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BACKGROUND: Autophagy is a highly regulated process involving the bulk degradation of cytoplasmic macromolecules and organelles in mammalian cells via the lysosomal system. Dysregulation of autophagy is implicated in the pathogenesis of many neurodegenerative diseases and integrity of the autophagosomal - lysosomal network appears to be critical in the progression of aging. Our aim was to survey the expression of autophagy markers and Amyloid precursor protein (APP) in aged bovine brains. For our study, we collected samples from the brain of old (aged 11-20 years) and young (aged 1-5 years) Podolic dairy cows. Formalin-fixed and paraffin embedded sections were stained with routine and special staining techniques. Primary antibodies for APP and autophagy markers such as Beclin-1 and LC3 were used to perform immunofluorescence and Western blot analysis. RESULTS: Histologically, the most consistent morphological finding was the age-related accumulation of intraneuronal lipofuscin. Furthermore, in aged bovine brains, immunofluorescence detected a strongly positive immunoreaction to APP and LC3. Beclin-1 immunoreaction was weak or absent. In young controls, the immunoreaction for Beclin-1 and LC3 was mild while the immunoreaction for APP was absent. Western blot analysis confirmed an increased APP expression and LC3-II/LC3-I ratio and a decreased expression of Beclin-1 in aged cows. CONCLUSIONS: These data suggest that, in aged bovine, autophagy is significantly impaired if compared to young animals and they confirm that intraneuronal APP deposition increases with age.
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Precursor de Proteína beta-Amiloide/metabolismo , Autofagia , Encéfalo/metabolismo , Bovinos/fisiologia , Lipofuscina/metabolismo , Envelhecimento/metabolismo , Animais , Proteína Beclina-1/metabolismo , Biomarcadores/metabolismo , Western Blotting , Feminino , Proteínas de Membrana/metabolismoRESUMO
Sarcopenia, the age-related loss of muscle mass and strength, is a multifactorial condition that represents a major healthcare concern for the elderly population. Although its morphologic features have been extensively studied in humans, animal models, and domestic and wild animals, only a few reports about spontaneous sarcopenia exist in other long-lived animals. In this work, muscle samples from 60 healthy Podolica-breed old cows (aged 15-23 years) were examined and compared with muscle samples from 10 young cows (3-6 years old). Frozen sections were studied through standard histologic and histoenzymatic procedures, as well as by immunohistochemistry, immunofluorescence, and Western blot analysis. The most prominent age-related myopathic features seen in the studied material included angular fiber atrophy (90% of cases), mitochondrial alterations (ragged red fibers, 70%; COX-negative fibers, 60%), presence of vacuolated fibers (75%), lymphocytic (predominantly CD8+) inflammation (40%), and type II selective fiber atrophy (40%). Immunohistochemistry revealed increased expression of major histocompatibility complex I in 36 cases (60%) and sarcoplasmic accumulations of ß-amyloid precursor protein-positive material in 18 cases (30%). In aged cows, muscle atrophy was associated with accumulation of myostatin. Western blot analysis indicated increased amount of both proteins-myostatin and ß-amyloid precursor protein-in muscles of aged animals compared with controls. These findings confirm the presence of age-related morphologic changes in cows similar to human sarcopenia and underline the possible role of amyloid deposition and subsequent inflammation in muscle senescence.
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Envelhecimento/patologia , Doenças dos Bovinos/patologia , Músculo Esquelético/patologia , Sarcopenia/veterinária , Animais , Bovinos , Feminino , Atrofia Muscular/patologia , Atrofia Muscular/veterinária , Miostatina/metabolismo , Sarcopenia/patologiaRESUMO
The understanding of mechanisms that provoke neurological diseases in humans and in animals has progressed rapidly in recent years, mainly due to the advent of new research instruments and our increasing liability to assemble large, complex data sets acquired across several approaches into an integrated representation of neural function at the molecular, cellular, and systemic levels. Nevertheless, morphology always represents the essential approaches that are crucial for any kind of interpretation of the lesions or to explain new molecular pathways in the diseases. This mini-review has been designed to illustrate the newest and also well-established principal methods for the nervous tissue collection and processing as well as to describe the histochemical and immunohistochemical staining tools that are currently most suitable for a neuropathological assessment of the central nervous system. We also present the results of our neuropathological studies covering material from 170 cases belonging to 10 different species of mammals. Specific topics briefly addressed in this paper provide a technical and practical guide not only for researchers that daily focus their effort on neuropathology studies, but also to pathologists who occasionally have to approach to nervous tissue evaluation to answer questions about neuropathology issues.
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Hepatitis B virus (HBV) infection represents a serious global health problem and persistent HBV infection is associated with an increased risk of cirrhosis, hepatocellular carcinoma and liver failure. Recently, the study of the role of microRNA (miRNA) in the pathogenesis of HBV has gained considerable interest as well as new treatments against this pathogen have been approved. A few studies have investigated the antiviral activity of vitamin E (VE) in chronic HBV carriers. Herein, we review the possible role of tocopherols in the modulation of host miRNA with potential anti-HBV activity. A systematic research of the scientific literature was performed by searching the MEDLINE, Cochrane Library and EMBASE databases. The keywords used were 'HBV therapy', 'HBV treatment', 'VE antiviral effects', 'tocopherol antiviral activity', 'miRNA antiviral activity' and 'VE microRNA'. Reports describing the role of miRNA in the regulation of HBV life cycle, in vitro and in vivo available studies reporting the effects of VE on miRNA expression profiles and epigenetic networks, and clinical trials reporting the use of VE in patients with HBV-related chronic hepatitis were identified and examined. Based on the clinical results obtained in VE-treated chronic HBV carriers, we provide a reliable hypothesis for the possible role of this vitamin in the modulation of host miRNA profiles perturbed by this viral pathogen and in the regulation of some cellular miRNA with a suggested potential anti-HBV activity. This approach may contribute to the improvement of our understanding of pathogenetic mechanisms involved in HBV infection and increase the possibility of its management and treatment.
Assuntos
Antivirais/uso terapêutico , Hepatite B Crônica/tratamento farmacológico , MicroRNAs/metabolismo , Tocoferóis/uso terapêutico , Genoma Viral , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/genética , Hepatite B Crônica/virologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , MicroRNAs/genética , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Replicação Viral/efeitos dos fármacosRESUMO
Osteosarcoma is the most common primary bone malignancy with a challenging prognosis marked by a high rate of metastasis. The limited success of current treatments may be partially attributed to an incomplete understanding of osteosarcoma pathophysiology and to the absence of reliable in vitro models to select the best molecules for in vivo studies. Among the natural compounds relevant for osteosarcoma treatment, Licochalcone A (Lic-A) and chalcone derivatives are particularly interesting. Here, Lic-A and selected derivatives have been evaluated for their anticancer effect on multicellular tumor spheroids from MG63 and 143B osteosarcoma cell lines. A metabolic activity assay revealed Lic-A, 1i, and 1k derivatives as the most promising candidates. To delve into their mechanism of action, caspase activity assay was conducted in 2D and 3D in vitro models. Notably, apoptosis and autophagic induction was generally observed for Lic-A and 1k. The invasion assay demonstrated that Lic-A and 1k possess the ability to mitigate the spread of osteosarcoma cells within a matrix. The effectiveness of chalcone as a natural scaffold for generating potential antiproliferative agents against osteosarcoma has been demonstrated. In particular, chalcones exert their antiproliferative activity by inducing apoptosis and autophagy, and in addition they are capable of reducing cell invasion. These findings suggest Lic-A and 1k as promising antitumor agents against osteosarcoma cells.
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BACKGROUND: Tumor genotyping is becoming crucial to optimize the clinical management of patients with advanced differentiated thyroid cancer (DTC); however, its implementation in clinical practice remains undefined. We herein report our single-center experience on molecular advanced DTC testing by next-generation sequencing approach, to better define how and when tumor genotyping can assist clinical decision making. MATERIALS AND METHODS: We retrospectively collected data on all adult patients with advanced DTC who received molecular profiling at the IRCSS Sant'Orsola-Malpighi Hospital from 2008 to 2022. The genetic alterations were correlated with radioactive iodide refractory (RAI-R), RAI uptake/disease status, and time to RAI resistance (TTRR) development. RESULTS: A significant correlation was found between RAI-R development and genetic alterations (P = 0.0001). About 48.7% of RAI-R cases were positive for TERT/TP53 mutations (as both a single event and comutations with other driver gene alterations, such as BRAF mutations, RAS mutations, or gene fusions), while the great majority of RAI-sensitive cases carried gene fusions (41.9%) or were wild type (WT; 41.9%). RAI uptake/disease status and time to TTRR were significantly associated with genetic alterations (P = 0.0001). In particular, DTC with TERT/TP53 mutations as a single event or as comutations displayed a shorter median TTRR of 35.4 months (range 15.0-55.8 months), in comparison to the other molecular subgroups. TERT/TP53 mutations as a single event or as comutations remained independently associated with RAI-R after Cox multivariate analysis (hazard ratio 4.14, 95% CI 1.51-11.32; P = 0.006). CONCLUSIONS: Routine testing for genetic alterations should be included as part of the clinical workup, for identifying both the subset of more aggressive tumors and the subset of tumors harboring actionable gene fusions, thus ensuring the appropriate management for all patients with advanced DTC.
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Adenocarcinoma , Neoplasias da Glândula Tireoide , Adulto , Humanos , Estudos Retrospectivos , Relevância Clínica , Neoplasias da Glândula Tireoide/genética , MutaçãoRESUMO
Chlamydia pneumoniae is responsible for respiratory tract infections and has been associated to chronic diseases such as atherosclerosis. The involvement of C. pneumoniae in chronic diseases may be correlated to its ability to induce persistent forms in which Chlamydiae remain viable but are not cultivable. The aim of our study is to investigate C. pneumoniae specific gene activities associated with the development of Chlamydial persistence in a cell culture system in the presence of penicillin G. Chlamydia-infected HEp 2 cells were incubated with or without penicillin G for up to 72 hours. The relative mRNA expression levels of early and late genes in treated and untreated cell cultures were determined by Real-time RT-PCR. Our results revealed a consistent down-regulation of Chlamydial hctA and hctB genes (p=0.012 and p=0.003 respectively) in association with up-regulation of htrA gene (p=0.002) during penicillin G-induced persistence suggesting these gene sets as leading candidate for in vivo investigation of the development of persistent Chlamydial infection. In conclusion, the Chlamydial expression pattern of hctA, hctB, and htrA genes may be helpful to identify target molecules to diagnose and treat Chlamydia-associated chronic diseases.
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Antibacterianos/farmacologia , Chlamydophila pneumoniae/efeitos dos fármacos , Penicilina G/farmacologia , Células Cultivadas , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/crescimento & desenvolvimento , Genes Bacterianos , Humanos , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Atypical protein kinase C (aPKC) and Lethal giant larvae (Lgl) regulate apical-basal polarity in Drosophila and mammalian epithelia. At the apical domain, aPKC phosphorylates and displaces Lgl that, in turn, maintains aPKC inactive at the basolateral region. The mutual exclusion of these two proteins seems to be crucial for the correct epithelial structure and function. Here we show that a cortical aPKC loading induces Lgl cytoplasmic release and massive overgrowth in Drosophila imaginal epithelia, whereas a cytoplasmic expression does not alter proliferation and epithelial overall structure. As two aPKC isoforms (iota and zeta) exist in humans and we previously showed that Drosophila Lgl is the functional homologue of the Human giant larvae-1 (Hugl-1) protein, we argued if the same mechanism of mutual exclusion could be impaired in human epithelial disorders and investigated aPKCiota, aPKCzeta and Hugl-1 localization in cancers deriving from ovarian surface epithelium. Both in mucinous and serous histotypes, aPKCzeta showed an apical-to-cortical redistribution and Hugl-1 showed a membrane-to-cytoplasm release, perfectly recapitulating the Drosophila model. Although several recent works support a causative role for aPKCiota overexpression in human carcinomas, our results suggest a key role for aPKCzeta in apical-basal polarity loosening, a mechanism that seems to be driven by changes in protein localization rather than in protein abundance.
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Citoplasma/metabolismo , Proteínas de Drosophila/metabolismo , Epitélio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica , Neoplasias Ovarianas/metabolismo , Proteína Quinase C/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Proliferação de Células , Drosophila melanogaster , Feminino , Humanos , Neoplasias Ovarianas/genética , Fenótipo , Proteína Quinase C/metabolismo , Asas de Animais/embriologiaRESUMO
Acid resistance (AR) in Escherichia coli is an important trait that protects this microorganism from the deleterious effect of low-pH environments. Reports on biofilm formation in E. coli K12 showed that the genes participating in AR were differentially expressed. Herein, we investigated the relationship between AR genes, in particular those coding for specific transcriptional regulators, and their biofilm-forming ability at the phenotypic level. The latter was measured in 96-well plates by staining the bacteria attached to the well, following 24-hour growth under static conditions, with crystal violet. The growth conditions were as follows: Luria Bertani (LB) medium at neutral and acidic pH, at 37°C or 25°C. We observed that the three major transcriptional regulators of the AR genes (gadX, gadE, gadW) only marginally affected biofilm formation in E. coli. However, a striking and novel finding was the different abilities of all the tested E. coli strains to form a biofilm depending on the temperature and pH of the medium: LB, pH 7.4, strongly supported biofilm formation at 25°C, with biofilm being hardly detectable at 37°C. On the contrary, LB, pH 5.5, best supported biofilm formation at 37°C. Moreover, we observed that when E. coli carried a plasmid, the presence of the plasmid itself affected the ability to develop a biofilm, typically by increasing its formation. This phenomenon varies from plasmid to plasmid, depends on growth conditions, and, to the best of our knowledge, remains largely uninvestigated.
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Escherichia coli glutamate decarboxylase (EcGad) is a homohexameric pyridoxal 5'-phosphate (PLP)-dependent enzyme. It is the structural component of the major acid resistance system that protects E. coli from strong acid stress (pH < 3), typically encountered in the mammalian gastrointestinal tract. In fact EcGad consumes one proton/catalytic cycle while yielding γ-aminobutyrate and carbon dioxide from the decarboxylation of l-glutamate. Two isoforms of Gad occur in E. coli (GadA and GadB) that are 99% identical in sequence. GadB is the most intensively investigated. Prompted by the observation that some transcriptomic and proteomic studies show EcGad to be expressed in conditions far from acidic, we investigated the structural organization of EcGadB in solution in the pH range 7.5-8.6. Small angle X-ray scattering, combined with size exclusion chromatography, and analytical ultracentrifugation analysis show that the compact and entangled EcGadB hexameric structure undergoes dissociation into dimers as pH alkalinizes. When PLP is not present, the dimeric species is the most abundant in solution, though evidence for the occurrence of a likely tetrameric species was also obtained. Trp fluorescence emission spectra as well as limited proteolysis studies suggest that PLP plays a key role in the acquisition of a folding necessary for the canonical catalytic activity.
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Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Glutamato Descarboxilase/química , Proteínas de Membrana/química , Multimerização Proteica , Concentração de Íons de Hidrogênio , Estrutura Quaternária de Proteína , Difração de Raios XRESUMO
One of the most important strategies that enteric bacteria adopt for maintaining the cytoplasmic pH neutral under acid stress involves the glutamate decarboxylase (Gad) system. The system works by the concerted action of a cytoplasmic, pyridoxal 5'-phosphate-dependent glutamate decarboxylase and a transmembrane antiporter, which imports glutamate and exports gamma-aminobutyrate (GABA), the decarboxylation product, thereby providing local buffering of the extracellular environment. Herein, we provide a preliminary biochemical characterization of GadX, an activator of the Gad system belonging to the AraC/XylS family of bacterial transcriptional regulators. The GadX protein has been purified as a chimeric MalE-GadX with a yield of 15-20 mg/l of bacterial culture. The fusion protein is fairly stable, although a conformational change occurs upon storage, which reduces the binding affinity by a factor of 2, without affecting the binding pattern. Partial removal of the MalE moiety from the fusion protein triggers the formation of a species which is likely to be a heterodimer, or a higher oligomer, of the type GadX/MalE-GadX. This experimental evidence is in line with the well-known tendency of AraC/XylS-like proteins to dimerize via their N-terminal domain.
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Fator de Transcrição AraC/química , Proteínas de Escherichia coli/química , Glutamato Descarboxilase/genética , Transativadores/química , Dimerização , Concentração de Íons de Hidrogênio , Proteínas Periplásmicas de Ligação/química , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
The peptide fraction extracted by methanol from the skin of Rana esculenta, a species widely distributed in Western Europe, was investigated. The pharmacological activity found in the extract is attributable to the presence of authentic bradykinin, together with a shorter, partially active version of this molecule, des-Arg9-bradykinin. Also the bradykinin fragment 1-7 has been isolated, but it was inactive in our bioassay system. Moreover, a family of hydrophobic peptides has been purified and characterized, which appeared devoid of pharmacological activities when tested on smooth muscle preparations, but were provided with hemolytic activities.
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Peptídeos/isolamento & purificação , Rana esculenta/metabolismo , Pele/análise , Sequência de Aminoácidos , Animais , Bradicinina/análogos & derivados , Bradicinina/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Hemólise/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Peptídeos/farmacologia , Conformação ProteicaRESUMO
gamma-Aminobutyric acid transaminase from pig liver, an alpha 2 dimeric enzyme of Mr 110,100, has been crystallized by the vapour diffusion method with polyethylene glycol as precipitant. The crystals are monoclinic, space group P2(1), unit cell dimensions a = 82.1 A, b = 230.0 A, c = 70.3 A, beta = 123.9 degrees and diffract to 2.5 A resolution. There are two dimers per asymmetric unit.
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4-Aminobutirato Transaminase/isolamento & purificação , Fígado/enzimologia , Animais , Cristalização , Polietilenoglicóis , Conformação Proteica , Suínos , Difração de Raios XRESUMO
INTRODUCTION: Glioblastoma (GBM) is the most frequent primary malignant brain tumour. Despite advances in treatment its prognosis remains poor. Histological features of GBM are well known. On the contrary histological description of recurrences is still not available. The aim of this study was to describe the morphological, immunohistochemical and molecular features of recurrent GBMs. METHODS: 25 recurrent GBMs, diagnosed after 2005, were collected. All patients had undergone an adjuvant treatment regimen (temozolomide and/or radiotherapy). All cases were immunostained using anti-GFAP, Olig2 and Nogo-A antisera. MGMT and IDH1 status was reassessed. Features of the recurrences were compared with those of primary GBMs, time of recurrence and survival. RESULTS: Recurrences were divided morphologically into three groups: 1) recurrences displaying the same features of primary GBM, were highly cellular, had the fastest progression and the worst prognosis; 2) recurrences changing dramatically morphological appearance, had a slightly longer survival, 3) poorly cellular recurrences, with sparse neoplastic cells intermingled with reactive and necrotic tissue, displayed the slowest progression and longer survival. MGMT and IDH1 status remained unchanged between primary tumours and recurrences. DISCUSSION: GBM histological subtypes display different reactions to adjuvant treatments, offering a possible role in predicting different recurrence and survival time.
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Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Recidiva Local de Neoplasia , Adulto , Idoso , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/química , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/terapia , Metilação de DNA , Análise Mutacional de DNA , Progressão da Doença , Feminino , Glioblastoma/química , Glioblastoma/genética , Glioblastoma/mortalidade , Glioblastoma/terapia , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Mutação , Valor Preditivo dos Testes , Fatores de TempoRESUMO
A homology model for the pig isozyme of the pyridoxal phosphate-dependent enzyme gamma-aminobutyrate (GABA) aminotransferase has been built based mainly on the structure of dialkylglycine decarboxylase and on a multiple sequence alignment of 28 evolutionarily related enzymes. The proposed active site structure is presented and analyzed. Hypothetical structures for external aldimine intermediates explain several characteristics of the enzyme. In the GABA external aldimine model, the pro-S proton at C4 of GABA, which abstracted in the 1,3-azaallylic rearrangement interconverting the aldimine and ketimine intermediates, is oriented perpendicular to the plane of the pyridoxal phosphate ring. Lys 329 is in close proximity and is probably the general base catalyst for the proton transfer reaction. The carboxylate group of GABA interacts with Arg 192 and Lys 203, which determine the specificity of the enzyme for monocarboxylic omega-amino acids such as GABA. In the proposed structure for the L-glutamate external aldimine, the alpha-carboxylate interacts with Arg 445. Glu 265 is proposed to interact with this same arginine in the GABA external aldimine, enabling the enzyme to act on omega-amino acids in one half-reaction and on alpha-amino acids in the other. The reactivities of inhibitors are well explained by the proposed active site structure. The R and S isomers of beta-substituted phenyl and p-chlorophenyl GABA would bind in very different modes due to differential steric interactions, with the reactive S isomer leaving the orientation of the GABA moiety relatively unperturbed compared to that of the natural substrate. In our model, only the reactive S isomer of the mechanism-based inhibitor vinyl-GABA, an effective anti-epileptic drug known clinically as Vigabatrin, would orient the scissile C4-H bond perpendicular to the coenzyme ring plane and present the proton to Lys 329, the proposed general base catalyst of the reaction. The R isomer would direct the vinyl group toward Lys 329 and the C4-H bond toward Arg 445. The active site model presented provides a basis for site-directed mutagenesis and drug design experiments.
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4-Aminobutirato Transaminase/química , Inibidores Enzimáticos/farmacologia , Isoenzimas/química , Modelos Moleculares , 4-Aminobutirato Transaminase/antagonistas & inibidores , 4-Aminobutirato Transaminase/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação de Hidrogênio , Substâncias Macromoleculares , Dados de Sequência Molecular , Fosfato de Piridoxal/química , Homologia de Sequência , Relação Estrutura-Atividade , Especificidade por Substrato , Suínos , Vigabatrina , Ácido gama-Aminobutírico/análogos & derivados , Ácido gama-Aminobutírico/farmacologiaRESUMO
Pyrroloquinoline quinone (PQQ) has been claimed to be a component of glutamate decarboxylase from Escherichia coli on the basis of a frequently used procedure in which the protein is extracted with hexanol. We demonstrate that if pyridoxal phosphate (PLP) is not added during the preparation, the apoenzyme prepared from glutamate decarboxylase contains no chromophore absorbing above 280 nm. Full enzyme activity and the original holoenzyme spectrum are restored by the addition of PLP alone. A 340 nm-absorbing band, similar to that which prompted analysis for PQQ, is produced by exposure of the enzyme to solutions of PLP.
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Coenzimas/química , Glutamato Descarboxilase/química , Quinolonas/química , Cofator PQQ , Fosfato de Piridoxal/química , Espectrofotometria UltravioletaRESUMO
The reaction between human 4-aminobutyrate aminotransferase and the anti-epileptic drug 4-aminohex-5-enoate, an irreversible inhibitor of the enzyme, has been studied using the radiolabelled compound. The inactivated enzyme was found to lose radiolabel over a period of a few days at 37 degrees C but even in the presence of the coenzyme, pyridoxal phosphate, no enzyme activity returned. At 4 degrees C the radiolabelled inhibitor remained stably bound. The amount of enzyme-bound 4-aminohex-5-enoate was significantly less than would be expected if one mol of inhibitor was bound per mol of active site. Reversed phase chromatography of a tryptic digest of the labelled enzyme showed that, apart from material eluting at the front of the chromatogram, all of the radioactivity was in a single fraction. This fraction contained a peptide, the sequence of which indicated that it included the lysine that binds the coenzyme and that the major release of radioactivity occurred in an Edman degradation cycle corresponding to this residue.
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4-Aminobutirato Transaminase/metabolismo , Aminocaproatos/metabolismo , 4-Aminobutirato Transaminase/antagonistas & inibidores , Sequência de Aminoácidos , Aminocaproatos/farmacologia , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Estabilidade Enzimática , Dados de Sequência Molecular , Ligação Proteica , VigabatrinaRESUMO
Six novel peptides belonging to the tachykinin and bombesin families were isolated and sequenced from extracts of the skin of the Australian myobatrachid frog Pseudophryne güntheri. One of these peptides (PG-L) was of the bombesin family and may be considered an N-elongation of the litorin/ranatensin molecule, with which it shares an identical spectrum of activity on isolated smooth muscle preparations. The other five peptides were of the tachykinin family with two of these peptides (PG-SPI and PG-SPII) related to substance P and three (PG-KI, PG-KII and PG-KIII) to kassinin. In contrast to the basic nature of substance P, the PG-SP peptides showed a clear acidic character and displayed a more potent and sustained action on isolated smooth muscle preparations and rat blood pressure than did substance P. Two of the three PG-K peptides were more potent than kassinin; PG-KIII was considerably less potent. PG-KI and PG-KII were also present in a deamidated, poorly active, form.
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Anuros , Bombesina/isolamento & purificação , Pele/análise , Taquicininas/isolamento & purificação , Sequência de Aminoácidos , Animais , Bombesina/análogos & derivados , Bombesina/farmacologia , Cromatografia/métodos , Cromatografia Líquida de Alta Pressão , Dados de Sequência Molecular , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Conformação Proteica , Ratos , Taquicininas/farmacologiaRESUMO
Inducible bacterial amino acid decarboxylases are expressed at the end of active cell division to counteract acidification of the extracellular environment during fermentative growth. It has been proposed that acid resistance in some enteric bacteria strictly relies on a glutamic acid-dependent system. The Escherichia coli chromosome contains distinct genes encoding two biochemically identical isoforms of glutamic acid decarboxylase, GadA and GadB. The gadC gene, located downstream of gadB, has been proposed to encode a putative antiporter implicated in the export of gamma-aminobutyrate, the glutamic acid decarboxylation product. In the present work, we provide in vivo evidence that gadC is co-transcribed with gadB and that the functional glutamic acid-dependent system requires the activities of both GadA/B and GadC. We also found that expression of gad genes is positively regulated by acidic shock, salt stress and stationary growth phase. Mutations in hns, the gene for the histone-like protein H-NS, cause derepressed expression of the gad genes, whereas the rpoS mutation abrogates gad transcription even in the hns background. According to our results, the master regulators H-NS and RpoS are hierarchically involved in the transcriptional control of gad expression: H-NS prevents gad expression during the exponential growth whereas the alternative sigma factor RpoS relieves H-NS repression during the stationary phase, directly or indirectly accounting for transcription of gad genes.