The molecular identities of functional chloride channels in hepatocytes are largely unknown. We examined the ClC-3 chloride channel in rat hepatocytes and found that mRNA for two different isoforms is present. A short form is identical to the previously reported sequence for rat ClC-3, and a long form contains a 176-bp insertion immediately upstream of the translation initiation site. This predicts a 58-amino acid NH(2) terminal insertion. Both long and short form mRNA was expressed in diverse tissues of the rat. Transient transfection of the long form in CHO-K1 cells resulted in currents with an I(-)>> B(-)>> Cl(-) selectivity sequence, outward rectification, and inactivation at positive voltages. Short form currents had identical ionic selectivity but displayed a more extreme outward rectification and showed no voltage-dependent inactivation. Immunofluorescence and immunoblots localized native ClC-3 preferentially but not exclusively to the canalicular membrane. We have therefore identified a new isoform of rat ClC-3 and shown that expression of both isoforms produces functional channels. In hepatocytes, ClC-3 is located in association with the canalicular membrane.

WNK kinases are a small group of unique serine/threonine protein kinases that are conserved among multicellular organisms. Mutations in WNK1-4 cause pseudohypoaldosteronism type II-a form of hypertension. WNKs have been linked to the STE20 kinases and ion carriers, but the underlying molecular mechanisms by which WNKs regulate cellular processes in whole animals are unknown. The Caenorhabditis elegans WNK-like kinase WNK-1 interacts with and phosphorylates germinal centre kinase (GCK)-3--a STE20-like kinase--which is known to inactivate CLH-3, a CIC chloride channel. The wnk-1 or gck-3 deletion mutation causes an Exc phenotype, a defect in the tubular extension of excretory canals. Expression of the activated form of GCK-3 or the clh-3 deletion mutation can partly suppress wnk-1 or gck-3 defects, respectively. These results indicate that WNK-1 controls the tubular formation of excretory canals by activating GCK-3, resulting in downregulation of CIC channel activity.

Members of the ClC chloride channel family participate in several physiological processes and are linked to human genetic diseases. The physiological role of ClC-4 is unknown and previous detailed characterizations of recombinant human ClC-4 (hClC-4) have provided conflicting results. To re-examine the hClC-4 phenotype, recombinant hClC-4 was expressed in three distinct mammalian cell lines and characterized using patch-clamp techniques. In all cells, the expression of hClC-4 generated strongly outward-rectifying Cl(-) currents with the conductance sequence: SCN(-)>>) NO(3)(-)>>) Cl(-)>> Br(-) approximate I(-)>>) aspartate. Continuous activity of hClC-4 was sustained to different degrees by internal nucleotides: ATP approximately ATPgammaS>>) AMP-PNP approximate GTP>> ADP. Although non-hydrolysable nucleotides are sufficient for channel function, ATP hydrolysis is required for full activity. Changing the extracellular (2 mM or nominal Ca(2+)-free) or intracellular Ca(2+) (25 or 250 nM) concentration did not alter hClC-4 currents. Acidification of external pH (pH(o)) inhibited hClC-4 currents (half-maximal inhibition approximate 6.19), whereas neither external alkalinization to pH 8.4 nor internal acidification to pH 6.0 reduced current levels. Single-channel recordings demonstrated a Cl(-) channel active only at depolarizing potentials with a slope conductance of approximately 3 pS. Acidic pH(o) did not alter single-channel conductance. We conclude that recombinant hClC-4 encodes a small-conductance, nucleotide-dependent, Ca(2+)-independent outward-rectifying chloride channel that is inhibited by external acidification. This detailed characterization will be highly valuable in comparisons of hClC-4 function with native chloride channel activities and for future structure-function correlations.

OBJECTIVE

We have shown that the chloride-proton antiporter chloride channel-3 (ClC-3) is required for endosome-dependent signaling by the Nox1 NADPH oxidase in SMCs. In this study, we tested the hypothesis that ClC-3 is necessary for proliferation of smooth muscle cells (SMCs) and contributes to neointimal hyperplasia following vascular injury.

RESULTS

Studies were performed in SMCs isolated from the aorta of ClC-3-null and littermate control (wild-type [WT]) mice. Thrombin and tumor necrosis factor-α (TNF-α) each caused activation of both mitogen activated protein kinase extracellular signal-regulated kinases 1 and 2 and the matrix-degrading enzyme matrix metalloproteinase-9 and cell proliferation of WT SMCs. Whereas responses to thrombin were preserved in ClC-3-null SMCs, the responses to TNF-α were markedly impaired. These defects normalized following gene transfer of ClC-3. Carotid injury increased vascular ClC-3 expression, and compared with WT mice, ClC-3-null mice exhibited a reduction in neointimal area of the carotid artery 28 days after injury.

CONCLUSIONS

ClC-3 is necessary for the activation of SMCs by TNF-α but not thrombin. Deficiency of ClC-3 markedly reduces neointimal hyperplasia following vascular injury. In view of our previous findings, this observation is consistent with a role for ClC-3 in endosomal Nox1-dependent signaling. These findings identify ClC-3 as a novel target for the prevention of inflammatory and proliferative vascular diseases.

ClC-3 is a ubiquitously expressed chloride transport protein that is present in synaptic vesicles and endosome/lysosome compartments. It is largely intracellular but has been observed at the plasma membrane as well. The aim of this study was to identify the pathways and regulation of ClC-3 trafficking to intracellular sites. At the steady state, approximately 94% of transfected ClC-3 was localized intracellularly, and only 6% was at the plasma membrane. Pulse labeling with [(35)S]methionine and biotinylation demonstrated that about 25% of newly synthesized ClC-3 traffics through the plasma membrane. We used both immunofluorescence microscopy and biotinylation assays to assess the trafficking of ClC-3. Plasma membrane ClC-3 was rapidly endocytosed (t((1/2)) approximately 9 min); a portion entered a recycling pool that returned to the cell surface after internalization, and the remainder trafficked to more distal intracellular compartments. ClC-3 associated with clathrin at the plasma membrane. Coimmunoprecipitation and glutathione S-transferase pulldown assays demonstrated that the N terminus of ClC-3 binds to clathrin. Alanine replacement of a dileucine acidic cluster within the cytosolic N terminus (amino acids 13-19) resulted in a molecule that had decreased endocytosis and increased surface expression. This replacement also abolished interaction with clathrin as assessed both by coimmunoprecipitation and glutathione S-transferase pulldown assays. We conclude that ClC-3 is primarily an intracellular transport protein that is transiently inserted into the plasma membrane where it is rapidly endocytosed. Internalization of ClC-3 depends on the interaction between an N-terminal dileucine cluster and clathrin.

Epithelial cells of the epididymis and vas deferens establish an optimum luminal environment in which spermatozoa mature and are stored. This is achieved by active transepithelial transport of various ions including Cl(-) and H(+). We investigated the localization of three closely related members of the ClC family, ClC-3, ClC-4, and ClC-5, in the epididymis and vas deferens. RT-PCR using mRNA isolated by laser capture microdissection (LCM)-detected ClC-3 and ClC-5 transcripts but did not detect any ClC-4-specific transcript. Western blot and immunofluorescence analysis demonstrated that ClC-3 and ClC-5 proteins are present in all regions of the epididymis and in the vas deferens. ClC-5 is expressed exclusively in H(+)-ATPase-rich cells (narrow and clear cells). Confocal microscopy showed that ClC-5 partially colocalizes with the H(+)-ATPase in the subapical pole of clear cells. ClC-3 is strongly expressed in the apical membrane of principal cells of the caput epididymidis and the vas deferens and is less abundant in principal cells of the body and cauda epididymidis. These findings are consistent with a potential role for ClC-3 in transepithelial chloride transport by principal cells and for ClC-5 in the acidification of H(+)-ATPase-containing vesicles in narrow and clear cells. ClC-5 might facilitate endosome trafficking in the epididymis, as has been proposed in the kidney.

The CLC-K1 chloride channel is a kidney-specific CLC chloride channel expressed in the thin ascending limb of Henle's loop (tAL). Recently, we determined that Clcnk1-/- mice show nephrogenic diabetes insipidus (NDI). To investigate the pathogenesis of impaired urinary concentrating ability, we analyzed renal functions of Clcnk1-/- mice in more detail. The osmolar clearance-to-creatinine clearance ratio was not significantly different between Clcnk1+/- and Clcnk1+/+ mice. Fractional excretion of sodium, chloride, and urea was also not significantly affected in Clcnk1-/- mice. These results indicate that the polyuria observed in Clcnk1-/- mice was water diuresis and not osmotic diuresis. The papillary osmolarity in Clcnk1-/- mice was significantly lower than that in Clcnk1+/+ mice under a hydrated condition, and it did not increase even after water deprivation. Sodium and chloride contents in the inner medulla in Clcnk1-/- mice were at about one-half the levels observed in Clcnk1+/+ mice. Furthermore, the accumulation of urea was also impaired in Clcnk1-/- mice, suggesting that the overall countercurrent system was impaired by a defect of its single component, chloride transport in the tAL. The aldose reductase mRNA abundance in Clcnk1-/- mice was decreased, further evincing that inner medullary tonicity is decreased in Clcnk1-/- mice. We concluded that NDI in Clcnk1-/- mice resulted from an impairment in the generation of inner medullary hypertonicity by a dysfunction of the countercurrent systems.

Proteins of the CLC family are comprised of two subunits, each with its own fast-gated protopore, both of these being regulated simultaneously by a slower common gate. Based on the X-ray crystal structure of a bacterial CLC, the carboxyl side chain of glutamate residue E232 has been proposed as the fast gate of hClC-1, swinging into each pore to close it and competing with chloride. We now show, using hClC-1 mutants expressed in whole-cell patch-clamped HEK293 cells, that elimination of this side chain in the E232Q mutation prevents fast gate closure at all voltages but common gating is also eliminated suggesting that E232 could be the final effector of both fast and common gating. We hypothesise that the conformational information essential for common gating flows between the two E232 protopore residues across the intra-membrane interface, rather than via any cytoplasmic carboxyl-tail interface, to drive common gating. Informed by in silico modelling, we have produced five site-directed mutants that increase the volumes of residues which might be involved in allosteric transfer (A272V, A272L, S289L, V292L and T293L) and assessed them for effects on gating. These mutations could be expected to increase molecular forces between, or torques around, the intimate L287-L287 and I290-I290 contacts that form the pseudo-asymmetric axis of the hClC-1 dimer. Common gating is practically eliminated in V292L and open probability is shifted to more depolarised potentials in A272V, S289L and T293L mainly by altering the voltage dependence of common gating.

The voltage-gated Cl(-) channel (CLC) family comprises cell surface Cl(-) channels and intracellular Cl(-)/H(+) exchangers. CLCs in organelle membranes are thought to assist acidification by providing a passive chloride conductance that electrically counterbalances H(+) accumulation. Following recent descriptions of Cl(-)/H(+) exchange activity in endosomal CLCs we have re-evaluated their role. We expressed human CLC-5 in HEK293 cells, recorded currents under a range of Cl(-) and H(+) gradients by whole-cell patch clamp, and examined the contribution of CLC-5 to endosomal acidification using a targeted pH-sensitive fluorescent protein. We found that CLC-5 only conducted outward currents, corresponding to Cl(-) flux into the cytoplasm and H(+) from the cytoplasm. Inward currents were never observed, despite the range of intracellular and extracellular Cl(-) concentrations and pH used. Endosomal acidification in HEK293 cells was prevented by 25 microm bafilomycin-A1, an inhibitor of vacuolar-type H(+)-ATPase (v-ATPase), which actively pumps H(+) into the endosomal lumen. Overexpression of CLC-5 in HEK293 cells conferred an additional bafilomycin-insensitive component to endosomal acidification. This effect was abolished by making mutations in CLC-5 that remove H(+) transport, which result in either no current (E268A) or bidirectional Cl(-) flux (E211A). Endosomal acidification in a proximal tubule cell line was partially sensitive to inhibition of v-ATPase by bafilomycin-A1. Furthermore, in the presence of bafilomycin-A1, acidification was significantly reduced and nearly fully ablated by partial and near-complete knockdown of endogenous CLC-5 by siRNA. These data suggest that CLC-5 is directly involved in endosomal acidification by exchanging endosomal Cl(-) for H(+).

JAK2 (Janus kinase-2) is activated by cell shrinkage and may thus participate in cell volume regulation. Cell volume regulatory ion channels include the small conductance Cl(-) channels ClC-2. The present study thus explored whether JAK2 influences ClC-2 activity. To this end, ClC-2 was expressed in Xenopus oocytes with or without wild type JAK2, active (V617F)JAK2 or inactive (K882E)JAK2 and the Cl(-) channel activity determined by dual electrode voltage clamp. Expression of ClC-2 was followed by a marked increase of cell membrane conductance. The conductance was significantly decreased following coexpression of JAK2 or (V617F)JAK2, but not by coexpression of (K882E)JAK2. Exposure of the oocytes expressing ClC-2 together with (V617F)JAK2 to the JAK2 inhibitor AG490 (40 μM) resulted in a gradual increase of the conductance. According to chemiluminescence JAK2 decreased the channel protein abundance in the cell membrane. The decline of conductance in ClC-2 and (V617F)JAK2 coexpressing oocytes following inhibition of channel protein insertion by brefeldin A (5 μM) was similar in oocytes expressing ClC-2 with (V617F)JAK2 and oocytes expressing ClC-2 alone, indicating that (V617F)JAK2 might slow channel protein insertion into rather than accelerating channel protein retrieval from the cell membrane. In conclusion, JAK2 down-regulates ClC-2 activity and thus counteracts Cl(-) exit, an effect which may impact on cell volume regulation.

Bacterial autolysins are endogenous enzymes that specifically cleave covalent bonds in the cell wall. These enzymes show both substrate and bond specificities. The former is related to their interaction with the insoluble substrate whereas the latter determine their site of action. The bond specificity allows their classification as muramidases (lysozymes), glucosaminidases, amidases, and endopeptidases. To demonstrate that the autolysin (LYC muramidase) of Clostridium acetobutylicum ATCC824 presents a domainal organization, a chimeric gene (clc) containing the regions coding for the catalytic domain of the LYC muramidase and the choline-binding domain of the pneumococcal phage CPL1 muramidase has been constructed by in vitro recombination of the corresponding gene fragments. This chimeric construction codes for a choline-binding protein (CLC) that has been purified using affinity chromatography on DEAE-cellulose. Several biochemical tests demonstrate that this rearrangement of domains has generated an enzyme with a choline-dependent muramidase activity on pneumococcal cell walls. Since the parental LYC muramidase was choline-independent and unable to degrade pneumococcal cell walls, the formation of this active chimeric enzyme by exchanging protein domains between two enzymes that specifically hydrolyse cell walls of bacteria belonging to different genera shows that a switch on substrate specificity has been achieved. The chimeric CLC muramidase behaved as an autolytic enzyme when it was adsorbed onto a live autolysin-defective mutant of Streptococcus pneumoniae. The construction described here provides experimental support for the theory of modular evolution which assumes that novel proteins have evolved by the assembly of preexisting polypeptide units.

Transepithelial Cl(-) transport in salivary gland ducts is a major component of the ion reabsorption process, the final stage of saliva production. It was previously demonstrated that a Cl(-) current with the biophysical properties of ClC-2 channels dominates the Cl(-) conductance of unstimulated granular duct cells in the mouse submandibular gland. This inward-rectifying Cl(-) current is activated by hyperpolarization and elevated intracellular Cl(-) concentration. Here we show that ClC-2 immunolocalized to the basolateral region of acinar and duct cells in mouse salivary glands, whereas its expression was most robust in granular and striated duct cells. Consistent with this observation, nearly 10-fold larger ClC-2-like currents were observed in granular duct cells than the acinar cells obtained from submandibular glands. The loss of inward-rectifying Cl(-) current in cells from Clcn2(-/-) mice confirmed the molecular identity of the channel responsible for these currents as ClC-2. Nevertheless, both in vivo and ex vivo fluid secretion assays failed to identify significant changes in the ion composition, osmolality, or salivary flow rate of Clcn2(-/-) mice. Additionally, neither a compensatory increase in Cftr Cl(-) channel protein expression nor in Cftr-like Cl(-) currents were detected in Clcn2 null mice, nor did it appear that ClC-2 was important for blood-organ barrier function. We conclude that ClC-2 is the inward-rectifying Cl(-) channel in duct cells, but its expression is not apparently required for the ion reabsorption or the barrier function of salivary ductal epithelium.

OBJECTIVE

Combined hepatocellular-cholangiocarcinoma (cHC-CC), a malignant liver tumour with poor prognosis, is composed of hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and diverse components with intermediate features between HCC and CC, which correspond to hepatic progenitor cells. According to the WHO classification 2010, we surveyed the prevalence and clinicopathological significance of each subtype with stem-cell features [SC subtype; typical subtype (TS), intermediate cell subtype (INT) and cholangiolocellular type (CLC)] in cHC-CC and HCC.

METHODS

Sixty-two patients with cHC-CC (19 women and 43 men) and 26 patients with HCC (all men) were examined. The prevalence of each component was histologically assessed with assistance of mucin and immunohistochemical stainings.

RESULTS

SC subtypes were observed in all cHC-CCs in various amount and combination. The prevalence of each SC subtype in cHC-CC was as follows: TS, 10 (16.1%); INT, 53 (83.9%) and CLC, 44 (71.0%). The proportion of INT was significantly correlated with gender (female-dominant) (P < 0.05), tumour size (P < 0.05), histological grading of HCC (P < 0.01) and inversely correlated with the degree of stromal fibrosis (P < 0.05). The proportion of CLC was significantly correlated with the degree of fibrosis (P < 0.01) and inflammation (P < 0.01), and inversely correlated with tumour size (P < 0.01) and histological grading of HCC (P < 0.05). The proportion of TS was significantly inversely correlated with the degree of inflammation (P < 0.01). Histological diversity score was significantly correlated with vascular invasion and the positivity for α-foetoprotein.

CONCLUSIONS

The proportion of each SC subtype was significantly associated with certain clinicopathological factors, suggesting different properties of each SC subtypes.

In order to clarify the mechanism underlying the reduction of resting membrane chloride conductance (gCl) during aging, the levels of mRNA encoding the principal skeletal muscle chloride channel, ClC-1, were measured. Total RNA samples isolated from tibialis anterior muscles of aged (24-29 months old) and adult (3-4 months old) rats were examined for ClC-1 expression using Northern blot analysis, and macroscopic gCl was recorded from extensor digitorum longus muscle fibers from each adult and aged rat in vitro using a two intracellular microelectrode technique. Although interindividual variability was observed, aged rats exhibited a parallel reduction of both gCl and ClC-1 mRNA expression as compared to adult rats. A linear correlation exists between individual values of ClC-1 mRNA and gCl. These results provide evidence that ClC-1 is the main determinant of sarcolemmal gCl and demonstrate that the decrease of gCl observed during aging is associated with a down-regulation of ClC-1 expression in muscle.

Whether ClC-3 encodes volume-sensitive organic osmolyte and anion channels (VSOACs) remains controversial. We have shown previously that native VSOACs in some cardiac and vascular myocytes were blocked by a commercial anti-ClC-3 carboxy terminal antibody (Alm C592-661 antibody), although recent studies have raised questions related to the specificity of Alm C592-661 antibody. Therefore, we have developed three new anti-ClC-3 antibodies and investigated their functional effects on native VSOACs in freshly isolated canine pulmonary artery smooth muscle cells (PASMCs) and guinea pig cardiac myocytes. These new antibodies produced a common prominent immunoreactive band with an apparent molecular mass of 90-92 kDa in the guinea pig heart and PASMCs, and a similar molecular mass immunoreactive band was observed in the brain from homozygous Clcn3+/+ mice but not from homozygous Clcn3-/- mice. VSOACs elicited by hypotonic cell swelling in PASMCs and guinea pig atrial myocytes were nearly completely abolished by intracellular dialysis with two new anti-ClC-3 antibodies specifically targeting the ClC-3 carboxy (C670-687 antibody) and amino terminus (A1-14 antibody). This inhibition of native VSOACs can be attributed to a specific interaction with endogenous ClC-3, because 1) preabsorption of the antibodies with corresponding antigens prevented the inhibitory effects, 2) extracellular application of a new antibody raised against an extracellular epitope (Ex133-148) of ClC-3 failed to inhibit native VSOACs in PASMCs, 3) intracellular dialysis with an antibody targeting Kv1.1 potassium channels failed to inhibit native VSOACs in guinea pig atrial myocytes, and 4) anti-ClC-3 C670-687 antibody had no effects on swelling-induced augmentation of the slow component of the delayed rectifying potassium current in guinea pig ventricular myocytes, although VSOACs in the same cells were inhibited by the antibody. These results confirm that endogenous ClC-3 is an essential molecular entity responsible for native VSOACs in PASMCs and guinea pig cardiac myocytes.

Volume regulation is essential for cell function, but it is unknown which channels are involved in a regulatory volume decrease (RVD) in human gastric epithelial cells. Exposure to a hypotonic solution caused the increase in AGS cell volume, followed by the activation of a current. The reversal potential of the swelling-induced current suggested that Cl- was the primary charge carrier. The selectivity sequence for different anions was I->> Br->> Cl->> F->> gluconate. This current was inhibited by flufenamate, DIDS, tamoxifen, and 5-nitro-2-(3-phenylpropylamino)benzoate. Intracellular dialysis of three different anti-ClC-3 antibodies abolished or attenuated the Cl- current and disrupted RVD, whereas the current and RVD was unaltered by anti-ClC-2 antibody. Immunoblot studies demonstrated the presence of ClC-3 protein in Hela and AGS cells. RT-PCR analysis detected expression of ClC-3, MDR-1, and pICln mRNA in AGS cells. These results suggest a fundamental role of endogenous ClC-3 in the swelling-activated Cl- channels function and cell volume regulation in human gastric epithelial cells.

BACKGROUND

This study aimed to compare the short-term outcomes of single-access laparoscopic cholecystectomy (SALC) and conventional laparoscopic cholecystectomy (CLC).

METHODS

In a prospective study, patients with symptomatic cholelithiasis were randomized to SALC or CLC with follow-up at 1 week, 1 and 6 months. The primary end point of this study was to assess the total outcomes of quality of life using the EuroQoL EQ-5D questionnaire. The secondary end points were postoperative pain, analgesia requirement and duration of use, operative time, perioperative complications, estimated blood loss, hospital stay, cosmesis outcome, and number of days required to return to normal activities.

RESULTS

A total of 269 patients were prospectively randomized into two groups (125 in each group after excluding 19 patients for various reasons). The SALC procedure was done safely without intraoperative or major postoperative complications. In four SALC patients, an extra epigastric port was inserted to enhance exposure. There was no open conversion in either group. SALC patients reported better results among four of the EuroQoL EQ-5D dimensions (mobility, self-care, activity, and pain/discomfort) at 1 week after surgery, an improved pain profile at 4, 12, and 24 h, better cosmetic outcome at 1 and 6 months (P ≤ 0.01), shorter duration of need for analgesia (P ≤ 0.02), and earlier return to normal activities (P ≤ 0.026). Operative times, hospital stay, QOL at 1 and 6 months postoperatively, and estimated blood loss were similar for both procedures.

CONCLUSIONS

This study supports other studies that show that SALC is a feasible and promising alternative to traditional laparoscopic cholecystectomy in selected patients with better cosmesis, QOL, and improved postoperative pain results, and it can be performed with the existing laparoscopic instruments.

We clinicopathologically studied 6 resected cases of cholangiolocarcinoma (CLC) including 2 referred cases from other hospitals. The frequency of CLC was 0.56% of the 708 consecutively resected cases of primary liver cancer and the mean age of CLC cases was 66 years. Three of the 6 cases (50%) were hepatitis C virus antibody (HCVab) positive, one (17%) was hepatitis B virus surface antigen (HBsAg) positive, and 2 (33%) were negative to both HCVAb and HBsAg. Serum levels of alpha-fetoprotein were slightly elavated only in 1 case. Clinically, 4 cases were diagnosed as hepatocellular carcinoma (HCC) and 2 cases as cholangiocellular carcinoma (CCC). Grossly, CLCs were whitish in color and solid, not encapsulated, and resembled CCC. Histologically, the tumor cells had eosinophilic cytoplasm with ovoid nuclei, and mild atypia. The tumor proliferated in an anastomosing pattern of Hering's canal-like small glands with an abundant fibrous stroma. Four of the 6 tumors (83%) consisted of only CLC and other 2 tumors contained CCC-like area and HCC-like area in a part of the nodules, respectively. Immunohistochemically, all tumors were positive to cytokeratin (CK) 7. CK8 were also positive in all of 6 cases. These results revealed that CLC had the clinical features resembling HCC but the morphologic features resembling CCC. It is suggested that CLC cells might be derived from Hering's canal or stem cells which have the intermediate features between hepatocytes and bile duct epithelium.

Avian influenza virus (AIV) is a type A virus of the family Orthomyxoviridae. Avian influenza virus infection can cause significant economic losses to the poultry industry, and raises a great public health threat due to potential host jump from animals to humans. To develop more effective intervention strategies to prevent and control AIV infection in poultry, it is essential to elucidate molecular mechanisms of host response to AIV infection in chickens. The objective of this study was to identify genes and signal pathways associated with resistance to AIV infection in 2 genetically distinct highly inbred chicken lines (Fayoumi, relatively resistant to AIV infection, and Leghorn, susceptible to AIV infection). Three-week-old chickens were inoculated with 10(7) EID50 of low pathogenic H5N3 AIV, and lungs and trachea were harvested 4 d postinoculation. Four cDNA libraries (1 library each for infected and noninfected Leghorn, and infected and noninfected Fayoumi) were prepared from the lung samples and sequenced by Illumina Genome Analyzer II, which yielded a total of 116 million, 75-bp single-end reads. Gene expression levels of all annotated chicken genes were analyzed using CLC Genomics Workbench. DESeq was used to identify differentially expressed transcripts between infected and noninfected birds and between genetic lines (false discovery rate < 0.05 and fold-change>> 2). Of the expressed transcripts in a total of 17,108 annotated chicken genes in Ensembl database, 82.44 and 81.40% were identified in Leghorn and Fayoumi birds, respectively. The bioinformatics analysis suggests that the hemoglobin family genes, the functional involvements for oxygen transportation and circulation, and cell adhesion molecule signaling pathway play significant roles in disease resistance to AIV infection in chickens. Further investigation of the roles of these candidate genes and signaling pathways in the regulation of host-AIV interaction can lead new directions for the development of antiviral drugs or vaccines in poultry.

OBJECTIVE

In families with idiopathic generalized epilepsy (IGE), multiple IGE subsyndromes may occur. We performed a genetic study of IGE families to clarify the genetic relation of the IGE subsyndromes and to improve understanding of the mode(s) of inheritance.

METHODS

Clinical and genealogic data were obtained on probands with IGE and family members with a history of seizures. Families were grouped according to the probands' IGE subsyndrome: childhood absence epilepsy (CAE), juvenile absence epilepsy (JAE), juvenile myoclonic epilepsy (JME), and IGE with tonic-clonic seizures only (IGE-TCS). The subsyndromes in the relatives were analyzed. Mutations in genes encoding alpha1 and gamma 2 gamma-aminobutyric acid (GABA)-receptor subunits, alpha1 and beta1 sodium channel subunits, and the chloride channel CLC-2 were sought.

RESULTS

Fifty-five families were studied. 122 (13%) of 937 first- and second-degree relatives had seizures. Phenotypic concordance within families of CAE and JME probands was 28 and 27%, respectively. JAE and IGE-TCS families had a much lower concordance (10 and 13%), and in the JAE group, 31% of relatives had CAE. JME was rare among affected relatives of CAE and JAE probands and vice versa. Mothers were more frequently affected than fathers. No GABA-receptor or sodium or chloride channel gene mutations were identified.

CONCLUSIONS

The clinical genetic analysis of this set of families suggests that CAE and JAE share a close genetic relation, whereas JME is a more distinct entity. Febrile seizures and epilepsy with unclassified tonic-clonic seizures were frequent in affected relatives of all IGE individuals, perhaps representing a nonspecific susceptibility to seizures. A maternal effect also was seen. Our findings are consistent with an oligogenic model of inheritance.

OBJECTIVE

The fluid secretion model predicts that intestinal obstruction disorders can be alleviated by promoting epithelial Cl(-) secretion. The adenosine 3',5'-cyclic monophosphate (cAMP)-activated anion channel CFTR mediates Cl(-)-dependent fluid secretion in the intestine. Although the role of the ClC-2 channel has not been determined in the intestine, this voltage-gated Cl(-) channel might compensate for the secretory defects observed in patients with cystic fibrosis and other chronic constipation disorders. We investigated whether mice that lack ClC-2 channels (Clcn2(-/-)) have defects in intestinal ion transport.

METHODS

Immunolocalization and immunoblot analyses were used to determine the cellular localization and the amount of ClC-2 expressed in mouse early distal colon (EDC) and late distal colon (LDC). Colon sheets from wild-type and Clcn2(-/-) littermates were mounted in Ussing chambers to determine transepithelial bioelectrical parameters and Na(+), K(+), and Cl(-) fluxes.

RESULTS

Expression of ClC-2 was higher in the basolateral membrane of surface cells in the EDC compared with the LDC, with little expression in crypts. Neither cAMP nor Ca(2+)-induced secretion of Cl(-) was affected in the EDC or LDC of Clcn2(-/-) mice, whereas the amiloride-sensitive short-circuit current was increased approximately 3-fold in Clcn2(-/-) EDC compared with control littermates. Conversely, electroneutral Na(+), K(+), and Cl(-) absorption was dramatically reduced in colons of Clcn2(-/-) mice.

CONCLUSIONS

Basolateral ClC-2 channels are required for colonic electroneutral absorption of NaCl and KCl. The increase in the amiloride-sensitive short-circuit current in Clcn2(-/-) mice revealed a compensatory mechanism that is activated in the colons of mice that lack the ClC-2 channel.

BACKGROUND

We conducted genomic sequencing to identify Epstein Barr Virus (EBV) genomes in 2 human peripheral blood B lymphocytes that underwent spontaneous immortalization promoted by mycoplasma infections in culture, using the high-throughput sequencing (HTS) Illumina MiSeq platform. The purpose of this study was to examine if rapid detection and characterization of a viral agent could be effectively achieved by HTS using a platform that has become readily available in general biology laboratories.

RESULTS

Raw read sequences, averaging 175 bps in length, were mapped with DNA databases of human, bacteria, fungi and virus genomes using the CLC Genomics Workbench bioinformatics tool. Overall 37,757 out of 49,520,834 total reads in one lymphocyte line (# K4413-Mi) and 28,178 out of 45,335,960 reads in the other lymphocyte line (# K4123-Mi) were identified as EBV sequences. The two EBV genomes with estimated 35.22-fold and 31.06-fold sequence coverage respectively, designated K4413-Mi EBV and K4123-Mi EBV (GenBank accession number KC440852 and KC440851 respectively), are characteristic of type-1 EBV.

CONCLUSIONS

Sequence comparison and phylogenetic analysis among K4413-Mi EBV, K4123-Mi EBV and the EBV genomes previously reported to GenBank as well as the NA12878 EBV genome assembled from database of the 1000 Genome Project showed that these 2 EBVs are most closely related to B95-8, an EBV previously isolated from a patient with infectious mononucleosis and WT-EBV. They are less similar to EBVs associated with nasopharyngeal carcinoma (NPC) from Hong Kong and China as well as the Akata strain of a case of Burkitt's lymphoma from Japan. They are most different from type 2 EBV found in Western African Burkitt's lymphoma.

Loss of the lysosomal ClC-7/Ostm1 2Cl(-)/H(+) exchanger causes lysosomal storage disease and osteopetrosis in humans and additionally changes fur colour in mice. Its conversion into a Cl(-) conductance in Clcn7(unc/unc) mice entails similarly severe lysosomal storage, but less severe osteopetrosis and no change in fur colour. To elucidate the basis for these phenotypical differences, we generated Clcn7(td/td) mice expressing an ion transport-deficient mutant. Their osteopetrosis was as severe as in Clcn7(-/-) mice, suggesting that the electric shunt provided by ClC-7(unc) can partially rescue osteoclast function. The normal coat colour of Clcn7(td/td) mice and their less severe neurodegeneration suggested that the ClC-7 protein, even when lacking measurable ion transport activity, is sufficient for hair pigmentation and that the conductance of ClC-7(unc) is harmful for neurons. Our in vivo structure-function analysis of ClC-7 reveals that both protein-protein interactions and ion transport must be considered in the pathogenesis of ClC-7-related diseases.

It has been found that nonsense mutation R419X of cereblon (CRBN) is associated with autosomal recessive non-syndromic mental retardation. Further experiments showed that CRBN binds to the cytosolic C-terminus of large-conductance Ca(++) activated potassium channel (BK(Ca)) α-subunit and the cytosolic C-terminus of a voltage-gated chloride channel-2 (ClC-2), suggesting that CRBN may play a role in memory and learning via regulating the assembly and surface expression of BK(Ca) and ClC-2 channels. In addition, it has also been found that CRBN directly interacts with the α1 subunit of AMP-activated protein kinase (AMPK) and prevents formation of a functional holoenzyme with regulatory subunits β and γ. Since AMPK is a master sensor of energy balance that inhibits ATP-consuming anabolic pathways and increases ATP-producing catabolic pathways, binding of CRBN with α1 subunit of AMPK may play a role in these pathways by regulating the function of AMPK. Furthermore, CRBN interacts with damaged DNA binding protein 1 and forms an E3 ubiquitin ligase complex with Cullin 4 where it functions as a substrate receptor in which the proteins recognized by CRBN might be ubiquitinated and degraded by proteasomes. Proteasome-mediated degradation of unneeded or damaged proteins plays a very important role in maintaining regular function of a cell, such as cell survival, dividing, proliferation and growth. Intriguingly, a new role for CRBN has been identified, i.e, the binding of immunomodulatory drugs (IMiDs), e.g. thalidomide, to CRBN has now been associated with teratogenicity and also the cytotoxicity of IMiDs, including lenalidomide, which are widely used to treat multiple myeloma patients. CRBN likely plays an important role in binding, ubiquitination and degradation of factors involved in maintaining function of myeloma cells. These new findings regarding the role of CRBN in IMiD action will stimulate intense investigation of CRBN's downstream factors involved in maintaining regular function of a cell.