Antineoplastic and antibacterial activity of some mononuclear Ru(II) complexes

These ligands (L) show a bidentate behavior, forming octahedral ruthenium complexes. The title complexes were subjected to in-vivo anticancer activity tests against a transplantable murine tumor cell line, Ehrlich's Ascitic Carcinoma (EAC) and in-vitro antibacterial activity against several Gram positive and Gram negative bacterial strains. [Ru(bpy)2(ihqs)]Cl2 and [Ru(bpy)2 (hc)]Cl2 (where bpy = 2,2'-bipyridine, ihqs = 7-iodo-8hydroxy quinoline-5-sulphonic acid and hc = 3-hydroxy coumarin) showed promising antitumor activity. Treatment with these complexes prolonged the life span of EAC bearing mice as well as decreased their tumor volume and viable ascitic cell count. All the tested complexes exhibited mild to moderate antibacterial activity.     

Dendritic cells from chronic lymphocytic leukemia patients are normal regardless of Ig V gene mutation status

Patients with B-type chronic lymphocytic leukemia (B-CLL) segregate into 2 subgroups based on the mutational status of the immunoglobulin (Ig) V genes and the patients in these subgroups follow very different clinical courses. To examine whether dendritic cells (DCs) generated from CLL patients can be candidates for immune therapy, we compared the phenotypic and functional capacities of DCs generated from patients of the 2 CLL subgroups (normal age-matched subjects [normal-DCs]). Our data show that immature DCs from B-CLL patients (B-CLL-DCs) have the same capacity to take up antigen as those from normal controls. Furthermore, B-CLL-DCs generated from the 2 CLL subgroups up-regulated MHC-II, CD80, CD86, CD83, CD40, and CD54 and down-regulated CD206 in response to stimulation with a cocktail of cytokines (CyC) and secreted increased levels of tumor necrosis factor alpha, interleukin (IL)-8, IL-6, IL-12 (p70), and RANTES in a manner typical of mature normal-DCs. Interestingly, CD54 was significantly more up-regulated by CyC in B-CLL-DCs compared with normal-DCs. Except for CD54, no significant differences in surface molecule expression were observed between normal-DCs and B-CLL-DCs. B-CLL-DCs from both subgroups, including 6 patients with VH1-69, that usually fare poorly, presented tetanus toxoid to autologous T cells in vitro similar to normal- DCs. Our data show that DCs generated from the B-CLL subgroup with unmutated Ig V genes are functionally normal. These results are very promising for the use of DCs from patients with poor prognosis for immunotherapy.     

Defining the structure-function relationships of bluetongue virus helicase protein VP6

The VP6 protein of bluetongue virus possesses a number of activities, including nucleoside triphosphatase, RNA binding, and helicase activity (N. Stauber, J. Martinez-Costas, G. Sutton, K. Monastyrskaya, and P. Roy, J. Virol. 71:7220-7226, 1997). Although the enzymatic functions of the protein have been documented, a detailed structure and function study has not been completed and the oligomeric form of the protein in solution has not been described. In this study, we have characterized VP6 activity by creating site-directed mutations in the putative functional helicase domains. Mutant proteins were expressed at high levels in an insect cell by using recombinant baculoviruses purified and analyzed for ATP binding, ATP hydrolysis, and RNA unwinding activities. UV cross-linking experiments indicated that the lysine residue in the conserved motif AXXGXGK(110)V is directly involved in ATP binding, whereas mutant R(205)Q in the arginine-rich motif ER(205)XGRXXR bound ATP at a level comparable to that of the wild-type protein. The RNA binding activity was drastically altered in the R(205)Q mutant and was also affected in the K(110)N mutant. Helicase activity was altered in both mutants. The mutation E(157)N in the DEXX sequence, presumed to act as a Walker B motif, showed an intermediate activity, implying that this motif does not play a crucial role in VP6 function. Purified protein demonstrated stable oligomers with a ring-like morphology in the presence of nucleic acids similar to those shown by other helicases. Gel filtration chromatography, native gel electrophoresis, and glycerol gradient analysis clearly indicated multiple oligomeric forms of VP6.     

Genetic characterization of mutants resistant to the antiauxin p-chlorophenoxyisobutyric acid reveals that AAR3, a gene encoding a DCN1-like protein, regulates responses to the synthetic auxin 2,4-dichlorophenoxyacetic acid in Arabidopsis roots

To isolate novel auxin-responsive mutants in Arabidopsis (Arabidopsis thaliana), we screened mutants for root growth resistance to a putative antiauxin, p-chlorophenoxyisobutyric acid (PCIB), which inhibits auxin action by interfering the upstream auxin-signaling events. Eleven PCIB-resistant mutants were obtained. Genetic mapping indicates that the mutations are located in at least five independent loci, including two known auxin-related loci, TRANSPORT INHIBITOR RESPONSE1 and Arabidopsis CULLIN1. antiauxin-resistant mutants (aars) aar3-1, aar4, and aar5 were also resistant to 2,4-dichlorophenoxyacetic acid as shown by a root growth assay. Positional cloning of aar3-1 revealed that the AAR3 gene encodes a protein with a domain of unknown function (DUF298), which has not previously been implicated in auxin signaling. The protein has a putative nuclear localization signal and shares homology with the DEFECTIVE IN CULLIN NEDDYLATION-1 protein through the DUF298 domain. The results also indicate that PCIB can facilitate the identification of factors involved in auxin or auxin-related signaling.     

Mineral proximity influences mechanical response of proteins in biological mineral-protein hybrid systems

The organic phase of nacre, which is composed primarily of proteins, has an extremely high elastic modulus as compared to that of bulk proteins, and also undergoes large deformation before failure. One reason for this unusually high modulus could be the mineral-organic interactions. In this work, we elucidate the specific role of mineral proximity on the structural response of proteins in biological structural composites such as nacre through molecular modeling. The "glycine-serine" domain of a nacre protein Lustrin A has been used as a model system. It is found that the amount of work needed to unfold is significantly higher when the GS domain is pulled in the proximity of aragonite. These results indicate that the proximity of aragonite has a significant effect on the unfolding mechanisms of proteins when pulled. These results will provide very useful information in designing synthetic biocomposites, as well as further our understanding of mechanical response in structural composites in nature.     

Degradation pathway, toxicity and kinetics of 2,4,6-trichlorophenol with different co-substrate by aerobic granules in SBR

The present study deals with cultivation of 2,4,6-trichlorophenol (TCP) degrading aerobic granules in two SBR systems based on glucose and acetate as co-substrate. Biodegradation of TCP containing wastewater starting from 10 to 360 mg L⁻¹ with more than 90% efficiency was achieved. Sludge volume index decreases as the operation proceeds to stabilize at 35 and 30 mL g⁻¹ while MLVSS increases from 4 to 6.5 and 6.2 g L⁻¹ for R1 (with glucose as co-substrate) and R2 (with sodium acetate as co-substrate), respectively. FTIR, GC and GC/MS spectral studies shows that the biodegradation occurred via chlorocatechol pathway and the cleavage may be at ortho-position. Haldane model for inhibitory substrate was applied to the system and it was observed that glucose fed granules have a high specific degradation rate and efficiency than acetate fed granules. Genotoxicity studies shows that effluent coming from SBRs was non-toxic.     

Morphology and transfection study of human microvascular endothelial cell angiogenesis: an in vitro three-dimensional model

Microvascular endothelial cells from human neonatal foreskin were grown in vitro until a three-dimensional network of capillary-like structures was formed. All stages of the angiogenic cascade could be observed in this in vitro model, including the formation of an internal lumen. The microscopy focused on morphology, formation of an internal lumen, role of the extracellular matrix, polarity of the cells, and the time-course of the angiogenic cascade. Bright-field microscopy revealed cells arranged circularly side by side and the internal lumen of capillary-like structures was verified by electron microscopy. Immunolabeling revealed a peritubular localization of collagen IV. Reporter gene expression after the formation of capillary-like structures was marginally higher than control expression, but clearly lower than the expression of cells at the stage of proliferation. Highest transfection efficiencies were obtained using vectors with the CMV promoter and the long fragment of the Ets-1 promoter. This is a first study of transfection efficiencies mapped for stages of in vitro angiogenesis. We describe here the morphological features of a long-term in vitro model of angiogenesis of human microvascular endothelial cells that could be used for transfection studies, without the provision of an extracellular matrix substrate. The cells self-create their own extracellular matrix to proliferate and form a three-dimensional network of capillary-like structures with an internal lumen.     

Active site structure of the aa3 quinol oxidase of Acidianus ambivalens

The membrane bound aa(3)-type quinol:oxygen oxidoreductase from the hyperthermophilic archaeon, Acidianus ambivalens, which thrives at a pH of 2.5 and a temperature of 80 degrees C, has several unique structural and functional features as compared to the other members of the heme-copper oxygen reductase superfamily, but shares the common redox-coupled, proton-pumping function. To better understand the properties of the heme a(3)-Cu(B) catalytic site, a resonance Raman spectroscopic study of the enzyme under a variety of conditions and in the presence of various ligands was carried out. Assignments of several heme vibrational modes as well as iron-ligand stretching modes are made to serve as a basis for comparing the structure of the enzyme to that of other oxygen reductases. The CO-bound oxidase has conformations that are similar to those of other oxygen reductases. However, the addition of CO to the resting enzyme does not generate a mixed valence species as in the bovine aa(3) enzyme. The cyanide complex of the oxidized enzyme of A. ambivalens does not display the high stability of its bovine counterpart, and a redox titration demonstrates that there is an extensive heme-heme interaction reflected in the midpoint potentials of the cyanide adduct. The A. ambivalens oxygen reductase is very stable under acidic conditions, but it undergoes an earlier alkaline transition than the bovine enzyme. The A. ambivalens enzyme exhibits a redox-linked reversible conformational transition in the heme a(3)-Cu(B) center. The pH dependence and H/D exchange demonstrate that the conformational transition is associated with proton movements involving a group or groups with a pK(a) of approximately 3.8. The observed reversibility and involvement of protons in the redox-coupled conformational transition support the proton translocation model presented earlier. The implications of such conformational changes are discussed in relation to general redox-coupled proton pumping mechanisms in the heme-copper oxygen reductases.     

Theoretical study on the mechanism of catalytic reduction of hydrazine to ammonia mediated by vanadium (III) thiolate complexes

DFT calculations on the free energy profile for the catalytic reduction of hydrazine to ammonia, the late stage of nitrogen fixation, mediated by vanadium (III) thiolate complexes VPS3 (1) and VNS3 (7) were carried out. The calculated energy profile revealed that all the reduction steps were exergonic while the protonation steps were endergonic. The generation of first equivalent of ammonia and the reduction of the cationic complex [V-NH₃]⁺ to the neutral V-NH₃ species were found to be the most exergonic of all the steps. Based on the calculated energy profile, both VPS3 and VNS3 were found to be catalytically active for the reduction of hydrazine to ammonia, although some quantitative differences in free energy profile had been observed.