BACKGROUND

Despite increasing data regarding clinical outcomes following transvaginal hybrid NOTES cholecystectomy (TVC), a consensus regarding safety based on comparative studies has yet to be reached. The aim of this systematic review and meta-analysis was to compare safety and clinical outcomes of TVC with conventional laparoscopic cholecystectomy (CLC) for the treatment of benign gallstone disease.

METHODS

A comprehensive search for published studies comparing TVC and CLC was performed. Review of each study was conducted and data were extracted. All pooled outcome measures were determined using random-effects models.

RESULTS

Data were retrieved from 14 studies describing 1,145 patients. There was no difference in total complications (POR = 0.68; 95 % CI 0.40-1.14; P = 0.14), incidence of bile duct injury (POR = 1.33; 95 % CI 0.31-5.66; P = 0.70), Clavien-Dindo Grade II (POR = 0.48; 95 % CI 0.14-1.60; P = 0.23) or Grade III (POR = 0.63; 95 % CI 0.24-1.65; P = 0.34) complications between TCV and CLC. Time of return to normal activities was significantly reduced in the TVC group (WMD = -4.86 days; 95 % CI -9.33 to -0.39; P = 0.03), and there was a non-significant reduction in postoperative pain on days 1 (WMD = -0.80; 95 % CI -1.60 to 0.01; P = 0.05) and 3 (WMD = -0.89; 95 % CI -1.77 to -0.01; P = 0.05).

CONCLUSIONS

TVC is safe when performed by appropriately trained surgeons and may be associated with a faster return to normal activities and decreased postoperative pain.

Functional characterization of intracellular transporters is hampered by the inaccessibility of animal endomembranes to standard electrophysiological techniques. Here, we used Arabidopsis mesophyll protoplasts as a novel heterologous expression system for the lysosomal chloride–proton exchanger CLC-7 from rat. Following transient expression of a rCLC-7:EGFP construct in isolated protoplasts, the fusion protein efficiently targeted to the membrane of the large central vacuole, the lytic compartment of plant cells. Membrane currents recorded from EGFP-positive vacuoles were almost voltage independent and showed time-dependent activation at elevated positive membrane potentials as a hallmark. The shift in the reversal potential of the current induced by a decrease of cytosolic pH was compatible with a 2Cl(-)/1H(+) exchange stoichiometry. Mutating the so-called gating glutamate into alanine (E245A) uncoupled chloride fluxes from the movement of protons, transforming the transporter into a chloride channel-like protein. Importantly, CLC-7 transport activity in the vacuolar expression system was recorded in the absence of the auxiliary subunit Ostm1, differently to recent data obtained in Xenopus oocytes using a CLC-7 mutant with partial plasma membrane expression. We also show that plasma membrane-targeted CLC-7(E245A) is non-functional in Xenopus oocytes when expressed without Ostm1. In summary, our data suggest the existence of an alternative CLC-7 operating mode, which is active when the protein is not in complex with Ostm1. The vacuolar expression system has the potential to become a valuable tool for functional studies on intracellular ion channels and transporters from animal cells.

Using the combination method with PCR phylogrouping and fimH SNPs analysis, this study investigates the epidemiology of Extra-intestinal pathogenic E. coli in China. 116 E. coli strains including (74 from Urine, 39 from other extra-intestinal sources and 3 references strains) were collected. The bacteria Genomic DNA were extracted; phylogroup and the fimH gene amplifications were determined by two-step triplex PCR-based phylogrouping and simple PCR amplification assay respectively. Finally the fimH SNPs analysis and phylogenetic analysis and construction of tree were carried out using DNAMAN Version 6.0.3.93 and MEGA4, ClustalW and CLC Bio software respectively for 50 E. coli strains isolated from clinical sample and 3 references; K-12 E. coli strain was used as reference comparison. For E. coli strains phylogroup, 25% (28/113) were observed to belong to the group A, 15% (17/113) to the group B1, 14% (16/113) to the group B2, and 46% (52/113) to the group D. 75% (85/113) were fimH positive. fimH SNPs analysis for 50 isolated from clinical sample and 3 references found 60 SNPs at 57 polymorphic sites. The number of amino-acid variants and silent SNPs were observed more in UPEC strains than in other extra-intestinal E. coli strains. Most of the UPEC strains with the same amino-acid variants were belong to the same phylogroup. This combination method could serve as a rapid, highly reproducible typing test for epidemiological studies of ExPEC. Large collection data could be compared with other clinical laboratories that the sequence data are accessible.

ClC-4 is a secondary active transporter that exchanges Cl(-) ions and H(+) with a 2:1 stoichiometry. In external SCN(-), ClC-4 becomes uncoupled and transports anions with high unitary transport rate. Upon voltage steps, the number of active transporters varies in a time-dependent manner, resembling voltage-dependent gating of ion channels. We here investigated modification of the voltage dependence of uncoupled ClC-4 by protons and anions to quantify association of substrates with the transporter. External acidification shifts voltage dependence of ClC-4 transport to more positive potentials and leads to reduced transport currents. Internal pH changes had less pronounced effects. Uncoupled ClC-4 transport is facilitated by elevated external [SCN(-)] but impaired by internal Cl(-) and I(-). Block by internal anions indicates the existence of an internal anion-binding site with high affinity that is not present in ClC channels. The voltage dependence of ClC-4 coupled transport is modulated by external protons and internal Cl(-) in a manner similar to what is observed under uncoupling conditions. Our data illustrate functional differences but also similarities between ClC channels and transporters.

BACKGROUND

Salt tolerance in grapevine is associated with chloride (Cl-) exclusion from shoots; the rate-limiting step being the passage of Cl- between the root symplast and xylem apoplast. Despite an understanding of the physiological mechanism of Cl- exclusion in grapevine, the molecular identity of membrane proteins that control this process have remained elusive. To elucidate candidate genes likely to control Cl- exclusion, we compared the root transcriptomes of three Vitis spp. with contrasting shoot Cl- exclusion capacities using a custom microarray.

RESULTS

When challenged with 50 mM Cl-, transcriptional changes of genotypes 140 Ruggeri (shoot Cl- excluding rootstock), K51-40 (shoot Cl- including rootstock) and Cabernet Sauvignon (intermediate shoot Cl- excluder) differed. The magnitude of salt-induced transcriptional changes in roots correlated with the amount of Cl- accumulated in shoots. Abiotic-stress responsive transcripts (e.g. heat shock proteins) were induced in 140 Ruggeri, respiratory transcripts were repressed in Cabernet Sauvignon, and the expression of hypersensitive response and ROS scavenging transcripts was altered in K51-40. Despite these differences, no obvious Cl- transporters were identified. However, under control conditions where differences in shoot Cl- exclusion between rootstocks were still significant, genes encoding putative ion channels SLAH3, ALMT1 and putative kinases SnRK2.6 and CPKs were differentially expressed between rootstocks, as were members of the NRT1 (NAXT1 and NRT1.4), and CLC families.

CONCLUSIONS

These results suggest that transcriptional events contributing to the Cl- exclusion mechanism in grapevine are not stress-inducible, but constitutively different between contrasting varieties. We have identified individual genes from large families known to have members with roles in anion transport in other plants, as likely candidates for controlling anion homeostasis and Cl- exclusion in Vitis species. We propose these genes as priority candidates for functional characterisation to determine their role in chloride transport in grapevine and other plants.

The field of molecular physiology of ClC chloride channels has witnessed a tremendous surge in knowledge over the past few years; however, fundamental issues such as the stoichiometry of ClC channels and the identification of pore-lining sequences have only recently begun to be addressed. New studies have also provided important insights into the role of ClC channels in cell volume regulation and their function in intracellular organelles.

Cl(-) channel activities vary during the cell cycle and are thought to play various roles including regulation of cell volume. We have shown previously that ClC-2 channels are directly phosphorylated and functionally regulated by the M phase-specific cyclin-dependent kinase p34(cdc2)/cyclin B. We investigate here to determine whether the expression levels of ClC-2 channel protein vary during the cell cycle. Immunoblot and immunocytochemical analyses of cells cycle-synchronized by serum depletion/replenishment reveal that ClC-2 channel protein is expressed predominantly at M phase in cells with two nuclei and a clear constriction ring, whereas RNA blot analysis shows that ClC-2 mRNA expression does not change during the cell cycle. Ubiquitin assays reveal that the ClC-2 channels are ubiquitinated at M phase, whereas the magnitude of ubiquitination is suppressed by incubation with olomoucine, an inhibitor of p34(cdc2)/cyclin B, and it is almost completely abolished in ClC-2 channels having an S632A mutation, which cannot be phosphorylated by p34(cdc2)/cyclin B, indicating that ubiquitination of ClC-2 channels requires phosphorylation by M phase-specific p34(cdc2)/cyclin B. Regulation at the post-transcriptional level, including phosphorylation-dependent ubiquitination, may contribute to M phase-specific expression of ClC-2 channels. Cell cycle-dependent regulation of expression at the protein level in addition to the regulation of function suggests that the ClC-2 channel plays a physiological role in the cell cycle.

OBJECTIVE

To evaluate the safety and feasibility of single-incision laparoscopic cholecystectomy (SILS-C) compared with conventional laparoscopic cholecystectomy (CLC).

METHODS

Sixty-five patients (SILS-C: 35, CLC: 30) were prospectively enrolled and operated with conventional straight instruments. The postoperative pain scores at 6, 24 hours, and 1 week, nausea, vomiting, commencement of oral intake, hospital stay, resumption of normal activities and work and satisfaction levels were noted.

RESULTS

Twenty-eight percent (10/35) SILS-C patients required introduction of additional trocars to complete the procedure. No patient required conversion to open. All the morbidity parameters were similar in both the groups, except that the seroma formation in the wound was significantly higher in the SILS-C group [SILS-C: 17% (6/35)/CLC: 0%, P=0.038]. One patient in SILS-C had a major bile duct injury.

CONCLUSIONS

SILS-C is safe and feasible with conventional instruments. However, caution needs to be exercised in view of a major bile duct injury and a higher rate of seroma formation in the wound.

We investigated whether voltage-dependent chloride channels (ClC-1, ClC-2 and ClC-3) are expressed in outer hair cells (OHCs) of rat cochlea using a single-cell reverse transcription-polymerase chain reaction (RT-PCR) technique. The OHCs were isolated from rat cochlea and the cytoplasm of each OHC was suctioned into a glass pipette containing RT-PCR reaction buffer with RNase inhibitor. RT-PCR revealed the presence of transcripts of ClC-1, ClC-2 and ClC-3, which were verified by DNA sequencing. The possible roles of these chloride channels in OHCs are discussed.

ACE.2 mice lack all tissue angiotensin-converting enzyme (ACE) but have 33% of normal plasma ACE activity. They exhibit the urine-concentrating defect and hyperkalemia present in mice that lack all ACE, but in contrast to the complete knockout, ACE.2 mice have normal medullary histology and creatinine clearance. To explore the urine-concentrating defect in ACE.2 mice, renal medullary transport proteins were analyzed using Western blot analysis. In the inner medulla, UT-A1, ClC-K1, and aquaporin-1 (AQP1) were significantly reduced to 28 +/- 5, 6 +/- 6, and 39 +/- 5% of the level in wild-type mice, respectively, whereas AQP2 and UT-B were unchanged. In the outer medulla, Na(+)-K(+)-2Cl(-) cotransporter (NKCC2/BSC1) and AQP1 were significantly reduced to 56 +/- 11 and 29 +/- 6%, respectively, whereas Na(+)-K(+)-ATPase, UT-A2, UT-B, and AQP2 were unchanged, and renal outer medullary potassium channel was significantly increased to 711 +/- 187% of the level in wild-type mice. The abnormal expression of these transporters was similar in ACE.2 mice backcrossed onto a C57BL/6 or a Swiss background and was not rescued by ANG II infusion. We conclude that the urine-concentrating defect in ACE.2 mice is associated with, and may result from, downregulation of some or all of these key urea, salt, and water transport proteins.

BACKGROUND

Two Bartter syndrome phenotypes have been described, and molecular analyses demonstrate mutations in 1 of 3 genes encoding ascending limb of Henle transporters. We report phenotypic observations in 5 African American children with Bartter syndrome in the context of a distinct genotype.

METHODS

Mutation analyses were performed in 5 unrelated African American children with Bartter syndrome. These results were correlated to clinical and laboratory data. Calcium metabolism was evaluated with a bone disk bioassay.

RESULTS

Mutation analyses demonstrated homozygous deletion of the ClC-Kb gene in all children. Two children had polyhydramnios and premature birth; the others were born at term and presented with failure to thrive or dehydration. All receive indomethacin, spironolactone, and potassium chloride with improved but borderline hypokalemia. Growth has improved with therapy, but height SD scores range from -3.9- to -1.4. Urinary calcium excretion is normal, and bone disk bioassay shows no abnormal calciotropic activity. No patient had nephrocalcinosis, but renal sonograms show loss of corticomedullary differentiation.

CONCLUSIONS

African Americans with Bartter syndrome genotyped to date have homozygous deletion of ClC-Kb Clinical observations in our patients include partial correction of hypokalemia and suboptimal growth despite therapy. Abnormal calciotropic activity and nephrocalcinosis are not seen, but renal ultrasounds are abnormal.

Age-related skeletal muscle decline is characterized by the modification of sarcolemma ion channels important to sustain fiber excitability and to prevent metabolic dysfunction. Also, calcium homeostasis and contractile function are impaired. In the aim to understand whether these modifications are related to oxidative damage and can be reverted by antioxidant treatment, we examined the effects of in vivo treatment with an waste water polyphenolic mixture (LACHI MIX HT) supplied by LACHIFARMA S.r.l. Italy containing hydroxytirosol (HT), gallic acid, and homovanillic acid on the skeletal muscles of 27-month-old rats. After 6-week treatment, we found an improvement of chloride ClC-1 channel conductance, pivotal for membrane electrical stability, and of ATP-dependent potassium channel activity, important in coupling excitability with fiber metabolism. Both of them were analyzed using electrophysiological techniques. The treatment also restored the resting cytosolic calcium concentration, the sarcoplasmic reticulum calcium release, and the mechanical threshold for contraction, an index of excitation-contraction coupling mechanism. Muscle weight and blood creatine kinase levels were preserved in LACHI MIX HT-treated aged rats. The antioxidant activity of LACHI MIX HT was confirmed by the reduction of malondialdehyde levels in the brain of the LACHI MIX HT-treated aged rats. In comparison, the administration of purified HT was less effective on all the parameters studied. Although muscle function was not completely recovered, the present study provides evidence of the beneficial effects of LACHI MIX HT, a natural compound, to ameliorate skeletal muscle functional decline due to aging-associated oxidative stress.

Inner ear physiology is reviewed with emphasis on features common to renal physiology. Genetic disorders in transporters/channels for chloride (ClC-K), bicarbonate (Cl(-)/HCO(3)(-) exchanger), protons (H(+)-ATPase), sodium (ENaC, NKKC1, NBC3, NHE3), potassium (KCNQ1/KCNE1, Kcc4), and water (AQP4) in the inner ear and their relation to the kidney are discussed. Based on data from human disorders (with or without mouse counterparts) and mouse models (without human counterparts) this article focuses on the involvement of these transporters/channels in hearing loss.

BACKGROUND

Recent reports have suggested that single-incision laparoscopic cholecystectomy (SILC) is technically feasible. We present our initial retrospective comparative study between SILC and conventional laparoscopic cholecystectomy (CLC) with respect to perioperative outcomes.

METHODS

The authors reviewed 100 SILC and 100 CLC performed by a single surgeon from May 2009 to July 2010 at the Shengjing Hospital of China Medical University. All the procedures were completed by using the standard trocars and rigid laparoscopic instruments. Demographic data, operating time, estimated blood loss, analgesics requirements, days to oral food intake, and complications were compared.

RESULTS

Of the attempted SILC cases, 99 cases (99%) were successfully performed, with 1 case requiring three additional trocars for safe dissection because of existence of accessory bile duct. In the CLC group, all the procedures were successfully completed (three trocars) without conversion to open cholecystectomy. Compared with the CLC group, there was a lower mean estimated blood loss (17.9 ± 15.8 mL versus 27.5 ± 13.9 mL; P = .000) and analgesic requirement (10 versus 23; P = .024) in the SILC group. However, there was no difference between SILC and CLC in operating time (53.5 ± 24.0 minutes versus 49.2 ± 13.8 minutes; P = .163), days to oral food intake (1.8 ± 0.8 days versus 1.8 ± 0.7 days; P = .873), and postoperative complication rate (2% versus 0%; P = .155).

CONCLUSIONS

SILC is feasible using the standard trocars and rigid laparoscopic instruments, and it is an effective alternative to CLC in selected patients. However, further clinical studies are necessary to confirm its real benefits.

BACKGROUND

Conventional laparoscopic cholecystectomy (CLC) is now accepted as the gold standard surgical treatment for gallbladder stone disease. Single-incision laparoscopic technology has recently been introduced into laparoscopic clinical practice in order to reduce the invasiveness of this procedure. A meta-analysis of randomized clinical trials (RCTs) was performed to compare single-incision laparoscopic cholecystectomy (SICL) and CLC.

METHODS

Medline, Embase, ISI Web of Knowledge CPCI-S and the Cochrane Library were searched and the methodological quality of the included trials was evaluated. Outcomes evaluated were adverse events, conversion rate, post-operative hospital stay, blood loss, post-operative pain, total wound length, operative time and wound satisfaction score. Results were pooled in meta-analyses as odds ratio and weighted mean differences (WMD).

RESULTS

Five RCTs on 264 patients qualified for the meta-analysis, 139 being allocated to SILC and 125 to CLC. There was no significant difference between SICL and CLC for adverse events, conversion rate, post-operative hospital stay, blood loss, post-operative pain and total wound length; however, operative time was significantly longer in SICL than in CLC (WMD 7.72 (95% confidence interval (CI): 1.38-14.07) min; P = 0.02, χ(2) P = 0.02, I(2) = 69%). Furthermore, wound satisfaction score was significantly higher in SICL than in CLC (WMD 1.40 (95% CI: 1.19-1.61) min; P < 0.00001, χ(2) P = 0.19, I(2) = 42%).

CONCLUSIONS

SILC may be superior to CLC in terms of cosmetic outcome, but not in operative time. Currently, SILC is a safe procedure for proper patients in experienced surgeons.

Despite similar stimulatory actions on the epithelial sodium channel (ENaC)-mediated sodium reabsorption in the distal tubule, insulin promotes kaliuresis, whereas insulin-like growth factor-1 (IGF-1) causes a reduction in urinary potassium levels. The factors contributing to this phenomenon remain elusive. Electrogenic distal nephron ENaC-mediated Na(+) transport establishes driving force for Cl(-) reabsorption and K(+) secretion. Using patch-clamp electrophysiology, we document that a Cl(-) channel is highly abundant on the basolateral plasma membrane of intercalated cells in freshly isolated mouse cortical collecting duct (CCD) cells. The channel has characteristics attributable to the ClC-K2: slow gating kinetics, conductance ∼10 pS, voltage independence, Cl(->>NO3 (-) anion selectivity, and inhibition/activation by low/high pH, respectively. IGF-1 (100 and 500 nM) acutely stimulates ClC-K2 activity in a reversible manner. Inhibition of PI3-kinase (PI3-K) with LY294002 (20 μM) abrogates activation of ClC-K2 by IGF-1. Interestingly, insulin (100 nM) reversibly decreases ClC-K2 activity in CCD cells. This inhibitory action is independent of PI3-K and is mediated by stimulation of a mitogen-activated protein kinase-dependent cascade. We propose that IGF-1, by stimulating ClC-K2 channels, promotes net Na(+) and Cl(-) reabsorption, thus reducing driving force for potassium secretion by the CCD. In contrast, inhibition of ClC-K2 by insulin favors coupling of Na(+) reabsorption with K(+) secretion at the apical membrane contributing to kaliuresis.

Previous studies have reported the mechanisms of ion absorption and secretion by diverse membrane transport proteins in gills of various teleostean species. To date, however, the chloride channel expressed in the basolateral membrane of mitochondrion-rich (MR) cells for Cl(-) uptake in freshwater (FW) fish is still unknown. In this study, the combination of bioinformatics tools [i.e. National Center for Biotechnology Information (NCBI) database, Tetraodon nigroviridis (spotted green pufferfish) genome database (Genoscope), BLAT and BLASTn] were used to identify the gene of ClC-3 (TnClC-3), a member of the CLC chloride channel family in the T. nigroviridis genome. RT-PCR analysis revealed that the gene encoding for the ClC-3 protein was widely expressed in diverse tissues (i.e. gill, kidney, intestine, liver and brain) of FW- and seawater (SW)-acclimated pufferfish. In whole-mount double immunofluorescent staining, branchial ClC-3-like immunoreactive protein was localized to the basolateral membrane of Na(+)/K(+)-ATPase (NKA) immunoreactive cells in both the FW- and SW-acclimated pufferfish. In response to salinity, the levels of transcript of branchial TnClC-3 were similar between FW and SW fish. Moreover, the membrane fraction of ClC-3-like protein in gills was 2.7-fold higher in FW compared with SW pufferfish. To identify whether the expression of branchial ClC-3-like protein specifically responded to lower environmental [Cl(-)], the pufferfish were acclimated to artificial waters either with a normal (control) or lower Cl(-) concentration (low-Cl). Immunoblotting of membrane fractions of gill ClC-3-like protein showed the expression was about 4.3-fold higher in pufferfish acclimated to the low-Cl environment than in the control group. Furthermore, branchial ClC-3-like protein was rapidly elevated in response to acute changes of environmental salinity or [Cl(-)]. Taken together, pufferfish ClC-3-like protein was expressed in the basolateral membrane of gill MR cells, and the protein amounts were stimulated by hyposmotic and low-Cl environments. The enhancement of ClC-3-like protein may trigger the step of basolateral Cl(-) absorption of the epithelium to carry out iono- and osmoregulatory functions of euryhaline pufferfish gills.

BACKGROUND

Pendred syndrome is caused by mutations in the gene coding for pendrin, an apical Cl-/I- exchanger.

OBJECTIVE

To analyze intrathyroidal compensatory mechanisms when pendrin is lacking, we investigated the thyroid of a patient with Pendred syndrome. The expression of proteins involved in thyroid hormone synthesis, markers of oxidative stress (OS), cell proliferation, apoptosis, and antioxidant enzymes were analyzed.

RESULTS

Three morphological zones were identified: nearly normal follicles with iodine-rich thyroglobulin in the colloid (zone 1.a), small follicles without iodine-rich thyroglobulin in lumina (zone 1.b), and destroyed follicles (zone 2). In zones 1.a, dual oxidase (Duox) and thyroid peroxidase (TPO) were localized at the apical pole, OS and cell apoptosis were absent, but ClC-5 expression was strongly increased. In zones 1.b, Duox and TPO were aberrantly present and increased in the cytosol and associated with high OS, apoptosis, cell proliferation, and increased expression of peroxiredoxin-5, catalase, and dehalogenase-1 but moderate ClC-5 expression.

CONCLUSIONS

In conclusion, the absence of pendrin is accompanied by increased ClC-5 expression that may transiently compensate for apical iodide efflux. In more affected follicles, Duox and TPO are relocated in the cytosol, leading to abnormal intracellular thyroid hormone synthesis, which results in cell destruction presumably because intracellular OS cannot be buffered by antioxidant defenses.

The properties of the inward-rectifying Cl- conductance in rat choroid plexus epithelial cells were investigated to allow comparisons to be made with ClC-2. All experiments were performed using the whole-cell configuration of the patch-clamp method. The conductance was transiently activated using an electrode solution which contained 375 nM catalytic subunit of protein kinase A (PKA). PKA failed to activate the conductance, however, when cells were pre-incubated with phorbol esters, which activate protein kinase C [1 microM phorbol 12-myristate 13-acetate (PMA) and 1 microM phorbol 12,13-dibutyrate (PDBu)]. Sustained activation of the conductance by PKA was observed in Ca2+-free conditions (5 mM BAPTA in the electrode solution), or when 100 nM calphostin C, a PKC inhibitor, was added to the electrode solution. The inward-rectifying Cl- conductance in choroid plexus is therefore similar to ClC-2 in that it is inhibited by PKC. The inward-rectifying conductance was blocked when Cd2+ (30 and 300 microM) and Zn2+ (1, 30 and 300 microM) were added to the bath solution. ClC-2 channels are also blocked by Zn2+ and Cd2+. The magnitude of the inward conductance was dependent on the concentration of ATP in the electrode solution. The conductance was not observed when ATP in the electrode was replaced with non-hydrolysable ATP analogues [adenosine 5'-O-(3-thiotriphosphate) (ATP[gamma-S]) and 5'-adenylylimidodiphosphate (AMP-PNP)), but it was supported by UTP and GTP. These data contrast with those of previous studies in which ClC-2 channels were activated in the absence of ATP. In conclusion, the inward-rectifying Cl- channel in rat choroid plexus shares some properties with ClC-2 (inhibition by PKC and block by divalent cations), but differs in that it depends on intracellular ATP.

Transition metals block the muscle Cl- channel ClC-1, which belongs to a large family of double-barreled Cl- channels and transporters. In the Torpedo Cl- channel ClC-0, Zn2+ block is closely related to the common gating mechanism that opens and closes both pores of the channel simultaneously, and the mutation C212S, which locks the common gate open, also eliminates the block. In ClC-1, however, previous results suggested that Zn2+ block is independent of gating, and that the cysteine residues involved in Zn2+ binding are in different positions to those that confer Zn2+ sensitivity on ClC-0. In this work, we show that Zn2+ block of ClC-1 is faster at hyperpolarized potentials where the channel is more likely to be in the closed state. Mutation C277S, equivalent to C212S in ClC-0, which locks the common gate in ClC-1 open, virtually eliminates Zn2+ block. A mutation, V321A, which reduces open probability of the common gate, facilitated Zn2+ block. These results demonstrate that Zn2+ block is state dependent, acting on the common gate. The extent of the block, however, is not a simple function of the open probability of the common gate. The Q10 of approximately 13 of the time course of Zn2+ block, which is significantly higher than the Q10 of common gating transitions in WT ClC-1, suggests that Zn2+ binds to a very high temperature-dependent low-probability closed substate of the common gate, which has not yet been characterized in this channel.

Electron microscopy, ultrastructural cytochemistry, and postembedding immunogold ultrastructural immunocytochemistry were used to study a papular cutaneous lesion from a patient with the hypereosinophilic syndrome. Peroxidase activity was detected cytochemically in 40-microns sections of skin utilizing the substrate diaminobenzidine; Charcot-Leyden crystal (CLC) protein was detected immunocytochemically in skin utilizing a postembedding immunogold technique; and a combined method was used where postembedding immunogold staining of CLC protein was performed on sections previously prepared to detect peroxidase activity. We describe a unique, eosinophil-rich inflammatory process in involved skin which contained extraordinary numbers of morphologically activated macrophages. Electron microscopy demonstrated (a) widespread eosinophil necrosis, (b) interstitial CLC formation, (c) macrophage activation, endocytosis, and phagocytosis, and (d) CLC formation in phagosomes of activated macrophages. Peroxidase activity was present as follows: (a) in the matrix of eosinophil specific granules in eosinophil cytoplasm, in membrane-bound specific granules released into interstitial tissues from dying eosinophils, being phagocytized by activated macrophages, and within macrophage phagosomes; (b) as amorphous interstitial debris; (c) in cytoplasm and nuclei of damaged eosinophils in the dermal tissues as well as in macrophage phagosomes; and (d) in endocytotic vesicles and vacuoles of macrophages and in CLC-containing phagosomes of macrophages. CLC protein was localized by immunocytochemistry to (a) eosinophil primary granules, (b) cytoplasm and nuclei of damaged eosinophils located in the interstitial tissues or within macrophage phagosomes, (c) CLC located in interstitial tissues adjacent to necrotic eosinophils and in macrophage phagosomes, and (d) aggregates of amorphous protein bound to macrophage surfaces; endocytotic vesicles and vacuoles of macrophages; amorphous protein aggregates in macrophage lysosomes.(ABSTRACT TRUNCATED AT 400 WORDS)

There is controversy regarding the ability of short term (2 to 3 days) cultured epidermal Langerhans cells (cLC) to process and present intact protein Ag to primed T cells. Some studies have shown that cLC are potent APC for both haptens and intact protein Ag, whereas in others cLC have been unable to process and present intact protein Ag. In an attempt to resolve this controversy, we tested the ability of Langerhans cells from several strains of mice to process and present intact protein Ag to T cell clones and T cell hybridomas. We found that both cLC and freshly prepared Langerhans cells from various Iak mice, including BALB.k mice, process and present intact protein antigens (i.e., hen egg lysozyme, cytochrome c, and OVA) to T cells. These functions are retained in cLC cultured for 7 days. In contrast, cLC from Iad mice do not process intact protein Ag, such as hen egg lysozyme and myoglobin, although they can present relevant peptides to specific T cells and are potent stimulators of allogeneic responses. Furthermore, cLC from (Iak x Iad)F1 mice process and present intact protein Ag to Iak-restricted T cells, but not to Iad-restricted T cells. Although cLC that processed and presented intact protein Ag to T cells exhibited enhanced class II MHC expression, they were, on a per cell basis, somewhat less efficient than were fresh Langerhans cells. Finally, we found that if Iad Langerhans cells are pulsed with intact protein Ag and then cultured for 3 days, they are then fully capable of inducing Ag- and MHC-specific T cell proliferation.

OBJECTIVE

Acute kidney injury associated with reduced urinary concentration is a frequent and severe complication during sepsis. The present study addressed the effect of endotoxemia on the functional and molecular mechanisms that determine urinary concentrating ability. Efficient urinary concentration depends on, amongst other factors, the expression of the Cl channel kidney-specific chloride channel 1 and its subunit Barttin, the urea transporter-A1, and the water channel aquaporin 2, all of which are regulated by the transcription factor TonEBP/NFAT5.

METHODS

Experimental animal and cell culture model.

METHODS

University laboratory.

METHODS

Wistar rats and Madin-Darby canine kidney cells.

METHODS

Rats were injected with lipopolysaccharide (5 mg/kg bodyweight intraperitoneal) or vehicle (phosphate-buffered saline) as control. After 24 hrs, urine, blood, and tissue samples from various kidney zones were analyzed for parameters that determine urinary concentration ability. Madin-Darby canine kidney cells were treated under isotonic or hypertonic conditions with the nitric oxide donor S-nitroso-N-acetylpenicillamine.

RESULTS

In rats injected with lipopolysaccharide, urine osmolality was reduced by ~40%, along with medullary induction of inducible nitric oxide synthase and a dramatic increase in urinary nitric oxide degradation products nitrite/nitrate. Concomitantly, expressions of ClC-K1, Barttin, urea transporter-A1, and aquaporin 2 were significantly lower. This was associated with the appearance of S-nitrosylated TonEBP/NFAT5, as monitored by the biotin-switch assay and immunoprecipitation, and reduced TonEBP/NFAT5 DNA binding activity in the renal inner medulla. These results were confirmed in Madin-Darby canine kidney cells transfected with a reporter construct driven by the urea transporter-A promoter, in which the nitric oxide donor S-nitroso-N-acetylpenicillamine reduces urea transporter-A reporter activity under isotonic and hypertonic conditions.

CONCLUSIONS

The present data demonstrate that lipopolysaccharide increases medullary nitric oxide production by iNOS induction, resulting in impairment of the transcriptional activity of TonEBP/NFAT5 by S-nitrosylation. The consequence thereof is reduced expression of TonEBP/NFAT5 target genes ClC-K1, Barttin, urea transporter-A1, and aquaporin 2 that are required for urinary concentration. Our findings may provide further insight into the molecular mechanisms underlying the urinary concentration defect in sepsis.

Oxidation and reduction (redox) are known to modulate the function of a variety of ion channels. Here, we report a redox regulation of the function of ClC-0, a chloride (Cl(-)) channel from the Torpedo electric organ. The study was motivated by the occasional observation of oocytes with hyperpolarization-activated Cl(-) current when these oocytes expressed ClC-0. We find that these atypical recording traces can be turned into typical ClC-0 current by incubating the oocyte in millimolar concentrations of reducing agents, suggesting that the channel function is regulated by oxidation and reduction. The redox control apparently results from an effect of oxidation on the slow (inactivation) gating: oxidation renders it more difficult for the channel to recover from the inactivated states. Introducing the point mutation C212S in ClC-0 suppresses the inactivation state, and this inactivation-suppressed mutant is no longer sensitive to the inhibition by oxidizing reagents. However, C212 is probably not the target for the redox reaction because the regulation of the inactivation gating by oxidation is still present in a pore mutant (K165C/K165 heterodimer) in which the C212S mutation is present. Taking advantage of the K165C/K165 heterodimer, we further explore the oxidation effect in ClC-0 by methane thiosulfonate (MTS) modifications. We found that trimethylethylammonium MTS modification of the introduced cysteine can induce current in the K165C/K165 heterodimer, an effect attributed to the recovery of the channel from the inactivation state. The current induction by MTS reagents is subjected to redox controls, and thus the extent of this current induction can serve as an indicator to report the oxidation state of the channel. These results together suggest that the inactivation gating of ClC-0 is affected by redox regulation. The finding also provides a convenient method to "cure" those atypical recording traces of ClC-0 expressed in Xenopus oocytes.