Expression of several domains of twitchin and myorod in the ontogenesis of mussel Mytilus trossulus

The expression of MLCK- and PEVK-domains of twitchin, as well as the unique N-terminal domain of myorod in early development of the mussel Mytilus trossulus has been studied. The MLCK-domain of twitchin and the unique N-terminal domain of myorod appear at the early stages of development, whereas the PEVK-domain of twitchin is present only in muscles of adult mussel. The sizes of genes of the N-terminal domain of myorod, obtained at the blastula stage and from the adult animal are similar, but the proteins have significant differences in the amino acid sequences. Consequently, myorod and twitchin appear at early stages of larval mussels before the formation of "adult" muscles capable of catch contraction, and at these stages both proteins are isoforms, which differ from the isoforms of adult animals. It is possible that the MLCK-domain in the "larval" isoform of twitchin is necessary for regulating the formation of the contractile apparatus of molluscan smooth muscles, while the PEVK-domain is important for the regulation of the catch state in muscles of adult animals.     

Characterization of six microsatellite loci in the Baltic blue mussel <Emphasis Type=Italic>Mytilus trossulus</Emphasis> and cross-species amplification in North Sea <Emphasis Type=Italic>Mytilus edulis</Emphasis>

The blue mussel Mytilus trossulus occurs in the Pacific and in the North Atlantic. We developed and characterized six microsatellite loci for Baltic M. trossulus. Seventeen microsatellite loci were screened, of which six were polymorphic. The number of alleles among 50 individuals ranged from 3 to 13 and the observed and expected heterozygosity were 0.09–0.46 and 0.34–0.86, respectively. The loci were also tested for cross amplification in M. edulis, in which four of the six microsatellite loci were successfully amplified.     

Appearance of Muscle Proteins in Ontogenesis of the Mussel <Emphasis Type=Italic>Mytilus trossulus</Emphasis> (Bivalvia)

The appearance of muscle proteins in the contractile apparatus of the mussel Mytilus trossulus was subjected to comparative analysis during ontogenesis. It was established, with the use of Western blot analysis and electrophoresis in polyacrylamid gel in the presence of sodium dodecylsulfate, that proteins of the contractile apparatus of mussel muscles express long before the formation of the first functionally active muscle system of the veliger larvae. Paramyosin is present in egg cells; twitchin, myorod, and actin appear at the stage of blastula (12 h after fertilization), and myosin appears at the trochophore stage (17 h after fertilization). The quantitative relation of muscle proteins was studied in actomyosin extracts of larvae obtained from different developmental stages. It was shown that the ratios actin/myosin and paramyosin/myosin at the veliger stage (96 h after fertilization) were found to be similar to those in the striated muscles of invertebrates.     

Attenuation of fibrosis <Emphasis Type=Italic>in vitro</Emphasis> and <Emphasis Type=Italic>in vivo</Emphasis> with <Emphasis Type=Italic>SPARC</Emphasis> siRNA

Introduction  

SPARC is a matricellular protein, which, along with other extracellular matrix components including collagens, is commonly over-expressed in fibrotic diseases. The purpose of this study was to examine whether inhibition of SPARC can regulate collagen expression in vitro and in vivo, and subsequently attenuate fibrotic stimulation by bleomycin in mouse skin and lungs.     

Time Course of <Emphasis Type=Italic>c-fos</Emphasis>, <Emphasis Type=Italic>vasopressin</Emphasis> and <Emphasis Type=Italic>oxytocin</Emphasis> mRNA Expression in the Hypothalamus Following Long-Term Dehydration

Summary 1. This study presents a time course analysis of the messenger RNA (mRNA) levels of c-fos, vasopressin (VP), and oxytocin (OT) in the paraventricular (PVN) and supraoptic nucleus (SON), following acute and chronic dehydration by water deprivation. 2. Male Wistar rats were separated into five groups: nondehydrated (control group) and dehydrated for 6, 24, 48 and 72 h. Following water deprivation, animals were decapitated, their blood was collected for hematocrit, osmolality, and plasma sodium measurements, and brains were removed for dissection of both PVN and SON. 3. As expected, the hematocrit, osmolality, plasma sodium, and weight loss were increased after water deprivation. In SON, a significant increase in both VP and OT mRNA expression was observed 6 h after dehydration reaching a peak at 24 h and returning to basal levels of expression at 72 h. In the PVN, an increase in both VP and OT mRNA expression occurred 24 h after dehydration. At 72 h the VP and OT mRNA expression levels had decreased but they were still at higher levels than those detected in control animals. 4. These results suggest that SON is the first nucleus to respond to the dehydration stimulus. Additionally, we also observed an increase in c-fos mRNA expression in both PVN and SON 6 h after water deprivation, which progressively decreased 24, 48, and 72 h after the onset of water deprivation. Therefore, it is possible that c-fos may be involved in the modulation of VP and OT genes, regulating the mRNA expression levels on a temporally distinct basis within the PVN and SON.     

Cryopreservation of <Emphasis Type=Italic>Robinia</Emphasis><Emphasis Type=Italic>pseudoacacia</Emphasis>

Cryopreservation of Robinia pseudoacacia explants by vitrification achieved 78% survival following the stepwise preculture of shoot tips in (0.3 + 0.5 + 0.7 M) sucrose with a 80 min incubation in PVS2; compared to 87% survival after desiccation of explants to 30% water content, following 3 days alginate bead (with glycerol and sucrose treatments) preculture in 0.7 M sucrose.     

Probiotic <Emphasis Type=Italic>Lactobacillus</Emphasis> strains: <Emphasis Type=Italic>in vitro</Emphasis> and <Emphasis Type=Italic>in vivo</Emphasis> studies

Three Lactobacillus strains (LOCK 0900, LOCK 0908, LOCK 0919) out of twenty-four isolates were selected according to their antagonistic activity against pathogenic bacteria, resistance to low pH and milieu of bile salts. Intragastric administration of a mixture of these strains to Balb/c mice affected cytokine T_H1-T_H2 balance toward nonallergic T_H1 response. Spleen cells, isolated from lactobacilli-treated mice and re-stimulated in vitro with the mixture of heat-inactivated tested strains, produced significantly higher amounts of anti-allergic tumor necrosis factor- and interferon-γ than control animals whereas the level of pro-allergic interleukin-5 was significantly lower. Lactobacillus cells did not translocate through the intestinal barrier into blood, liver and spleen; a few Lactobacillus cells found in mesenteric lymph nodes could create antigenic reservoir activating the immune system. The mixture of Lactobacillus LOCK 0900, LOCK 0908 and LOCK 0919 strains represents a probiotic bacterial preparation with possible use in prophylaxis and/or therapy of allergic diseases.     

Efficient separation and purification of allophycocyanin from <Emphasis Type=Italic>Spirulina</Emphasis> (<Emphasis Type=Italic>Arthrospira</Emphasis>) <Emphasis Type=Italic>platensis</Emphasis>

Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0). In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A ₆₅₀/A ₂₈₀ of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g⁻¹ wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from 5.6 to 4.0. The absorbance ratio A ₆₅₀/A ₂₈₀ of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g⁻¹ wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE.     

Regulation of the <Emphasis Type=Italic>ALBINO3</Emphasis>-mediated transition to flowering in <Emphasis Type=Italic>Arabidopsis</Emphasis> depends on the expression of <Emphasis Type=Italic>CO</Emphasis> and <Emphasis Type=Italic>GA1</Emphasis>

ALBINO3, a homologue of PPF1 in Arabidopsis, encodes a chloroplast protein, and is essential for chloroplast differentiation. In the present study, ALBINO3(−) transgenic plants exhibited a significant decrease in both the number of rosette leaves at bolting and the days before bolting, suggesting the important roles of ALBINO3 in regulating flowering during non-inductive short-day photoperiods. ALBINO3 mRNA was apparently accumulated in shoot apical meristem and floral meristems around the shoot apical meristem in wild-type plants. ALBINO3 might be predominantly involved in inducing the floral repression pathway by activating the expression of TFL1, and by suppressing the expression of LFY, respectively, in the shoot apical meristem. Moreover, the function of ALBINO3 in regulating flowering transition depended on the expression of CO and GA1, because ALBINO3 might function in the downstream integration of the photoperiod-dependent and the photoperiod-independent pathways. These results suggest that ALBINO3 may have an important integrative function in the flowering process in Arabidopsis.     

Interaction of <Emphasis Type=Italic>Bdellovibrio bacteriovorus</Emphasis> with bacteria <Emphasis Type=Italic>Campylobacter jejuni</Emphasis> and <Emphasis Type=Italic>Helicobacter pylori</Emphasis>

Interaction of Bdellovibrio bacteriovorus 100NCJB with bacteria Campylobacter jejuni (strains 1, 2, 3, 4, and 5) and Helicobacter pylori, strain TX30a, was confirmed. The results indicate that lytic activity of bdellovibrios both in liquid media and cells attached to a surface was observed. The potential use of the antimicrobial activity of predatory bacteria for environmental bioprotection and public health is discussed.     

<Emphasis Type=Italic>In vitro</Emphasis> organogenesis of <Emphasis Type=Italic>Passiflora alata</Emphasis>

Passiflora alata in vitro organogenesis was studied based on explant type, culture medium composition, and incubation conditions. The results indicated that the morphogenic process occurred more efficiently when hypocotyl segment-derived explants were cultured in media supplemented with cytokinin and AgNO₃ incubated under a 16-h photoperiod. The shoot bud elongation and plant development were obtained by transferring the material to MSM culture medium supplemented with GA₃ and incubated in flasks with vented lids. Histological analyses of the process revealed that the difficulties in obtaining plants could be related to the development of protuberances and leaf primordia structures, which did not contain shoot apical meristem. Roots developed easily by transferring elongated shoots to 1/2 MSM culture medium. Plant acclimatization occurred successfully, and somaclonal variation was not visually detected. The efficiency of this organogenesis protocol will be evaluated for genetic transformation of this species to obtain transgenic plants expressing genes that can influence the resistance to Cowpea aphid borne mosaic virus.     

High-Level Expression of Heme-Dependent Catalase Gene <Emphasis Type=Italic>katA</Emphasis> from <Emphasis Type=Italic>Lactobacillus Sakei</Emphasis> Protects <Emphasis Type=Italic>Lactobacillus Rhamnosus</Emphasis> from Oxidative Stress

Lactic acid bacteria (LAB) are generally sensitive to hydrogen peroxide (H₂O₂), Lactobacillus sakei YSI8 is one of the very few LAB strains able to degrade H₂O₂ through the action of a heme-dependent catalase. Lactobacillus rhamnosus strains are very important probiotic starter cultures in meat product fermentation, but they are deficient in catalase. In this study, the effect of heterologous expression of L. sakei catalase gene katA in L. rhamnosus on its oxidative stress resistance was tested. The recombinant L. rhamnosus AS 1.2466 was able to decompose H₂O₂ and the catalase activity reached 2.85 μmol H₂O₂/min/10⁸ c.f.u. Furthermore, the expression of the katA gene in L. rhamnosus conferred enhanced oxidative resistance on the host. The survival ratios after short-term H₂O₂ challenge were increased 600 and 10⁴-fold at exponential and stationary phase, respectively. Further, viable cells were 100-fold higher in long-term aerated cultures. Simulation experiment demonstrated that both growth and catalase activity of recombinant L. rhamnosus displayed high stability under environmental conditions similar to those encountered during sausage fermentation.     

Presence of <Emphasis Type=Italic>C. albidus</Emphasis>, <Emphasis Type=Italic>C. laurentii</Emphasis> and <Emphasis Type=Italic>C. uniguttulatus</Emphasis> in Crop and Droppings of Pigeon Lofts (<Emphasis Type=Italic>Columba livia</Emphasis>)

Columba livia is an important reservoir and carrier of Cryptococcus neoformans, Cryptococcus uniguttulatus, Cryptococcus laurentii and Cryptococcus albidus. Upper digestive tract of this species is also known as a habitat for Cryptococcus neoformans. Given the increasing clinical interest of this microorganism, 331 swabs from crop and 174 dropping samples from pigeon lofts in Grand Canary Island have been studied. The obtained results show an extensive presence samples 81 positive (24.47%) of Cryptococcus spp. in analysed crops: 32 (9.66%) for C. neoformans, 24 (7.2%) for C. uniguttulatus, 23 (6.9%) for C. albidus and 2 (0.6%) for C. laurentii. In the same way, Cryptococcus spp was also isolated in 82 (47.13%), dropping samples: C. neoformans in 59 (33.9%), C. uniguttulatus, in 9 (5.17%), C. laurentii in 8 (4.59%) and C. albidus in 6 (3.44%) of the investigated samples, respectively. The cryptococcosis produced by species of cryptococci other than C. neoformans has become more important during the last decade, supporting the study on the role of pigeon in the epidemiology of this disease.     

Distribution of <Emphasis Type=Italic>Hordoindoline</Emphasis> genes in the genus <Emphasis Type=Italic>Hordeum</Emphasis>

Hordoindoline (Hin) genes, which are known to comprise Hina, Hinb-1, and Hinb-2, are associated with grain hardness in barley. However, the interspecific variation in the Hin genes in the genus Hordeum has not been studied in detail. We examined the variation in Hin genes and used it to infer the phylogenetic relationships between the genes found in two H. vulgare subspecies (cultivated barley and H. vulgare subsp. spontaneum) and 10 wild relatives (H. bogdanii, H. brachyantherum, H. bulbosum, H. chilense, H. comosum, H. marinum, H. murinum, H. patagonicum, H. pusillum, and H. roshevitzii). The Hina and Hinb genes of these species were amplified by PCR. We found two Hinb genes in three wild species (H. bogdanii, H. brachyantherum, and H. roshevitzii) and preliminarily named them Hinb-A and Hinb-B. Cluster analysis showed that the 17 Hinb genes present in Hordeum formed two distinct clusters (named A and B). Seven Hinb genes were included in Cluster-A, and 10 Hinb genes were included in Cluster-B. All Hinb-A genes were included in Cluster-A, while all of the Hinb-B genes were included in Cluster-B. In contrast, the Hinb-1 and Hinb-2 genes in H. vulgare were included in Cluster-B. These results suggest that the Hinb genes duplicated during the early stages of diversification in the genus Hordeum. On the other hand, the Hinb-1 and Hinb-2 genes in H. vulgare seem to have been generated by a duplication of the Hinb gene after the split of the lineages leading to H. vulgare and H. bulbosum.     

Control of pyrimidine nucleotide formation in <Emphasis Type=Italic>Pseudomonas</Emphasis><Emphasis Type=Italic>fulva</Emphasis>

Control of pyrimidine formation was examined in Pseudomonas fulva ATCC 31418. Pyrimidine supplementation lowered pyrimidine biosynthetic pathway enzyme activities in cells grown on glucose or succinate as a carbon source indicating possible repression of enzyme synthesis. Pyrimidine limitation experiments were conducted using an orotidine 5′-monophosphate decarboxylase mutant strain isolated in this study. Compared to uracil-supplemented, glucose-grown mutant cells, pyrimidine limitation of this strain caused aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase activities to increase about 6-, 13-, 3-, 15-fold, respectively, which confirmed regulation of enzyme synthesis by pyrimidines. At the level of enzyme activity, transcarbamoylase activity in Ps. fulva was strongly inhibited by pyrophosphate, CTP, GTP and GDP under saturating substrate concentrations.