Matrilin-like molecules produced by circulating hemocytes of the zebra mussel (Dreissena polymorpha) upon stimulation

Since invading the Great Lakes basin, USA, in 1980s, the zebra mussel (Dreissena polymorpha) has caused significant economic and ecologic devastation. The absence of major diseases led to the belief that zebra mussel may have extraordinary host defense mechanisms. This study was undertaken in order to better understand zebra mussel hemocyte functions. A suppressive subtraction hybridization (SSH) cDNA library was constructed from naïve and stimulated hemocytes. Stimulation was performed using a mixture of lipopolysacchride (LPS), peptidoglycan (PGN), and zymosan (ZYM). In the stimulated hemocyte SHH cDNA library, 55 assembled ESTs were differentially expressed. These ESTs contained two putative immune- related molecules, namely, matrilin and agglutinin, three house-keeping genes, six cell metabolism/development genes, and 44 ESTs without putative functions. One of the putative adhesive molecules (CN-29, accession number: AM503947) was predicted to have homology with matrilins including the Von Willebrand Factor A (VWA) domain, which was identical to a matrilin molecule recently reported from the freshwater snail, Biomphalaria glabrata. Preliminary evidence suggests that the zebra mussel matrilin-like molecule is inducible upon hemocyte stimulation.     

Deficiency of peroxisome assembly in a mutant of the methylotrophic yeast Hansenula polymorpha

Summary We have isolated a mutant of the metholotrophic yeast Hansenula polymorpha defective in peroxisomal biosynthesis. The mutant strain has been derived by a selection procedure from cells of a high-copy number transformant that overproduces the major peroxisomal enzyme methanol oxidase (MOX) and forms enlarged peroxisomes. In contrast to the parental strain the mutant lacks intact peroxisomes in thin sections, but exhibits electron-dense particles that are devoid of intact membranes and crystalloid cores. Consequently, peroxisomal enzymes show severe proteolytic degradation in crude cell lysates. Complementation of this, and analogous mutations, will offer the possibility to identify genes that are required for peroxisome assembly.     

Highly-efficient electrotransformation of the yeast Hansenula polymorpha

A highly-efficient method for transformation of the methylotrophic yeast Hansenula polymorpha has been developed. Routinely, transformation frequencies of up to 1.7×10⁶/g plasmid DNA were obtained by applying an electric pulse of the exponential decay type of 7.5 kV/cm to a highly-concentrated cell mixture during 5 ms. Efficient transformation was dependent on: (1) pretreatment of the cells with the reducing agent dithiotreitol, (2) the use of sucrose as an osmotic stabilizer in an ionic electroporation buffer, and (3) the use of cells grown to the mid-logarithmic phase. Important parameters for optimizing the transformation frequencies were field strength, pulse duration, and cell concentration during the electric pulse. In contrast to electrotransformation protocols described for Saccharomyces cerevisiae and Candida maltosa, transformation frequencies (transformants per g DNA) for H. polymorpha remained high when large amounts (up to 10g) of plasmid DNA were added. This feature renders this procedure pre-eminently advantageous for gene cloning experiments when high numbers of transformants are needed.     

Evidence for autonomous replication and stabilization of recombinant plasmids in the transformants of yeast Hansenula polymorpha

Summary For the transformation of the yeast Hansenula polymorpha we have constructed a set of hybrid plasmids carrying the LEU2 gene of Saccharomyces cerevisiae as a selective marker and fragments of mitochondrial DNA of Candida utilis and H. polymorpha or chromosomal DNA fragments of H. polymorpha as replicator sequences. The replication properties of chimeric plasmids in the yeast H. polymorpha were investigated. We showed that for plasmids propagated autonomously in this yeast the plasmid monomers could be detected in the transformants only during the immediate time after the transformation event. Further growth under selective conditions led to the selection of polymeric forms of plasmid DNA as it was clearly shown for transformants carrying cosmid pL2 with mtDNA fragment of C. utilis. Such transformants carrying polymerized plasmids showed a remarkably increased stability of the transformed phenotype. Cosmid pL2 was able to shuttle between Escherichia coli, S. cerevisiae and H. polymorpha, whereas plasmids with DNA fragments from H. polymorpha did not transform S. cerevisiae effectively.     

Biosynthesis of the peroxisomal dihydroxyacetone synthase from Hansenula polymorpha in Saccharomyces cerevisiae induces growth but not proliferation of peroxisomes

Summary The DAS gene of Hansenula polymorpha was expressed in Saccharomyces cerevisiae under the control of different promoters. The heterologously synthesized dihydroxyacetone synthase (DHAS), a peroxisomal enzyme in H. polymorpha, shows enzymatic activity in baker's yeast. The enzyme was imported into the peroxisomes of S. cerevisiae not only under the appropriate physiological conditions for peroxisome proliferation (oleic acid media), but also in glucose-grown cells where it induced the enlargement of the few peroxisomes present. This growth process was not accompanied by an increase in the number of microbodies, which suggests a separate control mechanism for peroxisomal proliferation.     

Additional locus of rDNA sequence specific to the X chromosome of the liverwort, Marchantia polymorpha

The molecular cytogenetic organization of 17S ribosomal RNA genes (17S rDNA), a part of the 45S rDNA repeat, was investigated on the chromosomes of the liverwort Marchantia polymorpha using fluorescence in-situ hybridization (FISH). The numbers of 17S rDNA loci visualized in female and male chromosomes were ten and nine, respectively. This heterogeneous localization was due to the presence of an additional 17S rDNA locus on the X chromosome and its absence on the Y chromosome. The signal on the X chromosome covered almost the entire region of its long arm. The other nine signals were observed on the same loci of respective autosomes in both sexes. Southern hybridization analysis revealed an additional band including 17S rDNA exclusively on EcoRI digested female genomic DNA supporting the existence of an additional 17S rDNA locus on the X chromosome.     

The development of daughter sporocysts inside the mother sporocyst ofSchistosoma mansoni with special reference to the ultrastructure of the body wall

The development of the mother sporocyst and the differentiation of the daughter sporocyst ofSchistosoma mansoni inBiomphalaria pfeifferi are described. The tegumental structure of the mother sporocyst, consisting of an outer layer connected to internally situated nucleated cell bodies, forms extensions which enwrap the germinal cells. The parenchyma cells, in which the germinal cells were embedded before, degenerate. When daughter sporocyst embryos develop from germinal cells they are enveloped by a primitive epithelium which is formed by fusion of the extensions of the tegumental structure of the mother sporocyst. Somatic cells located peripherally in the developing daughter sporocyst expand and coalesce beneath the primitive epithelium to form the future outer layer of the tegumental structure of the daughter sporocyst. The primitive epithelium degenerates, the newlyformed layer looses its nuclei, and becomes connected to internally situated nucleated cell bodies. Further developments in the tegumental structure of the daughter sporocyst include the formation of microvillus-like projections, a surface coat, spines, and a basement membrane.     

Delayed damaging effects of anthracycline antibiotics on the reproductive system of rats

Destructive changes and disturbances of spermatogenesis are found to occur in the testes of Wistar rats 1 and 3 months after a single injection of the anthracycline antibiotic pharmorubicin in the maximum permissible dose. The morphological picture normalizes 6 months postinjection. Disturbances in reproductive function are observed as early as 3 months after treatment. The number of dominant lethal mutations rises one month postinjection. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, N ^o 1, pp. 55–58, January, 1996     

In vivo exposure to microcystins induces DNA damage in the haemocytes of the zebra mussel, Dreissena polymorpha, as measured with the comet assay

The Comet assay was used to investigate the potential of the biotoxin microcystin (MC) to induce DNA damage in the freshwater zebra mussel, Dreissena polymorpha. Mussels maintained in the laboratory were fed daily, over a 21-day period, with one of four strains of the cyanobacterium, Microcystis aeruginosa. Three of the strains produced different profiles of MC toxin, while the fourth strain did not produce MCs. The mussels were sampled at 0, 7, 14, and 21 days by withdrawing haemocytes from their adductor muscle. In addition, a positive control was performed by exposing a subsample of the mussels to water containing cadmium chloride (CdCl(2)). Cell viability, measured with the Fluorescein Diacetate/Ethidium Bromide test, indicated that the MC concentrations, to which the mussels were exposed, were not cytotoxic to the haemocytes. The Comet assay performed on the haemocytes indicated that exposure to CdCl(2) produced a dose-responsive increase in DNA damage, demonstrating that mussel haemocytes were sensitive to DNA-damaging agents. DNA damage, measured as percentage tail DNA (%tDNA), was observed in mussels exposed to the three toxic Microcystis strains, but not in mussels exposed to the nontoxic strain. Toxin analysis of the cyanobacterial cultures confirmed that the three MC-producing strains exhibit different toxin profiles, with the two MC variants detected being MC-LF and MC-LR. Furthermore, the DNA damage that was observed appeared to be strain-specific, with high doses of MC-LF being associated with a higher level of genotoxicity than low concentrations of MC-LR. High levels of MC-LF also seemed to induce relatively more persistent DNA damage than small quantities of MC-LR. This study is the first to demonstrate that in vivo exposure to MC-producing strains of cyanobacteria induces DNA damage in the haemocytes of zebra mussels and confirms the sublethal toxicity of these toxins.     

Development ofTrichobilharzia ocellata inLymnaea stagnalis and the effects of infection on the reproductive system of the host

Juvenile specimens ofLymnaea stagnalis were exposed to 0 or 4 miracidia ofTrichobilharzia ocellata. Highly productive infections developed in all infected snails. Qualitative and/or quantitative light microscopic observations were performed at various intervals between day 0 and 83 post exposure (p.e.). Gonadal maturation started between day 4 and 7 p.e. in all snails, but in infected animals gonadal growth was retarded considerably after day 7 and was blocked from day 19 onwards. This resulted in very small gonads (10% of the volumes of those of controls on day 83 p.e.) in infected snails, in which however, gametogenesis was initially normal. Due to the small gonad volume gamete production was highly reduced. From day 33 onwards spermatogenesis appeared to be inhibited and around day 83 oocyte maturation seemed to be impaired. The accessory sex organs (ASO) of infected snails showed a similar reduced growth. The cellular differentiation and production of secretory granules in these small ASO was normal albeit delayed in both the male and female organs. The severe reduction in growth of the gonad and the ASO started in the presence of immature primary sporocysts, the later effects on gametogenesis were simultaneous with the production of massive numbers of cercariae. These results are discussed in view of the available data on the endocrine control of reproduction inL. stagnalis. It is suggested that either the secretion of gonadotrophic hormones or the response of their targets is reduced by humoral effects of the parasites.     

Effects of anthracyclines on reproductive function in rats

A single injection of antitumor anthracycline antibiotic in a maximum tolerated dose decreased mating capacity in male rats (doxorubicin, farmorubicin) and decreased the efficiency of mating (farmorubicin). In female rats mating capacity did not decrease, but some of them became sterile. The majority of toxic effects were reversible. The incidence of embryonal death before implantation was increased in intact females mated with males treated with farmorubicin. In females treated with anthracycline antibiotics high incidence of pre- and postimplantation embryonal deaths was noted. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 130, No. 11, pp. 527–530, November, 2000     

Effect of single total irradiation on the reproductive system and vitamin content in rat progeny

Reduction in the epididymis weight and in the spermatozoid concentration together with marked morphological changes in the seminiferous tubules were observed in the progeny of irradiated rats. Vitamin E content in the testes increased 2.8-fold, while that of vitamin B₂ decreased 1.6-fold. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 12, pp. 652–654, December, 1998.     

Effect of experimental hyperprolactinemia on the sexual cycle and folliculogenesis

It is found that hyperprolactinemia results in the development of stable anovulation, in which the disruption of folliculogenesis sets in as early as at the stage of late preantral (graafian) follicles and manifests itself in a decrease of the mitotic activity in cells of the membrana granulosa. The concentration of follicle-stimulating hormone thereupon changes to different extents or remains at the control level. A direct influence of prolactin on intrafollicular estrogen production and on cell division is postulated. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 9, pp. 326–328, September, 1994     

Evolution of ribosomal DNA unit on the X chromosome independent of autosomal units in the liverwort Marchantia polymorpha

In the haploid dioecious liverwort, Marchantia polymorpha, the X chromosome, but not the Y, carries a cluster of ribosomal RNA genes (rDNAs). Here we show that sequences of 5S, 17S, 5.8S and 26S rDNAs are highly conserved (>99% identity) between the X chromosomal and autosomal rDNA repeat units, but the intergenic spacer sequences differ considerably. The most prominent difference is the presence of a 615-bp DNA fragment in the intergenic spacer, X615, which has accumulated predominantly in the rDNA cluster of the X chromosome. These observations suggest that the rDNA repeat unit on the X chromosome evolved independently of that on autosomes, incorporating sex chromosome-specific sequences. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effects of joint stress on sticklebacks

The effects on the three-spined stickleback, (Gasterosteus aculeatus L.), of various combinations of stresses due to parasitism bySchistocephalus solidus, dietary restriction, and exposure to cadmium (0.032 mg l^–1) were investigated. Sticklebacks exposed to a combination of all three stressors died first with a median period of survival significantly shorter than that for fish subjected to fewer stressors. Compared with fish on a restricted diet alone, parasitism plus dietary restriction also reduced survival; however, the combination of cadmium exposure and restricted diet did not. A possible explanation for this finding is proposed.     

Immediate and remote effects of antitumor drug on the morphology of rat liver

Experiments performed on rats have shown that a single intravenous injection of the antitumor preparations platidiam and pharmorubicine in the maximum tolerated dose cause morphological changes in the liver which are preserved for 3 months after platidiam and for 6 moths after pharmorubicine. Instability of reparative processes in the liver was revealed when the rats were poisoned with CCl₄, 1, 3, and 6 months after administration of the cytostatics. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 11, pp. 561–564, November, 1998     

Different effects of glucocorticoids on the density of β-adrenoreceptors in the lungs and cerebral cortex

Tissue specificity of the effect of glucocorticoids on the density of β-adrenoreceptors is shown. The hormone increases the number of receptors in the lungs, but has no effect on their density in the cerebral cortex either in the norm or after down-regulation. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 119, N ^o 3, pp. 328–330, March, 1995 Presented by V. A. Trufakin, Member of the Russian Academy of Medical Sciences     

Comparative effects of tranquilizers and nootropes on the elaboration and functional impairment of the avoidance response

Experiments with rats show that pyracetam, phenazepam, and gidazepam accelerate the development of an avoidance response and diminish its functional impairment through unexpected exposure to electric current applied notwithstanding the relations established between stimuli, the response, and its sequelae. The drugs under study increase the reproducibility of avoidance responses in the rats after the indicated procedure, by preventing or diminishing the increase in intersignal reactions, i.e., exerted equivalent effects despite differences in the pharmacological spectra of the drugs. The results of this study show that the substances used affect emotional tension but do so by different mechanisms. Thus, the tranquilizers decrease emotional tension by acting on the emotional sphere directly, whereas pyracetam decreases it in an indirect way, by activating cognitive and mnestic processes. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 8, pp. 152–155, August, 1996     

Morphological study of the effects of textile-immobilized enzymes on an experimental purulent wound

Morphological changes in an experimental purulent wound in a rat model is studied for application of surgical gauze with immobilized enzymes: trypsin, lysozyme, collitin, or co-immobilized trypsin and lysozyme. Comparison of the times of wound cleansing and healing shows that immobilized enzymes are more effective than native preparations, and the therapeutic effect of gauze with the enzyme complex is higher than that of gauze with individually immobilized enzymes. Morphological studies confirm that immobilized trypsin-lysozyme complex and collitin are the most efficient in hastening and potentiating reparative procevesses in a purulent wound. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o , 9, pp. 323–325, September, 1994