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Biomedical subjects

T Hunt

Publications and source records attributed to T Hunt.

At least 109 records · Page 6Linked to original sources
PubMed1991

A comparative study of the 'FDA' and 'USDA' methods for the detection of Listeria monocytogenes in foods.

Nineteen laboratories across Canada took part in a comparative study of the 'FDA' and 'USDA' methods for the detection of Listeria monocytogenes in foods and environmental samples. The results show that the enrichment period of the FDA method can be shortened from 7 to 2 days without substantially reducing the number of positive samples. With a limited number of samples, the USDA method proved to be slightly more efficient in isolating L. monocytogenes than the FDA method. Fraser broth, in principle, proved to be useful as a screening tool but is not very selective. Oxford agar and lithium chloride-phenylethanol-moxalactam medium were better than modified McBride's agar in isolating this microorganism.

Agriculture↗
PubMed1991

On the synthesis and destruction of A- and B-type cyclins during oogenesis and meiotic maturation in Xenopus laevis.

We have measured the levels of cyclin mRNAs and polypeptides during oogenesis, progesterone-induced oocyte maturation, and immediately after egg activation in the frog, Xenopus laevis. The mRNA for each cyclin is present at a constant level of approximately 5 x 10(7) molecules per oocyte from the earliest stages of oogenesis until after fertilization. The levels of polypeptides show more complex patterns of accumulation. The B-type cyclins are first detectable in stage IV and V oocytes. Cyclin B2 polypeptide is present at approximately 2 x 10(9) molecules (150 pg) per oocyte by stage VI. The amount increases after progesterone treatment, but returns to its previous level after GVBD and undergoes no further change until it is destroyed at fertilization. Cyclin B1 is present at 4 x 10(8) molecules per oocyte in stage VI oocytes, and rises steadily during maturation, ultimately reaching similar levels to cyclin B2 in unfertilized eggs. Unlike the B-type cyclins, cyclin A is barely detectable in stage VI oocytes, and only starts to be made in significant amounts after oocytes are exposed to progesterone. A portion of all the cyclins are destroyed after germinal vesicle breakdown (GVBD), and cyclins B1 and B2 also experience posttranslational modifications during oocyte maturation. Progesterone strongly stimulates both cyclin and p34cdc2 synthesis in these oocytes, but whereas cyclin synthesis continues in eggs and after fertilization, synthesis of p34cdc2 declines strongly after GVBD. The significance of these results is discussed in terms of the activation and inactivation of maturation-promoting factor.

Animals↗
PubMed1991

Xenopus oocyte maturation does not require new cyclin synthesis.

Progesterone induces fully grown, stage VI, Xenopus oocytes to pass through meiosis I and arrest in metaphase of meiosis II. Protein synthesis is required twice in this process: in order to activate maturation promoting factor (MPF) which induces meiosis I, and then again after the completion of meiosis I to reactivate MPF in order to induce meiosis II. We have used antisense oligonucleotides to destroy maternal stores of cyclin mRNAs, and demonstrate that new cyclin synthesis is not required for entry into either meiosis I or II. This finding is consistent with the demonstration that stage VI oocytes contain a store of B-type cyclin polypeptides (Kobayashi, H., J. Minshull, C. Ford, R. Golsteyn, R. Poon, and T. Hunt. 1991. J. Cell Biol. 114:755-765). Although approximately 70% of cyclin B2 is destroyed at first meiosis, the surviving fraction, together with a larger pool of surviving cyclin B1, must be sufficient to allow the reactivation of MPF and induce entry into second meiotic metaphase. Since stage VI oocytes do not contain any cyclin A, our results show that cyclin A is not required for meiosis in Xenopus. We discuss the possible nature of the proteins whose synthesis is required to induce meiosis I and II.

Animals↗
PubMed1991

Cyclins and their partners: from a simple idea to complicated reality.

The cyclins comprise a family of proteins which combine with protein kinase subunits encoded by members of the cdc2 family. The active protein kinase thus formed phosphorylates target proteins in the cell and promotes certain cell cycle transitions. Cyclins A and B show the unusual property of sudden and specific proteolysis shortly before the metaphase-anaphase transition during mitosis. The destruction of the cyclins leads to rapid loss of activity of their kinase companions. The 'simple idea' referred to in the title of this article was that accumulation of cyclin formed maturation promoting factor (MPF), the enzyme that promotes the G2-M transition, and cyclin destruction turned off MPF. The complexity refers to additional controls of cdc2 activity, such as its reversible phosphorylation. Furthermore, many new members of the cyclin family have recently been discovered that may play a role in the regulation of the G1-S transition, and in higher organisms, the cdc2 family is also more numerous than was at first appreciated. Cyclins make important contributions both to subcellular localization and the substrate specificity of their companion kinase subunits. It is too early to say if the entire range of cyclin-like and cdc2-like protein kinases are involved in the control of the cell cycle.

Amino Acid Sequence↗
PubMed1990

Triggering of cyclin degradation in interphase extracts of amphibian eggs by cdc2 kinase.

The cell cycles of early Xenopus embryos consist of a rapid succession of alternating S and M phases. These cycles are controlled by the activity of a protein kinase complex (cdc2 kinase) which contains two subunits. One subunit is encoded by the frog homologue of the fission yeast cdc2+ gene, p34cdc2 and the other is a cyclin. The concentration of cyclins follows a sawtooth oscillation because they accumulate in interphase and are destroyed abruptly during mitosis. The association of cyclin and p34cdc2 is not sufficient for activation of cdc2 kinase, however; dephosphorylation of key tyrosine and threonine residues of p34cdc2 is necessary to turn on its kinase activity. The activity of cdc2 kinase is thus regulated by a combination of translational and post-translational mechanisms. The loss of cdc2 kinase activity at the end of mitosis depends on the destruction of the cyclin subunits. It has been suggested that this destruction is induced by cdc2 kinase itself, thereby providing a negative feedback loop to terminate mitosis. Here we report direct experimental evidence for this idea by showing that cyclin proteolysis can be triggered by adding cdc2 kinase to a cell-free extract of interphase Xenopus eggs.

Amino Acid Sequence↗
PubMed1990

Cyclin is a component of maturation-promoting factor from Xenopus.

Highly purified maturation-promoting factor (MPF) from Xenopus eggs contains both cyclin B1 and cyclin B2 as shown by Western blotting and immunoprecipitation using Xenopus anti-B-type cyclin antibodies. Immunoprecipitates with these antibodies display the histone H1 kinase activity characteristic of MPF, for which exogenously added B1 and B2 cyclins are both substrates. Protein kinase activity against cyclin oscillates in maturing oocytes and activated eggs with the same kinetics as p34cdc2 kinase activity. These data indicate that B-type cyclin is the other component of MPF besides p34cdc2.

Adenosine Triphosphate↗
PubMed1990

The A- and B-type cyclin associated cdc2 kinases in Xenopus turn on and off at different times in the cell cycle.

Cyclins play a key role in the induction of mitosis. In this paper we report the isolation of a cyclin A cDNA clone from Xenopus eggs. Its cognate mRNA encodes a protein that shows characteristic accumulation and destruction during mitotic cell cycles. The cyclin A polypeptide is associated with a protein that cross-reacts with an antibody against the conserved 'PSTAIR' epitope of p34cdc2, and the cyclin A-cdc2 complex exhibits protein kinase activity that oscillates with the cell cycle. This kinase activity rises more smoothly than that of the cyclin B-cdc2 complexes and reaches a peak earlier in the cell cycle; indeed, cyclin A is destroyed before nuclear envelope breakdown. None of the cyclin-cdc2 complexes show simple relationships between the concentration of the cyclin moiety and the kinase activity. All three cyclin associated kinases (A, B1 and B2) phosphorylate identical sites on histones with the consensus XSPXK/R, although they show significant differences in their substrate preferences. We discuss possible models for the different roles of the A- and B-type cyclins in the control of cell division.

Amino Acid Sequence↗
PubMed1990

A contingent, conditioned suppression of eating following chronic benzodiazepine-induced hyperphagia.

While the hyperphagic effect of chloradizepoxide (CDP) has been reported by some to be enhanced with chronic drug treatment, the processes underlying this phenomenon are not well understood. In the present study, it was predicted that following chronic exposure to CDP-induced hyperphagia, animals given a placebo in place of their usual drug injection might be expected to exhibit evidence of a conditioned, drug-like response. Such a finding would then be consistent with an underlying process of behavioral sensitization. In Experiment 1a, Male Sprague-Dawley rats were randomly assigned to one of two groups receiving intraperitoneal (IP) injections of either 5 mg/kg CDP (Group CDP) or physiological saline (1 ml/kg; Group SAL) administered over 15 drug treatment days. Thirty minutes after each injection, all animals were given 30 min access to sweetened condensed milk. A significant enhancement of CDP-induced hyperphagia was observed over treatment sessions, confirming an earlier report. Unexpectedly, in the Placebo Test, the CDP animals exhibited a supression of milk consumption relative to that of the SAL group. Using the same animals, this finding was successfully replicated in Experiment 1b. In Experiment 2, it was hypothesized that if this conditioned, drug-opposite response were to reflect the involvement of some underlying compensatory, homeostatic mechanism, then it should only be observable under food-contingent conditions of chronic drug treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Maternal mRNA from clam oocytes can be specifically unmasked in vitro by antisense RNA complementary to the 3'-untranslated region.

Clam oocytes display a striking difference in the pattern of protein synthesis on fertilization, which is maintained when cell-free extracts are assayed in the rabbit reticulocyte lysate. The mRNAs encoding ribonucleotide reductase and cyclin A (the major masked messages in the oocyte extracts) can be translationally activated by gel filtration in 0.5 M KCl, presumably by removal of repressor protein(s). When synthetic RNAs corresponding to different segments of ribonucleotide reductase and cyclin A mRNAs were added to oocyte extracts in 0.5 M KCl in a "competitive unmasking" assay, specific and complete unmasking of these mRNAs occurred on addition of antisense 3'-noncoding transcripts. The unmasking region in ribonucleotide reductase mRNA maps to a region of 134 nucleotides centered one-third of the way down the 3'-noncoding region.

Animals↗
PubMed1990

Control of translation of masked mRNAs in clam oocytes.

The pattern of protein synthesis changes soon after fertilization of clam oocytes. The most abundant of the mRNAs whose translation increases at this time encode ribonucleotide reductase and the A- and B-type cyclins. These mRNAs have been cloned and sequenced, yet their sequences do not show regions of similarity that could explain the masking mechanism. However, these mRNAs retain their 'masked' state in cell-free translation assays and their translation can be activated by gel filtration in high salt, which probably removes repressor proteins. A 'competitive unmasking' assay was used to identify the protein-binding regions of each mRNA. This involved adding short segments of antisense RNA that annealed to the mRNA and displaced the repressors. The unmasking regions in ribonucleotide reductase and cyclin A mRNAs revealed by this assay are 120-140 nt long and are located in the central portions of the 3' non-coding regions.

Animals↗
PubMed1989

Translation of cyclin mRNA is necessary for extracts of activated xenopus eggs to enter mitosis.

The cyclins are a family of proteins encoded by maternal mRNA. Cyclin polypeptides accumulate during interphase and are destroyed during mitosis at about the time of entry into anaphase. We show here that Xenopus oocytes contain mRNAs encoding two cyclins that are major translation products in a cell-free extract from activated eggs. Cutting these mRNAs with antisense oligonucleotides and endogenous RNAase H blocks entry into mitosis in a cell-free egg extract. The extracts can enter mitosis if either of the cyclin mRNAs is left intact. We conclude that the synthesis of these cyclins is necessary for mitotic cell cycles in cleaving Xenopus embryos.

Amino Acid Sequence↗
PubMed1989

Cytotoxic T lymphocyte recognition of HLA-A2 antigens in normal and HLA-Cw3-transgenic mice.

It is well established that a large fraction of murine cytotoxic T cells can recognize allogeneic major histocompatibility complex (MHC) antigens, and that the majority of this response are not restricted by H-2 antigens of the responding host. In contrast, the murine response against the xenogeneic HLA class I antigens is relatively weak. Moreover, a large proportion of the responding murine T cells do not recognize the HLA antigen per se but only in an H-2-restricted manner, probably as an HLA peptide bound to H-2. Considerable evidence suggests that in mice the T cell repertoire is selected by thymic H-2 antigens. Therefore, we asked the question whether in transgenic mice expressing an HLA class I antigen the T cell repertoire would be shaped toward a more effective recognition of other HLA alleles. Normal C57BL/6 (B6) and HLA-Cw3-transgenic B6 mice were immunized with a B6-derived cell line transfected with HLA-A2. The resulting A2-specific CTL were tested on L cells transfected with either A2 alone, which should identify the H-2-unrestricted CTL, and on L cells transfected with A2 plus H-2b genes, which should identify the sum of H-2b-restricted and unrestricted CTL. Both bulk culture and limiting dilution experiments showed that the CTL precursor frequencies for A2-specific CTL were not increased in the transgenic mice, and that both strains produced comparable proportions of H-2b-restricted and of unrestricted A2-specific CTL. The B6.Cw3 mice seemed to respond less well to HLA-A2 than the normal B6 mice, suggesting the possibility of tolerance for peptides shared by the Cw3 and A2 molecules. In conclusion, the T cells in the B6.Cw3 transgenic mice did not seem to be selected towards a stronger and more unrestricted recognition of an allo-HLA antigen. The possible reasons are discussed.

Animals↗
PubMed1989

Cyclin is a component of the sea urchin egg M-phase specific histone H1 kinase.

A so-called 'growth-associated' or 'M-phase specific' histone H1 kinase (H1K) has been described in a wide variety of eukaryotic cell types; p34cdc2 has previously been shown to be a catalytic subunit of this protein kinase. In fertilized sea urchin eggs the activity of H1K oscillates during the cell division cycle and there is a striking temporal correlation between H1K activation and the accumulation of a phosphorylated form of cyclin. H1K activity declines in parallel with proteolytic cyclin destruction of the end of the first cell cycle. By virtue of the high affinity of the fission yeast p13suc1 for the p34cdc2 protein, H1K strongly binds to p13-Sepharose beads. Cyclin, p34cdc2 and H1K co-purify on this affinity reagent as well as through several conventional chromatographic procedures. Anticyclin antibodies immunoprecipitate the M-phase specific H1K in crude extracts or in purified fractions. Sea urchin eggs appear to contain much less cyclin than p34cdc2, suggesting that p34cdc2 may interact with other proteins. These results demonstrate that cyclin and p34cdc2 are major components of the M-phase specific H1K.

Animals↗
PubMed1989

A post-ribosomal supernatant from activated Xenopus eggs that displays post-translationally regulated oscillation of its cdc2+ mitotic kinase activity.

A cell-free extract prepared from activated Xenopus eggs by high-speed centrifugation displays one spontaneous cycle of activation and inactivation of histone H1 kinase and MPF activity that is largely attributable to Xenopus p32cdc2. The timing of the oscillation closely follows that observed in intact eggs, is associated with large changes in endogenous protein phosphorylation and depends entirely on post-translational events. The extract can be fractionated into soluble and particulate material, both of which components are required for the oscillatory behaviour. Kinase activation does not require Mg+ ATP, but its rapid inactivation, which coincides with the destruction of cyclin, is inhibited both by EDTA and the protein kinase inhibitor 6-dimethylaminopurine. This suggests that protein phosphorylation is required for cyclin destruction and kinase inactivation.

Adenine↗