (5 or 20 μM for 24 h). Moreover, the expression levels of BDNF and its receptors TrkB and p75 were examined in MK-801-treated astrocyte cultures. MK-801 treatment enhanced GFAP expression in the rat hippocampus and also increased the levels of GFAP protein and mRNA in hippocampal astrocytes in vitro. Treatment of cultured hippocampal astrocytes with MK-801 enhanced protein and mRNA levels of BDNF, TrkB, and p75. Collectively, our results suggest that hippocampal astrocytes may contribute to the pathophysiology of schizophrenia symptoms associated with NMDA receptor hypofunction by reactive transformation and altered BDNF signaling. PMID:26700309

Site specific incorporation of keto amino acids into proteins

Science.gov (United States)

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

2008-10-07

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

Astrocytic Insulin Signaling Couples Brain Glucose Uptake with Nutrient Availability

NARCIS (Netherlands)

García-Cáceres, Cristina; Quarta, Carmelo; Varela, Luis; Gao, Yuanqing; Gruber, Tim; Legutko, Beata; Jastroch, Martin; Johansson, Pia; Ninkovic, Jovica; Yi, Chun-Xia; Le Thuc, Ophelia; Szigeti-Buck, Klara; Cai, Weikang; Meyer, Carola W.; Pfluger, Paul T.; Fernandez, Ana M.; Luquet, Serge; Woods, Stephen C.; Torres-Alemán, Ignacio; Kahn, C. Ronald; Götz, Magdalena; Horvath, Tamas L.; Tschöp, Matthias H.

2016-01-01

We report that astrocytic insulin signaling co-regulates hypothalamic glucose sensing and systemic glucose metabolism. Postnatal ablation of insulin receptors (IRs) in glial fibrillary acidic protein (GFAP)-expressing cells affects hypothalamic astrocyte morphology, mitochondrial function, and

Residue-specific incorporation of noncanonical amino acids for protein engineering

NARCIS (Netherlands)

van Eldijk, Mark B.; van Hest, Jan C.M.; Lemke, E.A.

2018-01-01

The incorporation of noncanonical amino acids has given protein chemists access to an expanded repertoire of amino acids. This methodology has significantly broadened the scope of protein engineering allowing introduction of amino acids with non-native functionalities, such as bioorthogonal reactive

Uric acid contributes greatly to hepatic antioxidant capacity besides protein.

Science.gov (United States)

Mikami, T; Sorimachi, M

2017-12-20

Uric acid is the end-product of purine nucleotide metabolism and an increase in uric acid concentration in the body results in hyperuricemia, ultimately leading to gout. However, uric acid is a potent antioxidant and interacts with reactive oxygen species (ROS) to be non-enzymatically converted to allantoin. Uric acid accounts for approximately 60 % of antioxidant capacity in the plasma; however, its contribution to tissue antioxidant capacity is unknown. In this study, the contribution of uric acid to tissue antioxidant capacity and its conversion to allantoin by scavenging ROS in tissue were examined. The results showed that a decrease in hepatic uric acid content via allopurinol administration significantly reduced hepatic total-radical trapping antioxidant parameter (TRAP) content in protein-free cytosol. Additionally, treating protein-free cytosol with uricase led to a further reduction of hepatic TRAP content. Allantoin was also detected in the solution containing protein-free cytosol that reacted with ROS. These findings suggest that in the absence of protein, uric acid contributes greatly to antioxidant capacity in the liver, where uric acid is converted to allantoin by scavenging ROS.

Rewiring protein synthesis: From natural to synthetic amino acids.

Science.gov (United States)

Fan, Yongqiang; Evans, Christopher R; Ling, Jiqiang

2017-11-01

The protein synthesis machinery uses 22 natural amino acids as building blocks that faithfully decode the genetic information. Such fidelity is controlled at multiple steps and can be compromised in nature and in the laboratory to rewire protein synthesis with natural and synthetic amino acids. This review summarizes the major quality control mechanisms during protein synthesis, including aminoacyl-tRNA synthetases, elongation factors, and the ribosome. We will discuss evolution and engineering of such components that allow incorporation of natural and synthetic amino acids at positions that deviate from the standard genetic code. The protein synthesis machinery is highly selective, yet not fixed, for the correct amino acids that match the mRNA codons. Ambiguous translation of a codon with multiple amino acids or complete reassignment of a codon with a synthetic amino acid diversifies the proteome. Expanding the genetic code with synthetic amino acids through rewiring protein synthesis has broad applications in synthetic biology and chemical biology. Biochemical, structural, and genetic studies of the translational quality control mechanisms are not only crucial to understand the physiological role of translational fidelity and evolution of the genetic code, but also enable us to better design biological parts to expand the proteomes of synthetic organisms. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

Injury-induced inhibition of small intestinal protein and nucleic acid synthesis

International Nuclear Information System (INIS)

Carter, E.A.; Hatz, R.A.; Yarmush, M.L.; Tompkins, R.G.

1990-01-01

Small intestinal mucosal weight and nutrient absorption are significantly diminished early after cutaneous thermal injuries. Because these intestinal properties are highly dependent on rates of nucleic acid and protein synthesis, in vivo incorporation of thymidine, uridine, and leucine into small intestinal deoxyribonucleic acid, ribonucleic acid, and proteins were measured. Deoxyribonucleic acid synthesis was markedly decreased with the lowest thymidine incorporation in the jejunum (p less than 0.01); these findings were confirmed by autoradiographic identification of radiolabeled nuclei in the intestinal crypts. Protein synthesis was decreased by 6 h postinjury (p less than 0.01) but had returned to normal by 48 h. Consistent with a decreased rate of protein synthesis, ribonucleic acid synthesis was also decreased 18 h postinjury (p less than 0.01). These decreased deoxyribonucleic acid, ribonucleic acid, and protein synthesis rates are not likely a result of ischemia because in other studies of this injury model, intestinal blood flow was not significantly changed by the burn injury. Potentially, factors initiating the acute inflammatory reaction may directly inhibit nucleic acid and protein synthesis and lead to alterations in nutrient absorption and intestinal barrier function after injury

A Soluble, Folded Protein without Charged Amino Acid Residues

DEFF Research Database (Denmark)

Højgaard, Casper; Kofoed, Christian; Espersen, Roall

2016-01-01

Charges are considered an integral part of protein structure and function, enhancing solubility and providing specificity in molecular interactions. We wished to investigate whether charged amino acids are indeed required for protein biogenesis and whether a protein completely free of titratable...... side chains can maintain solubility, stability, and function. As a model, we used a cellulose-binding domain from Cellulomonas fimi, which, among proteins of more than 100 amino acids, presently is the least charged in the Protein Data Bank, with a total of only four titratable residues. We find...

Dietary fatty acids and membrane protein function.

Science.gov (United States)

Murphy, M G

1990-02-01

In recent years, there has been growing public awareness of the potential health benefits of dietary fatty acids, and of the distinction between the effects of the omega6 and omega3 polyunsaturated fatty acids that are concentrated in vegetable and fish oils, respectively. A part of the biologic effectiveness of the two families of polyunsaturated fatty acids resides in their relative roles as precursors of the eicosanoids. However, we are also beginning to appreciate that as the major components of the hydrophobic core of the membrane bilayer, they can interact with and directly influence the functioning of select integral membrane proteins. Among the most important of these are the enzymes, receptors, and ion channels that are situated in the plasma membrane of the cell, since they carry out the communication and homeostatic processes that are necessary for normal cell function. This review examines current information regarding the effects of diet-induced changes in plasma membrane fatty acid composition on several specific enzymes (adenylate cyclase, 5'-nucleotidase, Na(+)/K(+)-ATPase) and cell-surface receptors (opiate, adrenergic, insulin). Dietary manipulation studies have demonstrated a sensitivity of each to a fatty acid environment that is variably dependent on the nature of the fatty acid(s) and/or source of the membrane. The molecular mechanisms appear to involve fatty acid-dependent effects on protein conformation, on the "fluidity" and/or thickness of the membrane, or on protein synthesis. Together, the results of these studies reinforce the concept that dietary fats have the potential to regulate physiologic function and to further our understanding of how this occurs at a membrane level.

Spinal translocator protein (TSPO) modulates pain behavior in rats with CFA-induced monoarthritis.

Science.gov (United States)

Hernstadt, Hayley; Wang, Shuxing; Lim, Grewo; Mao, Jianren

2009-08-25

Translocator protein 18 kDa (TSPO), previously known as the peripheral benzodiazepine receptor (PBR), is predominantly located in the mitochondrial outer membrane and plays an important role in steroidogenesis, immunomodulation, cell survival and proliferation. Previous studies have shown an increased expression of TSPO centrally in neuropathology, as well as in injured nerves. TSPO has also been implicated in modulation of nociception. In the present study, we examined the hypothesis that TSPO is involved in the initiation and maintenance of inflammatory pain using a rat model of Complete Freund's Adjuvant (CFA)-induced monoarthritis of the tibio-tarsal joint. Immunohistochemistry was performed using Iba-1 (microglia), NeuN (neurons), anti-Glial Fibrillary Acidic Protein, GFAP (astrocytes) and anti-PBR (TSPO) on Days 1, 7 and 14 after CFA-induced arthritis. Rats with CFA-induced monoarthritis showed mechanical allodynia and thermal hyperalgesia on the ipsilateral hindpaw, which correlated with the increased TSPO expression in ipsilateral laminae I-II on all experimental days. Iba-1 expression in the ipsilateral dorsal horn was also increased on Days 7 and 14. Moreover, TSPO was colocalized with Iba-1, GFAP and NeuN within the spinal cord dorsal horn. The TSPO agonist Ro5-4864, given intrathecally, dose-dependently retarded or prevented the development of mechanical allodynia and thermal hyperalgesia in rats with CFA-induced monoarthritis. These findings provide evidence that spinal TSPO is involved in the development and maintenance of inflammatory pain behaviors in rats. Thus, spinal TSPO may present a central target as a complementary therapy to reduce inflammatory pain.

Laser-based optical activity detection of amino acids and proteins

Energy Technology Data Exchange (ETDEWEB)

Reitsma, B.H.

1987-08-01

The optical activity detector (OAD) for HPLC is a selective detector for optically active substances including amino acids and proteins. Four free amino acids were resolved using cation-exchange chromatography followed by detection with refractive index detector (RI) for proline and threonine and the OAD to an ultraviolet absorbance detector (uv) for tyrosine and phenylalanine. Amino acid detection by refractive index is not sensitive and uv absorbance detects only three amino acids. Derivatization of amino acids to make them detectable by uv absorbance enhances the applicability of OA/uv for the determination of enantiomeric ratios. The separation of 16 dansyl-L-amino acids by RP-HPLC with detection by OA/uv is illustrated. Calculation of the specific rotation of 22 dansyl-L-amino acids shows that derivatization enhances the OA detectability of some amino acids but degrades that of others. RP-HPLC of proteins is a rapidly developing technique. Several researchers have reported the detection of multiple peaks when a pure protein is subjected to HPLC under certain conditions. These multiple peaks have been determined to be different conformations of the same protein. Since proteins are optically active, OA is a suitable detector. The RP-HPLC separation of conformers of soybean trypsin inhibitor is illustrated. Detection by OA/uv provides insights from the chromatogram unavailable from uv absorbance detection alone. In addition, identification of impurities is simplified with OA/uv. Specific rotations of the separated protein fractions show no significant change accompanying change in conformation. 163 refs., 13 figs., 9 tabs.

The multiple roles of Fatty Acid Handling Proteins in brain

Directory of Open Access Journals (Sweden)

Valentine SF Moullé

2012-09-01

Full Text Available Lipids are essential components of a living organism as energy source but also as constituent of the membrane lipid bilayer. In addition fatty acid (FA derivatives interact with many signaling pathways. FAs have amphipathic properties and therefore require being associated to protein for both transport and intracellular trafficking. Here we will focus on several fatty acid handling proteins, among which the fatty acid translocase/CD36 (FAT/CD36, members of fatty acid transport proteins (FATPs, and lipid chaperones fatty acid-binding proteins (FABPs. A decade of extensive studies has helped decipher the mechanism of action of these proteins in peripheral tissue with high lipid metabolism. However, considerably less information is available regarding their role in the brain, despite the high lipid content of this tissue. This review will primarily focus on the recent studies that have highlighted the crucial role of lipid handling proteins in brain FA transport, neuronal differentiation and development, cognitive processes and brain diseases. Finally a special focus will be made on the recent studies that have revealed the role of FAT/CD36 in brain lipid sensing and nervous control of energy balance.

Towards an understanding of Mesocestoides vogae fatty acid binding proteins' roles.

Directory of Open Access Journals (Sweden)

Gabriela Alvite

Full Text Available Two fatty acid binding proteins, MvFABPa and MvFABPb were identified in the parasite Mesocestoides vogae (Platyhelmithes, Cestoda. Fatty acid binding proteins are small intracellular proteins whose members exhibit great diversity. Proteins of this family have been identified in many organisms, of which Platyhelminthes are among the most primitive. These proteins have particular relevance in flatworms since de novo synthesis of fatty acids is absent. Fatty acids should be captured from the media needing an efficient transport system to uptake and distribute these molecules. While HLBPs could be involved in the shuttle of fatty acids to the surrounding host tissues and convey them into the parasite, FABPs could be responsible for the intracellular trafficking. In an effort to understand the role of MvFABPs in fatty acid transport of M. vogae larvae, we analysed the intracellular localization of both MvFABPs and the co-localization with in vivo uptake of fatty acid analogue BODIPY FL C16. Immunohistochemical studies on larvae sections using specific antibodies, showed a diffuse cytoplasmic distribution of each protein with some expression in nuclei and mitochondria. MvFABPs distribution was confirmed by mass spectrometry identification from 2D-electrophoresis of larvae subcellular fractions. This work is the first report showing intracellular distribution of MvFABPs as well as the co-localization of these proteins with the BODIPY FL C16 incorporated from the media. Our results suggest that fatty acid binding proteins could target fatty acids to cellular compartments including nuclei. In this sense, M. vogae FABPs could participate in several cellular processes fulfilling most of the functions attributed to vertebrate's counterparts.

Scale-free behaviour of amino acid pair interactions in folded proteins

DEFF Research Database (Denmark)

Petersen, Steffen B.; Neves-Petersen, Maria Teresa; Mortensen, Rasmus J.

2012-01-01

The protein structure is a cumulative result of interactions between amino acid residues interacting with each other through space and/or chemical bonds. Despite the large number of high resolution protein structures, the ‘‘protein structure code’’ has not been fully identified. Our manuscript...... presents a novel approach to protein structure analysis in order to identify rules for spatial packing of amino acid pairs in proteins. We have investigated 8706 high resolution non-redundant protein chains and quantified amino acid pair interactions in terms of solvent accessibility, spatial and sequence...... which amino acid paired residues contributed to the cells with a population above 50, pairs of Ala, Ile, Leu and Val dominate the results. This result is statistically highly significant. We postulate that such pairs form ‘‘structural stability points’’ in the protein structure. Our data shows...

Characterization of fatty acid binding by the P2 myelin protein

International Nuclear Information System (INIS)

Gudaitis, P.G.; Weise, M.J.

1987-01-01

In recent years, significant sequence homology has been found between the P2 protein of peripheral myelin and intracellular retinoid- and fatty acid-binding proteins. They have found that salt extracts of bovine intradural nerve roots contain the P2 basic protein in association with free fatty acid. Preliminary results from quantitative analyses showed a ratio of 0.4-1.1 fatty acid (mainly oleate and palmitate) per P2 molecule. P2/ligand interactions were partially characterized using ( 3 H)-oleate in gel permeation assays and binding studies using lipidex to separated bound and free fatty acid. Methyloleate was found to displace ( 3 H)-oleate from P2, indicating that ligand binding interactions are predominantly hydrophobic in nature. On the other hand, myristic acid and retinol did not inhibit the binding of oleate to the protein, results consistent with a decided affinity for long chain fatty acids but not for the retinoids. The binding between P2 and oleic acid showed an apparent Kd in the micromolar range, a value comparable to those found for other fatty acid-binding proteins. From these results they conclude that P2 shares not only structural homology with certain fatty acid binding proteins but also an ability to bind long chain fatty acids. Although the significance of these similarities is not yet clear, they may, by analogy, expect P2 to have a role in PNS lipid metabolism

Modifications of proteins by polyunsaturated fatty acid peroxidation products

DEFF Research Database (Denmark)

Refsgaard, Hanne; Tsai, Lin; Stadtman, Earl

2000-01-01

The ability of unsaturated fatty acid methyl esters to modify amino acid residues in bovine serum albumin (BSA), glutamine synthetase, and insulin in the presence of a metal-catalyzed oxidation system [ascorbate/Fe(lll)/O-2] depends on the degree of unsaturation of the fatty acid. The fatty acid......-dependent generation of carbonyl groups and loss of lysine residues increased in the order methyl linoleate fatty acids were oxidized in the presence...... in the formation of protein carbonyls, These results are consistent with the proposition that metal-catalyzed oxidation of polyunsaturated fatty acids can contribute to the generation of protein carbonyls by direct interaction of lipid oxidation products (alpha,beta-unsaturated aldehydes) with lysine residues...

Utilizing knowledge base of amino acids structural neighborhoods to predict protein-protein interaction sites.

Science.gov (United States)

Jelínek, Jan; Škoda, Petr; Hoksza, David

2017-12-06

Protein-protein interactions (PPI) play a key role in an investigation of various biochemical processes, and their identification is thus of great importance. Although computational prediction of which amino acids take part in a PPI has been an active field of research for some time, the quality of in-silico methods is still far from perfect. We have developed a novel prediction method called INSPiRE which benefits from a knowledge base built from data available in Protein Data Bank. All proteins involved in PPIs were converted into labeled graphs with nodes corresponding to amino acids and edges to pairs of neighboring amino acids. A structural neighborhood of each node was then encoded into a bit string and stored in the knowledge base. When predicting PPIs, INSPiRE labels amino acids of unknown proteins as interface or non-interface based on how often their structural neighborhood appears as interface or non-interface in the knowledge base. We evaluated INSPiRE's behavior with respect to different types and sizes of the structural neighborhood. Furthermore, we examined the suitability of several different features for labeling the nodes. Our evaluations showed that INSPiRE clearly outperforms existing methods with respect to Matthews correlation coefficient. In this paper we introduce a new knowledge-based method for identification of protein-protein interaction sites called INSPiRE. Its knowledge base utilizes structural patterns of known interaction sites in the Protein Data Bank which are then used for PPI prediction. Extensive experiments on several well-established datasets show that INSPiRE significantly surpasses existing PPI approaches.

Influence of protein source on amino acid uptake patterns and protein utilization in rainbow trout Oncorhynchus mykiss

DEFF Research Database (Denmark)

Rolland, Marine; Holm, Jørgen; Dalsgaard, Anne Johanne Tang

induces reduced growth performances that remain partly unexplained. The aim of the current study was to investigate the effect of exchanging the protein source on protein utilization. Marine (fish meal) and vegetable (pea protein) sources were used with or without supplementation of crystalline amino......Matrixes of different protein sources (fish and plant products) combined with the use of crystalline amino acids allow for formulation of diets that meet fish requirements with little or no effect on protein digestibility and/or feed intake. Despite this, a total or partial replacement of fish meal...... acids to the fishmeal diet level (see Table 1). Amino acid uptake patterns were assessed by the appearance of amino acids in the blood stream following the ingestion of a meal, while dietary protein utilization was evaluated by examining the metabolic response to digestion and ammonium and urea...

Influence of irradiation on protein and amino acids in laboratory rodent diet

International Nuclear Information System (INIS)

Ford, D.J.

1979-01-01

The effect of irradiation treatment on the protein quality and constituent amino acids of laboratory rodent diets is reviewed and compared with other methods of sterilization - autoclaving and ethylene oxide fumigation. Gamma irradiation has been shown to have minimal influence on total protein, protein quality and total and available amino acid levels. Autoclaving reduces amino acid availability and consequently protein quality. Limited evidence shows reduction of certain available amino acids following ethylene oxide fumigation. (author)

Protein and Amino Acid Composition of Water Melon ( Citrullus ...

African Journals Online (AJOL)

The protein and amino acids composition of seeds and pulp of watermelon, Citrullus lanatus were analyzed using Kjeldahl method and ion-exchange chromatography (IEC) respectively. The protein contents (% dry matter) of seeds and pulp were found to be 24.23 and 1.05% respectively. The results of amino acids ...

Enhanced Bio-hydrogen Production from Protein Wastewater by Altering Protein Structure and Amino Acids Acidification Type

Science.gov (United States)

Xiao, Naidong; Chen, Yinguang; Chen, Aihui; Feng, Leiyu

2014-01-01

Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type via pH control was investigated. The hydrogen production reached 205.2 mL/g-protein when protein wastewater was pretreated at pH 12 and then fermented at pH 10. The mechanism studies showed that pH 12 pretreatment significantly enhanced protein bio-hydrolysis during the subsequent fermentation stage as it caused the unfolding of protein, damaged the protein hydrogen bonding networks, and destroyed the disulfide bridges, which increased the susceptibility of protein to protease. Moreover, pH 10 fermentation produced more acetic but less propionic acid during the anaerobic fermentation of amino acids, which was consistent with the theory of fermentation type affecting hydrogen production. Further analyses of the critical enzymes, genes, and microorganisms indicated that the activity and abundance of hydrogen producing bacteria in the pH 10 fermentation reactor were greater than those in the control. PMID:24495932

Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type.

Science.gov (United States)

Xiao, Naidong; Chen, Yinguang; Chen, Aihui; Feng, Leiyu

2014-02-05

Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type via pH control was investigated. The hydrogen production reached 205.2 mL/g-protein when protein wastewater was pretreated at pH 12 and then fermented at pH 10. The mechanism studies showed that pH 12 pretreatment significantly enhanced protein bio-hydrolysis during the subsequent fermentation stage as it caused the unfolding of protein, damaged the protein hydrogen bonding networks, and destroyed the disulfide bridges, which increased the susceptibility of protein to protease. Moreover, pH 10 fermentation produced more acetic but less propionic acid during the anaerobic fermentation of amino acids, which was consistent with the theory of fermentation type affecting hydrogen production. Further analyses of the critical enzymes, genes, and microorganisms indicated that the activity and abundance of hydrogen producing bacteria in the pH 10 fermentation reactor were greater than those in the control.

Heart-type fatty-acid-binding protein (FABP3 is a lysophosphatidic acid-binding protein in human coronary artery endothelial cells

Directory of Open Access Journals (Sweden)

Ryoko Tsukahara

2014-01-01

Full Text Available Fatty-acid-binding protein 3, muscle and heart (FABP3, also known as heart-type FABP, is a member of the family of intracellular lipid-binding proteins. It is a small cytoplasmic protein with a molecular mass of about 15 kDa. FABPs are known to be carrier proteins for transporting fatty acids and other lipophilic substances from the cytoplasm to the nucleus, where these lipids are released to a group of nuclear receptors such as peroxisome proliferator-activated receptors (PPARs. In this study, using lysophosphatidic acid (LPA-coated agarose beads, we have identified FABP3 as an LPA carrier protein in human coronary artery endothelial cells (HCAECs. Administration of LPA to HCAECs resulted in a dose-dependent increase in PPARγ activation. Furthermore, the LPA-induced PPARγ activation was abolished when the FABP3 expression was reduced using small interfering RNA (siRNA. We further show that the nuclear fraction of control HCAECs contained a significant amount of exogenously added LPA, whereas FABP3 siRNA-transfected HCAECs had a decreased level of LPA in the nucleus. Taken together, these results suggest that FABP3 governs the transcriptional activities of LPA by targeting them to cognate PPARγ in the nucleus.

Altered expression of sphingosine kinase 1 and sphingosine-1-phosphate receptor 1 in mouse hippocampus after kainic acid treatment

Energy Technology Data Exchange (ETDEWEB)

Lee, Dong Hoon; Jeon, Byeong Tak; Jeong, Eun Ae [Department of Anatomy and Neurobiology, Institute of Health Sciences, Medical Research Center for Neural Dysfunction, Biomedical Center (BK21), Gyeongsang National University School of Medicine, Jinju, Gyeongnam 660-751 (Korea, Republic of); Kim, Joon Soo; Cho, Yong Woon [Department of Neurosurgery, Masan Samsung Hospital, Sungkyunkwan University School of Medicine, Masan, Gyeongnam 630-723 (Korea, Republic of); Kim, Hyun Joon; Kang, Sang Soo; Cho, Gyeong Jae; Choi, Wan Sung [Department of Anatomy and Neurobiology, Institute of Health Sciences, Medical Research Center for Neural Dysfunction, Biomedical Center (BK21), Gyeongsang National University School of Medicine, Jinju, Gyeongnam 660-751 (Korea, Republic of); Roh, Gu Seob, E-mail: anaroh@gnu.ac.kr [Department of Anatomy and Neurobiology, Institute of Health Sciences, Medical Research Center for Neural Dysfunction, Biomedical Center (BK21), Gyeongsang National University School of Medicine, Jinju, Gyeongnam 660-751 (Korea, Republic of)

2010-03-12

Kainic acid (KA) induces hippocampal cell death and astrocyte proliferation. There are reports that sphingosine kinase (SPHK)1 and sphingosine-1- phosphate (S1P) receptor 1 (S1P{sub 1}) signaling axis controls astrocyte proliferation. Here we examined the temporal changes of SPHK1/S1P{sub 1} in mouse hippocampus during KA-induced hippocampal cell death. Mice were killed at 2, 6, 24, or 48 h after KA (30 mg/kg) injection. There was an increase in Fluoro-Jade B-positive cells in the hippocampus of KA-treated mice with temporal changes of glial fibrillary acidic protein (GFAP) expression. The lowest level of SPHK1 protein expression was found 2 h after KA treatment. Six hours after KA treatment, the expression of SPHK1 and S1P{sub 1} proteins steadily increased in the hippocampus. In immunohistochemical analysis, SPHK1 and S1P{sub 1} are more immunoreactive in astrocytes within the hippocampus of KA-treated mice than in hippocampus of control mice. These results indicate that SPHK1/S1P{sub 1} signaling axis may play an important role in astrocytes proliferation during KA-induced excitotoxicity.

Altered expression of sphingosine kinase 1 and sphingosine-1-phosphate receptor 1 in mouse hippocampus after kainic acid treatment

International Nuclear Information System (INIS)

Lee, Dong Hoon; Jeon, Byeong Tak; Jeong, Eun Ae; Kim, Joon Soo; Cho, Yong Woon; Kim, Hyun Joon; Kang, Sang Soo; Cho, Gyeong Jae; Choi, Wan Sung; Roh, Gu Seob

2010-01-01

Kainic acid (KA) induces hippocampal cell death and astrocyte proliferation. There are reports that sphingosine kinase (SPHK)1 and sphingosine-1- phosphate (S1P) receptor 1 (S1P 1 ) signaling axis controls astrocyte proliferation. Here we examined the temporal changes of SPHK1/S1P 1 in mouse hippocampus during KA-induced hippocampal cell death. Mice were killed at 2, 6, 24, or 48 h after KA (30 mg/kg) injection. There was an increase in Fluoro-Jade B-positive cells in the hippocampus of KA-treated mice with temporal changes of glial fibrillary acidic protein (GFAP) expression. The lowest level of SPHK1 protein expression was found 2 h after KA treatment. Six hours after KA treatment, the expression of SPHK1 and S1P 1 proteins steadily increased in the hippocampus. In immunohistochemical analysis, SPHK1 and S1P 1 are more immunoreactive in astrocytes within the hippocampus of KA-treated mice than in hippocampus of control mice. These results indicate that SPHK1/S1P 1 signaling axis may play an important role in astrocytes proliferation during KA-induced excitotoxicity.

Acceleration of astrocytic differentiation in neural stem cells surviving X-irradiation.

Science.gov (United States)

Ozeki, Ayumi; Suzuki, Keiji; Suzuki, Masatoshi; Ozawa, Hiroki; Yamashita, Shunichi

2012-03-28

Neural stem cells (NSCs) are highly susceptible to DNA double-strand breaks; however, little is known about the effects of radiation in cells surviving radiation. Although the nestin-positive NSCs predominantly became glial fibrillary acidic protein (GFAP)-positive in differentiation-permissive medium, little or no cells were GFAP positive in proliferation-permissive medium. We found that more than half of the cells surviving X-rays became GFAP positive in proliferation-permissive medium. Moreover, localized irradiation stimulated differentiation of cells outside the irradiated area. These results indicate for the first time that ionizing radiation is able to stimulate astrocyte-specific differentiation of surviving NSCs, whose process is mediated both by the direct activation of nuclear factor-κB and by the indirect bystander effect induced by X-irradiation.

Cellular plasticity in the supraoptic and paraventricular nuclei after prolonged dehydration in the desert rodent Meriones shawi: Vasopressin and GFAP immunohistochemical study.

Science.gov (United States)

Gamrani, Halima; Elgot, Abdeljalil; El Hiba, Omar; Fèvre-Montange, Michelle

2011-02-23

Supraoptic (SON) and paraventricular (PVN) nuclei are part of the hypothalamic-neurohypophysial system, they constitute the main source for vasopressin and they represent also obvious examples of activity-dependent neuroglial plasticity. Certain physiological conditions such as dehydration are accompanied by a structural remodeling of the neurons, their synaptic inputs and their surrounding glia. In the present work, an adult Meriones shawi (a rodent adapted to desert life) is used as an animal model. Using GFAP and vasopressin expressions as indicators successively of astrocytes and neuronal activations, the effect of a prolonged episode of water deprivation on the SON and PVN, hypothalamus nuclei were examined. We studied the immunoreactivity of GFAP and vasopressin in various hydration states (total deprivation of drinking water for 1 and 2months compared to hydrated animals). Prolonged dehydration produces an important decrease of GFAP immunoreactivity in both SON and PVN after 1 and 2months of water restriction. This decrease is accompanied by increased vasopressin immunoreactivity following the same periods of water deprivation. These findings may explain a real communication between vasopressin neurons and their surrounding astrocytes, thus the retraction of astrocytes and their processes is accompanied by an enhancement of vasopressin neuron density and their projecting fibers in response to this osmotic stress situation. Furthermore, these data could open further investigations concerning the possible involvement of the communication between astrocytes and vasopressin neurons in both PVN and SON in the regulation of Meriones hydrous balance and resistance to dehydration. Copyright © 2010. Published by Elsevier B.V.

Site-specific incorporation of redox active amino acids into proteins

Science.gov (United States)

Alfonta, Lital [San Diego, CA; Schultz, Peter G [La Jolla, CA; Zhang, Zhiwen [San Diego, CA

2009-02-24

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

Site-specific incorporation of redox active amino acids into proteins

Energy Technology Data Exchange (ETDEWEB)

Alfonta, Lital; Schultz, Peter G.; Zhang, Zhiwen

2017-10-10

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

Crystal structure of axolotl (Ambystoma mexicanum) liver bile acid-binding protein bound to cholic and oleic acid.

Science.gov (United States)

Capaldi, Stefano; Guariento, Mara; Perduca, Massimiliano; Di Pietro, Santiago M; Santomé, José A; Monaco, Hugo L

2006-07-01

The family of the liver bile acid-binding proteins (L-BABPs), formerly called liver basic fatty acid-binding proteins (Lb-FABPs) shares fold and sequence similarity with the paralogous liver fatty acid-binding proteins (L-FABPs) but has a different stoichiometry and specificity of ligand binding. This article describes the first X-ray structure of a member of the L-BABP family, axolotl (Ambystoma mexicanum) L-BABP, bound to two different ligands: cholic and oleic acid. The protein binds one molecule of oleic acid in a position that is significantly different from that of either of the two molecules that bind to rat liver FABP. The stoichiometry of binding of cholate is of two ligands per protein molecule, as observed in chicken L-BABP. The cholate molecule that binds buried most deeply into the internal cavity overlaps well with the analogous bound to chicken L-BABP, whereas the second molecule, which interacts with the first only through hydrophobic contacts, is more external and exposed to the solvent. (c) 2006 Wiley-Liss, Inc.

A Soluble, Folded Protein without Charged Amino Acid Residues

DEFF Research Database (Denmark)

Højgaard, Casper; Kofoed, Christian; Espersen, Roall

2016-01-01

side chains can maintain solubility, stability, and function. As a model, we used a cellulose-binding domain from Cellulomonas fimi, which, among proteins of more than 100 amino acids, presently is the least charged in the Protein Data Bank, with a total of only four titratable residues. We find......Charges are considered an integral part of protein structure and function, enhancing solubility and providing specificity in molecular interactions. We wished to investigate whether charged amino acids are indeed required for protein biogenesis and whether a protein completely free of titratable...... that the protein shows a surprising resilience toward extremes of pH, demonstrating stability and function (cellulose binding) in the pH range from 2 to 11. To ask whether the four charged residues present were required for these properties of this protein, we altered them to nontitratable ones. Remarkably...

Electricity-free, sequential nucleic acid and protein isolation.

Science.gov (United States)

Pawlowski, David R; Karalus, Richard J

2012-05-15

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable (1). The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment (2). The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis. Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters (3). CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation(4). By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification

Site-specific labeling of proteins with NMR-active unnatural amino acids

International Nuclear Information System (INIS)

Jones, David H.; Cellitti, Susan E.; Hao Xueshi; Zhang Qiong; Jahnz, Michael; Summerer, Daniel; Schultz, Peter G.; Uno, Tetsuo; Geierstanger, Bernhard H.

2010-01-01

A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling of proteins for NMR studies. In this perspective, we discuss these opportunities including new photocaged unnatural amino acids, outline usage of metal chelating and spin-labeled unnatural amino acids and expand the approach to in-cell NMR experiments.

The primary structure of fatty-acid-binding protein from nurse shark liver. Structural and evolutionary relationship to the mammalian fatty-acid-binding protein family.

Science.gov (United States)

Medzihradszky, K F; Gibson, B W; Kaur, S; Yu, Z H; Medzihradszky, D; Burlingame, A L; Bass, N M

1992-02-01

The primary structure of a fatty-acid-binding protein (FABP) isolated from the liver of the nurse shark (Ginglymostoma cirratum) was determined by high-performance tandem mass spectrometry (employing multichannel array detection) and Edman degradation. Shark liver FABP consists of 132 amino acids with an acetylated N-terminal valine. The chemical molecular mass of the intact protein determined by electrospray ionization mass spectrometry (Mr = 15124 +/- 2.5) was in good agreement with that calculated from the amino acid sequence (Mr = 15121.3). The amino acid sequence of shark liver FABP displays significantly greater similarity to the FABP expressed in mammalian heart, peripheral nerve myelin and adipose tissue (61-53% sequence similarity) than to the FABP expressed in mammalian liver (22% similarity). Phylogenetic trees derived from the comparison of the shark liver FABP amino acid sequence with the members of the mammalian fatty-acid/retinoid-binding protein gene family indicate the initial divergence of an ancestral gene into two major subfamilies: one comprising the genes for mammalian liver FABP and gastrotropin, the other comprising the genes for mammalian cellular retinol-binding proteins I and II, cellular retinoic-acid-binding protein myelin P2 protein, adipocyte FABP, heart FABP and shark liver FABP, the latter having diverged from the ancestral gene that ultimately gave rise to the present day mammalian heart-FABP, adipocyte FABP and myelin P2 protein sequences. The sequence for intestinal FABP from the rat could be assigned to either subfamily, depending on the approach used for phylogenetic tree construction, but clearly diverged at a relatively early evolutionary time point. Indeed, sequences proximately ancestral or closely related to mammalian intestinal FABP, liver FABP, gastrotropin and the retinoid-binding group of proteins appear to have arisen prior to the divergence of shark liver FABP and should therefore also be present in elasmobranchs

Protein and amino acid metabolism in skeletal muscle

Energy Technology Data Exchange (ETDEWEB)

Wu, Guoyao.

1989-01-01

Isolated chick extensor digitorum communis (EDC) muscles and, in some experiments, rat skeletal muscles were used to study a number of aspects of protein and amino acid metabolism. (1) Chick EDC muscles synthesize and release large amounts of alanine and glutamine, which indirectly obtain their amino groups from branched-chain amino acids (BCAA). (2) Acetoacetate or DL-{beta}-hydroxybutyrate (4 mM) decrease (P < 0.01) alanine synthesis and BCAA transamination in EDC muscles from 24-h fasted chicks by decreasing (P < 0.01) intracellular concentrations of pyruvate due to inhibition of glycolysis. (3) Glutamine is extensively degraded in skeletal muscles from both chicks and rats, thus challenging the traditional view that glutamine oxidation is negligible in skeletal muscle. The cytosolic glutamine aminotransferases L and K in the rat and the mitochondrial phosphate-activated glutaminase in the chick play important roles in the conversion of glutamine to {alpha}-ketoglutarate for further oxidation. (4) Although methionine has been reported to be extensively transaminated in rat skeletal muscle preparations in the absence of other amino acids, transamination of methionine is absent or negligible in chick and rat skeletal muscles in the presence of physiological concentrations of amino acids. (5) Glutamine at 1.0-15 mM increases (P < 0.01) protein synthesis ({sup 3}H-phenylalanine incorporation), and at 10.0-15.0 mM decreases (P < 0.05) protein degradation ({sup 3}H-phenylalanine release from prelabelled protein in vivo) in EDC muscles from fed chicks as compared to muscles incubated in the absence of glutamine. (6) Acetoacetate or DL-{beta}-hydroxybutyrate (4 mM) has a small but significant inhibitory effect (P < 0.05) on the rate of protein synthesis, but has no effect (P > 0.05) on the rate of protein degradation in EDC muscles from fed chicks.

Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running

Science.gov (United States)

Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine

2016-01-01

Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR) at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d3-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control) was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise. PMID:27367725

Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running

Directory of Open Access Journals (Sweden)

Hiroyuki Kato

2016-06-01

Full Text Available Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d3-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise.

Neuromuscular electrical stimulation prior to presleep protein feeding stimulates the use of protein-derived amino acids for overnight muscle protein synthesis.

Science.gov (United States)

Dirks, Marlou L; Groen, Bart B L; Franssen, Rinske; van Kranenburg, Janneau; van Loon, Luc J C

2017-01-01

Short periods of muscle disuse result in substantial skeletal muscle atrophy. Recently, we showed that both neuromuscular electrical stimulation (NMES) as well as presleep dietary protein ingestion represent effective strategies to stimulate muscle protein synthesis rates. In this study, we test our hypothesis that NMES can augment the use of presleep protein-derived amino acids for overnight muscle protein synthesis in older men. Twenty healthy, older [69 ± 1 (SE) yr] men were subjected to 24 h of bed rest, starting at 8:00 AM. In the evening, volunteers were subjected to 70-min 1-legged NMES, while the other leg served as nonstimulated control (CON). Immediately following NMES, 40 g of intrinsically l-[1- 13 C]-phenylalanine labeled protein was ingested prior to sleep. Blood samples were taken throughout the night, and muscle biopsies were obtained from both legs in the evening and the following morning (8 h after protein ingestion) to assess dietary protein-derived l-[1- 13 C]-phenylalanine enrichments in myofibrillar protein. Plasma phenylalanine concentrations and plasma l-[1- 13 C]-phenylalanine enrichments increased significantly following protein ingestion and remained elevated for up to 6 h after protein ingestion (P protein-bound l-[1- 13 C]-phenylalanine enrichments (MPE) increased to a greater extent in the stimulated compared with the control leg (0.0344 ± 0.0019 vs. 0.0297 ± 0.0016 MPE, respectively; P protein-derived amino acids in the NMES compared with CON leg. In conclusion, application of NMES prior to presleep protein feeding stimulates the use of dietary protein-derived amino acids for overnight muscle protein synthesis in older men. Neuromuscular electrical stimulation (NMES) as well as presleep dietary protein ingestion represent effective strategies to stimulate muscle protein synthesis rates. Here we demonstrate that in older men after a day of bed rest, the application of NMES prior to presleep protein feeding stimulates the use of

Zebrafish: an exciting model for investigating the spatio-temporal pattern of enteric nervous system development.

LENUS (Irish Health Repository)

Doodnath, Reshma

2012-02-01

AIM: Recently, the zebrafish (Danio rerio) has been shown to be an excellent model for human paediatric research. Advantages over other models include its small size, externally visually accessible development and ease of experimental manipulation. The enteric nervous system (ENS) consists of neurons and enteric glia. Glial cells permit cell bodies and processes of neurons to be arranged and maintained in a proper spatial arrangement, and are essential in the maintenance of basic physiological functions of neurons. Glial fibrillary acidic protein (GFAP) is expressed in astrocytes, but also expressed outside of the central nervous system. The aim of this study was to investigate the spatio-temporal pattern of GFAP expression in developing zebrafish ENS from 24 h post-fertilization (hpf), using transgenic fish that express green fluorescent protein (GFP). METHODS: Zebrafish embryos were collected from transgenic GFP Tg(GFAP:GFP)(mi2001) adult zebrafish from 24 to 120 hpf, fixed and processed for whole mount immunohistochemistry. Antibodies to Phox2b were used to identify enteric neurons. Specimens were mounted on slides and imaging was performed using a fluorescent laser confocal microscope. RESULTS: GFAP:GFP labelling outside the spinal cord was identified in embryos from 48 hpf. The patterning was intracellular and consisted of elongated profiles that appeared to migrate away from the spinal cord into the periphery. At 72 and 96 hpf, GFAP:GFP was expressed dorsally and ventrally to the intestinal tract. At 120 hpf, GFAP:GFP was expressed throughout the intestinal wall, and clusters of enteric neurons were identified using Phox2b immunofluorescence along the pathway of GFAP:GFP positive processes, indicative of a migratory pathway of ENS precursors from the spinal cord into the intestine. CONCLUSION: The pattern of migration of GFAP:GFP expressing cells outside the spinal cord suggests an organized, early developing migratory pathway to the ENS. This shows for the

Roseville Nursing Home, 49 Meath Road, Bray, Wicklow.

LENUS (Irish Health Repository)

Doodnath, Reshma

2012-02-01

AIM: Recently, the zebrafish (Danio rerio) has been shown to be an excellent model for human paediatric research. Advantages over other models include its small size, externally visually accessible development and ease of experimental manipulation. The enteric nervous system (ENS) consists of neurons and enteric glia. Glial cells permit cell bodies and processes of neurons to be arranged and maintained in a proper spatial arrangement, and are essential in the maintenance of basic physiological functions of neurons. Glial fibrillary acidic protein (GFAP) is expressed in astrocytes, but also expressed outside of the central nervous system. The aim of this study was to investigate the spatio-temporal pattern of GFAP expression in developing zebrafish ENS from 24 h post-fertilization (hpf), using transgenic fish that express green fluorescent protein (GFP). METHODS: Zebrafish embryos were collected from transgenic GFP Tg(GFAP:GFP)(mi2001) adult zebrafish from 24 to 120 hpf, fixed and processed for whole mount immunohistochemistry. Antibodies to Phox2b were used to identify enteric neurons. Specimens were mounted on slides and imaging was performed using a fluorescent laser confocal microscope. RESULTS: GFAP:GFP labelling outside the spinal cord was identified in embryos from 48 hpf. The patterning was intracellular and consisted of elongated profiles that appeared to migrate away from the spinal cord into the periphery. At 72 and 96 hpf, GFAP:GFP was expressed dorsally and ventrally to the intestinal tract. At 120 hpf, GFAP:GFP was expressed throughout the intestinal wall, and clusters of enteric neurons were identified using Phox2b immunofluorescence along the pathway of GFAP:GFP positive processes, indicative of a migratory pathway of ENS precursors from the spinal cord into the intestine. CONCLUSION: The pattern of migration of GFAP:GFP expressing cells outside the spinal cord suggests an organized, early developing migratory pathway to the ENS. This shows for the

Value of heart-type fatty acid-binding protein (H-FABP) for ...

African Journals Online (AJOL)

Key Words: heart-type fatty acid-binding protein, acute coronary syndrome, biomarker. ... is essential to prevent major complications and death. Routinely used biomarkers such ..... fatty acid binding proteins: their function and physiological sig-.

Degradation of protein translation machinery by amino acid starvation-induced macroautophagy

DEFF Research Database (Denmark)

Gretzmeier, Christine; Eiselein, Sven; Johnson, Gregory R.

2017-01-01

, unbiased approaches relying on quantitative mass spectrometry-based proteomics. Macroautophagy is induced by rapamycin treatment, and by amino acid and glucose starvation in differentially, metabolically labeled cells. Protein dynamics are linked to image-based models of autophagosome turnover. Depending...... on the inducing stimulus, protein as well as organelle turnover differ. Amino acid starvation-induced macroautophagy leads to selective degradation of proteins important for protein translation. Thus, protein dynamics reflect cellular conditions in the respective treatment indicating stimulus-specific pathways...

Predicting nucleic acid binding interfaces from structural models of proteins.

Science.gov (United States)

Dror, Iris; Shazman, Shula; Mukherjee, Srayanta; Zhang, Yang; Glaser, Fabian; Mandel-Gutfreund, Yael

2012-02-01

The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However, the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three-dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared with patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. Copyright © 2011 Wiley Periodicals, Inc.

Lipid recognition propensities of amino acids in membrane proteins from atomic resolution data

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Morita Mizuki

2011-12-01

Full Text Available Abstract Background Protein-lipid interactions play essential roles in the conformational stability and biological functions of membrane proteins. However, few of the previous computational studies have taken into account the atomic details of protein-lipid interactions explicitly. Results To gain an insight into the molecular mechanisms of the recognition of lipid molecules by membrane proteins, we investigated amino acid propensities in membrane proteins for interacting with the head and tail groups of lipid molecules. We observed a common pattern of lipid tail-amino acid interactions in two different data sources, crystal structures and molecular dynamics simulations. These interactions are largely explained by general lipophilicity, whereas the preferences for lipid head groups vary among individual proteins. We also found that membrane and water-soluble proteins utilize essentially an identical set of amino acids for interacting with lipid head and tail groups. Conclusions We showed that the lipophilicity of amino acid residues determines the amino acid preferences for lipid tail groups in both membrane and water-soluble proteins, suggesting that tightly-bound lipid molecules and lipids in the annular shell interact with membrane proteins in a similar manner. In contrast, interactions between lipid head groups and amino acids showed a more variable pattern, apparently constrained by each protein's specific molecular function.

Lipid recognition propensities of amino acids in membrane proteins from atomic resolution data

International Nuclear Information System (INIS)

Morita, Mizuki; Katta, AVSK Mohan; Ahmad, Shandar; Mori, Takaharu; Sugita, Yuji; Mizuguchi, Kenji

2011-01-01

Protein-lipid interactions play essential roles in the conformational stability and biological functions of membrane proteins. However, few of the previous computational studies have taken into account the atomic details of protein-lipid interactions explicitly. To gain an insight into the molecular mechanisms of the recognition of lipid molecules by membrane proteins, we investigated amino acid propensities in membrane proteins for interacting with the head and tail groups of lipid molecules. We observed a common pattern of lipid tail-amino acid interactions in two different data sources, crystal structures and molecular dynamics simulations. These interactions are largely explained by general lipophilicity, whereas the preferences for lipid head groups vary among individual proteins. We also found that membrane and water-soluble proteins utilize essentially an identical set of amino acids for interacting with lipid head and tail groups. We showed that the lipophilicity of amino acid residues determines the amino acid preferences for lipid tail groups in both membrane and water-soluble proteins, suggesting that tightly-bound lipid molecules and lipids in the annular shell interact with membrane proteins in a similar manner. In contrast, interactions between lipid head groups and amino acids showed a more variable pattern, apparently constrained by each protein's specific molecular function

Optimization of Glial Fibrillary Acidic Protein Immunoreactivity in Formalin-fixed, Paraffin-Embedded Guinea Pig Brain Sections

Science.gov (United States)

2003-09-01

fixed, paraffin-embedded guinea pig brain sections using a variety of commercially available GFAP antibody clones. Of the 7 clones tested for cross...determining neuropathological consequences in the guinea pig following exposure to chemical warfare nerve agent.

Optimizing scoring function of protein-nucleic acid interactions with both affinity and specificity.

Directory of Open Access Journals (Sweden)

Zhiqiang Yan

Full Text Available Protein-nucleic acid (protein-DNA and protein-RNA recognition is fundamental to the regulation of gene expression. Determination of the structures of the protein-nucleic acid recognition and insight into their interactions at molecular level are vital to understanding the regulation function. Recently, quantitative computational approach has been becoming an alternative of experimental technique for predicting the structures and interactions of biomolecular recognition. However, the progress of protein-nucleic acid structure prediction, especially protein-RNA, is far behind that of the protein-ligand and protein-protein structure predictions due to the lack of reliable and accurate scoring function for quantifying the protein-nucleic acid interactions. In this work, we developed an accurate scoring function (named as SPA-PN, SPecificity and Affinity of the Protein-Nucleic acid interactions for protein-nucleic acid interactions by incorporating both the specificity and affinity into the optimization strategy. Specificity and affinity are two requirements of highly efficient and specific biomolecular recognition. Previous quantitative descriptions of the biomolecular interactions considered the affinity, but often ignored the specificity owing to the challenge of specificity quantification. We applied our concept of intrinsic specificity to connect the conventional specificity, which circumvents the challenge of specificity quantification. In addition to the affinity optimization, we incorporated the quantified intrinsic specificity into the optimization strategy of SPA-PN. The testing results and comparisons with other scoring functions validated that SPA-PN performs well on both the prediction of binding affinity and identification of native conformation. In terms of its performance, SPA-PN can be widely used to predict the protein-nucleic acid structures and quantify their interactions.

The construction of an amino acid network for understanding protein structure and function.

Science.gov (United States)

Yan, Wenying; Zhou, Jianhong; Sun, Maomin; Chen, Jiajia; Hu, Guang; Shen, Bairong

2014-06-01

Amino acid networks (AANs) are undirected networks consisting of amino acid residues and their interactions in three-dimensional protein structures. The analysis of AANs provides novel insight into protein science, and several common amino acid network properties have revealed diverse classes of proteins. In this review, we first summarize methods for the construction and characterization of AANs. We then compare software tools for the construction and analysis of AANs. Finally, we review the application of AANs for understanding protein structure and function, including the identification of functional residues, the prediction of protein folding, analyzing protein stability and protein-protein interactions, and for understanding communication within and between proteins.

The relationship between amino acid and protein content of yellow ...

African Journals Online (AJOL)

feed industry are the relationships between isoleucine, leucine, lysine and arginine with crude protein content. Equations to predict the content of these amino acids from the amount of crude protein in maize are given. The remaining amino acids can be estimated without loss of accuracy from their mean value expressed as ...

Soy-dairy protein blend and whey protein ingestion after resistance exercise increases amino acid transport and transporter expression in human skeletal muscle

Science.gov (United States)

Reidy, P. T.; Walker, D. K.; Dickinson, J. M.; Gundermann, D. M.; Drummond, M. J.; Timmerman, K. L.; Cope, M. B.; Mukherjea, R.; Jennings, K.; Volpi, E.

2014-01-01

Increasing amino acid availability (via infusion or ingestion) at rest or postexercise enhances amino acid transport into human skeletal muscle. It is unknown whether alterations in amino acid availability, from ingesting different dietary proteins, can enhance amino acid transport rates and amino acid transporter (AAT) mRNA expression. We hypothesized that the prolonged hyperaminoacidemia from ingesting a blend of proteins with different digestion rates postexercise would enhance amino acid transport into muscle and AAT expression compared with the ingestion of a rapidly digested protein. In a double-blind, randomized clinical trial, we studied 16 young adults at rest and after acute resistance exercise coupled with postexercise (1 h) ingestion of either a (soy-dairy) protein blend or whey protein. Phenylalanine net balance and transport rate into skeletal muscle were measured using stable isotopic methods in combination with femoral arteriovenous blood sampling and muscle biopsies obtained at rest and 3 and 5 h postexercise. Phenylalanine transport into muscle and mRNA expression of select AATs [system L amino acid transporter 1/solute-linked carrier (SLC) 7A5, CD98/SLC3A2, system A amino acid transporter 2/SLC38A2, proton-assisted amino acid transporter 1/SLC36A1, cationic amino acid transporter 1/SLC7A1] increased to a similar extent in both groups (P protein blend resulted in a prolonged and positive net phenylalanine balance during postexercise recovery compared with whey protein (P protein synthesis increased similarly between groups. We conclude that, while both protein sources enhanced postexercise AAT expression, transport into muscle, and myofibrillar protein synthesis, postexercise ingestion of a protein blend results in a slightly prolonged net amino acid balance across the leg compared with whey protein. PMID:24699854

Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

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Gangi Setty, Thanuja [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Cho, Christine [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Govindappa, Sowmya [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Apicella, Michael A. [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Ramaswamy, S., E-mail: ramas@instem.res.in [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India)

2014-07-01

Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

International Nuclear Information System (INIS)

Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

2014-01-01

Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states

Phosphorylation of acidic ribosomal proteins from rabbit reticulocytes by a ribosome-associated casein kinase

DEFF Research Database (Denmark)

Issinger, O G

1977-01-01

Two acidic proteins from 80-S ribosomes were isolated and purified to homogeneity. The purified acidic proteins could be phosphorylated by casein kinase using [gamma-32P]ATP and [gamma-32P]GTP as a phosphoryl donor. The proteins became phosphorylated in situ, too. Sodium dodecyl sulfate polyacryl......Two acidic proteins from 80-S ribosomes were isolated and purified to homogeneity. The purified acidic proteins could be phosphorylated by casein kinase using [gamma-32P]ATP and [gamma-32P]GTP as a phosphoryl donor. The proteins became phosphorylated in situ, too. Sodium dodecyl sulfate...

Nanopore biosensors for detection of proteins and nucleic acids

NARCIS (Netherlands)

Maglia, Giovanni; Soskine, Mikhael

2014-01-01

Described herein are nanopore biosensors based on a modified cytolysin protein. The nanopore biosensors accommodate macromoiecules including proteins and nucleic acids, and may additionally comprise ligands with selective binding properties.

Relationship between Acute Phase Proteins and Serum Fatty Acid Composition in Morbidly Obese Patients

Science.gov (United States)

Fernandes, Ricardo; Beserra, Bruna Teles Soares; Cunha, Raphael Salles Granato; Hillesheim, Elaine; Camargo, Carolina de Quadros; Pequito, Danielle Cristina Tonello; de Castro, Isabela Coelho; Fernandes, Luiz Cláudio; Nunes, Everson Araújo; Trindade, Erasmo Benício Santos de Moraes

2013-01-01

Background. Obesity is considered a low-grade inflammatory state and has been associated with increased acute phase proteins as well as changes in serum fatty acids. Few studies have assessed associations between acute phase proteins and serum fatty acids in morbidly obese patients. Objective. To investigate the relationship between acute phase proteins (C-Reactive Protein, Orosomucoid, and Albumin) and serum fatty acids in morbidly obese patients. Methods. Twenty-two morbidly obese patients were enrolled in this study. Biochemical and clinical data were obtained before bariatric surgery, and fatty acids measured in preoperative serum. Results. Orosomucoid was negatively correlated with lauric acid (P = 0.027) and eicosapentaenoic acid (EPA) (P = 0.037) and positively with arachidonic acid (AA) (P = 0.035), AA/EPA ratio (P = 0.005), and n-6/n-3 polyunsaturated fatty acids ratio (P = 0.035). C-Reactive Protein (CRP) was negatively correlated with lauric acid (P = 0.048), and both CRP and CRP/Albumin ratio were negatively correlated with margaric acid (P = 0.010, P = 0.008, resp.). Albumin was positively correlated with EPA (P = 0.027) and margaric acid (P = 0.008). Other correlations were not statistically significant. Conclusion. Our findings suggest that serum fatty acids are linked to acute phase proteins in morbidly obese patients. PMID:24167354

The clinical significance of fatty acid binding proteins

Directory of Open Access Journals (Sweden)

Barbara Choromańska

2011-11-01

Full Text Available Excessive levels of free fatty acids are toxic to cells. The human body has evolved a defense mechanism in the form of small cytoplasmic proteins called fatty acid binding proteins (FABPs that bind long-chain fatty acids (LCFA, and then refer them to appropriate intracellular disposal sites (oxidation in mitochondria and peroxisomes or storage in the endoplasmic reticulum. So far, nine types of these proteins have been described, and their name refers to the place in which they were first identified or where they can be found in the greatest concentration. The most important FABPs were isolated from the liver (L-FABP, heart (H-FABP, intestine (I-FABP, brain (B-FABP, epidermis (E-FABP and adipocytes (A-FABP. Determination of H-FABP is used in the diagnosis of myocardial infarction, and L-FABP in kidney lesions of different etiologies. It is postulated that FABPs play an important role in the pathogenesis of metabolic diseases. Elevated levels of A-FABP have been found in the pericardial fat tissue and were associated with cardiac dysfunction in obese people. A rise in A-FABP has been observed in patients with type II diabetes. I-FABP is known as a marker of cell damage in the small intestine. Increased concentration of B-FABP has been associated with human brain tumors such as glioblastoma and astrocytoma, as well as with neurodegenerative diseases (Alzheimer’s, Parkinson’s and other disorders of cognitive function. The aim of this work was to present current data on the clinical significance of fatty acid binding proteins.

Role of Protein and Amino Acids in Infant and Young Child Nutrition: Protein and Amino Acid Needs and Relationship with Child Growth.

Science.gov (United States)

Uauy, Ricardo; Kurpad, Anura; Tano-Debrah, Kwaku; Otoo, Gloria E; Aaron, Grant A; Toride, Yasuhiko; Ghosh, Shibani

2015-01-01

Over a third of all deaths of children under the age of five are linked to undernutrition. At a 90% coverage level, a core group of ten interventions inclusive of infant and young child nutrition could save one million lives of children under 5 y of age (15% of all deaths) (Lancet 2013). The infant and young child nutrition package alone could save over 220,000 lives in children under 5 y of age. High quality proteins (e.g. milk) in complementary, supplementary and rehabilitation food products have been found to be effective for good growth. Individual amino acids such as lysine and arginine have been found to be factors linked to growth hormone release in young children via the somatotropic axis and high intakes are inversely associated with fat mass index in pre-pubertal lean girls. Protein intake in early life is positively associated with height and weight at 10 y of age. This paper will focus on examining the role of protein and amino acids in infant and young child nutrition by examining protein and amino acid needs in early life and the subsequent relationship with stunting.

Molecular cloning and expression of a novel keratinocyte protein (psoriasis-associated fatty acid-binding protein [PA-FABP]) that is highly up-regulated in psoriatic skin and that shares similarity to fatty acid-binding proteins

DEFF Research Database (Denmark)

Madsen, Peder; Rasmussen, H H; Leffers, H

1992-01-01

termed PA-FABP (psoriasis-associated fatty acid-binding protein). The deduced sequence predicted a protein with molecular weight of 15,164 daltons and a calculated pI of 6.96, values that are close to those recorded in the keratinocyte 2D gel protein database. The protein comigrated with PA-FABP...... as determined by 2D gel analysis of [35S]-methionine-labeled proteins expressed by transformed human amnion (AMA) cells transfected with clone 1592 using the vaccinia virus expression system and reacted with a rabbit polyclonal antibody raised against 2D gel purified PA-FABP. Structural analysis of the amino...... acid sequence revealed 48%, 52%, and 56% identity to known low-molecular-weight fatty acid-binding proteins belonging to the FABP family. Northern blot analysis showed that PA-FABP mRNA is indeed highly up-regulated in psoriatic keratinocytes. The transcript is present in human cell lines of epithelial...

Effects of Long-Term Protein Restriction on Meat Quality, Muscle Amino Acids, and Amino Acid Transporters in Pigs.

Science.gov (United States)

Yin, Jie; Li, Yuying; Zhu, Xiaotong; Han, Hui; Ren, Wenkai; Chen, Shuai; Bin, Peng; Liu, Gang; Huang, Xingguo; Fang, Rejun; Wang, Bin; Wang, Kai; Sun, Liping; Li, Tiejun; Yin, Yulong

2017-10-25

This study aimed to investigate the long-term effects of protein restriction from piglets to finishing pigs for 16 weeks on meat quality, muscle amino acids, and amino acid transporters. Thirty-nine piglets were randomly divided into three groups: a control (20-18-16% crude protein, CP) and two protein restricted groups (17-15-13% CP and 14-12-10% CP). The results showed that severe protein restriction (14-12-10% CP) inhibited feed intake and body weight, while moderate protein restriction (17-15-13% CP) had little effect on growth performance in pigs. Meat quality (i.e., pH, color traits, marbling, water-holding capacity, and shearing force) were tested, and the results exhibited that 14-12-10% CP treatment markedly improved muscle marbling score and increased yellowness (b*). pH value (45 min) was significantly higher in 17-15-13% CP group than that in other groups. In addition, protein restriction reduced muscle histone, arginine, valine, and isoleucine abundances and enhanced glycine and lysine concentrations compared with the control group, while the RT-PCR results showed that protein restriction downregulated amino acids transporters. Mechanistic target of rapamycin (mTOR) signaling pathway was inactivated in the moderate protein restricted group (17-15-13% CP), while severe protein restriction with dietary 14-12-10% CP markedly enhanced mTOR phosphorylation. In conclusion, long-term protein restriction affected meat quality and muscle amino acid metabolism in pigs, which might be associated with mTOR signaling pathway.

Experimental Evolution of a Green Fluorescent Protein Composed of 19 Unique Amino Acids without Tryptophan

Science.gov (United States)

Kawahara-Kobayashi, Akio; Hitotsuyanagi, Mitsuhiro; Amikura, Kazuaki; Kiga, Daisuke

2014-04-01

At some stage of evolution, genes of organisms may have encoded proteins that were synthesized using fewer than 20 unique amino acids. Similar to evolution of the natural 19-amino-acid proteins GroEL/ES, proteins composed of 19 unique amino acids would have been able to evolve by accumulating beneficial mutations within the 19-amino-acid repertoire encoded in an ancestral genetic code. Because Trp is thought to be the last amino acid included in the canonical 20-amino-acid repertoire, this late stage of protein evolution could be mimicked by experimental evolution of 19-amino-acid proteins without tryptophan (Trp). To further understand the evolution of proteins, we tried to mimic the evolution of a 19-amino-acid protein involving the accumulation of beneficial mutations using directed evolution by random mutagenesis on the whole targeted gene sequence. We created active 19-amino-acid green fluorescent proteins (GFPs) without Trp from a poorly fluorescent 19-amino-acid mutant, S1-W57F, by using directed evolution with two rounds of mutagenesis and selection. The N105I and S205T mutations showed beneficial effects on the S1-W57F mutant. When these two mutations were combined on S1-W57F, we observed an additive effect on the fluorescence intensity. In contrast, these mutations showed no clear improvement individually or in combination on GFPS1, which is the parental GFP mutant composed of 20 amino acids. Our results provide an additional example for the experimental evolution of 19-amino-acid proteins without Trp, and would help understand the mechanisms underlying the evolution of 19-amino-acid proteins. (236 words)

Age-related changes of structures in cerebellar cortex of cat

Indian Academy of Sciences (India)

We studied the structures of the cerebellar cortex of young adult and old cats for age-related changes, which were statistically analysed. Nissl staining was used to visualize the cortical neurons. The immunohistochemical method was used to display glial fibrillary acidic protein (GFAP)-immunoreactive (IR) astrocytes and ...

Dependence of intestinal amino acid uptake on dietary protein or amino acid levels

International Nuclear Information System (INIS)

Karasov, W.H.; Solberg, D.H.; Diamond, J.M.

1987-01-01

To understand how intestinal amino acid (AA) transport is regulated by dietary substrate levels, the authors measured uptake of seven radioactively-labelled AAs and glucose across the jejunal brush-border membrane of mice kept on one of three isocaloric rations differing in nitrogen content. In the high-protein ration, uptake increased by 77-81% for the nonessential, less toxic AAs, proline, and aspartate but only by 32-61% for the more toxic essential AAs tested. In the nitrogen-deficient ration, uptake decreased for the nonessential aspartate and proline but stayed constant or increased for essential AAs and for the nonessential alanine. These patterns imply independent regulation of the intestine's various AA transporters. With decreasing dietary AA (or protein), the imino acid and acidic AA private transporters are repressed, while activities of the basic AA transporter and the neutral AA public transporter decrease to an asymptote or else go through a minimum. These regulatory patterns can be understood as a compromise among conflicting constraints imposed by protein's multiple roles as a source of calories, nitrogen, and essential AAs and by the toxicity of essential AAs at high concentrations

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

Science.gov (United States)

Wang, Jiangyun; Schultz, Peter G.

2010-09-07

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lipidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

Fatty acid-binding protein in liver and small intestine of the preruminant calf

International Nuclear Information System (INIS)

Jenkins, K.J.

1986-01-01

Cytosol obtained from differential centrifugation of homogenates from liver and small intestine mucosa was incubated with 1-[ 14 C] oleic acid or 1-[ 14 C] palmitic acid and filtered through Sephadex G-75. Elution profiles for both tissues showed radioactivity in two main peaks, the first corresponding to binding of fatty acid to high molecular weight proteins and the second to a protein fraction with a molecular weight of approximately 12,000 daltons. The low molecular weight fraction had high fatty acid-binding activity, which was greater for oleic than palmitic acid. The findings demonstrate the presence of fatty acid-binding protein in liver and intestinal mucosa of the preruminant calf

Serial Sampling of Serum Protein Biomarkers for Monitoring Human Traumatic Brain Injury Dynamics: A Systematic Review.

Science.gov (United States)

Thelin, Eric Peter; Zeiler, Frederick Adam; Ercole, Ari; Mondello, Stefania; Büki, András; Bellander, Bo-Michael; Helmy, Adel; Menon, David K; Nelson, David W

2017-01-01

The proteins S100B, neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), and neurofilament light (NF-L) have been serially sampled in serum of patients suffering from traumatic brain injury (TBI) in order to assess injury severity and tissue fate. We review the current literature of serum level dynamics of these proteins following TBI and used the term "effective half-life" ( t 1/2 ) in order to describe the "fall" rate in serum. Through searches on EMBASE, Medline, and Scopus, we looked for articles where these proteins had been serially sampled in serum in human TBI. We excluded animal studies, studies with only one presented sample and studies without neuroradiological examinations. Following screening (10,389 papers), n  = 122 papers were included. The proteins S100B ( n  = 66) and NSE ( n  = 27) were the two most frequent biomarkers that were serially sampled. For S100B in severe TBI, a majority of studies indicate a t 1/2 of about 24 h, even if very early sampling in these patients reveals rapid decreases (1-2 h) though possibly of non-cerebral origin. In contrast, the t 1/2 for NSE is comparably longer, ranging from 48 to 72 h in severe TBI cases. The protein GFAP ( n  = 18) appears to have t 1/2 of about 24-48 h in severe TBI. The protein UCH-L1 ( n  = 9) presents a t 1/2 around 7 h in mild TBI and about 10 h in severe. Frequent sampling of these proteins revealed different trajectories with persisting high serum levels, or secondary peaks, in patients with unfavorable outcome or in patients developing secondary detrimental events. Finally, NF-L ( n  = 2) only increased in the few studies available, suggesting a serum availability of >10 days. To date, automated assays are available for S100B and NSE making them faster and more practical to use. Serial sampling of brain-specific proteins in serum reveals different temporal trajectories that should be

Serial Sampling of Serum Protein Biomarkers for Monitoring Human Traumatic Brain Injury Dynamics: A Systematic Review

Directory of Open Access Journals (Sweden)

Eric Peter Thelin

2017-07-01

Full Text Available BackgroundThe proteins S100B, neuron-specific enolase (NSE, glial fibrillary acidic protein (GFAP, ubiquitin carboxy-terminal hydrolase L1 (UCH-L1, and neurofilament light (NF-L have been serially sampled in serum of patients suffering from traumatic brain injury (TBI in order to assess injury severity and tissue fate. We review the current literature of serum level dynamics of these proteins following TBI and used the term “effective half-life” (t1/2 in order to describe the “fall” rate in serum.Materials and methodsThrough searches on EMBASE, Medline, and Scopus, we looked for articles where these proteins had been serially sampled in serum in human TBI. We excluded animal studies, studies with only one presented sample and studies without neuroradiological examinations.ResultsFollowing screening (10,389 papers, n = 122 papers were included. The proteins S100B (n = 66 and NSE (n = 27 were the two most frequent biomarkers that were serially sampled. For S100B in severe TBI, a majority of studies indicate a t1/2 of about 24 h, even if very early sampling in these patients reveals rapid decreases (1–2 h though possibly of non-cerebral origin. In contrast, the t1/2 for NSE is comparably longer, ranging from 48 to 72 h in severe TBI cases. The protein GFAP (n = 18 appears to have t1/2 of about 24–48 h in severe TBI. The protein UCH-L1 (n = 9 presents a t1/2 around 7 h in mild TBI and about 10 h in severe. Frequent sampling of these proteins revealed different trajectories with persisting high serum levels, or secondary peaks, in patients with unfavorable outcome or in patients developing secondary detrimental events. Finally, NF-L (n = 2 only increased in the few studies available, suggesting a serum availability of >10 days. To date, automated assays are available for S100B and NSE making them faster and more practical to use.ConclusionSerial sampling of brain-specific proteins in serum reveals

AFAL: a web service for profiling amino acids surrounding ligands in proteins

Science.gov (United States)

Arenas-Salinas, Mauricio; Ortega-Salazar, Samuel; Gonzales-Nilo, Fernando; Pohl, Ehmke; Holmes, David S.; Quatrini, Raquel

2014-11-01

With advancements in crystallographic technology and the increasing wealth of information populating structural databases, there is an increasing need for prediction tools based on spatial information that will support the characterization of proteins and protein-ligand interactions. Herein, a new web service is presented termed amino acid frequency around ligand (AFAL) for determining amino acids type and frequencies surrounding ligands within proteins deposited in the Protein Data Bank and for assessing the atoms and atom-ligand distances involved in each interaction (availability: http://structuralbio.utalca.cl/AFAL/index.html). AFAL allows the user to define a wide variety of filtering criteria (protein family, source organism, resolution, sequence redundancy and distance) in order to uncover trends and evolutionary differences in amino acid preferences that define interactions with particular ligands. Results obtained from AFAL provide valuable statistical information about amino acids that may be responsible for establishing particular ligand-protein interactions. The analysis will enable investigators to compare ligand-binding sites of different proteins and to uncover general as well as specific interaction patterns from existing data. Such patterns can be used subsequently to predict ligand binding in proteins that currently have no structural information and to refine the interpretation of existing protein models. The application of AFAL is illustrated by the analysis of proteins interacting with adenosine-5'-triphosphate.

Fatty Acid Binding Proteins in Prostate Cancer

National Research Council Canada - National Science Library

Jett, Marti

2000-01-01

We have shown that there is a distinct pattern of fatty acid binding protein (FAEP) expression in prostate cancer vs normal cells and that finding has be confirmed in patient samples of biopsy specimens...

Microwave-assisted acid and base hydrolysis of intact proteins containing disulfide bonds for protein sequence analysis by mass spectrometry.

Science.gov (United States)

Reiz, Bela; Li, Liang

2010-09-01

Controlled hydrolysis of proteins to generate peptide ladders combined with mass spectrometric analysis of the resultant peptides can be used for protein sequencing. In this paper, two methods of improving the microwave-assisted protein hydrolysis process are described to enable rapid sequencing of proteins containing disulfide bonds and increase sequence coverage, respectively. It was demonstrated that proteins containing disulfide bonds could be sequenced by MS analysis by first performing hydrolysis for less than 2 min, followed by 1 h of reduction to release the peptides originally linked by disulfide bonds. It was shown that a strong base could be used as a catalyst for microwave-assisted protein hydrolysis, producing complementary sequence information to that generated by microwave-assisted acid hydrolysis. However, using either acid or base hydrolysis, amide bond breakages in small regions of the polypeptide chains of the model proteins (e.g., cytochrome c and lysozyme) were not detected. Dynamic light scattering measurement of the proteins solubilized in an acid or base indicated that protein-protein interaction or aggregation was not the cause of the failure to hydrolyze certain amide bonds. It was speculated that there were some unknown local structures that might play a role in preventing an acid or base from reacting with the peptide bonds therein. 2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.

Stabilization of Proteins and Noncovalent Protein Complexes during Electrospray Ionization by Amino Acid Additives.

Science.gov (United States)

Zhang, Hua; Lu, Haiyan; Chingin, Konstantin; Chen, Huanwen

2015-07-21

Ionization of proteins and noncovalent protein complexes with minimal disturbance to their native structure presents a great challenge for biological mass spectrometry (MS). In living organisms, the native structure of intracellular proteins is commonly stabilized by solute amino acids (AAs) accumulated in cells at very high concentrations. Inspired by nature, we hypothesized that AAs could also pose a stabilizing effect on the native structure of proteins and noncovalent protein complexes during ionization. To test this hypothesis, here we explored MS response for various protein complexes upon the addition of free AAs at mM concentrations into the electrospray ionization (ESI) solution. Thermal activation of ESI droplets in the MS inlet capillary was employed as a model destabilizing factor during ionization. Our results indicate that certain AAs, in particular proline (Pro), pose considerable positive effect on the stability of noncovalent protein complexes in ESI-MS without affecting the signal intensity of protein ions and original protein-ligand equilibrium, even when added at the 20 mM concentration. The data suggest that the degree of protein stabilization is primarily determined by the osmolytic and ampholytic characteristics of AA solutes. The highest stability and visibility of noncovalent protein complexes in ESI-MS are achieved using AA additives with neutral isoelectric point, moderate proton affinity, and unfavorable interaction with the native protein state. Overall, our results indicate that the simple addition of free amino acids into the working solution can notably improve the stability and accuracy of protein analysis by native ESI-MS.

Mental retardation-related protease, motopsin (prss12), binds to the BRICHOS domain of the integral membrane protein 2a.

Science.gov (United States)

Mitsui, Shinichi; Osako, Yoji; Yuri, Kazunari

2014-01-01

Motopsin (prss12), a mosaic serine protease secreted by neuronal cells, is believed to be important for cognitive function, as the loss of its function causes severe nonsyndromic mental retardation. To understand the molecular role of motopsin, we identified the integral membrane protein 2a (Itm2a) as a motopsin-interacting protein using a yeast two-hybrid system. A pull-down assay showed that the BRICHOS domain of Itm2a was essential for this interaction. Motopsin and Itm2a co-localized in COS cells and in cultured neurons when transiently expressed in these cells. Both proteins were co-immunoprecipitated from lysates of these transfected COS cells. Itm2a was strongly detected in a brain lysate prepared between postnatal day 0 and 10, during which period motopsin protein was also enriched in the brain. Immunohistochemistry detected Itm2a as patchy spots along endothelial cells of brain capillaries (which also expressed myosin II regulatory light chain [RLC]), and on glial fibrillary acidic protein (GFAP)-positive processes in the developing cerebral cortex. The data raise the possibility that secreted motopsin interacts with endothelial cells in the developing brain. © 2013 International Federation for Cell Biology.

Expression of a fatty acid-binding protein in yeast

International Nuclear Information System (INIS)

Scholz, H.

1991-06-01

The unicellular eukaryotic microorganism, Saccharomyces cerevisiae, transformed with a plasmid containing a cDNA fragment encoding bovine heart fatty acid-binding protein (H-FABP C ) under the control of the inducible yeast GAL10 promoter, expressed FABP during growth on galactose. The maximum level of immunoreactive FABP, identical in size and isoelectric point to native protein, was reached after approximately 16 hours of induction. In contrast, transcription of the gene was induced within half an hour. Both, protein and mRNA were unstable and degraded within 1 h after repression of transcription. Analysis of subcellular fractions showed that FABP was exclusively associated with the cytosol. FABP expressed in yeast cells was functional as was demonstrated by its capacity to bind long chain fatty acids in an in vitro assay. Growth of all transformants on galactose as the carbon source showed no phenotype at temperatures up to 37 deg C, but the growth of FABP-expressing cells at 37 deg C was significantly retarded. Among the biochemical effects of FABP expression on lipid metabolism is a marked reduction of chain elongation and desaturation of exogenously added 14 C-palmitic acid. This effect is most pronounced in triacylglycerols and phospholipids when cells grow at 30 deg C and 37 deg C, respectively. In an in vitro assay determining the desaturation of palmitoyl CoA by microsomal membranes cytosol with or without exo- or endogenous FABP showed the same stimulation of the reaction. The desaturation of exogenously added 14 C-stearic acid, the pattern of unlabelled fatty acids (saturated vs. unsaturated) and the distribution of exogenously added radioactive fatty acids (palmitic, stearic or oleic acid) among lipid classes was not significantly affected. Using high concentrations (1 mM) the uptake of fatty acids was first stimulated and then inhibited when FABP was expressed. (author)

Myristic acid, a rare fatty acid, is the lipid attached to the transforming protein of Rous sarcoma virus and its cellular homolog

International Nuclear Information System (INIS)

Buss, J.E.; Sefton, B.M.

1985-01-01

The lipid bound to p60/sub src/, the transforming protein of Rous sarcoma virus, has been identified by gas and thin-layer chromatography as the 14-carbon saturated fatty acid, myristic acid. The protein can be labeled biosynthetically with either [ 3 H]myristic acid or [ 3 H]palmitic acid. Incorporation of [ 3 H]myristic acid was noticeably greater than incorporation of [ 3 H]palmitic acid. All of the [ 3 H]myristic acid-derived label in p60/sub src/ was present as myristic acid. In contrast, none of the radioactivity derived from [ 3 H]palmitic acid was recovered as palmitic acid. Instead, all 3 H incorporated into p60/sub src/ from [ 3 H]palmitic acid arose by metabolism to myristic acid. The cellular tyrosine kinase, p60c-/sub src/ also contains myristic acid. By comparison of the extent of myristylation of p60v-/sub src/ with that of the Moloney murine leukemia virus structural protein precursor, Pr65gag, the authors estimate that greater than 80% of the molecules of p60v-/sub src/ contain one molecule of this fatty acid. Myristylation is a rare form of protein modification. p60v-/sub src/ contains 10 to 40% of the myristic acid bound to protein in cells transformed by Rous sarcoma virus and is easily identified in total cell lysates when [ 3 H]myristic acid-labeled proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the amount of [ 3 H]myristic acid-labeled p60/sub src/ in total cell lysates and in immunoprecipitates suggests that immunoprecipitation with rabbit anti-Rous sarcoma virus tumor sera detects ca. 25% of the p60/sub src/ present in cells

Relationship of Quantity of Citric Acid and Protein Content of Mycelia during Citric Acid Production by Three Strains of Aspergillus niger

International Nuclear Information System (INIS)

Abdullah-Al-Mahin; Alamgir Z. Chowdhury; Rehana Begum

2006-01-01

The amount of protein in the surface grown mycelia of three strains of Aspergillus niger (CA16,79/20 and 318) was found to decrease with the increase of citric acid production in sucrose based fermentation medium. Throughout the study period of 6 to 10 days of fermentation, highest amount of citric acid was produced by Aspergillus niger 318 although the amount of protein in mycelia was lowest for this strain. On the other hand, lowest amount of citric acid was produced by the strain CA 16 which in tern produced highest amount of mycelial protein. Aspergillus niger 79/20 produced both intermediate level of protein and citric acid. The Protein was estimated by three commonly used methods namely: Kjeldahl, Biuret and Lowry methods. Kjeldahl and Lowry method gave the highest and lowest results respectively for protein determination in all cases.(authors)

Glutamic Acid - Amino Acid, Neurotransmitter, and Drug - Is Responsible for Protein Synthesis Rhythm in Hepatocyte Populations in vitro and in vivo.

Science.gov (United States)

Brodsky, V Y; Malchenko, L A; Konchenko, D S; Zvezdina, N D; Dubovaya, T K

2016-08-01

Primary cultures of rat hepatocytes were studied in serum-free media. Ultradian protein synthesis rhythm was used as a marker of cell synchronization in the population. Addition of glutamic acid (0.2 mg/ml) to the medium of nonsynchronous sparse cultures resulted in detection of a common protein synthesis rhythm, hence in synchronization of the cells. The antagonist of glutamic acid metabotropic receptors MCPG (0.01 mg/ml) added together with glutamic acid abolished the synchronization effect; in sparse cultures, no rhythm was detected. Feeding rats with glutamic acid (30 mg with food) resulted in protein synthesis rhythm in sparse cultures obtained from the rats. After feeding without glutamic acid, linear kinetics of protein synthesis was revealed. Thus, glutamic acid, a component of blood as a non-neural transmitter, can synchronize the activity of hepatocytes and can form common rhythm of protein synthesis in vitro and in vivo. This effect is realized via receptors. Mechanisms of cell-cell communication are discussed on analyzing effects of non-neural functions of neurotransmitters. Glutamic acid is used clinically in humans. Hence, a previously unknown function of this drug is revealed.

Gut luminal endogenous protein: implications for the determination of ileal amino acid digestibility in humans.

Science.gov (United States)

Moughan, Paul J; Rutherfurd, Shane M

2012-08-01

The true ileal digestibility assay provides the most informative measure of digestibility to assess bioavailability of amino acids in foods for humans. To determine 'true' estimates of ileal amino acid digestibility, requires that endogenous amino acids present in digesta at the terminal ileum be quantified. The amounts of endogenous amino acids in ileal digesta can be determined after feeding an animal or human a protein-free diet (traditional approach) or by various methods after giving a protein-containing diet. When the protein-free method has been applied with adult human subjects an overall mean value (three separate studies) for endogenous ileal nitrogen flow of 800 mg N/d has been reported. This value is considerably lower than a comparable value obtained after feeding protein of 1852 mg N/d (mean of four separate studies), and thus endogenous ileal N and amino acids should be measured under conditions of protein alimentation. There is some confusion concerning the terminology used to define digestibility, with the term "true" digestibility having different adopted meanings. Here, true amino acid digestibility is defined as apparent amino acid digestibility corrected for the basal amino acid losses determined after giving either a protein-free or a protein-containing diet. Basal losses should be determined at a defined dry-matter and protein intake. The protein-free diet approach to determining endogenous amino acids is considered unphysiological and basal losses refer to ileal endogenous amino acid flows associated with digesta dry-matter flow, and not including "specific" effects of dietary factors such as non starch polysaccharides and anti nutritional factors. Arguments are advanced that the enzyme hydrolysed protein/ultra filtration method may be suitable for routine application with a cannulated pig model, to obtain physiologically-valid basal estimates of ileal endogenous amino acids to allow calculation of true ileal amino acid digestibility in the

Heat-stable proteins and abscisic acid action in barley aleurone cells

International Nuclear Information System (INIS)

Jacobsen, J.V.; Shaw, D.C.

1989-01-01

[ 35 S]Methionine labeling experiments showed that abscisic acid (ABA) induced the synthesis of at least 25 polypeptides in mature barley (Hordeum vulgare) aleurone cells. The polypeptides were not secreted. Whereas most of the proteins extracted from aleurone cells were coagulated by heating to 100 degree C for 10 minutes, most of the ABA-induced polypeptides remained in solution (heat-stable). ABA had little effect on the spectrum of polypeptides that were synthesized and secreted by aleurone cells, and most of these secreted polypeptides were also heat-stable. Coomassie blue staining of sodium dodecyl sulfate polyacrylamide gels indicated that ABA-induced polypeptides already occurred in high amounts in mature aleurone layers having accumulated during grain development. About 60% of the total protein extracted from mature aleurone was heat stable. Amino acid analyses of total preparations of heat-stable and heat-labile proteins showed that, compared to heat-labile proteins, heat-stable intracellular proteins were characterized by higher glutamic acid/glutamine (Glx) and glycine levels and lower levels of neutral amino acids. Secreted heat-stable proteins were rich in Glx and proline. The possibilities that the accumulation of the heat-stable polypeptides during grain development is controlled by ABA and that the function of these polypeptides is related to their abundance and extraordinary heat stability are considered

Influence of the Amino Acid Sequence on Protein-Mineral Interactions in Soil

Science.gov (United States)

Chacon, S. S.; Reardon, P. N.; Purvine, S.; Lipton, M. S.; Washton, N.; Kleber, M.

2017-12-01

The intimate associations between protein and mineral surfaces have profound impacts on nutrient cycling in soil. Proteins are an important source of organic C and N, and a subset of proteins, extracellular enzymes (EE), can catalyze the depolymerization of soil organic matter (SOM). Our goal was to determine how variation in the amino acid sequence could influence a protein's susceptibility to become chemically altered by mineral surfaces to infer the fate of adsorbed EE function in soil. We hypothesized that (1) addition of charged amino acids would enhance the adsorption onto oppositely charged mineral surfaces (2) addition of aromatic amino acids would increase adsorption onto zero charged surfaces (3) Increase adsorption of modified proteins would enhance their susceptibility to alterations by redox active minerals. To test these hypotheses, we generated three engineered proxies of a model protein Gb1 (IEP 4.0, 6.2 kDA) by inserting either negatively charged, positively charged or aromatic amino acids in the second loop. These modified proteins were allowed to interact with functionally different mineral surfaces (goethite, montmorillonite, kaolinite and birnessite) at pH 5 and 7. We used LC-MS/MS and solution-state Heteronuclear Single Quantum Coherence Spectroscopy NMR to observe modifications on engineered proteins as a consequence to mineral interactions. Preliminary results indicate that addition of any amino acids to a protein increase its susceptibility to fragmentation and oxidation by redox active mineral surfaces, and alter adsorption to the other mineral surfaces. This suggest that not all mineral surfaces in soil may act as sorbents for EEs and chemical modification of their structure should also be considered as an explanation for decrease in EE activity. Fragmentation of proteins by minerals can bypass the need to produce proteases, but microbial acquisition of other nutrients that require enzymes such as cellulases, ligninases or phosphatases

Genetic regulation by amino acids of specific membrane protein biosynthesis in isolated rat hepatocytes

International Nuclear Information System (INIS)

Chiles, T.C.; Handlogten, M.E.; Kilberg, M.S.

1986-01-01

Rat Hepatocytes in primary culture were incubated in amino acid-free (AAF) medium or amino acid-supplemented (AAS) medium for 2-6 hr. The effect of amino acid starvation on the synthesis of specific membrane proteins was monitored by including 3 H-leucine during the incubation. A crude plasma membrane fraction was prepared and then analyzed by 2-D gel electrophoresis followed by fluorography. Amino acid deprivation caused an induction of the synthesis of 5 of the 30 proteins studied. The ratio (AAF/-AAS) of cpm incorporated into the remaining 25 proteins was 0.8 +/- 0.2, whereas the ratio for the 5 proteins that showed amino acid-dependent synthesis ranged from 1.5 to 2.5. The presence of 4 μM actinomycin in the AAF medium completely blocked the starvation-induced synthesis of the 5 proteins tested, but did not alter significantly the ratio of cpm incorporated into the other 25 proteins. Binding studies involving ConA suggested a plasma membrane location for the 5 proteins. The molecular weight values of the starvation-induced proteins are 70, 66, 66, 67, and 45kD. Surface-labelling of intact cells and preparation of antibodies against the 5 proteins will be used to establish the subcellular location and to describe the amino acid-dependent synthesis of each in more detail

Heterologous protein display on the cell surface of lactic acid bacteria mediated by the s-layer protein

Directory of Open Access Journals (Sweden)

Han Lanlan

2011-10-01

Full Text Available Abstract Background Previous studies have revealed that the C-terminal region of the S-layer protein from Lactobacillus is responsible for the cell wall anchoring, which provide an approach for targeting heterologous proteins to the cell wall of lactic acid bacteria (LAB. In this study, we developed a new surface display system in lactic acid bacteria with the C-terminal region of S-layer protein SlpB of Lactobacillus crispatus K2-4-3 isolated from chicken intestine. Results Multiple sequence alignment revealed that the C-terminal region (LcsB of Lb. crispatus K2-4-3 SlpB had a high similarity with the cell wall binding domains SA and CbsA of Lactobacillus acidophilus and Lb. crispatus. To evaluate the potential application as an anchoring protein, the green fluorescent protein (GFP or beta-galactosidase (Gal was fused to the N-terminus of the LcsB region, and the fused proteins were successfully produced in Escherichia coli, respectively. After mixing them with the non-genetically modified lactic acid bacteria cells, the fused GFP-LcsB and Gal-LcsB were functionally associated with the cell surface of various lactic acid bacteria tested. In addition, the binding capacity could be improved by SDS pretreatment. Moreover, both of the fused proteins could simultaneously bind to the surface of a single cell. Furthermore, when the fused DNA fragment of gfp:lcsB was inserted into the Lactococcus lactis expression vector pSec:Leiss:Nuc, the GFP could not be secreted into the medium under the control of the nisA promoter. Western blot, in-gel fluorescence assay, immunofluorescence microscopy and SDS sensitivity analysis confirmed that the GFP was successfully expressed onto the cell surface of L. lactis with the aid of the LcsB anchor. Conclusion The LcsB region can be used as a functional scaffold to target the heterologous proteins to the cell surfaces of lactic acid bacteria in vitro and in vivo, and has also the potential for biotechnological

Glutamic Acid as Enhancer of Protein Synthesis Kinetics in Hepatocytes from Old Rats.

Science.gov (United States)

Brodsky, V Y; Malchenko, L A; Butorina, N N; Lazarev Konchenko, D S; Zvezdina, N D; Dubovaya, T K

2017-08-01

Dense cultures of hepatocytes from old rats (~2 years old, body weight 530-610 g) are different from similar cultures of hepatocytes from young rats by the low amplitude of protein synthesis rhythm. Addition of glutamic acid (0.2, 0.4, or 0.6 mg/ml) into the culture medium with hepatocytes of old rats resulted in increase in the oscillation amplitudes of the protein synthesis rhythm to the level of young rats. A similar action of glutamic acid on the protein synthesis kinetics was observed in vivo after feeding old rats with glutamic acid. Inhibition of metabotropic receptors of glutamic acid with α-methyl-4-carboxyphenylglycine (0.01 mg/ml) abolished the effect of glutamic acid. The amplitude of oscillation of the protein synthesis rhythm in a cell population characterizes synchronization of individual oscillations caused by direct cell-cell communications. Hence, glutamic acid, acting as a receptor-dependent transmitter, enhanced direct cell-cell communications of hepatocytes that were decreased with aging. As differentiated from other known membrane signaling factors (gangliosides, norepinephrine, serotonin, dopamine), glutamic acid can penetrate into the brain and thus influence the communications and protein synthesis kinetics that are disturbed with aging not only in hepatocytes, but also in neurons.

Protein haze formation in wines revisited. The stabilising effect of organic acids

OpenAIRE

Batista, L.; Monteiro, L.; Loureiro, V.; Teixeira, A.R.; Ferreira, R.B.

2010-01-01

The effect on the wine protein haze potential of five organic acids commonly encountered in wines (L(+)- tartaric, L( )-malic, citric, succinic and gluconic acids) was assessed. All five acids, tested at 20 mM, reduced dramatically the haze potential of proteins, either in wine or dissolved in water, throughout the range of pH values typical of wines (i.e., from 2.8 through 3.8). Subtle differences among the acid effects did not correlate with the number of their carboxyl groups, ...

Analysis of the protein-protein interactions between the human acidic ribosomal P-proteins: evaluation by the two hybrid system

DEFF Research Database (Denmark)

Tchórzewski, M; Boldyreff, B; Issinger, O

2000-01-01

The surface acidic ribosomal proteins (P-proteins), together with ribosomal core protein P0 form a multimeric lateral protuberance on the 60 S ribosomal subunit. This structure, also called stalk, is important for efficient translational activity of the ribosome. In order to shed more light...... forms the 60 S ribosomal stalk: P0-(P1/P2)(2). Additionally, mutual interactions among human and yeast P-proteins were analyzed. Heterodimer formation could be observed between human P2 and yeast P1 proteins....

Monosodium Luminol for Improving Brain Function in Gulf War Illness

Science.gov (United States)

2015-10-01

of BrdU+ cells (i.e. newly born cells), doublecortin (DCX, a marker of newly born neurons ), glial fibrillary acidic protein, (GFAP, a marker of...can reduce hydrogen peroxide and short chain organic, fatty acid , and phospholipid hydroperoxides. It is also believed to play a role in the...gene encoding thioredoxin reductase 1, which reduces thioredoxins as well as other substrates, and plays a role in selenium metabolism and protection

Membrane fractionation of herring marinade for separation and recovery of fats, proteins, amino acids, salt, acetic acid and water

DEFF Research Database (Denmark)

Fjerbæk Søtoft, Lene; Lizarazu, Juncal Martin; Razi Parjikolaei, Behnaz

2015-01-01

In the production of marinated herring, nearly one ton of acidic saline marinade is produced per 1.5 tons herring fillet. This spent marinade contains highly valuable compounds such as proteins and amino acids. Membranes are suited to recover these substances. In this work, six membrane stages...... containing sugars, amino acids and smaller peptides and a NF permeate containing salt and acetic acid ready for reuse. 42% of the spent marinade is recovered to substitute fresh water and chemicals. The Waste water amount is reduced 62.5%. Proteins are concentrated 30 times, while amino acids and smaller...

Development of a Small Molecule P2X7R Antagonist as a Treatment for Acute SCI

Science.gov (United States)

2012-10-01

for glial fibrillary acid protein (GFAP, SMI21, 1:000, overnight; Covance ) or III-tubulin/TUJ1 (1:1000, overnight), fol- lowed by isotype-specific...CONTRACTING ORGANIZATION : University of Rochester Rochester, NY 14611...5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBER 9

Uric acid demonstrates neuroprotective effect on Parkinson's disease mice through Nrf2-ARE signaling pathway.

Science.gov (United States)

Huang, Ting-Ting; Hao, Dong-Lin; Wu, Bo-Na; Mao, Lun-Lin; Zhang, Jin

2017-12-02

Uric acid has neuroprotective effect on Parkinson's disease (PD) by inhibiting oxidative damage and neuronal cell death. Our previous study has shown that uric acid protected dopaminergic cell line damage through inhibiting accumulation of NF-E2-related factor 2 (Nrf2). This study aimed to investigate its in vivo neuroprotective effect. PD was induced by MPTP intraperitoneally injection for 7 d in male C57BL/6 mice. Mice were treated with either uric acid (intraperitoneally injection 250 mg/kg) or saline for a total of 13 d. We showed that uric acid improved behavioral performances and cognition of PD mice, increased TH-positive dopaminergic neurons and decreased GFAP-positive astrocytes in substantia nigra (SN). Uric acid increased mRNA and protein expressions of Nrf2 and three Nrf2-responsive genes, including γ-glutamate-cysteine ligase catalytic subunit (γ-GCLC), heme oxygenase-1 (HO-1) and NQO1. Uric acid significantly increased superoxide dismutase (SOD), CAT, glutathione (GSH) levels and decreased malondialdehyde (MDA) level in SN regions of MPTP-treated mice. Uric acid inhibited the hippocampal expression of IL-1β and decreased serum and hippocampus levels of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α). In conclusion, uric acid demonstrates neuroprotective properties for dopaminergic neurons in PD mice through modulation of neuroinflammation and oxidative stress. Copyright © 2017. Published by Elsevier Inc.

Amino acid starvation has opposite effects on mitochondrial and cytosolic protein synthesis.

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Mark A Johnson

Full Text Available Amino acids are essential for cell growth and proliferation for they can serve as precursors of protein synthesis, be remodelled for nucleotide and fat biosynthesis, or be burnt as fuel. Mitochondria are energy producing organelles that additionally play a central role in amino acid homeostasis. One might expect mitochondrial metabolism to be geared towards the production and preservation of amino acids when cells are deprived of an exogenous supply. On the contrary, we find that human cells respond to amino acid starvation by upregulating the amino acid-consuming processes of respiration, protein synthesis, and amino acid catabolism in the mitochondria. The increased utilization of these nutrients in the organelle is not driven primarily by energy demand, as it occurs when glucose is plentiful. Instead it is proposed that the changes in the mitochondrial metabolism complement the repression of cytosolic protein synthesis to restrict cell growth and proliferation when amino acids are limiting. Therefore, stimulating mitochondrial function might offer a means of inhibiting nutrient-demanding anabolism that drives cellular proliferation.

Inhibition of reactive astrocytosis in established experimental autoimmune encephalomyelitis favors infiltration by myeloid cells over T cells and enhances severity of disease

DEFF Research Database (Denmark)

Toft-Hansen, Henrik; Füchtbauer, Laila; Owens, Trevor

2011-01-01

encephalomyelitis (EAE), an animal model of multiple sclerosis. We made use of transgenic mice, which express herpes simplex virus-derived thymidine kinase under control of a glial fibrillary acidic protein promotor (GFAP HSV-TK mice). Treatment of these mice with ganciclovir leads to inhibition of reactive......-associated molecules TNFα, MMP-12 and TIMP-1 was elevated in spinal cord of GFAP HSV-TK mice treated with ganciclovir. Relative expression of CD3ε was downregulated, and expression levels of IFNγ, IL-4, IL-10, IL-17, and Foxp3 were not significantly changed. mRNA expression of CCL2 was upregulated, and CXL10...

Altered hypothalamic protein expression in a rat model of Huntington's disease.

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Wei-na Cong

Full Text Available Huntington's disease (HD is a neurodegenerative disorder, which is characterized by progressive motor impairment and cognitive alterations. Changes in energy metabolism, neuroendocrine function, body weight, euglycemia, appetite function, and circadian rhythm can also occur. It is likely that the locus of these alterations is the hypothalamus. We used the HD transgenic (tg rat model bearing 51 CAG repeats, which exhibits similar HD symptomology as HD patients to investigate hypothalamic function. We conducted detailed hypothalamic proteome analyses and also measured circulating levels of various metabolic hormones and lipids in pre-symptomatic and symptomatic animals. Our results demonstrate that there are significant alterations in HD rat hypothalamic protein expression such as glial fibrillary acidic protein (GFAP, heat shock protein-70, the oxidative damage protein glutathione peroxidase (Gpx4, glycogen synthase1 (Gys1 and the lipid synthesis enzyme acylglycerol-3-phosphate O-acyltransferase 1 (Agpat1. In addition, there are significant alterations in various circulating metabolic hormones and lipids in pre-symptomatic animals including, insulin, leptin, triglycerides and HDL, before any motor or cognitive alterations are apparent. These early metabolic and lipid alterations are likely prodromal signs of hypothalamic dysfunction. Gaining a greater understanding of the hypothalamic and metabolic alterations that occur in HD, could lead to the development of novel therapeutics for early interventional treatment of HD.

Region-specific maturation of cerebral cortex in human fetal brain: diffusion tensor imaging and histology

International Nuclear Information System (INIS)

Trivedi, Richa; Gupta, Rakesh K.; Saksena, Sona; Husain, Nuzhat; Srivastava, Savita; Rathore, Ram K.S.; Sarma, Manoj K.; Malik, Gyanendra K.; Das, Vinita; Pradhan, Mandakini; Pandey, Chandra M.; Narayana, Ponnada A.

2009-01-01

In this study, diffusion tensor imaging (DTI) and glial fibrillary acidic protein (GFAP) immunohistochemical analysis in different cortical regions in fetal brains at different gestational age (GA) were performed. DTI was performed on 50 freshly aborted fetal brains with GA ranging from 12 to 42 weeks to compare age-related fractional anisotropy (FA) changes in different cerebral cortical regions that include frontal, parietal, occipital, and temporal lobes at the level of thalami. GFAP immunostaining was performed and the percentage of GFAP-positive areas was quantified. The cortical FA values in the frontal lobe peaked at around 26 weeks of GA, occipital and temporal lobes at around 20 weeks, and parietal lobe at around 23 weeks. A significant, but modest, positive correlation (r=0.31, p=0.02) was observed between cortical FA values and percentage area of GFAP expression in cortical region around the time period during which the migrational events are at its peak, i.e., GA ≤ 28 weeks for frontal cortical region and GA≤22 weeks for rest of the lobes. The DTI-derived FA quantification with its GFAP immunohistologic correlation in cortical regions of the various lobes of the cerebral hemispheres supports region-specific migrational and maturational events in human fetal brain. (orig.)

Region-specific maturation of cerebral cortex in human fetal brain: diffusion tensor imaging and histology

Energy Technology Data Exchange (ETDEWEB)

Trivedi, Richa; Gupta, Rakesh K.; Saksena, Sona [Sanjay Gandhi Post Graduate Institute of Medical Sciences, Department of Radiodiagnosis, Lucknow, UP (India); Husain, Nuzhat; Srivastava, Savita [CSM Medical University, Department of Pathology, Lucknow (India); Rathore, Ram K.S.; Sarma, Manoj K. [Indian Institute of Technology, Department of Mathematics and Statistics, Kanpur (India); Malik, Gyanendra K. [CSM Medical University, Department of Pediatrics, Lucknow (India); Das, Vinita [CSM Medical University, Department of Obstetrics and Gynecology, Lucknow (India); Pradhan, Mandakini [Sanjay Gandhi Postgraduate Institute of Medical Sciences, Department of Medical Genetics, Lucknow (India); Pandey, Chandra M. [Sanjay Gandhi Postgraduate Institute of Medical Sciences, Department of Biostatistics, Lucknow (India); Narayana, Ponnada A. [University of Texas Medical School at Houston, Department of Diagnostic and Interventional Imaging, Houston, TX (United States)

2009-09-15

In this study, diffusion tensor imaging (DTI) and glial fibrillary acidic protein (GFAP) immunohistochemical analysis in different cortical regions in fetal brains at different gestational age (GA) were performed. DTI was performed on 50 freshly aborted fetal brains with GA ranging from 12 to 42 weeks to compare age-related fractional anisotropy (FA) changes in different cerebral cortical regions that include frontal, parietal, occipital, and temporal lobes at the level of thalami. GFAP immunostaining was performed and the percentage of GFAP-positive areas was quantified. The cortical FA values in the frontal lobe peaked at around 26 weeks of GA, occipital and temporal lobes at around 20 weeks, and parietal lobe at around 23 weeks. A significant, but modest, positive correlation (r=0.31, p=0.02) was observed between cortical FA values and percentage area of GFAP expression in cortical region around the time period during which the migrational events are at its peak, i.e., GA {<=} 28 weeks for frontal cortical region and GA{<=}22 weeks for rest of the lobes. The DTI-derived FA quantification with its GFAP immunohistologic correlation in cortical regions of the various lobes of the cerebral hemispheres supports region-specific migrational and maturational events in human fetal brain. (orig.)

P-aminobenzoic acid and tritiated cyanoborohydride for the detection of pyruvoyl residues in proteins

International Nuclear Information System (INIS)

van Poelje, P.D.; Snell, E.E.

1987-01-01

A procedure for the detection of covalently bound pyruvic acid in purified proteins or in crude extracts is described. The dialyzed sample is first treated with sodium cyanoborohydride to reduce any Schiff bases present and then incubated with p-aminobenzoic acid and sodium [ 3 H]cyanoborohydride. Derivatized proteins are visualized by fluorography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel slices containing the labeled proteins are hydrolyzed, and, after removal of polyacrylic acid, the hydrolysate is subjected to ion-exchange high-performance liquid chromatography. The presence of pyruvic acid is established by the detection of a tritiated, 280-nm absorbing compound with a retention time corresponding to that of synthetic N-(p-carboxyphenyl)alanine. The procedure is capable of detecting protein-bound pyruvic acid in the picomolar range and is easily modified to screen for other covalently bound keto acids

Amyloid beta(1-40-induced astrogliosis and the effect of genistein treatment in rat: a three-dimensional confocal morphometric and proteomic study.

Directory of Open Access Journals (Sweden)

Maryam Bagheri

Full Text Available Astrocytes are highly involved in regulation and homeostasis of the extracellular environment in the healthy brain. In pathological conditions, these cells play a major role in the inflammatory response seen in CNS tissues, which is called reactive astrogliosis and includes hypertrophy and proliferation of astrocytes. Here, we performed 3D confocal microscopy to evaluate the morphological response of reactive astrocytes positive for glial fibrillary acidic protein (GFAP in rats, to the presence of Aβ(1-40 in the rat brain before and after treatment with genistein. In 50 astrocytes per animal, we measured the volume and surface area for the nucleus, cell body, the entire cell, the tissue covered by single astrocytes and quantified the number and length of branches, the density of the astrocytes and the intensity of GFAP immunoreactivity. Injecting Aβ(1-40 into the brain of rats caused astrogliosis indicated by increased values for all measured parameters. Mass spectrometric analysis of hippocampal tissue in Aβ(1-40-injected brain showed decreased amounts of tubulins, enolases and myelin basic protein, and increased amounts of dihydropyrimidinase-related protein 2. In Aβ(1-40-injected rats pretreated with genistein, GFAP intensity was decreased to the sham-operated group level, and Aβ(1-40-induced astrogliosis was significantly ameliorated.

Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running

OpenAIRE

Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine

2016-01-01

Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential a...

Values for digestible indispensable amino acid scores (DIAAS) for some dairy and plant proteins may better describe protein quality than values calculated using the concept for protein digestibility-corrected amino acid scores (PDCAAS).

Science.gov (United States)

Mathai, John K; Liu, Yanhong; Stein, Hans H

2017-02-01

An experiment was conducted to compare values for digestible indispensable amino acid scores (DIAAS) for four animal proteins and four plant proteins with values calculated as recommended for protein digestibility-corrected amino acid scores (PDCAAS), but determined in pigs instead of in rats. Values for standardised total tract digestibility (STTD) of crude protein (CP) and standardised ileal digestibility (SID) of amino acids (AA) were calculated for whey protein isolate (WPI), whey protein concentrate (WPC), milk protein concentrate (MPC), skimmed milk powder (SMP), pea protein concentrate (PPC), soya protein isolate (SPI), soya flour and whole-grain wheat. The PDCAAS-like values were calculated using the STTD of CP to estimate AA digestibility and values for DIAAS were calculated from values for SID of AA. Results indicated that values for SID of most indispensable AA in WPI, WPC and MPC were greater (P<0·05) than for SMP, PPC, SPI, soya flour and wheat. With the exception of arginine and tryptophan, the SID of all indispensable AA in SPI was greater (P<0·05) than in soya flour, and with the exception of threonine, the SID of all indispensable AA in wheat was less (P<0·05) than in all other ingredients. If the same scoring pattern for children between 6 and 36 months was used to calculate PDCAAS-like values and DIAAS, PDCAAS-like values were greater (P<0·05) than DIAAS values for SMP, PPC, SPI, soya flour and wheat indicating that PDCAAS-like values estimated in pigs may overestimate the quality of these proteins.

Representation of protein-sequence information by amino acid subalphabets

DEFF Research Database (Denmark)

Andersen, C.A.F.; Brunak, Søren

2004-01-01

-sequence information, using machine learning strategies, where the primary goal is the discovery of novel powerful representations for use in AI techniques. In the case of proteins and the 20 different amino acids they typically contain, it is also a secondary goal to discover how the current selection of amino acids...

A nine-country study of the protein content and amino acid composition of mature human milk

Directory of Open Access Journals (Sweden)

Ping Feng

2016-08-01

Full Text Available Background: Numerous studies have evaluated protein and amino acid levels in human milk. However, research in this area has been limited by small sample sizes and study populations with little ethnic or racial diversity. Objective: Evaluate the protein and amino acid composition of mature (≥30 days human milk samples collected from a large, multinational study using highly standardized methods for sample collection, storage, and analysis. Design: Using a single, centralized laboratory, human milk samples from 220 women (30–188 days postpartum from nine countries were analyzed for amino acid composition using Waters AccQ-Tag high-performance liquid chromatography and total nitrogen content using the LECO FP-528 nitrogen analyzer. Total protein was calculated as total nitrogen×6.25. True protein, which includes protein, free amino acids, and peptides, was calculated from the total amino acids. Results: Mean total protein from individual countries (standard deviation [SD] ranged from 1,133 (125.5 to 1,366 (341.4 mg/dL; the mean across all countries (SD was 1,192 (200.9 mg/dL. Total protein, true protein, and amino acid composition were not significantly different across countries except Chile, which had higher total and true protein. Amino acid profiles (percent of total amino acids did not differ across countries. Total and true protein concentrations and 16 of 18 amino acid concentrations declined with the stage of lactation. Conclusions: Total protein, true protein, and individual amino acid concentrations in human milk steadily decline from 30 to 151 days of lactation, and are significantly higher in the second month of lactation compared with the following 4 months. There is a high level of consistency in the protein content and amino acid composition of human milk across geographic locations. The size and diversity of the study population and highly standardized procedures for the collection, storage, and analysis of human milk support

Shifts in renin-angiotensin system components, angiogenesis, and oxidative stress-related protein expression in the lamina cribrosa region of streptozotocin-induced diabetic mice.

Science.gov (United States)

Qian, Xiaobing; Lin, Leilei; Zong, Yao; Yuan, Yongguang; Dong, Yanmin; Fu, Yue; Shao, Wanwen; Li, Yujie; Gao, Qianying

2018-03-01

This study aimed to analyse shifts in renin-angiotensin system (RAS) components, angiogenesis, and oxidative stress-related protein expression in the lamina cribrosa (LC) region in streptozotocin (STZ)-induced diabetic mice. Six months after diabetes induction, the retinal vessels of male C57BL/6 J mice were observed by colour photography, fundus fluorescein angiography (FFA), and immunofluorescent staining following incubation with CD31. Immunofluorescence for glial fibrillary acidic protein (GFAP), alpha-smooth muscle actin (α-SMA),and NG2 was also performed. Angiotensin-converting enzyme 1 (ACE1), angiotensin II type I receptor (AT1R), renin, hypoxia-inducible factor 1-alpha (HIF-1α), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR2), and haeme oxygenase 1 (HO-1) expression levels were confirmed by immunohistochemical and western blotting analyses. Compared with control mice, diabetic mice had significantly higher blood glucose concentrations (p diabetic mice; however, immunostaining of whole-mount retinas revealed an increased number of retinal vessels. Furthermore, histopathological staining showed significant reduction in the whole retinal thickness. GFAP expression was slightly higher, whereas fewer NG2 + pericytes were observed in diabetic mice than in control mice. ACE1, AT1R, renin, HIF-1α, VEGF, VEGFR2, and HO-1 expression were up-regulated in the LC of the STZ-induced diabetic mice. Collectively, ACE 1, AT1R, HIF-1α, VEGF, VEGFR2, and HO-1 activation in the LC region in diabetic mice may be involved in diabetes via the RAS and induction of angiogenesis and oxidative stress.

Distinct expression/function of potassium and chloride channels contributes to the diverse volume regulation in cortical astrocytes of GFAP/EGFP mice

Czech Academy of Sciences Publication Activity Database

Benešová, Jana; Rusňáková, Vendula; Honsa, Pavel; Pivoňková, Helena; Džamba, Dávid; Kubista, Mikael; Anděrová, Miroslava

2012-01-01

Roč. 7, č. 1 (2012), e29725 E-ISSN 1932-6203 R&D Projects: GA ČR GAP303/10/1338 Grant - others:GA ČR(CZ) GD309/08/H079 Institutional research plan: CEZ:AV0Z50390703; CEZ:AV0Z50520701 Institutional support: RVO:86652036 ; RVO:68378041 Keywords : cell volume regulation * GFAP/EGFP * ischemia Subject RIV: FH - Neurology; FH - Neurology (BTO-N) Impact factor: 3.730, year: 2012

Enamel proteins mitigate mechanical and structural degradations in mature human enamel during acid attack

Science.gov (United States)

Lubarsky, Gennady V.; Lemoine, Patrick; Meenan, Brian J.; Deb, Sanjukta; Mutreja, Isha; Carolan, Patrick; Petkov, Nikolay

2014-04-01

A hydrazine deproteination process was used to investigate the role of enamel proteins in the acid erosion of mature human dental enamel. Bright field high resolution transmission electron micrographs and x-ray diffraction analysis show no crystallographic changes after the hydrazine treatment with similar nanoscale hydroxyapatite crystallite size and orientation for sound and de-proteinated enamel. However, the presence of enamel proteins reduces the erosion depth, the loss of hardness and the loss of structural order in enamel, following exposure to citric acid. Nanoindentation creep is larger for sound enamel than for deproteinated enamel but it reduces in sound enamel after acid attack. These novel results are consistent with calcium ion-mediated visco-elasticty in enamel matrix proteins as described previously for nacre, bone and dental proteins. They are also in good agreement with a previous double layer force spectroscopy study by the authors which found that the proteins electrochemically buffer enamel against acid attack. Finally, this suggests that acid attack, and more specifically dental erosion, is influenced by ionic permeation through the enamel layer and that it is mitigated by the enamel protein matrix.

Enamel proteins mitigate mechanical and structural degradations in mature human enamel during acid attack

International Nuclear Information System (INIS)

Lubarsky, Gennady V; Lemoine, Patrick; Meenan, Brian J; Deb, Sanjukta; Mutreja, Isha; Carolan, Patrick; Petkov, Nikolay

2014-01-01

A hydrazine deproteination process was used to investigate the role of enamel proteins in the acid erosion of mature human dental enamel. Bright field high resolution transmission electron micrographs and x-ray diffraction analysis show no crystallographic changes after the hydrazine treatment with similar nanoscale hydroxyapatite crystallite size and orientation for sound and de-proteinated enamel. However, the presence of enamel proteins reduces the erosion depth, the loss of hardness and the loss of structural order in enamel, following exposure to citric acid. Nanoindentation creep is larger for sound enamel than for deproteinated enamel but it reduces in sound enamel after acid attack. These novel results are consistent with calcium ion-mediated visco-elasticty in enamel matrix proteins as described previously for nacre, bone and dental proteins. They are also in good agreement with a previous double layer force spectroscopy study by the authors which found that the proteins electrochemically buffer enamel against acid attack. Finally, this suggests that acid attack, and more specifically dental erosion, is influenced by ionic permeation through the enamel layer and that it is mitigated by the enamel protein matrix. (papers)

Factor VII and protein C are phosphatidic acid-binding proteins.

Science.gov (United States)

Tavoosi, Narjes; Smith, Stephanie A; Davis-Harrison, Rebecca L; Morrissey, James H

2013-08-20

Seven proteins in the human blood clotting cascade bind, via their GLA (γ-carboxyglutamate-rich) domains, to membranes containing exposed phosphatidylserine (PS), although with membrane binding affinities that vary by 3 orders of magnitude. Here we employed nanodiscs of defined phospholipid composition to quantify the phospholipid binding specificities of these seven clotting proteins. All bound preferentially to nanobilayers in which PS headgroups contained l-serine versus d-serine. Surprisingly, however, nanobilayers containing phosphatidic acid (PA) bound substantially more of two of these proteins, factor VIIa and activated protein C, than did equivalent bilayers containing PS. Consistent with this finding, liposomes containing PA supported higher proteolytic activity by factor VIIa and activated protein C toward their natural substrates (factors X and Va, respectively) than did PS-containing liposomes. Moreover, treating activated human platelets with phospholipase D enhanced the rates of factor X activation by factor VIIa in the presence of soluble tissue factor. We hypothesize that factor VII and protein C bind preferentially to the monoester phosphate of PA because of its accessibility and higher negative charge compared with the diester phosphates of most other phospholipids. We further found that phosphatidylinositol 4-phosphate, which contains a monoester phosphate attached to its myo-inositol headgroup, also supported enhanced enzymatic activity of factor VIIa and activated protein C. We conclude that factor VII and protein C bind preferentially to monoester phosphates, which may have implications for the function of these proteases in vivo.

Cytosolic fatty acid-binding proteins: subjects and tools in metabolic research

Energy Technology Data Exchange (ETDEWEB)

Binas, B. [Max Delbrueck Center for Molecular Medicine, Berlin-Buch (Germany)

1998-12-31

Fatty acid-binding proteins (FABPs) are major targets for specific binding of fatty acids in vivo. They constitute a widely expressed family of genetically related, small cytosolic proteins which very likely mediate intracellular transport of free long chain fatty acids. Genetic inhibition of FABP expression in vivo should therefore provide a useful tool to investigate and engineer fatty acid metabolism. (orig.) [Deutsch] Fettsaeurebindungsproteine (FABPs) sind wichtige Bindungsstellen fuer Fettsaeuren in vivo; sie bilden eine breit exprimierte Familie genetisch verwandter kleiner Zytosoleiweisse, die sehr wahrscheinlich den intrazellulaeren Transport unveresterter langkettiger Fettsaeuren vermitteln. Die genetische Hemmung der FABP-Expanssion in vivo bietet sich deshalb als Werkzeug zur Erforschung und gezielten Veraenderung des Fettsaeurestoffwechsels an. (orig.)

Experiment K-6-18. Study of muscarinic and gaba (benzodiazepine) receptors in the sensory-motor cortex, hippcampus and spinal code

Science.gov (United States)

Daunton, N.; Damelio, F.; Krasnov, I.

1990-01-01

Frontal lobe samples of rat brains flown aboard Cosmos 1887 were processed for the study of muscarinic (cholinergic) and GABA (benzodiazepine) receptors and for immunocytochemical localization of the neurotransmitter gamma-aminobutyric acid (GABA) and glial fibrillary acidic protein (GFAP). Although radioactive labeling of both muscarinic cholinergic and GABA (benzodiazepine) receptors proved to be successful with the techniques employed, distinct receptor localization of individual laminae of the frontal neocortex was not possible since the sampling of the area was different in the various groups of animals. In spite of efforts made for proper orientation and regional identification of laminae, it was found that a densitometric (quantitation of autoradiograms) analysis of the tissue did not contribute to the final interpretation of the effects of weightlessness on these receptors. As to the immunocytochemical studies the use of both markers, GFAP and GABA antiserum, confirmed the suitability of the techniques for use in frozen material. However, similar problems to those encountered in the receptor studies prevented an adequate interpretation of the effects of micro-G exposure on the localization and distribution of GABA and GFAP. This study did, however, confirm the feasibility of investigating neurotransmitters and their receptors in future space flight experiments.

Proteins and amino acids are fundamental to optimal nutrition support in critically ill patients

NARCIS (Netherlands)

Weijs, P.J.M.; Cynober, L.; DeLegge, M.; Kreymann, G.; Wernerman, J.; Wolfe, R.R.

2014-01-01

Proteins and amino acids are widely considered to be subcomponents in nutritional support. However, proteins and amino acids are fundamental to recovery and survival, not only for their ability to preserve active tissue (protein) mass but also for a variety of other functions. Understanding the

Correlation of secreted protein acidic and rich in cysteine with diabetic nephropathy

OpenAIRE

Li, Lei; Song, Hai-Yan; Liu, Kai; An, Meng-Meng

2015-01-01

To detect the serum concentrations of secreted protein acidic and rich in cysteine (SPARC) in patients with diabetic nephropathy and SPARC mRNA and protein expressions in renal tissue of db/db mice (C57BL/KsJ, diabetic nephropathy mice), thus preliminary exploration on the role of secreted protein acidic riches in cysteine in the development of diabetic nephropathy were carried out. Serum SPARC levels in normal subjects, patients with type 2 diabetes mellitus (without diabetic nephropathy), c...

Gross and true ileal digestible amino acid contents of several animal body proteins and their hydrolysates.

Science.gov (United States)

Cui, J; Chong, B; Rutherfurd, S M; Wilkinson, B; Singh, H; Moughan, P J

2013-07-01

Amino acid compositions of ovine muscle, ovine myofibrillar protein, ovine spleen, ovine liver, bovine blood plasma, bovine blood globulins and bovine serum albumin and the amino acid compositions and in vivo (laboratory rat) true ileal amino acid digestibilities of hydrolysates (sequential hydrolysis with Neutrase, Alcalase and Flavourzyme) of these protein sources were determined. True ileal amino acid digestibility differed (Pprotein hydrolysates. The ovine myofibrillar protein and liver hydrolysates were the most digestible, with a mean true ileal digestibility across all amino acids of 99%. The least digestible protein hydrolysate was bovine serum albumin with a comparable mean true ileal digestibility of 93%. When the digestible amino acid contents were expressed as proportions relative to lysine, considerable differences, across the diverse protein sources, were found in the pattern of predicted absorbed amino acids. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effect of a keto acid-amino acid supplement on the metabolism and renal elimination of branched-chain amino acids in patients with chronic renal insufficiency on a low protein diet.

Science.gov (United States)

Teplan, V; Schück, O; Horácková, M; Skibová, J; Holecek, M

2000-10-27

The aim of our study was to evaluate the effect of a low-protein diet supplemented with keto acids-amino acids on renal function and urinary excretion of branched-chain amino acids (BCAA) in patients with chronic renal insufficiency (CRI). In a prospective investigation 28 patients with CRI (16 male, 12 female, aged 28-66 yrs, CCr 18.6 +/- 10.2 ml/min) on a low-protein diet (0.6 g of protein /kg BW/day and energy intake 140 kJ/kg BW/day) for a period of one month were included. Subsequently, this low protein diet was supplemented with keto acids-amino acids at a dose of 0.1 g/kg BW/day orally for a period of 3 months. Examinations performed at baseline and at the end of the follow-up period revealed significant increase in the serum levels of BCAA leucine (p Keto acid-amino acid administration had no effect on renal function and on the clearance of inulin, para-aminohippuric acid. Endogenous creatinine and urea clearance remained unaltered. A significant correlation between fractional excretion of sodium and leucine (p diet the supplementation of keto acids-amino acids does not affect renal hemodynamics, but is associated--despite increases in plasma concentrations--with a reduction of renal amino acid and protein excretion suggesting induction of alterations in the tubular transport mechanisms.

One-Pot Procedure for Recovery of Gallic Acid from Wastewater and Encapsulation within Protein Particles.

Science.gov (United States)

Nourbakhsh, Himan; Madadlou, Ashkan; Emam-Djomeh, Zahra; Wang, Yi-Cheng; Gunasekaran, Sundaram; Mousavi, Mohammad E

2016-02-24

A whey protein isolate solution was heat-denatured and treated with the enzyme transglutaminase, which cross-linked ≈26% of the amino groups and increased the magnitude of the ζ-potential value. The protein solution was microemulsified, and then the resulting water-in-oil microemulsion was dispersed within a gallic acid-rich model wastewater. Gallic acid extraction by the outlined microemulsion liquid membrane (MLM) from the exterior aqueous phase (wastewater) and accumulation within the internal aqueous nanodroplets induced protein cold-set gelation and resulted in the formation of gallic acid-enveloping nanoparticles. Measurements with a strain-controlled rheometer indicated a progressive increase in the MLM viscosity during gallic acid recovery corresponding to particle formation. The mean hydrodynamic size of the nanoparticles made from the heat-denatured and preheated enzymatically cross-linked proteins was 137 and 122 nm, respectively. The enzymatic cross-linking of whey proteins led to a higher gallic acid recovery yield and increased the glass transition enthalpy and temperature. A similar impact on glass transition indices was observed by the gallic acid-induced nanoparticulation of proteins. Scanning electron microscopy showed the existence of numerous jammed/fused nanoparticles. It was suggested on the basis of the results of Fourier transform infrared spectroscopy that the in situ nanoparticulation of proteins shifted the C-N stretching and C-H bending peaks to higher wavenumbers. X-ray diffraction results proposed a decreased β-sheet content for proteins because of the acid-induced particulation. The nanoparticles made from the enzymatically cross-linked protein were more stable against the in vitro gastrointestinal digestion and retained almost 19% of the entrapped gallic acid after 300 min sequential gastric and intestinal digestions.

Additive effects of lupin protein and phytic acid on aortic calcification in ApoE deficient mice.

Science.gov (United States)

Schutkowski, Alexandra; Hirche, Frank; Geissler, Stefanie; Radtke, Juliane; Stangl, Gabriele I

2015-03-01

Lupin proteins have repeatedly been shown to exhibit lipid lowering properties and reduce aortic calcification in atherosclerosis models. Despite many efforts on its identification, the component which is responsible for the observed effects is still under debate. Phytic acid which is generally associated with lupin protein isolates has currently been described as bioactive plant compound. The objective of the study was to determine the role of associated phytic acid for the described lupin protein effects. A two-factorial study with ApoE knockout mice was conducted in which mice received lupin protein isolate or casein with or without phytase. Phytic acid was added to the casein diets to a final concentration identical to the lupin protein diets. Here we show that the serum concentrations of cholesterol, lathosterol and desmosterol were lower and the faecal bile acid excretion was higher in the groups fed lupin proteins than in the groups fed casein ( p  < 0.05). Mice that received the lupin protein diet containing phytic acid were characterized by a lower aortic calcification than mice of the other three groups ( p  < 0.05). In conclusion, our results show that the cholesterol lowering properties of lupin protein isolate were not caused by phytic acid. However, the hypocalcific action of lupin proteins appears to depend on the combination of lupin proteins and phytic acid.

Compositional changes of proteins and amino acids in germinating coffee seeds

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Milton Massao Shimizu

2000-01-01

Full Text Available Endosperm is the main reserve tissue in coffee seeds. Coffee (Coffea arabica L. seeds were germinated for six weeks and qualitative and quantitative changes in amino acids and proteins were investigated. The total content of free amino acids were reduced during germination, however, protein content remained constant. SDS-PAGE profiles showed that legumin-like proteins became less stained in the last weeks. Asparagine, glutamic acid, aspartic acid, alanine and lysine were the major free amino acids, although serine and glutamine were also significant. Except for tyrosine, which increased with germination, all other amino acids were reduced. Analysis of the amino acid composition of the total soluble protein showed glutamic acid/glutamine and glycine as the main amino acids. However, other amino acids such as leucine, aspartic acid/asparagine, alanine, lysine, serine were also found in reasonable amounts.Endosperma é o principal tecido de reserva em sementes de café. Sementes de café (Coffea arabica L. foram germinadas por seis semanas e as alterações qualitativas e quantitativas de aminoácidos e proteínas foram investigadas. O conteúdo total de aminoácidos livres reduziu durante a germinação, no entanto, o conteúdo de proteínas permaneceu constante. Perfis eletroforéticos de proteínas em SDS-PAGE mostraram que proteínas do tipo legumina foram menos coradas nas últimas semanas. Asparagina, ácido glutâmico, ácido aspártico, alanina e lisina foram os principais aminoácidos, apesar de que serina e glutamina também estavam presentes em quantidades significativas. Exceto tirosina, a qual aumentou durante a germinação, todos os outros aminoácidos tiveram redução em sua concentração. A análise aminoacídica da fração de proteína solúvel total mostrou que ácido glutâmico/glutamina e glicina eram os principais aminoácidos presentes. No entanto, outros aminoácidos, tais como leucina, ácido asp

Effect of whey protein and a free amino acid mixture simulating whey protein on measures of satiety in normal-weight women.

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Chungchunlam, Sylvia M S; Henare, Sharon J; Ganesh, Siva; Moughan, Paul J

2016-11-01

Dietary protein is considered more satiating than carbohydrate, and whey protein is more satiating than other protein sources. The purported satiating effect of whey protein may be due to direct effects of the unique mixture of proteins in whey, due to the effects of peptides released upon digestion and/or its amino acid composition. The objective of the present study was to compare the satiating effects of intact whey protein isolate (WPI) or a free amino acid mixture (AAM) simulating the amino acid composition of the WPI. A single-blind completely randomised block design included twenty, healthy, adult women (age 24·2 (sem 0·8) years) of normal weight (BMI 22·7 (sem 0·4) kg/m2). Following consumption of isoenergetic (approximately 1800 kJ) preload meals enriched (52 g amino acid equivalent) with WPI or AAM, consumption of an ad libitum test meal 120 min later and subjective feelings of appetite using visual analogue scales (VAS) were determined. There were no significant differences (P=0·24) in the ad libitum test meal intakes between the WPI (268·5 (sem 27·3) g) and the AAM (238·4 (sem 22·7) g) preload meals. Subjective VAS ratings of appetite did not differ significantly between the WPI and the AAM preload meals (P>0·05). Intact whey protein and a free AAM simulating the whey protein showed similar effects on satiety. This suggests that the satiating effect of whey protein may be related to its specific amino acid composition.

Comparison of Bile Acids and Acetaminophen Protein Adducts in Children and Adolescents with Acetaminophen Toxicity.

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Laura James

Full Text Available Metabolomics approaches have enabled the study of new mechanisms of liver injury in experimental models of drug toxicity. Disruption of bile acid homeostasis is a known mechanism of drug induced liver injury. The relationship of individual bile acids to indicators of oxidative drug metabolism (acetaminophen protein adducts and liver injury was examined in children with acetaminophen overdose, hospitalized children with low dose exposure to acetaminophen, and children with no recent exposure to acetaminophen. Nine bile acids were quantified through targeted metabolomic analysis in the serum samples of the three groups. Bile acids were compared to serum levels of acetaminophen protein adducts and alanine aminotransferase. Glycodeoxycholic acid, taurodeoxycholic acid, and glycochenodeoxycholic acid were significantly increased in children with acetaminophen overdose compared to healthy controls. Among patients with acetaminophen overdose, bile acids were higher in subjects with acetaminophen protein adduct values > 1.0 nmol/mL and modest correlations were noted for three bile acids and acetaminophen protein adducts as follows: taurodeoxycholic acid (R=0.604; p<0.001, glycodeoxycholic acid (R=0.581; p<0.001, and glycochenodeoxycholic acid (R=0.571; p<0.001. Variability in bile acids was greater among hospitalized children receiving low doses of acetaminophen than in healthy children with no recent acetaminophen exposure. Compared to bile acids, acetaminophen protein adducts more accurately discriminated among children with acetaminophen overdose, children with low dose exposure to acetaminophen, and healthy control subjects. In children with acetaminophen overdose, elevations of conjugated bile acids were associated with specific indicators of acetaminophen metabolism and non-specific indicators of liver injury.

Adaptive Evolution of Eel Fluorescent Proteins from Fatty Acid Binding Proteins Produces Bright Fluorescence in the Marine Environment.

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David F Gruber

Full Text Available We report the identification and characterization of two new members of a family of bilirubin-inducible fluorescent proteins (FPs from marine chlopsid eels and demonstrate a key region of the sequence that serves as an evolutionary switch from non-fluorescent to fluorescent fatty acid-binding proteins (FABPs. Using transcriptomic analysis of two species of brightly fluorescent Kaupichthys eels (Kaupichthys hyoproroides and Kaupichthys n. sp., two new FPs were identified, cloned and characterized (Chlopsid FP I and Chlopsid FP II. We then performed phylogenetic analysis on 210 FABPs, spanning 16 vertebrate orders, and including 163 vertebrate taxa. We show that the fluorescent FPs diverged as a protein family and are the sister group to brain FABPs. Our results indicate that the evolution of this family involved at least three gene duplication events. We show that fluorescent FABPs possess a unique, conserved tripeptide Gly-Pro-Pro sequence motif, which is not found in non-fluorescent fatty acid binding proteins. This motif arose from a duplication event of the FABP brain isoforms and was under strong purifying selection, leading to the classification of this new FP family. Residues adjacent to the motif are under strong positive selection, suggesting a further refinement of the eel protein's fluorescent properties. We present a phylogenetic reconstruction of this emerging FP family and describe additional fluorescent FABP members from groups of distantly related eels. The elucidation of this class of fish FPs with diverse properties provides new templates for the development of protein-based fluorescent tools. The evolutionary adaptation from fatty acid-binding proteins to fluorescent fatty acid-binding proteins raises intrigue as to the functional role of bright green fluorescence in this cryptic genus of reclusive eels that inhabit a blue, nearly monochromatic, marine environment.

Radiation chemistry of amino acids, peptides and proteins in relation to the radiation sterilization of high-protein foods

International Nuclear Information System (INIS)

Garrison, W.M.

1981-12-01

An important source of information on the question of whether or not toxic or other deleterious substances are formed in the radiation sterilization of foods is the chemical study of reaction products and reaction mechanisms in the radiolysis of individual food components. The present evaluation of the radiation chemistry of amino acids, peptides, and proteins outlines the various radiation-induced processes which lead to amino acid degradation and to the synthesis of amino acid derivatives of higher molecular weight. Among the latter are the α,α'-diamino dicarboxylic acids which are formed as major products in the radiolysis of peptides both in aqueous solution and in the solid state. The α,α'-diamino acids are of particular interest as irradiation products because they represent a class of compounds not normally encountered in plant and animal protein sources. Such compounds have, however, been isolated from certain types of bacteria and bacterial products. All of the available data strongly suggest that the α,α'-diamino acids are produced in significant yield in the radiation sterilization of high protein foods. The importance of initiating extensive chemical and biological studies of these and of other high molecular weight products in irradiated food is emphasized

Radiation chemistry of amino acids, peptides and proteins in relation to the radiation sterilization of high-protein foods

International Nuclear Information System (INIS)

Garrison, W.M.

1979-03-01

An important source of information on the question of whether or not toxic or other deleterious substances are formed in the radiation sterilization of foods is the chemical study of reaction products and reaction mechanisms in the radiolysis of individual food components. The present evaluation of the radiation chemistry of amino acids, peptides and proteins outlines the various radiation-induced processes which lead to amino acid degradation and to the synthesis of amino acid derivatives of higher molecular weight. Among the latter are the α,α'-diamino dicarboxylic acids which are formed as major products in the radiolysis of peptides both in aqueous solution and in the solid state. The α,α'-diamino acids are of particular interest as irradiation products because they represent a class of compounds not normally encountered in plant and animal protein sources. Such compounds have, however, been isolated from certain types of bacteria and pathogenic toxins. All of the available data strongly suggest that the α,α'-diamino acids are produced in significant yield in the radiation sterilization of high protein foods. The importance of initiating extensive chemical and biological studies of initiating extensive chemical and biological studies of these and of other high molecular weight products in irradiated food is emphasized

Radiation chemistry of amino acids, peptides and proteins in relation to the radiation sterilization of high-protein foods

Energy Technology Data Exchange (ETDEWEB)

Garrison, W. M.

1981-12-01

An important source of information on the question of whether or not toxic or other deleterious substances are formed in the radiation sterilization of foods is the chemical study of reaction products and reaction mechanisms in the radiolysis of individual food components. The present evaluation of the radiation chemistry of amino acids, peptides, and proteins outlines the various radiation-induced processes which lead to amino acid degradation and to the synthesis of amino acid derivatives of higher molecular weight. Among the latter are the ..cap alpha..,..cap alpha..'-diamino dicarboxylic acids which are formed as major products in the radiolysis of peptides both in aqueous solution and in the solid state. The ..cap alpha..,..cap alpha..'-diamino acids are of particular interest as irradiation products because they represent a class of compounds not normally encountered in plant and animal protein sources. Such compounds have, however, been isolated from certain types of bacteria and bacterial products. All of the available data strongly suggest that the ..cap alpha..,..cap alpha..'-diamino acids are produced in significant yield in the radiation sterilization of high protein foods. The importance of initiating extensive chemical and biological studies of these and of other high molecular weight products in irradiated food is emphasized.

Additive effects of lupin protein and phytic acid on aortic calcification in ApoE deficient mice

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Alexandra Schutkowski

2015-03-01

A two-factorial study with ApoE knockout mice was conducted in which mice received lupin protein isolate or casein with or without phytase. Phytic acid was added to the casein diets to a final concentration identical to the lupin protein diets. Here we show that the serum concentrations of cholesterol, lathosterol and desmosterol were lower and the faecal bile acid excretion was higher in the groups fed lupin proteins than in the groups fed casein (p < 0.05. Mice that received the lupin protein diet containing phytic acid were characterized by a lower aortic calcification than mice of the other three groups (p < 0.05. In conclusion, our results show that the cholesterol lowering properties of lupin protein isolate were not caused by phytic acid. However, the hypocalcific action of lupin proteins appears to depend on the combination of lupin proteins and phytic acid.

Laser-based optical activity detection of amino acids and proteins

Energy Technology Data Exchange (ETDEWEB)

Reitsma, B.H.

1987-01-01

The optical activity detector (OAD) for HPLC is a selective detector for optically active substances including amino acids and proteins. This study illustrates the use of the OAD in three related areas. Section I illustrates the separation of four free amino acids using cation-exchange chromatography. Detection by coupling the OAD to a refractive index detector (RI) for proline and threonine and the OAD to an ultraviolet absorbance detector (UV) for tyrosine and phenylalanine allows the calculation of enantiomeric (D/L) ratios of these amino acids without physical separation. Specific rotations of these four amino acids are also reported. Section II illustrates the separation of 16 dansyl-L-amino acids by RP-HPLC with detection by OA/UV. Section III illustrates the RP-HPLC separation of conformers of soybean trypsin inhibitor. Detection by OA/UV provides insights from the chromatogram unavailable for UV absorbance detection alone. In addition, identification of impurities is simplified with OA/UV. Specific rotations of the separated protein fractions show no significant change accompanying change in conformation.

Assessment of the neuroprotective effects of Lavandula angustifolia extract on the contusive model of spinal cord injury in Wistar rats

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Gholamreza eKaka

2016-02-01

Full Text Available IntroductionSpinal cord injury (SCI involves a primary trauma and secondary cellular processes that can lead to severe damage to the nervous system, resulting in long-term spinal deficits. At the cellular level, SCI causes astrogliosis, of which glial fibrillary acidic protein (GFAP is a major index. ObjectiveThe aim of this study was to investigate the neuroprotective effects of Lavandula angustifolia (Lav on the repair of spinal cord injuries in Wistar rats.Materials and MethodsForty-five female rats were randomly divided into six groups of seven rats each: the intact, sham, control (SCI, Lav 100, Lav 200, and Lav 400 groups. Every week after SCI onset, all animals were evaluated for behavior outcomes by the Basso, Beattie, and Bresnahan (BBB score. H&E staining was performed to examine the lesions post-injury. GFAP expression was assessed for astrogliosis. Somatosensory evoked potential (SEP testing was performed to detect the recovery of neural conduction.Results BBB scores were significantly increased and delayed responses on sensory tests were significantly decreased in the Lav 200 and Lav 400 groups compared to the control group. The greatest decrease of GFAP was evident in the Lav 200 and Lav 400 groups. EMG results showed significant improvement in the hindlimbs in the Lav 200 and Lav 400 groups compared to the control group. Cavity areas significantly decreased and the number of ventral motor neurons significantly increased in the Lav 200 and Lav 400 groups.ConclusionLav at doses of 200 mg/kg and 400 mg/kg can promote structural and functional recovery after SCI. The neuroprotective effects of L. angustifolia can lead to improvement in the contusive model of spinal cord injury in Wistar rats.Keywords Spinal cord injury (SCI; Lavandula angustifolia; neuroprotection; Basso, Beattie, and Bresnahan (BBB; glial fibrillary acidic protein (GFAP; somatosensory evoked potential (SEP

The fragile X mental retardation protein has nucleic acid chaperone properties.

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Gabus, Caroline; Mazroui, Rachid; Tremblay, Sandra; Khandjian, Edouard W; Darlix, Jean-Luc

2004-01-01

The fragile X syndrome is the most common cause of inherited mental retardation resulting from the absence of the fragile X mental retardation protein (FMRP). FMRP contains two K-homology (KH) domains and one RGG box that are landmarks characteristic of RNA-binding proteins. In agreement with this, FMRP associates with messenger ribonucleoparticles (mRNPs) within actively translating ribosomes, and is thought to regulate translation of target mRNAs, including its own transcript. To investigate whether FMRP might chaperone nucleic acid folding and hybridization, we analysed the annealing and strand exchange activities of DNA oligonucleotides and the enhancement of ribozyme-directed RNA substrate cleavage by FMRP and deleted variants relative to canonical nucleic acid chaperones, such as the cellular YB-1/p50 protein and the retroviral nucleocapsid protein HIV-1 NCp7. FMRP was found to possess all the properties of a potent nucleic acid chaperone, requiring the KH motifs and RGG box for optimal activity. These findings suggest that FMRP may regulate translation by acting on RNA-RNA interactions and thus on the structural status of mRNAs.

A biotin enrichment strategy identifies novel carbonylated amino acids in proteins from human plasma

DEFF Research Database (Denmark)

Havelund, Jesper F; Wojdyla, Katarzyna; Davies, Michael J

2017-01-01

Protein carbonylation is an irreversible protein oxidation correlated with oxidative stress, various diseases and ageing. Here we describe a peptide-centric approach for identification and characterisation of up to 14 different types of carbonylated amino acids in proteins. The modified residues...... in vitro metal ion-catalysed oxidation. Furthermore, we assigned 133 carbonylated sites in 36 proteins in native human plasma protein samples. The optimised workflow enabled detection of 10 hitherto undetected types of carbonylated amino acids in proteins: aldehyde and ketone modifications of leucine...

Consumer perception of astringency in clear acidic whey protein beverages.

Science.gov (United States)

Childs, Jessica L; Drake, MaryAnne

2010-01-01

Acidic whey protein beverages are a growing component of the functional food and beverage market. These beverages are also astringent, but astringency is an expected and desirable attribute of many beverages (red wine, tea, coffee) and may not necessarily be a negative attribute of acidic whey protein beverages. The goal of this study was to define the consumer perception of astringency in clear acidic whey protein beverages. Six focus groups (n=49) were held to gain understanding of consumer knowledge of astringency. Consumers were presented with beverages and asked to map them based on astringent mouthfeel and liking. Orthonasal thresholds for whey protein isolate (WPI) in water and flavored model beverages were determined using a 7-series ascending forced choice method. Mouthfeel/basic taste thresholds were determined for WPI in water. Acceptance tests on model beverages were conducted using consumers (n=120) with and without wearing nose clips. Consumers in focus groups were able to identify astringency in beverages. Astringency intensity was not directly related to dislike. The orthonasal threshold for WPI in water was lower (P astringent mouthfeel and that both flavor and astringency should be the focus of ongoing studies to improve the palatability of these products. © 2010 Institute of Food Technologists®

Utilization of alimentary protein and amino acids in satisfying the nitrogen requirements of monogastric mammals

International Nuclear Information System (INIS)

Pion, R.; Arnal, M.

1976-01-01

The nitrogenous matter in the food of monogastric animals consists mainly of proteins, which are rapidly hydrolized in the intestinal tract when they have left the gastric reservoir. The digestive tube has several roles: it provides for hydrolysis of the food proteins and for a supply of endogenous nitrogen; it enables a certain digestive function to be performed by the intestinal flora and permits the transport of amino acids into the blood, selecting those which are needed for protein synthesis. The digestion products appear mainly in the form of free amino acids in the portal blood. A large proportion of these amino acids is taken up by the liver, so that intense protein synthesis takes place in the latter, coupled with a decrease in catabolism leading to a rhythmic increase in the liver content of proteins and RNA. The labile proteins retained are mainly enzymes, which catabolize the amino acids, and the liver is the site of the catabolism of most of the excess amino acids except those with chain branching. Alimentary deficiencies do not markedly reduce protein synthesis in this organ, since the rate of re-utilization of the amino acids is increased and the liver thus plays a regulatory role. The utilization of amino acids in muscle also follows a certain rhythm, partly connected with feeding, and under hormonal control. The muscle is the seat of catabolism of a large part of the branched chain amino acids, and like the liver it contributes to the energy utilization of amino acids. The rate of utilization of certain essential amino acids can be measured by metabolic criteria, including determination of blood and muscle concentrations and excretion of 14 CO 2 labels in the exhaled air or of 35 S labels in urine. (author)

Acid stress response and protein induction in Campylobacter jejuni isolates with different acid tolerance

DEFF Research Database (Denmark)

Birk, Tina; Wik, Monica Takamiya; Lametsch, René

2012-01-01

with MALDI-TOF-TOF. The most acid-sensitive isolate was C. jejuni 327, followed by NCTC 11168 and isolate 305 as the most tolerant. Overall, induction of five proteins was observed within the pI range investigated: 19 kDa periplasmic protein (p19), thioredoxin-disulfide (TrxB), a hypothetical protein Cj0706......RT-PCR. In this transcriptomic analysis, only up-regulation of trxB and p19 was observed. CONCLUSIONS: A defined medium that supports the growth of a range of Campylobacter strains and suitable for proteomic analysis was developed. Mainly proteins normally involved in iron control and oxidative stress defence were induced...

PLASMA PROTEIN AND HEMOGLOBIN PRODUCTION : DELETION OF INDIVIDUAL AMINO ACIDS FROM GROWTH MIXTURE OF TEN ESSENTIAL AMINO ACIDS. SIGNIFICANT CHANGES IN URINARY NITROGEN.

Science.gov (United States)

Robscheit-Robbins, F S; Miller, L L; Whipple, G H

1947-02-28

Given healthy dogs fed abundant iron and protein-free or low protein diets with sustained anemia and hypoproteinemia, we can study the capacity of these animals to produce simultaneously new hemoglobin and plasma protein. Reserve stores of blood protein-building materials are measurably depleted and levels of 6 to 8 gm. per cent for hemoglobin and 4 to 5 gm. per cent for plasma protein can be maintained for weeks or months depending upon the intake of food proteins or amino acid mixtures. These dogs are very susceptible to infection and various poisons. Dogs tire of these diets and loss of appetite terminates many experiments. Under these conditions (double depletion) standard growth mixtures of essential amino acids are tested to show the response in blood protein output and urinary nitrogen balance. As a part of each tabulated experiment one of the essential amino acids is deleted from the complete growth mixture to compare such response with that of the whole mixture. Methionine, threonine, phenylalanine, and tryptophane when singly eliminated from the complete amino acid mixture do effect a sharp rise in urinary nitrogen. This loss of urinary nitrogen is corrected when the individual amino acid is replaced in the mixture. Histidine, lysine, and valine have a moderate influence upon urinary nitrogen balance toward nitrogen conservation. Leucine, isoleucine, and arginine have minimal or no effect upon urinary nitrogen balance when these individual amino acids are deleted from the complete growth mixture of amino acids during 3 to 4 week periods. Tryptophane and to a less extent phenylalanine and threonine when returned to the amino acid mixture are associated with a conspicuous preponderance of plasma protein output over the hemoglobin output (Table 4). Arginine, lysine, and histidine when returned to the amino acid mixture are associated with a large preponderance of hemoglobin output. Various amino acid mixtures under these conditions may give a positive

Sequential activation of microglia and astrocyte cytokine expression precedes increased Iba-1 or GFAP immunoreactivity following systemic immune challenge.

Science.gov (United States)

Norden, Diana M; Trojanowski, Paige J; Villanueva, Emmanuel; Navarro, Elisa; Godbout, Jonathan P

2016-02-01

Activation of the peripheral immune system elicits a coordinated response from the central nervous system. Key to this immune to brain communication is that glia, microglia, and astrocytes, interpret and propagate inflammatory signals in the brain that influence physiological and behavioral responses. One issue in glial biology is that morphological analysis alone is used to report on glial activation state. Therefore, our objective was to compare behavioral responses after in vivo immune (lipopolysaccharide, LPS) challenge to glial specific mRNA and morphological profiles. Here, LPS challenge induced an immediate but transient sickness response with decreased locomotion and social interaction. Corresponding with active sickness behavior (2-12 h), inflammatory cytokine mRNA expression was elevated in enriched microglia and astrocytes. Although proinflammatory cytokine expression in microglia peaked 2-4 h after LPS, astrocyte cytokine, and chemokine induction was delayed and peaked at 12 h. Morphological alterations in microglia (Iba-1(+)) and astrocytes (GFAP(+)), however, were undetected during this 2-12 h timeframe. Increased Iba-1 immunoreactivity and de-ramified microglia were evident 24 and 48 h after LPS but corresponded to the resolution phase of activation. Morphological alterations in astrocytes were undetected after LPS. Additionally, glial cytokine expression did not correlate with morphology after four repeated LPS injections. In fact, repeated LPS challenge was associated with immune and behavioral tolerance and a less inflammatory microglial profile compared with acute LPS challenge. Overall, induction of glial cytokine expression was sequential, aligned with active sickness behavior, and preceded increased Iba-1 or GFAP immunoreactivity after LPS challenge. © 2015 Wiley Periodicals, Inc.

Effect of dietary proteins on the incorporation of amino acids in plasma proteins of ruminants

International Nuclear Information System (INIS)

Mehra, Usha R.; Singh, U.B.; Kumar, S.

1979-01-01

Experiments were conducted on nine male calves (Hariana x Holstein) of about one and a half years of age and fed different amounts of crude protein. 14 C-DL-leucine was injected into the blood of the animals and specific radioactivity of plasma protein measured. There was linear correlation between nitrogen ingested, digested and retained by the animals and the specific radioactivity of total plasma proteins. The experiments suggest the possible use of the incorporation of amino acids into plasma proteins as an index of nutritional status of the animals. (auth.)

Curcumin decreases astrocytic reaction after gliotoxic injury in the rat brainstem

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Eduardo Bondan

Full Text Available ABSTRACT Recent studies have demonstrated that curcumin (Cur has antioxidant, anti-inflammatory and anti-fibrotic effects. Ethidium bromide (EB injections into the central nervous system (CNS are known to induce local oligodendroglial and astrocytic loss, resulting in primary demyelination and neuroinflammation. Peripheral astrogliosis is seen around the injury site with increased immunoreactivity to glial fibrillary acidic protein (GFAP. This investigation aimed to evaluate the effect of Cur administration on astrocytic response following gliotoxic injury. Wistar rats were injected with EB into the cisterna pontis and treated, or not, with Cur (100 mg/kg/day, intraperitoneal route during the experimental period. Brainstem sections were collected at 15, 21 and 31 days after EB injection and processed for GFAP immunohistochemical staining. Astrocytic reactivity was measured in a computerized system for image analysis. In Cur-treated rats, the GFAP-stained area around the lesion was significantly smaller in all periods after EB injection compared to untreated animals, showing that Cur reduces glial scar development following injury.

[Activity of glial cells in trigeminal nervous system in rats with experimental pulpitis].

Science.gov (United States)

Gu, Bin; Liu, Na; Liu, Hongchen

2014-04-29

To observe the activity change of astrocyte in related nucleus caused by acute pulpitis in rats. Rat acute pulpitis model was induced by lipopolysaccharides (LPS). And, according to processing time, a total of 30 rats were divided into 5 groups of control, 6, 12, 24 and 48 h. Immunohistochemistry and Western blot were employed to detect the dynamic expression of glial fibrillary acidic protein (GFAP) in spinal nucleus of trigeminal nerve (Vc). The relative gray value of ipsilateral Vc GFAP expression in experimental groups was 153 ± 11 at 12 h. And it significantly increased versus the control group (100 ± 4)(P pulpitis model, activated glial cells are probably involved in the processes of pulpitis and hyperalgesia.

Apicoplast lipoic acid protein ligase B is not essential for Plasmodium falciparum.

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Svenja Günther

2007-12-01

Full Text Available Lipoic acid (LA is an essential cofactor of alpha-keto acid dehydrogenase complexes (KADHs and the glycine cleavage system. In Plasmodium, LA is attached to the KADHs by organelle-specific lipoylation pathways. Biosynthesis of LA exclusively occurs in the apicoplast, comprising octanoyl-[acyl carrier protein]: protein N-octanoyltransferase (LipB and LA synthase. Salvage of LA is mitochondrial and scavenged LA is ligated to the KADHs by LA protein ligase 1 (LplA1. Both pathways are entirely independent, suggesting that both are likely to be essential for parasite survival. However, disruption of the LipB gene did not negatively affect parasite growth despite a drastic loss of LA (>90%. Surprisingly, the sole, apicoplast-located pyruvate dehydrogenase still showed lipoylation, suggesting that an alternative lipoylation pathway exists in this organelle. We provide evidence that this residual lipoylation is attributable to the dual targeted, functional lipoate protein ligase 2 (LplA2. Localisation studies show that LplA2 is present in both mitochondrion and apicoplast suggesting redundancy between the lipoic acid protein ligases in the erythrocytic stages of P. falciparum.

Isotopomer distributions in amino acids from a highly expressed protein as a proxy for those from total protein

Energy Technology Data Exchange (ETDEWEB)

Shaikh, Afshan; Shaikh, Afshan S.; Tang, Yinjie; Mukhopadhyay, Aindrila; Keasling, Jay D.

2008-06-27

{sup 13}C-based metabolic flux analysis provides valuable information about bacterial physiology. Though many biological processes rely on the synergistic functions of microbial communities, study of individual organisms in a mixed culture using existing flux analysis methods is difficult. Isotopomer-based flux analysis typically relies on hydrolyzed amino acids from a homogeneous biomass. Thus metabolic flux analysis of a given organism in a mixed culture requires its separation from the mixed culture. Swift and efficient cell separation is difficult and a major hurdle for isotopomer-based flux analysis of mixed cultures. Here we demonstrate the use of a single highly-expressed protein to analyze the isotopomer distribution of amino acids from one organism. Using the model organism E. coli expressing a plasmid-borne, his-tagged Green Fluorescent Protein (GFP), we show that induction of GFP does not affect E. coli growth kinetics or the isotopomer distribution in nine key metabolites. Further, the isotopomer labeling patterns of amino acids derived from purified GFP and total cell protein are indistinguishable, indicating that amino acids from a purified protein can be used to infer metabolic fluxes of targeted organisms in a mixed culture. This study provides the foundation to extend isotopomer-based flux analysis to study metabolism of individual strains in microbial communities.

[Evaluation of the adjusted amino acid score by digestibility for estimating the protein quality and protein available in food and diets].

Science.gov (United States)

Pak, N; Vera, G; Araya, H

1985-03-01

The purpose of the present study was to evaluate the amino acid score adjusted by digestibility to estimate protein quality and utilizable protein in foods and diets, considering net protein utilization (NPU) as a biological reference method. Ten foods of vegetable origin and ten of animal origin, as well as eight mixtures of foods of vegetable and animal origin were studied. When all the foods were considered, a positive (r = 0.83) and highly significant correlation (p less than 0.001) between NPU and the amino acid score adjusted by digestibility was found. When the foods were separated according to their origin, this correlation was positive only for the foods of vegetable origin (r = 0.93) and statistically significant (p less than 0.001). Also, only in those foods were similar values found between NPU and amino acid score adjusted by digestibility, as well as in utilizable protein estimated considering both methods. Caution is required to interpret protein quality and utilizable protein values of foods of animal origin and mixtures of foods of vegetable and animal origin when the amino acid score method adjusted by digestibility, or NPU, are utilized.

New insights into structure and function of the different types of fatty acid-binding protein

NARCIS (Netherlands)

Zimmerman, Augusta Wilhelmina

2002-01-01

Fatty acid binding proteins (FABPs) are small cytosolic proteins with virtually identical backbone structures that facilitate the solubility and intracellular transport of fatty acids. They may also modulate the effect of fatty acids on various metabolic enzymes and receptors and on cellular

Accelerated protein digestion and amino acid absorption after Roux-en-Y gastric bypass

DEFF Research Database (Denmark)

Bojsen-Møller, Anna Kirstine; Jacobsen, Siv H; Dirksen, Carsten

2015-01-01

BACKGROUND: Roux-en-Y gastric bypass (RYGB) involves exclusion of major parts of the stomach and changes in admixture of gastro-pancreatic enzymes, which could have a major impact on protein digestion and amino acid absorption. OBJECTIVE: We investigated the effect of RYGB on amino acid appearance......: RYGB accelerates caseinate digestion and amino acid absorption, resulting in faster and higher but more transient postprandial elevation of plasma amino acids. Changes are likely mediated by accelerated intestinal nutrient entry and clearly demonstrate that protein digestion is not impaired after RYGB...

Plant Proteins and Synthetic Amino Acids in the Nutrition of Non-Ruminants

Energy Technology Data Exchange (ETDEWEB)

Lewis, D. [Department of Applied Biochemistry and Nutrition, University Of Nottingham, Nottingham (United Kingdom)

1968-07-01

It is to be emphasized that in formulating diets for farm animals other than ruminants it is important to meet the requirements for individual essential amino acids and not merely to give regard to over-ail protein quality. The protein component serves to meet the needs for essential amino acids and also supplies material to synthesize those amino acids that are individually dispensable. In arranging for efficient formulation it is important to have available amino acid requirement standards to meet a particular production objective and data on the quantity of amino acids supplied by the various ingredients available. In considering the amino acid content of ingredients it is important to pay due regard to the problems of availability. Efforts to define amino acid requirements for the pig and chick have given somewhat variable results: it is possible to account for some of this variability. It is recognized that under certain circumstances non-amino nitrogen can be utilized by such species as the chick and the pig. The mechanisms involved are briefly considered. Some experimental work has shown that non-amino nitrogen can support growth, but it is difficult to establish a situation in which the non-essential amino acid levels are sufficiently low to take advantage of this fact. Extensive use of synthetic essential amino acids could change this situation. The case for the use of synthetic amino acids in the diets of farm animals is essentially an economic one. It is no longer necessary to demonstrate that free dietary amino acids can meet the needs of the animal. The only question is whether the needs of the animal are more effectively met by the addition of amino acids or more intact protein. The place of alternative protein sources to such attractive commodities as fish meal or soyabean meal must be considered in terms of amino acid supply. Whilst synthetic methionine and lysine are available there is a developing case for the use of such products as sunflower

Analysis of Protein Amino Acids in Tobacco Using Microwave Digestion of Plant Material

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Moldoveanu SC

2014-12-01

Full Text Available This paper describes a technique using microwave digestion and gas chromatography-mass spectrometry (GC-MS, which makes possible the analysis of protein amino acids in tobacco. The technique involves first the measurement of free amino acids, a hydrolysis using microwave digestion, and a measurement of total resulting amino acids. The content of protein amino acids is determined from the difference of total and free amino acids. The digestion is performed with aqueous 6 N HCl (with 1% phenol for two hours in a microwave at 120°C in sealed vials. The GC-MS analysis is performed after the amino acids are derivatized with N-methyl-N-(t-butyldimethylsilyltrifluoroacetamide (MTBSTFA. The technique provides reliable results with less than 10% relative standard deviation (RSD for most amino acids. Only the determination of very low level amino acids is affected by larger errors. The method provides results for free amino acids that are in very good agreement with those obtained by high performance liquid chromatography (HPLC, and also results for protein levels in tobacco in agreement with data previously reported in the literature. Results are given for several single grade tobaccos and for tobacco blends from four Kentucky reference cigarettes.

Abscisic acid-regulated protein degradation causes osmotic stress-induced accumulation of branched-chain amino acids in Arabidopsis thaliana.

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Huang, Tengfang; Jander, Georg

2017-10-01

Whereas proline accumulates through de novo biosynthesis in plants subjected to osmotic stress, leucine, isoleucine, and valine accumulation in drought-stressed Arabidopsis thaliana is caused by abscisic acid-regulated protein degradation. In response to several kinds of abiotic stress, plants greatly increase their accumulation of free amino acids. Although stress-induced proline increases have been studied the most extensively, the fold-increase of other amino acids, in particular branched-chain amino acids (BCAAs; leucine, isoleucine, and valine), is often higher than that of proline. In Arabidopsis thaliana (Arabidopsis), BCAAs accumulate in response to drought, salt, mannitol, polyethylene glycol, herbicide treatment, and nitrogen starvation. Plants that are deficient in abscisic acid signaling accumulate lower amounts of BCAAs, but not proline and most other amino acids. Previous bioinformatic studies had suggested that amino acid synthesis, rather than protein degradation, is responsible for the observed BCAA increase in osmotically stressed Arabidopsis. However, whereas treatment with the protease inhibitor MG132 decreased drought-induced BCAA accumulation, inhibition of BCAA biosynthesis with the acetolactate synthase inhibitors chlorsulfuron and imazapyr did not. Additionally, overexpression of BRANCHED-CHAIN AMINO ACID TRANSFERASE2 (BCAT2), which is upregulated in response to osmotic stress and functions in BCAA degradation, decreased drought-induced BCAA accumulation. Together, these results demonstrate that BCAA accumulation in osmotically stressed Arabidopsis is primarily the result of protein degradation. After relief of the osmotic stress, BCAA homeostasis is restored over time by amino acid degradation involving BCAT2. Thus, drought-induced BCAA accumulation is different from that of proline, which is accumulated due to de novo synthesis in an abscisic acid-independent manner and remains elevated for a more prolonged period of time after removal of

Site specific incorporation of heavy atom-containing unnatural amino acids into proteins for structure determination

Science.gov (United States)

Xie, Jianming [San Diego, CA; Wang, Lei [San Diego, CA; Wu, Ning [Boston, MA; Schultz, Peter G [La Jolla, CA

2008-07-15

Translation systems and other compositions including orthogonal aminoacyl tRNA-synthetases that preferentially charge an orthogonal tRNA with an iodinated or brominated amino acid are provided. Nucleic acids encoding such synthetases are also described, as are methods and kits for producing proteins including heavy atom-containing amino acids, e.g., brominated or iodinated amino acids. Methods of determining the structure of a protein, e.g., a protein into which a heavy atom has been site-specifically incorporated through use of an orthogonal tRNA/aminoacyl tRNA-synthetase pair, are also described.

Astrócitos imunorreativos à proteína glial fibrilar ácida (GFAP em SNC de bovinos normais e de bovinos com raiva. I. Hipocampo e giro dentato

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Gisele Fabrino Machado

1997-12-01

Células nervosas da região estudada não apresentavam alterações importantes. Os resultados colaboram para o conhecimento da distribuição e da morfologia de astrócitos imunorreativos à GFAP, em bovinos normais e em bovinos com raiva.

Astrócitos imunorreativos à proteína glial fibrilar ácida (GFAP em sistema nervoso central de equinos normais e de equinos com leucoencefalomalácia

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Lemos Karen Regina

1999-01-01

Full Text Available A proteína glial fibrilar ácida (GFAP, subunidade dos filamentos intermediários do citoesqueleto celular, está presente no citoplasma de astrócitos. Técnicas imunohistoquímicas com anticorpos primários anti-GFAP são geralmente empregadas para identificar astrócitos no sistema nervoso, permitindo verificar também sua hipertrofia. Vários estudos mostram a distribuição, a morfologia e a citoarquitetura de astrócitos em várias regiões do SNC do homem e de animais de laboratório. No entanto, em animais domésticos e, especialmente em equinos, poucas informações estão disponíveis. No presente trabalho, verificou-se a densidade e a morfologia de astrócitos imunorreativos à GFAP na substância branca da córtex cerebral de equinos com leucoencefalomalácia (LEM comparando-se esses aspectos com o de equinos normais. Animais com LEM apresentaram hipertrofia de astrócitos em áreas próximas às lesões, representada pelo aumento do corpo celular, do núcleo e dos prolongamentos citoplasmáticos. O número de astrócitos apresentou-se reduzido e a imunorreatividade foi mais acentuada. Nos animais normais, verificou-se distribuição constante de astrócitos imunorreagentes com características de fibrosos. Alterações vasculares nos animais com LEM, como por exemplo degeneração de endotélio vascular, também foram observadas, podendo estar associadas às alterações astrocíticas.

A method for analysing small samples of floral pollen for free and protein-bound amino acids.

Science.gov (United States)

Stabler, Daniel; Power, Eileen F; Borland, Anne M; Barnes, Jeremy D; Wright, Geraldine A

2018-02-01

Pollen provides floral visitors with essential nutrients including proteins, lipids, vitamins and minerals. As an important nutrient resource for pollinators, including honeybees and bumblebees, pollen quality is of growing interest in assessing available nutrition to foraging bees. To date, quantifying the protein-bound amino acids in pollen has been difficult and methods rely on large amounts of pollen, typically more than 1 g. More usual is to estimate a crude protein value based on the nitrogen content of pollen, however, such methods provide no information on the distribution of essential and non-essential amino acids constituting the proteins.Here, we describe a method of microwave-assisted acid hydrolysis using low amounts of pollen that allows exploration of amino acid composition, quantified using ultra high performance liquid chromatography (UHPLC), and a back calculation to estimate the crude protein content of pollen.Reliable analysis of protein-bound and free amino acids as well as an estimation of crude protein concentration was obtained from pollen samples as low as 1 mg. Greater variation in both protein-bound and free amino acids was found in pollen sample sizes amino acids in smaller sample sizes, we suggest a correction factor to apply to specific sample sizes of pollen in order to estimate total crude protein content.The method described in this paper will allow researchers to explore the composition of amino acids in pollen and will aid research assessing the available nutrition to pollinating animals. This method will be particularly useful in assaying the pollen of wild plants, from which it is difficult to obtain large sample weights.

Acidic ribosomal proteins and histone H3 from Leishmania present a high rate of divergence

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Ysabel Montoya

2000-08-01

Full Text Available Another additional peculiarity in Leishmania will be discussed about of the amino acid divergence rate of three structural proteins: acidic ribosomal P1 and P2b proteins, and histone H3 by using multiple sequence alignment and dendrograms. These structural proteins present a high rate of divergence regarding to their homologous protein in Trypanosoma cruzi. At this regard, L. (V. peruviana P1 and T. cruzi P1 showed 57.4% of divergence rate. Likewise, L. (V. braziliensis histone H3 and acidic ribosomal P2 protein exhibited 31.8% and 41.7% respectively of rate of divergence in comparison with their homologous in T. cruzi.

White clover fractions as protein source for monogastrics: dry matter digestibility and protein digestibility-corrected amino acid scores.

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Stødkilde, Lene; Damborg, Vinni K; Jørgensen, Henry; Laerke, Helle N; Jensen, Søren K

2018-05-01

The present study aimed to evaluate the use of white clover as an alternative protein source for monogastrics. White clover plant and leaves were processed using a screw-press resulting in a solid pulp and a juice from which protein was acid-precipitated. The chemical composition of all fractions was determined and digestibility of dry matter (DM) and protein was assessed in an experiment with growing rats. Protein concentrates were produced with crude protein (CP) contents of 451 g kg -1 and 530 g kg -1 DM for white clover plant and leaves, respectively, and a pulp with CP contents of 313 and 374 g kg -1 DM from plant and leaves, respectively. The amino acid composition ranged from 4.72 to 6.49 g per 16 g of nitrogen (N) for lysine, 1.82-2.6 g per 16 g N for methionine and cysteine, and 3.66-5.24 g per 16 g N for threonine. True faecal digestibility of protein varied from 0.81 to 0.88, whereas DM digestibility was in the range 0.72-0.80. Methionine and cysteine were found to be limiting in all fractions, regardless of the reference group used. A high digestibility of white clover protein was found irrespective of the physical fractionation. Together with a well-balanced amino acid composition, this makes white clover a promising protein source for monogastrics. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Amino Acid Composition, Molecular Weight Distribution and Gel Electrophoresis of Walnut (Juglans regia L. Proteins and Protein Fractionations

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Xiaoying Mao

2014-01-01

Full Text Available As a by-product of oil production, walnut proteins are considered as an additional source of plant protein for human food. To make full use of the protein resource, a comprehensive understanding of composition and characteristics of walnut proteins are required. Walnut proteins have been fractionated and characterized in this study. Amino acid composition, molecular weight distribution and gel electrophoresis of walnut proteins and protein fractionations were analyzed. The proteins were sequentially separated into four fractions according to their solubility. Glutelin was the main component of the protein extract. The content of glutelin, albumin, globulin and prolamin was about 72.06%, 7.54%, 15.67% and 4.73% respectively. Glutelin, albumin and globulin have a balanced content of essential amino acids, except for methionine, with respect to the FAO pattern recommended for adults. SDS-PAGE patterns of albumin, globulin and glutelin showed several polypeptides with molecular weights 14.4 to 66.2 kDa. The pattern of walnut proteins in two-dimension electrophoresis (2-DE showed that the isoelectric point was mainly in the range of 4.8–6.8. The results of size exclusion chromatogram indicated molecular weight of the major components of walnut proteins were between 3.54 and 81.76 kDa.

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

Interactions of myelin basic protein with mixed dodecylphosphocholine/palmitoyllysophosphatidic acid micelles

International Nuclear Information System (INIS)

Mendz, G.L.; Brown, L.R.; Martenson, R.E.

1990-01-01

The interactions of myelin basic protein and peptides derived from it with detergent micelles of lysophosphatidylglycerol, lysophosphatidylserine, palmitoyllysophosphatidic acid, and sodium lauryl sulfate, and with mixed micelles of the neutral detergent dodecylphosphocholine and the negatively charged detergent palmitoyllysophosphatidic acid, were investigated by 1 H NMR spectroscopy and circular dichroic spectropolarimetry. The results with single detergents suggested that there are discrete interaction sites in the protein molecule for neutral and anionic detergent micelles and that at least some of these sites are different for each type of detergent. The data on the binding of the protein and peptides to mixed detergent micelles suggested that intramolecular interactions in the intact protein and in one of the longer peptides limited the formation of helices and also that a balance between hydrophobic and ionic forces is achieved in the interactions of the peptides with the detergents. At high detergent/protein molar ratios, hydrophobic interactions appeared to be favored

Using analyses of amino Acid coevolution to understand protein structure and function.

Science.gov (United States)

Ashenberg, Orr; Laub, Michael T

2013-01-01

Determining which residues of a protein contribute to a specific function is a difficult problem. Analyses of amino acid covariation within a protein family can serve as a useful guide by identifying residues that are functionally coupled. Covariation analyses have been successfully used on several different protein families to identify residues that work together to promote folding, enable protein-protein interactions, or contribute to an enzymatic activity. Covariation is a statistical signal that can be measured in a multiple sequence alignment of homologous proteins. As sequence databases have expanded dramatically, covariation analyses have become easier and more powerful. In this chapter, we describe how functional covariation arises during the evolution of proteins and how this signal can be distinguished from various background signals. We discuss the basic methodology for performing amino acid covariation analysis, using bacterial two-component signal transduction proteins as an example. We provide practical suggestions for each step of the process including assembly of protein sequences, construction of a multiple sequence alignment, measurement of covariation, and analysis of results. Copyright © 2013 Elsevier Inc. All rights reserved.

Synthesis, physicochemical and biological properties of poly-α-amino acids - the simplest of protein models

International Nuclear Information System (INIS)

Katchalski-Katzir, Ephraim

1996-01-01

During the 1950s, linear and multichain poly-α-amino acids were synthesized by polymerization of the corresponding N-carboxy-amino acid anhydrides in solution in the presence of suitable catalysts. The resulting homo- and heteropolymers have since been widely employed as simple protein models. Under appropriate conditions, poly-α-amino acids, in the solid state and in solution, were found to acquire conformations of an α-helix and β-parallel and antiparallel pleased sheets, or to exist as random coils. Their use in experimental and theoretical investigations of helix-coil transitions helped to shed new light on the mechanisms involved in protein denaturation. Poly-α-amino acids played an important role in the deciphering of the genetic code. In addition, analysis of the antigenicity of poly-α-amino acids led to the clucidation of the factors determining the antigenicity of proteins and peptides. Interest in the biological and physicochemical characteristics of poly-α-amino acids was recently renewed because of the reported novel finding that some copolymers of amino acids are effective as drugs in multiple sclerosis, and that glutamine repeats and reiteration of other amino acids occur in inherited neurodegenerative diseases. The presence of repeating sequences of amino acids in proteins, and of nucleotides in DNA, raises many interesting questions about their respective roles in determining protein structure and function, and gene performance and regulation. (author). 35 refs, 3 figs, 2 tabs

Value of eight-amino-acid matches in predicting the allergenicity status of proteins: an empirical bioinformatic investigation

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ThirumalaiswamySekhar Arvind

2009-10-01

Full Text Available Abstract The use of biotechnological techniques to introduce novel proteins into food crops (transgenic or GM crops has motivated investigation into the properties of proteins that favor their potential to elicit allergic reactions. As part of the allergenicity assessment, bioinformatic approaches are used to compare the amino-acid sequence of candidate proteins with sequences in a database of known allergens to predict potential cross reactivity between novel food proteins and proteins to which people have become sensitized. Two criteria commonly used for these queries are searches over 80-amino-acid stretches for >35% identity, and searches for 8-amino-acid contiguous matches. We investigated the added value provided by the 8-amino-acid criterion over that provided by the >35%-identity-over-80-amino-acid criterion, by identifying allergens pairs that only met the former criterion, but not the latter criterion. We found that the allergen-sequence pairs only sharing 8-amino-acid identity, but not >35% identity over 80 amino acids, were unlikely to be cross reactive allergens. Thus, the common search for 8-amino-acid identity between novel proteins and known allergens appears to be of little additional value in assessing the potential allergenicity of novel proteins.

Effect of whey protein on plasma amino acids in diabetic mice

OpenAIRE

HAN, TING; CAI, DONGLIAN; GENG, SHANSHAN; WANG, YING; ZHEN, HUI; WU, PEIYING

2013-01-01

The aim of this study was to investigate the effect of whey protein on plasma amino acid levels in a mouse model of type II diabetes, using high-performance liquid chromatography (HPLC). The composition and content of amino acids in the whey proteins were analyzed using HPLC. Type I and type II diabetic mouse models were prepared using streptozotocin (STZ) and normal mice were used as a control. The ICR mice in each group were then randomly divided into four subgroups, to which 0, 10, 20 and ...

Properties of whey protein isolates extruded under acidic and alkaline conditions.

Science.gov (United States)

Onwulata, C I; Isobe, S; Tomasula, P M; Cooke, P H

2006-01-01

Whey proteins have wide acceptance and use in many products due to their beneficial nutritional properties. To further increase the amount of whey protein isolates (WPI) that may be added to products such as extruded snacks and meats, texturization of WPI is necessary. Texturization changes the folding of globular proteins to improve interaction with other ingredients and create new functional ingredients. In this study, WPI pastes (60% solids) were extruded in a twin-screw extruder at 100 degrees C with 4 pH-adjusted water streams: acidic (pH 2.0 +/- 0.2) and alkaline (pH 12.4 +/- 0.4) streams from 2 N HCl and 2 N NaOH, respectively, and acidic (pH 2.5 +/- 0.2) and alkaline (pH 11.5 +/- 0.4) electrolyzed water streams; these were compared with WPI extruded with deionized water. The effects of water acidity on WPI solubility at pH 7, color, microstructure, Rapid Visco Analyzer pasting properties, and physical structure were determined. Alkaline conditions increased insolubility caused yellowing and increased pasting properties significantly. Acidic conditions increased solubility and decreased WPI pasting properties. Subtle structural changes occurred under acidic conditions, but were more pronounced under alkaline conditions. Overall, alkaline conditions increased denaturation in the extruded WPI resulting in stringy texturized WPI products, which could be used in meat applications.

Protein and Essential Amino Acids to Protect Musculoskeletal Health during Spaceflight: Evidence of a Paradox?

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Kyle J. Hackney

2014-07-01

Full Text Available Long-duration spaceflight results in muscle atrophy and a loss of bone mineral density. In skeletal muscle tissue, acute exercise and protein (e.g., essential amino acids stimulate anabolic pathways (e.g., muscle protein synthesis both independently and synergistically to maintain neutral or positive net muscle protein balance. Protein intake in space is recommended to be 12%–15% of total energy intake (≤1.4 g∙kg−1∙day−1 and spaceflight is associated with reduced energy intake (~20%, which enhances muscle catabolism. Increasing protein intake to 1.5–2.0 g∙kg−1∙day−1 may be beneficial for skeletal muscle tissue and could be accomplished with essential amino acid supplementation. However, increased consumption of sulfur-containing amino acids is associated with increased bone resorption, which creates a dilemma for musculoskeletal countermeasures, whereby optimizing skeletal muscle parameters via essential amino acid supplementation may worsen bone outcomes. To protect both muscle and bone health, future unloading studies should evaluate increased protein intake via non-sulfur containing essential amino acids or leucine in combination with exercise countermeasures and the concomitant influence of reduced energy intake.

Tetrahymena thermophila acidic ribosomal protein L37 contains an archaebacterial type of C-terminus

DEFF Research Database (Denmark)

Hansen, T S; Andreasen, P H; Dreisig, H

1991-01-01

We have cloned and characterized a Tetrahymena thermophila macronuclear gene (L37) encoding the acidic ribosomal protein (A-protein) L37. The gene contains a single intron located in the 3'-part of the coding region. Two major and three minor transcription start points (tsp) were mapped 39 to 63 ...... by protein sequencing. The T. thermophila L37 clearly belongs to the P1-type family of eukaryotic A-proteins, but the C-terminal region has the hallmarks of archaebacterial A-proteins.......We have cloned and characterized a Tetrahymena thermophila macronuclear gene (L37) encoding the acidic ribosomal protein (A-protein) L37. The gene contains a single intron located in the 3'-part of the coding region. Two major and three minor transcription start points (tsp) were mapped 39 to 63...... nucleotides upstream from the translational start codon. The uppermost tsp mapped to the first T in a putative T. thermophila RNA polymerase II initiator element, TATAA. The coding region of L37 predicts a protein of 109 amino acid (aa) residues. A substantial part of the deduced aa sequence was verified...

Potential Role of Amino Acid/Protein Nutrition and Exercise in Serum Albumin Redox State

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Yasuaki Wada

2017-12-01

Full Text Available Albumin is the major protein in the serum of mammals. It is synthesized exclusively in the liver, before being secreted into the circulation. Similar to skeletal muscle protein, albumin synthesis is stimulated by dietary amino acids and proteins as well as exercise. Albumin has three isoforms based on the redox states of the free cysteine residue at position 34. The redox state of serum albumin has long been extensively investigated in terms of oxidative stress-related chronic diseases, with the redox state of serum albumin having been regarded as a marker of systemic oxidative stress. However, according to recent animal studies, the redox state of serum albumin is modulated by albumin turnover and may also reflect amino acid/protein nutritional status. Furthermore, as the redox state of serum albumin is modulated by exercise training, measuring the pre- and post-exercise redox states of serum albumin in athletes may be useful in assessing amino acid/protein nutritional status and exercise-induced oxidative stress, which are closely associated with skeletal muscle adaptive responses. This article extensively reviews serum albumin and the redox state of albumin in the context of amino acid/protein nutritional status and exercise training.

Pyridostigmine Bromide, the Enteric Nervous System, and Functional Gastrointestinal Disorders in Gulf War Illness

Science.gov (United States)

2017-10-01

coming year. Major Task 3: Effects of reversing gliosis with PEA on gut function and neuroinflammation. Milestone(s) Achieved: Evaluation of a...neurochemical coding. We did not observe differences in glial fibrillary acidic protein (GFAP) expression that would suggest reactive gliosis in day...to start experiments from major task 3 that will test the beneficial effects of the anti-inflammatory drug palmitoylethanolamide ( PEA

Fed levels of amino acids are required for the somatotropin-induced increase in muscle protein synthesis.

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Wilson, Fiona A; Suryawan, Agus; Orellana, Renán A; Nguyen, Hanh V; Jeyapalan, Asumthia S; Gazzaneo, Maria C; Davis, Teresa A

2008-10-01

Chronic somatotropin (pST) treatment in pigs increases muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin could not account for the pST-induced increase in muscle protein synthesis when amino acids were maintained at fasting levels. This study aimed to determine whether the pST-induced increase in insulin promotes skeletal muscle protein synthesis when amino acids are provided at fed levels and whether the response is associated with enhanced translation initiation factor activation. Growing pigs were treated with pST (0 or 180 microg x kg(-1) x day(-1)) for 7 days, and then pancreatic-glucose-amino acid clamps were performed. Amino acids were raised to fed levels in the presence of either fasted or fed insulin concentrations; glucose was maintained at fasting throughout. Muscle protein synthesis was increased by pST treatment and by amino acids (with or without insulin) (P<0.001). In pST-treated pigs, fed, but not fasting, amino acid concentrations further increased muscle protein synthesis rates irrespective of insulin level (P<0.02). Fed amino acids, with or without raised insulin concentrations, increased the phosphorylation of S6 kinase (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1), decreased inactive 4EBP1.eIF4E complex association, and increased active eIF4E.eIF4G complex formation (P<0.02). pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of muscle protein synthesis requires fed amino acid levels, but not fed insulin levels. However, under the current conditions, the response to amino acids is not mediated by the activation of translation initiation factors that regulate mRNA binding to the ribosomal complex.

Nucleic acid programmable protein array a just-in-time multiplexed protein expression and purification platform.

Science.gov (United States)

Qiu, Ji; LaBaer, Joshua

2011-01-01

Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. Copyright © 2011 Elsevier Inc. All rights reserved.

Nutritional analyses for proteins and amino acids in beans (Phaseolus sp.

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Wathelet B.

1999-01-01

Full Text Available The chemical index is a good estimator of seed protein quality of Phaseolus beans. In order to estimate this value, a protein hydrolysis and amino acid quantification are realised. The problems inherent to these techniques are presented.

Processing and fatty acid acylation of RAS1 and RAS2 proteins in Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Fujiyama, A.; Tamanoi, F.

1986-01-01

The authors demonstrate the pathway for the biosynthesis of RAS1 and RAS2 gene products of Saccharomyces cerevisiae leading to their localization in membranes. The primary translation products of these genes are detected in a soluble fraction. Shortly after synthesis, these precursor molecules are converted to forms that migrate slightly faster than the precursor forms on a NaDodSO 4 /polyacrylamide gel. These processed proteins are further modified by fatty acid acylation, which is detected by [ 3 H]palmitic acid labeling. The acylated derivatives are found exclusively in cell membranes, indicating the translocation of the RAS proteins from cytosol to membranes during maturation process. The attached fatty acids can be released by mild alkaline hydrolysis, suggesting that the linkage between the fatty acid and the protein is an ester bond. The site of the modification by fatty acid is presumably localized to the COOH-terminal portion of the RAS proteins. Fraction of the membranes by sucrose gradient demonstrates that a majority of the fatty-acylated RAS proteins are localized in plasma membrane

Poly-gamma-glutamic acid a substitute of salivary protein statherin

International Nuclear Information System (INIS)

Qamar, Z.; Rahim, Z.B.H.A.; Fatima, T.

2016-01-01

The modus operandi of salivary proteins in reducing the kinetics of enamel dissolution during simulated caries challenges is thought to be associated with interaction of glutamic acid residues with human teeth surfaces. Japanese traditional food stuff natto is rich with chain of repeating glutamic acid residues linked by gamma-peptide bond and hence, named poly-gamma-glutamic acid (PGGA). It is a naturally occurring polypeptide and may therefore perform similar caries inhibitory functions as statherin. (author)

Noncoded amino acids in protein engineering: Structure-activity relationship studies of hirudin-thrombin interaction.

Science.gov (United States)

De Filippis, Vincenzo; Acquasaliente, Laura; Pontarollo, Giulia; Peterle, Daniele

2018-01-01

The advent of recombinant DNA technology allowed to site-specifically insert, delete, or mutate almost any amino acid in a given protein, significantly improving our knowledge of protein structure, stability, and function. Nevertheless, a quantitative description of the physical and chemical basis that makes a polypeptide chain to efficiently fold into a stable and functionally active conformation is still elusive. This mainly originates from the fact that nature combined, in a yet unknown manner, different properties (i.e., hydrophobicity, conformational propensity, polarizability, and hydrogen bonding capability) into the 20 standard natural amino acids, thus making difficult, if not impossible, to univocally relate the change in protein stability or function to the alteration of physicochemical properties caused by amino acid exchange(s). In this view, incorporation of noncoded amino acids with tailored side chains, allowing to finely tune the structure at a protein site, would facilitate to dissect the effects of a given mutation in terms of one or a few physicochemical properties, thus much expanding the scope of physical organic chemistry in the study of proteins. In this review, relevant applications from our laboratory will be presented on the use of noncoded amino acids in structure-activity relationships studies of hirudin binding to thrombin. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

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.

Urinary excretion of fatty acid-binding proteins in idiopathic membranous nephropathy.

NARCIS (Netherlands)

Hofstra, J.M.; Deegens, J.K.J.; Steenbergen, E.; Wetzels, J.F.M.

2008-01-01

BACKGROUND: It is suggested that proteinuria contributes to progressive renal failure by inducing tubular cell injury. The site of injury is unknown. Most studies have used markers of proximal tubular cell damage. Fatty acid-binding proteins (FABPs) are intracellular carrier proteins with different

Tsetse salivary gland proteins 1 and 2 are high affinity nucleic acid binding proteins with residual nuclease activity.

Directory of Open Access Journals (Sweden)

Guy Caljon

Full Text Available Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2 display DNA/RNA non-specific, high affinity nucleic acid binding with K(D values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents.

Comparative genome analysis to identify SNPs associated with high oleic acid and elevated protein content in soybean.

Science.gov (United States)

Kulkarni, Krishnanand P; Patil, Gunvant; Valliyodan, Babu; Vuong, Tri D; Shannon, J Grover; Nguyen, Henry T; Lee, Jeong-Dong

2018-03-01

The objective of this study was to determine the genetic relationship between the oleic acid and protein content. The genotypes having high oleic acid and elevated protein (HOEP) content were crossed with five elite lines having normal oleic acid and average protein (NOAP) content. The selected accessions were grown at six environments in three different locations and phenotyped for protein, oil, and fatty acid components. The mean protein content of parents, HOEP, and NOAP lines was 34.6%, 38%, and 34.9%, respectively. The oleic acid concentration of parents, HOEP, and NOAP lines was 21.7%, 80.5%, and 20.8%, respectively. The HOEP plants carried both FAD2-1A (S117N) and FAD2-1B (P137R) mutant alleles contributing to the high oleic acid phenotype. Comparative genome analysis using whole-genome resequencing data identified six genes having single nucleotide polymorphism (SNP) significantly associated with the traits analyzed. A single SNP in the putative gene Glyma.10G275800 was associated with the elevated protein content, and palmitic, oleic, and linoleic acids. The genes from the marker intervals of previously identified QTL did not carry SNPs associated with protein content and fatty acid composition in the lines used in this study, indicating that all the genes except Glyma.10G278000 may be the new genes associated with the respective traits.

Constraints and consequences of the emergence of amino acid repeats in eukaryotic proteins.

Science.gov (United States)

Chavali, Sreenivas; Chavali, Pavithra L; Chalancon, Guilhem; de Groot, Natalia Sanchez; Gemayel, Rita; Latysheva, Natasha S; Ing-Simmons, Elizabeth; Verstrepen, Kevin J; Balaji, Santhanam; Babu, M Madan

2017-09-01

Proteins with amino acid homorepeats have the potential to be detrimental to cells and are often associated with human diseases. Why, then, are homorepeats prevalent in eukaryotic proteomes? In yeast, homorepeats are enriched in proteins that are essential and pleiotropic and that buffer environmental insults. The presence of homorepeats increases the functional versatility of proteins by mediating protein interactions and facilitating spatial organization in a repeat-dependent manner. During evolution, homorepeats are preferentially retained in proteins with stringent proteostasis, which might minimize repeat-associated detrimental effects such as unregulated phase separation and protein aggregation. Their presence facilitates rapid protein divergence through accumulation of amino acid substitutions, which often affect linear motifs and post-translational-modification sites. These substitutions may result in rewiring protein interaction and signaling networks. Thus, homorepeats are distinct modules that are often retained in stringently regulated proteins. Their presence facilitates rapid exploration of the genotype-phenotype landscape of a population, thereby contributing to adaptation and fitness.

Neuronal plasticity and astrocytic reaction in Down syndrome and Alzheimer disease

DEFF Research Database (Denmark)

Jørgensen, Ole Steen; Brooksbank, B W; Balázs, R

1990-01-01

Proteins relatively enriched in neurons (neural cell adhesion molecule (NCAM) and D3-protein) or in glia (glutamine synthetase, glial fibrillary acidic protein (GFAP) and S100) were measured by quantitative immunochemical methods in autopsy samples of the cerebral cortex of subjects with Alzheimer...... disease (AD) and adults with Down syndrome (DS), the latter also presenting manifest signs of Alzheimer type of neuropathology. The trend of changes was similar in AD and DS, but more marked in the latter. The biochemical make-up of astrocytes was differentially affected: in both the frontal and DS...

Statistical potential-based amino acid similarity matrices for aligning distantly related protein sequences.

Science.gov (United States)

Tan, Yen Hock; Huang, He; Kihara, Daisuke

2006-08-15

Aligning distantly related protein sequences is a long-standing problem in bioinformatics, and a key for successful protein structure prediction. Its importance is increasing recently in the context of structural genomics projects because more and more experimentally solved structures are available as templates for protein structure modeling. Toward this end, recent structure prediction methods employ profile-profile alignments, and various ways of aligning two profiles have been developed. More fundamentally, a better amino acid similarity matrix can improve a profile itself; thereby resulting in more accurate profile-profile alignments. Here we have developed novel amino acid similarity matrices from knowledge-based amino acid contact potentials. Contact potentials are used because the contact propensity to the other amino acids would be one of the most conserved features of each position of a protein structure. The derived amino acid similarity matrices are tested on benchmark alignments at three different levels, namely, the family, the superfamily, and the fold level. Compared to BLOSUM45 and the other existing matrices, the contact potential-based matrices perform comparably in the family level alignments, but clearly outperform in the fold level alignments. The contact potential-based matrices perform even better when suboptimal alignments are considered. Comparing the matrices themselves with each other revealed that the contact potential-based matrices are very different from BLOSUM45 and the other matrices, indicating that they are located in a different basin in the amino acid similarity matrix space.

Effects of medium-chain fatty acids and oleic acid on blood lipids, lipoproteins, glucose, insulin, and lipid transfer protein activities

DEFF Research Database (Denmark)

Tholstrup, T.; Ehnholm, C.; Jauhiainen, M.

2004-01-01

Background: Dietary medium-chain fatty acids (MCFAs) are of nutritional interest because they are more easily absorbed from dietary medium-chain triacylglycerols (MCTs) than are long-chain fatty acids from, for example, vegetable oils. It has generally been claimed that MCFAs do not increase plasma...... cholesterol, although this claim is poorly documented. Objective: We compared the effects of a diet rich in either MCFAs or oleic acid on fasting blood lipids, lipoproteins, glucose, insulin, and lipid transfer protein activities in healthy men. Design: In a study with a double-blind, randomized, crossover...... plasma total triacylglycerol (P = 0.0361), and higher plasma glucose (P = 0.033). Plasma HDL-cholesterol and insulin concentrations and activities of cholesterol ester transfer protein and phospholipid transfer protein did not differ significantly between the diets. Conclusions: Compared with fat high...

Computational mining for hypothetical patterns of amino acid side chains in protein data bank (PDB)

Science.gov (United States)

Ghani, Nur Syatila Ab; Firdaus-Raih, Mohd

2018-04-01

The three-dimensional structure of a protein can provide insights regarding its function. Functional relationship between proteins can be inferred from fold and sequence similarities. In certain cases, sequence or fold comparison fails to conclude homology between proteins with similar mechanism. Since the structure is more conserved than the sequence, a constellation of functional residues can be similarly arranged among proteins of similar mechanism. Local structural similarity searches are able to detect such constellation of amino acids among distinct proteins, which can be useful to annotate proteins of unknown function. Detection of such patterns of amino acids on a large scale can increase the repertoire of important 3D motifs since available known 3D motifs currently, could not compensate the ever-increasing numbers of uncharacterized proteins to be annotated. Here, a computational platform for an automated detection of 3D motifs is described. A fuzzy-pattern searching algorithm derived from IMagine an Amino Acid 3D Arrangement search EnGINE (IMAAAGINE) was implemented to develop an automated method for searching of hypothetical patterns of amino acid side chains in Protein Data Bank (PDB), without the need for prior knowledge on related sequence or structure of pattern of interest. We present an example of the searches, which is the detection of a hypothetical pattern derived from known structural motif of C2H2 structural pattern from zinc fingers. The conservation of particular patterns of amino acid side chains in unrelated proteins is highlighted. This approach can act as a complementary method for available structure- and sequence-based platforms and may contribute in improving functional association between proteins.

Process for the separation of proteins from acid whey

Energy Technology Data Exchange (ETDEWEB)

Mirabel, B

1980-01-01

Acid whey from cheese or casein manufacture (pH less than 4.6) and containing about 5.2 g protein/l is passed through a cation exchange resin (of silica coated with a copolymer of styrene/vinyltriethoxysilane carrying SO/sub 3/H functional groups). The proteins adsorbed on the resin (alpha-lactalbumin, beta-lactoglobulin, serum albumin and immunoglobulins) are eluated with an 0.1 M ammonia solution, concentrated under vacuum and freeze-dried, obtaining a final product with 88% undenatured protein. The products are for use in the food and pharmaceutical industries and for dietetic and veterinary purposes.

Wen-Luo-Tong Prevents Glial Activation and Nociceptive Sensitization in a Rat Model of Oxaliplatin-Induced Neuropathic Pain.

Science.gov (United States)

Deng, Bo; Jia, Liqun; Pan, Lin; Song, Aiping; Wang, Yuanyuan; Tan, Huangying; Xiang, Qing; Yu, Lili; Ke, Dandan

2016-01-01

One of the main dose-limiting complications of the chemotherapeutic agent oxaliplatin (OXL) is painful neuropathy. Glial activation and nociceptive sensitization may be responsible for the mechanism of neuropathic pain. The Traditional Chinese Medicine (TCM) Wen-luo-tong (WLT) has been widely used in China to treat chemotherapy induced neuropathic pain. However, there is no study on the effects of WLT on spinal glial activation induced by OXL. In this study, a rat model of OXL-induced chronic neuropathic pain was established and WLT was administrated. Pain behavioral tests and morphometric examination of dorsal root ganglia (DRG) were conducted. Glial fibrillary acidic protein (GFAP) immunostaining was performed, glial activation was evaluated, and the excitatory neurotransmitter substance P (SP) and glial-derived proinflammatory cytokine tumor necrosis factor-α (TNF-α) were analyzed. WLT treatment alleviated OXL-induced mechanical allodynia and mechanical hyperalgesia. Changes in the somatic, nuclear, and nucleolar areas of neurons in DRG were prevented. In the spinal dorsal horn, hypertrophy and activation of GFAP-positive astrocytes were averted, and the level of GFAP mRNA decreased significantly. Additionally, TNF-α mRNA and protein levels decreased. Collectively, these results indicate that WLT reversed both glial activation in the spinal dorsal horn and nociceptive sensitization during OXL-induced chronic neuropathic pain in rats.

Hypoxia during pregnancy in rats leads to the changes of the cerebral white matter in adult offspring

International Nuclear Information System (INIS)

Wang, Lingxing; Cai, Ruowei; Lv, Guorong; Huang, Ziyang; Wang, Zhenhua

2010-01-01

The aim of the present study is to evaluate the effect of reduced fetal oxygen supply on cerebral white matter in the adult offspring and further assess its susceptibility to postnatal hypoxia and high-fat diet. Based on a 3 x 2 full factorial design consisting of three factors of maternal hypoxia, postnatal high-fat diet, and postnatal hypoxia, the ultrastructure of myelin, axon and capillaries were observed, and the expression of myelin basic protein (MBP), neurofilament-H+L(NF-H+L), and glial fibrillary acidic protein (GFAP) was analyzed in periventricular white matter of 16-month-old offspring. Demyelination, injured axon and damaged microvasculars were observed in maternal hypoxia offspring. The main effect of maternal hypoxia lead to decreased expression of MBP or NF-H+L, and increased expression of GFAP (all P < 0.05). Moreover, there was positive three-way interaction among maternal hypoxia, high-fat diet and postnatal hypoxia on MBP, NF-H+L or GFAP expression (all P < 0.05). In summary, our results indicated that maternal hypoxia during pregnancy in rats lead to changes of periventricular white matter in adult offspring, including demyelination, damaged axon and proliferated astroglia. This effect was amplified by high-fat diet and postnatal hypoxia.

Amino acids augment muscle protein synthesis in neonatal pigs during acute endotoxemia by stimulating mTOR-dependent translation initiation.

Science.gov (United States)

Orellana, Renán A; Jeyapalan, Asumthia; Escobar, Jeffery; Frank, Jason W; Nguyen, Hanh V; Suryawan, Agus; Davis, Teresa A

2007-11-01

In skeletal muscle of adults, sepsis reduces protein synthesis by depressing translation initiation and induces resistance to branched-chain amino acid stimulation. Normal neonates maintain a high basal muscle protein synthesis rate that is sensitive to amino acid stimulation. In the present study, we determined the effect of amino acids on protein synthesis in skeletal muscle and other tissues in septic neonates. Overnight-fasted neonatal pigs were infused with endotoxin (LPS, 0 and 10 microg.kg(-1).h(-1)), whereas glucose and insulin were maintained at fasting levels; amino acids were clamped at fasting or fed levels. In the presence of fasting insulin and amino acids, LPS reduced protein synthesis in longissimus dorsi (LD) and gastrocnemius muscles and increased protein synthesis in the diaphragm, but had no effect in masseter and heart muscles. Increasing amino acids to fed levels accelerated muscle protein synthesis in LD, gastrocnemius, masseter, and diaphragm. LPS stimulated protein synthesis in liver, lung, spleen, pancreas, and kidney in fasted animals. Raising amino acids to fed levels increased protein synthesis in liver of controls, but not LPS-treated animals. The increase in muscle protein synthesis in response to amino acids was associated with increased mTOR, 4E-BP1, and S6K1 phosphorylation and eIF4G-eIF4E association in control and LPS-infused animals. These findings suggest that amino acids stimulate skeletal muscle protein synthesis during acute endotoxemia via mTOR-dependent ribosomal assembly despite reduced basal protein synthesis rates in neonatal pigs. However, provision of amino acids does not further enhance the LPS-induced increase in liver protein synthesis.

Perceptron learning of pairwise contact energies for proteins incorporating the amino acid environment

Science.gov (United States)

Heo, Muyoung; Kim, Suhkmann; Moon, Eun-Joung; Cheon, Mookyung; Chung, Kwanghoon; Chang, Iksoo

2005-07-01

Although a coarse-grained description of proteins is a simple and convenient way to attack the protein folding problem, the construction of a global pairwise energy function which can simultaneously recognize the native folds of many proteins has resulted in partial success. We have sought the possibility of a systematic improvement of this pairwise-contact energy function as we extended the parameter space of amino acids, incorporating local environments of amino acids, beyond a 20×20 matrix. We have studied the pairwise contact energy functions of 20×20 , 60×60 , and 180×180 matrices depending on the extent of parameter space, and compared their effect on the learnability of energy parameters in the context of a gapless threading, bearing in mind that a 20×20 pairwise contact matrix has been shown to be too simple to recognize the native folds of many proteins. In this paper, we show that the construction of a global pairwise energy function was achieved using 1006 training proteins of a homology of less than 30%, which include all representatives of different protein classes. After parametrizing the local environments of the amino acids into nine categories depending on three secondary structures and three kinds of hydrophobicity (desolvation), the 16290 pairwise contact energies (scores) of the amino acids could be determined by perceptron learning and protein threading. These could simultaneously recognize all the native folds of the 1006 training proteins. When these energy parameters were tested on the 382 test proteins of a homology of less than 90%, 370 (96.9%) proteins could recognize their native folds. We set up a simple thermodynamic framework in the conformational space of decoys to calculate the unfolded fraction and the specific heat of real proteins. The different thermodynamic stabilities of E.coli ribonuclease H (RNase H) and its mutants were well described in our calculation, agreeing with the experiment.

Analysis of Protein–Protein Interactions in MCF-7 and MDA-MB-231 Cell Lines Using Phthalic Acid Chemical

Directory of Open Access Journals (Sweden)

Shih-Shin Liang

2014-11-01

Full Text Available Phthalates are a class of plasticizers that have been characterized as endocrine disrupters, and are associated with genital diseases, cardiotoxicity, hepatotoxicity, and nephrotoxicity in the GeneOntology gene/protein database. In this study, we synthesized phthalic acid chemical probes and demonstrated differing protein–protein interactions between MCF-7 cells and MDA-MB-231 breast cancer cell lines. Phthalic acid chemical probes were synthesized using silicon dioxide particle carriers, which were modified using the silanized linker 3-aminopropyl triethoxyslane (APTES. Incubation with cell lysates from breast cancer cell lines revealed interactions between phthalic acid and cellular proteins in MCF-7 and MDA-MB-231 cells. Subsequent proteomics analyses indicated 22 phthalic acid-binding proteins in both cell types, including heat shock cognate 71-kDa protein, ATP synthase subunit beta, and heat shock protein HSP 90-beta. In addition, 21 MCF-7-specific and 32 MDA-MB-231 specific phthalic acid-binding proteins were identified, including related proteasome proteins, heat shock 70-kDa protein, and NADPH dehydrogenase and ribosomal correlated proteins, ras-related proteins, and members of the heat shock protein family, respectively.

Murine protein H is comprised of 20 repeating units, 61 amino acids in length

DEFF Research Database (Denmark)

Kristensen, Torsten; Tack, B F

1986-01-01

A cDNA library constructed from size-selected (greater than 28 S) poly(A)+ RNA isolated from the livers of C57B10. WR mice was screened by using a 249-base-pair (bp) cDNA fragment encoding 83 amino acid residues of human protein H as a probe. Of 120,000 transformants screened, 30 hybridized......, 448 bp of 3'-untranslated sequence, and a polyadenylylated tail of undetermined length. Murine pre-protein H was deduced to consist of an 18-amino acid signal peptide and 1216 residues of H-protein sequence. Murine H was composed of 20 repetitive units, each about 61 amino acid residues in length...

Isolation of progenitor cells from GFP-transgenic pigs and transplantation to the retina of allorecipients

DEFF Research Database (Denmark)

Klassen, Henry; Warfvinge, Karin; Schwartz, Philip H

2008-01-01

to survival as allografts and integrate into the host retinal architecture, we isolated donor cells from fetal green fluorescent protein (GFP)-transgenic pigs. Cultures were propagated from the brain, retina, and corneo-scleral limbus. GFP expression rapidly increased with time in culture, although lower...... in conjunction with photoreceptor markers and glial fibrillary acid protein (GFAP), thus suggesting downregulation of GFP during differentiation. Following transplantation, GFP expression allowed histological visualization of integrated cells and extension of fine processes to adjacent plexiform layers. GFP...

INDUCTION OF GLIAL FIBRILLARY ACIDIC PROTEIN IMMUNOREACTIVITY IN THE RAT DENTATE GYRUS AFTER ADRENALECTOMY - COMPARISON WITH NEURODEGENERATIVE CHANGES USING SILVER IMPREGNATION

NARCIS (Netherlands)

KRUGERS, HJ; MEDEMA, RM; POSTEMA, F; KORF, J

In the present study we performed a light microscopic anatomical comparison of adrenalectomy (ADX)-induced neurodegeneration using silver impregnation and reaction of astroglial cells using GFAP immunocytochemistry in the hippocampus of the rat. Three survival times following ADX were studied: 24

Thermophysical properties of starch and whey protein composite prepared in presence of organic acid and esters

Science.gov (United States)

Previously, we prepared starch and protein composite by reactive mixing in presence of various organic acids and found that use of these acid esters resulted in composites with good mechanical properties. In this study, concentration (% w/w) of acid citrates in the starch-protein composites were var...

Measurement of the incorporation rates of four amino acids into proteins for estimating bacterial production.

Science.gov (United States)

Servais, P

1995-03-01

In aquatic ecosystems, [(3)H]thymidine incorporation into bacterial DNA and [(3)H]leucine incorporation into proteins are usually used to estimate bacterial production. The incorporation rates of four amino acids (leucine, tyrosine, lysine, alanine) into proteins of bacteria were measured in parallel on natural freshwater samples from the basin of the river Meuse (Belgium). Comparison of the incorporation into proteins and into the total macromolecular fraction showed that these different amino acids were incorporated at more than 90% into proteins. From incorporation measurements at four subsaturated concentrations (range, 2-77 nm), the maximum incorporation rates were determined. Strong correlations (r > 0.91 for all the calculated correlations) were found between the maximum incorporation rates of the different tested amino acids over a range of two orders of magnitude of bacterial activity. Bacterial production estimates were calculated using theoretical and experimental conversion factors. The productions calculated from the incorporation rates of the four amino acids were in good concordance, especially when the experimental conversion factors were used (slope range, 0.91-1.11, and r > 0.91). This study suggests that the incorporation of various amino acids into proteins can be used to estimate bacterial production.

Alexander disease as a cause of nocturnal vomiting in a 7-year-old girl

Energy Technology Data Exchange (ETDEWEB)

Niinikoski, Harri [University of Turku, Department of Paediatrics, Turku (Finland); Haataja, Leena [University of Turku, Department of Paediatrics, Turku (Finland); University of Turku, Department of Pediatric Neurology, Turku (Finland); Brander, Antti [University of Tampere, Department of Radiology, Tampere (Finland); Valanne, Leena [University of Helsinki, Department of Radiology, Helsinki (Finland); Blaser, Susan [Hospital for Sick Children, Department of Radiology, Toronto (Canada)

2009-08-15

Alexander disease is a rare form of leukodystrophy with a highly variable clinical course. Occasionally night-time nausea and vomiting are the first symptoms of juvenile Alexander disease. A 7-year-old girl had recurrent night-time vomiting and her growth and weight gain had deteriorated after her sixth birthday. Cranial MRI demonstrated two small, symmetrical focal areas of abnormally high signal intensity in the dorsal medulla oblongata on T2-W and FLAIR images. These were suggestive of juvenile Alexander disease, and subsequent sequencing of the glial fibrillary acidic protein (GFAP) gene revealed a heterogeneous missense mutation in the GFAP gene in exon 6. Alexander disease should be considered in young patients with atypical anorexia nervosa-type symptoms. (orig.)

Alexander disease as a cause of nocturnal vomiting in a 7-year-old girl

International Nuclear Information System (INIS)

Niinikoski, Harri; Haataja, Leena; Brander, Antti; Valanne, Leena; Blaser, Susan

2009-01-01

Alexander disease is a rare form of leukodystrophy with a highly variable clinical course. Occasionally night-time nausea and vomiting are the first symptoms of juvenile Alexander disease. A 7-year-old girl had recurrent night-time vomiting and her growth and weight gain had deteriorated after her sixth birthday. Cranial MRI demonstrated two small, symmetrical focal areas of abnormally high signal intensity in the dorsal medulla oblongata on T2-W and FLAIR images. These were suggestive of juvenile Alexander disease, and subsequent sequencing of the glial fibrillary acidic protein (GFAP) gene revealed a heterogeneous missense mutation in the GFAP gene in exon 6. Alexander disease should be considered in young patients with atypical anorexia nervosa-type symptoms. (orig.)

Effect of 12-Day Spaceflight on Brain of Thick-Toed Geckos

Science.gov (United States)

Proshchina, A. E.; Karlamova, A. S.; Barabanovet, V. M.; Godovalova, O. S.; Guilimova, V. I.; Krivova, Y. S.; Makarov, A. N.; Nikitin, V. B.; Savelieva, E. S.; Saveliev, S. V.

2008-06-01

In the frames of Russian-American joint space experiment onboard Foton-M3 satellite there was undertaken a study of spaceflight influence on brain of the thick-toed gecko (Pachydactylus turneri Gray, 1864). Serial brain sections were stained according to Nissl and also the immunohistochemical method with antibodies to NGF-receptor (p75NGFR), CD95 (also known as Fas and APO-1), glial fibrillary acidic protein (GFAP) and transferrin-receptor (CD71). Detailed examination of the sections of rhombencephalon revealed cytological changes in the neuron bodies of vestibular nuclei inside the flight group. Immunohistochemicaly we found the increase density of CD95 and p75NGFR and decrease of GFAP expression in medial cortex and epithalamus in flight group compared both control.

Two classes of astrocytes in the adult human and pig retina in terms of their expression of high affinity NGF receptor (TrkA).

Science.gov (United States)

Ruiz-Ederra, Javier; Hitchcock, Peter F; Vecino, Elena

2003-02-13

Astrocytes have been implicated in axon guidance and synaptic regeneration in the retina and these processes involve activation of the high affinity nerve growth factor receptor, known as the tyrosine kinase A (TrkA) receptor. The purpose of the present study was to characterize the expression of TrkA in astrocytes of the adult pig and human retina. To this end, sections of human and pig retinas were immunolabeled with a combination of antibodies to glial fibrillary acidic protein (GFAP) and TrkA. Our study revealed that most of the GFAP-positive cells express TrkA, whereas a rare, novel subpopulation of astrocytes was found to be devoid of TrkA. Our results support the idea that astrocytes play an important neurotrophic role in the retina.

Structure of the human-heart fatty-acid-binding protein 3 in complex with the fluorescent probe 1-anilinonaphthalene-8-sulphonic acid

Energy Technology Data Exchange (ETDEWEB)

Hirose, Mika; Sugiyama, Shigeru, E-mail: sugiyama@chem.eng.osaka-u.ac.jp [Lipid Active Structure Project, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Osaka University, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Ishida, Hanako; Niiyama, Mayumi [Lipid Active Structure Project, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Osaka University, 2-1 Yamadaoka, Suita 565-0871 (Japan); Matsuoka, Daisuke; Hara, Toshiaki [Lipid Active Structure Project, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Osaka University, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Mizohata, Eiichi [Osaka University, 2-1 Yamadaoka, Suita 565-0871 (Japan); Murakami, Satoshi [Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama, Kanagaw 226-8501 (Japan); Inoue, Tsuyoshi [Osaka University, 2-1 Yamadaoka, Suita 565-0871 (Japan); Matsuoka, Shigeru; Murata, Michio [Lipid Active Structure Project, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan); Osaka University, 1-1 Machikaneyama-cho, Toyonaka 560-0043 (Japan)

2013-11-01

The crystal structure of human-heart-type fatty-acid-binding protein in complex with anilinonaphthalene-8-sulfonate was solved at 2.15 Å resolution revealing the detailed binding mechanism of the fluorescent probe 1-anilinonaphthalene-8-sulfonate. Heart-type fatty-acid-binding protein (FABP3), which is a cytosolic protein abundantly found in cardiomyocytes, plays a role in trafficking fatty acids throughout cellular compartments by reversibly binding intracellular fatty acids with relatively high affinity. The fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) is extensively utilized for examining the interaction of ligands with fatty-acid-binding proteins. The X-ray structure of FABP3 was determined in the presence of ANS and revealed the detailed ANS-binding mechanism. Furthermore, four water molecules were clearly identified in the binding cavity. Through these water molecules, the bound ANS molecule forms indirect hydrogen-bond interactions with FABP3. The adipocyte-type fatty-acid-binding protein (FABP4) exhibits 67% sequence identity with FABP3 and its crystal structure is almost the same as that of FABP3. However, FABP4 can bind with a higher affinity to ANS than FABP3. To understand the difference in their ligand specificities, a structural comparison was performed between FABP3–ANS and FABP4–ANS complexes. The result revealed that the orientation of ANS binding to FABP3 is completely opposite to that of ANS binding to FABP4, and the substitution of valine in FABP4 to leucine in FABP3 may result in greater steric hindrance between the side-chain of Leu115 and the aniline ring of ANS.

Structure of the human-heart fatty-acid-binding protein 3 in complex with the fluorescent probe 1-anilinonaphthalene-8-sulphonic acid

International Nuclear Information System (INIS)

Hirose, Mika; Sugiyama, Shigeru; Ishida, Hanako; Niiyama, Mayumi; Matsuoka, Daisuke; Hara, Toshiaki; Mizohata, Eiichi; Murakami, Satoshi; Inoue, Tsuyoshi; Matsuoka, Shigeru; Murata, Michio

2013-01-01

The crystal structure of human-heart-type fatty-acid-binding protein in complex with anilinonaphthalene-8-sulfonate was solved at 2.15 Å resolution revealing the detailed binding mechanism of the fluorescent probe 1-anilinonaphthalene-8-sulfonate. Heart-type fatty-acid-binding protein (FABP3), which is a cytosolic protein abundantly found in cardiomyocytes, plays a role in trafficking fatty acids throughout cellular compartments by reversibly binding intracellular fatty acids with relatively high affinity. The fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) is extensively utilized for examining the interaction of ligands with fatty-acid-binding proteins. The X-ray structure of FABP3 was determined in the presence of ANS and revealed the detailed ANS-binding mechanism. Furthermore, four water molecules were clearly identified in the binding cavity. Through these water molecules, the bound ANS molecule forms indirect hydrogen-bond interactions with FABP3. The adipocyte-type fatty-acid-binding protein (FABP4) exhibits 67% sequence identity with FABP3 and its crystal structure is almost the same as that of FABP3. However, FABP4 can bind with a higher affinity to ANS than FABP3. To understand the difference in their ligand specificities, a structural comparison was performed between FABP3–ANS and FABP4–ANS complexes. The result revealed that the orientation of ANS binding to FABP3 is completely opposite to that of ANS binding to FABP4, and the substitution of valine in FABP4 to leucine in FABP3 may result in greater steric hindrance between the side-chain of Leu115 and the aniline ring of ANS

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....

Cartilage Acidic Protein 2 a hyperthermostable, high affinity calcium-binding protein.

Science.gov (United States)

Anjos, Liliana; Gomes, Ana S; Melo, Eduardo P; Canário, Adelino V; Power, Deborah M

2013-03-01

Cartilage Acidic Protein 2 (CRTAC2) is a novel protein present from prokaryotes to vertebrates with abundant expression in the teleost fish pituitary gland and an isoform of CRTAC1, a chondrocyte marker in humans. The two proteins are non-integrins containing N-terminal integrin-like Ca(2+)-binding motifs and their structure and function remain to be assigned. Structural studies of recombinant sea bream (sb)CRTAC2 revealed it is composed of 8.8% α-helix, 33.4% β-sheet and 57.8% unordered protein. sbCRTAC2 bound Ca(2+) with high affinity (K(d)=1.46nM) and favourable Gibbs free energy (∆G=-12.4kcal/mol). The stoichiometry for Ca(2+) bound to sbCRTAC2 at saturation indicated six Ca(2+) ligand-binding sites exist per protein molecule. No conformational change in sbCRTAC2 occurred in the presence of Ca(2+). Fluorescence emission revealed that the tertiary structure of the protein is hyperthermostable between 25°C and 95°C and the fully unfolded state is only induced by chemical denaturing (4M GndCl). sbCRTAC has a widespread tissue distribution and is present as high molecular weight aggregates, although strong reducing conditions promote formation of the monomer. sbCRTAC2 promotes epithelial cell outgrowth in vitro suggesting it may share functional homology with mammalian CRTAC1, recently implicated in cell-cell and cell-matrix interactions. Copyright © 2012 Elsevier B.V. All rights reserved.

Functional Properties and Amino Acid Profile of Spirulina Platensis Protein Isolates

International Nuclear Information System (INIS)

Bashir, S.; Sharif, M. K.; Butt, M. S.; Shahid, M.

2016-01-01

Protein malnutrition and food insecurity represent serious obstructions to sustainable development, poverty reduction and food quality throughout the world. The present study has been designed to evaluate the Spirulina platensis (SP) as a protein alternative source for the utilization in food products. A protein isolate was prepared from S. platensis powder through extraction with 0.1N NaOH, precipitation at pH 3, neutralization of the dispersed precipitate to pH 6.8-7.0, and subsequent freeze drying. The S. platensis isolate amino acids compositions revealed that the total essential amino acids contribution was comparatively higher in SPI (31.16±1.43 g/100 g) as compared with SP (27.75±1.21 g/100 g). Moreover, oil and water absorption capacities, foaming and emulsifying properties, surface hydrophobicity and nitrogen solubility index were found better functional properties under laboratory conditions except emulsion properties. Conclusively, SP and its isolates might be used in various food products to curtail protein energy malnutrition. (author)

Novel humic acid-bonded magnetite nanoparticles for protein immobilization

Energy Technology Data Exchange (ETDEWEB)

Bayrakci, Mevlut, E-mail: mevlutbayrakci@gmail.com [Ulukisla Vocational School, Nigde University, 51100 Ulukisla, Nigde (Turkey); Gezici, Orhan [Department of Chemistry, Nigde University, 51100 Nigde (Turkey); Bas, Salih Zeki; Ozmen, Mustafa; Maltas, Esra [Department of Chemistry, Selcuk University, 42031 Konya (Turkey)

2014-09-01

The present paper is the first report that introduces (i) a useful methodology for chemical immobilization of humic acid (HA) to aminopropyltriethoxysilane-functionalized magnetite iron oxide nanoparticles (APS-MNPs) and (ii) human serum albumin (HSA) binding to the obtained material (HA-APS-MNPs). The newly prepared magnetite nanoparticle was characterized by using Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and elemental analysis. Results indicated that surface modification of the bare magnetite nanoparticles (MNPs) with aminopropyltriethoxysilane (APS) and HA was successfully performed. The protein binding studies that were evaluated in batch mode exhibited that HA-APS-MNPs could be efficiently used as a substrate for the binding of HSA from aqueous solutions. Usually, recovery values higher than 90% were found to be feasible by HA-APS-MNPs, while that value was around 2% and 70% in the cases of MNPs and APS-MNPs, respectively. Hence, the capacity of MNPs was found to be significantly improved by immobilization of HA. Furthermore, thermal degradation of HA-APS-MNPs and HSA bonded HA-APS-MNPs was evaluated in terms of the Horowitz–Metzger equation in order to determine kinetic parameters for thermal decomposition. Activation energies calculated for HA-APS-MNPs (20.74 kJ mol{sup −1}) and HSA bonded HA-APS-MNPs (33.42 kJ mol{sup −1}) implied chemical immobilization of HA to APS-MNPs, and tight interactions between HA and HA-APS-MNPs. - Highlights: • A new magnetite nanoparticle based humic acid was prepared for the first time. • Protein binding studies of magnetite nanoparticle based humic acid were performed. • Kinetic parameters of protein and/or humic acid bonded nanoparticles were evaluated.

Fast computational methods for predicting protein structure from primary amino acid sequence

Science.gov (United States)

Agarwal, Pratul Kumar [Knoxville, TN

2011-07-19

The present invention provides a method utilizing primary amino acid sequence of a protein, energy minimization, molecular dynamics and protein vibrational modes to predict three-dimensional structure of a protein. The present invention also determines possible intermediates in the protein folding pathway. The present invention has important applications to the design of novel drugs as well as protein engineering. The present invention predicts the three-dimensional structure of a protein independent of size of the protein, overcoming a significant limitation in the prior art.

Neural Mobilization Treatment Decreases Glial Cells and Brain-Derived Neurotrophic Factor Expression in the Central Nervous System in Rats with Neuropathic Pain Induced by CCI in Rats

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Aline Carolina Giardini

2017-01-01

Full Text Available Background. Glial cells are implicated in the development of chronic pain and brain-derived neurotropic factor (BDNF released from activated microglia contributes to the nociceptive transmission. Neural mobilization (NM technique is a method clinically effective in reducing pain sensitivity. Here we examined the involvement of glial cells and BDNF expression in the thalamus and midbrain after NM treatment in rats with chronic constriction injury (CCI. CCI was induced and rats were subsequently submitted to 10 sessions of NM, every other day, beginning 14 days after CCI. Thalamus and midbrain were analyzed for glial fibrillary acidic protein (GFAP, microglial cell OX-42, and BDNF using Immunohistochemistry and Western blot assays. Results. Thalamus and midbrain of CCI group showed increases in GFAP, OX-42, and BDNF expression compared with control group and, in contrast, showed decreases in GFAP, OX-42, and BDNF after NM when compared with CCI group. The decreased immunoreactivity for GFAP, OX-42, and BDNF in ventral posterolateral nucleus in thalamus and the periaqueductal gray in midbrain was shown by immunohistochemistry. Conclusions. These findings may improve the knowledge about the involvement of astrocytes, microglia, and BDNF in the chronic pain and show that NM treatment, which alleviates neuropathic pain, affects glial cells and BDNF expression.

Effect of Maillard browning reaction on protein utilization and plasma amino acid response by rainbow trout (Salmo gairdneri).

Science.gov (United States)

Plakas, S M; Lee, T C; Wolke, R E; Meade, T L

1985-12-01

The effect of the Maillard browning reaction in the diet of rainbow trout (Salmo gairdneri) on growth and amino acid availability was investigated. Chemical and enzymatic hydrolysis methods were applied for the detection of the losses of amino acids in a model protein browning system. Arginine and lysine exhibited the greatest losses in the mixture of fish protein isolate and glucose stored for 40 d at 37 degrees C. The apparent digestibility and absorption of individual amino acids, particularly lysine, was lower in trout fed browned protein than in those fed the control protein. Plasma lysine levels were significantly depressed, while the plasma levels of glucose and most other amino acids were elevated in relation to the loss in nutritive value of dietary protein after browning. The early Maillard reaction derivative of lysine, epsilon-deoxy-fructosyl-lysine, was recovered from browned protein (by using the in vitro enzymatic hydrolysis procedure) and from the plasma of trout fed browned protein. Analysis of plasma free amino acids provided an indication of lysine bioavailability and identified lysine as the first-limiting amino acid in the diets containing browned protein.

The insulin-like growth factors I and II stimulate proliferation of different types of Schwann cells

DEFF Research Database (Denmark)

Sondell, M; Svenningsen, Åsa Fex; Kanje, M

1997-01-01

in combination with BrdU immunocytochemistry showed that around 93% of the proliferating cells in the nerve segments were Schwann cells. Immunostaining for BrdU and GFAP (glial fibrillary acid protein) showed that IGF-II enhanced proliferation of Schwann cells surrounding unmyelinated nerve fibres. In contrast......, truncated IGF-I promoted proliferation of Schwann cells of myelinated nerve fibres while insulin increased proliferation of both cell types....

Fluorescent Proteins for Investigating Biological Events in Acidic Environments

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Hajime Shinoda

2018-05-01

Full Text Available The interior lumen of acidic organelles (e.g., endosomes, secretory granules, lysosomes and plant vacuoles is an important platform for modification, transport and degradation of biomolecules as well as signal transduction, which remains challenging to investigate using conventional fluorescent proteins (FPs. Due to the highly acidic luminal environment (pH ~ 4.5–6.0, most FPs and related sensors are apt to lose their fluorescence. To address the need to image in acidic environments, several research groups have developed acid-tolerant FPs in a wide color range. Furthermore, the engineering of pH insensitive sensors, and their concomitant use with pH sensitive sensors for the purpose of pH-calibration has enabled characterization of the role of luminal ions. In this short review, we summarize the recent development of acid-tolerant FPs and related functional sensors and discuss the future prospects for this field.

Concentration and entry rate of amino acids in buffalo calves fed on two planes of crude protein

International Nuclear Information System (INIS)

Verma, D.N.; Singh, U.B.; Varma, A.; Ranjhan, S.K.

1974-01-01

Amino acid entry rates into the body pool have been estimated in buffalo calves using a single injection isotope dilution technique. The animals received 2 levels of crude protein, 13 percent lower and 19 percent higher than NRC recommendation. The concentrations of free amino acid in plasma were 5.49 and 7.17 mg/100 ml in animals fed on low and high crude protein diet, respectively. There was significant differences in the plasma amino acid concentration and entry rates between the groups. Amino acid entry rates were 79.17 and 117.78 mg per min in groups fed on low and high plane of crude protein respectively, showing that availability of amino acid is better in animals given ratio high in crude protein contents. (author)

Amino acid alphabet reduction preserves fold information contained in contact interactions in proteins.

Science.gov (United States)

Solis, Armando D

2015-12-01

To reduce complexity, understand generalized rules of protein folding, and facilitate de novo protein design, the 20-letter amino acid alphabet is commonly reduced to a smaller alphabet by clustering amino acids based on some measure of similarity. In this work, we seek the optimal alphabet that preserves as much of the structural information found in long-range (contact) interactions among amino acids in natively-folded proteins. We employ the Information Maximization Device, based on information theory, to partition the amino acids into well-defined clusters. Numbering from 2 to 19 groups, these optimal clusters of amino acids, while generated automatically, embody well-known properties of amino acids such as hydrophobicity/polarity, charge, size, and aromaticity, and are demonstrated to maintain the discriminative power of long-range interactions with minimal loss of mutual information. Our measurements suggest that reduced alphabets (of less than 10) are able to capture virtually all of the information residing in native contacts and may be sufficient for fold recognition, as demonstrated by extensive threading tests. In an expansive survey of the literature, we observe that alphabets derived from various approaches-including those derived from physicochemical intuition, local structure considerations, and sequence alignments of remote homologs-fare consistently well in preserving contact interaction information, highlighting a convergence in the various factors thought to be relevant to the folding code. Moreover, we find that alphabets commonly used in experimental protein design are nearly optimal and are largely coherent with observations that have arisen in this work. © 2015 Wiley Periodicals, Inc.

Study of Stationary Phase Metabolism Via Isotopomer Analysis of Amino Acids from an Isolated Protein

Energy Technology Data Exchange (ETDEWEB)

Shaikh, AfshanS.; Tang, YinjieJ.; Mukhopadhyay, Aindrila; Martin, Hector Garcia; Gin, Jennifer; Benke, Peter; Keasling, Jay D.

2009-09-14

Microbial production of many commercially important secondary metabolites occurs during stationary phase, and methods to measure metabolic flux during this growth phase would be valuable. Metabolic flux analysis is often based on isotopomer information from proteinogenic amino acids. As such, flux analysis primarily reflects the metabolism pertinent to the growth phase during which most proteins are synthesized. To investigate central metabolism and amino acids synthesis activity during stationary phase, addition of fully 13C-labeled glucose followed by induction of green fluorescent protein (GFP) expression during stationary phase was used. Our results indicate that Escherichia coli was able to produce new proteins (i.e., GFP) in the stationary phase, and the amino acids in GFP were mostly from degraded proteins synthesized during the exponential growth phase. Among amino acid biosynthetic pathways, only those for serine, alanine, glutamate/glutamine, and aspartate/asparagine had significant activity during the stationary phase.

Backbone and sidechain 1H, 13C and 15N resonance assignments of the human brain-type fatty acid binding protein (FABP7) in its apo form and the holo forms binding to DHA, oleic acid, linoleic acid and elaidic acid

DEFF Research Database (Denmark)

Oeemig, Jesper S; Jørgensen, Mathilde L; Hansen, Mikka S

2009-01-01

In this manuscript, we present the backbone and side chain assignments of human brain-type fatty acid binding protein, also known as FABP7, in its apo form and in four different holo forms, bound to DHA, oleic acid, linoleic acid and elaidic acid.......In this manuscript, we present the backbone and side chain assignments of human brain-type fatty acid binding protein, also known as FABP7, in its apo form and in four different holo forms, bound to DHA, oleic acid, linoleic acid and elaidic acid....

PrPC has nucleic acid chaperoning properties similar to the nucleocapsid protein of HIV-1.

Science.gov (United States)

Derrington, Edmund; Gabus, Caroline; Leblanc, Pascal; Chnaidermann, Jonas; Grave, Linda; Dormont, Dominique; Swietnicki, Wieslaw; Morillas, Manuel; Marck, Daniel; Nandi, Pradip; Darlix, Jean-Luc

2002-01-01

The function of the cellular prion protein (PrPC) remains obscure. Studies suggest that PrPC functions in several processes including signal transduction and Cu2+ metabolism. PrPC has also been established to bind nucleic acids. Therefore we investigated the properties of PrPC as a putative nucleic acid chaperone. Surprisingly, PrPC possesses all the nucleic acid chaperoning properties previously specific to retroviral nucleocapsid proteins. PrPC appears to be a molecular mimic of NCP7, the nucleocapsid protein of HIV-1. Thus PrPC, like NCP7, chaperones the annealing of tRNA(Lys) to the HIV-1 primer binding site, the initial step of retrovirus replication. PrPC also chaperones the two DNA strand transfers required for production of a complete proviral DNA with LTRs. Concerning the functions of NCP7 during budding, PrPC also mimices NCP7 by dimerizing the HIV-1 genomic RNA. These data are unprecedented because, although many cellular proteins have been identified as nucleic acid chaperones, none have the properties of retroviral nucleocapsid proteins.

Characterization of hapten-protein conjugates: antibody generation and immunoassay development for chlorophenoxyacetic acid pesticides.

Science.gov (United States)

Boro, Robin C; Singh, K Vikas; Suri, C Raman

2009-01-01

The generation of specific and sensitive antibodies against small molecules is greatly dependent upon the characteristics of the hapten-protein conjugates. In this study, we report a new fluorescence-based method for the characterization of hapten-protein conjugates. The method is based on an effect promoted by hapten-protein conjugation density upon the fluorescence intensity of the intrinsic tryptophan chromophore molecules of the protein. The proposed methodology is applied to quantify the hapten-protein conjugation density for two different chlorophenoxyacetic acid pesticides, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenoxybutyric acid (2,4-DB), coupled to carrier protein. Highly sensitive anti-2,4-D and anti-2,4-DB antibodies were obtained using these well-characterized hapten-protein conjugates. The generated antibodies were used in an immunoassay format demonstrating inhibitory concentration (IC50) values equal to 30 and 7 ng/mL for 2,4-D and 2,4-DB, respectively. Linearity was observed in the concentration range between 0.1-500 nglmL with LODs around 4 and 3 ng/mL for 2,4-D and 2,4-DB, respectively, in standard water samples. The proposed method was successfully applied for the determination of the extent of hapten-protein conjugation to produce specific antibodies for immunoassay development against pesticides.

Xanthurenic acid translocates proapoptotic Bcl-2 family proteins into mitochondria and impairs mitochondrial function

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Hess Otto M

2004-04-01

Full Text Available Abstract Background Xanthurenic acid is an endogenous molecule produced by tryptophan degradation, produced in the cytoplasm and mitochondria. Its accumulation can be observed in aging-related diseases, e.g. senile cataract and infectious disease. We previously reported that xanthurenic acid provokes apoptosis, and now present a study of the response of mitochondria to xanthurenic acid. Results Xanthurenic acid at 10 or 20 μM in culture media of human aortic smooth muscle cells induces translocation of the proteins Bax, Bak, Bclxs, and Bad into mitochondria. In 20 μM xanthurenic acid, Bax is also translocated to the nucleus. In isolated mitochondria xanthurenic acid leads to Bax and Bclxs oligomerization, accumulation of Ca2+, and increased oxygen consumption. Conclusion Xanthurenic acid interacts directly with Bcl-2 family proteins, inducing mitochondrial pathways of apoptosis and impairing mitochondrial functions.

Preparation, crystallization and preliminary X-ray diffraction analysis of two intestinal fatty-acid binding proteins in the presence of 11-(dansylamino)undecanoic acid

International Nuclear Information System (INIS)

Laguerre, Aisha; Wielens, Jerome; Parker, Michael W.; Porter, Christopher J. H.; Scanlon, Martin J.

2011-01-01

Intestinal fatty-acid binding proteins from human and rat have been crystallized in complex with the fluorescent probe 11-(dansylamino)undecanoic acid. Diffraction data for the crystals were collected to 1.8 Å resolution (human) and 1.6 Å resolution (rat). Fatty-acid binding proteins (FABPs) are abundantly expressed proteins that bind a range of lipophilic molecules. They have been implicated in the import and intracellular distribution of their ligands and have been linked with metabolic and inflammatory responses in the cells in which they are expressed. Despite their high sequence identity, human intestinal FABP (hIFABP) and rat intestinal FABP (rIFABP) bind some ligands with different affinities. In order to address the structural basis of this differential binding, diffraction-quality crystals have been obtained of hIFABP and rIFABP in complex with the fluorescent fatty-acid analogue 11-(dansylamino)undecanoic acid

Applications of hydrophilic interaction chromatography to amino acids, peptides, and proteins.

Science.gov (United States)

Periat, Aurélie; Krull, Ira S; Guillarme, Davy

2015-02-01

This review summarizes the recent advances in the analysis of amino acids, peptides, and proteins using hydrophilic interaction chromatography. Various reports demonstrate the successful analysis of amino acids under such conditions. However, a baseline resolution of the 20 natural amino acids has not yet been published and for this reason, there is often a need to use mass spectrometry for detection to further improve selectivity. Hydrophilic interaction chromatography is also recognized as a powerful technique for peptide analysis, and there are a lot of papers showing its applicability for proteomic applications (peptide mapping). It is expected that its use for peptide mapping will continue to grow in the future, particularly because this analytical strategy can be combined with reversed-phase liquid chromatography, in a two-dimensional setup, to reach very high resolving power. Finally, the interest in hydrophilic interaction chromatography for intact proteins analysis is less evident due to possible solubility issues and a lack of suitable hydrophilic interaction chromatography stationary phases. To date, it has been successfully employed only for the characterization of membrane proteins, histones, and the separation of glycosylated isoforms of an intact glycoprotein. From our point of view, the number of hydrophilic interaction chromatography columns compatible with intact proteins (higher upper temperature limit, large pore size, etc.) is still too limited. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

TMV-Cg Coat Protein stabilizes DELLA proteins and in turn negatively modulates salicylic acid-mediated defense pathway during Arabidopsis thaliana viral infection.

Science.gov (United States)

Rodriguez, Maria Cecilia; Conti, Gabriela; Zavallo, Diego; Manacorda, Carlos Augusto; Asurmendi, Sebastian

2014-08-03

Plant viral infections disturb defense regulatory networks during tissue invasion. Emerging evidence demonstrates that a significant proportion of these alterations are mediated by hormone imbalances. Although the DELLA proteins have been reported to be central players in hormone cross-talk, their role in the modulation of hormone signaling during virus infections remains unknown. This work revealed that TMV-Cg coat protein (CgCP) suppresses the salicylic acid (SA) signaling pathway without altering defense hormone SA or jasmonic acid (JA) levels in Arabidopsis thaliana. Furthermore, it was observed that the expression of CgCP reduces plant growth and delays the timing of floral transition. Quantitative RT-qPCR analysis of DELLA target genes showed that CgCP alters relative expression of several target genes, indicating that the DELLA proteins mediate transcriptional changes produced by CgCP expression. Analyses by fluorescence confocal microscopy showed that CgCP stabilizes DELLA proteins accumulation in the presence of gibberellic acid (GA) and that the DELLA proteins are also stabilized during TMV-Cg virus infections. Moreover, DELLA proteins negatively modulated defense transcript profiles during TMV-Cg infection. As a result, TMV-Cg accumulation was significantly reduced in the quadruple-DELLA mutant Arabidopsis plants compared to wild type plants. Taken together, these results demonstrate that CgCP negatively regulates the salicylic acid-mediated defense pathway by stabilizing the DELLA proteins during Arabidopsis thaliana viral infection, suggesting that CgCP alters the stability of DELLAs as a mechanism of negative modulation of antiviral defense responses.

Synthesis of acid-soluble spore proteins by Bacillus subtilis.

OpenAIRE

Leventhal, J M; Chambliss, G H

1982-01-01

The major acid-soluble spore proteins (ASSPs) of Bacillus subtilis were detected by immunoprecipitation of radioactively labeled in vitro- and in vivo-synthesized proteins. ASSP synthesis in vivo began 2 h after the initiation of sporulation (t2) and reached its maximum rate at t7. This corresponded to the time of synthesis of mRNA that stimulated the maximum rate of ASSP synthesis in vitro. Under the set of conditions used in these experiments, protease synthesis began near t0, alkaline phos...

Radiation chemistry of amino acids, peptides and proteins in relation to the radiation sterilization of high-protein foods. [106 references

Energy Technology Data Exchange (ETDEWEB)

Garrison, W.M.

1979-03-01

An important source of information on the question of whether or not toxic or other deleterious substances are formed in the radiation sterilization of foods is the chemical study of reaction products and reaction mechanisms in the radiolysis of individual food components. The present evaluation of the radiation chemistry of amino acids, peptides and proteins outlines the various radiation-induced processes which lead to amino acid degradation and to the synthesis of amino acid derivatives of higher molecular weight. Among the latter are the ..cap alpha..,..cap alpha..'-diamino dicarboxylic acids which are formed as major products in the radiolysis of peptides both in aqueous solution and in the solid state. The ..cap alpha..,..cap alpha..'-diamino acids are of particular interest as irradiation products because they represent a class of compounds not normally encountered in plant and animal protein sources. Such compounds have, however, been isolated from certain types of bacteria and pathogenic toxins. All of the available data strongly suggest that the ..cap alpha..,..cap alpha..'-diamino acids are produced in significant yield in the radiation sterilization of high protein foods. The importance of initiating extensive chemical and biological studies of initiating extensive chemical and biological studies of these and of other high molecular weight products in irradiated food is emphasized.

Identification of proteins regulated by ferulic acid in a middle cerebral artery occlusion animal model-a proteomics approach.

Science.gov (United States)

Sung, Jin-Hee; Cho, Eun-Hae; Cho, Jae-Hyeon; Won, Chung-Kil; Kim, Myeong-Ok; Koh, Phil-Ok

2012-11-01

Ferulic acid plays a neuroprotective role in cerebral ischemia. The aim of this study was to identify the proteins that are differentially expressed following ferulic acid treatment during ischemic brain injury using a proteomics technique. Middle cerebral artery occlusion (MCAO) was performed to induce a focal cerebral ischemic injury in adult male rats, and ferulic acid (100 mg/kg) or vehicle was administered immediately after MCAO. Brain tissues were collected 24 hr after MCAO. The proteins in the cerebral cortex were separated using two-dimensional gel electrophoresis and were identified by mass spectrometry. We detected differentially expressed proteins between vehicle- and ferulic acid-treated animals. Adenosylhomocysteinase, isocitrate dehydrogenase [NAD(+)], mitogen-activated protein kinase kinase 1 and glyceraldehyde-3-phosphate dehydrogenase were decreased in the vehicle-treated group, and ferulic acid prevented the injury-induced decreases in these proteins. However, pyridoxal phosphate phosphatase and heat shock protein 60 were increased in the vehicle-treated group, while ferulic acid prevented the injury-induced increase in these proteins. It is accepted that these enzymes are involved in cellular metabolism and differentiation. Thus, these findings suggest evidence that ferulic acid plays a neuroprotective role against focal cerebral ischemia through the up- and down-modulation of specific enzymes.

Amino acid size, charge, hydropathy indices and matrices for protein structure analysis

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Biro JC

2006-03-01

Full Text Available Abstract Background Prediction of protein folding and specific interactions from only the sequence (ab initio is a major challenge in bioinformatics. It is believed that such prediction will prove possible if Anfinsen's thermodynamic principle is correct for all kinds of proteins, and all the information necessary to form a concrete 3D structure is indeed present in the sequence. Results We indexed the 200 possible amino acid pairs for their compatibility regarding the three major physicochemical properties – size, charge and hydrophobicity – and constructed Size, Charge and Hydropathy Compatibility Indices and Matrices (SCI & SCM, CCI & CCM, and HCI & HCM. Each index characterized the expected strength of interaction (compatibility of two amino acids by numbers from 1 (not compatible to 20 (highly compatible. We found statistically significant positive correlations between these indices and the propensity for amino acid co-locations in real protein structures (a sample containing total 34630 co-locations in 80 different protein structures: for HCI: p We tried to predict or reconstruct simple 2D representations of 3D structures from the sequence using these matrices by applying a dot plot-like method. The location and pattern of the most compatible subsequences was very similar or identical when the three fundamentally different matrices were used, which indicates the consistency of physicochemical compatibility. However, it was not sufficient to choose one preferred configuration between the many possible predicted options. Conclusion Indexing of amino acids for major physico-chemical properties is a powerful approach to understanding and assisting protein design. However, it is probably insufficient itself for complete ab initio structure prediction.

Amino acids digestibility of pelleted microparticle protein of fish meal and soybean meal in broiler chickens

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N. Suthama

2018-05-01

Full Text Available Commom protein sources for poultry, fish meal and soybean meal, were ground to obtain reduced particle size. The particle was then dissolved in distilled water (1 : 4 w/v, and added with 2 mL virgin coconut oil for every 500 mL solution prior to ultrasound transducer (ultrasonic bath treatment to obtain protein microparticle. Reducing particle size is one possible way to increase protein utilization.180 birds were used for forced feeding and 10 other birds were plotted for endogenous correction, when they were one month and a half old. Microparticle protein of both ingredients were tested separately in either mash or pelleted forms and compared to intact protein. Completely randomized design with 3 treatments (intact, mash, and pellet and 6 replications (10 bidrs each was arranged for the respective ingredient. Protein and essential amino acid digestibilities, and calcium retention were the parameters measured. Analysis of variance continued to Duncan test were applied to statistically evaluate the data. Pelleted microparticle protein of fish meal and soybean meal, respectively, resulted in significantly (P<0.05 highest protein and amino acids digestibilities, and Ca retention although lower disgestibility of fewer amino acids was found in mash form. In conclusion, pelleted form of microparticle protein of either fish meal or soybean meal improve protein and mostly amino acids digestibilities, and calcium retention in broiler.

Chemical composition and ruminal degradation kinetics of crude protein and amino acids, and intestinal digestibility of amino acids from tropical forages

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Lidia Ferreira Miranda

2012-03-01

Full Text Available The objective of this research was to determine the chemical composition and ruminal degradation of the crude protein (CP, total and individual amino acids of leaves from tropical forages: perennial soybean (Neonotonia wightii, cassava (Manihot esculenta, leucaena (Leucaena leucocephala and ramie (Boehmeria nivea, and to estimate the intestinal digestibility of the rumen undegradable protein (RUDP and individual amino acids of leaves from the tropical forages above cited, but including pigeon pea (Cajanus cajan. Three nonlactating Holstein cows were used to determine the in situ ruminal degradability of protein and amino acids from leaves (6, 18 and 48 hours of ruminal incubation. For determination of the intestinal digestibility of RUDP, the residue from ruminal incubation of the materials was used for 18 hours. A larger concentration of total amino acids for ramie and smaller for perennial soybean were observed; however, they were very similar in leucaena and cassava. Leucine was the essential amino acid of greater concentration, with the exception of cassava, which exhibited a leucine concentration 40.45% smaller. Ramie showed 14.35 and 22.31% more lysine and methionine, respectively. The intestinal digestibility of RUDP varied from 23.56; 47.87; 23.48; 25.69 and 10.86% for leucaena, perennial soybean, cassava, ramie and pigeon pea, respectively. The individual amino acids of tropical forage disappeared in different extensions in the rumen. For the correct evaluation of those forages, one should consider their composition of amino acids, degradations and intestinal digestibility, once the amino acid composition of the forage does not reflect the amino acid profiles that arrived in the small intestine. Differences between the degradation curves of CP and amino acids indicate that degradation of amino acids cannot be estimated through the degradation curve of CP, and that amino acids are not degraded in a similar degradation profile.

Quantitation of glial fibrillary acidic protein in human brain tumours

DEFF Research Database (Denmark)

Rasmussen, S; Bock, E; Warecka, K

1980-01-01

The glial fibrillary acidic protein (GFA) content of 58 human brain tumours was determined by quantitative immunoelectrophoresis, using monospecific antibody against GFA. Astrocytomas, glioblastomas, oligodendrogliomas, spongioblastomas, ependymomas and medulloblastomas contained relatively high...

Glioblastoma Cell Malignancy and Drug Sensitivity Are Affected by the Cell of Origin

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Yiwen Jiang

2017-01-01

Full Text Available The identity of the glioblastoma (GBM cell of origin and its contributions to disease progression and treatment response remain largely unknown. We have analyzed how the phenotypic state of the initially transformed cell affects mouse GBM development and essential GBM cell (GC properties. We find that GBM induced in neural stem-cell-like glial fibrillary acidic protein (GFAP-expressing cells in the subventricular zone of adult mice shows accelerated tumor development and produces more malignant GCs (mGC1GFAP that are less resistant to cancer drugs, compared with those originating from more differentiated nestin- (mGC2NES or 2,′3′-cyclic nucleotide 3′-phosphodiesterase (mGC3CNP-expressing cells. Transcriptome analysis of mouse GCs identified a 196 mouse cell origin (MCO gene signature that was used to partition 61 patient-derived GC lines. Human GC lines that clustered with the mGC1GFAP cells were also significantly more self-renewing, tumorigenic, and sensitive to cancer drugs compared with those that clustered with mouse GCs of more differentiated origin.

Alterations of proliferation and differentiation of hippocampal cells in prenatally stressed rats.

Science.gov (United States)

Sun, Hongli; Su, Qian; Zhang, Huifang; Liu, Weimin; Zhang, Huiping; Ding, Ding; Zhu, Zhongliang; Li, Hui

2015-06-01

To clarify the alterations of proliferation and differentiation of hippocampal cells in prenatally stressed rats. We investigated the impact of prenatal restraint stress on the hipocampal cell proliferation in the progeny with 5-bromo-2'-deoxyuridine (BrdU), which is a marker of proliferating cells and their progeny. In addition, we observed the differentiation of neural stem cells (NSCs) with double labeling of BrdU/neurofilament (NF), BrdU/glial fibrillary acidic protein (GFAP) in the hipocampus. Prenatal stress (PS) increased cell proliferation in the dentate gyrus (DG) only in female and neuron differentiation of newly divided cells in the DG and CA4 in both male and female. Moreover, the NF and GFAP-positive cells, but not the BrdU-positive cells, BrdU/NF and BrdU/GFAP-positive cells, were found frequently in the CA3 and CA1 in the offspring of each group. These results possibly suggest a compensatory adaptive response to neuronal damage or loss in hippocampus induced by PS. Copyright © 2014 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Melamine and Cyanuric Acid do not interfere with Bradford and Ninhydrin assays for protein determination.

Science.gov (United States)

Field, Anjalie; Field, Jeffrey

2010-08-01

In the fall of 2007 pet food contaminated with melamine and cyanuric acid caused kidney stones in thousands of animals. In the summer of 2008, a more serious outbreak of adulterated dairy food caused the deaths of six infants and sickened about 290,000 children in China. In all cases, melamine was likely added to inflate the apparent protein content of the foods. To determine if we could measure protein without interference from melamine and cyanuric acid we tested these compounds in the Bradford and Ninhydrin assays, two common dye-based assays for protein, as well as by ammonia release, the most common assay used in the food industry. Neither compound was detected in the Ninhydrin and Bradford assays at concentrations of >100 μg/ml. The ammonia assay detected melamine but was inconclusive with respect to cyanuric acid. To develop an accurate test for food that would not detect either chemical as a protein, assays were run on cat food and reconstituted milk powder. The Bradford assay readily measured the protein content of each food, and importantly, the addition of melamine or cyanuric acid to reconstituted milk did not affect the readings. The protein concentrations obtained for reconstituted milk powder were as expected, but those for the cat food were 10 to 30-fold lower, due to its low solubility. We conclude that dye-binding assays can be employed to detect protein in food without interference from melamine and cyanuric acid, thus reducing the incentive to use them as additives.

Nucleic acid-binding properties of the RRM-containing protein RDM1

International Nuclear Information System (INIS)

Hamimes, Samia; Bourgeon, Dominique; Stasiak, Alicja Z.; Stasiak, Andrzej; Van Dyck, Eric

2006-01-01

RDM1 (RAD52 Motif 1) is a vertebrate protein involved in the cellular response to the anti-cancer drug cisplatin. In addition to an RNA recognition motif, RDM1 contains a small amino acid motif, named RD motif, which it shares with the recombination and repair protein, RAD52. RDM1 binds to single- and double-stranded DNA, and recognizes DNA distortions induced by cisplatin adducts in vitro. Here, we have performed an in-depth analysis of the nucleic acid-binding properties of RDM1 using gel-shift assays and electron microscopy. We show that RDM1 possesses acidic pH-dependent DNA-binding activity and that it binds RNA as well as DNA, and we present evidence from competition gel-shift experiments that RDM1 may be capable of discrimination between the two nucleic acids. Based on reported studies of RAD52, we have generated an RDM1 variant mutated in its RD motif. We find that the L 119 GF → AAA mutation affects the mode of RDM1 binding to single-stranded DNA

Lactic acid induces aberrant amyloid precursor protein processing by promoting its interaction with endoplasmic reticulum chaperone proteins.

Directory of Open Access Journals (Sweden)

Yiwen Xiang

Full Text Available BACKGROUND: Lactic acid, a natural by-product of glycolysis, is produced at excess levels in response to impaired mitochondrial function, high-energy demand, and low oxygen availability. The enzyme involved in the production of β-amyloid peptide (Aβ of Alzheimer's disease, BACE1, functions optimally at lower pH, which led us to investigate a potential role of lactic acid in the processing of amyloid precursor protein (APP. METHODOLOGY/PRINCIPAL FINDINGS: Lactic acid increased levels of Aβ40 and 42, as measured by ELISA, in culture medium of human neuroblastoma cells (SH-SY5Y, whereas it decreased APP metabolites, such as sAPPα. In cell lysates, APP levels were increased and APP was found to interact with ER-chaperones in a perinuclear region, as determined by co-immunoprecipitation and fluorescence microscopy studies. Lactic acid had only a very modest effect on cellular pH, did increase the levels of ER chaperones Grp78 and Grp94 and led to APP aggregate formation reminiscent of aggresomes. CONCLUSIONS/SIGNIFICANCE: These findings suggest that sustained elevations in lactic acid levels could be a risk factor in amyloidogenesis related to Alzheimer's disease through enhanced APP interaction with ER chaperone proteins and aberrant APP processing leading to increased generation of amyloid peptides and APP aggregates.

Far UV irradiation of DNA in the presence of proteins, amino acids or peptides

International Nuclear Information System (INIS)

Larcom, L.L.; Rains, C.A.

1985-01-01

The DNA of bacteriophage SPO2c12 was subjected to 254 nm irradiation in solutions containing lysozyme or histone. The sensitivity of phage DNA to biological inactivation by UV increased as the amount of lysozyme bound per DNA strand increased. Although binding constants could not be measured for the DNA-histone interaction, this protein had a protective effect which was greater under conditions which cause enhanced binding. No crosslinking of either protein could be detected. Irradiation was also performed in the presence of various amino acids and short peptides. These were chosen to include amino acids which: (1) are positively charged, (2) absorb UV of this wavelength or (3) form UV-induced crosslinks to DNA. None of the amino acids tested affected sensitivity of the DNA to biological inactivation. Peptides containing a UV-absorbing amino acid and a positively charged amino acid enhanced sensitivity. For each of these peptides, a mixture of the constituent amino acids had the same effect as the peptide itself. Under the conditions used, no evidence for formation of DNA-amino acid crosslinks was found. The results indicate that proteins and peptides can sensitize DNA to UV inactivation by mechanisms other than covalent crosslink formation. (author)

Muscle protein degradation and amino acid metabolism during prolonged knee-extensor exercise in humans

DEFF Research Database (Denmark)

Van Hall, Gerrit; Saltin, B; Wagenmakers, A J

1999-01-01

to a substantial increase in net muscle protein degradation, and that a lowering of the starting muscle glycogen content leads to a further increase. The carbon atoms of the branched-chain amino acids (BCAA), glutamate, aspartate and asparagine, liberated by protein degradation, and the BCAA and glutamate......The aim of this study was to investigate whether prolonged one-leg knee-extensor exercise enhances net protein degradation in muscle with a normal or low glycogen content. Net amino acid production, as a measure of net protein degradation, was estimated from leg exchange and from changes...... in the concentrations of amino acids that are not metabolized in skeletal muscle. Experiments were performed at rest and during one-leg knee-extensor exercise in six subjects having one leg with a normal glycogen content and the other with a low glycogen content. Exercise was performed for 90 min at a workload of 60...

Tetrahymena thermophila acidic ribosomal protein L37 contains an archaebacterial type of C-terminus.

Science.gov (United States)

Hansen, T S; Andreasen, P H; Dreisig, H; Højrup, P; Nielsen, H; Engberg, J; Kristiansen, K

1991-09-15

We have cloned and characterized a Tetrahymena thermophila macronuclear gene (L37) encoding the acidic ribosomal protein (A-protein) L37. The gene contains a single intron located in the 3'-part of the coding region. Two major and three minor transcription start points (tsp) were mapped 39 to 63 nucleotides upstream from the translational start codon. The uppermost tsp mapped to the first T in a putative T. thermophila RNA polymerase II initiator element, TATAA. The coding region of L37 predicts a protein of 109 amino acid (aa) residues. A substantial part of the deduced aa sequence was verified by protein sequencing. The T. thermophila L37 clearly belongs to the P1-type family of eukaryotic A-proteins, but the C-terminal region has the hallmarks of archaebacterial A-proteins.

A comparative study of ribosomal proteins: linkage between amino acid distribution and ribosomal assembly

International Nuclear Information System (INIS)

Lott, Brittany Burton; Wang, Yongmei; Nakazato, Takuya

2013-01-01

Assembly of the ribosome from its protein and RNA constituents must occur quickly and efficiently in order to synthesize the proteins necessary for all cellular activity. Since the early 1960’s, certain characteristics of possible assembly pathways have been elucidated, yet the mechanisms that govern the precise recognition events remain unclear. We utilize a comparative analysis to investigate the amino acid composition of ribosomal proteins (r-proteins) with respect to their role in the assembly process. We compared small subunit (30S) r-protein sequences to those of other housekeeping proteins from 560 bacterial species and searched for correlations between r-protein amino acid content and factors such as assembly binding order, environmental growth temperature, protein size, and contact with ribosomal RNA (rRNA) in the 30S complex. We find r-proteins have a significantly high percent of positive residues, which are highly represented at rRNA contact sites. An inverse correlation between the percent of positive residues and r-protein size was identified and is mainly due to the content of Lysine residues, rather than Arginine. Nearly all r-proteins carry a net positive charge, but no statistical correlation between the net charge and the binding order was detected. Thermophilic (high-temperature) r-proteins contain increased Arginine, Isoleucine, and Tyrosine, and decreased Serine and Threonine compared to mesophilic (lower-temperature), reflecting a known distinction between thermophiles and mesophiles, possibly to account for protein thermostability. However, this difference in amino acid content does not extend to rRNA contact sites, as the proportions of thermophilic and mesophilic contact residues are not significantly different. Given the significantly higher level of positively charged residues in r-proteins and at contact sites, we conclude that ribosome assembly relies heavily on an electrostatic component of interaction. However, the binding order of

Effect of low protein diet supplemented with or without amino acids ...

African Journals Online (AJOL)

Jane

2011-08-31

Aug 31, 2011 ... Methionine acts as lipotropic agent through its role as an amino acid in balancing crude protein (Hesabi et al., 2006). It is well known that crude protein and lysine interaction is considered to be an important factor which affects performance and carcass quality of growing chicks; so the dietary requirement of.

Phospholipase D and phosphatidic acid in plant defence response: from protein-protein and lipid-protein interactions to hormone signalling.

Science.gov (United States)

Zhao, Jian

2015-04-01

Phospholipase Ds (PLDs) and PLD-derived phosphatidic acids (PAs) play vital roles in plant hormonal and environmental responses and various cellular dynamics. Recent studies have further expanded the functions of PLDs and PAs into plant-microbe interaction. The molecular diversities and redundant functions make PLD-PA an important signalling complex regulating lipid metabolism, cytoskeleton dynamics, vesicle trafficking, and hormonal signalling in plant defence through protein-protein and protein-lipid interactions or hormone signalling. Different PLD-PA signalling complexes and their targets have emerged as fast-growing research topics for understanding their numerous but not yet established roles in modifying pathogen perception, signal transduction, and downstream defence responses. Meanwhile, advanced lipidomics tools have allowed researchers to reveal further the mechanisms of PLD-PA signalling complexes in regulating lipid metabolism and signalling, and their impacts on jasmonic acid/oxylipins, salicylic acid, and other hormone signalling pathways that essentially mediate plant defence responses. This review attempts to summarize the progress made in spatial and temporal PLD/PA signalling as well as PLD/PA-mediated modification of plant defence. It presents an in-depth discussion on the functions and potential mechanisms of PLD-PA complexes in regulating actin filament/microtubule cytoskeleton, vesicle trafficking, and hormonal signalling, and in influencing lipid metabolism-derived metabolites as critical signalling components in plant defence responses. The discussion puts PLD-PA in a broader context in order to guide future research. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Protein and Amino Acid Profile of Filial Etawah Crossbred and Castrated Filial Boer Crossbred Goat Meat

Directory of Open Access Journals (Sweden)

Hari Purnomo

2012-03-01

Full Text Available The aim of this study was to know the protein content and amino acid profile of filial Etawah and castrated Boer goat meat. The results were expected to be used as information about protein content and amino acid composition of filial Etawah and filial castrated Boer goat meat and  as a reference for further experiment about different livestock. The material of the research were loin meat, front  and back thigh of filial Etawah and filial castrated Boer goat meat. Data were analysed with t-test. The results showed that castrated filial Boer goat meat had significantly higher protein content  and 7 essensial amino acids namely lysine, leucine, arginine, phenylalanine, isoleucine, valine and histidine compared to the one from filial Etawah goat meat. Key words: protein, amino acid profiles, goat  meat

Temperature effects on protein depolymerization and amino acid immobilization rates in soils.

Science.gov (United States)

Noll, Lisa; Hu, Yuntao; Zhang, Shasha; Zheng, Qing; Wanek, Wolfgang

2017-04-01

Increasing N deposition, land use change, elevated atmospheric CO2 concentrations and global warming have altered soil nitrogen (N) cycling during the last decades. Those changes affected ecosystem services, such as C and N sequestration in soils, which calls for a better understanding of soil N transformation processes. The cleavage of macromolecular organic N by extracellular enzymes maintains an ongoing flow of new bioavailable organic N into biotic systems and is considered to be the bottle neck of terrestrial N cycling in litter and soils. Recent studies showed that protein depolymerization is susceptible to changes in C and N availabilities. Based on general biological observations the temperature sensitivity of soil organic N processes is expected to depend on whether they are rather enzyme limited (i.e. Q10=2) or diffusion limited (i.e. Q10= 1.0 - 1.3). However, temperature sensitivities of protein depolymerization and amino acid immobilization are still unknown. We therefore here report short-term temperature effects on organic N transformation rates in soils differing in physicochemical parameters but not in climate. Soil samples were collected from two geologically distinct sites close to the LFZ Raumberg-Gumpenstein, Styria, Austria, each from three different management types (arable land, grassland, forest). Four replicates of mineral soil were taken from every site and management type. The area provides a unique opportunity to study geological and management controls in soils without confounding effects of climate and elevation. The soils differ in several soil chemical parameters, such as soil pH, base saturation, soil C: N ratio and SOM content as well as in soil physical parameters, such as soil texture, bulk density and water holding capacity. Soils were pre-incubated at 5, 15 and 25˚ C for one day. Protein depolymerization rates and amino acid immobilization rates were assessed by an isotope pool dilution assay with 15N labeled amino acids at

A histological and functional study on hippocampal formation of normal and diabetic rats [v1; ref status: indexed, http://f1000r.es/y9

Directory of Open Access Journals (Sweden)

Shaimaa N Amin

2013-07-01

Full Text Available Background: The hippocampus is a key brain area for many forms of learning and memory and is particularly sensitive to changes in glucose homeostasis. Aim of the work: To investigate in experimentally induced type 1 and 2 diabetes mellitus in rat model the effect of  diabetes mellitus on cognitive functions and related markers of hippocampal synaptic plasticity, and the possible impact of blocking N-methyl-D-aspartic acid (NMDA receptors by memantine. Materials and methods: Seven rat groups were included: non-diabetic control and non-diabetic receiving memantine; type-1 diabetic groups - untreated, treated with insulin alone and treated with insulin and memantine; and type 2 diabetic groups - untreated and memantine treated. Cognitive functions were assessed by the Morris Water Maze and passive avoidance test. Biochemical analysis was done for serum glucose, serum insulin and insulin resistance. Routine histological examination was done, together with immunohistochemistry for detection of the hippocampal learning and memory plasticity marker, namely activity regulated cytoskeletal-associated protein (Arc, and the astrocytes reactivity marker, namely glial fibrillary acidic protein (GFAP.  Results: Both type 1 and 2 untreated diabetic groups showed significantly impaired cognitive performance compared to the non-diabetic group. Treating the type 1 diabetic group with insulin alone significantly improved cognitive performance, but significantly decreased GFAP and Arc compared to the untreated type 1 group. In addition, the type 2 diabetic groups showed a significant decrease in hippocampus GFAP and Arc compared to the non-diabetic groups. Blocking NMDA receptors by memantine significantly increased cognitive performance, GFAP and Arc in the type 1 insulin-memantine group compared to the type 1-insulin group and significantly increased Arc in the type 2-memantine group compared to the untreated type 2 diabetic group. The non-diabetic group

Isolation and characterization of undenatured chlorogenic acid free sunflower (Helianthus annuus) Proteins

NARCIS (Netherlands)

Gonzales-Perez, S.; Merck, K.B.; Vereijken, J.M.; Koningsveld, van G.A.; Gruppen, H.; Voragen, A.G.J.

2002-01-01

A method for obtaining sunflower protein (SFP) isolate, nondenatured and free of chlorogenic acid (CGA), has been developed. During the isolating procedure, the extent of CGA removal and protein denaturation was monitored. The defatted flour contained 2.5 percent CGA as the main phenolic compound.

Contribution of buried aspartic acid to the stability of the PDZ2 protein

International Nuclear Information System (INIS)

Jayasimha, Pruthvi; Shanmuganathan, Aranganathan; Suladze, Saba; Makhatadze, George I.

2012-01-01

Highlights: ► Buried Asp residues on average form 2.5 to 3 hydrogen bonds and/or 0.8 salt bridges. ► Contribution of buried Asp to stability was estimated using model protein PDZ2. ► The energetic contribution of Asp56 to PDZ2 stability estimated to be 18 kJ · mol −1 . ► Findings are discussed in terms of contribution of Asp residues to protein stability. - Abstract: Statistical analysis of protein structures shows that buried aspartic acid residues on average form 2.5 to 3 hydrogen bonds and/or 0.8 potential ionic interactions with other protein groups. To estimate the energetic contribution of such buried groups to the Gibbs free energy of proteins, we measured the effects of amino acid substitutions of D56 in a model protein PDZ2 on its stability. We used temperature-induced unfolding monitored by DSC and denaturant-induced unfolding monitored by the changes in fluorescence intensity. We find that all substitutions of D56 lead to protein unfolding, thus suggesting that this buried hydrogen bonded aspartic acid has a significant contribution to the stability. To quantify the changes in the Gibbs free energy, one of the variants, D56N was stabilized by addition of the protective osmolyte TMAO. Comparison of the stability of the D56N variant with the wild-type PDZ2 in the presence and absence of TMAO allowed us to estimate the contribution of D56 to the protein stability to be 18 kJ · mol −1 . These findings are discussed in terms of contribution of buried ionizable groups to protein stability.

Structure and behaviour of proteins, nucleic acids and viruses from vibrational Raman optical activity

DEFF Research Database (Denmark)

Barron, L.D.; Blanch, E.W.; McColl, I.H.

2003-01-01

stacking arrangement and the mutual orientation of the sugar and base rings around the C-N glycosidic link. The ROA spectra of intact viruses provide information on the folds of the coat proteins and the nucleic acid structure. The large number of structure-sensitive bands in protein ROA spectra...... is especially favourable for fold determination using pattern recognition techniques. This article gives a brief account of the ROA technique and presents the ROA spectra of a selection of proteins, nucleic acids and viruses that illustrate the applications of ROA spectroscopy in biomolecular research....

Determination of proteins induced in response to jasmonic acid and salicylic acid in resistant and susceptible cultivars of tomato.

Science.gov (United States)

Afroz, Amber; Khan, Muhammad Rashid; Komatsu, Setsuko

2010-07-01

Jasmonic acid (JA) and salicylic acid (SA) are signaling molecules that play key roles in the regulation of metabolic processes, reproduction, and defense against pathogens. The proteomics approach was used to identify proteins that are induced by JA and SA in the tomato cultivars Roma and Pant Bahr, which are susceptible and resistant to bacterial wilt, respectively. Threonine deaminase and leucine amino peptidase were upregulated, and ribulose-1,5-bisphosphate carboxylase/oxygenase small chain was downregulated by time-course application of JA. Translationally controlled tumor protein was upregulated by time-course application of SA. Protein disulfide isomerase was upregulated by application of either JA or SA. Proteins related to defense, energy, and protein destination/storage are suspected to be responsible for the susceptibility or resistance of the cultivars. Furthermore, in Roma, iron ABC transporter was upregulated by JA and down-regulated by SA. Iron ABC transporter plays a part in the signal transduction of both JA and SA in cultivars of tomato that are resistant to bacterial wilt.

White clover fractions as protein source for monogastrics - Dry matter digestibility and Protein Digestibility-Corrected Amino Acid Scores

DEFF Research Database (Denmark)

Stødkilde, Lene; Damborg, Vinni K; Jørgensen, Henry

2018-01-01

BACKGROUND: The aim was to evaluate white clover as an alternative protein source for monogastrics. White clover plant and leaves were processed using a screw-press resulting in a solid pulp and a juice from which protein was acid-precipitated. The chemical composition of all fractions...... was determined and digestibility of dry matter (DM) and protein was assessed in an experiment with growing rats. RESULTS: Protein concentrates were produced with crude protein (CP) content of 451 g/kg DM and 530 g/kg DM for white clover plant and leaves, respectively and a pulp with CP content of 313 and 374 g...

Influence of Bleaching on Flavor of 34% Whey Protein Concentrate and Residual Benzoic Acid Concentration in Dried Whey Proteins

Science.gov (United States)

Previous studies have shown that bleaching negatively affects the flavor of 70% whey protein concentrate (WPC70), but bleaching effects on lower-protein products have not been established. Benzoyl peroxide (BP), a whey bleaching agent, degrades to benzoic acid (BA) and may elevate BA concentrations...

Bacillus anthracis Overcomes an Amino Acid Auxotrophy by Cleaving Host Serum Proteins

Science.gov (United States)

Terwilliger, Austen; Swick, Michelle C.; Pflughoeft, Kathryn J.; Pomerantsev, Andrei; Lyons, C. Rick; Koehler, Theresa M.

2015-01-01

ABSTRACT Bacteria sustain an infection by acquiring nutrients from the host to support replication. The host sequesters these nutrients as a growth-restricting strategy, a concept termed “nutritional immunity.” Historically, the study of nutritional immunity has centered on iron uptake because many bacteria target hemoglobin, an abundant circulating protein, as an iron source. Left unresolved are the mechanisms that bacteria use to attain other nutrients from host sources, including amino acids. We employed a novel medium designed to mimic the chemical composition of human serum, and we show here that Bacillus anthracis, the causative agent of anthrax disease, proteolyzes human hemoglobin to liberate essential amino acids which enhance its growth. This property can be traced to the actions of InhA1, a secreted metalloprotease, and extends to at least three other serum proteins, including serum albumin. The results suggest that we must also consider proteolysis of key host proteins to be a way for bacterial pathogens to attain essential nutrients, and we provide an experimental framework to determine the host and bacterial factors involved in this process. IMPORTANCE The mechanisms by which bacterial pathogens acquire nutrients during infection are poorly understood. Here we used a novel defined medium that approximates the chemical composition of human blood serum, blood serum mimic (BSM), to better model the nutritional environment that pathogens encounter during bacteremia. Removing essential amino acids from BSM revealed that two of the most abundant proteins in blood—hemoglobin and serum albumin—can satiate the amino acid requirement for Bacillus anthracis, the causative agent of anthrax. We further demonstrate that hemoglobin is proteolyzed by the secreted protease InhA1. These studies highlight that common blood proteins can be a nutrient source for bacteria. They also challenge the historical view that hemoglobin is solely an iron source for

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.

Analysis of nucleic acid chaperoning by the prion protein and its inhibition by oligonucleotides.

Science.gov (United States)

Guichard, Cécile; Ivanyi-Nagy, Roland; Sharma, Kamal Kant; Gabus, Caroline; Marc, Daniel; Mély, Yves; Darlix, Jean-Luc

2011-10-01

Prion diseases are unique neurodegenerative illnesses associated with the conversion of the cellular prion protein (PrP(C)) into the aggregated misfolded scrapie isoform, named PrP(Sc). Recent studies on the physiological role of PrP(C) revealed that this protein has probably multiple functions, notably in cell-cell adhesion and signal transduction, and in assisting nucleic acid folding. In fact, in vitro findings indicated that the human PrP (huPrP) possesses nucleic acid binding and annealing activities, similarly to nucleic acid chaperone proteins that play essential roles in cellular DNA and RNA metabolism. Here, we show that a peptide, representing the N-terminal domain of huPrP, facilitates nucleic acid annealing by two parallel pathways nucleated through the stem termini. We also show that PrP of human or ovine origin facilitates DNA strand exchange, ribozyme-directed cleavage of an RNA template and RNA trans-splicing in a manner similar to the nucleocapsid protein of HIV-1. In an attempt to characterize inhibitors of PrP-chaperoning in vitro we discovered that the thioaptamer 5'-GACACAAGCCGA-3' was extensively inhibiting the PrP chaperoning activities. At the same time a recently characterized methylated oligoribonucleotide inhibiting the chaperoning activity of the HIV-1 nucleocapsid protein was poorly impairing the PrP chaperoning activities.

Protein and amino acid intakes in a rural area of Bangladesh.

Science.gov (United States)

Heck, Julia E; Nieves, Jeri W; Chen, Yu; Parvez, Faruque; Brandt-Rauf, Paul W; Howe, Geoffrey R; Ahsan, Habibul

2010-06-01

Few studies have described protein and amino acid intakes in rural Bangladesh, a country with considerable undernutrition. The purpose of this population-based study was to assess and describe protein and amino acid intakes in Araihazar, Bangladesh. The study participants were 11,170 adult men and women who participated in the Health Effects of Arsenic Longitudinal Study (HEALS), which had a 98% participation rate. Dietary exposures were assessed by a food-frequency questionnaire that had been designed and validated for the HEALS study population. The mean body mass index (BMI) was 19.7 among all participants, and 34.9% of women and 44.4% of men had a BMI below 18.5. The average caloric intake was 2142 and 2394 kcal/day among women and men, respectively, and the mean protein intake was 67.5 and 78.2 g/day. The largest sources of protein were from rice and fish. Greater protein intake was related to younger age and several socioeconomic measures, including more years of education, land and television ownership, and employment in business, farming, or as a laborer (for men) or as a homemaker (for women). This study found a high prevalence of underweight among study participants. Nonetheless, most participants had adequate protein intake according to Food and Agriculture Organization standards for body weight.

Comparison of the amino acid and peptide composition and postprandial response of beef, hydrolyzed chicken, and whey protein nutritional preparations

Directory of Open Access Journals (Sweden)

Christopher J. Detzel

2016-10-01

Full Text Available Background: Increasing dietary protein intake synergistically improves the effect of exercise to stimulate muscle protein synthesis. The purpose of this study was to evaluate the plasma amino acid response of two novel protein nutritional preparations, beef protein isolate (BeefISO™ and hydrolyzed chicken protein isolate (MyoCHX™. Methods: The postprandial plasma amino acid response over 3 hours was monitored in young adults (n=6 following consumption of 23 grams of WPC, BeefISO™, or MyoCHX™. Amino acid compositional analysis and molecular weight distributions of each protein were performed by HPLC. Statistical analyses were performed using one-way or two-way ANOVA where appropriate and corrected for multiple comparisons to account for the cross-over design. Results: Compositional evaluations revealed similar levels of essential and branched-chain amino acids for WPC and MyoCHX™. While the results of this study predictably demonstrated plasma amino acids levels increased following consumption of the different proteins, the kinetics of the postprandial response was unique to each protein source. WPC and MyoCHX™ were rapidly absorbed with maximum plasma amino acid concentrations observed at 30 and 15 min, respectively. The slightly faster absorption of MyoCHX™ was associated with the increased peptide content of MyoCHX™ (greater than 76% of protein is <2kDa. BeefISO™ exhibited sustained release characteristics as evidenced by increased post prandial amino acid concentrations after 3 hours. Conclusions: The protein preparations studied each had different amino acid profiles and absorption kinetics. WPC and MyoCHX™ contained a higher essential amino acid content and were rapidly absorbed with plasma amino acid concentrations peaking within 30 minutes following consumption. BeefISO™ contained a higher proportion of conditionally essential amino acids that steadily increased in plasma over 3 hours, indicating a sustained release

Preclinical Studies of Induced Pluripotent Stem Cell-Derived Astrocyte Transplantation in ALS

Science.gov (United States)

2014-12-01

antibodies used: Human nuclear antigen (HuNA) (Millipore #MAB1281, 1:100), Tuj1 ( Covance #MRB-435P, 1:500), Glial Fibrillary Acidic Protein (GFAP...Nicholas J. Maragakis, M.D. CONTRACTING ORGANIZATION : Johns Hopkins University School of Medicine REPORT DATE: December 2014 TYPE OF REPORT: Final...Hongjun Song, Ph.D. 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS

Analysis on Protein Profile and Amino Acid of Edible Bird's Nest (Collocalia Fuchiphaga) From Painan

OpenAIRE

Elfita, Lina

2014-01-01

This study was aimed to analyze protein profile and amino acid composition of bird nest from Painan, Pesisir Selatan Distric, West Sumatra. Protein analysis was performed by Sodium Dodecyl Sulphate Polyacrilamide Gel Electrophoresis (SDS-PAGE), meanwhile High Performance Liquid Chromatography (HPLC) was used for analysis of amino acid. Analysis on water extract of bird nest by SDS-PAGE showed six bands which correspond to molecular protein which had molecular weight of 147.2; 142.6; 133.4; 73...

Opioid-dependent growth of glial cultures: Suppression of astrocyte DNA synthesis by met-enkephalin

International Nuclear Information System (INIS)

Stiene-Martin, A.; Hauser, K.F.

1990-01-01

The action of met-enkephalin on the growth of astrocytes in mixed-glial cultures was examined. Primary, mixed-glial cultures were isolated from 1 day-old mouse cerebral hemispheres and continuously treated with either basal growth media, 1 μM met-enkephalin, 1 μM met-enkephalin plus the opioid antagonist naloxone, or naloxone alone. Absolute numbers of neural cells were counted in unstained preparations, while combined [ 3 H]-thymidine autoradiography and glial fibrillary acid protein (GFAP) immunocytochemistry was performed to identify specific changes in astrocytes. When compared to control and naloxone treated cultures, met-enkephalin caused a significant decrease in both total cell numbers, and in [ 3 H]-thymidine incorporation by GFAP-positive cells with flat morphology. These results indicate that met-enkephalin suppresses astrocyte growth in culture

Opioid-dependent growth of glial cultures: Suppression of astrocyte DNA synthesis by met-enkephalin

Energy Technology Data Exchange (ETDEWEB)

Stiene-Martin, A.; Hauser, K.F. (Univ. of Kentucky, Lexington (USA))

1990-01-01

The action of met-enkephalin on the growth of astrocytes in mixed-glial cultures was examined. Primary, mixed-glial cultures were isolated from 1 day-old mouse cerebral hemispheres and continuously treated with either basal growth media, 1 {mu}M met-enkephalin, 1 {mu}M met-enkephalin plus the opioid antagonist naloxone, or naloxone alone. Absolute numbers of neural cells were counted in unstained preparations, while combined ({sup 3}H)-thymidine autoradiography and glial fibrillary acid protein (GFAP) immunocytochemistry was performed to identify specific changes in astrocytes. When compared to control and naloxone treated cultures, met-enkephalin caused a significant decrease in both total cell numbers, and in ({sup 3}H)-thymidine incorporation by GFAP-positive cells with flat morphology. These results indicate that met-enkephalin suppresses astrocyte growth in culture.

Immunocytochemical detection of the microsomal glucose-6-phosphatase in human brain astrocytes.

Science.gov (United States)

Bell, J E; Hume, R; Busuttil, A; Burchell, A

1993-10-01

Using an antibody raised against the catalytic subunit of glucose-6-phosphatase, this enzyme was immunolocalized in many astrocytes in 20 normal human brains. Double immunofluorescence studies showed co-localization of glial fibrillary acidic protein (GFAP) with glucose-6-phosphatase in astrocytes. However, not all GFAP-positive cells were also glucose-6-phosphatase positive, indicating that some astrocytes do not contain demonstrable expression of this enzyme. Reactive astrocytes in a variety of abnormal brains were strongly glucose-6-phosphatase positive, but neoplastic astrocytes were often only weakly positive. Expression of the enzyme could not be demonstrated in radial glia, neurons or oligodendroglia. Astrocytes normally contain glycogen and the demonstration that some astrocytes also contain glucose-6-phosphatase indicates that they are competent for both glycogenolysis and gluconeogenesis, which may be critical for neuronal welfare.

Seasonal variations in the amino acid profile and protein nutritional value of Saccharina latissima cultivated in a commercial IMTA system

DEFF Research Database (Denmark)

Silva Marinho, Goncalo; Holdt, Susan Løvstad; Angelidaki, Irini

2015-01-01

. Aspartic and glutamic acids dominated the amino acid profile, accounting for up to 49 % of the total. Greatest seasonal differences in amino acid composition occurred in July, with leucine contributing most (22.7–26.7 %) of the observed differences. A maximal essential amino acid (EAA) score of 68......Seaweeds have potential for the provision of biomass for food and feed supplements. The demand is increasing especially for proteins as ingredients; however, the amino acid profile is essential for evaluation of the nutritional value of proteins. The year-round protein concentration and amino acid.......9 % (based on WHO/FAO/UNU requirements) was achieved in November 2013. The presence of epiphytes in July to November changed neither the amino acid content nor the EAA score. S. latissima is comparable with wheat as a protein ingredient for fish feed and appears to be a suitable protein/amino acid source...

Peroxisome proliferator-binding protein: identification and partial characterization of nafenopin-, clofibric acid-, and ciprofibrate-binding proteins from rat liver.

Science.gov (United States)

Lalwani, N D; Alvares, K; Reddy, M K; Reddy, M N; Parikh, I; Reddy, J K

1987-01-01

Peroxisome proliferators (PP) induce a highly predictable pleiotropic response in rat and mouse liver that is characterized by hepatomegaly, increase in peroxisome number in hepatocytes, and induction of certain peroxisomal enzymes. The PP-binding protein (PPbP) was purified from rat liver cytosol by a two-step procedure involving affinity chromatography and ion-exchange chromatography. Three PP, nafenopin and its structural analogs clofibric acid and ciprofibrate, were used as affinity ligands and eluting agents. This procedure yields a major protein with an apparent Mr of 70,000 on NaDodSO4/PAGE in the presence of reducing agent and Mr 140,000 (Mr 140,000-160,000) on gel filtration and polyacrylamide gradient gel electrophoresis under nondenaturing conditions, indicating that the active protein is a dimer. This protein has an acidic pI of 4.2 under nondenaturing conditions, which rises to 5.6 under denaturing conditions. The isolation of the same Mr 70,000 protein with three different, but structurally related, agents as affinity ligands and the immunological identity of the isolated proteins constitute strong evidence that this protein is the PPbP capable of recognizing PP that are structurally related to clofibrate. The PPbP probably plays an important role in the regulation of PP-induced pleiotropic response. Images PMID:3474650

Ascorbic acid glycation of lens proteins produces UVA sensitizers similar to those in human lens

International Nuclear Information System (INIS)

Ortwerth, B.J.; Linetsky, Mikhail; Olesen, P.R.

1995-01-01

Soluble calf lens proteins were extensively glycated during a 4 week incubation with ascorbic acid in the presence of oxygen. Amino acids analysis of the dialyzed proteins removed at weekly intervals showed an increasing loss of lysine, arginine and histidine, consistent with the extensive protein cross-linking observed. Irradiation of the dialyzed samples with UVA light (1.0 kJ/cm 2 total illumination through a 338 nm cutoff filter) caused an increasing loss of tryptophan, an additional loss of histidine and the production of micromolar concentrations of hydrogen peroxide. No alteration in amino acid content and no photolytic effects were seen in proteins incubated without ascorbic acid in proteins incubated with glucose for 4 weeks. The rate of hydrogen peroxide formation was linear with each glycated sample with a maximum production of 25 nmol/mg protein illuminated. The possibility that the sensitizer activity was due to an ascorbate-induced oxidation of tryptophan was eliminated by the presence of a heavy metal ion chelator during the incubation and by showing equivalent effects with ascorbate-incubated ribonuclease A, which is devoid of tryptophan. The ascorbate-incubated samples displayed increasing absorbance at wavelengths above 300 nm and increasing fluorescence (340/430) as glycation proceeded. The spectra of the 4 week glycated proteins were identical to those obtained with a solubilized water-insoluble fraction from human lens, which is known to have UVA sensitizer activity. (Author)

Bile Acids Trigger GLP-1 Release Predominantly by Accessing Basolaterally Located G Protein-Coupled Bile Acid Receptors

DEFF Research Database (Denmark)

Brighton, Cheryl A.; Rievaj, Juraj; Kuhre, Rune E.

2015-01-01

Bile acids are well-recognized stimuli of glucagon-like peptide-1 (GLP-1) secretion. This action has been attributed to activation of the G protein-coupled bile acid receptor GPBAR1 (TGR5), although other potential bile acid sensors include the nuclear farnesoid receptor and the apical sodium......-coupled bile acid transporter ASBT. The aim of this study was to identify pathways important for GLP-1 release and to determine whether bile acids target their receptors on GLP-1-secreting L-cells from the apical or basolateral compartment. Using transgenic mice expressing fluorescent sensors specifically in L...... to either TLCA or TDCA. We conclude that the action of bile acids on GLP-1 secretion is predominantly mediated by GPBAR1 located on the basolateral L-cell membrane, suggesting that stimulation of gut hormone secretion may include postabsorptive mechanisms....

Intracerebroventricular administration of okadaic acid induces hippocampal glucose uptake dysfunction and tau phosphorylation.

Science.gov (United States)

Broetto, Núbia; Hansen, Fernanda; Brolese, Giovana; Batassini, Cristiane; Lirio, Franciane; Galland, Fabiana; Dos Santos, João Paulo Almeida; Dutra, Márcio Ferreira; Gonçalves, Carlos-Alberto

2016-06-01

Intraneuronal aggregates of neurofibrillary tangles (NFTs), together with beta-amyloid plaques and astrogliosis, are histological markers of Alzheimer's disease (AD). The underlying mechanism of sporadic AD remains poorly understood, but abnormal hyperphosphorylation of tau protein is suggested to have a role in NFTs genesis, which leads to neuronal dysfunction and death. Okadaic acid (OKA), a strong inhibitor of protein phosphatase 2A, has been used to induce dementia similar to AD in rats. We herein investigated the effect of intracerebroventricular (ICV) infusion of OKA (100 and 200ng) on hippocampal tau phosphorylation at Ser396, which is considered an important fibrillogenic tau protein site, and on glucose uptake, which is reduced early in AD. ICV infusion of OKA (at 200ng) induced a spatial cognitive deficit, hippocampal astrogliosis (based on GFAP increment) and increase in tau phosphorylation at site 396 in this model. Moreover, we observed a decreased glucose uptake in the hippocampal slices of OKA-treated rats. In vitro exposure of hippocampal slices to OKA altered tau phosphorylation at site 396, without any associated change in glucose uptake activity. Taken together, these findings further our understanding of OKA neurotoxicity, in vivo and vitro, particularly with regard to the role of tau phosphorylation, and reinforce the importance of the OKA dementia model for studying the neurochemical alterations that may occur in AD, such as NFTs and glucose hypometabolism. Copyright © 2016 Elsevier Inc. All rights reserved.

Structure and expression of an unusually acidic matrix protein of pearl oyster shells

International Nuclear Information System (INIS)

Tsukamoto, Daiki; Sarashina, Isao; Endo, Kazuyoshi

2004-01-01

We report identification and characterization of the unusually acidic molluscan shell matrix protein Aspein, which may have important roles in calcium carbonate biomineralization. The Aspein gene (aspein) encodes a sequence of 413 amino acids, including a high proportion of Asp (60.4%), Gly (16.0%), and Ser (13.2%), and the predicted isoelectric point is 1.45; this is the most acidic of all the molluscan shell matrix proteins sequenced so far, or probably even of all known proteins on earth. The main body of Aspein is occupied by (Asp) 2-10 sequences punctuated with Ser-Gly dipeptides. RT-PCR demonstrated that the transcript of aspein is expressed at the outer edge of the mantle, corresponding to the calcitic prismatic layer, but not at the inner part of the mantle, corresponding to the aragonitic nacreous layer. Our findings and previous in vitro experiments taken together suggest that Aspein is responsible for directed formation of calcite in the shell of the pearl oyster Pinctada fucata

Fish protein hydrolysates: proximate composition, amino acid composition, antioxidant activities and applications: a review.

Science.gov (United States)

Chalamaiah, M; Dinesh Kumar, B; Hemalatha, R; Jyothirmayi, T

2012-12-15

The fish processing industry produces more than 60% by-products as waste, which includes skin, head, viscera, trimmings, liver, frames, bones, and roes. These by-product wastes contain good amount of protein rich material that are normally processed into low market-value products, such as animal feed, fish meal and fertilizer. In view of utilizing these fish industry wastes, and for increasing the value to several underutilised fish species, protein hydrolysates from fish proteins are being prepared by several researchers all over the world. Fish protein hydrolysates are breakdown products of enzymatic conversion of fish proteins into smaller peptides, which normally contain 2-20 amino acids. In recent years, fish protein hydrolysates have attracted much attention of food biotechnologists due to the availability of large quantities of raw material for the process, and presence of high protein content with good amino acid balance and bioactive peptides (antioxidant, antihypertensive, immunomodulatory and antimicrobial peptides). Copyright © 2012 Elsevier Ltd. All rights reserved.

Comparison of femtosecond laser and continuous wave UV sources for protein-nucleic acid crosslinking.

Science.gov (United States)

Fecko, Christopher J; Munson, Katherine M; Saunders, Abbie; Sun, Guangxing; Begley, Tadhg P; Lis, John T; Webb, Watt W

2007-01-01

Crosslinking proteins to the nucleic acids they bind affords stable access to otherwise transient regulatory interactions. Photochemical crosslinking provides an attractive alternative to formaldehyde-based protocols, but irradiation with conventional UV sources typically yields inadequate product amounts. Crosslinking with pulsed UV lasers has been heralded as a revolutionary technique to increase photochemical yield, but this method had only been tested on a few protein-nucleic acid complexes. To test the generality of the yield enhancement, we have investigated the benefits of using approximately 150 fs UV pulses to crosslink TATA-binding protein, glucocorticoid receptor and heat shock factor to oligonucleotides in vitro. For these proteins, we find that the quantum yields (and saturating yields) for forming crosslinks using the high-peak intensity femtosecond laser do not improve on those obtained with low-intensity continuous wave (CW) UV sources. The photodamage to the oligonucleotides and proteins also has comparable quantum yields. Measurements of the photochemical reaction yields of several small molecules selected to model the crosslinking reactions also exhibit nearly linear dependences on UV intensity instead of the previously predicted quadratic dependence. Unfortunately, these results disprove earlier assertions that femtosecond pulsed laser sources provide significant advantages over CW radiation for protein-nucleic acid crosslinking.

Myeloperoxidase-mediated protein lysine oxidation generates 2-aminoadipic acid and lysine nitrile in vivo.

Science.gov (United States)

Lin, Hongqiao; Levison, Bruce S; Buffa, Jennifer A; Huang, Ying; Fu, Xiaoming; Wang, Zeneng; Gogonea, Valentin; DiDonato, Joseph A; Hazen, Stanley L

2017-03-01

Recent studies reveal 2-aminoadipic acid (2-AAA) is both elevated in subjects at risk for diabetes and mechanistically linked to glucose homeostasis. Prior studies also suggest enrichment of protein-bound 2-AAA as an oxidative post-translational modification of lysyl residues in tissues associated with degenerative diseases of aging. While in vitro studies suggest redox active transition metals or myeloperoxidase (MPO) generated hypochlorous acid (HOCl) may produce protein-bound 2-AAA, the mechanism(s) responsible for generation of 2-AAA during inflammatory diseases are unknown. In initial studies we observed that traditional acid- or base-catalyzed protein hydrolysis methods previously employed to measure tissue 2-AAA can artificially generate protein-bound 2-AAA from an alternative potential lysine oxidative product, lysine nitrile (LysCN). Using a validated protease-based digestion method coupled with stable isotope dilution LC/MS/MS, we now report protein bound 2-AAA and LysCN are both formed by hypochlorous acid (HOCl) and the MPO/H 2 O 2 /Cl - system of leukocytes. At low molar ratio of oxidant to target protein N ε -lysine moiety, 2-AAA is formed via an initial N ε -monochloramine intermediate, which ultimately produces the more stable 2-AAA end-product via sequential generation of transient imine and semialdehyde intermediates. At higher oxidant to target protein N ε -lysine amine ratios, protein-bound LysCN is formed via initial generation of a lysine N ε -dichloramine intermediate. In studies employing MPO knockout mice and an acute inflammation model, we show that both free and protein-bound 2-AAA, and in lower yield, protein-bound LysCN, are formed by MPO in vivo during inflammation. Finally, both 2-AAA and to lesser extent LysCN are shown to be enriched in human aortic atherosclerotic plaque, a tissue known to harbor multiple MPO-catalyzed protein oxidation products. Collectively, these results show that MPO-mediated oxidation of protein lysyl

Application of infrared portable sensor technology for predicting perceived astringency of acidic whey protein beverages.

Science.gov (United States)

Wang, Ting; Tan, Siow-Ying; Mutilangi, William; Plans, Marcal; Rodriguez-Saona, Luis

2016-12-01

Formulating whey protein beverages at acidic pH provides better clarity but the beverages typically develop an unpleasant and astringent flavor. Our aim was to evaluate the application of infrared spectroscopy and chemometrics in predicting astringency of acidic whey protein beverages. Whey protein isolate (WPI), whey protein concentrate (WPC), and whey protein hydrolysate (WPH) from different manufacturers were used to formulate beverages at pH ranging from 2.2 to 3.9. Trained panelists using the spectrum method of descriptive analysis tested the beverages providing astringency scores. A portable Fourier transform infrared spectroscopy attenuated total reflectance spectrometer was used for spectra collection that was analyzed by multivariate regression analysis (partial least squares regression) to build calibration models with the sensory astringency scores. Beverage astringency scores fluctuated from 1.9 to 5.2 units and were explained by pH, protein type (WPC, WPI, or WPH), source (manufacturer), and their interactions, revealing the complexity of astringency development in acidic whey protein beverages. The WPC and WPH beverages showed an increase in astringency as the pH of the solution was lowered, but no relationship was found for WPI beverages. The partial least squares regression analysis showed strong relationship between the reference astringency scores and the infrared predicted values (correlation coefficient >0.94), giving standard error of cross-validation ranging from 0.08 to 0.12 units, depending on whey protein type. Major absorption bands explaining astringency scores were associated with carboxylic groups and amide regions of proteins. The portable infrared technique allowed rapid prediction of astringency of acidic whey protein beverages, providing the industry a novel tool for monitoring sensory characteristics of whey-containing beverages. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

A single extracellular amino acid in Free Fatty Acid Receptor 2 defines antagonist species selectivity and G protein selection bias

DEFF Research Database (Denmark)

Sergeev, Eugenia; Hansen, Anders Højgaard; Bolognini, Daniele

2017-01-01

selectivity and mutational swap studies confirmed this hypothesis. Extending these studies to agonist function indicated that although the lysine - arginine variation between human and mouse orthologs had limited effect on G protein-mediated signal transduction, removal of positive charge from this residue...... produced a signalling-biased variant of Free Fatty Acid Receptor 2 in which Gi-mediated signalling by both short chain fatty acids and synthetic agonists was maintained whilst there was marked loss of agonist potency for signalling via Gq/11 and G12/13 G proteins. A single residue at the extracellular face...

Seasonal changes in amino acids, protein and total nitrogen in needles of fertilized Scots pine trees.

Science.gov (United States)

Näsholm, T; Ericsson, A

1990-09-01

Seasonal changes in amino acids, protein and total nitrogen in needles of 30-year-old, fertilized Scots pine (Pinus sylvestris L.) trees growing in Northern Sweden were investigated over two years in field experiments. The studied plots had been fertilized annually for 17 years with (i) a high level of N, (ii) a medium level of N, or (iii) a medium level of N, P and K. Trees growing on unfertilized plots served as controls. In control trees, glutamine, glutamic acid, gamma-aminobutyric acid, aspartic acid and proline represented 50-70% of the total free amino acids determined. Arginine was present only in low concentrations in control trees throughout the year, but it was usually the most abundant amino acid in fertilized trees. Glutamine concentrations were high during the spring and summer in both years of study, whereas proline concentrations were high in the spring but otherwise low throughout the year. In the first year of study, glutamic acid concentrations were high during the spring and summer, whereas gamma-aminobutyric acid was present in high concentrations during the winter months. This pattern was less pronounced in the second year of investigation. The concentrations of most amino acids, except glutamic acid, increased in response to fertilization. Nitrogen fertilization increased the foliar concentration of arginine from trees to a maximum of 110 micromol g(dw) (-1). Trees fertilized with nitrogen, phosphorus and potassium had significantly lower arginine concentrations than trees fertilized with the same amount of nitrogen only. Protein concentrations were similar in all fertilized trees but higher than those in control trees. For all treatments, protein concentrations were high in winter and at a minimum in early spring. In summer, the protein concentration remained almost constant except for a temporary decrease which coincided with the expansion of new shoots. Apart from arginine, the amino acid composition of proteins was similar in all

Photo-induced formation of nitrous acid (HONO) on protein surfaces

Science.gov (United States)

Meusel, Hannah; Elshorbany, Yasin; Bartels-Rausch, Thorsten; Selzle, Kathrin; Lelieveld, Jos; Ammann, Markus; Pöschl, Ulrich; Su, Hang; Cheng, Yafang

2014-05-01

The study of nitrous acid (HONO) is of great interest, as the photolysis of HONO leads to the OH radical, which is the most important oxidant in the troposphere. HONO is directly emitted by combustion of fossil fuel and from soil biogenic nitrite (Su et al., 2011), and can also be formed by gas phase reactions of NO and OH and heterogeneous reactions of NO2. Previous atmospheric measurements have shown unexpectedly high HONO concentrations during daytime. Measured mixing ratios were about one order of magnitude higher than model simulations (Kleffmann et al. 2005, Vogel et al. 2003). The additional daytime source of HONO might be attributed to the photolysis of adsorbed nitric acid or heterogeneous photochemistry of NO2 on organic substrates, such as humic acids or polyphenolic compounds (Stemmler et al., 2006), or indirectly through nitration of phenols and subsequent photolysis of nitrophenols (Sosedova et al., 2011, Bejan et al., 2006). An important reactive surface for the heterogeneous formation of HONO could involve proteins, which are ubiquitous in the environment. They are part of coarse biological aerosol particles like pollen grains, fine particles (fragments of pollen, microorganism, plant debris) and dissolved in rainwater, soil and road dust (Miguel et al. 1999). In this project a thin film of bovine serum albumin (BSA), a model protein with 67 kDa and 21 tyrosine residues per molecule, is irradiated and exposed to nitrogen dioxide in humidified nitrogen. The formation of HONO is measured with long path absorption photometry (LOPAP). The generated HONO is in the range of 100 to 1100 ppt depending on light intensity, NO2 concentration and film thickness. Light induced HONO formation on protein surfaces is stable over the 20-hours experiment of irradiation and exposure. On the other hand, light activated proteins reacting with NO2 form nitrated proteins, as detected by liquid chromatography (LC-DAD). Our experiments on tetranitromethane (TNM) nitrated

Fluoxetine requires the endfeet protein aquaporin-4 to enhance plasticity of astrocyte processes