RESUMO
Synaptic vesicles are proposed to dock at the presynaptic plasma membrane through the interaction of two integral membrane proteins of synaptic vesicles, VAMP and synaptotagmin, and two plasma membrane proteins, syntaxin and SNAP-25. We have characterized the binding properties of these proteins and observed SNAP-25 potentiation of VAMP 2 binding to syntaxins 1a and 4 but not syntaxins 2 or 3. n-sec1, a neuron-specific syntaxin-binding protein, bound syntaxin with nanomolar affinity, forming a complex that is distinct from the previously identified 7S and 20S syntaxin-containing complexes. This suggests that syntaxin exists in at least three states: bound to n-sec1, in a 7S particle, and in a 20S particle. Recombinant n-sec1 inhibited VAMP or SNAP-25 binding to syntaxin. We propose that the specific associations of VAMP, SNAP-25, and syntaxin mediate vesicle docking and that a syntaxin/n-sec1 complex precedes and/or regulates formation of these complexes.
Assuntos
Exocitose , Proteínas Fúngicas/fisiologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Vesículas Sinápticas/fisiologia , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Animais , Técnicas In Vitro , Substâncias Macromoleculares , Dados de Sequência Molecular , Proteínas Munc18 , Peptídeos/química , Ligação Proteica , Proteínas R-SNARE , Ratos , Proteínas Recombinantes de Fusão , Proteína 25 Associada a SinaptossomaRESUMO
Cysteine string proteins (CSPs) are secretory vesicle proteins bearing a "J domain" and a palmitoylated cysteine-rich "string" region that are critical for neurotransmitter release. The precise role of CSP in neurotransmission is controversial. Here, we demonstrate a novel interaction between CSP, receptor-coupled trimeric GTP binding proteins (G proteins), and N-type Ca2+ channels. G. subunits interact with the J domain of CSP in an ATP-dependent manner; in contrast, Gbetagamma subunits interact with the C terminus of CSP in both the presence and absence of ATP. The interaction of CSP with both G proteins and N-type Ca2+ channels results in a tonic G protein inhibition of the channels. In view of the crucial importance of N-type Ca2+ channels in presynaptic vesicle release, our data attribute a key role to CSP in the fine tuning of neurotransmission.
Assuntos
Canais de Cálcio Tipo N/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Membrana/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N/efeitos dos fármacos , Linhagem Celular , Reagentes de Ligações Cruzadas/farmacologia , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/farmacologia , Proteínas de Choque Térmico HSP40 , Hipocampo/química , Hipocampo/metabolismo , Humanos , Immunoblotting , Técnicas In Vitro , Transporte de Íons/efeitos dos fármacos , Proteínas de Membrana/efeitos dos fármacos , Modelos Moleculares , Técnicas de Patch-Clamp , Cloreto de Potássio/farmacologia , Ligação Proteica/efeitos dos fármacos , Subunidades Proteicas , RatosRESUMO
We recently reported the identification and initial characterization of the human myeloperoxidase (MPO) promoter. The minimal or basic MPO promoter lies within the proximal 128 bp of the 5'-flanking region of the MPO gene. Plasmids containing progressively larger segments of the 5'-flanking region show correspondingly greater MPO promoter activity and increased tissue specificity compared with smaller promoter fragments. These findings suggested the presence of a multiple important regulatory cis-elements in the 5'-flanking region of the MPO gene. We now report results of studies which reveal the presence of seven discrete nuclear protein binding sites (DP1-DP7) within the proximal 600 bp of 5'-flanking MPO DNA. DNase I footprinting and gel shift analyses indicate tissue-specific and/or maturation-specific differences in nuclear protein binding to most of these sites, suggesting that they play a role in transcriptional regulation. Mutation of site DP7 stimulates the activity of a 594-bp MPO promoter construct in transfection studies, whereas mutation of any of the six other sites (DP1-DP6) reduces promoter activity. These results indicate that oligonucleotides DP1-DP7 constitute cis-elements which contribute to the activity of the human MPO promoter.
Assuntos
Peroxidase/genética , Regiões Promotoras Genéticas/genética , Sequência de Bases , Sítios de Ligação , Pegada de DNA , Regulação Enzimológica da Expressão Gênica , Humanos , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/metabolismo , Oligonucleotídeos/genética , Peroxidase/metabolismo , Plasmídeos , TransfecçãoRESUMO
Synaptic vesicles are the secretory organelles responsible for the regulated secretion of neurotransmission. Proteins associated with or integral components of the lipid bilayer likely represent important components in regulated secretion. CSP (cysteine string protein) is a 34 kDa protein which copurifies with cholinergic synaptic vesicles from the marine ray Torpedo californica. In Drosophila melanogaster deletion of the CSP gene causes impaired presynaptic neuromuscular transmission. A rat brain complementary DNA (cDNA) encoding CSP was isolated and sequence analysis predicts a protein with 86% identity to Torpedo CSP. Rat CSP contains a "J domain" as well as a cysteine rich "string" region. The J domain "fingerprints" the CSP family as members of the universally conserved DnaJ/hsp40 (heat shock protein) chaperone family. Polyclonal antisera raised against a seventeen amino acid peptide representing the carboxy terminus of rat CSP detected a 35 kDa immunoreactive protein in a rat brain synaptic vesicle enriched preparation. A 35 kDa immunoreactive protein that comigrates electrophoretically with rat brain CSP was also detected in zymogen granule membranes. These results establish the presence of a CSP in rat brain and in the zymogen granule of the rat pancreas and suggest that CSPs have a role in exocrine and neural secretion.
Assuntos
Grânulos Citoplasmáticos/química , Proteínas de Membrana , Proteínas do Tecido Nervoso/análise , Vesículas Sinápticas/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Química Encefálica , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico/química , Masculino , Dados de Sequência Molecular , Pâncreas/química , Ratos , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The organization and outcome of influenza immunization programs were studied in 67 randomly or systematically selected nursing homes (8354 residents) in six states during the autumn of 1982 and/or 1983. In each home, influenza vaccine was usually offered to all residents on a voluntary basis, independent of their age, level of required nursing care, or underlying medical conditions. However, the proportion of residents who were vaccinated ranged from 8 to 98% (mean, 62% overall), with significantly lower rates in homes that also required consent from relatives (usually by return mail) than in homes that did not (P less than .00001; median, 57 versus 90%, respectively). These observations suggest that distribution of educational materials about the risks and benefits of influenza vaccine and systematic follow-up of relatives who fail to return the consent form may be useful strategies to further increase the number of nursing home residents who are immunized.
Assuntos
Vacinas contra Influenza , Influenza Humana/prevenção & controle , Casas de Saúde , Vacinação/estatística & dados numéricos , Idoso , Atitude Frente a Saúde , Família , Humanos , Vacinas contra Influenza/efeitos adversos , Consentimento Livre e Esclarecido , Pessoa de Meia-Idade , Estados UnidosRESUMO
Fas ligand (FasL) is involved in the pathogenesis of inflammatory diseases and immune privilege. We examined the expression of FasL in the enteric nervous system (ENS) in murine colitis and guinea-pig ileitis. We studied FasL immunoreactivity, functional integrity of the ENS, severity of colitis, and distribution of neutrophils in wild type and B6/gld mice that lack functional FasL. In ileitis, the distribution of FasL, CD4+ and CD8+ T cells was examined. FasL expression was increased in the ENS of wild type mice with colitis, but decreased labelling of nerve fibres was noted in B6/gld mice. Neutrophils were more abundant and widely distributed in B6/gld mice. Colitis was more severe and persistent in B6/gld mice 7 days after induction. Functional parameters of intestinal secretion and motility in B6/gld mice were the same as controls. In ileitis, FasL expression was increased in the guinea-pig ENS and returned to control levels following the resolution of inflammation. While T cells were not present in the ENS of controls, they were observed during inflammation, but were excluded from ganglia. The number of enteric neurons was unchanged over the course of inflammation. The expression of FasL is altered in intestinal inflammation and contributes to its resolution in experimental colitis.
Assuntos
Inflamação/metabolismo , Intestinos/fisiologia , Glicoproteínas de Membrana/biossíntese , Plexo Mientérico/metabolismo , Animais , Western Blotting , Colite/induzido quimicamente , Colite/imunologia , Colite/metabolismo , Modelos Animais de Doenças , Proteína Ligante Fas , Trânsito Gastrointestinal/fisiologia , Cobaias , Ileíte/induzido quimicamente , Ileíte/imunologia , Ileíte/metabolismo , Imuno-Histoquímica , Inflamação/imunologia , Intestinos/fisiopatologia , Masculino , Camundongos , Camundongos Transgênicos , Plexo Mientérico/imunologia , Neutrófilos/imunologia , Ácido Trinitrobenzenossulfônico/toxicidadeRESUMO
Oxygenated suspensions of M13 bacteriophages, containing single-stranded M13mp10 DNA, were gamma-irradiated followed by infection of E. coli cells. Mutants in the mutational target sequence, which consists of the lac promoter /operator region, the lacZ alpha gene, and a 144 bp inframe insert in the lacZ alpha gene, were selected and characterized. Except for three one-base deletions, all of the 51 mutations characterized were base substitutions. All base substitutions appeared to involve guanines and cytosines and none affect adenines and thymines. Since most of the known repair systems do not act on single-stranded DNA, the conclusion can be drawn that radiation induces under these conditions only mutagenic damages on guanine and cytosine. Although all possible G- and C-transversions and transitions were found, there is a strong preference for G-->C and G-->T transversions (21 and 25% of all base substitutions, respectively) and C-->T transitions (48% of all base substitutions). These results indicate, that the G/C-->C/G and G/C-->T/A transversions, found after irradiation of double-stranded M13 DNA, are mainly due to radiation guanine products, whereas cytosine damage is mainly responsible for G/C-->A/T transitions.
Assuntos
Bacteriófago M13/genética , Bacteriófago M13/efeitos da radiação , Dano ao DNA , DNA de Cadeia Simples/efeitos da radiação , DNA Viral/efeitos da radiação , Raios gama , Mutação , Sequência de Bases , Elementos de DNA Transponíveis , DNA de Cadeia Simples/genética , DNA Viral/genética , Infecções por Escherichia coli/genética , Óperon Lac/efeitos da radiação , Dados de Sequência MolecularRESUMO
Sulfhydryl compounds can protect DNA against free-radical-induced DNA damages not only by scavenging of radicals, but also by chemical non-enzymatic repair or modification of such damages by hydrogen-donation. To investigate the influence of chemical repair and modification on mutations, induced by gamma-radiation-generated free radicals (.OH, .H), phosphate-buffered aqueous solutions of double-stranded (ds) M13 DNA were exposed to gamma-rays under N2 in the presence of 5 mM cysteamine. The exposed DNA was subsequently transfected to wild-type E. coli and mutations in the mutational target were characterized. This target in fact contains three different target sequences, i.e., the lac promoter/operator, the lacZ alpha gene and a 144 bp inframe insert. The mutation spectrum obtained was compared with those in the absence of cysteamine under N2 and N2O. In the latter case, the ratio of .OH and .H available for reacting with DNA is about the same as under N2 + cysteamine. The results show that chemical repair and/or modification by cysteamine of potentially lethal lesions takes place, leading to a much higher survival of ds M13 DNA in the presence of cysteamine than could be expected on basis of scavenging of .OH and .H alone. This higher survival appeared to be accompanied with a higher mutation induction. However, the N2 + cysteamine mutation spectrum shows a remarkable resemblance with the N2O-spectrum. This holds for the total mutation target, as well as each of the three targets, although the mutations obtained in each of the three targets under the same irradiation conditions are quite different. Thus, it can be concluded that cysteamine is mainly effective on radiation-induced potentially lethal DNA lesions, and not so much on (pre)mutagenic damages. Moreover, the type of mutation appeared to be strongly dependent on the mutational target sequence.
Assuntos
Bacteriófago M13/genética , Cisteamina/farmacologia , Reparo do DNA/efeitos dos fármacos , Raios gama , Protetores contra Radiação/farmacologia , Sequência de Bases , Análise Mutacional de DNA , DNA Viral/genética , DNA Viral/efeitos da radiação , Escherichia coli/genética , Radicais Livres , Dados de Sequência Molecular , Mutagênese/efeitos da radiação , Mutação , Nitrogênio/farmacologia , Óxido Nitroso/farmacologiaRESUMO
To get more insight into the possible mutagenic consequences of DNA damage induced by radiation-generated H radicals (.H), a nitrogen-saturated solution of double-stranded (ds) M13mp10 DNA in phosphate buffer was irradiated with gamma-rays. Under these conditions 55% of the DNA-damaging species consists of H radicals and 45% of OH radicals (.OH). The mutations were investigated in a 144-bp mutational target sequence inserted into the lacZ alpha gene. A very specific mutation spectrum was obtained with respect to the type of mutations. Twenty out of the 28 radiation-induced mutations were C/G to A/T transversions; the remaining 8 mutations were 4 C/G to G/C transversions, 2 C/G to T/A transitions, one T/A to A/T transversion and only one -1 bp deletion. The mutations were rather randomly distributed along the 144-bp mutation target sequence with no clear mutational hot spots. When these results are compared with those we have obtained previously after irradiation of ds M13mp10 DNA under O2 (100% .OH) or N2O (90% .OH; 10% .H) (Hoebee et al., 1988, 1989), the data strongly suggest that H radicals may be responsible for the observed C/G to A/T transversions but not for -1 bp deletions.
Assuntos
DNA Viral/efeitos da radiação , Raios gama , Mutagênese , Nitrogênio/toxicidade , Mutação Puntual , Bacteriófago M13/genética , Radioisótopos de Cobalto/toxicidade , Dano ao DNA , Análise Mutacional de DNA , Escherichia coli/genética , Radicais Livres , Hidrogênio/toxicidade , Óperon Lac/efeitos da radiação , Mutagênese Sítio-Dirigida , Mutagênicos/toxicidade , Óxido Nitroso/toxicidade , Radioquímica , Espécies Reativas de Oxigênio/toxicidade , TransfecçãoRESUMO
In this paper we describe our studies on the mutagenic consequences of oxidative DNA damage introduced by radiation-induced OH radicals (.OH) and by exposure to singlet oxygen (1O2), released by thermo-dissociation of the endoperoxide 3,3'-(1,4-naphthalidene) dipropionate (NDPO2). We have made use of M13mp10 bacteriophage and pUC18 plasmid DNA, containing a 144 base pair (bp) insert in the lacZ alpha gene. This 144 bp insert was used as a mutational target sequence. When dilute aqueous solutions of double-stranded (ds) M13mp10 (plus 144 bp insert) were gamma-irradiated in the presence of oxygen (O2; 100% .OH) or nitrous oxide (N2O; 90% .OH, 10% .H), very specific mutation spectra were found. Mainly bp substitutions were observed, of which C/G to G/C transversions are the predominant type. Moreover, the mutations are for the most part concentrated into two mutational hot spots: a minor and major one. Differences between the oxic (O2) and anoxic (N2O) mutation spectra could also be observed. Under N2O-1 bp deletions were detected, which are absent in the presence of O2, and in the anoxic spectrum more C/G to A/T transversions are present. To investigate whether these differences were due to the small amount of H radicals, which are formed under N2O, ds M13mp10 (plus 144 bp insert) was exposed to gamma-rays in phosphate buffer under nitrogen (55% .H, 45% .OH). Under these conditions a remarkable shift was observed from C/G-->G/C to C/G-->A/T transversions, while the mutations were far more scattered along the 144 bp sequence and no -1 bp deletions were detected. These results strongly suggest that H radicals do not cause -1 bp deletions, but may be responsible for the observed C/G to A/T transversions. The kind of bp substitution not only appeared to be dependent on the type of the water radicals, but also appeared to be strongly influenced by the replicon in which the target sequence is incorporated. When an oxygenated solution of pUC18 plasmid DNA (plus 144 bp insert) is irradiated, mainly C/G to A/T transversions were found at the same major hot spot instead of C/G to G/C transversions when the 144 bp sequence is part of M13mp10 DNA. Finally, in agreement with the observation that 1O2 reacts preferentially with guanine in DNA, a guanine is involved in most of the mutations scored after exposure of single-stranded (ss) M13mp10 DNA to NDPO2-generated 1O2.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Dano ao DNA , DNA Bacteriano/efeitos da radiação , DNA de Cadeia Simples/efeitos da radiação , DNA Viral/efeitos da radiação , DNA/efeitos da radiação , Hidróxidos , Mutagênese , Oxigênio , Sequência de Bases , DNA/genética , DNA Bacteriano/genética , DNA de Cadeia Simples/genética , DNA Viral/genética , Relação Dose-Resposta à Radiação , Escherichia coli/genética , Radicais Livres , Raios gama , Genes Bacterianos , Radical Hidroxila , Dados de Sequência Molecular , Fotoquímica , Regiões Promotoras Genéticas , Oxigênio Singlete , beta-Galactosidase/genéticaRESUMO
To investigate a possible role of nucleotide excision repair (NER) of E. coli in the removal of gamma-radiation-induced DNA lesions, double-stranded M13mp10 DNA, which contains a part of the lac operon, including the promoter/operator region, the lacZ alpha gene and a 144 basepair (bp) inframe insert in the lacZ alpha gene, as mutational target was gamma-irradiated in a phosphate buffer under N2. Subsequently, the radiation-exposed DNA was transfected to wild-type or NER-deficient (uvrA-) E. coli, mutants in the mutational target selected, followed by characterization of the mutants by sequence analysis. Both the mutations obtained from wild-type and uvrA- E. coli appeared to consist mainly of bp substitutions. However, in contrast to wild-type cells, a relatively large proportion of the mutations obtained from the NER-deficient cells (about 25%) is represented by -1 bp deletions, indicating that NER may be responsible for the removal of lesions which cause this particular type of frameshift. Comparison of the bp substitutions between both E. coli strains showed considerable differences. Thirty per cent of all bp substitutions in the NER-deficient host are T/A-->C/G transitions which are virtually absent in wild-type E. coli. This indicates that NER is involved in the elimination of lesions responsible for these transitions. This may also be true for a part of the lesions which cause C/G-->T/A transitions, which make up 52% of the bp substitutions in uvrA- cells versus 17% in wild-type cells. Strikingly, C/G-->G/C transversions appeared to be only formed in wild-type, where they make up 22% of all bp substitutions, and not in the NER-deficient E. coli. This result suggests, that due to the action of NER, a particular type of mutation may be introduced. A similar indication holds for C/G-->A/T transversions, which are predominant in wild-type (58%) and in the minority in uvrA- cells (15%).
Assuntos
Colífagos/efeitos da radiação , Reparo do DNA , Escherichia coli/genética , Mutagênese , Sequência de Bases , DNA Viral/efeitos da radiação , Raios gama , Dados de Sequência Molecular , Mutagênese/efeitos da radiação , Nitrogênio , Deleção de SequênciaRESUMO
BACKGROUND: Hepatitis C virus (HCV) is a major cause of acute and chronic hepatitis in the United States and abroad. HCV antibody prevalences ranging from 10 to 90% have been reported in intravenous drug abusers, hemodialysis patients, and persons suffering from other liver diseases, whereas HCV seropositivity rates for volunteer-blood donor populations are generally under 1%. However no information has been available concerning the prevalence of HCV in general hospital populations in the United States. METHODS: We examined the rate of HCV seropositivity in 530 patients admitted to the Atlanta VA Medical Center between November 1993 and November 1994. The test population consisted of 400 random hospital admissions, 100 successive admissions to the surgical service, and 30 random admissions to the gastrointestinal service. Serum samples were assayed for HCV antibodies by a second generation EIA, and all repeat reactives were re-examined using a supplemental research assay to confirm the presence of HCV antibodies. Complete chart reviews were carried out on all HCV seropositive patients and on 100 HCV seronegative patients. RESULTS: Sixty-two of the 530 patients tested (11.7%) were repeatedly positive for HCV antibodies. Of these 62 repeat reactives, 56 (90.3%) were positive and 3 others (4.8%) indeterminate by the supplemental assay. The HCV seropositivity rate after supplemental testing was 11.8% for random admissions, 5.0% for surgical admissions, and 13.3% for patients admitted to the gastroenterology service. HCV-associated risk factors in HCV seropositive patients included a history of intravenous drug abuse, current or previous alcohol abuse, previous or concurrent liver disease, previous blood transfusions, hemodialysis, and multiple sex partners or unsafe sex. CONCLUSIONS: HCV infection may be more prevalent among hospitalized VA patients (and among other US hospital populations) than previously expected.
Assuntos
Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/sangue , Hepatite C/epidemiologia , Hepatite C/imunologia , Hospitalização , Veteranos/estatística & dados numéricos , Adulto , Idoso , Feminino , Georgia/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , Estados Unidos/epidemiologiaRESUMO
Incubation of cardiac myocytes from rat heart with low-molecular weight heparin (LMWH; Mr approx. 3 kDa) for 30 min resulted in a concentration-dependent release of lipoprotein lipase (LPL) activity into the incubation medium. The release of lipoprotein lipase from cardiac myocytes isolated from both control and diabetic rat hearts induced by LMWH (10 micrograms/mL) following incubation times of 10 or 30 min was significantly greater than that produced by unfractionated heparin (10 and 30 micrograms/mL) or decavanadate (1 mM). Since LMWH released more LPL activity into the incubation medium than unfractionated heparin following a short (10 min) incubation time. LMWH is probably more effective in displacing LPL bound to heparan sulfate proteoglycan binding sites on the cell surface of cardiac myocytes.
Assuntos
Heparina de Baixo Peso Molecular/farmacologia , Lipase Lipoproteica/metabolismo , Miocárdio/enzimologia , Animais , Sítios de Ligação , Coração/efeitos dos fármacos , Heparina/farmacologia , Técnicas In Vitro , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
The purpose of this study was to determine the blood lead levels (Pb-B) of urban pregnant women with low incomes and/or living in areas with heavily traveled roads, dilapidated housing, and industrial plants. We measured blood lead in 1,055 pregnant Minneapolis-area women at entry to prenatal care and in one-third of the sample during the second half of pregnancy. The mean Pb-B level of the first sample (n = 1,055) was 1.83 +/- 1.83 micrograms/dL; of the second sample (n = 375), 1.99 +/- 1.92 micrograms/dL. Only one woman had a Pb-B level greater than 12.0 micrograms/dL, which was the result of occupational exposure. The low lead levels found in this study indicate that it is not necessary to routinely screen pregnant women for elevated Pb-B levels in our geographic area. Rather, women should be screened via an environmental questionnaire to ascertain the risk of lead exposure.
Assuntos
Intoxicação por Chumbo/sangue , Chumbo/farmacocinética , Complicações na Gravidez/sangue , Diagnóstico Pré-Natal , Adolescente , Adulto , Feminino , Humanos , Recém-Nascido , Intoxicação por Chumbo/prevenção & controle , Minnesota , Gravidez , Complicações na Gravidez/prevenção & controle , Fatores de Risco , Fatores SocioeconômicosRESUMO
J proteins are chief regulators of the Hsp70 family, a highly conserved family of ATPases that mediate conformational changes in a broad range of proteins. The J protein family has been the central focus of numerous prokaryote and eukaryote biologists. Common questions that arise include: How does the J protein/Hsp70 machinery support protein folding? What role do J proteins play in protein misfolding and neurodegenerative disorders? Can the J protein/ Hsp70 machinery be harnessed to provide a rational basis for recombinant protein production? The current progress that has resulted from the convergence of biochemistry with Escherichia coli and Saccharomyces cerevisiae genetics has accelerated the pace at which these questions are being elucidated. We are beginning to gain some insights into the neuronal network of J proteins. Here, we highlight recent advances in our understanding of how select J proteins harness Hsp70 s for fundamentally important conformational work in neurons.
Assuntos
Proteínas de Choque Térmico HSP40/metabolismo , Neurônios/metabolismo , Sequência de Aminoácidos , Animais , Citoproteção , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP40/química , Resposta ao Choque Térmico , Humanos , Dados de Sequência MolecularRESUMO
Using high-resolution resonance-enhanced two-photon ionization spectroscopy in combination with genetic-algorithm-based computer-aided rotational fit analysis and ab initio quantum chemistry calculations we determined the conformational structure and transition moment orientation in 2-phenylethanol and its 1:1 clusters with argon. The results clearly demonstrate that the gauche structure of 2-phenylethanol, which is stabilized by the intramolecular pi-hydrogen bond between the folded side chain and the benzene ring, is the most abundant in the cold molecular beam. In this conformer the transition moment is rotated by 18 degrees from the short axis of the aromatic ring. Two distinct 1:1 complexes of 2-phenylethanol with argon in a cis- and trans-configuration with respect to the side chain have been found. Employing the Kraitchman [Am. J. Phys. 21, 17 (1953)] analysis we have found that the structure of the 2-phenylethanol moiety and the orientation of the transition moment do not change after the complexation with argon within the experimental accuracy. From the measured band intensities we conclude that in addition to the dispersion interaction of the argon atom with the aromatic ring a hydrogen-bond-type interaction with the terminal -OH group of the side chain stabilizes the cis-structure of the 1:1 complex of 2-phenylethanol with argon.
RESUMO
Ab initio calculations predict four stable conformational structures of the singly hydrated cluster of p-fluorostyrene: two out of plane with pi- and two in plane with sigma-type intermolecular hydrogen bonding between p-fluorostyrene and water. We employed mass-selective resonance-enhanced two-photon ionization high-resolution (70-MHz FWHM laser bandwidth) spectroscopy to partially resolve the rotational structure of the 0(0) (0) origin band of the S(1) <--S(0) electronic transition. A computer-aided fit based on genetic algorithms was used to analyze the experimental high-resolution spectrum and to determine the observed conformational structure. The good agreement between the experimental and the simulated spectra of the 0(0) (0) band and the assignment of the other prominent bands as inter- and intramolecular vibrational progressions clearly demonstrates that the anti in-plane conformer is the most abundant one in the molecular beam. The existence of the sigma-type hydrogen bond between p-fluorostyrene and water manifests that the electron attracting effect of fluorine dominates over the releasing mesomeric effect of the vinyl group and thus a pi-type hydrogen bonding with the aromatic ring is not favored in this case.
RESUMO
Streptozotocin-induced diabetes reduced cellular lipoprotein lipase (LPL) activity in cardiac myocytes from rat hearts and decreased the heparin-induced release of LPL into the medium. This effect of diabetes was rapidly reversed by in vivo treatment with insulin (5 U iv for 1 h); administration of insulin in vivo to control rats also increased heparin-releasable LPL activity. In contrast, in vitro addition of insulin to control and diabetic myocytes did not alter either cellular or heparin-releasable LPL activities. Insulin stimulated glucose oxidation and protein synthesis in control and diabetic myocytes. Decavanadate (0.05-1 mM) or vanadyl ion (0.5 mM) enhanced the release of LPL into the medium. Heparin- and decavanadate-induced release of LPL was not additive, and heparin pretreatment reduced the subsequent release of LPL by decavanadate. Decavanadate displaced LPL bound to heparin-Sepharose and increased LPL release into the perfusate of hearts. Therefore, decavanadate can mimic heparin in its effect on LPL. The absence of a direct in vitro effect of insulin on LPL in cardiac myocytes suggests that insulin may require some other in vivo factor or that diabetes-induced changes in LPL activity are secondary to some other metabolic factor.
Assuntos
Diabetes Mellitus Experimental/enzimologia , Insulina/farmacologia , Lipase Lipoproteica/metabolismo , Miocárdio/enzimologia , Vanadatos/farmacologia , Animais , Separação Celular , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Glucose/metabolismo , Heparina/farmacologia , Técnicas In Vitro , Masculino , Miocárdio/metabolismo , Miocárdio/patologia , Oxirredução , Perfusão , Biossíntese de Proteínas , Ratos , Ratos Endogâmicos , Valores de ReferênciaRESUMO
We have identified synaptic protein complexes in intact rat hippocampal slices using the rapid chemical cross-linking reagent paraformaldehyde. Cellular proteins were rapidly cross-linked, solubilized, separated electrophoretically by SDS-PAGE, and then identified immunologically. Multiple complexes containing syntaxin, the synaptosomal-associated protein of 25 kDa (SNAP25), and vesicle-associated membrane protein (VAMP) were observed to coexist in a single hippocampal slice including a 100 kDa cross-linked protein complex that exhibited the same electrophoretic migration as a member of the previously identified SDS-resistant soluble N-ethylmaleimide-sensitive fusion attachment protein receptor "core" of the 20 S complex. A VAMP-synaptophysin complex, reported previously in vitro, was also observed in the hippocampal slices. This study links biochemical and physiological studies involving presynaptic proteins implicated in secretion and confirms that these proteins that have been studied extensively previously in the presence of detergent do form "bona fide" cellular complexes. Importantly, we have also detected additional novel protein complexes that do not correspond to complexes identified previously in vitro. After the induction of persistent synaptic potentiation, an abundant 40 kDa SNAP25-caveolin1 complex was observed. The SNAP25-caveolin1 complex was not abundant in control slices and, therefore, represents the first demonstration of a reorganization of protein complexes in intact hippocampal slices during the induction of synaptic potentiation. The interaction between caveolin1 and SNAP25 was confirmed biochemically by demonstration of the association of caveolin with recombinant-immobilized SNAP25 and by the coimmunoprecipitation of SNAP25 using caveolin-specific antisera. Caveolin1, like SNAP25, was observed to be abundant in isolated hippocampal nerve terminals (synaptosomes). Immunofluorescent studies demonstrated that both SNAP25 and caveolin1 are present in neurons and colocalize in axonal varicosities. These results suggest that a short-lasting SNAP25-caveolin interaction may be involved in the early phase of synaptic potentiation.
Assuntos
Caveolinas , Hipocampo/metabolismo , Potenciação de Longa Duração/fisiologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Sinapses/metabolismo , Animais , Axônios/fisiologia , Caveolina 1 , Detergentes/farmacologia , Estimulação Elétrica , Hipocampo/ultraestrutura , Técnicas In Vitro , Masculino , Proteínas de Membrana/química , Memória/fisiologia , Proteínas do Tecido Nervoso/química , Neurônios/ultraestrutura , Estrutura Terciária de Proteína/efeitos dos fármacos , Proteínas Qa-SNARE , Proteínas R-SNARE , Ratos , Ratos Sprague-Dawley , Dodecilsulfato de Sódio/farmacologia , Solubilidade , Sinapses/ultraestrutura , Membranas Sinápticas/metabolismo , Proteína 25 Associada a SinaptossomaRESUMO
Incubation of isolated cardiac myocytes from rat hearts with heparin or phosphatidylinositol-specific phospholipase C (PLC) resulted in the release of lipoprotein lipase (LPL) into the medium. The release of LPL by the combination of heparin and PLC was not additive, and preincubation of cardiac myocytes with heparin eliminated the release of LPL in a subsequent incubation with PLC. This evidence suggests that LPL may be bound ionically to heparan sulfate proteoglycans that are covalently linked to the cell surface of cardiac myocytes by a phosphatidylinositol-glycan membrane anchor; a second pool of LPL may also be bound to proteoglycans attached directly to the myocardial cell surface. The induction of diabetes by the administration of streptozotocin (100 mg/kg for 3-4 days) to rats resulted in a decrease in the initial cellular activity of LPL and a marked reduction in the heparin-induced secretion of LPL into the medium of cardiac myocytes. The intravenous administration of insulin (5 U for 1 h) in diabetic rats reversed the effects of diabetes on cellular and heparin-releasable LPL activities. Diabetes also reduced the PLC-induced release of LPL. The reduction in the release of LPL from diabetic cardiac myocytes could result in a decrease in functional LPL activity at the capillary endothelium of whole hearts.