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Biomedical subjects

L Shapiro

Publications and source records attributed to L Shapiro.

At least 19 recordsLinked to original sources
PubMed2001

The presynaptic particle web: ultrastructure, composition, dissolution, and reconstitution.

We report the purification of a presynaptic "particle web" consisting of approximately 50 nm pyramidally shaped particles interconnected by approximately 100 nm spaced fibrils. This is the "presynaptic grid" described in early EM studies. It is completely soluble above pH 8, but reconstitutes after dialysis against pH 6. Interestingly, reconstituted particles orient and bind PSDs asymmetrically. Mass spectrometry of purified web components reveals major proteins involved in the exocytosis of synaptic vesicles and in membrane retrieval. Our data support the idea that the CNS synaptic junction is organized by transmembrane adhesion molecules interlinked in the synaptic cleft, connected via their intracytoplasmic domains to the presynaptic web on one side and to the postsynaptic density on the other. The CNS synaptic junction may therefore be conceptualized as a complicated macromolecular scaffold that isostatically bridges two closely aligned plasma membranes.

Animals↗
PubMed2001

A moving DNA replication factory in Caulobacter crescentus.

The in vivo intracellular location of components of the Caulobacter replication apparatus was visualized during the cell cycle. Replisome assembly occurs at the chromosomal origin located at the stalked cell pole, coincident with the initiation of DNA replication. The replisome gradually moves to midcell as DNA replication proceeds and disassembles upon completion of DNA replication. Although the newly replicated origin regions of the chromosome are rapidly moved to opposite cell poles by an active process, the replisome appears to be an untethered replication factory that is passively displaced towards the center of the cell by the newly replicated DNA. These results are consistent with a model in which unreplicated DNA is pulled into the replication factory and newly replicated DNA is bidirectionally extruded from the complex, perhaps contributing to chromosome segregation.

Bacterial Proteins↗
PubMed2001

Interleukin-18 inhibits human immunodeficiency virus type 1 production in peripheral blood mononuclear cells.

Interleukin (IL)-18 is an interferon (IFN)-gamma-inducing factor and contributes to the Th1 immune response. IL-18 added after infection of peripheral blood mononuclear cells (PBMC) with monocyte-tropic human immunodeficiency virus type 1 (HIV-1) inhibited p24 antigen production by a maximum of 72%. IFN-gamma levels in these cultures were increased, and a significant inverse relationship between HIV-1 production and IFN-gamma levels was observed. A neutralizing anti-IFN-gamma antibody reversed the IL-18 inhibitory effect. Preincubation of PBMC with IL-18 before infection inhibited p24 without additional IL-18 (64%). However, compared with the degree of IL-18 inhibition observed after a 4-day culture, no additional IL-18 inhibitory effect was observed during days 5-13. IL-18 also reduced cell surface expression of the HIV-1 receptor CD4. These results demonstrate that IL-18 inhibited HIV-1 production in PBMC through intermediate IFN-gamma. Furthermore, inhibition was present during the early stages of viral infection and was associated with reduced HIV-1 receptor expression.

CD4 Antigens↗
PubMed2001

G-protein signaling through tubby proteins.

Dysfunction of the tubby protein results in maturity-onset obesity in mice. Tubby has been implicated as a transcription regulator, but details of the molecular mechanism underlying its function remain unclear. Here we show that tubby functions in signal transduction from heterotrimeric GTP-binding protein (G protein)-coupled receptors. Tubby localizes to the plasma membrane by binding phosphatidylinositol 4,5-bisphosphate through its carboxyl terminal "tubby domain." X-ray crystallography reveals the atomic-level basis of this interaction and implicates tubby domains as phosphorylated-phosphatidyl- inositol binding factors. Receptor-mediated activation of G protein alphaq (Galphaq) releases tubby from the plasma membrane through the action of phospholipase C-beta, triggering translocation of tubby to the cell nucleus. The localization of tubby-like protein 3 (TULP3) is similarly regulated. These data suggest that tubby proteins function as membrane-bound transcription regulators that translocate to the nucleus in response to phosphoinositide hydrolysis, providing a direct link between G-protein signaling and the regulation of gene expression.

Active Transport, Cell Nucleus↗
PubMed2001

Complete genome sequence of Caulobacter crescentus.

The complete genome sequence of Caulobacter crescentus was determined to be 4,016,942 base pairs in a single circular chromosome encoding 3,767 genes. This organism, which grows in a dilute aquatic environment, coordinates the cell division cycle and multiple cell differentiation events. With the annotated genome sequence, a full description of the genetic network that controls bacterial differentiation, cell growth, and cell cycle progression is within reach. Two-component signal transduction proteins are known to play a significant role in cell cycle progression. Genome analysis revealed that the C. crescentus genome encodes a significantly higher number of these signaling proteins (105) than any bacterial genome sequenced thus far. Another regulatory mechanism involved in cell cycle progression is DNA methylation. The occurrence of the recognition sequence for an essential DNA methylating enzyme that is required for cell cycle regulation is severely limited and shows a bias to intergenic regions. The genome contains multiple clusters of genes encoding proteins essential for survival in a nutrient poor habitat. Included are those involved in chemotaxis, outer membrane channel function, degradation of aromatic ring compounds, and the breakdown of plant-derived carbon sources, in addition to many extracytoplasmic function sigma factors, providing the organism with the ability to respond to a wide range of environmental fluctuations. C. crescentus is, to our knowledge, the first free-living alpha-class proteobacterium to be sequenced and will serve as a foundation for exploring the biology of this group of bacteria, which includes the obligate endosymbiont and human pathogen Rickettsia prowazekii, the plant pathogen Agrobacterium tumefaciens, and the bovine and human pathogen Brucella abortus.

Adaptation, Biological↗
PubMed2001

Dynamic localization of a cytoplasmic signal transduction response regulator controls morphogenesis during the Caulobacter cell cycle.

We present evidence that a bacterial signal transduction cascade that couples morphogenesis with cell cycle progression is regulated by dynamic localization of its components. Previous studies have implicated two histidine kinases, DivJ and PleC, and the response regulator, DivK, in the regulation of morphogenesis in the dimorphic bacterium Caulobacter crescentus. Here, we show that the cytoplasmic response regulator, DivK, exhibits a dynamic, cyclical localization that culminates in asymmetric distribution of DivK within the two cell types that are characteristic of the Caulobacter cell cycle; DivK is dispersed throughout the cytoplasm of the progeny swarmer cell and is localized to the pole of the stalked cell. The membrane-bound DivJ and PleC histidine kinases, which are asymmetrically localized at the opposite poles of the predivisional cell, control the temporal and spatial localization of DivK. DivJ mediates DivK targeting to the poles whereas PleC controls its release from one of the poles at times and places that are consistent with the activities and location of DivJ and PleC in the late predivisional cell. Thus, dynamic changes in subcellular location of multiple components of a signal transduction cascade may constitute a novel mode of prokaryotic regulation to generate and maintain cellular asymmetry.

Bacterial Proteins↗
PubMed2001

Alpha-1-antitrypsin inhibits human immunodeficiency virus type 1.

Several observations suggest the existence of potent endogenous suppressors of human immunodeficiency virus type 1 (HIV-1) production, and inhibitors of serine proteases may participate in this effect. Alpha-1-antitrypsin (AAT) is the most abundant circulating serine protease inhibitor. Physiological AAT concentrations inhibited HIV-1 production in chronically infected U1 monocytic cells, reduced virus replication in freshly infected peripheral blood mononuclear cells, and blocked infection of permissive HeLa cells. In U1 cells, AAT suppressed activation of the HIV-1-inducing transcription factor NF-kappaB. Similar results were obtained using CE-2072, a synthetic inhibitor of host serine proteases. HIV-1 did not replicate in blood obtained from healthy volunteers, but marked replication was observed in blood from individuals with hereditary AAT deficiency. These results identify AAT as a candidate circulating HIV-1 inhibitor in vivo. Two different mechanisms of AAT-induced HIV-1 inhibition were identified, including reduced HIV-1 infectivity and blockade of HIV-1 production. A novel host-pathogen interaction is suggested, and an alternative strategy to treat HIV-1-related disease may be possible.

Adult↗
PubMed2001

The CcrM DNA methyltransferase of Agrobacterium tumefaciens is essential, and its activity is cell cycle regulated.

DNA methylation is now recognized as a regulator of multiple bacterial cellular processes. CcrM is a DNA adenine methyltransferase found in the alpha subdivision of the proteobacteria. Like the Dam enzyme, which is found primarily in Escherichia coli and other gamma proteobacteria, it does not appear to be part of a DNA restriction-modification system. The CcrM homolog of Agrobacterium tumefaciens was found to be essential for viability. Overexpression of CcrM is associated with significant abnormalities of cell morphology and DNA ploidy. Mapping of the transcriptional start site revealed a conserved binding motif for the global response regulator CtrA at the -35 position; this motif was footprinted by purified Caulobacter crescentus CtrA protein in its phosphorylated state. We have succeeded in isolating synchronized populations of Agrobacterium cells and analyzing their progression through the cell cycle. We demonstrate that DNA replication and cell division can be followed in an orderly manner and that flagellin expression is cyclic, consistent with our observation that motility varies during the cell cycle. Using these synchronized populations, we show that CcrM methylation of the chromosome is restricted to the late S phase of the cell cycle. Thus, within the alpha subdivision, there is a conserved cell cycle dependence and regulatory mechanism controlling ccrM expression.

Agrobacterium tumefaciens↗
PubMed2001

A homolog of the CtrA cell cycle regulator is present and essential in Sinorhizobium meliloti.

During development of the symbiotic soil bacterium Sinorhizobium meliloti into nitrogen-fixing bacteroids, DNA replication and cell division cease and the cells undergo profound metabolic and morphological changes. Regulatory genes controlling the early stages of this process have not been identified. As a first step in the search for regulators of these events, we report the isolation and characterization of a ctrA gene from S. meliloti. We show that the S. meliloti CtrA belongs to the CtrA-like family of response regulators found in several alpha-proteobacteria. In Caulobacter crescentus, CtrA is essential and is a global regulator of multiple cell cycle functions. ctrA is also an essential gene in S. meliloti, and it is expressed similarly to the autoregulated C. crescentus ctrA in that both genes have complex promoter regions which bind phosphorylated CtrA.

Amino Acid Sequence↗
PubMed2001

Conserved promoter motif is required for cell cycle timing of dnaX transcription in Caulobacter.

Cells use highly regulated transcriptional networks to control temporally regulated events. In the bacterium Caulobacter crescentus, many cellular processes are temporally regulated with respect to the cell cycle, and the genes required for these processes are expressed immediately before the products are needed. Genes encoding factors required for DNA replication, including dnaX, dnaA, dnaN, gyrB, and dnaK, are induced at the G(1)/S-phase transition. By analyzing mutations in the dnaX promoter, we identified a motif between the -10 and -35 regions that is required for proper timing of gene expression. This motif, named RRF (for repression of replication factors), is conserved in the promoters of other coordinately induced replication factors. Because mutations in the RRF motif result in constitutive gene expression throughout the cell cycle, this sequence is likely to be the binding site for a cell cycle-regulated transcriptional repressor. Consistent with this hypothesis, Caulobacter extracts contain an activity that binds specifically to the RRF in vitro.

Bacterial Proteins↗
PubMed2001

Should celecoxib be contraindicated in patients who are allergic to sulfonamides? Revisiting the meaning of 'sulfa' allergy.

Celecoxib, a selective cyclo-oxygenase-2 inhibitor, is a diaryl-substituted pyrazole derivative containing a sulfonamide substituent. Because of this structural component, celecoxib is contraindicated for use in patients who have demonstrated allergic reactions to sulfonamides. However, there is a lack of data demonstrating cross-reactivity among sulfonamide medications. A sulfonamide is any compound with an SO2NH2 moiety. The major difference between sulfonamide antimicrobials and other sulfonamide-containing medications such as furosemide, thiazide diuretics and celecoxib, is that sulfonamide antimicrobials contain an aromatic amine group at the N4 position. This allows for division of the sulfonamides into 2 groups: aromatic amines (i.e., sulfonamide antimicrobials) and nonaromatic amines. In addition, sulfonamide antimicrobials contain a substituted ring at the N1-position; this group is not found with nonaromatic amine-containing sulfonamides. Adverse reactions to sulfonamide antimicrobials include type I, or immunoglobulin (Ig) E-mediated reactions, hypersensitivity syndrome reactions, and severe skin reactions such as toxic epidermal necrolysis. The aromatic amine portion of the sulfonamide antimicrobial is considered to be critical in the development of latter 2 reactions. In susceptible individuals, the hydroxylamine metabolite is unable to be detoxified leading to a cascade of cytotoxic and immunological events that eventually results in the adverse reaction. Since celecoxib does not contain the aromatic amine, adverse reactions such as hypersensitivity syndrome reactions and toxic epidermal necrolysis would not be expected to occur at the same frequency as they do with sulfonamide antimicrobials. Similarly, for IgE-mediated reactions, the N1-substituent and not the sulphonamide moiety is important in determining specificity to antibodies. Celecoxib and other nonaromatic amine-containing sulfonamide medications do not contain the N1-substituent. Cross-reactivity among the various sulfonamide-containing medications has also not been substantiated by published case reports. In fact, conflicting information exists in the literature. Reports showing lack of cross-reactivity balance the few case reports suggesting cross-reactivity. Cross-reactivity between sulfonamide medications should be based on scientific data, including chemistry, metabolism, immune responses and clinical data. Based on the current information, there is no documentation for cross-reactivity between sulfonamide antimicrobials and other sulfonamide medications, such as celecoxib.

Anti-Infective Agents↗
PubMed2000

Global analysis of the genetic network controlling a bacterial cell cycle.

This report presents full-genome evidence that bacterial cells use discrete transcription patterns to control cell cycle progression. Global transcription analysis of synchronized Caulobacter crescentus cells was used to identify 553 genes (19% of the genome) whose messenger RNA levels varied as a function of the cell cycle. We conclude that in bacteria, as in yeast, (i) genes involved in a given cell function are activated at the time of execution of that function, (ii) genes encoding proteins that function in complexes are coexpressed, and (iii) temporal cascades of gene expression control multiprotein structure biogenesis. A single regulatory factor, the CtrA member of the two-component signal transduction family, is directly or indirectly involved in the control of 26% of the cell cycle-regulated genes.

Bacterial Proteins↗
PubMed2000

A multicenter, randomized, double-blind, placebo-controlled trial of recombinant human interleukin-10 in HIV-infected subjects.

OBJECTIVE: To determine the effect of multiple subcutaneous doses of recombinant human interleukin (rhuIL)-10 on plasma HIV RNA levels and CD4 T-cell counts, and to evaluate its safety and tolerability in HIV-infected subjects. DESIGN: Prospective, randomized, double-blind, placebo-controlled, multicenter trial. SUBJECTS: Thirty-nine HIV-infected subjects with CD4 T-cell counts > 200 x 10(6)/l, plasma HIV RNA concentrations > or = 3.18 log10 copies/ml and on stable antiretroviral therapy were recruited from six centers. INTERVENTION: Subjects received (subcutaneously) rhuIL-10 1 microg/kg daily, 4 microg/kg daily, 8 microg/kg three times per week, placebo daily or placebo three times per week for 4 weeks. MAIN OUTCOME MEASURES: Prospectively defined outcomes included safety and tolerability, plasma HIV RNA levels and CD4 T-cell counts. Outcomes were assessed at baseline, weeks 1, 2, 3 and 4 during treatment and weeks 2 and 4 following completion of therapy. RESULTS: Baseline characteristics were similar in all groups. Compared to baseline, no significant change in plasma HIV RNA concentrations or CD4 T-cell counts was observed in any of the groups. RhuIL-10 was generally well tolerated. Two patients receiving rhuIL-10 4 microg/kg required discontinuation due to thrombocytopenia. One patient receiving rhuIL-10 4 microg/kg who had chronic hepatitis B and C infections discontinued drug because of elevated liver function tests. One patient receiving placebo discontinued study drug because of depression. CONCLUSION: The lack of a demonstrable virological benefit, as assessed by plasma viral load, with 4 weeks of rhuIL-10 does not support the development of this immune-based therapy for treatment of HIV infection.

Adult↗
PubMed2000

Bioengineered cardiac grafts: A new approach to repair the infarcted myocardium?

BACKGROUND: The myocardium is unable to regenerate because cardiomyocytes cannot replicate after injury. The heart is therefore an attractive target for tissue engineering to replace infarcted myocardium and enhance cardiac function. We tested the feasibility of bioengineering cardiac tissue within novel 3-dimensional (3D) scaffolds. METHODS AND RESULTS: We isolated and grew fetal cardiac cells within 3D porous alginate scaffolds. The cell constructs were cultured for 4 days to evaluate viability and morphology before implantation. Light microscopy revealed that within 2 to 3 days in culture, the dissociated cardiac cells form distinctive, multicellular contracting aggregates within the scaffold pores. Seven days after myocardial infarction, rats were randomized to biograft implantation (n=6) or sham-operation (n=6) into the myocardial scar. Echocardiography study was performed before and 65+/-5 days after implantation to assess left ventricular (LV) remodeling and function. Hearts were harvested 9 weeks after implantation. Visual examination of the biograft revealed intensive neovascularization from the neighboring coronary network. Histological examination revealed the presence of myofibers embedded in collagen fibers and a large number of blood vessels. The specimens showed almost complete disappearance of the scaffold and good integration into the host. Although control animals developed significant LV dilatation accompanied by progressive deterioration in LV contractility, in the biograft-treated rats, attenuation of LV dilatation and no change in LV contractility were observed. CONCLUSIONS: Alginate scaffolds provide a conducive environment to facilitate the 3D culturing of cardiac cells. After implantation into the infarcted myocardium, the biografts stimulated intense neovascularization and attenuated LV dilatation and failure in experimental rats compared with controls. This strategy can be used for regeneration and healing of the infarcted myocardium.

Alginates↗
PubMed2000

The lipopolysaccharide-activated toll-like receptor (TLR)-4 induces synthesis of the closely related receptor TLR-2 in adipocytes.

The central regulatory role of the adipocyte in whole body energy homeostasis is well established. However, recent findings suggest that preadipocytes and adipocytes may play an important physiological role in the regulation of both the innate and adaptive immune response. To systematically characterize the molecular machinery of the adipocyte that mediates the recognition of pathogens, we have focused our analysis on the recently identified Toll-like receptors (TLRs). These receptors have been implicated as mediators of the cellular response to bacterial lipopolysacharides (LPSs). Here, we report the cloning and functional characterization of mouse TLR-2 from 3T3-L1 adipocytes. TLR-2 synthesis is strongly induced in the adipocyte by LPS, TNFalpha, and the yeast cell wall extract zymosan. TLR-2 undergoes a lengthy intracellular maturation process with a half-life of exit from the ER of approximately 3 h. Furthermore, LPS treatment of adipocytes results in dramatic changes at the level of gene expression, including the synthesis of a distinct set of secretory proteins such as interleukin-6. Our studies demonstrate the presence of a fully intact pathway of innate immunity in the adipocyte that can be activated by LPS binding to the cell surface and results in the secretion of immunomodulatory molecules.

3T3 Cells↗