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Biomedical subjects

M Ruta

Publications and source records attributed to M Ruta.

18 recordsLinked to original sources
PubMed2000

Nice snake, shame about the legs.

Snakes are one of the most extraordinary groups of terrestrial vertebrates, with numerous specializations distinguishing them from other squamates (lizards and their allies). Their musculoskeletal system allows creeping, burrowing, swimming and even gliding, and their predatory habits are aided by chemo- and thermoreceptors, an extraordinary degree of cranial kinesis and, sometimes, powerful venoms. Recent discoveries of indisputable early fossil snakes with posterior legs are generating intense debate about the evolutionary origin of these reptiles. New cladistic analyses dispute the precise significance and phylogenetic placement of these fossils. These conflicting hypotheses imply radically different scenarios of snake origins and relationships with wide biological implications.

Journal Article↗
PubMed1991

Kinetics of early HIV-1 gene expression in infected H9 cells assessed by PCR.

The time course of viral gene expression in H9 cells acutely infected with HIV-1 was analysed by the polymerase chain reaction (PCR). Virus-specific sequences were first detected in genomic DNA of H9 cells 1-2 h after infection. RNA for the regulatory genes such as the tat and nef appeared 2-3 h post-infection and RNA for the gag and env at 3 h. The results demonstrate that viral DNA synthesis occurs rapidly after infection of target cells followed by synthesis of viral RNA. Cell-associated reverse transcriptase activity increased after 24 h, while culture supernatant enzyme activity increased later, between 1-5 days. The delay in virus release after rapid integration and transcriptional activity suggests the involvement of additional factors, perhaps both cellular and viral, that control the formation and budding of mature virions.

Cell Line↗
PubMed1990

Negative regulation of human immune deficiency virus replication in monocytes. Distinctions between restricted and latent expression in THP-1 cells.

In THP-1 monocytoid cells infected with HIV, viral expression can be regulated in several ways: (a) latency (no viral expression); (b) restricted expression (chronic low-level viral expression with little or no detectable virus released); and (c) continuous production. In cells with restricted HIV expression, nuclear factor(s) were found that blocked tat-associated DNA binding complex formation, suggesting that initiation of transcription was negatively regulated. Also, viral particles were seen budding into and accumulating within intracytoplasmic vacuoles with little virus released, suggesting multiple levels of regulation. These cells with restricted expression had no detectable viral antigens on the cell surface and were not lysed by IL-2-activated large granular lymphocytes. However, they could cause viral-mediated T cell cytolysis in cell-cell assays, suggesting viral transmission through cell contact. In addition, cells with latent HIV were identified and could still produce infectious virus after 5-azacytidine exposure 10 mo later. LPS and other treatments could increase viral production in cells with restricted but not latent expression, suggesting they occur by distinct mechanisms. These infected cells provide a reservoir for viral transmission to uninfected T cells that itself is not detected by immune surveillance mechanisms.

Antigens, Viral↗
PubMed1990

Cloning and expression of two distinct high-affinity receptors cross-reacting with acidic and basic fibroblast growth factors.

The fibroblast growth factor (FGF) family consists of at least seven closely related polypeptide mitogens which exert their activities by binding and activation of specific cell surface receptors. Unanswered questions have been whether there are multiple FGF receptors and what factors determine binding specificity and biological response. We report the complete cDNA cloning of two human genes previously designated flg and bek. These genes encode two similar but distinct cell surface receptors comprised of an extracellular domain with three immunoglobulin-like regions, a single transmembrane domain, and a cytoplasmic portion containing a tyrosine kinase domain with a typical kinase insert. The expression of these two cDNAs in transfected NIH 3T3 cells led to the biosynthesis of proteins of 150 kd and 135 kd for flg and bek, respectively. Direct binding experiments with radiolabeled acidic FGF (aFGF) or basic FGF (bFGF), inhibition of binding with native growth factors, and Scatchard analysis of the binding data indicated that bek and flg bind either aFGF or bFGF with dissociation constants of (2-15) x 10(-11) M. The high affinity binding of two distinct growth factors to each of two different receptors represents a unique double redundancy without precedence among polypeptide growth factor-receptor interactions.

Amino Acid Sequence↗
PubMed1990

Assessment by gene amplification and serological markers of transmission of HIV-1 from hemophiliacs to their sexual partners and secondarily to their children.

The transmission of HIV-1 infection from men with hemophilia A to their female sex partners and secondarily to their children was studied by serological markers including antibody, antigen, and HIV genome as detected by the polymerase chain reaction (PCR). Among 27 sex partners of 26 seropositive hemophiliacs, 5 were seropositive-PCR positive (active), 11 were seronegative-PCR positive (latent), and 11 were negative for both. These results were confirmed by testing serial serum samples and paired samples of DNA from peripheral blood mononuclear cells (PBMCs) and serum from seronegative women. PCR negativity in exposed women was correlated with the use of condoms (p less than 0.01). Eight children from five couples were seronegative. However, HIV-1 infection as detected by PCR was transmitted to 60% of exposed children, including one from a seronegative-PCR positive mother.

Adult↗
PubMed1989

Receptor for acidic fibroblast growth factor is related to the tyrosine kinase encoded by the fms-like gene (FLG).

We have previously isolated a human gene from an endothelial cell cDNA library encoding a putative tyrosine kinase; we have designated this gene the fms-like gene (FLG). To analyze the gene product(s) of FLG, we have generated rabbit polyclonal antibodies directed against a synthetic peptide from FLG and used it to immunoprecipitate biosynthetically labeled FLG protein from a variety of human cell lines. These antibodies specifically recognized glycoprotein(s) of 100, 120, and 135 kDa with protein cores of 90 and 110 kDa. Acidic fibroblast growth factor (aFGF) stimulated tyrosine kinase activity of FLG in vitro and in living cells, suggesting that FLG encodes the membrane receptor for aFGF. Further supporting evidence came from cross-linking experiments on intact cells with the covalent cross-linking agent disuccinimidyl suberate and 125I-labeled aFGF as a specific probe. The cross-linked 125I-labeled aFGF-aFGF receptor complex was specifically immunoprecipitated with FLG antipeptide antibodies. It appears, therefore, that the receptor(s) for aFGF is related to the FLG gene product.

Animals↗
PubMed1989

Co-amplification of multiple regions of the HIV-1 genome by the polymerase chain reaction: potential use in multiple diagnosis.

We have used the polymerase chain reaction (PCR) to detect by co-amplification, multiple regions of the HIV-1 genome in infected cells. Genomic RNA and DNA from productively infected H9 cells were independently extracted and amplified in reactions with and without reverse transcriptase respectively using primer pairs to the gag, env, tat and nef regions of the viral genome in the same reaction mixture. PCR-products were analysed by liquid hybridization with end labelled oligonucleotide probes followed by gel-electrophoresis (oligomer hybridization). The primer pairs were capable of detecting as few as 10 copies of RNA and 10-20 copies of integrated proviral DNA. The ability to co-amplify multiple target regions in the same incubation mixture provides a method for detecting and confirming the presence of HIV-1 in samples for which limited nucleic acid is available. In addition, in reconstitution experiments, the same method was used to detect HIV-1 and HTLV-I simultaneously with comparable sensitivity (20-40 gene copies each). This offers the possibility of simultaneous diagnosis of multiple viral infections, such as those that occur in AIDS, on the same sample preparation.

DNA, Viral↗
PubMed1986

Nucleotide sequence of the two rat cellular rasH genes.

We present the nucleotide sequence of the coding region of the rat c-rasH-1 gene and a partial sequence analysis of the rat c-rasH-2 gene. By comparing these sequences with the Harvey murine sarcoma virus ras gene, we predict that the p21 protein encoded by the Harvey virus differs from the cellular c-rasH-1-encoded p21 at only two amino acids; those at positions 12 and 59. Alterations at each of these positions may play a role in activating the viral p21 protein. The c-rasH-2 gene is likely to be a nonfunctional pseudogene because it lacks introns, cannot be activated to transform NIH 3T3 cells, and differs in sequence from both c-rasH-1 and v-rasH at several base pair positions.

Amino Acid Sequence↗
PubMed1983

Loss of leukemogenicity caused by mutations in the membrane glycoprotein structural gene of Friend spleen focus-forming virus.

Friend virus infection of mice causes progressive leukemogenesis--a rapid splenic erythroblastosis that develops weeks later into a disseminating erythroleukemia. Furthermore, the replication-defective Friend spleen focus-forming virus (F-SFFV) encodes a membrane glycoprotein with an apparent Mr of 55,000 (designated gp55), which is structurally and immunologically related to the membrane envelope glycoproteins of dual tropic murine leukemia viruses. We now have isolated three spontaneous F-SFFV mutants that encode abnormally sized gp55-related glycoproteins with apparent Mrs of 40,000, 54,000, and 58,000, respectively. RNA blot and Southern blot analyses indicate that the mutant nucleic acids do not have substantial deletions or insertions in their glycoprotein gene regions. Protein fragmentation patterns indicate that the mutations affect nonoverlapping domains of the glycoprotein. Furthermore, these mutant glycoproteins seem to be defective in their processing to the plasma membranes. Although transmitted efficiently between cultured cells, the mutants have dramatically reduced leukemogenicities compared with the same titers of wild-type F-SFFV. We conclude that the gp55 structural gene is necessary for initiating the erythroblast proliferative phase of Friend disease and that changes in membranes can be primary causes rather than only secondary consequences of tumor progression.

Animals↗
PubMed1981

Mutant cells that abnormally process plasma membrane glycoproteins encoded by murine leukemia virus.

Wild-type normal rat kidney fibroblasts infected with the Friend strain of murine leukemia virus (MuLV) contain two virus-encoded glycoproteins on the outer surfaces of their plasma membranes: an envelope glycoprotein with an apparent molecular weight of 70,000 (gp70), and a glycoprotein that reacts with antisera to the major virion internal core proteins p30, p15, p12 and p10 and has an apparent molecular weight of 93,000 (gp93gag). To analyze the functions of these glycoproteins and to develop a model system for studying genetics of membrane synthesis, we used an immunoselection method to isolate variant cell clones defective in processing these glycoproteins into their plasma membranes. Several lines of evidence, including complementation of glycoprotein processing defects by fusion with uninfected wild-type cells, indicate that the immunoselected variants have stably inherited membrane synthesis abnormalities that are encoded by cellular rather than by viral genes. The H-4 cell line, which was selected by use of antiserum to gp70, has metabolic defects that interfere with processing of both gp70 and gp93gag into its plasma membranes. Nevertheless, this cell line releases noninfectious MuLV. Furthermore, two cell lines (2 and 5), which were selected by use of antiserum to the virion core protein p30, specifically lack detectable cell surface or intracellular gp93gag but contain cell surface gp70 and release infectious MuLV. These results suggest that MuLV particles can bud efficiently from cells that lack known virus-encoded plasma membrane constituents.

Animals↗
PubMed1980

Immunoselection of mutants deficient in cell surface glycoproteins encoded by murine erythroleukemia viruses.

We have described a heterogeneously processed glycoprotein with an apparent molecular weight of 55,000 (gp55) that is encoded by the Friend spleen focus-forming virus, an acute erythroleukemia virus [Dresler, S., Ruta, M., Murray, M.J. & Kabat, D. (1976) J. Virol. 30, 564-573]. Several lines of evidence suggest that a small proportion of the gp55 in infected cells is located on the surface membranes. First, different nonproducer cell lines infected with cloned Friend spleen focus-forming virus were efficiently killed in the presence of complement by cytotoxic antisera that react with gp55. Furthermore, a clone of cells selected for resistance to the cytotoxic antibody synthesized an altered intracellular form of gp55. This immunoselection procedure appears to also be useful for isolating glycoprotein mutants of other RNA tumor viruses. Analysis of cell surface proteins labeled with [125I]iodine supported the idea that gp55 occurs on the plasma membranes. However, the cell surface gp55 had more highly processed oligosaccharides than the majority of the gp55, which occurs within the infected cells. In addition, we have found that leukemia cells from mice infected with the erythroleukemic Rauscher virus complex contain a membrane glycoprotein that appears similar to the gp55 encoded by Friend spleen focus-forming virus. The encoding of similar glycoproteins by independently isolated acute erythroleukemia viruses suggests that these glycoproteins may be important in leukemogenesis.

Animals↗
PubMed1980

Plasma membrane glycoproteins encoded by cloned Rauscher and Friend spleen focus-forming viruses.

Rauscher spleen focus-forming virus (SFFV) was cloned free of its helper virus into normal rat kidney and mouse fibroblasts, and the resulting nonproducer fibroblast clones were analyzed. Our results suggested that Rauscher SFFV encodes a glycoprotein with an apparent Mr of 54,000 (gp54) that reacts with antisera made to the envelope glycoprotein (gp70) of ecotropic murine leukemia viruses, as well as with a rat antiserum that reacts with the gp70's of dual-tropic mink cell focus-inducing and HIX viruses but not with the gp70's of ecotropic viruses. In these respects and in its tryptic peptide map, Rauscher SFFV-encoded gp54 is nearly identical to the gp55 glycoprotein which we previously reported to be encoded by Friend SFFV (Dresler et al., J. Virol. 30:564--575, 1979). However, gp54 is slightly smaller, and it lacks one methionine-containing tryptic peptide that occurs in gp55. Studies with cytotoxic antiserum in the presence of complement and with a rosetting technique which employed sheep erythrocytes coupled to protein A suggested that the gp54 and gp55 glycoproteins are weakly expressed on the surface membranes of SFFV-infected cells. In addition, the Rauscher SFFV genome also encodes gag polyproteins which appear to be identical to the gag polyproteins encoded by helper Rauscher murine leukemia virus, but differ from the antigenically related polyproteins encoded by some but not all clones of Friend SFFV. Furthermore, the glycosylated gag polyproteins encoded by Rauscher SFFV and by some Friend SFFVs also appear to be expressed on the surface membranes of infected cells. These results suggest that similar env gene recombination and partial deletion events were involved in the independent origins of two different strains of acute erythroleukemia virus.

Animals↗
PubMed1979

A murine leukemia virus mutant with a temperature-sensitive defect in membrane glycoprotein synthesis.

Cells infected with a temperature-sensitive mutant (ts-26) of Rauscher murine leukemia virus (R-MuLV) or with wild-type virus were labeled with 35S-methionine, and cell extracts were examined for radioactive polypeptides which could be precipitated by monospecific antisera to viral proteins. When shifted from permissive (31 degrees C) to nonpermissive (39 degrees C) temperature, cells infected with ts-26 rapidly begin to accumulate gPr90enr, the glycoprotein precursor to the membrane envelope glycoprotein gp70 and to the membrane-associated protein p15E. Simultaneously, formation of these mature virion proteins ceases. In addition, lactoperoxidase-catalyzed surface labeling with 125I--iodine indicates that the plasma membrane of cells infected with ts-26 becomes depleted of gp70 antigens at 39 degrees C. Nevertheless, at 39 degrees C these cells release defective MuLVs which lack gp70 and p15E but contain an outer membrane. The released particles also contain an aberrantly processed form of the major virion core protein p30, and many of these virion cores have an unusual immature crescent shape. It has previously been reported that cells infected with the ts-26 mutant of R-MuLV process a 65,000 dalton precursor (Pr65gag) of the virion core proteins more slowly at 39 degrees C than do cells infected with wild-type virus (Stephenson, Tronick and Aaronson, 1975). Although we have confirmed these results, this effect is relatively small and it is known that various alterations of MuLV assembly can lead secondarily to inhibited processing of Pr65gag. We propose that the ts-26 mutant has a primary temperature-sensitive defect in membrane glycoprotein synthesis and that this change causes pleiotropic effects on core morphogenesis.

Genes↗
PubMed1979

Glycoprotein encoded by the Friend spleen focus-forming virus.

The Friend spleen focus-forming virus (F-SFFV) released from cultured erythroleukemia cells (cell line F4-6/K) was cloned free of its helper lymphatic leukemia virus (F-MuLV). After allowing adsorption to Sc-1 fibroblasts at a low multiplicity of infection, the cells were seeded individually into wells of a microtitier test plate and the resulting colonies were grown into large cultures. Among 14 of these cell cultures that have been analyzed thoroughly, 6 contained F-SFFV alone, 1 contained F-MuLV plus F-SFFV, and 7 were uninfected. Each of the Sc-1 cell lines which had been infected with cloned F-SFFV contained a glycoprotein with an apparent molecular weight of 55,000 (gp55) that was absent from the cell lines that lacked F-SFFV. gp55 was also present in Friend erythroleukemia cells and in fibroblasts infected with an F-SFFV that had been doubly cloned in another laboratory. These results indicate that gp55 is encoded by the F-SFFV genome. gp55 has the following additional properties. It can be immunoprecipitated with antiserum made to the F-MuLV virion envelope glycoprotein (gp75). Its unglycosylated polypeptide, formed in cells treated with 2-deoxy-D-glucose, has a molecular weight of approximately 45,000. Its tryptic peptide map contains peptides in common with F-MuLV gp75 but it also contains unique peptides. It appears to be absent or present in only low concentrations in erythroleukemia cell plasma membranes as determined by lactoperoxidase-catalyzed iodination, and it accumulates intracellularly in large amounts. In addition, it is absent from released virions. The majority of the cellular gp55 has an isoelectric point of 8.5 to 9.0. These results are consistent with the idea that an env gene recombination event was involved in the origin of F-SFFV.

Cell Line↗
PubMedDate not supplied

Brief review of the stylophoran debate.

The "calcichordate" theory interprets an extinct group of calcite-plated invertebrates, the stylophorans, as chordates. In this theory, comute stylophorans are interpreted as stem chordates, whereas mitrate stylophorans are primitive members of the acraniates, tunicates, and craniates. However, this theory discounts major synapomorphies between comutes and mitrates. These groups constitute a natural, monophyletic group which is here argued to lie within the echinoderm radiation. The "calcichordate" theory is, therefore, rejected because it relies on assumption-driven hypotheses of character transformation which are supported by ambiguous, poor, or missing fossil evidence. Stylophorans may lie at the base of the echinoderm clade and primitively lack pentameral symmetry, therefore casting light on the near-ancestral body organization of the phylum.

Animals↗