Carboxypeptidase from Streptomyces bikiniensis: primary structure, isolation, and properties.

A metallocarboxypeptidase produced by Streptomyces bikiniensis 27 strain (VKPM Ac-1783) (CPSb) was purified and characterized. The enzyme cleaves both basic and hydrophobic C-terminal amino acid residues from synthetic peptides, that is, it possesses specificity of mammalian carboxypeptidases A and B. The enzyme also hydrolyzes peptides bearing glutamic acid at the C-end. CPSb exhibits its maximal activity at pH 7.0-7.6 and 55°C. The nucleotide sequence encoding the mature CPSb in S. bikiniensis 27 (VKPM Ac-1783) genome (Accession No. GU362077) was determined. It is shown that the primary structure of the mature enzyme has a moderate degree of identity with orthologs from Streptomyces griseus (79% identity) and Streptomyces avermitilis (85% identity).

[Intellectual, psychomotor and speech development of children born to mothers with epilepsy].

Intellectual, psychomotor and speech development of 50 children born to mothers with epilepsy were studied. The sample has been stratified into 2 subgroups: children aged from 6 months to 4 years and children aged from 4 to 16 years. This group included 4 (8%) children with children absence epilepsy, juvenile myoclonic and unclassified epilepsy; one patient had a diagnosis of epileptic encephalopathy without epileptic seizures. Video-EEG monitoring revealed the epileptiform activity on EEG in 9 (18%) children from subgroup 2, four of them suffered from epilepsy. The subclinical epileptiform activity on EEG was observed in 5 (10%) children. The Wechsler test showed that general IQ scores were low (<95%) in 4 (20%) children of this subgroup. No significant difference was found in the performance on the Bayley-III scales between children from subgroup 1 and children born to healthy mothers (controls) though the scores on the fine motor scale and speech development scale were lower in 3 (10%) and 5 (16%) children of the index group, respectively.

[Structure and functions of bacterial proteinase precursors]. / Struktura i funktsii predshestvennikov bakterial'nykh proteinaz.

The data on the precursors of bacterial proteases were generalized. The structure and special features of processing of the precursors of bacillary subtilisins, the alpha-lytic protease from Lysobacter enzymogenes and the related chymotrypsin-like proteases from Streptomyces griseus, and the metalloproteases from bacilli and Pseudomonas aeruginosa were discussed. The approaches to producing the precursors and the protease propeptides and to in vitro characterizing them were particularly analyzed. The following physiological functions of the propeptides within the protease precursors were considered probable: (a) inhibition of the proteases to protect the host cells from the proteolytic damage; (b) participation in the folding of the mature enzyme; and (c) providing for the protease interaction with the bacterial cell surveillance mechanisms, including protease translocation through the cell wall.

[Deletion analysis of the structural-functional organization of metalloendopeptidase precursor in B. amyloliquefaciens]. / Izuchenie strukturno-funktsional'noi organizatsii predshestvennika metalloéndopeptidazy B. amyloliquefaciens metodom deletsionnogo analiza.

Functional destination of propeptides and precursors in bacillar secretory proteases remains uncertain. Formerly deletion assay demonstrated folding and secretion of subtilisin E, chymotrypsin-like protease SGPB from S. griseus and B. cereus metalloprotease to depend on full-length propeptide in the precursors. Actually an artificial B. amyloliquefaciens metalloprotease gene with deletion of 51 amino acid residues from N-terminus was constructed with regard to carry out functional mapping of secretory metalloprotease propeptides. B. subtilis wprA gene 5'-terminal region spanning promoter and secretory leader was coupled to provide transcription to the truncated gene and secretion to its product. B. subtilis clones bearing a plasmid with the modified gene synthesised an active mature metalloprotease.

[Isolation of glutamylendopeptidase precursor from B. licheniformis and its processing in vitro]. / Poluchenie predshestvennika glutamiléndopeptidazy B. licheniformis i izuchenie ego protsessinga in vitro.

A secretory system based on L-form cells of Proteus mirabilis was developed for production of native Bacillus licheniformis glutamylendopeptidase precursor never formerly available. The produced precursor was stable per se under physiological conditions and in presence of trypsin and glutamylendopeptidase from B. intermedius. Complete conversion of the precursor to the mature glutamylendopeptidase was performed by bacillar metalloproteases and subtilisin. The artificially processed glutamylendopeptidase was purified by affinity chromatography on bacitracin-sepharose. Native tertiary structure in the purified glutamylendopeptidase was confirmed by demonstrating its activity towards a specific chromogenous peptide substrate.

Expression of bacillar glutamyl endopeptidase genes in Bacillus subtilis by a new mobilizable single-replicon vector pLF.

The pLF1311 natural plasmid from Lactobacillus fermentum 1311 was used to construct a single-replicon vector suitable for rapid cloning in a wide range of gram-positive hosts and Escherichia coli. The new vector is capable of conjugative mobilization from E. coli to various hosts by conjugal transfer. The final vector (3.4 kb) showed a high segregational and structural stability and a high copy number. Glutamyl endopeptidase genes from Bacillus licheniformis (gseBL) and B. intermedius (gseBI) were cloned in both pLF9 and pLF14 vectors and introduced to B. subtilis. The yield of enzymes in the pLF-derived producers was 6- to 30-fold more than in the natural producers and reached 100-150 mg/L of mature protease.

Introduction of a mini-gene encoding a five-amino acid peptide confers erythromycin resistance on Bacillus subtilis and provides temporary erythromycin protection in Proteus mirabilis.

A 15-bp mini-gene was introduced into Bacillus subtilis and into stable protoplast-like L-forms of Proteus mirabilis. This mini-gene encoded the peptide MVLFV and modeled a fragment of Escherichia coli 23S rRNA responsible for E. coli erythromycin (Ery) resistance. Expression of the introduced mini-gene conferred permanent Ery resistance on B. subtilis. In L-forms of P. mirabilis, the Ery-protective effect was maintained in the course of several generations. Herewith, the mechanism of Ery resistance mediated by expression of specific short peptides was shown to exist in evolutionary distant bacteria. Three new plasmids were constructed containing the gene under study transcriptionally fused with the genes encoding glutamylendopeptidase of Bacillus licheniformis or delta-endotoxin of Bacillus thuringiensis. The Ery resistance pentapeptide (E-peptide) mini-gene served as an efficient direct transcriptional reporter and allowed to select bacillar glutamylendopeptidase with improved productivity. The mini-genes encoding E-peptides may be applied as selective markers to transform both Gram-positive and Gram-negative bacteria. The small size of the E-peptide mini-genes makes them attractive selective markers for vector construction.

Propeptide of the metalloprotease of Brevibacillus brevis 7882 is a strong inhibitor of the mature enzyme.

A metalloprotease gene of Brevibacillus brevis (npr) was expressed in Escherichia coli in a soluble form as native Npr precursor. A significant fraction of the precursor was spontaneously processed, producing the N-terminal propeptide and the mature enzyme. A strong inhibition of the mature Npr by its own propeptide in the crude lysate was observed even in the absence of the covalent linkage between them. Pure precursor, propeptide and the mature Npr were isolated and kinetic parameters of the mature enzyme inhibition by the propeptide were determined. The inhibition is of the tight-binding competitive type with Ki 0.17 nM. Inhibition of metalloproteases from Brevibacillus megaterium and thermolysine by the heterologous propeptide of the Npr from B. brevis was much weaker or none.

Expression of the carboxypeptidase T gene from Thermoactinomyces vulgaris in stable protoplast type L-forms of Proteus mirabilis.

The structural gene of the carboxypeptidase T (cpt) was successfully expressed in cell wall-less L-form cells of Proteus mirabilis. The DNA sequence encoding the PhoA leader peptide was fused with a truncated cpt gene encoding the mature enzyme. The modified gene in a pUC-based kanamycin resistance vector under the control of the lac promoter was transformed into L-form cells of P. mirabilis. They were able to produce the recombinant CpT both as a secretory and as a cell-bound insoluble form. The co-secretory processing of the PhoA leader peptide was quite efficient. The yield of the secreted CpT was not less than 20 mg l-1 and should be improvable.

[Conservative treatment of acute pancreatitis using intralesional electrophoresis]. / Konservativnoe lechenie ostrogo pankreatita s primeneniem vnutri- tkanevogo élektroforeza.

Experimental data are discussed and experience in complex treatment of 102 patients with acute pancreatitis with the application of intralesional+ electrophoresis is analysed. It was demonstrated in experiments on 90 albino rats that in intralesional+ electrophoresis of radioactive++ 5-fluorouracil (5-FU), its concentration in the pancreas was 2.4-3-fold that in the group of animals in which only intraperitoneal injections of the agent were given. Intralesional+ 5-FU, antibiotic, and contrykal electrophoresis was applied in the clinic in 75 patients with edematous and in 27 patients with destructive pancreatitis. Intralesional+ electrophoresis had a favourable effect on the course of acute pancreatitis, but its efficacy was lower in destructive pancreatitis. Intralesional+ electrophoresis made it possible to lower the general therapeutic doses of these agents and to reduce the number of injections per 24 hours. Three fatal outcomes occurred.

[Differential diagnosis of ischemic syndrome of the lower extremities at an angiology department of a polyclinic]. / Differentsial'naia diagnostika ishemicheskogo sindroma nizhnikh konechnostei v usloviiakh angiologicheskogo kabineta polikliniki.

A total of 1545 patients, referred with the diagnosis of obliterating endarteritis to the angiologic unit of the Irkutsk Vascular Centre from various other medical institutions, are examined. Differential diagnosis of the ischemic lower limb syndrome, rooted in vascular and other diseases, is shown to be possible, using noninvasive diagnostic procedures on an outpatient basis. The diagnosis of obliterating endarteritis was only verified in 18.2% of the patients. Atherosclerosis was detected in 41.2% of the patients, aortoarteritis, in 4.7%, diabetic angiopathy, in 5.6%, and nonvascular pathology, in 30.3%. A schedule is proposed for differential diagnosis of the lower limb ischemic syndrome.

[Clinical variants of chronic ischemic syndrome of the lower extremities]. / Klinicheskoe varianty khronicheskogo ishemicheskogo sindroma nizhnikh konechnostei.

Analysis is made of 1,014 cases with occlusive lesion of the bifurcation of the aorta and iliofemoral segment in patients with chronic ischemic syndrome of the lower limbs. The cause of high arterial occlusion with atherosclerosis in 95.5% of patients and nonspecific aortitis in 12 cases. A group of 20 patients with selective lesion of the femoral segment, which is distinguished from atherosclerosis obliterans, endarteritis with ascending thrombosis, and nonspecific aortitis by its clinico-angiographic and morphological manifestations, was analyzed in detail. This form of arterial occlusion is called "segmental thromboarteritis". The results of reconstructive operations in most patients with "segmental thromboarteritis" proved unfavourable.