Isolation and characteristics of Bacillus intermedius glutamyl endopeptidase secreted by a recombinant strain of Bacillus subtilis at various growth phases

The recombinant strain of Bacillus subtilis bearing B. intermedius glutamyl endopeptidase gene in multicopy plasmid delta58.21 secretes the enzyme to the medium at the phase of slowing of growth and the stationary growth phase with accumulation maxima at 24 and 48 h. Enzyme samples were isolated from the culture liquid after 24 and 48 h of culturing of and were purified up to homogeneity by ion exchange chromatography on carboxymethyl cellulose and HPLC on a MonoS column. The molecular weight of the corresponding proteins was 29 kDa. Both preparations had identical structure, but differed in affinity to the specific substrate Z-Glu-pNA. The effects of Ca+ ions and specific low-molecular and protein inhibitors on the activity of the enzyme corresponding to various growth phases has been studied.     

Isolation and characteristics of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> glutamyl endopeptidase secreted by a recombinant strain of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> at various growth phases

The recombinant strain of Bacillus subtilis bearing B. intermedius glutamyl endopeptidase gene in multicopy plasmid Δ58.21 secretes the enzyme to the medium at the phase of slowing of growth and the stationary growth phase with accumulation maxima at 24 and 48 h. Enzyme samples were isolated from the culture liquid after 24 and 48 h of culturing of and were purified up to homogeneity by ion exchange chromatography on carboxymethyl cellulose and HPLC on a MonoS column. The molecular weight of the corresponding proteins was 29 kDa. Both preparations had identical structure, but differed in affinity to the specific substrate Z-Glu-pNA. The effects of Ca²⁺ ions and specific low-molecular and protein inhibitors on the activity of the enzyme corresponding to various growth phases has been studied.     

Heterologous expression of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> gene of glutamyl endopeptidase in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains defective in regulatory proteins

Expression of the gene of glutamyl endopeptidase from Bacillus intermedius (gseBi) cloned on the plasmid pV has been studied in Bacillus subtilis recombinant strains with mutations of the regulatory proteins involved in sporogenesis and spore germination. It has been established that inactivation of the regulatory protein Spo0A involved in sporulation initiation resulted in a decrease in the expression of the gseBi gene by 65% on average. A mutation in the gene of the sensor histidine kinase kinA had no effect on the biosynthesis of the enzyme. Inactivation of Ger proteins regulating bacterial spore germination resulted in a 1.5–5-fold decrease in glutamyl endopeptidase activity. It has been concluded that expression of the B. intermedius glutamyl endopeptidase gene from plasmid pV in recombinant cells of B. subtilis is under impaired control by the regulatory system of Spo0F/Spo0A phosphorelay, which participates in sporulation initiation. The regulatory Ger proteins responsible for spore germination also affect expression of the gene of this enzyme.     

ZnT3 mRNA levels are reduced in Alzheimer's disease post-mortem brain

Extensive genetic, biochemical, and histological evidence has implicated the amyloid-β peptide (Aβ) in Alzheimer's disease pathogenesis, and several mechanisms have been suggested, such as metal binding, reactive oxygen species production, and membrane pore formation. However, recent evidence argues for an additional role for signaling mediated by the amyloid precursor protein, APP, in part via the caspase cleavage of APP at aspartate 664. Here we review the effects and implications of this cleavage event, and propose a model of Alzheimer's disease that focuses on the critical nature of this cleavage and its downstream effects.     

Transport lattice models of heat transport in skin with spatially heterogeneous,temperature-dependent perfusion

Background  

Investigation of bioheat transfer problems requires the evaluation of temporal and spatial distributions of temperature. This class of problems has been traditionally addressed using the Pennes bioheat equation. Transport of heat by conduction, and by temperature-dependent, spatially heterogeneous blood perfusion is modeled here using a transport lattice approach.     

Phylogenetic relationships of bolitoglossine salamanders: a demonstration of the effects of combining morphological and molecular data sets

We analyzed sequence data for 555 bp of the mitochondrial gene cytochrome b in plethodontid salamanders, taken from 18 ingroup (tribe Bolitoglossini) and 4 outgroup (tribe Plethodontini) taxa. There were 257 variable sites, of which 219 were phylogenetically informative. Sequence differences among taxa exceeded 20%, and there were up to 15% amino acid differences among the sequences. We also analyzed 37 morphological (including karyological) characters, taken from the literature. Data were analyzed separately and then combined using parsimony and likelihood approaches. There is little conflict between the morphological and DNA data, and that which occurs is at nodes that are weakly supported by one or both of the data sets. Treated separately, the morphological and DNA data provide strong support for some nodes but not for others. The combined data act synergistically so that good support is obtained for nearly all of the nodes in the tree. Recent divergences are supported by silent transitions, and older divergences are supported by a combination of morphological, karyological, DNA transversion, and amino acid changes. Eliminating silent changes from the DNA data improves the consistency index and improves some bootstrap and decay index values for several deeper branches in the tree. However, the combined data set with all characters included provides a better supported tree overall. Maximum likelihood and parsimony with all of the data give not only the same topology but also remarkably similar branch lengths. Results of this analysis support the monopoly of the supergenera Hydromantes and Batrachoseps, and of a sister group relationship of Batrachoseps and the supergenus Bolitoglossa (represented in this study one species of the genus Bolitoglossa).      

Salivary and serum procalcitonin and C-reactive protein as biomarkers of periodontitis in United States veterans with osteoarthritis or rheumatoid arthritis

Serum procalcitonin (ProCT) is elevated in response to bacterial infections, whereas high sensitivity C-reactive protein (hsCRP) is a nonspecific inflammatory marker that is increased by excess adipose tissue. We examined the efficacy of ProCT and hsCRP as biomarkers of periodontitis in the saliva and serum of patients with arthritis, which is characterized by variable levels of systemic inflammation that potentially can confound the interpretation of inflammatory biomarkers. Blood and unstimulated whole saliva were collected from 33 patients with rheumatoid arthritis (RA) and 50 with osteoarthritis (OA). Periodontal status was assessed by full mouth examination and patients were categorized as having no/mild, moderate or severe periodontitis by standard parameters. Salivary and serum ProCT and hsCRP concentrations were compared. BMI, diabetes, anti-inflammatory medications and smoking status were ascertained from the patient records. Differences between OA and RA in proportionate numbers of patients were compared for race, gender, diabetes, adiposity and smoking status. Serum ProCT was significantly higher in arthritis patients with moderate to severe and severe periodontitis compared with no/mild periodontitis patients. There were no significant differences in salivary ProCT or salivary or serum hsCRP in RA patients related to periodontitis category. Most of the OA and RA patients were middle aged or older, 28.9% were diabetic, 78.3% were overweight or obese, and slightly more than half were either current or past smokers. The OA and RA groups differed by race, but not gender; blacks and males were predominant in both groups. The OA and RA groups did not differ in terms of controlled or uncontrolled diabetes, smoking status or BMI. The RA patients had been prescribed more anti-inflammatory medication than the OA patients. Our results demonstrate that circulating ProCT is a more discriminative biomarker for periodontitis than serum hsCRP in patients with underlying arthritis. Any elevation in salivary and serum hsCRP due to periodontitis apparently was overshadowed by differences among these patients in factors that influence CRP, such as the extent of inflammation between RA and OA, the extent of adipose tissue, the use of anti- inflammatory medications and smoking status. Although our study showed no differences in salivary ProCT related to severity of periodontitis, this biomarker also may be useful with further refinement.     

Effect of the regulation system of metabolic nitrogen exchange on biosynthesis of serine proteinases from <Emphasis Type="Italic">Bacillus intermedius</Emphasis>

The regulatory link between biosynthesis of Bacillus intermedius subtilisin-like serine proteinase and nitrogen metabolism in B. intermedius cells was determined. The level of the enzyme biosynthesis by the recombinant strain of Bacillus subtilis in the medium containing ammonium ions was three- to fivefold less than that in the medium with poorly utilized sodium nitrate. Accumulation of glutamyl endopeptidase in a culture liquid of this microorganism did not depend on the source of nitrogen present in the medium. During cultivation in the rich medium, the productivity of subtilisin-like proteinase in the recombinant B. subtilis strain carrying a mutation in the NrgB sensor protein was demonstrated to increase threefold compared to that of the control strain. In the minimal culture medium, mutation in the nrgB gene abolished the effect of a nitrogen source on the level of the subtilisin-like proteinase gene expression. At the same time, this mutation did not affect glutamyl endopeptidase biosynthesis. Thus, expression of the gene coding for subtilisin-like proteinase from B.intermedius is suggested to be positively regulated by the regulatory system of nitrogen metabolism.     

Recent evolutionary history of American Onchocerca volvulus, based on analysis of a tandemly repeated DNA sequence family

Polymerase chain reaction (PCR) products were characterized for a repeated sequence family (designated "O-150") of the human filarial parasite Onchocerca volvulus. In phylogenetic inferences, the O-150 sequences clustered into closely related groups, suggesting that concerted evolution maintains sequence homology in this family. Using a novel mathematical model based on a nested application of an analysis of variance, we demonstrated that African rainforest and savannah strain parasite populations are significantly different. In contrast, parasites collected in the New World are indistinguishable from African savannah strains of O. volvulus. This finding supports the hypothesis that onchocerciasis was recently introduced into the New World, possibly as a result of the slave trade.