Synthesis, spectroscopic and structural characterization of the high-spin Fe(II) cyanato-N and thiocyanato-N “picket fence” porphyrin complexes

Two new iron(II) five-coordinated porphyrin complexes [Na(2,2,2-crypt)] [Fe^II(TpivPP)(NCO)] (1) (TpivPP = α,α,α,α-tetrakis(o-pivalamidophenyl) porphyrin known as picket fence porphyrin and 2,2,2-crypt is the cryptand-222) and [K(2,2,2-crypt)][Fe^II(TpivPP)(NCS)] (2) have been prepared and characterized. The UV-Vis and IR spectroscopic data are consistent with a cyanato-N and thiocyanato-N ferrous porphyrinates. The Mössbauer data and the X-ray structural analysis indicate that the Fe(II) cation in 1 and 2 is high-spin (S = 2) and has the (d_xy)²(d_xz)¹(d_yz)¹(d_z²)¹(d_x²-y²)¹ ground state electronic configuration.For complex 1, the average equatorial iron-pyrrole N bond length (Fe-N_p = 2.120(2) Å), the distance between the iron and the 24-atom mean plane of the porphyrin ring (Fe-P_C = 0.6805(7) Å) and the distance between the iron and the plane made by the four pyrrole nitrogens (Fe-P_N = 0.5923(12) Å) are longer than those of complex 2 and similar five-coordinated Fe(II) high-spin porphyrinates. This is probably due to the significant electronic repulsion of the d_x²-y² and d_xy orbitals by the negative charge of the pyrrole N atoms in case of 1.     

Systematic search for putative new domain families in Mycoplasma gallisepticum genome

Background

Stem cell factor (SCF) receptor c-Kit is recognized as a key signaling molecule, which transduces signals for the proliferation, differentiation and survival of stem cells. Binding of SCF to its receptor triggers transactivation, leading to the recruitment of kinases and phosphatases to the docking platforms of c-Kit catalytic domain. Tyrosine phosphatase-1 (Shp-1) deactivates/attenuates 'Kit' kinase activity. Whereas, Asp816Val mutation in the Kit activation loop transforms kinase domain to a constitutively activated state (switch off-to-on state), in a ligand-independent manner. This phenomenon completely abrogates negative regulation of Shp-1. To predict the possible molecular basis of interaction between c-Kit and Shp-1, we have performed an in silico protein-protein docking study between crystal structure of activated c-Kit (phosphorylated c-Kit) and full length crystal structure of Shp-2, a close structural counterpart of Shp-1.

Findings

Study revealed a stretch of conserved amino acids (Lys818 to Ser821) in the Kit activation domain, which makes decisive H-bonds with N-sh2 and phosphotyrosine binding pocket residues of the phosphatase. These H-bonds may impose an inhibitory steric hindrance to the catalytic domain of c-Kit, there by blocking further interaction of the activation loop molecules with incoming kinases. We have also predicted a phosphotyrosine binding pocket in SH2 domains of Shp-1, which is found to be predominantly closer to a catalytic groove like structure in c-Kit kinase domain.

Conclusions

This study predicts that crucial hydrogen bonding between N-sh2 domain of Shp-1 and Kit activation loop can modulate the negative regulation of c-Kit kinase by Shp-1. Thus, this finding is expected to play a significant role in designing suitable gain-of-function c-Kit mutants for inducing conditional proliferation of hematopoietic stem cells.     

In vitro inhibition of human influenza A virus replication by chloroquine

Inhibition of the human cytomegalovirus UL97 kinase by maribavir is thought to be responsible for the antiviral activity of this compound. Some mutations that confer resistance to maribavir map to UL97, however additional mutations that also confer resistance to the drug were mapped to UL27. These open reading frames share a low level of homology, yet the function of pUL27 remains unknown. A recombinant virus with a deletion in the UL27 open reading frame was reported previously to exhibit a slight replication deficit, but a more important function in vivo was hypothesized given its homology to the UL97 kinase. The potential for an important function in vivo was investigated by determining if these knockout viruses could replicate in human tissue implanted in SCID mice. None of the AD169 derived viruses replicated well in the implanted thymus/liver tissue, and is consistent with previous observations, although all of the viruses replicated to some degree in retinal tissue implants. Replication of the parent viruses was observed at 7 days post inoculation, whereas no replication was detected with any of the recombinant viruses with deletions in UL27. By day 14, replication was detected in two of the three knockout viruses and in all of the viruses by day 42. These data are consistent with minimal defects observed in cell culture, but are not consistent with an important role for UL27 in vivo. We conclude that UL27 is not required for viral replication in vivo.     

Unusual pattern of bacterial ice nucleation gene evolution

Bacterial ice nucleation activity (INA+ phenotype) can be traced to the product of a single gene, ina. A remarkably sparse distribution of this phenotype within three bacterial genera indicates that the ina gene may have followed an unusual evolutionary path. Southern blot analyses, coupled with assays for ice-nucleating ability, revealed that within four bacterial species an ina gene is present in some strains but absent from others. Results of hybridization experiments using DNA fragments that flank the ina gene suggested that the genotypic dimorphism of ina may be anomalous. A phylogenetic analysis of 16S ribosomal RNA gene sequences from a total of 14 ina+ and ina- bacterial strains indicated that the ina+ bacteria are not monophyletic but instead phylogenetically interspersed among ina- bacteria. The relationships of ina+ bacteria inferred from ina sequence did not coincide with those inferred from the 16S data. These results suggest the possibility of horizontal transfer in the evolution of bacterial ina genes.      

β-Decay and the origin of optical purity

Improving Keszthelyis simple model the evolutionary appearance of concentration difference of enanthiomeric compounds due to their differential decomposition by -rays is investigated taking into account the racemization as well. It is shown that if the difference in the cross sections is very small then the resulting concentration difference will never exceed the statistical fluctuations, while in the case of a sufficiently large difference in the cross sections the concentration difference can overgrow the statistical fluctuations in an evolutionary reasonable period of time. The relative difference of the concentrations, however, will be generally much smaller than that of the cross sections. Therefore some other, amplifying mechanism must be postulated in order to explain the optical purity of living beings.     

Production and purification of a calcium-dependent protease from Bacillus cereus BG1

The production and purification of a calcium-dependent protease by Bacillus cereus BG1 were studied. The production of the protease was found to depend specifically on the calcium concentration in the culture medium. This suggests that this metal ion is essential for the induction of protease production and/or stabilisation of the enzyme after synthesis. The calcium requirement is highly specific since other metal ions (such as Mg²⁺ and Ba²⁺, which both activate the enzyme) are not able to induce protease production. The most appropriate medium for growth and protease production comprises (g L^–1) starch 5, CaCl₂ 2, yeast extract 2, K₂HPO₄ 0.2 and KH₂PO₄ 0.2. The protease of BG1 strain was purified to homogeneity by ultrafiltration, heat treatment, gel filtration on Sephacryl S-200, ion exchange chromatography on DEAE-cellulose and, finally, a second gel filtration on Sephacryl S-200, with a 39-fold increase in specific activity and 23% recovery. The molecular weight was estimated to be 34 kDa on SDS-PAGE. The optimum temperature and pH of the purified enzyme were determined to be 60°C and 8.0, respectively, in 100 mM Tris-HCl buffer + 2 mM CaCl₂.     

Effects of ICI 182780 on estrogen receptor expression,fluid absorption and sperm motility in the epididymis of the bonnet monkey

Background  

The importance of estrogen in regulation of fluid absorption and sperm maturation in the rodent epididymis has been established from studies on estrogen receptor-alpha knockout mice. However, functional studies on the role of estrogen in primate epididymis have been few. The main objective of this study was therefore to extend these observations and systematically analyze the presence and function of estrogen receptors in modulating the function of the primate epididymis, using the bonnet monkey (Macaca radiata) as a model system.     

Purification and characterization of an alkaline protease from Pseudomonas aeruginosa MN1

An alkaline protease produced by Pseudomonas aeruginosa MN1, isolated from an alkaline tannery waste water, was purified and characterized. The enzyme was purified 25-fold by gel filtration and ion exchange chromatography to a specific activity of 82350 U mg⁻¹. The molecular weight of the enzyme was estimated to be 32000 daltons. The optimum pH and temperature for the proteolytic activity were pH 8.00 and 60°C, respectively. Enzyme activity was inhibited by EDTA suggesting that the preparation contains a metalloprotease. Enzyme activity was strongly inhibited by Zn²⁺, Cu²⁺ and Hg²⁺(5 mM), while Ca²⁺ and Mn²⁺ resulted in partial inhibition. The enzyme is different from other Pseudomonas aeruginosa alkaline proteases in its stability at high temperature; it retained more than 90% and 66% of the initial activity after 15 and 120 min incubation at 60°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 291–295. Received 09 June 1999/ Accepted in revised form 24 January 2000     

Locomotor behaviour in males,females and groups of Orchestia montagui (Amphipoda,Talitridae) in the supralittoral zone of Bizerte lagoon

Locomotor activity rhythm of Orchestia montagui was investigated under constant darkness in a population collected in spring from the supralittoral zone of Bizerte lagoon (northern region of Tunisia) at Menzel Jmil in spring. This rhythm was recorded in individual and groups of animal by infrared actography every 20 min by multichannel data loggers, at a constant temperature of 18 ± 0.5 °C. According to double-plotted actograms, wave forms and periodogram analysis, results revealed different locomotor pattern. However, locomotor activity rhythm of males was more stable than females. Furthermore, the mortality was statistically higher in unmixed groups than in mixed groups.     

Effects of microwave (2.45 GHz) irradiation on some biological characters of Salmonella typhimurium

The present study was carried out to evaluate the effects of sub-lethal doses of microwave radiation on some biological characteristics in Salmonella typhimurium. The aim was to show the relationship between this treatment and the development of radiotolerance in this pathogen because there is a need for more information on physiological responses of pathogens to sub-lethal doses of microwave radiation. So, the bacterial strain was treated with a dose of 3600 J (40-s exposure with power P = 90 W) to cause cellular damage. The results have shown that the exposure of bacteria to microwaves resulted in a significant inhibition of cellular growth. This treatment has notably increased the effectiveness of the most tested antibiotics by the amelioration or the appearance of sensitivity in exposed bacteria. Gas chromatography (GC) analysis was performed to demonstrate the modification of the fatty acids (FA) composition. Results obtained have shown that this treatment had a significant effect on the FA content with an increase of unsaturated FA percentage. The acquisition of sensitivity to the sodium deoxycholate and the significant increase in the amount of extracellular proteins in exposed bacteria has confirmed the weakening of the bacterial membrane by microwaves. This study represents one of the few demonstrating the modifications on the bacterial membrane as a cellular response to survive the non-ionising radiation stress.