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M Johnston

Publications and source records attributed to M Johnston.

At least 91 records · Page 5Linked to original sources
PubMed1999

The nuclear exportin Msn5 is required for nuclear export of the Mig1 glucose repressor of Saccharomyces cerevisiae.

BACKGROUND: Mig1 is a transcriptional repressor responsible for glucose repression of many genes in the budding yeast Saccharomyces cerevisiae. Glucose regulates Mig1 function by affecting its phosphorylation, which is catalyzed by the Snf1 protein kinase. Phosphorylation alters the subcellular localization of Mig1, causing it to be nuclear when glucose is present, and cytoplasmic when glucose is absent. RESULTS: Here, we report that Msn5, a member of the importin beta family of nuclear transport receptors, is required to export Mig1 from the nucleus when glucose is removed. Mig1 and Msn5 interacted in a yeast two-hybrid assay. Within the portion of Mig1 that regulates its nuclear transport, we found a region that directed its nuclear export. Within this region, two leucine-rich sequences similar to known nuclear export signals were not required for Mig1 export. The corresponding domain of the yeast Kluyveromyces lactis Mig1 conferred glucose-regulated Msn5-dependent protein export from the nucleus in S. cerevisiae. Sequence comparison with S. cerevisiae Mig1 revealed short patches of homology in K. lactis and K. marxianus Mig1 that might be Msn5-interaction domains. These regions overlapped with the serine residues predicted to be Snf1 phosphorylation sites, suggesting that Msn5 and Snf1 recognize similar sequences in Mig1. Altering these serines abolished glucose-dependent phosphorylation of Mig1 and caused it to be a constitutive repressor that was retained in the nucleus. CONCLUSIONS: Mig1 contains a new nuclear export signal that is phosphorylated by Snf1 upon glucose removal, causing it to be recognized by the nuclear exportin Msn5 and carried out of the nucleus into the cytoplasm where it contributes to derepression of glucose-repressed genes.

Amino Acid Sequence↗
PubMed1999

Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

Culture Media↗
PubMed1999

Binding of the glucose-dependent Mig1p repressor to the GAL1 and GAL4 promoters in vivo: regulationby glucose and chromatin structure.

Binding of the MIG1 repressor to the glucose-repressible GAL1 and GAL4 promoters was analyzed in vivo by cyclobutane dimer footprinting in two yeast strains that show different glucose repression responses. Mig1p binding to the two promoters in both strains was glucose-induced. In cells subject to rapid and stringent glucose repression (S288c), long-term Mig1p binding in glucose-grown cells was inhibited by the formation of a competing chromatin structure. Under conditions where glucose repression was only partially effective (gal80 - or low glucose), the chromatin structure did not form and long-term Mig1p binding was observed The same long-term binding of Mig1p was seen in cells of a different strain (W303A) that shows only partial glucose repression of the GAL1 promoter. We conclude from these experiments that Mig1p binding to glucose-repressed promoters is glucose-dependent but transient. We suggest that Mig1p functions at an early step in repression, but is not required to maintain the repressed state.

Base Sequence↗
PubMed1999

Vasoflux, a new anticoagulant with a novel mechanism of action.

BACKGROUND: Heparin and direct thrombin inhibitors, such as hirudin, have limitations in the treatment of acute coronary syndromes. Heparin does not inactivate fibrin-bound thrombin, whereas hirudin fails to block thrombin generation. In contrast, Vasoflux is a novel anticoagulant that inactivates fibrin-bound thrombin and attenuates factor Xa generation. METHODS AND RESULTS: Vasoflux is prepared by depolymerization of heparin, restricting molecular size to between 3000 and 8000 Da, and reducing antithrombin affinity by periodate oxidation. Vasoflux catalyzes fibrin-bound thrombin inactivation by heparin cofactor II (HCII) and inhibits factor IXa activation of factor X independently of antithrombin and HCII. Compared with other anticoagulants in a thrombogenic extracorporeal circuit, Vasoflux maintains filter patency at concentrations that produce an activated clotting time (ACT) of 220 seconds. In contrast, to maintain filter patency, heparin, low-molecular-weight heparin (LMWH), and hirudin require concentrations that produced an ACT of 720, 415, and >1500 seconds, respectively, whereas dermatan sulfate was ineffective at concentrations that produced an ACT of 360 seconds. CONCLUSIONS: Vasoflux is more effective than heparin and LMWH because it inactivates fibrin-bound thrombin and is superior to hirudin and dermatan sulfate because it also blocks factor Xa generation.

Anticoagulants↗
PubMed1999

Systematic analysis of S. cerevisiae chromosome VIII genes.

To begin genome-wide functional analysis, we analysed the consequences of deleting each of the 265 genes of chromosome VIII of Saccharomyces cerevisiae. For 33% of the deletion strains a growth phenotype could be detected: 18% of the genes are essential for growth on complete glucose medium, and 15% grow significantly more slowly than the wild-type strain or exhibit a conditional phenotype when incubated under one of 20 different growth conditions. Two-thirds of the mutants that exhibit conditional phenotypes are pleiotropic; about one-third of the mutants exhibit only one phenotype. We also measured the level of expression directed by the promoter of each gene. About half of the promoters direct detectable transcription in rich glucose medium, and most of these exhibited only low or medium activity. Only 1% of the genes are expressed at about the same level as ACT1. The number of active promoters increased to 76% upon growth on a non-fermentable carbon source, and to 93% in minimal glucose medium. The majority of promoters fluctuated in strength, depending on the medium.

DNA Primers↗
PubMed1999

Pericardial fluid absorption into lymphatic vessels in sheep.

We estimated the volumetric lymphatic clearance rate of pericardial fluid in sheep. In the first group of studies, 125I-human serum albumin (HSA) was injected into the pericardial cavity and after 4 h, various lymph nodes and tissues were excised and counted for radioactivity. Several lymphatic drainage pathways existed defined by elevated 125I-HSA in the middle and caudal mediastinal, intercostal, and the cardiac nodes located near the root of the aorta. In a second group of experiments, the plasma recovery of intrapericardially administered tracer was compared in sheep with intact lymphatics and in animals in which thoracic duct lymph was diverted and other relevant lymphatics ligated. The 4-h plasma recoveries were reduced significantly from an average of 12.2 +/- 3. 4% injected dose in the lymph-intact group to 3.0 +/- 1.1% injected dose in the diverted/ligated group (an inhibition of approximately 75%). In order to estimate the volumetric clearance of pericardial fluid through lymphatics in conscious sheep, 125I-HSA was administered into the pericardial cavity to serve as the lymph flow marker. 131I-HSA was injected intravenously to permit calculation of plasma tracer loss and tracer recirculation into lymphatics. From mass balance equations, total pericardial clearance into lymphatics averaged 1.50 +/- 0.43 ml/h or approximately 1.13 ml/h if one was to assume that the average 25% recovered plasma tracer in lymph diverted/ligated animals was due to nonlymphatic transport. In conclusion, mediastinal lymphatic pathways remove a volume equivalent to the pericardial volume (8.1 +/- 1.1 ml) every 5.4 to 7. 2 h.

Absorption↗
PubMed1999

Feasting, fasting and fermenting. Glucose sensing in yeast and other cells.

Glucose is the primary fuel for most cells. Because the amount of available glucose can fluctuate wildly, organisms must sense the amount available to them and respond appropriately. Altering gene expression is one of the major effects glucose has on cells. Two different glucose sensing and signal transduction pathways in the yeast S. cerevisiae--one for repression, and one for induction of gene expression--have recently come into focus. What we have learned about these glucose sensing and signaling mechanisms might shed light on how other cells sense and respond to glucose.

Adenosine Triphosphate↗
PubMed1999

Extracranial drainage of cerebrospinal fluid: a study of beta-transferrins in nasal and lymphatic tissues.

OBJECTIVE: To determine whether an alternative route of cerebrospinal fluid (CSF) drainage exists through the nasal mucosa and the cervical lymphatic system. STUDY DESIGN: A prospective study was carried out on 18 patients at a university teaching hospital. METHODS: Ten patients undergoing routine endoscopic sinus surgeries and eight patients undergoing neck dissections were recruited for this study. Tissues were sampled from the middle turbinate, nasopharynx, and upper septum in the first group; jugulodigastric lymph nodes and nasopharyngeal tissues were obtained from the second group. Specimens were subjected to immunofixation electrophoresis in an attempt to identify the presence of beta-1 and beta-2 transferrins. Serum samples were obtained from each subject to serve as controls. RESULTS: All tissue specimens contained beta-1 transferrin; none showed evidence of beta-2 transferrin. CONCLUSION: Using this technique, an alternate route of CSF drainage through the nose and the cervical lymphatic system could not be confirmed. Nevertheless, a new technique of performing immunofixation in solid tissues for the purpose of beta-transferrin identification is described.

Cerebrospinal Fluid Rhinorrhea↗
PubMed1999

Function and regulation of yeast hexose transporters.

Glucose, the most abundant monosaccharide in nature, is the principal carbon and energy source for nearly all cells. The first, and rate-limiting, step of glucose metabolism is its transport across the plasma membrane. In cells of many organisms glucose ensures its own efficient metabolism by serving as an environmental stimulus that regulates the quantity, types, and activity of glucose transporters, both at the transcriptional and posttranslational levels. This is most apparent in the baker's yeast Saccharomyces cerevisiae, which has 20 genes encoding known or likely glucose transporters, each of which is known or likely to have a different affinity for glucose. The expression and function of most of these HXT genes is regulated by different levels of glucose. This review focuses on the mechanisms S. cerevisiae and a few other fungal species utilize for sensing the level of glucose and transmitting this information to the nucleus to alter HXT gene expression. One mechanism represses transcription of some HXT genes when glucose levels are high and works through the Mig1 transcriptional repressor, whose function is regulated by the Snf1-Snf4 protein kinase and Reg1-Glc7 protein phosphatase. Another pathway induces HXT expression in response to glucose and employs the Rgt1 transcriptional repressor, a ubiquitin ligase protein complex (SCF(Grr1)) that regulates Rgt1 function, and two glucose sensors in the membrane (Snf3 and Rgt2) that bind glucose and generate the intracellular signal to which Rgt1 responds. These two regulatory pathways collaborate with other, less well-understood, pathways to ensure that yeast cells express the glucose transporters best suited for the amount of glucose available.

Gene Expression Regulation, Fungal↗
PubMed1999

On becoming non-judgmental: some difficulties for an ethics of counselling.

The growth in the availability of counselling services has been accompanied by growing concern about the conduct of counsellors, which in turn has led to the expressed need for an ethics of counselling. This paper will argue that there is an inherent tension between this need and the central tenets of one variety of counselling, client-centred counselling. The tension is identifiable on the basis of an inquiry into the nature of moral judgment which results in the recognition of the implicit value base in client-centred counselling. It is only when this value base is made explicit that any adequate ethics of counselling becomes a viable possibility.

Attitude of Health Personnel↗
PubMed1999

Contribution of extracranial lymphatics and arachnoid villi to the clearance of a CSF tracer in the rat.

The objective of this study was to determine the relative roles of arachnoid villi and cervical lymphatics in the clearance of a cerebrospinal fluid (CSF) tracer in rats. 125I-labeled human serum albumin (125I-HSA; 100 micrograms) was injected into one lateral ventricle, and an Evans blue dye-rat protein complex was injected intravenously. Arterial blood was sampled for 3 h. Immediately after this, multiple cervical vessels were ligated in the same animals, and plasma recoveries were monitored for a further 3 h after the intracerebroventricular injection of 100 micrograms 131I-HSA. Tracer recovery in plasma at 3 h averaged (%injected dose) 0.697 +/- 0.042 before lymphatic ligation and dropped significantly to 0.357 +/- 0. 060 after ligation. Estimates of the rate constant associated with the transport of the CSF tracer to plasma were also significantly lower after obstruction of cervical lymphatics (from 0.584 +/- 0. 072/h to 0.217 +/- 0.056/h). No significant changes were observed in sham-operated animals. Assuming that the movement of the CSF tracer to plasma in lymph-ligated animals was a result of arachnoid villi clearance, we conclude that arachnoid villi and extracranial lymphatic pathways contributed equally to the clearance of the CSF tracer from the cranial vault.

Animals↗
PubMed1999

Relationship between intracranial pressure and cervical lymphatic pressure and flow rates in sheep.

Previous reports from our group demonstrated that about one-half of the total volume of cerebrospinal fluid (CSF) removed from the cranial vault in sheep is transported into extracranial lymphatics, especially cervical lymphatic vessels in the neck. In this study, we tested the hypothesis that an elevation of intracranial pressure (ICP) would increase cervical lymphatic pressure and lymph flow rates in anesthetized sheep. Catheters were inserted into both lateral ventricles, the cisterna magna, cervical lymphatics, and the jugular vein. A ventriculo-cisternal perfusion system was employed to regulate ICP. Mean (P = 0.008), peak (P = 0.007), and baseline (P = 0.013) cervical lymphatic pressures increased as ICP was elevated from 10 to 70 cmH2O in 20-cmH2O increments. Similarly, cervical lymph flow rates increased (P < 0.001), with flows at 70 cmH2O ICP observed to be approximately fourfold higher than those at 10 cmH2O ICP. No changes were observed in mesenteric lymph flow rates (vessels not expected to drain CSF). We conclude that cervical lymphatic vessels play an important role in the transport of CSF from the cranial vault when ICP is elevated.

Animals↗
PubMed1999

The management of syncope in a British emergency department compared to recent American guidelines.

This descriptive study aims to determine current clinical practice in an emergency department when assessing patients presenting with syncope and then to compare this with recent guidelines from the American College of Physicians. Key points on history, examination, investigation and disposition of patients from the department were measured. Of 100 patients recruited, few had relevant symptoms documented. Seventy-five patients had an electrocardiogram performed, in which 18 were defined as abnormal. 40% of those with a history of organic heart disease were sent home. 28% of those with an abnormal electrocardiogram were discharged. 44% of those aged over 70 were discharged. A total of 45 patients were admitted. An extra 17 to 26 patients may have been admitted if the American guidelines had been adhered to. This study highlights deficiencies in assessment and a difference in current practice when compared with the recommendations from the American College of Physicians. Further research is required to produce guidelines on disposition applicable to emergency departments in the United Kingdom.

Emergency Medical Services↗
PubMed1999

[Resection of lung metastases: long-term results and prognostic analysis based on 5206 cases--the International Registry of Lung Metastases].

The International Registry of Lung Metastases was established in 1991 to asses the long-term results of pulmonary metastasectomy. The Registry has accrued 5206 cases of lung metastasectomy, from 18 departments of thoracic surgery in Europe (n = 13), USA (n = 4) and Canada (n = 1). Of these patients 4572 (88%) underwent complete surgical resection. The primary tumor was epithelial in 2260 (43%), sarcoma in 2173 (42%), germ cell in 363 (7%), and melanoma in 328 (6%) patients. The disease-free interval was 0 to 11 months in 1729 (33%) cases, 12 to 35 months in 1857 (36%) and more than 36 months in 1620 (31%). Single metastases accounted for 2383 (46%) cases and multiple lesions for 2726 (52%). Mean follow up was 46 months. Analysis was performed by Kaplan-Meier estimates of survival, relative risk of death and multivariate Cox model. The actuarial survival after complete metastasectomy was 36% at 5 years, 26% at 10 years and 22% at 15 years (median 35 months); the corresponding values for incomplete resection were 13% at 5 years and 7% at 10 years (median 15 months). Among complete resections, the 5-year survival was 33% for patients with a disease free-interval of 0 to 11 months and 45% for those with a disease-free interval of more than 36 months; 43% for single lesions and 27 for four or more lesions. Multivariate analysis showed a better prognosis for patients with germ cell tumors, disease-free interval of 36 months and more and single metastases. These results confirm that lung metastasectomy is a safe and potentially curative procedure.

Adolescent↗
PubMed1999

The influence of developmental haemostasis on the laboratory diagnosis and management of haemostatic disorders during infancy and childhood.

The physiology of the hemostatic system in children is different from that in adults. Reference ranges for components of the hemostatic system are age dependent. During early childhood, physiologic values may overlap with values observed in congenital deficiencies of acquired pathological conditions. Physiologically decreased plasma concentrations of hemostatic components also influence the response to antithrombotic therapy. The relevance of developmental hemostasis for the laboratory diagnosis and management of hemorrhagic and thrombotic disorders during childhood is discussed.

Adolescent↗
PubMed1999

Multicenter evaluation of lyophilized and deep-frozen plasmas for assignment of the International Normalized Ratio.

The interlaboratory variation of the International Normalized Ratio (INR) in various external quality assessment schemes is still relatively high. This is partly caused by inaccuracy of manufacturers' stated International Sensitivity Index (ISI) and/or local instrumentation effects. The interlaboratory variation and accuracy of INR determinations may be improved by a local calibration procedure based on lyophilized plasmas with assigned INRs. The purpose of the present study was to determine INR values for different types of lyophilized plasmas to be used for local calibration. A total of 13 lyophilized plasmas (one normal, six from coumarin-treated patients, six artificially depleted) were analyzed by 10 laboratories, each using five calibrated prothrombin time (PT) systems. INRs were calculated for each plasma using each laboratory's specific ISI and mean normal prothrombin time values. In the same way, five deep-frozen pooled plasmas from coumarin-treated patients were analyzed. There were significant INR differences for the lyophilized plasmas between the prothrombin time systems. The differences were relatively small for the deep-frozen coumarin plasmas (CV 2.6-3.3%) and three lyophilized coumarin plasmas from one manufacturer (CV 3.7-4.8%). Important INR differences were observed for three lyophilized coumarin plasmas from another manufacturer (CV 9.5-14.1%) and several artificially depleted plasmas (CV 5.3-12.8%). The citrate concentrations in the artificially depleted plasmas were lower than those in the normal and coumarin plasmas. These differences should be considered in the selection and certification of plasmas as calibrants for local calibration of PT systems. The lyophilized plasmas' INR values obtained in the present study will be used for a field study of local PT calibration to assess their efficacy.

Animals↗
PubMed1999

Influence of three types of automated coagulometers on the international sensitivity index (ISI) of rabbit, human, and recombinant human tissue factor preparations--a multicenter study.

Five tissue factor reagents and three types of automated instruments for prothrombin time (PT) determination were studied in an international multicenter collaborative exercise. The purpose of this work was to determine the international sensitivity index (ISI) for each combination of reagent and instrument against the international reference preparation RBT/90. Each type of instrument was used by 3 or 4 centers to assess the interlaboratory variation of the ISI. The interlaboratory variation of the ISI for each combination of reagent and instrument ranged between 0.4% and 7.8% coefficient of variation. For three reagents, the mean ISI values for ACL (nephelometric) and STA (mechanical) were practically identical, but the mean ISI values for MLA (photo-optical) were at least 10% higher. For two other reagents prepared from rabbit tissue, the mean ISI values increased in the order ACL, STA, MLA. The widest range of mean ISI values was noted with one rabbit tissue factor reagent: 1.68 for ACL and 2.00 for MLA. Exclusion of patient specimens with INR <1.5 and INR >4.5 determined by the international reference preparation resulted in a slight decrease of the mean ISI. The interlaboratory variation of the International Normalized Ratio (INR) was assessed from the results obtained with common lyophilized and deep-frozen plasmas. The use of instrument-specific ISI values resulted in reduced interlaboratory variation of the INR. It is recommended that thromboplastin manufacturers provide instrument-specific ISI values.

Animals↗