Increased expression of anion transporter SLC26A9 delays diabetes onset in cystic fibrosis.

Diabetes is a common complication of cystic fibrosis (CF) that affects approximately 20% of adolescents and 40%-50% of adults with CF. The age at onset of CF-related diabetes (CFRD) (marked by clinical diagnosis and treatment initiation) is an important measure of the disease process. DNA variants associated with age at onset of CFRD reside in and near SLC26A9. Deep sequencing of the SLC26A9 gene in 762 individuals with CF revealed that 2 common DNA haplotypes formed by the risk variants account for the association with diabetes. Single-cell RNA sequencing (scRNA-Seq) indicated that SLC26A9 is predominantly expressed in pancreatic ductal cells and frequently coexpressed with CF transmembrane conductance regulator (CFTR) along with transcription factors that have binding sites 5' of SLC26A9. These findings were replicated upon reanalysis of scRNA-Seq data from 4 independent studies. DNA fragments derived from the 5' region of SLC26A9-bearing variants from the low-risk haplotype generated 12%-20% higher levels of expression in PANC-1 and CFPAC-1 cells compared with the high- risk haplotype. Taken together, our findings indicate that an increase in SLC26A9 expression in ductal cells of the pancreas delays the age at onset of diabetes, suggesting a CFTR-agnostic treatment for a major complication of CF.

Structure-function analysis of the DNA-binding domain of a transmembrane transcriptional activator.

The transmembrane DNA-binding protein CadC of E. coli, a representative of the ToxR-like receptor family, combines input and effector domains for signal sensing and transcriptional activation, respectively, in a single protein, thus representing one of the simplest signalling systems. At acidic pH in a lysine-rich environment, CadC activates the transcription of the cadBA operon through recruitment of the RNA polymerase (RNAP) to the two cadBA promoter sites, Cad1 and Cad2, which are directly bound by CadC. However, the molecular details for its interaction with DNA have remained elusive. Here, we present the crystal structure of the CadC DNA-binding domain (DBD) and show that it adopts a winged helix-turn-helix fold. The interaction with the cadBA promoter site Cad1 is studied by using nuclear magnetic resonance (NMR) spectroscopy, biophysical methods and functional assays and reveals a preference for AT-rich regions. By mutational analysis we identify amino acids within the CadC DBD that are crucial for DNA-binding and functional activity. Experimentally derived structural models of the CadC-DNA complex indicate that the CadC DBD employs mainly non-sequence-specific over a few specific contacts. Our data provide molecular insights into the CadC-DNA interaction and suggest how CadC dimerization may provide high-affinity binding to the Cad1 promoter.

Evidence of estrone-sulfate uptake modification in young and middle-aged rat prostate.

High plasma exposure to estrogens is often associated with prostate cancer. Reducing this phenomenon may present therapeutic benefits. The involvement of estrone sulphate (E1S), the most abundant circulating estrogen in men, has been partially studied in this age-related pathology. To investigate the consequences of plasma E1S overload on blood and prostate sex steroid levels and inflammatory tissue responses, young and middle-aged male rats were treated with E1S with or without steroid sulfatase (STS) inhibitor STX64 for 21 consecutive days. A plasma and prostate tissue steroid profile was determined. STS activity, mRNA expression of E1S organic anion transporting polypeptides (slco1a2, slco2b1, slco4a1) and pro-inflammatory cytokines (Il1-beta, Il6, TNF-alpha) were evaluated in prostate tissue according to age and treatment group. A significant correlation between plasma and prostate steroid levels related to hormone treatment was observed in all rat age groups. However, while the E1S level in prostate tissue increased in middle-aged treated rats (p<0.0001), no significant variation was observed in young treated rats. The protective effect of STX64 during E1S infusion was observed by the maintenance of low free estrogen concentrations in both plasma and tissue. However, this protection was not associated with mRNA expression stability of pro-inflammatory cytokines in older rat prostate. These results suggest that E1S uptake in rat prostate cells increases during aging. Therefore, if a similar phenomenon existed in men, preventively reducing the STS activity could be of interest to limit uptake of estrogens in prostate when high E1S plasma level is assayed.

Expression and functional analysis of putative vacuolar Ca2+-transporters (CAXs and ACAs) in roots of salt tolerant and sensitive rice cultivars.

Vacuolar Ca2+-transporters could play an important role for salt tolerance in rice (Oryza sativa L.) root. Here, we compared the expression profiles of putative vacuolar cation/H+ exchanger (CAX) and calmodulin-regulated autoinhibited Ca2+-ATPase (ACA) in rice roots of salt tolerant cv. Pokkali and salt sensitive cv. IR29. In addition to five putative vacuolar CAX genes in the rice genome, a new CAX gene (OsCAX4) has been annotated. In the present study, we isolated the OsCAX4 gene and showed that its encoded protein possesses a unique transmembrane structure and is potentially involved in transporting not only Ca2+ but also Mn2+ and Cu2+. These six OsCAX genes differed in their mRNA expression pattern in roots of tolerant versus sensitive rice cultivars exposed to salt stress. For example, OsCAX4 showed abundant expression in IR29 (sensitive) upon prolonged salt stress. The mRNA expression profile of four putative vacuolar Ca2+-ATPases (OsACA4-7) was also examined. Under control conditions, the mRNA levels of OsACA4, OsACA5, and OsACA7 were relatively high and similar among IR29 and Pokkali. Upon salt stress, only OsACA4 showed first a decrease in its expression in Pokkali (tolerant), followed by a significant increase. Based on these results, a role of vacuolar Ca2+ transporter for salt tolerance in rice root was discussed.

Antibiotic treatment of Pseudomonas aeruginosa biofilms stimulates expression of the magnesium transporter gene mgtE.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen with the capacity to cause serious disease, including chronic biofilm infections in the lungs of cystic fibrosis (CF) patients. These infections are treated with high concentrations of antibiotics. Virulence modulation is an important tool utilized by P. aeruginosa to propagate infection and biofilm formation in the CF airway. Many different virulence modulatory pathways and proteins have been identified, including the magnesium transporter protein MgtE. We have recently found that isogenic deletion of mgtE leads to increased cytotoxicity through effects on the type III secretion system. To explore the role of the CF lung environment in MgtE activity, we investigated mgtE transcriptional regulation following antibiotic treatment. Utilizing quantitative real-time-PCR, we have demonstrated an increase in mgtE transcript levels following antibiotic treatment with most of the 12 antibiotics tested. To begin to determine the regulatory network governing mgtE expression, we screened a transposon-mutant library of P. aeruginosa to look for mutants with potentially altered mgtE activity, using cytotoxicity as a readout. In this screen, we observed that AlgR, which regulates production of the biofilm polysaccharide alginate, alters MgtE-mediated cytotoxicity. This cross-talk between MgtE and AlgR suggests that AlgR is involved in linking external inducing signals (e.g. antibiotics) to mgtE transcription and downstream virulence and biofilm activities. Analysing such interactions may lead to a better understanding of how the CF lung environment shapes P. aeruginosa biofilm infections.

Expression of anion exchangers in cultured human endolymphatic sac epithelia.

HYPOTHESIS: Pendrin acts as a Cl-/HCO3- exchanger and is responsible for endolymphatic fluid volume and pH homeostasis in human endolymphatic sac epithelial cells. BACKGROUND: The endolymphatic sac (ES) is part of the membranous labyrinth in the inner ear that plays an important role in maintaining homeostasis of the endolymphatic fluid system. However, the exact mechanism of fluid volume and pH regulation is not fully understood yet. We aimed to demonstrate the expression of various anion exchangers (AEs), including pendrin, in cultured human endolymphatic sac epithelial (HESE) cells. METHODS: Endolymphatic sac specimens were harvested during acoustic neuroma surgery (n = 24) using the translabyrinthine approach and then subcultured with high epidermal growth factor (EGF) (25 ng/ml) media and differentiated using low-EGF (0.5 ng/ml) media. The cultured cells were classified according to the morphology on TEM. The Cl-/HCO3- exchanger activity was assessed by pHi measurement using pH sensitive dye 2', 7'-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF/AM). We performed reverse transcriptase-polymerase chain reaction and immunohistochemical staining for AEs. RESULTS: We determined that 7.3 ± 6.7% of cells differentiated into mitochodria-rich cells and 50.2 ± 15.1 of cells differentiated into ribosome-rich cells. bAE3, AE4, SLC26A4, SLC26A6, and SLC26A11 were also expressed in cultured HESE cells. The cultured cells had Cl-/HCO3- and Cl-/formate exchange activity on the luminal membrane, which is sensitive to anion channel inhibitors (DIDS 500 µM). Furthermore, we showed that pendrin (SLC26A4) was expressed in cultured HESE cell membranes. CONCLUSION: Our results suggest that AEs, including pendrin, are expressed in epithelia of ES and may have role in maintaining ionic homeostasis, and the HESE culture system are useful for uncovering the functional role of ES epithelial cells.

Impaired mucin synthesis and bicarbonate secretion in the colon of NHE8 knockout mice.

Sodium/hydrogen exchanger 8 (NHE8), the newest member of the SLC9 family, is expressed at the apical membrane of the epithelial cells in the intestine and the kidney. Although NHE8 has been shown to be an important player for intestinal sodium absorption early in development, its physiological role in the intestine remains unclear. Here, we successfully created a NHE8 knockout (NHE8(-/-)) mouse model to study the function of this transporter in the intestinal tract. Embryonic stem cells containing interrupted NHE8 gene were injected into mouse blastocyst to produce NHE8(+/-) chimeras. NHE8(-/-) mice showed no lethality during embryonic and fetal development. These mice had normal serum sodium levels and no signs of diarrhea. Apically expressed NHE2 and NHE3 were increased in the small intestine of the NHE8(-/-) mice in compensation. The number of goblet cells and mucin (MUC)-positive cells in the colon was reduced in NHE8(-/-) mice along with mucosal pH, MUC2 expression as well as downregulated in adenoma (DRA) expression. Therefore, the role of NHE8 in the intestine involves both sodium absorption and bicarbonate secretion.

New insights into the signaling mechanism of the pH-responsive, membrane-integrated transcriptional activator CadC of Escherichia coli.

The membrane-integrated transcriptional regulator CadC of Escherichia coli activates expression of the cadBA operon at low external pH with concomitantly available lysine, providing adaptation to mild acidic stress. CadC is a representative of the ToxR-like proteins that combine sensory, signal transduction, and DNA-binding activities within a single polypeptide. Although several ToxR-like regulators such as CadC, as well as the main regulator of Vibrio cholerae virulence, ToxR itself, which activate gene expression at acidic pH, have been intensively investigated, their molecular activation mechanism is still unclear. In this study, a structure-guided mutational analysis was performed to elucidate the mechanism by which CadC detects acidification of the external milieu. Thus, a cluster of negatively charged amino acids (Asp-198, Asp-200, Glu-461, Glu-468, and Asp-471) was found to be crucial for pH detection. These amino acids form a negatively charged patch on the surface of the periplasmic domain of CadC that stretches across its two subdomains. The results of different combinations of amino acid replacements within this patch indicated that the N-terminal subdomain integrates and transduces the signals coming from both subdomains to the transmembrane domain. Alterations in the phospholipid composition did not influence pH-dependent cadBA expression, and therefore, interplay of the acidic surface patch with the negatively charged headgroups is unlikely. Models are discussed according to which protonation of these acidic amino acid side chains reduces repulsive forces between the two subdomains and/or between two monomers within a CadC dimer and thereby enables receptor activation upon lowering of the environmental pH.

The liver and kidney expression of sulfate anion transporter sat-1 in rats exhibits male-dominant gender differences.

The sulfate anion transporter (sat-1, Slc26a1) has been cloned from rat liver, functionally characterized, and localized to the sinusoidal membrane in hepatocytes and basolateral membrane (BLM) in proximal tubules (PT). Here, we confirm previously described localization of sat-1 protein in rat liver and kidneys and report on gender differences (GD) in its expression by immunochemical, transport, and excretion studies in rats. The approximately 85-kDa sat-1 protein was localized to the sinusoidal membrane in hepatocytes and BLM in renal cortical PT, with the male-dominant expression. However, the real-time reverse-transcription polymerase chain reaction data indicated no GD at the level of sat-1 mRNA. In agreement with the protein data, isolated membranes from both organs exhibited the male-dominant exchange of radiolabeled sulfate for oxalate, whereas higher oxalate in plasma and 24-h urine indicated higher oxalate production and excretion in male rats. Furthermore, the expression of liver, but not renal, sat-1 protein was: unaffected by castration, upregulated by ovariectomy, and downregulated by estrogen or progesterone treatment in males. Therefore, GD (males > females) in the expression of sat-1 protein in rat liver (and, possibly, kidneys) are caused by the female sex-hormone-driven inhibition at the posttranscriptional level. The male-dominant abundance of sat-1 protein in liver may conform to elevated uptake of sulfate and extrusion of oxalate, causing higher plasma oxalate in males. Oxalate is then excreted by the kidneys via the basolateral sat-1 (males > females) and the apical CFEX (Slc26a6; GD unknown) in PT and eliminated in the urine (males > females), where it may contribute to the male-prevailing development of oxalate urolithiasis.

Expression, purification and characterisation of the C-terminal STAS domain of the SLC26 anion transporter prestin.

The membrane protein prestin is the voltage-sensitive molecular motor underlying somatic electromotility of outer hair cells. In order to produce adequate quantities to perform structural and functional studies, we cloned and expressed in bacterial systems three variants of the cytosolic C-terminal STAS domain of prestin from Rattus norvegicus. While the expression level of the longer form of the C-terminal domain (fragment [505-744]) was very low or absent, we succeeded in the overexpression of two shorter fragment of the STAS domain (fragments [529-744], PreCD(L), and [529-720], PreCD(S)). These two polypeptides were purified to homogeneity and characterised by circular dichroism, fluorescence spectroscopy and dynamic light scattering. The two proteins possess a three-dimensional structure and show a great tendency to aggregate. In particular, PreCD(L) is present in solution mainly as dimers and tetramers. These data correlate with that of full-length prestin that forms stable tetramers, suggesting that the C-terminal domain play an important role in modulating the properties of the entire prestin.

Kinetic analysis of butyrate transport in human colon adenocarcinoma cells reveals two different carrier-mediated mechanisms.

Butyrate has antitumorigenic effects on colon cancer cells, inhibits cell growth and promotes differentiation and apoptosis. These effects depend on its intracellular concentration, which is regulated by its transport. We have analysed butyrate uptake kinetics in human colon adenocarcinoma cells sensitive to the apoptotic effects of butyrate (BCS-TC2, Caco-2 and HT-29), in butyrate-resistant cells (BCS-TC2.BR2) and in normal colonic cells (FHC). The properties of transport were analysed with structural analogues, specific inhibitors and different bicarbonate and sodium concentrations. Two carrier-mediated mechanisms were detected: a low-affinity/high-capacity (K(m)=109+/-16 mM in BCS-TC2 cells) anion exchanger and a high-affinity/low-capacity (K(m)=17.9+/-4.0 microM in BCS-TC2 cells) proton-monocarboxylate co-transporter that was energy-dependent and activated via PKCdelta (protein kinase Cdelta). All adenocarcinoma cells analysed express MCT (monocarboxylate transporter) 1, MCT4, ancillary protein CD147 and AE2 (anion exchanger 2). Silencing experiments show that MCT1, whose expression increases with butyrate treatment in butyrate-sensitive cells, plays a key role in high-affinity transport. Low-affinity uptake was mediated by a butyrate/bicarbonate antiporter along with a possible contribution of AE2 and MCT4. Butyrate treatment increased uptake in a time- and dose-dependent manner in butyrate-sensitive but not in butyrate-resistant cells. The two butyrate-uptake activities in human colon adenocarcinoma cells enable butyrate transport at different physiological conditions to maintain cell functionality. The high-affinity/low-capacity transport functions under low butyrate concentrations and may be relevant for the survival of carcinoma cells in tumour regions with low glucose and butyrate availability as well as for the normal physiology of colonocytes.

MT1-MMP down-regulates the glucose 6-phosphate transporter expression in marrow stromal cells: a molecular link between pro-MMP-2 activation, chemotaxis, and cell survival.

Bone marrow-derived stromal cells (BMSC) are avidly recruited by experimental vascularizing tumors, which implies that they must respond to tumor-derived growth factor cues. In fact, BMSC chemotaxis and cell survival are regulated, in part, by the membrane type-1 matrix metalloproteinase (MT1-MMP), an MMP also involved in pro-MMP-2 activation and in degradation of the extracellular matrix (ECM). Given that impaired chemotaxis was recently observed in bone marrow cells isolated from a glucose 6-phosphate transporter-deficient (G6PT-/-) mouse model, we sought to investigate the potential MT1-MMP/G6PT signaling axis in BMSC. We show that MT1-MMP-mediated activation of pro-MMP-2 by concanavalin A (ConA) correlated with an increase in the sub-G1 cell cycle phase as well as with cell necrosis, indicative of a decrease in BMSC survival. BMSC isolated from Egr-1-/- mouse or MT1-MMP gene silencing in BMSC with small interfering RNA (siMT1-MMP) antagonized both the ConA-mediated activation of pro-MMP-2 and the induction of cell necrosis. Overexpression of recombinant full-length MT1-MMP triggered necrosis and this was signaled through the cytoplasmic domain of MT1-MMP. ConA inhibited both the gene and protein expression of G6PT, while overexpression of recombinant G6PT inhibited MT1-MMP-mediated pro-MMP-2 activation but could not rescue BMSC from ConA-induced cell necrosis. Cell chemotaxis in response to the tumorigenic growth factor sphingosine 1-phosphate was significantly abrogated in siMT1-MMP BMSC and in chlorogenic acid-treated BMSC. Altogether, we provide evidence for an MT1-MMP/G6PT signaling axis that regulates BMSC survival, ECM degradation, and mobilization. This may lead to optimized clinical applications that use BMSC as a platform for the systemic delivery of therapeutic or anti-cancer recombinant proteins in vivo.

Cooperative action of glutamate transporters and cystine/glutamate antiporter system Xc- protects from oxidative glutamate toxicity.

Oxidative glutamate toxicity in the neuronal cell line HT22 is a model for cell death by oxidative stress. In this paradigm, an excess of extracellular glutamate blocks the glutamate/cystine-antiporter system Xc-, depleting the cell of cysteine, a building block of the antioxidant glutathione. Loss of glutathione leads to the accumulation of reactive oxygen species and eventually cell death. We selected cells resistant to oxidative stress, which exhibit reduced glutamate-induced glutathione depletion mediated by an increase in the antiporter subunit xCT and system Xc- activity. Cystine uptake was less sensitive to inhibition by glutamate and we hypothesized that glutamate import via excitatory amino acid transporters and immediate re-export via system Xc- underlies this phenomenon. Inhibition of glutamate transporters by l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) and DL-threo-beta-benzyloxyaspartic acid (TBOA) exacerbated glutamate-induced cell death. PDC decreased intracellular glutamate accumulation and exacerbated glutathione depletion in the presence of glutamate. Transient overexpression of xCT and the glutamate transporter EAAT3 cooperatively protected against glutamate. We conclude that EAATs support system Xc- to prevent glutathione depletion caused by high extracellular glutamate. This knowledge could be of use for the development of novel therapeutics aimed at diseases associated with depletion of glutathione like Parkinson's disease.

Expression of SLC26A3, CFTR and NHE3 in the human male reproductive tract: role in male subfertility caused by congenital chloride diarrhoea.

Congenital chloride diarrhoea (CLD) is a rare inherited disease caused by mutations in the solute carrier family 26 member 3 (SLC26A3) gene. Disruption of intestinal Cl(-)/HCO(3)(-) exchange causes watery Cl(-) rich diarrhoea from birth, and recently male subfertility was observed as a novel manifestation. Expression of SLC26A3, together with interacting proteins cystic fibrosis transmembrane conductance regulator (CFTR) and Na(+)/H(+) exchanger 3 (NHE3), was studied using immunohistochemistry in the testis (n = 2) and efferent ducts (ED) (n = 1) of patients with CLD (V317del genotype) and in the testis and epididymis (n = 11), seminal vesicle (n = 9) and prostate (n = 4) of the controls. SLC26A3 was immunolocalized in the head of the elongating spermatids (stages III-VI) and CFTR in the elongating spermatids (stages III and IV) and pachytene (stages III-V) and diplotene spermatocytes. In the non-ciliated cells of the ED, apical expression of all three proteins was observed, but only SLC26A3 and CFTR were detected on the luminal border of the apical mitochondria-rich cells (AMRC) of the ductus epididymis and in the epithelium of the seminal vesicle. Only CFTR was present in the epithelium of the prostatic duct. In the patient with CLD, the expression of both SLC26A3 and CFTR was absent in the ED, but testicular expression was identical to that of the controls. These results suggest a primary role for SLC26A3 in male reproduction. Tissue-specific co-expression with CFTR and NHE3 supports diverse functions of SLC26A3 and may have an impact on pathophysiology of male subfertility both in CLD and in cystic fibrosis (CF), as well as spermatoceles.

T-Cell activation by t(9;22) acute lymphoblastic leukemia-derived dendritic-like cells is associated with increased tapasin expression.

Dendritic-like cells from t(9;22) acute lymphoblastic leukemia (ALL) blasts can activate T cells, while the original unmodified leukemic blasts cannot. To determine whether these functional differences were associated with differences in antigen-processing machinery (APM) component expression, we have measured the level of APM component expression in unmodified blasts and ALL-derived dendritic-like cells. Seven t(9;22) ALL patient samples and one cell line were studied for APM component expression utilizing a unique panel of recently developed monoclonal antibodies and a recently developed intracellular staining technique. In addition, the HLA class I antigen cell surface expression was measured. HLA class I antigens were similarly expressed on the unmodified blasts and on the autologous dendritic-like cells. Intracellular HLA class I antigen and tapasin expression (P=0.03 for both) were upregulated in all t(9;22) ALL-derived dendritic-like cells, in comparison to the unmodified blasts. These results provide a potential mechanism for the ability of t(9;22) ALL-derived dendritic-like cells to induce T-cell activation and, suggest that tapasin upregulation may serve as a marker to standardize and monitor the quality of the dendritic-like cells used in immunotherapy.

Restoration of the expression of transporters associated with antigen processing in lung carcinoma increases tumor-specific immune responses and survival.

A wide variety of human carcinomas have low expression of tumor-associated antigen presentation in the context of MHC class I antigens due to defects in the antigen presentation pathway. This immune evasion mechanism renders many tumors unrecognizable by host immune surveillance mechanisms. The present study examines the expression of HLA, tapasin, transporter associated with antigen processing 1 (TAP1), and beta2 microglobulin in human small cell lung carcinoma and non-small cell lung carcinoma. Immunohistochemical staining showed severe impairment of the antigen presentation pathway in all patients. In order to recover tumor immunogenicity, a nonreplicating adenovirus expressing human TAP1 (AdhTAP1) was used to restore the expression of TAP1 in the antigen presentation pathway-deficient mouse lung carcinoma cell line, CMT.64. Infection of CMT.64 cells with AdhTAP1 increased MHC class I antigen surface expression, antigen presentation, and susceptibility to antigen-specific CTLs. Fluorescence-activated cell sorting and ELISPOT analysis showed that AdhTAP1 treatment significantly increased dendritic cell cross-presentation and cross-priming of tumor antigens. Furthermore, ex vivo and in vivo AdhTAP1 treatment significantly retarded tumor growth and increased survival of mice bearing CMT.64 tumors. Fluorescence-activated cell sorting analysis and immunohistochemical staining showed a significant increase in CD8+ and CD4+ T cells and CD11c+ dendritic cells infiltrating the tumors. The results show that TAP should be considered as a part of the immunotherapies for various cancers because it is likely to provide a general method for increasing immune responses against tumors regardless of the antigenic composition of the tumor or the MHC haplotypes of the host.

Qualitative and quantitative differences in peptides bound to HLA-B27 in the presence of mouse versus human tapasin define a role for tapasin as a size-dependent peptide editor.

Tapasin (Tpn) is a chaperone of the endoplasmic reticulum involved in peptide loading to MHC class I proteins. The influence of mouse Tpn (mTpn) on the HLA-B*2705-bound peptide repertoire was analyzed to characterize the species specificity of this chaperone. B*2705 was expressed on Tpn-deficient human 721.220 cells cotransfected with human (hTpn) or mTpn. The heterodimer to beta(2)-microglobulin-free H chain ratio on the cell surface was reduced with mTpn, suggesting lower B*2705 stability. The B*2705-bound peptide repertoires loaded with hTpn or mTpn shared 94-97% identity, although significant differences in peptide amount were observed in 16-17% of the shared ligands. About 3-6% of peptides were bound only with either hTpn or mTpn. Nonamers differentially bound with mTpn had less suitable anchor residues and bound B*2705 less efficiently in vitro than those loaded only with hTpn or shared nonamers. Decamers showed a different pattern: those found only with mTpn had similarly suitable residues as shared decamers and bound B*2705 with high efficiency. Peptides differentially presented by B*2705 on human or mouse cells showed an analogous pattern of residue suitability, suggesting that the effect of mTpn on B*2705 loading is comparable in both cell types. Thus, mTpn has quantitative and qualitative effects on the B*2705-bound peptide repertoire, impairing presentation of some suitable ligands and allowing others with suboptimal anchor residues and lower affinity to be presented. Our results favor a size-dependent peptide editing role of Tpn for HLA-B*2705 that is species-dependent and suboptimally performed, at least for nonamers, by mTpn.

Expression of prostaglandin transporter in the bovine uterus and fetal membranes during pregnancy.

Uteroplacental prostaglandins (PGs) play pivotal roles in the maintenance and termination of pregnancy in mammals. In the present study, we have characterized the expression of prostaglandin transporter (PGT) in placentome caruncles, intercaruncular tissues, fetal membranes, and utero-ovarian plexus during pregnancy in cattle. Pregnant bovine uteri were collected and classified into six groups covering the entire gestational length. In caruncles and intercaruncular tissues, PGT mRNA (also known as SLC02A1) and PGT protein were highly expressed at the late stage of pregnancy compared to the early and mid stages, whereas the level of expression is constant and low in fetal membranes throughout pregnancy. PGT mRNA and PGT protein were expressed at a constant level in the utero-ovarian plexus both ipsilateral and contralateral to corpus luteum throughout the course of pregnancy. Overall, the relative expression of PGT mRNA and PGT protein were higher in caruncles than in intercaruncular tissue and fetal membranes, whereas no differences were detected between intercaruncular tissues and fetal membranes at any stage of gestation. Immunohistochemistry indicated that PGT was preferentially expressed in caruncular epithelial cells of placentomes and endometrial luminal epithelial and myometrial smooth muscle cells of the intercaruncular regions. The level of PGT expression was comparatively higher in maternal components than in fetal components. In conclusion, differential spatiotemporal tissue-specific expression of PGT in uterine and intrauterine tissues suggests a role for this transporter in the exchange of PGs between the maternal and the fetal compartments, as well as for intrauterine metabolism of PGs during pregnancy.

Expression of AtMHX, an Arabidopsis vacuolar metal transporter, is repressed by the 5' untranslated region of its gene.

AtMHX is an Arabidopsis tonoplast transporter that can exchange protons with Mg2+ and Zn2+ ions. This transporter, which may play a role in ion homeostasis of plants, is encoded by a single gene in Arabidopsis. The molecular mechanisms that regulate the expression of this transporter are practically unknown. While AtMHX transcript can be easily visualized, expression of the corresponding protein is apparently low. To understand whether AtMHX expression is repressed at the translational level, the 5' untranslated region (5' UTR) of this gene was fused to reporter genes. In vitro analyses showed that the 5' UTR of AtMHX can repress the translation of downstream coding sequences. The major cause of the repression was efficient initiation at an upstream open-reading-frame (uORF) included in the 5' UTR. Although the sequence context of the upstream AUG (uAUG) codon was highly unfavourable, it was recognized by over 90% of the scanning ribosomes both in vitro and in vivo. The inhibitory effect of the uORF was mediated by imposing the need for reinitiation and not by ribosome stalling, as the inhibition was not dependent on the amino-acid sequence of the uORF peptide. The efficiency of reinitiation was low. The in vivo studies, carried out with transiently transformed tobacco plants, indicated that alternations in the Mg2+ or Zn2+ levels did not affect the rate of translation. These data suggest that AtMHX expression is repressed by the 5' UTR of its gene.

Transport of prostaglandin E1 across the blood-brain barrier in rats.

The transport of prostaglandin E(1) (PGE(1)) across the blood-brain barrier (BBB) was characterized using an in-situ rat brain perfusion technique. The uptake of [(3)H]PGE(1) was not affected by shortchain monocarboxylic acids (butyric acid and valeric acid). On the other hand, uptake of [(3)H]PGE(1) was significantly inhibited by medium-chain monocarboxylic acids such as hexanoic acid, enanthic acid and octanoic acid. These medium-chain monocarboxylic acids showed a more potent inhibitory effect on [(3)H]PGE(1) uptake with increasing number of carbon atoms. In contrast, there was no decrease in [(3)H]PGE(1) transport by any dicarboxylic acids with 5-8 carbon atoms. Valproic acid decreased [(3)H]PGE(1) uptake, whereas p-aminohippuric acid, a substrate for the organic anion transporter family, did not inhibit [(3)H]PGE(1) transport. Bromocresol green, an inhibitor of prostaglandin transporter (PGT), strongly decreased [(3)H]PGE(1) transport across the BBB. In addition, digoxin and taurocholate, substrates for organic anion transporting polypeptide subtype 2 (Oatp2), significantly inhibited [(3)H]PGE(1) uptake. RT-PCR analysis revealed that PGT mRNA and Oatp2 mRNA are expressed in a capillary-rich fraction from rat brain. Thus, it is suggested that PGE(1) transport across the BBB is mediated by some specific transport systems, possibly by the members of the Oatp family.