Streptococcal beta-hemolysins: genetics and role in disease pathogenesis

A zone of β-hemolysis surrounding colonies on blood-agar media is a hallmark phenotypic feature of the pathogens group A Streptococcus (GAS) and group B Streptococcus (GBS). In each case, lysis of red blood cells reflects the action of a potent protein exotoxin. Although these toxins have been the subjects of numerous investigations over the years, their purification and molecular identification have proven elusive. These difficulties reflect the instability of hemolytic activity, as both toxins function only in the context of the bacterial surface or certain high molecular weight ‘stabilizer’ molecules. This review highlights the recent discoveries of two markedly distinct genetic loci, necessary and sufficient for the β-hemolytic phenotypes of GAS and GBS, respectively. The generation of isogenic GAS and GBS β-hemolysin-deficient mutants and their analysis using in vitro and in vivo model systems has shown that both toxins function as virulence factors in the pathogenesis of invasive infections.     

Directional Modifications of Resibufogenin by Mucor subtilissimus and Pseudomonas aeruginosa

Directional modifications of resibufogenin 1 by Mucor subtilissimus and Pseudomonas aeruginosa were carried out. The substrate was hydroxylated at C-12 by M. subtilissimus AS 3.2454, from which a major product 12-hydroxyresibufogenin 2 was obtained. Then product 2 was dehydrogenated by P. aeruginosa AS 1.860, which resulted in a new compound 12β-hydroxy-3-keto-resibufogenin 3.     

Highly selective affinity labelling of RNA polymerase B (II) from wheat germ

DNA-dependent RNA polymerase B (II) from wheat germ was modified by incubation with 4-[N-(β-hydroxyethyl)-N-methyl]benzaldehyde esters of AMP, ADP or ATP, followed by reduction with NaBH₄. Reaction of the modified enzyme with [-³²P]UTP in the presence of various DNA templates led to a highly selective affinity labelling of the subunit with M_r 140000 by covalently linked ApU. Labelling was inhibited by 1μg/ml -amanitin.     

The role of cytokines in the regulation of Leydig cell P450c17 gene expression

Cytokines produced by immune-activated testicular interstitial macrophages (TIMs) may play a fundamental role in the local control mechanisms of testosterone biosynthesis in Leydig cells. We investigated whether in vivo immune-activation of TIMs can modulate Leydig cell steroidogenesis. To immune activate TIMs in vivo, mice were injected intraperitoneally (i.p.) with lipopolysaccharide (LPS, 6 mg/kg). TIMs and Leydig cells were purified for RNA analysis. LPS treatment resulted in a 47-fold increase in interleukin-1β (IL-1β) mRNA in TIMs. P450c17 mRNA levels in the Leydig cells from the same animals, decreased to less than 10% compared to control. The effect of LPS on IL-1β and P450c17 mRNA levels was reversible on both TIMs and Leydig cells, respectively. To determine if the effect of LPS on P450c17 was mediated by a possible decrease in pituitary LH secretion, mice were co-injected with LPS and hCG. Treatment with hCG did not change the effect observed with LPS alone, in TIMs or in Leydig cells. In vitro, LPS treatment of TIMs resulted in marked induction of IL-1β mRNA expression. In parallel, in vitro treatment of Leydig cells with recombinant IL-1 resulted in a dose-dependent inhibition of P450c17 mRNA expression and testosterone production. These data demonstrate that LPS treatment, in vivo and in vitro, induced IL-1 gene expression in TIMs, and that IL-1 inhibits P450c17 mRNA in vitro. Therefore, we suggest that immune-activation of TIMs might have caused the observed inhibition of P450c17 gene expression in Leydig cells in vivo.     

Functional expression of cDNAs for bovine 11 beta-hydroxylase-aldosterone synthases, P450(11 beta)-2 and -3 and their chimeras.

Two molecular species of bovine P450(11β), P450(11β)-2 and P450(11β)-3 have been identified, in which the amino acid differences were found at the 6th, 36th and 82nd positions from the NH₂-termini of the mature proteins. They catalyzed the 11β-, 18- and 19-hydroxylation and aldosterone formation from 11-deoxycorticosterone, and the rate of production of 18-hydroxycorticosterone and aldosterone by P450(11β)-3 was greater than that by P450(11β)-2 [Morohashi et al., J. Biochem. 107 (1990) 635–640].

In this study, chimeric clones were constructed whose 6th, 36th and 82nd amino acid residues were exchanged with each other. Two original clones and six chimeric clones were expressed in COS-7 cells, and their steroidogenic activities studied. The ratio of aldosterone or 18-hydroxycorticosterone production to corticosterone production by one clone was compared with that of the other. The ratios for the four clones having Gly36 [P450(11β)-3 type] were 0.08–0.22, whereas those for the clones having Ser36 [P450(11β)-2 type] were 0.03–0.05, suggesting that the Gly36 structure is important for aldosterone production.     

The temperature and pH properties of the extracellular hemicellulose-Degrading enzymes of aureobasidium pullulans NRRL Y 2311-1

Aureobasidium pullulans grown on arabinoxylan accumulates β-xylanase, p-nitrophenyl xylosidase, - -arabinofuranosidase and acetyl esterase activity in the culture fluid. The pH and temperature optima of these arabinoxylan-degrading enzymes were determined. The temperature optima of β-xylanase and p-nitrophenyl xylosidase were between 45 and 50°C whereas the optima for acetyl esterase and - -arabinofuranosidase were 55 and 60°C, respectively. β-xylanase, p-nitrophenyl xylosidase and - -arabinofuranosidase were stable over 3 h at 35°C, 35°C and 60°C, respectively, whereas acetyl esterase remained stable at 55°C for h. The enzymes were inactivated at higher temperatures. The pH optima for β-xylanase, p-nitrophenyl xylosidase and - -arabinofuranosidase were pH 4·0, between 4·0 and 7·0 and 5·0, respectively. β-xylanase, p-nitrophenyl xylosidase, - -arabinofuranosidase and acetyl esterase were most stable at pH 5·0 4·0–5·0, 6·0 and 5·0–6·0, respectively. The most suitable conditions for the use of the enzymes together to hydrolyze arabinoxylan would be 35 °C and pH 5.     

Beta2-adrenoceptor agonists induce the mammalian clock gene, hPer1, mRNA in cultured human bronchial epithelium cells in vitro

The mammalian Per1 gene is one of the most important components of circadian clock function of the suprachiasmatic nucleus and peripheral tissues. We examined whether the β₂-adrenoceptor agonists, procaterol and fenoterol, induce human Per1 mRNA expression in human bronchial epithelium. The in vitro stimulation of β₂-adrenoceptor agonists in BEAS-2B cells led to a remarkable increase in the level of hPer1 mRNA. Moreover, fenoterol or procaterol induced the phosphorylation of CREB in BEAS-2B cells as verified by immunoblot analysis. β₂-adrenoceptor agonists induced human Per1 mRNA expression by the signaling pathways of cAMP-CREB in BEAS-2B cells.     

Organometallic flavonoid derivatives as spectroscopic probes

Derivatives of naringenin have been synthesized with organometalcarbonyl reporting groups for IR spectroscopy attached at C-2, C-3′, or C-6, and the products have been tested for the induction of nod gene expression using a Rhizobium leguminosarum strain which contains the Escherichia coli lacZ (β-galactosidase) gene fused to nodABC. Derivatives with an OMe substituent within the reporting group moiety showed residual gene induction activity.     

Cloning and identification of the promoter of the tobacco Sar8.2b gene,a gene involved in systemic acquired resistance

Expression of the Sar8.2 gene family is induced by salicylic acid (SA) in tobacco during induction of systemic acquired resistance. Expression of Sar8.2b, one member of this 12-member family, was detected as early as 12 h after treatment with SA and was maximal 36 h after SA treatment. In NahG transgenic tobacco plants, benzothiadiazole and dichloroisonicotinic acid induced expression of Sar8.2b but SA did not, suggesting that expression of the Sar8.2b gene is SA-dependent. Several putative cis-acting elements were found in the Sar8.2b gene promoter region, including an as-1 element and GT-1 and Dof binding sequences. We constructed a series of progressive deletion mutations in the Sar8.2b promoter region linked to the β-glucuronidase (GUS) coding region and analyzed GUS activities by stable expression in transformants of Arabidopsis thaliana. Deletions between −728 and −927 bp or between −351 and −197 bp of the promoter region resulted in a significant reduction in GUS activity induced by SA treatment as shown in stable transformants of A. thaliana. The −197 bp fragment of the promoter region was found to confer a relatively low level of GUS activity induced by SA treatment in stable expression of transformants in A. thaliana. The results suggest that 927 bp of the Sar8.2b gene promoter confers full promoter activity and that cis-acting elements required for high-level SA-inducible expression of the Sar8.2b gene may exist within the regions −728 to −927 bp and −197 to −351 bp.     

Vaccination with a Plasmodium chabaudi adami multivalent DNA vaccine cross-protects A/J mice against challenge with P. c. adami DK and virulent Plasmodium chabaudi chabaudi AS parasites

A current goal of malaria vaccine research is the development of vaccines that will cross-protect against multiple strains of malaria. In the present study, the breadth of cross-reactivity induced by a 30K multivalent DNA vaccine has been evaluated in susceptible A/J mice (H-2a) against infection with the Plasmodium chabaudi adami DK strain and a virulent parasite subspecies, Plasmodium chabaudi chabaudi AS. Immunized A/J mice were significantly protected against infection with both P. c. adami DK (31–40% reduction in cumulative parasitemia) and P. c. chabaudi AS parasites, where a 30–39% reduction in cumulative parasitemia as well as enhanced survival was observed. The 30K vaccine-induced specific IFN-γ production by splenocytes in response to native antigens from both P. c. chabaudi AS and P. c. adami DK. Specific antibodies reacting with surface antigens expressed on P. c. adami DS and P. c. chabaudi AS infected red blood cells, and with opsonizing properties, were detected. These results suggest that multivalent vaccines encoding conserved antigens can feasibly induce immune cross-reactivity that span Plasmodium strains and subspecies and can protect hosts of distinct major histocompatibility complex haplotypes.     

Trichoderma reesei acetyl esterase catalyzes transesterification in water

Partially purified Trichoderma reesei RUT-C30 acetyl esterase preparation was found to catalyze acyl transfer reactions in organic solvents, mixtures of organic solvents with water and even in water. Using different acyl donors, the best results for acetyl transfer in water were obtained using vinyl acetate. As acetyl acceptors, a variety of hydroxyl bearing compounds in aqueous solutions were used. Degree of conversion and the number of newly formed acetates varied according to the acceptor used. Conversions over 50% were observed for the majority of several common monosaccharides, their methyl and deoxy derivatives and oligosaccharides. In several cases, the transesterification reaction exhibited strict regioselectivity, leading to only one acetyl derivative. Preparative potential of the transesterification in water was demonstrated by acetylation of methyl β- -glucopyranoside, 4-nitrophenyl β- -glucopyranoside and kojic acid, yielding 56.4% of methyl 3-O-acetyl β- -glucopyranoside, 70.2% of 4-nitrophenyl 3-O-acetyl β- -glucopyranoside and 30.9% of 7-O-acetyl-kojic acid as the only reaction products.

This enzymatically catalyzed transacetylation in water, which is applied to transformation of saccharides for the first time, opens a new area in chemoenzymatic synthesis. Its major advantages are simplicity, highly regioselective esterification of polar compounds, high yields, low enzyme consumption and elimination of the need to use toxic organic solvents.     

Improving enzyme characteristics by gene shuffling; application to β-glucosidase

The genes of family 3 β-glucosidase enzymes consist of five distinct regions; the N-terminal residues, an N-terminal catalytic domain, a nonhomologous region, a C-terminal domain of unknown function and the C-terminal residues. The β-glucosidase genes derived from Cellvibrio gilvus (CG) and Agrobacterium tumefaciens (AT) have been subjected to gene deletion, truncation and shuffling. The folding information was found to be distributed unevenly across the different regions based on the gene manipulation results. Chimeric enzymes with improved enzyme characteristics were obtained only by gene shuffling at the C-terminal domain.     

Relationship between cellular RecA protein concentration and untargeted mutagenesis in Escherichia coli

We measured the production of untargeted mutations in the cI and cII genes of untreated λ phage undergoing a lytic cycle in UV-irradiated bacterial hosts. As previously shown, treatment with 4 μg/ml of rifampicin during post-irradiation incubation inhibited amplification of the RecA protein in these cells. In addition, we observed a decreased mutation rate compared to the untreated, irradiated bacteria. Treatment with 4 μg/ml or 8 μg/ml rifampicin did not prevent the UV induction of the umuDC operon, as judged by assay of β-galactosidase activity in a umuC-lacZ fusion strain. In contrast, the UV-induction of β-galactosidase in the sulA-lacZ fusion strain was decreased by 4 μg/ml rifampicin. The inhibition of untargeted mutagenesis by this drug treatment was also observed in a strain constitutive for SOS functions (lexA (Def)) as well as ina RecA-overproducing plasmid strain, that blocks induction of heat-shock proteins, factor(s) in wild-type recA⁺ cells. An htpR165-carrying strain, that blocks induction of heat-shock proteins, exhibited normal UV-promoted mutagenesis. A correlation was observed between the cellular concentration of RecA protein, increased spontaneously by a temperature shift in a lexA(Ts) strain, and the extent of UV-promoted untargeted mutagenesis. These results suggest a mechanistic role of RecA protein in this process.     

Effect of a nuclepolyhedrovirus of Autographa californica expressing the enhancin gene of Trichoplusia ni granulovirus on T. ni larvae

A recombinant Autographa californica nucleopolyhedrovirus (AcMNPV) strain showing higher virulence against Trichoplusia ni larvae than the wild-type virus was developed. The 'enhancin' (VEF) gene of T. ni granulovirus (TnGV) and the AcMNPV polyhedrin gene were cloned into the baculovirus transfer vector pAcUW31. This plasmid and AcMNPV BacPAK6 DNA were co-transfected into the BTI-Tn5B1-4 cell line. A recombinant AcMNPV strain (BacVEFPol) was purified, amplified, and bioassayed against T. ni first instar larvae. Its estimated LC₅₀ (0.184 OB/mm²) was 2.18 times lower than the LC₅₀ estimated for the wild-type AcMNPV (0.402 OB/mm²). Likewise, an LT₅₀ of 67.7 h was estimated for the recombinant AcMNPV strain while the LT₅₀ of wild-type AcMNPV was estimated at 81.9 h. This indicates a 17.4% reduction of the time required to kill the larvae. The higher virulence of the recombinant strain, evidenced by its LC₅₀ and LT₅₀ values being lower than those of the wild-type strain, indicates that the VEF protein is expressed properly and may be occluded in the OBs.     

Stereoselective reduction of keto esters: thermophilic bacteria and microalgae as new biocatalysts

This review covers the possibility of aerobic thermophilic bacteria (Bacillus strains and thermophilic actinomycetes) and microalgae (Chlorella strains and marine algae) as new biocatalysts for the stereoselective reduction of - and β-keto esters. The mechanistic interpretation of the reduction by a thermophilic actinomycete is also delineated.