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At least 1,279 records · Page 71Linked to original sources
PubMed1985

Sequences in the gag-pol-5'env region of avian leukosis viruses confer the ability to induce osteopetrosis.

DNA sequences encoding the genomes of three subgroup E avian leukosis viruses have been molecularly cloned. Virus recovered from one of these cloned DNAs (pRAV-0) caused no osteopetrosis while virus recovered from the second (lambda NY203) caused late onset osteopetrosis and virus recovered from the third (lambda NTRE-2) caused intermediate onset osteopetrosis. Restriction endonuclease fragments of the cloned viral DNAs were used to construct recombinant viruses that could be used to test for the role of gag-pol-5'env and 3'env-LTR sequences in the induction of osteopetrosis. The results of the pathogenicity tests indicate that gag-pol-5'env sequences confer the ability to induce osteopetrosis while 3'env-LTR sequences influence the time of onset and the severity of osteopetrosis.

Animals↗
PubMed1994

Four cross-linked HIV Gag peptides prime the immune response to HIV proteins in mice.

The functional help provided by four cross-linked synthetic peptides from HIV-1 Gag structural proteins was investigated in the mouse model. These peptides, selected upon non-self-criteria, are not predicted as T epitopes by classical prediction methods such as the Rothbard consensus or the amphipathy rule. Priming mice with these peptides allows the enhancement of the antibody response to HIV-1 Gag proteins (p55, p18, p24) given in the viral particle form. Furthermore, all of them also induce spleen and lymph node cells from primed mice to proliferate in vitro, in a MHC class II restricted context. This approach may help to identify relevant immunogenic viral epitopes that may be involved in a vaccinal strategy.

Amino Acid Sequence↗
PubMed1986

Phase I study of alpha-difluoromethylornithine and methyl-GAG.

alpha-Difluoromethylornithine (DFMO) is a potent inhibitor of the synthesis of putrescine (pu) and spermidine (sd) in some benign and malignant tissues. Intracellular deprivation of pu and sd has been shown to induce an enhanced uptake of polyamine-analogs such as methyl-GAG (MGBG). The purpose of this study was to investigate the tolerance and the toxicity of the combination of DFMO and MGBG. Thirty-six patients received 4 X 2 g of DFMO/day orally and every 2 weeks 250-500 mg/m2 of MGBG as a 2-hr infusion, starting on day 14. Besides the well known acute and late side-effects of methyl-GAG, dose-limiting toxicity consisted also of thrombocytopenia, leucopenia, dyspnea, hemolysis and jaundice. The maximal tolerated dose of MGBG for one course was 350 mg/m2 and for repeated courses 250 mg/m2, due to cumulative toxicity. Furthermore, after 8 weeks of continuous administration of DFMO 70% of the patients had a severe hearing loss, which was reversible after a treatment delay of 4-6 weeks. Since the hearing loss prohibited the continuous use of DFMO, two different schedules of intermittent DFMO-administration together with two different infusion periods of MGBG have been investigated in 15 patients. In none of these patients did hearing loss occur. The schedule of continuous administration of 4 X 2 g of DFMO/day orally for 21 days and 250 mg/m2 of MGBG as a 24-hr infusion on days 7, 14 and 21, repeated on day 42, was tolerated best. In 28 evaluable patients two partial remissions were seen. Pretreatment with DFMO significantly enhanced the toxicity of MGBG and the combination of both drugs produced side-effects not seen with either drug alone.

Adolescent↗
PubMed2004

Activation of c-myb by 5' retrovirus promoter insertion in myeloid neoplasms is dependent upon an intact alternative splice donor site (SD') in gag.

Alternative splicing in Mo-MuLV recruits a splice donor site, SD', within the gag that is required for optimal replication in vitro. Remarkably, this SD' site was also found to be utilized for production of oncogenic gag-myb fusion RNA in 100% of murine-induced myeloid leukemia (MML) in pristane-treated BALB/c mice. Therefore, we investigated the influence of silent mutations of SD' in this model. Although there was no decrease in the overall incidence of disease, there was a decrease in the incidence of myeloid leukemia with a concomitant increase in lymphoid leukemia. Importantly, there was a complete lack of myeloid tumors associated with 5' insertional mutagenic activation of c-myb, suggesting the specific requirement of the SD' site in this mechanism.

Animals↗
PubMed2002

Acid-labile formylation of amino terminal proline of human immunodeficiency virus type 1 p24(gag) was found by proteomics using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

HIV-1(LAV-1) particles were collected by ultracentrifugation, treated with subtilisin, and then purified by Sepharose CL-4B column chromatography to remove microvesicles. The lysate of the purified human immunodeficiency virus type 1 (HIV-1) particles was subjected to two-dimensional (2D) gel electrophoresis and stained, and the stained spots were excised and digested with trypsin. The resulting peptide fragments were characterized by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Twenty-five proteins were identified as the proteins inside the virion and the acid-labile formyl group of an amino terminal proline residue of HIV-1(LAV-1) p24(gag) was determined by MALDI-TOF MS before and after weak-acid treatments (0.6 N hydrochloric acid) and confirmed by post-source decay (PSD) of the N-formylated N-terminal tryptic peptide (N-formylated Pro(1)-Arg(18)). The role of formylation has been unclear so far, but it is surmised that the acid-labile formylation of HIV-1(LAV-1) p24(gag) may play a critical role in the formation of the HIV-1 core for conferring HIV-1 infectivity.

Amino Acid Sequence↗
PubMed2000

Contractile behavior of smooth muscle actin-containing osteoblasts in collagen-GAG matrices in vitro: implant-related cell contraction.

The contraction of connective tissue cells may facilitate their production and maintenance of extracellular matrix architecture and can benefit healing through wound closure. However, aberrant cell contraction can result in pathological contracture. Implants may stimulate this process leading to contracture of the surrounding fibrous capsule. In the case of compliant porous matrices used for tissue engineering the cell contraction may cause the constriction of pores and the distortion of the implant. The objective of this study was to determine if osteoblasts expressed a specific muscle actin, alpha-smooth muscle actin (SMA), that could provide for their contraction. Immunohistochemistry revealed the presence of SMA in some cells in all of three human and three canine trabecular bone specimens. The majority of the cells of an osteoblastic cell line, MC3T3-E 1, also expressed this actin isoform in two-dimensional culture and when seeded into a collagen-glycosaminoglycan (GAG) matrix. These SMA-containing cells were found to cause contraction of the collagen-GAG analog of extracellular matrix. These findings demonstrate that osteoblasts can display contractile behavior that might help to explain the mechanism by which they impart architecture to bone matrix, including that at implant interfaces. An understanding of this process could also guide the development of matrices for bone tissue engineering.

3T3 Cells↗
PubMed1999

c-Src association with and phosphorylation of p58gag, a membrane- and microfilament-associated retroviral Gag-like protein in a xenotransplantable rat mammary tumor.

The retroviral Gag-like protein p58gag expressed in a highly metastatic ascites rat mammary adenocarcinoma has been implicated in cell surface changes contributing to xenotransplantability. p58gag is present in the cells in a plasma membrane- and microfilament-associated signal transduction particle containing Src and is phosphorylated on tyrosine. Overlay analyses and affinity chromatography with glutathione S-transferase (GST) fusion proteins of Src homology-3 (SH3) domains showed direct binding of the Src but not the Crk SH3 domain to p58gag. This association was confirmed by co-immunoprecipitation of partially purified p58gag from ascites cell lysates with platelet Src. Further, a GST-p58gag fusion protein bound full length c-Src from either platelets or c-Src-expressing insect cells. The GST-p58gag fusion protein, but not GST, was phosphorylated by platelet or insect cell-expressed c-Src, but not by a kinase negative c-Src variant. The binding of GST-p58gag to c-Src was almost completely abolished by a 50-fold excess of the GST-SH3 domain of Src, and a parallel decrease in tyrosine phosphorylation of p58gag was observed. These results demonstrate that p58gag is tyrosine-phosphorylated as a consequence of its specific association with c-Src via its SH3 domain. These observations suggest a mechanism by which Gag proteins may contribute to retroviral maturation or pathogenesis through binding and relocalization of SH3 domain-containing proteins such as Src-like tyrosine kinases to sites of association of microfilaments with the plasma membrane.

Adenocarcinoma↗
PubMed1996

Role of glycosaminoglycans (GAGs) in regulation of the immunogenicity of human vascular endothelial cells.

Heparan sulphate is a common glycosaminoglycan component of proteoglycans present on the luminal surface of vascular endothelium. It has been proposed that an important function of these molecules is the sequestration of a range of proinflammatory and proadhesive cytokines. Such cytokines play a vital role during lymphocyte recruitment from the blood at sites of inflammation. In this study it is shown that the effects of interferon-gamma (IFN-gamma), but not of tumour necrosis factor-alpha (TNF-alpha), are inhibited by treatment with soluble heparin. Specifically, heparin was shown to inhibit the induction of class II MHC antigens and the up-regulation of intercellular adhesion molecule-1 (ICAM-1) produced by treatment of cultured human endothelial cells with IFN-gamma. Furthermore, it was shown that heparin blocked the enhanced adhesion of T lymphocytes to IFN-gamma-treated endothelial cells. Investigation of the inhibitory effects of other GAG molecules demonstrated a requirement for heparin-like structural domains as chondroitin sulphate was unable to inhibit the function of IFN-gamma. These results may explain reported immunosuppressive properties of heparin, and are consistent with the model that heparin may compete with cell surface GAGs to bind IFN-gamma, thereby reducing effective biological activity.

Cell Adhesion↗
PubMed1977

Identification of Rauscher murine leukemia virus-specific mRNAs for the synthesis of gag- and env-gene products.

Polyadenylylated mRNA isolated from cells infected with Rauscher murine leukemia virus was fractionated by centrifugation in in a denaturing sucrose gradient into different sizes. Each RNA fraction was injected into oocytes of Xenopus laevis and the virus-specific products were analyzed by immunoprecipitation with polyvalent and monospecific antisera against polypeptides of Rauscher murine leukemia virus, and then by gel electrophoresis and scintillation autoradiography. It was shown that a 35S mRNA species directs the synthesis of a precursor of the internal or group-specific antigens of the virion (the gag-gene products). A 22S mRNA species directs the synthesis of two viral envelope polypeptides and their precursor polypeptide (env-gene products). The results indicate that the gag- and env-related polypeptides of Rauscher murine leukemia virus are synthesized uncoordinately and provide evidence for open and closed cistrons on the virus-specific mRNAs.

Antigen-Antibody Reactions↗
PubMed2001

Mapping of hiv-1 Gag epitopes recognized by polyclonal antibodies using gene-fragment phage display system.

Phage display has emerged as a powerful technique for mapping epitopes recognised by monoclonal and polyclonal antibodies. We have recently developed a simple gene-fragment phage display system and have shown its utility in mapping epitope recognised by a monoclonal antibody. In the present study, we have employed this system in mapping epitopes recognised by polyclonal antibodies raised against HIV-1 capsid protein, p24 which is derived from proteolytic cleavage of Gag polyprotein. HIV-1 gag DNA was fragmented by DNase I and the fragments (50-250 bp) were cloned into gene-fragment phage display vector to construct a library of phages displaying peptides. This phage library was used for affinity selection of phages displaying epitopes recognised by rabbit anti-p24 polyclonal antibodies. Selected phages contained sequences from two discrete regions of p24, demonstrating the presence of two antigenic regions. The DNA sequences encoding these regions were also cloned and expressed as GST fusion proteins. The immunoreactivity of these epitopes as GST fusion proteins, or as phage-displayed peptides, was comparable in ELISA system using same anti-p24 polyclonal antibodies. The results indicate that the gene-fragment based phage display system can be used efficiently to identify epitopes recognised by polyclonal antibodies, and phage displayed epitopes can be directly employed in ELISA to detect antibodies.

Animals↗
PubMed1994

Betaglycan can act as a dual modulator of TGF-beta access to signaling receptors: mapping of ligand binding and GAG attachment sites.

Betaglycan, also known as the TGF-beta type III receptor, is a membrane-anchored proteoglycan that presents TGF-beta to the type II signaling receptor, a transmembrane serine/threonine kinase. The betaglycan extracellular region, which can be shed by cells into the medium, contains a NH2-terminal domain related to endoglin and a COOH-terminal domain related to uromodulin, sperm receptors Zp2 and 3, and pancreatic secretory granule GP-2 protein. We identified residues Ser535 and Ser546 in the uromodulin-related region as the glycosaminoglycan (GAG) attachment sites. Their mutation to alanine prevents GAG attachment but does not interfere with betaglycan stability or ability to bind and present TGF-beta to receptor II. Using a panel of deletion mutants, we found that TGF-beta binds to the NH2-terminal endoglin-related region of betaglycan. The remainder of the extracellular domain and the cytoplasmic domain are not required for presentation of TGF-beta to receptor II; however, membrane anchorage is required. Soluble betaglycan can bind TGF-beta but does not enhance binding to membrane receptors. In fact, recombinant soluble betaglycan acts as potent inhibitor of TGF-beta binding to membrane receptors and blocks TGF-beta action, this effect being particularly pronounced with the TGF-beta 2 isoform. The results suggest that release of betaglycan into the medium converts this enhancer of TGF-beta action into a TGF-beta antagonist.

Animals↗
PubMed1980

Amplified env and gag products on AKR cells. Origin from different murine leukemia virus genomes.

Thymocytes of AKR mice express two species of gp70, the envelope glycoprotein of murine leukemia virus (MuLV), encoded by the env gene. One is denoted Ec+ gp70 in reference to the type-antigen Ec and association with ecotropic virus. The other, Ec- gp70, resembles gp70 found also on thymocytes of mouse strains that are not overt producers of MuLV, and has no evident relation to ecotropic virus. Expression of Ec- gp70 type, but not of Ec+ gp70 type, is amplified with age on AKR thymocytes. In contrast, viral core polyproteins, encoded by the gag gene and simultaneously amplified with age, appear to be related to ecotropic virus. These observations imply selective amplification of products of env and gag genes from two sorts of provirus, a phenomenon which may be connected to the dual genetic origin of recombinant mink-cell-focus inducing viruses in AKR mice.

Animals↗
PubMed2000

Articular cartilage chondrocytes in type I and type II collagen-GAG matrices exhibit contractile behavior in vitro.

Natural healing of articular cartilage defects generally does not occur, and untreated lesions may predispose the joint to osteoarthritis. To promote healing of cartilage defects, many researchers are turning toward a tissue engineering approach involving cultured cells and/or porous, resorbable matrices. This study investigated the contractile behavior of cultured canine chondrocytes seeded in a porous collagen-glycosaminoglycan (GAG) scaffold. Chondrocytes isolated from the knee joints of adult canines and expanded in monolayer culture were seeded into porous collagen-GAG scaffolds. Scaffolds were of two different compositions, with the predominant collagen being either type I or type II collagen, and of varying pore diameters. Over the 4-week culture period, the seeded cells contracted all of the type I and type II collagen-based matrices, despite a wide range of stiffness (145 +/- 23 Pa, for the type I scaffold, to 732 +/- 35 Pa, for the type II material). Pore diameter (25-85 microm, type I; and 53-257 microm, type II) did not affect cell-mediated contraction. Immunohistochemical staining revealed the presence of alpha-smooth muscle actin, an isoform responsible for contraction of smooth muscle cells and myofibroblasts, in the cytoplasm of the seeded cells and in chondrocytes in normal adult canine articular cartilage.

Animals↗
PubMed2006

Effects of cross-linking type II collagen-GAG scaffolds on chondrogenesis in vitro: dynamic pore reduction promotes cartilage formation.

Articular cartilage tissue-engineering investigations often implement bioassays for chondrogenesis in vitro using articular chondrocytes or mesenchymal stem cells in cell pellets that contract with time in culture, suggesting an association between the processes of contraction of the cell pellet and cartilage formation. The objective of the present study was to investigate this relationship further using adult canine articular chondrocyte-seeded type II collagen-GAG scaffolds. The collagen-GAG scaffolds were chemically cross-linked to achieve a range of cross-link densities. Chondrocyte-seeded scaffolds of varying cross-link densities were then cultured for 2 weeks to evaluate the effect of crosslink density on scaffold contraction and chondrogenesis. Scaffolds with low cross-link densities experienced cell-mediated contraction, increased cell number densities, a greater degree of chondrogenesis (viz., chondrocytic morphology of cells, synthesis of type II collagen), and an apparent increase in the rate of degradation of the scaffold compared to more highly cross-linked scaffolds that resisted cellular contraction. The results of this study suggest the promise of "dynamic pore reduction" for scaffolds for articular cartilage tissue engineering. In this approach, scaffolds would have an initial pore diameter large enough to facilitate cell seeding and a mechanical stiffness low enough to allow for cell-mediated contraction to yield a reduced pore volume to favor chondrogenesis. This approach may provide a useful alternative to traditional means of increasing cell number density and retention of synthesized molecules that promote cartilage formation in tissue-engineered constructs.

Animals↗
PubMed1985

Phase II trial of methylglyoxal-bis-guanylhydrazone (methyl-GAG) in patients with soft-tissue sarcomas.

A phase II study of Methylglyoxal-bis-guanylhydrazone (Methyl-GAG) was conducted on 20 previously treated patients with soft-tissue sarcomas. No major responses were seen among 18 adequately treated patients. Toxicity including severe fatigue, muscle pains, and pharyngitis was noted in most patients. Methyl-GAG does not have significant antitumor activity in previously treated patients with soft-tissue sarcomas.

Adolescent↗
PubMed1982

Self-inflicted hanging with bound wrists and a gag.

This is a report of an unusual suicidal hanging by a 30-year-old male. He was found in his bedroom by friends, suspended from a ceiling light fixture by an electrical cord around his neck. His hands were bound behind his back and a sock gag was in his mouth. A paperback book was lying on the bed headboard. The book contained a detailed but totally inaccurate description of a fantasized hanging which may have contributed to the unusual binding and gagging. Homicide and autoerotic accident had to be considered. A series of suicide notes, however, simplified the case.

Adult↗
PubMed1997

Primary proliferative responses to peptides of HIV Gag p24.

Primary proliferative responses can be initiated by adding dendritic cells pulsed with antigen to autologous T cells in 20-microliter hanging drop cultures. To identify primary T-cell epitopes of HIV gag, a series of 23 overlapping peptides, 15 amino acids long, spanning the p24 region were used. Significant proliferative responses were induced in cells from healthy HIV-negative donors by 11 of these peptides. One of two peptides that bound human leukocyte antigen (HLA)-A *0201 in a peptide-binding assay using the antigen-processing defective cell line T2 also induced a primary proliferative response. Primary T-cell proliferation was seen in response to some peptides of gag that have not previously been identified as T-cell epitopes in cells from infected individuals. These epitopes might be useful not only for vaccines in antigenically naive individuals but also might increase the breadth of immune responses in seropositive patients.

Amino Acid Sequence↗
PubMed1993

Molecular basis and haematological characterization of beta-thalassaemia major in Taiwan, with a mutation of IVS-1 3' end TAG-->GAG in a Chinese patient.

We studied 41 patients with beta-thalassaemia major and their parents by using a combination of polymerase chain reaction (PCR) amplification, slot-blot hybridization of allele-specific oligonucleotide (ASO), and direct genomic sequencing. Eight different point mutations were characterized. C to T substitution at nucleotide (nt) 654 of intervening sequences (IVS) 2, accounting for 46.3% of mutant beta-globin genes, is the most common mutation in Taiwan, followed by frameshift codons 41/42 with four nucleotides (TCTT) deletion for 31.7%, A to G substitution at position -28 of promotor area for 8.5%, A to T substitution at codon 17 for 6.1%, frameshift codons 27/28 (insertion of C) for 2.4%, G to T substitution at nucleotide 1 of IVS-1 for 2.4%, frameshift codons 71/72 (insertion of A) and IVS-1 3 end TAG-->GAG for 1.2%. The former four mutations showed no obvious difference between two major ethnic groups in Taiwan. As to mutations in each individual of beta-thalassaemia major, the incidence of compound heterozygotes of two different mutations is much higher than homozygotes of single mutation, 78.0% v 22.0%. Compound heterozygotes of C to T substitution at nt 654 of IVS-2 and frameshift codons 41/42 with four nucleotides deletion is the most common pattern of beta-thalassaemia mutations in each individual (41.5%). The results are somewhat different from other documented reports concerning the mutations of beta-thalassaemia in southern China. This is the first report of mutation of IVS-1 3' end TAG-->GAG which causes consensus change in Chinese people. Patients with heterozygotes of beta zero and -28 beta(+)-thalassaemia mutations would have a greater delay in initial transfusion in comparison to patients with homozygotes of both beta zero-thalassaemia mutation, but their initial clinical manifestation might be aggravated when combined with a glucose-6-phosphate dehydrogenase (G-6-PD) deficiency and an insult such as exposure to infection and certain drugs.

Adolescent↗