Development of an inducible and enhancible expression and secretion system in Bacillus subtilis.

A set of inducible secretion vectors has been developed in Bacillus subtilis based on the regulatory region and the signal peptide sequence of the sacB gene encoding an extracellular enzyme, levansucrase. The expression of the inserted foreign gene (bla) encoding TEM beta-lactamase (Bla), can be induced by the addition of sucrose to the medium. Either the installation of a sacQ expression cassette into the same secretion vector, or the use of a sacUh two-protease-deficient strain (WB30), can significantly enhance expression of the bla gene.

Repeated prevalence surveys for monitoring effectiveness of hospital infection control.

In a 1400-bedded teaching hospital single-day prevalence surveys of hospital infection were done every six months for 3 years. The prevalence of community-acquired infection remained constant; but, after the introduction of a general infection-control policy, the prevalence of hospital-acquired infection (HAI) fell linearly from 10.5% in the second survey to 5.

Developmental patterns of antioxidant defense mechanisms in human erythrocytes.

To obtain a profile of erythrocyte antioxidant defense potential during late fetal development, we studied selected antioxidant parameters in blood samples from 65 neonates with birth wt between 520 and 4210 g and from 12 healthy adults. Erythrocyte superoxide dismutase activity did not change significantly with maturation and no significant differences were observed among preterm infants grouped in increasing birth wt categories, term neonates, and adults. Erythrocyte catalase and glutathione peroxidase, as well as plasma vitamin E levels, showed highly significant positive correlations (p less than 0.

Bacillus subtilis subtilisin gene (aprE) is expressed from a sigma A (sigma 43) promoter in vitro and in vivo.

In vitro studies demonstrated that the Bacillus subtilis subtilisin gene (aprE) could be transcribed by RNA polymerase holoenzyme reconstituted from core and sigma A factor obtained from vegetative cells. Upstream deletions (from -45) reduced the amount of transcription from the promoter. A deletion downstream of the promoter that overlapped a putative downstream minor promoter did not affect transcription from the sigma A promoter, which indicated that the putative downstream promoter is not utilized in vivo.

Vitamin E and selenium in preterm infants: lack of effect on clinical patency of ductus arteriosus.

Levels of antioxidant defenses, which include vitamin E and Se-dependent GSH-Px, are generally relatively low in the fetus and neonate. Se-dependent GSH-Px and vitamin E are known to modulate biosynthesis of eicosanoids and therefore could have the potential for affecting patency or closure of the ductus arteriosus after birth. We evaluated some indices of antioxidant defense in sick LBW infants in relationship to one another as well as to clinical PDA, and investigated the effects of producing a rapid rise in serum vitamin E levels in some of these infants.

Cloning and nucleotide sequence of senN, a novel 'Bacillus natto' (B. subtilis) gene that regulates expression of extracellular protein genes.

A new 'Bacillus natto' gene, senN, that regulates the expression of several extracellular proteins in B. subtilis has been cloned and sequenced. senN codes for a small, highly basic protein with an amino acid sequence different from the products coded by the regulatory genes sacQ, sacV, prtR and hpr.

Expression and secretion of human atrial natriuretic alpha-factor in Bacillus subtilis using the subtilisin signal peptide.

Using the signal peptide of the Bacillus subtilis subtilisin gene (aprE) and a synthetic cDNA corresponding to the mature region of the human atrial natriuretic alpha-factor (hANF), we have constructed a secretion vector. B. subtilis cells, when transformed with this vector, secrete immunoreactive hANF peptides into the medium at about 500 micrograms/liter.

Use of the Bacillus subtilis subtilisin signal peptide for efficient secretion of TEM beta-lactamase during growth.

We report the development of an efficient Bacillus subtilis secretory system, with the secreted product stably maintained in the medium for 100 h. The system is based on characterization of the subtilisin signal peptidase cleavage site and promoters, catabolite repression of sporulation, presence of a vegetative secreting mechanism, and availability of a protease-deficient strain.

Determination of the signal peptidase cleavage site in the preprosubtilisin of Bacillus subtilis.

Bacillus subtilis subtilisin is predicted to be synthesized as a preproenzyme according to the sequence analysis of its gene. We have synthesized the [35S] methionine-labeled preprosubtilisin in vitro and processed the precursor to prosubtilisin by the addition of membrane vesicles derived from vegetative cells of B. subtilis and Triton X-100.

Promoter analysis of a soybean nuclear gene coding for nodulin-23, a nodule-specific polypeptide involved in symbiosis with Rhizobium.

Soybean nodulin-23 gene, induced in nodules formed due to symbiosis with Rhizobium, was found to contain multiple sequences capable of acting as eucaryotic and procaryotic promoters. The transcription start sites of this gene were localized by S1 nuclease mapping, primer extension with nodule mRNA and in vitro run-off transcription analysis. A major transcription start site was observed by S1 mapping; however, the the primer extension revealed a second start site.

Postnatal decline in pyridoxal phosphate and riboflavin. Accentuation by phototherapy.

Riboflavin is a cofactor in the conversion of pyridoxine (vitamin B6) to pyridoxal phosphate (PALP), an essential coenzyme in numerous metabolic pathways, including neurotransmitter synthesis. Riboflavin and pyridoxine are light sensitive in vitro, and conflicting results have been reported on the in vivo effects of phototherapy on riboflavin. We studied 25 full-term neonates receiving phototherapy and 16 healthy controls to evaluate their riboflavin and PALP status.

Utilization of a Bacillus subtilis sigma 37 promoter by Escherichia coli RNA polymerase in vivo.

The promoter region of Bacillus subtilis subtilisin E was found to be composed of two overlapping promoters with their transcription starting sites separated from each other by 15 base pairs (Wong, S.-L., Price, C.

The subtilisin E gene of Bacillus subtilis is transcribed from a sigma 37 promoter in vivo.

A cloned Bacillus subtilis gene (sprE) expressed only during the stationary growth phase is shown to encode the subtilisin E protease, an enzyme associated with sporulation. We have determined the DNA sequence of the sprE promoter region and the promoter-proximal half of the structural gene. The sprE gene codes for a putative 29-residue signal peptide and a 77-residue leader peptide preceding the mature subtilisin sequence.

A temporally regulated promoter from Bacillus subtilis is transcribed only by an RNA polymerase with a 37,000 dalton sigma factor.

A 1,250 base pair Bacillus subtilis chromosomal HindIII restriction fragment (S fragment) has been cloned into the B. subtilis expression-probe plasmid pGR71. The S fragment induces the expression of the pGR71 chloramphenicol resistance gene shortly after the initiation of sporulation.

Peptide mapping of Bacillus subtilis RNA polymerase alpha factors and core-associated polypeptides.

An analysis of the peptide maps of the sigma factors and core-associated subunits of Bacillus subtilis RNA polymerase has revealed that all the sigma factors ad core-associated polypeptides are derived from separate genes and are not proteolytically modified products of the major 55,000-dalton sigma factor. A comparison of the peptide pattern of the major B. subtilis and Escherichia coli sigma factors revealed limited homology between them.

Hematologic and selenium status of low-birth-weight infants fed formulas with and without iron.

Preterm infants with birth weights between 1,001 and 1,600 gm were randomly assigned at one week of age to three groups and fed a standard milk-based formula, the same formula with iron, or a soy-based formula with iron. Hematologic values and selenium status were then studied prospectively for five weeks. Rates of decline in hematocrit and hemoglobin did not differ significantly among the three groups and did not correlate with red cell selenium values or glutathione peroxidase activity.

Aging in the retinas and the lateral geniculate bodies of the albino mice.

Aging of the retinas and the lateral geniculate bodies were studied by biochemical, morphometrical, and ultrastructural means. A decrease in the phagosome number in the central retinas were recorded in the aged mice. In the lateral geniculate bodies of the aged animals, the neurons showed a decrease in nuclear volume and the microglia cells were active.