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At least 289 records · Page 16Linked to original sources
PubMed1985

A primary in vitro antibody assay for antigen-specific T-suppressor factor: cross-suppression of TNP-specific antibody responses by TMA-specific TsF1.

We have previously shown that phenyltrimethylammonium (TMA)-specific, first-order suppressor T cells (Ts1) and soluble factors extracted from these cells (TsF1) can suppress delayed-type hypersensitivity (DTH) responses. The TsF1, as monitored in the DTH system, was characterized and found to be a single-chain, antigen-binding, I-J+, and Id+ molecule. To monitor TsF1 in an efficient manner, an in vitro antibody system was developed. The studies show that in vitro stimulation of naive A/J spleen cells with the thymic-independent antigen, Brucella abortus, to which TMA and trinitrophenol (TNP) or fluorescein (FL) are coupled (TMA-BA-TNP or TMA-BA-FL), induces significant numbers of anti-TNP or anti-FL plaque-forming cell (PFC) responses. The addition of TMA-specific TsF1 results in the cross-suppression of 30-50% of the total anti-TNP and FL PFC responses. This activity is antigen (TMA) dependent since suppression occurs only when the TMA ligand is present in the culture media. Analysis of the TNP-specific PFC responses in nonsuppressed cultures revealed that 20-35% of the PFC bear the cross-reactive idiotype(s) (CRI) normally associated with anti-TMA antibodies. In cultures containing TMA-TsF1, CRI+PFC are suppressed by 90-100% while the CRI-PFC are suppressed only by 10-30%. Our studies further show that an induction-phase, antigen-binding, CRI+, and I-J+ single-chain factor is responsible for the observed in vitro suppression. The possibility of utilizing this assay to monitor a variety of antigen-specific suppressor factors is discussed.

Animals↗
PubMed1985

Regulation of hapten-specific T-cell response. II. Functional analysis of helper T cells and cytotoxic T cells in animals suppressed by azobenzenearsonate (ABA)-specific suppressor T cells.

The administration of azobenzenearsonate-modified syngeneic spleen cells (ABA-SC) intravenously induces a population of first order hapten-specific inducer suppressor T cells (Ts1), which downregulate various aspects of T-cell-mediated immune responses via a well defined suppressor-T-cell pathway. In this study, we investigated the effects of these suppressor cells on the generation of ABA-specific cytolytic T lymphocytes (CTL) and helper T cells (Th) in vivo. We found evidence for functional impairment of ABA-activated Th and ABA-specific CTL precursors (CTLp) in the suppressed animals by a number of different in vitro criteria. Functional analysis of ABA-specific CTLp and ABA-activated Th in suppressed animals revealed that ABA-specific Ts inhibit the generation of CTL by impairing the antigen-specific activation of Th, which may in turn, prevent the clonal expansion of antigen-specific CTLp. The significance of these findings in relationship to our understanding of the cellular interactions necessary for the generation of CTL and the mode of action and mechanisms of suppressor T cells is discussed.

Animals↗
PubMed1996

Association of Drosophila cysteine string proteins with membranes.

Cysteine string proteins are putative synaptic vesicle proteins that lack a transmembrane domain. Our analysis shows that Drosophila cysteine string proteins are extensively modified by hydroxylamine-sensitive fatty acylation. This modification could be responsible for association of csp's with membranes. Extensive deacylation of Dcsp's by a 20 h incubation in 1 M hydroxylamine, pH 7.0, or methanolic KOH produces a protein of 6-7 kDa lower mass than untreated Dcsp's. Surprisingly, the hydroxylamine treatment does not cause release of Dcsp's from membranes. On the other hand, alkaline stripping of membranes isolated from Drosophila brain by 0.1 M sodium carbonate, pH 11.5, causes a significant release of Dcsp's from membranes into the cytosol. These results indicate that fatty acylation may not form the main anchor of Dcsp's in membranes. Taking advantage of the endocytotic block in the Drosophila mutant shibire ts1, we analyzed the acylation states of Dcsp's in two stages during synaptic vesicle recycling and found no evidence for an acylation/deacylation cycle of Dcsp's in the brain nerve terminals.

Acylation↗
PubMed1976

Mutation induction by MNNG in a bacteriophage of Haemophilus influenzae.

Three temperature-sensitive mutants of the Haemophilus influenzae phage HP1c1 were tested for reversion to wild type (ts leads to ts+). Treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) produced revertants at levels up to 0.1% of the total progeny phage from treated lysogens. Cells treated with MNNG after infection with whole ts phage produced progeny phage with similar reversion frequencies, but when the uninfected cells or the phage were treated alone no reversion was induced. Fixation of premutational lesions was shown to occur with no evidence for host-cell DNA synthesis, indicating that phage DNA synthesis may be responsible for fixation of mutation in phage DNA. Evidence is given which shows that prophage DNA replicating by the cells' replicating system after treatment and before induction, produces the same number of revertants per survivor as phage DNA which is replicated outside the host genome. Two of the phage mutants (ts1 and ts2) reverted at similar frequencies, while one of the mutants (ts3) exhibited a much lower induced reversion frequency.

Bacteriophages↗
PubMed1983

A group of temperature-sensitive mutants of Moloney leukemia virus which is defective in cleavage of env precursor polypeptide in infected cells also induces hind-limb paralysis in newborn CFW/D mice.

A group of temperature sensitive mutants of Moloney murine leukemia virus (MoMuLV) designated as ts1, ts7, and ts11 rapidly and invariably induce hind-limb paralysis ranging from 28 to 52 days postinjection of neonatal CFW/D mice. These temperature-sensitive mutants are defective in the processing of the precursor of the env protein, gPr80env in infected cells, resulting in the accumulation of gPr80env in the infected cell and production of virions with reduced amounts of gp70, p15E, and p12E when compared to that of the wild-type virion. In contrast, two nonparalytogenic ts mutants, ts3 and ts10, like the wild-type virus, show normal processing of gPr80env in infected cells and production of virions with a similar amount of env proteins to that of the wild-type virion.

Animals↗
PubMed1986

The effect of overproduction of nonstructural proteins on alphavirus plus-strand and minus-strand RNA synthesis.

We determined the effect of the overproduction of viral nonstructural proteins on alphavirus plus-strand and minus-strand RNA synthesis. Because alphavirus minus-strand synthesis ceases normally at 3 to 4 hr postinfection and requires continuous protein synthesis [D. L. Sawicki and S. G. Sawicki, J. Virol. 34, 108-118 (1980); D. L. Sawicki, S. G. Sawicki, S. Keranen, and L. Kaariainen, J. Virol. 39, 348-358 (1981a)], we determined if the cessation of minus-strand synthesis was the result of the failure to continue synthesis of viral nonstructural proteins after 3-4 hr postinfection and if the overproduction of viral nonstructural proteins would increase the rate of plus-strand synthesis. Cells infected with ts1, an RNA-positive mutant of Semliki Forest virus (SFV) which overproduced the viral nonstructural proteins and underproduced the viral structural proteins at the nonpermissive temperature, did not cause the synthesis of increased amounts of viral minus strands relative to parental SFV and did not affect the time at which minus-strand synthesis ceased. All four viral nonstructural proteins were synthesized at early and late times after infection in the same relative proportions. The overproduction and the continued synthesis of nonstructural proteins late in infection did not increase the maximal rate of plus-strand synthesis above that in wild-type SFV-infected cells.

Alphavirus↗
PubMed1992

Studies on compartmentation and turnover of murine retrovirus envelope proteins.

Several aspects of turnover and degradation of cell membrane proteins were studied in an NIH 3T3 cell clone expressing the env gene of Moloney murine leukemia virus ts1. Both internalization and shedding of the extracellular domain of the envelope protein gp70 occurred at the cell surface, albeit, in the case of shedding, only a very small fraction of gp70 was shed. The turnover rate of gp70 at the cell surface was similar to that of the same protein in the postendoplasmic reticulum intracellular compartment. In the presence of L-methionine methyl ester, the transmembrane domain of the envelope protein Prp15E was degraded faster than gp70.

3T3 Cells↗
PubMed1981

Identification of the gene and mRNA for the adenovirus terminal protein precursor.

The precursor of the 55K adenovirus terminal protein is an 87K protein that is covalently linked to viral DNA. This protein is likely to be identical to the 80,000 dalton protein described by Challberg et al. (1980). The mRNA for the 87K terminal protein precursor, like that for the E2-72K DNA binding protein, is detectable at both early and late times of infection, and its production is sensitive to protein synthesis inhibition (Lewis and Mathews, 1980). The 87K protein, together with proteins of 105,000 and 75,000 daltons, are translated from leftward transcribed (1-strand) messenger RNAs that are complementary to the viral genome between positions 11.2 and 31.5. Additional hybridization to the region between coordinates 37.3 and 41 suggests that the RNA body is spliced to sequences mapping farther right in the genome. Electron microscopic heteroduplex analysis has revealed a family of 1-strand RNAs that probably encode these proteins. The RNA bodies extend from coordinated 30, 26 and 23 to 11.1, with leaders at 39, 68.5 and 75 map units, defining a new adenovirus early region. These RNAs and region E2 RNAs share the first leader and presumably the same promoter, and may be coordinately expressed. Virions of the protease-deficient adenovirus 2 mutant ts1 grown at the restrictive temperature contain only the 87K form; when grown at the permissive temperature they contain both the 87K and 55K forms, and an additional 62K form; wild-type virions contain only the 55K form. Peptide analysis shows all these proteins to be related. The DNA-protein complex containing the 87K form is active as a template for viral DNA replication in vitro. This data supports a model of adenovirus DNA replication in which the 87K terminal protein precursor is the primary translation product and primes DNA synthesis. The 87K precursor is processed curing virus maturation to the 55K terminal protein, possibly via a 62K intermediate form, by the virus-specified Ad2ts1 protease.

Adenoviruses, Human↗
PubMed1994

A simple method for obtaining highly viable virus from culture supernatant.

Traditionally density-gradient methods are used to purify viruses. However, these procedures are not only time consuming and cumbersome, recovery of viable viruses are often quite low. In this report, a single-step concentration technique was used to concentrate a mutant of Moloney murine leukemia virus (ts1) virus from culture supernatants by ultrafiltration. A special ultrafiltration unit with a 100,000 mol wt cut-off was able to concentrate viruses about 30-fold without losing any infectivity. In comparison, traditional sucrose density gradient purified viruses lost a significant portion of their infectivity. This technique could be used for concentrating other viruses for many useful purposes where more viable viruses are needed, e.g., study of virus-cell binding.

Cell Line↗
PubMed1991

The overexpression of the 3' terminal region of the CDC25 gene of Saccharomyces cerevisiae causes growth inhibition and alteration of purine nucleotides pools.

The CDC25 gene is transcribed at a very low level in S. cerevisiae cells. We have studied the effects of an overexpression of this regulatory gene by cloning either the whole CDC25 open reading frame (pIND25-2 plasmid) or its 3' terminal portion (pIND25-1 plasmid) under the control of the inducible strong GAL promoter. The strain transformed with pIND25-2 produced high levels of CDC25 specific mRNA, induced by galactose. This strain does not show any apparent alteration of growth, both in glucose and in galactose. Instead the yeast cells transformed with pIND25-1, that overexpress the 3' terminal part of CDC25 gene, grow very slowly in galactose medium, while they grow normally in glucose medium. The nucleotides were extracted from transformed cells, separated by HPLC and quantitated. The ATP/ADP and GTP/GDP ratios were almost identical in control and in pIND25-2 transformed strains growing in glucose and in galactose, while the strain that overexpresses the 3' terminal portion of CDC25 gene showed a decrease of ATP/ADP ratio and a partial depletion of the GTP pool. The disruption of RAS genes was only partially able to 'cure' this phenotype. A ras2-ts1, ras1::URA3 strain, transformed with pIND25-1 plasmid, was able to grow in galactose at 36 degrees C. These results suggest that the carboxy-terminal domain of the CDC25 protein could stimulate an highly unregulated GTPase activity in yeast cells by interacting not only with RAS gene products but also with some other yeast G-proteins.

Cell Cycle Proteins↗
PubMed1983

In vitro cleavage specificity of the adenovirus type 2 proteinase.

Two in vitro proteinase assay systems were developed and used to study the peptide bond specificity and substrate specificity of the adenovirus endoproteinase. Five adenovirus precursor proteins (PVI, PVII, PVIII, 87K, 11K), all found in the virion of the ts1 mutant grown at the nonpermissive temperature, were digested by the proteinase. All, except 11K, were cleaved to their mature counterparts. Some of the proteins, particularly the 87K terminal protein, were processed via cleavage intermediates similar to those found in vivo. The data suggest that the proteinase specifically hydrolyses Gly-Ala bonds. The high specificity for the natural substrates and the failure to cleave foreign proteins suggest that cleavage activity is determined not only by primary sequence but also by other physical features of the substrate. Enzyme activity was inhibited by diisopropylfluorophosphate, showing that it is a serine proteinase.

Adenoviruses, Human↗
PubMed1996

Analysis of choline acetyltransferase protein in temperature sensitive mutant flies using newly generated monoclonal antibody.

The protein, choline acetyltransferase (ChAT; EC 2.3.1.6), was analyzed in wild-type and two different temperature-sensitive ChAT mutants of Drosophila (Cha(ts1) and Cha(ts2)) using newly generated monoclonal antibodies. In all of the three genotypes, Western blots of crude fly head extracts showed a band stained at approximately the 80-kDa position, supporting the hypothesis that these temperature-sensitive mutants were generated by point mutation in the structural gene. The staining intensity of the bands indicated that these mutants have a lesser amount of ChAT protein than wild-type, even when they are reared at the permissive temperature (18 degrees C).

Amino Acid Sequence↗
PubMed1986

Possible role of ADP-ribosylation of adenovirus core proteins in virus infection.

We have investigated the role of poly(ADP)-ribosylation of adenoviral proteins in virus infection. Viral core proteins V and the precursor to protein VII were shown to be in vivo and in vitro acceptors of ADP-ribose. In vivo ADP-ribosylation was restricted to viral proteins as the histones were not labeled during the late phase of infection. The ADP-ribosylated core proteins were assembled into mature virus particles. In vitro ADP-ribosylation of adenoviral core proteins performed with purified poly(ADP-ribose) polymerase led to relaxation of the chromatin structure of both ts1 and wild type pyridine cores and pentonless particles and triggered the complete dissociation of wild type particles. A critical role for poly(ADP)-ribosylation in virus infection was confirmed by measuring the effect of the inhibitors 3-aminobenzamide and nicotinamide on virus particle yield and infectivity. Both inhibitors depressed particle yield by up to 9-fold, but infectivity was reduced by up to 10(4)-fold. These results suggest that ADP-ribosylation of adenovirus core proteins may have a role in virus decapsidation.

Adenosine Diphosphate Ribose↗
PubMed1985

Haemagglutinin transport mutants.

Two mutants (ts1 and ts651) with a temperature sensitive defect in the intracellular transport of the haemagglutinin from the rough endoplasmic reticulum to the plasma membrane have been analysed. Nucleotide sequencing of the haemagglutinin revealed with each mutant two point mutations that are located in the stem region of the molecule.

Base Sequence↗
PubMed1996

Repression of thrombospondin-1 expression, a natural inhibitor of angiogenesis, in polyoma middle T transformed NIH3T3 cells.

Thrombospondin-1 (TSI) is a modular extracellular matrix glycoprotein and expressed by many cell types in culture. Thrombospondin-1 inhibits angiogenesis and its expression inversely correlates with the degree of invasiveness and metastasis in tumor cell lines. Here, we demonstrate that expression of Polyoma middle T oncogene in NIH3T3 cells results not only in transformation but also represses expression of three thrombospondin isoforms, TS1, TS2, and TS3. Similar results were observed in ras, and to a lesser extent in src transformed NIH3T3 cells. Middle T and ras transformed cells expressed higher levels of c-jun mRNA, while the src transformed cells expressed higher levels of junB mRNA when compared to control cells. Thus, repression of thrombospondin levels appears to play an important role in establishment and maintenance of a malignant phenotype. This is mediated, at least in part, by alteration in c-jun activity in middle T and ras transformed NIH3T3 cells.

3T3 Cells↗
PubMed1989

Pharmacological characterization of sodium channels in the primary culture of individual Drosophila embryos: neurons of a mutant deficient in a putative sodium channel gene.

Sodium channels in Drosophila embryonic neurons were characterized pharmacologically in the primary culture of individual gastrulae. In normal cultures, presence of sodium channels was demonstrated by neuronal degeneration in the presence of veratridine and ouabain, which was inhibited by tetrodotoxin. Embryonic neurons of Df(2R)M-c33a homozygotes that lack a putative sodium channel gene at 60E region showed normal neurotoxin sensitivity. Therefore, sodium channel genes other than 60E must be functional at this developmental stage. We also examined para(ts1) and nap(ts) mutants and found that they were also sensitive to the neurotoxins. To determine the genotypes of single embryo cultures by histochemical staining, we utilized a special chromosome bearing a hsp70-lacZ fusion gene. The lacZ expression in the culture was studied in detail.

Animals↗
PubMed2004

Temperature may influence and regulate NF-YB expression in toad oocyte.

Fully grown oocytes derived from Bufo gargarizans maintained at relatively low temperatures (4 degrees C, LTE-oocytes) acquire the competence to resume normal meiosis. In contrast, fully grown oocytes derived from toads maintained at relatively high temperatures (28 degrees C, HTE-oocytes) never acquire maturation competence. By suppression subtractive hybridization, we obtained 18 ESTs preliminarily thought to be preferentially expressed in LTE-oocytes; of these, TS1-4 shared homology with the human and mouse NF-YB genes. We cloned the full-length toad NF-YB gene by RACE and identified three alternatively spliced transcripts: tNF-YB1, tNF-YB2, and tNF-YB3 (GenBank Accession Nos. AY442015, AY442016, and AY442017, respectively). Toad NF-YB was differentially transcribed and translated in LTE-oocytes versus HTE-oocytes, likely resulting in differential CCAAT-binding and/or transcriptional activity of NF-Y. Furthermore, toad cyclin B2 was differentially transcribed at high and low temperatures. Taken together, this report of the differential expression of toad NF-YB at different temperatures is the first evidence that temperature may influence and regulate NF-YB expression.

Alternative Splicing↗
PubMed2004

Characterization of a Saccharomyces cerevisiae thermosensitive lytic mutant leads to the identification of a new allele of the NUD1 gene.

To improve our understanding of the factors involved in the osmotic stability of yeast cells, a search for novel conditional Saccharomyces cerevisiae cell lysis mutants was performed. Ten temperature-sensitive (ts) mutant strains of S. cerevisiae were isolated that lyse at the restrictive temperature on hypotonic, but not on osmotically supported medium. The ten mutants fell into four complementation groups: ts1 to ts4. To clone the wild-type gene corresponding to the ts4 mutation, a strategy aimed at complementing the thermosensitive phenotype-using low-copy and high-copy DNA libraries--was followed, but only two extragenic suppressors were identified. Another approach, in which classic genetic methods were combined with the use of yeast artificial chromosomes and traditional cloning procedures, allowed the identification of the NUD1 gene--which codes for a component of the spindle-pole body-as the wild-type gene corresponding to the ts4 mutation. Cloning and sequencing of the defective allele from the chromosome of the mutant cells resulted in the identification of a point mutation that produces a single amino acid change in the protein: a Gly-to-Glu change at position 585 (the nud1-G585E allele). Further analysis revealed that cells carrying this allele show a thermosensitive growth defect. At the restrictive temperature, the cells arrest with large buds, elongated spindles, and duplicated nuclei. In addition, with longer incubation times they are unable to maintain cellular integrity and lyse. Our results have allowed the identification of the first single amino acid mutation in NUD1, and suggest a link between cell cycle progression and cellular integrity.

Alleles↗