Crystal structure and conformation of 5-fluorouridine: conformational preferences for 5-fluorinated pyranosides

Crystals of 5-fluorouridine (5FUrd) have unit cell dimensions a = 7.716(1), b = 5.861(2), c = 13.041(1)A, alpha = gamma = 90 degrees, beta = 96.70 degrees (1), space group P2(1), Z = 2, rho obs = 1.56 gm/c.c and rho calc = 1574 gm/c.c The crystal structure was determined with diffractometric data and refined to a final reliability index of 0.042 for the observed 2205 reflections (I > or = 3sigma). The nucleoside has the anti conformation [chi = 53.1(4) degrees] with the furanose ring in the favorite C2'-endo conformation. The conformation across the sugar exocyclic bond is g+, with values of 49.1(4) and -69.3(4) degrees for phi(theta c) and phi (infinity) respectively. The pseudorotational amplitude tau(m) is 34.5 (2) with a phase angle of 171.6(4) degrees. The crystal structure is stabilized by a network of N-H...O and O-H...O involving the N3 of the uracil base and the sugar 03' and 02' as donors and the 02 and 04 of the uracil base and 03' oxygen as acceptors respectively. Fluorine is neither involved in the hydrogen bonding nor in the stacking interactions. Our studies of several 5-fluorinated nucleosides show the following preferred conformational features: 1) the most favored anti conformation for the nucleoside [chi varies from -20 to + 60 degrees] 2) an inverse correlation between the glycosyl bond distance and the chi angle 3) a wide variation of conformations of the sugar ranging froni C2'-endo through C3'-endo to C4'-exo 4) the preferred g+ across the exocyclic C4'-C5' bond and 5) no role for the fluorine atom in the hydrogen bonding or base stacking interactions.     

Real-time observation of DNA translocation by the type I restriction modification enzyme EcoR124I

Type I restriction enzymes bind sequence-specifically to unmodified DNA and subsequently pull the adjacent DNA toward themselves. Cleavage then occurs remotely from the recognition site. The mechanism by which these members of the superfamily 2 (SF2) of helicases translocate DNA is largely unknown. We report the first single-molecule study of DNA translocation by the type I restriction enzyme EcoR124I. Mechanochemical parameters such as the translocation rate and processivity, and their dependence on force and ATP concentration, are presented. We show that the two motor subunits of EcoR124I work independently. By using torsionally constrained DNA molecules, we found that the enzyme tracks along the helical pitch of the DNA molecule. This assay may be directly applicable to investigating the tracking of other DNA-translocating motors along their DNA templates.     

Characterization of serotonin in protein and membrane mimetic environments: a spectroscopic study

As a first step toward using the photophysical properties of serotonin to probe its interactions with biological target sites, we have examined its interactions with human serum albumin (HSA), chosen as a surrogate for the actual receptor proteins in physiological systems, and with sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/heptane/water reverse micelles, chosen as a biomembrane mimetic environment for the transmembrane portion of the receptor protein. Although the emission maximum of serotonin is relatively insensitive to the polarity of the local environment, which is attributed to lack of solvent dipolar reorientation of the 5-hydroxyindole chromophore, the fluorescence anisotropy (r) served as a useful and sensitive parameter from which the binding constants (K) and Gibbs energy changes (deltaG) were estimated for serotonin-HSA and serotonin-AOT reverse micellar interactions. Fluorescence-decay studies of serotonin show double-exponential kinetics in homogeneous aqueous solvent due to the structural heterogeneity arising from different rotamers of serotonin. In contrast, upon binding to HSA, a single-exponential fluorescence-decay profile was observed indicating the occurrence of a single structural species of serotonin in the protein environment. Furthermore, far-UV-circular-dichroism (CD) spectroscopic data indicate that the secondary structural features of HSA remain essentially intact after binding to serotonin. This preliminary research can be expected to open the door to extensive future studies on interactions of serotonin with relevant target proteins and associated cell membranes involved in its diverse physiological functions.     

Isolation and characterization of nickel-resistant microflora from serpentine soils of Andaman

Serpentine soils of Andaman Islands, India characteristically contain high levels of nickel, cobalt and chromium and are colonized by indigenous nickel-hyperaccumulating plants. Attempts have been made to isolate and characterize nickel-resistant microorganisms from these hitherto unexplored naturally nickel-percolated soils. The majority of the nickel-resistant organisms showed a minimum inhibitory concentration (MIC) of Ni²⁺ ranging from 300 to 400 mg/l and about 3.4% of the total 89 isolates representing bacterial strains were able to grow at 400 mg/l Ni²⁺. The potent Ni²⁺-resistant strains AND305 and AND603 were tentatively identified as Pseudomonas spp. and strain AND408 as Bacillus sp. following detailed analysis of morphological and physio-biochemical characteristics. Growth kinetics of these Ni²⁺-resistant bacteria showed a prolonged lag phase in Ni²⁺-containing media, which extended with increasing nickel concentration. In addition to Ni²⁺, these isolates were also resistant to Co²⁺, Cd²⁺, Cr⁶⁺, Fe³⁺, Cu²⁺, Mg²⁺, Mn²⁺(50–200 mg/l) and Hg²⁺ (0.5–2.0 mg/l) and the multiple metal-resistance of the isolates were also associated with the resistance to antibiotics ampicillin, cycloserine and penicillin G.     

In vivo and in vitro oxidative regulation of rat aryl sulfotransferase IV (AST IV)

Sulfotransferase catalyzed sulfation is important in the regulation of different hormones and the metabolism of hydroxyl containing xenobiotics. In the present investigation, we examined the effects of hyperoxia on aryl sulfotransferase IV in rat lungs in vivo. The enzyme activity of aryl sulfotransferase IV increased 3- to 8-fold in >95% O2 treated rat lungs. However, hyperoxic exposure did not change the mRNA and protein levels of aryl sulfotransferase IV in lungs as revealed by Western blot and RT-PCR. This suggests that oxidative regulation occurs at the level of protein modification. The increase of nonprotein soluble thiol and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios in treated lung cytosols correlated well with the aryl sulfotransferase IV activity increase. In vitro, rat liver cytosol 2-naphthol sulfation activity was activated by GSH and inactivated by GSSG. Our results suggest that Cys residue chemical modification is responsible for the in vivo and in vitro oxidative regulation. The molecular modeling structure of aryl sulfotransferase IV supports this conclusion. Our gel filtration chromatography results demonstrated that neither GSH nor GSSG treatment changed the existing aryl sulfotransferase IV dimer status in cytosol, suggesting that oxidative regulation of aryl sulfotransferase IV is not caused by dimer-monomer status change.     

Molecular evidence of genetic modification of Sinorhizobium meliloti: enhanced PCB bioremediation

The genome of the nitrogen-fixing soil bacterium Sinorhizobium meliloti does not possess genes for bioremediation of aromatic pollutants. It has the well-known ability to interact specifically with the leguminous alfalfa plant, Medicago sativa. Our previous work has shown enhanced degradation of the nitroaromatic compound 2,4-dinitrotoluene (DNT) when a plasmid containing degradative genes was introduced in it. In this study we report molecular evidence of the transfer of a polychlorinated biphenyl (PCB)-biodegradative plasmid pE43 to S. meliloti strain USDA 1936. Several standard analytical tests and plant growth chamber studies were conducted to test the ability of S. meliloti to degrade 2',3,4-PCB congener. Alfalfa plant alone was able to degrade 30% of PCBs compared with control. No enhanced dechlorination was noted when alfalfa plant was grown with wild-type S. meliloti, and when alfalfa plant was grown with the S. meliloti electrotransformants (genetically modified) dechlorination of PCBs was more than twice that when alfalfa plant was grown with wild-type S. meliloti. When alfalfa plant was grown with uncharacterized mixed culture (containing nodule formers), almost equally significant PCB degradation was observed. The significance of this work is that the naturally occurring nitrogen-fixing soil bacterium S. meliloti (genetically modified) has the ability to enhance fertility of soil in association with the leguminous alfalfa plant while simultaneously enhancing bioremediation of PCB-contaminated soils. Enhanced bioremediation of PCB and robust alfalfa plant growth was also noted when uncharacterized mixed cultures containing alfalfa plant nodule formers were used.     

Compositional variation in bacterial genes and proteins with potential expression level

Usage of guanine and cytosine at three codon sites in eubacterial genes vary distinctly with potential expressivity, as predicted by Codon Adaptation Index (CAI). In bacteria with moderate/high GC-content, G(3) follows a biphasic relationship, while C(3) increases with CAI. In AT-rich bacteria, correlation of CAI is negative with G(3), but non-specific with C(3). Correlations of CAI with residues encoded by G-starting codons are positive, while with those by C-starting codons are usually negative/random. Average Size/Complexity Score and aromaticity of gene-products decrease with CAI, confirming general validity of cost-minimization principle in free-living eubacteria. Alcoholicity of bacterial gene-products usually decreases with expressivity.     

Experimental therapeutic intervention with ascorbic acid in ethanol induced testicular injuries in rats

Ascorbic acid treatment significantly increased the activities of testicular delta5, 3beta-HSD and 17beta-HSD. Moreover, the treatment was also associated with significant decrease in oxidative stress in the testis. Ethanol induced oxidative stress and decreased steroidogenesis can be reversed by treatment with ascorbic acid.     

Sheep erythrocyte demonstrated better effect than IL-2 and IFN-gamma as biological response modifier against glioma in experimental model

The significant insights into the immunobiology of central nervous system (CNS) and brain tumor have opened up the feasibility of applying 'Immunotherapy' as an alternative to the poor prognosis of malignant brain tumor with conventional therapeutic approaches. Though cytokines like IL-2 and IFN-gamma used against glioma showed some favorable results by eliciting Th1 type immune response, a proper immunotherapeutic agent is still to be searched for. Sheep erythrocyte (SRBC), a corpuscular antigen showed a better therapeutic efficacy in terms of enhanced survival and augmentation of cell mediated immunity (CMI) in a glioma model developed by chemical carcinogen ethyl nitrosourea. Histological findings revealed most efficient glioma rejection in SRBC and combination biological response modifier (BRM) treated groups. Simultaneously E-rosetting, cytotoxicity of lymphocytes, phagocytosis and antigen presenting capacity of myeloid cells established the better therapeutic efficacy of SRBC alone than other BRMs viz. IL-2 and IFN-gamma. Even the effect of combination therapy of different BRMs showed marginal differences in facilitating glioma reduction than the single use of SRBC. These findings emphasized the application of SRBC as an exogenous BRM having the potential as a rational therapeutic adjunct against glioma.