[Nascent Polypeptide-Associated Complex as Tissue-Specific Cofactor during Germinal Cell Differentiation in Drosophila Testes].

During the process of spermatogenesis, the proliferation of spermatogonia (stem cell descendants) is replaced by their differentiation in growing spermatocytes responsible for the preparation to meiosis, which is accompanied by a cardinal change in transcriptional programs. We have demonstrated that, in drosophila, this process is accompanied by a splash of the expression of ß-subunit of nascent polypeptide-associated complex (NAC) associated by ribosomes. Nascent polypeptide-associated complex is known as a chaperone involved in co-translational protein folding. This is the first case of the detection of tissue-specific co-translational NAC cofactor in multicellular eukaryotes. It is proposed that spermatocyte specific NAC is involved in the modulation of the expression of the proteins that provide the functioning of subsequent stages of spermatogenesis.

[Multifunctional protein complex NAC (nascent polypeptide associated complex].

The functions of the evolutionary conservative complex NAC (Nascent polypepetide Associated Complex) and its subunits are discussed. The heterodimeric NAC protein contains alpha- and beta-subunits and is found to be reversibly bounded to the ribosome in all eukaryotes, from yeast to humans. NAC contacts the nascent polypeptide and protects it from proteolysis. NAC participates in polypeptide chain folding and modulates protein secretion and transmembrane protein formation. Mutations and deletions of genes, encoding NAC subunits are lethal in early development of multicellular eukaryotes. NAC is involved in the ribosome biogenesis. The beta-subunit interacts with caspase-3 and may be involved in the regulation of the apoptotic pathway. The variants of NAC proteins can be considered as chaperone complexes, involved in the response of the cell and the organism to stress factors, as well as regulators of apoptosis. The genes encoding beta-subunits are rapidly evolved, their duplications cause the formation of tissue specific beta-subunit variants with a different number of putative caspase cleavage sites. The homodimer of alpha-subunits is shown to be the RNA/DNA binding protein and acts as a transcriptional cofactor. The diversity in the functioning of NAC is a prime example of a protein that performs a variety of biological functions (moonlighting protein).

[Molecular variability of testes-specific betaNACtes genes in Drosophila melanogaster genome].

Family of betaNACtes genes in the Drosophila melanogaster genome is a model to investigate the mechanisms of molecular evolution of recently evolved genes. The betaNACtes genes encode proteins, homologous to beta subunit of nascent polypeptide-associated complex (NAC), are expressed in testes and localized on the X chromosome as two two-gene clusters and one separate copy. We collected population polymorphism data for betaNACtes genes using several wild-type stocks of D. melanogaster and compared betaNACtes paralogs with each other. We have shown heterogeneous pattern of betaNACtes genes polymorphism: genes in 3' region of two-gene clusters are low polymorphic, whereas separate betaNACtesl gene is most variable. 5'betaNACtes copies in two-gene tandems are practically identical, whereas 3'betaNACtes copies are highly diverged. Thus, we propose local gene conversion providing selective homogenization of 5'genes. Comparison of betaNACtes paralogs has shown that majority of amino acid differences are in N-terminal part of proteins, containing betaNAC domain. McDonald-Kreitman test of betaNACtes paralog divergence shows the involvement of positive selection in the course of betaNACtes gene family evolution.

Reduction of genotoxic effects of N-nitrosomorpholine in human hepatoma cells and hamster lung cells by carboxymethyl chitin-glucan.

N-nitrosomorpholine (NMOR) belongs to the group of N-nitrosamines and represents a known hepatocarcinogen. Exposure to this compound is considered to be a potential health hazard to humans. There is, however, considerable evidence that the effect of many carcinogenic agents can be markedly influenced or altered by various natural substances. The objective of this study was to assess the DNA-protective and anticlastogenic effects of the derivative of a natural compound, carboxymethyl chitin-glucan (CM-CG), against genotoxicity of N-nitrosomorpholine (NMOR) in human hepatoma cells HepG2 and hamster lung cells V79 cultured in vitro. The exponentially growing cells were pre-treated during 24 h with three different concentrations of CM-CG (150, 750 and 1500 mg/ml) and then treated with different concentrations of NMOR. DNAprotective effects of CM-CG were evaluated by single-cell gel electrophoresis (SCGE, comet assay) and anticlastogenic effects by chromosomal aberration assay. At the SCGE assay a short-term (30 min) and at the chromosomal aberration assay a continuous treatment with NMOR was used. In both HepG2 and V79 cells pre-treated with CM-CG, a significant decrease of the percentage of DNA lesions induced by NMOR was observed along with a reduction of NMOR-induced chromosomal aberrations. We did not find any substantial differences between the genotoxic effects of NMOR on HepG2 and V79 cells, which have different histopathological origins and different levels of metabolizing enzymes. Three different concentrations of CM-CG exerted a similar protective effect against NMOR-induced DNA lesions and chromosomal aberrations in both HepG2 and V79 cells.

Yeast cell wall polysaccharides as antioxidants and antimutagens: can they fight cancer?

Polysaccharides represent the major part of the yeast cell wall dry weight and build the skeletal carcass defining cell wall stability and cell morphology (beta-D-glucans) or constitute amorphous matrix and cell surface fibrous material (mannans and mannoproteins). It is known that yeast cell wall beta-D-glucans reveal immunomodulating properties, which allows for their application in anti-infective and antitumor therapy. Recent data also suggest that polysaccharides reveal antioxidant activity that can result in their protective function as antioxidants, antimutagens, and antigenotoxic agents. The paper provides a review of our continuing research involving water-soluble derivatives of beta-D-glucan isolated from the baker's yeast Saccharomyces serevisiae and of a glucomannan isolated from the industrial yeast Candida utilis. The results are confronted with the available literature data. The derivatives of beta-D-glucan demonstrated potent inhibitory effect on lipid peroxidation comparable to that of the known antioxidants and exerted DNA protection from oxidative damage. The free radical scavenging activity was confirmed by spin-trap electron paramagnetic resonance. Antimutagenic and antigenotoxic activity of the yeast polysaccharides was demonstrated using yeast, bacterial, and algal models. The derivatives of beta-D-glucan exerted potent enhancement of tumor necrosis factor alpha (TNF-alpha) released from murine macrophages and revealed synergistic effect with cyclophosphamide in the treatment of Lewis lung carcinoma and two types of lymphosarcoma in murine models. The results indicate significant protective antioxidant, antimutagenic, and antigenotoxic activities of the yeast polysaccharides and imply their potential application in anticancer prevention/therapy.

Evaluation of follow-up of therapy with fenbendazole incorporated into stabilized liposomes and immunomodulator glucan in mice infected with Toxocara canis larvae.

Anthelmintic activity of benzimidazole carbamate anthelmintics is low against dormant Toxocara canis larvae during late infections in paratenic hosts. The present study was conducted to examine the efficacy of pure fenbendazole, or drug incorporated into sterically stabilized liposomes (SL-FBZ) administered to T. canis-infected mice alone and after its co-administration with the immunomodulator (1-->3)-beta-D-glucan against larvae localized in muscles and brains. Therapy with either drug forms (in total 250 mg/kg in 10 doses) commenced on day 28 post-infection (p.i.) and the efficacy of treatment, examined on day 30 after the last dose of drug, was the highest in groups of mice treated with SL-FBZ in combination with glucan (89.5+/-5.8% in the muscles, 66.1+/-8.1% in brains). During 56 days of follow-up after termination of therapy, serum levels of anti-TES IgG antibodies, circulating IgG-TES immune complexes (CIC) as well as IgG antibodies to the most immunogenic part of recombinant myosin antigen of T. canis larvae were investigated. In contrast to anti-TES IgG antibodies, levels of CIC and anti-myosin antibodies were in the linear correlation with the efficacy of treatments beginning from day 38 post-therapy. We also showed that the serum levels of CIC as well as anti-myosin IgG antibodies seem to be the suitable serological markers for the monitoring of progress in larval destruction and TES resorption from the tissues.

Degradation of high-molar-mass hyaluronan by an oxidative system comprising ascorbate, Cu(II), and hydrogen peroxide: inhibitory action of antiinflammatory drugs--naproxen and acetylsalicylic acid.

Changes in dynamic viscosity of the solutions of a high-molar-mass hyaluronan (HA) were monitored using a rotational viscometer. The degradative conditions generated in the HA solutions by a system comprising ascorbate plus Cu(II) plus H(2)O(2) were studied either in the presence or absence of a drug--naproxen or acetylsalicylic acid. Continual decrease of the dynamic viscosity of HA solution was indicative of the polymer degradation. Addition of the drug retarded/inhibited the HA degradation in a concentration-dependent manner. The characteristics of the fragmented polymers were investigated by FT-IR spectroscopy and by two different liquid chromatographic techniques, namely by size-exclusion chromatography equipped with a multi-angle light scattering photometric detector and by high-performance liquid chromatography connected on-line to a spectrofluorometer.

Carboxymethylated (1 --> 3)-beta-D-glucan protects liposomes against ultraviolet light-induced lipid peroxidation.

In this study we have analyzed antioxidant capabilities of the carboxymethylated (1 --> 3)-beta-D-glucan (M(w) = 5.88 x 10(5)) against lipid peroxidation induced by ultraviolet (UV) radiation--UVA (320-400 nm), which is known to produce mainly singlet oxygen, (1)O(2) . Lipid peroxidation was monitored by measuring the absorption spectra of the conjugated dienes and quantified by Klein oxidation index. The results imply that the (1 --> 3)-beta-D-glucan derivative studied is an antioxidant with the scavenging ability lying between alpha-tocopherol and hyaluronic acid. Thus, glucan as a potential safe and effective dietary supplement may be used for a prolonged time for a systemic photoprotection of humans.

[Synthesis of oligosaccharide fragments of mannan from Candida albicans cell wall and their BSA conjugates].

3-Aminopropyl glycosides of alpha-D-mannopyranosyl-(1-->2)-alpha-D-mannopyranosyl-(1-->2)-alpha-D-mannopyra-nosyl-(1-->2)-alpha-D-mannopyranose, alpha-D-mannopyranosyl-(1-->3)-alpha-D-mannopyranosyl-(1-->2)-alpha-D-mannopyranosyl-(l -- 2)-alpha-D-mannopyranose, and alpha-D-mannopyranosyl-(1-->2)-[alpha-D-mannopyranosyl-(1-->3)]-alpha-D-mannopyranosyl-(1-->2)-alpha-D-mannopyranosyl-(1-->2)-alpha-D-mannopyranose were efficiently synthesized starting from ethyl 2-O-acetyl(benzoyl)-3,4,6-tri-O-benzyl-l-thio-alpha-D-mannopyranoside, ethyl 4,6-di-O-benzyl-2-O-benzoyl-1-thio-alpha-D-mannopyranoside, ethyl 4,6-di-O-benzyl-2,3-di-O-benzoyl-l-thio-alpha-D-mannopyranoside, and 2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl bromide. The oligosaccharide chains synthesized correspond to the three structural types of side chains of mannan from Candida albicans cell wall. A conjugate of the third pentasaccharide with bovine serum albumin was prepared using the squarate method.

[Distorted heterochromatin replication in Drosophila melanogaster polytene chromosomes as a result of euchromatin-heterochromatin rearrangements].

Studies of the position effect resulting from chromosome rearrangements in Drosophila melanogaster have shown that replication distortions in polytene chromosomes correlate with heritable gene silencing in mitotic cells. Earlier studies mostly focused on the effects of euchromatin--heterochromatin rearrangements on replication and silencing of euchromatic regions adjacent to the heterochromatin breakpoint. This review is based on published original data and considers the effect of rearrangements on heterochromatin: heterochromatin blocks that are normally underrepresented or underreplicated in polytene chromosomes are restored. Euchromatin proved to affect heterochromatin, preventing its underreplication. The effect is opposite to the known inactivation effect, which extends from heterochromatin to euchromatin. The trans-action of heterochromatin blocks on replication of heterochromatin placed within euchromatin is discussed. Distortions of heterochromatin replication in polytene chromosomes are considered to be an important characteristic associated with the functional role of the corresponding genome regions.

Carboxymethyl chitin-glucan enriched diet exhibits protective effects against oxidative DNA damage induced in freshly isolated rat cells.

The connection between dietary intake of carboxymethyl chitin-glucan (CM-CG, approximately 200 mg/kg body weight, during 21 days) and the response of freshly isolated rat cells to genotoxic treatment with a combination of photosensitizer Methylene Blue and visible light (MB+VL) was evaluated in presented study. Blood lymphocytes, testicular cells, and hepatocytes were isolated from rats fed by a standard or CM-CG enriched diet and in ex vivo conditions challenged with oxidative agent. Induced DNA damage was assessed using a modified comet assay. When added to the diet, CM-CG itself did not induce any negative effect on the health condition of animals or on level of DNA breaks in rat cells. Moreover, the cells isolated from CM-CG fed animals were more resistant to oxidative stress induced by visible light-excited Methylene Blue. In conclusion, we have demonstrated that carboxymethyl chitin-glucan represents a natural fungal polysaccharide that is able to exert antimutagenic properties upon application in diet.

Natural microbial polysaccharide sulphoethyl glucan as antigenotoxic and cancer preventing agent.

Naturally occurring polysaccharides isolated from the yeasts are the substances with versatile intriguing biomodulatory activities. One of the novel derivatives prepared from the (1 --> 3)-beta-D-glucan isolated from the cell walls of baker's yeast Saccharomyces cerevisiae is sulfoethyl glucan (SEG). Its DNA-protective, antimutagenic, anticlastogenic and cytotoxic/cytostatic enhancing effect was evaluated using five eukaryotic systems. SEG showed bioprotective effect in recombination- repair-deficient strain of alga Chlamydomonas reinhardtii against methyl methanesulfonate-induced genotoxicity, antimutagenic effect against ofloxacin-induced genetic changes in yeast Saccharomyces cerevisiae assay and anticlastogenic activity in plants Vicia sativa and Vicia faba assays against maleic hydrazide-induced clastogenicity. In the combined application with cytostatic drug vumon, SEG exerted enhancement of the drug's cytotoxic/cytostatic effect in the cell revitalization assay using mouse leukemia cells. The study sheds light on the possible mechanisms of actions and utilization of this microbial polysaccharide derivative in the cancer prevention and therapy.

Heterochromatic Stellate gene cluster in Drosophila melanogaster: structure and molecular evolution.

The 30-kb cluster comprising close to 20 copies of tandemly repeated Stellate genes was localized in the distal heterochromatin of the X chromosome. Of 10 sequenced genes, nine contain undamaged open reading frames with extensive similarity to protein kinase CK2 beta-subunit; one gene is interrupted by an insertion. The heterochromatic array of Stellate repeats is divided into three regions by a 4.5-kb DNA segment of unknown origin and a retrotransposon insertion: the A region (approximately 14 Stellate genes), the adjacent B region (approximately three Stellate genes), and the C region (about four Stellate genes). The sequencing of Stellate copies located along the discontinuous cluster revealed a complex pattern of diversification. The lowest level of divergence was detected in nearby Stellate repeats. The marginal copies of the A region, truncated or interrupted by an insertion, escaped homogenization and demonstrated high levels of divergence. Comparison of copies in the B and C regions, which are separated by a retrotransposon insertion, revealed a high level of diversification. These observations suggest that homogenization takes place in the Stellate cluster, but that inserted sequences may impede this process.

Molecular cloning of double-stranded cDNA from the eye lens of the frog Rana temporaria: construction of the cDNA clonotheque and identification of a clone containing the nucleotide sequences of the lambda-crystallin gene.

Poly(A)+ RNA from the lens of the frog Rana temporaria contains three components (1200 +/- 50, 1000 +/- 50, and 900 +/- 50 bp in size) and a more heterogeneous RNA species with a length of 650-750 nucleotides. This RNA was used as a template for the AMV reverse transcriptase and Escherichia coli DNA-polymerase I and the total cDNA obtained was cloned in the PstI site of the pBR322 plasmid vector. Recombinant plasmids corresponding to abundant poly(A)+ RNA classes contain cDNA inserts from less than or equal to 200 to 1200 nucleotides in length. Part of the library (clonotheque) was divided into classes differing in the presence of absence of the restriction sites for BamHI, EcoRI and HindIII restriction endonucleases. The clones belonging to each of the five classes were characterized by the hybridization-translation test. The translation product of mRNA hybridizing with the clone pRT(1)294 has an M4 of about 22 000 and is specifically precipitated by the antiserum to lambda-crystallins of Rana temporaria. The size of the cDNA present in pRT(1)294, equal to 580 +/- 20 bp, is sufficient for coding the greater part of the lambda-crystallin amino acid sequence. On the basis of these data, we conclude that the clone pRT(1)294 codes for one of the frog lambda-crystallins.

Structure of the Escherichia coli 0104 polysaccharide and its identity with the capsular K9 polysaccharide.

The 0104 antigen (lipopolysaccharide, LPS) of Escherichia coli has an acidic O specific polysaccharide. From the aqueous phase of a phenol water extraction of E. coli O104: K-, a fraction was obtained by ultracentrifugation and Cetavlon precipitation of the supernatant, which was enriched in long-chain LPS. Compositional analysis, NMR spectroscopy, periodate oxidation and methylation analysis showed that the polysaccharide chain of O104 LPS II consisted of galactose, N-acetylgalactosamine and neuraminic acid and acetate in the molar ratio of 2:1:1:1 and contained 3-beta Gal, 3-beta GalNAc, 4-alpha Gal, and 4-alpha(9-OAc-NeuNAc) in linear sequence. The same results were obtained with the capsular K9 polysaccharide from E. coli O9:K9, as presented here and reported previously (Dutton et al. (1987) Carbohydr. Res. 170, 193-206).

Protective effect of the yeast glucomannan against cyclophosphamide-induced mutagenicity.

Glucomannan (GM) isolated from Candida utilis with molecular weight 30 kDa was administered either intraperitoneally or orally prior to cyclophosphamide (CP) injection and its effect on the frequency of micronuclei was evaluated in polychromatic erythrocytes of mouse bone marrow. GM administration by either route decreased significantly (p<0.002) the clastogenic effect of CP. The protective effect was concentration-dependent, with a higher decrease achieved by 200 mg/kg than by 100 mg/kg b. wt. (body weight). The fact that GM was effective also at oral administration is indicative of the passage of GM molecules through the wall of the gastrointestinal tract. The important characteristics of GM isolated from C. utilis, such as good water solubility, relatively small molecular weight (30 kDa), and antimutagenic effect exerted also at oral administration, appear to be promising features for its prospective use as a natural protective agent.

Comparative DNA protectivity and antimutagenicity studies using DNA-topology and Ames assays.

Two experimental techniques, the DNA-topology assay and the Ames assay, were proved to be suitable for monitoring compounds with a genotoxic potential and/or with an antimutagenic effect. Both procedures were used in assaying the acid-mine water (AMW) containing toxic metals and sulfoethyl chitin-glucan (SE-Ch-G), a derivative of chitin-glucan, in which bioprotective activities were detected earlier. It was shown that after toxic metal concentrations were decreased due to AMW dilution to the limits that correspond with those set by the Slovak Technical Norm (STN) for drinking water, AMW was not genotoxic in the Ames assay. As it is possible to detect any single-strand DNA (ssDNA) break in the DNA-topology assay, the SE-Ch-G protective effect against the ssDNA breaks induced by Fe(2+) in the DNA-topology assay was recorded. SE-Ch-G exhibited the antimutagenic potential after its application simultaneously with diagnostic mutagens in the Ames assay. These results demonstrate the complementarity of both experimental systems.

Structural studies of mannans from the cell walls of the pathogenic yeasts Candida albicans serotypes A and B and Candida parapsilosis.

A comparative study of three cell-wall mannans, of Candida albicans serotypes A and B and Candida parapsilosis, by means of methylation analysis supports a model of yeast mannans as having an alpha-(1----6)-linked backbone with some units (depending on the origin of the mannan) being substituted at O-2 with oligosaccharides joined by alpha-(1---2) and, to a lesser extent, by alpha-(1----3) glycosidic bonds. Branching points in the side chains of Candida albicans mannans were found in substantial proportions for the first time, and the corresponding branched hexasaccharides were isolated by means of acetolysis and subsequent gel filtration. 13C-N.m.r. spectroscopy of the mannans, as well as a 1H-n.m.r. spectroscopic study of the oligosaccharides obtained on acetolysis of the mannans, led to results that agreed with those of methylation analysis.

Novel structure of the cellular mannan of the pathogenic yeast Candida krusei.

On the basis of methylation analysis, 13C- and 1H-n.m.r. spectroscopic data, and the results of immunological methods, a new structure for the cell-wall alpha-D-mannan of the pathogenic yeast Candida krusei is proposed. In contrast to the alpha-D-mannans of other pathogenic yeasts of the Candida species, which have (1----6)-linked main chains and many (1----2)- and/or (1----3)-linked side chains, the C. krusei mannan is lightly branched and contains (1----2) and (1----6) linkages in the ratio 3:1.

Structure of the O56 antigen of Escherichia coli, a polysaccharide containing 7-substituted alpha-N-acetylneuraminic acid.

The O56 polysaccharide moiety of the O56 antigen (LPS) consists of D-glucose, D-galactose, 2-acetamido-2-deoxy-D-glucose, and N-acetylneuraminic acid in the molar ratios 1:1:1:1. Methylation analysis, periodate oxidation, mild acid hydrolysis, as well as 1H and 13C NMR spectroscopy showed that the O56 polysaccharide has the primary structure [formula: see text]