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G Matlashewski

Publications and source records attributed to G Matlashewski.

54 records · Page 3Linked to original sources
PubMed1991

Leishmania donovani lipophosphoglycan selectively inhibits signal transduction in macrophages.

The effect of purified lipophosphoglycan (LPG) of Leishmania donovani on signal transduction and gene expression in murine bone marrow-derived macrophages was investigated. LPG stimulated the rapid expression of both c-fos and TNF genes within 30 min after exposure, suggesting that the interaction of LPG with its receptor stimulated a specific signal transduction pathway. Macrophages pretreated with LPG for 3 h became unresponsive to subsequent stimulation with LPS and the activators of protein kinase C, 1-oleoyl-2-acetyl-glycerol, and calcium ionophore A23187. Moreover, LPG induced a rapid down-modulation of TNF receptors. In contrast, the ability of macrophages to express the c-fos gene in response to the cAMP analogue, dibutyryl cAMP, was not impaired by LPG. Fragmentation of LPG revealed that the inhibitory activity of LPG required both the repeating phosphorylated disaccharides and the phosphosaccharide core. Collectively, these data demonstrate that LPG selectively impaired signal transduction in macrophages and suggest a role for this molecule in the survival of the parasite within the macrophage.

Animals↗
PubMed1991

Expression of the human papillomavirus E7 oncogene during cell transformation is sufficient to induce susceptibility to lysis by activated macrophages.

Human papillomaviruses (HPV), and in particular HPV type 16, are etiologic agents in the development of cervical cancer, which is the second most common form of cancer in women worldwide. Mammalian cells are susceptible to transformation in vitro by the E6 and E7 oncogenes derived from the HPV-16 genome. NIH-3T3 cells transfected with the HPV-16 E7 oncogene were found to exhibit cytolytic susceptibility to murine-activated macrophages. In comparison, E6 oncogene-expressing cells were not susceptible to lysis by activated macrophages. The E7 oncoprotein is multifunctional, being capable of complexing with the retinoblastoma tumor suppressor gene (anti-oncogene) product, stimulating DNA synthesis, and causing cell transformation in vitro. Macrophage killing assays performed on cell lines expressing E7 mutants revealed that the ability to complex the retinoblastoma tumor suppressor gene product and stimulate DNA synthesis did not induce susceptibility to activated macrophages, whereas the ability of E7 to cause transformation was required to induce susceptibility to activated macrophages. These data suggest that cell transformation is a more important prerequisite for inducing susceptibility to activated macrophages than is the loss of tumor suppressor gene function. This study also provides an initial link between HPV-16 oncogene expression and the ability of activated macrophages to selectively recognize and destroy HPV-16-associated neoplastic cells.

Amino Acid Sequence↗
PubMed1991

Characterization of a beta-tubulin gene and a beta-tubulin gene products of Brugia pahangi.

A genomic clone containing a beta-tubulin gene from the parasitic nematode Brugia pahangi was isolated. This gene was sequenced to determine its size, structural organization, and corresponding primary amino acid sequence. The coding sequence of the beta-tubulin gene spans 3.8 kb, is organized into 9 exons and expresses an mNRA of 1.8 kb which codes for a protein of 448 amino acids. The predicted beta-tubulin amino acid sequence is 89%, 94%, 90% and 88% identical to the chicken beta 2, and the Caenorhabditis elegans ben-1, tub-1 and mec-7 gene products, respectively. Southern hybridization analyses demonstrated that there is only one copy of this gene isotype but that other distinct beta-tubulin genes may exist in the Brugia pahangi genome. A nematode specific antipeptide rabbit antiserum raised against the predicted amino acid sequence of the extreme carboxy-terminal region of the B. pahangi beta-tubulin was used to identify beta-tubulin isoforms in adult nematodes and microfilariae. Isoforms detected by this nematode-specific antipeptide antiserum were identical in both adult worms and microfilariae and did not differ from the isoform patterns detected by a monoclonal antibody recognizing a conserved beta-tubulin epitope. This suggests that this carboxy-terminal peptide is highly represented in the beta-tubulin isoforms of B. pahangi.

Amino Acid Sequence↗
PubMed1990

Regulation of tumor necrosis factor gene expression and protein synthesis in murine macrophages treated with recombinant tumor necrosis factor.

The effect of murine rTNF-alpha on c-fos and TNF mRNA accumulation and protein synthesis was investigated in bone marrow-derived macrophages to examine the mechanism(s) by which TNF modulates macrophage activity. A rapid and transient expression of the c-fos gene was induced by murine rTNF. This was blocked by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, suggesting that the murine rTNF stimulated a protein kinase C-dependent signal transduction pathway. Although LPS induced the accumulation of one TNF mRNA species, murine rTNF induced the synthesis of two distinct TNF mRNA species. Both LPS- and murine rTNF-induced TNF mRNA accumulation was equally enhanced by pretreatment with mouse rIFN-gamma. In contrast, cycloheximide pretreatment had little effect on murine rTNF-induced TNF mRNA accumulation, whereas this treatment increased LPS-induced TNF mRNA by sevenfold. These results argue that TNF mRNA accumulation can be modulated in macrophages by distinct mechanisms. As assessed by Western blot and immunoprecipitation analysis, LPS stimulated the synthesis of both cell-associated and secreted forms of TNF protein. In comparison, newly synthesized TNF protein was not detected when macrophages were treated with murine rTNF alone or in combination with murine rIFN-gamma. This demonstrates that although murine rTNF stimulated the synthesis of two distinct TNF mRNA species, additional signal(s) are necessary for their translation into protein and that such signals are present after LPS stimulation.

Animals↗
PubMed1989

c-fos and tumor necrosis factor gene expression in Leishmania donovani-infected macrophages.

Leishmania donovani is an obligate intracellular protozoan which resides in macrophages and impairs a number of macrophage functions. We have undertaken to study this host cell-parasite interaction by examining the ability of L. donovani to impair the transmission of information from the cell surface to the nucleus and thus influence normal gene expression. We demonstrate that, in response to lipopolysaccharide, expression of both the c-fos and tumor necrosis factor genes was impaired in L. donovani-infected macrophages. Indomethacin reversed the parasite-mediated downregulation of the tumor necrosis factor gene but not the c-fos gene, suggesting that the impaired expression of these two genes occurred through different mechanisms. Direct stimulation of protein kinase C with oleoyl-2-acetoyl-3-glycerol did not abrogate inhibition of c-fos gene expression by L. donovani; however, L929 cell-conditioned medium induced a similar level of c-fos gene expression in both infected and noninfected macrophages. Impairment of c-fos gene expression by L. donovani thus appeared to be selective, depending on the external stimuli used to induce its expression. These data argue that L. donovani was capable of impairing macrophage gene expression in a selective rather than a general manner.

Animals↗
PubMed1988

Transformation of primary human fibroblast cells with human papillomavirus type 16 DNA and EJ-ras.

Human papillomavirus type 16 (HPV type 16) has been implicated as an etiological agent in human cervical cancer. This virus contains sequences which, under the proper transcriptional control, can increase the tumorigenicity of established mouse cells and cooperate with EJ-ras in transforming primary baby rat kidney (BRK) cells. These data argue that this virus contains oncogenic sequences. Because this is a human virus, it was important to study the effect on primary human cells of HPV type-16 DNA in both the presence and absence of EJ-ras. We now present data which demonstrate that HPV type-16 DNA, under the transcriptional control of Moloney murine leukemia viral long terminal repeats (MoMuLV-LTR), can extend the life span of primary human fibroblast cells in culture. Co-transfection of the HPV type-16 DNA containing plasmid together with an activated EJ-ras oncogene gives rise to transformed cells which grow faster, are morphologically different from and more aneuploid than cells established with only HPV type-16 DNA. Molecular analysis of these established and partially transformed human cells reveals that they contain and express the transfected DNA. These data argue that primary human cells can be transformed with HPV type-16 and EJ-ras sequences, whereas cells harboring only HPV type 16 DNA are largely normal but have an extended life span.

Animals↗
PubMed1987

Human papillomavirus type 16 DNA cooperates with activated ras in transforming primary cells.

The close association of human papillomavirus type 16 DNA with a majority of cervical carcinomas implies some role for the virus in this type of cancer. To define the transforming properties of HPV-16 DNA in vitro we have now performed transfection experiments on baby rat kidney cells using HPV-16 DNA in conjunction with an activated ras gene. We have demonstrated that a 6.6-kb DNA fragment, containing the early genes of HPV-16 under the control of Moloney murine leukaemia virus long terminal repeats (MoMuLV-LTRs), cooperates with EJ-ras in transforming these cells. Both DNAs are required and neither alone is effective. The cooperating activity appears to reside in a protein or proteins derived from the E6/E7 region of the HPV-16 genome.

Animals↗
PubMed1987

Expression of human papillomavirus type 6 and type 16 capsid proteins in bacteria and their antigenic characterization.

The L1 and L2 capsid proteins encoded by human papillomavirus types 6 and 16 (HPV-6 and HPV-16) have been synthesized in bacteria. Antisera were raised against the HPV-6 L1- and L2-beta-galactosidase fusion proteins and against an HPV-16 L1 C-terminal peptide which was 14 amino acids long. The HPV-16 L1 peptide antibodies have been shown to be highly reactive with the HPV-16 L1-beta-galactosidase protein but not against the equivalent HPV-6 L1-beta-galactosidase fusion protein. The effectiveness of these antibodies was compared with commercially available antibovine papillomavirus type 1 (BPV-1) antibodies and the results demonstrated that the anti-BPV-1 antibodies reacted well against HPV-6 L1-beta-galactosidase but not against HPV-16 L1-beta-galactosidase. In addition, the L2 portion of the HPV-6 L2-beta-galactosidase fusion protein appeared particularly immunogenic, since antibodies raised against this fusion protein were predominantly reactive with the L2 moiety. The HPV-16 L1 peptide antibodies described here will be preferred reagents for the specific detection of HPV-16 capsid antigens, which may be particularly important in early diagnosis of HPV-16 infection.

Antibodies, Viral↗
PubMed1987

Identification of human papillomavirus type 18 E6 polypeptide in cells derived from human cervical carcinomas.

We recently reported the expression of human papillomavirus type 18 (HPV-18) E6 protein in bacteria and the production of anti-E6 polyclonal antibodies. This work has now been extended with the production of a panel of monoclonal antibodies against the HPV-18 E6 protein. These antibodies demonstrate that there is little antigenic conservation in the E6 protein between HPV-16 and HPV-18, with only one antibody recognizing a cross-reactive epitope. We have used both the monoclonal and the polyclonal antibodies to look for E6 expression in a number of HPV DNA-containing cell lines. These reagents specifically detected a 16.5K mol. wt. polypeptide in cells derived from a human cervical carcinoma.

Antibodies, Monoclonal↗
PubMed1987

Alternative splicing of human p53 transcripts.

The human p53 gene is expressed in both normal and transformed human cells as a family of polypeptides. One type of heterogeneity, which results in variation in electrophoretic mobility, is found at amino acid residue 72 (Matlashewski et al, 1987). A second type of heterogeneity is described here which involves an alteration in the sequence of the open reading frame at the 5' end of the p53 mRNA. This heterogeneity is generated by alternative splicing at the acceptor site in intron 2 resulting in an altered 5' end coding region. This study represents the first description of an altered human p53 transcript arising by differential splicing.

Amino Acid Sequence↗
PubMed1986

Isolation of human-p53-specific monoclonal antibodies and their use in the studies of human p53 expression.

The isolation and construction of a complete human p53 cDNA and subsequent expression in monkey cells is described. A set of new anti-(human p53) monoclonal antibodies has also been obtained and used to show the expression of the human p53 cDNA in cos-l cells. These antibodies enable the specific detection of human p53, which is synthesised in the presence of p53 from other species. Fusion proteins of p53 with beta-galactosidase were used firstly as antigen and secondly, in conjunction with competition assays, to localise the determinants recognized by the antibodies. At least two previously unrecognized epitopes are involved and two of the antibodies are human-p53-specific. The epitopes are denaturation-resistant and the antibodies are, therefore, valuable for immunoblotting as well as immunoprecipitation and enzyme-linked immunoassay. Transfection of plasmids containing complete human p53 cDNA into monkey (cos-l) cells cause expression of human p53 recognized by the monoclonal antibodies. Control plasmids did not induce immunoreactive protein.

Animals↗
PubMed1986

Analysis of human p53 proteins and mRNA levels in normal and transformed cells.

p53 mRNA and proteins were examined in a variety of human transformed cells and in normal human foreskin fibroblast cells. Both the steady-state and translatable levels of p53 mRNA were the same in normal and transformed human cells. In vitro synthesized p53, programmed by mRNA from normal and transformed human cells, revealed that there was heterogeneity in the primary structure of p53 from these cells. Pulse labeling of cells and immunoprecipitation analysis with a panel of human reactive anti-p53 antibodies demonstrated that the types of p53 synthesized in vitro corresponded to the types made in vivo from SV80 and COLO 320 cells. No p53 was detectable by similar pulse-labeling analysis of HeLa and normal foreskin fibroblast cells. Since it was necessary to use anti-p53 sera from cancer patients to carry out much of the immunoprecipitation analysis in this study we therefore further characterised these sera to determine if they reacted with one or more than one epitope. p53-beta-galactosidase fusion proteins were synthesized in Escherichia coli and used to analyse the anti-p53 antibodies produced by cancer patients. We demonstrate that the antisera contain antibodies directed against epitopes in both the N-terminal and C-terminal regions of the p53 molecule.

Antibody Specificity↗
PubMed1986

The expression of human papillomavirus type 18 E6 protein in bacteria and the production of anti-E6 antibodies.

Human papillomavirus type 18 (HPV-18) has recently been closely linked with human malignant cervical lesions. The early region of the genome of the related bovine papillomavirus (BPV) has been shown to be important for the production of the transformed phenotype. BPV E6 has been shown to be a transforming protein. We report the primary structure of the HPV-18 E6 open reading frame and its predicted amino acid sequence. Both E6 protein and E6-beta-galactosidase fusion protein were synthesized in bacteria. Antisera were raised against the E6-beta-galactosidase fusion protein and against an E6 N-terminal peptide which was 14 amino acids long. We show that these antisera reacted on Western blots against E6 synthesized in bacteria. The HPV E6 antigen and antibodies described here will be useful in understanding HPV expression and its association with human malignancies and may also be diagnostically useful.

Amino Acid Sequence↗
PubMed1984

Isolation and characterization of a human p53 cDNA clone: expression of the human p53 gene.

A cDNA clone for human p53 cellular tumor antigen has been isolated and characterized. This clone contains the complete 3'-untranslated region and most of the open reading frame for the protein. Nucleotide sequence analysis revealed that p53 mRNA contains an Alu repeat in the 3'-untranslated region. Hybridization selection experiments showed this clone was capable of selectively binding p53 mRNA. In vitro translation of SV80 mRNA resulted in the synthesis of two immunoreactive p53 polypeptide species. Northern blot analysis showed that human p53 mRNA was 2.8 kb in length and was present in cell lines containing high and low levels of p53 protein. There appears to be only a single p53 gene in human cells and Southern blot analysis demonstrated no major genomic rearrangements or amplification of the p53 gene in the transformed cell lines examined.

Antigens, Neoplasm↗
PubMedDate not supplied

Control of interferon signaling in human papillomavirus infection.

Human papillomaviruses (HPV) infect mucosal and cutaneous epithelium resulting in several types of pathologies, most notably, cervical cancer. Persistent infection with sexually transmitted oncogenic HPV types represents the major risk factor for the development of cervical cancer. The development of HPV-associated cervical cancer has been closely linked to the expression of the viral oncogenes E6 and E7 in the tumor cells. The major viral oncoproteins, E6 and E7, target the cellular tumor suppressor gene products p53 and Rb, respectively. As detailed within, these interactions result in the stimulation of proliferation and the inhibition of apoptosis, thus representing major oncogenic insults to the infected cell. In addition to mediating transformation, the E6 and E7 genes also play significant roles in altering the immune response against infected cells by suppressing interferon (IFN) expression and signaling. At the clinical level, IFNs have been used in the treatment of HPV-associated cervical intraepithelial neoplasia (CIN) or cervical cancers with mixed results. The success of the treatment is largely dependent on the subtype of HPV and the immune response of the patients. Despite this inefficiency, the increasing knowledge about the regulation of IFN signaling pathways at molecular level may hold a promise for the use of new therapeutic strategies against HPV infection. Studies on the regulation of the function of IFN-inducible gene products by the E6 and E7 may lead to the development of new therapeutic approaches based on strategies that modify the function of the HPV oncoproteins and restore IFN-signaling pathways through endogenous control mechanisms.

Animals↗
PubMedDate not supplied

Molecular analysis of different allelic variants of wild-type human p53.

The p53 tumour suppressor gene is intensively studied because mutations in this gene are the most common genetic alteration so far identified in human cancer. Considerable emphasis has thus been placed on characterizing the biological differences between mutant and wild-type p53 protein. This has led to the realization that in cultured cells, mutant p53 behaves like an oncogene, whereas wild-type p53 is a tumor suppressor gene. The p53 protein is also a target for the tumour virus oncogene products SV40 large T, adenovirus E1B, and human papillomavirus type 16 E6, which are all capable of forming complexes to the p53 protein. Although p53 represents an extremely important cellular regulatory molecule which is well conserved, there exists two allelic variants of wild-type human p53 that differ both in primary and confirmational structure. One variant contains an arginine at amino acid 72 (p53Arg), whereas the other form contains a proline at this residue (p53Pro). The possible implications for more than one allelic variant of wild-type human p53 in the general population is unknown. The present study was undertaken to compare some of the biological features of the different wild-type p53 variants. We present data demonstrating that there was a post-transcriptional selection against accumulation of both variants of wild-type human p53 in 3T3-A31 cells, arguing that both forms are proliferation inhibitory in these cells. Both variants of human p53 were stabilized by SV40 large T, but did not displace mouse p53 from SV40 large T. Neither allelic variant of human p53 was able to reduce significantly SV40-mediated anchorage-independent growth of 3T3-A31 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMedDate not supplied

The cell biology of human papillomavirus transformed cells.

Viruses are becoming increasingly recognized as a major etiological agent in the development of numerous forms of human cancer. Human papillomaviruses (HPVs) have been associated with a number of neoplastic lesions, most notably cervical cancer which is one of the major forms of cancer world wide. Of the over 50 types of identified HPVs, HPV types 16, 18, 31 and 33 are the types most commonly associated with malignant carcinomas. These viruses contain double stranded DNA which code for about eight gene products, some of which are oncogenic when introduces into cultured rodent or human cells. In particular, both the E6 and E7 gene products have different oncogenic capabilities and these genes are selectively retained within the genome of cervical carcinoma derived cells. The E7 gene product has immortalizing capabilities in primary cells and is able to cooperate with an activated ras oncogene to fully transform primary rodent cells. The E7 gene product from HPV type 16 is also capable of complexing in vitro to the anti-oncogene product, Rb. Similar complexes occur with Adenovirus E1A and SV40 large T proteins which may suggest a shared mechanism of transformation used by HPV type 16, Adenovirus and SV40. Transformation studies using primary human cells and nontumorigenic HeLa/fibroblast hybrid cells have also suggested that chromosome 11 may be important in suppressing the HPV transformed phenotype. The transformed phenotype may therefore also involve an impaired intracellular control of persisting HPV oncogenic sequences. Although there exists no solid evidence that a cytotoxic T-lymphocyte reaction is mounted against HPV transformed cells, there is evidence that both NK cells and activated macrophages can preferentially kill HPV transformed cells in vitro. Future studies are required to identify possible targets present on HPV transformed cells which are absent on normal cells.

Animals↗