前炎症细胞因子对人结肠腺癌细胞5-羟色胺转运体表达的影响

目的 研究前炎症细胞因子白细胞介素(IL)-1β和肿瘤坏死因子(TNF)-α对人结肠癌细胞(Caco-2细胞)5-羟色胺转运体(SERT)表达的影响.方法 Caco-2细胞系体外培养5d后,分为对照组(新鲜培养基)、IL-1β孵育组(50ng/ml)和TNF-α孵育组(50ng/ml),采用RT-PCR和Western印迹法分别观察各组2、24和48h时Caco-2细胞SERT mRNA的表达和24、48和72h时SERT蛋白的表达情况.结果对照组在2、24、48h时SERT mRNA相对表达水平分别为2.282±1.367、1.586±0.421和1.86±0.496;IL-1β孵育组分别为1.393±1.184、1.064±0.625和1.013±0.415,较对照组明显降低(P<0.05);TNF-α孵育组分别为1.000±0.000、0.829±0.162和0.945±0.147,较对照组明显降低(P<0.01),三组间比较差异有统计学意义(P<0.01).IL-1β孵育组和TNF-α孵育组于24、48、72h时SERT蛋白表达水平均低于对照组.结论 IL-1β和TNF-α在体外对结肠细胞SERT表达具有抑制作用,提示IL-1β和TNF-α可通过改变5-羟色胺系统的活性,影响内脏敏感性.     

Effect of increased pancreatic islet norepinephrine,dopamine and serotonin concentration on insulin secretion in the Golden hamster

Summary The purpose of this study was to determine if increased concentrations of pancreatic islet norepinephrine, dopamine, or serotonin alter insulin secretion. Golden hamsters received intraperitoneal injections of the norepinephrine precursor DL-threo-dihydroxyphenylserine, the dopamine precursor L-3,4-dihydroxyphenylalanine, or the serotonin precursor 5-hydroxytryptophan with and without pretreatment of the hamsters with the monoamine oxidase inhibitor tranylcypromine. Administration of the monoamine precursors to animals pretreated with tranylcypromine resulted in a mean increase in plasma glucose of 192% and a mean decrease in plasma insulin of 58%. Using a collagenase isolation technique, islets from control and treated animals were evaluated for monoamine content and insulin secretory capacity. The monoamine concentrations in control islets, in mol/kg wet weight, were: norepinephrine 42±8; dopamine 8±2; and serotonin 26±9. Administration of the appropriate precursor to control hamsters resulted in a 1.9-fold (norepinephrine), 6-fold (dopamine), and 22-fold (serotonin) increase in monoamines. There was no alteration in the glucose (16.3 mmol/l)-stimulated in vitro insulin secretion from islets obtained from these hamsters. Administration of the precursors to hamsters pretreated with tranylcypromine resulted in a 3.5-fold (norepinephrine), 22-fold (dopamine), and 59-fold (serotonin) increase in monoamines. Glucose-stimulated in vitro insulin secretion from islets obtained from these hamsters was completely blocked. This study suggests that high concentrations of norepinephrine, dopamine, and serotonin in the pancreatic islets can decrease glucose-stimulated insulin secretion.     

肺动脉高压的5-羟色胺/5-羟色胺转运体机制研究进展

肺动脉高压是临床常见的以肺血管阻力进行性增加并伴有不可逆的血管构型重塑为特征的疾病.5-羟色胺作为一种血管活性物质,可以通过5-羟色胺转运体介导,诱导肺动脉平滑肌细胞增殖,促进肺中小动脉构型重塑.因此,揭示5-羟色胺及5-羟色胺转运体在肺动脉高压形成发展中的重要作用,探讨其成为抗肺动脉高压药物治疗新靶点的可能性具有重要意义.     

Alleles of a functional serotonin transporter promoter polymorphism are associated with major depression in alcoholics

BACKGROUND: Serotonergic neurotransmission has been implicated in the pathogenesis of both alcohol dependence and mood disorders and may therefore be important in understanding the pathophysiology of comorbid alcohol dependence and major depression. Studies of the association of these disorders with a functional polymorphism in the promoter region of the gene encoding the serotonin transporter protein (locus SLC6A4) have yielded inconsistent results. Because the convergence of these disorders may provide a refined phenotype, we examined the association of serotonin (5-HT) transporter linked polymorphic region (5-HTTLPR) alleles to comorbid alcohol dependence and major depression. METHODS: A sample of 296 European American and 16 African American patients with comorbid alcohol dependence and major depression was recruited from treatment studies. The control group included 260 European Americans and 43 African Americans; all were screened to exclude the presence of a mood or substance use disorder. DNA isolated from whole blood was polymerase chain reaction-amplified, and genotypes were assigned on the basis of agarose gel size fractionation. RESULTS: The frequency of the short allele in the patient group was in the range of those previously reported for samples with unipolar depression but was significantly more common than among controls (short allele frequency of cases, 45.8%; controls, 39.8%; chi(2)(1) = 4.02; p = 0.045). CONCLUSIONS: With respect to the frequency of the short allele at the SLC6A4 locus (5-HTTLPR), major depression in alcoholics is similar to major depression in nonalcoholics. Further efforts to characterize depressed alcoholics and to examine genetic predictors of response to antidepressant treatment seem warranted.     

Structure of a pantothenate transporter and implications for ECF module sharing and energy coupling of group II ECF transporters

Energy-coupling factor (ECF) transporters are a unique group of ATP-binding cassette (ABC) transporters responsible for micronutrient uptake from the environment. Each ECF transporter is composed of an S component (or EcfS protein) and T/A/A′ components (or EcfT/A/A′ proteins; ECF module). Among the group II ECF transporters, several EcfS proteins share one ECF module; however, the underlying mechanism remains unknown. Here we report the structure of a group II ECF transporter–pantothenate transporter from Lactobacillus brevis (LbECF-PanT), which shares the ECF module with the folate and hydroxymethylpyrimidine transporters (LbECF-FolT and LbECF-HmpT). Structural and mutational analyses revealed the residues constituting the pantothenate-binding pocket. We found that although the three EcfS proteins PanT, FolT, and HmpT are dissimilar in sequence, they share a common surface area composed of the transmembrane helices 1/2/6 (SM1/2/6) to interact with the coupling helices 2/3 (CH2/3) of the same EcfT. CH2 interacts mainly with SM1 via hydrophobic interactions, which may modulate the sliding movement of EcfS. CH3 binds to a hydrophobic surface groove formed by SM1, SM2, and SM6, which may transmit the conformational changes from EcfA/A′ to EcfS. We also found that the residues at the intermolecular surfaces in LbECF-PanT are essential for transporter activity, and that these residues may mediate intermolecular conformational transmission and/or affect transporter complex stability. In addition, we found that the structure of EcfT is conformationally dynamic, which supports its function as a scaffold to mediate the interaction of the ECF module with various EcfS proteins to form different transporter complexes.In recent years, a family of ATP-binding cassette (ABC) transporters, the energy-coupling factor (ECF) transporters, has been identified in bacteria and archaea. These ECF transporters are responsible for micronutrient uptake from the environment (1–3). Compared with the classical ABC importers, ECF transporters lack periplasmic solute-binding proteins (SBPs) and instead use membrane-embedded EcfS proteins (S components) for specific substrate binding. Besides the EcfS protein, each ECF transporter contains an energy-coupling module (ECF module) comprised of two cytosolic ATPases EcfA and EcfA′ (A/A′ components) and another transmembrane protein, EcfT (T component). Based on the unique features of the ECF modules, ECF transporters have been classified into two groups: group I, in which each S component has a dedicated ECF module, and group II, in which several S components share a common ECF module (4, 5).In 1970s, several vitamin-associated transporters were found to contain different membrane-embedded substrate-binding proteins but to share a common module for energy coupling, and thus were termed ECF transporters (6). The exact molecular components of these transporters remained unclear, however, until the identification and characterization of the Co²⁺ and Ni²⁺ transporters CbiMNQO and NikMNQO (1). These transporters are now classified as members of group I ECF transporters, and the constituting MN, Q, and O proteins correspond to the EcfS, EcfT, and EcfA/A′ proteins, respectively (3). A subsequent detailed study on another group I ECF transporter, BiMNY (in which the constituting M, N, and Y proteins correspond to the EcfA/A′, EcfT, and EcfS proteins, respectively), identified the tripartite protein complex as a high-efficiency biotin transporter, and found that the solitary BioY can also bind and transport biotin in Escherichia coli (2, 7). In contrast, the EcfS proteins of group II ECF transporters—RibU, ThiT, and FolT—were found to tightly bind the substrate riboflavin, thiamine, and folate, respectively (8-10), but to have no transport activity in their solitary state (3, 11). In addition, for group I ECF transporters, the EcfT protein can form a stable subcomplex with the EcfA protein (2), whereas such a subcomplex can hardly be obtained for group II ECF transporters (11, 12). Different EcfS proteins of group II ECF transporters can form functional complexes with a common ECF module, however (3, 12). These results indicate that group I and II ECF transporters have some distinct features.In the past few years, a number of structural and functional studies have been performed to elucidate the underlying mechanisms of ECF transporters. The crystal structures of three different EcfS proteins of group II ECF transporters—Staphylococcus aureus RibU in complex with riboflavin, and Lactococcus lactis ThiT and BioY in complexes with thiamine and biotin, respectively—have been determined in the substrate-bound state (11, 13, 14). Although the sequences of these EcfS proteins are very dissimilar, they have a common structural fold of a six-helix bundle; however, the structures of the substrate-binding site are quite different. To accommodate for the chemically and structurally different substrates, the orientations of the transmembrane helices of EcfS (SM, especially SM4–6) and the residues constituting the substrate-binding pocket are substantially different, accounting for distinct substrate specificities of different EcfS proteins (15). Based on the biochemical and structural studies of individual EcfS and EcfA/A′ proteins, the conserved AxxxA motif of different EcfS proteins was suggested to form the binding site for the ECF module (13), and several possible working models of transport were proposed (11, 13, 16). The recently determined structures of the folate and hydroxymethylpyrimidine transporters (LbECF-FolT and LbECF-HmpT) revealed the structure of the T component; the interactions among the S, T, and A/A′ components; and the architecture of group II ECF transporter complexes, leading to a proposed more accurate working model of transport (17, 18). The detailed transport mechanism remains illusive, however; in particular, the molecular basis for ECF module sharing, the coupling between EcfT and EcfA/A′, and the conformational transmission between EcfS and EcfT remain unclear (15, 19).In this paper, we report the crystal structure of a group II pantothenate ECF transporter complex from Lactobacillus brevis (LbECF-PanT) at 3.25-Å resolution. A structural comparison of LbECF-PanT with LbECF-FolT and LbECF-HmpT revealed a common interacting surface of different EcfS proteins with the same EcfT protein. We evaluated the functional roles of the residues involved in the intermolecular interactions by functional studies. Taken together, the structural and functional data provide the molecular basis for how different S components share a common ECF module, and shed new light on the transport mechanism of group II ECF transporters.     

选择性5-羟色胺再摄取抑制剂治疗肠易激综合征疗效的系统评价

目的:系统评价选择性5-羟色胺再摄取抑制剂(selective serotonin reuptake inhibitor,SSRI)治疗肠易激综合征(irritable bowel syndrome,IBS)的有效性及安全性.方法:计算机检索PubMed、Embase、Cochrane图书馆临床对照试验数据库、中国生物医学文献数据库(CBM)、中国期刊全文数据库(CNKI),查找SSRI治疗肠易激综合征的所有随机对照试验(randomized controlledtrials,RCTs),应用Cochrane协作网提供的Revman5.0软件进行分析评价.结果:共纳入5项试验,共300例患者.治疗使用的药物包括氟西汀、西酞普兰、帕罗西汀.RCT表明,SSRI改善IBS总体症状缓解率无统计学意义[RR=2.09,95%CI=(0.97,4.47),P<0.05];严重不良反应的发生率与安慰剂组相比无明显统计学差异[RR=1.38,95%CI=(0.68,2.82),P>0.05].结论:SSRI对缓解IBS患者的总体症状与安慰剂相比未见明显疗效差别,不良事件发生率无统计学意义.     

Molecular determinants of inhibition of the human proton channel hHv1 by the designer peptide C6 and a bivalent derivative

The human voltage-gated proton channel (hHv1) is important for control of intracellular pH. We designed C6, a specific peptide inhibitor of hHv1, to evaluate the roles of the channel in sperm capacitation and in the inflammatory immune response of neutrophils [R. Zhao et al., Proc. Natl. Acad. Sci. U.S.A. 115, {"type":"entrez-nucleotide-range","attrs":{"text":"E11847-E11856","start_term":"E11847","end_term":"E11856","start_term_id":"22025469","end_term_id":"22025478"}}E11847-E11856 (2018)]. One C6 binds with nanomolar affinity to each of the two S3–S4 voltage-sensor loops in hHv1 in cooperative fashion so that C6-bound channels require greater depolarization to open and do so more slowly. As depolarization drives hHv1 sensors outwardly, C6 affinity decreases, and inhibition is partial. Here, we identified residues essential to C6–hHv1 binding by scanning mutagenesis, five in the hHv1 S3–S4 loops and seven on C6. A structural model of the C6–hHv1 complex was then generated by molecular dynamics simulations and validated by mutant-cycle analysis. Guided by this model, we created a bivalent C6 peptide (C6₂) that binds simultaneously to both hHv1 subunits and fully inhibits current with picomolar affinity. The results help delineate the structural basis for C6 state-dependent inhibition, support an anionic lipid-mediated binding mechanism, and offer molecular insight into the effectiveness of engineered C6 as a therapeutic agent or lead.

Human voltage-gated proton channels (hHv1s) are expressed in many human tissues, including innate and adaptive immune cells, cancer cells, and sperm (1, 2). hHv1 channels comprise two identical subunits, each with 273 residues and 4 transmembrane spans (S1–S4), that resemble the voltage-sensor domains (VSDs) in conventional voltage-gated ion channels (3, 4). In hHv1s, there are two H⁺-selective conduction pathways, one in each VSD (5). The activation of hHv1 depends on both the transmembrane potential and the pH gradient across the membrane (1). Upon membrane depolarization, hHv1 S4 segments move outward, leading to conformational changes that open the H⁺ conduction pathways (1).hHv1 has been implicated in many aspects of health and disease (1, 2). In the absence of known high-affinity and specific inhibitors of the channel, we designed a selective inhibitor of hHv1, the C6 peptide, and used it to demonstrate that H⁺ efflux via the channel is required in human sperm to induce intracellular alkalization and Ca²⁺ influx to initiate capacitation. Furthermore, we showed that it also operates in human neutrophils to maintain cytoplasmic pH during the respiratory burst, allowing sustained reactive oxygen species (ROS) production (6, 7). We identified C6 using a high-throughput, phage-display strategy whereby ∼1 million novel peptides were fabricated on an inhibitor cysteine knot (ICK) toxin scaffold and sorted on purified hHv1 protein. Phagemids expressing C6 were selected by their capacity to bind to hHv1 protein (6). C6 has 41 residues, including 6 cysteines, that form three intramolecular disulfide bonds (SI Appendix, Fig. S1).Before the development of C6, known hHv1 inhibitors were pharmacologically promiscuous or of low affinity (8–10). Synthesized C6 inhibits hHv1 by binding with positive cooperatively to the external channel residues linking the S3 and S4 transmembrane spans (S3–S4 loop), one peptide on each loop, and holds the voltage sensor in a conformation that favors channel closure (Fig. 1A) (6). As a result, the channel passes fewer H⁺ ions because more positive voltages are required to open the pores. C6 does not inhibit Ciona intestinalis Hv1 (CiHv1) and other voltage-gated K⁺, Na⁺, and Ca²⁺ channels that we tested (6). Single-molecule total internal reflection fluorescent (smTIRF) microscopy was used to show that C6 binds to hHv1 expressed in HEK293T cells with a dissociation constant (K_d) of 0.75 nM at resting membrane potential (−49 mV) (6). C6 affinity for hHv1 decreases with depolarization, achieving just 50% inhibition with 1 μM C6 at +40 mV with a partial block inhibition constant (K_i) of 31 nM (6).Open in a separate windowFig. 1.State-dependent inhibition of C6 on hHv1 channels. hHv1, hHv1-R205N, and hHv1-R211S channels were expressed in HEK293T cells and studied by whole-cell patch clamp to assess blocking parameters with a holding voltage of −60 mV, 1.5-s test pulses, and 10-s interpulse intervals, with pH_i = 6.5 and pH_o = 7.5. Values are mean ± SEM; n = 3 to 6 cells for each condition. (A) Cartoon showing two C6 peptides binding on two subunits of a hHv1 channel. (B) Representative H⁺ current traces for hHv1 channels before (Left) and in the presence of 250 nM C6 (Right) with steps of 20 mV from −60 to +40 mV. The peak current at the end of the step was used to determine the extent of block. (C) Current–voltage relationships for hHv1 in the absence (black) or presence (red) of 250 nM C6. C6 inhibition was greater at more hyperpolarized potentials with a maximal blockade of 90% at −20 mV, which decreased to 77% at 0 mV. (D) Dose–response relationships for C6 inhibition of hHv1 studied at +40 mV (black), +20 mV (dark gray), and 0 mV (light gray). The inhibition constant K_i of C6 for hHv1 channels at +40 mV, +20 mV, and 0 mV were estimated from the fit to Eq. 1 to be 30.9 ± 3.4 nM (with a Hill coefficient of h = 0.48 ± 0.04), 4.9 ± 0.8 nM (h = 0.69 ± 0.05), and 1.5 ± 0.2 nM (h = 1.22 ± 0.17), respectively (Eqs. 3 and 4, relationships derived for bimolecular binding reactions, because the kinetics were well-fitted with single exponentials (Materials and Methods). The V_1/2 of G–V of channels are reported in SI Appendix, Table S1. (H) Dose–response relationship for C6 inhibition of hHv1-R205N studied at +40 mV. (H, Inset) Representative current traces with 1 μM C6 (red) or without C6 (black). (I) Dose–response relationship for C6 inhibition of hHv1-R211S studied at +40 mV. (I, Inset) Representative current traces with 1 μM C6 (red) or without C6 (black).Here, we demonstrate that, as with natural gating modifier toxins, C6 dissociation is accelerated by depolarization (11, 12). As expected, mutations in the S4 of hHv1 that favor the open-channel state show a lower affinity for C6, while mutants that favor channel closure enhance C6 inhibition. Using point mutations, we identified five residues in the hHv1 S3–S4 loop that alter the free energy of blockade (ΔΔG) by more than 2 kcal/mol and that we therefore characterize as important for C6 binding. Taking advantage of a membrane-tethered toxin method (13), we scanned 35 noncysteine residues in tethered C6 (T-C6) and identified 7 that also significantly decrease affinity. We also show that C6 partitions most readily into lipid membranes that contain negatively charged phospholipids. Based on these screening results, molecular dynamics (MD) simulations were carried out to predict energetically important residue–residue interactions in the binding of C6 to hHv1 and the potential role of the lipid bilayer in inhibition. Three predicted C6–hHv1 interaction pairs were confirmed by thermodynamic mutant-cycle analysis, supporting the presented structural model of the complex. This model underpins a gating-modification mechanism through which binding of C6 holds the S4 helix of hHv1 in the closed “down” conformation. hHv1 operates differently than well-studied voltage-gated channels like those for K⁺, Na⁺, and Ca²⁺ ions that have one central pore and four peripheral voltage sensors. Our findings assess the operation and ICK toxin inhibition of H⁺ channels that are dimeric, with two pores, one within each voltage sensor.Natural bivalent toxin peptides, such as DkTx and Hi1a, isolated from spider venoms have been shown to modify the function of TRPV1 and ASIC1a channels, respectively. The natural toxins act with superior affinity due to their extremely slow dissociation rate (14, 15) and comprise two nonidentical ICK motifs that target two adjacent binding epitopes in the channel (16). Guided by the C6–hHv1 complex model, we constructed a homobivalent C6 (C6₂) linking two C6 peptides via a 10-residue linker and show that it fully inhibits hHv1 at +40 mV with a K_i of 630 pM. The importance of blocking the channel at positive potentials is clear when considering neutrophils that depolarize during the inflammatory respiratory burst to +58 mV (17), a voltage where monomeric C6 has low affinity. C6₂ offers a powerful tool for basic studies of hHv1 and serves with the state-dependent blocker C6 as a lead for studying treatment of diseases where the channel contributes to pathology, including pulmonary damage by leukocytes in pneumonia and acute respiratory distress syndrome (an inflammatory lung disease that is lethal in 40% of patients) (18), ischemic stroke (19), cancer (20), and both neuropathic and inflammatory pain (21, 22).     

Molecular defects in mucosal serotonin content and decreased serotonin reuptake transporter in ulcerative colitis and irritable bowel syndrome

BACKGROUND & AIMS: Serotonin (5-HT) is a critical signaling molecule in the gut. 5-HT released from enterochromaffin cells initiates peristaltic, secretory, vasodilatory, vagal, and nociceptive reflexes. Despite being pathophysiologically divergent, ulcerative colitis (UC) and irritable bowel syndrome (IBS) are both associated with clinical symptoms that include alterations in the normal patterns of motility, secretion, and sensation. Our aim was to test whether enteric 5-HT signaling is defective in these disorders. METHODS: Rectal biopsy specimens were obtained from healthy controls and patients with UC, IBS with diarrhea (IBS-D), and IBS with constipation (IBS-C). Key elements of 5-HT signaling, including measures of 5-HT content, release, and reuptake, were analyzed with these samples. RESULTS: Mucosal 5-HT, tryptophan hydroxylase 1 messenger RNA, serotonin transporter messenger RNA, and serotonin transporter immunoreactivity were all significantly reduced in UC, IBS-C, and IBS-D. The enterochromaffin cell population was decreased in severe UC samples but was unchanged in IBS-C and IBS-D. When 5-HT release was investigated under basal and mechanical stimulation conditions, no changes were detected in any of the groups relative to controls. CONCLUSIONS: These data show that UC and IBS are associated with similar molecular changes in serotonergic signaling mechanisms. While UC and IBS have distinct pathophysiologic properties, these data suggest that shared defects in 5-HT signaling may underlie the altered motility, secretion, and sensation. These findings represent the first demonstration of significant molecular alterations specific to the gut in patients with IBS and support the assertion that disordered gastrointestinal function in IBS involves changes intrinsic to the bowel.     

Characterization of oxytocin receptors and serotonin transporters in mast cells

Oxytocin (OT) inhibits the uptake of serotonin (5HT) into uterine mast cells. This may modulate 5HT bioavailability in the myometrium. Because 5HT is an important endogenous uterotonic compound, it has been postulated that this effect of OT may contribute to its potency as a labor inducer. This also predicts the presence of oxytocin receptors (OTRs) and transducing signals that will interact with 5HT transporters (SERT) in mast cells. In this study, OTR and SERT were characterized in murine peritoneal mast cells by radioligandbinding studies. Saturation assays for OTR showed no changes in K _d along the estrous cycle (6.95±2.76 nM in estrus and 4.07±1.73 nM in diestrus) but an increase in B _max in estrus (0.71±0.08 pmol/10⁶ cells and 0.37±0.05 pmol/10⁶ cells in estrus and diestrus, respectively). B _max and K _d for SERT were not affected along the estrous cycle. The signaling between the OTR and the SERT was analyzed by measuring the extent of inhibition of OT and PMA (activator of protein kinase C on 5HT uptake and the capability of Ro318220 (specific inhibitor of PKC) to increase 5HT uptake and block the effect of the above compounds in mast cells. The results showed that in murine peritoneal mast cells in vitro (1) ovarian hormones modulate OTR but not SERT expression, (2) the magnitude of OT action on 5HT uptake depends on the number of OTRs expressed in mast cells, and (3) the signaling between OTR and the SERT is mediated through the activation of protein kinase C. It is concluded that the ovarian hormones have a modulatory action on 5HT uptake which involves OT-mediated mechanism.     

Hemorrhagic syndromes related to selective serotonin reuptake inhibitor (SSRI) antidepressants. Seven case reports and review of the literature

PURPOSE: Selective serotonin reuptake inhibitors (SSRIs) are widely prescribed. Since their release unexpected adverse effects such as bleeding disorders have been described. METHODS: Thirty patients with either hematoma or muco-cutaneous bleeding have been selected from case reports of the Saint-Etienne Pharmacovigilance center and from a literature review. RESULTS: The female/male sex-ratio was 3:4 and the mean age 42 years. Two newborns who had been exposed in utero to SSRIs were also included in the study. Eleven patients presented an underlying disease or were at risk. Various adverse effects such as bruising, hematoma, petechiae or purpura, epistaxis, and more rarely intestinal hemorrhage, ocular bleeding or cerebral hemorrhage were encountered. Symptoms were sometimes associated with prolonged bleeding time and platelet aggregation disorders and usually resolved within two days to four months after treatment discontinuation. CONCLUSION: Hematoma and muco-cutaneous bleeding would therefore be related to treatment, including selective serotonin reuptake inhibitors. However, these adverse effects are still poorly known and rarely reported. The main mechanism suggested would be a decrease in serotonin platelet leading to a defect in platelet aggregation. However, an increase in capillary fragility or susceptibility related to the patient's condition might be involved. Study of hemostasis history in patients requiring treatment with SSRIs might be of value.     

Clinical and genetic determinants of urinary glucose excretion in patients with diabetes mellitus

Aims/IntroductionGlucosuria is a representative symptom in diabetes patients with poor glycemic control and in those treated with sodium–glucose cotransporter 2 inhibitors. Renal threshold levels of glucose excretion are known to vary among individuals, but factors contributing to glucosuria are not well characterized. The present study aimed to clarify clinical and genetic determinants of glucosuria in individuals with diabetes mellitus.Materials and MethodsThe 24‐h urinary glucose excretion was measured in 135 hospitalized patients on admission, with continuous measurement for five consecutive days in 75 patients. Genetic and clinical factors contributing to glucosuria were studied. As a genetic factor, SLC5A2 polymorphism was genotyped. A total of 476 participants (266 participants with type 2 diabetes and 210 healthy controls) were additionally genotyped for the association study of SLC5A2 with type 2 diabetes. A meta‐analysis was carried out with the present study and previous association studies.ResultsMultiple regression analysis showed that the independent variables of average blood glucose (β = 0.41, P = 1.4 × 10⁻⁷), estimated glomerular filtration rate (β = 0.28, P = 6.0 × 10⁻⁵), sex (β = 0.28, P = 5.7 × 10⁻⁵) and SLC5A2 rs9934336 polymorphism (β = 0.17, P = 0.02) were significantly correlated with urinary glucose excretion. The frequency of the A allele of rs9934336 tended to be lower in participants with type 2 diabetes than in controls (odds ratio 0.78, 95% confidence interval 0.53–1.13, not significant), and meta‐analysis showed a significant association between the A allele and type 2 diabetes (summary odds ratio for minor allele [A] 0.86, 95% confidence interval 0.78–0.94, P < 0.002).ConclusionsBlood glucose, estimated glomerular filtration rate, sex and SLC5A2 polymorphism were independent determinants of glucosuria in diabetes mellitus.     

Alterations in expression of p11 and SERT in mucosal biopsy specimens of patients with irritable bowel syndrome

BACKGROUND & AIMS: The pathophysiology of irritable bowel syndrome (IBS) remains enigmatic; abnormalities in serotonin metabolism have been implicated. Two proteins that influence the function of serotonin and serotonergic receptors are serotonin transporter protein (SERT or soluble carrier protein, SLC6A4) and p11 (S-100A10, or calpactin I light chain). Both proteins are reported to be associated with depression-like states, a frequent comorbid condition in IBS. We explored the hypothesis that expression of these 2 proteins in colonic and rectal mucosa is abnormal in patients with IBS as compared with healthy controls. METHODS: Messenger RNA (mRNA) expression of SLC6A4 and p11 was measured in sigmoid and rectal mucosal biopsy specimens. Genotype of the promoter for SLC6A4 was also assessed in all participants. Validation studies explored reproducibility of 2 biopsy specimens taken from the same region and biopsy specimens taken an average of approximately 3 months apart. RESULTS: We found normal colonic mucosal expression of SLC6A4 in diarrhea (IBS-D)- or constipation-predominant IBS (IBS-C). On the other hand, p11 expression was increased in IBS. No significant effect on p11 mRNA expression in sigmoid colon or rectum was noted from antidepressant treatment in any of the analyzed subgroups. CONCLUSIONS: Colonic mucosal expression of SLC6A4 in IBS is normal. Given that overexpression of p11 can increase serotonergic receptor functions (eg, 5-HT(1B) receptors), these data support the need for further study of the interaction between p11 expression in health and disease and its role in the therapeutic response to serotonergic agents, including antidepressants.     

Molecular determinants of human T-lymphotropic virus type 1 transmission and spread

Human T-lymphotrophic virus type-1 (HTLV-1) infects approximately 15 to 20 million people worldwide, with endemic areas in Japan, the Caribbean, and Africa. The virus is spread through contact with bodily fluids containing infected cells, most often from mother to child through breast milk or via blood transfusion. After prolonged latency periods, approximately 3 to 5% of HTLV-1 infected individuals will develop either adult T-cell leukemia/lymphoma (ATL), or other lymphocyte-mediated disorders such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The genome of this complex retrovirus contains typical gag, pol, and env genes, but also unique nonstructural proteins encoded from the pX region. These nonstructural genes encode the Tax and Rex regulatory proteins, as well as novel proteins essential for viral spread in vivo such as, p30, p12, p13 and the antisense encoded HBZ. While progress has been made in the understanding of viral determinants of cell transformation and host immune responses, host and viral determinants of HTLV-1 transmission and spread during the early phases of infection are unclear. Improvements in the molecular tools to test these viral determinants in cellular and animal models have provided new insights into the early events of HTLV-1 infection. This review will focus on studies that test HTLV-1 determinants in context to full length infectious clones of the virus providing insights into the mechanisms of transmission and spread of HTLV-1.     

Genetic variants in the serotonin transporter influence the efficacy of bupropion and nortriptyline in smoking cessation

Aims We investigated whether variants in the serotonin transporter gene (SLC6A4) influence smoking cessation rates using antidepressant therapy (i.e. bupropion and nortriptyline). Design Pharmacogenetic (secondary) analysis of a randomized, placebo‐controlled efficacy trial of bupropion and nortriptyline for smoking cessation. Setting Single‐centre study, Maastricht University, the Netherlands. Participants A total of 214 of 255 (84%) current daily smokers participating in a randomized controlled efficacy trial. Measurements Subjects were genotyped for three functional variants in SLC6A4 (5‐HTTLPR, STin2, rs25531). Primary outcome measures were prolonged abstinence from weeks 4–12, 4–26 and 4–52. Secondary outcome measures included 7‐day point prevalence abstinence at weeks 4, 12, 26 and 52. Findings Carriers of the 5‐HTTLPR high‐activity L‐variant had higher prolonged cessation rates with bupropion than placebo [odds ratio (OR) = 1.44, 95% confidence interval (CI) = 1.01–2.05, P = 0.04]. Combining the three variants resulted in increased prolonged cessation rates for both bupropion and nortriptyline among carriers of four to five high‐activity variants (bupropion: OR = 2.00, 95% CI = 1.21–3.29, P = 0.01; nortriptyline: OR = 1.91, 95% CI = 1.02–3.56, P = 0.04). Similar results were found for point prevalence abstinence. Conclusions Bupropion and nortriptyline seem to be more effective in smoking cessation among SLC6A4 high‐activity variant carriers, probably by blocking the increased serotonin transporter activity, thereby increasing serotonin levels. Prospective studies have to assess if this can improve cessation rates when treatment is targeted at individuals based on their genotypes.     

Expression and function of renal and hepatic organic anion transporters in extrahepatic cholestasis

Obstructive jaundice occurs in patients suffering from cholelithiasis and from neoplasms affecting the pancreas and the common bile duct. The absorption, distribution and elimination of drugs are impaired during this pathology. Prolonged cholestasis may alter both liver and kidney function. Lactam antibiotics, diuretics, non-steroidal anti-inflammatory drugs, several antiviral drugs as well as endogenous compounds are classified as organic anions. The hepatic and renal organic anion transport pathways play a key role in the pharmacokinetics of these compounds. It has been demonstrated that acute extrahepatic cholestasis is associated with increased renal elimination of organic anions. The present work describes the molecular mechanisms involved in the regulation of the expression and function of the renal and hepatic organic anion transporters in extrahepatic cholestasis, such as multidrug resistance-associated protein 2, organic anion transporting polypeptide 1, organic anion transporter 3, bilitranslocase, bromosulfophthalein/bilirubin binding protein, organic anion transporter 1 and sodium dependent bile salt transporter. The modulation in the expression of renal organic anion transporters constitutes a compensatory mechanism to overcome the hepatic dysfunction in the elimination of organic anions.     

Orchestration of three transporters and distinct vascular structures in node for intervascular transfer of silicon in rice

Requirement of mineral elements in different plant tissues is not often consistent with their transpiration rate; therefore, plants have developed systems for preferential distribution of mineral elements to the developing tissues with low transpiration. Here we took silicon (Si) as an example and revealed an efficient system for preferential distribution of Si in the node of rice (Oryza sativa). Rice is able to accumulate more than 10% Si of the dry weight in the husk, which is required for protecting the grains from water loss and pathogen infection. However, it has been unknown for a long time how this hyperaccumulation is achieved. We found that three transporters (Lsi2, Lsi3, and Lsi6) located at the node are involved in the intervascular transfer, which is required for the preferential distribution of Si. Lsi2 was polarly localized to the bundle sheath cell layer around the enlarged vascular bundles, which is next to the xylem transfer cell layer where Lsi6 is localized. Lsi3 was located in the parenchyma tissues between enlarged vascular bundles and diffuse vascular bundles. Similar to Lsi6, knockout of Lsi2 and Lsi3 also resulted in decreased distribution of Si to the panicles but increased Si to the flag leaf. Furthermore, we constructed a mathematical model for Si distribution and revealed that in addition to cooperation of three transporters, an apoplastic barrier localized at the bundle sheath cells and development of the enlarged vascular bundles in node are also required for the hyperaccumulation of Si in rice husk.Plants have different requirement for mineral elements depending on organs and tissues. For examples, developing tissues such as new leaves and reproductive organs require more mineral elements for their active growth (1). However, these tissues usually have low transpiration; therefore, transpiration-dependent distribution of mineral elements is not sufficient to meet their high requirements. Plants must have developed systems for preferential distribution of mineral elements depending on the requirements. However, the molecular mechanisms for these systems are poorly understood. Recently, several studies have shown that nodes function as a hub for distribution of mineral elements in graminaceous plants (2).Node is a key component of the phytomer in graminaceous plants, which is connected with a leaf, a tiller (or a tiller bud), and crown roots (or these primordia) to the culm or panicle (2, 3). Therefore, graminaceous plants are composed of repeated nodes. Nodes have apparently complicated but regularly organized vascular bundles (VBs). No VBs are continuously connected from the bottom to the top of the culm, but a unit of VB is repeated with a two-thirds overlapped rule; one axial VB is connected with three nodes and a leaf and specialized at different nodes as diffuse VB (DVB), transit VB (TVB), and enlarged VB (EVB) from basal to apical (2, 3). In a node, three different axial VBs with different phases coexist and connected transversely by nodal vascular anastomosis (NVA). TVB is a passage phase, but its appearance and function within the node are similar to one of the multiple DVBs. After EVB phase, the VB is continued to a VB of the leaf associating the node (2–4). As an exception, DVBs in the uppermost two nodes (nodes I and II), in which VBs have no corresponding leaves, become VBs in the panicle without EVB phase as detailed in ref. 2. Based on these repetitive vascular constructions, intervascular transfers between axial VBs are required for preferential delivery of nutrients to apical tissues such as developing leaf or panicle (2). Furthermore, the node also shows distinct structures at cellular and subcellular level, which are characterized by (i) expanded xylem area of EVBs; (ii) differentiation of xylem transfer cells (XTCs), which have expanded cell surface due to cell wall ingrowth facing to the xylem vessel; (iii) increased number of DVBs around the EVBs; and (iv) parenchyma cell bridge (PCB) with dense plasmodesmata between EVB and DVB (2, 4, 5). However, the role of these structures in intervascular transfer of nutrients has not been examined.In the present study, we examined the preferential distribution system of silicon (Si) in rice. Silicon, the most abundant mineral element, shows beneficial effects for plant growth (6). The beneficial effects are characterized by protecting plants from abiotic and biotic stresses (6). Silicon is especially important for high and sustainable production of rice (Oryza sativa), a typical Si accumulating species. Rice is able to accumulate Si up to 10% of the shoot dry weight, which is severalfold more than macronutrients such as nitrogen, phosphorus, and potassium (7). Low accumulation of Si in rice results in significant reduction of the yield (8); therefore, Si fertilization has been considered as a practice of rice production in many countries, especially under heavy application of nitrogen fertilizers (7).Silicon in soil solution is present in the form of silicic acid, an uncharged molecule (7). Silicic acid is taken by the roots through two different transporters: Lsi1 and Lsi2 in rice (9, 10). Lsi1 belongs to NIP subgroup of aquaporin and preamble to silicic acid, whereas Lsi2 is an efflux transporter of silicic acid (9, 10). Both Lsi1 and Lsi2 are localized at the exodermis and endodermis cells of rice roots, but in contrast to Lsi1, which is localized at the distal side, Lsi2 is localized at the proximal side (9, 10). Knockout of either Lsi1 or Lsi2 results in defect of Si uptake (9, 10). Recently, in silico mathematical modeling revealed that in addition to cooperation of Lsi1 and Lsi2, presence of the Casparian strips at the exodermis and endodermis is also required for efficient Si uptake and high accumulation (11).After uptake, more than 95% of Si is rapidly translocated from the roots to the shoots (7). Silicon is also present in the form of silicic acid in the xylem sap (12), which is unloaded by Lsi6, a homolog of Lsi1 (13). Lsi6 is polarly localized in the adaxial side of the xylem parenchyma cells in the leaf sheaths and leaf blades (13). With water loss due to transpiration, silicic acid is gradually concentrated and polymerized to amorphous silica, which is deposited beneath the cuticle, forming cuticle–silica double layers, and inside of particular cells of leaf epidermis (motor cells and short silica cells) (7). Knockout of Lsi6 results in leakage of large amount of silicic acid into the leaf guttation and altered deposition of Si in the leaves (13).At the reproductive stage, higher Si is finally deposited in the husk, which can reach more than 10% of the dry weight (7). Silicon in the husk is also deposited between the epidermal cell wall and the cuticle, forming a cuticle–silica double layer (7). This heavy deposition of Si in the husk is very important for rice grain fertility because it prevents water loss and pathogen (e.g., panicle blast) infection (6–8). However, the question arises on how Si is highly deposited in the husk. The rice husk does not have stoma at the outside. Furthermore, compared with the leaves, the surface area is much smaller. Therefore, transpiration-dependent distribution is unlikely responsible for the high Si accumulation in the husk.Previously, Lsi6 highly expressed in the nodes was reported to be involved in the intervascular transfer for preferential distribution of Si. Lsi6 in nodes is polarly localized at the xylem parenchyma cells (mainly XTCs) of EVBs (14). Knockout of Lsi6 resulted in decreased Si accumulation in the husk but increased Si in the flag leaf (14). However, Lsi6 is a channel-type passive transporter, which transports silicic acid following concentration gradient (13, 15). Therefore, Lsi6 alone cannot explain hyperaccumulation of Si in the husk. In the present study, we found that the other two active efflux transporters are also involved in the intervascular transfer required for high accumulation of Si in the husk. Furthermore, our mathematical modeling revealed that the distinct nodal structures including EVBs, XTCs, and apoplastic barrier are also required for the efficient intervascular transfer of Si in rice.