Use of p-aminobenzamidine to monitor activation of trypsin-like serine proteases

The fluorescent compound p-aminobenzamidine was used to monitor activation of the trypsin-like serine proteases trypsin, thrombin, and blood coagulation factors IXa and Xa. p-Aminobenzamidine, when bound to the activated forms of these proteases but not the corresponding zymogens, displayed an increase in fluorescence. This fluorescence increase was coincident with activation as measured by synthetic substrate hydrolysis, physiological coagulation activity, and the appearance of activation products on gel electrophoresis. The activation of proteolytically modified factor X was also monitored. These results suggest that following p-aminobenzamidine fluorescence is a convenient procedure for monitoring activation of trypsin-like serine proteases.     

Identification of proapoa-I in rat lymph and plasma: metabolic conversion to "mature" apoA-I

Rat apoA-I polymorphism has been analyzed in lymph and plasma. Two major proteins were present and their relative distribution was different in lymph and plasma lipoproteins. The basic protein (pI 5.60) was quantitatively most abundant among plasma lipoproteins and the acidic protein (pI 5.50) was predominant in lymph chylomicrons and lipoproteins. Microsequence amino acid analysis of the two proteins isolated by preparative isoelectrofocusing revealed that pI 5.50 apoA-I was proapoA-I with six additional amino acids (H2N-Ser-Glu-Phe-Trp-Gln-Gln) at the N-terminal end of "mature" apoA-I (pI 5.60 apoA-I). When radioiodinated proapoA-I was injected in rats, a conversion to "mature" apoA-I was observed and the process reached 92% completion in six hours. These data demonstrate the origin of apoA-I polymorphism in vivo.     

Further studies of the β-lactamases ofBacteroides species

The -lactamases of individual strains ofBacteroides fragilis, B. thetaiotaomicron, andB. melaninogenicus were examined to characterize their enzymatic activity and the relation between the periplasmic and cytoplasmic forms of the enzymes. K_m and V_max values indicate that all strains examined were very similar in terms of enzymatic activity with the antibiotics tested. Electrophoretic analysis and treatment with phospholipase D suggest the presence of a cytoplasmic form of the enzyme that is modified upon entry into the periplasmic space.     

Penicillinase activity in three strains ofClostridium butyricum

Three strains ofClostridium butyricum exhibited elevated minimal inhibitory concentrations (MICs) to penicillin (64–1,024 g/ml), ampicillin (32–256 g/ml), carbenicillin (128–1,024 g/ml), and oxacillin (32–64 g/ml). Cephalosporin/cephamycin agents were more active than penicillin drugs. All isolates were found to possess a -lactamase. The -lactamases were primarily cell associated during the logarithmic phase of growth. Stationary-phase cells released most of the enzyme into the culture medium. Cephalothin supplementation of broth cultures with concentrations equivalent to one-eighth of the MIC significantly increased the quantity of -lactamase synthesized. The -lactamases produced by these three isolates exhibited greatest activity with penicillin followed by ampicillin>cephaloridine>carbenicillin and oxacillin. No enzymatic activity was observed using cephalothin, cephalexin, cefazolin, cefoxitin, or cephamandole substrates. The -lactamases were inhibited by clavulanic acid and para-chloromurcuribenzoate and not inhibited by cloxacillin. Each enzyme exhibited an isoelectric point of 4.2.     

Correlation of low and high density lipoprotein binding in vivo with rates of lipoprotein degradation in the rat. A comparison of lipoproteins of rat and human origin

These studies were done in the rat to correlate the ability of low and high density lipoproteins of rat (rLDL and rHDL) and human (hLDL and hHDL) origin to bind in vivo to specific tissues with the rates at which these same lipoprotein fractions were cleared from the circulation. The adrenal gland and liver manifested the greatest amounts of rLDL binding in vivo, but activity also was found in spleen, lung, kidney, ovary, and intestine. In contrast, little or no such binding was found utilizing either methyl-rLDL or hLDL. rHDL containing E apoprotein bound to the same group of tissues although in lesser amounts, except in the case of ovary and adrenal gland which bound disproportionately greater amounts of rHDL than rLDL. In keeping with these marked differences in tissue binding, the clearance of rLDL from the plasma equaled 847 +/- 36 microliters/h/100 g of rat while that of methyl-rLDL and hLDL was only 368 +/- 8 and 363 +/- 11 microliters/h/100 g of rat, respectively. When the steady state plasma level of rLDL was raised 2.5-fold, the clearance decreased slightly to 705 +/- 20 microliters/h/100 g of rat. The clearance of hLDL remained constant, however, at about 350 microliters/h/100 g of rat even when the plasma hLDL level was raised to very high values. The clearance of rHDL and hHDL equaled 644 +/- 16 and 408 +/- 13 microliters/h/100 g of rat, respectively, reflecting the more similar rate of binding of rHDL and hHDL to the tissues of the rat. Rates of whole animal sterol synthesis were lowered from 28 mumol/h to 8.8 mumol/h or 13 mumol/h by fasting and cholesterol feeding, respectively, and stimulated to 71 mumol/h by cholestyramine treatment. Under these same conditions, hepatic cholesterol synthesis could be lowered from the normal rate of 15 mumol/h to 4.2 mumol/h and raised to 50 mumol/h. None of these treatments, however, affected the plasma clearance of rLDL and rHDL. In contrast, treatment with ethinyl estradiol increased by 3-fold both the hepatic binding and the whole animal plasma clearance of rLDL. Following resection of approximately two-thirds of the liver under carefully controlled metabolic conditions, there was no change in the rate of hepatic cholesterol synthesis or rLDL binding in the remaining liver, but the clearance of chylomicrons, rLDL, and rHDL diminished by 67%, 26%, and 17%, respectively, suggesting that in the rat the liver was responsible for the degradation of approximately 97%, 39%, and 27%, respectively, of these lipoprotein fractions.     

Comparative effects of Aroclor 1254 (polychlorinated biphenyls) and phenanthrene on glucose uptake by freshwater microbial populations.

The effects of polychlorinated biphenyl (PCB) and phenanthrene stress on glucose uptake by natural microbial populations were examined by the heterotrophic potential technique. Temporal and spatial distributions in glucose uptake velocities were examined for natural samples as well as PCB- and phenanthrene-stressed samples. Statistical analysis indicated significant variability among the various samples. It was demonstrated that the environmental variables contributed significantly to the variability in uptake kinetics. Although general trends indicated a PCB-induced stimulation in uptake velocities, these trends were in part masked by sample variability. Data analysis indicated no statistically significant PCB or phenanthrene effect on either total glucose uptake velocities or the proportion of 14CO2 evolved, as compared to natural unstressed samples.     

Infection of human dendritic cells by a sindbis virus replicon vector is determined by a single amino acid substitution in the E2 glycoprotein

The ability to target antigen-presenting cells with vectors encoding desired antigens holds the promise of potent prophylactic and therapeutic vaccines for infectious diseases and cancer. Toward this goal, we derived variants of the prototype alphavirus, Sindbis virus (SIN), with differential abilities to infect human dendritic cells. Cloning and sequencing of the SIN variant genomes revealed that the genetic determinant for human dendritic cell (DC) tropism mapped to a single amino acid substitution at residue 160 of the envelope glycoprotein E2. Packaging of SIN replicon vectors with the E2 glycoprotein from a DC-tropic variant conferred a similar ability to efficiently infect immature human DC, whereupon those DC were observed to undergo rapid activation and maturation. The SIN replicon particles infected skin-resident mouse DC in vivo, which subsequently migrated to the draining lymph nodes and upregulated cell surface expression of major histocompatibility complex and costimulatory molecules. Furthermore, SIN replicon particles encoding human immunodeficiency virus type 1 p55(Gag) elicited robust Gag-specific T-cell responses in vitro and in vivo, demonstrating that infected DC maintained their ability to process and present replicon-encoded antigen. Interestingly, human and mouse DC were differentially infected by selected SIN variants, suggesting differences in receptor expression between human and murine DC. Taken together, these data illustrate the tremendous potential of using a directed approach in generating alphavirus vaccine vectors that target and activate antigen-presenting cells, resulting in robust antigen-specific immune responses.     

Radioimmunoassay of rat apolipoprotein A-IV

We have developed a specific and sensitive radioimmunoassay for rat apolipoprotein A-IV (apoA-IV). The protocol includes treatment of the samples for 1 h at 60 degrees C with 0.7% Tween 20. Under these conditions, linear logit-log plots have been obtained for apoA-IV in lymph and plasma lipoprotein fractions as well as for purified apoA-IV. The sensitivity of the assay is to 20 ng. Absolute mass values obtained with the assay were validated by comparison with values obtained with an independent method of colorimetric reading of apoA-IV separated by polyacrylamide gel electrophoresis from plasma high density lipoproteins. The concentration of apoA-IV in fasting plasma averaged 10.2 mg/dl and in the mesenteric duct lymph 15.8 and 12.6 mg/dl during the fasting and the fat absorption states, respectively.     

Microbial translocation into amniotic fluid of vervet monkeys is common and unrelated to adverse infant outcomes

Amniotic fluid was collected from pregnant female African green monkeys (n = 20). Analyses indicate microbial translocation into amniotic fluid during pregnancy is typical, and microbial load reduces across gestation. Microbial translocation does not relate to infant outcome or maternal factors. Lastly, we demonstrate that sample contamination is easily introduced and detectable.