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L Shapiro

Publications and source records attributed to L Shapiro.

At least 91 records · Page 5Linked to original sources
PubMed1997

Translation of the leaderless Caulobacter dnaX mRNA.

The expression of the Caulobacter crescentus homolog of dnaX, which in Escherichia coli encodes both the gamma and tau subunits of the DNA polymerase III holoenzyme, is subject to cell cycle control. We present evidence that the first amino acid in the predicted DnaX protein corresponds to the first codon in the mRNA transcribed from the dnaX promoter; thus, the ribosome must recognize the mRNA at a site downstream of the start codon in an unusual but not unprecedented fashion. Inserting four bases in front of the AUG at the 5' end of dnaX mRNA abolishes translation in the correct frame. The sequence upstream of the translational start site shows little homology to the canonical Shine-Dalgarno ribosome recognition sequence, but the region downstream of the start codon is complementary to a region of 16S rRNA implicated in downstream box recognition. The region downstream of the dnaX AUG, which is important for efficient translation, exhibits homology with the corresponding region from the Caulobacter hemE gene adjacent to the replication origin. The hemE gene also appears to be translated from a leaderless mRNA. Additionally, as was found for hemE, an upstream untranslated mRNA also extends into the dnaX coding sequence. We propose that translation of leaderless mRNAs may provide a mechanism by which the ribosome can distinguish between productive and nonproductive templates.

Amino Acid Sequence↗
PubMed1997

Identification of the fliI and fliJ components of the Caulobacter flagellar type III protein secretion system.

Caulobacter crescentus is motile by virtue of a polar flagellum assembled during the predivisional stage of the cell cycle. Three mutant strains in which flagellar assembly was blocked at an early stage were isolated. The mutations in these strains mapped to an operon of two genes, fliI and fliJ, both of which are necessary for motility. fliI encodes a 50-kDa polypeptide whose sequence is closely related to that of the Salmonella typhimurium FliI protein, an ATPase thought to energize the export of flagellar subunits across the cytoplasmic membrane through a type III protein secretion system. fliJ encodes a 16-kDa hydrophilic protein of unknown function. Epistasis experiments demonstrated that the fliIJ operon is located in class II of the C. crescentus flagellar regulatory hierarchy, suggesting that the gene products act at an early stage in flagellar assembly. The expression of fliIJ is induced midway through the cell cycle, coincident with other class II operons, but the FliI protein remains present throughout the cell cycle. Subcellular fractionation showed that FliI is present both in the cytoplasm and in association with the membrane. Mutational analysis of FliI showed that two highly conserved amino acid residues in a bipartite ATP binding motif are necessary for flagellar assembly.

Amino Acid Sequence↗
PubMed1997

The CcrM DNA methyltransferase is widespread in the alpha subdivision of proteobacteria, and its essential functions are conserved in Rhizobium meliloti and Caulobacter crescentus.

The Caulobacter crescentus DNA methyltransferase CcrM (M.CcrMI) methylates the adenine residue in the sequence GANTC. The CcrM DNA methyltransferase is essential for viability, but it does not appear to be part of a DNA restriction-modification system. CcrM homologs are widespread in the alpha subdivision of gram-negative bacteria. We have amplified and sequenced a 258-bp region of the cerM gene from several of these bacteria, including Rhizobium meliloti, Brucella abortus, Agrobacterium tumefaciens, and Rhodobacter capsulatus. Alignment of the deduced amino acid sequences revealed that these proteins constitute a highly conserved DNA methyltransferase family. Isolation of the full-length ccrM genes from the aquatic bacterium C. crescentus, the soil bacterium R. meliloti, and the intracellular pathogen B. abortus showed that this sequence conservation extends over the entire protein. In at least two alpha subdivision bacteria, R. meliloti and C. crescentus, CcrM-mediated methylation has important cellular functions. In both organisms, CcrM is essential for viability. Overexpression of CcrM in either bacterium results in defects in cell division and cell morphology and in the initiation of DNA replication. Finally, the C. crescentus and R. meliloti ccrM genes are functionally interchangeable, as the complemented strains are viable and the chromosomes are methylated. Thus, in both R. meliloti and C. crescentus, CcrM methylation is an integral component of the cell cycle. We speculate that CcrM-mediated DNA methylation is likely to have similar roles among alpha subdivision bacteria.

Amino Acid Sequence↗
PubMed1997

Isolation and characterization of a xylose-dependent promoter from Caulobacter crescentus.

An inducible promoter is a useful tool for the controlled expression of a given gene. Accordingly, we identified, cloned, and sequenced a chromosomal locus, xylX, from Caulobacter crescentus which is required for growth on xylose as the sole carbon source and showed that transcription from a single site is dependent on the presence of xylose in the growth medium. P(xylX) promoter activity was determined as a function of the composition of the growth medium both in single copy and on a plasmid using different reporter genes. One hundred micromolar exogenously added xylose was required for maximal induction of P(xylX) in a strain that is unable to metabolize xylose. P(xylX) activity was induced immediately after the addition of xylose and repressed almost completely when xylose was removed from the growth medium. In addition to the strong transcriptional control, the expression of xylX is also regulated on the translational level.

Amino Acid Sequence↗
PubMed1997

Transcription of genes encoding DNA replication proteins is coincident with cell cycle control of DNA replication in Caulobacter crescentus.

DNA replication in the dimorphic bacterium Caulobacter crescentus is tightly linked to its developmental cell cycle. The initiation of chromosomal replication occurs concomitantly with the transition of the motile swarmer cell to the sessile stalked cell. To identify the signals responsible for the cell cycle control of DNA replication initiation, we have characterized a region of the C. crescentus chromosome containing genes that are all involved in DNA replication or recombination, including dnaN, recF, and gyrB. The essential dnaN gene encodes a homolog of the Escherichia coli beta subunit of DNA polymerase III. It is transcribed from three promoters; one is heat inducible, and the other two are induced at the transition from swarmer to stalked cell, coincident with the initiation of DNA replication. The single gyrB promoter is induced at the same time point in the cell cycle. These promoters, as well as those for several other genes encoding DNA replication proteins that are induced at the same time in the cell cycle, share two sequence motifs, suggesting that they represent a family whose transcription is coordinately regulated.

Amino Acid Sequence↗
PubMed1997

Randomized placebo-controlled clinical trial of high-dose interleukin-2 in combination with a soluble p75 tumor necrosis factor receptor immunoglobulin G chimera in patients with advanced melanoma and renal cell carcinoma.

PURPOSE: A randomized, double-blind, placebo-controlled trial was performed to compare the toxicity and biologic effects of treatment with high-dose intravenous (IV) bolus interleukin-2 (IL-2) plus the recombinant human soluble p75 tumor necrosis factor (TNF) receptor immunoglobulin G (IgG) chimera (rhuTNFR:Fc) with high-dose IL-2 alone in patients with advanced melanoma and renal cell carcinoma. PATIENTS AND METHODS: Twenty patients with advanced melanoma or renal cell carcinoma were randomized to receive IL-2 (Chiron, Emeryville, CA) 600,000 IU/kg every 8 hours on days 1 to 5 and 15 to 19 (maximum, 28 doses) combined with placebo or the rhuTNFR:Fc fusion protein (Immunex, Seattle, WA) 10 mg/m2 on days 1 and 15 and 5 mg/m2 on days 3, 5, 17, and 19. The impact of rhuTNFR:Fc on IL-2 toxicity and biologic effects was evaluated. RESULTS: No clinically significant difference in toxicity was observed in the two treatment arms. The adjusted median number of IL-2 doses administered during cycle 1 was 24.5 (range, seven to 28) and 21.5 (range, five to 27) for the placebo and rhuTNFR:Fc arms, respectively (P = .544). IL-2-induced TNF bioactivity, neutrophil chemotactic defect, and serum IL-6, IL-8, and IL-1 receptor antagonist (IL-1RA) induction were suppressed by rhuTNFR:Fc. Two of nine assessable patients (22%) on IL-2/placebo and three of 10 patients (30%) on IL-2/rhuTNFR:Fc responded. CONCLUSION: Despite evidence of in vitro neutralization of TNF functional activity and partial inhibition of other secondary biologic effects of IL-2, rhuTNFR:Fc does not reduce the clinical toxicity associated with high-dose IL-2 therapy. These results suggest that the toxicity and antitumor effects of IL-2 treatment are independent of circulating TNF.

Adult↗
PubMed1997

Bicalutamide for advanced prostate cancer: the natural versus treated history of disease.

PURPOSE: To determine the therapeutic effects of bicalutamide 200 mg in patients with prostate cancers of different hormone sensitivities. METHODS: Patients with progressive prostate cancer were treated with bicalutamide 200 mg daily. Before treatment, patients' tumors were classified on the basis of prior hormone exposure and by serum testosterone levels into androgen-dependent and androgen-independent groups. Prior exposure to flutamide and response to flutamide withdrawal was also considered. Outcomes were reported independently on the basis of posttherapy changes in prostate-specific antigen (PSA), measurable disease, and radionuclide bone scans. RESULTS: Outcomes varied by prior hormone exposure as a higher proportion of patients with progression of androgen-dependent tumors showed posttherapy PSA decreases of more than 50% or more than 80%, measurable disease regression, and improvement on radionuclide bone scans than did patients with androgen-independent progression. Within the category of androgen-independent progression, clinical benefit was observed in patients who had previously progressed on flutamide, independent of the response to flutamide withdrawal. Patients who had progressed on a gonadotropin-releasing hormone (GnRH) analog alone had a low response proportion, whereas those who progressed after two or more hormone therapies did not respond. Overall, the drug was well tolerated. After progression on bicalutamide monotherapy, one third of patients with androgen-dependent progression responded to medical castration with a GnRH analog. CONCLUSION: Classifying patient tumors on the basis of prior hormone exposure permits a more precise estimate of the potential benefit of a specific hormone therapy for the individual patient. The precision is further increased by reporting the effects of a drug on each parameter of disease independently. The difference in outcomes for patients with androgen-independent progression suggests that the specific hormone therapy administered and the response to that therapy can influence the biology of the relapsing tumor and the sensitivity to subsequent therapies. The sensitivity to bicalutamide after progression on flutamide deserves further study.

Aged↗
PubMed1996

A novel promoter motif for Caulobacter cell cycle-controlled DNA replication genes.

Caulobacter crescentus contains a single chromosome that is replicated once during a defined period in the cell cycle. The onset of replication coincides with the stimulation of transcription of several genes involved in the replication process. Analysis of the C. crescentus homolog of dnaX, which in Escherichia coli encodes both the gamma and tau subunits of the DNA polymerase III holoenzyme, identified the dnaX transcription start site and showed that activity from the dnaX promoter is stimulated fourfold at the onset of DNA replication. We have identified a conserved sequence motif that is present in the promoter of dnaX and several other genes involved in the replication of DNA, all of which show an induction of transcription at the onset of chromosome replication. Independent mutations in the conserved sequence that lies between the -10 and -35 regions increased transcription, suggesting that a repressor may bind at this site. We propose that the coincident transcriptional activation of several dna genes at the swarmer to stalked cell transition occurs in response to cell cycle regulatory factors, in a manner analogous to the transient transcriptional regulation of flagellar and DNA methylation genes later in the cell cycle.

Amino Acid Sequence↗
PubMed1996

Delivering the payload. Bacterial pathogenesis.

A specialized protein secretion pathway is used by some Gram-negative bacterial pathogens for delivery of virulence factors directly into mammalian host cells. This pathway is parallel to, and probably evolved from, a system used for construction of the bacterial flagellum.

Bacterial Proteins↗
PubMed1996

Caulobacter Lon protease has a critical role in cell-cycle control of DNA methylation.

CcrM, an adenine DNA methyltransferase, is essential for viability in Caulobacter crescentus. The CcrM protein is present only in the predivisional stage of the cell cycle, resulting in cell-cycle-dependent variation of the DNA methylation state of the chromosome. The availability of CcrM is controlled in two ways: (1) the ccrM gene is transcribed only in the predivisional. cell, and (2) the CcrM protein is rapidly degraded prior to cell division. We demonstrate here that CcrM is an important target of the Lon protease pathway in C. crescentus. In a lon null mutant, ccrM transcription is still temporally regulated, but the CcrM protein is present throughout the cell cycle because of a dramatic increase in its stability that results in a fully methylated chromosome throughout the cell cycle. Because the Lon protease is present throughout the cell cycle, it is likely that the level of CcrM in the cell is controlled by a dynamic balance between temporally varied transcription and constitutive degradation. We have shown previously that restriction of CcrM to the C. crescentus predivisional cell is essential for normal morphogenesis and progression through the cell cycle. Comparison of the lon null mutant strain with a strain whose DNA remains fully methylated as a result of constitutive expression of ccrM suggests that the effect of Lon on DNA methylation contributes to several developmental defects observed in the lon mutant. These defects include a frequent failure to complete cell division and loss of precise cell-cycle control of initiation of DNA replication. Other developmental abnormalities exhibited by the lon null mutant, such as the formation of abnormally long stalks, appear to be unrelated to altered chromosome methylation state. The Lon protease thus exhibits pleiotropic effects in C. crescentus growth and development.

ATP-Dependent Proteases↗
PubMed1996

Cell cycle-controlled proteolysis of a flagellar motor protein that is asymmetrically distributed in the Caulobacter predivisional cell.

Flagellar biogenesis and release are developmental events tightly coupled to the cell cycle of Caulobacter crescentus. A single flagellum is assembled at the swarmer pole of the predivisional cell and is released later in the cell cycle. Here we show that the MS-ring monomer FliF, a central motor component that anchors the flagellum in the cell membrane, is synthesized only in the predivisional cell and is integrated into the membrane at the incipient swarmer cell pole, where it initiates flagellar assembly. FliF is proteolytically turned over during swarmer-to-stalked cell differentiation, coinciding with the loss of the flagellum, suggesting that its degradation is coupled to flagellar release. The membrane topology of FliF was determined and a region of the cytoplasmic C-terminal domain was shown to be required for the interaction with a component of the motor switch. The very C-terminal end of FliF contains a turnover determinant, required for the cell cycle-dependent degradation of the MS-ring. The cell cycle-dependent proteolysis of FliF and the targeting of FliF to the swarmer pole together contribute to the asymmetric localization of the MS-ring in the predivisional cell.

Amino Acid Sequence↗
PubMed1996

A cell cycle-regulated bacterial DNA methyltransferase is essential for viability.

The CcrM adenine DNA methyltransferase, which specifically modifies GANTC sequences, is necessary for viability in Caulobacter crescentus. To our knowledge, this is the first example of an essential prokaryotic DNA methyltransferase that is not part of a DNA restriction/modification system. Homologs of CcrM are widespread in the alpha subdivision of the Proteobacteria, suggesting that methylation at GANTC sites may have important functions in other members of this diverse group as well. Temporal control of DNA methylation state has an important role in Caulobacter development, and we show that this organism utilizes an unusual mechanism for control of remethylation of newly replicated DNA. CcrM is synthesized de novo late in the cell cycle, coincident with full methylation of the chromosome, and is then subjected to proteolysis prior to cell division.

Alleles↗
PubMed1996

Cell cycle control by an essential bacterial two-component signal transduction protein.

Dividing cells must coordinate cell cycle events to ensure genetic stability. Here we identify an essential two-component signal transduction protein that controls multiple events in the Caulobacter cell cycle, including cell division, stalk synthesis, and cell cycle-specific transcription. This protein, CtrA, is homologous to response regulator transcription factors and controls transcription from a group of cell cycle-regulated promoters critical for DNA replication, DNA methylation, and flagellar biogenesis. CtrA activity in the cell cycle is controlled both transcriptionally and by phosphorylation. As purified CtrA binds an essential DNA sequence motif found within its target promoters, we propose that CtrA acts in a phosphorelay signal transduction system to control bacterial cell cycle events directly at the transcriptional level.

Amino Acid Sequence↗
PubMed1996

Clinical, hematologic, and immunologic effects of interleukin-10 in humans.

We conducted a double-blind, placebo-controlled study to investigate the safety, pharmacokinetics, and immunological properties of interleukin-10 (IL-10) administration in healthy humans. Volunteers received a single intravenous bolus injection of recombinant human IL-10 (1, 10, or 25 micrograms/kg) or placebo. Cytokine production in whole blood and peripheral blood mononuclear cells (PBMC) was assessed before and 3, 6, 24, and 48 hr after the injection. Peak serum concentrations of IL-10 (15 +/- 1.1, 208 +/- 20.1, and 505 +/- 22.3 ng/ml) occurred after 2-5 min for 1, 10, and 25 micrograms/kg IL-10, respectively. The terminal-phase half-life was 3.18 hr. A transient leukocytosis (24-63% above baseline) was observed 6 hr after injection, which coincided with a dose-dependent decrease (12-24%) in neutrophil superoxide generation. There was a marked inhibition (60-95%) of endotoxin-induced IL-6 production from whole blood in each group receiving IL-10. Production of IL-8 in endotoxin-stimulated blood was reduced in the 10 micrograms/kg group. In PBMC stimulated with phytohemagglutinin and phorbol ester, there was a decrease (72-87%) in interferon-gamma (IFN gamma) production 6 hr after IL-10 with a return to pre-IL-10 levels after 24 hr. This reduction was only partially associated with a decrease in the number of CD2-bearing cells. We conclude that IL-10 administration into humans is without significant side effects, and a single injection reduces ex vivo production of IL-6, IL-8, and IFN gamma.

Adolescent↗
PubMed1996

Dapsone in prevention of recurrent neutrophilic eccrine hidradenitis.

Neutrophilic eccrine hidradenitis has been described in patients with acute myelogenous leukemia and other malignant diseases, usually during chemotherapy. We describe a 46-year-old man with Hodgkin's disease in whom neutrophilic eccrine hidradenitis developed after each of the first two treatments with lomustine. Dapsone, 100 mg daily, was initiated 48 hours before the patient's third treatment with lomustine and was continued for 14 days. This regimen was successful in suppressing the reaction during the first course and three subsequent courses of lomustine.

Antineoplastic Agents↗
PubMed1996

Crystal structure of the extracellular domain from P0, the major structural protein of peripheral nerve myelin.

P0, the major protein of peripheral nerve myelin, mediates membrane adhesion in the spiral wraps of the myelin sheath. We have determined the crystal structure of the extracellular domain from P0 (P0ex) at 1.9 A resolution. P0ex is folded like a typical immunoglobulin variable-like domain; five residues at the C-terminus are disordered, suggesting a flexible linkage to the membrane. The requirements for crystallization of P0ex are similar to those for maintaining the native extracellular spacing of adjacent myelin lamellae; thus, given the self-adhesive character of P0ex, the crystal itself may reveal some of the natural interactions that occur between P0 molecules in myelin. The structure leads to the suggestion that P0 extracellular domains may emanate from the membrane surface as tetramers that link to tetramers on the opposing membrane surface, to result in the formation of networks of molecules. We report analytical ultracentrifugation data for P0ex that support this idea.

Animals↗
PubMed1996

The control of temporal and spatial organization during the Caulobacter cell cycle.

The Caulobacter cell cycle exhibits time-dependent expression of differentiation events. These include the morphological transition of a swarmer cell to a replication-competent stalked cell and the subsequent polarized distribution of specific gene products that results in an asymmetric predivisional cell. Cell division then yields a new swarmer cell and a stem-cell-like stalked cell. Two-component signal transduction proteins involved in cell cycle control and proteins required for cell division and flagellar biogenesis have been shown to be regulated temporally and spatially during the cell cycle. The mechanisms underlying this regulation include protein phosphorylation and proteolysis.

Bacterial Proteins↗