两株乳杆菌对牙鲆体表及消化道粘液的粘附特性

目的利用从牙鲆肠道分离的1株鼠李糖乳杆菌P15和1株干酪乳杆菌(Lc),研究它们在牙鲆粘液中的粘附特性以及不同pH和盐度对其粘附的影响。方法用异硫氰酸荧光素(FITC)标记法。结果P15和Lc对牙鲆的体表粘液和消化道粘液均有粘附作用,2株菌对消化道粘液的粘附效果好于体表粘液,且对胃的粘附百分率最高,分别是48.18%和63.0%。这2株菌在各部位的粘附受pH和盐度的影响,在弱酸性环境中和32左右的盐度中具有较强的粘附能力。结论在海水盐度条件下,P15和Lc在消化道粘附率较高。     

Tissue expression of glandular kallikrein and its response to 17 beta-estradiol in the acclimatized carp

Cyprinus carpio skeletal muscle kallikrein was isolated to apparent homogeneity, and a polyclonal antiserum against the purified protein was generated. Glandular kallikrein expression and tissue distribution were assessed using both Western blots and immunohistochemistry. A 39-kDa protein was detected in skeletal muscle, the gill, kidney, and pituitary gland, where an additional 72-kDa immunoreactive band was observed. Immunohistochemistry revealed immunoreactive kallikrein in the intermuscle tissue, epithelial gill cells, apical portion of distal and proximal tubular cells in the kidney, mucus and epithelial cells of the skin, intestinal tube, and prolactin-producing cells of the pituitary gland. In addition, the effect of 17beta-estradiol on kallikrein expression was analyzed in three different tissues of winter- and summer-acclimatized male carps. A 2.5-fold (p<0.05) increase in kallikrein immunoreactivity due to estrogen treatment was observed in winter-acclimatized carp muscle, but not in summer-acclimatized fish. In contrast, the gill responded differently, since a 2-fold (p<0.05) increase was found only in summer-acclimatized carps. Kallikrein immunoreactivity in the kidney increased both in summer- (2.5 fold) and in winter-acclimatized carps (1.5 fold). The signals obtained demonstrate the existence of tissue-specific variable responses to estrogen treatment in vivo, between winter and summer-acclimatized carp.     

The fine structure of the gastric epithelium of the rainbow trout, Salmo gairdneri, Richardson

Fine structural characteristics of the columnar mucus cells lining the surface and pits of the gastric mucosa, oxyntic cells lining the glands and the gastric endocrine cells were studied. The surface mucus cells, in addition to their primary involvement in production of mucus, showed structural adaptations. Release of the mucus vesicles was achieved by exocytosis. Transition from pit gland cells was abrupt since no mucus neck cells were observed. The oxyntic cells possessed apical and basal microvillous processes, a well developed tubulovesicular system, zymogen granules and extensive RER associated with many large mitochondria. When stimulated by distension of the stomach, the apical cytoplasm was converted into a labyrinth of cytoplasmic processes, while annular lamellae, each of which showed a short peripheral linear density appeared in the basal cytoplasm. The endocrine cells showed apical modifications as microvilli, cilia and reduced glycocalyx covering. Three types were distinguished on the basis of their granular morphology.     

STUDIES ON SMALL INTESTINAL CRYPT EPITHELIUM : I. The Fine Structure of the Crypt Epithelium of the Proximal Small Intestine of Fasting Humans

Small intestinal crypt epithelium obtained from normal fasting humans by peroral biopsy of the mucosa was studied with the electron microscope. Paneth cells were identified at the base of the crypts by their elaborate highly organized endoplasmic reticulum, large secretory granules, and small lysosome-like dense bodies within the cytoplasm. Undifferentiated cells were characterized by smaller cytoplasmic membrane-bounded granules which were presumed to be secretory in nature, a less elaborate endoplasmic reticulum, many unattached ribosomes and, in some cells, the presence of glycogen. Some undifferentiated cells at the base of the crypts contained lobulated nuclei and striking paranuclear accumulations of mitochondria. Membrane-bounded cytoplasmic fragments, probably originating from undifferentiated and Paneth cells, were frequently apparent within crypt lumina. Of the goblet cells, some were seen actively secreting mucus. In these, apical mucus appeared to exude into the crypt lumen between gaps in the microvilli. The membrane formerly surrounding the apical mucus appeared to fuse with and become part of the plasma membrane of the cell, suggesting a merocrine secretory mechanism. Enterochromaffin cells were identified by their location between the basal regions of other crypt cells and by their unique intracytoplasmic granules.     

Osmoregulation in Saccharomyces cerevisiae. Studies on the osmotic induction of glycerol production and glycerol-3-phosphate dehydrogenase (NAD+)

Production of glycerol and a key enzyme in glycerol production, glycerol 3-phosphate dehydrogenase (NAD+) (GPD), was studied in Saccharomyces cerevisiae cultured in basal media or media of high salinity, with glucose, raffinose or ethanol as the sole carbon source. At high salinity, glycerol production was stimulated with all carbon sources and glycerol was accumulated to high intracellular concentration in cells grown on glucose and raffinose. Cells grown on ethanol accumulated glycerol to a lower level but showed an increased content of trehalose at high salinity. However, the trehalose concentration corresponded only to about 20% of the glycerol level, and did not compensate for the shortfall in intracellular osmolyte content. Immunoblot analysis demonstrated an increased production of GPD at high salinity. This increase was osmotically mediated but was lower when glycerol was substituted for NaCl or sorbitol as the stress-solute. The enzyme also appeared to be subject to glucose repression; the specific activity of GPD was significantly lower in cells grown on glucose, than on raffinose or ethanol.     

Characteristics of bacteria showing high denitrification activity in saline wastewater

AIMS: Denitrification efficiency at 10% salinity was compared with that at 2% salinity. The characteristics of bacterial strains isolated from the denitrification system, where an improvement of denitrification efficiency was observed at a high salinity were investigated. METHODS AND RESULTS: Two continuous feeding denitrification systems for saline solutions of 2% and 10% salinity, were operated. Denitrification efficiency at 10% salinity was higher than that at 2% salinity. The bacterial strains were isolated using the trypticase soy agar (TSA) medium at 30 degrees C. The phylogenetic analysis of 16S rRNA gene sequences of isolates indicated that halophilic species were predominant at 10% salinity. CONCLUSIONS: The improvement of denitrification efficiency at a high salinity was demonstrated. The strains isolated from the denitrifying system with 10% salinity were halophilic bacteria, Halomonas sp. and Marinobacter sp., suggesting that these bacteria show a high denitrifying activity at 10% salinity. SIGNIFICANCE AND IMPACT OF THE STUDY: The long-term acclimated sludge used in this study resulted in high denitrification performance at a high salinity, indicating that the design of a high-performance denitrification system for saline wastewater will be possible.     

Characteristics and Living Patterns of Marine Myxobacterial Isolates

The growth, morphology, and life cycle of two marine myxobacterial isolates, halotolerant Myxococcus fulvus strain HW-1 and halophilic Haliangium ochraceum strain SMP-2, were studied as models to determine the living patterns of myxobacteria in the ocean. The growth, morphology, and development of halotolerant strain HW-1 shifted in response to salinity. The optimal seawater concentration for growth of HW-1 was 0 to 80% (salinity, 0.1 to 2.9%), and the strain grew poorly in media with a salinity of more than 4%. The cells became shorter as the seawater concentration increased. The fruiting body structure was complete only on agar prepared with low concentrations of seawater or salts (less than 60% seawater; salinity, 2.1%), and rudimentary structures or even simple cell mounds appeared as the seawater concentration increased. In contrast, the halophilic strain SMP-2 was unable to grow without NaCl. The cell length and the morphology of the fruiting body-like structure did not change in response to salts. In seawater liquid medium, the cells of both strains were confirmed to be able to form myxospores directly from vegetative cells, but they could not do so in medium containing a low seawater concentration (10% or less). HW-1 cells from medium containing a high concentration of seawater grew independent of cell density, while cells from medium containing a low concentration of seawater (10% or less) showed density-dependent growth. SMP-2 cells showed density-dependent growth under all salinity conditions. The results suggest that the halotolerant myxobacteria are the result of degenerative adaptation of soil myxobacteria to the marine environment, while the halophilic myxobacteria form a different evolutionary group that is indigenous to the ocean.     

Characteristics and living patterns of marine myxobacterial isolates

The growth, morphology, and life cycle of two marine myxobacterial isolates, halotolerant Myxococcus fulvus strain HW-1 and halophilic Haliangium ochraceum strain SMP-2, were studied as models to determine the living patterns of myxobacteria in the ocean. The growth, morphology, and development of halotolerant strain HW-1 shifted in response to salinity. The optimal seawater concentration for growth of HW-1 was 0 to 80% (salinity, 0.1 to 2.9%), and the strain grew poorly in media with a salinity of more than 4%. The cells became shorter as the seawater concentration increased. The fruiting body structure was complete only on agar prepared with low concentrations of seawater or salts (less than 60% seawater; salinity, 2.1%), and rudimentary structures or even simple cell mounds appeared as the seawater concentration increased. In contrast, the halophilic strain SMP-2 was unable to grow without NaCl. The cell length and the morphology of the fruiting body-like structure did not change in response to salts. In seawater liquid medium, the cells of both strains were confirmed to be able to form myxospores directly from vegetative cells, but they could not do so in medium containing a low seawater concentration (10% or less). HW-1 cells from medium containing a high concentration of seawater grew independent of cell density, while cells from medium containing a low concentration of seawater (10% or less) showed density-dependent growth. SMP-2 cells showed density-dependent growth under all salinity conditions. The results suggest that the halotolerant myxobacteria are the result of degenerative adaptation of soil myxobacteria to the marine environment, while the halophilic myxobacteria form a different evolutionary group that is indigenous to the ocean.     

Behavioural signs of estrus and their relationship to time of ovulation in Zebu (Sahiwal) cattle

The present study reports the behavioural signs of estrus, their temporal distribution and duration of expression and their relationship with the time of ovulation in zebu cattle in order to identify the reliable sign(s) of estrus that could fairly predict the ovulation time. The onset, intensity and expression of various signs of estrus were continuously recorded till ovulation in 60 Sahiwal cows. Time of ovulation was determined by ultrasound examinations at 2h interval. Estruses were mostly of moderate (52%) or weak (34%) intensity. Mucus discharge, tumefaction of vulva and reddening of vulvar mucus membrane appeared early in relation to the ovulation time (31.27±1.97, 31.05±2.98 and 30.79±2.53h, respectively) in comparison to mounting (27.67±2.33h) and standing to be mounted (25.37±2.11h). Mucus discharge, tumefaction of vulva and reddening of vulvar mucus membrane persisted significantly more duration (P<0.01) than mounting and standing to be mounted. Further these cardinal signs appeared early in relation to time of ovulation, persisted for longer duration and expressed intensely. We conclude that mucus discharge, tumefaction of vulva and reddening of vulvar mucus membrane can be good predictor ovulation in this breed of cattle.     

Diploid radiation gynogenesis in carp. I. Massive production of diploid gynogenetic progeny]

Mass lost of diploid gynogenetic carp offspring were obtained with the use of fish-farm method of reproduction. The average yield of gynogenetic diploids in usual experiments was 0.1% (from fertilized eggs). The cooling of unfertilized spawn (at the stage of metaphase II) to 8--10 degrees C during 3.5--4.5 hours in 50% of cases permitted to increase by tens of times (in the most successful experiments up to 8%) the yield of gynogenetic diploids. The rate of survival of carps remained relatively low during the first two years of life, particularly during the first hibernation that is a critical period. No specific depression of growth in gynogenetic diploid carps was observed. A high yield of gynogenetic diploids (3.9% of fertilized eggs) and their relatively high rate of survival were observed in the second generation of artificially induced gynogenesis.     

Morphology of the prairie dog gallbladder: normal characteristics and changes during early lithogenesis

Studies were undertaken to describe the normal structure of the prairie dog gallbladder and adjacent cystic duct, and then to determine sequential changes that occurred as abnormalities in bile composition developed during high cholesterol feeding. Control animals were fed a diet with trace cholesterol, while experimental animals were fed a diet enriched with 1.2% cholesterol for 1, 2, 3, or 4 weeks. Light microscopy and scanning and transmission electron microscopy were used to characterize morphologic changes at each time interval. Biliary lipid composition was altered in all experimental groups, evidenced by significant decreases in bile-acid-to-cholesterol ratios. Cholesterol crystals appeared in experimental bile at 1 and 2 weeks, while stones formed at 3 and 4 weeks. The cystic duct and neck of the gallbladder occasionally displayed goblet cells. Little mucus was demonstrable in principal cells of the gallbladder, but much more in those lining the cystic duct. After 2 weeks of lithogenic diet, there was an increase in mucus content and secretion from all areas, as well as an influx of polymorphonuclear and mononuclear leukocytes. Accumulation of plasma cells in the lamina propria was an especially prominent feature of experimental tissues. These results suggest that 1) there is regional heterogeneity in the mucus content of the gallbladder and cystic duct of the prairie dog, and 2) both regions respond to lithogenesis with mucus hypersecretion and acute and chronic inflammatory changes prior to the appearance of cholesterol gallstones.     

Influence of salts and sodium chloride on the recovery ofEscherichia coli from seawater

The objective of this study was to evaluate the influence of seawater salts, and more specially sodium chloride, on the recovery ofEscherichia coli cells after exposure to natural seawater in laboratory microcosms, and the possible adaptation in this bacterium to high salinity. The recovery efficiency of a complex organic medium supplemented with sodium chloride largely depended on the strain and varied with starvation time and salinity. Moreover, cells previously grown on salted medium appeared more able to survive after exposure to seawater. It is assumed that, withinE. coli populations, some cells are able to adapt to seawater in the presence of both salts and organic matter.     

Isolation and characterization of glycoproteins from canine tracheal pouch secretions.

Canine tracheal pouch secretions were solubilized with 1% sodium dodecyl sulfate and visualized by sodium dodecyl sulfate-agarose-acrylamide gel electrophoresis. Intact mucus, and water-soluble and insoluble fractions of mucus were shown to be composed of high molecular weight glycoproteins (Mr greater than or equal to 3 . 10(6)) and three major classes of proteins of lower molecular weight (Mr approximately 4 . 10(5), 2 . 10(5), and 6 . 10(4)). When the mucus secretions were further treated with a reducing agent, the glycoproteins were dissociated into subunits which appeared on the gel as three discrete bands. Separation of the high molecular weight glycoproteins from the other proteins was achieved by gel filtration on Biogel A-15m in the presence of 1% dodecyl sulfate following reduction and alkylation of mucus. These glycoproteins were further resolved, using DEAE cellulose chromatography in the presence of 6 M urea, into two protein fractions. Both fractions contained approximately 87% carbohydrate, high amounts of serine and threonine but differed significantly in contents of N-acetyl glucosamine and sialic acid; their mobility on gel electrophoresis was also different. Significant contents of cysteine were noted in both fractions. Results of this study indicate that the canine tracheal pouch preparations provide normal tracheal secretions which bear similarity in structure to the tracheobronchial secretions obtained from human patients.     

In vitro response of Ichthyophthirius multifiliis to sera from immune channel catfish

Sera from channel catfish rendered immune to the protozoan pathogen Ichthyophthirius multifiliis were screened for activity against live parasites. Cells in the infective stage (tomites) were incubated in doubling dilutions of immune and pre-immune sera from fish that had been immunized by exposure to sublethal infections. When examined by light microscopy, tomites were found to agglutinate in the presence of immune sera. While the stength of individual sera varied, agglutination of cells occurred at dilutions as high as 1:128. Cells showed little tendency to agglutinate in pre-immune sera, and virtually no effects were seen with dilutions of pre-immune sera greater than 1:16. Agglutination was usually accompanied by release of mucus from cells, and while tomites appeared to be immobilized, their cilia continued to beat. Low dilutions of immune sera appeared to be toxic. Similar effects on tomites were seen with rabbit antisera prepared against Ichthyophthirius cilia. The involvement of humoral antibodies in agglutination and protective immunity is discussed.     

盐度对异育银鲫呼吸和氨氮排泄生理的影响

研究了异育银鲫(Carassius auratus gibeliovar. E′erqisi)从淡水向盐度1.5‰、3‰、6‰、9‰、12‰突变与适应过程中的呼吸和氨氮排泄生理的变化规律。结果表明:盐度突变开始时,异育银鲫的耗氧率和排氨率随外界盐度的升高而增大,随盐度作用时间延长,耗氧率和排氨率升到最大值后又开始下降并最终维持在稳定水平,但开始下降的时间和下降的幅度以及最终的稳定水平与外界盐度有关。盐度1.5‰和3‰处理组在作用12h时耗氧率升到最大值,此后下降,于第3天后保持在比淡水对照组略低的稳定水平,但二者差异不显著(P0.05);盐度6‰和9‰处理组在作用到第3天后才开始缓慢下降,并分别于第10、15天时保持在显著高于淡水对照的稳定水平(P0.05);盐度12‰处理组也在第3天后下降,到第10天后保持在比淡水对照组略低的稳定水平,但二者差异不显著(P0.05)。各盐度处理组的排氨率均在盐度作用24h时达到最大值,其中盐度1.5‰处理组的变化不显著(P0.05),其他各组均显著升高(P0.05),并都于第10天时下降到稳定水平,其中盐度3‰处理组的稳定水平略低于对照组(P0.05),盐度6‰、9‰、12‰处理组的稳定水平显著高于对照组(P0.05)。     

In situ study on effect of food competition on diet shifts and growth of silver and bighead carps in large biomanipulation fish pens in Meiliang Bay, Lake Taihu

Silver and bighead carps were cultured in large fish pens to reduce the risks of cyanobacterial bloom outbreaks in Meiliang Bay, Lake Tauhu in 2004 and 2005. Diet compositions and growth rates of the carps were studied from April to November each year. Both carp species fed mainly on zooplankton (>50% in diet) in 2004 when competition was low, but selected more phytoplankton in 2005 when competition was high. Silver carp had a broader diet breadth than did bighead carp. Higher densities and fewer food resources increased diet breadths but decreased the diet overlap in both types of carps. It can be predicted that silver and bighead carps would be released from diet competition and shift to feed mainly on zooplankton at low densities, decreasing the efficiency of controlling cyanobacterial blooms. Conclusively, when silver and bighead carps are used to control cyanobacterial blooms, a sufficiently high stocking density is very important for a successful practice.     

Survival of Vibrio anguillarum and Vibrio salmonicida at different salinities

The fish pathogenic bacteria Vibrio anguillarum and V. salmonicida showed the capacity to survive for more than 50 and 14 months, respectively, in seawater microcosms. A salinity of 5% proved lethal to V. anguillarum harvested in the late-exponential growth phase, whereas a salinity of 9% was lethal to the bacterium after it had been starved at a salinity of 30% for 67 days. The lethal salinity for V. salmonicida harvested in the late-exponential growth phase was probably in the vicinity of 10%. V. anguillarum and V. salmonicida were very sensitive to nalidixic acid. Direct determination of viable cells after incubation with nalidixic acid was not possible, since the cells did not elongate. Samples of V. salmonicida were double stained with fluorescein isothiocyanate-labeled antibodies and 4',6-diamidino-2-phenylindole. After 3 or 4 days of starvation, there was a discrepancy between the total numbers of cells as determined by immunofluorescence versus by staining with 4',6-diamidino-2-phenylindole. The immunofluorescence counts remained high, which indicated the presence of intact cell envelopes but leakage of DNA and other cytoplasm components. After 2 weeks of starvation, for some of the cells, the region stained with 4',6-diamidino-2-phenylindole (i.e., DNA) was markedly smaller than the cell envelope. I attributed this to a shrinkage of the cytoplasm or a confined nucleoid or both. V. anguillarum lost its exoproteolytic activity before 11 days of starvation.     

Survival of Vibrio anguillarum and Vibrio salmonicida at different salinities.

The fish pathogenic bacteria Vibrio anguillarum and V. salmonicida showed the capacity to survive for more than 50 and 14 months, respectively, in seawater microcosms. A salinity of 5% proved lethal to V. anguillarum harvested in the late-exponential growth phase, whereas a salinity of 9% was lethal to the bacterium after it had been starved at a salinity of 30% for 67 days. The lethal salinity for V. salmonicida harvested in the late-exponential growth phase was probably in the vicinity of 10%. V. anguillarum and V. salmonicida were very sensitive to nalidixic acid. Direct determination of viable cells after incubation with nalidixic acid was not possible, since the cells did not elongate. Samples of V. salmonicida were double stained with fluorescein isothiocyanate-labeled antibodies and 4',6-diamidino-2-phenylindole. After 3 or 4 days of starvation, there was a discrepancy between the total numbers of cells as determined by immunofluorescence versus by staining with 4',6-diamidino-2-phenylindole. The immunofluorescence counts remained high, which indicated the presence of intact cell envelopes but leakage of DNA and other cytoplasm components. After 2 weeks of starvation, for some of the cells, the region stained with 4',6-diamidino-2-phenylindole (i.e., DNA) was markedly smaller than the cell envelope. I attributed this to a shrinkage of the cytoplasm or a confined nucleoid or both. V. anguillarum lost its exoproteolytic activity before 11 days of starvation.     

Kinetics of adhesion of selected fish-pathogenic Vibrio strains of skin mucus of gilt-head sea bream (Sparus aurata L.).

The kinetics of adhesion of Vibrio strains isolated from diseased fish to skin mucus of gilt-head sea bream was studied. A modified Langmuir adsorption isotherm was calculated, and the results obtained indicate that the strains tested (Vibrio alginolyticus DP1HE4 and Vibrio anguillarum-like DC12R8 and DC12R9) showed a saturation kinetics except for V. alginolyticus (CAN), which showed a proportional adsorption kinetics. The adhesive capability for skin mucus does not seem to be an essential virulence factor of pathogenic strains of Vibrio, since this specific interaction depended on several environmental factors, temperature and salinity being the most important. However, the absence of an inhibitory effect of mucus on the pathogenic microorganisms, and the capability of the Vibrio strains to utilize mucus as a carbon source, could favor their settlement on the skin with a potential for infection of cultured, stressed fish.