[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.

[The family 28 carbohydrate-binding module of the thermostable endo-1,4-beta-glucanase CelD Caldicellulosiruptor bescii maximizes the enzyme's activity and binds irreversibly to amorphous cellulose].

The nucleotide sequence of a chromosome fragment of the thermophilic anaerobic bacterium Caldicellulosiruptor bescii (syn. Anaerocellum thermophilum) has been determined. The fragment contains four open reading frames with the second one of 749 aa encoding a multimodular endo-1,4-beta-glucanase CelD (85019 Da). N-terminal region of the protein includes the signal peptide and the catalytic module of glycoside hydrolase family 5 (GH5), followed by the substrate-binding module of family 28 (CBM28). The C-terminal region bears three SLH modules. The recombinant endoglucanase and its two separate modules, the catalytic one and CBM28, were produced in E. coli cells and purified to homogeneity. Analysis of the catalytic properties showed CelD to be endo-1,4-beta-glucanase whose maximum activity was exhibited on beta-glucan of barley at pH 6.2 and 70 degrees C. The enzyme was stable at 50 degrees C for 30 days. Upon removal of the C-terminal CBM28, the activity of GH5 decreased on cellulose substrates, and its thermostability was dropped. Binding of CBM28 to amorphous cellulose was almost irreversible as it could not be removed from this substrate in a range of pH 4-11, temperatures--of 0-75 degrees C, and NaCl concentration--of 0-5 M. Only 100% formamide or 1% SDS were able to remove the protein.

[The properties of four C-terminal carbohydrate-binding modules (CBM4) of laminarinase Lic16A of Clostridium thermocellum].

At the C-terminus of multimodular laminarinase Lic16A Clostridium thermocellum four carbohydrate-binding modules (CBM), belonging to family 4, were found. The isolated CBM - CBM4_1, CBM4_2, CBM4_3, CBM4_4 and the tandem CBM4_(1-4) were obtained. None of the recombinant proteins did have the affinity to soluble beta-1,3-1,4-glucans--laminarin and lihenan--the main specific substrates of Licl6A. All modules, except CBM4_4, had the ability to bind bacterial crystalline cellulose, that was atypical for the family 4 CBMs. We found that all CBMs 4 of Licl6A had affinity for xylan, chitin, beta-glucan from yeast cell wall and Avicel, while CBM4_3 and CBM4_4 had additional affinity to chitosan. The tandem CBM4_(1-4) had the highest affinity to yeast cell wall beta-glucan, avicel and pustulan. The binding constants for these substrates were about 100 times higher than that of the individual modules, suggesting a synergy in the process of absorption to these polysaccharides. This finding helps to explain the evolutionary process of CBM multiplication.

Chimeric lactase capable of spontaneous and strong immobilization on cellulose and development of a continuous-flow system for lactose hydrolysis at high temperatures.

Recombinant plasmids containing fusion proteins composed of two different modules were constructed and expressed in Escherichia coli. The modules encoded the lactase LacA (LacZ) from the thermophilic bacterium Thermoanaerobacter ethanolicus and the cellulase CelD, a cellulose-binding module (CBM) from Anaerocellum thermophilum. The CelD CBM provides a spontaneous and strong sorption of the fusion proteins onto a cellulose carrier. The enzymatic activities of both the free LacA protein and LacA-CelD CBM fusion proteins immobilized onto the cellulose carrier were assessed. The LacA activity of the fusion protein was dependent upon its position with respect to the CBM. The highest level of lactase activity and stability was observed when the lactase domain was localized at its N terminus. A continuous-flow column reactor of lactase immobilized on a cellulose carrier was constructed, and its activity was assessed. The lactose hydrolysis rate for a 150 mM (5%) solution at a flow rate of 1 reactor volume per min was 75%, which is a value optimal for further whey transformation into glucose/galactose syrup.

[Substrate-binding properties of family 54 module of laminarinase Lic16A of Clostridium thermocelwnum].

Endo beta-1.3-1.4-glucanase Lic16A of the moderate thermophilic anaerobe Clostridium thermocellum has a complex multimodular structure. In addition to the catalytic module it contains 8 auxiliary modules, 5 of which are substrate binding modules. The new family 54 substrate binding module CBM54 (25.2 kDa), localized at the N-terminus of the enzyme, owns a cleavage site, in its N-terminal part manifested by the appearance of a shortened module CBM54C (17.2 kDa) in vivo and in vito. CBM54C was cloned in Escherichia coli cells and purified to electrophoretic homogeneity. The binding constants of CBM54C to xylan, chitin, insoluble B-glucan of yeast cell wall and bacterial crystalline cellulose were in the same order of magnitude as for CBM54. However CBM54C, unlike CBM54, did not bind pustulan, avicel, and chitosan. Nevertheless the presence of calcium ions restored the ability of CBM54C to bind the latter three carbohydrates. CBM54 substrate binding promiscuity permits to suggest the presence of multiple binding sites, some of them calcium dependent. The collected data allow to localize Ca2+ -independent sites for avicel, pustulan an d chitosan binding inthe spontaneously split-off CBM54 N-terminal area (8 kDa). In this report the localization scheme of Ca2+ -dependent and Ca2+ -independent binding sites for various substrates has been suggested.

[Expression of the genes CelA and XylA isolated from a fragment of metagenomic DNA in Escherichia coli].

The glycosyl hydrolase genes cel5A and xyl3A previously isolated by ourselves within a fragment of DNA from the methagenomic library of cow rumen microflora DNA were sub-cloned and expressed in E. coli. The recombinant proteins Cel5A and Xyl3A were purified and characterized. Cellulase Cel5A belongs to the Family 5 glycosyl hydrolases and is a one-module 38.2 kDa enzyme that hydrolyses the 1,4-glycoside bonds of soluble cellulose substrates and amorphous cellulose, showing its maximal activity (31200 u/mg) on lichenan, a soluble substrate with mixed (beta-1,3-1,4) bonds. The end product of the amorphous cellulose hydrolysis is cellobiose. Cel5A is inactive toward the crystal forms of cellulose. Cel5A is an endoglucanase capable of exohydrolysis. The molecular mass of beta-xylosidase Xyl3A belonging to the Family 3 glycosyl hydrolases is 83.7 kDa. The enzyme is active only on xylooligosaccharides, with the maximal activity shown on xylobiose, the end product of the reaction being xylose. No activity on xylane was hitherto observed. Recombinant Cel5A and Xyl3A are stable over a wide range of pH and temperatures, their maximal activity being observed at pH 6.5 and at 55 degrees C.

[Cloning and characterization of a large metagenomic DNA fragment containing glycosyl-hydrolase genes].

The problem of search for and characterization of enzymes synthesized by non-cultivated microorganisms is presently being settled by creating metagenomic libraries. A 6000-clone library with the average size of its inserts amounting to 15 bp has been constructed on the basis of total DNA isolated from cow rumen microorganisms. As the result of the screening of the library on plates with different substrates, a clone was selected that efficiently hydrolyzed lichenan and carboxymethylcellulose. The clone contained the recombinant plasmid pBlue-13 bearing a 12071 bp.-long metagenomic fragment carrying ten open reading frames, two of them being identified as glycosyl hydrolase genes. No homology of the metagenomic DNA with any known microorganism genomes was revealed. The amino acid sequence, deduced on the basis of frame 4 and denoted by Xyl3A, bears resemblance with beta-xylosidases of glycosyl hydrolase Family 3. Frame 6 encodes polypeptide Cel5A homologous to cellulases of glycosyl hydrolase Family 5. The amino acid sequences deduced on the basis of seven out of ten open reading frames were homologous to proteins of microorganisms belonging to the Bacteroides sp. family, the bacteria inhabiting mammalian intestines.

[Thermoanaerobacter ethanolicus gene cluster containing the alpha- and beta-galactosidases genes melA and lacA, and properties of recombinant lacA].

The nucleotide sequence of a 4936 bp Thermoanaerobacter ethanolicus genomic DNA fragment containing the thermostable beta-galactosidase gene lacA and two incomplete open reading frames has been determined. The product of the first frame is highly homologous to alpha-galactosidases (melibiases), the product of the third frame is homologous to the alpha-D-mannosidases. The terminal area of the lacA, immediately following the stop-codon, harbors presumably a transcription termination site. Based on the location of the putative alpha-galactosidase gene melA and of the beta-galactosidase gene lacA on the T. ethanolicus chromosome, their combined transcription could be presumed. The calculated molecular mass of LacA is 86 kDa. LacA belongs to GH family 2 (GH2). Maximal activity of the purified recombinant enzyme was observed between pH values of 5.7 and 6.0 and temperatures of 75-80 degrees C. The highest activity, 480 units mg(-1), was found on lactose (Km 30 mM), the activities on pNPhGal and oNPhGal amounting to 330 and 420 units mg(-1), respectively. Immobilization on aldehyde silochrome increases the thermostability of the enzyme and keeps its high activity.

[Prospects for practical application of substrate-binding modules of glycosyl hydrolases (A review)]. / Perspektivy prakticheskogo primeneniia substratsviazyvaiushchikh modulei glikozilgidrolaz (Obzor).

The properties of substrate-binding modules of glycosyl hydrolases have been reviewed. Variation of the properties of these modules makes them promising as components of chimeric proteins, which is a rapidly developing field of biotechnology. Examples of applying substrate-binding modules of glycosyl hydrolases to immobilization of proteins and whole cells on polysaccharides and purification of proteins are described. Promising methods for (1) detecting various compounds using hybrids of substrate-binding modules with antibodies and (2) locating polysaccharides in live tissues are reviewed as well.

The effect of three-dimensional structure on the solid state isotope exchange of hydrogen in polypeptides with spillover hydrogen.

The effect of the three-dimensional structure of polypeptides and proteins on their ability to undergo isotopic exchange under the action of spillover hydrogen (SH) in the high temperature solid state catalytic isotope exchange reaction (HSCIE) was theoretically and experimentally studied. The HSCIE reaction in the beta-galactosidase protein from Thermoanaerobacter ethanolicus (83kDa) was studied. The influence of the beta-galactosidase structure on isotopic exchange as peptide fragments with spillover tritium was studied. The most accessible peptide fragment, which does not contribute to alpha-helix and beta-strand formations (KEMQKE215-220), had the largest relative reactivity. The one located in the contact area between the subunits (YLRDSE417-422) showed the smallest relative reactivity. The relative reactivities of these peptides differ more than 150 times. Data collected during a study devoted to the HSCIE reaction of the beta-galactosidase protein indicate that the HSCIE reaction might be employed for acquiring information about their three-dimensional structure and protein-protein interactions. The results of ab initio calculations have shown that alpha-helix formation in polypeptides decreases the reactivity in HSCIE. Hydrogen exchange in the alpha-helical fragment Trp1-Leu8 of zervamycin IIB was also analyzed using theoretical methods. It was shown by ab initio quantum-chemical calculations that the high degree of substitution of C(alpha)H for tritium in Gln3 might be associated with the participation of electron donor O and N atoms in transition state stabilization in the HSCIE reaction.

[Thermotoga neopolitina gene cluster, participating in degradation of starch and maltodextrins: expression of aglB and aglA gene in Escherichia coli, properties of recombinant enzymes]. / Klaster genov Thermotoga neapolitana, uchastvuiushchikh v degradatsii krakhmala i mal'todekstrinov: éksptressiia genov aglB i aglA v Escherichia coli, svoistva rekombinantykh fermentov.

The aglB and aglA genes from the starch/maltodextrin utilization gene cluster of Thermotoga neapolitana were subcloned into pQE vectors for expression in Escherichia coli. The recombinant proteins AglB and AglA were purified to homogeneity and characterized. Both enzymes are hyperthermostable, the highest activity was observed at 85 degrees C. AglB is an oligomer of identical 55-kDa subunits capable of aggregation. This protein hydrolyses cyclodextrins and linear maltodextrins to glucose and maltose by liberating glucose from the reducing end of the molecules, and it is a cyclodextrinase with alpha-glucosidase activity. The pseudo-tetrasaccharide acarbose, a potent alpha-amylase and alpha-glucosidase inhibitor, does not inhibit AglB but, on the contrary, acarbose is degraded quantitatively by AglB. Recombinant AglB is activated in the presence of CaCl2, KCl, and EDTA, as well as after heating of the enzyme. AglA is a dimer of two identical 54-kDa subunits, and it hydrolyses the alpha-glycoside bonds of disaccharides and short maltooligosaccharides, acting on the substrate from the non-reducing end of the chain. It is a cofactor-dependent alpha-glucosidase with a wide action range, hydrolysing both oligoglucosides and galactosides with alpha-link. Thereby, the enzyme is not specific with respect to the configuration at the C4 position of its substrate. For the enzyme to be active, the presence of NAD+, DTT, and Mn2+ is required. Enzymes AglB and AglA supplement one another in substrate specificity and ensure complete hydrolysis to glucose for the intermediate products of starch degradation.

[Thermotoga neapolitana gene clusters participating in degradation of starch and maltodextins: molecular structure of the locus]. / Klaster genov Thermotoga neapolitana, uchastvuiushchikh v degradatsii krakhmala i mal'todekstrinov: molekuliarnaia struktura lokusa.

A 5451-bp genome fragment of the hyperthermophilic anaerobic eubacterium Thermotoga neapolitana has been cloned and sequenced. The fragment contains one truncated and three complete open reading frames highly homologous to the starch/maltodextrin utilization gene cluster from Thermotoga maritima whose genome sequence is known. The incomplete product of the first frame is highly homologous to MalG, the E. coli protein of starch and maltodextrin transport. The product of the second frame, AglB, is highly homologous to cyclomaltodextrinase with the alpha-glucosidase activity TMG belonging to family 13 of glycosyl hydrolases (GH13). The product of the third frame, AglA, is homologous to the Thermotoga maritima cofactor-dependent alpha-glucosidase from the GH4 family. The two enzymes form a separate branch on the phylogenetic tree of the family. The AglA and AglB proteins supplement each other in substrate specificity and can ensure complete hydrolysis to glucose of cyclic and linear maltodextrins, the intermediate products of starch degradation. The product of the fourth reading frame has sequence similarity with the riboflavin-specific deaminase RibD from T. maritima. The homologous locus of this bacterium, between the aglA and ribD genes, has five open reading frames missing in T. neapolitana. The nucleotide sequences of two frames are homologous to transposase genes. The deletion size is 2.9 kb.

Beet yellows closterovirus: complete genome structure and identification of a leader papain-like thiol protease.

The sequence of 8734 nucleotides (nt) from the 5'-end of the beet yellows closterovirus (BYV) RNA was determined to complete the 15,480-nt sequence of the virus genome. The 5'-terminal two-thirds of the sequence are occupied by two overlapping open reading frames (ORFs) 1a and 1b, encoding products with calculated M(r) of 295K and 48K, respectively. The RNA sequence surrounding the stop codon in ORF 1a shows structural elements typical of ribosomal frameshifting signals in a number of animal and plant viruses. It is predicted that the ORF 1b product is expressed via a +1 ribosomal frameshifting as the 348K ORF 1a/1b fusion protein. This putative protein contains the array of methyltransferase, RNA helicase, and RNA-dependent RNA polymerase domains that is conserved in the Sindbis-like supergroup of positive-strand RNA viruses. The 348K protein of BYV is longer than the putative replicases of the most closely related viruses (tobra- and tobamoviruses) by about 1300 amino acids distributed between two unique regions, one at the N-terminus, and the other in the central portion. The N-terminal domain showed sequence similarity to the helper component papain-like protease of potyviruses. By using in vitro translation of the T7 transcripts encoding the N-terminal 92K peptide of the BYV ORF 1a product, we found that the N-terminal fragment of 588 amino acids is released from the translation product by cleavage at the Gly-Gly dipeptide. Site-directed mutagenesis of either of the predicted catalytic residues Cys-509 and His-569 or of the Gly-588 at the cleavage site completely abolished the cleavage. The central unique region of the 348K protein contains a domain distantly resembling the aspartic protease of HIV and other lentiviruses. As shown previously, the 3'-terminal portion of the BYV genome encompasses seven more ORFs, one of which codes for a protein related to the HSP70 cell heat shock proteins, whereas two others encode the capsid protein and its diverged copy. Thus, despite the apparent evolutionary relationship with Sindbis-like viruses, BYV comprises a collection of genomic modules absorbed from different sources and has a unique expression strategy.

Poa semilatent virus, a hordeivirus having no internal polydisperse poly(A) in the 3' non-coding region of the RNA genome.

RNA from the Hungarian isolate of poa semilatent virus (PSLV) directed in vitro synthesis of 120K, 75K, 25K (coat protein) and 20K polypeptides. In vitro translation of PSLV RNA was blocked by the cap analogue, m7Gpp, thus suggesting that the virus RNA was capped. PSLV RNA could be aminoacylated with [14C]tyrosine in vitro. The sequence of 1.5 kb from the 3' end of the PSLV RNA gamma component revealed two open reading frames (ORFs) separated by a uridine-rich intergenic region. The putative product of the incomplete 5'-proximal ORF showed a close amino acid sequence similarity with the C-terminal segment of the gamma a protein (putative RNA replicase) encoded in the barley stripe mosaic virus (BSMV) RNA gamma, and the 20K product of the 3'-proximal ORF was found to be related to the 17K gamma b product of BSMV. The sequence of 0.8 kb from the 3' end of PSLV RNA beta encompassed two (incomplete) overlapping ORFs whose putative products are related to the beta c and beta d proteins encoded in the similarly arranged ORFs of BSMV RNA beta. Nucleotide sequence homology between the respective parts of the two hordeivirus genomes was restricted to the ORF for gamma a, the spacer between the ORFs for gamma a and gamma b, and the 3' non-coding region, particularly the 95 nucleotide segment at the 3' end representing a tRNA-like structure. Despite limited sequence conservation beyond this segment, the entire 3' non-coding region of PSLV RNA could be folded in a tight pseudoknotted structure closely resembling that of BSMV RNA. Surprisingly, the 'signature' sequence typical for BSMV RNA, internal polydisperse poly(A) intercalated between the coding part of the 3' tRNA-like structure, was not detected in the PSLV genome. Instead, the virus RNA contained several oligoadenylate stretches spaced by other residues, close to the junction of its coding and 3' non-coding portions.

Nucleotide sequence of the 3'-terminal half of beet yellows closterovirus RNA genome: unique arrangement of eight virus genes.

The sequence of 6746 nucleotides representing the 3'-proximal half of the beet yellows closterovirus (BYV) genome was determined. In the direction 5' to 3', the sequence was composed of eight open reading frames (ORFs) potentially encoding proteins of 6.4K (ORF2), 65K (ORF3), 64K (ORF4), 24K (ORF5), 22K (ORF6), 20K (ORF7) and 21K (ORF8). An incomplete ORF, ORF1, encoded the C-terminal part of a putative RNA-dependent RNA polymerase, most closely related to polymerases of tricornaviruses; the putative product of ORF3, 65K, was found to be a homologue of the hsp70 family of cell heat-shock proteins. ORF2 potentially encoded a small hydrophobic 6.4K protein, apparently homologous to small hydrophobic proteins of potex- and carlaviruses. ORF6 encoded the viral coat protein, as indicated by its deduced Mr and amino acid composition. The products of ORFs 4, 5, 7 and 8 showed no significant similarities with protein sequences in the database and there are therefore no justifiable speculations concerning their possible functions. BYV RNA contains a 3'-terminal non-coding region of 181 nucleotides, with two stem-loop structures potentially folded within the 86 nucleotide sequence at the extreme 3' end. Analysis of the primary and secondary structure of this region together with the absence of aminoacylation and adenylylation in vitro showed that the BYV genome is devoid of a tRNA-like structure at its 3' end.

[Dosimetric characteristics of a californium neutron source]. / Dozimetricheskie kharakteristiki kalifornievogo istochnika neitronov.

The spectrum of energy linear transmission (ELT) and a coefficient of neutron quality were measured in the air in the clinical use of sources by means of a set of low efficacy Geiger counters to find a solution to a problem of the standardization of working conditions for persons dealing with 252Cf sources. The ELT mean values LN = 27 +/- 3 keV/micron and LD = 51 +/- 5 KeV/micron and a coefficient of quality Q = 8.9 +/- 1.0 were obtained in measurements with sources in the air. Values obtained in measurements on a patient receiving interstitial therapy with the use of 252Cf were as follows: 33 +/- 5; 57 +/- 8 keV/micron and 9.3 +/- 1.3 respectively. The value Q-7 is recommended for the evaluation of an equivalent dose of mixed 252Cf gamma-neutron radiation. Using 237Np track detectors for various distances and azimuthal angles which are of the main interest to the therapeutic use of 252Cf neutron sources, spatial distribution of the specific standard rate of neutrons was measured in the air (in geometry without scattering) and in the water phantom (in geometry with maximum scattering and accumulation). The results obtained made it possible to deduce regularities of neutron field formation in the vicinity of a single source and to evaluate the reliability of data obtained with calculation methods. They can be used for the planning of application, interstitial and intracavitary tumor therapy, for a neutron activation analysis and radiography.

A comparative study on the in vitro translation products of individual RNAs from two-, three-, and four-component strains of barley stripe mosaic virus.

Passaging through plants of the three-component barley stripe mosaic virus (BSMV) strain Norwich (Norwich III) yielded a two-component isolate (Norwich II). A study was made of the sets of polypeptides translated in a rabbit reticulocyte and wheat embryo cell-free systems from individual RNAs of (1) the natural three-component strain Norwich III, (2) a two-component isolate derived from the former (Norwich II), and (3) an isolate intermediate between the three- and two-component ones, Norwich;. Translation of RNA 1 from all three variants of the Norwich strain in vitro yields a single major product with a molecular weight (Mr) of 120,000 (p120). RNA 2 from Norwich III in vitro produces two polypeptides: the viral coat protein (p23) and, in certain ionic conditions, a polypeptide of 25,000 M(r) (p25). RNA 3 from Norwich III is translated into a protein with Mr of 75,000 (p75). Conversion of Norwich III into Norwich II is accompanied by the changes in the coding specificity of RNA 2; beside the coat protein and p25, a protein of 85,000 M(r) (p85) is formed upon its translation-a feature characteristic of RNA 2 of the naturally occurring bipartite BSMV strains (Russian, type). With the Norwich(i) variety, which is marked by a significantly reduced portion of RNA 3 in the total virion RNA preparation, RNA 2 in addition to the coat protein, p25, and p85 directs the synthesis of another product with M(r) of about 78,000. Thus, in conversion of the three-component BSMV into a two-component one, the loss of RNA 3 is concomitant with the actuation in RNA 2 of a sequence coding for p85. RNAs 1-3 of the quadripartite Argentina Mild (AM) BSMV strain code in vitro for the same polypeptides as RNAs 1-3 of Norwich III. AM RNA 4 is translated as a monocistronic template into a polypeptide with Mr of 55,000 (p55). Amino acid sequences of p85, p75, and p55 are shown to overlap among these polypeptides but not to appreciably overlap with p120 sequences. Data presented here allowed a tentative structure to be proposed for genome of two-, three-, and four-component BSMV strains.