RESUMO
BACKGROUND AND OBJECTIVES: The effect of leukoreduction and storage periods on the accumulation of bioactive lysophospholipids and substances in human autologous blood (AB units) has not been fully investigated. MATERIALS AND METHODS: The accumulation of bioactive lysophospholipids such as sphingosine 1-phosphate (S1P) and lysophosphatidylserine (LysoPS) in AB units during the storage was investigated. The time-dependent changes and the effect of the filtration in pre-storage leuckoreduction (LR) and unmodified samples derived from 46 AB units were analysed. Additionally, the changes of lysophospholipids and platelet releasate, namely ß-thromboglobulin (ß-TG), induced by exposure of whole blood (WB) or platelet-rich plasma (PRP) to the filter material were analysed. RESULTS: LysoPS, but not S1P levels, time-dependently and significantly increased in both unmodified and LR samples. LysoPS significantly decreased in LR compared with unmodified samples, whereas S1P increased in LR compared with unmodified samples. In addition, exposure of WB and/or PRP to the filter material in vitro resulted in increased levels of S1P, LysoPS and ß-TG. CONCLUSIONS: LR effectively reduced the accumulation of LysoPS in AB units. On the other hand, it increased concentrations of S1P due to platelet activation by exposure to the filter material. These suggest that increases of S1P levels in LR and LysoPS in the unmodified samples were mainly caused by the leukocytes and/or platelets and that LR was effective in inhibiting the accumulation of LysoPS.
Assuntos
Preservação de Sangue , Transfusão de Sangue Autóloga , Procedimentos de Redução de Leucócitos , Lisofosfolipídeos/sangue , Esfingosina/análogos & derivados , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Esfingosina/sangueRESUMO
BACKGROUND: There may be uncertainty as to whether enlarged abdominal lymph nodes (LNs) in children are normal or abnormal. OBJECTIVE: To compare, by ultrasonography (US), enlarged abdominal LNs in healthy children with those in children with acute abdominal pain or acute gastroenteritis. MATERIALS AND METHODS: One hundred and twenty-two asymptomatic children were selected by questionnaire and compared with 44 children with acute abdominal pain of unknown origin and 27 children with acute gastroenteritis. The number of LNs, their location, their shape and the presence of tenderness as detected by finger compression of each LN were evaluated. The children were divided into four groups according to age: 0-2, 3-6, 7-10, and 11-15 years. RESULTS: LNs were detected in the ileo-caecal and/or para-aortic areas in almost all of the asymptomatic children. The number of large LNs ( > 10 mm) in the para-aortic areas was higher in the older children (>/= 7 years of age) than in the younger children ( = 6 years of age) (P < 0.05). The number of spindle-shaped LNs (ratio of long- to short-axis diameter >/= 2.0) was increased in the older children. The number of LNs was not increased in the children with acute abdominal pain. The size of the LNs was largest in the children with acute gastroenteritis, followed by the children with acute abdominal pain and the asymptomatic children (P < 0.001). Although the shape of the LNs was no different among the three groups of children, the frequency of round-shaped LNs (ratio of long- to short-axis diameter < 2.0) was greater in the older children with acute abdominal pain or acute gastroenteritis than in the asymptomatic children (P < 0.01). The number of LNs with tenderness detected by finger compression was significantly greater in the children with acute abdominal pain and acute gastroenteritis than in the asymptomatic children (P < 0.0001). CONCLUSION: The number of large and round-shaped LNs with tenderness tended to be increased in the children with acute gastroenteritis and acute abdominal pain. There is no clear specificity of LN enlargement in the children with acute abdominal pain, and the main challenge is to diagnose or estimate the organic pathology by US, regardless of the presence of lymphadenopathy.
Assuntos
Abdome/diagnóstico por imagem , Linfonodos/diagnóstico por imagem , Doenças Linfáticas/diagnóstico por imagem , Dor Abdominal/diagnóstico por imagem , Doença Aguda , Adolescente , Análise de Variância , Distribuição de Qui-Quadrado , Criança , Pré-Escolar , Diagnóstico Diferencial , Estudos de Avaliação como Assunto , Gastroenterite/diagnóstico por imagem , Humanos , Lactente , UltrassonografiaRESUMO
HCV encoding serine proteinase was expressed in E. coli as a fused form with maltose binding protein (MBP) and a six histidine tag. The enzyme was partially purified by using affinity chromatography for these fused peptides. Proteolytic cleavage activity of the partially purified enzyme was detected by means of an assay using both a recombinant protein and a synthetic peptide substrate which had an amino acid sequence corresponding to the most efficient cleavage site in vivo, the NS5A-NS5B junction. The cleavage occurred at the same site that was reported before.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Escherichia coli , Hepacivirus/metabolismo , Proteínas de Transporte de Monossacarídeos , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/biossíntese , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Escherichia coli , Histidina , Maltose/metabolismo , Proteínas Ligantes de Maltose , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Sitios de Sequências Rotuladas , Serina Endopeptidases/biossíntese , Serina Endopeptidases/isolamento & purificação , Especificidade por SubstratoRESUMO
We developed a novel method for analysis of hepatitis C viral proteinase activity in cultured cells, in which the proteinase activity was measured as the enhancement of reporter gene expression. In this system, plasmids encoding a reporter gene, the enzyme gene, and the substrate gene were simultaneously transfected into COS-1 cells. The reporter plasmid contains chloramphenicol acetyltransferase (CAT) gene downstream of an enhancer/promoter sequence derived from the human T-cell leukemia virus type-1 (HTLV-I) long-terminal repeat (LTR). The substrate expression plasmid was a triple chimera; HCV nonstructural protein 2 (NS2) and the Tax1 protein of HTLV-I sandwiched the substrate polypeptide, which was inserted upstream of Tax1. This method assumes that since the HCV NS2 appears to be located in the lipid bilayer of endoplasmic reticulum (ER) membranes, the Tax1 of the chimeric substrate was trapped on the surface of the ER in the absence of HCV proteinase activity. After release from the chimera by HCV proteinase-dependent cleavage, Tax1 could transactivate the expression of the CAT gene through the enhancer sequence of HTLV-I LTR. This system should enable us to simply and safely screen the potential antiviral activity of proteinase inhibitors in vivo, although this system may be limited to proteinase inhibitors that are permeable to the plasma membrane.
Assuntos
Endopeptidases/biossíntese , Hepacivirus/enzimologia , Transfecção/métodos , Animais , Sequência de Bases , Linhagem Celular , Cloranfenicol O-Acetiltransferase/análise , Cloranfenicol O-Acetiltransferase/biossíntese , Chlorocebus aethiops , Primers do DNA , Endopeptidases/genética , Elementos Facilitadores Genéticos , Imunofluorescência , Hepacivirus/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Immunoblotting/métodos , Rim , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Plasmídeos , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Sequências Repetitivas de Ácido Nucleico , Mapeamento por RestriçãoRESUMO
Proteins of hepatitis C virus (HCV) are produced from a polyprotein precursor by post-translational processing. Production of HCV proteins was analyzed with in vitro translation, as well as plasmid-based transient gene expression, in mammalian cell lines. A minimum of three different processing pathways yielded at least 10 viral proteins from the polyprotein precursor. One pathway depended on signal protease processing, and the other two pathways utilized viral proteinases. The signal peptidase cleaved the viral structural proteins, and two viral activities broke up the viral nonstructural proteins. With staggered cleavages, the signal peptidase produced two E2 products from the E2 region, gp70 type A and type B, differing in the C-terminal structure. Two viral proteinases partially overlapped in the N-terminal region of NS3; the functional amino acid residues required for those two activities differed. Most of the processed viral proteins bound together; some of the associated proteins were membrane bound.
Assuntos
Hepacivirus , Peptídeo Hidrolases/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Cães , Regulação Viral da Expressão Gênica/fisiologia , Técnicas de Transferência de Genes , Técnicas In Vitro , Dados de Sequência Molecular , Biossíntese de Proteínas , CoelhosRESUMO
Processing of the putative nonstructural (NS) proteins, p70(NS3), p4(NS4A), p27(NS4B), p58/56(NS5A), and p66(NS5B), of Japanese type hepatitis C virus (HCV) in insect cells was analyzed by using a baculovirus expression system. Products processed by the HCV serine proteinase (Cpro-2) were essentially identical to those found in mammalian cultured cells transiently producing the NS region of the HCV precursor polyprotein. A series of internal and carboxy (C)-terminal deletion experiments coupled with epitope scanning analysis showed that efficient cleavage at the Cpro-2-dependent processing sites, except at the p4(NS4A)/p27(NS4B) site, is not significantly influenced by those mutations. Efficient cleavage at p4(NS4A)/p27(NS4B) required about 40% of the NS5A N-terminal region. Estimation of the processing sites by determination of the N-terminal amino acid sequences of the processed products revealed that all the Cpro-2-dependent cleavages occurred at essentially identical sites to those reported for another HCV genotype, suggesting that Cpro-2 is a possible target for the development of a strain-independent anti-HCV agent.
Assuntos
Endopeptidases/metabolismo , Vetores Genéticos , Hepacivirus/genética , Nucleopoliedrovírus/genética , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/metabolismo , Serina Endopeptidases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Endopeptidases/genética , Expressão Gênica , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Sequências Reguladoras de Ácido Nucleico , Deleção de Sequência , Serina Endopeptidases/genética , Spodoptera/metabolismo , Proteínas não Estruturais Virais/genéticaRESUMO
Hepatitis C virus (HCV) serine proteinase (Cpro-2) is responsible for the processing of HCV nonstructural (NS) protein processing. To clarify the mechanism of Cpro-2-dependent processing, pulse-chase and mutation analyses were performed by using a transient protein production system in cultured cells. Pulse-chase study revealed the sequential production of HCV-NS proteins. Production of p70(NS3) and p66(NS5B) were rapid. An 89-kDa processing intermediate protein (p89) was observed during the early part of the chase. p89 seemed to be cleaved first into a 31-kDa protein (p31) and a p58/56(NS5A). p31 was further processed into p4(NS4A) and p27(NS4B). Mutation analysis of cleavage sites of NS4A/4B, NS4B/5A, and NS5A/5B revealed that cleavage at each site is essentially independent from cleavage occurring at the other site.
Assuntos
Hepacivirus/metabolismo , Mutagênese Sítio-Dirigida , Processamento de Proteína Pós-Traducional , Serina Endopeptidases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Eletroforese em Gel de Poliacrilamida , Hepacivirus/genética , Rim , Cinética , Metionina/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , Fatores de Tempo , Transfecção , Proteínas não Estruturais Virais/biossíntese , Proteínas não Estruturais Virais/isolamento & purificaçãoRESUMO
A serine proteinase, Cpro-2, encoded in the hepatitis C virus (HCV) genome, is considered to be located in the N-terminal part of HCV p70, one of the putative nonstructural (NS) proteins of HCV. Cpro-2 is suggested to be responsible for producing several kinds of NS proteins by processing of the HCV precursor polyprotein. We identified the active domain of Cpro-2 and clarified the mechanism of HCV polyprotein processing; various HCV mutants deleted around this serine proteinase structure were cosynthesized with unprocessed HCV polypeptides containing Cpro-2-dependent cleavage sites in COS-1 cells. We showed that Cpro-2 cleaved the HCV precursor polyprotein intermolecularly (trans) and that Cpro-2 domain which is necessary and sufficient for that cleavage mapped to within 167 aa, from Gly1049 to Ser1215 of the HCV precursor polyprotein.
Assuntos
Endopeptidases/genética , Endopeptidases/metabolismo , Hepacivirus/enzimologia , Animais , Sequência de Bases , Células Cultivadas , Análise Mutacional de DNA , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Especificidade por Substrato , Proteínas não Estruturais Virais/biossínteseRESUMO
Two proteinase activities, encoded by hepatitis C virus (HCV), Cpro-1 and Cpro-2. Cpro-1 and Cpro-2 appear to process the precursor polyprotein from which they originate. Mutant HCV polypeptides containing the region for these proteinases were produced in Escherichia coli as fusion proteins. The N- and C-terminal ends of the HCV polypeptides were fused with the E. coli maltose-binding protein (MBP) and E. coli dihydrofolate reductase (DHFR), respectively. The proteinase activities cleaved the fusion polypeptides by the same processing pathway used in eukaryotic protein production systems. The N-terminal amino acid (aa) sequences of the processed fusion proteins were determined. A comparison of those N-terminal sequences with the aa sequence of the HCV precursor polyprotein showed that the N-terminal and C-terminal cleavage sites of p70(NS3), one of the HCV nonstructural (NS) proteins, were the same as those identified in other processing studies: cleavages were estimated to be between aa 1026 and 1027 and between aa 1657 and 1658 of the HCV precursor protein, which are known to be cleaved by Cpro-1 and Cpro-2, respectively. Cpro-1 and Cpro-2 both functioned in E. coli and possessed authentic characteristic features.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Endopeptidases/metabolismo , Proteínas de Escherichia coli , Hepacivirus/enzimologia , Proteínas de Transporte de Monossacarídeos , Processamento de Proteína Pós-Traducional , Proteínas não Estruturais Virais/metabolismo , Sequência de Bases , Proteínas de Transporte/genética , Análise Mutacional de DNA , Endopeptidases/genética , Escherichia coli/genética , Proteínas Ligantes de Maltose , Dados de Sequência Molecular , Precursores de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Tetra-Hidrofolato Desidrogenase/genética , Proteínas não Estruturais Virais/genéticaRESUMO
By using a plasmid-based transient protein expression system in cultured cells and an in vitro transcription/translation system, we analyzed the proteolytic processing of the putative nonstructural protein region of the precursor polyprotein from a Japanese type of hepatitis C virus. In addition to the previously reported viral proteins, p21 and p70, we identified products of 4 kDa (p4), 27 kDa (p27), 56 kDa (p56), 58 kDa (p58), and 66 kDa (p66). These products were produced in a viral serine proteinase (proteinase 2)-dependent manner from the region downstream of p70 in the precursor polyprotein and were arranged as NH2-p70-p4-p27-p58(p56)-p66-COOH as determined with region-specific antibodies. We showed that p56 was an N-terminally truncated form of p58, which suggested that a small polypeptide of 2 kDa (p2) was produced from the N-terminal part of p58. Cleavage between p4 and p27 was inefficient in vitro and we saw the 31-kDa precursor polypeptide (p31) accumulate. Furthermore, efficient cleavage at this site in vivo required the presence of p58/p56. Immunoprecipitation analysis in vitro also suggested the mutual interaction of those nonstructural protein products. An especially close association of p4 with p70 may contribute to association of p70 with microsomal membranes.
Assuntos
Hepacivirus/metabolismo , Proteínas de Membrana/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Clonagem Molecular , Primers do DNA/química , Epitopos , Técnicas In Vitro , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Deleção de SequênciaRESUMO
Processing of HCV viral precursor protein requires at least two viral proteinases, Cpro-1 and Cpro-2, in addition to cellular proteinases. The HCV polypeptide that covers the region for the two viral proteinase domains was expressed in insect cells using a baculovirus expression system. The two proteinase activities were demonstrated in the infected cells. The Cpro-1-dependent cleavage site was estimated from the amino acid sequence of the N-terminus of the processed product. Analyses of the susceptibilities of various mutants altered at position P1 and P1' of the putative cleavage site suggested that amino acid residues at these positions is not essential for recognition and cleavage by Cpro-1-dependent activity.