Statistical Studies on Protein Polymorphism in Natural Populations II. Gene Differentiation between Populations

With the aim of testing the validity of the mutation-drift hypothesis, we examined the pattern of genetic differentiation between populations by using data from Drosophila, fishes, reptiles, and mammals. The observed relationship between genetic identity and correlation of heterozygosities of different populations or species was generally in good agreement with the theoretical expectations from the mutation-drift theory, when the variation in mutation rate among loci was taken into account. In some species of Drosophila, however, the correlation was unduly high. The relationship between the mean and variance of genetic distance was also in good agreement with the theoretical prediction in almost all organisms. We noted that both the distribution of heterozygosity within species and the pattern of genetic differentiation between species can be explained by the same set of genetic parameters in each group of organisms. Alternative hypotheses for explaining these observations are discussed.     

Potentialities of induced pluripotent stem (iPS) cells for treatment of diseases

Induced pluripotent stem (iPS) cell research has been growing a new height throughout the world due to its potentialities in medical applications. We can explore several therapeutic applications through the iPS cell research. In this review, we have first discussed the development of iPS cells, reprogramming factors, and effectiveness of iPS cells. Then we have emphasized the potential applications of iPS cells in pharmaceutical and medical sectors, such as, study of cellular mechanisms for spectrum of disease entities, disease-specific iPS cell lines for drugs discovery and development, toxicological studies of drugs development, personalized medicine, and regenerative medicine.     

Macrophage Mannosyl Fucosyl Receptor: Its Role in Invasion of Virulent and Avirulent L. Donovani Promastigotes

The interaction of leishmania parasites with macrophages is known to be receptor mediated. Previous study from this laboratory (J. Parasitol. 82:632, 1996) showed the significant involvement of LPG and gp63 receptors in the recognition of virulent strains onto the macrophages. The role of carbohydrate receptors--the other major receptors besides LPG and gp63 receptors, in the recognition of both virulent (strains AG83 and GE1) and avirulent (strain UR6) leishmania onto the host macrophages has been the major focus of the present investigation. Various neoglycoproteins were used as efficient ligands to preblock the carbohydrate receptors on the macrophage surface. Similarly, various sugar specific lectins were used to preblock the corresponding carbohydrate ligands on the parasite surface. When these preblocked macrophages or parasites were used to study their mode of recognition, it was obvious from the findings that avirulent leishmania promastigotes possibly use the mannosyl fucosyl receptors (MFR) more avidly for their initial attachment and subsequent internalization into the macrophages whereas the virulent leishmania exhibits limited use of this receptor. When a macrophage-like cell line (J774), lacking in MFR, was purposely selected to test the previous findings, as expected, the attachment of avirulent promastigotes (UR6) onto the cell line was found to be negligible when compared to the peritoneal macrophages. Thus, it appears that avirulent leishmania promastigotes probably utilize MFR significantly for their initial recognition and subsequent internalization by macrophages.     

Radio-attenuated leishmanial parasites as immunoprophylactic agent against experimental murine visceral leishmaniasis

The present study intends to evaluate the role of radio-attenuated leishmania parasites as immunoprophylactic agents for experimental murine visceral leishmaniasis. BALB/c mice were immunized with gamma (γ)-irradiated Leishmania donovani. A second immunization was given after 15 days of first immunization. After two immunizations, mice were infected with virulent L. donovani promastigotes. Protection against Kala-azar (KA) was estimated from spleen and liver parasitic burden along with the measurement of nitrite and superoxide anion generation by isolation of splenocytes and also by T-lymphocyte helper 1(Th1) and T-lymphocyte helper 2(Th2) cytokines release from the experimental groups. It was observed that BALB/c mice having prior immunization with radio-attenuated parasites showed protection against L. donovani infection through higher expression of Th1 cytokines and suppression of Th2 cytokines along with the generation of protective free radicals. The group of mice without prior priming with radio-attenuated parasites surrendered to the disease. Thus it can be concluded that radio-attenuated L. donovani may be used for.     

Extrapolating the effect of deleterious nsSNPs in the binding adaptability of flavopiridol with CDK7 protein: a molecular dynamics approach

Background

Recent reports suggest the role of nonsynonymous single nucleotide polymorphisms (nsSNPs) in cyclin-dependent kinase 7 (CDK7) gene associated with defect in the DNA repair mechanism that may contribute to cancer risk. Among the various inhibitors developed so far, flavopiridol proved to be a potential antitumor drug in the phase-III clinical trial for chronic lymphocytic leukemia. Here, we described a theoretical assessment for the discovery of new drugs or drug targets in CDK7 protein owing to the changes caused by deleterious nsSNPs.

Methods

Three nsSNPs (I63R, H135R, and T285M) were predicted to have functional impact on protein function by SIFT, PolyPhen2, I-Mutant3, PANTHER, SNPs&GO, PhD-SNP, and screening for non-acceptable polymorphisms (SNAP). Furthermore, we analyzed the native and proposed mutant models in atomic level 10 ns simulation using the molecular dynamics (MD) approach. Finally, with the aid of Autodock 4.0 and PatchDock, we analyzed the binding efficacy of flavopiridol with CDK7 protein with respect to the deleterious mutations.

Results

By comparing the results of all seven prediction tools, three nsSNPs (I63R, H135R, and T285M) were predicted to have functional impact on the protein function. The results of protein stability analysis inferred that I63R and H135R exhibited less deviation in root mean square deviation in comparison with the native and T285M protein. The flexibility of all the three mutant models of CDK7 protein is diverse in comparison with the native protein. Following to that, docking study revealed the change in the active site residues and decrease in the binding affinity of flavopiridol with mutant proteins.

Conclusion

This theoretical approach is entirely based on computational methods, which has the ability to identify the disease-related SNPs in complex disorders by contrasting their costs and capabilities with those of the experimental methods. The identification of disease related SNPs by computational methods has the potential to create personalized tools for the diagnosis, prognosis, and treatment of diseases.

Lay abstract

Cell cycle regulatory protein, CDK7, is linked with DNA repair mechanism which can contribute to cancer risk. The main aim of this study is to extrapolate the relationship between the nsSNPs and their effects in drug-binding capability. In this work, we propose a new methodology which (1) efficiently identified the deleterious nsSNPs that tend to have functional effect on protein function upon mutation by computational tools, (2) analyze d the native protein and proposed mutant models in atomic level using MD approach, and (3) investigated the protein-ligand interactions to analyze the binding ability by docking analysis. This theoretical approach is entirely based on computational methods, which has the ability to identify the disease-related SNPs in complex disorders by contrasting their costs and capabilities with those of the experimental methods. Overall, this approach has the potential to create personalized tools for the diagnosis, prognosis, and treatment of diseases.

     

Eukaryotic expression plasmid transfer from the intracellular bacterium Listeria monocytogenes to host cells

The facultative intracellular, Gram-positive bacterium Listeria monocytogenes invades phagocytic and non-phagocytic cells from the tissues and organs of a wide variety of animals and humans. Here, we report the use of these bacteria as vehicles for gene transfer. Eukaryotic expression plasmids were introduced into the nucleus of host cells following lysis of the intracytosolic, plasmid-carrying bacteria with antibiotics. Cell lines of different tissues and species could be transfected in this way. We examined bacterial properties required for delivery of the expression plasmids and found that this was strictly dependent on the ability of these bacteria to both invade eukaryotic cells and egress from the vacuole into the cytosol of the infected host cells. Macrophage-like cell lines or primary, peritoneal macrophages proved to be almost refractory to Listeria ‐mediated gene transfer. Thus, attenuated L. monocytogenes represents a serious candidate for consideration as a DNA-transfer vehicle for in vivo somatic gene therapy. The potential for oral administration of L. monocytogenes and the ease in producing and cultivating recombinant strains are further attributes that make its use as a gene transfer vehicle attractive.     

Pollen Transport in the Dark: Hawkmoths Prefer Non Crop Plants to Crop Plants in an Agricultural Landscape

In many previous studies hawkmoths have been found to be involved in nocturnal pollination of many plants. But their role in crop pollination is still unexplored. This study tried to evaluate their potential to carry pollens of crop. Hawkmoths were collected by light traps and slides were made from the pollen attached to their body. The hawkmoths and the pollens were identified. A pollen transport network was created using the data. The species level indices of each plant species in the network were estimated. Only a small fraction of pollens were made by the crops among the total types of pollens carried by the hawkmoths. Rest of the pollens belonged to that of the non crops. The species level indices suggest the crop pollens to be relatively less important in the network as reflected in the lower values of species degree and species strength. The higher species level specialisation values of crop pollens suggested they are restricted to be carried by few hawkmoth species only. It is clearly revealed that hawkmoths carry mostly non crop pollens, not crop pollens.     

Structure of internalin,a major invasion protein of Listeria monocytogenes,in complex with its human receptor E-cadherin

Listeria monocytogenes, a food-borne bacterial pathogen, enters mammalian cells by inducing its own phagocytosis. The listerial protein internalin (InlA) mediates bacterial adhesion and invasion of epithelial cells in the human intestine through specific interaction with its host cell receptor E-cadherin. We present the crystal structures of the functional domain of InlA alone and in a complex with the extracellular, N-terminal domain of human E-cadherin (hEC1). The leucine rich repeat (LRR) domain of InlA surrounds and specifically recognizes hEC1. Individual interactions were probed by mutagenesis and analytical ultracentrifugation. These include Pro16 of hEC1, a major determinant for human susceptibility to L. monocytogenes infection that is essential for intermolecular recognition. Our studies reveal the structural basis for host tro-pism of this bacterium and the molecular deception L. monocytogenes employs to exploit the E-cadherin system.     

Enhanced intrinsic migration of aggressive breast cancer cells by inhibition of Rac1 GTPase

Rac GTPases are known to play a crucial role in regulating cytoskeletal changes necessary for cell migration. Migration has been shown to be positively regulated by Rac in most cell types. However, there is also a large body of conflicting evidence in some other cell types with respect to the role of Rac in migration, suggesting that Rac GTPases regulate cell migration in a cell type-dependent manner. In the present study, we have characterized the effects of Rac1 GTPase inhibition on the migratory abilities of a number of breast cancer cell lines with differential degrees of tumorigenic and metastatic potentials. We show that Rac1 inhibition in non-metastatic (MCF-7, T-47D) or moderately metastatic (Hs578T) cell lines results in inhibition of migration, whereas in highly metastatic cell lines (MDA-MB-435, MDA-MB-231, and C3L5) Rac1 inhibition results in stimulation of migration. This stimulation of migration following Rac1 inhibition is also accompanied by the enhanced RhoA activity, suggesting a possible existence of a dominating role of RhoA over Rac1 in regulating intrinsic migration of the highly metastatic breast cancer cells.     

Altered N-glycosylation in macrophage x melanoma fusion hybrids.

It was recently reported that a majority of hybrids generated in vitro between weakly metastatic mouse Cloudman S91 melanoma cells and human or mouse macrophages showed enhanced metastatic potential (Rachkovsky et al., 1998). With few exceptions, hybrids with enhanced metastatic potential also had elevated basal melanin content, enhanced chemotactic responses to fibroblast-conditioned media, and stronger responsiveness to MSH compared to parental cells. Analyses revealed that altered N-glycosylation in metastatic hybrids could explain the multiple phenotypic changes. Tyrosinase, TRP-2 and LAMP-1 from hybrids migrated more slowly on gels compared to the same proteins from parental melanoma cells, consistent with increased glycosylation. Migration of LAMP-1 from hybrids was similar to that from peritoneal macrophages which also appeared to be more heavily glycosylated than LAMP-1 from Cloudman cells. The incorporation of 3H-glucosamine, as a marker of N-glycosylation, into tyrosinase and LAMP-1 was found to be elevated in hybrids, suppressed by N-glycosylation inhibitors and stimulated by MSH to a greater degree in hybrids compared to parental cells. These results indicate N-glycosylation as an important regulatory pathway for MSH-induced melanogenesis, and further suggest that altered N-linked glycosylation may be an underlying mechanism for regulation of both melanogenesis and metastasis in macrophage x melanoma hybrids.