[Study of genetic diversity in potato cultivars using PCR analysis of organelle DNA].

The genetic diversity of 98 potato cultivars of Russian and foreign breeding was studied using of PCR with organelle-specific primers. The polymorphism of both plastid (atpE, trnG/trnK) and mitochondrial (rps, atp6) loci was revealed. Eight different haplotypes were detected in the sample of cultivars studied. Comparatively low of polymorphism of organelle DNA in the potato cultivars was demonstrated: most cultivars (91 or 92.9%) possessed only two haplotypes (I and II); 62 cultivars of them had similar "cultural" cytoplasmic type (haplotype I). The breeding cultivars of the Russian and foreign origin did not differ from each other in frequency of basic haplotypes.

Toxin-binding proteins from midgut epithelium membranes of Anopheles stephensi larvae.

Proteins of 65 and 57 kD were isolated from the apical membranes of midgut epithelium of Anopheles stephensi larvae by affinity chromatography. These proteins can specifically bind endotoxin Cry11A and activate toxin Cry4B (Cry4B-tox) under conditions of ligand blotting, and both Cry proteins compete for this binding. At least in the case of Cry4B-tox, the binding with 65 and 57 kD proteins is reversible. The ability of the products of limited proteolysis of Cry11A and Cry4B to bind the 65 and 57 kD proteins correlates with their toxicity to A. stephensi larva. The N-terminal amino acid sequence of the 57 kD protein is unique and absent in the NCBI GenBank. The proteins of 65 and 57 kD share most of the properties studied with Aedes aegypti toxin-binding proteins. It is possible that they altogether represent a novel class (or classes) of delta-endotoxin receptors.

Reconstruction of Bacillus thuringiensis ssp. israelensis Cry11A endotoxin from fragments corresponding to its N- and C-moieties restores its original biological activity.

Subtilisin hydrolyzes Cry11A endotoxin (of 70 kD) produced by Bacillus thuringiensis ssp. israelensis to fragments of 33- and 36-kD, which correspond to N- and C-terminal halves of the endotoxin molecule. Thermitase (a serine protease from Thermoactinomyces vulgaris) and insect gut proteases from Diptera and Lepidoptera exhibit the same hydrolytic effect on Cry11A. Hydrolyzates maintain high toxicity with respect to larvae of Aedes aegypti, Anopheles stephensi, and Culex pipiens. The 33- and 36-kD Cry11A endotoxin components purified by ion-exchange chromatography from the subtilisin hydrolyzate were inactive; however, equimolar mixture of these proteins exhibited almost the same activity as the initial hydrolyzate.

Interaction of 65- and 62-kD proteins from the apical membranes of the Aedes aegypti larvae midgut epithelium with Cry4B and Cry11A endotoxins of Bacillus thuringiensis.

A protein with the molecular weight of 65 kD is the only component of Aedes aegypti larvae BBM capable to specifically bind mosquitocidal toxins Cry4B and Cry11A of Bacillus thuringiensis. This protein lacks the leucine aminopeptidase activity which is characteristic for the toxin-binding proteins from the membranes of caterpillars. Cry-toxins inactive against A. aegypti larvae either fail to bind to the 65-kD protein and to a putative product of its proteolysis with the molecular weight of 62 kD (Cry1Ab), or bind but do not compete for this binding with mosquitocidal proteins (Cry9A). The proteolytic splitting out of the first five alpha-helices in the Cry4B toxin molecule does not affect its binding to the 65- and 62-kD proteins, but an additional removal of 20-30 amino acids from the C-terminal of the molecule sharply spoils this binding. Monosaccharide residues are not involved in the binding of the 65- and 62-kD proteins with Cry4B, Cry11A, and Cry9A.

Membrane proteins of Aedes aegypti larvae bind toxins Cry4B and Cry11A of Bacillus thuringiensis ssp. israelensis.

Proteins of molecular weight 65 and 62 kD and having affinity for toxins Cry4B and Cry11A produced by Bacillus thuringiensis ssp. israelensis have been isolated from brush border membranes of Aedes aegypti larvae using affinity chromatography. Using a ligand blotting technique, we show that the binding of these proteins to the biotinylated toxins is reversible and that the two toxins compete for binding to the two proteins. These proteins are likely to be Cry4B and Cry11A toxin receptors in gut epithelial cells of Aedes aegypti larvae.

Limited proteolysis of Bacillus thuringiensis CryIG and CryIVB delta-endotoxins leads to formation of active fragments that do not coincide with the structural domains.

Bacillus thuringiensis "true" toxins consist of three domains: the N-terminal, alpha-helical domain followed by two beta-structural domains. Their limited proteolysis does not proceed at the domain boundaries, but is directed to the loops within the domains. There are at least two patterns of the limited proteolysis of "true" toxins. The first pattern, observed for CryIA and CryIVD delta-endotoxins, results in the proteolysis of the loops connecting beta-strands of the second domain. The second pattern, detected for CryIG and CryIVB proteins, consists in the cleavage of the loop connecting the fifth and the sixth alpha-helices of the first domain. The choice between the routes depends on the size, sequence, and dynamics of the loop that define its accessibility to a proteinase. Bioassay of CryIG and CryIVB delta-endotoxin fragments indicates that only two alpha-helices, the sixth and the seventh within the first domain, followed by the two beta-structural domains are sufficient for the insecticidal activity.

Production of multiple delta-endotoxins by Bacillus thuringiensis: delta-endotoxins produced by strains of the subspecies galleriae and wuhanensis.

A method was developed to assess the number of delta-endotoxins contained in Bacillus thuringiensis entomocidal crystals. It utilized proteolytic conversion of 130-kDa protoxin into 60- to 65-kDa "true" toxin via limited proteolysis with trypsin and separation of stable N-terminal domains by fast-performance liquid chromatography. Immunodiffusion experiments and N-terminal sequence determination (applied to the major component isolated by SDS-PAGE) completed the analysis of the crystal protein composition. The application of this approach to crystals produced by cells of B. thuringiensis subsp. galleriae and wuhanensis allowed us to identify at least seven and eight different delta-endotoxins, respectively. Among those delta-endotoxins assigned to previously described families, CryIA, CryID, CryIF, and CryIG were found, as well as crystal proteins, which possess N-terminal amino acid sequences very different from those of all known delta-endotoxins. Possible functional consequences of delta-endotoxin multiplicity are discussed.

Subdomain organization of Bacillus thuringiensis entomocidal proteins' N-terminal domains.

N-Terminal domain (65 kD) of delta-endotoxin produced by Bacillus thuringiensis ssp. alesti, as shown by limited proteolysis, consists of two subdomains of molecular mass 30 and 33 kD that correspond, respectively, to conservative and variable regions of the delta-endotoxin primary structure. Furthermore, proteolysis of these subdomains leads to their conversion into at least two fragments of molecular mass 10 kD stable to proteinase action. Such a pattern of molecular organization appears to be common for several structurally related delta-endotoxins that belong to the kurstaki group. Entomicidal protein produced by ssp. israelensis (70 kD), which differs strongly from alesti and other kurstaki group delta-endotoxins, retains a similar type of molecular organization and consists of two subdomains with molecular mass of approximately 35 kD. Apparently, the characteristic pattern of the delta-endotoxins' molecular structure reflects separation of functions (e.g., host recognition and toxicity per se) between domains and subdomains of these proteins.

Crystal-forming proteins of Bacillus thuringiensis. The limited hydrolysis by endogeneous proteinases as a cause of their apparent multiplicity.

The crystals of the entomocidal protein of Bacillus thuringiensis are admixed with proteinases that in the course of their dissolution cause gradual degradation of the "genuine" crystal-forming protein components (i.e. the primary biosynthetic products) to products of lower molecular weight. This phenomenon might explain at least partially the contradictory data on the molecular parameters of the crystal-forming proteins. Preliminary inactivation of the proteinases adsorbed on the crystals allowed us to eliminate this source of the artefacts and to gain more reliable data on the protein composition of the crystals formed by various strains of B. thuringiensis. It has been shown that the crystals formed by all serotypes of B. thuringiensis, with the exception of the serotype V, contain only one protein with a mol. wt. of 145000, 135000 or 130000, depending on the strain. The majority of the strains that belong to the serotype V form crystals consisting of two proteins with mol. wts. of 135000 and 130000, but some of them also have a third component with a mol. wt. of 65000.

[Proteolytic enzymes bound to Bac. thuringiensis crystals]. / Proteinazy, Sviazannye s kristallami Bac. thuringiensis.

It was demonstrated that crystals of entomopathogenic protein from Bac. thuringiensis contain admixture of proteinase either adhered to their surface on inconponated into crystal lattice defects. A proteolytic action, particularly when enhanced by crystal dissolution, causes progressive degradation of crystal proteins with molecular weights of 140 000--129 000 down to the components with smaller molecular weights. This may, at least, partially account for the contradictions in the literature data on crystal composition. Using synthetic peptide substrates and specific inhibitors, it was shown that the enzymes incorporated into crystals belong to serine and metalloproteases. The presence of leucine aminopeptidase was also noted. A method for enzyme separation from crystal has been developed.

[Proteins of Bacillus thuringiensis delta-endotoxin crystals]. / Belki kristallov delta-endotoksina Bac. thuringiensis.

Pure crystals (at least 99% purification) of sigma-endotoxin were isolated from Bac. thuringiensis var. galleriae. The complete dissolution of crystals might be achieved by the increase of pH up to 12 and higher or by a combined action of S = S-reducing and denaturing agents. Electrophoresis of the solubilized crystal proteins in 5% polyacrylamide gels containing 0,1% sodium dodecyl sulfate and 8 M urea reveals two major bands corresponding to molecular weights of 120000--140000 (65%) and 65000 (8-10%), and some minor components whose molecular weights varied from 65000 to 340000. Urea (3--8 M) causes to partial dissolution of the crystals; the component with molecular weight of 65000 is mainly found in the solution (component A). In dithioerythritol extracts at pH 9 the major component of the crystal is the protein with molecular weight 120000--140000 (component B). The crystals, alkali-soluble components and proteins isolated from crystals by selective extraction (3--8 M urea or 0.01 M dithioerythrytol, pH 9) were found toxic for the larvae of Galleria mellonella.

[Differential diagnosis between pyometra and tumor of the uterine body after radiotherapy of cervical cancer]. / Differentsial'nyi diagnoz mezhdu piometroi i opukho l'iu tela matki posle luchevoi terapii raka sheiki matki.

Clinico-morphological data on 25 patients are presented, in whom within the terms from 7 months to 25 years following radiotherapy for cervical cancer an increased size of the uterine body was noted, the latter would practically be induced either by recurrent tumor growth (in 13 patients), or primary-multiple tumor-adenocarcinoma (in 6 patients), or mixed mesodermal tumor (5 patients).