Effects of rhamnolipid-biosurfactant on cell surface of Pseudomonas aeruginosa.

The effect of rhamnolipid-biosurfactant produced by Pseudomonas sp. PS-17 on cell surface structures of Pseudomonas aeruginosa NBIMCC 1390 was studied. The results demonstrated that the rhamnolipid at concentrations below and above CMC provoked a multi-component response of the bacterial cells without affecting their growth and viability. Above CMC, the rhamnolipid caused reduction of total cellular LPS content of 22%, which can be associated with an increase in cell hydrophobicity to 31% adherence. The rhamnolipid-biosurfactant at concentration below CMC did not affect the LPS component of the bacterial outer membrane but caused changes in OMP composition of P. aeruginosa. Examination of the OMP profiles revealed that the amount of major proteins (Opr F, Opr D, Opr J and Opr M) markedly decreased. To our knowledge this is the first report on the rhamnolipid-biosurfactant interactions with bacterial cells showing changes in outer membrane proteins of P. aeruginosa. In both concentrations, the biosurfactant caused changes in cell surface morphology. The results indicate that the rhamnolipid-biosurfactant from Pseudomonas sp. PS-17 has a potential application in the relatively new field of biomedicine.

Rhamnolipid-biosurfactant permeabilizing effects on gram-positive and gram-negative bacterial strains.

The potential of biosurfactant PS to permeabilize bacterial cells of Pseudomonas aeruginosa, Escherichia coli, and Bacillus subtilis on growing (in vivo) and resting (in vitro) cells was studied. Biosurfactant was shown to have a neutral or detrimental effect on the growth of Gram-positive strains, and this was dependent on the surfactant concentration. The growth of Gram-negative strains was not influenced by the presence of biosurfactant in the media. Cell permeabilization with biosurfactant PS was shown to be more effective with B. subtilis resting cells than with Pseudomonas aeruginosa. Scanning-electron microscopy observations showed that the biosurfactant PS did not exert a disruptive action on resting cells such that it was detrimental to the effect on growing cells of B. subtilis. Low critical micelle concentrations, tender action on nongrowing cells, and neutral effects on the growth of microbial strains at low surfactant concentrations make biosurfactant PS a potential candidate for application in different industrial fields, in environmental bioremediation, and in biomedicine.

Scanning electron microscopy investigations on bis(2-ethylhexyl)phthalate treated Mycobacterium cells.

Comparative investigation of steroid transforming activity and ultrastructural changes of bis(2-ethylhexyl)phthalate (BEHP, phthalate) treated Mycobacterium sp. NRRL B-3805 cells was carried out. Transformation of beta-sitosterol into androstenedione (AD) and androstadienedione (ADD) was performed in phthalate medium by resting cells preincubated in the organic solvent for a period from 3 to 24 h. It was observed that a preincubation greater than 12 h leads to the development of dense formations on the cells surface, reduction in the cell turgor, disruption in the cell walls, and formation of zones with reduced electron density. The preincubation for 24 h causes deeper changes in the cell ultrastructure but the treated cells retain their steroid transforming activity, allowing up to 80% of the substrate to be converted into AD and ADD. A preincubation of the resting Mycobacterium cells in BEHP for 6 h might be recommended as it leads to an achievement of stoichiometrical transformation of the substrate into AD and ADD and slightly higher initial rate of the reaction performed.

Ultrastructural localization of succinate dehydrogenase in some bacteria, after treatment with Lubrol W1.

The localization of succinate dehydrogenase in some gram-negative and gram-positive bacteria (Salmonella typhimurium, Pseudomonas pseudomallei, Pseudomonas aeruginosa and Listeria monocytogenes) treated with the surface membrane active agent, Lubrol W1, was studied by a cytochemical method combined with electron microscopy.

Production and properties of a bacterial thermostable exo-inulinase.

Inulinase and Invertase Activities, Thermophilic Bacilli, Enzyme Thermostability Enzyme production of newly isolated thermophilic inulin-degrading Bacillus sp. 11 strain was studied by batch cultivation in a fermentor. The achieved inulinase and invertase activities after a short growth time (4.25 h) were similar or higher compared to those reported for other mesophilic aerobic or anaerobic thermophilic bacterial producers and yeasts. The investigated enzyme belonged to the exo-type inulinases and splitted-off inulin, sucrose and raffinose. It could be used at temperatures above 65 degrees C and pH range 5.5-7.5. The obtained crude enzyme preparation possessed high thermostability. The residual inulinase and invertase activities were 92-98% after pretreatment at 65 degrees C for 60 min in the presence of substrate inulin.

Yeast permeabilization as a tool for measurement of in situ enzyme activity: localization of alkaline phosphatase.

The biochemical and ultracytochemical localization of alkaline phosphatases in permeabilized cells of Saccharomyces cerevisiae 257 has been studied. The treatment with non-ionic surfactant Triton X-100 allows the penetration of the substrate into intact yeast cells and thus provides detailed detection of the enzyme activity in ultracytochemical studies.

Comparative investigation of a streptovaricin-producing strain of Streptomyces spectabilis and its selectant.

Some differences were found in the ultrastructural, cultural and physiological-biochemical properties between the parent strain Streptomyces spectabilis 1000 and its natural selectant S. spectabilis 1011-10, producers of streptovaricin.

Ultrastructural localization of acid phosphatase in some bacteria, after treatment with Lubrol W1.

The ultracytochemical localization of acid phosphatase from some bacteria (Listeria monocytogenes, Salmonella typhimurium, Pseudomonas pseudomallei and Pseudomonas aeruginosa) was dependent on the changes in the lipoprotein content of the membranes as a result of the action of the Lubrol W1.

Thermostable alpha-amylase production by immobilized Bacillus licheniformis cells in agar gel and on acrylonitrile/acrylamide membranes.

Immobilized cells of Bacillus licheniformis 44MB82-G were used for the production of thermostable alpha-amylase. The immobilization was carried out by entrapment in agar gel or by binding to formaldehyde-activated acrylonitrile/acrylamide membranes. The alpha-amylase production after 144 h of cultivation of membrane immobilized cells was 40% higher in comparison with the free cells. The respective value for the agar-entrapped cells was 22%. Similar trends were observed in the repeated batch fermentations performed with the immobilized cells. The scanning electron micrographs (SEM) of the immobilized cells gave additional information about their binding to the respective carriers.

Ultrastructural characteristics of some bacteria after treatment with Lubrol W.

Specific ultrastructural changes occurred mainly in the cell wall and cytoplasmic membrane of Listeria monocytogenes, Salmonella typhimurium, Pseudomonas pseudomallei and Pseudomonas aeruginosa bacteria when treated with 0.5% and 1% Lubrol W1 by means of transmission and scanning electron microscopy.

[Effect of sugars on the biological properties of bull spermatozoa after cryopreservation]. / Vliianie na niakoi zakhari vurkhu biologichnite kachestva na spermatozoidi ot bik sled kriokonservatsiia.

An investigation was carried out on the protective qualities of some type of sugars as ingredients of the g X22l diluent with regard to bull spermatozoa upon freezing after the granule technology. It was ascertained that sugars varied in effect. On the base of the results obtained a new diluent was developed under the conditional name of g X22A, with better cryoprotective qualities than those of the currently used diluents. The optimum effect of semen dilution with the granule technology of freezing of g X22A proved within the range of 1 + 4 to 1 + 8.

[Cryoprotective properties of the components of diluents for bull seminal fluid]. / Krioprotektivni kachestva na niakoi sustavki na razrediteli za semenna technost ot bik.

Studied were the protective qualities of lipovitellinine with regard to spermatozoa at cryoconservation. It was found that following centrifugation at 2000 G of the GH22L diluent for 20 min the electrophoretic investigations on Cellogel revealed a strong drop of lipovitellinine from 25.33 +/- 4.21 to 2.33 +/- 0.24 per cent. With the Nagaze-Niva diluent the drop was from 24.20 +/- 4.56 to 14.10 +/- 2.95 per cent. The changes in the percent ratio of the protein fractions in dilution of bull semen at 1 + 4 were shown to be considerable with the GH22L diluent after the separation of part of the lipovitellinine. With the Nagaze-Niva diluent these changes were negligible. The separation of part of the lipovitellinine with GH22L led to an essential drop of spermatozoid motility and of the heat resistance of semen following thawing. With the Nagaze-Niva diluent the changes in the qualities of spermatozoa were not substantial after separating part of the lipovitellinine. It was also found that the GH22L diluent produced a better cryoprotective effect on spermatozoa than the Nagaze-Niva diluent.

[Viscosity, pH and the protective properties of the diluents of bull seminal fluid after cryoconservation]. / Prouchvaniia vurkhu viskoziteta, pH i protektivnite kachestva na razrediteli za semenna technost ot bik sled kriokonservatsiia.

The changes in the values of viscosity and pH of some diluents in centrifugation and heat treatment and their protective qualities on bull semen at cryoconservation were followed up. It was found that following centrifugation of the gamma X22 pi diluent at 2,000 G for 20 min viscosity considerably dropped (from 2.240 +/- 0.1170 to 1.234 +/- 0.1153 centipoises). With the Nagaze Niva diluent treated under the same conditions the drop of viscosity was not reliable (from 1.368 +/- 0.1063 to 1.258 +/- 0.147). Following thermal treatment at 70 degrees C for 60 min viscosity in the first case considerably rose (from 2.440 +/- 0.1170 to 2.839 +/- 0.1319 centipoises), while in the second case the difference was not reliable (from 1.368 +/- 0.1063 to 1.706 +/- 0.2579 centipoises). After centrifugation at 2,000 G for 20 min and thermal treatment at 70 degrees C for 60 min the values of the two diluents' pH did not change. The drop of viscosity following centrifugation of the gamma X22 pi diluent was found to be associated a with a drop of its protective qualities on the spermatozoa too; and vice versa--the rise of viscosity of the same diluent following thermal treatment increased its protective qualities with regard to spermatozoa following cryoconservation. The centrifugation of the Nagaze Niva diluent at 2,000 G for 20 min and its thermal treatment at 70 degrees C for 60 min did not lead to statistically reliable changes in its viscosity. Neither were statistically reliable the changes in its protection of spermatozoa after cryoconservation.

[Relation between the denaturation changes in the diluents and the freezability of bull spermatozoa]. / Zavisimost mezhdu denaturatsionnite promeni v niakoi razrediteli i zamraziaemostta na spermatozoidite pri bik.

Studied were the changes taking place in the proteins of diluted and frozen semen of bulls following heat treatment. Studied was also the effect of heat-treated diluents on some of the qualitative indices of bull semen at cryo-conservation. It was found that at the dilution of semen with extenders with or without heat treatment there set in various changes in the percent distribution of proteins. Most strongly expressed were the changes in proteins when the gkh22l diluent was used. They were associated with the various degree of dilution with different extenders. The heat treatment of gkh22l improved the heat resistance of spermatozoa after thawing; the Nagaze Niva diluent was shown to slightly deteriorate this index. The cumulation of more proteins in the region of the 2nd fraction following the heat treatment of gkh22l at 70 degrees C for 60 min and dilution of 1+6 rendered more favourable conditions for the spermatozoa at freezing. The drop of the proteins in the region of the 1st fraction at dilution of 1+2 with Nagaze Niva led to the drop of its protective properties.

[Protein fractions in diluted bull seminal fluid with various biological indices after thermal treatment]. / Izsledvaniia vurkhu beltuchnite fraktsii na razredena semenna technost ot bik s razlichni biologichni pokazateli sled termichna obrabotka.

Studied were electrophoretically the protein fractions of bull semen after heat treatment of the GH22L extender. Studied were also the changes in the protein fractions of diluted semen of various biologic indices. It was found that after heat treatment of the extender at 70 degrees C for 60 min. most stable proved the fractions that were at the front and the start. Negligible changes underwent the proteins of the 3rd and 4th fraction. The changes in the percent distribution of the protein fractions of diluted qualitative and unqualitative bull semen at 1+2 degree with thermically untreated GH22L extender were best manifested with the semen of lower quality. Weaker were the changes taking place with dilution of qualitative and unqualitative semen at 1+6 degree. These changes were observed also with dilution at 1+2 with thermically treated extender (70 degrees C for 60 min.). Dilution of qualitative semen with thermically treated (at 1+6) extender most strongly changed the proteins of the 1st, 2nd, and 3rd fraction. The proteins of the start showed high stability. Nonsignificant were the changes with unqualitative semen with which all fractions showed great heat stability.

[Preservation of ram seminal fluid in different diluents]. / Prouchvaniia vurkhu sukhranenieto na semenna technost ot kochove v razlichni razrediteli.

Studied was the conformation of the protein macromolecules with the dilution of ram semen to a various extent, using three yolk diluents -- gkh18, zhgts, and Zlatno rouno. Studied was also the effect of some yolk extenders used in this country's practice on some qualitative indices of ram semen under storing conditions. It was found that following dilution with the media cited above various conformational changes of the proteins take place with the semen of rams. The different rates of dilution had no effect on the primary motility of spermatozoa. Statistically significant was the difference in favour of the gkh18 diluent in so far as the heat resistance of spermatozoa was concerned. Most effective dilution (in terms of retaining the motility of spermatozoa at storing) proved 1 + 3, followed by 1 + 5 with the use of gkh18, and 1 + 1 with zhgts and Zlatno rouno. gkh18 was shown to produce a varying blocking effect on motility of spermatozoa. All results concerning the conformation of protein macromolecules, quality indices, and particularly the resistance to heat in evaluating the storing capacity of semen showed that the cumulation of more proteins of higher electrophoretic mobility in III group developed more favourable conditions in preserving the vitality of spermatozoa at storage.

[Thermal stability study of yolk extenders for the cryopreservation of semen]. / Prouchvaniia vurkhu termichnata stabilnost na niakoi zhultuchni razrediteli za kriokonservatsiia na semennia technost.

Investigated was the fate of proteins in some yolk extenders--NN (Nagasse--Niva), GH18 (after Kichev), Zr (after Zlatarev), and ZhGTs (after Milovanov)--following heat treatment. Centrifugation at 14 000 rpm of untreated extenders (NN, GH18, Zr, and ZhGTs was found to lead to the strong drop of proteins of the start with NN and GH18, whereas there was no change in the proteins of the start with Zr and ZhGTs. The heat denaturation of the four extenders at 55 degrees C, 60 degrees C, and 65 degrees C for 30 min. and their centrifugation at 14 000 for 40 min. resulted in the change of proteins--most strongly expressed with the proteins of the start and those immediately in front of it with Zr and ZhGTs. The changes in the protein macromolecules of the four extenders were best expressed at the heat denaturation for 30 min. at 70 degrees C. The densitometric profiles were strongly simplified with Zr and ZhGTs, at which first group proteins (of the front) completely disappeared. NN and GH18 changed but slightly. Second and third group proteins of NN and GH18 showed high stability as against the remaining proteins. With Zr and ZhGTs most stable proved the proteins of the second group. The strong drop of the percent share of the start proteins of NN and GH18 and the disappearance of first group proteins of Zr and ZhGTs was associated with essential denaturation changes that affected adversely the structure of protein macromolecules.