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.

IPSC-Derived Corneal Endothelial-like Cells Act as an Appropriate Model System to Assess the Impact of SLC4A11 Variants on Pre-mRNA Splicing.

Purpose: To report molecular genetic findings in six probands with congenital hereditary endothelial dystrophy (CHED) variably associated with hearing loss (also known as Harboyan syndrome). Furthermore, we developed a cellular model to determine if disease-associated variants induce aberrant SLC4A11 pre-mRNA splicing. Methods: Direct sequencing of the entire SLC4A11 coding region was performed in five probands. In one individual, whole genome sequencing was undertaken. The effect of c.2240+5G>A on pre-mRNA splicing was evaluated in a corneal endothelial-like (CE-like) cell model expressing SLC4A11. CE-like cells were derived from autologous induced pluripotent stem cells (iPSCs) via neural crest cells exposed to B27, PDGF-BB, and DKK-2. Total RNA was extracted, and RT-PCR was performed followed by Sanger and a targeted next generation sequencing (NGS) approach to identify and quantify the relative abundance of alternatively spliced transcripts. Results: In total, 11 different mutations in SLC4A11 evaluated as pathogenic were identified; of these, c.1237G>A, c.2003T>C, c.1216+1G>A, and c.2240+5G>A were novel. The c.2240+5G>A variant was demonstrated to result in aberrant pre-mRNA splicing. A targeted NGS approach confirmed that the variant introduces a leaky cryptic splice donor site leading to the production of a transcript containing an insertion of six base pairs with the subsequent introduction of a premature stop codon (p.Thr747*). Furthermore, a subset of transcripts comprising full retention of intron 16 also were observed, leading to the same functionally null allele. Conclusions: This proof-of-concept study highlights the potential of using CE-like cells to investigate the pathogenic consequences of SLC4A11 disease-associated variants.

Role of AcrAB-TolC multidrug efflux pump in drug-resistance acquisition by plasmid transfer.

Drug-resistance dissemination by horizontal gene transfer remains poorly understood at the cellular scale. Using live-cell microscopy, we reveal the dynamics of resistance acquisition by transfer of the Escherichia coli fertility factor-conjugation plasmid encoding the tetracycline-efflux pump TetA. The entry of the single-stranded DNA plasmid into the recipient cell is rapidly followed by complementary-strand synthesis, plasmid-gene expression, and production of TetA. In the presence of translation-inhibiting antibiotics, resistance acquisition depends on the AcrAB-TolC multidrug efflux pump, because it reduces tetracycline concentrations in the cell. Protein synthesis can thus persist and TetA expression can be initiated immediately after plasmid acquisition. AcrAB-TolC efflux activity can also preserve resistance acquisition by plasmid transfer in the presence of antibiotics with other modes of action.

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.

Increased salt tolerance with overexpression of cation/proton antiporter 1 genes: a meta-analysis.

Cation/proton antiporter 1 (CPA1) genes encode cellular Na+ /H+ exchanger proteins, which act to adjust ionic balance. Overexpression of CPA1s can improve plant performance under salt stress. However, the diversified roles of the CPA1 family and the various parameters used in evaluating transgenic plants over-expressing CPA1s make it challenging to assess the complex functions of CPA1s and their physiological mechanisms in salt tolerance. Using meta-analysis, we determined how overexpression of CPA1s has influenced several plant characteristics involved in response and resilience to NaCl stress. We also evaluated experimental variables that favour or reduce CPA1 effects in transgenic plants. Viewed across studies, overexpression of CPA1s has increased the magnitude of 10 of the 19 plant characteristics examined, by 25% or more. Among the ten moderating variables, several had substantial impacts on the extent of CPA1 influence: type of culture media, donor and recipient type and genus, and gene family. Genes from monocotyledonous plants stimulated root K+ , root K+ /Na+ , total chlorophyll, total dry weight and root length much more than genes from dicotyledonous species. Genes transformed to or from Arabidopsis have led to smaller CPA1-induced increases in plant characteristics than genes transferred to or from other genera. Heterogeneous expression of CPA1s led to greater increases in leaf chlorophyll and root length than homologous expression. These findings should help guide future investigations into the function of CPA1s in plant salt tolerance and the use of genetic engineering for breeding of resistance.

In vitro membrane protein synthesis inside Sec translocon-reconstituted cell-sized liposomes.

Protein synthesis using an in vitro transcription-translation system (IVTT) inside cell-sized liposomes has become a valuable tool to study the properties of biological systems under cell-mimicking conditions. However, previous liposome systems lacked the machinery for membrane protein translocation. Here, we reconstituted the translocon consisting of SecYEG from Escherichia coli inside cell-sized liposomes. The cell-sized liposomes also carry the reconstituted IVTT, thereby providing a cell-mimicking environment for membrane protein synthesis. By using EmrE, a multidrug transporter from E. coli, as a model membrane protein, we found that both the amount and activity of EmrE synthesized inside the liposome is increased approximately three-fold by incorporating the Sec translocon. The topological change of EmrE induced by the translocon was also identified. The membrane integration of 6 out of 9 E. coli inner membrane proteins that was tested was increased by incorporation of the translocon. By introducing the Sec translocon, the membrane integration efficiency of the membrane protein of interest was increased, and enabled the integration of membrane proteins that otherwise cannot be inserted. In addition, this work represents an essential step toward the construction of an artificial cell through a bottom-up approach.

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.

Mechanisms of Cl(-) uptake in rainbow trout: cloning and expression of slc26a6, a prospective Cl(-)/HCO3(-) exchanger.

In fresh waters, fishes continuously acquire ions to offset diffusive losses to a more dilute ambient environment and to maintain acid-base status. The objectives of the present study were to clone slc26a6, a prospective Cl(-)/HCO3(-) exchanger from rainbow trout, investigate its expression patterns in various tissues, at different developmental stages and after differential salinity exposure, and probe the mechanisms of Cl(-) uptake in rainbow trout embryos during development using a pharmacological inhibitor approach combined with (36)Cl(-) unidirectional fluxes. Results showed that the cloned gene encoded a 783 amino acid protein with conserved domains characteristic of the SLC26a family of anion exchange proteins. Phylogenetic analysis of this sequence against all subfamilies of the SLC26a family demonstrated that this translated protein shared a common ancestor with other actinopterygii and mammalian SLC26a6 isoforms and thus confirmed the identity of the cloned gene. Expression of slc26a6 was detected in all tissues and developmental stages assayed but was highest in the gill of juvenile trout. In trout embryos, Cl(-) uptake increased significantly post-hatch and was demonstrated to be mediated via an anion exchanger specific (DIDS sensitive) pathway that was also sensitive to hypercapnia. This parallels well with the predicted function of slc26a6, and the detection of the transcript in embryos and tissues of trout. In conclusion, this study is the first report of slc26a6 in rainbow trout and functional and expression analyses indicate its likely involvement in Cl(-)/HCO3(-) exchange in two life stages of rainbow trout.

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.

Altered pharmacokinetics of cimetidine caused by down-regulation of renal rat organic cation transporter 2 (rOCT2) after liver ischemia-reperfusion injury.

The renal tubular secretion of cationic drugs is dominated by basolateral organic cation transporter 2 (rOCT2/SLC22A2) and luminal multidrug and toxin extrusion 1 (rMATE1/SLC47A1). Little is known about the variation in the expression of these renal transporters after liver ischemia-reperfusion (I/R) injury. Here, we examined the pharmacokinetics of a cationic drug, cimetidine, and renal rOCT2 and rMATE1 levels as well as their regulation after liver I/R. Rats were subjected to 60 min of liver ischemia followed by 12 h of reperfusion. The antioxidant Trolox was administered intravenously 5 min before reperfusion. The systemic and tubular secretory clearances of cimetidine (78% and 55%) as well as renal rOCT2 and rMATE1 levels (67% and 61%) in I/R rats were decreased compared with those in sham-operated rats, respectively. However, the renal tissue-to-plasma concentration ratio but not the renal tissue-to-urine clearance ratio of cimetidine was decreased after liver I/R. Moreover, Trolox prevented the decreases in renal rOCT2 levels and systemic clearance of cimetidine after liver I/R. These results demonstrate that liver I/R decreases the tubular secretion of cimetidine, mainly because of the decreased rOCT2 level in the kidney, and that oxidative stress should be responsible in part for decreased renal rOCT2 after liver I/R injury.

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.

Expression of the sodium/calcium/potassium exchanger, NCKX4, in ameloblasts.

Transcellular calcium transport is an essential activity in mineralized tissue formation, including dental hard tissues. In many organ systems, this activity is regulated by membrane-bound sodium/calcium (Na(+)/Ca(2+)) exchangers, which include the NCX and NCKX [sodium/calcium-potassium (Na(+)/Ca(2+)-K(+)) exchanger] proteins. During enamel maturation, when crystals expand in thickness, Ca(2+) requirements vastly increase but exactly how Ca(2+) traffics through ameloblasts remains uncertain. Previous studies have shown that several NCX proteins are expressed in ameloblasts, although no significant shifts in expression were observed during maturation which pointed to the possible identification of other Ca(2+) membrane transporters. NCKX proteins are encoded by members of the solute carrier gene family, Slc24a, which include 6 different proteins (NCKX1-6). NCKX are bidirectional electrogenic transporters regulating Ca(2+) transport in and out of cells dependent on the transmembrane ion gradient. In this study we show that all NCKX mRNAs are expressed in dental tissues. Real-time PCR indicates that of all the members of the NCKX group, NCKX4 is the most highly expressed gene transcript during the late stages of amelogenesis. In situ hybridization and immunolocalization analyses clearly establish that in the enamel organ, NCKX4 is expressed primarily by ameloblasts during the maturation stage. Further, during the mid-late maturation stages of amelogenesis, the expression of NCKX4 in ameloblasts is most prominent at the apical poles and at the lateral membranes proximal to the apical ends. These data suggest that NCKX4 might be an important regulator of Ca(2+) transport during amelogenesis.

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.

Depletion of SLC4A11 causes cell death by apoptosis in an immortalized human corneal endothelial cell line.

PURPOSE: To investigate the effects of SLC4A11 gene depletion in human corneal endothelial cells. METHODS: To achieve stable downregulation of SLC4A11 gene expression in immortalized human corneal endothelial cells (HCECs), short-hairpin RNA (shRNA) targeted against SLC4A11 was used. Cell growth and viability were determined using the real-time cell analyzer and trypan blue staining respectively. Apoptosis was investigated by Annexin V and TUNEL assays. Alterations in apoptotic gene expression following SLC4A11 silencing were determined using the RT(2)Profiler PCR array for human apoptosis while activation of the apoptotic pathway was ascertained by western analysis. RESULTS: SLC4A11 silencing in HCECs could be achieved by stable expression of shRNA targeted against SLC4A11. SLC4A11 knockdown suppressed HCEC growth and reduced HCEC viability compared to the control. This reduction in cell growth is associated with increased apoptosis in SLC4A11-silenced cells. CONCLUSIONS: Our data suggest that the reduction of cell number with time in SLC4A11-depleted HCECs is due to an increase in cell death by apoptosis. This suggests that SLC4A11 is necessary for cell survival and may explain the pathologic corneal endothelial cell loss in endotheliopathies due to SLC4A11 mutations.

NHE1 activity contributes to migration and is necessary for proliferation of human gastric myofibroblasts.

Myofibroblasts play central roles in wound healing, deposition of the extracellular matrix and epithelial function. Their functions depend on migration and proliferation within the subepithelial matrix, which results in accelerated cellular metabolism. Upregulated metabolic pathways generate protons which need to be excreted to maintain intracellular pH (pH(i)). We isolated human gastric myofibroblasts (HGMs) from surgical specimens of five patients. Then we characterized, for the first time, the expression and functional activities of the Na(+)/H(+) exchanger (NHE) isoforms 1, 2 and 3, and the functional activities of the Na(+)/HCO(3)(-) cotransporter (NBC) and the anion exchanger (AE) in cultured HGMs using microfluorimetry, immunocytochemistry, reverse transcription polymerase chain reaction and immunoblot analysis. We showed that NHE1-3, NBC and AE activities are present in HGMs and that NHE1 is the most active of the NHEs. In scratch wound assays we also demonstrated (using the selective NHE inhibitor HOE-642) that carbachol and insulin like growth factor II (IGF-II) partly stimulate migration of HGMs in a NHE1-dependent manner. EdU incorporation assays revealed that IGF-II induces proliferation of HGMs which is inhibited by HOE-642. The results indicate that NHE1 is necessary for IGF-II-induced proliferation response of HGMs. Overall, we have characterized the pH(i) regulatory mechanisms of HGMs. In addition, we demonstrated that NHE1 activity contributes to both IGF-II- and carbachol-stimulated migration and that it is obligatory for IGF-II-induced proliferation of HGMs.

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.

Distribution of NBCn2 (SLC4A10) splice variants in mouse brain.

The five known Na-coupled HCO(3)(-) transporters (NCBTs) of the solute carrier 4 (SLC4) family play important roles in pH regulation and transepithelial HCO(3)(-) transport. Nearly all of the NCBTs have multiple splice variants. One particular NCBT, the electroneutral Na/HCO(3)(-) cotransporter NBCn2 (SLC4A10), which is predominantly expressed in brain, has three known splice variants-NBCn2-A, -B, and -C-as well as a potential variant-D. It is important to know the tissue-specific expression of the splice variants for understanding the physiological roles of NBCn2 in central nervous system. In the present study, we developed three novel rabbit polyclonal antibodies against NBCn2: (1) anti-ABCD, which recognizes all four variants; (2) anti-BD, which recognizes NBCn2-B and -D; (3) anti-CD, which recognizes NBCn2-C and -D. By western blotting, we examined the expression and distribution of NBCn2 splice variants in five brain regions: cerebral cortex, subcortex, cerebellum, hippocampus, and medulla. The expression pattern revealed with anti-ABCD is distinct from those revealed with anti-BD and anti-CD. Moreover, by using immunoprecipitation in combination with western blotting, we demonstrate that NBCn2-D does indeed exist and that it is predominantly expressed in subcortex, to a lesser extent in medulla, but at very low levels in cortex, cerebellum, and hippocampus. NBCn2-A may be the dominant variant in mouse brain as a whole, and may also dominate in cerebral cortex, cerebellum, and hippocampus. Immunohistochemistry with anti-ABCD shows that NBCn2 is highly expressed in choroid plexus, cortex, molecular layer of cerebellum, hippocampus, and some specific regions of the brainstem.

Ductal plate malformation-like arrays in early explants after a Kasai procedure are independent of splenic malformation complex (heterotaxy).

Biliary atresia has at least 2 proposed forms, the common perinatal and the less common embryonic subtype with earlier onset and/or extrahepatic developmental anomalies. Histologic evidence of ductal plate malformation (DPM)-like change in liver has been proposed both as a marker for the embryonic type and as a predictor of poor outcome after Kasai portoenterostomy. We investigated the prevalence of DPM-like change in liver explants in usual biliary atresia (BA) and in BA with splenic malformation syndrome (BASM). Liver sections from 8 patients with BA and 6 with BASM, all of whom had a Kasai procedure followed by explant before age 2 years, were analyzed using hematoxylin and eosin, trichrome, CK7, and AE1/AE3 stains. Each block was scored for inflammation and fibrosis. We estimated the number of portal areas per block and counted the number of definite and possible examples of DPM-like change, defined as a circumferential duct complex arranged around a fibrovascular core. We assessed whether the frequency per portal area was related to low and high scores for either inflammation or fibrosis. Definite and possible examples of DPM-like arrays were present in about 10% of portal areas in both patient groups, but these were unevenly distributed. There was no statistical difference between BA and BASM in terms of the number of examples per portal area. No correlation existed between degree of fibrosis and the intensity of portal inflammation and the number of DPM-like arrays. Ductal plate malformation-like arrays do not distinguish perinatal BA from BA associated with heterotaxy in liver explants after a failed Kasai procedure.

Structural plasticity and distinct drug-binding modes of LfrR, a mycobacterial efflux pump regulator.

The TetR-like transcriptional repressor LfrR controls the expression of the gene encoding the Mycobacterium smegmatis efflux pump LfrA, which actively extrudes fluoroquinolones, cationic dyes, and anthracyclines from the cell and promotes intrinsic antibiotic resistance. The crystal structure of the apoprotein form of the repressor reveals a structurally asymmetric homodimer exhibiting local unfolding and a blocked drug-binding site, emphasizing the significant conformational plasticity of the protein necessary for DNA and multidrug recognition. Crystallographic and calorimetric studies of LfrR-drug complexes further confirm the intrinsic flexibility of the homodimer, which provides a dynamic mechanism to broaden multidrug binding specificity and may be a general property of transcriptional repressors regulating microbial efflux pump expression.