Physical and structural characterization of yersiniophore, a siderophore produced by clinical isolates of Yersinia enterocolitica

Clinical isolates of Yersinia enterocolitca, which belong to mouse-lethal serotypes, produce the siderophore yersiniophore. Siderophore production was shown to be iron regulated and to reach maximum production in late log phase. Yersiniophore is a fluorescent siderophore with maximum excitation at 270 nm and a major emission peak at 428 nm. Absorption maxima were seen at 210 and 250 nm with a low broad peak from 280 to 320 nm. Purification of unchelated yersiniophore for structural analysis was made difficult by low yields (1–2 mg mg^-1), and susceptibility to acid hydrolysis, oxidation and possibly polymerization. Yersinophore was therefore purified as an Al³⁺ chelate, which was found to be stable in solution for several weeks. To purify Al³⁺-yersiniophore, unchelated yersiniophore was first extracted from culture supernatants with dichloromethane, concentrated by rotary evaporation and adsorbed to a DEAE-sephacel column. Al³⁺-yersiniophore was eluted with 0.01 m AlCl₃ and further purified by HPLC. The structure was established by a combination of elemental analysis, high resolution mass spectrometry and two-dimensional NMR experiments. Yersiniophore is a phenolate-thiazole siderophore with the formula C₂₁H₂₄N₃O₄S₃Al and a molecular weight of 505.07404 when chelated to Al³⁺. The structure of yersiniophore was determined to be closely related to the structures of pyochelin, produced by Pseudomonas aeruginosa, and anguibactin, produced by Vibrio anguillarum.     

Dietary use and conservation concern of edible wetland plants at indo-burma hotspot: a case study from northeast India

Background

The wetlands of the North East India fall among the global hotspots of biodiversity. However, they have received very little attention with relation to their intrinsic values to human kind; therefore their conservation is hardly addressed. These wetlands are critical for the sustenance of the tribal communities.

Methods

Field research was conducted during 2003 to 2006 in seven major wetlands of four districts of Manipur state, Northeast India (viz. Imphal-East, Imphal-West, Thoubal, and Bishnupur). A total of 224 wetland-plant-collectors were interviewed for the use and economics of species using semi-structured questionnaires and interview schedules. Imphal, Bishenpur and Thoubal markets were investigated in detail for influx and consumption pattern of these plants. The collectors were also inquired for medicinal use of wetland species. Nutritive values of 21 species were analyzed in laboratory. The vouchers were collected for all the species and deposited in the CSIR-NEIST (Formerly Regional Research Laboratory), Substation, Lamphelpat, Imphal, Manipur, India.

Results

We recorded 51 edible wetland species used by indigenous people for food and medicinal purposes. Thirty eight species had high medicinal values and used in the traditional system to treat over 22 diseases. At least 27 species were traded in three markets studied (i.e. Imphal, Thoubal and Bishenpur), involving an annual turnover of 113 tons of wetland edible plants and a gross revenue of Rs. 907, 770/- (US$1 = Rs. 45/-). The Imphal market alone supplies 60% of the total business. Eighty per cent of the above mentioned species are very often used by the community. The community has a general opinion that the availability of 45% species has depleted in recent times, 15 species need consideration for conservation while another 7 species deserved immediate protection measures. The nutrient analysis showed that these species contribute to the dietary balance of tribal communities.

Conclusions

Considering the importance of wild wetland plants in local sustenance, it is suggested to protect their habitats, develop domestication protocols of selected species, and build programs for the long-term management of wetland areas by involving local people. Some medicinal plants may also be used to develop into modern medicines.

     

High-resolution recombination patterns in a region of human chromosome 21 measured by sperm typing

For decades, classical crossover studies and linkage disequilibrium (LD) analysis of genomic regions suggested that human meiotic crossovers may not be randomly distributed along chromosomes but are focused instead in "hot spots." Recent sperm typing studies provided data at very high resolution and accuracy that defined the physical limits of a number of hot spots. The data were also used to test whether patterns of LD can predict hot spot locations. These sperm typing studies focused on several small regions of the genome already known or suspected of containing a hot spot based on the presence of LD breakdown or previous experimental evidence of hot spot activity. Comparable data on target regions not specifically chosen using these two criteria is lacking but is needed to make an unbiased test of whether LD data alone can accurately predict active hot spots. We used sperm typing to estimate recombination in 17 almost contiguous ~5 kb intervals spanning 103 kb of human Chromosome 21. We found two intervals that contained new hot spots. The comparison of our data with recombination rates predicted by statistical analyses of LD showed that, overall, the two datasets corresponded well, except for one predicted hot spot that showed little crossing over. This study doubles the experimental data on recombination in men at the highest resolution and accuracy and supports the emerging genome-wide picture that recombination is localized in small regions separated by cold areas. Detailed study of one of the new hot spots revealed a sperm donor with a decrease in recombination intensity at the canonical recombination site but an increase in crossover activity nearby. This unique finding suggests that the position and intensity of hot spots may evolve by means of a concerted mechanism that maintains the overall recombination intensity in the region.     

Influence of Iron on Yields of Extracellular Products in Pseudomonas aeruginosa Cultures

The effect of the iron content of the medium on the yields of extracellular products by seven distinct strains of Pseudomonas aeruginosa was examined. All strains showed at least an 85% decrease in toxin A yields when grown in medium containing 5.0 mug of iron per ml (high iron) as compared to 0.05 mug/ml (low iron), whereas bacterial growth increased approximately twofold. During the course of examining extracellular products produced by P. aeruginosa, we found many strains that produced an extracellular factor which agglutinated erythrocytes. This hemagglutinin was nondialyzable, heat stable, and resistant to Pronase and trypsin. The effect of iron on extracellular yields of hemagglutinin was strain dependent; four of seven strains showed decreases in hemagglutinin yields in high-iron medium. Similarly, the effect of increasing the iron concentration of the growth medium on yields of total extracellular proteases or on elastase was strain dependent. The amount of total extracellular protein was decreased by at least 31% in the high-iron medium for all strains of P. aeruginosa examined. Detailed studies on one strain (WR-9) showed that, in the presence of increasing amounts of iron in the medium, the extracellular yields of toxin A, protease, and hemagglutinin were decreased in a similar manner. In addition, the kinetics of release of these extracellular products were similar at a given iron concentration. Thus it appears that the yields of other extracellular products of P. aeruginosa besides toxin A are influenced by the concentration of iron in the growth medium.     

Lenalidomide Induces Lipid Raft Assembly to Enhance Erythropoietin Receptor Signaling in Myelodysplastic Syndrome Progenitors

Anemia remains the principal management challenge for patients with lower risk Myelodysplastic Syndromes (MDS). Despite appropriate cytokine production and cellular receptor display, erythropoietin receptor (EpoR) signaling is impaired. We reported that EpoR signaling is dependent upon receptor localization within lipid raft microdomains, and that disruption of raft integrity abolishes signaling capacity. Here, we show that MDS erythroid progenitors display markedly diminished raft assembly and smaller raft aggregates compared to normal controls (p = 0.005, raft number; p = 0.023, raft size). Because lenalidomide triggers raft coalescence in T-lymphocytes promoting immune synapse formation, we assessed effects of lenalidomide on raft assembly in MDS erythroid precursors and UT7 cells. Lenalidomide treatment rapidly induced lipid raft formation accompanied by EpoR recruitment into raft fractions together with STAT5, JAK2, and Lyn kinase. The JAK2 phosphatase, CD45, a key negative regulator of EpoR signaling, was displaced from raft fractions. Lenalidomide treatment prior to Epo stimulation enhanced both JAK2 and STAT5 phosphorylation in UT7 and primary MDS erythroid progenitors, accompanied by increased STAT5 DNA binding in UT7 cells, and increased erythroid colony forming capacity in both UT7 and primary cells. Raft induction was associated with F-actin polymerization, which was blocked by Rho kinase inhibition. These data indicate that deficient raft integrity impairs EpoR signaling, and provides a novel strategy to enhance EpoR signal fidelity in non-del(5q) MDS.     

Oral administration of probiotic bacteria (E. coli nissle,E. coli O83,Lactobacillus casei) influences the severity of dextran sodium sulfate-induced colitis in BALB/c mice

Our study examined whether repeated preventive oral administration of live probiotic bacterial strains Escherichia coli O83:K24:H31 (Ec O83), Escherichia coli Nissle 1917 O6:K5:H1 (Ec Nis) and Lactobacillus casei DN 114001 (Lc) can protect mice against dextran sodium sulfate (DSS)-induced colitis. A significant decrease in average symptom score was observed in Ec O83-, Ec Nis- and Lc-pretreated group (p < 0.05). Significant differences in body mass loss between Lc pretreated mice with DSS-induced colitis were found when compared with nontreated mice (p < 0.05). PBS pretreated mice had a significantly shorter colon than Ec O83-, Ec Nis- and Lc-pretreated mice (p < 0.05). Administration of Lc significantly decreased the severity of DSS induced histological marks of inflammation (p < 0.05). A significant difference (p < 0.05) was also found in specific IgA level against given probiotic in enteral fluid between colitic mice and healthy mice pretreated with Ec 083 and Ec Nis.     

An examination of the electrostatic interactions between the N-terminal tail of the Brome Mosaic Virus coat protein and encapsidated RNAs

The coat protein of positive-stranded RNA viruses often contains a positively charged tail that extends toward the center of the capsid and interacts with the viral genome. Electrostatic interaction between the tail and the RNA has been postulated as a major force in virus assembly and stabilization. The goal of this work is to examine the correlation between electrostatic interaction and amount of RNA packaged in the tripartite Brome Mosaic Virus (BMV). Nanoindentation experiment using atomic force microscopy showed that the stiffness of BMV virions with different RNAs varied by a range that is 10-fold higher than that would be predicted by electrostatics. BMV mutants with decreased positive charges encapsidated lower amounts of RNA while mutants with increased positive charges packaged additional RNAs up to ～900 nt. However, the extra RNAs included truncated BMV RNAs, an additional copy of RNA4, potential cellular RNAs, or a combination of the three, indicating that change in the charge of the capsid could result in several different outcomes in RNA encapsidation. In addition, mutant with specific arginines changed to lysines in the capsid also exhibited defects in the specific encapsidation of BMV RNA4. The experimental results indicate that electrostatics is a major component in RNA encapsidation but was unable to account for all of the observed effects on RNA encapsidation. Thermodynamic modeling incorporating the electrostatics was able to predict the approximate length of the RNA to be encapsidated for the majority of mutant virions, but not for a mutant with extreme clustered positive charges. Cryo-electron microscopy of virions that encapsidated an additional copy of RNA4 revealed that, despite the increase in RNA encapsidated, the capsid structure was minimally changed. These results experimentally demonstrated the impact of electrostatics and additional restraints in the encapsidation of BMV RNAs, which could be applicable to other viruses.     

Systematic analysis of the physiological importance of deubiquitinating enzymes

Deubiquitinating enzymes (DUBs) are proteases that control the post-translational modification of proteins by ubiquitin and in turn regulate diverse cellular pathways. Despite a growing understanding of DUB biology at the structural and molecular level, little is known about the physiological importance of most DUBs. Here, we systematically identify DUBs encoded by the genome of Drosophila melanogaster and examine their physiological importance in vivo. Through domain analyses we uncovered 41 Drosophila DUBs, most of which have human orthologues. Systematic knockdown of the vast majority of DUBs throughout the fly or in specific cell types had dramatic consequences for Drosophila development, adult motility or longevity. Specific DUB subclasses proved to be particularly necessary during development, while others were important in adults. Several DUBs were indispensable in neurons or glial cells during developmental stages; knockdown of others perturbed the homeostasis of ubiquitinated proteins in adult flies, or had adverse effects on wing positioning as a result of neuronal requirements. We demonstrate the physiological significance of the DUB family of enzymes in intact animals, find that there is little functional redundancy among members of this family of proteases, and provide insight for future investigations to understand DUB biology at the molecular, cellular and organismal levels.