The molecular genetics of differentiation.
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Cell Differentiation↗
Biomedical subjects
L Shapiro
Publications and source records attributed to L Shapiro.
Positioning of gene products during Caulobacter cell differentiation.
Caulobacter crescentus has one of the simplest known developmental programs that exhibits both temporal and spatial organization. A hallmark of the Caulobacter cell cycle is that the progeny cells that result from each cell division differ from one another with respect to structure and developmental program. The process of establishing asymmetry prior to cell division requires that a number of gene products be targeted to a pole of the predivisional cell and consequently segregated to one of the two progeny. Several products involved in flagellar biogenesis and the chemotaxis machinery are segregated to the swarmer cell. Evidence suggests that the protein product of some fla and che genes is targeted to the incipient swarmer cell pole. In the case of other flagellar genes, it is the mRNA that is apparently segregated to the swarmer cell. Two heat shock proteins, DnaK and Lon are specifically segregated to the progeny stalked cell.
Bacterial Proteins↗
Organization and temporal expression of a flagellar basal body gene in Caulobacter crescentus.
Caulobacter crescentus assembles a single polar flagellum at a defined time in the cell cycle. The protein components of the flagellar hook and filament are synthesized just prior to their assembly. We demonstrated that the expression of a gene, flaD, that is involved in the formation of the flagellar basal body is under temporal control and is transcribed relatively early in the cell cycle, before the hook and flagellin genes are transcribed. Thus, the order of flagellar gene transcription reflects the order of assembly of the protein components. A mutation in the flaD gene results in the assembly of a partial basal body which is missing the outermost P and L rings as well as the external hook and filament (K.M. Hahnenberger and L. Shapiro, J. Mol. Biol. 194:91-103, 1987). The flaD gene was cloned and characterized by nucleotide sequencing and S1 nuclease protection assays. In contrast to the protein components of the hook and filament, the protein encoded by the flaD gene contains a hydrophobic leader peptide. The predicted amino acid sequence of the leader peptide of flaD is very similar to the leader peptide of the flagellar basal body P ring of Salmonella typhimurium (M. Homma, Y. Komeda, T. Iino, and R.M. Macnab, J. Bacteriol. 169:1493-1498, 1987).
Amino Acid Sequence↗
Continuous recording of coronary sinus oxygen saturation during atrial pacing in patients with coronary artery disease or with syndrome X.
Coronary sinus oxygen saturation was measured continuously during incremental atrial pacing in 34 patients undergoing cardiac catheterisation. In eleven patients with normal coronary arteriograms, negative exercise tests, and no ST segment depression on the electrocardiogram, an increase in the rate of atrial pacing transiently decreased coronary sinus oxygen saturation but within 20 s oxygen saturation returned to the control value. In six patients with coronary artery disease ST segment depression developed during atrial pacing. The coronary sinus oxygen saturation fell and remained reduced until pacing was discontinued. The size of the fall of coronary sinus oxygen saturation increased with increasing heart rate. In seven patients with coronary artery disease the ST segments were unaltered during atrial pacing and coronary sinus oxygen saturation did not fall. Ten patients with syndrome X were studied. In six ST segment depression developed on atrial pacing. In five, three of whom developed ST segment depression, the changes in coronary sinus oxygen saturation during atrial pacing were similar to those observed in patients without any evidence of coronary artery disease. In three, all of whom developed ST segment depression, coronary sinus oxygen saturation gradually increased throughout the period of atrial pacing. In two patients coronary sinus oxygen saturation fell in a manner similar to that observed in patients with obstructive coronary artery disease who developed ST segment depression on pacing. Thus regulation of coronary blood flow in normal persons in response to an increase of heart rate is rapid. Oxygen extraction across the coronary bed can increase by up to 30% and a persistent increase in oxygen extraction is an indicator of myocardial ischaemia. The term "syndrome X" does not describe a homogeneous group of patients but in the majority coronary sinus oxygen saturation does not fall despite symptoms and changes on the electrocardiogram, indicating that inadequate coronary blood flow is not the dominant mechanism.
Adult↗
Silent myocardial ischaemia in chronic stable angina: a study of its frequency and characteristics in 150 patients.
One hundred and fifty unselected patients with documented coronary artery disease were studied to establish the frequency and characteristics of silent myocardial ischaemia. Patients underwent ambulatory ST segment monitoring off all routine antianginal treatment (total 6264 hours) and exercise testing (n = 146). Ninety one patients (61%) had a total of 598 episodes of significant ST segment change, of which 446 (75%) were asymptomatic. Twenty seven patients (18%) had only painless episodes; 14 (9%) patients only painful episodes; 50 patients (33%) had both painless and painful episodes. The mean number of ST segment changes per day was 2.58 (1.95 silent); however, 11 patients (7%) had 50% of all silent episodes, and 48 patients (32%) had 91% of all silent episodes. Fifty nine patients (39%) had no ST segment changes on ambulatory monitoring, and 73 patients (49%) had no evidence of silent ischaemia. Episodes of silent ischaemia occurred with a similar circadian distribution to that of painful ischaemia, predominantly between 0730 and 1930. There was a similar mean rise in heart rate at the onset of both silent and painful episodes of ischaemia. Silent ischaemia was significantly more frequent in patients with three vessel disease than in those with single vessel disease, and was also significantly related to both time to 1 mm ST depression and maximal exercise duration on exercise testing. There was a highly significant relation between the mean number and duration of episodes of silent ischaemia in patients with positive exercise tests when compared with those with negative tests. No episode of ventricular tachycardia was recorded in association with silent ischaemic change.
Adult↗
Lack of effect of warfarin on the restenosis rate or on clinical outcome after balloon coronary angioplasty.
Between September 1985 and April 1987, 110 consecutive patients who had successful coronary angioplasty were included in a randomised prospective controlled evaluation of the effects of warfarin on restenosis. The warfarin (n = 56) and the control (n = 54) groups were not different in terms of age, sex, previous coronary bypass surgery or coronary balloon angioplasty, severity of symptoms, and frequency of multivessel disease or of total coronary occlusions. Warfarin was started on the day of the procedure and the dosage was adjusted to maintain the thromboplastin international normalised ratio greater than or equal to 2.5. One hundred and five (96%) of the patients were given verapamil and other antianginal drugs were prescribed as needed. Low molecular weight dextran and heparin were given during the procedure and heparin was continued for 24 hours in all patients. One hundred and eight (98%) of patients were followed up clinically after a median of five months (range 1-20). Eighty five (77%) had follow up angiography at five months. In the warfarin group symptoms improved in 46 (85%) patients by at least 1 angina class and 31 (57%) were symptom free; the exercise test remained positive in 20 (36%) patients and the angiographic restenosis rate was 25% per lesion and 29% per patient. There were no major bleeding complications. In the control group 46 (85%) patients were improved by at least 1 angina class and 31 (57%) were symptom free; the exercise test was positive in 11 (21%) patients and the angiographic restenosis rate was 33% per lesion and 37% per patient. Although the incidence of angiographic restenosis tended to be lower with warfarin, none of these differences was significant. These data suggest that the combination of verapamil and warfarin, in the absence of aspirin, is not significantly better than verapamil alone in preventing symptom recurrence or angiographic restenosis after coronary angioplasty.
Adult↗
A simplified scheme for the investigation of thyroid function in chronic liver disease.
The investigation of thyroid disease in the presence of chronic liver disease (CLD) is difficult. Conventional tests are influenced by chronic illness and abnormal concentrations of binding proteins. A three tier system is normally used; thyroxine (T4), then T3 or TSH and an index for binding proteins. The recently introduced TSH immunoradiometric assays (IRMA) offer the potential of a single test assessment of thyroid function. This was assessed by the measurement of T4, T3, THBC and the free thyroxine indices in subjects with CLD. Conventional tests showed a high number of abnormal T4 values but binding indices were normal. The TSH-IRMA results were all normal. TSH-IRMA assays are rapid, easy to use and cheap. They remove the uncertainties in assessing thyroid function in CLD and may be the test of choice.
Adolescent↗
Clinical and sociodemographic risk factors for readmission of Medicare beneficiaries.
In a random sample of Medicare beneficiaries, multiple logistic regression was used to examine clinical, sociodemographic, and insurance coverage risk factors for readmission within 60 days of discharge. The patients most likely to be readmitted were those with poor health status or with chronic diseases and those who had not had surgery. Age, marital status, living situation, and having insurance to supplement Medicare were not independently predictive of readmission risk. The dominant roles of health status, diagnosis, and surgery as predictors of readmission provide evidence that risk-adjusted readmission rates can be equitably used for quality of care studies.
Demography↗
Galactosemia detection from phenylketonuria screening.
We describe a case of classical galactosemia in which the diagnosis was first suggested by the finding of a moderately increased blood-spot phenylalanine concentration. The child was clinically unaffected at six days when the initial sample was collected. Prompt institution of dietary management averted a serious metabolic crisis.
Female↗
Asymmetric segregation of heat-shock proteins upon cell division in Caulobacter crescentus.
Three Caulobacter crescentus heat-shock proteins were shown to be immunologically related to the Escherichia coli heat-shock proteins GroEL, Lon and DnaK. A fourth heat-shock protein was detected with antibody to the C. crescentus RNA polymerase. This 37,000 Mr heat-shock protein might be related to the E. coli 32,000 Mr heat-shock sigma subunit. The synthesis of the major C. crescentus RNA polymerase sigma factor was not induced by heat shock. The E. coli GroEL protein and the related protein from C. crescentus were also induced by treatment with hydrogen peroxide. Like some of the proteins in the heat-shock protein families of Drosophila and yeast, the four heat-shock proteins in C. crescentus were found to be regulated developmentally under normal conditions. All four proteins were synthesized in the predivisional cell, but the progeny showed cell type-specific bias in the level of enhanced synthesis after heat shock. The 92,000 Mr Lon homolog and the 37,000 Mr RNA polymerase subunit were preferentially synthesized in the stalked cell, whereas the synthesis of the 62,000 Mr GroEL homolog was enhanced in the progeny swarmer cell. Furthermore, the four heat-shock proteins synthesized in the predivisional cell were partitioned in a specific manner upon cell division. The stalked cell, which initiates chromosome replication immediately upon division, received the Lon homolog, the DnaK homolog and the 37,000 Mr RNA polymerase subunit. The GroEL homolog, however, was distributed equally to both the stalked cell and the swarmer cell. These results provide access to the functions of C. crescentus heat-shock proteins under both normal and stress conditions. They also allow an investigation of the regulatory signals that modulate the asymmetric distribution of proteins and their subsequent cell type-specific expression in the initial stages of a developmental program.
Autoradiography↗
Cascade regulation of Caulobacter flagellar and chemotaxis genes.
The assembly of a functional flagellum in the bacterium Caulobacter crescentus requires the protein products of approximately 30 genes expressed in a temporally discrete and spatially distinct manner. Our current understanding of this system has been limited by the fact that purified protein products are available for only about one-fifth of these genes. A genetically engineered transposon promoter probe, Tn5-VB32, containing a promoterless gene encoding neomycin phosphotransferase II (NPTase II) was used to generate a series of non-motile (fla-), kanamycin resistant strains of C. crescentus. These transcription-fusions allow the expression of NPTase II to be controlled by flagellar promoters, and thus questions of temporal regulation of flagellar genes can be addressed without the need to obtain purified protein products. The flagellar promoters accessed by Tn5-VB32 exhibited temporal regulation analogous to the known flagellar and chemotaxis gene products. The expression of NPTase II in these mutants is read from a chimeric mRNA that initiates in a chromosomal fla promoter and continues through the inserted NPTase II gene. Thus, temporal regulation is controlled by modulating either the initiation of transcription, or transcript turnover, at specific times in the cell cycle. Epistatic interactions between the genes accessed by the promoter probe and other flagellar loci were studied in double fla mutants generated by transducing the promoter-probe mutations into spontaneously derived second-site fla-mutant backgrounds. The synthesis of both natural fla gene products and the accessed NPTase II was assayed in these strains using antisera to purified components of the flagellum and to purified NPTase II. On the basis of these interactions, a trans-acting hierarchy of flagellar and chemotaxis gene expression is proposed.
Chemotaxis↗
Identification of a gene cluster involved in flagellar basal body biogenesis in Caulobacter crescentus.
The bacterial flagellum is a complex structure composed of a transmembrane basal body, a hook, and a filament. In Caulobacter crescentus the biosynthesis and assembly of this structure is under temporal and spatial control. To help to define the order of assembly of the flagellar components and to identify the genes involved in the early steps of basal body construction, mutants defective in basal body formation have been analyzed. Mutants in the flaD flaB flaC gene cluster were found to be unable to assemble a complete basal body. The flaD BC motC region was cloned and the genes were localized by subcloning and complementation analysis. A series of Tn5 insertion mutations in the flaD BC region were mapped. Complementation analysis of the Tn5 insertion mutants indicated the existence of at least four transcriptional units in the region and identified the presence of two new genes designated flbN and flbO. Mutants in flbN, flaB, flaC and flbO were unable to assemble any basal body structure and are likely to be involved in the early steps of basal body formation. The flaD mutant, however, was found to contain a partially assembled basal body consisting of the rod and three hook-distal rings. All of the mutants in this cluster exhibited pleiotropic effects on the expression of other flagellar and chemotaxis functions, including the level of synthesis of flagellins, the hook protein and hook protein precursor, and the level of chemotaxis methylation.
Bacterial Proteins↗
Separation of temporal control and trans-acting modulation of flagellin and chemotaxis genes in Caulobacter.
The genes involved in the biogenesis of the flagellum and the chemotaxis machinery are temporally regulated during the Caulobacter crescentus cell cycle. Using plasmid complementation, we have mapped the extent of the flaY and flaE genes. These genes function in trans to regulate the expression of the flagellin genes and the chemotaxis genes. We have found that the trans regulation that modulates the amount of the flagellins and the chemotaxis proteins can be separated from the temporal control of fla and che gene expression. This conclusion is based on two observations: the low level of synthesis of flagellins and chemotaxis proteins in flaY and flaE mutant strains occurred at the correct time in the cell cycle, and complementation with plasmids containing intact flaY and flaE genes resulted in the synthesis of normal levels of flagellins and chemotaxis gene products with the maintenance of temporal cell cycle control.
Bacteria↗
DNA and RNA: NMR studies of conformations and dynamics in solution.
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DNA↗
Control of synthesis and positioning of a Caulobacter crescentus flagellar protein.
The Caulobacter crescentus flagellum is assembled during a defined time period in the cell cycle. Two genes encoding the major components of the flagellar filament, the 25K and the 27.5K flagellins, are expressed coincident with flagellar assembly. A third gene, flgJ, is also temporally regulated. The synthesis of the product of flgJ, the 29K flagellin, occurs prior to the synthesis of the other flagellin proteins. We demonstrate here that the time of initiation of flgJ expression is independent of chromosomal location but is dependent upon cis-acting sequences present upstream of the flgJ structural gene. Evidence that there is transcriptional control of flgJ expression includes the following: (1) The initial appearance of flgJ message was coincident with the onset of 29K flagellin protein synthesis, and (2) expression of an NPT II reporter gene driven by the flgJ promoter was temporally correct. Post-transcriptional regulation might contribute to the control of expression, because the flgJ mRNA persisted for a longer period of time than did the synthesis of the 29K protein. The 29K flagellin was found only in the progeny swarmer cell after cell division. In a mutant strain that failed to assemble a flagellum, the 29K flagellin still segregated to the presumptive swarmer cell, demonstrating that positioning of the protein is independent of filament assembly. Analysis of a chimeric flgJ-NPT II transcriptional fusion showed that the flgJ regulatory sequences do not control the segregation of the 29K flagellin to the swarmer cell progeny, suggesting that correct segregation depends on the protein product.
Bacteria↗
Coronary balloon angioplasty for elderly patients with severe angina.
From February 1983 to December 1986, 65 angioplasty procedures were attempted in 51 patients aged 65 or more. All had angina refractory to medical treatment and 89% of procedures were performed in patients in Canadian Cardiovascular Society angina class III or IV. Sixty eight per cent of patients had multivessel disease. Angioplasty was attempted on a single vessel in 52 instances (80%) and on multiple vessels in 13 (20%). The initial success rate was 75% for stenosis and 44% for occlusion. No attempt was made to treat all visible lesions in every case. At least one major complication occurred in 6.2% of the procedures: Q wave infarction in 4.6%, emergency surgery in 4.6%, and death in 4.6%. After a median follow up of four months (range 1-31) 37% of the patients in whom the procedure was initially successful were asymptomatic and 76% were improved by at least one Canadian Cardiovascular Society angina class. Angiographic restenosis occurred in 28% of treated lesions and 39% of patients had at least one recurrent stenosis. Among the patients with an initially successful procedure, 11% needed surgical revascularisation during the follow up period compared with 50% of those with a failed angioplasty. Balloon coronary angioplasty can be an effective form of treatment for the relief of severe angina in elderly patients.
Aged↗
Nuclear magnetic resonance and distance geometry studies of DNA structures in solution.
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Base Sequence↗
Thyroid function screening of psychiatric inpatient admissions: a worthwhile procedure?
A thyroid function screening programme of admissions to a general psychiatric service was established. Over a 22-week period 588 patients were admitted and thyroid function tests were performed on 336, a screening rate of 57.1%. Initial investigation showed that 21.3% had a total thyroxine outside the range 75-130 nmol/l. On further investigation only 9 patients (1 thyrotoxic, 8 hypothyroid) had confirmed thyroid disease. In 7 of the 9 patients the disease had been clinically suspected. Thus the screening programme identified only 2 unsuspected cases of thyroid dysfunction and we conclude that this level of detection does not warrant a formal screening programme.
Adult↗