Biomedical subjects
Publications and source records attributed to M C Walsh.
Persistent pulmonary hypertension of the newborn is a common disorder among near-term gestation newborns. Persistent pulmonary hypertension of the newborn is characterized by hypoxemia that is frequently refractory to conventional management. This article describes the pathophysiologic basis of the disorder and the current therapy that is based on this knowledge.
A large proportion of the 6,000 genes present in the genome of Saccharomyces cerevisiae, and of those sequenced in other organisms, encode proteins of unknown function. Many of these genes are "silent, " that is, they show no overt phenotype, in terms of growth rate or other fluxes, when they are deleted from the genome. We demonstrate how the intracellular concentrations of metabolites can reveal phenotypes for proteins active in metabolic regulation. Quantification of the change of several metabolite concentrations relative to the concentration change of one selected metabolite can reveal the site of action, in the metabolic network, of a silent gene. In the same way, comprehensive analyses of metabolite concentrations in mutants, providing "metabolic snapshots," can reveal functions when snapshots from strains deleted for unstudied genes are compared to those deleted for known genes. This approach to functional analysis, using comparative metabolomics, we call FANCY-an abbreviation for functional analysis by co-responses in yeast.
Lipoteichoic acid (LTA) is associated with the cell envelope of most gram-positive bacteria. Although previously thought to act mainly as a virulence factor by virtue of its adhesive nature, evidence is now provided that LTA can also suppress the function of interleukin-2 (IL-2), an autocrine growth factor for T cells. LTA from four separate bacterial strains lowered the levels of detectable IL-2 during a peripheral blood mononuclear cell response to the antigen tetanus toxoid (TT). T-cell proliferation in response to TT was similarly inhibited by LTA. In contrast, levels of detectable gamma interferon increased. In addition, LTA inhibited IL-2 detection by enzyme-linked immunosorbent assay (ELISA) and blocked the proliferative response of an IL-2-dependent T-cell line to soluble IL-2. Further studies using ELISA demonstrated that LTA blocks IL-2 detection and function by binding directly to IL-2. Flow cytometric analysis revealed that IL-2 binding to T cells is inhibited in the presence of purified LTA but not LTA plus anti-LTA monoclonal antibody. In summary, these studies demonstrate a novel effect of LTA on the immune response through direct binding to IL-2 and inhibition of IL-2 function. Importantly, gram-positive organisms from which LTA is obtained not only play an important role in the pathology of diseases such as bacterial endocarditis, septic shock, acute respiratory distress syndrome, and multiple organ failure but also comprise a significant portion of commensal populations within the human host. Inhibition of IL-2 function by LTA may represent yet another mechanism by which gram-positive bacteria dampen the host immune response and facilitate survival. Thus, LTA provides a potential target for therapeutic intervention when gram-positive organisms are involved.
Primary T cell proliferative responses to TCR ligation plus CD28 costimulation are surprisingly heterogeneous. Many cells that enter G1 fail to progress further through the cell cycle, and some of these cells subsequently fail to divide upon restimulation, even in the presence of IL-2. Such IL-2-refractory anergy is distinct from the IL-2-reversible anergy induced by TCR occupancy in the absence of CD28 costimulation. Here, we focus on the contributions of cell cycle progression and costimulatory (CD28/CTLA-4) signals in the regulation of anergy. We show that CD28 costimulation is not sufficient for anergy avoidance and that activated T cells must progress through the cell cycle in order to escape anergy. Induction of this "division-arrest" form of anergy requires CTLA-4 signaling during the primary response. Also, cell division per se is not sufficient for anergy avoidance: the few T cells that undergo multiple rounds of cell division during overt CD28 costimulatory blockade do not escape the ultimate induction of clonal anergy. Anergy avoidance by primary T cells is thus a multistep process: in order to participate in a productive immune response, an individual T cell activated through its antigen receptor must receive CD28 costimulation and progress through the cell cycle. Anergy may be induced either through a combination of CTLA-4 signaling and the failure of cell cycle progression, or through a proliferation-independent mechanism in which TCR ligation occurs in the absence of CD28.
We have shown previously that T cells activated by optimal TCR and CD28 ligation exhibit marked proliferative heterogeneity, and approximately 40% of these activated cells fail entirely to participate in clonal expansion. To address how prior cell division influences the subsequent function of primary T cells at the single cell level, primary CD4+ T cells were subjected to polyclonal stimulation, sorted based on the number of cell divisions they had undergone, and restimulated by ligation of TCR/CD28. We find that individual CD4+ T cells exhibit distinct secondary response patterns that depend upon their prior division history, such that cells that undergo more rounds of division show incrementally greater IL-2 production and proliferation in response to restimulation. CD4+ T cells that fail to divide after activation exist in a profoundly hyporesponsive state that is refractory to both TCR/CD28-mediated and IL-2R-mediated proliferative signals. We find that this anergic state is associated with defects in both TCR-coupled activation of the p42/44 mitogen-activated protein kinase (extracellular signal-related kinase 1/2) and IL-2-mediated down-regulation of the cell cycle inhibitor p27kip1. However, these defects are selective, as TCR-mediated intracellular calcium flux and IL-2R-coupled STAT5 activation remain intact in these cells. Therefore, the process of cell division or cell cycle progression plays an integral role in anergy avoidance in primary T cells, and may represent a driving force in the formation of the effector/memory T cell pool.
An overview is presented of the problems and challenges facing the analyst who is asked to provide data to support the law. Factors that the analyst should consider include: the legal reasons for performing the analysis; situations where analytical data should and should not be used in support of litigation; the scientific basis for the legislation; and the sample source and method of sampling.
This paper examines whether the in vivo behavior of yeast glycolysis can be understood in terms of the in vitro kinetic properties of the constituent enzymes. In nongrowing, anaerobic, compressed Saccharomyces cerevisiae the values of the kinetic parameters of most glycolytic enzymes were determined. For the other enzymes appropriate literature values were collected. By inserting these values into a kinetic model for glycolysis, fluxes and metabolites were calculated. Under the same conditions fluxes and metabolite levels were measured. In our first model, branch reactions were ignored. This model failed to reach the stable steady state that was observed in the experimental flux measurements. Introduction of branches towards trehalose, glycogen, glycerol and succinate did allow such a steady state. The predictions of this branched model were compared with the empirical behavior. Half of the enzymes matched their predicted flux in vivo within a factor of 2. For the other enzymes it was calculated what deviation between in vivo and in vitro kinetic characteristics could explain the discrepancy between in vitro rate and in vivo flux.
The rate of glucose transport across the plasma membrane of the bloodstream form of Trypanosoma brucei was modulated by titration of the hexose transporter with the inhibitor phloretin, and the effect on the glycolytic flux was measured. A rapid glucose uptake assay was developed to measure the transport activity independently of the glycolytic flux. Phloretin proved a competitive inhibitor. When the effect of the intracellular glucose concentration on the inhibition was taken into account, the flux control coefficient of the glucose transporter was between 0.3 and 0.5 at 5 mM glucose. Because the flux control coefficients of all steps in a metabolic pathway sum to 1, this result proves that glucose transport is not the rate-limiting step of trypanosome glycolysis. Under physiological conditions, transport shares the control with other steps. At glucose concentrations much lower than physiological, the glucose carrier assumed all control, in close agreement with model predictions.
The mechanisms of allograft tolerance have been classified as deletion, anergy, ignorance and suppression/regulation. Deletion has been implicated in central tolerance, whereas peripheral tolerance has generally been ascribed to clonal anergy and/or active immunoregulatory states. Here, we used two distinct systems to assess the requirement for T-cell deletion in peripheral tolerance induction. In mice transgenic for Bcl-xL, T cells were resistant to passive cell death through cytokine withdrawal, whereas T cells from interleukin-2-deficient mice did not undergo activation-induced cell death. Using either agents that block co-stimulatory pathways or the immunosuppressive drug rapamycin, which we have shown here blocks the proliferative component of interleukin-2 signaling but does not inhibit priming for activation-induced cell death, we found that mice with defective passive or active T-cell apoptotic pathways were resistant to induction of transplantation tolerance. Thus, deletion of activated T cells through activation-induced cell death or growth factor withdrawal seems necessary to achieve peripheral tolerance across major histocompatibility complex barriers.
A glucose-sensing mechanism has been described in Saccharomyces cerevisiae that regulates expression of glucose transporter genes. The sensor proteins Snf3 and Rgt2 are homologous to the transporters they regulate. Snf3 and Rgt2 are integral plasma membrane proteins with unique carboxy-terminal domains that are predicted to be localized in the cytoplasm. In a recent paper Ozcan and colleagues [Ozcan S, et al. EMBO J 1998; 17:2556-2773 (Ref. 1)] present evidence that the cytoplasmic domains of Snf3 and Rgt2 are required to transmit a glucose signal. They provide additional evidence to support their earlier assertion [Ozcan S, et al. Proc Natl Acad Sci USA 1996;93:12428-12432 (Ref. 2)] that glucose transport via Snf3 and Rgt2 is not involved in glucose sensing but, rather, that these proteins behave like glucose receptors. Other examples of transporter homologs with regulatory functions have recently been described in fungi as well [Madi L, et al. Genetics 1997; 146:499-508 (Ref. 3). and Didion T, et al. Mol Microbiol 1998;27:643-650 (Ref. 4)]. The identification of this class of nutrient sensors is an important step in elucidating the complex of regulatory mechanisms that leads to adaptation of fungi to different environments.
Many catabolic pathways begin with an ATP-requiring activation step, after which further metabolism yields a surplus of ATP. Such a 'turbo' principle is useful but also contains an inherent risk. This is illustrated by a detailed kinetic analysis of a paradoxical Saccharomyces cerevisiae mutant; the mutant fails to grow on glucose because of overactive initial enzymes of glycolysis, but is defective only in an enzyme (trehalose 6-phosphate synthase) that appears to have little relevance to glycolysis. The ubiquity of pathways that possess an initial activation step, suggests that there might be many more genes that, when deleted, cause rather paradoxical regulation phenotypes (i.e. growth defects caused by enhanced utilization of growth substrate).
In Saccharomyces cerevisiae cells exhibiting high-affinity glucose transport, the glucose consumption rate at extracellular concentrations above 10 mM was only half of the zero trans-influx rate. To determine if this regulation of glucose transport might be a consequence of intracellular free glucose we developed a new method to measure intracellular glucose concentrations in cells metabolizing glucose, which compares glucose stereoisomers to correct for adhering glucose. The intracellular glucose concentration was 1.5 mM, much higher than in most earlier reports. We show that for the simplest model of a glucose carrier, this concentration is sufficient to reduce the glucose influx by 50%. We conclude that intracellular glucose is the most likely candidate for the observed regulation of glucose import and hence glycolysis. We discuss the possibility that intracellular glucose functions as a primary signal molecule in these cells.
Glucose uptake in Saccharomyces cerevisiae is believed to consist of two kinetically distinguishable components, the affinity of which is modulated during growth on glucose. It has been reported that triple hexose-kinase deletion mutants do not exhibit high-affinity glucose uptake. This raises the question of whether and how high-affinity glucose uptake is related to the presence of glucose-phosphorylating enzymes. In this study the kinetics of glucose uptake in both wild-type cells and cells of hexose-kinase deletion mutants, grown on either glycerol or galactose, were determined using a rapid-uptake method. In wild-type cells glucose uptake measured over either 5 s or 200 ms exhibited high affinity. In contrast, in cells of hexose-kinase deletion mutants the apparent affinity of glucose uptake was dependent on the time scale during which uptake was measured. Measurements on the 5-s scale showed apparent low-affinity uptake whereas measurements on the 200-ms scale showed high-affinity uptake. The affinity and maximal rate of the latter were comparable to those in wild-type cells. Using a simple model for a symmetrical facilitator, it was possible to simulate the experimentally determined relation between apparent affinity and the time scale used. The results suggest that high-affinity glucose transport is not necessarily dependent on the presence of glucose-phosphorylating enzymes. Apparent low-affinity uptake kinetics can arise as a consequence of an insufficient rate of removal of intracellular free glucose by phosphorylation. This study underlines the need to differentiate between influences of the translocator and of metabolism on the apparent kinetics of sugar uptake in yeast.
The kinetics of glucose transport in a number of different mutants of Saccharomyces cerevisiae with multiple deletions in the glucose transporter gene family were determined. The deletions led to differences in maximal rate and affinity for glucose uptake by the cells, dependent on the growth conditions. At the same time, there were changes in glucose repression, as determined by expression of invertase activity. Only in the strain with genes HXT1-4 and SNF3 deleted but carrying HXT6/7 were glucose uptake kinetics and invertase activity independent of the presence or concentration of glucose in the growth medium. Some degree of glucose sensitivity was recovered if the SNF3 or HXT2 gene was present in the multiple-deletion background. It is hypothesized that during growth on glucose, both modulation of the kinetics of glucose uptake and derepression of invertase activity require the presence of more than one active gene of the glucose transporter family.
The Escherichia coli fabH gene encoding 3-ketoacyl-acyl carrier protein synthase III (KAS III) was isolated and the effect of overproduction of bacterial KAS III was compared in both E. coli and Brassica napus. The change in fatty acid profile of E. coli was essentially the same as that reported by Tsay et al. (J Biol Chem 267 (1992) 6807-6814), namely higher C14:0 and lower C18:1 levels. In our study, however, an arrest of cell growth was also observed. This and other evidence suggests that in E. coli the accumulation of C14:0 may not be a direct effect of the KAS III overexpression, but a general metabolic consequence of the arrest of cell division. Bacterial KAS III was expressed in a seed- and developmentally specific manner in B. napus in either cytoplasm or plastid. Significant increases in KAS III activities were observed in both these transformation groups, up to 3.7 times the endogenous KAS III activity in mature seeds. Only the expression of the plastid-targeted KAS III gene, however, affected the fatty acid profile of the storage lipids, such that decreased amounts of C18:1 and increased amounts of C18:2 and C18:3 were observed as compared to control plants. Such changes in fatty acid composition reflect changes in the regulation and control of fatty acid biosynthesis. We propose that fatty acid biosynthesis is not controlled by one rate-limiting enzyme, such as acetyl-CoA carboxylase, but rather is shared by a number of component enzymes of the fatty acid biosynthetic machinery.
Incubation of starved galactose-grown S. cerevisiae cells with cyanide reduced glucose uptake as measured over a 5-s period. The Vmax for glucose uptake was decreased by over a factor of two but the apparent affinity for glucose doubled. When measured in the sub-second time scale, however, there was no significant inhibition of glucose uptake, by cyanide, up to 200-ms, clearly demonstrating that, in cyanide treated cells, glucose uptake was not linear for the first 5-s. After a 200-ms exposure of untreated cells to radio-labelled glucose, less than 10% of the intracellular label resided in soluble uncharged compounds. In cyanide-treated cells up to 43% of the labelled compounds were uncharged, with a concurrent reduction of intracellular label residing in anionic compounds. The results suggest that, in the presence of 10 mM cyanide when respiration is inhibited, a reduction in the cellular ATP concentration causes a reduction in hexose-kinase activity which results in an accumulation of internal free glucose, which in turn causes a reduction in net glucose transport.
In a multiple deletion mutant hxt1deltahxt2deltahxt3delta hxt4deltasnf3delta of Saccharomyces cerevisiae growing on a 2% glucose, high-affinity glucose-uptake (low Km) was exhibited throughout growth on glucose in contrast to the wild-type, which exhibited the usual low-affinity to high-affinity transition as the glucose in the medium was consumed. Elevated levels of invertase activity throughout growth on glucose, in this mutant as compared to the wild-type, indicate that glucose repression may be impaired. However, in a mutant containing only the HXT2 gene (hxt1deltahxt3deltahxt4deltasnf3delta), invertase levels were similar to those in the wild-type. It is likely, therefore, that some of these putative glucose transporters, such as HXT2, also have regulatory roles in cellular metabolism. In triple hexose-kinase mutants, rapid (200-ms) measurements of initial glucose-uptake revealed high-affinity glucose uptake (Km approx. 2 mmol/L) while measurements on the slower 5-s scale clearly demonstrate that uptake is not linear over this longer period. These results suggest that this high-affinity component does not require a functional hexose-kinase.
By using a modified technique to measure glucose uptake in Saccharomyces cerevisiae, potential uncertainties have been identified in previous determinations. These previous determinations had led to the proposal that S. cerevisiae contained a constitutive low-affinity glucose transporter and a glucose-repressible high-affinity transporter. We show that, upon transition from glucose-repressed to -derepressed conditions, the maximum rate of glucose transport is constant and only the affinity for glucose changes. We conclude that the transporter or group of transporters is constitutive and that regulation of glucose transport occurs via a factor that modifies the affinity of the transporters and not via the synthesis of different kinetically independent transporters. Such a mechanism could, for instance, be accommodated by the binding of kinases causing a change in affinity for glucose.