PubMed Health⌕ Search

Biomedical subjects

M J Stark

Publications and source records attributed to M J Stark.

At least 19 recordsLinked to original sources
PubMed2004

A prospective study of household smoking bans and subsequent cessation related behaviour: the role of stage of change.

OBJECTIVE: To assess the degree to which smokers living with a full household ban on smoking change their cessation related behaviour. DESIGN, SETTING, AND PARTICIPANTS: Prospective cohort study; follow up of a population based cohort of 1133 smokers, identified from a 1997 telephone survey of adult Oregonians. After a median of 21 months, 565 were located and reinterviewed. MAIN OUTCOME MEASURES: Quit attempts, time until relapse, and smoking cessation, defined as seven day and 90 day sustained abstinence at follow up. RESULTS: A full ban at baseline was associated with a doubling of the odds of a subsequent quit attempt (odds ratio (OR) = 2.0, 95% confidence interval (CI), 1.0 to 3.9). Among respondents in the preparation stage at baseline (intention to quit in the next month with a quit attempt in the previous year), a full ban was associated with a lower relapse rate (hazard ratio = 0.5 (95% CI, 0.2 to 0.9)), while for those in precontemplation/contemplation (no intention to quit or intention to quit within the next six months, respectively), there was no significant association between full ban and relapse rate. For respondents in preparation, those with a full ban had over four times the odds of being in cessation for seven or more days before the follow up call (OR = 4.4 (1.1 to 18.7)), but for those in precontemplation/contemplation, full bans were unrelated to cessation. CONCLUSIONS: Full household bans may facilitate cessation among smokers who are preparing to quit by increasing quit attempts. They may also prolong time to relapse among those smokers.

Adolescent↗
PubMed2001

The Saccharomyces cerevisiae phosphotyrosyl phosphatase activator proteins are required for a subset of the functions disrupted by protein phosphatase 2A mutations.

In Saccharomyces cerevisiae, PTPA is encoded by two genes, YPA1 and YPA2. In order to examine the biological role of PTPA as potential regulator of protein phosphatase 2A (PP2A), we compared the phenotypes of the ypaDelta mutants with these of PP2A-deficient strains. While deletion of both YPA genes is lethal, deletion of YPA1 alone results in a phenotype resembling that of PP2A-deficient strains in specific aspects such as aberrant bud morphology, abnormal actin distribution, and similar growth defects under various growth conditions. These phenotypes were even more pronounced when YPA1 was deleted in a pph21Delta genetic background. Moreover, ypaDelta mutants are hypersensitive to nocodazole and show inappropriate mitotic spindle formation as previously described for mutants in the catalytic subunit of PP2A, suggesting that Ypa, like PP2A, has a function in mitotic spindle formation. These results are consistent with an in vivo role of Ypa as a regulator of PP2A. However, unlike a PP2A-deficient strain, ypaDelta mutants do not show a G2 arrest. Therefore, Ypa does not seem to play a role in the regulation of PP2A at this stage of the cell cycle. These results imply that Ypa regulates a specific subset of PP2A functions, possibly by controlling the subunit composition of PP2A.

Actins↗
PubMed2001

Saccharomyces cerevisiae cell wall chitin, the Kluyveromyces lactis zymocin receptor.

The exozymocin secreted by Kluyveromyces lactis causes sensitive yeast cells, including Saccharomyces cerevisiae, to arrest growth in the G(1) phase of the cell cycle. Despite its heterotrimeric (alpha beta gamma) structure, intracellular expression of its smallest subunit, the gamma-toxin, is alone responsible for the G(1) arrest. The alpha subunit, however, has a chitinase activity that is essential for holozymocin action from the cell exterior. Here we show that sensitive yeast cells can be rescued from zymocin treatment by exogenously applying crude chitin preparations, supporting the idea that chitin polymers can compete for binding to zymocin with chitin present on the surface of sensitive yeast cells. Consistent with this, holozymocin can be purified by way of affinity chromatography using an immobilized chitin matrix. PCR-mediated deletions of chitin synthesis (CHS) genes show that most, if not all, genetic scenarios that lead to complete loss (chs3 Delta), blocked export (chs7 Delta) or reduced activation (chs4 Delta), combined with mislocalization (chs4 Delta chs5 Delta; chs4 Delta chs6 Delta; chs4 Delta chs5 Delta chs6 Delta) of chitin synthase III activity (CSIII), render cells refractory to the inhibitory effects of exozymocin. In contrast, deletions in CHS1 and CHS2, which code for CSI and CSII, respectively, have no effect on zymocin sensitivity. Thus, CSIII-polymerized chitin, which amounts to almost 90% of the cell's chitin resources, appears to be the carbohydrate receptor required for the initial interaction of zymocin with sensitive cells.

Amino Acid Sequence↗
PubMed2001

Kluyveromyces lactis zymocin mode of action is linked to RNA polymerase II function via Elongator.

The putative Kluyveromyces lactis zymocin target complex, TOT, from Saccharomyces cerevisiae comprises five Tot proteins, four of which are RNA polymerase II (RNAP II) Elongator subunits. Recently, two more Elongator subunit genes, ELP6 (TOT6) and ELP4 (TOT7), have been identified. Deletions of both TOT6 and TOT7 result in the complex tot phenotype, including resistance to zymocin, thermosensitivity, slow growth and hypersensitivity towards drugs, thus reinforcing the notion that TOT/Elongator may be crucial in signalling zymocicity. Mutagenesis of ELP3/TOT3, the Elongator histone acetyltransferase (HAT) gene, revealed that zymocin sensitivity could be uncoupled from Elongator wild-type function, indicating that TOT interacts genetically with zymocin. To test the possibility that zymocin functions by affecting RNAP II activity in a TOT/Elongator-dependent manner, global poly(A)+ mRNA levels were found to decline drastically on zymocin treatment. Moreover, cells overexpressing Fcp1p, the RNAP II carboxy-terminal domain phosphatase, acquired partial zymocin resistance, whereas cells underproducing RNAP II became zymocin hypersensitive. This suggests that zymocin may convert TOT/Elongator into a cellular poison toxic for RNAP II function and eventually leading to the observed G1 cell cycle arrest.

Acetyltransferases↗
PubMed2001

Sit4p protein phosphatase is required for sensitivity of Saccharomyces cerevisiae to Kluyveromyces lactis zymocin.

We have identified two Saccharomyces cerevisiae genes that, in high copy, confer resistance to Kluyveromyces lactis zymocin, an inhibitor that blocks cells in the G(1) phase of the cell cycle prior to budding and DNA replication. One gene (GRX3) encodes a glutaredoxin and is likely to act at the level of zymocin entry into sensitive cells, while the other encodes Sap155p, one of a family of four related proteins that function positively and interdependently with the Sit4p protein phosphatase. Increased SAP155 dosage protects cells by influencing the sensitivity of the intracellular target and is unique among the four SAP genes in conferring zymocin resistance in high copy, but is antagonized by high-copy SAP185 or SAP190. Since cells lacking SIT4 or deleted for both SAP185 and SAP190 are also zymocin resistant, our data support a model whereby high-copy SAP155 promotes resistance by competition with the endogenous levels of SAP185 and SAP190 expression. Zymocin sensitivity therefore requires a Sap185p/Sap190p-dependent function of Sit4p protein phosphatase. Mutations affecting the RNA polymerase II Elongator complex also confer K. lactis zymocin resistance. Since sit4Delta and SAP-deficient strains share in common several other phenotypes associated with Elongator mutants, Elongator function may be a Sit4p-dependent process.

Amino Acid Sequence↗
PubMed2001

LET versus EMLA for pretreating lacerations: a randomized trial.

OBJECTIVE: To compare the anesthetic efficacy of EMLA cream (eutectic mixture of local anesthetics) with that of LET solution (lidocaine, epinephrine, tetracaine) for pretreating lacerations prior to lidocaine injection. METHODS: This was a randomized, double-blind clinical trial in a convenience sample of 60 patients aged 1 to 59 years with traumatic lacerations. Eligible wounds were uncomplicated, clean lacerations < or = 6 hours old. Finger and toe lacerations were excluded. At the time of initial presentation to triage, patients were randomized to LET or EMLA. A nurse applied the topical anesthetic into the laceration with a 5-mL syringe. A physician assessed the laceration edges for the presence of blanching and adequacy of anesthesia to a 27-gauge needlestick. Supplemental lidocaine was then infiltrated through the wound edges and the pain of infiltration was recorded by the patient (or guardian) on a 100-mm visual analog scale marked "most pain" at the high end. A sample of 44 patients had 90% power to detect a 20-mm difference in injection pain (two-tailed alpha = 0.05). RESULTS: Sixty patients were randomized to LET (29) or EMLA (31). Median age was 8.5 years; 23% were female. Most lacerations were facial and closed with sutures. There was no difference in baseline characteristics between groups. More wounds treated with LET were anesthetic to a needlestick than wounds treated with EMLA (73% vs 40%, p = 0.01); however, there was no between-group difference in the median pain of lidocaine infiltration (LET-12 mm vs EMLA-13 mm, p = 0.89). CONCLUSIONS: Pretreatment of simple lacerations with LET or EMLA at the time of patient presentation results in similar amounts of pain of subsequent local infiltration of lidocaine

Adolescent↗
PubMed2000

Mutations in SPC110, encoding the yeast spindle pole body calmodulin-binding protein, cause defects in cell integrity as well as spindle formation.

The 110 kDa spindle pole body component, Spc110p, is an essential target of calmodulin in budding yeast. Cells with mutations which reduce calmodulin binding to Spc110p are unable to form a mitotic spindle and die. Here we show that these effects can be overcome either directly by increasing extracellular calcium or calmodulin expression, which reverse the primary spindle defect, or indirectly through increased extracellular osmolarity or high dosage of MID2 or SLG1/HCS77/WSC1 which preserve viability. We propose that overcoming a cell integrity defect associated with the mitotic arrest enables the defective spindle pole bodies to provide sufficient function for proliferation of a large proportion of mutant cells. Our findings demonstrate a role for calcium in the Spc110p-calmodulin interaction in vivo and have important general implications for the interpretation of genetic interactions involving cell integrity genes.

Calcium↗
PubMed2000

Evaluation of the CaMAL2 promoter for regulated expression of genes in Candida albicans.

An expression vector (CIp10-MAL2p) for use in Candida albicans has been constructed in which a gene of interest can be placed under the control of the CaMAL2 maltase promoter and stably integrated at the CaRP10 locus. Using this vector to express the Candida URA3 gene from the CaMAL2 promoter, we have demonstrated tight regulation of CaURA3 expression by carbon source. Thus under conditions when the CaMAL2 promoter is not induced, expression of Candida URA3 was unable either to complement a C. albicans ura3 mutation or to confer sensitivity to 5-fluoroorotic acid, a compound which is highly toxic to URA3 strains. Since Candida albicans is an obligate diploid organism, analysis of gene function requires manipulation of both copies of any gene of interest. Our expression vector provides a strategy by which the remaining copy of a gene of interest can be placed under CaMAL2 promoter control in a strain where the first copy has been deleted, permitting analysis of gene function by manipulation of carbon source. CIp10-MAL2p should therefore provide a useful means for functional analysis of genes in C. albicans. We have used this strategy with C. albicans DPB2 to demonstrate that the gene is essential and that loss of function leads cells to adopt a hypha-like morphology as they cease proliferation.

Antigens, Fungal↗
PubMed2000

A novel 'two-component' protein containing histidine kinase and response regulator domains required for sporulation in Aspergillus nidulans.

We have characterised a-novel Aspergillus nidulans gene encoding a 'two-component' signalling protein (tcsA). tcsA encodes both a histidine kinase domain and a response regulator domain similar to those found in bacterial, lower eukaryotic and plant members of the two-component family of proteins, while two PAS domains in the amino-terminal region of the predicted tcsA product may monitor the signal which regulates a tcsA histidine kinase-response regulator phosphorelay. While tcsA is nonessential for vegetative growth, cells lacking the gene are unable to produce conidia on standard Aspergillus growth media. However, tcsA is not absolutely required for production since this defect is suppressed by growth on 1 M sorbitol.

Amino Acid Sequence↗
PubMed2000

Pretreatment of lacerations with lidocaine, epinephrine, and tetracaine at triage: a randomized double-blind trial.

OBJECTIVE: Pretreatment of lacerations with topical anesthetics reduces the pain of subsequent anesthetic injection yet requires time. This study was conducted to determine the ability of triage nurses to identify lacerations necessitating closure and pretreat them with a topical anesthetic and to compare the pain levels of lidocaine injection in lacerations pretreated with LET (lidocaine 2%, epinephrine 1:1,000, tetracaine 2%) vs a placebo. METHODS: This was a double-blind, randomized clinical trial that included consecutive emergency department patients aged > or = 1 year with clean, non-bite lacerations < or = 6 hours old. At triage the lacerations were randomized to LET solution or a placebo containing epinephrine 1:1,000. At examination an emergency practitioner assessed the laceration edges for the presence of blanching and adequacy of anesthesia to a 27-gauge needlestick. At the practitioner's discretion, supplemental lidocaine was infiltrated through the wound and the patient (or guardian) recorded the pain of infiltration on a 100-mm visual analog scale marked "most pain" at the high end. The mean levels of pain of lidocaine infiltration were compared between groups with a t-test and the proportions of adequately anesthetized wounds were compared with a chi2 test. A sample of 40 patients had 80% power to detect a 20-mm between-group difference in the pain of injection (alpha = 0.05). RESULTS: Of 43 patients enrolled (mostly children and males), 22 received LET and 21 placebo. The groups were similar for baseline characteristics. Lacerations in the LET group were more frequently adequately anesthetized (46% vs 14%, p = 0.03), and LET patients experienced less pain from injection than controls (22 mm vs 42 mm, p = 0.02). CONCLUSIONS: Application of LET by triage nurses is more effective than placebo in adequately anesthetizing simple lacerations in normal hosts and decreases the pain of local anesthetic infiltration.

Administration, Topical↗
PubMed2000

Dynamic, Rho1p-dependent localization of Pkc1p to sites of polarized growth.

In eukaryotes, the Rho GTPases and their effectors are key regulators of the actin cytoskeleton, membrane trafficking and secretion, cell growth, cell cycle progression and cytokinesis. Budding yeast Pkc1p, a protein kinase C-like enzyme involved in cell wall biosynthesis and cytoskeletal polarity, is structurally and functionally related to the Rho-associated kinases (PRK/ROCK) of mammalian cells. In this study, localization of Pkc1p was monitored in live cells using a GFP fusion (Pkc1p-GFP). Pkc1p-GFP showed dynamic spatial and temporal localization at sites of polarized growth. Early in the cell cycle, Pkc1p-GFP was found at the pre-bud site and bud tips, becoming delocalized as the cell progressed further and finally relocalizing around the mother-daughter bud neck in an incomplete ring, which persisted until cell separation. Bud localization was actin-dependent but stability of Pkc1p-GFP at the neck was actin-independent, although localization at both sites required functional Rho1p. In addition, Pkc1p-GFP showed rapid relocalization after cell wall damage. These results suggest that the roles of Pkc1p in both polarized growth and the response to cell wall stress are mediated by dynamic changes in its localization, and suggest an additional potential role in cytokinesis.

Actins↗
PubMed2000

Type 1 protein phosphatase is required for maintenance of cell wall integrity, morphogenesis and cell cycle progression in Saccharomyces cerevisiae.

GLC7 encodes the catalytic subunit of type 1 protein serine/threonine phosphatase (PP1) in the yeast Saccharomyces cerevisiae. Here we have characterized the temperature-sensitive glc7-10 allele, which displays aberrant bud morphology and an abnormal actin cytoskeleton at the restrictive temperature. At 37 degrees C glc7-10 strains accumulated a high proportion of budded cells with an unmigrated nucleus, duplicated spindle pole bodies, a short spindle, delocalized cortical actin and 2C DNA content, indicating a cell cycle block prior to the metaphase to anaphase transition. glc7-10 was suppressed by growth on high osmolarity medium and exhibited temperature-sensitive cell lysis upon hypo-osmotic stress. Pkc1p, the yeast protein kinase C homolog which is thought to regulate the Mpk1p MAP kinase pathway involved in cell wall remodelling and polarized cell growth, was found to act as a dosage suppressor of glc7-10. Although neither activation of BCK1 (MEKK) by the dominant BCK1-20 mutation nor increased dosage of MKK1 (MEK) or MPK1 (MAP kinase) mimicked PKC1 as a glc7-10 dosage suppressor, extra copies of genes encoding upstream components of the Pkc1p pathway such as ROM2, RHO2, HCS77/WSC1/SLG1 and MID2 also suppressed glc7-10 effectively. Conversely, mpk1delta glc7-10 and bck1delta glc7-10 double mutants displayed a synthetic cell lysis defect compared with each single mutant and glc7-10 was hypersensitive to reduced PKC1 function, displaying highly aberrant morphologies and inviability even at the normally permissive temperature of 26 degrees C. Dephosphorylation by PP1 therefore functions positively to promote cell integrity, bud morphology and polarization of the actin cytoskeleton and glc7-10 cells require higher levels of Pkc1p activity to sustain these functions.

Amino Acid Substitution↗
PubMed2000

A recQ family DNA helicase gene from Aspergillus nidulans.

We have identified an Aspergillus nidulans gene encoding a RecQ family helicase which we have therefore named recQ. The A. nidulans recQ protein is most closely related in sequence to human recQ helicase 5. Like the latter polypeptide, A. nidulans recQ consists of little more than the conserved helicase domain, lacking the long amino- and carboxy-terminal extensions seen in other recQ family members such as BLM and WRN and in the sole RecQ family helicase of the yeast Saccharomyces cerevisiae (Sgs1p). By analogy with other eukaryotic RecQ helicases, A. nidulans recQ helicase is likely to play an important role in the maintenance of genomic integrity.

Adenosine Triphosphatases↗
PubMed1999

Control of the terminal step of intracellular membrane fusion by protein phosphatase 1.

Intracellular membrane fusion is crucial for the biogenesis and maintenance of cellular compartments, for vesicular traffic between them, and for exo- and endocytosis. Parts of the molecular machinery underlying this process have been identified, but most of these components operate in mutual recognition of the membranes. Here it is shown that protein phosphatase 1 (PP1) is essential for bilayer mixing, the last step of membrane fusion. PP1 was also identified in a complex that contained calmodulin, the second known factor implicated in the regulation of bilayer mixing. The PP1-calmodulin complex was required at multiple sites of intracellular trafficking; hence, PP1 may be a general factor controlling membrane bilayer mixing.

Biological Transport↗
PubMed1999

Regulation of Saccharomyces cerevisiae kinetochores by the type 1 phosphatase Glc7p.

We have investigated the role of protein phosphorylation in regulation of Saccharomyces cerevisiae kinetochores. By use of phosphatase inhibitors and a type 1 protein phosphatase mutant (glc7-10), we show that the microtubule binding activity, but not the centromeric DNA-binding activity, of the kinetochore complex is regulated by a balance between a protein kinase and the type 1 protein phosphatase (PP1) encoded by the GLC7 gene. glc7-10 mutant cells exhibit low kinetochore-microtubule binding activity in vitro and a high frequency of chromosome loss in vivo. Specifically, the Ndc10p component of the centromere DNA-binding CBF3 complex is altered by the glc7-10 mutation; Ndc10p is hyperphosphorylated in glc7-10 extracts. Furthermore, addition of recombinant Ndc10p reconstitutes the microtubule-binding activity of a glc7-10 extract to wild-type levels. Finally, the glc7-10-induced mitotic arrest is abolished in spindle checkpoint mutants, suggesting that defects in kinetochore-microtubule interactions caused by hyperphosphorylation of kinetochore proteins activate the spindle checkpoint.

Chromosome Segregation↗
PubMed1999

Understanding HIV risks of chronic drug-using men who have sex with men.

Focus groups and individual structured interviews were conducted in six cities with 98 predominantly street-recruited men who had a recent history of smoking crack or injecting drugs and who reported having had sex with other men (MSM) in the past year. Twenty-six focus groups explored the cultural and social context of participant's drug use and sexual activity and addressed outreach and HIV prevention issues pertinent to this population. Narrative summaries developed from verbatim focus group transcripts identified seven themes: (a) sexual orientation and gender identity; (b) interactions within and between MSM networks; (c) drug use, sexual activity and personal relationships; (d) HIV transmission bridges; (e) preferred HIV information sources; (f) HIV knowledge, prevention practices and risk behaviours; and (g) availability of HIV and drug-related services. Of the 98 MSM drug users, 42% identified publicly as gay or homosexual; 35% identified publicly, but only 21% privately, as heterosexual. A total of 51% had one or more female sex partners in the past year. There was a high frequency of unprotected sex in conjunction with drug use and a distinct preference for having sex when high. For most participants, drug use rather than sexual orientation formed the core of personal identity. Participants reported associating primarily with other drug users, usually MSM, and had limited contact with people who did not use drugs and the mainstream gay community. Participants' sexual and drug-injecting activities were judged to be a bridge for transmission of HIV to both people who used drugs and those who did not.

Adolescent↗