Glycogen synthesis from glucose and fructose in hepatocytes from diabetic rats

In rat hepatocytes, the basal glycogen synthase activation state is decreased in the fed and diabetic states, whereas glycogen phosphorylase a activity decreases only in diabetes. Diabetes practically abolishes the time- and dose-dependent activation of glycogen synthase to glucose especially in the fed state. Fructose, however, is still able to activate this enzyme. Glycogen phosphorylase response to both sugars is operative in all cases. Cell incubation with the combination of 20 mM glucose plus 3 mM fructose produces a great activation of glycogen synthase and a potentiated glycogen deposition in both normal and diabetic conditions. Using radiolabeled sugars, we demonstrate that this enhanced glycogen synthesis is achieved from both glucose and fructose even in the diabetic state. Therefore, the presence of fructose plays a permissive role in glycogen synthesis from glucose in diabetic animals. Glucose and fructose increase the intracellular concentration of glucose 6-phosphate and fructose reduces the concentration of ATP. There is a close correlation between the ratio of the intracellular concentrations of glucose 6-phosphate and ATP (G6-P/ATP) and the activation state of glycogen synthase in hepatocytes from both normal and diabetic animals. However, for any given value of the G6-P/ATP ratio, the activation state of glycogen synthase in diabetic animals is always lower than that of normal animals. This suggests that the system that activates glycogen synthase (synthase phosphatase activity) is impaired in the diabetic state. The permissive effect of fructose is probably exerted through its capacity to increase the G6-P/ATP ratio which may partially increase synthase phosphatase activity, rendering glycogen synthase active.     

Glycogen synthesis from pyruvate in the periportal and from glucose in the perivenous zone in perfused livers from fasted rats

The isolated liver of 24 h fasted rats was perfused in a non-recirculating manner in the orthograde or retrograde direction with media containing glucose and/or gluconeogenic precursors. Glycogen formation was determined biochemically and demonstrated histochemically. With glucose as the only exogenous substrate glycogen was formed exclusively in the perivenous area during both orthograde and retrograde perfusion. With gluconeogenic precursors as the exogenous substrates glycogen was deposited in the periportal zone during orthograde perfusion and in the intermediate zone during retrograde perfusion. Supply of glucose and gluconeogenic substrates initiated glycogen synthesis only in the upstream region, i.e. in the periportal zone during orthograde and in the perivenous zone during retrograde perfusion. This localization of glycogen synthesis was probably due to an unavoidable, insufficient oxygen supply of the respective downstream area. In general, the results confirm the hypothesis that periportal and perivenous glycogen was synthesized from different substrates.     

Recovery from acidification in boreal lakes inferred from macroinvertebrates and subfossil chironomids

Acidification of waters and soils caused by emissions and the long-range transport of air pollutants has been a serious worldwide problem during the last decades. The extent of the acidification problem in Finnish acid-sensitive forest lakes was examined in the Acidification Research Project (HAPRO) in the mid-1980s. The recent decline in the emissions of air pollutants has resulted in the chemical recovery of watersheds in many regions, and the present work on the recovery processes in acidified Finnish headwater lakes (REPRO) was launched to examine whether the chemical recovery has already been accompanied by biological recovery. The patterns of recovery were studied by re-sampling littoral macrozoobenthos in a subset of the previously sampled HAPRO lakes. Paleolimnological samples were taken in order to assess the possible dependence of lacustrine chironomid communities on the changing degree of acidification. Acid sensitive and moderately acid sensitive benthic species revealed slight recovery in the formerly most acidic (pH 5.5) but recently recovered lakes. The most significant factors affecting the response of benthic communities were increased mean lake pH and decreased labile aluminium concentration. Paleolimnological chironomid analysis revealed a slight response along the pH gradient, but also significant structural similarity between the present and pristine chironomid assemblages. This implies that no major changes in chironomid communities of these acidic lakes have occurred during the past centuries. The alternative future trends and threats to biological recovery in small headwater lakes are discussed.     

Extrapolations from species to species and from various cell types in assessing risks from chemical mutagens

Strategies for estimating human mutation frequencies can be based on extrapolations of dose: mutation ratios in other species.     

Phenotypic and genetic characterization of vancomycin-resistant enterococci from hospitalized humans and from poultry in Korea

Vancomycin resistant enterococci (VRE) isolates from humans (23 isolates) and poultry (20 isolates) were characterized by antibiotic susceptibility, vancomycin resistance transferability, pulsed-field gel electrophoresis (PFGE), and structural analysis of Tn1546-like elements. VRE isolates from humans and poultry showed different resistance patterns, transferability, and transfer rate. In addition to these phenotypic differences between humans and poultry VRE, PFGE and the structure of Tn1546-like elements were also distinct. Most poultry isolates (16/20) were identical to the prototype vanA transposon, Tn1546, while most human isolates (21/23) had multiple integrations of insertion sequence. The transmission of VRE and vancomycin resistance determinant between humans and poultry could not be demonstrated in this study.     

Lipogenesis from glucose and pyruvate in fat cells from genetically obese rats

Subcutaneous fat cells were isolated from genetically obese rats and from rats with obesity produced by hypothalamic lesions. Insulin did not augment the oxidation of fatty acids or their synthesis from glucose-1-(14)C or glucose-1-(3)H by fat cells from either group. Radioactivity from pyruvate-3-(14)C was incorporated into fatty acids to the same degree by fat cells from these two groups. The presence of 5 mm glucose in the incubation medium containing fat cells and pyruvate-3-(14)C or aspartate-3-(14)C stimulated the synthesis of fatty acids to a greater extent in cells of genetically obese rats. Fasting, in contrast, reduced the incorporation of radioactivity from pyruvate and glucose into fatty acids by fat cells from the genetically obese animals. In all experiments the fat cells from genetically obese rats converted more radioactivity into glyceride-glycerol relative to CO(2) than did fat cells from hypothalamic obese rats. Parabiosis between one thin and one genetically obese litter mate was performed in three pairs of rats without influencing growth of either rat. Thus in the present studies fat cells from genetically obese rats showed two differences from normal fat cells: they channeled more radioactivity from pyruvate into fatty acids in the presence of glucose, and they uniformly converted more radioactivity into glyceride-glycerol.     

Subtilase cytotoxin-coding genes in verotoxin-producing Escherichia coli strains from sheep and goats differ from those from cattle

Subtilase cytotoxin (SubAB) from verotoxin (VT)-producing Escherichia coli (VTEC) strains was first described in the 98NK2 strain and has been associated with human disease. However, SubAB has recently been found in two VT-negative E. coli strains (ED 591 and ED 32). SubAB is encoded by two closely linked, cotranscribed genes (subA and subB). In this study, we investigated the presence of subAB genes in 52 VTEC strains isolated from cattle and 209 strains from small ruminants, using PCR. Most (91.9%) VTEC strains from sheep and goats and 25% of the strains from healthy cattle possessed subAB genes. The presence of subAB in a high percentage of the VTEC strains from small ruminants might increase the pathogenicity of these strains for human beings. Some differences in the results of PCRs and in the association with some virulence genes suggested the existence of different variants of subAB. We therefore sequenced the subA gene in 12 strains and showed that the subA gene in most of the subAB-positive VTEC strains from cattle was almost identical (about 99%) to that in the 98NK2 strain, while the subA gene in most of the subAB-positive VTEC strains from small ruminants was almost identical to that in the ED 591 strain. We propose the terms subAB1 to describe the SubAB-coding genes resembling that in the 98NK2 strain and subAB2 to describe those resembling that in the ED 591 strain.     

Biogeography and Breeding in Gymnotiformes from Uruguay

This study was carried out in Uruguay (30–35°S), South America, with two complementary approaches. First, an extensive exploration of Uruguayan freshwaters allowed us to assess the distribution of the two major species of Gymnotiformes (out of 4) across sites. Gymnotus carapo was uniformly distributed in Uruguayan territory, whereas Brachyhypopomus pinnicaudatus was observed in the northern and eastern part of the country. There was a highly significant negative correlation between the relative abundance of Brachyhypopomus pinnicaudatus and pH and water conductivity. Moreover, these environmental factors are significant contributors to its spatial differences in relative abundance. Second, temperature, conductivity, photoperiod, and the structure of a Brachyhypopomus pinnicaudatus population were analyzed across seasons in a small lake over a two-year period. Water temperature and photoperiod exhibited important seasonal changes, whereas water conductivity remained low and relatively constant. The presence of sexually mature males, females, and the sudden increase of juveniles indicated the occurrence of the breeding season in November, December, and January, coinciding with high mean water temperatures and extreme photoperiod. These results agree with previous data that support the hypothesis of temperature as an important environmental factor for the onset of breeding in Gymnotiformes from the temperate zone.     

Differences and similarities in enzymes from the neopullulanase subfamily isolated from thermophilic species

Six glycoside hydrolase (GH) family 13 members, classified under the polyspecific neopullulanase subfamily GH13_20 (also termed cyclomaltodextrinase) were analysed. They originate from thermophilic bacterial strains (Anoxybacillus flavithermus, Laceyella sacchari, and Geobacillus thermoleovorans) or from environmental DNA, collected after in situ enrichments in Icelandic hot springs. The genes were isolated following the CODEHOP consensus primer strategy, utilizing the first two of the four conserved sequence regions in GH13. The typical domain structure of GH13_20, including an N-terminal domain (classified as CBM34), the catalytic module composed of the A-and B-domains, and a C-terminal domain, was found in five of the encoded enzymes (abbreviated Amy1, 89, 92, 98 and 132). These five enzymes degraded cyclomaltodextrins (CDs) and starch, while only three, Amy92 (L. sacchari), Amy98 (A. flavithermus) and Amy132 (environmental DNA), also harboured neopullulanase activity. The L. sacchari enzyme was monomeric, but with CD as the preferred substrate, which is an unusual combination. The sixth enzyme (Amy29 from environmental DNA), was composed of the ABC-domains only. Preferred substrate for Amy29 was pullulan, which was degraded to panose, and the enzyme had no detectable activity on CDs. In addition to its different activity profile and domain composition, Amy29 also displayed a different conservation (LPKF) in the fifth conserved region (MPKL) proposed to identify the subfamily. All enzymes had apparent temperature optima in the range 50–65°C, while thermostability varied, and was highest for Amy29 with a half-life of 480 min at 80°C. Calcium dependent activity or stability was monitored in four enzymes, but could not be detected for Amy29 or 98. Tightly bound calcium can, however, not be ruled out, and putative calcium ligands were conserved in Amy98.     

Offspring born from chimeras reconstructed from parthenogenetic and in vitro fertilized bovine embryos

Chimeric embryos were produced by aggregation of parthenogenetic (Japanese Red breed) and in vitro fertilized (Holstein breed) bovine embryos at the Yamaguchi Research Station in Japan and by aggregation of parthenogenetic (Red Angus breed) and in vitro fertilized (Holstein breed) embryos at the St. Gabriel Research Station in Louisiana. After embryo reconstruction, live offspring were produced at each station from transplanting these embryos. The objective of this joint study was to evaluate the developmental capacity of reconstructed parthenogenetic and in vitro fertilized bovine embryos. In experiment I, chimeric embryos were constructed: by aggregation of four 8‐cell (demi‐embryo) parthenogenetic and four 8‐cell stage (demi‐embryo) IVF‐derived blastomeres (method 1) and by aggregation of a whole parthenogenetic embryo (8‐cell stage) and a whole IVF‐derived embryo (8‐cell stage) (method 2). Similarly in experiment II, chimeric embryos were constructed by aggregating IVF‐derived blastomeres with parthenogenetic blsatomeres. In this experiment, three categories of chimeric embryos with different parthenogenetic IVF‐derived blastomere ratios (2:6; 4:4, and 6:2) were constructed from 8‐cell stage bovine embryos. In experiment III, chimeric embryos composed of four 8‐cell parthenogenetic and two 4‐cell IVF‐derived blastomeres or eight 16‐cell parthenogenetic and four 8‐cell IVF‐derived blastomeres were constructed. Parthenogenetic demi‐embryos were aggregated with sexed (male) IVF demi‐embryos to produce chimeric blastocysts (experiment IV). In the blastocyst stage, hatching and hatched embryos were karyotyped. In experiment V, chimeric embryos that developed to blastocysts (zona‐free) were cryopreserved in ethylene glycol (EG) plus trehalose (T) with different concentrations of polyvinylpyrrolidone (PVP; 5%, 7.5%, and 10%). In experiment I, the aggregation rate of the reconstructed demi‐embryos cultured in vitro without agar embedding was significantly lower than with agar embedding (53% for 0% agar, 93% for 1% agar, and 95% for 1.2% agar, respectively). The aggregation was also lower when the aggregation resulted from a whole parthenogenetic and IVF‐derived embryos cultured without agar than when cultured with agar (70% for 0% agar, 94% for 1% agar, and 93% for 1.2% agar, respectively). The development rate to blastocysts, however, was not different among the treatments. In experiment II, the developmental rates to the morula and blastocyst stages were 81%, 89%, and 28% for the chimeric embryos with parthenogenetic:IVF blastomere ratios of 2:6, 4:4, and 6:2, respectively. In experiment III, the developmental rate to the morula and blastocyst stages was 60% and 65% for the two 4‐cell and four 8‐cell chimeric embryos compared with 10% for intact 8‐cell parthenogenetic embryos and 15% for intact 16‐cell parthenogenetic embryos. To verify participation of parthenogenetic and the cells derived from the male IVF embryos in blastocyst formation, 51 embryos (hatching and hatched) were karyotyped, resulting in 27 embryos having both XX and XY chromosome plates in the same sample, 14 embryos with XY and 10 embryos with XX. The viability and the percentage of zona‐free chimeric embryos at 24 hr following cryopreservation in EG plus T with 10% PVP were significantly greater than those cryopreserved without PVP (89% vs. 56%). Pregnancies were diagnosed in both stations after the transfer of chimeric blastocysts. Twin male (stillbirths) and single chimeric calves were delivered at the Yamaguchi station, with each having both XX and XY chromosomes detected. Three pregnancies resulted from the transferred 40 chimeric embryos at the Louisiana station. Two pregnancies were lost prior to 4 months and one phenotypically‐ chimeric viable male calf was born. We conclude that the IVF‐derived blastomeres were able to stimulate the development of bovine parthenogenetic blastomeres and that the chimeric parthenogenetic bovine embryos were developmentally competent. Mol. Reprod. Dev. 53:159–170, 1999. © 1999 Wiley‐Liss, Inc.     

Steatitis in egrets and herons from Japan

More than 70 egrets and herons were found sick or dead at an agricultural water reservoir in Kanagawa Prefecture, Japan between September and October 2008. The birds showed weakness, lethargy, and inability to fly before death. Postmortem findings included large amounts of firm subcutaneous and cavitary fat comprised of necrotic adipose tissues with infiltrates of heterophils and macrophages. The birds were diagnosed with steatitis on the basis of the gross lesions and histopathology. Egrets with steatitis had low blood levels of vitamin E. High counts of cyanobacteria (Microcystis aeruginosa) were found in the reservoir concurrent with the outbreak of steatitis. No microcystin was detected in the reservoir water or the livers from the egrets. This is the first report of steatitis in wild birds in Japan.     

Fusarium species complex and mycotoxins in grain maize from maize hybrid trials and from grower's fields

Aims: To quantify and to compare the occurrence of Fusarium species in maize kernels and stalk pieces, to analyse mycotoxins in kernels and maize crop residues, to evaluate two approaches to obtain kernel samples and to compare two methods for mycotoxin analyses. Methods and Results: The occurrence of Fusarium species in maize kernels and stalk pieces from a three‐year maize hybrid trial and 12 kernel samples from grower’s fields was assessed. Nine to 16 different Fusarium species were detected in maize kernels and stalks. In kernels, F. graminearum, F. verticillioides and F. proliferatum were the most prevalent species whereas in stalks, they were F. equiseti, F. proliferatum and F. verticillioides. In 2006, 68% of the kernel samples exceeded the recommended limit for pig feed for deoxynivalenol (DON) and 42% for zearalenone (ZON), respectively. Similarly, 75% of the samples from grower’s fields exceeded the limits for DON and 50% for ZON. In maize crop residues, toxin concentrations ranged from 2·6 to 15·3 mg kg^?1 for DON and from 0·7 to 7·4 mg kg^?1 for ZON. Both approaches to obtain maize kernel samples were valid, and a strong correlation between mycotoxin analysis using ELISA and LC‐MS/MS was found. Conclusions: The contamination of maize kernels, stalk pieces and remaining crop residues with various mycotoxins could pose a risk not only to animal health but also to the environment. With the hand‐picked sample, the entire Fusarium complex can be estimated, whereas combine harvested samples are more representative for the mycotoxin contents in harvested goods. Significance and Impact of the Study: This is the first multi‐year study investigating mycotoxin contamination in maize kernels as well as in crop residues. The results indicate a high need to identify cropping factors influencing the infection of maize by Fusarium species to establish recommendations for growers.     

Leptospires in wildlife from Trinidad and Grenada

Serum samples from 894 wild animals (representing 31 species) from Trinidad and Grenada were examined by the microscopic agglutination test for leptospiral antibodies; 198 were positive. These included 39 bats, 88 mongooses, six opossums, 10 peridomestic rodents, 15 forest rodents, 10 lizards, and 30 toads. Thirteen pathogenic serogroups were involved. Thirty-nine Leptospira isolates were reported from mongooses, opossums, rodents and toads.     

南极中山站长城站土壤及海洋沉积物可培养细菌多样性

南极地区具有气候酷寒干燥、强紫外(UV)辐射和季节性光照与温度波动等恶劣的极端环境条件,孕育了大量微生物资源和潜在新物种。为了挖掘南极不同生境样品蕴含的微生物菌种资源,更好地认识和探索南极地区微生物资源多样性,利用低温寡营养富集结合梯度稀释涂布的方法,对南极长城站、中山站土壤和近海表层沉积物3个生境的样品进行细菌分离和16S rRNA基因序列相似性分析以确定南极细菌分类学地位。结果表明,共获得482株纯培养细菌,有54个属,142个种,分布于变形菌门(Proteobacteria,42%),放线菌门(Actinobacteria,40%),厚壁菌门(Firmicutes,12%),拟杆菌门(Bacteroidetes,4%)和异常球菌-栖热菌门(Deinococcus-Thermus,1%)。优势属为嗜冷杆菌属(Psychrobacter,12%)、假单胞属(Pseudomonas,10%)和节杆菌属(Arthrobacter,9%)。不同生境样品中纯培养细菌多样性存在较大差异。中山站干燥土壤样品中放线菌门比例明显高于变形菌门,有13个特有属,嗜冷杆菌属(Psychrobacter)、红球菌属(Rhodococcus)为优势菌;长城站近岸砂土样品中变形菌门菌株最多,拟杆菌门比例高于中山站样品,有高比例的假单胞菌,分离到12个特有属;近海沉积物样品细菌群落结构最简单,分离到9个特有属。经16S rRNA基因序列对比发现,有7株菌为潜在新种。     

Mutagens in feces from vegetarians and non-vegetarians

Mutagens in water extracts from feces of persons in 3 different diet groups were measured with the fluctuation test for weak mutagens using Salmonella typhimurium TA100 and TA98 as tester strains. The 3 diet groups were ovo-lacto vegetarians (N = 6), strict vegetarians (N = 11) and non-vegetarians (N = 12). All subjects were from the urban area of Vancouver, British Columbia, Canada. On TA100 ovo-lacto vegetarians and strict vegetarians had significantly lower levels of fecal mutagens than non-vegetarians (P less than or equal to 0.025 and P less than 0.010, resp.). The same pattern, although less significant, was obtained with TA98. Correlation studies between mutagenicity on TA100 and TA98 and between the pH of the fecal homogenate and mutagenicity indicate the presence of 2 or more major fecal mutagens.