Structure-based design of novel human Pin1 inhibitors (II)

Following the discovery of a novel series of phosphate-containing small molecular Pin1 inhibitors, the drug design strategy shifted to replacement of the phosphate group with an isostere with potential better pharmaceutical properties. The initial loss in potency of carboxylate analogs was likely due to weaker charge–charge interactions in the putative phosphate binding pocket and was subsequently recovered by structure-based optimization of ligand–protein interactions in the proline binding site, leading to the discovery of a sub-micromolar non-phosphate small molecular Pin1 inhibitor.     

The X Chromosomes of Mammals: Karyological Homology as Revealed by Banding Techniques

A comparison of the Giemsa-banding patterns of the X chromosomes in various mammalian species including man indicates that two major bands (A and B), which are resistant to trypsin and urea-treatments, are always present irrespective of the gross morphology of the X chromosomes. This is true in all mammalian species with the "original or standard type" X chromosomes (5-6% of the haploid genome) thus far analyzed. In the unusually large-sized X chromosomes the extra chromosomal material may be due either to the addition of genetically inert constitutive heterochromatin or to an X-autosome translocation. In these X chromosomes two major bands are present in the actual X-chromosome segment. Our data on C and G band patterns also support Ohno's hypothesis that the mammalian X chromosome is extremely conservative in its genetic content, in spite of its cytogenetic variability.     

From molecular signal activation to locomotion: an integrated, multiscale analysis of cell motility on defined matrices

The adhesion, mechanics, and motility of eukaryotic cells are highly sensitive to the ligand density and stiffness of the extracellular matrix (ECM). This relationship bears profound implications for stem cell engineering, tumor invasion and metastasis. Yet, our quantitative understanding of how ECM biophysical properties, mechanotransductive signals, and assembly of contractile and adhesive structures collude to control these cell behaviors remains extremely limited. Here we present a novel multiscale model of cell migration on ECMs of defined biophysical properties that integrates local activation of biochemical signals with adhesion and force generation at the cell-ECM interface. We capture the mechanosensitivity of individual cellular components by dynamically coupling ECM properties to the activation of Rho and Rac GTPases in specific portions of the cell with actomyosin contractility, cell-ECM adhesion bond formation and rupture, and process extension and retraction. We show that our framework is capable of recreating key experimentally-observed features of the relationship between cell migration and ECM biophysical properties. In particular, our model predicts for the first time recently reported transitions from filopodial to "stick-slip" to gliding motility on ECMs of increasing stiffness, previously observed dependences of migration speed on ECM stiffness and ligand density, and high-resolution measurements of mechanosensitive protrusion dynamics during cell motility we newly obtained for this study. It also relates the biphasic dependence of cell migration speed on ECM stiffness to the tendency of the cell to polarize. By enabling the investigation of experimentally-inaccessible microscale relationships between mechanotransductive signaling, adhesion, and motility, our model offers new insight into how these factors interact with one another to produce complex migration patterns across a variety of ECM conditions.     

Transcriptional status of known and novel genes tagged with consensus of 33.15 repeat loci employing minisatellite-associated sequence amplification (MASA) and real-time PCR in water buffalo, Bubalus bubalis

We conducted minisatellite-associated sequence amplification (MASA) with an oligo (5' CACCTCTCCACCTGCC 3') based on consensus of 33.15 repeat loci using cDNA from the testis, ovary, spleen, kidney, heart, liver, and lung of water buffalo Bubalus bubalis and uncovered 25 amplicons of six different sizes (1,263, 846/847, 602, 576, 487, and 324 base pairs). These fragments, cloned and sequenced, were found to represent several functional, regulatory, and structural genes. Blast search of all the 25 amplicons showed homologies with 43 transcribing genes across the species. Of these, the 846/847-bp fragment, having homology with the adenylate kinase gene, showed nucleotide changes at six identical places in the ovary and testis. The 1,263; 324; and 487-bp fragments showed homology with the secreted modular calcium binding protein (SMOC-1), leucine-rich repeat neuronal 6A (LRRN6A) mRNA, and human TTTY5 mRNA, respectively. Real-time PCR showed maximum expression of AKL, LRRN6A, and T-cell receptor gamma (TCR-gamma)-like genes in the testis, SMOC-1 in the liver, and the T-cell receptor-like (TCRL) gene in the spleen compared to those used as endogenous control. We construe that these genes have evolved from a common progenitor and conformed to various biological functions during the course of evolution. MASA approach coupled with real-time PCR has potentials to uncover accurate expression of a large number of genes within and across the species circumventing the screening of cDNA library.     

Identification of two distinct human immunodeficiency virus type 1 Vif determinants critical for interactions with human APOBEC3G and APOBEC3F

Human cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) inhibit replication of Vif-deficient human immunodeficiency virus type 1 (HIV-1). HIV-1 Vif overcomes these host restriction factors by binding to them and inducing their proteasomal degradation. The Vif-A3G and Vif-A3F interactions are attractive targets for antiviral drug development because inhibiting the interactions could allow the host defense mechanism to control HIV-1 replication. It was recently reported that the Vif amino acids D(14)RMR(17) are important for functional interaction and degradation of the previously identified Vif-resistant mutant of A3G (D128K-A3G). However, the Vif determinants important for functional interaction with A3G and A3F have not been fully characterized. To identify these determinants, we performed an extensive mutational analysis of HIV-1 Vif. Our analysis revealed two distinct Vif determinants, amino acids Y(40)RHHY(44) and D(14)RMR(17), which are essential for binding to A3G and A3F, respectively. Interestingly, mutation of the A3G-binding region increased Vif's ability to suppress A3F. Vif binding to D128K-A3G was also dependent on the Y(40)RHHY(44) region but not the D(14)RMR(17) region. Consistent with previous observations, subsequent neutralization of the D128K-A3G antiviral activity required substitution of Vif determinant D(14)RMR(17) with SEMQ, similar to the SERQ amino acids in simian immunodeficiency virus SIV(AGM) Vif, which is capable of neutralizing D128K-A3G. These studies are the first to clearly identify two distinct regions of Vif that are critical for independent interactions with A3G and A3F. Pharmacological interference with the Vif-A3G or Vif-A3F interactions could result in potent inhibition of HIV-1 replication by the APOBEC3 proteins.     

A unique and highly efficient non-viral DNA/siRNA delivery system based on PEI-bisepoxide nanoparticles

Delivery of DNA and siRNA into mammalian cells is a powerful technique in treating various diseases caused by single gene defects. Herein, we report a highly efficient delivery system using 1,4-butanediol diglycidyl ether (bisepoxide) crosslinked polyethylenimine (PEI) nanoparticles (PN). The nanoparticle/DNA complexes (nanoplexes) exibited approximately 2.5- to 5.0-fold gene transfer efficacy and decreased cytotoxicity in cultured cell lines, compared to the native PEI (25 kDa) (gold standard) and commercially available transfection agents such as Lipofectamine 2000 and Fugene. The bisepoxide crosslinking results in change in amine ratio in PEI; however, it retains the net charge on PN unaltered. A series of nanoparticles obtained by varying the degree of crosslinking was found to be in the size range of 69-77 nm and the zeta potential varying from +35 to 40 mV. The proposed system was also found to deliver siRNA efficiently into HEK cells, resulting in approximately 70% suppression of the targetted gene (GFP).     

Absence of ClC5 in knockout mice leads to glycosuria, impaired renal glucose handling and low proximal tubule GLUT2 protein expression.

Glycosuria is one of the well-documented characteristics in ClC-5 knockout (KO) mice and patients with Dent's disease. However, the underlying pathophysiology of its occurrence is unknown. In this study, we have compared ClC-5 KO mice with age and gender matched wild-type (WT) control mice to investigate if the underlying cause of manifested glycosuria is an impairment of glucose homeostasis and/or an alteration in expression levels of proximal tubule (PT) glucose transporters. We observed that, the blood glucose concentration (n=12, p<0.01) and the fractional excretion of glucose and insulin (n=6, p<0.05) were higher in KO mice. In contrast, the fasting blood glucose levels (n=7) were not significantly different in the two groups. Plasma glucose increased to a greater extent in KO mice (n=7, p<0.05) when challenged by an intraperitoneal injection of glucose. However, no peripheral tissue insulin resistance was observed following an intraperitoneal injection of insulin (n=9) in the KO mice. ELISA analysis demonstrated low plasma insulin concentrations after a 12 hour fasting period and also following glucose injection in KO mice. The total insulin released during a 2 hour period following glucose challenge was significantly lower in KO mice (n=6, p<0.05). By western blot, we observed a significant decrease in GLUT2 protein expression levels in isolated PT ((n=10, p<0.01)) of KO mice. This decrease in protein levels was corroborated by a significant decrease in GLUT2 mRNA levels estimated semi quantitatively by RT-PCR in isolated PT (n=10, p<0.01). No significant changes in mRNA expression levels of SGLT2, SGLT1 and GLUT1, as analyzed by RT-PCR, could be detected in the isolated PT (n=10). Also, we have shown by western blot analysis that expression of megalin is lower in the renal cortex of KO mice when compared to WT mice (n=3, p<0.05). Our results suggest that low plasma insulin concentration together with renal function changes observed in KO mice significantly contribute towards the glucose intolerance and documented glycosuria observed in this animal.     

Closing in on the resting state of the Shaker K(+) channel

Membrane depolarization causes voltage-gated ion channels to transition from a resting/closed conformation to an activated/open conformation. We used voltage-clamp fluorometry to measure protein motion at specific regions of the Shaker Kv channel. This enabled us to construct new structural models of the resting/closed and activated/open states based on the Kv1.2 crystal structure using the Rosetta-Membrane method and molecular dynamics simulations. Our models account for the measured gating charge displacement and suggest a molecular mechanism of activation in which the primary voltage sensors, S4s, rotate by approximately 180 degrees as they move "outward" by 6-8 A. A subsequent tilting motion of the S4s and the pore domain helices, S5s, of all four subunits induces a concerted movement of the channel's S4-S5 linkers and S6 helices, allowing ion conduction. Our models are compatible with a wide body of data and resolve apparent contradictions that previously led to several distinct models of voltage sensing.     

Enantioselective phenylacetylene addition to aldehydes and ketones catalyzed by recyclable polymeric Zn(salen) complex

New polymeric Zn(salen) complex was employed in the enantioselective phenylacetylene addition to aldehydes and ketones to produce corresponding chiral secondary propargylic alcohols with yields (up to 96%) and enantioselectivity (up to 72%) and tertiary propargylic alcohols with yields (up to 79%) and enantioselectivity (up to 68%) at room temperature, with added advantage of four times reuse with retention of enantioselectivity.