Effect of Heterologous Expression of Chemotaxis Proteins from Genus Thermotoga on the Growth Kinetics of Escherichia coli Cells.

In the process of optimization of heterologous expression of thermostable chemotaxis proteins CheW and CheY as industrially useful polypeptides, their direct influence on the cell growth kinetics and morphology of Escherichia coli was observed. CheW and CheY of bacteria of the genus Thermotoga, being expressed in recombinant form in E. coli cells, are involved in the corresponding signal pathways of the mesophilic microorganisms. The effects of such involvement in the metabolism of "host" cells are extremely diverse: from rapid aging of the culture to elongation of the stationary growth phase. We also discuss the mechanisms of the influence of the heterologous chemotaxis proteins on cells, their positive and negative effects, as well as potential applications in industry and biomedicine.

[The clinical features of cystic fibrosis in adult patients with carbohydrate metabolic disturbances].

AIM: To investigate the clinical features of cystic fibrosis (CF) in adult patients with carbohydrate metabolic disturbances (CMD). MATERIAL AND METHODS: CF was diagnosed on the basis of its clinical picture and a positive sweat test, and/or genetic study. Clinical, anthropometric, functional, microbiological data were compared with the results of imaging diagnosis in CF patients with and without CMD. RESULTS: The data of 350 patients were retrospectively analyzed. An oral glucose tolerance test was randomly performed in 154 CF patients without CMD. There was normal carbohydrate metabolism in 92 (59.7%) patients with CF, impaired glucose tolerance (IGT) in 44 (28.6%), CF-dependent diabetes mellitus (CFDDM) in 18 (11.70%). The latter had been previously diagnosed in 37 (10.6%) other patients with CF. Three groups of patients were formed: 1) 92 patients without CMD; 2) 44 with IGT, and 3) 55 with CFDDM. The patients with CFDDM had lower stature, weight, and lung function, significantly more common bronchiectases, a lower Staphylococcus aureus colonization with a tendency toward a higher Burkholderia cepacia colonization than those without CMD. As compared with the patients without CMD, those with this disorder were found to have a high rate of severe mutations; mild mutations were absent in the patients with CFDDM. CONCLUSION: CMD in CF is characterized by its high rates and latent course. The patients with CMD have retarded physical development, more pronounced morphofunctional disorders in the bronchopulmonary system, lower lung functional parameters, and more aggressive sputum microbial composition.

Physico-chemical and biological properties of solvent/detergent treated immunoglobuin G preparations.

Protein composition homogeneity, fraction composition, molecular weight composition, anticomplementary activity, anti-A and anti-B haemagglutinins, specific activity, and pyrogens were studied in intramuscular and intravenous immunoglobulin G preparations, treated and non-treated with solvent/detergent mixture for virus inactivation. Physico-chemical and biological properties retention is shown. Quality of immunoglobulin preparations was improved due to purification by the methods of anion- and cation- exchange preparative low pressure liquid chromatography.

[pSTS4--a new shuttle vector for cyanobacterium Plectonema boryanum CALU 465]. / pSTS4--novyi chelnochnyi vektor dlia tsianobakterii Plectonema boryanum CALU 465.

Three vector constructions--pSTS2, pSTS3 and pSTS4 have been obtained on the basis of the standard vector for Escherichia coli pBR 322, cyanobacterial plasmid pSM1 and its separate fragments. The structure of pSTS4, carrying the full-size plasmid pSM1 (cloned by the restriction site Hpa II), replicated both in the cells of E. coli and in the cells-transconjugants of the trichomal cyanobacterium Plectonema boryanum. Under these conditions resistance of P. boryanum cells to ampicillin increases three orders, and to tetracyclin--50 times. Structural integrity of pSTS4 is stably supported in conjugates during seven successive passages (time of the experiment) and preserved the transforming capacity in respect of the cells of E. coli. It is supposed that pSTS4 is a shuttle-vector with two active origins of replication; one of them provides for the replication of pSTS4 in E. coli cells, another--in P. boryanum cells.

[The nucleotide sequence of the rep gene of cyanobacterial plasmid pSM1]. / Nukleotidnaia posledovatel'nost' gena Rep-belka tsianobakterial'noi plazmidy pSM1.

The nucleotide sequence was established for the rep gene of plasmid pSM1 isolated from cyanobacterium Plectonema boryanum CALU 465. Both nucleotide sequence and the encoded amino acid sequences showed 98% homology to the corresponding sequences of small plasmids pPF1, pGL3, pPBS1, pBLX, and pPB1. An active center was identified in the replicative protein sequences.

[Isolation and purification of nonspecific nuclease of cyanobacterium Plectonema boryanum CALU 465]. / Vydelenie i ochistka nespetsificheskoi nukleazy tsianobakterii Plectonema boryanum CALU 465.

Nonspecific nuclease has been isolated from the cells of cyanobacterium Plectonema boryanum and purified to homogenic state. It has been established that the method of centrifugation of cell-free culture extract in the sucrose density gradient is efficient for the separation of pigment proteins and enzyme concentration. Under the successive use of two ion-exchangers the nuclease activity was determined in the concentration range of NaCl 0.065-0.085 M after separation of the cell-free cyanobacterium extract on the column with phosphocellulose in the range of 0.2-0.25 M, on the column with DEAE--Toyopearl respectively. The molecular mass of nuclease which is 40 kDa, has been determined by electrophoresis in polyacrylamide gel under denaturating conditions and gel-filtration on Sephadex G-100. It has been also established that the given enzyme is monosubunitary as to its structure.

[Construction of vector plasmids for the Plectonema boryanum cyanobacterium and creation, on such basis, of the vector-host system]. / Konstruirovanie vektornykh plazmid dlia tsianobakterii Plectonema boryanum i sozdanie na ikh osnove sistemy vektor-khoziain.

The plasmid composition of the Plectonema boryanum Gom. Cyanobacterium, strain CALU 465, was analyzed. A small pSM1 plasmid, size 1.5, was chosen for genetic engineering. The physical map, constructed for the plasmid, was used to create the pSTS series, i.e. vector construction for the P. boryanum cyanobacterium. A method was selected and tested for the introduction of the vector into host cells. The needed orientation of pSM1 cyanobacterium plasmid within the vector as well as a possible role of its separate elements in inheriting the construction by host were defined.

[Localization of DNAase activity in cyanobacterium Plectonema boryanum CALU 465]. / Lokalizatsiia DNKaznoi aktivnosti v kletkakh tsianobakterii Plectonema boryanum CALU 465.

It was shown in experiments carried out on cyanobacterium P. boryanum, that when cells are incubated in a buffer with low concentrations of nonion detergent (0.1%, Triton X-100) one can observe the DNAse activity release into the medium. The methods of supersound desintegration of cells and following gradient centrifugation of the cell-free extracts were used to obtain three fractions of the cell membranes identified by means of spectrophotometric analysis. It has been established that P. boryanum is characterised by high level of DNAse activity associated with the cytoplasmic cell membrane. As to its activity character the DNAse under study belongs to the class of nonspecific endonucleases.

[Physical mapping of plasmid pSM1 of the cyanobacterium Plectonema boryanum GOM. strain CALU 465]. / Fizicheskoe kartirovanie plazmidy pSM1 iz tsianobakterii Plectonema boryanum GOM. shtamm CALU 465.

A physical map of plasmid pSM1 has been compiled as a result of restriction analysis of pBSMI isolated from the cells of cyanobacterium Plectonema boryanum 465. A comparative analysis of the restriction sites localization in DNA of plasmid pSM1 with site topology of plasmids pGL3 of P. boryanum and pPF1 of Phormidium foveolarum indicates to high degree of these plasmids homology.

[Construction of the shuttle vector pSTS1 and development of the method of its introduction into the cells of the cyanbacterium Plectonema boryanum CALU 465]. / Konstruirovanie chelnochnogo vektora pSTS1 i otrabotka sposoba ego vvedeniia v kletki tsianobakterii Plectonema boryanum GOM. shtamm CALU 465.

It has been shown that cells of trichoma cyanobacterium Plectonema boryanum G o m. strain CALU 465 do not possess natural resistance to antibiotics ampicyllin, chloramphenicol, streptomycin and canamycin. A shuttle vector pSTS1 has been constructed on the basis of cyanobacterial plasmid pSM1 and vector pBR 322. Conditions have been developed and conjugative transfer of pSTS1 to the cells of cyanobacterium P. boryanum has been performed. As the result of expression of beta-lactamase gene pSTS1 the resistance of cells-conjugantes to ampicillin increased by three orders.

[The isolation, purification and cloning of the pSM1 plasmid from the cyanobacterium Plectonema boryanum]. / Vydelenie, ochistka i klonirovanie plazmidy pSM1 iz tsianobakterii Plectonema boryanum.

Three methods of plasmids isolation from the cells of cyanobacteria: Plectonema boryanum 465, P. nostocorum 636, P. nostocorum 638, P. edaphicum 262, Plectonema sp. 456, Phormidium uncinatum 528, Anabaena variabilis 458 and Synechococcus cedrorum 750 have been approved. The method of plasmids isolation by 1 M NaCl salting out was used for plasmid screening. All the strains studied contained large, above 55 KB plasmids, small plasmids (1.5 KB) were found in the cells of P. boryanum 465 and P. nostocorum 636. After the restriction analysis a small plasmid of P. boryanum 465 was chosen to be used in the experiments on the vector construction. The above plasmid was called pSM1 was cloned into the vector pBluescri ptIISK(+) on the unique ClaI site. The obtained construction pBSMI may be used as the basic one for creation of cyanobacterial vectors.

[Effectiveness of the new carbapenem antibiotic, meropenem, in adult patients with mucoviscidosis]. / Effektivnost' primeneniia novogo karbapenemnogo antibiotika-- meropenema u vzroslykh bol'nykh s mukovistsidozom.

Clinical efficacy of meropenem (meronem, Zeneca), a new carbapenem, was studied in the treatment of 4 adult patients with mucoviscidosis. The drug was administered as intravenous infusions 3 times a day in a daily dose of 60 mg/kg body weight for 10 days. A positive clinical effect was observed in 3 patients and in 1 patient stabilization of the clinical state was recorded. A significant decrease in the titre of the Pseudomonas aeruginosa colonies in 2 patients and that of the P. aeruginosa mucosa colonies in 3 patients was bacteriologically confirmed. Good tolerance and no side effects of meropenem in the doses used were stated. The study showed that meropenem may be recommended for the treatment of adult patients with mucoviscidosis.

[Analysis of the changes in the electrophysical and morphometric parameters in the growth and sporulation periods of Bacillus thuringiensis]. / Analiz izmenenii élektrofizicheskikh i morfometricheskikh parametrov v period rosta i sporuliatsii u Bacillus thuringiensis.

Changes in the electrophysical and morphometrical parameters of Bacillus thuringiensis cells were studied during their growth and differentiation using the electrophysical procedure with the frequency of an alternating electric field varying within a wide range. The appearing variations were interpreted on the basis of the data about changes in the concentration of vegetative cells and spores, the optical density and pH of the cultural broth, and the assimilation of glucose by the culture. The changes found in the electrophysical and morphometrical parameters were shown to accompany the main physiological processes in the cells. In principle, they can be used to control the process of cultivation.

[Plasmid preservation in Escherichia coli cells in batch and continuous cultivation]. / Sokhranenie plazmid v kletkakh kishechnoi palochki pri periodicheskom i nepreryvnom kul'tivirovanii.

The growth kinetics of plasmid and nonplasmid strains of 2 isogenic E. coli K12 culture lines has been studied in experiments on batch and continuous cultivation. Under the optimum conditions the growth rate of R+- and R- cells is the same; under the conditions of limited glucose supply the plasmid strains grown slower, as their energy is partly spent on maintaining plasmids in the cell.

[Effect of temperature on Candida utilis morphology and growth rate under limited and unlimited cultivation conditions]. / Vliianie temperatury na morfologiiu i skorost' rosta Candida utilis v limitirovannykh i nelimitirovannykh usloviiakh kul'tivirovaniia.

The effect of temperature on the growth rate, morphology and ultrastructure of the yeast Candida utilis was studied under the conditions of turbidostat and chemostat. The mean size of the yeast cell was found to increase at suboptimal and supraoptimal temperatures while the quantity of membranous structures was shown to decrease under the same conditions. The mean size of cells grown under the conditions of turbidostat was much greater than that of cells grown under the conditions limited with glucose. The mechanisms involved in morphocytological changes at suboptimal and supraoptimal temperatures appear to be different.