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Biomedical subjects

A Hall

Publications and source records attributed to A Hall.

At least 37 records · Page 2Linked to original sources
PubMed2001

Comparison of PCR to histology for the diagnosis of invasive candidiasis in a murine model.

The ability of a PCR method to diagnose invasive candidiasis in comparison to histology was determined using a murine model. One hundred and sixty mice were divided into 16 groups of 10 mice each. One group served as a negative control while 15 groups were infected with Candida albicans. Of the infected groups, 14 (A1-A7 and B1-B7) received fluconazole treatment. Surviving animals of group A1 were killed on day 3, and animals of successive groups were sacrificed every other day thereafter. Fluconazole treatment was stopped in the corresponding B group. All surviving mice were killed on day 43. One hundred and thirty-eight mice were evaluable. Yeast were seen in the kidney specimens of 77 mice; 66/77 of these, plus an additional 7 histologically-negative mice, were positive by PCR. The sensitivity and specificity of the PCR test compared with histology were 85.71% and 88.52%, respectively. In combination with blood cultures, PCR could be a useful diagnostic tool for invasive candidiasis in clinical settings.

Animals↗
PubMed2001

Circadian clock-regulated expression of phytochrome and cryptochrome genes in Arabidopsis.

Many physiological and biochemical processes in plants exhibit endogenous rhythms with a period of about 24 h. Endogenous oscillators called circadian clocks regulate these rhythms. The circadian clocks are synchronized to the periodic environmental changes (e.g. day/night cycles) by specific stimuli; among these, the most important is the light. Photoreceptors, phytochromes, and cryptochromes are involved in setting the clock by transducing the light signal to the central oscillator. In this work, we analyzed the spatial, temporal, and long-term light-regulated expression patterns of the Arabidopsis phytochrome (PHYA to PHYE) and cryptochrome (CRY1 and CRY2) promoters fused to the luciferase (LUC(+)) reporter gene. The results revealed new details of the tissue-specific expression and light regulation of the PHYC and CRY1 and 2 promoters. More importantly, the data obtained demonstrate that the activities of the promoter::LUC(+) constructs, with the exception of PHYC::LUC(+), display circadian oscillations under constant conditions. In addition, it is shown by measuring the mRNA abundance of PHY and CRY genes under constant light conditions that the circadian control is also maintained at the level of mRNA accumulation. These observations indicate that the plant circadian clock controls the expression of these photoreceptors, revealing the formation of a new regulatory loop that could modulate gating and resetting of the circadian clock.

Arabidopsis↗
PubMed2001

Lipopolysaccharide-induced activation of beta2-integrin function in macrophages requires Irak kinase activity, p38 mitogen- activated protein kinase, and the Rap1 GTPase.

Lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, is a potent activator of macrophages. Besides inducing many transcriptional pathways, LPS also elicits rapid morphological changes such as cell spreading. Here we have investigated the signaling pathway that controls macrophage beta2-integrin-dependent spreading in response to LPS. We show that inhibition of the adapter protein MyD88, the interleukin-1 receptor-associated kinase Irak, the p38 mitogen-activated protein kinase, or the Ras-like GTPase Rap1 blocks LPS-induced spreading. In addition, Irak activates p38 and stimulates p38-dependent spreading. The activation of p38 by Irak requires Irak's kinase activity. We find that p38 controls spreading independently of its role in transcription but rather through activation of Rap1. Together, our results suggest that beta2-integrin-dependent spreading of macrophages in response to LPS is controlled by a linear signaling pathway via MyD88, Irak, p38, and Rap1.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Alternatively spliced isoform of apical Na(+)-K(+)-Cl(-) cotransporter gene encodes a furosemide-sensitive Na(+)-Cl(-)cotransporter.

In the absence of vasopressin, medullary thick ascending limb cells express a K(+)-independent, furosemide-sensitive Na(+)-Cl(-) cotransporter that is inhibited by hypertonicity. The murine renal specific Na(+)-K(+)-2 Cl(-) cotransporter gene (SLC12A1) gives rise to six alternatively spliced isoforms. Three feature a long COOH-terminal domain that encodes the butmetanide-sensitive Na(+)-K(+)-2 Cl(-) cotransporter (BSC1-9/NKCC2), and three with a short COOH-terminal domain, known as mBSC1-A4, B4, or F4 (19). Here we have determined the functional characteristics of mBSC1-A4, as expressed in Xenopus laevis oocytes. When incubated at normal oocyte osmolarity (approximately 200 mosmol/kgH(2)O), mBSC1-4-injected oocytes do not express significant Na(+) uptake over H(2)O-injected controls, and immunohistochemical analysis shows that the majority of mBSC1-4 protein is in the oocyte cytoplasm and not at the plasma membrane. In contrast, when mBSC1-4 oocytes are exposed to hypotonicity (approximately 100 mosmol/kgH(2)O), a significant increase in Na(+) uptake but not in (86)Rb(+) uptake is observed. The increased Na(+) uptake is Cl(-) dependent, furosemide sensitive, and cAMP sensitive but K(+) independent. Sodium uptake increases with decreasing osmolarity between 120 and 70 mosmol/kgH(2)O (r = 0.95, P < 0.01). Immunohistochemical analysis shows that in hypotonic conditions mBSC1-A4 protein is expressed in the plasma membrane. These studies indicate that the mBSC1-A4 isoform of the SLC12A1 gene encodes a hypotonically activated, cAMP- and furosemide-sensitive Na(+)-Cl(-) cotransporter. Thus it is possible that alternative splicing of the BSC1 gene could provide the molecular mechanism enabling the Na(+)-Cl(-)-to-Na(+)-K(+)-2Cl(-) switching in thick ascending limb cells.

1-Methyl-3-isobutylxanthine↗
PubMed2001

Improving understanding of research consent disclosures among persons with mental illness.

OBJECTIVE: The objective of this study was to evaluate alternative procedures for improving the understanding of research consent disclosures by persons who have mental illness. METHODS: Three groups participated in the study: persons with schizophrenia (N=79), persons with depression (N=82), and a healthy control group (N=80). The participants were guided through an informed consent process in which two factors were manipulated. One was the structure of the disclosure form; either a typical disclosure form involving standard dense text was used, or a graphically enhanced form was used. The other was the interpersonal process: the presence or absence of a third-party facilitator, with iterative feedback given to participants for whom a facilitator was not present. Participants' understanding of the disclosure was assessed with the use of recall tests that involved paraphrasing and recognition tests that involved multiple choice. RESULTS: The mean understanding scores did not differ significantly between the depression and control groups, and the mean scores of the schizophrenia group were significantly lower than those of the other two groups. Neither the graphically enhanced consent disclosure form nor the presence of a third-party facilitator was associated with improved understanding. The use of iterative feedback was associated with improvement in comprehension scores in all groups. CONCLUSIONS: The use of a feedback procedure in the consent disclosure process during the recruitment of persons who are mentally ill may be a valuable safeguard for ensuring adequate understanding and appropriate participation in research.

Adult↗
PubMed2001

Analysis of R-Ras signalling pathways.

R-Ras has a high degree of sequence homology to Ras and to other members of the Ras subfamily including Rap, TC21 and M-Ras. Activated versions of Ras and TC21 are highly transforming in a variety of cell lines and mutated forms of both proteins have been found in human tumours. R-Ras interacts with many of the same proteins as Ras and TC21, including c-Raf1, and can induce transformed foci, although this activity is weak compared to Ras and appears to be cell-type specific. Here, we have investigated R-Ras signalling pathways in a variety of cell types. We find that microinjection of activated R-Ras into quiescent fibroblasts stimulates cell cycle progression through G(1) phase and subsequent DNA synthesis. However, unlike Ras, R-Ras does not activate the ERK MAP kinase pathway nor does it activate the JNK or p38/Mpk2 MAP kinase pathways. Microinjection of R-Ras into PC12 cells does not induce terminal differentiation, but instead causes extensive cell spreading, consistent with R-Ras having a role in integrin activation. Finally, in a macrophage cell line, R-Ras activates the alpha(M)beta(2) integrin via the small GTPase Rap1, leading to phagocytosis of opsonized red blood cells, whereas Ras does not. These results indicate that R-Ras has an important role in the regulation of cell growth and adhesion, but that this is mediated through downstream signals distinct from those used by Ras.

3T3 Cells↗
PubMed2001

Conditional circadian regulation of PHYTOCHROME A gene expression.

The phytochrome photoreceptors and the circadian clock control many of the same developmental processes, in all organs and throughout the growth of Arabidopsis plants. Phytochrome A (phyA) provides light input signals to entrain the circadian clock. The clock is known to rhythmically regulate its light input pathway, so we tested rhythmic regulation of phyA, using transgenic plants carrying a PHYA promoter fusion to the luciferase reporter (PHYA:LUC). We provide the first images of LUC activity with subcellular resolution in intact tissue. PHYA transcription and the accumulation of all three PHYA mRNAs were indeed clock controlled. PHYA is expressed throughout the seedling, so we tested whether circadian rhythms were observed in all PHYA-expressing organs and whether the rhythms were autonomously controlled by each organ. In contrast to our previous results using other clock controlled genes, the rhythmic pattern of PHYA expression varied markedly among isolated organs and between isolated organs and intact plants. High-amplitude rhythms were maintained for many days in isolated leaves in darkness, whereas the leaves of intact plants rapidly lost rhythmicity. Wounding the leaves of intact plants had no effect. The rhythmic pattern of PHYA expression is not organ autonomous but depends upon the physical continuity or isolation of the rhythmic tissues, consistent with the presence of a transmitted signal that controls the overt expression of circadian rhythms without necessarily affecting the underlying clock. A circadian system might be present in most, if not all, plant cells, but its effect on intracellular rhythms can be controlled by supracellular signaling.

Arabidopsis↗
PubMed2000

The ELF3 zeitnehmer regulates light signalling to the circadian clock.

The circadian system regulates 24-hour biological rhythms and seasonal rhythms, such as flowering. Long-day flowering plants like Arabidopsis thaliana, measure day length with a rhythm that is not reset at lights-off, whereas short-day plants measure night length on the basis of circadian rhythm of light sensitivity that is set from dusk, early flowering 3 (elf3) mutants of Arabidopsis are aphotoperiodic and exhibit light-conditional arrhythmias. Here we show that the elf3-7 mutant retains oscillator function in the light but blunts circadian gating of CAB gene activation, indicating that deregulated phototransduction may mask rhythmicity. Furthermore, elf3 mutations confer the resetting pattern of short-day photoperiodism, indicating that gating of phototransduction may control resetting. Temperature entrainment can bypass the requirement for normal ELF3 function for the oscillator and partially restore rhythmic CAB expression. Therefore, ELF3 specifically affects light input to the oscillator, similar to its function in gating CAB activation, allowing oscillator progression past a light-sensitive phase in the subjective evening. ELF3 provides experimental demonstration of the zeitnehmer ('time-taker') concept.

Arabidopsis↗
PubMed2000

Rac/Cdc42 and p65PAK regulate the microtubule-destabilizing protein stathmin through phosphorylation at serine 16.

We have identified a rapid protein phosphorylation event at residue serine 16 of stathmin using two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption/ionization mass spectrometry in combination with post-source decay analysis, which is induced by the epidermal growth factor receptor. Phosphorylation is specifically mediated by the small GTPases Rac and Cdc42 and their common downstream target, the serine/threonine kinase p65PAK. Both GTPases have previously been shown to regulate the dynamics of actin polymerization. Because stathmin destabilizes microtubules, and this process is inhibited by phosphorylation at residue 16, Rac and Cdc42 can potentially regulate both F-actin and microtubule dynamics.

Cell Line↗
PubMed2000

The health of enrolled and non enrolled children of school age in Tanga, Tanzania.

The health of 227 children enrolled at primary school was compared with that of 214 non enrolled children living in rural Tanga, Tanzania. No consistent difference was observed with respect to prevalence and intensity of parasitic infection (hookworm, T. trichiura, A. lumbricoides, S. haematobium and P. falciparum). Since enrolled children were as commonly and as heavily infected as non enrolled children, treatment of enrolled children would be effective in reducing transmission throughout the total population. Non enrolled children were more stunted (P=0.0001) and wasted (P=0.0001) than enrolled children and also tended to be more anaemic (P=0.080) showing that poor nutrition is not only associated with delayed enrolment but continues to be associated with non enrolment throughout the school age years. Given that treatment has the greatest impact on the most malnourished children, additional measures to extend treatment to non enrolled children would be justified.

Animals↗
PubMed2000

Functional independence of circadian clocks that regulate plant gene expression.

BACKGROUND: Circadian clocks regulate the gene expression, metabolism and behaviour of most eukaryotes, controlling an orderly succession of physiological processes that are synchronised with the environmental day/night cycle. Central circadian pacemakers that control animal behaviour are located in the brains of insects and rodents, but the location of such a pacemaker has not been determined in plants. Peripheral plant and animal tissues also maintain circadian rhythms when isolated in culture, indicating that these tissues contain circadian clocks. The degree of autonomy that the multiple, peripheral circadian clocks have in the intact organism is unclear. RESULTS: We used the bioluminescent luciferase reporter gene to monitor rhythmic expression from three promoters in transgenic Arabidopsis and tobacco plants. The rhythmic expression of a single gene could be set at up to three phases in different anatomical locations of a single plant, by applying light/dark treatments to restricted tissue areas. The initial phases were stably maintained after the entraining treatments ended, indicating that the circadian oscillators in intact plants are autonomous. This result held for all the vegetative plant organs and for promoters expressed in all major cell types. The rhythms of one organ were unaffected by entrainment of the rest of the plant, indicating that phase-resetting signals are also autonomous. CONCLUSIONS: Higher plants contain a spatial array of autonomous circadian clocks that regulate gene expression without a localised pacemaker. Circadian timing in plants might be less accurate but more flexible than the vertebrate circadian system.

Arabidopsis↗
PubMed2000

The GTPase Rap1 controls functional activation of macrophage integrin alphaMbeta2 by LPS and other inflammatory mediators.

BACKGROUND: beta2 integrins mediate many aspects of the inflammatory and immune responses, including adhesion of leukocytes to the endothelium, complement-mediated phagocytosis in macrophages and neutrophils, and antigen-specific conjugate formation between cytotoxic T cells and their targets. A variety of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha), platelet-activating factor (PAF), and lipopolysaccharide (LPS) and other bacterial products induce the functional activation of beta2 integrins, but the signaling events that link membrane receptors to integrin activation are poorly understood. RESULTS: We report here that expression of the constitutively active small GTPases Rap1 or R-ras, but not Ras or RalA, is sufficient for functional activation of alphaMbeta2, the complement receptor 3 (CR3), in macrophages, allowing phagocytosis of C3bi-opsonized targets. Inhibition of Rap1, but not other Ras-like or Rho-like small GTPases, abolishes activation of alphaMbeta2 induced by phorbol esters, LPS, TNF-alpha or PAF. Finally, Rap1 activation specifically controls the binding properties of alphaMbeta2 towards its physiological ligand, namely the complement-opsonized phagocytic targets. CONCLUSIONS: In macrophages, the Rap1 GTPase regulates activation of the alphaMbeta2 integrin in response to a wide variety of inflammatory mediators.

Animals↗
PubMed2000

Rho GTPases: molecular switches that control the organization and dynamics of the actin cytoskeleton.

The actin cytoskeleton plays a fundamental role in all eukaryotic cells it is a major determinant of cell morphology and polarity and the assembly and disassembly of filamentous actin structures provides a driving force for dynamic processes such as cell motility, phagocytosis, growth cone guidance and cytokinesis. The ability to reorganize actin filaments is a fundamental property of embryonic cells during development; the shape changes accompanying gastrulation and dorsal closure, for example, are dependent on the plasticity of the actin cytoskeleton, while the ability of cells or cell extensions, such as axons, to migrate within the developing embryo requires rapid and spatially organized changes to the actin cytoskeleton in response to the external environment. Work in mammalian cells over the last decade has demonstrated the central role played by the highly conserved Rho family of small GTPases in signal transduction pathways that link plasma membrane receptors to the organization of the actin cytoskeleton.

Actins↗
PubMed2000

Rho GTPases and their effector proteins.

Rho GTPases are molecular switches that regulate many essential cellular processes, including actin dynamics, gene transcription, cell-cycle progression and cell adhesion. About 30 potential effector proteins have been identified that interact with members of the Rho family, but it is still unclear which of these are responsible for the diverse biological effects of Rho GTPases. This review will discuss how Rho GTPases physically interact with, and regulate the activity of, multiple effector proteins and how specific effector proteins contribute to cellular responses. To date most progress has been made in the cytoskeleton field, and several biochemical links have now been established between GTPases and the assembly of filamentous actin. The main focus of this review will be Rho, Rac and Cdc42, the three best characterized mammalian Rho GTPases, though the genetic analysis of Rho GTPases in lower eukaryotes is making increasingly important contributions to this field.

Amino Acid Sequence↗
PubMed2000

Long-term ACE-inhibitor therapy in patients with heart failure or left-ventricular dysfunction: a systematic overview of data from individual patients. ACE-Inhibitor Myocardial Infarction Collaborative Group.

BACKGROUND: We undertook a prospective systematic overview based on data from individual patients from five long-term randomised trials that assessed inhibitors of angiotensin-converting enzyme (ACE) in patients with left-ventricular dysfunction or heart failure. METHODS: Three of the trials enrolled patients within a week after acute myocardial infarction. Data were combined by use of the Peto-Yusuf method. FINDINGS: Overall 12,763 patients were randomly assigned treatment or placebo and followed up for an average of 35 months. In the three post-infarction trials (n=5,966), mortality was lower with ACE inhibitors than with placebo (702/2995 [23.4%] vs 866/2971 [29.1%]; odds ratio 0.74 [95% CI 0.66-0-83]), as were the rates of readmission for heart failure (355 [11.9%] vs 460 [15.5%]; 0.73 [0.63-0.85]), reinfarction (324 [10.8%] vs 391 [13.2%]; 0.80 [0.69-0.94]), or the composite of these events (1049 [35.0%] vs 1244 [41.9%]; 0.75 [0.67-0.83]; all p<O.001). For all five trials the ACE inhibitor group had lower rates of death than the placebo group (1,467/6,391 [23.0%] vs 1,710/6,372 [26.8%]; 0.80 [0.74-0.87]) and lower rates of reinfarction (571 [8.9%] vs 703 [11.0%]; 0.79 [0.70-0.89]), readmission for heart failure (876 [13.7%] vs 1202 [18.9%]; 0.67 [0.61-0.74]), and the composite of these events (2161 [33.8%] vs 2610 [41.0%]; 0.72 [0.67-0.78]; all p<0.0001). The benefits were observed early after the start of therapy and persisted long term. The benefits of treatment on all outcomes were independent of age, sex, and baseline use of diuretics, aspirin, and beta-blockers. Although there was a trend towards greater reduction in risk of death or readmission for heart failure in patients with lower ejection fractions, benefit was apparent over the range examined.

Aged↗
PubMed2000

Phosphatidylinositol 4,5-bisphosphate induces actin-based movement of raft-enriched vesicles through WASP-Arp2/3.

BACKGROUND: Phosphatidylinositol 4,5-bisphosphate (PIP(2)) has been implicated in the regulation of the actin cytoskeleton and vesicle trafficking. It stimulates de novo actin polymerization by activating the pathway involving the Wiskott-Aldrich syndrome protein (WASP) and the actin-related protein complex Arp2/3. Other studies show that actin polymerizes from cholesterol-sphingolipid-rich membrane microdomains called 'rafts', in a manner dependent on tyrosine phosphorylation. Although actin has been implicated in vesicle trafficking, and rafts are sites of active phosphoinositide and tyrosine kinase signaling that mediate apically directed vesicle trafficking, it is not known whether phosphoinositide regulation of actin dynamics occurs in rafts, or if it is linked to vesicle movements. RESULTS: Overexpression of type I phosphatidylinositol phosphate 5-kinase (PIP5KI), which synthesizes PIP(2), promoted actin polymerization from membrane-bound vesicles to form motile actin comets. Pervanadate (PV), a tyrosine phosphatase inhibitor, induced comets even in the absence of PIP5KI overexpression. PV increased PIP(2) levels, suggesting that it induces comets by changing PIP(2) homeostasis and by increasing tyrosine phosphorylation. Platelet-derived growth factor (PDGF) enhanced PV-induced comet formation, and these stimuli together potentiated the PIP5KI effect. The vesicles at the heads of comets were enriched in PIP5KIs and tyrosine phosphoproteins. WASP-Arp2/3 involvement was established using dominant-negative WASP constructs. Endocytic and exocytic markers identified vesicles enriched in lipid rafts as preferential sites of comet generation. Extraction of cholesterol with methyl-beta-cyclodextrin reduced comets, establishing that rafts promote comet formation. CONCLUSIONS: Sphingolipid-cholesterol rafts are preferred platforms for membrane-linked actin polymerization. This is mediated by in situ PIP(2) synthesis and tyrosine kinase signaling through the WASP-Arp2/3 pathway. Actin comets may provide a novel mechanism for raft-dependent vesicle transport and apical membrane trafficking.

3T3 Cells↗