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Biomedical subjects

L Shapiro

Publications and source records attributed to L Shapiro.

At least 127 records · Page 7Linked to original sources
PubMed1995

The control of asymmetric gene expression during Caulobacter cell differentiation.

The dimorphic bacterium Caulobacter crescentus provides a simple model for cellular differentiation. Each cell division produces two distinct cell types: a swarmer cell and a stalked cell. These cells possess distinct functional morphologies and differential programs of transcription and DNA replication. The synthesis of a single polar flagellum is restricted to the swarmer pole of the predivisional cell by a genetic hierarchy comprising at least 50 genes whose transcription is regulated by novel and ubiquitous promoters, cognate sigma factors, and auxiliary transcriptional regulators. Chromosome replication is restricted to the stalked cell by a unique chromosome origin of replication that may be regulated by a novel cell-specific transcriptional control system. Phosphorylation signals, DNA methylation, differential chromosome structures, protein targeting, and selective protein degradation are also involved in establishing and maintaining cellular asymmetry. The molecular details of these universal cellular processes in C. crescentus will provide paradigms applicable to many general aspects of cellular differentiation.

Bacterial Proteins↗
PubMed1995

Cytokines in human renal interstitial fibrosis. I. Interleukin-1 is a paracrine growth factor for cultured fibrosis-derived kidney fibroblasts.

We studied the role of interleukin-1 beta (IL-1 beta) and basic fibroblast growth factor (bFGF) in the proliferative response of transformed human renal interstitial fibroblast cell lines established from either a kidney with glomerulonephritis and interstitial fibrosis or a normal kidney in comparison to primary human foreskin fibroblasts. Growth of fibrosis-derived renal fibroblasts was inhibited in the presence of IL-1 receptor antagonist (IL-1Ra) by 35% (P < 0.005), suggesting that these cells produce IL-1 and possess IL-1 receptors as part of paracrine growth. In contrast, spontaneous proliferation of fibroblasts derived from a normal kidney or normal skin were not inhibited by IL-1Ra. In fibrosis-derived but not in normal renal cells, fibronectin synthesis was increased 2.2-fold (P < 0.01) in the presence of IL-1Ra. Addition of exogenous IL-1 beta or bFGF stimulated proliferation of skin fibroblasts. In contrast, growth of fibrosis-derived renal fibroblasts was stimulated by IL-1 beta and unchanged by bFGF. Growth of normal kidney fibroblasts was unaffected by bFGF and inhibited by IL-1 beta. We conclude that compared to normal fibroblasts, fibrosis-derived renal fibroblasts have a different cytokine-response profile, are IL-1-dependent, produce IL-1 as a paracrine growth factor and do not proliferate to bFGF, a classical fibroblast growth factor.

Cell Count↗
PubMed1995

Regulation of the Caulobacter crescentus dnaKJ operon.

The bacterial heat shock proteins DnaK and DnaJ are members of a class of molecular chaperones that are required for a wide variety of cellular functions at normal growth temperatures. In Caulobacter crescentus, the expression of the dnaKJ operon is regulated both temporally during the normal cell cycle and by heat shock. Analysis of deletions and base substitutions in the 5' region of the operon established the presence of two functional promoters: a heat shock-inducible promoter, P1, with characteristics of a sigma 32 promoter, and an adjacent sigma 70-like promoter, P2. Transcription initiating at the sigma 70-like promoter is under strict temporal control, whereas transcription initiating at the heat shock promoter at 30 degrees C is not. Transcription of dnaKJ occurs during a short period in the cell cycle, concomitant with the onset of DNA replication. Deletions in the 5' region have also revealed that all cis-acting sites required for temporal control of transcription reside within 50 bases of the P2 start site. Transcripts initiating from either the P1 or the P2 promoter have an RNA leader sequence with a high probability of forming an extensive secondary structure. Deletion of this leader sequence resulted in an increased rate of expression in both transcriptional and translational fusions. Although the temporal control of expression at physiological temperatures is not affected by the presence or absence of the leader sequence, changes in mRNA secondary structure may contribute to the modulation of DnaK and DnaJ levels at normal temperatures and during heat shock.

Bacterial Proteins↗
PubMed1995

Caulobacter FliQ and FliR membrane proteins, required for flagellar biogenesis and cell division, belong to a family of virulence factor export proteins.

The Caulobacter crescentus fliQ and fliR genes encode membrane proteins that have a role in an early step of flagellar biogenesis and belong to a family of proteins implicated in the export of virulence factors. These include the MopD and MopE proteins from Erwinia carotovora, the Spa9 and Spa29 proteins from Shigella flexneri, and the YscS protein from Yersinia pestis. Inclusion in this family of proteins suggests that FliQ and FliR may participate in an export pathway required for flagellum assembly. In addition, mutations in either fliQ or fliR exhibit defects in cell division and thus may participate directly or indirectly in the division process. fliQ and fliR are class II flagellar genes residing near the top of the regulatory hierarchy that determines the order of flagellar gene transcription. The promoter sequence of the fliQR operon differs from most known bacterial promoter sequences but is similar to other Caulobacter class II flagellar gene promoter sequences. The conserved nucleotides in the promoter region are clustered in the -10, -20 to -30, and -35 regions. The importance of the conserved bases for promoter activity was demonstrated by mutational analysis. Transcription of the fliQR operon is initiated at a specific time in the cell cycle, and deletion analysis revealed that the minimal sequence required for transcriptional activation resides within 59 bp of the start site.

Amino Acid Sequence↗
PubMed1995

Coordinate cell cycle control of a Caulobacter DNA methyltransferase and the flagellar genetic hierarchy.

The expression of the Caulobacter ccrM gene and the activity of its product, the M.Ccr II DNA methyltransferase, are limited to a discrete portion of the cell cycle (G. Zweiger, G. Marczynski, and L. Shapiro, J. Mol. Biol. 235:472-485, 1994). Temporal control of DNA methylation has been shown to be critical for normal development in the dimorphic Caulobacter life cycle. To understand the mechanism by which ccrM expression is regulated during the cell cycle, we have identified and characterized the ccrM promoter region. We have found that it belongs to an unusual promoter family used by several Caulobacter class II flagellar genes. The expression of these class II genes initiates assembly of the flagellum just prior to activation of the ccrM promoter in the predivisional cell. Mutational analysis of two M.Ccr II methylation sites located 3' to the ccrM promoter suggests that methylation might influence the temporally controlled inactivation of ccrM transcription. An additional parallel between the ccrM and class II flagellar promoters is that their transcription responds to a cell cycle DNA replication checkpoint. We propose that a common regulatory system coordinates the expression of functionally diverse genes during the Caulobacter cell cycle.

Amino Acid Sequence↗
PubMed1995

Evaluation of the renal arteries in kidney donors: value of three-dimensional phase-contrast MR angiography with maximum-intensity-projection or surface rendering.

OBJECTIVE: Donors routinely undergo preoperative conventional arteriography to evaluate the renal arteries before nephrectomy. The purpose of this study was to assess the capability of three-dimensional phase-contrast MR angiograms postprocessed with maximum-intensity-projection and surface-rendering techniques to show the renal arteries of potential donors. MATERIALS AND METHODS: Postprocessed three-dimensional phase-contrast MR angiograms of 17 patients were retrospectively reviewed by two experienced radiologists for the number and length of renal arteries visualized. Conventional arteriograms were used as the reference standard. Coronal maximum-intensity-projection and surface-rendered MR angiograms were also compared with each other with regard to the delineation of renal arteries from overlapping vessels. RESULTS: MR angiograms showed all 34 single or dominant renal arteries but only eight of 10 accessory arteries seen on conventional arteriograms. One of the nonvisualized accessory arteries was located within the imaged volume, and the other one arose from the distal aorta beyond the imaged regions. Five of six arterial branches arising from the proximal 30-mm portions of the renal arteries were seen on MR angiograms. Postprocessing with either maximum-intensity projection or surface-rendering showed the same number of renal arteries, although surface rendering separated overlapping veins from the renal arteries better than the maximum-intensity-projection technique. CONCLUSION: These results suggest that three-dimensional MR angiography is a reliable method of imaging single or dominant renal arteries, but not for showing all accessory renal arteries and small arterial branches. Surface rendering may provide specific advantages over maximum-intensity-projection in delineating renal arteries from overlapping vessels.

Adult↗
PubMed1994

Interleukin-1 induces interleukin-8 secretion from endothelial cells by a juxtacrine mechanism.

Inflammation is characterized by migration of neutrophils through the endothelium, and the chemokine interleukin-8 (IL-8) appears to be involved. We asked whether adherence of cells bearing a membrane-form of interleukin 1 (IL-1) induces IL-8 secretion from human umbilical vein endothelial cells (HUVEC) and fibroblasts. Human peripheral blood mononuclear cells (PBMC) were stimulated with endotoxin for 12 hours and then fixed for 4 hours with paraformaldehyde. When these cells were added to HUVEC or fibroblasts, IL-8 production was induced. This stimulation by fixed PBMC was attributed to IL-1, because pretreatment of HUVEC or fibroblasts with IL-1 receptor antagonist (IL-1Ra) reduced the induction by 95% and 80%, respectively, P < .005. Using anti-IL-1 alpha monoclonal antibodies, reduction was complete, whereas anti-IL-1 beta had no effect. IL-1 alpha was shown on the surface of monocytes by fluorescence-activated cell sorter (FACS) analysis. Blockade of IL-1 receptors on PBMC did not affect the activity of membrane-associated IL-1 alpha, indicating that IL-1 is not anchored to the membrane through its receptors. However, PBMC treated with D-mannose before fixation resulted in a loss of activity; this loss of activity was associated with release of IL-1 alpha, not IL-1 beta, into the supernatant. Thus, anchoring of IL-1 alpha to the membrane may be via a lectin or mannose receptor-like interaction. Blockade of membrane IL-1 alpha required a 30-fold and fivefold excess of IL-1Ra compared with the amount required to block soluble IL-1 beta and IL-1 alpha, respectively. We conclude that the fixed PBMC IL-8 inducing activity is almost entirely caused by IL-1, that this represents IL-1 alpha bound to a surface lectin or mannose receptor on the monocyte, and that it functions in inflammation via juxtacrine interactions.

Cell Membrane↗
PubMed1994

Caulobacter flagellar function, but not assembly, requires FliL, a non-polarly localized membrane protein present in all cell types.

Caulobacter crescentus has a single polar flagellum, which is assembled in the predivisional cell. Known flagellar genes encode structural and regulatory components that are required for flagellar assembly and function. These genes are organized in several classes which form a transcriptional regulatory hierarchy. A member of the Class II genes, the fliLM operon, encodes homologs of the Escherichia coli flagellar switch protein, FliM, and a protein with a hitherto unknown function, FliL. We report here that flagellar rotation requires the FliL protein. In-frame deletions in the chromosomal copy of the fliL gene result in cells that form a flagellum but are non-motile. The FliL protein was found to be associated with the inner membrane and to be present in all cell types. This is the first report of a Caulobacter crescentus protein that is essential for motility but is not spatially restricted to the region of the flagellar basal body. Although FliL is required for flagellar function, it is not part of the transcriptional hierarchy, supporting the hypothesis that, as is the case for the enterics, the regulatory hierarchy responds to assembly cues rather than directly to the expression of flagellar proteins.

Amino Acid Sequence↗
PubMed1994

Induction of circulating antagonists to IL-1 and TNF by IL-2 administration and their effects on IL-2 induced cytokine production in vitro.

The objectives of this study were to determine whether intensive immunotherapy with IL-2 results in detectable levels of circulating IL-1 and TNF antagonists and whether the levels achieved in vivo are sufficient to affect the generation of secondary proinflammatory cytokines such as IL-1 beta and TNF-alpha. We also sought to determine the extent to which endogenous TNF mediates the generation of an IL-1 antagonist by IL-2-activated PBMCs. In patients undergoing high dose IL-2 immunotherapy, plasma IL-1 receptor antagonist (IL-1ra) levels rose dramatically after the first IL-2 injection, reaching a plateau of 11.03 +/- 0.92 ng/ml within 4 h. TNF-soluble receptor p55 (TNFsRp55) was also detected in the plasma shortly after initiating treatment, and the levels progressively increased throughout the treatment course. PBMCs exposed to IL-2 expressed IL-1ra mRNA and secreted the IL-1ra protein, but neither PBMCs nor neutrophils shed TNFsRp55 in response to IL-2 or supernatants from IL-2-activated PBMCs. IL-1ra at concentrations achieved in the plasma during IL-2 immunotherapy (approximately 10 ng/ml) inhibited the in vitro production of IL-1 beta and TNF-alpha by IL-2-activated PBMCs by 65% and 30%, respectively. Although the monomeric receptor TNFsRp55 at concentrations achieved in the plasma had no effect on the in vitro production of IL-1ra, TNF-alpha, or IL-1 beta, the bivalent TNFsRp75-Fc chimera suppressed the generation of TNF and IL-1. IL-1ra synthesis was unaffected. These results suggest that the amount of IL-1ra generated in response to IL-2 is most likely sufficient to down-modulate the production of proinflammatory cytokines.

Cells, Cultured↗
PubMed1994

A Caulobacter DNA methyltransferase that functions only in the predivisional cell.

Caulobacter crescentus was found to have a DNA methyltransferase, CcrM, that methylates the adenine base of the HinfI recognition sequence, GANTC. The ccrM gene was cloned, and DNA sequence analysis revealed that the predicted amino acid sequence has 49% identity with the Haemophilus influenzae methyltransferase HinfM. Expression of the ccrM gene was found to be restricted to the portion of the cell cycle immediately prior to cell division. At three separate chromosomal sites the CcrM recognition sequence is fully methylated in swarmer cells, becomes hemimethylated upon DNA replication in stalked cells, and does not become remethylated until just prior to cell division. The time of methyltransferase expression coincides with the time of methylation of these three chromosomal sites and of plasmid DNA in the predivisional cell. When ccrM gene expression is placed under control of a constitutive promoter, these chromosomal sites are fully methylated throughout the cell cycle. A high proportion of morphologically aberrant cells, and cells that have undergone an additional chromosome replication initiation, are found in this population. Thus, the temporal control of this methyltransferase appears to contribute to the accurate cell-cycle control of DNA replication and cellular morphology.

Amino Acid Sequence↗
PubMed1994

Ciliary neurotrophic factor combined with soluble receptor inhibits synthesis of proinflammatory cytokines and prostaglandin-E2 in vitro.

In human peripheral blood mononuclear cells, ciliary neurotrophic factor (CNTF) weakly suppressed endotoxin-induced interleukin (IL)-1 and prostaglandin E2(PGE2). Suppression of PGE2 and IL-8 synthesis was significantly greater (up to 42.6%, P < 0.05) by adding a 10-fold molar excess of soluble CNTF receptor (sCNTFR alpha). In cultured human fibroblasts, CNTF at 12 micrograms/ml did not suppress IL-1 alpha-induced IL-8. However, in the presence of a 10-fold excess of sCNTFR alpha, 300 ng/ml of CNTF suppressed IL-1 alpha-induced IL-8 by 44%. Therefore, sCNTFR alpha can confer to CNTF anti-inflammatory properties in vitro. IL-6 which, like CNTF, utilizes the gp130 signal transducer, possesses similar inhibitory effects. That CNTF and IL-6 share gp130 as a receptor component suggests that gp130 mediates these anti-inflammatory responses.

Cells, Cultured↗
PubMed1994

Induction of circulating soluble tumour necrosis factor receptor and interleukin 1 receptor antagonist following interleukin 1 alpha infusion in humans.

The aim of this study was to investigate circulating levels of tumour necrosis factor soluble receptor p55 (TNFsrp55) and interleukin 1 receptor antagonist (IL-1ra) in cancer patients undergoing treatment with IL-1 alpha. Patients were treated with 0.03 micrograms/kg IL-1 alpha administered intravenously over a 30 min interval daily for five consecutive days. Plasma TNFsrp55 levels rose dramatically and peaked (24.5 +/- 3.6 ng/ml) within 1 h after the first IL-1 alpha infusion. Thereafter, the levels rapidly declined and reached baseline levels within 24 h. The increases observed on days 3 and 5 of treatment were less pronounced but the reductions in peak levels were not statistically significant. IL-1ra levels increased less abruptly after an IL-1 alpha infusion than did TNFsrp55 levels and peaked (25.3 +/- 5.1 ng/ml) within 2 h of the start of the IL-1 alpha infusion. Levels then rapidly declined reaching baseline values within 24 h. As with TNFsrp55 levels, peak IL-1ra levels observed on days 3 and 5 of treatment were less than those measured on day 1. IL-1 alpha and IL-1 beta levels were consistently below the threshold of detection of the RIAs employed in these studies. Likewise, with the exception of a single time point in one of the four patients studied, TNF-alpha was undetectable in all plasma samples assayed.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclophosphamide↗
PubMed1994

Plasma levels of IL-1 beta, TNF alpha and their specific inhibitors in undialyzed chronic renal failure, CAPD and hemodialysis patients.

The presence of naturally occurring inhibitors of interleukin-1 (IL-1) and tumor necrosis factor (TNF) in a variety of diseases has been demonstrated. The IL-1 receptor antagonist (IL-1Ra) binds to IL-1 receptors and blocks the activity of IL-1, and a soluble form of the p55 TNF receptor (TNFsRp55) binds and neutralizes TNF. In the present study, plasma levels of IL-1 beta, IL-1Ra, TNF alpha and TNFsRp55 were measured in 29 undialyzed patients with chronic renal failure (CRF), 13 patients on continuous ambulatory peritoneal dialysis (CAPD), 42 patients on chronic hemodialysis (HD) and in 15 healthy controls. Of the 29 patients with CRF, 13 had end-stage renal disease (ESRD, estimated GFR < 10 ml/min). Among health controls, plasma levels of IL-1 beta, IL-1Ra and TNF alpha were at or below the limit of detection of the assay. In undialyzed patients with ESRD, or in patients on CAPD or HD, plasma levels of IL-1 beta were 428 +/- 134 pg/ml, 378 +/- 83 and 352 +/- 43 pg/ml, respectively. Although plasma levels of IL-1 beta in each group of patients were higher than those in healthy controls (< 160 pg/ml), these differences were not statistically significant. In contrast, plasma levels of IL-1Ra in undialyzed patients with ESRD (629 +/- 125 pg/ml, P = 0.03), CAPD (902 +/- 164 pg/ml, P < 0.0001) and HD patients (642 +/- 73 pg/ml, P = 0.004) were significantly higher than those in healthy controls (103 +/- 15).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Cell cycle arrest of a Caulobacter crescentus secA mutant.

Cell differentiation is an inherent component of the Caulobacter crescentus cell cycle. The transition of a swarmer cell, with a single polar flagellum, into a sessile stalked cell includes several morphogenetic events. These include the release of the flagellum and pili, the proteolysis of chemotaxis proteins, the biogenesis of the polar stalk, and the initiation of DNA replication. We have isolated a group of temperature-sensitive mutants that are unable to complete this process at the restrictive temperature. We show here that one of these strains has a mutation in a homolog of the Escherichia coli secA gene, whose product is involved in protein translocation at the cell membrane. This C. crescentus secA mutant has allowed the identification of morphogenetic events in the swarmer-to-stalked cell transition that require SecA-dependent protein translocation. Upon shift to the nonpermissive temperature, the mutant secA swarmer cell is able to release the polar flagellum, degrade chemoreceptors, and initiate DNA replication, but it is unable to form a stalk, complete DNA replication, or carry out cell division. At the nonpermissive temperature, the cell cycle blocks prior to the de novo synthesis of flagella and chemotaxis proteins that normally occurs in the predivisional cell. Although interactions between the chromosome and the cytoplasmic membrane are believed to be a functional component of the temporal regulation of DNA replication, the ability of this secA mutant to initiate replication at the nonpermissive temperature suggests that SecA-dependent events are not involved in this process. However, both cell division and stalk formation, which is analogous to a polar division event, require SecA function.

Adenosine Triphosphatases↗
PubMed1994

Expression of Caulobacter dnaA as a function of the cell cycle.

The initiation of DNA replication is under differential control in Caulobacter crescentus. Following cell division, only the chromosome in the progeny stalked cell is able to initiate DNA replication, while the chromosome in the progeny swarmer cell does not replicate until later in the cell cycle. We have isolated the dnaA gene in order to determine whether this essential and ubiquitous replication initiation protein also contributes to differential replication control in C. crescentus. Analysis of the cloned C. crescentus dnaA gene has shown that the deduced amino acid sequence can encode a 486-amino-acid protein that is 37% identical to the DnaA protein of Escherichia coli. The gene is located 2 kb from the origin of replication. Primer extension analysis revealed a single transcript originating from a sigma 70-type promoter. Immunoprecipitation of a DnaA'-beta-lactamase fusion protein showed that although expression occurs throughout the cell cycle, there is a doubling in the rate of expression just prior to the initiation of replication.

Amino Acid Sequence↗