Biomedical subjects

M J Stark

Publications and source records attributed to M J Stark.

At least 37 records · Page 2Linked to original sources

PubMed1998

Identification and characterization of the KlCMD1 gene encoding Kluyveromyces lactis calmodulin.

The KlCMD1 gene was isolated from a Kluyveromyces lactis genomic library as a suppressor of the Saccharomyces cerevisiae temperature-sensitive mutant spc110-124, an allele previously shown to be suppressed by elevated copy number of the S. cerevisiae calmodulin gene CMD1. The KlCMD1 gene encodes a polypeptide which is 95% identical to S. cerevisiae calmodulin and 55% identical to calmodulin from Schizosaccharomyces pombe. Complementation of a S. cerevisiae cdm1 deletion mutant by KlCMD1 demonstrates that this gene encodes a functional calmodulin homologue. Multiple sequence alignment of calmodulins from yeast and multicellular eukaryotes shows that the K. lactis and S. cerevisiae calmodulins are considerably more closely related to each other than to other calmodulins, most of which have four functional Ca2+-binding EF hand domains. Thus like its S. cerevisiae counterpart Cmd1p, the KlCMD1 product is predicted to form only three Ca2+-binding motifs.

Amino Acid Sequence↗

PubMed1998

Mass spectrometric analysis of the anaphase-promoting complex from yeast: identification of a subunit related to cullins.

Entry into anaphase and exit from mitosis depend on a ubiquitin-protein ligase complex called the anaphase-promoting complex (APC) or cyclosome. At least 12 different subunits were detected in the purified particle from budding yeast, including the previously identified proteins Apc1p, Cdc16p, Cdc23p, Cdc26p, and Cdc27p. Five additional subunits purified in low nanogram amounts were identified by tandem mass spectrometric sequencing. Apc2p, Apc5p, and the RING-finger protein Apc11p are conserved from yeast to humans. Apc2p is similar to the cullin Cdc53p, which is a subunit of the ubiquitin-protein ligase complex SCFCdc4 required for the initiation of DNA replication.

Amino Acid Sequence↗

PubMed1998

Psychosocial factors associated with the stages of change for condom use among women at risk for HIV and STDs: implications for intervention development.

This study examined the prevalence of consistent condom use among inner-city women at risk for HIV, measured the distribution of these women across the stages of change for condom use, determined psychosocial factors associated with the stages, and suggested intervention strategies based on the results. The 5-city sample of women aged 15-34 years consisted predominantly of African Americans. Only 18% reported consistent condom use with main partners and 45% with other partners. Logistic regressions compared women in each stage of change with those in higher stages for each partner type. Results indicated that women who practice or intend to practice consistent condom use were more likely to talk with others about condoms, acknowledge the advantages of condoms, have higher self-efficacy for condom use, and indicate that people important to them favored condom use. Intervention approaches are suggested for women in different stages of change for condom use.

Adolescent↗

PubMed1998

Violence and HIV sexual risk behaviors among female sex partners of male drug users.

OBJECTIVE: Violence and HIV are emerging as interconnected public health hazards among drug users and their families. The purposes of this study are to (1) determine the prevalence of sexual and physical abuse of non-drug-using female sex partners of male drug users, and (2) ascertain the association between such violence and HIV-related risk behaviors. METHODS: From 11/93 to 11/95, 208 female sex partners of injection drug or crack users in Collier County, FL, Tucson, AZ, and Portland, OR, were interviewed as part of a NIDA-funded HIV risk reduction project. Their mean age was 30 years (range 18-54); 21% were White, 6% African American, 7% Native American, and 63% Hispanic. RESULTS: Of the 208 women, 28% reported being sexually molested and 20% raped before age 13; 41% reported being raped at least once in their lifetime. Forty-two percent of the women were physically assaulted by their sex partners; 36% had been threatened with assaulted by their sex partners. Those who were raped or threatened with assault were more likely to have multiple sex partners and engage in unprotected anal sex; there was a trend for women who had been physically assaulted to be more likely to engage in unprotected anal sex. DISCUSSION: Rape, assault and the threat of assault are commonplace in the histories of female sex partners of male drug users. Experiences of violence and threats of violence are associated with heightened risk for the sexual transmission of HIV. Providers of HIV prevention need to understand the sequelae of violence, and design interventions which empower women to protect themselves from sexual transmission of HIV.

Adult↗

PubMed1997

Inhibition of pancreatic cancer growth by the dietary isoprenoids farnesol and geraniol.

Fruits and vegetables have protective effects against many human cancers, including pancreatic cancer. Isoprenoids are one class of phytochemicals which have antitumor activity, but little is known about their effects on cancer of the pancreas. We tested the hypothesis that isoprenoids would inhibit the growth of pancreatic tumor cells. Significant (60-90%) inhibition of the anchorage-independent growth of human MIA PaCa2 pancreatic tumor cells was attained with 25 microM farnesol, 25 microM geranylgeraniol, 100 microM perillyl amine, 100 microM geraniol, or 300 microM perillyl alcohol. We then tested the relative in vivo antitumor activities of dietary farnesol, geraniol, and perillyl alcohol against transplanted PC-1 hamster pancreatic adenocarcinomas. Syrian Golden hamsters fed geraniol or farnesol at 20 g/kg diet exhibited complete inhibition of PC-1 pancreatic tumor growth. Both farnesol and geraniol were more potent than perillyl alcohol, which inhibited tumor growth by 50% at 40 g/kg diet. Neither body weights nor plasma cholesterol levels of animals consuming isoprenoid diets were significantly different from those of pair-fed controls. Thus, farnesol, geraniol, and perillyl alcohol suppress pancreatic tumor growth without significantly affecting blood cholesterol levels. These dietary isoprenoids warrant further investigation for pancreatic cancer prevention and treatment.

Acyclic Monoterpenes↗

PubMed1997

Mutations in the Saccharomyces cerevisiae type 2A protein phosphatase catalytic subunit reveal roles in cell wall integrity, actin cytoskeleton organization and mitosis.

Temperature-sensitive mutations were generated in the Saccharomyces cerevisiae PPH22 gene that, together with its homologue PPH21, encode the catalytic subunit of type 2A protein phosphatase (PP2A). At the restrictive temperature (37 degrees), cells dependent solely on pph22 alleles for PP2A function displayed a rapid arrest of proliferation. Ts pph22 mutant cells underwent lysis at 37 degrees, showing an accompanying viability loss that was suppressed by inclusion of 1 M sorbitol in the growth medium. Ts pph22 mutant cells also displayed defects in bud morphogenesis and polarization of the cortical actin cytoskeleton at 37 degrees. PP2A is therefore required for maintenance of cell integrity and polarized growth. On transfer from 24 degrees to 37 degrees, Ts- pph22 mutant cells accumulated a 2N DNA content indicating a cell cycle block before completion of mitosis. However, during prolonged incubation at 37 degrees, many Ts- pph22 mutant cells progressed through an aberrant nuclear division and accumulated multiple nuclei. Ts- pph22 mutant cells also accumulated aberrant microtubule structures at 37 degrees, while under semi-permissive conditions they were sensitive to the microtubule-destabilizing agent benomyl, suggesting that PP2A is required for normal microtubule function. Remarkably, the multiple defects of Ts- pph22 mutant cells were suppressed by a viable allele (SSD1-v1) of the polymorphic SSD1 gene.

Actins↗

PubMed1997

Comparison of types of research articles published in emergency medicine and non-emergency medicine journals.

INTRODUCTION: As the specialty of emergency medicine (EM) matures, its journals should be publishing research of a quality similar to that which appears in other premier journals. OBJECTIVE: To compare the types of original research published in 4 EM vs 3 non-EM journals. METHODS: Retrospective review of all 1995 articles published in Academic Emergency Medicine, American Journal of Emergency Medicine, Annals of Emergency Medicine, Journal of Emergency Medicine, Annals of Internal Medicine, JAMA, and New England Journal of Medicine. Research articles were classified as longitudinal vs cross-sectional, prospective vs retrospective, and interventional vs observational. Other characteristics noted were number of subjects, randomization, blinding, control, and power calculations. Journals were reviewed by 4 investigators who received specific training in research classification, adhering to previously reported criteria for retrospective reviews. Interobserver reliability was independently validated. RESULTS: The authors reviewed 3,524 articles, of which 874 (24.8%) were original research. Compared with research reported in non-EM journals, EM journals contained fewer longitudinal studies (40.5% vs 60.4%, p < 0.0001) and fewer prospective studies (70.8% vs 78.7%, p = 0.008). Fewer EM journals had studies that were blinded (13.7% vs 18.9%, p = 0.047) or controlled (36.3% vs 50.0%, p = 0.003). Studies reported in EM journals had fewer subjects (138 vs 300, p < 0.001). Research reports in EM journals were less likely to have been funded, even after adjustment for the differences in study designs (adjusted odds ratio 7.0, 95% CI 5.1-9.7). CONCLUSION: Significant differences in types of research published in EM and non-EN journals were identified.

Authorship↗

PubMed1997

Two-step tuberculin skin testing of injection drug users recruited from community-based settings.

SETTING: Cross-sectional study of drug users recruited from street-based settings in four US cities: Denver, Portland, Oakland and San Francisco. OBJECTIVE: To evaluate responses to two-step tuberculin skin testing among HIV-positive and HIV-negative injection drug users. DESIGN: Subjects were recruited from existing studies of HIV and risk behaviors for tuberculin skin testing. Those with a negative initial tuberculin test were referred for a second skin test 1-3 weeks later. A positive tuberculin test was defined as > or = 10 mm, or > or = 5 mm if the subject was HIV-positive. RESULTS: Of 997 persons receiving an initial tuberculin test, 13% had a positive response. Of 644 persons receiving a second tuberculin test, 8% had a positive response, with rates as high as 14% among those from Oakland and 12% among African Americans. HIV-positive subjects were less likely to have skin test responses > or = 10 mm on the initial test (P = 0.03), or increases between the initial and second test of > or = 10 mm (P = 0.06). CONCLUSION: Boosting occurred in both HIV-positive and HIV-negative injection drug users. Two-step testing should be considered for this population, particularly those on whom repeat tuberculin testing will be performed.

Adult↗

PubMed1996

The phosphorylation state of the 110 kDa component of the yeast spindle pole body shows cell cycle dependent regulation.

The 110 kDa component of the yeast spindle pole body (SPB) encoded by the SPC110/NUF1 gene is an essential protein which is important for the generation of a functional mitotic spindle in the yeast Saccharomyces cerevisiae. Spc110p exists in a number of forms which differ in mobility upon separation by SDS-polyacrylamide gel electrophoresis. We show that this heterogeneity is due to differential phosphorylation on serine or threonine residues and that Spc110p phosphorylation varies throughout the cell cycle. Specifically, the phosphorylated form Spc110p appears as cells progress from S-phase and is coincident with the presence of preanaphase mitotic spindles. Thus phosphorylation of Spc110p occurs at the stage in the cell cycle where SPBs acquire the competence to organize a mitotic spindle and could therefore represent an important regulatory event.

Blotting, Western↗

PubMed1996

Yeast protein serine/threonine phosphatases: multiple roles and diverse regulation.

Since the isolation of the first yeast protein phosphatase genes in 1989, much progress has been made in understanding this important group of proteins. Yeast contain genes encoding all the major types of protein phosphatase found in higher eukaryotes and the ability to use genetic approaches will complement the wealth of biochemical information available from other systems. This review will summarize recent progress in understanding the structure, function and regulation of the PPP family of protein serine-threonine phosphatases, concentrating on the budding yeast Saccharomyces cerevisiae.

Fungal Proteins↗

PubMed1996

The Iowa CHMIS (Community Health Management Information System): work in progress.

The Iowa Community Health Management Information System (CHMIS) is a legislatively authorized system that is designed to meet Iowa's shared information needs and reduce administrative costs. A public/private partnership has been planning this system for over three years, implementation is scheduled to begin in July 1996. At that time, certified transaction networks will route claims data between providers and payors, a subset of that data will be stored by a centralized data repository. As the system evolves, provider access will be expanded and additional data elements will be captured.

Community Networks↗

PubMed1996

Mutations which block the binding of calmodulin to Spc110p cause multiple mitotic defects.

We have generated three temperature-sensitive alleles of SPC110, which encodes the 110 kDa component of the yeast spindle pole body (SPB). Each of these alleles carries point mutations within the calmodulin (CaM) binding site of Spc110p which affect CaM binding in vitro; two of the mutant proteins fail to bind CaM detectably (spc110-111, spc110-118) while binding to the third (spc110-124) is temperature-sensitive. All three alleles are suppressed to a greater or lesser extent by elevated dosage of the CaM gene (CMD1), suggesting that disruption of CaM binding is the primary defect in each instance. To determine the consequences on Spc110p function of loss of effective CaM binding, we have therefore examined in detail the progression of synchronous cultures through the cell division cycle at the restrictive temperature. In each case, cells replicate their DNA but then lose viability. In spc110-124, most cells duplicate and partially separate the SPBs but fail to generate a functional mitotic spindle, a phenotype which we term 'abnormal metaphase'. Conversely, spc110-111 cells initially produce nuclear microtubules which appear well-organised but on entry into mitosis accumulate cells with 'broken spindles', where one SPB has become completely detached from the nuclear DNA. In both cases, the bulk of the cells suffer a lethal failure to segregate the DNA.

Alleles↗

PubMed1995

Chemotherapy of pancreatic cancer with the monoterpene perillyl alcohol.

Perillyl alcohol has antitumor activity against rat mammary and liver cancer. Here, we report the chemotherapeutic effects of perillyl alcohol on pancreatic cancer. Perillyl alcohol reduced the growth of hamster pancreatic tumors to less than half that of controls (P < 0.025). Moreover, 16% of perillyl alcohol-treated pancreatic tumors completely regressed whereas no control tumors regressed (P < 0.05). Perillyl alcohol induced contact inhibition in cultured human pancreatic carcinoma cells and inhibited their anchorage-independent growth (P < 0.001). Thus, perillyl alcohol has antitumor activity against pancreatic carcinomas at non-toxic doses, and may be an effective chemotherapeutic agent for human pancreatic cancer.

Animals↗

PubMed1995

A regulated MET3-GLC7 gene fusion provides evidence of a mitotic role for Saccharomyces cerevisiae protein phosphatase 1.

Saccharomyces cerevisiae possesses a single essential gene (GLC7) encoding protein phosphatase 1 (PP1). Elevated expression of this gene from the GAL1 promoter is highly detrimental to the cell, causing a growth defect and aberrant bud morphology, which leads to cells exhibiting long, extended buds. By comparison, expression of GLC7 from the weaker MET3 promoter was without significant effect on either growth or morphology. However, repression of GLC7 expression from the MET3 promoter in cells where the MET3-GLC7 fusion was the sole source of PP1 resulted in a mitotic delay. Such cultures showed a massive decrease in the rate of proliferation in conjunction with a significant increase in the proportion of large, budded cells. 4'6-diamidino-2-phenylindole dihydrochloride (DAPI) staining and anti-tubulin immunofluorescence analysis of these cells revealed that many were blocked in mitosis, with a short spindle and DAPI-stained material stretched between the mother and daughter cell within the bud neck. These results support a role for PP1 in the completion of mitosis in S. cerevisiae.

Base Sequence↗

PubMed1995

The Saccharomyces cerevisiae gene SDS22 encodes a potential regulator of the mitotic function of yeast type 1 protein phosphatase.

In higher eukaryotes, the activity and specificity of the type 1 protein serine-threonine phosphatase (PP1) catalytic subunit is thought to be controlled by its association with a number of regulatory or targeting subunits. Here we describe the characterization of a gene encoding one such potential polypeptide in the yeast Saccharomyces cerevisiae. The gene which we have isolated (termed SDS22) encodes a product with a high degree of sequence identity to the fission yeast sds22 protein, a known regulator of the mitotic function of PP1 in Schizosaccharomyces pombe. Using two different criteria, we have demonstrated that Sds22p and the catalytic subunit of PP1 (Glc7p) interact in yeast cells. We have also generated a temperature-sensitive allele of GLC7 (glc7-12) which causes a block to the completion of mitosis at the restrictive temperature. Additional copies of SDS22 lead to allele-specific suppression of the glc7-12 mutant, strongly suggesting that the interaction between the two proteins is of functional significance. Sds22p is therefore likely to be the second example of a PP1 regulatory subunit identified in S. cerevisiae.

Amino Acid Sequence↗

PubMed1994

Interaction with calmodulin is required for the function of Spc110p, an essential component of the yeast spindle pole body.

NUF1/SPC110, encoding a nuclear filament-related protein which is a component of the yeast spindle pole body (SPB), has been identified in a screen designed to isolate genes encoding targets of yeast calmodulin. Spc110p interacts with calmodulin by two different criteria and the calmodulin interacting region has been localized within the C-terminus of the protein. Point mutations between residues 898 and 917 further define the calmodulin binding site within this region. Mutations in this domain which abolish calmodulin binding in vitro prevent Spc110p function in vivo, demonstrating that calmodulin binding by Spc110p has important functional consequences. In keeping with a role for calmodulin in Spc110p function, we show that calmodulin localizes to the yeast SPB when cells are prepared under appropriate conditions. Non-functional mutant Spc110 proteins which cannot bind calmodulin are present at lowered steady-state levels in the cell; when their level is increased by elevated gene dosage, partial recovery of Spc110p function is seen. Overexpression of calmodulin suppresses the defect(s) associated with the mutant Spc110 proteins, supporting the notion that Spc110p stability is a consequence of its ability to bind calmodulin and pointing to a direct role for calmodulin in Spc110p function.

Alleles↗

PubMed1994

Inositol trisphosphate metabolism in Saccharomyces cerevisiae: identification, purification and properties of inositol 1,4,5-trisphosphate 6-kinase.

Ins(1,4,5)P3 metabolism was examined in Saccharomyces cerevisiae extracts. S. cerevisiae contains readily detectable Ins(1,4,5)P3 kinase activity that is predominantly soluble, but phosphomonoesterase activity acting on Ins(1,4,5)P3 was not detected in either soluble or particulate preparations from this organism. We have purified the kinase activity approximately 685-fold in a rapid four-step process, and obtained a stable preparation. The enzyme has an apparent native molecular mass of approximately 40 kDa, and displays Michaelis-Menten kinetics with respect to its two substrates, ATP and Ins(1,4,5)P3. The Km for ATP was 2.1 mM, and that for Ins(1,4,5)P3 was 7.1 microM. The enzyme appeared to be the first step in the conversion of Ins(1,4,5)P3 into an InsP5, and the partially purified preparation contained another activity that converted the InsP4 product into an InsP5. The InsP4 product of the partially purified kinase was not metabolized by human erythrocyte ghosts and co-chromatographed with an Ins(3,4,5,6)P4 [L-Ins(1,4,5,6)P4] standard, identifying it as D-Ins(1,4,5,6)P4. The yeast enzyme is thus an Ins(1,4,5)P3 6-kinase. This activity may be an important step in the production of inositol polyphosphates such as InsP5 and InsP6 in S. cerevisiae.

Adenosine Triphosphate↗

PubMed1994

Genetic analyses of yeast protein serine/threonine phosphatases.

Protein phosphorylation is an important regulatory phenomenon in yeasts just as in other eukaryotic cells and controls a wide variety of cellular processes. The importance of protein phosphatases as well as protein kinases as key elements in such control is becoming increasingly clear. Over the past four years since the first yeast protein phosphatase gene was isolated, many more such genes have been described and the number of genes encoding protein phosphatase catalytic subunits in Saccharomyces cerevisiae has comfortably entered double figures. Given the genetic approaches available, yeasts offer powerful systems for addressing the cellular roles of these enzymes. This review summarises the results of genetic studies aimed at determining the functions of protein serine/threonine phosphatases in yeast.

Genes, Fungal↗