Cytidine is a novel substrate for wild-type concentrative nucleoside transporter 2

Nucleoside transporter (NT) plays key roles in the physiology of nucleosides and the pharmacology of its analogues in mammals. We previously cloned Na+/nucleoside cotransporter CNT2 from mouse M5076 ovarian sarcoma cells, the peptide encoded by it differing from that by the previously reported mouse CNT2 in five substitutions, and observed that the transporter can take up cytidine, like CNT1 and CNT3. In the present study, we examined which of the two aforementioned CNT2 is the normal one, and whether or not cytidine is transported via the previously reported CNT2. The peptide encoded by CNT2 derived from mouse intestine, liver, spleen, and ovary was identical to that previously reported. The uptake of [3H]cytidine, but not [3H]thymidine, by Cos-7 cells transfected with CNT2 cDNA obtained from mouse intestine was much greater than that by mock cells, as in the case of [3H]uridine, a typical substrate of NT. [3H]Cytidine and [3H]uridine were taken up via CNT2, in temperature-, extracellular Na+-, and substrate concentration-dependent manners. The uptake of [3H]cytidine and [3H]uridine mediated by CNT2 was significantly inhibited by the variety of nucleosides used in this study, except for thymidine, and inhibition of the [3H]uridine uptake by cytidine was competitive. The [3H]uridine uptake via CNT2 was significantly decreased by the addition of cytarabin or gemcitabine, antimetabolites of cytidine analogue. These results indicated that the previously reported mouse CNT2 is the wild-type one, and cytidine is transported mediated by the same recognition site on the CNT2 with uridine, and furthermore, cytidine analogues may be substrates for the transporter.     

Roles of adrenomedullin 2 in regulating the cardiovascular and sympathetic nervous systems in conscious rats

Recently, a new member of the calcitonin gene-related peptide (CGRP) family, adrenomedullin 2 (AM2) or intermedin (IMD), was identified. AM2/IMD has been shown to have a vasodilator effect in mice and rats and an effect on urine formation in rats. In the present study, we investigated the effects of intravenously infused rat AM2 (rAM2) on blood pressure (BP), heart rate (HR), renal sympathetic nerve activity (RSNA), and renal blood flow (RBF) in conscious unrestrained rats relative to the effects of rat adrenomedullin (rAM) and proadrenomedullin NH2-terminal 20 peptide (rPAMP). Intravenous infusion of rAM2 (5 nmol/kg) significantly decreased BP and increased HR, RSNA, and RBF. These hypotensive and sympathoexcitatory effects diminished after 20 min, and HR returned to control levels 30 min after cessation of the infusion. In contrast, a significant increase in RBF was still evident 60 min after cessation of the peptide infusion. The duration of BP, HR, and RSNA responses was longer with rAM (5 nmol/kg) than with rAM2 infusion, whereas the increases in RBF induced by rAM2 and rAM were similar in their amplitude and duration. Infusion of rPAMP (200 nmol/kg) increased HR and RSNA but had no effect on RBF. Baroreceptor denervation suppressed, but did not diminish, the increases in HR and RSNA to rAM2. These findings indicate that the physiological roles of rAM2 and rAM are similar and that rAM2 also has a long-lasting vasodilator action on the renal vascular bed.     

Heterogeneity in smooth muscle cell population accumulating in the neointimas and the media of poststenotic dilatation of the rabbit carotid artery.

Rabbit smooth muscles contain at least three types of myosin heavy chain (MHC) isoforms; SM1, SM2 and SMemb (NMHC-B), the expression of which is developmentally regulated. We have recently reported that smooth muscles with the embryonic phenotype accumulate in the neointimas produced by endothelial denudation or high-cholesterol feeding. In this study, we examined MHC isoform expression in the neointimas and the media of poststenotic dilatation of the rabbit carotid artery, and determined the phenotype of the smooth muscle cell in the dilated segment. We report here that neointimal cells in the dilated segment are smooth muscle cells with the embryonic phenotype as previously reported in our ballooning-injury study. The medial smooth muscles, however, are composed of heterogeneous population of smooth muscles which differ in stage of differentiation as determined by the MHC isoform expression. These results indicate that MHC isoforms are useful molecular markers to identify abnormally proliferating smooth muscles in diseased arteries and to understand the process of atherogenesis occurring following vascular injury.     

Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control

Genetically encoded fluorescent calcium indicator proteins (FCIPs) are promising tools to study calcium dynamics in many activity-dependent molecular and cellular processes. Great hopes—for the measurement of population activity, in particular—have therefore been placed on calcium indicators derived from the green fluorescent protein and their expression in (selected) neuronal populations. Calcium transients can rise within milliseconds, making them suitable as reporters of fast neuronal activity. We here report the production of stable transgenic mouse lines with two different functional calcium indicators, inverse pericam and camgaroo-2, under the control of the tetracycline-inducible promoter. Using a variety of in vitro and in vivo assays, we find that stimuli known to increase intracellular calcium concentration (somatically triggered action potentials (APs) and synaptic and sensory stimulation) can cause substantial and rapid changes in FCIP fluorescence of inverse pericam and camgaroo-2.     

6-O-sulfation of heparan sulfate differentially regulates various fibroblast growth factor-dependent signalings in culture

Heparan sulfate (HS) interacts with diverse heparin-binding growth factors and thereby regulates their bioactivities. These interactions depend on the structures characterized by the sulfation pattern and isomer of uronic acid residues. One of the biosynthetic modifications of HS, namely 6-O-sulfation, is catalyzed by three isoforms of HS6-O-sulfotransferase. We generated HS6ST-1- and/or HS6ST-2-deficient mice (6ST1-KO, 6ST2-KO, and double knock-out (dKO)) that exhibited different phenotypes. We examined the effects of HS 6-O-sulfation in heparin-binding growth factor signaling using fibroblasts derived from these mutant mice. Mouse embryonic fibroblasts (MEF) prepared from E14.5 dKO mice produced HS with little 6-O-sulfate, whereas 2-O-sulfation in HS from dKO-MEF (dKO-HS) was increased by 1.9-fold. HS6-O-sulfotransferase activity in the dKO-MEF was hardly detected, and HS2-O-sulfotransferase activity was 1.5-fold higher than that in wild type (WT)-MEFs. The response of dKO-MEFs to fibroblast growth factors (FGFs) was distinct from that of WT-MEFs; in dKO-MEFs, FGF-4- and FGF-2-dependent signalings were reduced to approximately 30 and 60% of WT-MEFs, respectively, and FGF-1-dependent signaling was moderately reduced compared with that of WT-MEFs but only at the lower FGF-1 concentrations. Analysis with a surface plasmon resonance biosensor demonstrated that the apparent affinity of dKO-HS for FGF-4 was markedly reduced and was also reduced for FGF-1. In contrast, the affinity of dKO-HS for FGF-2 was 2.5-fold higher than that of HS from WT-MEFs. Thus, 6-O-sulfate in HS may regulate the signalings of some of HB-GFs, including FGFs, by inducing different interactions between ligands and their receptors.     

Prevention by polyhydroxysteroids and saponins of Asterina pectinifera of the desquamation of stratum corneum cells

Two polyhydroxysteroids and three steroidal saponins (pectiniosides A, B and C) were isolated as bioactive substances from Asterina pectinifera. These compounds inhibited the release of guinea-pig skin stratum corneum cells by 48-67% (control, 0%) at 2 mg/ml. It is suggested that the bioactivity of these compounds is connected with the remedial and preventive effects of an aqueous extract of A. pectinifera on rough human skin.     

Novel proteinaceous toxins from the nematocyst venom of the Okinawan sea anemone Phyllodiscus semoni Kwietniewski

The Okinawan sea anemone Phyllodiscus semoni is known to cause cases of severe stinging. We isolated P. semoni toxins 60A and 60B (PsTX-60A and PsTX-60B; ca. 60 kDa) as the major toxins from the isolated nematocysts of this species for the first time. PsTX-60A and PsTX-60B showed lethal toxicity to the shrimp Palaemon paucidence when administered via intraperitoneal injection (LD(50) values: 800-900 and 800 microg/kg, respectively) and hemolytic activity toward a 0.8% suspension of sheep red blood cells (ED(50) values: 600 and 300 ng/ml, respectively). Furthermore, we sequenced the cDNA encoding PsTX-60A. The deduced amino acid sequence of PsTX-60A did not show any similarity to previously reported proteins. The N-terminal amino acid sequence of PsTX-60B showed homology with that of PsTX-60A. These toxins represent a novel class of cytolytic proteinaceous toxins.     

Synthesis and structure of poly(binaphthyl salen manganese complex) and its application to asymmetric epoxidation

Poly(binaphthyl salen manganese complex)es 3-Mn were synthesized from a 3,3'-diformylbinaphthol derivative, alpha,omega-diamines, and Mn(OAc)2. Their helical structures were well-supported by their IR, UV, and CD spectra. The catalysis of 3-Mn in an asymmetric epoxidation was investigated.     

An expeditious synthesis of tamoxifen, a representative SERM (selective estrogen receptor modulator), via the three-component coupling reaction among aromatic aldehyde, cinnamyltrimethylsilane, and beta-chlorophenetole

Two new synthetic pathways to the anti-cancer agent tamoxifen and its derivatives were developed. The first route involved the aldol reaction of benzyl phenyl ketone with acetaldehyde followed by Friedel–Crafts substitution with anisole in the presence of Cl₂Si(OTf)₂ to produce 1,1,2-triaryl-3-acetoxybutane, a precursor of the tamoxifen derivatives. The second one utilized the novel three-component coupling reaction among aromatic aldehydes, cinnamyltrimethylsilane, and aromatic nucleophiles using HfCl₄ as a Lewis acid catalyst to produce 3,4,4-triarylbutene, that is also a valuable intermediate of the tamoxifen derivatives. The former strategy requires a total of 10 steps from the aldol formation to the final conversion to tamoxifen, whereas the latter needs only three or four steps to produce tamoxifen and droloxifene including the installation of the side-chain moiety and the base-induced double-bond migration to form the tetra-substituted olefin structure. This synthetic strategy seems to serve as a new and practical pathway to prepare not only the tamoxifen derivatives but also the other SERMs (selective estrogen receptor modulators) including estrogen-dependent breast cancer and osteoporosis agents.