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Biomedical subjects

L Shapiro

Publications and source records attributed to L Shapiro.

At least 253 records · Page 14Linked to original sources
PubMed1982

In vitro transcription of the early region of Caulobacter phage phi Cd1 deoxyribonucleic acid by host RNA polymerase.

Transcription of the Caulobacter crescentus phage phi Cd1 genome requires both the host RNA polymerase and a phage-encoded, rifampicin-resistant RNA polymerase. Transcription of the early region of the phi Cd1 genome was examined in vitro with C. crescentus RNA polymerase. Four transcripts, A, B, C, and D, which ranged in size from 2.9 X 10(6) to 0.53 X 10(6) daltons, were synthesized in vitro by the holoenzyme. Transcript A appeared to be the major transcript since (a) it was the size of the entire 20% of the genome shown in vivo to code for the early phage mRNA, (b) it was one of the first transcripts synthesized at low enzyme-to-DNA molar ratios, and (c) it was synthesized in approximately 3 times the molar equivalent observed for the other transcripts. The A transcript initiated primarily with GTP although a portion was also labeled with ATP. The B, C, and D transcripts were present in equivalent molar ratios, were all smaller than transcript A, and were found to yield RNase III digestion products that were subsets of each other as well as of transcript A. Each of these transcripts proved to be a de novo transcript since (a) each could be pulse labeled during the initial 20 s of the reaction and (b) each transcript contained a triphosphate at its 5' terminus. Evidence is presented that suggests that the B and C transcripts initiate at or near the major A promoter but terminate at different termination or pause sites within the early region of the phage genome. Transcript D appears to initiate at a minor promoter within the terminally redundant region of the genome preceding the A promoter.

Bacteria↗
PubMed1982

Isolation of a Caulobacter gene cluster specifying flagellum production by using nonmotile Tn5 insertion mutants.

Caulobacter crescentus assembles a single polar flagellum from protein components synthesized at a specific time in the cell cycle. Of the 26 genes required for flagellum production, at least 4 of them-flaY, E, F, and G-map together in a single cluster. We have isolated DNA from this region of the chromosome by using a nonmotile mutant with a Tn5 insertion into flaE. C. crescentus DNA carrying the Tn5-flaE region and adjacent sequences was cloned into pBR325 and selected by transposon-encoded kanamycin resistance. The resulting plasmid was used as a probe to isolate the flaE region from a wild-type gene bank and to determine the chromosomal location of several deletion and insertion mutations within the flaY/E/F/G cluster. At least three promotors and three major transcripts were shown to originate from the cloned gene cluster. The role of these genes in flagellar biogenesis was examined by immunoprecipitation of mutant cell extracts with antiflagellin antibody. Deletions extending rightward into this gene cluster eliminated one of the two flagellin proteins normally synthesized by C. crescentus. Mutations mapping to the left permitted synthesis of both normal flagellins but at significantly decreased levels. These results suggest that the leftward end of this cluster contains a region that may function in a regulatory capacity whereas the rightward end may contain sequences overlapping a flagellin structural gene.

Journal Article↗
PubMed1982

Synthesis and utilization of fatty acids by wild-type and fatty acid auxotrophs of Caulobacter crescentus.

The fatty acid composition of the dimorphic bacterium Caulobacter crescentus was found to consist primarily of 16- and 18-carbon fatty acids, both saturated and monounsaturated, in agreement with the findings of Chow and Schmidt (J. Gen. Microbiol. 83:359-373, 1974). In addition, two minor but as yet unidentified fatty acids were detected. Chromatographic mobilities suggested that these fatty acids may be a cyclopropane and a branched-chain fatty acid. In addition, we demonstrated that the fatty acid composition of wild-type C. crescentus can be altered by growing the cells in medium supplemented with any one of a variety of unsaturated fatty acids. Linoleic acid, a diunsaturated fatty acid which is not synthesized by C. crescentus, was incorporated into phospholipids without apparent modification. In addition, we found that C. crescentus, like Escherichia coli, synthesizes vaccenic acid (18:1 delta 11,cis) rather than oleic acid (18:1 delta 9,cis). This result allowed us to deduce that the mechanism of fatty acid desaturation in C. crescentus is anaerobic, as it is in E. coli. Finally, we examined the fatty acid biosynthesis and composition of two unsaturated fatty acid auxotrophs of C. crescentus. Neither of these mutants resembled the E. coli unsaturated fatty acid auxotrophs, which have defined enzymatic lesions in fatty acid biosynthesis. Rather, the mutants appeared to have defects relating to the complex coordination of membrane biogenesis and cell cycle events in C. crescentus.

Bacteria↗
PubMed1981

Phospholipid biosynthesis is required for stalk elongation in Caulobacter crescentus.

Membrane phospholipid synthesis was inhibited in Caulobacter crescentus by growth of a glycerol auxotroph in the absence of glycerol or by treatment with the antibiotic cerulenin. It was observed that the final step in the swarmer cell-to-stalked cell transition, stalk elongation, was inhibited under these conditions. Since an early effect of inhibiting phospholipid synthesis in C. crescentus is the termination of deoxyribonucleic acid (DNA) replication (I. Contreras, R. Bender, A. Weissborn, K. Amemiya J. D. Mansour, S. Henry, and L. Shapiro, J. Mol. Biol. 138:401-410, 1980), we questioned whether the inhibition of stalk formation was due directly to the inhibition phospholipid synthesis or secondarily to the inhibition of DNA synthesis. Under conditions which inhibited DNA synthesis but permitted phospholipid synthesis, i.e., growth of a temperature-sensitive DNA elongation mutant at the restrictive temperature or treatment with hydroxy-urea, stalk elongation occurred normally. Therefore phospholipid synthesis is required for stalk elongation in C. crescentus.

Bacteria↗
PubMed1980

Differential membrane phospholipid synthesis during the cell cycle of Caulobacter crescentus.

The pattern of phospholipid synthesis during the cell cycle of Caulobacter crescentus has been determined. Although the phospholipid composition of swarmer and stalked cells was indistinguishable in continuously labeled cultures if the two cell types were pulse-labeled for a short time period, marked differences in the pattern of phospholipid synthesis were detected. Pulse-labeled swarmer cells exhibited a higher proportion of phosphatidic acid and a lower proportion of phosphatidylglycerol. In addition, minor phospholipids were detected in the swarmer cells that were not detected in stalked cells. Stalked cells that developed directly from swarmer cells showed that same phospholipid profile as the swarmer cells. The switch to the second phospholipid profile was observed to occur at the predivisional cell stage. Because cell division then yielded a swarmer cell with a different phospholipid profile than its sibling stalked cell, the cell division process may trigger a mechanism which alters the pattern of phospholipid synthesis.

Bacteria↗
PubMed1980

Physical map of Caulobacter crescentus bacteriophage phi Cd1 DNA.

A restriction map of the Caulobacter crescentus bacteriophage phi Cd1 genome was constructed by using the restriction endonucleases HindIII and HpaI. A total of 12 fragments, ranging in molecular weight from 7.7 X 10(6) to 0.25 X 10(6), were produced by HindIII, and 7 fragments, ranging in molecular weight from 9.0 X 10(6) to 0.24 X 10(6), were generated by HpaI. The molecular weight of the genome was estimated to be approximately 28.8 X 10(6) on the basis of the relative electrophoretic mobilities of the restriction fragments. The relative order of the cleavage fragments was determined by specific cleavage of isolated restriction fragments, terminal labeling of both the whole genome and isolated fragments, and hybridization of isolated fragments to restriction fragments generated by other restriction enzymes. The genome of phi Cd1 was found to be terminally repetitive, and analysis of previously determined in vivo and in vitro RNA transcripts showed that the restriction map could be oriented such that transcription began on the left and proceeded to the right end of the genome.

Bacteriophages↗
PubMed1980

Carbonic anhydrase activity of rabbit lungs.

Pulmonary carbonic anhydrase (CA) activity was studied in rabbit lungs perfused with solutions containing no CA. Measurements were made of the amount of 14CO2 appearing in the expired gas following injections of H14CO3(-), 14CO2, or a 20:1 mixture of each into the pulmonary artery. The fraction of the injected label in the expired gas was only 17% greater for 14CO2 than for the mixture, suggesting that equilibration between H14CO3(-) and 14CO2 was nearly complete during the capillary transit time. Inhibition of pulmonary CA decreased excretion of H14CO3(-) and the mixture by 40 and 49% and increased the excretion of 14CO2 by 96%. Addition of CA to the perfusate had no effect. Thus, CO2 exchange is not significantly limited by pulmonary CA if inhibitors are absent. Tissue binding of [3H]acetazolamide injected into the pulmonary artery was diminished by 50% when acetazolamide concentrations reached 0.13 x 10(-6) M. Each liter of extravascular lung water contained 1.25 x 10(-6) mol of receptors for acetazolamide that were accessible to plasma during a single circulation. Binding of [3H]acetazolamide was also observed in lungs of anesthetized rabbits, suggesting that pulmonary CA is accessible to plasma in vivo as well as in situ.

Acetazolamide↗
PubMed1980

Accelerated clearance of small solutes from the lungs in interstitial lung disease.

Solutions of 99mTc-diethylenetriaminepenta-acetate (99mTc-DTPA) and 99mTcO-4 were aerosolized and inhaled by subjects seated against a scintillation camera. Initial clearance rates of these radionuclides were determined over 6 posterior lung regions. Clearance of 99mTcO-4 (molecular weight, 163 dalton exceeded that of 99mTc-DTPA (molecular weight, 492 daltons) by an average factor of 3.3. Upper-lobe clearance of both radionuclides was greater than lower-lobe clearance in the normal subjects, but this gradient was abolished when the subjects exhaled against 7 cm of positive end-expiratory pressure. Twenty-one patients with clinical and roentgenographic evidence of interstitial lung disease (ILD) and diminished CO diffusion rates were studied with 99mTc-DTPA. Clearance of 99mTc-DTPA was increased in each of 5 patients with idiopathic pulmonary fibrosis, 4 of 8 with sarcoid, 2 of 5 with pneumoconiosis, and 2 of 3 patients with other forms of ILD. In contrast, the clearance of 99mTcO-4 was decreased in 4 patients with pulmonary alveolar proteinosis. Furthermore, no increase in 99mTc-DTPA clearance was found in 5 patients with chronic obstructive pulmonary disease. These studies suggest that the initial clearance of these aerosolized hydrophilic radionuclides is accomplished in part by diffusion through the epithelium of alveoli and respiratory bronchioles. Whereas radionuclide clearance is impaired by the presence of precipitated protein in these structures in pulmonary alveolar proteinosis, clearance is accelerated in ILD. This may indicate increased epithelial permeability in ILD related to injury and increased retractile forces.

Adult↗
PubMed1980

Sulcular exudate protein levels as an indicator of the clinical inflammatory response.

Protein concentrations in gingival fluid exudate were obtained in both the clinically minimal and severe inflammatory states. A comparison of the results indicated no significant differences in the two groups studied. Since total protein concentrations do not appear to reflect accurately the clinical inflammatory status of the gingival tissues, specific components of the protein molecule should be studied.

Gingiva↗
PubMed1980

Gingival fluid fucose to protein ratios as indicators of the severity of periodontal disease.

Total fucose and protein was measured in gingival fluid samples collected from 16 human subjects. A significantly higher total protein concentration was found for the control (clinically normal) group when compared to the experimental (severe clinical inflammation) group. This is probably due to the higher gingival flow rate found in the experimental group. The total fucose analyses showed no statistical difference between both groups, but when fucose to protein ratios were studied, a significantly higher ratio was found for the experimental group when compared to the controls. This probably represents the breakdown of plasma and tissue glycoproteins which may occur as a result of inflammation.

Adult↗