Genetic Characterization of Vibrio vulnificus Strains from Tilapia Aquaculture in Bangladesh

Outbreaks of Vibrio vulnificus wound infections in Israel were previously attributed to tilapia aquaculture. In this study, V. vulnificus was frequently isolated from coastal but not freshwater aquaculture in Bangladesh. Phylogenetic analyses showed that strains from Bangladesh differed remarkably from isolates commonly recovered elsewhere from fish or oysters and were more closely related to strains of clinical origin.Vibrio vulnificus causes severe wound infections and life-threatening septicemia (mortality, >50%), primarily in patients with underlying chronic diseases (10, 19, 23) and primarily from raw oyster consumption (21). This Gram-negative halophile is readily recovered from oysters (27, 35, 43) and fish (14) and was initially classified into two biotypes (BTs) based on growth characteristics and serology (5, 18, 39). Most human isolates are BT1, while BT2 is usually associated with diseased eels (1, 39). An outbreak of wound infections from aquacultured tilapia in Israel (6) revealed a new biotype (BT3). Phenotypic assays do not consistently distinguish biotypes (33), but genetic analyses have helped resolve relationships (20). A 10-locus multilocus sequence typing (MLST) scheme (8, 9) and a similar analysis of 6 loci (13) segregated V. vulnificus strains into two clusters. BT1 strains were in both clusters, while BT2 segregated into a single cluster and BT3 was a genetic mosaic of the two lineages. Significant associations were observed between MLST clusters and strain origin: most clinical strains (BT1) were in one cluster, and the other cluster was comprised mostly of environmental strains (some BT1 and all BT2). Clinical isolates were also associated with a unique genomic island (13).The relationship between genetic lineages and virulence has not been determined, and confirmed virulence genes are universally present in V. vulnificus strains from both clinical and environmental origins (19, 23). However, segregation of several polymorphic alleles agreed with the MLST analysis and correlated genotype with either clinical or environmental strain origin. Alleles include 16S rRNA loci (15, 26, 42), a virulence-correlated gene (vcg) locus (31, 41, 42), and repetitive sequence in the CPS operon (12). DiversiLab repetitive extrageneic palindromic (rep-PCR) analysis also confirmed these genetic distinctions and showed greater diversity among clinical strains (12).Wound infections associated with tilapia in Israel implicated aquaculture as a potential source of V. vulnificus in human disease (6, 40). Tilapia aquaculture is increasing rapidly, as shown by a 2.8-fold increase in tons produced from 1998 to 2007 (Food and Agriculture Organization; http://www.fao.org/fishery/statistics/en). Therefore, presence of V. vulnificus in tilapia aquaculture was examined in Bangladesh, a region that supports both coastal and freshwater sources of industrial-scale aquaculture. V. vulnificus strains were recovered from market fish, netted fish, and water samples, and the phylogenetic relationship among strains was examined relative to clinical and environmental reference strains collected elsewhere.     

Influence of the nucleoid and the early stages of DNA replication on positioning the division site in Bacillus subtilis

Although division site positioning in rod‐shaped bacteria is generally believed to occur through the combined effect of nucleoid occlusion and the Min system, several lines of evidence suggest the existence of additional mechanisms. Studies using outgrown spores of Bacillus subtilis have shown that inhibiting the early stages of DNA replication, leading up to assembly of the replisome at oriC, influences Z ring positioning. Here we examine whether Z ring formation at midcell under various conditions of DNA replication inhibition is solely the result of relief of nucleoid occlusion. We show that midcell Z rings form preferentially over unreplicated nucleoids that have a bilobed morphology (lowering DNA concentration at midcell), whereas acentral Z rings form beside a single‐lobed nucleoid. Remarkably however, when the DnaB replication initiation protein is inactivated midcell Z rings never form over bilobed nucleoids. Relieving nucleoid occlusion by deleting noc increased midcell Z ring frequency for all situations of DNA replication inhibition, however not to the same extent, with the DnaB‐inactivated strain having the lowest frequency of midcell Z rings. We propose an additional mechanism for Z ring positioning in which the division site becomes increasingly potentiated for Z ring formation as initiation of replication is progressively completed.     

Reactive oxygen species damaged human serum albumin in patients with type 1 diabetes mellitus: biochemical and immunological studies

The role of hydroxyl radical (.OH) damaged human serum albumin (HSA) in type 1 diabetes has been investigated in the present study. Hydroxyl radical induced modification on HSA has been studied by UV absorption spectroscopy, ANS fluorescence and carbonyl estimation. Hydroxyl radical modified HSA was found to be highly immunogenic in rabbits as compared to native HSA. The binding characteristics of circulating autoantibodies in type 1 diabetes patients against native and modified HSA were assessed. Diabetes patients (n=31) were examined by direct binding ELISA and the results were compared with healthy age-matched controls (n=22). High degree of specific binding by 54.8% of patients sera towards .OH modified HSA, in comparison to its native analogue (p<0.05) was observed. Sera from those type 1 diabetes patients having smoking history, high aging with high degree of disease showed substantially stronger binding to .OH modified HSA over native HSA in particular. Normal human sera showed negligible binding with either antigen. Competitive inhibition ELISA reiterates the direct binding results. Gel retardation assay further substantiated the enhanced recognition of modified HSA by circulating autoantibodies in diabetes patients. The increase in total serum protein carbonyl levels in the diabetes patients was largely due to an increase in oxidized albumin. HSA of diabetes mellitus patients (DM-HSA) and normal subjects (normal-HSA) were purified on a Sephacryl S-200 HR column. Spectroscopic analysis confirmed that the DM-HSA samples contained higher levels of carbonyls than normal-HSA (p<0.001). DM-HSA was conformationally altered, with more exposure of its hydrophobic regions. Collectively, the oxidation of plasma proteins, especially HSA, might enhance oxidative stress in type 1 diabetes mellitus patients.     

The influence of glucose and other sources of carbon on nitrate reduction rates in two temperate forest soils

The effects of different sources of organic carbon on the potential NO ₃ ⁻ reduction rates in an acid mull and an anmoor (anmoor is a type of soil which is rich in organic matter, remains water saturated most of the time and where organic and mineral fractions are closely associated) were studied under anaerobic incubation. The disappearance of NO ₃ ⁻ was higher in the anmoor than in the mull in all cases. This contradicts our previous findings where the apparent denitrification was higher in the mull than in the anmoor in the presence of added glucose. This seems to indicate that drying of soil samples might have caused the formation of stable soil aggregates in the case of anmoor (containing 16% of organic matter). Thus a favourable condition was created for securing improved diffusion of nutrients in the anmoor. Glucose, a readily available source of carbon, led to a very high degree of NO ₃ ⁻ disappearance. Among other sources, the cellulose caused higher reduction of NO ₃ ⁻ than litter and lignin. The accumulation although small, of NH ₄ ⁺ which was presumably formed (at least partially) during dissimilatory reduction of NO ₃ ⁻ , and its biological immobilization were more apparent in the anmoor than in the mull.     

The effect of weak auxins on the growth of blue-green algae

Summary 5-Hydroxy indole-3-acetic acid promoted the growth (increase in dry weight) of Anacystis nidulans, Chlorogloea fritschii, Phormidium foveolarum, Nostoc muscorum and Tolypothrix tenuis. 5-Hydroxy tryptamine stimulated the growth of Chlorogloea fritschii and Nostoc muscorum. Phenyl-acetic acid promoted the growth of Nostoc muscorum and Tolypothrix tenuis. Tryptophol stimulated the growth of Chlorogloea fritschii, it failed to stimulate the growth of Nostoc muscorum. Isatin promoted the growth of Anacystis nidulans and Chlorogloea fritschii 2, 3, 5-triidobenzoic acid inhibited the growth of Anacystis nidulans, Chlorogloea fritschii, Phormidium foveolarum, Nostoc muscorum and Tolypothrix tenuis.     

Food selection by Labeo Rohita (Ham.) and its feeding relationship with other major carps

Selectivity of food byLabeo rohita (Ham.) was studied in a stocking pond (Moat), by calculating an Electivity index (E) for each food organism as described byIvlev (1961). It was found thatL. rohita was definitely selective in its feeding. In case of fingerlings, there was a strong selection for zooplanktonic organisms (Arcella andDifflugia among protozoans,Keratella andBrachionus among rotifer andDaphnia andCyclops among crustaceans) and smaller algae (Cosmarium andClosterium among desmisd,Euglena andVolvox among phytoflagellates and algal spores and zygotes) while most of the phytoplanktonic organisms, belonging to green algae, diatoms and blue green algae, were avoided. In case of adults, a strong negative selection was observed for all zooplanktonic organisms and a strong positive selection for most of the green algae and diatoms (Ankistrodesmus, Zygnema, Spirogyra, Selenastrum, Pediastrum, Scenedesmus, Tetraspora, Stephanodiscus, Naviculla, Diatoma, Synedra andNitzchia). However, all blue green algae were avoided.The feeding relationship ofL. rohita with other major carps,Cirrhina mrigala (Ham.) andCatla catla (Ham.) was studied in two different habitats, pond and river. It was observed that in both habitats all three species were found to feed on almost similar types of food organisms, but the quantity of any food item eaten by adults differed markedly from one species to another and the food items which were dominant in one species, were of secondary importancce for the other two species. The adults ofL. rohita were found to feed mainly on phytoplankton and macrovegetation, the main food of adultC. mrigala was decayed organic matter, sand and mud supplimented by plankton, while the food of adultC. catla was chiefly composed of zooplankton, and some phytoplankton. Hence there was no true identity of feeding habits between the adults of any two species. However, there was an indication of competition for food between the fingerlings of all three species, because all of them feed mainly on zooplankton (Crustaceans, rotifers and protozoans). However, such feeding habits lasted a very short time only and as the fishes grew, their feeding habits diverged.     

Ultrastructure of embryogenic pollen ofNicotiana tabacum var. Badischer burley

Summary Pollen grains capable of embryogenesis were selectively isolated from (a) near-mature buds from plants induced to flower in short days and low temperature (8 hours light and 18 °C) and (b) young buds from these plants with an additional low temperature treatment (10 °C for 10 days) and fixed for electron microscopy. The pollen from the former formed embryos at a very low frequency in culture, and at the subcellular level showed different degrees of regression of cytoplasm and mitochondria. On the contrary, cold-treated pollen were characterized by a high frequency of embryogenesis, up to 25% of the cultured pollen. They did not show regression of cytoplasm or organelles but had an attenuated cytoplasm which was not rich in ribosomes. Another noteworthy feature of embryogenic grains was the condensed nature of mitochondria. These characteristics of embryogenic grains indicate that they are repressed for gametophytic differentiation. The embryogenic pollen did not differentiate from gametophytic pollen which were very distinctive, having a thick exine, and dense cytoplasm rich in ribosomes. The close similarity of embryogenic grains with young microspores in terms of thin exine and sparse cytoplasm is suggestive of an indeterminate state and that determination into gametophytic or sporophytic (embryogenic) type is probably the function of differential gene activity. Of interest, in this context, is the condensation of mitochondria in embryogenic grains. The relationship, if any, between mitochondrial condensation and embryogenesis remains to be resolved.     

Development of gametophores from isolated protoplasts of the mossAnoectangium thomsvnii Mitt.

Summary Protoplasts of the mossAnoectangium thomsonii Mitt. were isolated from preplasmolyzed protonemal filaments, grown in suspension cultures, after the digestion of the cell wall by the enzymes cellulase and macerozyme or driselase. Driselase was more effective than cellulase and macerozyme. After purification these protoplasts were plated in the form of small agar drops in modified Kofler's medium without hormones and incubated in the dark at 26 ± 2 °C. Cell walls regenerated within three days and cell divisions started seven days after the initiation of the cultures. When the regenerants were transferred to normal protonemal culture medium and illuminated by 3,000 lux continuous light, a multi-cellular protonema developed which formed leafy gametophores on salicylic acid supplemented medium.