Bulk tank milk failures.
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Animal Husbandry↗
Biomedical subjects
J G Cook
Publications and source records attributed to J G Cook.
Replication factors MCM2 and ORC1 interact with the histone acetyltransferase HBO1.
The minichromosome maintenance (MCM) proteins, together with the origin recognition complex (ORC) proteins and Cdc6, play an essential role in eukaryotic DNA replication through the formation of a pre-replication complex at origins of replication. We used a yeast two-hybrid screen to identify MCM2-interacting proteins. One of the proteins we identified is identical to the ORC1-interacting protein termed HBO1. HBO1 belongs to the MYST family, characterized by a highly conserved C2HC zinc finger and a putative histone acetyltransferase domain. Biochemical studies confirmed the interaction between MCM2 and HBO1 in vitro and in vivo. An N-terminal domain of MCM2 is necessary for binding to HBO1, and a C2HC zinc finger of HBO1 is essential for binding to MCM2. A reverse yeast two-hybrid selection was performed to isolate an allele of MCM2 that is defective for interaction with HBO1; this allele was then used to isolate a suppressor mutant of HBO1 that restores the interaction with the mutant MCM2. This suppressor mutation was located in the HBO1 zinc finger. Taken together, these findings strongly suggest that the interaction between MCM2 and HBO1 is direct and mediated by the C2HC zinc finger of HBO1. The biochemical and genetic interactions of MYST family protein HBO1 with two components of the replication apparatus, MCM2 and ORC1, suggest that HBO1-associated HAT activity may play a direct role in the process of DNA replication.
Acetyltransferases↗
Susceptibility of elk to lungworms from cattle.
Two studies were conducted to determine the infectivity of the lungworm, (Dictyocaulus viviparus) of cattle origin, in Rocky Mountain elk (Cervus elaphus nelsoni) or wapiti. In the first study, each of three 9-mo-old elk was administered 3,000 D. viviparus larvae from cattle using a nasogastric tube. In the second study, four 16-mo-old elk were each inoculated with 2,000 D. viviparus from cattle using a nasogastric tube. Elk were observed daily for signs of respiratory disease, and fecal samples were collected during the studies and evaluated for lungworm larvae using a modified Baermann technique. One elk was euthanatized during the patent period for recovery of adult lungworms, and three elk were euthanatized after larvae were no longer detected in feces. Lungworm larvae were not detected before inoculation in any of the 16-mo-old elk, but were detected 22 days after inoculation in one elk, 23 days after inoculation in two elk and 24 days after inoculation in all four elk. The prepatent period of this cattle isolate of D. viviparus in elk is therefore 22 to 24 days. The precise prepatent period was not determined in the three 9-mo-old elk, but larvae were detected in all three elk 25 days after inoculation. Numbers of larvae ranged from 1/ to 101/g feces with peak larval detection occurring 32 to 50 days after inoculation. Elk shed larvae from 22 to 83 days after inoculation, and patent periods of the parasite ranged from 24 to 62 days. Clinical signs of respiratory disease, with the exception of mild coughing after exercise, were not observed during the infections. Results from this experiment indicated that D. viviparus larvae of cattle origin can mature in elk and larvae can be passed in large numbers in feces, but this cattle isolate of D. viviparus was not highly pathogenic in elk.
Animals↗
Collaborative role of E2F transcriptional activity and G1 cyclindependent kinase activity in the induction of S phase.
A considerable body of evidence points to a role for both cyclin E/cyclin-dependent kinase (cdk)2 activity and E2F transcription activity in the induction of S phase. We show that overexpression of cyclin E/cdk2 in quiescent cells induces S phase, that this coincides with an induction of E2F activity, and that coexpression of E2F enhances the cyclin E/cdk2-mediated induction of S phase. Likewise, E2F overexpression can induce S phase and does so in the apparent absence of cyclin E/cdk2 activity. In addition, although the inhibition of cyclin E/cdk2 activity blocks the induction of S phase after growth stimulation of normal mouse embryo fibroblasts, inhibition of cyclin E/cdk2 does not block S phase induction in Rb-/- cells where E2F activity is deregulated. These results point to the important roles for E2F and cyclin E/cdk2 in the induction of S phase. Moreover, the nature of the E2F targets and the suspected targets for cyclin E/cdk2 suggests a potential molecular mechanism for the collaborative action of cyclin E/cdk2 and E2F in the induction of S phase.
Animals↗
Differential regulation of transcription: repression by unactivated mitogen-activated protein kinase Kss1 requires the Dig1 and Dig2 proteins.
Kss1, a yeast mitogen-activated protein kinase (MAPK), in its unphosphorylated (unactivated) state binds directly to and represses Ste12, a transcription factor necessary for expression of genes whose promoters contain filamentous response elements (FREs) and genes whose promoters contain pheromone response elements (PREs). Herein we show that two nuclear proteins, Dig1 and Dig2, are required cofactors in Kss1-imposed repression. Dig1 and Dig2 cooperate with Kss1 to repress Ste12 action at FREs and regulate invasive growth in a naturally invasive strain. Kss1-imposed Dig-dependent repression of Ste12 also occurs at PREs. However, maintenance of repression at PREs is more dependent on Dig1 and/or Dig2 and less dependent on Kss1 than repression at FREs. In addition, derepression at PREs is more dependent on MAPK-mediated phosphorylation than is derepression at FREs. Differential utilization of two types of MAPK-mediated regulation (binding-imposed repression and phosphorylation-dependent activation), in combination with distinct Ste12-containing complexes, contributes to the mechanisms by which separate extracellular stimuli that use the same MAPK cascade can elicit two different transcriptional responses.
Calcium-Calmodulin-Dependent Protein Kinases↗
Repression of yeast Ste12 transcription factor by direct binding of unphosphorylated Kss1 MAPK and its regulation by the Ste7 MEK.
The mitogen-activated protein kinase (MAPK) Kss1 has a dual role in regulating filamentous (invasive) growth of the yeast Saccharomyces cerevisiae. The stimulatory function of Kss1 requires both its catalytic activity and its activation by the MAPK/ERK kinase (MEK) Ste7; in contrast, the inhibitory function of Kss1 requires neither. This study examines the mechanism by which Kss1 inhibits invasive growth, and how Ste7 action overcomes this inhibition. We found that unphosphorylated Kss1 binds directly to the transcription factor Ste12, that this binding is necessary for Kss1-mediated repression of Ste12, and that Ste7-mediated phosphorylation of Kss1 weakens Kss1-Ste12 interaction and relieves Kss1-mediated repression. Relative to Kss1, the MAPK Fus3 binds less strongly to Ste12 and is correspondingly a weaker inhibitor of invasive growth. Analysis of Kss1 mutants indicated that the activation loop of Kss1 controls binding to Ste12. Potent repression of a transcription factor by its physical interaction with the unactivated isoform of a protein kinase, and relief of this repression by activation of the kinase, is a novel mechanism for signal-dependent regulation of gene expression.
Amino Acid Sequence↗
Changes in milk purchasing arrangements.
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Animals↗
One-sample pregnancy diagnosis in elk using fecal steroid metabolites.
Recent research has demonstrated the potential of pregnancy diagnosis in elk (Cervus elaphus nelsoni) using immunoassays of fecal steroid concentration. However, multiple samples are required to insure accurate results, limiting its utility for free-ranging animals. We attempted to develop an accurate one-sample pregnancy diagnosis using 153 fecal samples that were collected from free-ranging, radio-collared, adult female elk in Yellowstone National Park (Wyoming, USA) and from captive elk maintained at the Starkey Research Facility (La Grande, Oregon, USA) February through April 1992 and 1997. The pregnancy status of each animal was diagnosed using serum pregnancy-specific protein B (PSPB) assays providing fecal samples from 38 nonpregnant and 115 pregnant animals. Fecal radioimmunoassay (RIA) indicated that mean (+/- SD) progestagens (P4) were elevated significantly in pregnant (2.96 +/- 1.49 micrograms/gm) compared to nonpregnant (0.43 +/- 0.26 microgram/gm) individuals. Confidence intervals (1.96 +/- SE) for the two groups were widely separated (nonpregnant 0.34-0.51, pregnant 2.69-3.24) with little overlap in the range of concentrations measured for each group (nonpregnant 0.09-0.98, pregnant 0.90-8.29). These results indicate that fecal progestagens RIA provides a reliable method of noninvasive pregnancy diagnosis using single fecal samples collected from elk during late gestation. However, independent validation of the suggested discrimination criteria should be performed before routine application.
Animals↗
Antibody response of elk immunized with porcine zona pellucida.
Immunocontraception using porcine zona pellucida (PZP) vaccines is being explored as a nonlethal method of solving the problems of locally overabundant wildlife populations. This study characterized the immunological response of captive elk (Cervus elaphus nelsoni) to PZP challenge using 18 3-yr-old cows and was conducted from 14 September 1994 to 13 December 1995. All animals were given a single PZP inoculation and 1 mo later six of these animals were randomly chosen and received a booster inoculation. Blood samples were drawn from all animals at the time of the initial inoculation and 1, 2, 4, 6, 10, and 15 mo later. Immunological response was assessed by measuring anti-PZP antibody levels in serum. All animals demonstrated a strong immune response with no evidence that the booster enhanced antibody levels. Antibody levels rose from between 0 and 4 at the time of the initial injection to peak levels of 85 to 163 within 2 to 6 mo, followed by a noticeable decline by 15 mo post-vaccination. Limited data suggest that antibody levels > 100 may be required to effect contraception. High individual variability in immune response observed in this study suggests it may be difficult to predict the proportion of animals effectively treated. Disruption of seasonal synchrony in calving also could occur if antibody levels in individuals fall below effective levels while animals are still cycling. These results indicate that immunocontraception using PZP vaccines is possible for elk. However, carefully controlled population experiments will be required in order to assess the potential and limitations for management applications of this technique.
Animals↗
Inhibitory and activating functions for MAPK Kss1 in the S. cerevisiae filamentous-growth signalling pathway.
Mitogen-activated protein kinase (MAPK) cascades are conserved signalling modules that regulate responses to diverse extracellular stimuli, developmental cues and environmental stresses. A MAPK is phosphorylated and activated by a MAPK kinase (MAPKK), which is activated by an upstream protein kinase, such as Raf, Mos or a MAPKK kinase. Ste7, a MAPKK in the yeast Saccharomyces cerevisiae, is required for two developmental pathways: mating and invasive (filamentous) growth. Kss1 and Fus3, the MAPK targets of Ste7, are required for mating, but their role in invasive growth has been unclear. Because no other S. cerevisiae MAPK has been shown to function in invasive growth, it was proposed that Ste7 may have non-MAPK targets. We show instead that Kss1 is the principal target of Ste7 in the invasive-growth response in both haploids and diploids. We demonstrate further that Kss1 in its inactive form is a potent negative regulator of invasive growth. Ste7 acts to relieve this negative regulation by switching Kss1 from an inhibitor to an activator. These results indicate that this MAPK has a physiologically important function in its unactivated state. Comparison of normal and MAPK-deficient cells indicates that nitrogen starvation and activated Ras stimulate filamentous growth through both MAPK-independent and MAPK-dependent means.
Calcium-Calmodulin-Dependent Protein Kinases↗
Two novel targets of the MAP kinase Kss1 are negative regulators of invasive growth in the yeast Saccharomyces cerevisiae.
Haploid cells of budding yeast Saccharomyces cerevisiae respond to mating pheromones by inducing genes required for conjugation, arresting cell cycle progression, and undergoing morphological changes. The same cells respond to nutrient deprivation by altering budding pattern and inducing genes required for invasive growth. Both developmental alternatives to vegetative proliferation require the MAP kinase Kss1 and the transcriptional transactivator Ste12. Using a two-hybrid screen for gene products that interact with Kss1, two homologous and previously uncharacterized loci (DIG1 and DIG2, for down-regulator of invasive growth) were identified. DIG2 is pheromone-inducible, whereas DIG1 is constitutively expressed. Dig1 colocalizes with Kssl in the nucleus, coimmunoprecipitates with Kss1 from cell extracts in a pheromone-independent manner, and is phosphorylated by Kss1 in immune complexes in a pheromone-stimulated manner. Kss1 binds specifically to a GST-Dig1 fusion in the absence of any other yeast protein. Using the two-hybrid method, both Dig1 and Dig2 also interact with the other MAP kinase of the pheromone response pathway, Fus3. However, neither dig1 or dig2 single mutants, nor a dig1 dig2 double mutant, have a discernible effect on mating. In contrast, dig1 dig2 cells constitutively invade agar medium, whereas a dig1 dig2 ste12 triple mutant does not, indicating that Dig1 and Dig2 share a role in negatively regulating the invasive growth pathway. High-level expression of Dig1 suppresses invasive growth and also causes cells to appear more resistant to pheromone-imposed cell cycle arrest. Ste12 also binds specifically to GST-Dig1 in the absence of any other yeast protein. Collectively, these findings indicate that Dig1, and most likely Dig2, are physiological substrates of Kssl and suggest that they regulate Ste12 function by direct protein-protein interaction.
Amino Acid Sequence↗
Signaling in the yeast pheromone response pathway: specific and high-affinity interaction of the mitogen-activated protein (MAP) kinases Kss1 and Fus3 with the upstream MAP kinase kinase Ste7.
Kss1 and Fus3 are mitogen-activated protein kinases (MAPKs or ERKs), and Ste7 is their activating MAPK/ERK kinase (MEK), in the pheromone response pathway of Saccharomyces cerevisiae. To investigate the potential role of specific interactions between these enzymes during signaling, their ability to associate with each other was examined both in solution and in vivo. When synthesized by in vitro translation, Kss1 and Fus3 could each form a tight complex (Kd of approximately 5 nM) with Ste7 in the absence of any additional yeast proteins. These complexes were specific because neither Hog1 nor Mpk1 (two other yeast MAPKs), nor mammalian Erk2, was able to associate detectably with Ste7. Neither the kinase catalytic core of Ste7 nor the phosphoacceptor regions of Ste7 and Kss1 were necessary for complex formation. Ste7-Kss1 (and Ste7-Fus3) complexes were present in yeast cell extracts and were undiminished in extracts prepared from a ste5delta-ste11delta double mutant strain. In Ste7-Kss1 (or Ste7-Fus3) complexes isolated from naive or pheromone-treated cells, Ste7 phosphorylated Kss1 (or Fus3), and Kss1 (or Fus3) phosphorylated Ste7, in a pheromone-stimulated manner; dissociation of the high-affinity complex was shown to be required for either phosphorylation event. Deletions of Ste7 in the region required for its stable association with Kss1 and Fus3 in vitro significantly decreased (but did not eliminate) signaling in vivo. These findings suggest that the high-affinity and active site-independent binding observed in vitro facilitates signal transduction in vivo and suggest further that MEK-MAPK interactions may utilize a double-selection mechanism to ensure fidelity in signal transmission and to insulate one signaling pathway from another.
Base Sequence↗
Phosphorylation and localization of Kss1, a MAP kinase of the Saccharomyces cerevisiae pheromone response pathway.
Kss1 protein kinase, and the homologous Fus3 kinase, are required for pheromone signal transduction in Saccharomyces cerevisiae. In MATa haploids exposed to alpha-factor, Kss1 was rapidly phosphorylated on both Thr183 and Tyr185, and both sites were required for Kss1 function in vivo. De novo protein synthesis was required for sustained pheromone-induced phosphorylation of Kss1. Catalytically inactive Kss1 mutants displayed alpha-factor-induced phosphorylation on both residues, even in kss1 delta cells; hence, autophosphorylation is not obligatory for these modifications. In kss1 delta fus3 delta double mutants, Kss1 phosphorylation was elevated even in the absence of pheromone; thus, cross-phosphorylation by Fus3 is not responsible for Kss1 activation. In contrast, pheromone-induced Kss1 phosphorylation was eliminated in mutants deficient in two other protein kinases, Ste11 and Ste7. A dominant hyperactive allele of STE11 caused a dramatic increase in the phosphorylation of Kss1, even in the absence of pheromone stimulation, but required Ste7 for this effect, suggesting an order of function: Ste11-->Ste7-->Kss1. When overproduced, Kss1 stimulated recovery from pheromone-imposed G1 arrest. Catalytic activity was essential for Kss1 function in signal transmission, but not for its recovery-promoting activity. Kss1 was found almost exclusively in the particulate material and its subcellular fractionation was unaffected by pheromone treatment. Indirect immunofluorescence demonstrated that Kss1 is concentrated in the nucleus and that its distribution is not altered detectably during signaling.
Amino Acid Sequence↗
Signal propagation and regulation in the mating pheromone response pathway of the yeast Saccharomyces cerevisiae.
Extracellular signals can affect the rate of proliferation and the state of differentiation of eukaryotic cells. Signal transduction pathways have evolved to detect these signals at the plasma membrane, transmit them through the cytoplasm and into the nucleus, and thereby generate the appropriate changes in metabolism and transcription. Much attention has been focused recently on regulatory pathways of this sort that lead to activation of a family of protein kinases known as the mitogen- or messenger-activated, or extracellular signal-regulated protein kinases (MAPKs or ERKs) because this particular class of enzyme is highly conserved among eukaryotes, as is documented here and in the accompanying reviews in this issue. The mating pheromone response pathway in a unicellular microbe, the budding yeast Saccharomyces cerevisiae, is perhaps the best understood multicomponent signaling pathway known in any eukaryotic organism, especially at the genetic level. Furthermore, structural homologs and functional analogs of the components of the yeast pheromone response pathway are recapitulated in the signaling systems present in multicellular eukaryotes. This article emphasizes recent findings and common molecular themes for understanding the organization and regulation of MAPK-dependent signaling cascades that have emerged from biochemical and genetic analysis of the mating pheromone response pathway in yeast.
Cell Cycle↗
Sex- and age-specific lungworm infection in Rocky Mountain bighorn sheep during winter.
Sex- and age-specific fecal lungworm (Protostrongylus spp.) larvae concentrations in Rocky Mountain bighorn sheep (Ovis canadensis canadensis) from the Cinnabar Winter Range in southwestern Montana were determined. All 228 fecal samples collected from bighorn sheep of known sex and age class from November to April, 1984 to 1987 contained first-stage lungworm larvae. Fecal lungworm concentrations of ewes and rams declined significantly from late fall through early spring, whereas number of lungworms in lamb feces increased as winter progressed.
Age Factors↗
A study of the hypouricaemic effect of tiaprofenic acid.
Tiaprofenic acid is a non-steroidal anti-inflammatory drug which also has hypouriceamic effect. Studies involving 10 healthy volunteers were designed to investigate the mode of this effect. We postulate that the site of action of tiaprofenic acid is at the cell membrane, the mechanism being an interference with the transport of uric acid from intra- to extra-cellular fluid thus limiting its passage into the plasma. The same mechanism acting on renal tubular cells impedes reabsorption thereby increasing uric acid clearance.
Adult↗
Whole blood osmolality.
The osmolality of plasma and heparinised whole blood samples collected from hospital patients was estimated using measurement of the depression of freezing point. There was no clinically significant difference between osmolality measurement made on either whole blood, or plasma taken from the same patient. Neither cell volume nor haemolysis was found to affect the measurement. The reproducibility of whole blood measurements was similar to that for determinations carried out on plasma. Measurement of osmolality on whole blood is quicker and cheaper and needs a smaller specimen than if serum or plasma is used.
Blood↗