Immunocytochemical localization of a calmodulinlike protein in Bacillus subtilis cells.

To determine possible functions of the calmodulinlike protein of Bacillus subtilis, the time course of its expression during sporulation and its cellular localization were studied. The protein was expressed in a constitutive manner from the end of logarithmic growth through 8 h of sporulation as determined by antibody cross-reactivity immunoblots and enzyme-linked immunosorbent assays (ELISAs). In partially purified extracts, the immunopositive protein comigrated upon electrophoresis with a protein which selectively bound [(45)Ca]CaCl(2), ruthenium red, and Stains-all. Previous studies showed increased extractability of the calmodulinlike protein from B. subtilis cells when urea and 2-mercaptoethanol were used in breakage buffers, implying that the protein might be partially associated with the membrane fraction. This was confirmed by demonstrating that isolated membrane vesicles of B. subtilis also gave positive immunological tests with Western blotting and ELISAs. To more precisely locate the protein in cells, thin sections of late-log-phase cells, sporulating cells, and free spores were reacted first with bovine brain anticalmodulin specific antibodies and then with gold-conjugated secondary antibodies; the thin sections were examined by transmission electron microscopy. The calmodulinlike protein was found almost exclusively associated with the cell envelope of these fixed, sectioned cells. A possible function of the calmodulinlike protein in sensing calcium ions or regulating calcium ion transport is suggested.

Preparation of general proteinase substrates using 3,5-dinitrosalicylaldehyde.

To search for new proteinases in Bacillus subtilis we have developed a general method for synthesizing chromogenic proteinase substrates using 3,5-dinitrosalicylaldehyde (DNSA). Hammersten casein and soluble protein from extracts from B. subtilis cells were labeled with DNSA in the presence of NaBH4. After dialysis (pH 7.8), the resultant 3,5-dinitro-2-hydroxybenzyl-casein (DNHB-casein) and DNHB-bacterial cell protein solutions were a light orange color. A model compound, N-benzyl-3,5-dinitro-2-hydroxybenzylamine was synthesized and estimated to have a molar absorption coefficient of 14,100 M-1 cm-1 at 366 nm at pH 8, which was used to calculate dye loading on casein. Chromogenic substrates prepared in this way should retain positive charges on lysine residues. DNHB-casein and DNHB-bacterial cell protein were incubated with varying concentrations of subtilisin BPN' for varying times, precipitated with trichloroacetic acid and centrifuged. The acid-soluble supernatant fractions were made basic with NaOH and absorbances were measured at 366 nm, the absorption maximum. Color production was proportional to subtilisin concentration and times of incubation; under the assay conditions used, the limit of detection of subtilisin was about 100 ng. Five proteinase activities were detected in soluble extracts of B. subtilis using DNHB-labeled proteins as substrates.

Primary aortoesophageal fistula caused by an atherosclerotic thoracoabdominal aortic aneurysm: a case report and review of the literature.

A 55-year-old woman suffered an episode of massive hematemesis caused by an aortoesophageal fistula from an atherosclerotic thoracoabdominal aortic aneurysm. In situ grafting of the thoracic portion of the aneurysm was followed by sepsis and a sinus tract between the mid-esophagus and the aortic prosthesis. Graft removal, aortic closure, esophageal closure and axillobifemoral bypass allowed clearing of the sepsis and recovery. Severe hypertension followed aortic closure and extra-anatomic bypass and resulted in the eventual death of the patient 16 months later from dissection of the ascending aorta with pericardial tamponade. There are very few treated cases of aortoesophageal fistulas caused by atherosclerotic aneurysms reported in the literature. Furthermore, there are no reported cases where the aorta was closed at the level of the subclavian artery with extra-anatomic bypass to restore blood flow to the lower half of the body.

Structure of 4-carboxy-2-nitrobenzeneboronic acid.

4-(Dihydroxyboryl)-3-nitrobenzoic acid, C7H6BNO6, M(r) = 210.94, monoclinic, P2(1)/n, a = 10.542 (2), b = 6.411 (1), c = 13.105 (4) A, beta = 106.47 (2) degrees, V = 849.3 (4) A3, Z = 4, Dm = 1.65 (flotation in CCl4/1,2-dibromoethane), Dx = 1.649 Mg m-3, lambda(Mo K alpha) = 0.71073 A, mu = 0.135 mm-1, F(000) = 432, T = 293 K, R = 0.0530 for 1328 observed reflections with F > 2 sigma(F). The molecule is flat [the carboxy and nitro groups are rotated 5.8 (4) and 1.9 (4) degrees, respectively, out of the plane] with the boronic acid group almost normal to the plane of the benzene ring, 92.4 (3) degrees. The B atom and one O atom of the nitro group are separated by only 2.457 (4) A implying an interaction that is consistent with observed chemical behavior.

Comparison of pentoxifylline pharmacokinetics between smokers and nonsmokers.

Pentoxifylline is a synthetic xanthine derivative and is hepatically cleared. The natural dimethylxanthines theobromine and theophylline have been shown to have enhanced metabolism in smokers when compared with nonsmokers. Subsequently, the effect of smoking on pentoxifylline plasma concentrations was investigated. Twenty healthy volunteers (10 smokers and 10 nonsmokers) received pentoxifylline 400 mg as a controlled-release tablet every 8 hours for 17 doses. Several blood samples were collected for 8 hours after the final dose. These samples were assayed for pentoxifylline and its metabolites. The mean values of the smokers were compared with those of the nonsmokers. With respect to pentoxifylline, no statistically significant differences in maximum concentration and time of maximum concentration were observed between the two groups. Although no statistical differences in plasma concentrations and area-under-the-curve at steady state (AUCss) were observed, the oral clearance of pentoxifylline among the smokers (.22 +/- .08 L/minute/kg) was significantly greater (P < .05) than that among the nonsmokers (0.15 +/- 0.06 L/minute/kg) when corrected for body weight. With respect to the pentoxifylline metabolite 1-(5-hydroxy-hexyl)-3,7-dimethylxanthine (MI), the maximum concentration and AUCss of the smokers were significantly decreased when compared with the nonsmokers. The AUCss of the smokers was 1438 +/- 819 ng.hour/mL and of the nonsmokers was 2864 +/- 1375 ng.hour/mL (P < .02). The results of this trial suggest that smoking tends to reduce pentoxifylline plasma concentrations and significantly reduces MI plasma concentrations.

Boronic Acid matrices for the affinity purification of glycoproteins and enzymes.

Boronic acids immobilized on insoluble matrices have been used for over a decade to purify proteins and enzymes (1,2). Boronic acid matrices have been used to purify: 1. Glycoproteins, 2. Enzymes for which a boronic acid moiety is an inhibitor, and 3. Enzymes that bind to specific dial-containing cofactors (such as UTP or NADP(+)).

Purification and properties of an intracellular calmodulinlike protein from Bacillus subtilis cells.

Although calcium ions are crucial in a variety of bacterial processes, including spore development, reports of calmodulin in procaryotes have been few. We have purified to homogeneity a calmodulinlike protein (CaLP) from sporulating cells of Bacillus subtilis grown in a chemically defined sporulation medium; purification involved heat treatment, fractionation with ammonium sulfate, affinity chromatography, and gel filtration on high-performance columns. The protein was eluted from a phenothiazine affinity column in a calcium ion-dependent manner, stained poorly with Coomassie blue and silver stain dyes, bound poorly to nitrocellulose filters, and was not an inhibitor of the major intracellular serine proteinase. It stimulated bovine brain phosphodiesterase in a dose- and Ca2(+)-dependent manner and stimulated NAD kinase from peas in a dose-dependent manner. The B. subtilis calmodulin reacted with anti-bovine brain calmodulin antibodies in enzyme-linked immunoabsorbance assays. The amino acid composition data showed it to be distinctly different from eucaryotic calmodulins, having particularly high levels of serine and glycine. The pI of the protein was estimated to be 4.9 to 5.0. The molecular weight was estimated to be 23,000 or 25,000, based on amino acid composition and detergent gel electrophoresis, respectively. The protein reacted with rhodamine isothiocyanate, which blocked its enzyme-activating capacity and greatly increased its electrophoretic mobility and Coomassie dye-binding ability.

Energy and calcium ion dependence of proteolysis during sporulation of Bacillus subtilis cells.

Bacterial cells degrade intracellular proteins at elevated rates during starvation and can selectively degrade proteins by energy-dependent processes. Sporulating bacteria can degrade protein with apparent first-order rate constants of over 0.20 h-1. We have shown, with an optimized [14C]leucine-labeling and chasing procedure, in a chemically defined sporulation medium, that intracellular protein degradation in sporulating cells of Bacillus subtilis 168 (trpC2) is apparently energy dependent. Sodium arsenate, sodium azide, carbonyl cyanide m-chlorophenylhydrozone, and N,N'-dicyclohexylcarbodiimide, at levels which did not induce appreciable lysis (less than or equal to 10%) over 10-h periods of sporulation, inhibited intracellular proteolysis by 13 to 93%. Exponentially growing cells acquired arsenate resistance. In contrast to earlier reports, we found that chloramphenicol (100 micrograms/ml) strongly inhibited proteolysis (68%) even when added 6 h into the sporulation process. Restricting the calcium ion concentration (less than 2 microM) in the medium had no effect on rates or extent of vegetative growth, strongly inhibited sporulation (98%), and inhibited rates of proteolysis by 60% or more. Inhibitors of energy metabolism, at the same levels which inhibited proteolysis, did not affect the rate or degree of uptake of Ca2+ by cells, which suggested that the Ca2+ and metabolic energy requirements of proteolysis were independent. Restricting the Ca2+ concentration in the medium reduced by threefold the specific activity in cells of the major intracellular serine proteinase after 12 h of sporulation. Finally, cells of a mutant of B. subtilis bearing an insertionally inactivated gene for the Ca2(+)-dependent intracellular proteinase-1 degraded protein in chemically defined sporulation medium at a rate indistinguishable from that of the wild-type cells for periods of 8 h.

Boronic acids for affinity chromatography: spectral methods for determinations of ionization and diol-binding constants.

Arylboronic acids attached to solid matrices have proved useful for the diol-specific chromatography of biomolecules and affinity purification of enzymes by exchangeable-ligand chromatography. The latter use has been limited by the intrinsic ionization constant (pKa approximately 9) of the most common commercial products. The synthesis of several arylboronic acids with ionization constants near neutrality are described, and the application of a new general, spectral-difference method for determining acid ionization constants and formation constants with fructose is developed. In particular 4-(N-methyl) carboxamido-benzeneboronic acid was found to have a pKa of 7.86 and a formation constant with D-fructose of 8600. It was stable toward acid or base hydrolysis. We suggest that 4-carboxybenzeneboronic acid might be useful for preparing matrices for enzyme affinity chromatography.

Alteration of pentoxifylline pharmacokinetics by cimetidine.

Pentoxifylline, recently approved for the treatment of intermittent claudication, is hepatically cleared with a high degree of first-pass metabolism. Subsequently, the effect of cimetidine on pentoxifylline pharmacokinetics was studied in humans. Ten healthy subjects received, in random cross-over fashion, pentoxifylline 400 mg as a controlled-release tablet every 8 hours with and without cimetidine 300 mg four times a day for 7 days. Pentoxifylline and metabolite plasma concentrations over one dosing interval were measured on day 7 of each phase. The unavailability of an immediate-release pentoxifylline dosage form prevented a single dose trial. Cimetidine significantly increased (P less than .05) pentoxifylline area under the curve at steady state 26.2% from 675 +/- 97 (mean +/- SEM) to 852 +/- 108 ng. hr/mL. The average steady-state plasma concentration increased 27.4% from 84 +/- 12 to 107 +/- 14 ng/mL (P less than .05). Apparent oral clearance decreased 21.5% from 1309 +/- 304 to 1027 +/- 244 mL/min (P less than .02). Significant alterations in pentoxifylline metabolite concentrations were also observed. The results of this trial suggest cimetidine elevates pentoxifylline plasma concentrations, presumably by decreasing apparent oral clearance, although a reduction in total body clearance or an increase in gastric absorption could not be ruled out.

Protein turnover and proteolysis during sporulation of Bacillus subtilis.

A two-dimensional electrophoretic method was used to show that protein degradation occurs immediately after the end of exponential growth but that its occurrence is masked in the usual assay methods for a 2-h period and that degradation is apparently nonselective with respect to protein molar mass or charge. The results suggest that considerable reutilization of internal amino acids may occur during sporulation regardless of the size of the external chase. Finally, the levels of intracellular proteinase activities present even at the end of exponential phase growth, as measured in vitro, are sufficient to account for the maximum rates of protein degradation observed in vivo.

Vitamin B12 and monensin effects on performance, liver and serum vitamin B12 concentrations and activity of propionate metabolizing hepatic enzymes in feedlot lambs.

Monensin in ruminant diets increases production of propionic acid. We have tested the hypothesis that propionic acid may be elevated to such an extent by monensin that it cannot be optimally metabolized by the methyl malonyl-CoA pathway requiring vitamin B12 (B12) in the liver. Thus, the effects of weekly B12 injections (10 mg X head-1 X wk-1, intramuscularly) with and without dietary monensin (25 mg/kg diet) on average daily gain (ADG), dry matter intake (DMI), feed to gain ration (F/G), liver and serum B12 concentrations and liver activity of propionate metabolizing enzymes were examined in an 84-d trial. Sixteen lambs (27.5 kg average initial wt) were assigned randomly to one of four treatments in a factorial arrangement: monensin plus B12, monensin without B12, no monensin plus B12 and no monensin without B12. Lambs were fed an 80% concentrate diet and slaughtered at the end of the trial. Liver samples were obtained by biopsy on d 0 and at slaughter on d 84 to determine activity of propionate metabolizing enzymes and B12 concentrations. Serum samples were taken on d 0, 28, 56 and 84 to determine serum B12 concentration. Neither monensin nor B12 affected (P greater than .10) ADG, DMI or F/G. Lambs receiving B12 had higher (P less than .01) serum B12 concentrations, but this was not reflected (P greater than .10) in higher liver B12 concentrations. No difference (P greater than .10) in liver propionate metabolizing activity among treatments was detected; however, monensin decreased (P less than .05) fumarate and malate formation by liver homogenates. Liver B12 concentrations were highly correlated with endogenous propionate metabolizing activity at d 0 (r = .73, P less than .01) and d 84 (r = .51, P less than .05). Results suggest no advantage to providing supplemental B12 to lambs fed monensin-supplemented, high-concentrate diets.

Activation of intracellular serine proteinase in Bacillus subtilis cells during sporulation.

Cells of Bacillus subtilis 168 (trpC2) growing and sporulating in a single chemically defined medium carried out intracellular protein degradation and increased their levels of intracellular serine protease-1 in a manner very similar to what had previously been reported for cells sporulating in nutrient broth. The results were interpreted to mean that these processes are intrinsic to sporulation rather than medium dependent. To determine the cause of these increases in specific activity of proteinases, we purified the protease, prepared rabbit immunoglobulins directed against it, and monitored changes in protease antigen levels by performing rocket immunoelectrophoresis. In cells sporulating in nutrient broth, the protease antigen levels increased about 7-fold, whereas the specific activity increased about 150-fold, for an activation of about 20-fold. In cells sporulating in the single chemically defined sporulation medium, the protease antigen increased about 10-fold, whereas the specific activity increased at least 400-fold, for an activation of about 40-fold. These results were interpreted to mean that a posttranslational event activated the protease in vivo; a previously described endogenous proteinase inhibitor was confirmed to be present in the strain used. Chloramphenicol added to the cultures inhibited both the increases in antigen levels and in the specific activity of the proteinase.

Calmodulin-like protein from Bacillus subtilis.

The first example of a calmodulin-like activity in a Gram-positive bacterium, Bacillus subtilis, is reported. A calcium ion-dependent, 3', 5' cyclic-AMP phosphodiesterase-stimulating activity was found in the soluble fraction of cell-free extracts of cells sporulating in a chemically-defined medium; activation was reversed by trifluoperazine. The activity was heat stable, bound to phenothiazine-agarose in a calcium ion-dependent manner and was eluted therefrom with buffer containing EGTA, and displaced authentic beef brain calmodulin from its antibody in a radioimmunoassay.

Popliteal vein aneurysm causing pulmonary embolus.

A case of pulmonary embolus arising from a popliteal vein aneurysm is reviewed. These aneurysms are believed to be developmental in origin; with the exception of embolic phenomenon, these venous anomalies are generally asymptomatic. To our knowledge, only six cases have been previously reported. Physical examination was not helpful, and noninvasive studies were of no value in detecting the aneurysm. Venography was the only reliable diagnostic test. Because popliteal vein aneurysms are a potential source of emboli, surgical intervention is recommended. Venous aneurysmorrhaphy or excision of the aneurysm with a venous bypass are the surgical procedures most often carried out. Aneurysmorrhaphy appears to be associated with an unacceptably high rate of thrombosis; excision with placement of a vein graft may be a more satisfactory alternative.

Single, chemically defined sporulation medium for Bacillus subtilis: growth, sporulation, and extracellular protease production.

The composition and application of a single, chemically defined medium or growth and sporulation of Bacillus subtilis is described. At 37 degrees C cells grew with a doubling time of about 40 min; cultures attained near-maximal spore formation (70 to 80% by 12 h after the end of exponential growth and produced 1 X 10(9) to 2 X 10(9) heat-resistant free spores at 24 h. Dipicolinic acid production was completed between 7 and 11 h. Cells grown in the single, chemically defined medium excreted levels of serine and neutral proteases comparable to those excreted in nutrient broth medium.

Central venous thrombosis and embolism associated with peritoneovenous shunts.

During a five-year period from Aug 1, 1977 through Aug 1, 1982, 36 patients required 47 peritoneovenous shunt procedures (36 initial and 11 revisions) for the management of their intractable ascites. The results at six months showed 23 (63.9%) of 36 patients were dead, but in those living, 12 (92.3%) of 13, the ascites was satisfactorily controlled. Patency was measurably prolonged by appropriate revision of the shunt. The early and late complication rates were surprisingly high, 38.3% and 40.4%, respectively. The most serious complication was central venous thrombosis, 11 (23.4%) of 47 procedures, including one nonfatal and two fatal pulmonary emboli. Treatment included the use of fibrinolytic agents, anticoagulation, and shunt revisions. Careful attention to the details of shunt fabrication, insertion, and patient selection may help to reduce the occurrence of central venous thrombosis associated with peritoneovenous shunts.

Assaying proteinases with azocoll.

Azocoll, an insoluble, ground collagen to which a bright-red azodye is attached has been widely used for the assay of proteolytic enzymes. Earlier studies showed that hydrolysis of azocoll progressed linearly as a function of proteinase concentration but in an exponentially increasing manner as a function of time. No explanation for the latter behavior has been offered. We have found that assays of both crude extracts of Bacillus subtilis and commercial preparations of subtilisin BPN' gave linear rates of hydrolysis of azocoll as a function of protease concentration; however, both gave increasing rates of hydrolysis of azocoll as a function of time. In attempting to improve and standardize proteolytic assays using azocoll we have found: (a) the absorption maximum of solubilized azocoll at pH 7.8 is 516 nm and is not significantly altered at acid pH; (b) assays which are perfectly linear as a function of time can be obtained by using azocoll that has been vigorously prewashed with buffer; (c) the soluble filtrate removed by prewashing can regenerate the nonlinear time courses previously observed; and (d) the rate of hydrolysis of azocoll can be varied by a factor of 3 by varying the rates of agitation of the assay tubes. In summary, to obtain reproducible, linear assays it was essential to prewash commercial azocoll and agitate reaction tubes vigorously.

Kinetics of cytoplasmic aspartate aminotransferase from three genotypes of the deer mouse (Peromyscus maniculatus).

Three genotypic forms of the cytoplasmic enzyme, aspartate aminotransferase from the deer mouse (Peromyscus maniculatus) were each analyzed kinetically after partial purification. Kmapp and Vmax for aspartate decreased in value as temperature changed from 37.9 to 15 degrees C for all three forms. For the AA genotype, the binding enthalpies were highest at 37.9 degrees C and lowest at 25 degrees C, while for the A'A' form, they were lowest at 37.9 degrees and highest at 25 degrees C. Results for the heterozygote were generally intermediate reflecting the properties of both subunits. Inhibition by glyceraldehyde-3-phosphate was of a non-competitive type for all three genotypes.