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T J Kelly

Publications and source records attributed to T J Kelly.

At least 37 records · Page 2Linked to original sources
PubMed1997

Cyclin-dependent kinase and initiation at eukaryotic origins: a replication switch?

A growing body of evidence indicates that cyclin-dependent kinases (CDKs) regulate the activity of eukaryotic origins of replication both positively and negatively. Although the details of this control remain unclear, recent work suggests that CDKs act directly at origins, where they associate with and phosphorylate several key initiator proteins. These data suggest that a CDK-regulated replication switch operates at each origin to ensure that initiation occurs precisely once per cell cycle.

Animals↗
PubMed1997

The evolving health-care environment: new arguments for closer collaboration between cardiac surgical intensive-care nurses and clinical engineers.

"Not vain the weakest, if their force unite." Homer, Iliad (9th century, B.C.). In today's evolving health-care environment, the cardiac surgical intensive-care nurse is required to provide care to patients whose acuity levels are increasing. Simultaneously, these nurses are asked to use more technologically complex devices to deliver that care. In addition, practice protocols are being integrated into hospitals' clinical information systems. To meet these challenges, cardiac surgical intensive-care nurses must collaborate with clinical engineers on the evaluation and installation of new products and on the automation and refinement of clinical pathways and other outcomes measurement tools. Each discipline also must ensure that the other keeps pace with and maintains its levels of proficiency in the technology used to care for patients undergoing cardiac surgery.

Biomedical Engineering↗
PubMed1997

The Schizosaccharomyces pombe rad11+ gene encodes the large subunit of replication protein A.

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein present in all eukaryotes. In vitro studies have implicated RPA in simian virus 40 DNA synthesis and nucleotide excision repair, but little direct information is available about the in vivo roles of the protein. We report here the cloning of the largest subunit of RPA (rpa1+) from the fission yeast Schizosaccharomyces pombe. The rpa1+ gene is essential for viability and is expressed specifically at S phase of the cell cycle. Genetic analysis revealed that rpa1+ is the locus of the S. pombe radiation-sensitive mutation rad11. The rad11 allele exhibits pleiotropic effects consistent with an in vivo role for RPA in both DNA repair and DNA synthesis. The mutant is sensitive to both UV and ionizing radiation but is not defective in the DNA damage-dependent checkpoint, consistent with the hypothesis that RPA is part of the enzymatic machinery of DNA repair. When incubated in hydroxyurea, rad11 cells initially arrest with a 1C DNA content but then lose viability coincident with reentry into S phase, suggesting that DNA synthesis is aberrant under these conditions. A significant fraction of the mutant cells subsequently undergo inappropriate mitosis in the presence of hydroxyurea, indicating that RPA also plays a role in the checkpoint mechanism that monitors the completion of S phase. We propose that RPA is required to maintain the integrity of replication complexes when DNA replication is blocked. We further suggest that the rad11 mutation leads to the premature breakdown of such complexes, thereby preventing recovery from the hydroxyurea arrest and eliminating a signal recognized by the S-phase checkpoint mechanism.

Alleles↗
PubMed1996

The ATM homologue MEC1 is required for phosphorylation of replication protein A in yeast.

Replication protein A (RPA) is a highly conserved single-stranded DNA-binding protein, required for cellular DNA replication, repair, and recombination. In human cells, RPA is phosphorylated during the S and G2 phases of the cell cycle and also in response to ionizing or ultraviolet radiation. Saccharomyces cerevisiae exhibits a similar pattern of cell cycle-regulated RPA phosphorylation, and our studies indicate that the radiation-induced reactions occur in yeast as well. We have examined yeast RPA phosphorylation during the normal cell cycle and in response to environmental insult, and have demonstrated that the checkpoint gene MEC1 is required for the reaction under all conditions tested. Through examination of several checkpoint mutants, we have placed RPA phosphorylation in a novel pathway of the DNA damage response. MEC1 is similar in sequence to human ATM, the gene mutated in patients with ataxia-telangiectasia (A-T). A-T cells are deficient in multiple checkpoint pathways and are hypersensitive to killing by ionizing radiation. Because A-T cells exhibit a delay in ionizing radiation-induced RPA phosphorylation, our results indicate a functional similarity between MEC1 and ATM, and suggest that RPA phosphorylation is involved in a conserved eukaryotic DNA damage-response pathway defective in A-T.

Ataxia Telangiectasia↗
PubMed1996

Rum1 and Cdc18 link inhibition of cyclin-dependent kinase to the initiation of DNA replication in Schizosaccharomyces pombe.

Eukaryotic cells have evolved regulatory mechanisms to ensure the strict alternation of DNA replication and mitosis. Recent work has suggested that the mitotic form of cyclin-dependent kinase (Cdc2/cyclin B) has a role in preventing re-replication of the genome before mitosis, but the relevant targets of this inhibition are unknown. In this report we present evidence that the mitotic cyclin-dependent kinase affects DNA replication by inhibiting the accumulation and function of Cdc18, a critical regulator of S-phase entry. We found that the ruml+ gene efficiently suppresses the lethality of a conditional cdc18 mutant. Conversely, deletion of ruml+ increases the severity of the cdc18 mutant phenotype, resulting in inappropriate cell division and a rapid loss of viability. Biochemical experiments indicate that Ruml potently inhibits Cdc2 phosphorylation of histone H1 or a Cdc18 fusion protein by directly interacting with the Cdc2/cyclin B complex. Overexpression of Ruml under conditions that promote re-replication of the genome induces a striking accumulation of Cdc18 protein by a largely post-transcriptional mechanism. Overexpression of SIC1, an unrelated cyclin-dependent kinase inhibitor from budding yeast, causes a similar accumulation of Cdc18 and also leads to re-replication. Our data link a potent inhibitor of Cdc2 kinase to a key protein required for the initiation of DNA replication and strongly suggest that inhibition of Cdc18 by cyclin-dependent kinases has an important role in ensuring that the genome is duplicated precisely once each cell cycle.

CDC2 Protein Kinase↗
PubMed1996

cdc18+ regulates initiation of DNA replication in Schizosaccharomyces pombe.

In the fission yeast Schizosaccharomyces pombe the cdc18'+gene is required both for initiation of DNA replication and for coupling mitosis to the completion of S phase. Cells lacking Cdc18 fail to enter S phase but still undergo nuclear division. Expression of cdc18+ is sufficient to drive a G1-arrested cdc10ts mutant into the S phase of the cell cycle, indicating that cdc18+ represents a critical link between passage through START and the initiation of DNA replication. Here we show that Cdcl8 is a highly unstable protein that is expressed only once per cell cycle at the boundary between GI and S phase. De novo synthesis of Cdc18 is required before, but not after, the initiation of DNA replication, indicating that Cdc18 function is not necessary once the initiation event has occurred. Overproduction of the protein results in an accumulation of cells with DNA content of greater than 2C and delays mitosis, suggesting that Cdc18 is sufficient to cause reinitiation of DNA replication within a given cell cycle. Our data indicate that the synthesis of Cdc18 protein is a critical rate-limiting step in the initiation of DNA replication during each cell cycle. The extreme lability of the protein may contribute to the prevention of reinitiation.

Cell Cycle↗
PubMed1996

Sex-specific and hormone-controlled expression of a vitellogenin-encoding gene in the gypsy moth.

Microvitellogenin and vitellogenin cDNA from Manduca sexta (tobacco hornworm) were tested for use as molecular probes to investigate the expression of genes coding for vitellogenins in Spodoptera frugiperda (fall armyworm) and Lymantria dispar (gypsy moth). Cross-hybridization was not observed between the M. sexta cDNAs and S. frugiperda DNA and mRNA. Vitellogenin cDNA from M. sexta did not hybridize to L. dispar DNA or mRNA. However, the 834 bp microvitellogenin cDNA from M. sexta hybridized to an approximately 850 bp transcript in L. dispar mRNA. A 2.5 kb cDNA clone, pz64, was isolated from late last instar larvae of female L. dispar by differential screening. This clone has 38% amino acid sequence (deduced) and 55% nucleic acid sequence similarities with the 3'-end of high molecular weight vitellogenin in Bombyx mori (silkworm). When used as a probe in northern analysis of L. dispar mRNA, this cDNA hybridized to a 5.3 kb transcript in female last instar larvae, pupae, and adults, but not to male last instar larvae and adults. This cDNA did not hybridize to mRNA from M. sexta or S. frugiperda. Expression of the 5.3 kb vitellogenin transcript hybridizing to the 2.5 kb cDNA clone was suppressed in 5-day-old last instar larvae of female L. dispar treated on day 2 with doses of the juvenile hormone analog, methoprene, greater than 10 nmol. Apparently, the high in vivo titer of juvenile hormone during the first 2 days of the last instar represses the transcription of vitellogenin mRNA.

Amino Acid Sequence↗
PubMed1996

Serum concentrations of methanol after inhalation at 200 ppm.

Methanol has been proposed as an alternative automotive fuel to reduce pollution in the urban environment. Utilization of methanol will increase exposure to low levels of methanol vapors for the general public and in occupational settings. Information on absorption by inhalation and serum concentrations after low-level exposure would be important in evaluating the health impact of generalized methanol exposure. During a randomized double-blind study of the potential neurobehavioral effects of inhaled methanol at 200 ppm for 4 hours, 15 timed specimens from 22 subjects were obtained for methanol analysis by head-space gas chromatography. Methanol was rapidly absorbed by inhalation (absorption rate constant = 0.87 +/- 0.60 hours-1). Serum methanol concentrations were increased by more than fourfold at the end of the exposure period (6.5 +/- 2.7 vs 0.9 +/- 0.6 mg/L), as were urinary methanol excretion rates, although formate concentrations were not increased over background concentrations. The overall (n = 22) elimination half-life was 3.2 +/- 2.3 hours. Elimination from plasma fit a monoexponential model for only half of the subjects during the 4-hour postexposure follow-up period (mean half-life = 2.2 hours). Subjects with poor fits either showed greater variability or apparent slow (nonsignificant) declines in serum methanol concentrations, possibly because of the offsetting contributions of dietary intake or endogenous production, but more likely as a result of the limited number of sampling times and limited follow-up period.

Adult↗
PubMed1996

Expression of the HsOrc1 gene, a human ORC1 homolog, is regulated by cell proliferation via the E2F transcription factor.

The initiation of DNA replication in Saccharomyces cerevisiae requires the action of a multisubunit complex of six proteins known as the origin recognition complex (ORC). The identification of higher eukaryotic homologs of several ORC components suggests a universal role for this complex in DNA replication. We now demonstrate that the expression of one of these homologs is regulated by cell proliferation. Expression of the human Orc1 gene (HsOrc1) is low in quiescent cells, and it is then dramatically induced upon stimulation of cell growth. In contrast, expression of the HsOrc2 gene does not appear to be similarly regulated. We have isolated the promoter that regulates HsOrc1 transcription, and we show that the promoter confers cell growth-dependent expression. We also demonstrate that the cell growth control is largely the consequence of E2F-dependent negative transcription control in quiescent cells. Activation of HsOrc1 transcription following growth stimulation requires G1 cyclin-dependent kinase activity, and forced E2F1 expression can bypass this requirement. These results thus provide a direct link between the initiation of DNA replication and the cell growth regulatory pathway involving G1 cyclin-dependent kinases, the Rb tumor suppressor, and E2F.

Base Sequence↗
PubMed1996

Discrepancies in osmolal gaps and calculated alcohol concentrations.

OBJECTIVES: To estimate unexplained osmolal gaps (UEOG) during ethanol ingestions after accounting for measured ethanol concentrations and to explore the causes of observed discrepancies. DESIGN AND SPECIMENS: A random convenience sample of serum specimens with a request to perform osmometry for the detection of alcohols was studied. MAIN OUTCOME MEASURES: Serum concentrations of sodium, glucose, urea nitrogen, ethanol, osmolality, and ethanol were measured by two analytic methods to determine calculated osmolalities and osmolal gaps using two commonly used formulas. RESULTS: In 79 serum specimens submitted for osmometry, mean ethanol concentrations were 199 +/- 154 (SD) mg/dL; 15 specimens had no detectable ethanol. After regression of the calculated ethanol concentrations and the measured ethanol concentrations, the Dorwart and Chalmers formula showed a large constant bias of 45.217 +/- 6.414 (SE) mOsm/kg in predicting measured ethanol concentrations. The Smithline and Gardner formula was similarly as precise (r = .9786), but had a much smaller bias of -3.929 +/- 6.623 (SE) mOsm/kg. Actual mean differences between measured and calculated osmolalities (including ethanol osmols) gave an estimate of UEOG seen during ethanol ingestions. Using the less biased formula, the mean UEOG was 1.4 +/- 8.5 mOsm/kg, giving a 95% upper limit of the reference range of 18 mOsm/kg in this population with ethanol ingestions. Three significant outliers were identified and their UEOG could be explained by the presence of mannitol, isopropanol, or possibly a change in the assumed value for fractional water, normally a constant in these formulas for calculated osmolality. Other specific causes of osmolal gap and UEOG are discussed. CONCLUSION: The potential range of osmolal gaps seen during ethanol ingestions after accounting for ethanol is demonstrated to be greater than in previous studies, which have included a lower proportion of ethanol-intoxicated subjects in their study sample.

1-Propanol↗
PubMed1995

Orp1, a member of the Cdc18/Cdc6 family of S-phase regulators, is homologous to a component of the origin recognition complex.

cdc18+ of Schizosaccharomyces pombe is a periodically expressed gene that is required for entry into S phase and for the coordination of S phase with mitosis. cdc18+ is related to the Saccharomyces cerevisiae gene CDC6, which has also been implicated in the control of DNA replication. We have identified a new Sch. pombe gene, orp1+, that encodes an 80-kDa protein with amino acid sequence motifs conserved in the Cdc18 and Cdc6 proteins. Genetic analysis indicates that orp1+ is essential for viability. Germinating spores lacking the orp1+ gene are capable of undergoing one or more rounds of DNA replication but fail to progress further, arresting as long cells with a variety of deranged nuclear structures. Unlike cdc18+, orp1+ is expressed constitutively during the cell cycle. cdc18+, CDC6, and orp1+ belong to a family of related genes that also includes the gene ORC1, which encodes a subunit of the origin recognition complex (ORC) of S. cerevisiae. The products of this gene family share a 250-amino acid domain that is highly conserved in evolution and contains several characteristic motifs, including a consensus purine nucleotide-binding motif. Among the members of this gene family, orp1+ is most closely related to S. cerevisiae ORC1. Thus, the protein encoded by orp1+ may represent a component of an Sch. pombe ORC. The orp1+ gene is also closely related to an uncharacterized putative human homologue. It is likely that the members of the cdc18/CDC6 family play key roles in the regulation of DNA replication during the cell cycle of diverse species from archaebacteria to man.

Amino Acid Sequence↗
PubMed1995

Genetic analysis of an ARS element from the fission yeast Schizosaccharomyces pombe.

ARS (autonomously replicating sequence) elements are DNA fragments that can function as origins of DNA replication in yeast. We report the first fine-structure analysis of ars1, an ARS element of the fission yeast Schizosaccharomyces pombe. Characterization of a series of nested deletion mutations indicated that the minimal fragment of DNA encompassing ars1 is surprisingly large. No fragment < 650 bp retained significant ARS activity. Analysis of deletion and substitution mutations scanning the entire minimal ars1 identified a single essential 50 bp fragment (segment 1). Only one other 50 bp mutation reduced activity as much as 5-fold and most deletions were without effect. Thus, the minimal ars1 is composed of two general types of genetic elements, a small segment that is absolutely required for efficient ARS activity and a much larger region that is tolerant of internal structural alterations. Higher resolution analysis of segment 1 defined a critical 30 bp A/T-rich segment which appears to contain redundant genetic elements. Schizosaccharomyces pombe ars1 promoted high frequency transformation in the budding yeast S.cerevisiae but this heterologous activity was not dependent on segment 1. Our analysis indicates that the functional elements required for ARS function in S.pombe and S.cerevisiae are clearly different.

Base Sequence↗
PubMed1995

A novel low molecular weight ecdysiotropin in post-diapause, pre-hatch eggs of the gypsy moth, Lymantria dispar L. (Lepidoptera: Lymantriidae).

Extracts of post-diapause, pre-hatch eggs of the gypsy moth, Lymantria dispar L. were examined for prothoracicotropic hormone (PTTH)-like activity using an in vitro assay involving last-instar prothoracic glands (PGs). The eggs were extracted in water, eluted from a low-pressure C18-silica cartridge in 60% acetonitrile, and fractionated on a high-performance, size-exclusion column. The primary ecdysiotropic activity eluted with an estimated molecular weight of 2.1 kDa far below the 4-7 kDa size determined for the low molecular weight PTTHs (bombyxins). Dose-response analysis revealed that the maximum activation was reduced by 75% by organic solvent extraction, but the remaining activity retained the ability to maximally activate the PGs 10-fold in vitro. At least some of the ecdysiotropic activity in the post-diapause, pre-hatch egg is localized in the brain of the pharate larva, and this activity increases dramatically prior to hatch when eggs are incubated at 25 degrees C.

Animals↗
PubMed1995

Overexpression of Bombyx mori prothoracicotropic hormone using baculovirus vectors.

Recombinant baculoviruses were constructed that express the cDNA encoding the prothoracicotropic hormone (PTTH) of Bombyx mori. This hormone stimulates the production of ecdysteroids by the insect's prothoracic glands. Two groups of viruses were constructed, expressing either the entire cDNA encoding prepro-PTTH, or a synthetic chimeric gene encoding a signal peptide fused to the mature PTTH subunit. In both cases, the genes were expressed in wild-type Autographa californica nuclear polyhedrosis virus (AcMNPV) and in vEGTDEL, an ACMNPV mutant that lacks a functional egt gene. The egt gene is required for viral-mediated inactivation of host ecdysteroids. High levels of functional PTTH were produced only by viruses expressing the mature subunit cDNA. This recombinant PTTH resembled the native hormone by all criteria examined. The overproduction of B. mori PTTH induced higher than normal levels of haemolymph ecdysteroids but had no observable effects on the development of infected Spodoptera frugiperda larvae. However, expression of PTTH by AcMNPV was found to inhibit the pathogenicity of the virus. This effect was particularly marked in the case of viruses lacking a functional egt gene.

Amino Acid Sequence↗
PubMed1995

Concentrations of apolipoprotein AI, AII, and E in plasma and lipoprotein fractions of alcoholic patients: gender differences in the effects of alcohol.

Previous studies have shown that plasma levels of high-density lipoprotein (HDL) cholesterol and the two major protein components of HDLs, i.e., apolipoproteins AI and AII, were elevated in male alcoholic patients without serious liver injury. By contrast, alcohol effect on apolipoprotein E remains unclear. Apolipoprotein E is a major component of very low-density lipoprotein (VLDL) and a minor component of human high-density lipoprotein. It plays a critical role in lipoprotein metabolism through cellular lipoprotein receptors. Furthermore, previous works were carried out mostly with male subjects, whereas alcohol effects on serum apolipoproteins in female subjects have not yet been adequately addressed. In this study, we have raised antibodies specifically to recognize human apolipoprotein AI, AII, and E, respectively, to quantify apolipoprotein concentrations in plasma and lipoprotein fractions of male and female alcoholic patients. We have also measured plasma apolipoprotein concentrations in patients who had abstained from alcohol while in the hospital. Our results showed the following: (1) plasma concentrations of apolipoprotein AI and AII were significantly elevated yet plasma apolipoprotein E decreased (33%) significantly (P < .01) in male alcoholic patients; (2) apolipoprotein AI concentrations in female nondrinking control subjects were higher than in male controls, and the concentrations of apolipoprotein AI in female alcoholic patients were not significantly elevated over those of female controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗