DNA methylation and expression of estrogen receptor alpha in fathead minnows exposed to 17α-ethynylestradiol.

The gene expression response thought to underlie the negative apical effects resulting from estrogen exposure have been thoroughly described in fish. Although epigenetics are believed to play a critical role translating environmental exposures into the development of adverse apical effects, they remain poorly characterized in fish species. This study investigated alterations of DNA methylation of estrogen receptor alpha (esr1) in brain and liver tissues from 8 to 10 month old male fathead minnows (Pimephales promelas) after a 2d exposure to either 2.5 ng/L or 10 ng/L 17α-ethynylestradiol (EE2). Changes in the patterns of methylation were evaluated using targeted deep sequencing of bisulfite treated DNA in the 5' region of esr1. Methylation and gene expression were assessed at 2d of exposure and after a 7 and 14d depuration period. After 2d EE2 exposure, males exhibited significant demethylation in the 5' upstream region of esr1 in liver tissue, which was inversely correlated to gene expression. This methylation pattern reflected what was seen in females. No gene body methylation (GBM) was observed for liver of exposed males. Differential methylation was observed for a single upstream CpG site in the liver after the 14d depuration. A less pronounced methylation response was observed in the upstream region in brain tissue, however, several CpGs were necessarily excluded from the analysis. In contrast to the liver, a significant GBM response was observed across the entire gene body, which was sustained until at least 7d post-exposure. No differential expression was observed in the brain, limiting functional interpretation of methylation changes. The identification of EE2-dependent changes in methylation levels strongly suggests the importance of epigenetic mechanisms as a mediator of the organismal response to environmental exposures and the need for further characterization of the epigenome. Further, differential methylation following depuration indicates estrogenic effects persist well after the active exposure, which has implications for the risk posed by repeated exposures..

First Report of Neofusicoccum parvum Associated with Grapevine Trunk Diseases in Croatia.

In September 2010, during survey of diseased grapevines (Vitis vinifera L.) in vineyards at localities Zmajevac (BZ), Orahovica (SO), Cilipi (KC), and Novalja (PN), symptoms characteristic of grapevine trunk diseases (GTD) (3) were observed, showing on cross-sectioned cordons and trunks as brown, wedge-shaped perennial cankers and/or dark streaking of the wood. In Croatia, these symptoms were traditionally associated with Eutypa Tul. & C.Tul. and with fungi from Diaporthaceae (2). From affected grapevines (cvs. Grasevina, Pinot bijeli, Malvazija dubrovacka, and Gegic), samples of symptomatic cordons and trunks were collected (n ≥ 35). To isolate the causal agents from the samples, woodchips of symptomatic tissue, surface-sterilized in 2% sodium hypochlorite for 2 min, were placed on potato dextrose agar amended with streptomycin sulphate (50 µg/ml) and incubated for 7 days at 25°C in darkness. A percentage of samples (72, 15, 27, and 54% from BZ, SO, KC, and PN, respectively) yielded fungal colonies with abundant aerial mycelium, initially white, but turning olivaceous grey after 5 days. From these colonies, monohyphal isolates were obtained and pycnidial formation stimulated by cultivation on 2% water agar with stems of plant species Foeniculum vulgare Mill. at 25°C under diffuse light for 3 weeks. Pycnidia contained conidia that were hyaline, unicellular, ellipsoid with round apices and truncated bases, and thin walled with smooth surface. Dimensions of conidia (n ≥ 50) were (12.8) 15.3 ± 1.4 (17.6) × (5.4) 6.3 ± 0.8 (7.6) µm, with length/width ratio (2.0) 2.5 ± 0.5 (3.2). Based on morphological data, species Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips was suspected (1). For molecular identification, isolates BZ330, SO334, KC342, and PN121 were used for PCR to amplify internal transcribed spacer region and partial translation elongation factor 1-alpha gene, using primers ITS5/ITS4 and EF1-728F/EF1-986R, respectively. Obtained sequences were shown to be identical between the four isolates (GenBank: KF296318, KF296319) and when compared with sequences for reference N. parvum isolate CMW9080 (AY236942, AY236887) they showed >99% homology, confirming the isolates as species N. parvum. Pathogenicity tests were done by inoculation of detached green shoots (GS) and lignified canes (LC) (n = 5) of grapevine cv. Skrlet by either mycelial plugs of the same four isolates, or sterile agar plugs for the controls. Inoculated GS were kept in flasks with sterile water in a glasshouse for 10 days, and LC in humid dark chambers for 30 days, at 25°C. Resulting vascular necrosis measured 62 to 81 mm (GS) and 215 to 246 mm (LC), but was absent on controls. Koch's postulates were satisfied by successful reisolation of N. parvum only from plants inoculated with mycelial plugs. N. parvum has been recognized as a serious grapevine pathogen, causing similar symptoms worldwide (3). To our knowledge, this is the first report of N. parvum associated with GTD in Croatia, and due to its relatively high incidence at surveyed localities, it could present considerable threat, particularly for neighboring vine growing regions. Diplodia seriata De Not., a weak pathogen (3), was also identified from a percentage of samples in this survey. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) J. Kaliterna et al. Arh. Hig. Rada Toksikol. 63:471, 2012. (3) J. R. Urbez-Torres. Phytopathol. Mediterr. 50(Suppl.):S5, 2011.

First Report of Diplodia seriata as Causal Agent of Olive Dieback in Croatia.

In August 2010, a dieback of young olive (Olea europea L.) trees (cvs. Pendolino and Leccino) occurred in two orchards in Istria, Croatia. According to the producers, low temperatures during the winter severely damaged the plants and led to their decline. Distinctive symptoms, assumed fungal infection, were observed in internal tissue of stems and branches. Elongated brown necrosis, sometimes with black streaks, was visible under the bark, therefore Verticillium wilt was suspected. Of 1,086 trees in two orchards (4 ha), 165 (15%) showed symptoms. To isolate the causal agent, surface-sterilized wood chips of symptomatic tissue were placed on potato dextrose agar (PDA). Fungal colonies resembling Botryosphaeriaceae spp. grew from all wood fragments placed on PDA, and from these colonies, monohyphal isolates were obtained. For morphological identification, pycnidial formation was stimulated by growing the isolates on 2% water agar that included stems of plant species Foeniculum vulgare Mill. at room temperature under diffuse light. Pycnidia contained conidia that initially showed as hyaline, becoming light to dark brown as they matured, ovoid with truncated or rounded base and obtuse apex, aseptate, with wall moderately thick, externally smooth, roughened on the inner surface, and 22.8 to 23.5 × 9.6 to 10.5 µm. On the basis of these morphological characters, fungal species Diplodia seriata (teleomorph "Botryosphaeria" obtusa) was suspected (3). For molecular identification, four isolates (MN3, MN4, MN5, and MN6) were used for PCR to amplify the internal transcribed spacer (ITS) region and partial translation elongation factor 1-alpha (EF1-α) gene, using primers ITS4/ITS5 and EF1-728F/EF1-986R, respectively. Sequencing was performed with those amplified genes, then sequences were deposited in GenBank. Comparison of these sequences with GenBank sequences for referent D. seriata isolate CBS 112555 (AY259094 and AY573220) (3) showed 100% homology. On the basis of molecular data, the isolates were confirmed to be species D. seriata De Not. Pathogenicity tests were performed by inoculation of 2-year-old olive plants, six plants per tested cultivar (Pendolino and Leccino). For every cultivar, four plants were wounded and mycelium plugs from D. seriata cultures on PDA were placed on the wounds and sealed with Parafilm. Two control plants per tested cultivar were inoculated with sterile PDA plugs. After 2 months, six of eight inoculated plants wilted completely, and under the bark, brown necrosis was observed. D. seriata was constantly reisolated from the inoculated plants and fulfilled Koch's postulates and confirmed pathogenicity of D. seriata on olive as causal agent of olive dieback. Control plants showed no symptoms of the disease. This fungus has been recognized as the cause of fruit rot of olive (1) and branch canker or dieback in Spain (2). To our knowledge, this is the first report of D. seriata as a pathogen of olive in Croatia. Also, this is one of the first reports of D. seriata as the cause of olive dieback in the world, while Moral et al. (1,2) mostly reported it as the cause of olive fruit rot. Since the same symptoms of olive dieback were observed at other localities in Croatia, the disease could represent a serious threat, particularly for young olive orchards. References: (1) J. Moral et al. Plant Dis. 92:311, 2008. (2) J. Moral et al. Phytopathology 100:1340, 2010. (3) A. J. L. Phillips et al. Fungal Divers. 25:141, 2007.

Adenosine triphosphate levels in steelhead (Oncorhynchus mykiss) eggs: an examination of turnover, localization and role.

The dynamics of energy production and utilization in fish eggs before and shortly after fertilization may be critical for embryo survival. Therefore, the current study examined the turnover of adenosine triphosphate (ATP) as well as examined the possible role and localization of ATP in unfertilized steelhead (Oncorhynchus mykiss) eggs and early embryos. The mean ATP level in unfertilized steelhead eggs was 1.92+/-0.10 (mean+/-S.E.M., n=17) nmol ATP per egg. Exposure of the unfertilized egg to 10 degrees C water (water activation) and fertilization resulted in comparable and substantial decreases (approx. 20-50%) in egg ATP levels within 3 min. This suggests that the energy expended at fertilization is used in response to water activation rather than fertilization per se. Unfertilized eggs maintained in ovarian fluid for 9 days at 10 degrees C under air showed a progressive decline of fertility that reached zero after 6 days. In contrast, no significant changes were seen in ATP levels throughout this 9 days period. Thus, fertility does not positively correlate with egg ATP levels in stored eggs. In the unfertilized egg, the ATP stored in the yolk accounted for approximately 1.5% of the total egg ATP. After fertilization, the concentration of ATP in the yolk increased approximately seven-fold, with the yolk and blastoderm each now accounting for approximately 20% of the total remaining ATP. Finally, to estimate the changes in oxidative metabolism following fertilization, the cyanide (KCN)-sensitive decline in total ATP was determined for unfertilized eggs and 1 day embryos. In the presence of KCN, ATP levels declined to approximately 50% within 24 h in both unfertilized eggs as well as embryos; the rates of ATP decline were not different. Therefore, there was not a discernible increase in ATP generation by oxidative phosphorylation at the time of fertilization.

Comparison of mouse and human NASP genes and expression in human transformed and tumor cell lines.

We previously cloned and sequenced cDNAs encoding mouse NASP (mNASP), a cell cycle regulated histone H1 binding protein. Here we report the genomic sequence and organization for mNASP along with its 5' regulatory region and compare these with human NASP (hNASP). The mNASP gene contains 16 exons interrupted by 15 introns. The sequence encoding testis mNASP uses all 16 exons while the somatic form uses 13 exons by differential splicing. All the exons conform to the AG/GT splicing rule. Putative TATA box-containing transcription initiation sites are present for somatic NASP in human and mouse and for testis hNASP. Comparison of the promoter regions of mNASP and hNASP approximately 1 kb upstream of the transcription start sites for the two splice variants revealed a number of possible transcription factor binding sites relevant to specific patterns of NASP tissue expression. The presence of single bands on Southern blots of mouse genomic DNA suggests that mNASP is a single copy gene although pseudogenes exist in both the mouse and human genomes. Chromosome fluorescence by in situ hybridization revealed that mNASP is present on chromosome 4, in an area that corresponds to band 4D1, a region syntenic to the locus of hNASP on chromosome 1. Additionally, we report that human somatic and testis NASP mRNAs are expressed at varying levels in all the transformed cell lines and human tumors tested, further supporting NASP's role in the cell cycle of dividing cells.

Does CO2 enhance short-term storage success of Chinook salmon (Oncorhynchus tshawytscha) milt?

Successful short-term storage of salmonid milt depends on numerous factors, including temperature, fluid volume, and gaseous environment, with storage at low temperatures under an atmosphere of 100% O2 being the most common method. Salmonid sperm maintained in a storage environment with elevated carbon dioxide (CO2) levels, such as the approximately 4% CO2 in exhaled air, are not motile when activated. While these modest levels of CO2 inhibit sperm motility, the effect is reversible within hours after exposure to a CO2-free oxygenated environment. Therefore, the effect of CO2 (as a component gas in the storage environment) on chinook salmon (Oncorhynchus tshawytscha) sperm motility and viability was examined. The hypothesis of the current investigation was that CO2-exposure with subsequent CO2 removal would be beneficial during short-term chinook salmon milt storage. Milt samples were collected from mature (adult) and precocious (jack) male chinook salmon and stored under various CO2 and O2 levels at 3 to 4 degrees C for up to 14 days. Milt samples were then removed from the incubation environments and maintained under CO2-free humidified air with continuous mixing for 4 h at 10 degrees C before analysis of motility. The resultant motility of samples incubated under 3.5% or less CO2 was not different than controls during the 14 d incubation period; motility of samples stored under higher CO2 tensions were significantly lower. The motility of samples incubated under 3.5% CO2 reached the maximum recovered motility after 2 h exposure to CO2-free humidified air, while the motility of sperm incubated under 13.4% CO2 levels recovered no motility even after 6 h exposure to CO2-free humidified air. The motility of samples incubated under normoxia was significantly greater than that of samples incubated under hyperoxia (approximately 90% O2) at both 7 and 14 d, regardless of the CO2 level. Sperm viability was relatively unaltered by any of the incubation conditions examined. The results of this investigation suggest that there is no apparent advantage to storage of chinook salmon sperm in the presence of CO2 and that storage under hyperoxia negatively affects sperm function compared to storage under normoxia.

Ecto-ATPase activity of vertebrate blood cells.

Ecto-ATPase activity was measured for red blood cells, white blood cells, and whole blood from a variety of vertebrates. A large range of red blood cell ecto-ATPase activity was observed; for example, at 10 degrees C, red blood cells from a catastomid fish (Catostomus macrocheilus) and a newt (Taricha rivularis) had activities of 56 +/- 9 and 25,000,000 +/- 14,000,000 pmol ATP per 10(6) red blood cells per hour, respectively (mean +/- SD). Several control experiments verified that the measured ATPase activity was not the result of intracellular ATPases released due to cell damage or lysis nor due to the release of intracellular nucleoside triphosphate or uptake of extracellular ATP. Red blood cell ecto-ATPase activity was relatively low within the teleosts, was high within the reptiles, and had the greatest range and single highest value within the amphibians. Within the endotherms, avian red blood cell ecto-ATPase activities were greater than mammalian red blood cell ecto-ATPase activities, which were the lowest for all vertebrates examined. The lowest ecto-ATPase activities measured were for human and skunk red blood cells, which had activities of 13 +/- 1 and 11 +/- 2 pmol ATP per 10(6) red blood cells per hour, respectively, at 35 degrees C. Ecto-ATPase activity was measured in white blood cells of several vertebrate species and appeared generally high and less variable than red blood cell ecto-ATPase activity. Measured whole blood ecto-ATPase activity showed a range of three orders of magnitude and correlated positively with red blood cell ecto-ATPase activities. Ecto-ATPase activity was also determined for red blood cells from fetal, 1-3 d old neonatal, and pregnant garter snakes (Thamnophis elegans); these activities were not significantly different from the activity of red blood cells from nonpregnant adult females. Overall, the data from the present study demonstrate a wide range of red blood cell and whole blood ecto-ATPase activities among vertebrates and include some of the highest ecto-ATPase activities reported to date.

Stability of nucleoside triphosphate levels in the red cells of the snake

Nucleated red cells in the nonpregnant garter snake (Thamnophis elegans) contain relatively high concentrations of nucleoside triphosphate (NTP), largely in the form of ATP, which is found at concentrations of approximately 10 mmol l-1 relative to cell volume and 15 mmol l-1 relative to cell water. During pregnancy, levels of NTP in adult red cells rise by approximately 50 % concomitant with an increase in blood progesterone level. The stability of the NTP pool within these red cells was assessed by maintaining cells in vitro at 20 °C, without exogenous nutrients, and in the presence and absence of the metabolic inhibitors iodoacetate and/or cyanide. After 96 h, NTP levels in adult red cells not exposed to the inhibitors had decreased by only approximately 10 %, while in the presence of both inhibitors NTP levels had fallen by less than 50 %. Red cell NTP levels were not affected by acute exposure to high concentrations of progesterone either in vivo or in vitro. NTP levels were much more labile when the cells were maintained in vitro at either low or high pH. Maintenance of red cells at pH 6.0 for 24 h resulted in a decrease in NTP levels of approximately 50 % and at pH 10.0 the levels fell by approximately 80 %, while buffers containing only ATP caused no detectable degradation. Incubation at low or high pH promoted some cell swelling; however, the magnitude of the decreases in intracellular NTP concentration prompted by these pH levels could not be mimicked by incubation of red cells in hypotonic buffer. Total nonspecific ATPase activity at pH 6.0 was approximately 55 % greater than that at pH 7.4, while at pH 10.0 it was approximately 6 % of that at pH 7.4. The pH-dependent decrease in intracellular NTP levels cannot, therefore, be due to stimulation of ATPase activity, at least not at high pH. Overall, the data are consistent with the hypothesis that an appreciable portion of the NTP within these cells is compartmentalized in a stable, but pH-sensitive, pool sequestered from intracellular ATP-hydrolyzing processes.

The role of T-lymphocyte subpopulation in bronchoalveolar lavage in pulmonary parenchyma diseases.

Cellularity and T-lymphocyte to T-lymphocyte subpopulation ratio were examined in 37 cases of interstitial pulmonary disease as well as in two healthy subjects in bronchoalveolar lavage (BAL) using monoclonal antibody indirect immunofluorescence technique. In BAL, active sarcoidosis cases (n = 18) were found to have increased T-lymphocytes of helper phenotype (CD4) accompanied by a markedly heightened CD4/CD8 proportion. Conversely, T-lymphocytes of the suppressor cytotoxic phenotype (CD8) with markedly reduced CD4/CD8 proportion predominated in the BAL of cases with hypersensitive pneumonitis (HP). Case group having sarcoidosis (S) after corticosteroid therapy (n = 11) showed a reduction in the proportion of T-lymphocytes (CD4) as well as in that of CD4/CD8 in BAL. An analysis of T-lymphocytic subpopulation in BAL could be helpful in diagnosing pulmonary interstitial disease, and give an insight into disease activity.

[Pathogenic and clinical differences between stasis and chronic bronchitis]. / Patogenetske i klinicke razlike izmedu zastojnog i kronicnog bronhitisa.

There is often a misguide of chronic bronchitis and passive pulmonary hypertension. In 42 patients with chronic bronchitis a revision of the results was made and it was found that in 28 (67%) patients the former diagnosis of chronic bronchitis was confirmed, while in 8 (19%) patients a new diagnosis was established--a passive pulmonary hypertension and in 6 (14%) patients a combination of chronic bronchitis and passive pulmonary hypertension was found. It is considered that the symptoms like cough, sputum and dyspnea as well as the similar physical finding may lead the clinician to change the diagnoses.