Cloning, sequencing and expression of the CAMP factor gene of Streptococcus uberis

The gene coding for the CAMP factor from a strain of Streptococcus uberis (ATCC 9927) was cloned in Escherichia coli. Chromosomal DNA from Streptococcus uberis was used to construct a gene library in plasmid pTZ18R and six CAMP-reaction positive clones were obtained from a total of 10,000 transformants. One clone, pJLD21, was subcloned and the CAMP factor gene was located in a 3.2 kb BamHI fragment. The nucleotide sequence of Streptococcus uberis CAMP factor gene was determined and the deduced amino acid sequence is highly homologous to the corresponding Streptococcus agalactiae protein. Immunoblot analysis revealed that the recombinant strain pJLD21 expressed a protein with a molecular weight of 28 000. Antibodies raised against purified Streptococcus uberis CAMP factor cross-reacted with Streptococcus agalactiae protein B.     

Partial characterization of Streptococcus suis type 2 hemolysin

Streptococcus suis type 2 was evaluated for hemolysin production. Supernatants of S. suis type 2 grown in Todd-Hewitt broth were assayed for hemolytic activity by a photometric assay. Twenty-two additional serotypes of S. suis (1,3 to 22, and 1/2) were evaluated for hemolysin production; nine of them (1/2, 1, 4, 5, 14, 15, 17, 19, and 20) were positive. The effects of temperature, atmosphere, centrifugation, sonication, chemicals, bovine serum albumin, fetal calf serum, and enzymes on S. suis type 2 hemolysin activity were studied. Maximum hemolysis occurred after incubation in RPMI 1640 medium at 40 degrees C in 6% CO2 and after growth in Todd-Hewitt broth at 37 degrees C under anaerobic conditions. Hemolytic activity was absent after the addition of fetal calf serum and decreased after the addition of trypsin or amylase. However, treatment of erythrocytes with amylase or trypsin prior to incubation with supernatant also resulted in a decrease in hemolytic activity. The addition of bovine serum albumin caused increased hemolytic activity. Dipyridyl and EDTA had negligible effects on hemolysis. Hemolytic S. suis type 2 culture supernatant injected intraperitoneally failed to cause death in BALB/c mice. Data from our study indicate that S. suis type 2 hemolysin is a secreted or loosely cell bound, thermolabile molecule whose activity is growth condition dependent.     

Further studies on the efficacy of a live vaccine against mastitis caused by Streptococcus uberis

Three groups of dairy cows were immunized by subcutaneous (s.c.) administration of a preparation of live Streptococcus uberis (strain 0140J) and an intramammary infusion of a soluble surface extract derived from same the bacteria. Animals in Groups 1 and 2 received two s.c. vaccinations plus an intramammary inoculation. Animals in Group 3 received two s.c. vaccinations but did not receive the intramammary infusion. In addition to the vaccinated animals, each group also contained two non-vaccinated (control) animals. All animals were challenged experimentally by intramammary infusion (in two quarters per animal) of ca 100 c.f.u. of S. uberis (strain 0140J or C221) and monitored for clinical signs of disease, bacterial numbers in milk, somatic cell count in milk, and daily milk yield for the following 10 days. Animals in Group I were challenged with strain 0140J. Only one out of six challenged quarters of three vaccinated cows developed clinical disease compared to all (four out of four) quarters of non-vaccinated cows. Animals in Group 2 were challenged with strain C221. All challenged quarters of three vaccinated (six out of six) and two non-vaccinated (four out of four) cows developed clinical mastitis. Animals in Group 3 were challenged with strain 0140J. Five out of eight quarters on four vaccinated cows developed clinical mastitis but the onset was delayed in comparison with that in both non-vaccinated cows in which four out of four challenged quarters developed clinical mastitis. These results indicated that vaccination with live S. uberis protects against challenge with the homologous strain but was less effective against a heterologous strain. Reduced protection was also seen when the intramammary booster was omitted.     

Purification and characterization of thermophilin T, a novel bacteriocin produced by Streptococcus thermophilus ACA-DC 0040

Asialoglycoprotein receptor (ASGP-R), a hepatic lectin involved in the clearance of galactose-terminal glycoproteins, is also present in extrahepatic tissues, but its expression in renal cells is not well established. This study examines the presence of ASGP-R in cultured mesangial cells (MC), key cells involved in the removal of macromolecules deposited in the glomerulus. The binding of asialo-orosomucoid (ASOR) to rat MC was saturable and galactose-specific. In addition, MC internalized and degraded ASOR in a Ca(2+)-dependent manner. Parallel studies were performed in a homologous system (human MC), obtaining similar binding curve and competition with unlabeled ASOR and carbohydrates. The purified receptor from rat MC consisted of two proteins (41 and 55 kD) with similar size to the hepatic receptor. Both subunits were detected by mRNA expression analysis (ratio 2:1). Because the hepatic receptor presents avidity for the carbohydrates of IgA1, a protein deposited in the glomerulus of patients with IgA nephropathy, the interaction of IgA1 with the mesangial ASGP-R was explored. As for the interaction with ASOR, catabolism of IgA1 by rat and human MC was Ca(2+)-dependent and was reduced with galactose. In addition, the interaction of ASOR with rat MC was partially inhibited by incubation with IgA1 and its desialylated form, but not by IgA2, as demonstrated in binding experiments and in receptor purification. It is concluded that MC possess ASGP-R specific for galactose residues of several glycoproteins, including IgA1. These data could be important for a better understanding of the pathogenesis of IgA nephropathy.     

In vitro expression of adhesion receptors and diapedesis by polymorphonuclear neutrophils during experimentally induced Streptococcus uberis mastitis

The expression of adhesion receptors and diapedesis by polymorphonuclear neutrophils (PMN) were studied before and during experimentally induced Streptococcus uberis mastitis. Both quarters of the left half of the udders of five midlactation cows were inoculated with a suspension containing approximately 500 CFU of S. uberis 0140J. Clinical signs of an inflammatory reaction and leukocyte influx were observed 24 h after challenge. The expression of CD11b/CD18 adhesion receptors, determined by flow cytometry, was upregulated 24 h after challenge. A confluent monolayer of bovine secretory mammary epithelial cells on collagen-coated inserts was used to study PMN diapedesis. Bovine C5a was used as the chemoattractant. An 80% decrease in PMN diapedesis was observed 24 h after challenge. The decrease in diapedesis continued for 3 weeks after challenge.     

Decreased growth of Streptococcus uberis in milk from mammary glands of cows challenged with the same mastitis pathogen

Milk samples from mammary glands challenged with Streptococcus uberis and from unchallenged mammary glands were selected for analyses of bacterial growth, antibody response, and lactoperoxidase activity. All challenged mammary glands became infected with isolation of S. uberis and elevated somatic cell counts in milk during the first week after challenge. In vitro growth of the homologous challenge strain and a heterologous strain of S. uberis was significantly lower in milk from challenged mammary glands than in milk from control mammary glands at 3, 5, and 7 days after challenge. Removal of casein significantly reduced bacterial growth. In general, antibodies specific to S. uberis started to increase at day 3 post-challenge and were higher in milk from challenged mammary glands than in milk from control mammary glands. There was also a marked increase in total IgG in milk from challenged mammary glands. Growth of S. uberis increased following heat treatment at 56 degrees C of pooled milk or whey samples from challenged mammary glands. Growth of S. uberis correlated negatively with the specific antibody response to the bacteria (P < 0.001). Lactoperoxidase activity varied among cows and among different samples over time and did not appear to contribute to decreased growth of S. uberis. These results suggest that decreased growth of S. uberis in milk from challenged mammary glands in comparison to milk from control mammary glands could result from the interaction of antibodies with complement components.     

Partial reinforcement reduces the associative change produced by nonreinforcement.

Four autoshaping experiments with pigeons investigated the associative decrement produced by nonreinforcement of stimuli with a history of partial or continuous reinforcement. In each experiment, one keylight was reinforced on a 25% schedule and one on a 100% schedule. Then nonreinforced presentations of each stimulus were accompanied by different diffuse stimuli. In each experiment, the diffuse stimulus nonreinforced in the presence of the 100% excitor developed inhibition more rapidly than the diffuse stimulus nonreinforced in the presence of the 25% excitor. This inhibition was measured by transfer to another excitor reinforced on 100% (Experiment 1) or 25% (Experiment 2) schedule. The same difference was observed when the 25% excitor underwent a period of 100% reinforcement (Experiment 3) or was associatively stronger than the 100% excitor (Experiment 4). These results suggest that partial reinforcement acts in part to reduce the subsequent effectiveness of a nonreinforcement in producing associative change. This may contribute to the partial reinforcement extinction effect. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Purification and characterization of enterotoxin produced by Aeromonas sobria

We purified the toxin of Aeromonas sobria capable of inducing a positive response in the mouse intestinal loop assay. The purified toxin showed a positive response not only in the loop assay but also in a hemolytic assay. Subsequently, we cloned the toxin gene and demonstrated that the product of this gene possessed both hemolytic and enterotoxic activities. These results showed that the enterotoxin of A. sobria possesses hemolytic activity. Nucleotide sequence determination of the toxin gene and amino acid sequence analysis of the purified toxin revealed that it is synthesized as a precursor composed of 488 amino acid residues, and that the 24 amino-terminal amino acid residues of the precursor is removed in the mature toxin. As antiserum against the purified toxin neutralized the fluid accumulation induced by living cells not only of A. sobria but also of A. hydrophila, this and antigenically related toxin(s) are thought to play an essential role in the induction of diarrhea by these organisms. The toxin-injured Chinese hamster ovary (CHO) cells induced the release of intracellular lactose dehydrogenase (LDH). The release of LDH from CHO cells and the lysis of erythrocytes by the toxin were repressed by the addition of dextran to the reaction solution, indicating that the toxin forms pores in the membranes and that the cells were injured by the osmotic gradient developed due to pore formation. However, the histopathological examination of intestinal cells exposed to the toxin showed that it caused fluid accumulation in the mouse intestinal loop without causing cellular damage.     

Biochemical and functional characterization of recombinant von Willebrand factor produced on a large scale

Recombinant von Willebrand factor (r-vWF) was produced in serum-free medium on a large scale in recombinant Chinese hamster ovary cells and was purified from fermentation supernatant by a combination of anion exchange chromatography and heparin affinity chromatography. Heparin affinity chromatography yielded r-vWF polymers of different degrees of multimerization. r-vWF was analysed by qualitative and quantitative functional analysis. We could show that while binding of r-vWF to platelets did not depend on multimerization of the molecule, ristocetin-induced platelet aggregation, binding to collagen and binding to heparin correlated directly with the extent of multimerization. Binding of recombinant coagulation factor VIII (r-FVIII) to r-vWF was studied by real-time biospecific interaction analysis and surface plasmon technology. The data indicated that binding of r-FVIII did not depend on r-vWF multimerization. Real-time biospecific interaction analysis suggested a potential stoichiometry of 2 to 2.5 r-vWF subunits per r-FVIII molecule. Kinetic analysis of the r-vWF-r-FVIII interaction gave a binding rate constant of 3 x 10(6) M-1 s-1 and an association constant of 2.5 x 10(9) M-1. Reaction of r-vWF with carbohydrate-specific lectins demonstrated that r-vWF contained a high proportion of N-glycans composed of mannose, galactose, glucose, N-acetylglucosamine and terminal sialic acid. Carbohydrate moities were covalently bound to the protein structure and were quantitatively removed from r-vWF only after protein denaturation. The results demonstrated that r-vWF produced on large scale under serum-free culture conditions exhibited qualitative and quantitative functional properties comparable to human plasma-derived vWF.     

Kinetic characterization of the recombinant hyaluronan synthases from Streptococcus pyogenes and Streptococcus equisimilis

The two hyaluronan synthases (HASs) from Streptococcus pyogenes (spHAS) and Streptococcus equisimilis (seHAS) were expressed in Escherichia coli as recombinant proteins containing His6 tails. The accompanying paper has described the purification and lipid dependence of both HASs, their preference for cardiolipin, and their stability during storage (Tlapak-Simmons, V. L., Baggenstoss, B. A., Clyne, T., and Weigel, P. H. (1999) J. Biol. Chem. 274, 4239-4245). Kinetic characterization of the enzymes in isolated membranes gave Km values for UDP-GlcUA of 40 +/- 4 microM for spHAS and 51 +/- 5 microM for seHAS. In both cases, the Vmax profiles at various concentrations of UDP-GlcNAc were hyperbolic, with no evidence of cooperativity. In contrast, membrane-bound spHAS, but not seHAS, showed sigmoidal behavior as the UDP-GlcNAc concentration was increased, with a Hill number of approximately 2, indicating significant cooperativity. The Hill number for UDP-GlcNAc utilization by seHAS was 1, confirming the lack of cooperativity for UDP-GlcNAc in this enzyme. The Km values for UDP-GlcNAc were 60 +/- 7 microM for seHAS and 149 +/- 3 microM for spHAS in the isolated membranes. The kinetic characteristics of the two affinity-purified HAS enzymes were assessed in the presence of cardiolipin after 8-9 days of storage at -80 degreesC without cardiolipin. With increasing storage time, the enzymes showed a gradual increase in their Km values for both substrates and a decrease in Vmax. Even in the presence of cardiolipin, the detergent-solubilized, purified HASs had substantially higher Km values for both substrates than the membrane-bound enzymes. The KUDP-GlcUA for purified spHAS and seHAS increased 2-4-fold. The KUDP-GlcNAc for spHAS and seHAS increased 4- and 5-fold, respectively. Despite the higher Km values, the Vmax values for the purified HASs were only approximately 50% lower than those for the membrane-bound enzymes. Significantly, purified spHAS displayed the same cooperative interaction with UDP-GlcNAc (nH approximately 2), whereas purified seHAS showed no cooperativity.     

Partial characterization of in vivo chemotactic activity: comparison to human C5a

Spontaneous eosinophil chemotactic activity (SECA) can mediate the directed movement of human eosinophils and neutrophils. Preliminary characterization of SECA has been carried out. SECA is nondialyzable and heat-stable (56 degrees C, 30 min). Chromatography on Sephadex G-75 demonstrated that SECA had elutional and functional properties similar to C5a (prepared from endotoxin-activated normal sera). Polyacrylamide gel electrophoresis (PAGE) with the use of 15% bisacrylamide gels of lyophilized, chemotactically active column fractions demonstrated a single protein band of identical electrophoretic mobility from either SECA or C5a preparations. Enzymatic hydrolysis with carboxypeptidase B, a known inhibitor of C5a activity, significantly decreased chemotactic activities of C5a and SECA. The addition of purified anti-C5 to either SECA or C5a significantly inhibited chemotactic activity. SECA is naturally occurring chemotactic activity identical to human C5a. Thus C5a may be an important source of in vivo chemotactic activity in various inflammatory disorders.     

Analysis of the exudate produced by Streptococcus mutans SL-1 colonies of sucrose-containing agar media

The watery exudate produced by Streptococcus mutans SL-1 colonies on sucrose-containing agar media was found to contain about 7% (wt/vol) of a water-soluble, branched dextran, 4% sucrose, and smaller (less than 1%) amounts of fructose, Folin-phenol-positive material, and lactic acid.     

Purification and characterization of the glucoamylase produced by a strain of Aspergillus flavus

A strain of Aspergillus flavus isolated from an agricultural soil in Egypt produced a gluycoamylase which when purified had a molecular weight of 51,300 +/- 800 Daltons. The optimum pH for activity was 4 and the optimum temperature was 60 degrees C. The enzyme was stable at 70 degrees C for 15 min but denatured at 90 degrees C over 30 min. The Km value with soluble starch was 2.85 mg ml-1, and 10 mM HgCl2 inhibited the enzyme. It was possible to store the enzyme for at least 1 year at -20 degrees C without significant loss in activity.     

A novel factor produced by placental cells with activity against HIV-1

The factors controlling the dynamics of HIV-1 transmission from mother to infant are not clearly known. Previous studies have suggested the existence of maternal and placental protective mechanisms that inhibit viral replication in utero. Preliminary studies from our laboratory revealed that supernatant from placental stromal cells protected HIV-1-infected PBMC from virus-induced apoptosis and suppressed virus production. We have attempted to characterize the antiviral activity of this placental factor (PF) and delineate the stages of HIV-1 replication affected. This activity was not due to the presence of any known cytokine reported to have anti-HIV effect. Direct exposure to PF had no suppressive effect on the infectivity of cell-free HIV-1, and envelope-mediated membrane fusion appeared to be unaffected. Western blot analysis of HIV-1 from infected PBMC treated with PF revealed that expression of all viral proteins was reduced proportionately, both intracellularly and in released virions. However, exposure of HIV-1-infected cells to PF resulted in production of virions with 10-100-fold-reduced infectivity. PF-treated virions contained two- to threefold reduced ratios of cyclophilin A:Gag protein as compared with untreated virus. Reduced cyclophilin A content resulting in decreased binding of cyclophilin A to Gag could account, in part, for the observed reduction in infectivity. Our results suggest that placental cells produce an antiviral factor that protects the fetus during gestation and may have therapeutic potential.     

Note: purification and characterization of the bacteriocin PsVP-10 produced by Pseudomonas sp

A bacteriocin (bacteriocin PsVP-10) produced by Pseudomonas sp. R-10 was purified by a simple method that included an extraction of the bacteriocin with chloroform, followed by cation exchange chromatography. The purity of the bacteriocin was verified by RP-HPLC. It is a peptide of 2.4 kDa, very stable to heat, to proteolytic enzymes and to pH. It presents a very broad spectrum of antimicrobial activity against Gram-positive and Gram-negative bacteria.     

IgA protease produced by Streptococcus sanguis and antibody production against IgA protease in patients with Beh?et's disease

The production of IgA protease in twelve strains of Streptococcus sanguis isolated from patients with Behcet's disease (BD) was examined. Protease activity was detected in 10 out of 12 strains. The protease was purified from one representative strain, S. sanguis 113-20, by employing Rotofor and DEAE-Sephacel chromatography. The molecular mass of the purified protease was approximately 100 kDa, and it cleaved the proline-threonine site of the IgA. Both IgG and IgA titers against the cells (113-20) and the purified IgA protease in the sera of BD patients and healthy controls, 36 each, were assayed. The IgG titers against the cells and protease were not significant in the BD patients or controls, but the IgA titers against the cells and protease in the BD patients were significantly higher than those of the controls. These data indicate that the BD patients are infected with IgA protease-producing S. sanguis strains, which cause an increase of IgA titer against these organisms and IgA protease antigen. Since the organisms can proliferate in BD patients for a long period of time (years), it seems that IgA antibodies cannot effectively eliminate the organisms.     

Purification, characterization, and pathogenicity of urease produced by Vibrio parahaemolyticus

Little is known about the zinc content of breast milk in developing countries. Zinc content in breast milk was analyzed in 34 mothers of low socio-economic status; 17 were primiparae and 17 multiparae. Women in their 6th to 36th week of lactation provided 3 samples of breast milk at different times within a single day. The mean zinc concentration in breast milk (micrograms/ml) was 1.89 +/- 0.64 with a range from 0.17 to 4.38 micrograms/ ml. Zinc content in the morning, midday and evening samples were 2.1 +/- 0.84, 1.74 +/- 0.53, 1.84 +/- 0.69 respectively. There was significant variation between morning and midday samples (p = 0.038). Maternal age, parity, nutritional status or age of the child did not affect the zinc content of milk in the population studied.     

A comparison of the other-directed stigmatization produced by legal and illegal forms of affirmative action.

Recent studies have begun to show that the stigma of incompetence sometimes directed toward the beneficiaries of affirmative action may be significantly reduced as the preferences granted to women and minorities become more moderate. The author examined whether the stigmatization of African Americans would differ under hiring policies that represented legal and illegal levels of racial preference according to federal regulations. Participants were 178 students and 161 corporate employees who rated fictitious Black and White target employees working under (a) an illegal policy of selection of unequal candidates, (b) a legal policy of selection of comparable candidates, or (c) equal opportunity. Participants rated Black targets' achievement-related traits lower than White targets only under the illegal policy. Under the legal policy, no such stigmatization was observed. Additional dependent measures and theoretical implications were explored. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Purification and characterization of a cysteine protease produced by pathogenic luminous Vibrio harveyi

A histologic and biochemical comparison of interface membranes around femoral components of bipolar endoprostheses (n = 17) and total hip prostheses (n = 17) inserted without cement was conducted. The patients' profiles were similar in both groups with respect to age, sex, primary diagnosis, weight, and the interval between primary and revision arthroplasty. Macroscopically, marked circumferential abrasion of the polyethylene insert in the retrieved bipolar cups was noted. Histologic analysis revealed significantly larger amounts of polyethylene debris in the bipolar group. The membranes from the bipolar group also produced significantly greater amounts of prostaglandin E2 (P < .05). The inflammatory membranes associated with large amounts of polyethylene debris may have contributed to aseptic loosening and osteolysis in patients with a bipolar hip prosthesis.