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PubMed2000

Most env and gag subtype A HIV-1 viruses circulating in West and West Central Africa are similar to the prototype AG recombinant virus IBNG.

The genetic subtype was identified in gag and env of 219 HIV-1-positive samples collected in different African countries, 44 from Senegal, 55 from Cameroon, 82 from Gabon, and 38 from Djibouti. In total, 20 (9.1%) samples had discordant subtypes between gag and env, 6 of 44 (13.9%) in Senegal, 4 of 55 (7.2%) in Cameroon, 1 of 38 (2.6%) in Djibouti, and 10 of 82 (12.1%) in Gabon. Subtypes A and G were predominantly involved in the recombination events. Phylogenetic tree analysis of gag showed that an important number of the A sequences form a distinct subcluster with the AG-IBNG prototype strain (a complex A/G mosaic virus): 27 of 32 (84.3%) in Senegal, 12 of 17 (70.6%) in Nigeria, 24 of 39 (61.5%) in Cameroon, and 38 of 70 (54.3%) in Gabon. Full-length genome analysis of 3 and additional sequences in pol for 10 such strains confirmed that they have a similar complex A/G mosaic genomic structure. These data suggest that in West Africa, most probably between 60% and 84% of the subtype A viruses are recombinant AG-IBNG viruses. This finding has potential implications on future vaccine, diagnostic, and treatment strategies. The actual and future role of these viruses in the global pandemic must be monitored in all new molecular epidemiologic studies, a discrimination between subtype A and AG-IBNG-like viruses is necessary.

Amino Acid Sequence↗
PubMedDate not supplied

The substitution of glucose-antiglucose oxidase complex (GAG) for peroxidase-antiperoxidase (PAP) in immunohistochemical studies of skin.

The substitution of glucose-antiglucose oxidase complex (GAG) for peroxidase antiperoxidase (PAP) in the Sternberger three-layer unlabeled antibody method avoids the problems associated with the use of the chromogen 3,3,diaminobenzidine (DAB). The blue final reaction product obtained using the GAG complex is not obscured by melanin granules, which is often the case when using the PAP-DAB combination. The replacement of PAP with GAG complexes facilitates the detection of melanocytes in melanoma and blue nevi. The absence of endogenous glucose oxidase in mammals provides for greater image contrast, which is not possible using alternative chromogens and immunoperoxidase.

Glucose Oxidase↗
PubMed1977

Type C viral gag gene expression in chicken embryo fibroblasts and avian sarcoma virus-transformed mammalian cells.

Sensitive radioimmunoassays were developed for avian type C viral gag gene-coded proteins. These assays were used to examine the restriction to virus production by avian embryo cells and mammalian cells transformed by avian sarcoma viruses. The results indicate that although a high-molecular-weight primary translational product of the gag gene is expressed, its cleavage and processing are incomplete. Furthermore, analysis of intermediate cleavage products provided information regarding the order of sequences coding for the individual viral proteins within the avian type C viral gag gene.

Alpharetrovirus↗
PubMed1978

Proteins of Rous-associated virus 61, an avian retrovirus: common precursor for glycoproteins gp85 and gp35 and use of pactamycin to map translational order of proteins in the gag, pol, and env genes.

Cells infected by Rous-associated virus 61 (RAV-61) contained a precursor-like protein, pr90, that was specifically precipitated by antiserum directed against envelope glycoproteins, gp85 and gp35. Tryptic peptide mapping showed that pr90 contained tryptic sequences of both gp85 and gp35. Pactamycin mapping experiments indicated that the two glycoproteins are translated from the env-mRNA in the order (5') gp85--gp35. The pactamycin mapping experiments also indicated a translational order of p10--(p27, p12)--p15 for the gag proteins; this agreement with the order previously reported from tryptic mapping studies on precursor pr76 of avian myeloblastosis virus implied that the stoichiometry of the core proteins was unchanged when virions were assembled in the presence of pactamycin. The reverse transcriptase proteins, unlike those of the env and gag genes, fell on the right side of the pactamycin map. This result is in accord with the idea that most, if not all, of the reverse transcriptase protein is translated by read-through of the gag(pol) message rather than by translation of a hypothetical pol-mRNA devoted solely to synthesis of that protein.

Avian Leukosis Virus↗
PubMed1980

Novel p19-related protein in Rous-associated virus type 61: implications for avian gag gene order.

Virions of Rous-associated virus type 61 contain a previously unrecognized p19-related protein, called p19f, which comigrates with gag protein p12 during electrophoresis in sodium dodecyl sulfate-polyacrylamide gels but can be separated by gel filtration chromatography in 6 M guanidine hydrochloride. It is shown that the existence of p19f accounts for the earlier inability to order p27 and p12 by the pactamycin mapping procedure. Remapping with pactamycin by using methods which take this new protein into account yielded a gag gene order of NH2-p219-p27-p12-p15-COOH. It also confirmed earlier positions for the env and pol genes and placed unclassified protein p10 near a translational initiation site. The pactamycin-derived mapping position of p12 differs from reports based on tryptic analysis. An analysis of procedural shortcomings emphasizes the need for more definitive determinations of the avian gag gene order.

Avian Leukosis Virus↗
PubMed1986

Molecular cloning and characterization of gag-, pol-, and env-related gene sequences in the ev- chicken.

Using less stringent hybridization conditions and cloned viral DNA probes representing the avian sarcoma virus gag, pol, env, and long terminal repeat (LTR) gene sequences, we detected related sequences in two avian species purportedly lacking all endogenous avian leukosis viruses, the ev- chicken and the Japanese quail. The blot hybridization patterns obtained with the various probes suggest the presence of between 40 and 100 copies of retrovirus-related sequences in the genomes of these two species. An ev- chicken genomic DNA library was prepared and screened with gag-specific and pol-specific DNA probes. Several different clones were obtained from this library and characterized. Analysis of these clones revealed that the retrovirus-related gene sequences are linked in the order LTR-gag-pol-env-LTR, a structure indicative of a complete provirus. These data indicate the presence of previously unidentified endogenous retrovirus species in avian cells, suggesting that under the appropriate conditions of hybridization additional, more distantly evolved families of endogenous retrovirus genes may be identified in vertebrate species.

Animals↗
PubMed1998

Functional interaction of human immunodeficiency virus type 1 Vpu and Gag with a novel member of the tetratricopeptide repeat protein family.

Viral protein U (Vpu) is a protein encoded by human immunodeficiency virus type 1 (HIV-1) that promotes the degradation of the virus receptor, CD4, and enhances the release of virus particles from cells. We isolated a cDNA that encodes a novel cellular protein that interacts with Vpu in vitro, in vivo, and in yeast cells. This Vpu-binding protein (UBP) has a molecular mass of 41 kDa and is expressed ubiquitously in human tissues at the RNA level. UBP is a novel member of the tetratricopeptide repeat (TPR) protein family containing four copies of the 34-amino-acid TPR motif. Other proteins that contain TPR motifs include members of the immunophilin superfamily, organelle-targeting proteins, and a protein phosphatase. UBP also interacts directly with HIV-1 Gag protein, the principal structural component of the viral capsid. However, when Vpu and Gag are coexpressed, stable interaction between UBP and Gag is diminished. Furthermore, overexpression of UBP in virus-producing cells resulted in a significant reduction in HIV-1 virion release. Taken together, these data indicate that UBP plays a role in Vpu-mediated enhancement of particle release.

Amino Acid Sequence↗
PubMed2003

Potent, persistent induction and modulation of cellular immune responses in rhesus macaques primed with Ad5hr-simian immunodeficiency virus (SIV) env/rev, gag, and/or nef vaccines and boosted with SIV gp120.

Immunity elicited by multicomponent vaccines delivered by replication-competent Ad5hr-simian immunodeficiency virus (SIV) recombinants was systematically investigated. Rhesus macaques were immunized mucosally at weeks 0 and 12 with Ad5hr-SIV(smH4) env/rev, with or without Ad5hr-SIV(mac239) gag or Ad5hr-SIV(mac239) nef, or with all three recombinants. The total Ad5hr dosage was comparably adjusted among all animals with empty Ad5hr-DeltaE3 vector. The macaques were boosted with SIV gp120 in monophosphoryl A-stable emulsion adjuvant at 24 and 36 weeks. Controls received Ad5hr-DeltaE3 vector or adjuvant only. By ELISPOT analysis, all four SIV gene products elicited potent cellular immune responses that persisted 42 weeks post-initial immunization. Unexpectedly, modulation of this cellular immune response was observed among macaques receiving one, two, or three Ad5hr-SIV recombinants. Env responses were significantly enhanced throughout the immunization period in macaques immunized with Ad5hr-SIV env/rev plus Ad5hr-SIV gag and tended to be higher in macaques that also received Ad5hr-SIV nef. Macaques primed with all three recombinants displayed significant down-modulation in numbers of gamma interferon (IFN-gamma)-secreting cells specific for SIV Nef, and the Env- and Gag-specific responses were also diminished. Modulation of antibody responses was not observed. Down-modulation was seen only during the period of Ad5hr-recombinant priming, not during subunit boosting, although SIV-specific IFN-gamma-secreting cells persisted. The effect was not attributable to Ad5hr replication differences among immunization groups. Vaccine delivery via replication-competent live vectors, which can persistently infect new cells and continuously present low-level antigen, may be advantageous in overcoming competition among complex immunogens for immune recognition. Effects of current multicomponent vaccines on individual immune responses should be evaluated with regard to future vaccine design.

Adenoviridae↗
PubMed1982

An unlabeled antibody method using glucose oxidase-antiglucose oxidase complexes (GAG): a sensitive alternative to immunoperoxidase for the detection of tissue antigens.

Immunoenzyme staining for tissue antigens using glucose oxidase as marker enzyme was compared with immunoperoxidase techniques. Both the indirect, conjugated antibody method and the unlabeled antibody procedure employing preformed complexes of glucose oxidase-antiglucose oxidase (GAG) and peroxidase-antiperoxidase (PAP) were used to stain carcinoembryonic antigen (CEA) in paraffin tissue sections. The localization of CEA within a tissue specimen was the same in all cases, and background staining was minimal. The percentage of positive specimens detected with GAG and PAP was similar but was slightly greater with glucose oxidase compared to peroxidase conjugates. The glucose oxidase conjugates and GAG were similar to the comparable immunoperoxidase reagents in enzyme-antibody molar ratio, retention of antibody and enzyme activity, immunohistochemical staining dilutions, and stability. Immunoglucose oxidase and immunoperoxidase were combined to localize CEA and colon-specific antigen-p simultaneously. Excellent contrast and staining separation were shown between the enzymatic reaction products of the two systems. Since immunoglucose oxidase methods are as sensitive as the comparable immunoperoxidase techniques, they should be considered as a reliable alternative to the latter, especially when endogenous peroxidase activity may be a problem. Furthermore, the two methods can be conveniently combined for the simultaneous detection of two antigens.

Carcinoembryonic Antigen↗
PubMed2003

Cross-reactivity between HLA-A2-restricted FLU-M1:58-66 and HIV p17 GAG:77-85 epitopes in HIV-infected and uninfected individuals.

BACKGROUND: The matrix protein of the influenza A virus and the matrix and capsid proteins of the human immunodeficiency virus (HIV) share striking structural similarities which may have evolutionary and biological significance. These similarities led us to hypothesize the existence of cross-reactivity between HLA-A2-restricted FLU-M1:58-66 and HIV-1 p17 GAG:77-85 epitopes. METHODS: The hypothesis that these two epitopes are cross-reactive was tested by determining the presence and extent of FLU/GAG immune cross-reactivity in lymphocytes from HIV-seropositive and seronegative HLA-A2+ donors by cytotoxicity assays and tetramer analyses. Moreover, the molecular basis for FLU/GAG cross-reactivity in HIV-seropositive and seronegative donors was studied by comparing lymphocyte-derived cDNA sequences corresponding to the TCR-beta variable regions, in order to determine whether stimulation of lymphocytes with either peptide results in the expansion of identical T-cell clonotypes. RESULTS: Here, we report evidence of cross-reactivity between FLU-M1:58-66 and HIV-1 p17 GAG:77-85 epitopes following in vitro stimulation of PBMC derived from either HIV-seropositive or seronegative HLA-A2+ donors as determined by cytotoxicity assays, tetramer analyses, and molecular clonotyping. CONCLUSION: These results suggest that immunity to the matrix protein of the influenza virus may drive a specific immune response to an HLA-A2-restricted HIV gag epitope in HIV-infected and uninfected donors vaccinated against influenza.

Journal Article↗
PubMed2003

[Construction and Identification of DNA vaccine containing chimeric gene gag-gp120 of HIV-1].

AIM: To construct DNA vaccine expression plasmid containing the chimeric gene gag-gp120 of HIV-1. METHODS: The recombinant eukaryotic expression vector pVAXGE was constructed via inserting the chimeric gene gag-gp120 into the vector pVAX1. Hela cells had been transfected by recombinant plasmid via liposome. After 72 h, the transfected cells was detected by RT-PCR and analyzed by Dot-ELISA. RESULTS: The transcript products of target gene could be amplified from the cells transfected by recombinant plasmid. Dot-ELISA detection showed that the target gene was expressed in Hela cells. CONCLUSION: The DNA vaccine plasmid expressing chimeric gene gag-gp120 was successfully constructed, which lays the foundation for preparing DNA vaccine against HIV-1.

Genetic Vectors↗
PubMed1989

Bacterial expression of the p24 gag protein of the bovine leukaemia virus.

The possibility of expression of the gag gene of bovine leukaemia virus (BLV) in the bacterial system was investigated. The DNA fragment coding for the gag core 24 kDa protein of BLV was inserted into the pORF1 expression vector. The polypeptides expressed in E. coli were analysed by Western blotting. The bacterially synthesized antigens were detected by the serum of a BLV-infected cow and by mouse monoclonal antibodies against the native p24 gag protein.

Antibodies, Monoclonal↗
PubMed1983

Localization of lipid-protein and protein-protein interactions within the murine retrovirus gag precursor by a novel peptide-mapping technique.

In HTG2 hamster cells infected with the replication-defective Gazdar murine sarcoma virus only immature virus particles are formed, with the uncleaved gag precursor Pr65 as the only major protein in the virion. We have investigated the structure of these particles by using in situ cross-linking followed by chemical and enzymatic cleavages of Pr65 to localize sites of lipid-protein and protein-protein interactions. Lipid-protein cross-links were localized within a 10-kDa fragment in the p15 region of Pr65. Homotypic protein-protein cross-links between Pr65 units were localized within the p15 regions and also within the p10 regions of Pr65. Similar data for processed gag proteins in Rauscher murine leukemia virus, a prototype of a mature C-type virus, suggest that these interactions of the gag precursor are not altered during maturation. To identify the sites of cross-linking within Pr65, we have developed a two-dimensional peptide mapping technique that is based on nearest neighbor analysis of fragments released by cyanogen bromide treatment of partial cleavage products in gel slices. In conjunction with cross-linking, the peptide mapping technique is a powerful means for localizing specific interactions on a polypeptide backbone.

Animals↗
PubMed1983

Dialyzability of methyl-GAG.

Methyl-GAG at low doses was administered to an anephric male patient with metastatic hypernephroma, who required hemodialysis. No toxicity was observed. Measurement of serum levels of methyl-GAG showed that the drug is dialyzable. Thus, when methyl-GAG is considered for patients who are anephric and are being dialyzed, full doses are allowable. In addition, dialysis should be effective in rapidly eliminating the drug in patients who develop toxic effects.

Adenocarcinoma↗
PubMed1983

The effects of Methyl-GAG on a transplantable murine renal cell adenocarcinoma.

The effect of Methyl-GAG on a transplantable murine renal cell adenocarcinoma was evaluated. Inoculation of Balb/C mice with 1 X 10(5) renal adenocarcinoma cells under the renal capsule resulted in 100% tumor growth and death of the animal in 29 +/- 5 days. In 66% of the control animals lung metastases was observed. Intraperitoneal injections of Methyl-GAG at dose levels of 200 mg/m2, 150 mg/m2, and 100 mg/m2 administered weekly failed to demonstrate any effect on local growth of the tumor. More than 50% incidence of metastatic development was noted in the treated groups. Methyl-GAG had no apparent effect on primary tumor growth or the retardation of metastatic incidence.

Adenocarcinoma↗
PubMed1981

Effectiveness of methyl-GAG (methylglyoxal-bis[guanylhydrazone]) in patients with advanced malignant lymphoma.

We treated 51 patients with advanced malignant lymphoma refractory to conventional therapy with methyl-glyoxal-bis(guanylhydrazone) (methyl-GAG) at doses ranging from 400 to 800 mg/sq m. Therapy was started on a weekly schedule and was switched to every other week in responding patients at the onset of toxicity. Partial responses were observed in 6 of 13 evaluable patients with Hodgkin's disease (46%), 5 of 10 patients with diffuse poorly differentiated lymphocytic lymphoma (50%), 2 of 4 patients with nodular poorly differentiated lymphocytic lymphoma (50%), and 3 of 13 patients with diffuse histiocytic lymphoma (23%). Two of six patients with mycosis fungoides showed objective improvement in cutaneous disease. Toxicity was generally mild and included muscular weakness, myalgia, mucositis, and diarrhea; two patients developed bronchospasm following drug infusions. We conclude that methyl-GAG has major antitumor activity when administered on this schedule to patients with advanced malignant lymphoma. The low degree of toxicity, unique mechanism of action, and minimal myelosuppressive effects suggest that methyl-GAG will prove useful in future trials of combination chemotherapy regimens for the treatment of lymphoma.

Adult↗
PubMedDate not supplied

Phase I--II trial of methyl-GAG in the treatment of patients with metastatic renal adenocarcinoma.

Methyl-GAG, a polyamine synthesis inhibitor, was prospectively evaluated in the treatment of advanced renal adenocarcinoma. Twenty-five patients with measurable disease received methyl-GAG weekly at a starting dose of 500 mg/m2 iv, with dose escalation by 50 mg/m2/week (maximum dose, 825). All 25 patients are evaluable for response. Four of these patients (16%) achieved responses including three partial responses and one complete response, with a median duration of 9 weeks (range, 4--15). Nine patients (36%) remained stable and 12 (48%) had progressive disease. In the four responders, regression of disease occurred within the first 4 weeks of therapy. Toxic effects were generally mild and included nausea or vomiting (68%), myalgia (44%), mucositis (40%), neuralgia (40%), weight loss (32%), diarrhea (24%), skin rash (8%), leukopenia (8%), and genital ulcers (4%). We conclude that methyl-GAG has clear, albeit limited, activity against renal adenocarcinoma.

Adenocarcinoma↗
PubMedDate not supplied

Plasma levels and urinary excretion of methyl-GAG following iv infusion in man.

The pharmacokinetics of methyl-GAG was studied after nephrectomy in five patients with renal cell carcinoma and in one patient with breast cancer. Following rapid infusion (30 minutes) of 700 mg/m2 of methyl-GAG, terminal half-life of 136-224 hours. Drug levels declined to 6%-20% of the initial plasma concentration in the first 24 hours. Approximately 40% of the dose was recovered in the urine after 2 weeks. Extensive extravascular distribution of methyl-GAG is suggested.

Antineoplastic Agents↗