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1.
Virus Res ; 12(1): 11-8, 1989 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-2541578

RESUMO

An HCMV specific clone was isolated from a genomic library of human cytomegalovirus (HCMV) DNA cloned into the expression vector lambda gt11. This clone (lambda 111-1) expressed an HCMV/beta-galactosidase fusion protein which was reactive with rabbit antibody prepared against purified HCMV virions and dense bodies as well as human HCMV immune serum. By probing Western blots of HCMV virion proteins or HCMV-infected cells with antibody prepared against the fusion protein, the authentic gene product of clone lambda 111-1 was identified as a high molecular weight polypeptide of 140. Probing the restriction digests of HCMV DNA with insert DNA from the immunoreactive lambda gt11 clone permitted us to localize the coding sequence for the 140 kDa polypeptide to the long unique region (map coordinates of 0.16-0.18) on HCMV Towne and AD169 genomes.


Assuntos
Citomegalovirus/genética , DNA Viral/genética , Genes Virais , Proteínas da Matriz Viral , Proteínas Virais/genética , Southern Blotting , Western Blotting , Genes , Fosfoproteínas/genética , Proteínas Recombinantes de Fusão/genética
2.
J Virol Methods ; 25(3): 301-14, 1989 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-2555378

RESUMO

We have analyzed the ability to use in situ cytohybridization to distinguish between human cytomegalovirus (HCMV) DNA and RNA in human cells infected in vitro. Two different viral-specific probes were used, one for an abundantly expressed late gene, and one which includes at least two genes coding for immediate early (IE) proteins. In productively infected cells, hybridization of the late gene probe extended over both the nucleus and cytoplasm and was RNase sensitive, whereas hybridization of the IE probe was restricted to the nucleus and was DNase-sensitive. In nonproductively infected cells hybridization of the IE probe was localized to the cytoplasm and was RNase-sensitive. The specific nuclease sensitivities indicate that a cytoplasmic hybridization pattern correlates with detection of viral RNA sequences, whereas a nuclear pattern represents detection of viral DNA. These results demonstrate that in situ cytohybridization can potentially be used to determine the extent of HCMV infection in a particular tissue or cell type by distinguishing between transcription and replication of specific viral genes.


Assuntos
Citomegalovirus/genética , DNA Viral/análise , RNA Viral/análise , Autorradiografia , Células Cultivadas , Cicloeximida , Sondas de DNA , Desoxirribonucleases , Fibroblastos , Humanos , Ribonucleases , Transfecção
3.
J Bone Joint Surg Br ; 73(4): 618-20, 1991 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-2071646

RESUMO

We report the results of conservative treatment of stage III and stage IV avascular necrosis of bone (AVN) affecting the hip or knee in renal transplant patients. Twenty-nine patients were followed for a mean period of five years. Conservative management was successful in controlling symptoms in 40% of those with AVN of the hip and in 70% of those with AVN of the knee. Knowledge of the natural history of AVN is important because of the long survival times after renal transplantation.


Assuntos
Necrose da Cabeça do Fêmur/epidemiologia , Transplante de Rim/efeitos adversos , Articulação do Joelho , Osteonecrose/epidemiologia , Adulto , Idoso , Ciclosporinas/uso terapêutico , Feminino , Necrose da Cabeça do Fêmur/etiologia , Necrose da Cabeça do Fêmur/terapia , Seguimentos , Prótese de Quadril/normas , Humanos , Incidência , Londres/epidemiologia , Masculino , Pessoa de Meia-Idade , Osteonecrose/fisiopatologia , Osteonecrose/terapia , Osteotomia/normas
4.
J Bone Joint Surg Br ; 74(2): 272-4, 1992 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-1544968

RESUMO

We perfused 16 human femora with a 50% barium sulphate suspension and studied the intra-osseous vessels by microfocal radiography and histology. There were few anastomoses between the vessels of the greater trochanter and those of the adjacent cancellous bone of the shaft. Ischaemia of the trochanter may contribute to nonunion after trochanteric osteotomy.


Assuntos
Fêmur/irrigação sanguínea , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Fêmur/diagnóstico por imagem , Técnicas Histológicas , Humanos , Masculino , Pessoa de Meia-Idade , Perfusão/métodos , Radiografia
5.
J R Soc Med ; 84(10): 606-7, 1991 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-1744843

RESUMO

The intrinsic blood supplies of 13 human patellae with varying degrees of articular surface degeneration were studied by perfusion techniques. Disruption of the normal pattern of arterial arcades correlated with increasing surface wear.


Assuntos
Doenças das Cartilagens/patologia , Cartilagem Articular/patologia , Patela/irrigação sanguínea , Idoso , Idoso de 80 Anos ou mais , Humanos , Isquemia/patologia , Prótese do Joelho , Pessoa de Meia-Idade , Patela/patologia
7.
J Virol ; 37(3): 1103-6, 1981 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-6262532

RESUMO

All four capsid proteins of encephalomyocarditis virus and the precursor to two of these were resolved from purified virions with two-dimensional gel electrophoresis. In addition, all of the known stable virus-specific proteins found in infected cells, but not the primary and intermediate precursor proteins, could be resolved with these techniques.


Assuntos
Capsídeo/análise , Vírus da Encefalomiocardite/análise , Precursores de Proteínas/análise , Proteínas Virais/análise , Eletroforese em Gel de Poliacrilamida
8.
J R Coll Surg Edinb ; 37(3): 203-4, 1992 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-1404052

RESUMO

A total of 33 consecutive patients undergoing elective hand operations were randomly allocated 5 days after the operation either to have all dressings discarded or to continue with an occlusive bandage for a further 2 weeks. Assessments at 2 and 6 weeks showed that wound healing, hand function, symptoms and complications were identical in both groups. It was concluded that for selected patients early removal of hand dressings after surgery does not have any deleterious effects.


Assuntos
Mãos/cirurgia , Curativos Oclusivos , Cicatrização , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Resultado do Tratamento
9.
Ann Intern Med ; 87(6): 754-9, 1977 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-145198

RESUMO

In a retrospective analysis of bacterial endocarditis, 84 of 192 cases (44%) were found to have musculoskeletal manifestations of one or more types. Common manifestations were arthralgias (32 cases), arthritis (26 cases), low back pain (24 cases), diffuse myalgia (16 cases), and myalgias localized to the thigh or calf (11 cases). The joint manifestations typically were monarticular or oligoarticular, and the myalgias were commonly unilateral. No association was found between the pattern of rheumatic symptoms and other clinical manifestations, laboratory tests, or causative bacterial organisms. In 52 patients (27%), musculoskeletal complaints were the first or among the first symptoms of bacterial endocarditis. The frequency and character of these manifestations and their tendency to occur early in the course of the disease indicate that they are an important feature of endocarditis which, if not recognized, may cause a delay in the diagnosis by mimicking a rheumatic disease.


Assuntos
Doenças Ósseas/etiologia , Endocardite Bacteriana/complicações , Doenças Musculares/etiologia , Adolescente , Adulto , Idoso , Artrite/etiologia , Dor nas Costas/etiologia , Doenças Ósseas/imunologia , Endocardite Bacteriana/imunologia , Feminino , Humanos , Artropatias/etiologia , Masculino , Pessoa de Meia-Idade , Doenças Musculares/imunologia , Dor/etiologia , Estudos Retrospectivos , Fatores de Tempo
10.
Virology ; 178(1): 6-14, 1990 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-2167561

RESUMO

The major 64-kDa structural protein of human cytomegalovirus (pp64) was isolated from a guanidinium chloride extract of the virions and dense bodies of HCMV (Towne) by reverse-phase HPLC. Purified pp64 was reduced and alkylated followed by digestion with trypsin. The molecular mass of each of the tryptic peptides was determined by fast atom bombardment/mass spectrometry and compared with the predicted molecular mass of the fragments deduced from the corresponding DNA-derived peptide sequence of pp65 from HCMV (AD169). Microsequence analysis was employed to confirm selected peptides. Results of protein sequence analysis of pp64 from HCMV (Towne) are in complete agreement with the DNA-derived protein sequence of pp65 predicted for HCMV (AD169) with the following exceptions and modifications. The protein isolated from HCMV (Towne) was found to contain an Ala at position 448 instead of Ser448 reported for the protein from HCMV (AD169). We also identified Ser472 as a site of phosphorylation in pp64 from HCMV (Towne). Finally, on the basis of the sequence of HCMV (AD169) DNA fragment encoding the matrix protein and on S1 nuclease protection analysis, it has been predicted that one version of the matrix protein (possibly the lower matrix protein, Mr 65K) is encoded by an mRNA that is formed through splicing of a short intron. However, we have obtained peptides that contain sequences spanning through the splice-junction region, suggesting that in HCMV (Towne), the matrix protein is encoded by an unspliced message.


Assuntos
Citomegalovirus/análise , Fosfoproteínas/genética , Proteínas da Matriz Viral , Proteínas da Matriz Viral/genética , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , DNA Viral/metabolismo , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Mapeamento de Peptídeos , Fosfoproteínas/biossíntese , Fosforilação , Processamento de Proteína Pós-Traducional , RNA Mensageiro/biossíntese , Proteínas da Matriz Viral/biossíntese , Proteínas Estruturais Virais/biossíntese
11.
Virology ; 167(1): 306-10, 1988 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-2847421

RESUMO

We have determined the map position of a viral gene encoding a 32-kDa late structural protein of human cytomegalovirus (HCMV) using a murine monoclonal antibody. This monoclonal antibody was reactive with two protein bands of 32 and 27 kDa in HCMV-infected cell lysates and with a single 32-kDa protein band in HCMV virions as detected by immunoblot analysis. When purified HCMV envelope preparation was used for immunoblotting, the monoclonal antibody did not display a detectable band. We used this monoclonal antibody to screen a cDNA library that was constructed from poly(A)+ RNA of late HCMV-infected cells and cloned into the expression vector lambda gt11. A cDNA clone that expressed an immunoreactive epitope of the late HCMV protein fused to beta-galactosidase was identified. Probing the restriction digests of HCMV (Towne and AD169) DNA with insert DNA from the immunoreactive lambda gt11 clone permitted us to localize the coding sequence within the long unique region between map coordinates of 0.62 and 0.64 of HCMV Towne and AD169 genomes. Using the same probe, a single transcript of 1.4 kb was detected in total RNA from HCMV-infected cells at late times after infection.


Assuntos
Capsídeo/genética , Citomegalovirus/genética , Genes Virais , Animais , Anticorpos Monoclonais/imunologia , Northern Blotting , Southern Blotting , Capsídeo/imunologia , Centrifugação com Gradiente de Concentração , Clonagem Molecular , Sondas de DNA , Enzimas de Restrição do DNA , DNA Viral/análise , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Hibridomas , Immunoblotting , Hibridização de Ácido Nucleico , RNA Viral/análise
12.
J Virol ; 64(3): 1366-9, 1990 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-2154616

RESUMO

Human cytomegalovirus (HCMV) induces morphological changes in infected cells that are remarkably similar to those seen in oncogenically transformed cells. The molecular bases of these phenotypic alterations are not known but their occurrence in some transformed cells can be associated with abnormal fibronectin (FN) expression. In this report, we have compared FN levels in normal and HCMV-infected cells. In these studies, the HCMV-infected fibroblasts exhibited a progressive loss of cellular FN. Northern (RNA) blot analysis revealed that the decrease in FN levels resulted from a lowering of FN mRNA levels in HCMV-infected cells. We detected an initial decrease in FN mRNA of 25 to 30% at immediate-early and early times, whereas at late times after infection the levels of FN mRNA were lowered by greater than 80%. These results indicated that the HCMV-induced decrease in FN expression is due to a decrease in the quantity of FN mRNA and suggested that HCMV-encoded and/or -induced functions may be involved in producing these alterations.


Assuntos
Transformação Celular Viral , Citomegalovirus/genética , Fibronectinas/genética , Regulação da Expressão Gênica , Genes , Sequência de Bases , Northern Blotting , Células Cultivadas , Humanos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Pele , Transcrição Gênica
13.
J Infect Dis ; 155(3): 501-9, 1987 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-3027201

RESUMO

DNA-DNA hybridization was compared with culture for virus and histopathology for the detection and quantitation of human cytomegalovirus (HCMV) in lungs from bone marrow transplant recipients. The hybridization assay detected 100 pg of HCMV DNA or greater than 520 plaque-forming unit equivalents. Compared with culture for virus, the hybridization assay had a sensitivity of 90.6% and a specificity of 77.8%. The quantity of HCMV DNA and the infectivity titer were directly correlated. Quantitation of HCMV DNA by hybridization was more reproducible than were infectivity titers for frozen specimens. Histopathologic examination of lung specimens detected HCMV inclusions in 60% (15 of 25) of patients with HCMV-associated interstitial pneumonia, whereas hybridization results were positive for 96% (24 of 25) of these patients. A significant inverse correlation was demonstrated between quantitative values for HCMV DNA and the duration of pneumonia.


Assuntos
Transplante de Medula Óssea , Infecções por Citomegalovirus/diagnóstico , Citomegalovirus/isolamento & purificação , DNA Viral/análise , Pulmão/microbiologia , Fibrose Pulmonar/diagnóstico , Citomegalovirus/genética , Humanos , Corpos de Inclusão Viral/ultraestrutura , Pulmão/patologia , Hibridização de Ácido Nucleico , Pneumonia Viral/diagnóstico
14.
J Gen Virol ; 73 ( Pt 9): 2367-74, 1992 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-1328492

RESUMO

The gene encoding the highly antigenic 28K (pp28) tegument phosphoprotein of human cytomegalovirus (HCMV) was expressed in insect cells utilizing a recombinant baculovirus. The mature intracellular form of the recombinant-derived pp28 had mobility on SDS-polyacrylamide gels similar to that of native pp28 from HCMV strain Towne-infected human foreskin fibroblasts (HFFs). In vitro labelling of recombinant Autographa california nuclear polyhedrosis virus-infected Spodoptera frugiperda cells or of HCMV-infected HFFs with [32P]orthophosphate followed by immunoprecipitation showed that both the insect cell-derived and HCMV strain Towne-infected fibroblast-derived pp28 were phosphorylated. The mobility of pp28 derived from these two sources as well as from extracellular HCMV virions indicated the existence of multiple charged forms of the protein, and a difference in the relative amounts of these forms expressed in HCMV-infected HFFs and recombinant baculovirus-infected insect cells. The recombinant pp28 expressed in insect cells was readily and specifically recognized by antibodies to native pp28, including HCMV-seropositive human serum, and was used in an ELISA to screen human sera for seropositivity.


Assuntos
Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Citomegalovirus/imunologia , Fosfoproteínas/biossíntese , Fosfoproteínas/imunologia , Proteínas Virais/biossíntese , Proteínas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Formação de Anticorpos , Reações Antígeno-Anticorpo , Células Cultivadas , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Mariposas , Fosforilação , Proteínas Recombinantes/biossíntese
15.
J Clin Microbiol ; 28(12): 2602-7, 1990 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1980680

RESUMO

The human cytomegalovirus (HCMV) a-sequence (a-seq) is located in the joining region between the long (L) and short (S) unique sequences of the virus (L-S junction), and this hypervariable junction has been used to differentiate HCMV strains. The purpose of this study was to investigate whether there are differences among strains of human cytomegalovirus which could be characterized by polymerase chain reaction (PCR) amplification of the a-seq of HCMV DNA and to compare a PCR method of strain differentiation with conventional restriction fragment length polymorphism (RFLP) methodology by using HCMV junction probes. Laboratory strains of HCMV and viral isolates from individuals with HCMV infection were characterized by using both RFLPs and PCR. The PCR assay amplified regions in the major immediate-early gene (IE-1), the 64/65-kDa matrix phosphoprotein (pp65), and the a-seq of the L-S junction region. HCMV laboratory strains Towne, AD169, and Davis were distinguishable, in terms of size of the amplified product, when analyzed by PCR with primers specific for the a-seq but were indistinguishable by using PCR targeted to IE-1 and pp65 sequences. When this technique was applied to a characterization of isolates from individuals with HCMV infection, selected isolates could be readily distinguished. In addition, when the a-seq PCR product was analyzed with restriction enzyme digestion for the presence of specific sequences, these DNA differences were confirmed. PCR analysis across the variable a-seq of HCMV demonstrated differences among strains which were confirmed by RFLP in 38 of 40 isolates analyzed. The most informative restriction enzyme sites in the a-seq for distinguishing HCMV isolates were those of MnlI and BssHII. This indicates that the a-seq of HCMV is heterogeneous among wild strains, and PCR of the a-seq of HCMV is a practical way to characterize differences in strains of HCMV.


Assuntos
Citomegalovirus/genética , Sequência de Bases , Citomegalovirus/classificação , Citomegalovirus/isolamento & purificação , DNA Viral/genética , Amplificação de Genes , Genes Virais , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição
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