Phosphorylation-independent activity of the response regulators AlgB and AlgR in promoting alginate biosynthesis in mucoid Pseudomonas aeruginosa

Overproduction of the capsular polysaccharide alginate appears to confer a selective advantage for Pseudomonas aeruginosa in the lungs of cystic fibrosis patients. The regulators AlgB and AlgR, which are both required as positive activators in alginate overproduction, have homology with the regulator class of two-component environmental responsive proteins which coordinate gene expression through signal transduction mechanisms. Signal transduction in this class of proteins generally occurs via autophosphorylation of the sensor kinase protein and phosphotransfer from the sensor to a conserved aspartate residue, which is present in the amino terminus of the response regulator. Recently, kinB was identified downstream of algB and was shown to encode the cognate histidine protein kinase that efficiently phosphorylates AlgB. However, we show here that a null mutation in kinB in a mucoid cystic fibrosis isolate, P. aeruginosa FRD1, did not block alginate production. The role of the conserved aspartate residue in the phosphorylation of AlgB was examined. The predicted phosphorylation site of AlgB (D59) was mutated to asparagine (N), and a derivative of an AlgB lacking the entire amino-terminal phosphorylation domain (AlgB delta1-145) was constructed. A hexahistidine tag was included at the amino terminus of the wild-type (H-AlgB), H-AlgB delta1-145, and mutant (H-AlgB.59N) AlgB proteins. These derivatives were purified by Ni2+ affinity chromatography and examined for in vitro phosphorylation by the purified sensor kinase protein, KinB. The results indicated that while KinB efficiently phosphorylated H-AlgB, no phosphorylation of H-AlgB delta1-145 or H-AlgB.D59N was apparent. An allelic exchange system was developed to transfer mutant algB alleles onto the chromosome of a P. aeruginosa algB mutant to examine the effect on alginate production. Despite the defect in AlgB phosphorylation, P. aeruginosa strains expressing AlgB.D59N or H-AlgB delta1-145 remained mucoid. The roles of the conserved aspartate residues in the phosphorylation of AlgR were also examined. As seen with AlgB, mutations in the predicted phosphorylation site of AlgR (AlgR.D54N and AlgR.D85N) did not affect alginate production. These results indicate that in vivo phosphorylation of AlgB and AlgR are not required for their roles in alginate production. Thus, the mechanism by which these response regulators activate alginate genes in mucoid P. aeruginosa appears not to be mediated by conventional phosphorylation-dependent signal transduction.     

Rapid p-Nitroaniline Test for Assessing Microbial Quality of Refrigerated Meat

The p-nitroaniline test, a rapid method to estimate microbial quality of refrigerated meat, was applied to many retail meat samples from several animal species to confirm its applicability. This method allows determination of psychotroph levels between 10⁴ and 10⁸ CFU/cm² in about 2.5 hr. A good correlation was found (r = 0.88, p < 0.001) between this method and the standard plate count technique. Because of the yellow color of the p-nitroaniline released during reaction, bacterial loads higher than 10^s CFU/cm² develop a color intense enough to be detected visually.     

Sleep complaints in chronic postconcussion syndrome

This study was designed to identify the incidence of sleep complaints in 39 patients with chronic postconcussion syndrome compared to those of a control group of 27 patients with orthopedic injuries. Patients with orthopedic injuries were selected as a comparison group to control for the effects of traumatic injury or chronic pain. There were no differences in age, sex distribution, or time from injury between the patient groups. Type and frequency of sleep disturbances in a mean two-year postinjury period were evaluated. Patients with chronic postconcussion syndrome reported more difficulty in initiating and maintaining sleep at night and greater difficulty with sleepiness during the day.     

Expression of an exogenous eukaryotic DNA methyltransferase gene induces transformation of NIH 3T3 cells

Abnormal regional increases in DNA methylation, which have potential for causing gene inactivation and chromosomal instability, are consistently found in immortalized and tumorigenic cells. Increased DNA methyltransferase activity, which is also a characteristic of such cells, is a candidate to mediate these abnormal DNA methylation patterns. We now show that, in NIH 3T3 mouse fibroblasts, constitutive overexpression of an exogenous mouse DNA methyltransferase gene results in a marked increase in overall DNA methylation which is accompanied by tumorigenic transformation. These transformation changes can also be elicited by dexamethasone-inducible expression of an exogenous DNA methyltransferase gene. Our findings provide strong evidence that the increase in DNA methyltransferase activity associated with tumor progression could be a key step in carcinogenesis and provide a model system that can be used to further study this possibility.     

Coronary revascularization after myocardial infarction in the very elderly: outcomes and long-term follow-up

OBJECTIVE: To determine the outcome of very elderly patients who had coronary revascularization during hospitalization for an acute myocardial infarction. DESIGN: Retrospective cohort study. SETTING: Community-based tertiary-care teaching hospital. PATIENTS: A total of 1215 consecutive patients 80 years and older were hospitalized with a myocardial infarction between 1985 and 1990. The study sample included all 93 patients (8%) who had cardiac catheterization before discharge and had not been excluded from study because of the following: severe valvular disease, absence of significant coronary disease, or death before a decision about revascularization could be made. MEASUREMENTS: Survival, quality of life, and functional status at least 1 year after discharge. RESULTS: After catheterization, 41 patients had angioplasty, 18 had coronary artery bypass surgery, and 34 did not have revascularization. Among the patients alive at discharge, those who had revascularization had a high likelihood of achieving a good or excellent quality of life (angioplasty, 86% [31 of 36]; surgery, 89% [16 of 18]; medical therapy, 44% [11 of 25]) and of being able to care for themselves (angioplasty, 89% [32 of 36], surgery, 89% [16 of 18], medical therapy, 52% [13 of 25]). Mortality rates at 1 year were 24% (95% CI, 15% to 47%) for the angioplasty group, 6% (CI, 0% to 27%) for the surgery group, and 44% (CI, 27% to 62%) for the medical therapy group. In a Cox proportional hazards model that adjusted for clinical, demographic, hemodynamic, and anatomic differences between the groups, the performance of coronary revascularization was associated with increased survival (hazard ratio, 0.42; CI, 0.18 to 0.98). CONCLUSIONS: A small percentage of very elderly patients with complicated acute myocardial infarctions, selected by their physicians for invasive cardiovascular procedures, can tolerate these procedures, avoid serious complications, return to independent living, and have excellent probability of survival. Although our results suggest that coronary revascularization may have benefited these patients, the study design did not permit definite conclusions, and future studies are needed to resolve this important question.     

Splenic lymphoid tissue of mice after irradiation with fast carbon ions

Reorganization of cellular content of splenic functional zones was studied morphometrically in mice after single irradiation with fast carbon ions. It was demonstrated that irradiation does not cause significant changes in microtopography of splenic structures but is accompanied with changes in cytoarchitecture and rearrangement of lymphoid cells throughout the experiment. Two arbitrary stages are determined in lymphoid tissue response. The first one (30 hrs-7 d) is characterized with destructive processes intensification and decrease of small lymphocytes number in all functional zones of the organ. Strong plasmocellular reaction was observed in the organ except for the lymphoid nodules mantle zone in this period as well. On the second stage (15-60 d) orientation of plasmocellular reaction changes along with the tendency of small lymphocytes to restore population (up to 57-79% from the control level on d 60 of the experiment. As a result plasma cell content decreases sharply in all the organ structures. Low level of lymphocytopoiesis was demonstrated in the course of the experiment resulting from cell mitotic activity decline following the irradiation. At the distant period (d 60 following the irradiation) another decrease of cell lymphoid population was noted, which indicates that irradiation with fast carbon ions is suppressive to the animal immune system.