Antibacterial,tyrosinase, and DNA photocleavage studies of some triazolylnucleosides

In order to explore the biological potential, some synthesized triazolylnucleosides were evaluated for their antibacterial, tyrosinase and DNA photocleavage activities. Triazolylnucleosides (5–12) were screened against Staphylococcus aureus (ATCC 6538), gram-positive and Escherichia coli (ATCC 10536), gram-negative bacterial strains. Among the series, compound 9 exhibited a significant level of antibacterial activity against both strains at higher concentration in reference to the standard drug, Levofloxacin. Tyrosinase activity and inhibition of these compounds were also studied, and it has been found that compounds 8 and 11 displayed more than 50% inhibitory activity. In addition, six compounds (7–12) were evaluated for their DNA photocleavage activity. The compounds 8 and 12 exhibited excellent DNA photocleavage activity at a concentration of 10 μg and may be used as template for antitumor drugs in the future.     

Induction of a novel class of diacylglycerol acyltransferases and triacylglycerol accumulation in Mycobacterium tuberculosis as it goes into a dormancy-like state in culture

Mycobacterium tuberculosis enters the host by inhalation of an infectious aerosol and replicates in the alveolar macrophages until the host's immune defense causes bacteriostasis, which leads the pathogen to go into nonreplicative drug-resistant dormancy. The dormant pathogen can survive for decades till the host's immune system is weakened and active tuberculosis develops. Even though fatty acids are thought to be the major energy source required for the persistence phase, the source of fatty acids used is not known. We postulate that the pathogen uses triacylglycerol (TG) as a storage form of fatty acids. Little is known about the biosynthesis of TG in M. tuberculosis. We show that 15 mycobacterial genes that we identified as putative triacylglycerol synthase (tgs) when expressed in Escherichia coli showed TGS activity, and we report some basic catalytic characteristics of the most active enzymes. We show that several tgs genes are induced when the pathogen goes into the nonreplicative drug-resistant state caused by slow withdrawal of O(2) and also by NO treatment, which is known to induce dormancy-associated genes. The gene (Rv3130c) that shows the highest TGS activity when expressed in E. coli shows the highest induction by hypoxia and NO treatment. Biochemical evidence shows that TG synthesis and accumulation occur under both conditions. We conclude that TG may be a form of energy storage for use during long-term dormancy. Therefore, TG synthesis may be an appropriate target for novel antilatency drugs that can prevent the organism from surviving dormancy and thus assist in the control of tuberculosis.     

On the disulfide-linker strategy for designing efficacious cationic transfection lipids: an unexpected transfection profile

Herein, employing a previously reported disulfide-linker strategy, we have designed and synthesized a novel cationic lipid 2 with a disulfide-linker and its non-disulfide control analog lipid 1. The relative efficacies of lipids 1 and 2 in transfecting CHO, COS-1 and MCF-7 cells were measured using both reporter gene and whole cell histochemical staining assays. In stark contrast to the expectation based on the disulfide-linker strategy, the control non-disulfide cationic lipid 1 showed phenomenally superior in vitro transfection efficacies to its essentially transfection incompetent disulfide counterpart lipid 2. Results in DNase I protection experiments and the electrophoretic gel patterns in the presence of glutathione, taken together, are consistent with the notion that the success of the disulfide-linker strategy may depend more critically on the DNase I sensitivity of the lipoplexes than on the efficient DNA release induced by intracellular glutathione pool.     

Genetic structure and gene flow in Gran Chaco populations of Argentina: evidence from Y-chromosome markers

The Gran Chaco region of central South America has been settled by humans for only the last 4,000-5,000 years. To investigate population structure and variation in this region's indigenous population, we scored males from tribes of the Argentinean part of the Gran Chaco (Pilagá, Wichí, and Toba, representing two major language groups, the Mataco and Guaycurú) for a number of Y-chromosome polymorphisms. The markers included eight microsatellites (DYS19, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, and DYS439) and the unique native American single nucleotide polymorphism, DYS199. Sixty males (77%) from the total sample carried the DYS199T chromosome, and these were the focus of the present analysis. Unlike most other native Americans, Gran Chaco males show a moderate level of diversity at the DYS19 locus but still less than that seen in non-native Americans. The FST value for Y-chromosome markers in Gran Chaco was 0.107, a value that is more than double that found for mtDNA haplogroups in the same tribes but is not particularly high compared with other Y-chromosome studies. Phylogenetic trees based on all eight microsatellites showed relatively poor correlation of the tribes with either geography or language, and this is possibly explained by their ecology. They are seasonal hunters living in small bands, and under such circumstances drift will be a powerful evolutionary force. An UPGMA tree based on five microsatellites (DYS19, DYS390, DYS391, DYS392, and DYS393), however, showed a more positive relationship, suggesting that DYS437, DYS438, and DYS439 may behave differently from the other microsatellites. No association was found between maternal and paternal lineage distributions. The time to the most recent common ancestor of the DYS199T chromosome is calculated to lie between 13,000 and 26,000 years. This range is consistent with estimates from other Y-chromosome studies as well as with estimates from mtDNA and the archeology of the colonization of South America. We conclude that the male lineages present in the contemporary Gran Chaco population reflect the level of diversity found in South America and that the region's male founders did not carry a restricted gene pool.     

Larval productivity of Fasciola gigantica in two lymnaeid snails

Two groups of Galba truncatula and two groups of Lymnaea natalensis were experimentally infected with Fasciola gigantica to determine if snail species had an influence on the redial burden and cercarial shedding of this trematode when snails of both species were infected with the same isolate of miracidia. In the two groups used for the study of redial burden, the total number of free rediae was significantly higher at day 49 post-exposure in L. natalensis than in G. truncatula. In the groups used for cercarial shedding, the life-span of cercaria-shedding snails and those of infected snails which died without cercarial emission, and the duration of the prepatent period were significantly longer in L. natalensis than those noted in G. truncatula. However, the mean numbers of shed cercariae did not significantly differ and showed no differences in their daily distribution throughout the shedding period. These results demonstrate that G. truncatula might be the principal intermediate host of F. gigantica in Egypt, at least in the areas where this lymnaeid species lives.     

Mechanics and hemodynamics of esophageal varices during peristaltic contraction

Our hypothesis states that variceal pressure and wall tension increase dramatically during esophageal peristaltic contractions. This increase in pressure and wall tension is a natural consequence of the anatomy and physiology of the esophagus and of the esophageal venous plexus. The purpose of this study was to evaluate variceal hemodynamics during peristaltic contraction. A simultaneous ultrasound probe and manometry catheter was placed in the distal esophagus in nine patients with esophageal varices. Simultaneous esophageal luminal pressure and ultrasound images of varices were recorded during peristaltic contraction. Maximum variceal cross-sectional area and esophageal luminal pressures at which the varix flattened, closed, and opened were measured. The esophageal lumen pressure equals the intravariceal pressure at variceal flattening due to force balance laws. The mean flattening pressures (40.11 +/- 16.77 mmHg) were significantly higher than the mean opening pressures (11.56 +/- 25.56 mmHg) (P < or = 0.0001). Flattening pressures >80 mmHg were generated during peristaltic contractions in 15.5% of the swallows. Variceal cross-sectional area increased a mean of 41% above baseline (range 7-89%, P < 0.0001) during swallowing. The peak closing pressures in patients that experience future variceal bleeding were significantly higher than the peak closing pressures in patients that did not experience variceal bleeding (P < 0.04). Patients with a mean peak closing pressure >61 mmHg were more likely to bleed. In this study, accuracy of predicting future variceal bleeding, based on these criteria, was 100%. Variceal models were developed, and it was demonstrated that during peristaltic contraction there was a significant increase in intravariceal pressure over baseline intravariceal pressure and that the peak intravariceal pressures were directly proportional to the resistance at the gastroesophageal junction. In conclusion, esophageal peristalsis in combination with high resistance to blood flow through the gastroesophageal junction leads to distension of the esophageal varices and an increase in intravariceal pressure and wall tension.     

Mutation of a single CTCF target site within the H19 imprinting control region leads to loss of Igf2 imprinting and complex patterns of de novo methylation upon maternal inheritance

The differentially methylated imprinting control region (ICR) region upstream of the H19 gene regulates allelic Igf2 expression by means of a methylation-sensitive chromatin insulator function. We have previously shown that maternal inheritance of mutated (three of the four) target sites for the 11-zinc finger protein CTCF leads to loss of Igf2 imprinting. Here we show that a mutation in only CTCF site 4 also leads to robust activation of the maternal Igf2 allele despite a noticeably weaker interaction in vitro of site 4 DNA with CTCF compared to other ICR sites, sites 1 and 3. Moreover, maternally inherited sites 1 to 3 become de novo methylated in complex patterns in subpopulations of liver and heart cells with a mutated site 4, suggesting that the methylation privilege status of the maternal H19 ICR allele requires an interdependence between all four CTCF sites. In support of this conclusion, we show that CTCF molecules bind to each other both in vivo and in vitro, and we demonstrate strong interaction between two CTCF-DNA complexes, preassembled in vitro with sites 3 and 4. We propose that the CTCF sites may cooperate to jointly maintain both methylation-free status and insulator properties of the maternal H19 ICR allele. Considering many other CTCF targets, we propose that site-specific interactions between various DNA-bound CTCF molecules may provide general focal points in the organization of looped chromatin domains involved in gene regulation.     

Inhibition of a mitotic motor protein: where, how, and conformational consequences

We report here the first inhibitor-bound structure of a mitotic motor protein. The 1.9 A resolution structure of the motor domain of KSP, bound with the small molecule monastrol and Mg2+ x ADP, reveals that monastrol confers inhibition by "induced-fitting" onto the protein some 12 A away from the catalytic center of the enzyme, resulting in the creation of a previously non-existing binding pocket. The structure provides new insights into the biochemical and mechanical mechanisms of the mitotic motor domain. Inhibition of KSP provides a novel mechanism to arrest mitotic spindle formation, a target of several approved and investigative anti-cancer agents. The structural information gleaned from this novel pocket offers a new angle for the design of anti-mitotic agents.     

Inhibition of 1,4-beta-D-xylan xylanohydrolase by the specific aspartic protease inhibitor pepstatin: probing the two-step inhibition mechanism

This is the first report that describes the inhibition mechanism of xylanase from Thermomonospora sp. by pepstatin A, a specific inhibitor toward aspartic proteases. The kinetic analysis revealed competitive inhibition of xylanase by pepstatin A with an IC50 value 3.6 +/- 0.5 microm. The progress curves were time-depended, consistent with a two-step slow tight binding inhibition. The inhibition followed a rapid equilibrium step to form a reversible enzyme-inhibitor complex (EI), which isomerizes to the second enzyme-inhibitor complex (EI*), which dissociated at a very slow rate. The rate constants determined for the isomerization of EI to EI* and the dissociation of EI* were 15 +/- 1 x 10(-5) and 3.0 +/- 1 x 10(-8) s(-1), respectively. The Ki value for the formation of EI complex was 1.5 +/- 0.5 microm, whereas the overall inhibition constant Ki* was 28.0 +/- 1 nm. The conformational changes induced in Xyl I by pepstatin A were monitored by fluorescence spectroscopy, and the rate constants derived were in agreement with the kinetic data. Thus, the conformational alterations were correlated to the isomerization of EI to EI*. Pepstatin A binds to the active site of the enzyme and disturbs the native interaction between the histidine and lysine, as demonstrated by the abolished isoindole fluorescence of o-phthalaldehyde-labeled xylanase. Our results revealed that the inactivation of xylanase is due to the interference in the electronic microenvironment and disruption of the hydrogen-bonding network between the essential histidine and other residues involved in catalysis, and a model depicting the probable interaction between pepstatin A with xylanase has been proposed.