Structural flexibility of the calmodulin-binding locus in Bordetella pertussis adenylate cyclase. Reconstitution of catalytically active species from fragments or inactive forms of the enzyme

The catalytic domain of Bordetella pertussis adenylate cyclase, a calmodulin-activated enzyme with toxic properties, is a modular construct cleaved by trypsin into two subdomains of 224 (T25) and 175 (T18) amino acids. The calmodulin-binding locus of the bacterial enzyme consists of approximately 70 amino acids and overlaps the C-terminus of T25 and the N-terminus of T18. This region, exposed to the solvent or proteases, also exhibits an unusual high flexibility and allows, as demonstrated in this study, reconstitution in the presence of calmodulin of active species of adenylate cyclase from overlapping inactive fragments of the enzyme. Moreover, several combinations of inactive variants of the bacterial enzyme obtained by site-directed mutagenesis can yield active species. Heterodimers, resulting from a few selected combinations of inactive species of adenylate cyclase, exhibit specific activity similar to that of the native enzyme. Productive complementation from inactive fragments is a unique phenomenon among calmodulin-activated enzymes and represents a new and helpful tool in the understanding of the molecular mechanism of activation of B. pertussis adenylate cyclase upon binding of calmodulin.     

Identification of cardiovascular abnormalities in children with empyema thoracis by two-dimensional and Doppler echocardiography

To our knowledge, echocardiographic assessment of children with empyema has not been reported previously in the literature. Two-dimensional and Doppler echocardiography were performed in 47 children with acute (n = 23) and chronic (n = 24) empyema and 34 control subjects. Echocardiography demonstrated pericardial effusion in 11 of 47 patients (23 percent). Those with acute empyema had significantly thicker pericardium (p < 0.009) than control subjects. Tricuspid regurgitation was present in 21 of 47 patients (45 percent). The mean right ventricular internal dimension in diastole was significantly larger in patients with acute (p < 0.00002) and chronic (p < 0.006) empyema than that of control subjects. The mean tricuspid pressure gradients indicated an elevated mean right ventricular systolic pressure with increased calculated mean pulmonary arterial systolic pressures of children with acute empyema (38.5 +/- 6.4 mm Hg) and chronic (39.8 +/- 5.6 mm Hg) empyema than the normal mean (20 +/- 4 mm Hg). Children with chronic empyema had significantly less mean left ventricular internal dimension in diastole (p < 0.005) and left ventricular internal dimension in systole (p < 0.02) than control subjects. Strikingly, their mean left ventricular mass was also significantly less (p < 0.05) than that of subjects with either acute empyema or control subjects. These results provide baseline data for follow-up of children with acute and chronic empyema.     

The replacement of accessory T-lymphocytes by synthetic peptides during the formation of splenic hematopoietic colonies

Whether accessory T cells can be replaced by the synthetic immunomodulators thymogen (Glu-Trp) and thymohexin (Arg-Lys-Asp-Val-Tyr-Arg) was studied. The latter immunomodulator was found to show a 3-fold increase in splenic colony formation after incubation of bone marrow cells with rabbit antimouse brain serum (RAMBS). The former preparation failed to show the same action. Its effect was close to that of thymocytes. When the recipients exposed to lethal irradiation were administered the RAMBS-treated bone marrow cells and one of the peptides, it was shown that in concomitant administration, thymohexin and thymocytes lost their ability to restore colony formation by RAMBS-treated bone marrow. Thymogen did not suppress the stimulating activity of thymocytes. It is suggested that the differences observed between the tested peptides in their ability to recover colony formation were determined by their structure.     

A flow cytometric method for counting very low levels of white cells in blood and blood components

Reduction of white cells (WBCs) in blood components may reduce the risk of virus transmission and HLA alloimmunization. Filtration provides a means by which to achieve high-efficiency WBC reduction. A method has been developed using flow cytometry to quantitate the number of WBCs in WBC-reduced packed red cells or platelet concentrates. This method uses a detergent and propidium iodide (PI) solution to label the WBC nuclei and incorporates a known amount of fluorescein isothiocyanate (FITC)-labeled chicken red cells (cRBCs) into the mixture as an indicator of the volume examined. The number of observed WBCs per mL is calculated as follows: Number of PI WBC nuclei events/Number of FITC cRBC events x Number of FITC cRBCs added to mixture/Volume of blood in mixture. The method may allow the detection of WBCs at a concentration as low as 0.01 per microliters (10/mL) in a blood sample. It is an efficient method of collecting data, as it requires less than 10 minutes per sample. This flow cytometric technique is suitable for research purposes and for quality control of WBC-reduced blood components, because it is precise and can be used to quantitate WBCs in large or small numbers in a sample.     

'The dwindles'. Failure to thrive in older patients

Geriatric failure to thrive has three elements: deterioration in the biological, psychological, and social domains; weight loss or undernutrition; and lack of any obvious explanation for the condition. It results from the combined effects of normal aging, malnutrition, and specific physical, social, or psychological precipitants (eg, chronic disease, dementia, medication, dysphagia, depression, social isolation). Failure to thrive can be managed with a commonsense approach by primary care physicians and healthcare providers such as social workers and dietitians; extensive referral is not necessary. The key to effective care is to identify all of the precipitants and intervene early to prevent progression.     

Stabilization criteria for thermally explosive materials

Summary A previously obtained condition on the boundary temperature for the existence of a unique steady state solution of the zeroth order Arrhenius reaction equation is shown to be sufficient to guarantee decay of the temperature in the corresponding transient problem.For the heat equation we obtain a bound on the size of the initial data. The bound depends upon domain size and geometry, which may be used to ascertain when disturbances decay to zero in time.     

Cloning and expression of a plasmid pBS195 gene, determining oxygenase activity, in Escherichia coli cells

Plasmid pBS195, detected in a strain of Lactobacillus sp. isolated from long-living persons, has a broad host range, including Gram-positive and Gram-negative microorganisms [1]. Plasmid-harboring colonies of the strain Escherichia coli HB101 give a color reaction with catechol. This indicates that genes mediating the activity of oxygenase are present in this plasmid. The high activity level of this enzyme, mediated by pBS195, and substrate specificity, which has not bee detected in any known metapyrocatechases, were found in cells of E. coli. Hybridization with a 32P-labeled fragment containing the NahC gene revealed a region of homology with a 1.6-kb EcoR I- BamH I fragment of plasmid pBS195. Deletion variants of this plasmid that lost oxygenase activity confirmed the location of the oxygenase gene in this region. The gene responsible for oxygenase activity in the plasmid was cloned on the pUC19 vector in E. coli cells. The expression of the cloned gene is controlled by the lac promoter of this vector. Physical, hybridization, and deletion analyses as well as analysis of polypeptides, which are synthesized in E. coli mini-cells, showed that this activity requires the participation of a polypeptide with molecular mass of 34 kDa.