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1.
Osteoporos Int ; 30(8): 1617-1625, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31127317

RESUMO

INTRODUCTION: Both cardiovascular diseases (CVD) and osteoporosis are common comorbidities in rheumatoid arthritis (RA) patients. Although accumulating evidence indicates a link between CVD and osteoporotic fracture, whether CVD contributes to osteoporotic fracture risk in RA has yet to be explored. We examined the incidence rate and risk factors of osteoporotic vertebral fracture in RA patients with new-onset CVD (RA-CVD) and evaluated the effects of medications on such fracture risk. METHODS: A retrospective study was conducted using a nationwide database from 2000 to 2010: 1267 RA-CVD and 1267 non-CVD patients were enrolled from 30,507 patients with newly diagnosed RA. The main outcome was the development of osteoporotic vertebral fracture. After being adjusted for age, gender, and comorbidities, the Cox proportional hazard model was used to identify independent factors contributing to osteoporotic vertebral fracture. RESULTS: The adjusted hazard ratio (aHR) of developing osteoporotic vertebral fracture was 1.47-fold greater in RA-CVD group than in non-CVD group (95% confidence interval 1.19-1.81, p < 0.001). Both the age above 40 years and female gender were significant risk factors for developing osteoporotic vertebral fracture in RA-CVD patients. Using patients not taking medication as a reference group, the aHR of osteoporotic vertebral fracture was significantly lower in those receiving statins (0.50), low-dose corticosteroids (0.57), or hydroxychloroquine (0.12). CONCLUSIONS: The risk of osteoporotic vertebral fracture was significantly increased in RA-CVD patients, particularly women above 40 years of age, and could be reduced by statin therapy. However, the protective effect of low-dose corticosteroids or hydroxychloroquine on osteoporotic vertebral fracture risk needs further validation.


Assuntos
Artrite Reumatoide/epidemiologia , Doenças Cardiovasculares/epidemiologia , Fraturas por Osteoporose/epidemiologia , Adulto , Idoso , Artrite Reumatoide/complicações , Doenças Cardiovasculares/complicações , Comorbidade , Bases de Dados Factuais , Feminino , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Incidência , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Fraturas por Osteoporose/etiologia , Fraturas por Osteoporose/prevenção & controle , Estudos Retrospectivos , Medição de Risco/métodos , Fatores de Risco , Fraturas da Coluna Vertebral/epidemiologia , Fraturas da Coluna Vertebral/etiologia , Fraturas da Coluna Vertebral/prevenção & controle , Taiwan/epidemiologia
2.
Artigo em Chinês | MEDLINE | ID: mdl-28635225

RESUMO

Mutations in the mitochondrial DNA have been certified to be one of the most important causes of maternally inherited sensorineural hearing loss. Among these, mitochondrial 12S rRNA1555A>G, 1494C>T and other mutations are associated with both nonsyndromic and drug induced hearing loss caused by aminoglycosides. Individuals carrying 1555A>G or 1494C>T mutation have a variety of clinical manifestations, which implies that the 1555A>G or 1494C>T mutation is a chief factor underlying the development of deafness but insufficient to produce the clinical phenotype. Therefore other modifier factors, such as aminoglycosides, mitochondrial haplotypes, secondary mutation or nuclear modifier genes, may play an important role in the phenotypic expression of the deafness-associated mitochondrial 12S rRNA1555A>G or 1494C>T mutation. In this review, the modifier factors for the phenotypic expression of deafness-associated mitochondrial 12S rRNA1555A>G or 1494C>T mutations were summarized and proposed the pathogenesis of maternally inherited deafness.


Assuntos
DNA Mitocondrial , Perda Auditiva/etiologia , Herança Materna , Mutação , Fenótipo , Aminoglicosídeos/efeitos adversos , Surdez , Família , Haplótipos , Perda Auditiva/genética , Perda Auditiva Neurossensorial/genética , Humanos
3.
Osteoporos Int ; 28(4): 1377-1384, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28188454

RESUMO

This study sought to determine the minimal serum 25-hydroxyvitamin D [25(OH)D] concentration required to maintain bone health in postmenopausal women with low bone mass. A serum 25(OH)D concentration of 20 ng/mL rather than 30 ng/mL was appropriate for bone health. INTRODUCTION: There is no consensus on the minimal serum 25-hydroxyvitamin D [25(OH)D] concentration required to maintain bone health. The aim of this study was to investigate the relationship between 25(OH)D measured via liquid chromatography-mass spectrometry (LC-MS/MS), which is the current gold standard, and biochemical markers of bone turnover, PTH, and bone mineral densitometry (BMD). METHODS: The medical records of 750 postmenopausal women newly diagnosed with osteoporosis or osteopenia at Samsung Medical Center from 2009 to 2014 were investigated. Subjects were divided into four groups according to serum 25(OH)D concentration: <10, 10-20, 20-30, and ≥30 ng/mL. Serum concentrations of bone-specific alkaline phosphatase (BS-ALP), carboxy-terminal cross-linking telopeptide of type 1 collagen (CTx), intact PTH (iPTH), and BMD were compared among the four groups using analysis of covariance. Thresholds of 25(OH)D were then assessed using spline plots and locally weighted regression smoothing (LOESS) plots. RESULTS: 25(OH)D was negatively correlated with serum BS-ALP, CTx, and iPTH. Only femur neck and total femur BMD had significant positive relationships with 25(OH)D. Cutoff values of 11.9 and 9.7 ng/mL were estimated from the spline plots of femur neck and total femur BMD, respectively. For iPTH, the LOESS plot showed a steep decrease to a serum 25(OH)D concentration of about 20 ng/mL, followed by a plateau. CONCLUSIONS: According to this study, a serum 25(OH)D concentration of 20 ng/mL, rather than 30 ng/mL, was appropriate for bone health.


Assuntos
Densidade Óssea/fisiologia , Osteoporose Pós-Menopausa/sangue , Deficiência de Vitamina D/sangue , Vitamina D/análogos & derivados , Idoso , Biomarcadores/sangue , Remodelação Óssea/fisiologia , Cromatografia Líquida/métodos , Feminino , Fêmur/fisiopatologia , Colo do Fêmur/fisiopatologia , Humanos , Pessoa de Meia-Idade , Osteoporose Pós-Menopausa/etiologia , Osteoporose Pós-Menopausa/fisiopatologia , Hormônio Paratireóideo/sangue , Espectrometria de Massas em Tandem/métodos , Vitamina D/sangue , Deficiência de Vitamina D/complicações , Deficiência de Vitamina D/fisiopatologia
5.
Chem Commun (Camb) ; 50(79): 11757-9, 2014 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-25144878

RESUMO

The forces required for the detachment of ferrocene (Fc) from ß-cyclodextrin (ßCD) in a single host (ßCD)-guest (Fc) complex were investigated using force spectroscopy under electrochemical conditions. The redox state of the guest Fc moiety as well as the structure of the supporting matrix was found to decisively affect the nanomechanical properties of the complex.

6.
Genet Mol Res ; 12(4): 5335-47, 2013 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-24301794

RESUMO

Chrysanthemums are well known for their esthetic and medicinal values. Characterization of chrysanthemums is vital for their conservation and management as well as for understanding their genetic relationships. We found 12 simple sequence repeat markers (SSRs) of 100 designed primers to be polymorphic. These novel SSR markers were used to evaluate 95 accessions of chrysanthemums (3 indigenous and 92 cultivated accessions). Two hundred alleles were identified, with an average of 16.7 alleles per locus. KNUCRY-77 gave the highest polymorphic information content value (0.879), while KNUCRY-10 gave the lowest (0.218). Similar patterns of grouping were observed with a distance-based dendrogram developed using PowerMarker and model-based clustering with Structure. Three clusters with some admixtures were identified by model-based clustering. These newly developed SSR markers will be useful for further studies of chrysanthemums, such as taxonomy and marker-assisted selection breeding.


Assuntos
Chrysanthemum/classificação , Chrysanthemum/genética , DNA de Plantas , Repetições de Microssatélites , Filogenia , Alelos , Cruzamento , Análise por Conglomerados , Frequência do Gene , Genética Populacional , Genótipo , Polimorfismo Genético , República da Coreia
7.
Heart Vessels ; 25(6): 493-9, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20878168

RESUMO

Disruption of the myocardial architecture in left ventricular noncompaction (LVNC) may alter myocardial deformation. We evaluated LV myocardial deformation and tested the hypothesis that tight systolic-diastolic coupling occurs in LVNC. Longitudinal and circumferential strain and strain rates (SRs) as determined by speckle tracking echocardiography in nine children aged 5.6 ± 5.5 years was compared with those in nine controls. Left ventricular systolic myocardial deformation parameters were correlated with ejection fraction and indices of diastolic deformation. Compared with controls, patients had lower global LV systolic longitudinal strain (P = 0.008), systolic SR (P = 0.05) and early diastolic SR (P < 0.001). Similarly, LV systolic circumferential strain (base, P = 0.04; papillary muscle level, P = 0.01; apex, P = 0.04), systolic SR (base, P = 0.04) and early diastolic SR (papillary muscle level, P = 0.004, apex, P = 0.02) were lower in patients than in controls. Among patients, the LV ejection fraction correlated with global longitudinal systolic strain and SR and circumferential systolic strain and SR at all levels (all P < 0.05). Positive correlations existed between early diastolic and systolic SRs in corresponding dimensions (longitudinal r = 0.80, P = 0.01; circumferential at base, r = 0.91, P = 0.001; papillary muscle level, r = 0.96, P < 0.001; apex r = 0.98, P = <0.001). In conclusion, LV myocardial deformation is reduced in the longitudinal and circumferential dimensions and manifests tight systolic-diastolic coupling in children with LVNC.


Assuntos
Acoplamento Excitação-Contração , Ventrículos do Coração/fisiopatologia , Miocárdio Ventricular não Compactado Isolado/fisiopatologia , Contração Miocárdica , Função Ventricular Esquerda , Estudos de Casos e Controles , Criança , Pré-Escolar , China , Diástole , Ecocardiografia Tridimensional , Feminino , Ventrículos do Coração/diagnóstico por imagem , Humanos , Lactente , Recém-Nascido , Miocárdio Ventricular não Compactado Isolado/diagnóstico por imagem , Masculino , Volume Sistólico , Sístole
8.
Minim Invasive Neurosurg ; 49(5): 296-301, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17163344

RESUMO

OBJECTIVE: The surgical treatments for unilateral cervical radiculopathy have been performed by either the anterior or posterior approach. The anterior approach has usually been used more than the posterior approach. The authors compared the results of newly advanced upper vertebral transcorporeal (UVTC) approach with those of the original transuncal (TU) approach in the anterior approach. METHODS: The anterior cervical microforaminotomy was performed for 60 patients (male:female=40:20) from June, 2000 to October, 2003. 40 patients were treated by the TU approach while 20 patients were operated on by the new UVTC approach. The authors analyzed postoperative changes of disc height, the spinal instability, the average length of hospital stay, the degree of patients' satisfaction and complications from each approach. The mean follow-up period was 9.5 months. RESULTS: In the TU approach, postoperative intervertebral disc height was decreased from 7.1+/-0.65 mm to 6.2+/-0.61 mm. In the UVTC approach, postoperative intervertebral disc height was decreased from 6.6+/-0.43 mm to 6.3+/-0.41 mm. The average length of hospital stay was 5.2 days for the TU approach and 3.4 days for the UVTC approach. In the TU approach, 28 patients experienced excellent results, 11 patients experienced good results, one patient who experienced a fair result was operated by anterior cervical fusion because of a recurrent herniated disc. In the UVTC approach, 16 patients had excellent results and four patients experienced good results. CONCLUSIONS: This comparative study demonstrates that the UVTC approach is a better surgical technique than the TU approach considering the preservation of disc height, spinal stability, length of hospital stay, degree of satisfaction and complications.


Assuntos
Procedimentos Cirúrgicos Minimamente Invasivos/métodos , Procedimentos Neurocirúrgicos/métodos , Radiculopatia/cirurgia , Coluna Vertebral/cirurgia , Atividades Cotidianas , Adulto , Feminino , Humanos , Tempo de Internação , Masculino , Microcirurgia/métodos , Pessoa de Meia-Idade , Satisfação do Paciente , Complicações Pós-Operatórias/prevenção & controle , Raízes Nervosas Espinhais/cirurgia , Resultado do Tratamento
9.
Virus Genes ; 23(1): 89-95, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11556407

RESUMO

The complete genomic sequence of a Singapore isolate of the hepatitis C virus (HCV) was obtained from serum of an individual chronically infected with HCV. Nine overlapping cDNA clones covering the entire viral genome was amplified by reverse-transcription-polymerase chain reaction (RT-PCR), This isolate (HCV-S1) comprised 9,609 nucleotides (nt), including 341 nt of the complete 5' untranslated region (5' UTR), a single open reading frame of 3,011 amino acids (aa) and 235 nt of the complete 3' UTR. Its genotype was identified as type lb from analyses of its sequences in the 5' UTR, NS3 and NS5B regions. When compared against nine reported HCV isolates, the overall aa homology of HCV-SI was closest with an Australian strain, HCV-A (94%) and a Japanese strain, HCV-JT (93.9%). Phylogenetic analysis revealed that it was most closely related to the Taiwan strain, HCV-TW and another Japanese strain, HCV-K1-R1.


Assuntos
Genoma Viral , Hepacivirus/genética , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Sequência de Bases , DNA Viral , Hepacivirus/classificação , Hepacivirus/isolamento & purificação , Hepatite C Crônica/sangue , Hepatite C Crônica/virologia , Humanos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Singapura
10.
J Virol ; 75(1): 205-14, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11119590

RESUMO

Interaction between viral proteins is necessary for viral replication and viral particle assembly. We used the yeast two-hybrid assay to identify interactions among all the mature proteins of the hepatitis C virus. The interaction between NS3 and NS3 was one of the strongest viral protein-protein interactions detected. The minimal region required for this interaction was mapped to a specific subdomain of 174 amino acids in the N terminus of the helicase region. Random mutations in the minimal region were generated by PCR, and mutants that failed to interact with a wild-type minimal fragment were isolated using the yeast two-hybrid assay as a screen. Three of these mutations resulted in a reduction or a loss of interaction between helicases. Analytical gel filtration showed that in the presence of an oligonucleotide, wild-type helicases form dimers whereas the mutants remain mostly monomeric. All three mutants were partially or almost inactive when assayed for helicase activity in vitro. Mixing a mutant helicase (Y267S) with wild-type helicase did not dramatically affect helicase activity. These data indicate that dimerization of the helicase is important for helicase activity. The mutations that reduce self-association of the helicase may define the key residues involved in NS3-NS3 dimerization.


Assuntos
RNA Helicases/química , Proteínas não Estruturais Virais/química , Sequência de Aminoácidos , Animais , Células COS , Dimerização , Dados de Sequência Molecular , Mutação , RNA Helicases/metabolismo , Proteínas não Estruturais Virais/metabolismo
11.
Virology ; 290(2): 224-36, 2001 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-11883187

RESUMO

Viral proteins interact with one another during viral replication, assembly, and maturation. Systematic interaction assays of the hepatitis C virus (HCV) proteins using the yeast two-hybrid method have uncovered a novel interaction between core and NS5A. This interaction was confirmed by in vitro binding assays, and coimmunoprecipitation in mammalian cells. Core and NS5A are also colocalized in COS-7 cells. Interestingly, NS5A is cleaved to give specific-size fragments, when core is coexpressed in mammalian cells. Overexpression of core produced many dying and rounded cells and effects such as DNA laddering and the truncation of poly(ADP-ribose) polymerase 1 (PARP1), both indicators of apoptosis. These observations led us to investigate the link between the induction of apoptosis by core and the cleavage of NS5A. The proteolysis of NS5A and these apoptotic events can be inhibited by caspase inhibitor, Z-VAD, indicating that core induces apoptosis and the cleavage of NS5A by caspases. In cells infected by the HCV, core may provide the intrinsic apoptotic signal, which produces truncated forms of NS5A. The biological function of core-NS5A interaction and the downstream effect of NS5A cleavage are discussed.


Assuntos
Caspases/metabolismo , Hepacivirus/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas do Core Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Células COS , Inibidores de Caspase , Chlorocebus aethiops , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Endopeptidases/metabolismo , Ativação Enzimática , Hepacivirus/genética , Humanos , Cetonas/farmacologia , Proteínas do Core Viral/genética , Proteínas não Estruturais Virais/genética
12.
J Biol Chem ; 266(29): 19710-6, 1991 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-1680866

RESUMO

Dipeptidyl peptidase IV (DPPIV) is a membrane glycoprotein with type II orientation. It is predominantly localized to the apical surface in epithelial cells. Previous studies (Bantles, J. P., Feracci, H. M., Shinger, B., and Hubbard, A. L. (1987) J. Cell Biol. 105, 1241-1251) using cellular fractionation and immunoprecipitation in rat liver suggest that DPPIV is targeted to the apical surface by an indirect pathway through transient appearance in the basolateral surface followed by specific transcytosis to the apical domain. In transfected Madin-Darby canine kidney (MDCK) cells using domain-selective biotinylation and streptavidin absorption, it was, however, shown that DPPIV is directly sorted to the apical surface (Low, S. H., Wong, S. H., Tang, B. L. Subramaniam, V. N., and Hong, W. (1991) J. Biol. Chem, 266, 13391-13396). These studies suggest that the sorting pathway for DPPIV may be cell type-specific, but it cannot be ruled out that the observed difference in the DPPIV sorting pathway may be due to different methods employed for dissecting the sorting pathway. In this study, we have expressed rat DPPIV, using an expression system driven by the Rous sarcoma virus enhancer and the SV40 early promoter region, in another epithelial cell line, LLC-PK1. As in MDCK cells, DPPIV is preferentially (about 90%) localized to the apical surface. Employing identical methods used previously in MDCK cells, it was found that both direct and transcytotic pathways are involved in the apical surface localization of DPPIV in this epithelial cell type. These observations clearly illustrate that the sorting pathway of rat DPPIV is cell type-specific.


Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Transfecção , Animais , Sequência de Bases , Biotina/metabolismo , Fracionamento Celular , Linhagem Celular , Dipeptidil Peptidase 4 , Cães , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Microscopia de Fluorescência , Dados de Sequência Molecular , Plasmídeos , Testes de Precipitina , Ratos , Suínos
13.
J Biol Chem ; 266(20): 13391-6, 1991 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-1677006

RESUMO

Dipeptidyl peptidase IV (DPPIV) is a type II membrane glycoprotein that is predominantly localized to the apical plasma membrane in various epithelial cells. In order to understand in more detail the biogenesis and sorting of DPPIV, the cDNA for rat DPPIV was inserted into a mammalian plasmid expression vector so that DPPIV expression was driven by a control region composed of the SV40 early promoter region fused to the enhancer of the Rous sarcoma virus. Madin-Darby canine kidney cells transfected with this construct were found to express the DPPIV protein. In these transfected cells, the majority of DPPIV was present on the apial cell surface. This observation suggests that the information for apical surface localization is inherent in the DPPIV molecule itself and that this sorting information is decipherable in the epithelial cells of a different species. DPPIV is transported efficiently from the endoplasmic reticulum to the Golgi apparatus as assessed by pulse-chase experiments. Furthermore, evidence is presented which suggests that the majority of DPPIV is sorted intracellularly to the apical cell surface. The same protein has, however, been reported to be sorted by an indirect pathway through transcytosis from the basolateral to the apical cell surface in hepatocytes (Bartles, J.R., Feracci, H., M., Stinger, B., and Hubbard, A.L. (1987) J. Cell Biol. 105, 1241-1251). This study suggests that the same protein can take two different pathways in different cell types for its correct apical cell surface localization.


Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Animais , Linhagem Celular , Membrana Celular/enzimologia , Dipeptidil Peptidase 4 , Dipeptidil Peptidases e Tripeptidil Peptidases/biossíntese , Dipeptidil Peptidases e Tripeptidil Peptidases/isolamento & purificação , Cães , Imunofluorescência , Rim , Cinética , Plasmídeos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Transfecção
14.
Biochem J ; 273 ( Pt 3): 497-502, 1991 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-1705112

RESUMO

Dipeptidyl peptidase IV (DPPIV) is a serine exoproteinase expressed at high levels in epithelial cells of kidney, liver and small intestine. Recently Watanabe, Kohima & Fujimoto [(1987) Experientia 43, 400-401] and Gossrau et al. [(1990) Histochem. J. 22, 172-173] reported that Fischer 344 rats are deficient in this enzyme. We have examined DPPIV expression in Fischer 344 rats available from U.S. and German suppliers and find that livers of the U.S. Fischer rats, in contrast with their German counterparts, express active DPPIV (D+). Northern analysis of liver RNA showed comparable levels of 3.4 kb and 5.6 kb DPPIV transcripts in both D+ rats from the U.S. and German (D-) rats. Monoclonal antibody (MAb) 236.3 to DPPIV immunoprecipitated at 150 kDa enzymically active (105 kDa, denatured) protein from surface-labelled D+ hepatocytes and reacted with canalicular and sinusoidal membranes (as shown by immunofluorescence microscopy). MAb 236.3 failed to immunoprecipitate a labelled peptide from D- cell extract or to stain D- liver sections. Polyclonal antibody (PAb) specific for DPPIV immunoprecipitated an enzymically active peptide from D+ hepatocyte extracts and a smaller, inactive peptide from D- hepatocyte extracts. Peptide maps of DPPIV immunoprecipitated from D+ extracts with MAb 236.3 and PAb were identical, but differed from that of the D- hepatocyte component recognized by PAb. The molecular basis of the DPPIV deficiency in the D- rats thus appears to be the translation of an enzymically inactive protein missing the epitope recognized by MAb 236.3. We have exploited these D- rats as hosts for syngeneic transplantation of liver cells from D+ Fischer rats. DPPIV expression is stable in the transplanted cells and allows them to be readily distinguished from the surrounding D- tissue.


Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Transplante de Fígado/fisiologia , Fígado/enzimologia , Biossíntese de Proteínas , Transcrição Gênica , Animais , Anticorpos Monoclonais , Northern Blotting , Células Cultivadas , Dipeptidil Peptidase 4 , Dipeptidil Peptidases e Tripeptidil Peptidases/deficiência , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Feminino , Histocitoquímica , Masculino , RNA/genética , RNA/isolamento & purificação , Ratos , Ratos Endogâmicos F344 , Transplante de Tecidos
15.
J Cell Biol ; 111(2): 323-8, 1990 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-1974258

RESUMO

Dipeptidyl peptidase IV (DPPIV) is a membrane glycoprotein with a type II orientation in the plasma membrane. As shown in a cell-free translation system, the amino-terminal 34 amino acids of rat DPPIV are involved in translocating nascent polypeptide across the membrane of microsomes and in anchoring the translocated polypeptide in the microsomal membrane. The amino-terminal sequence performing this dual function is composed of: a central hydrophobic core of 22 amino acid residues; 6 amino-terminal residues preceding the hydrophobic core (MKTPWK); and 6 residues following the hydrophobic core. The six residues preceding the hydrophobic core are exposed on the outside (cytoplasmic side) of the microsomal membrane. Site-directed mutagenesis studies show that deletion of this cytoplasmic domain, excluding the amino-terminal initiating methionine, does not affect translocation of nascent DPPIV polypeptide, but does affect significantly anchoring of the translocated polypeptide in the microsomal membrane. In contrast, changing the two cytoplasmic Lys to Glu residues or shortening of the hydrophobic core from 22 to 15 residues or converting the last 11e of the shortened hydrophobic core into Ala affects neither translocation across nor anchoring of the DPPIV polypeptide in the microsomal membrane. These and other structural features of the DPPIV amino-terminal signal-anchor sequences are discussed along with other types of sequences for their role in targeting nascent polypeptides to the RER.


Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Sinais Direcionadores de Proteínas/genética , Sequência de Aminoácidos , Animais , Membrana Celular/enzimologia , Deleção Cromossômica , Clonagem Molecular , Citoplasma/enzimologia , Dipeptidil Peptidase 4 , Membranas Intracelulares/enzimologia , Glicoproteínas de Membrana/genética , Microssomos/enzimologia , Dados de Sequência Molecular , Mutação , Pâncreas/enzimologia , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Ratos , Mapeamento por Restrição
16.
Exp Cell Res ; 187(2): 299-308, 1990 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-1969354

RESUMO

The expression of four integral membrane glycoproteins was examined in detail utilizing monospecific antibodies during liver development. These included asialoglycoprotein receptor, a hepatocyte glycoprotein residing in the sinusoidal domain, and three bile canalicular glycoproteins, leucine aminopeptidase, dipeptidyl peptidase IV, and a Mr 110,000 glycoprotein denoted GP 110. It was observed that asialoglycoprotein receptor, GP 110, and dipeptidyl peptidase IV were present in low amounts in fetal liver and reached adult levels between 1 to 3 weeks. In contrast, leucine aminopeptidase was present in nearly adult amounts in 18-day-old fetal livers. These observations were qualitatively confirmed by indirect immunofluorescent staining of frozen thin liver sections obtained from fetal and adult rats. Further, in fetal livers it was found that leucine aminopeptidase was not localized to typical bile canalicular areas. Immunoprecipitation studies performed in the presence of proteolytic inhibitors using detergent-solubilized extracts of metabolically labeled liver minces revealed that GP 110 was present in low amounts as Mr 110,000 and Mr 105,000 polypeptides in 17-day fetal livers but by 21 days of gestation the larger polypeptide was the major synthesis product. Conversely, the apparent molecular weights of leucine aminopeptidase and dipeptidyl peptidase IV were not altered during development. Experiments determining relative rates of synthesis using excess amounts of antibodies showed that the concentrations of the three bile canalicular glycoproteins in liver during ontogeny reflect their rates of synthesis. These results underscore that plasma membrane constituents of the hepatocyte undergo dramatic changes in expression and localization as the liver changes its physiological role at birth.


Assuntos
Fígado/crescimento & desenvolvimento , Glicoproteínas de Membrana/biossíntese , Envelhecimento , Animais , Anticorpos Monoclonais , Receptor de Asialoglicoproteína , Membrana Celular/ultraestrutura , Dipeptidil Peptidase 4 , Dipeptidil Peptidases e Tripeptidil Peptidases/análise , Feto , Imunofluorescência , Homeostase , Soros Imunes , Imunoglobulina G , Leucil Aminopeptidase/análise , Fígado/embriologia , Fígado/enzimologia , Glicoproteínas de Membrana/análise , Peso Molecular , Ratos , Ratos Endogâmicos BUF , Receptores Imunológicos/análise
17.
Biochemistry ; 28(21): 8474-9, 1989 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-2605197

RESUMO

Dipeptidyl peptidase IV (DPPIV) is a cell surface membrane glycoprotein expressed in many tissues. We have subcloned the coding region of a full-length cDNA for DPPIV into the inducible eukaryotic expression vector pMSG. The resulting construct was used to transfect Chinese hamster ovary (CHO) cells. Stable transformants were found to express DPPIV, and the expression is enhanced by dexamethasone. Metabolic labeling of the transfected cells with [35S]Met followed by immunoprecipitation revealed the presence of two specific products of apparent Mr 100,000 (100-kDa form) and 110,000 (110-kDa form), respectively. Pulse-chase experiments demonstrated that the 100-kDa form can be chased into the 110-kDa form, suggesting the 100-kDa form is the precursor of the 110-kDa form. Further studies with endo H treatment demonstrated that the carbohydrate structures are of the high-mannose type, and of the complex type for the 100- and 110-kDa forms, respectively. The 110-kDa form is present at the cell surface as shown by its accessibility to cell surface iodination. The DPPIV expressed on the cell surface is resistant to digestion by relatively high concentrations of trypsin. Studies also demonstrated that the surface DPPIV is fairly stable with a half-life for turnover of about 40 h. Furthermore, the DPPIV produced in the transfected cells displays specific dipeptidyl peptidase activity. The stably transfected cells that express enzymatically active DPPIV in an inducible manner will provide an excellent system for further biochemical, functional, and cell biological characterizations of DPPIV.


Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/biossíntese , Transfecção , Animais , Transporte Biológico , Northern Blotting , Linhagem Celular , Membrana Celular/enzimologia , Clonagem Molecular , Cricetinae , Dexametasona/farmacologia , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Indução Enzimática , Feminino , Plasmídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Mapeamento por Restrição , Transformação Genética , Tripsina/metabolismo
18.
J Biol Chem ; 264(22): 12758-64, 1989 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-2753883

RESUMO

We have isolated a 3922-base pair (bp) cDNA clone for rat nonerythroid alpha-spectrin from a rat kidney lambda gt11 cDNA library. Sequence analysis revealed that this cDNA contains an open reading frame of 3090 bp encoding for the C-terminal 1030 amino acid sequence of rat kidney alpha-spectrin. The 3'-untranslated region (including a 38-bp poly(A+) tail) contains an 832-bp sequence. A single mRNA of about 8 kilobase pairs was detected in rat liver, kidney, brain, heart, intestine, lung, testis, stomach, spleen, and muscle with varying abundances, which is consistent with and further confirms the presence of spectrins in nonerythroid tissues as demonstrated previously by immunoblot analysis. Southern blot analysis suggested that there is a single gene for nonerythroid alpha-spectrin. The derived amino acid sequence contains sequence from the spectrin 106-residue internal repeat 12 to the C terminus of rat kidney alpha-spectrin. Sequence comparison with human and chicken nonerythroid alpha-spectrin showed that nonerythroid alpha-spectrin is well conserved during evolution. The rat kidney alpha-spectrin sequence, when compared to rat brain alpha-spectrin, contains an extra 76-amino-acid sequence at the C terminus. Sequence comparison of all the internal repeats available revealed that the internal repeat 3, 4, 5, 6, 7, and 8 has highest sequence similarity with internal repeat 12, 13, 14, 15, 16, and 17, respectively. Therefore, internal repeats 3-8 and 12-17 are most likely derived from an ancestral gene through gene duplication, suggesting that the spectrin gene is derived from a half-spectrin gene by gene duplication and divergence during evolution.


Assuntos
DNA/isolamento & purificação , Rim/análise , Espectrina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Clonagem Molecular/métodos , Dados de Sequência Molecular , Ratos , Ratos Endogâmicos , Mapeamento por Restrição , Espectrina/isolamento & purificação
19.
Exp Cell Res ; 182(1): 256-66, 1989 May.
Artigo em Inglês | MEDLINE | ID: mdl-2565820

RESUMO

Dipeptidyl peptidase IV (DPPIV) is a serine peptidase that cleaves N-terminal dipeptides from polypeptides when the second residue is a proline or an alanine. We have recently cloned cDNAs for rat gp110, a membrane glycoprotein with Mr of 110,000 isolated initially from rat liver. Studies reported here establish that the gp110 for which we have cloned cDNAs is DPPIV. Using the antibodies against and cDNA for DPPIV, we have assessed the tissue distribution of DPPIV by molecular approaches. Immunoblot analysis demonstrated that DPPIV is present in the kidney, lung, and small intestine at high levels, in the liver and spleen at moderate levels, and in the heart at low levels. The highest levels of mRNA for DPPIV were detected in the kidney and small intestine as compared to moderate levels found in the lung, liver, and spleen. The lowest levels of DPPIV mRNA were found in the stomach, testis, and heart. No detectable DPPIV protein and mRNA were found in brain or muscle. LDPPIV protein and mRNA are present at much lower levels in fetal livers as compared to the adult liver. Indirect immunofluorescence microscopy demonstrated that DPPIV is localized in the bile canaliculus of hematocytes and in the apical membrane domains of kidney tubule and small intestine. Further studies by Southern blot analysis indicate that DPPIV is encoded by a single gene.


Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Animais , Southern Blotting , Clonagem Molecular , Dipeptidil Peptidase 4 , Regulação da Expressão Gênica , Genes , Técnicas Imunológicas , Intestino Delgado/enzimologia , Rim/enzimologia , Fígado/enzimologia , Fígado/crescimento & desenvolvimento , Glicoproteínas de Membrana/genética , RNA Mensageiro/genética , Ratos , Distribuição Tecidual
20.
Yi Chuan Xue Bao ; 16(3): 238-43, 1989.
Artigo em Chinês | MEDLINE | ID: mdl-2629917

RESUMO

The nucleoid sedimentation technique was developed to analyze DNA repair capacity in 108 cancer patients (esophageal cancer 34, lung cancer 24 and ovarian cancer 50) and 139 normal persons. After exposing lymphocytes to UV in radiation at the dose of 2.5 microJ/mm2, the cells were incubated for different periods of time at 37 degrees C for repairing the damaged DNA. The nucleoid sedimentation distance which corresponds to DNA repair capacity was determined. It was found that most normal persons finished the process of DNA repair in II hours while the cancer patients could not do so even 17 hours after incubation. This study showed that decreased DNA repair capacity may be a component of the genetically determined susceptibility to cancer.


Assuntos
Reparo do DNA , Neoplasias/genética , Adolescente , Adulto , Idoso , Sedimentação Sanguínea , Núcleo Celular , Suscetibilidade a Doenças , Feminino , Humanos , Linfócitos , Masculino , Pessoa de Meia-Idade
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