Expression of chromosomally inserted bacillus thuringiensis israelensis toxin genes in bacillus sphaericus

Bacillus thuringiensis israelensis delta-endotoxin genes were inserted into transposon Tn917 in plasmid pTV51Ts and cloned into the chromosome of Bacillus sphaericus 2362. Many of the transformants reacted with antibody to the 135-, 128-, 65-, and 28-kDa B.t.israelensis toxin proteins and were approximately 10 times more toxic to A. aegypti larvae than the untransformed host. Some of the transformants differed physiologically and morphologically from the wild-type B. sphaericus. The toxicity of the transformed phenotype was maintained through many transfers in the absence of selective pressure. Copyright 1998 Academic Press.

Increased neutrophil function induced by bile duct ligation in a rat model.

Neutrophil function was studied in rats with common bile duct ligation. Superoxide production stimulated by phorbol myristate acetate, opsonized zymosan or formyl-methionyl-leucyl-phenylalanine; phagocytosis; and chemotaxis were significantly greater in neutrophils from rats with common bile duct ligation than in sham-operated control rats. Enhanced neutrophil activity was observed within 12 hr of bile duct ligation; it remained increased during the 15-day study. Preincubation of neutrophils from control rats with sera of rats with common bile duct ligation did not increase superoxide generation. This suggests that the high superoxide production observed in neutrophils of rats with common bile duct ligation was not an immediate effect of the serum. Neutrophils of rats with portal vein ligation exhibited normal activity, indicating that portal systemic shunting per se is not the underlying mechanism for increased activity. The elevated levels of AST and alkaline phosphatase, indicating liver damage, that appeared within 12 hr of bile duct ligation correlated with the increased superoxide generation.

Membrane-protein phosphorylation in the Bacillus subtilis cell cycle.

Sphingosine, an inhibitor of Ca(2+)-dependent protein kinases in eukaryotic cells, inhibited initiation of DNA replication in Bacillus subtilis at a concentration of 10 microM, without inhibiting elongation. The tumor promoter 12-tetradecanoyl 13-phorbol acetate, (TPA), an activator of protein kinase C in eukaryotic cells, partially counteracted the inhibition of initiation by sphingosine. Phosphorylation of polypeptides was observed in vivo at initiation of DNA replication in B. subtilis. Sphingosine, TPA, and vancomycin affected this protein phosphorylation.

Bromosulfophthalein disposition in chronically bile duct obstructed rats.

The disposition of bromosulfophthalein was studied in chronically bile duct obstructed rats. In this model a catheter was inserted into the common bile duct and the distal tip was sealed. Resumption of bile flow was achieved with great ease. Obstruction of bile duct for 18 days in rats resulted in elevated bilirubin, ALT, AST, and alkaline phosphatase levels. Portal hypertension developed within this period (11.6 +/- 0.5 in obstructed rats vs. 8.6 +/- 0.6 mm Hg in sham-operated group). After the bile duct obstruction was opened, the half-life time for elimination of bromosulfophthalein (42.30 +/- 6.47 min) was longer than in sham-operated rats (21.23 +/- 3.34 min). Plasma clearance was reduced by 70% in bile duct obstructed rats. In spite of increased bile flow rate, biliary excretion of the dye was reduced by 40% in chronically bile duct obstructed rats. Hepatic glutathione levels were significantly reduced by 20% in this model. The specific activity of glutathione S-transferase with chlorodinitrobenzene and styrene oxide, as substrates, was reduced by 50% and 30%, respectively. However, the percent of conjugated bromosulfophthalein in bile was similar to that of sham-operated rats.

Environmental and genetic factors of hypertension in a biracial Beduin population.

This study attempts to understand the various factors involved in the pathophysiology of hypertension in black Beduins. Parameters known to differentiate US black from white hypertensives were examined. Sixty Beduin families (thirty families each of black and white, total of 205 subjects) were evaluated for environmental risk factors: a traditional nomad shepherd life-style compared with working in a city, living in tents or in western style housing and dietary habits related to cardiovascular risk factors. Blood pressure, body mass index (BMI), sodium-lithium counter transport rate and 24 hour urinary sodium excretion (UNa) were measured and the data obtained were compared between normotensives and hypertensives, within each racial group. The mean value of the BMI of the white population was greater than that of the black population while the BMI of hypertensives was greater than that of the normotensives in each of the racial groups. The mean systolic BP of black hypertensives was greater than that of the corresponding whites. There were no significant differences in UNa between the four groups. Sodium-lithium countertransport was significantly higher in the hypertensive whites compared with the normotensive population (0.46 versus 0.22 mmol Li efflux/IRBC/hr). The countertransport rate for black hypertensives was lower than that of white hypertensives (0.20 versus 0.46). Black families had lower socio-economic scores than did white families and families with a hypertensive member scored lower than did families with a normotensive history. These results demonstrate some similarities between the American and Beduin black hypertensive populations, in spite of entirely different life-styles, indicating that in these populations genetic factors, rather than environmental influences, appear to be dominant in the pathophysiology of hypertension.

Risk indicators after envenomation in humans by Echis coloratus (mid-east saw scaled viper).

To determine the frequency, severity and predictors of bleeding and azotemia after envenomation in humans by Echis coloratus, a retrospective survey of 68 cases in Israel between 1970 and 1989 was carried out. We used univariate and multivariate analyses of clinical variables on admission for the outcome variables of bleeding, hemoglobin and platelet levels, and blood urea. Within hours or days after envenomation, a major bleeding episode occurred in 18% of the victims, a drop in hemoglobin to 10 g/dliter or less in 14%, and an increase in blood urea to 9 mmole/liter or more in 15%. These complications correlated with time interval between envenomation and hospital admission, and the following admission variables: degree of bleeding, hemoglobin level, platelet and white blood cell counts, blood urea and proteinuria. Complications were unlikely in patients who were presented with all of the following: a hemoglobin level of 13 g/dliter or more, a platelet count of 100,000/mm3 or more, a blood urea level of 7 mmole/liter or less, no proteinuria and no bleeding. Treatment on admission with a specific monovalent antiserum was associated with a shorter duration of hemostatic failure and a reduced incidence of anemia and thrombopenia. Infusion of fresh frozen plasma on admission did not appear to be effective in preventing complications.

The possible involvement of trypsin-like enzymes in germination of spores of Bacillus cereus T and Bacillus subtilis 168.

Germination of spores of Bacillus cereus T and Bacillus subtilis 168 was inhibited by the trypsin inhibitors leupeptin and tosyllysine chloromethyl ketone (TLCK) and by the substrates tosylarginine methyl ester (TAME), benzoyl-L-arginine-p-nitroanilide (L-BAPNA) and D-BAPNA. Potencies of these inhibitory compounds were estimated by finding the concentration which inhibited 50% germination (ID50), as measured by events occurring early (loss of heat resistance), at an intermediate stage [dipicolinic acid (DPA) release], and late in germination (decrease in optical density). In B. cereus T, all the compounds inhibited early and late events with the same ID50. In B. subtilis, TAME inhibited early and late events at the same ID50, but all other inhibitors had a lower ID50 for late events than for early events. This suggests that a trypsin-like enzyme activity is involved at two sequential stages in the germination of B. subtilis spores, one occurring at or before the loss of heat resistance and one at or before the decrease in optical density. Different trypsin-like activities were detected in broken dormant spores and germinated spores of B. cereus T and in germinated spores of B. subtilis by means of three chromogenic substrates: benzoyl-L-phenylalanyl-L-valyl-L-arginine-p-nitroanilide (L-PheVA), L-BAPNA and D-BAPNA. Separation of extracts of germinated spores on non-denaturing polyacrylamide gels showed that in both species the substrates were hydrolysed by three distinct enzymes with different electrophoretic mobilities. The three enzymes had different Ki values for the above inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and expression of Bacillus thuringiensis israelensis delta-endotoxin DNA in B. sphaericus.

Bacillus thuringiensis israelensis delta-endotoxin genes were cloned into Bacillus sphaericus 2362, producing stable transformants reacting with antibody to the 28- and 65-kDa B. thuringiensis israelensis crystal proteins and approximately 10 times more toxic to Aedes mosquito larvae than the original host strain. The LC50 after 48 hr of exposure of Aedes larvae to the most active transformed clone was 0.19 microgram/ml, compared with an LC50 of 1.9 microgram/ml for B. sphaericus 2362 and less than 0.1 microgram/ml for B. thuringiensis israelensis. The cloning vector, plasmid pPL603E, was also effective in transforming B. subtilis 1E20 with B. thuringiensis israelensis DNA, producing highly toxic clones with less stable gene expression than the clones of B. sphaericus.

Hemodynamic characterization of conscious and ketamine-anesthetized bile duct-ligated rats.

The present study was conducted to characterize the hemodynamic alterations in common bile duct-ligated (CBDL) rats under ketamine anesthesia and in the awake restrained state. Hemodynamic studies using the radioactive microspheres technique were performed 17.6 +/- 0.6 (SE) days after bile duct ligation or sham operation. CBDL rats had lower mean arterial pressure, reduced systemic and renal resistance, and increased renal blood flow compared with sham-operated rats. This was found both in the conscious and anesthetized states. Anesthetized CBDL rats had higher portal pressure (13.2 +/- 0.5 vs. 9.2 +/- 0.4 mmHg; P less than 0.001) and lower splanchnic arteriolar resistance (15.4 +/- 1.3 vs 26.8 +/- 4.6 mmHg.ml-1.min.100 g body wt; P less than 0.05) than sham-operated rats. Portosystemic shunting was 52.3 +/- 11.7% in CBDL and negligible in sham-operated rats. The last three parameters could not be measured in conscious animals. Total peripheral resistance was lower in the conscious than in the anesthetized state, diverting a higher fraction of cardiac output at the expense of splanchnic organs and leading to a significant reduction of portal venous inflow in sham-operated but not in CBDL rats [3.36 +/- 0.47 vs. 5.38 +/- 0.65 (P less than 0.05) and 5.33 +/- 0.58 vs. 6.34 +/- 0.37 ml.min-1.100 g body wt-1 (P = NS), respectively]. These findings indicate that CBDL and normal rats respond differently to anesthesia and restraint. Because the restrained state is stressful and studies in anesthetized animals are technically simpler, provide additional information such as portal pressure and portosystemic shunting, and diminish animal suffering, we suggest that hemodynamic studies in rats, using the microsphere technique, should be preferably performed under ketamine anesthesia.

Abnormalities in bilirubin and liver enzyme levels in adult patients with bacteremia. A prospective study.

Eighty-four patients with bacteremia were surveyed prospectively for biochemical markers of liver damage. Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and bilirubin levels were elevated in 44 (53%), 39 (47%), 45 (54%), and 5 (6%) of the patients on the first determination (2.0 +/- 0.1 days after onset of fever) and in 11 (13%), 17 (20%), 26 (31%), and 1 (1%) on the second determination (5.4 +/- 0.2 days after onset of fever), respectively. The elevation rarely exceeded three times the upper limit of normal. One patient had severe jaundice. An abnormality of at least one of these values was found in 55 patients (65%). There were no differences in site of infection, bacteria isolated, and outcome between patients with and without biochemical abnormalities. We conclude that in adult patients with bacteremia, elevation of liver enzymes and bilirubin is common, usually mild, of short duration, and of no prognostic significance.

Dead Sea water poisoning.

A unique chemical intoxication by Dead Sea water and its resultant physiological effects of combined severe hypercalcemia and hypermagnesemia are described. Of 48 adult patients, 16 had at least one severe clinical manifestation of either disturbed sensorium or a syndrome similar to adult respiratory distress syndrome. The patients had tachyarrhythmias (11 of 38, 29%), conduction disturbance (nine of 37, 24%), and a normal QTc interval in the presence of severe hypercalcemia. Combined respiratory and metabolic acidosis and concomitant extreme hypercalcemia and hypermagnesemia, reaching serum levels up to 28.8 and 33 mg/dL, respectively, were responsible for most of the clinical findings. The adult mortality was 19%. Four pediatric patients with variable degrees of intoxication survived. Discriminant function analysis determined that admission serum calcium concentration of more than 15.5 mg/dL was the best predictor of mortality. In patients with severe intoxication, supportive medical treatment, including forced diuresis, was disappointing. Early dialysis for severe cases merits further study.

Bacteria of the genus Bacillus have a hydrolase stereospecific to the D isomer of benzoyl-arginine-p-nitroanilide.

A stereospecific enzyme activity capable of cleaving the amide bond of the synthetic substrate N-benzoyl-D-arginine-p-nitroanilide (D-BAPA) has been found in all aerobic and anaerobic members of the family Bacillaceae tested by us. Cells of nonsporeforming gram-positive or gram-negative bacteria contain a hydrolase activity stereospecific to N-benzoyl-L-arginine-p-nitroanilide. The D-BAPA-hydrolyzing enzymes (D-BAPAases) of mid-logarithmic-phase cells of Bacillus subtilis 168 and B. cereus T were compared. These enzymes had the same molecular weight of approximately 66,000 in gel filtration and the same electrophoretic mobility after electrophoresis on polyacrylamide gels. The D-BAPAases of B. subtilis 168 and B. cereus T differed in the effect of inhibitors on enzymatic activity. While both hydrolases were inhibited by tosyl-L-lysine chloromethyl ketone and tosyl-L-arginine-methyl ester as well as leupeptin, only the D-BAPAase of B. cereus T was inhibited by p-chloromercuribenzene sulfonic acid. The D-BAPAases of B. subtilis and B. cereus T had a Michaelis constant for D-BAPA of 2.9 x 10(-5) M and 1.4 x 10(-4) M, respectively. D-BAPAase is an intracellular enzyme localized in the protoplast (80 to 90% in soluble form in the cytoplasm). The ability to cleave D-BAPA is suggested as an additional chemotaxonomic characteristic of sporeforming bacteria of the genera Bacillus and Clostridium.

"Intermittent" and transient ST-segment elevation following direct current cardioversion.

Transient ST-segment elevation immediately following direct current cardioversion is a clearly documented occurrence in a small percentage of cases. Two main explanations have been suggested: myocardial injury or coronary vasospasm. We report two cases of "intermittent" and transient ST-segment elevation after cardioversion. Such intermittent elevation of the ST segment has not been reported previously. We hypothesize that this electrocardiographic observation is explained by a timing difference induced by electroshock in the action potential between the epicardium and endocardium.

Membrane binding and release of Bacillus subtilis DNA as a function of the cell cycle.

The presence of origin-region DNA in preparations containing bacterial cell wall and cytoplasmic membrane is well established, but little is known about the relationship between this association and events of the cell cycle. We have observed, during renewed growth of stationary-phase cultures of Bacillus subtilis, an association of DNA, including newly synthesized regions, with a specific region of the plasma membrane. Attachment was transitory, occurring once per replication cycle, and was prevented by inhibitors of cell wall synthesis.