[Cancer-Associated Fibroblasts: Heterogeneity and Bimodality in Oncogenesis].

Cancer-associated fibroblasts (CAFs) often form a major component of the tumor microenvironment (TMA), providing conditions for cancer cells to thrive. CAFs may contribute to tumor growth, invasion, metastasis, and resistance to therapy. However, clinical trials of treatment strategies targeting CAFs have largely failed. Moreover, there is evidence that CAFs are capable of inhibiting tumor development. The review considers the current data on the functional heterogeneity of CAFs and their bimodality in tumor development and progression. Understanding the tumor-promoting and tumor-inhibiting activities of CAFs can help to develop new diagnostic and therapeutic approaches.

Effect of inactivation of luxS gene on the properties of Serratia proteamaculans 94 strain.

The luxS gene is responsible for the synthesis of AI-2 (autoinducer-2), a signaling molecule that participates in quorum sensing regulation in a large number of bacteria. In this work, we investigated which phenotypes are regulated by luxS gene in Serratia proteamaculans 94, psychrotrophic strain isolated from spoiled refrigerated meat. AI-2 was identified in S. proteamaculans 94, and the luxS gene involved in its synthesis was cloned and sequenced. A mutant with the inactivated luxS gene was constructed. Inactivation of the luxS gene was shown to lead to the absence of AI-2 synthesis, chitinolytic activity, swimming motility, suppression of the growth of fungal plant pathogens Rhizoctonia solani and Helminthosporium sativum by volatile compounds emitted by S. proteamaculans 94 strain, and to a decrease of extracellular proteolytic activity. The knockout of the luxS gene did not affect synthesis of N-acyl-homoserine lactones, lipolytic, and hemolytic activities of S. proteamaculans 94.

Organism-Level Tumor Models in Zebrafish Danio rerio.

Development and implementation of adequate organism-level models is one of the key elements in biomedical research that focuses on experimental oncology. Over the last decade, studies using Zebrafish (Danio rerio) have gained in popularity in this area of research. This review describes the various approaches that have been used in developing highly effective models for oncological (clinical term, better cancer or tumor) studies based on D. rerio. Priority is given to transplantation models of cancer and their application to optically transparent D. rerio lines, including clonal ones, and utilization tumors of various origins bearing fluorescent labels. The combination of tumor transplantation at organism-level models in transparent clonal D. rerio lines with fluorescent microscopy, FACS-fractionation of tumor cell subsets, and transcription analysis can result in one of the most promising research approaches in providing new information on tumor formation and growth.

Glutamyl Endopeptidases: The Puzzle of Substrate Specificity.

Glutamyl endopeptidases (GEPases) are chymotrypsin-like enzymes that preferentially cleave the peptide bonds of the α-carboxyl groups of glutamic acid. Despite the many years of research, the structural determinants underlying the strong substrate specificity of GEPases still remain unclear. In this review, data concerning the molecular mechanisms that determine the substrate preference of GEPases is generalized. In addition, the biological functions of and modern trends in the research into these enzymes are outlined.

Oligopeptidase B from Serratia proteamaculans. II. Enzymatic characteristics: substrate analysis, influence of calcium ions, pH and temperature dependences.

Enzymatic properties of a novel oligopeptidase B from psychrotolerant gram-negative microorganism Serratia proteamaculans (PSP) were studied. The substrate specificity of PSP was analyzed using p-nitroanilide substrates, and the influence of calcium ions on the enzyme activity was studied. Hydrolysis of oligopeptides by PSP was studied using melittin as the substrate. Optimal conditions for the PSP activity (pH and temperature) have been established. It was found that PSP shares some properties with oligopeptidases B from other sources containing two Asp/Glu residues in the S2 site, but it differs significantly in some characteristics. The S2 site of PSP contains only one Asp460 residue. The secondary specificity of PSP has a number of specific features: an unusual substrate inhibition by peptides with hydrophobic residues at the P2 position, as well as the drastic influence of calcium ions on substrate characteristics of the enzyme. It is assumed that the PSP molecule contains a large hydrophobic substrate-binding site, and significant conformational rearrangements of the enzyme active site are induced by Ca(2+) binding and by the formation of the enzyme-substrate complex. The temperature characteristics of PSP (high activity at low temperature as well as low apparent temperature optimum (25°C)) confirm that PSP is a psychrophilic enzyme.

Oligopeptidase B from Serratia proteamaculans. I. Determination of primary structure, isolation, and purification of wild-type and recombinant enzyme variants.

A novel trypsin-like protease (PSP) from the psychrotolerant gram-negative microorganism Serratia proteamaculans was purified by ion-exchange chromatography on Q-Sepharose and affinity chromatography on immobilized basic pancreatic trypsin inhibitor (BPTI-Sepharose). PSP formed a tight complex with GroEL chaperonin. A method for dissociating the GroEL-PSP complex was developed. Electrophoretically homogeneous PSP had molecular mass of 78 kDa; the N-terminal amino acid sequence 1-10 was determined, and mass-spectral analysis of PSP tryptic peptides was carried out. The enzyme was found to be the previously unknown oligopeptidase B (OpdB). The S. proteamaculans 94 OpdB gene was sequenced and the producer strain Escherichia coli BL-21(DE3) pOpdB No. 22 was constructed. The yield of expressed His(6)-PSP was 1.5 mg/g biomass.

Probing for actinase activity of protealysin.

The ability of protealysin, a thermolysin-like metallopeptidase from Serratia proteamaculans 94, to cleave actin and matrix metalloprotease MMP2 is reported. In globular actin, protealysin and S. proteamaculans 94 cell extracts are shown to hydrolyze the Gly42-Val43 peptide bond within the DNase-binding loop and the Gly63-Ile64 and Thr66-Ile67 peptide bonds within the nucleotide cleft of the molecule. At enzyme/substrate mass ratio of 1 : 50 and below, a 36 kDa-fragment produced by the cleavage between Gly42 and Val43 was virtually resistant to further breakdown. Judging from the results of zymography, protealysin transforms proMMP2 into a 66 kDa polypeptide characteristic of mature MMP2, indicating that protealysin can activate MMP2. Upon incubation of S. proteamaculans 94 with human larynx carcinoma Hep-2 cells intracellular bacteria were detected in about 10% of Hep-2 cells, this being the first evidence for invasion of eukaryotic cells with bacteria of this species. Thus, S. proteamaculans 94 turned out to be one more bacterial strain in which synthesis of actin-specific metalloprotease is coupled with bacterial invasion. These results are consistent with the idea of the actinase activity of bacterial metalloproteases being a factor that may promote bacterial invasion of eukaryotic cells.

Hetero- and auto-activation of recombinant glutamyl endopeptidase from Bacillus intermedius.

Glutamyl endopeptidase from Bacillus intermedius (BIGEP) is a secretory serine proteinase specifically hydrolyzing peptide bonds involving alpha-carboxyl groups of glutamic and aspartic acids. In this work, different BIGEP forms (full-length precursor, precursor without signal peptide and mature part) were expressed in Escherichia coli and the process of enzyme maturation was studied in vitro. BIGEP precursor renaturation leads to autocatalytic hydrolysis of the propeptide at Glu(-16). At the same time, the enzyme activation requires the complete removal of the prosequence by other proteinases. The mature part of BIGEP cannot be activated, which indicates that the propeptide is required for the active protein formation. The data obtained allowed us to apply directed mutagenesis of the processing site to obtain a BIGEP form that matured autocatalytically. This approach makes it possible to produce the enzyme without extrinsic proteinases, which is a prerequisite for using it in limited hydrolysis of proteins and peptides.

[Isolation and properties of cysteine protease from Serratia proteamaculans 94].

A new cysteine protease (SpCP) with a molecular mass of about 50 kDa and optimal functioning at pH 8.0 was isolated from the culture medium of a Serratia proteamaculans 94 psychrotolerant strain using affinity and gel permeation chromatography. The enzyme N terminal amino acid sequence (SPVEEAEGDGIVLDV-) exhibits a reliable similarity to N terminal sequences of gingipains R, cysteine proteases from Polphyromonas gingivalis. Unlike gingipains R, SpCP displays a double substrate specificity and cleaves bonds formed by carboxylic groups of Arg, hydrophobic amino acid residues (Val, Leu, Ala, Tyr, and Phe), Pro, and Gly. SpCP can also hydrolyze native collagen. The enzyme catalysis is effective in a wide range of temperatures. Kinetic studies of Z-Ala-Phe-Arg-pNA hydrolysis catalyzed by the protease at 4 and 37 degrees C showed that a decrease in temperature by more than 30 degrees C causes a 1.3-fold increase in the kcat/Km ratio. Thus, SpCP is an enzyme adapted to low positive temperatures. A protease displaying such properties was found in microorganisms of the Serratia genus for the first time and may serve as a virulent factor for these bacteria.

[Oligomeric organization of recombinant human neurotrophins expressed in Escherichia coli cells].

Genes of human neurotrophins NGF, BDNF, NT-3 were cloned, and the corresponding proteins and their fragments were expressed in Escherichia coli BL-21 (DE3lambda) cells. Their intracellular localization was determined. The conditions for isolation and purification of the target recombinant proteins and for folding of BDNF and NT-3 precursors were selected. The recombinant proprecursors of human neurotrophines have been shown to possess complex oligomeric structure.

[Start codon in the serine proteinase gene from Bacillus intermedius].

The translation initiation site in the extracellular serine subtilisin-like proteinase gene from Bacillus intermedius (aprBi) (AN AY754946) secreting at the stationary growth phase was established. The analysis of aprBi open reading frame revealed three putative translation start sites (TTG, GTG, ATG). Using SignalP online freeware program we have determined the functional activity probability of each of them. To identify the translation start point the modified subtilisin-like protease genes carrying nucleotide replacements in supposed start codons were developed using oligonucleotide-directed mutagenesis. We have investigated the expression of these genetic constructions in protease-deficient strain B. subtilis AJ73. According our results it was concluded that the translation in aprBi gene starts from GTG kodon.

[Heterologous expression of Bacillus intermedius gene of glutamyl endopeptidase in Bacillus subtilis strains defective in regulatory proteins].

Expression of the gene of glutamyl endopeptidase from Bacillus intermedius (gseBi) cloned on the plasmid pV has been studied in Bacillus subtilis recombinant strains with mutations of the regulatory proteins involved in sporogenesis and spore germination. It has been established that inactivation of the regulatory protein Spo0A involved in sporulation initiation resulted in a decrease in the expression of the gseBi gene by 65% on average. A mutation in the gene of the sensor histidine kinase kinA had no effect on the biosynthesis of the enzyme. Inactivation of Ger proteins regulating bacterial spore germination resulted in a 1.5-5-fold decrease in glutamyl endopeptidase activity. It has been concluded that expression of the B. intermedius glutamyl endopeptidase gene from plasmid pV in recombinant cells of B. subtilis is under impaired control by the regulatory system of Spo0F/Spo0A phosphorelay, which participates in sporulation initiation. The regulatory Ger proteins responsible for spore germination also affect expression of the gene of this enzyme.

The expression of Bacillus intermedius glutamyl endopeptidase gene in Bacillus subtilis recombinant strains.

The gene encoding for B. intermedius glutamyl endopeptidase (gseBi) has previously been cloned and its nucleotide sequence analyzed. In this study, the expression of this gene was explored in protease-deficient strain B. subtilis AJ73 during stationary phase of bacterial growth. We found that catabolite repression usually involved in control of endopeptidase expression during vegetative growth was not efficient at the late stationary phase. Testing of B. intermedius glutamyl endopeptidase gene expression with B. subtilis spo0-mutants revealed slight effect of these mutations on endopeptidase expression. Activity of glutamyl endopeptidase was partly left in B. subtilis ger-mutants. Probably, gseBi expression was not connected with sporulation. This enzyme might be involved in outgrowth of the spore, when germinating endospore converts into the vegetative cell. These data suggest complex regulation of B. intermedius glutamyl endopeptidase gene expression with contribution of several regulatory systems and demonstrate changes in control of enzyme biosynthesis at different stages of growth.

[Biosynthesis of the subtilisin-like serine proteinase of Bacillus intermedius under salt stress conditions].

The biosynthesis of the subtilisin-like serine proteinase of Bacillus intermedius 3-19 by the recombinant strain Bacillus subtilis AJ73(pCS9) was found to be enhanced under salt stress conditions (growth in a medium containing 1 M NaCl and 0.25 M sodium citrate). In a recombinant strain of B. subtilis deficient in the regulatory proteins DegS and DegU, which control the synthesis of degradative enzymes, the expression of the proteinase gene was inhibited. In contrast, in the strain B. subtilis degU32 (Hy), which provides for the over-synthesis of proteins positively regulated by the DegS-DegU system, the biosynthesis of the subtilisin-like proteinase of B. intermedius 3-19 increased by 6-10 times. These data suggest that the DegS-DegU system is involved in the positive regulation of the expression of the subtilisin-like B. intermedius proteinase gene in recombinant B. subtilis strains.

[Growth conditions and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant Bacillus subtilis strain].

The effect of the components of the nutrient medium on growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. The production of proteinase was found to be dependent on the composition of the nutrient medium and showed two peaks, at the 28th and 48th h of growth. The concentrations of the main components of the nutrient medium (peptone and inorganic phosphate) optimal for the biosynthesis of subtilisin-like serine proteinase at the 28th and 48th h of growth were determined in factorial experiments. Complex organic substances, casein at concentrations of 0.5-1%, gelatin at concentrations of 0.5-1%, and yeast extract at a concentration of 0.5%, stimulated the production of subtilisin-like serine proteinase by the recombinant strain. The study of the sporulation dynamics in this strain showed that the proteinase peaks at the 28th and 48th h of growth correspond, respectively, to the initial stage of sporulation and to the terminal stages of endospore formation (V-VII stages of sporulation).

[Biosynthesis of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant Bacillus subtilis strain].

The effect of certain nutrients on the growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. Glucose was found to inhibit the synthesis of proteinase in the early (28 h of growth) but not in the late stationary phase (48 h of growth). The inhibitory effect of the other mono- and disaccharides studied was less pronounced. Casamino acids added to the medium at concentrations of 0.1-1% as an additional carbon and nitrogen source stimulated enzyme biosynthesis. Individual amino acids (cysteine, asparagine, glutamine, tryptophan, histidine, and glutamate) also stimulated enzyme biosynthesis in the early stationary phase by 25-30%, whereas other amino acids (valine, leucine, alanine, and aspartate) were ineffective or even slightly inhibitory to enzyme production. The stimulatory effect of the first group of amino acids on the synthesis of proteinase in the late stationary phase was negligible. In contrast, the bivalent ions Ca2+, Mg2+, and Mn2+ stimulated biosynthesis of proteinase in the late stationary phase (by 20-60%) and not in the early stationary phase. The data indicate that there are differences in the biosyntheses of proteinase by the recombinant B. subtilis strain during the early and late periods of the stationary phases.

Psychrophilic trypsin-type protease from Serratia proteamaculans.

A preparative method for purification of a novel protease from the psychrotolerant Gram-negative microorganism Serratia proteamaculans (PSP) was developed using affinity chromatography on BPTI-Sepharose. It yielded electrophoretically homogeneous PSP preparation of 60 kD. The PSP properties (temperature and pH stability, high catalytic efficiency) indicate that this enzyme can be defined as a psychrophilic protease. Inhibitory analysis together with substrate specificity indicates that the studied PSP exhibits properties of serine trypsin-like and Zn-dependent protease.

[Biosynthesis of Bacillus intermedius glutamyl endopeptidase in recombinant Bacillus subtilis cells during the stationary growth phase].

We studied the biosynthesis of Bacillus intermedius glutamyl endopeptidase in the recombinant Bacillus subtilis strain AJ73 delta58.21 during the stationary growth phase. We optimized the composition of the culture medium to favor effective enzyme production during the stationary growth phase, and found that the nutritional requirements for glutamyl endopeptidase synthesis were different in the stationary phase and growth retardation phase. Proteinase accumulation was activated by complex organic substrates (casein and gelatin). During final stages of the culture growth, the enzyme production was stimulated by Ca2+, Mn2+, and Co2+ and inhibited by Zn2+, Fe2+, and Cu2+. The synthesis of glutamyl endopeptidase in the late stationary phase was not inhibited by glucose, unlike that in the trophophase during proliferation. We conclude that the regulatory mechanisms of proteinase synthesis during vegetative growth and sporulation are different.

[Investigation of the process of personal hygiene items biodegradation by cellulose-fermenting microorganisms]. / Issledovanie protsessa biodegradatsii sredstv lichnoi gigieny mikroogranizmami-tseliulolitikami.

Cellulose-containing wastes are one of the heaviest and biggest ingredients of solid domestic wastes piling up during spaceflight. For the most part these are disposable personal hygiene items used in large quantities in the absence of shower. These wastes contain human body products which are very dangerous from the sanitary-epidemiological standpoint. The purpose was to explore potentiality of microbial biodegradation of cellulose-containing hygiene items anaerobically with dry mass transformation into liquid and biogas. Among specific objectives were test cultivation of active strains of reference cultures of cellulose-fermenting anaerobic thermophilic bacteria on hygiene items as the only source of carbon, evaluation of ways and need of pretreatment of gauze pads to stimulate biodegradation, and chemical analysis of resulting biogas. From the investigation it was concluded that gauze pads are susceptible to biodegradation by anaerobic bacteria producing a low toxicity gas fraction. Therefore, the proposed technology can be considered as a candidate for integration into the spacecrew life support system.

[The regulation of Bacillus intermedius glutamyl endopeptidase biosynthesis in the recombinant Bacillus subtilis strain during sporulation]. / Reguliatsiia biosinteza vnekletochnoi glutamiléndopeptidazy Bacillus intermedius v rekombinantnom shtamme Bacillus subtilis na stadii sporoobrazovaniia.

The growth of the recombinant Bacillus subtilis strain AJ73 carrying the Bacillus intermedius 3-19 glutamyl endopeptidase gene on a multicopy plasmid and the effect of some nutrients on the efficiency of extracellular glutamyl endopeptidase production in the stationary growth phase were studied. In this phase, the concentration of glutamyl endopeptidase in the culture liquid peaked at the 48th and 78th h of cultivation and depended on the composition of the cultivation medium. Unlike the synthesis of glutamyl endopeptidase in the trophophase (i.e., during vegetative growth), which was suppressed by glucose, the synthesis of this enzyme during sporulation was resistant to glucose present in the cultivation medium. A multifactorial experimental design allowed optimal proportions between the concentrations of major nutrients (peptone and inorganic phosphate) to be determined. Inorganic phosphate and ammonium ions augmented the production of glutamyl endopeptidase by 30-150%, and complex organic substrates, such as casein and gelatin, enhanced the production of glutamyl endopeptidase by 50-100%. During sporulation, the production of glutamyl endopeptidase was stimulated by some bivalent cations (Ca2+, Mg2+, and Co2+) and inhibited by others (Zn2+, Fe2+, and Cu2+). The inference is drawn that the regulatory mechanisms of glutamyl endopeptidase synthesis during vegetative growth and sporulation are different.