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1.
J Virol ; 71(4): 2765-71, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9060630

ABSTRACT

An unusual set of 3' coterminal, spliced mRNAs transcribed through the BamHI A fragment have been previously identified in nasopharyngeal carcinoma (NPC) tissues. These RNAs have also been detected at low levels in Burkitt's lymphoma (BL) cell lines and Epstein-Barr virus (EBV)-transformed lymphocytes. Sequence analyses of clones from a cDNA library derived from the C15 NPC tumor indicated that the primary transcripts are differentially spliced, giving rise to a family of related transcripts, all of which encompass the BARF0 open reading frame (ORF) at the 3' end of the transcripts. One cDNA was identified that extended the BARF0 ORF at the 5' end, forming the RK-BARF0 ORF. In this study, a rabbit antiserum to a synthetic peptide representing an amino acid sequence encoded by the BARF0 ORF was prepared. This antiserum detected a glutathione S-transferase-BARF0 fusion protein and both BARF0 and RK-BARF0 proteins expressed from transfected constructs in H1299 cells. The serum also immunoprecipitated the 20-kDa BARF0 and 30-kDa RK-BARF0 in vitro-translated proteins. Immunoblot analyses identified a protein doublet of 30 and 35 kDa in all of the EBV-infected cell lines tested. Cellular fractionation studies revealed that the proteins were membrane associated. The sizes of the proteins detected in cell lines and their association with membranes suggests that they are likely encoded by the RK-BARF0 transcript, which is predicted to contain a membrane localization signal. The proteins were also detected in protein extracts prepared from NPC biopsies and a BL biopsy but not from hairy leukoplakia, a permissive EBV infection. These results reveal that the rightward RNA transcripts from the BamHI A region of EBV encode one or more proteins that are expressed in latently infected cells and in tumor tissue.


Subject(s)
Herpesvirus 4, Human/genetics , Viral Proteins/genetics , Amino Acid Sequence , Animals , Antibodies, Viral/biosynthesis , Deoxyribonuclease BamHI/metabolism , Herpesvirus 4, Human/metabolism , Herpesvirus 4, Human/physiology , Humans , Molecular Sequence Data , Nasopharyngeal Neoplasms , Open Reading Frames , Rabbits , Tumor Cells, Cultured , Viral Proteins/metabolism , Virus Replication
2.
J Virol ; 69(7): 4577-81, 1995 Jul.
Article in English | MEDLINE | ID: mdl-7769727

ABSTRACT

The complete 5' sequence of the Epstein-Barr virus 3.5-kb latent membrane protein 1 (LMP1) mRNA, expressed in nasopharyngeal carcinoma, has been determined. The transcript initiates from heterogeneous start sites within the first terminal repeat (TR) of the viral genome. This region is TATA-less, consistent with heterogeneous starting, but contains multiple GC-rich elements which potentially interact with the Sp1 transcription factor. Expression of the 3.5-kb mRNA was consistently detected in nasopharyngeal carcinoma samples and in additional cell types, including a Burkitt's lymphoma. This is the first identification of an Epstein-Barr virus mRNA containing TR sequence and the first report of the ability of the TR to function as a transcriptional promoter.


Subject(s)
Herpesvirus 4, Human/genetics , Promoter Regions, Genetic , RNA, Messenger/analysis , Repetitive Sequences, Nucleic Acid , TATA Box , Viral Matrix Proteins/genetics , Base Sequence , Cell Line , Humans , Molecular Sequence Data , Nasopharyngeal Neoplasms/virology
3.
J Virol ; 69(2): 1132-41, 1995 Feb.
Article in English | MEDLINE | ID: mdl-7815492

ABSTRACT

Epstein-Barr virus (EBV) gene expression in nasopharyngeal carcinoma (NPC) includes abundant rightward transcription of the BamHI A fragment, consisting of mRNAs ranging in size from approximately 4.0 to 8.0 kb. These transcripts include several distinctly spliced forms which are 3'-end coterminal and contain the BamHI A rightward frame 0 (BARF0) open reading frame (ORF) in the final exon. BamHI A transcription is detected at a lower level of expression in EBV-infected lymphoid cells. In this study, cDNA cloning, reverse transcription-based PCR, and Northern (RNA) blotting were used to further define the structures of the BamHI A transcripts and to characterize their expression in different EBV-infected tissues. Three BamHI A cDNAs isolated from a passaged NPC represent previously unidentified mRNAs that contain BARF0 and additional ORFs encoded by multiple exons, including one which extends the size of the BARF0 ORF from 174 to 279 codons. The distinct exons were detected in multiple, differently sized mRNAs, indicating that these transcripts have complex patterns of alternate splicing. In support of this finding, 5'-end analysis confirmed the presence of a previously reported start site and also identified a subset of transcripts of 4.8 kb and larger that initiate further 5' to this site. In addition, 3'-end analysis identified heterogeneous 3'-end processing in all of the BamHI A mRNAs, resulting in transcripts that either contain the entire BARF0 ORF or are cleaved and polyadenylated 5' of the stop codon. Finally, the expression of multiple, distinctly spliced BamHI A transcripts was consistently detected in a wide range of EBV-infected samples, including NPC, Burkitt's lymphoma, and parotid carcinoma biopsy samples, and in type I and type III Burkitt's lymphoma lines and type III lymphoblastoid cell lines. This complex pattern of start site selection, alternate splicing, and heterogeneous 3'-end processing is likely to regulate the expression in vivo of the ORFs encoded by the EBV BamHI A transcripts.


Subject(s)
DNA-Cytosine Methylases/genetics , Herpesvirus 4, Human/genetics , RNA, Messenger/chemistry , RNA, Viral/chemistry , Alternative Splicing , Amino Acid Sequence , Base Sequence , Cell Line , Cloning, Molecular , Humans , Molecular Sequence Data , Open Reading Frames , Polymerase Chain Reaction
4.
J Virol ; 67(10): 6303-8, 1993 Oct.
Article in English | MEDLINE | ID: mdl-8396685

ABSTRACT

Epstein-Barr virus (EBV) DNA has been detected in peripheral T-cell lymphomas. In this study, analysis of the EBV termini indicated that the infection was clonal and nonpermissive. Analysis of viral expression detected the processed, spliced mRNAs representing EBNA1, LMP1, LMP2, and BamHI A transcripts in all EBV-positive peripheral T-cell lymphomas. The LMP1 protein was detected by immunofluorescence in a single specimen. In contrast, neither the EBNA2 protein nor the spliced EBNA2 mRNA were detected. These data indicate that EBV-infected T-cell lymphomas are clonal expansions of a single EBV-infected cell with a pattern of gene expression which is distinct from that detected in Burkitt's lymphomas or posttransplant lymphomas but similar to viral expression in nasopharyngeal carcinomas.


Subject(s)
Antigens, Viral/biosynthesis , DNA-Binding Proteins/biosynthesis , Gene Expression , Herpesvirus 4, Human/isolation & purification , Herpesvirus 4, Human/metabolism , Lymphoma, T-Cell, Peripheral/microbiology , Viral Matrix Proteins/biosynthesis , Antibodies, Monoclonal , Antigens, Viral/analysis , Base Sequence , DNA-Binding Proteins/analysis , Epstein-Barr Virus Nuclear Antigens , HeLa Cells , Herpesvirus 4, Human/immunology , Humans , Molecular Sequence Data , Oligodeoxyribonucleotides , Polymerase Chain Reaction , RNA Splicing , RNA, Messenger/biosynthesis , RNA, Messenger/metabolism , Restriction Mapping , Transcription, Genetic , Viral Matrix Proteins/analysis
5.
J Neurosci Res ; 30(4): 616-24, 1991 Dec.
Article in English | MEDLINE | ID: mdl-1787538

ABSTRACT

Experimental allergic encephalomyelitis is characterized by invasion of lymphocytes and macrophages into the central nervous system resulting in inflammation, edema, and demyelination. Sera from Lewis rats from 7-95 days after immunization with purified guinea pig CNS myelin were examined with respect to their ability to opsonize myelin. This was correlated with the appearance of antibody components and the relative amounts of antibody to myelin basic protein (MBP) and proteolipid protein (PLP). Sera from rats 10-95 days after immunization preincubated with purified myelin induced phagocytosis of myelin by cultured macrophages with the resulting production of cholesterol ester. This opsonization activity as measured by the percentage of cholesterol esterified reached a peak at 26-27 days after immunization but remained significantly elevated up to 95 days post-immunization compared to the activity of serum from the Freund's adjuvant-injected controls. Immunoblots of the sera revealed a gradual increase in antibody activity against myelin components. ELISA assays for MBP and PLP antibody showed a similar pattern. Antibody to galactocerebroside (GC) was not detected by immunostains nor by the ELISA assay. Areas of demyelination were observed histologically by luxol-fast blue stained spinal cords up to 60 days post-immunization. These results indicate that antibodies to myelin protein when given access to myelin through or within the blood brain barrier could initiate or enhance the phagocytic response by peripheral or resident macrophages.


Subject(s)
Antibodies/immunology , Demyelinating Diseases/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Myelin Sheath/immunology , Animals , Antibody Formation , Encephalomyelitis, Autoimmune, Experimental/blood , Enzyme-Linked Immunosorbent Assay , Male , Myelin Proteins/analysis , Myelin Sheath/physiology , Phagocytosis , Rats , Rats, Inbred Lew
6.
J Neurosci Res ; 27(3): 298-306, 1990 Nov.
Article in English | MEDLINE | ID: mdl-1711126

ABSTRACT

We have previously shown that antisera to whole CNS myelin, whole PNS myelin, galactocerebroside (GC), and myelin basic protein (MBP) promote the uptake of CNS myelin by cultured macrophages, and stimulate the conversion of myelin lipids to cholesterol ester and triglycerides. Here we report the results of similar studies using PNS myelin purified from the rat sciatic nerve. Antisera to whole CNS myelin, whole PNS myelin, GC, and MBP preincubated with 14C-labeled PNS myelin increased the production of radioactive cholesterol ester by macrophages in culture to a level about twice that with preimmune serum, and five to six times that of untreated myelin. The amounts of [14C]triglyceride were similarly increased with these antisera, whole P0 and P2 antisera had little or no effect. IgG prepared from the antisera stimulated lipid metabolism to almost the same extent, while heating the antisera did not decrease the stimulatory effect, indicating that myelin was opsonized by IgG, but not likely by complement. With a few exceptions, the four active sera and their IgGs promoted the macrophage metabolism of CNS and PNS myelin almost equally. The cultured macrophages converted about 3% of untreated CNS myelin and about 6% PNS myelin cholesterol to cholesterol ester. Under phase contrast microscopy it was noted that vesicles of CNS myelin appeared to bind individually to macrophages, whereas PNS myelin vesicles tended to self-associate to form large clumps which were found to macrophages. Binding studies showed PNS myelin to be bound more firmly to macrophages than CNS myelin.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Myelin Basic Protein/immunology , Phagocytosis , Animals , Antibodies/immunology , Cells, Cultured , Central Nervous System/chemistry , Cholesterol Esters/metabolism , Lipid Metabolism , Macrophages/physiology , Myelin Basic Protein/isolation & purification , Peripheral Nerves/chemistry , Rats
7.
Trans R Soc Trop Med Hyg ; 81(3): 437-40, 1987.
Article in English | MEDLINE | ID: mdl-3120369

ABSTRACT

A laboratory technician developed fever, malaise, headache and non-tender erythematous swelling proximal to the site of accidental inoculation of his thumb, 24 days earlier, with a needle contaminated with Trypanosoma cruzi. Findings included a characteristic rash, remarkable fever, relative bradycardia and leukopaenia--T lymphopaenia with maintenance of a normal helper/suppressor ratio. Trypanosomes were not detected in blood concentrates or in biopsies of an enlarged lymph node and a skin lesion. T. cruzi antibody was first detected 33 days after the laboratory accident, when parasites were first isolated. Therapy with nifurtimox was well tolerated and the patient's serology became negative 9 months after the accident.


Subject(s)
Chagas Disease/pathology , Laboratory Infection , Adult , Chagas Disease/parasitology , Chagas Disease/transmission , Humans , Male
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