Isolation of the 66-kilodalton polypeptide from promastigotes of Leishmania donovani as a ligand molecule for binding to macrophages.

A 66-kDa major plasma membrane-associated molecule of promastigotes of Leishmania donovani (UR6) was purified by affinity chromatography. The immunoreactivity of the 66-kDa molecule was lost upon exposure to heat or treatment with trypsin. The metaperiodate oxidation significantly reduced its immunoreactivity. The 66-kDa molecule is, therefore, glycoprotein in nature. With a fluorescent probe, the 66-kDa molecule was found to be located on the tip of flagellum and on the kinetoplast. The exposure of promastigotes of L. donovani to monospecific anti-66-kDa antibodies significantly reduced the percentage of macrophages with attached promastigotes in the cultured cell line (J774G8). The data suggested that promastigotes of L. donovani utilize the 66-kDa molecule in recognizing and as ligand for binding to macrophages.     

Acid phosphatase activity of promastigotes of Leishmania donovani: a marker of virulence

Seven cloned lines of promastigotes of Leishmania donovani (UR 6) were isolated by limiting dilution. One clone, UR6-C25, failed to multiply inside the macrophages of line J774G8 and thus was labelled as avirulent. Another, UR6-C24, multiplied inside macrophages, had a virulence index as high as 93 +/- 9.8 and was thus labelled as highly virulent. The other five clones had variable degree of virulence indices ranging from 46.4 +/- 5.8 to 67.6 +/- 3.5. No significant difference in the degrees of attachment of virulent and avirulent populations of promastigotes to macrophages was observed, suggesting no difference in the ligand utilised by these populations for attachment to the macrophages. Acid phosphatase activity of cloned promastigotes correlated with the degree of virulence. These data suggest that acid phosphatase activity could be used as a marker to differentiate avirulent from virulent populations of promastigotes of L. donovani.     

Analysis of transfer RNA during the early embryogenesis of the freshwater teleost,Heteropneustes fossilis

Total RNA as well as transfer RNA were quantified from mature ova apart from four different embryonic stages namely mid-cleavage, early gastrula, mid-gastrula and organogenesis of the freshwater teleostHeteropneustes fossilis. Total RNA as well as transfer RNA quantity follow a similar variation pattern, being maximum during mid-gastrulation. When analysed by total amino acid acceptance capacity, transfer RNA shows its maximum activity during mid-gastrulation. This coincides with the higher ratio of tRNA to total RNA at this stage. The relative aminoacylation capacity for Ser, Gly, Asn and Thr are found to be higher (9–34%) compared to that for other amino acids. Total tRNA, resolved into three peaks upon HPLC fractionation, shows a high cumulative peak area during mid-gastrulation and organogenesis. These results indicate a switch over of maternal to embryonic translation machinery during gastrulation.Abbreviations tRNA transfer RNA - aaRS aminoacyl tRNA synthetase - HPLC high pressure liquid chromatography - AUF absorption unit full scale     

Effect of Terminalia arjuna on antioxidant defense system in cancer

Constant production of reactive oxygen species (ROS) during aerobic metabolism is balanced by antioxidant defense system of an organism. Although low level of ROS is important for various physiological functions, its accumulation has been implicated in the pathogenesis of age-related diseases such as cancer and coronary heart disease and neurodegenerative disorders such as Alzheimer’s disease. It is generally assumed that frequent consumption of phytochemicals derived from vegetables, fruits, tea and herbs may contribute to shift the balance towards an adequate antioxidant status. The present study is aimed to investigate the effect of aqueous extract of medicinal plant Terminalia arjuna on antioxidant defense system in lymphoma bearing AKR mice. Antioxidant action of T. arjuna is monitored by the activities of catalase, superoxide dismutase and glutathione S transferase which constitute major antioxidant defense system by scavenging ROS. These enzyme activities are low in lymphoma bearing mice indicating impaired antioxidant defense system. Oral administration of different doses of aqueous extract of T. arjuna causes significant elevation in the activities of catalase, superoxide dismutase and glutathione S transferase. T. arjuna is found to down regulate anaerobic metabolism by inhibiting the activity of lactate dehydrogenase in lymphoma bearing mice, which was elevated in untreated cancerous mice. The results indicate the antioxidant action of aqueous extract of T. arjuna, which may play a role in the anti carcinogenic activity by reducing the oxidative stress along with inhibition of anaerobic metabolism.     

The Genetic Basis of Laboratory Adaptation in Caulobacter crescentus

The dimorphic bacterium Caulobacter crescentus has evolved marked phenotypic changes during its 50-year history of culture in the laboratory environment, providing an excellent system for the study of natural selection and phenotypic microevolution in prokaryotes. Combining whole-genome sequencing with classical molecular genetic tools, we have comprehensively mapped a set of polymorphisms underlying multiple derived phenotypes, several of which arose independently in separate strain lineages. The genetic basis of phenotypic differences in growth rate, mucoidy, adhesion, sedimentation, phage susceptibility, and stationary-phase survival between C. crescentus strain CB15 and its derivative NA1000 is determined by coding, regulatory, and insertion/deletion polymorphisms at five chromosomal loci. This study evidences multiple genetic mechanisms of bacterial evolution as driven by selection for growth and survival in a new selective environment and identifies a common polymorphic locus, zwf, between lab-adapted C. crescentus and clinical isolates of Pseudomonas aeruginosa that have adapted to a human host during chronic infection.Colonization of new environments or changes in resource availability, predatory regime, or climate can drive adaptive evolution. Determining the genetic basis of these changes informs our understanding of the evolution of diversity and the nature of selection. Domestication of crop plants, adaptive radiations, and in-host evolution during chronic microbial infection are characterized by the evolution of a suite of phenotypes that are advantageous in the new environment. Recent work has successfully identified several of the polymorphisms responsible for this type of adaptive evolution in a variety of species (3, 7, 11, 12, 15, 22, 25, 35-37). With comparative genome sequencing emerging as a powerful tool for identifying genetic polymorphism (5, 14, 23), these studies are becoming faster and easier. Still, large genome sizes and countless sequence differences between individuals, isolates, strains, and species have made comprehensive analyses intractable.Upon isolation and introduction into the laboratory, model research organisms experience extreme environmental changes, with associated selection pressures. Indeed, adaptation to life in captivity has been observed in a wide range of domesticated and model research organisms (2) and in zoo populations of endangered species (31). Many phenotypes that evolve in these nonnative environments do so repeatedly and become common features of human-cultured, -raised, or -cultivated organisms (2), providing evidence of positive selection. Likewise, the aquatic bacterium Caulobacter crescentus has evolved marked phenotypic changes during the 50 years it has been cultured in the laboratory environment. At least six phenotypic differences (Fig. ​(Fig.1)1) between two closely related strains (NA1000 and CB15) derived from the same common ancestor have evolved over decades of laboratory cultivation. It is presumed that these phenotypes evolved in response to the dynamic culture conditions and associated selection pressures experienced by bacteria in the laboratory environment. However, the extent of genetic divergence between these strains was uncharacterized, and it was not known whether the phenotypes could be explained by a few single nucleotide polymorphisms (SNPs), insertions/deletions, or genome rearrangements or by the accumulation of many mutations, each with a small contribution to particular phenotypes. In an effort to comprehensively characterize their divergence, we identified the genetic basis of all known phenotypic differences between two laboratory strains (NA1000 and CB15) of C. crescentus.Open in a separate windowFIG. 1.Evolved phenotypic differences between CB15 (Crosson₂) and NA1000 (Crosson₁). (A) Caulobacter cells divide asymmetrically to yield a swarmer and a stalked cell, which are mixed in culture. NA1000 stalked and predivisional cells (light gray) pellet less efficiently than swarmer cells (dark gray), allowing them to be physically separated. Synchrony capacity is quantified by calculating the proportion of cultured cells remaining in suspension. Error bars are ±standard errors of the mean (SEM). (B) When patched and grown on high-sugar media, NA1000 colonies develop a mucoid morphology, while CB15 colonies do not. (C) The transducing phage φCR30 efficiently infects and lyses CB15 cells, resulting in clear plaques, while infection of NA1000 with the same phage lysate results in fewer plaques that are visually turbid. (D) Holdfast-mediated attachment to a surface can be measured using a crystal violet assay. CB15 cells attach, resulting in robust staining, while NA1000 exhibits negligible adherence. (E) Upon continued aeration and incubation of stationary-phase Caulobacter cultures, NA1000 (▪) loses viability more rapidly than CB15 (○). Error bars are ±SEM. (F) In glucose minimal medium, NA1000 generation time is 20% shorter than that of CB15. Error bars are ±SEM.Our study revealed 11 coding, noncoding, and insertion/deletion polymorphisms between these two strains, five of which completely account for the evolved differences between the strains. The results presented herein provide insight into prokaryotic evolution driven by selection for growth and survival in a research laboratory and demonstrate the utility of combining whole-genome sequencing and alignment with molecular genetic tools to reveal the genetic basis of multiple derived phenotypes. Our work demonstrates that rapid adaptation of C. crescentus to the laboratory environment occurred in both strain lineages and is characterized by relatively few genetic changes, including nonsynonymous mutation, noncoding regulatory changes, acquisition of new genes, and inactivation of existing genes, each with a large phenotypic effect.     

Mutational Insights into the Roles of Amino Acid Residues in Ligand Binding for Two Closely Related Family 16 Carbohydrate Binding Modules

Carbohydrate binding modules (CBMs) are specialized proteins that bind to polysaccharides and oligosaccharides. Caldanaerobius polysaccharolyticus Man5ACBM16-1/CBM16-2 bind to glucose-, mannose-, and glucose/mannose-configured substrates. The crystal structures of the two proteins represent the only examples in CBM family 16, and studies that evaluate the roles of amino acid residues in ligand binding in this family are lacking. In this study, we probed the roles of amino acids (selected based on CBM16-1/ligand co-crystal structures) on substrate binding. Two tryptophan (Trp-20 and Trp-125) and two glutamine (Gln-81 and Gln-93) residues are shown to be critical in ligand binding. Additionally, several polar residues that flank the critical residues also contribute to ligand binding. The CBM16-1 Q121E mutation increased affinity for all substrates tested, whereas the Q21G and N97R mutants exhibited decreased substrate affinity. We solved CBM/substrate co-crystal structures to elucidate the molecular basis of the increased substrate binding by CBM16-1 Q121E. The Gln-121, Gln-21, and Asn-97 residues can be manipulated to fine-tune ligand binding by the Man5A CBMs. Surprisingly, none of the eight residues investigated was absolutely conserved in CBM family 16. Thus, the critical residues in the Man5A CBMs are either not essential for substrate binding in the other members of this family or the two CBMs are evolutionarily distinct from the members available in the current protein database. Man5A is dependent on its CBMs for robust activity, and insights from this study should serve to enhance our understanding of the interdependence of its catalytic and substrate binding modules.     

New effective inhibitors of the Abelson kinase

The effects of substituents on the aryl ring were studied by the preparation and testing of several PD173955 analogs. Inserting a single carbon atom into the C–N bond in the aniline subunit (PDC) reduced the kinase inhibition by a factor of 200. Despite its decreased affinity for Abl compared with PD173955, PDC exhibits a Ki very similar to that reported for Imatinib. Increased water solubility is also gained by replacing the thiomethyl group with an amino or glycyl moiety. For both PD173955 and PDC, the analogs with amino groups in place of the methylthio group are 10 times more inhibitory than the parent molecules. Two molecules were identified with Kis about three orders of magnitude lower than reported for Imatinib.