Modulation of PPAR subtype selectivity. Part 2: Transforming PPARα/γ dual agonist into α selective PPAR agonist through bioisosteric modification

A novel series of oxime containing benzyl-1,3-dioxane-r-2-carboxylic acid derivatives (6a-k) were designed as selective PPARα agonists, through bioisosteric modification in the lipophilic tail region of PPARα/γ dual agonist. Some of the test compounds (6a, 6b, 6c and 6f) showed high selectivity towards PPARα over PPARγ in vitro. Further, highly potent and selective PPARα agonist 6c exhibited significant antihyperglycemic and antihyperlipidemic activity in vivo, along with its improved pharmacokinetic profile. Favorable in-silico interaction of 6c with PPARα binding pocket correlate its in vitro selectivity profile toward PPARα over PPARγ. Together, these results confirm discovery of novel series of oxime based selective PPARα agonists for the safe and effective treatment of various metabolic disorders.     

Genetic control of leaf-blade morphogenesis by the <Emphasis Type="Italic">INSECATUS</Emphasis> gene in <Emphasis Type="Italic">Pisum sativum</Emphasis>

To understand the role of INSECATUS (INS) gene in pea, the leaf blades of wild-type, ins mutant and seven other genotypes, constructed by recombining ins with uni-tac, af, tl and mfp gene mutations, were quantitatively compared. The ins was inherited as a recessive mutant allele and expressed its phenotype in proximal leaflets of full size leaf blades. In ins leaflets, the midvein development was arrested in distal domain and a cleft was formed in lamina above this point. There was change in the identity of ins leaflets such that the intercalary interrupted midvein bore a leaf blade. Such adventitious blades in ins, ins tl and ins tl mfp were like the distal segment of respective main leaf blade. The ins phenotype was not seen in ins af and ins af uni-tac genotypes. There was epistasis of uni-tac over ins. The ins, tl and mfp mutations interacted synergistically to produce highly pronounced ins phenotype in the ins tl mfp triple mutant. The role(s) of INS in leaf-blade organogenesis are: positive regulation of vascular patterning in leaflets, repression of UNI activity in leaflet primordia for ectopic growth and in leaf-blade primordium for indeterminate growth of rachis, delimitation of proximal leaflet domain and together with TL and MFP homeostasis for meristematic activity in leaflet primordia. The variant apically bifid shape of the affected ins leaflets demonstrated that the leaflet shape is dependent on the venation pattern.     

Apolipoprotein B-containing lipoprotein assembly in microsomal triglyceride transfer protein-deficient McA-RH7777 cells

Microsomal triglyceride transfer protein (MTP) is required for the assembly and secretion of apolipoprotein (apo) B-containing lipoproteins. Previously, we demonstrated that the N-terminal 1,000 residues of apoB (apoB:1000) are necessary for the initiation of apoB-containing lipoprotein assembly in rat hepatoma McA-RH7777 cells and that these particles are phospholipid (PL) rich. To determine if the PL transfer activity of MTP is sufficient for the assembly and secretion of primordial apoB:1000-containing lipoproteins, we employed microRNA-based short hairpin RNAs (miR-shRNAs) to silence Mttp gene expression in parental and apoB:1000-expressing McA-RH7777 cells. This approach led to 98% reduction in MTP protein levels in both cell types. Metabolic labeling studies demonstrated a drastic 90–95% decrease in the secretion of rat endogenous apoB100-containing lipoproteins in MTP-deficient McA-RH7777 cells compared with cells transfected with negative control miR-shRNA. A similar reduction was observed in the secretion of rat endogenous apoB48 under the experimental conditions employed. In contrast, MTP absence had no significant effect on the synthesis, lipidation, and secretion of human apoB:1000-containing particles. These results provide strong evidence in support of the concept that in McA-RH7777 cells, acquisition of PL by apoB:1000 and initiation of apoB-containing lipoprotein assembly, a process distinct from the conventional first-step assembly of HDL-sized apoB-containing particles, do not require MTP. This study indicates that, in hepatocytes, a factor(s) other than MTP mediates the formation of the PL-rich primordial apoB:1000-containing initiation complex.     

Synthesis of an Fmoc-threonine bearing core-2 glycan: A building block for PSGL-1 via Fmoc-assisted solid-phase peptide synthesis

Selectins (L, E, and P) are vascular endothelial molecules that play an important role in the recruitment of leukocytes to inflamed tissue. In this regard, P-Selectin glycoprotein-1 (PSGL-1) has been identified as a ligand for P-Selectin. PSGL-1 binds to P-Selectin through the interaction of core-2 O-glycan expressing sialyl Lewis^x oligosaccharide and the three tyrosine sulfate residues. Herein, we report the synthesis of threonine-linked core-2 O-glycan as an amino acid building block for the synthesis of PSGL-1. This building block was further incorporated in the Fmoc-assisted solid-phase peptide synthesis to provide a portion of the PSGL-1 glycopeptide.     

Recruitment of memory B cells to lymph nodes remote from the site of immunization requires an inflammatory stimulus

Successful recall Ab responses require recruitment of quiescent memory B cells to secondary lymphoid organs. However, the cellular dynamics of memory cells responding to local antigenic challenge at lymphoid sites distal from the initial Ag encounter are not well understood. We show in this study that memory B cells generated following s.c. immunization in one footpad generate secondary responses to soluble Ag given i.p. but not to Ag given s.c. in the contralateral footpad unless LPS is coadministered. Memory B cells do not express CD62L, and CD62L(-ve) cells cannot enter lymph nodes unless LPS-mediated inflammation is induced there. Functional TLR4 is required on the B cells, as well as on non-B cells, in the lymph node to achieve full recruitment. Furthermore, splenectomized mice fail to respond to such inflammatory s.c. challenge in contralateral footpads, unlike lymphadenectomized mice lacking the original draining lymph nodes. Splenectomized mice also fail to respond to i.p. challenge with soluble Ag. Together, these data indicate that, unlike the central memory pool of T cells, which circulates through resting lymph nodes, the majority of long-lived memory B cells are spleen resident and require inflammatory signals for mounting recall responses at distal challenge sites.     

Changing Museum Visitors’ Conceptions of Evolution

We examined whether a single visit to an evolution exhibition contributed to conceptual change in adult (n?=?30), youth, and child (n?=?34) museum visitors?? reasoning about evolution. The exhibition included seven current research projects in evolutionary science, each focused on a different organism. To frame this study, we integrated a developmental model of visitors?? understanding of evolution, which incorporates visitors?? intuitive beliefs, with a model of free-choice learning that includes personal, sociocultural, and contextual variables. Using pre- and post-measures, we assessed how visitors?? causal explanations about biological change, drawn from three reasoning patterns (evolutionary, intuitive, and creationist), were modified as a result of visiting the exhibition. Whatever their age, background beliefs, or prior intuitive reasoning patterns, visitors significantly increased their use of explanations from the evolutionary reasoning pattern across all measures and extended this reasoning across diverse organisms. Visitors also increased their use of one intuitive reasoning pattern, need-based (goal-directed) explanations, which, we argue, may be a step toward evolutionary reasoning. Nonetheless, visitors continued to use mixed reasoning (endorsing all three reasoning patterns) in explaining biological change. The personal, socio-cultural, and contextual variables were found to be related to these reasoning patterns in predictable ways. These findings are used to examine the structure of visitors?? reasoning patterns and those aspects of the exhibition that may have contributed to the gains in museum visitors?? understanding of evolution.     

Mechanistic and Structural Understanding of Uncompetitive Inhibitors of Caspase-6

Inhibition of caspase-6 is a potential therapeutic strategy for some neurodegenerative diseases, but it has been difficult to develop selective inhibitors against caspases. We report the discovery and characterization of a potent inhibitor of caspase-6 that acts by an uncompetitive binding mode that is an unprecedented mechanism of inhibition against this target class. Biochemical assays demonstrate that, while exquisitely selective for caspase-6 over caspase-3 and -7, the compound’s inhibitory activity is also dependent on the amino acid sequence and P1’ character of the peptide substrate. The crystal structure of the ternary complex of caspase-6, substrate-mimetic and an 11 nM inhibitor reveals the molecular basis of inhibition. The general strategy to develop uncompetitive inhibitors together with the unique mechanism described herein provides a rationale for engineering caspase selectivity.     

Localization of PBP3 in Caulobacter crescentus is highly dynamic and largely relies on its functional transpeptidase domain

In rod-shaped bacteria, septal peptidoglycan synthesis involves the late recruitment of the ftsI gene product (PBP3 in Escherichia coli) to the FtsZ ring. We show that in Caulobacter crescentus, PBP3 accumulates at the new pole at the beginning of the cell cycle. Fluorescence recovery after photobleaching experiments reveal that polar PBP3 molecules are, constantly and independently of FtsZ, replaced by those present in the cellular pool, implying that polar PBP3 is not a remnant of the previous division. By the time cell constriction is initiated, all PBP3 polar accumulation has disappeared in favour of an FtsZ-dependent localization near midcell, consistent with PBP3 function in cell division. Kymograph analysis of time-lapse experiments shows that the recruitment of PBP3 to the FtsZ ring is progressive and initiated very early on, shortly after FtsZ ring formation and well before cell constriction starts. Accumulation of PBP3 near midcell is also highly dynamic with a rapid exchange of PBP3 molecules between midcell and cellular pools. Localization of PBP3 at both midcell and pole appears multifactorial, primarily requiring the catalytic site of PBP3. Collectively, our results suggest a role for PBP3 in pole morphogenesis and provide new insights into the process of peptidoglycan assembly during division.