Morphological changes in an acidophilic bacterium induced by heavy metals

The Acidiphilium strains inhabit acidic mine regions where they are subjected to occasional environmental stresses such as high and low temperatures, exposure to various heavy metals, etc. Change in morphology is one of the strategies that bacteria adopt to cope with environmental stresses; however, no study on this aspect has been reported in the case of Acidiphilium sp. This work is an attempt using the acidophilic heterotrophic bacterium Acidiphilium symbioticum H8. It was observed that the maximum alterations in size occurred when the bacterium was exposed to sub-inhibitory concentrations of Cu and Cd. Loosely packed coccobacillus-type normal cells formed characteristic chains of coccoidal lenticular shape with constrictions at the junctions between them in the presence of Cd; Cu induced transformation of cells to become round shaped; Ni caused the cells to aggregate, but Zn showed no effect. Respective metal depositions on the cell surface were confirmed by scanning electron microscopy equipped with energy dispersive X-ray analysis. Cell bound Ca²⁺ ions were replaced by these metal ions and measured by inductively coupled plasma mass spectrometry from the culture filtrate. Cell shape changed only after the addition of sub-inhibitory concentrations of the metals, but in growth inhibitory concentrations it was similar to the normal cells.     

Backbone dynamics of apocytochrome b5 in its native, partially folded state

The backbone dynamics in the native state of apocytochrome b5 were studied using 15N nuclear magnetic spin relaxation measurements. The field (11.7 and 14.1 T) and temperature (10-25 degrees C) dependence of the relaxation parameters (R1, R2, and R1rho) and the 1H-15N NOE established that the protein undergoes multiple time scale internal motions related to the secondary structure. The relaxation data were analyzed with the reduced spectral density mapping approach and within the extended model-free framework. The apoprotein was confirmed to contain a disordered heme-binding loop of approximately 30 residues with dynamics on the sub-nanosecond time scale (0.6 < S2 < 0.7, 100 ps < taue < 500 ps). This loop is attached to a structured hydrophobic core, rigid on the picosecond time scale (S2 > 0.75, taue < 50 ps). The inability to fit the data for several residues with the model-free protocol revealed the presence of correlated motion. An exchange contribution was detected in the transverse relaxation rate (R2) of all residues. The differential temperature response of R2 along the backbone supported slower exchange rates for residues in the loop (tauex > 300 micros) than for the folded polypeptide chain (tauex < 150 micros). The distribution of the reduced spectral densities at the 1H and 15N frequencies followed the dynamic trend and predicted the slowing of the internal motions at 10 degrees C. Comparison of the dynamics with those of the holoprotein [Dangi, B., Sarma, S., Yan, C., Banville, D. L., and Guiles, R. D. (1998) Biochemistry 37, 8289-8302] demonstrated that binding of the heme alters the time scale of motions both in the heme-binding loop and in the structured hydrophobic core.     

QSAR study on some pyridoacridine ascididemin analogues as anti-tumor agents

Pyridoacridine ascididemin analogues have been reported as anticancer agents for their interesting antitumor activity against human cancer cells. A quantitative structure–activity relationship (QSAR) analysis of ascididemin analogues was attempted using the physicochemical parameters and the electrotopological state atom (ETSA) indices. This study indicates that the electron withdrawing substituents with higher MR (molar refractivity) value at R₁ position favor the anti-tumor activity and the presence of NHR (R is hydrogen or alkyl group) at the R₃ position has contribution to the anti-tumor activity. ETSA indices have been incorporated as independent variable in the QSAR model with physicochemical parameters. It clearly suggests the importance of atoms 2, 3, 4, 5, 6 and 7 to the anti-tumor activity.     

Metabolic burden as reflected by maintenance coefficient of recombinant Escherichia coli overexpressing target gene

Metabolic burden as a consequence of overexpression of target gene in a recombinant strain of E. coli 1727 has been analyzed with respect to maintenance energy coefficient (m). The values of m for the host, uninduced recombinant and IPTG induced recombinant were determined to be 0.12, 0.17 and 0.32 g.g^-1.h^-1 respectively. Transient plasmid instability and nearly 33% fall in maximum specific growth rate were observed under conditions of enhanced requirements for maintenance energy.     

A Comparison of Exogenous Promoter Activity at the ROSA26 Locus Using a PhiC31 Integrase Mediated Cassette Exchange Approach in Mouse ES Cells

The activities of nine ubiquitous promoters (ROSA26, CAG, CMV, CMVd1, UbC, EF1α, PGK, chicken β-actin and MC1) have been quantified and compared in mouse embryonic stem cells. To avoid the high variation in transgene expression which results from uncontrolled copy number and chromosomal position effects when using random insertion based transgenic approaches, we have adopted a PhiC31 integrase mediated cassette exchange method for the efficient insertion of transgenes at single copy within a defined and well characterized chromosomal position, ROSA26. This has enabled the direct comparison of constructs from within the same genomic context and allows a systematic and quantitative assessment of the strengths of the promoters in comparison with the endogenous ROSA26 promoter. The behavior of these exogenous promoters, when integrated at ROSA26 in both sense and antisense orientations, reveals a large variation in their levels of activity. In addition, a subset of promoters, EF1α, UbC and CAG, show an increased activity in the sense orientation as a consequence of integration. Transient transfection experiments confirmed these observations to reflect integration dependent effects and also revealed significant differences in the behaviour of these promoters when delivered transiently or stably. As well as providing an important reference which will facilitate the choice of an appropriate promoter to achieve the desired level of expression for a specific research question, this study also demonstrates the suitability of the cassette exchange methodology for the robust and reliable expression of multiple variant transgenes in ES cells.     

Arginine metabolic pathways determine its therapeutic benefit in experimental heatstroke: Role of Th1/Th2 cytokine balance

We have demonstrated that therapeutic administration of L-arginine (L-arg) (120 mg/kg) at +2 h of whole body hyperthermia (WBH) could rescue the mice from heatstroke-induced death. Studies were undertaken to elucidate the role of L-arg in the immunomodulation of the heat-stressed mice. Administration of L-arginine (L-arg), (120 mg/kg, i.p.), at +2 h of WBH, rescued the mice from heat-induced death and reduced the hypothermia. At +4 and +24 h of WBH, levels of IL-1beta, IFN-gamma, nitrite, TNF-alpha, IL-4, TGF-beta1, inducible form of nitric oxide synthase (iNOS), and corticosterone significantly increased compared to the sham group. The elevated levels of Th(1) cytokines, namely TNF-alpha, IL-1beta, IFN-gamma, nitrite, and iNOS, decreased significantly both at +4 and +24 h of WBH, following L-arg administration. However, L-arg administration did not reduce the increased levels of Th(2) cytokines, namely IL-4 and TGF-beta1, in WBH mice at +4 h of WBH. L-arg administration significantly increased the levels of Th(2) cytokines at +24 h of WBH, compared to the saline-treated WBH mice. L-arg administration significantly increased both the splenic and hepatic arginase activity at +4 and +24 h of WBH compared to the saline-treated WBH mice. L-NAME treatment at +2 h of WBH and anti-TGF-beta antibody treatment at 0 h of WBH significantly increased the mortality compared to the saline-treated WBH mice. Altered liver histopathology was attenuated following the administration of L-arg at +2 h of WBH. These results suggest that therapeutic administration of L-arg at appropriate concentration and time attenuates the acute inflammatory response, leading to the rescue of mice from heatstroke.     

High Prevalence of Posterior Polymorphous Corneal Dystrophy in the Czech Republic; Linkage Disequilibrium Mapping and Dating an Ancestral Mutation

Posterior polymorphous corneal dystrophy (PPCD) is a rare autosomal dominant genetically heterogeneous disorder. Nineteen Czech PPCD pedigrees with 113 affected family members were identified, and 17 of these kindreds were genotyped for markers on chromosome 20p12.1- 20q12. Comparison of haplotypes in 81 affected members, 20 unaffected first degree relatives and 13 spouses, as well as 55 unrelated controls, supported the hypothesis of a shared ancestor in 12 families originating from one geographic location. In 38 affected individuals from nine of these pedigrees, a common haplotype was observed between D20S48 and D20S107 spanning approximately 23 Mb, demonstrating segregation of disease with the PPCD1 locus. This haplotype was not detected in 110 ethnically matched control chromosomes. Within the common founder haplotype, a core mini-haplotype was detected for D20S605, D20S182 and M189K2 in all 67 affected members from families 1–12, however alleles representing the core mini-haplotype were also detected in population matched controls. The most likely location of the responsible gene within the disease interval, and estimated mutational age, were inferred by linkage disequilibrium mapping (DMLE+2.3). The appearance of a disease-causing mutation was dated between 64–133 generations. The inferred ancestral locus carrying a PPCD1 disease-causing variant within the disease interval spans 60 Kb on 20p11.23, which contains a single known protein coding gene, ZNF133. However, direct sequence analysis of coding and untranslated exons did not reveal a potential pathogenic mutation. Microdeletion or duplication was also excluded by comparative genomic hybridization using a dense chromosome 20 specific array. Geographical origin, haplotype and statistical analysis suggest that in 14 unrelated families an as yet undiscovered mutation on 20p11.23 was inherited from a common ancestor. Prevalence of PPCD in the Czech Republic appears to be the highest worldwide and our data suggests that at least one other novel locus for PPCD also exists.