Streptomyces tsukubensis VKM Aс-2618D-an Effective Producer of Tacrolimus.

The Streptomyces sp. VKM Ac-2618D strain has been identified, and its morphological and physiological features have been studied in relation to the production of the immunosuppressant tacrolimus. The phenotypic variability of the strain was analyzed, and a dissociant with a high level of tacrolimus production was selected. Based on a comprehensive study of morphological, physiological, and chemotaxonomic properties and on phylogenetic analysis, the strain was named Streptomyces tsukubensis VKM Ac-2618D. The strain genome contains the full version of the tacrolimus biosynthetic gene cluster. The advantages of fed-batch cultivation mode for tacrolimus biosynthesis are shown. The results broaden the understanding of the characteristics of polyketide biosynthesis and can be used in the development of technology for tacrolimus production.

[Structural, Functional, and Evolutionary Characteristics of Proteins with Repeats].

The review summarizes and systematizes the data on the classification, taxonomic distribution, structural features, and functions of proteins with structural repeats. Modern approaches to the identification of structural repeats in proteins are considered. Features of specialized databases of protein domains are described. The review discusses the prospects of using repeat-containing proteins as scaffolds for drug design. The role of proteins with structural repeats in the pathogenesis of various diseases is considered. Various modern approaches to understanding the mechanisms of the evolutionary development of proteins with repeats are described and analyzed.

Use of biocompatible redox-active polymers based on carbon nanotubes and modified organic matrices for development of a highly sensitive BOD biosensor.

This work investigated the use of redox-active polymers based on bovine serum albumin and chitosan, covalently bound to mediators neutral red and ferrocene and containing carbon nanotubes, for immobilization of Paracoccus yeei VKM B-3302 bacteria. The structures of produced polymers were studied by IR spectroscopy and scanning electron microscopy. Cyclic voltammetry and impedance spectroscopy found the electrochemical characteristics of the investigated systems: the heterogeneous electron transfer rate constant, the constant of the rate of interaction with P. yeei bacteria and the impedance. The systems containing carbon nanotubes and ferrocene-based redox-active polymer proved to be the most promising. Biosensors formed using the hybrid polymers had a high sensitivity with the lower boundary of 0.1 mg/dm3 of the detected BOD5 concentrations and a high correlation (R = 0.9916) with the standard BOD assay of surface water samples.

Lysis of cells of diverse bacteria by l,d-peptidases of Escherichia coli bacteriophages RB43, RB49 and T5.

AIMS: The objective of this work was to study the antibacterial specificity and antibacterial effect of endolysins isolated from colibacteriophages RB43, RB49 and T5-as manifested on the exponential and stationary cell cultures of diverse bacteria depending on the growth stage, structure of peptidoglycan (PG) and antibiotic resistance. METHODS AND RESULTS: Enzyme activity was assayed by the spectrophotometric method. Antimicrobial activity was estimated by the number of colony forming units (CFUs), with the results represented as logarithmic units. Morphological examination of bacterial cells was conducted using phase-contrast and scanning electron microscopy. The enzymes EndoT5, endolysin of bacteriophage T5, EndoRB43, endolysin of bacteriophage RB43 and EndoRB49, endolysin of bacteriophage RB49 turned out to be much less bacteriospecific than the corresponding Escherichia coli phages; they lysed bacteria of the genera Bacillus, Cellulomonas and Sporosarcina, whose PGs had different structures (A1γ, A4α and A4ß) and chemical modifications (amidation). The specific lytic activity of phage enzymes was independent of the antibiotic resistance of bacterial cells and was higher when the cells were in the exponential, rather than stationary, growth phase. The analysis of morphological changes showed that the intermediate stage of the endolysin-induced lysis of bacterial cells was the formation of spheroplasts and protoplasts. CONCLUSIONS: Endolysins of colibacteriophages RB49, RB43 and T5 have a wide spectrum of antibacterial action, which includes a number of diverse micro-organisms with different PG structures. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a study of the bacterial selectivity of enzymes degrading bacterial cell wall in relation to the chemical structure of PG. It is shown that endolysins of bacteriophages RB49 and RB43 efficiently lyse cell wall of Gram-positive bacteria of the genus Bacillus and Gram-negative bacteria of the genus Pseudomonas (including an antibiotic-resistant strain). The number of bacterial cells is reduced by 3-6 orders of magnitude, which indicates good prospects for using these enzymes in biotechnology.

Registration of BOD using Paracoccus yeei bacteria isolated from activated sludge.

This work investigated the properties of Paracoccus yeei VKM B-3302 bacteria isolated from activated sludge and immobilized in an N-vinylpyrrolidone-modified poly(vinyl alcohol) matrix. The developed hydrogel formed a network structure to enable the entrapment of microbial cells with their viability and biocatalytic properties preserved, which ensured the technological possibility of replicating expendable biosensor receptor elements. A new ratio of the components for the synthesis selected in this work enabled producing a copolymer of an earlier undescribed chemical structure, which can be efficiently used for immobilization of highly sensitive P. yeei bacteria. A biological oxygen demand (BOD) biosensor with these bacteria and matrix was shown to possess a long-time stability exceeding that described earlier, to have a broad substrate specificity and to exceed approximately tenfold the nearest analogues by its sensitivity and the lower boundary value of 0.05 mg/dm3. The biosensor enabled assays of water samples initially attributed to pure samples (the BOD range, 0.05-5.0 mg/dm3). BOD assays of water samples from various sources showed the use of the receptor element of this composition to enable the data that closely correlated with the standard method (R 2 = 0.9990).

In vitro study of the antibacterial effect of the bacteriophage T5 thermostable endolysin on Escherichia coli cells.

AIMS: This study aimed to evaluate lysis of Escherichia coli stationary cell cultures induced by the combined action of bacteriophage T5 endolysin (l-alanyl-d-glutamate peptidase) and low doses of various cationic agents permeabilizing the outer membrane of Gram-negative bacteria (polymyxin B, gramicidin D, poly-l-lysine, chlorhexidine and miramistin). METHODS AND RESULTS: The enzyme activity was assayed with the turbidimetric method. Antimicrobial activity was assessed through the number of colony-forming units (CFUs); the results of calculation were represented as logarithmic units. The optical microscopy examination of bacterial cells was conducted in the phase-contrast mode. The use of bacteriophage T5 endolysin in combination with polymyxin B (0·4 µg ml-1 ) or chlorhexidine (0·5 µg ml-1 ) made it possible to reduce the number of CFUs by five orders of magnitude; and in combination with poly-l-lysine (80 µg ml-1 ) by four orders, as compared to control. The endolysin was found to be a thermostable protein: it retained ~65% of its initial activity after heating for 30 min at 90°C. Examining the curves of its thermal denaturation revealed the half-transition temperature to be 56·3 ± 1·0°C. Circular dichroism spectra showed that after recooling the protein restored up to 80% of its native structure. CONCLUSIONS: A substantial synergistic effect of the bacteriophage T5 endolysin and membrane-permeabilizing compounds was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: The study of thermal stability of the bacteriophage T5 endolysin and the quantified assessment of its antimicrobial activity have been done for the first time. The approach examined lays foundations for designing a two-component preparation which would effectively lyse cells of Gram-negative pathogens from outside.

The structure-forming role of magnesium pyrophosphate in the formation of DNA-containing microparticles during PCR.

This work is devoted to studying the mechanisms of formation of DNA-containing microparticles (MPs) during PCR. It was found that pyrophosphate, a byproduct of DNA synthesis, and magnesium cations are required for their formation, as evidenced by the results of biochemical and electron microscopy studies.

Yeast-based self-organized hybrid bio-silica sol-gels for the design of biosensors.

The methylotrophic Pichia angusta VKM Y-2559 and the oleaginous Cryptococcus curvatus VKM Y-3288 yeast cells were immobilized in a bimodal silica-organic sol-gel matrix comprised of tetraethoxysilane (TEOS), the hydrophobic additive methyltriethoxysilane (MTES) and the porogen polyethylene glycol (PEG). Under carefully optimized experimental conditions, employing basic catalysts, yeast cells have become the nucleation centers for a silica-organic capsule assembled around the cells. The dynamic process involved in the formation of the sol-gel matrix has been investigated using optical and scanning electron microscopic techniques. The results demonstrated the influence of the MTES composition on the nature of the encapsulation of the yeast cells, together with the architecture of the three-dimensional (3D) sol-gel biomatrix that forms during the encapsulation process. A silica capsule was found to form around each yeast cell when using 85 vol% MTES. This capsule was found to protect the microorganisms from the harmful effects that result from exposure to heavy metal ions and UV radiation. The encapsulated P. angusta BKM Y-2559 cells were then employed as a biosensing element for the detection of methanol. The P. angusta-based biosensor is characterized by high reproducibility (Sr, 1%) and operational stability, where the biosensor remains viable for up to 28 days.

[transversion of cell polarity from Bi- to multipolarity is the mechanism determining multiple spore formation in Anaerobacter polyendosporus PS-1T].

The number of spores formed in a single cell ofAnaerobacterpolyendosporus PS-1T is significantly influenced by the composition of nutrient media. Depending on carbohydrate concentration in synthetic medium, the number of spores may vary from one-two to five-seven. Investigation of spore formation by fluorescence and electron microscopy revealed that on media with 0.5-1.0% glucose or galactose most of the vegetative cells remained rod-shaped after cessation of cell division in the culture. Their nucleoids were localized at cell poles close to the polar site of the cytoplasmic membrane. Forespores were formed at one or both of these poles. A satellite nucleoid (operator) was detected close to each forespore. In the variant with bipolar organization of mother cells only one or two spores per cell were formed. In the second variant of cultivation, when the cells grew at low galactose concentrations (0.1-0.3%), most of the vegetative cells increased in volume and became oval or spherical after cessation of cell division in the culture. Epifluorescence microscopy with nucleic acids-specific fluorochromes (DAPI and acridine orange) revealed the presence of multiple (six to nine) nucleoids in these cells. The nucleoids were located at the cell periphery in close contact with the cytoplasmic membrane. These nucleoids became the centers (poles) for forespore formation. Thus, in the early stationary phase transversion from bipolar to multipolar cells occurred during the early stationary phase. Cessation of cell division combined with continuing replication of the nucleoids resulted in formation on multinuclear cells. The multiplicity of nucleoides and multipolarity of these cells were prerequisites determining endogenous polysporogenesis, occurring as synchronous formation of three to seven twin spores in a number of the oval and spherical cells.

[Investigation of site-specific DNA binding with nicking endonuclease Nt.BspD6I at single molecule level by atomic force microscopy].

Nicking endonuclease Nt.BspD6I is a heterodimeric restriction endonuclease, one subunit of which exhibits specific nicking activity. It gets bound to double-stranded DNA and makes a break (nick) in one chain at a distance of 4 nucleotides from the binding site. In this work, for visualization of the specific binding and protein landing site an atomic force microscopy was used. In five minutes after incubation of DNA solution with nicking endonuclease, the DNA molecules with associated proteins which located at the expected binding site and "shared" DNA strand into two segments (approximately, 1/3 and 2/3 of length) were observed in the images. In addition, near the binding site DNA molecule had a height corresponding to a single-stranded DNA molecule, which was in good agreement with single-stranded cleavage by nickase in the course of complex formation.

[Study of phototrophic purple bacterium Rhodobacter sphaeroides cell morphology of wild-type and ipt-transformant by atomic force and electron microscopy].

A comparative study of phototrophic purple bacterium Rhodobacter sphaeroides cell morphology of wild-type and ipt-transformant was done by atomic force and electron microscopy. It was shown that transformation led to a decrease in the number or total disappearance of the flagella, as well as to changes in the structure of the outer membrane of the bacteria cell wall. On the wild-type cell surface phage-like structures were found, and in transformed cells at their places hollows were identified. This study significantly extends an understanding of the changes occurring in the ipt-transformants of phototrophic purple bacterium Rhodobacter sphaeroides. This investigation not only confirmed earlier obtained data about the differences in the wild-type and ipt-transformant phototrophic purple bacteria cell wall, but also showed fine changes in the structure of its outer membrane.

[Family of ribosomal proteins S1 contains unique conservative domain].

Different representatives of bacteria have different number of amino acid residues in the ribosomal proteins S1. This number varies from 111 (Spiroplasma kunkelii) to 863 a.a. (Treponema pallidum). Traditionally and for lack of this protein three-dimensional structure, its architecture is represented as repeating S1 domains. Number of these domains depends on the protein's length. Domain's quantity and its boundaries data are contained in the specialized databases, such as SMART, Pfam and PROSITE. However, for the same object these data may be very different. For search of domain's quantity and its boundaries, new approach, based on the analysis of dicted secondary structure (PsiPred), was used. This approach allowed us to reveal structural domains in amino acid sequences of S1 proteins and at that number varied from one to six. Alignment of S1 proteins, containing different domain's number, with the S1 RNAbinding domain of Escherichia coli PNPase elicited a fact that in family of ribosomal proteins SI one domain has maximal homology with S1 domain from PNPase. This conservative domain migrates along polypeptide chain and locates in proteins, containing different domain's number, according to specified pattern. In this domain as well in the S1 domain from PNPase, residues Phe-19, Phe-22, His-34, Asp-64 and Arg-68 are clustered on the surface and formed RNA binding site.

[Metabolic monitoring of heart surgery patients during the immediate postoperative period]. / Monitorización metabólica durante el período inmediato postoperatorio de pacientes cardioquirúrgicos.

The situation of aggression is accompanied by metabolic alterations with an important neuro-endocrinal context. Extracorporeal surgery is an especially aggressive situation which involves both surgery and the added metabolic state of hypothermia. In 48 patients subjected to extracorporeal circulation with hypothermia, the metabolic parameters of Oxygen Consumption (VO2), Production of carbon dioxide (VCO2), Respiratory Quotient (RQ) and Energy Expenditure (GE) were studied. It was observed that the patients could be divided into two subgroups, depending on their metabolic response. Studying the RQ parameter, it was seen that this divided patients into two subgroups depending on their posterior evolution. Patients in whom RQ did not exceed 0.8 showed more favourable evolution than those in whom RQ was higher than 0.8. The correct metabolic response was regarded as a parameter indicative of the evolution, directly correlated to the hemodynamic parameters studied. In one patient, creatine phosphate was infused at a dosage of 30 mg/kg for 60 min, and an improvement was noted in GE and Cardiac Index. It was concluded that the intravenous intake of creatine phosphate in patients undergoing extracorporeal surgery improved both metabolic parameters and the myocardiac function.

[Moradol (butorphanol tartrate) as the analgesic component of current combination general anesthesia]. / Moradol (butorfanol-tartrat) v kachestve sredstva, obespechivaiushchego analgeticheskii komponent sovremennoi kombinirovannoi obshchei anestezii.

A technique is described for the use of moradol as an agent ensuring analgesia in modern combined general anesthesia during abdominal and thoracic surgery, cardiac surgery, cardiopulmonary bypass included, and diagnostic manipulations. Moradol was particularly effective for long-term surgery. The drug was also useful for premedication (0.06 mg/kg) and needed no combination with any tranquilizers or analgesics. Agonist opioid activity of moradol was maximum 10 min after its intravenous administration, therefore a 5 to 10 min exposition upon moradol injection prior to hypnotic drug administration is suggested. Bolus drug dose (150 mg/kg) for induction to anesthesia was an adequate protection against pain impulsation in surgical trauma in the course of 3-4 hours of surgical intervention.

[Use of dalargin in complex anesthesiological protection during operations on the abdominal organs]. / Primenenie dalargina v komplekse anesteziologicheskoi zashchity pri operatsiiakh na organakh briushnoi polosti.

The authors analyse experience in the use of the synthetic analogue of ++leu enkephalin dalargin in multicomponent balanced anesthesia in operations on the abdominal organs in 36 patients. With the use of the suggested method the intraoperative expenditure of narcotic analgesics is 6 times less but the efficacy of the anesthesia remains the same. Study of the activity of the liver-specific enzymes (histidase and urokinase) in blood plasma and biopsy material taken from the liver and the content of malonic dialdehyde in the hepatic biopsy specimen revealed hepatoprotective properties of dalargin. The decrease of the total peripheral resistance and increase of the elasticity of the arterial reservoir++ in the main group as compared to these values in patients treated by the traditional method were considered by the authors to be the consequence of the autonomic priming effect of the opiopeptide used. It is concluded that the use of synthetic analogues of endogenous opioids (dalargin) in the anesthesiological protection complex in operations on the abdominal organs is expedient.

[Dalargin--a basic means of intraoperative protection of a patient during correction of an atrial septal defect under conditions of artificial circulation: a new method of anesthesia]. / Dalargin--osnovnoe sredstvo intraoperatsionnoi zashchity bol'nogo pri korrektsii defekta mezhpredserdnoi peregorodki v usloviiakh iskusstvennogo krovoobrashcheniia: novyi metod anestezii.

The paper presents results of application of the Soviet synthetic neuropeptide Dalargin as a major agent of anesthesiologic protection in the correction of a secondary atrial septal defect under general artificial circulation. From the findings obtained from examinations of central and peripheral hemodynamic parameters and blood oxygen transport function it was concluded that the anesthesiological protection was adequate at all the stages of an operation. The authors also give data on the optimization of the afterload of the right ventricle and preload of the left ventricle under anesthesia in which dalargin was used by directly affecting the elasticity of pulmonary vessels. The nature of changes in the ventilation-perfusion relationships, in the postoperative period, in particular, also enables the author to recommend the preferable employment of the described method for anesthesia while correcting this type of heart disease.