The role of ion antiporters in the maintenance of intracellular pH in rat vascular smooth muscle cells

Vascular smooth muscle intracellular pH is maintained by the Na⁺/H⁺ and Cl^–/HCO ₃ ^– antiporters. The Na⁺/H⁺ exchanger is a major route of H⁺ extrusion in most eukaryotic cells and is present in vascular smooth muscle cells in a similar capacity. It extrudes H^– into the extracellular space in exchange for Na⁺. The Cl^–/HCO ₃ ^– exchanger plays an analogous role to lower the pH of vascular smooth muscle cells when increases in intracellular pH occur. Its activity has also been demonstrated in A7r5 and A10 vascular smooth muscle cells. The Na⁺/H⁺ exchanger is regulated by a number of agents which act through inositol trisphosphate/diacylglycerol, to stimulate the antiporter. Calcium-calmodulin dependent protein kinase may also activate the antiporter in vivo. Phosphorylation of the Cl^–/HCO ₃ ^– exchanger has also been observed but its physiological role is not known. Both these antiporters exist in the plasma membrane as integral proteins with free acidic cytoplasmic termini. These regions may be important in sensing changes in intracellular pH, to which these antiporters respond.Abbreviations CaM Calmodulin - DCCD Dicylohexyl-Carbodiimide - DG Diacylglycerol - DIDS-4 4-Diisthiocyanostilbene-2,2-Disulfonic Acid - IP₃ Inositol Trisphosphate - PKC protein Kinase C - SITS-4 4-Acetamido-4-Isothiocyanstilbene-2,2-Disulfonate - VSMC Vascular Smooth Muscle Cell     

P78-T Solid-Phase Strategy for Phosphorylation/ Glycosylation Site Mapping

Chemically induced β-elimination of phosphate from serine and threonine coupled with Michael addition has emerged as a chemical strategy to address both the ion suppression and the gas-phase lability of the phosphate group. In previous work, we have adopted the chemistry to solid-phase derivatization on C18 ZipTip pipette tips using barium hydroxide as elimination base and 2-amino-ethanethiol as nucleophile.¹ The utility of the protocol for improved phosphopeptide detection by signal enhancement was demonstrated with low-level amounts of tryptic protein digests, and the resultant increased MS/MS spectral information content greatly facilitated mapping of the site of phosphorylation.In this report, we have focused on chemistry optimization of O-phospho and O-GlcNAc modified peptides reported as resistant to β-elimination, i.e., those containing the modified residues followed by proline. Conclusive mapping of these phosphorylation sites has become increasingly important in view of the fact that phosphorylated Ser/Thr-Pro motifs are substrates for prolyl cis/trans isomerase Pin1.² Similarly, unambiguous site determination of O-GlcNAc modifications has more recently attracted considerable interest because of the global and often site-specific reciprocal relationship between O-GlcNAc and O-phosphate in many cellular responses.³ We have used a panel of model peptides to define the optimal reaction conditions for both concurrent and consecutive elimination/Michael addition reactions and employed carbon/C18 mixed-phase ZipTips to afford efficient binding of small hydrophilic peptides.     

The adaptive significance of mandibular symphyseal fusion in mammals

The mandibular symphyseal joint is remarkably variable across major mammalian clades, ranging in adults from unfused (amphiarthrosis) to partially fused (synarthrosis) to completely ossified (synostosis). Experimental work conducted on primates suggests that greater ossification of the symphysis is a response to increased recruitment of the balancing-side (i.e. nonchewing side) jaw-adductor muscles during forceful unilateral biting and chewing, with increased fusion strengthening the symphysis against correspondingly elevated joint stresses. It is thus expected that species with diets composed primarily of foods that require high-magnitude bite forces and/or repetitive loading to process will be characterized by greater degrees of symphyseal ossification than species with relatively easy-to-process diets (i.e. food items typified by low toughness and/or low stiffness). However, comparative support for this idea is limited. We tested this hypothesis in four dietarily diverse mammalian clades characterized by variation in symphyseal fusion - the Strepsirrhini, Marsupialia, Feliformia, and Caniformia. We scored fusion in adult specimens of 292 species, assigned each to a dietary category based on literature accounts, and tested for an association between these two variables using Pagel's test for the correlated evolution of binary characters. Results indicate that greater fusion is associated with diets composed of resistant items in strepsirrhines, marsupials, and feliforms, providing some support for the hypothesis. However, no such relationship was detected in caniforms, suggesting that factors other than dietary mechanical properties influence symphyseal ossification. Future work should focus on such factors, as well as those that favour an unfused mandibular symphysis.     

Distinct subcellular localization for constitutive and agonist-modulated palmitoylation of the human delta opioid receptor

Protein palmitoylation is a reversible lipid modification that plays important roles for many proteins involved in signal transduction, but relatively little is known about the regulation of this modification and the cellular location where it occurs. We demonstrate that the human delta opioid receptor is palmitoylated at two distinct cellular locations in human embryonic kidney 293 cells and undergoes dynamic regulation at one of these sites. Although palmitoylation could be readily observed for the mature receptor (Mr 55,000), [3H]palmitate incorporation into the receptor precursor (Mr 45,000) could be detected only following transport blockade with brefeldin A, nocodazole, and monensin, indicating that the modification occurs initially during or shortly after export from the endoplasmic reticulum. Blocking of palmitoylation with 2-bromopalmitate inhibited receptor cell surface expression, indicating that it is needed for efficient intracellular transport. However, cell surface biotinylation experiments showed that receptors can also be palmitoylated once they have reached the plasma membrane. At this location, palmitoylation is regulated in a receptor activation-dependent manner, as was indicated by the opioid agonist-promoted increase in the turnover of receptor-bound palmitate. This agonist-mediated effect did not require receptor-G protein coupling and occurred at the cell surface without the need for internalization or recycling. The activation-dependent modulation of receptor palmitoylation may thus contribute to the regulation of receptor function at the plasma membrane.     

Effects of ezetimibe and simvastatin on apolipoprotein B metabolism in males with mixed hyperlipidemia

Sixteen hyperlipidemic men were enrolled in a randomized, placebo-controlled, double-blind, cross-over study to evaluate the effect of ezetimibe 10 mg and simvastatin 40 mg, coadministered and alone, on the in vivo kinetics of apolipoprotein (apo) B-48 and B-100 in humans. Subjects underwent a primed-constant infusion of a stable isotope in the fed state. The coadministration of simvastatin and ezetimibe significantly reduced plasma concentrations of cholesterol (−43.0%), LDL-C (−53.6%), and triglycerides (−44.0%). Triglyceride-rich lipoproteins (TRL) apoB-48 pool size (PS) was significantly decreased (−48.9%) following combination therapy mainly through a significant reduction in TRL apoB-48 production rate (PR) (−38.0%). The fractional catabolic rate (FCR) of VLDL and LDL apoB-100 were significantly increased with all treatment modalities compared with placebo, leading to a significant reduction in the PS of these fractions. We also observed a positive correlation between changes in TRL apoB-48 PS and changes in TRL apoB-48 PR (r = 0.85; P < 0.0001) with combination therapy. Our results indicate that treatment with simvastatin plus ezetimibe is effective in reducing plasma TRL apoB-48 levels and that this effect is most likely mediated by a reduction in the intestinal secretion of TRL apoB-48. Our study also indicated that the reduction in LDL-C concentration following combination therapy is mainly driven by an increase in FCR of apoB-100 containing lipoproteins.     

Phylogeography of the marine macroalga Sargassum hemiphyllum (Phaeophyceae,Heterokontophyta) in northwestern Pacific

Sargassum hemiphyllum is commonly found in Japan and Korea, with a variety, var. chinense, that is found distributed in the southern Chinese coast. We previously reported distinct genetic differentiation between the two taxa based on the PCR‐RFLP data of plastid RubiscoL‐S spacer. The present study aims at elucidating the phylogeographic pattern of S. hemiphyllum based on more markers in the nuclear and extranuclear genomes, with a view to reveal the occurrence of hybridization. The two allopatrically distributed taxa were found to be genetically distinct in nuclear ITS2, plastidial Rubisco (Rbc) and mitochondrial TrnW_I (Trn) spacers. Their divergence was postulated to be attributable to the vicariant event which resulted from the isolation of the Sea of Japan during the late Miocene (6.58–11.25 Mya). Divergence within both S. hemiphyllum and the chinense variety was observed based on Trn spacer, while the divergence in S. hemiphyllum was further confirmed in Rbc spacer. This divergence appears to correspond to the separation of the Japanese populations between the Sea of Japan and the Pacific that occurred around 0.92–2.88 Mya (the early Pleistocene). The presence of an ITS2 clone resembling var. chinense sequences in a Japanese population of S. hemiphyllum (JpNS) raises the possibility of the introgression of var. chinense individuals into S. hemiphyllum population. Compared to that between S. hemiphyllum and the chinense variety, hybridization among the Japanese and Korean populations of S. hemiphyllum is highly probable as all these individuals share a pool of nuclear ITS2 sequences, possibly attributable to incomplete concerted evolution of ITS2.     

Macroevolutionary pattern of sexual size dimorphism in geckos corresponds to intraspecific temperature‐induced variation

Many animal lineages exhibit allometry in sexual size dimorphism (SSD), known as ‘Rensch’s rule’. When applied to the interspecific level, this rule states that males are more evolutionary plastic in body size than females and that male‐biased SSD increases with body size. One of the explanations for the occurrence of Rensch’s rule is the differential‐plasticity hypothesis assuming that higher evolutionary plasticity in males is a consequence of larger sensitivity of male growth to environmental cues. We have confirmed the pattern consistent with Rensch’s rule among species of the gecko genus Paroedura and followed the ontogeny of SSD at three constant temperatures in a male‐larger species (Paroedura picta). In this species, males exhibited larger temperature‐induced phenotypic plasticity in final body size than females, and body size and SSD correlated across temperatures. This result supports the differential‐plasticity hypothesis and points to the role phenotypic plasticity plays in generating of evolutionary novelties.