Recent studies have identified several chloride (Cl-) channel genes in the heart, including CFTR, ClC-2, ClC-3, CLCA, Bestrophin, and TMEM16A. Gene targeting and transgenic techniques have been used to delineate the functional role of cardiac Cl- channels in the context of health and disease. It has been shown that Cl- channels may contribute to cardiac arrhythmogenesis, myocardial hypertrophy and heart failure, and cardioprotection against ischaemia-reperfusion. The study of physiological or pathophysiological phenotypes of cardiac Cl- channels, however, may be complicated by the compensatory changes in the animals in response to the targeted genetic manipulation. Alternatively, tissue-specific conditional or inducible knockout or knockin animal models may be more valuable in the phenotypic studies of specific Cl- channels by limiting the effect of compensation on the phenotype. The integrated function of Cl- channels may involve multi-protein complexes of the Cl- channel subproteome and similar phenotypes can be attained from alternative protein pathways within cellular networks, which are influenced by genetic and environmental factors. Therefore, the phenomics approach, which characterizes phenotypes as a whole phenome and systematically studies the molecular changes that give rise to particular phenotypes achieved by modifying the genotype (such as gene knockouts or knockins) under the scope of genome/proteome/phenome, may provide a more complete understanding of the integrated function of each cardiac Cl- channel in the context of health and disease.

The inactivation of the ClC-0 chloride channel is very temperature sensitive and is greatly facilitated by the binding of a zinc ion (Zn2+) from the extracellular side, leading to a Zn2+-induced current inhibition. To further explore the relation of Zn2+ inhibition and the ClC-0 inactivation, we mutated all 12 cysteine amino acids in the channel and assayed the effect of Zn2+ on these mutants. With this approach, we found that C212 appears to be important for the sensitivity of the Zn2+ inhibition. Upon mutating C212 to serine or alanine, the inactivation of the channel in macroscopic current recordings disappears and the channel does not show detectable inactivation events at the single-channel level. At the same time, the channel's sensitivity to Zn2+ inhibition is also greatly reduced. The other two cysteine mutants, C213G and C480S, as well as a previously identified mutant, S123T, also affect the inactivation of the channel to some degree, but the temperature-dependent inactivation process is still present, likewise the high sensitivity of the Zn2+ inhibition. These results further support the assertion that the inhibition of Zn2+ on ClC-0 is indeed due to an effect on the inactivation of the channel. The absence of inactivation in C212S mutants may provide a better defined system to study the fast gating and the ion permeation of ClC-0.

The voltage-gated Cl- channel from Torpedo electroplax was purified in functional form by an immunoaffinity procedure. Channel activity was assayed by 36Cl- uptake into reconstituted liposomes and by direct recording after insertion into planar lipid bilayers. The purified channel displays the same "double-barreled" gating kinetics observed with native membranes, as well as the correct single-channel permeation characteristics. Preparations of active channels consist of a 90-kDa polypeptide, as expected from the known cDNA sequence. No associated subunits are present in the purified material. Direct protein sequencing confirms the absence of a cleavable signal sequence and demonstrates an N-terminus at Ser-2 of the cDNA-derived sequence. This "ClC-0" protein is lightly glycosylated, losing only approximately 2 kDa of sugar upon treatment with endoglycosidase H or N-glycanase. Most if not all of this glycosylation is found on Asn-365. This result necessitates revision of current transmembrane topology proposals, which have placed this residue on the cytoplasmic side of the membrane. Sedimentation in sucrose density gradients under activity-preserving conditions suggests the ClC-0 channel is slightly larger than the Na/K-ATPase alpha/beta-protomer (approximately equal to 150 kDa) and substantially smaller than the reduced form of the nicotinic acetylcholine receptor (approximately equal to 300 kDa). The detergent-solubilized ClC-0 channel, which invariably displays two Cl- diffusion pores in the active complex, is therefore built most likely as a homodimer of the 90-kDa protein purified here.

Genes for the degradation of organic pollutants have usually been allocated to plasmid DNAs in bacteria or considered non-mobile when detected in the chromosome. New discoveries have shown that catabolic genes can also be part of so-called integrative and conjugative elements (ICElands), a group of mobile DNA elements also known as genomic islands and conjugative transposons. One such ICEland is the clc element for chlorobenzoate and chlorocatechol degradation in Pseudomonas sp. strain B13. Genome comparisons and genetic data on integrase functioning reveal that the clc element and several other unclassified ICElands belong to a group of elements with conserved features. The clc element is unique among them in carrying the genetic information for several degradation pathways, whereas the others give evidence for pathogenicity functions. Many more such elements may exist, bridging the gap between pathogenicity and degradation functions.

Retinal degeneration is a major cause of severe visual impairment or blindness. Understanding the underlying molecular mechanisms is a prerequisite to develop therapeutic approaches for human patients. We show in three mouse models that induced and inherited retinal degeneration induces LIF and CLC as members of the interleukin (IL)-6 family of proteins, activates proteins of the Jak-STAT signaling pathway, and up-regulates suppressors of cytokine signaling as a negative feedback loop. Inhibition of Jak2 leads to protection of photoreceptors in a model of induced but not in a model of inherited retinal degeneration. Differential activation of Akt suggests alternative pathways for cell death and/or survival in different models. Proteins induced during photoreceptor degeneration are not mainly expressed in photoreceptors but in cells of other retinal layers. This suggests a model in which photoreceptor injury is signaled to cells of the inner retina, which in turn initiate a response either to support viability or accelerate death of injured cells.

The manner in which vasa recta function and contribute to the concentrating mechanism depends on their three-dimensional relationships to each other and to tubular elements. We have examined the three-dimensional architecture of vasculature relative to tubular structures in the central region of rat kidney inner medulla from the base through the first 3 mm by combining immunohistochemistry and semiautomated image acquisition techniques with graphical modeling software. Segments of descending vasa recta (DVR), ascending vasa recta (AVR), descending thin limb (DTL), ascending thin limb (ATL), and collecting duct (CD) were identified with antibodies against segment-specific proteins associated with solute and water transport (urea channel B, PV-1, aquaporin-1, ClC-K1, aquaporin-2, respectively) by immunofluorescence. Results indicate: 1) DVR, like DTLs, are excluded from CD clusters that we have previously shown to be the organizing element for the inner medulla; 2) AVR, like ATLs, are nearly uniformly distributed transversely across the entire inner medulla outside of and within CD clusters; 3) DVR and AVR outside CD clusters appear to be sufficiently juxtaposed to permit good countercurrent exchange; 4) within CD clusters, about four AVR closely abut each CD, surrounding it in a highly symmetrical fashion; and 5) AVR abutting each CD and ATLs within CD clusters form repeating nodal interstitial spaces bordered by a CD on one side, one or more ATLs on the opposite side, and one AVR on each of the other two sides. These relationships may be highly significant for both establishing and maintaining the inner medullary osmotic gradient.

Alpha-helical bundles and beta-barrel proteins represent the two basic types of architecture known for integral membrane proteins. Irregular structural motifs have been revealed with the growing number of structures determined. "Discontinuous" helices are present in membrane proteins that actively transport ions. In the Ca(2+)-ATPase, a primary active transporter, and in the secondary transporters NhaA, LeuT(Aa), ClC H(+)/Cl(-) exchanger and Glt(Ph), the helical structure of two membrane segments is interrupted and the interjacent polypeptide chain forms an extended peptide. The discontinuous helices are integrated in the membrane either as transmembrane-spanning or hairpin-type segments. In addition, the secondary transporters have inverted internal duplication domains, which are only weakly correlated with their amino acid sequence. The symmetry comprises either parts of or the complete molecule, but always includes the discontinuous helices. The helix-peptide-helix motif is correlated with the ion translocation function. The extended peptides with their backbone atoms, the helix termini and the polar/charged amino acid residues in close vicinity provide the basis for ion recognition, binding and translocation.

BACKGROUND

Up to 60% of pheochromocytoma (PCC) and paraganglioma (PGL) are associated with either somatic or germline mutations in established PCC and PGL susceptibility loci. Most unexplained cases are characterized by an increased activity of the RAS/RAF/ERK signaling pathway. Mutations in RAS subtypes H, K, and N are common in human cancers; however, previous studies have been inconsistent regarding the mutational status of RAS in PCC and PGL.

OBJECTIVE

The aim of this study was to identify novel disease causing genes in PCC and PGL tumors.

METHODS

Four benign and sporadic PCC and PGL tumors were subjected to whole exome sequencing using the Illumina HiSeq Platform. Sequences were processed by CLC genomics 4.9 bioinformatics software and the acquired list of genetic variants was filtered against the Catalogue of Somatic Mutations in Cancer database. Findings were validated in an additional 78 PCC and PGL tumor lesions.

RESULTS

Exome sequencing identified 2 cases with somatic mutations in the H-RAS. In total, 6.9% (n = 4/58) of tumors negative for mutations in major PCC and PGL loci had mutations in H-RAS: G13R, Q61K, and Q61R. There were 3 PCC and 1 PGL; all had sporadic presentation with benign tumor characteristics and substantial increases in norepinephrine and/or epinephrine. H-RAS tumors were exclusively found in male patients (P = .007).

CONCLUSIONS

We identified recurrent somatic H-RAS mutations in pheochromocytoma and paraganglioma. Tumors with H-RAS mutations had activation of the RAS/RAF/ERK signaling pathway and were associated with male PCC patients having benign and sporadic disease characteristics. H-RAS could serve as a prognostic and predictive marker as well as a novel therapeutic target.

BACKGROUND

To assess the potential of different types of sequence data combined with de novo and hybrid assembly approaches to improve existing draft genome sequences.

RESULTS

Illumina, 454 and PacBio sequencing technologies were used to generate de novo and hybrid genome assemblies for four different bacteria, which were assessed for quality using summary statistics (e.g. number of contigs, N50) and in silico evaluation tools. Differences in predictions of multiple copies of rDNA operons for each respective bacterium were evaluated by PCR and Sanger sequencing, and then the validated results were applied as an additional criterion to rank assemblies. In general, assemblies using longer PacBio reads were better able to resolve repetitive regions. In this study, the combination of Illumina and PacBio sequence data assembled through the ALLPATHS-LG algorithm gave the best summary statistics and most accurate rDNA operon number predictions. This study will aid others looking to improve existing draft genome assemblies.

METHODS

All assembly tools except CLC Genomics Workbench are freely available under GNU General Public License.

BACKGROUND

brownsd@ornl.gov

BACKGROUND

Supplementary data are available at Bioinformatics online.

ClC-1 is a dimeric, double-pored chloride channel that is present in skeletal muscle. Mutations of this channel can result in the condition myotonia, a muscle disorder involving increased muscle stiffness. It has been shown that the dominant form of myotonia often results from mutations that affect the so-called slow, or common, gating process of the ClC-1 channel. Mutations causing dominant myotonia are seen to cluster at the interface of the ClC-1 channel monomers. This study has investigated the role of the H, I, P, and Q helices, which lie on this interface, as well as the G helix, which is situated immediately behind the H and I helices, on ClC-1 gating. 11 mutant ClC-1 channels (T268M, C277S, C278S, S289A, T310M, S312A, V321S, T539A, S541A, M559T, and S572V) were produced using site-directed mutagenesis, and gating properties of these channels were investigated using electrophysiological techniques. Six of the seven mutations in G, H, and I, and two of the four mutations in P and Q, caused shifts of the ClC-1 open probability. In the majority of cases this was due to alterations in the common gating process, with only three of the mutants displaying any change in fast gating. Many of the mutant channels also showed alterations in the kinetics of the common gating process, particularly at positive potentials. The changes observed in common gating were caused by changes in the opening rate (e.g. T310M), the closing rate (e.g. C277S), or both rates. These results indicate that mutations in the helices forming the dimer interface are able to alter the ClC-1 common gating process by changing the energy of the open and/or closed channel states, and hence altering transition rates between these states.

The cytoplasmic domains of ClC chloride channels and transporters are ubiquitously found in eukaryotic family members and have been suggested to be involved in the regulation of ion transport. All cytoplasmic ClC domains share a conserved scaffold that contains a pair of CBS motifs. Here we describe the structure of the cytoplasmic component of the human chloride channel ClC-Ka at 1.6 A resolution. The structure reveals a dimeric organization of the domain that is unusual for CBS motif containing proteins. Using a biochemical approach combining mutagenesis, crosslinking, and analytical ultracentrifugation, we demonstrate that the interaction interface is preserved in solution and that the distantly related channel ClC-0 likely exhibits a similar structural organization. Our results reveal a conserved interaction interface that relates the cytoplasmic domains of ClC proteins and establish a structural relationship that is likely general for this important family of transport proteins.

ClC-2 is localized to the apical membranes of secretory epithelia where it has been hypothesized to play a role in fluid secretion. Although ClC-2 is clearly the inwardly rectifying anion channel in several tissues, the molecular identity of the hyperpolarization-activated Cl(-) current in other organs, including the salivary gland, is currently unknown. To determine the nature of the hyperpolarization-activated Cl(-) current and to examine the role of ClC-2 in salivary gland function, a mouse line containing a targeted disruption of the Clcn2 gene was generated. The resulting homozygous Clcn2(-/-) mice lacked detectable hyperpolarization-activated chloride currents in parotid acinar cells and, as described previously, displayed postnatal degeneration of the retina and testis. The magnitude and biophysical characteristics of the volume- and calcium-activated chloride currents in these cells were unaffected by the absence of ClC-2. Although ClC-2 appears to contribute to fluid secretion in some cell types, both the initial and sustained salivary flow rates were normal in Clcn2(-/-) mice following in vivo stimulation with pilocarpine, a cholinergic agonist. In addition, the electrolytes and protein contents of the mature secretions were normal. Because ClC-2 has been postulated to contribute to cell volume control, we also examined regulatory volume decrease following cell swelling. However, parotid acinar cells from Clcn2(-/-) mice recovered volume with similar efficiency to wild-type littermates. These data demonstrate that ClC-2 is the hyperpolarization-activated Cl(-) channel in salivary acinar cells but is not essential for maximum chloride flux during stimulated secretion of saliva or acinar cell volume regulation.

The structural determinants of electrostatics of ion stabilization within EcClC, a ClC-type chloride channel homologue from Escherichia coli, are studied using a continuum dielectric approximation. Specifically, the ion occupancy is investigated in the wild-type protein and a mutant thereof, and the contribution to the electrostatic binding free energy of local and non-local interactions is characterized at the single-residue level. This analysis shows that, in spite of the desolvation cost and the strong ion-ion repulsion, all previously reported binding sites can be occupied simultaneously. The stabilizing effect of the protein arises from hydrogen bonding as well as from longer-range favorable interactions, such as with the strictly conserved Lys131 side-chain. The latter is involved in the stabilization of the conserved GSGIP motif that delimits two of the binding sites. Interestingly, an additional low-affinity binding site, mediated by a structurally analogous motif including the side-chain of Arg340, can be identified on the extracellular side of the permeation pathway. Finally, it is shown that, in contrast to K-channels, and in analogy to the SBP/PBP sulfate/phosphate-binding proteins, the contribution of helix macrodipoles to chloride binding in EcClC is only marginal.

Barttin is an accessory subunit of a subgroup of ClC-type chloride channels expressed in renal and inner ear epithelia. In this study, we examined the effects of barttin on two ClC-K channel isoforms, rat ClC-K1 and human ClC-Kb, using heterologous expression, patch clamping, confocal imaging, and flow cytometry. In the absence of barttin, only a small percentage of rClC-K1 and hClC-Kb channels are inserted into the plasma membrane. Coexpression of barttin enhances surface membrane insertion and furthermore modifies permeation and gating of ClC-K channels. hClC-Kb channels are nonfunctional without barttin and require the coexpressed accessory subunit to become anion conducting. In contrast, rClC-K1 channels are active without barttin, but at the cost of reduced unitary conductance as well as altered voltage dependence of activation. We mapped the separate functions of barttin to structural domains by a deletion analysis. Whereas the transmembrane core is necessary and sufficient to promote ClC-K channel exit from the endoplasmic reticulum, a short cytoplasmic segment following the second transmembrane helix modifies the unitary conductance. The entire cytoplasmic carboxyl terminus affects the open probability of ClC-K channels. The multiple functions of barttin might be necessary for a tight adjustment of epithelial Cl(-) conductances to ensure a precise regulation of body salt content and endocochlear potential.

BACKGROUND

Airway inflammation is associated with asthma exacerbation risk, treatment response, and disease mechanisms.

OBJECTIVE

This study aimed to identify and validate a sputum gene expression signature that discriminates asthma inflammatory phenotypes.

METHODS

An asthma phenotype biomarker discovery study generated gene expression profiles from induced sputum of 47 asthmatic patients. A clinical validation study (n = 59 asthmatic patients) confirmed differential expression of key genes. A 6-gene signature was identified and evaluated for reproducibility (n = 30 asthmatic patients and n = 20 control subjects) and prediction of inhaled corticosteroid (ICS) response (n = 71 asthmatic patients). Receiver operating characteristic curves were calculated, and area under the curve (AUC) values were reported.

RESULTS

From 277 differentially expressed genes between asthma inflammatory phenotypes, we identified 23 genes that showed highly significant differential expression in both the discovery and validation populations. A signature of 6 genes, including Charcot-Leydon crystal protein (CLC); carboxypeptidase A3 (CPA3); deoxyribonuclease I-like 3 (DNASE1L3); IL-1β (IL1B); alkaline phosphatase, tissue-nonspecific isozyme (ALPL); and chemokine (C-X-C motif) receptor 2 (CXCR2), was reproducible and could significantly (P < .0001) discriminate eosinophilic asthma from other phenotypes, including patients with noneosinophilic asthma (AUC, 89.6%), paucigranulocytic asthma (AUC, 92.6%), or neutrophilic asthma (AUC, 91.4%) and healthy control subjects (AUC, 97.6%), as well as discriminating patients with neutrophilic asthma from those with paucigranulocytic asthma (AUC, 85.7%) and healthy control subjects (AUC, 90.8). The 6-gene signature predicted ICS response (>12% change in FEV1; AUC, 91.5%). ICS treatment reduced the expression of CLC, CPA3, and DNASE1L3 in patients with eosinophilic asthma.

CONCLUSIONS

A sputum gene expression signature of 6 biomarkers reproducibly and significantly discriminates inflammatory phenotypes of asthma and predicts ICS treatment response. This signature has the potential to become a useful diagnostic tool to assist in the clinical diagnosis and management of asthma.

We cloned two novel members of the CLC chloride channel family from rat and human brain. ClC-6 is a 97-kDa protein, and ClC-7 a 89-kDa protein roughly 45% identical with ClC-6. Together they define a new branch of this gene family. Both genes are very broadly expressed, e.g. in brain, testes, muscle and kidney. In mouse embryos, both genes are expressed as early as day 7. While the human gene for ClC-6 is located on human chromosome 1p36 and shares this region with hClC-Ka and hClC-Kb, ClC-7 is on 16p13. ClC-6 has a highly conserved glycosylation site between transmembrane domains D8 and D9, while ClC-7 is the only known eukaryotic ClC protein which lacks this site. Hydropathy analysis indicates that domain D4 cannot serve as a transmembrane domain. Both ClC-6 and ClC-7 cannot be expressed as chloride channels in Xenopus oocytes, either singly or in combination.

Ion binding to secondary active transporters triggers a cascade of conformational rearrangements resulting in substrate translocation across cellular membranes. Despite the fundamental role of this step, direct measurements of binding to transporters are rare. We investigated ion binding and selectivity in CLC-ec1, a H(+)-Cl(-) exchanger of the CLC family of channels and transporters. Cl(-) affinity depends on the conformation of the protein: it is highest with the extracellular gate removed and weakens as the transporter adopts the occluded configuration and with the intracellular gate removed. The central ion-binding site determines selectivity in CLC transporters and channels. A serine-to-proline substitution at this site confers NO(3)(-) selectivity upon the Cl(-)-specific CLC-ec1 transporter and CLC-0 channel. We propose that CLC-ec1 operates through an affinity-switch mechanism and that the bases of substrate specificity are conserved in the CLC channels and transporters.

A point mutation (D136G) predicting the substitution of glycine for aspartate in position 136 of the human muscle Cl- channel (hClC-1) causes recessive generalized myotonia. Heterologous expression of a recombinant D136G produces functional Cl- channels with profound alterations in voltage-dependent gating, without concomitant changes in pore properties. The mutant exhibits slowly activating current upon hyperpolarization, in contrast to wild-type channels, which display time-dependent current decay (deactivation) at negative membrane potentials. Steady-state activation of D136G depends upon the transmembrane Cl- gradient, reaching zero at voltages positive to the Cl- reversal potential in physiological Cl- distribution. This explains the reduced sarcolemmal Cl- conductance that causes myotonia. The functional disturbances exhibited by D136G may stem from a defect in the ClC-1 voltage sensor.

The chloride channel from the Torpedo electric organ, ClC-0, is the best studied member of a large gene-family (Jentsch, T.J. 1996. Curr. Opin. Neurobiol. 6:303-310.). We investigate the temperature dependence of both the voltage- and chloride-dependent fast gate and of the slow gate of the "double-barreled" ClC-0 expressed in Xenopus oocytes. Kinetics of the fast gate exhibit only a moderate temperature dependence with a Q10 of 2.2. Steady-state P(open) of the fast gate is relatively independent of temperature. The slow gate, in contrast, is highly temperature sensitive. Deactivation kinetics at positive voltages are associated with a Q10 of approximately 40. Steady-state open probability of the slow gate (P(open)slow(V)) can be described by a Boltzmann distribution with an apparent gating valence of approximately 2 and a variable "offset" at positive voltages. We note a positive correlation of this offset (i.e., the fraction of channels that are not closed by the slow gate) with the amount of expression. This offset is also highly temperature sensitive, being drastically decreased at high temperatures. Paradoxically, the maximum degree of activation of the slow gate also decreases at higher temperatures. The strong temperature dependence of the slow gate was also observed at the single channel level in inside-out patches. The results imply that within a Markovian-type description at least two open and two closed states are needed to describe slow gating. The strong temperature dependence of the slow gate explains the phenotype of several ClC-0 point-mutants described recently by Ludewig et al. (Ludewig, U., T.J. Jentsch, and M. Pusch. 1996. J. Physiol. (Lond.). In press). The large Q10 of slow gating kinetics points to a complex rearrangement. This, together with the correlation of the fraction of noninactivating channels with the amount of expression and the fact that the slow gate closes both protochannels simultaneously suggests that the slow gate is coupled to subunit interaction of the multimeric ClC-0 channel.

Genetic disorders of acid-base transporters involve plasmalemmal and organellar transporters of H(+), HCO3(-), and Cl(-). Autosomal-dominant and -recessive forms of distal renal tubular acidosis (dRTA) are caused by mutations in ion transporters of the acid-secreting Type A intercalated cell of the renal collecting duct. These include the AE1 Cl(-)/HCO3(-) exchanger of the basolateral membrane and at least two subunits of the apical membrane vacuolar (v)H(+)-ATPase, the V1 subunit B1 (associated with deafness) and the V0 subunit a4. Recessive proximal RTA with ocular disease arises from mutations in the electrogenic Na(+)-bicarbonate cotransporter NBC1 of the proximal tubular cell basolateral membrane. Recessive mixed proximal-distal RTA accompanied by osteopetrosis and mental retardation is associated with mutations in cytoplasmic carbonic anhydrase II. The metabolic alkalosis of congenital chloride-losing diarrhea is caused by mutations in the DRA Cl(-)/HCO3(-) exchanger of the ileocolonic apical membrane. Recessive osteopetrosis is caused by deficient osteoclast acid secretion across the ruffled border lacunar membrane, the result of mutations in the vH(+)-ATPase V0 subunit or in the CLC-7 Cl(-) channel. X-linked nephrolithiasis and engineered deficiencies in some other CLC Cl(-) channels are thought to represent defects of organellar acidification. Study of acid-base transport disease-associated mutations should enhance our understanding of protein structure-function relationships and their impact on the physiology of cell, tissue, and organism.

Pseudomonas sp. strain B13 carries the clcRABDE genes encoding chlorocatechol-degradative enzymes on the self-transmissible 105-kb clc element. The element integrates site and orientation specifically into the chromosomes of various bacterial recipients, with a glycine tRNA structural gene (glyV) as the integration site. We report here the localization and nucleotide sequence of the integrase gene and the activity of the integrase gene product in mediating site-specific integration. The integrase gene (int-B13) was located near the right end of the clc element. It consisted of an open reading frame (ORF) of maximally 1,971 bp with a coding capacity for 657 amino acids (aa). The full-length protein (74 kDa) was observed upon overexpression and sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation. The N-terminal 430 aa of the predicted Int-B13 protein had substantial similarity to integrases from bacteriophages of the P4 family, but Int-B13 was much larger than P4-type integrases. The C-terminal 220 aa of Int-B13 were homologous to an ORF flanking a gene cluster for naphthalene degradation in Pseudomonas aeruginosa PaK1. Similar to the bacteriophages phiR73 and P4, the clc element integrates into the 3' end of the target tRNA gene. This target site was characterized from four different recipient strains into which the clc element integrated, showing sequence specificity of the integration. In Pseudomonas sp. strain B13, a circular form of the clc element, which carries an 18-bp DNA sequence identical to the 3'-end portion of glyV as part of its attachment site (attP), could be detected. Upon chromosomal integration of the clc element into a bacterial attachment site (attB), a functional glyV was reconstructed at the right end of the element. The integration process could be demonstrated in RecA-deficient Escherichia coli with two recombinant plasmids, one carrying the int-B13 gene and the attP site and the other carrying the attB site of Pseudomonas putida F1.

Myotonia is a symptom of many different acquired and genetic muscular conditions that impair the relaxation phase of muscular contraction. Myotonia congenita is a specific inherited disorder of muscle membrane hyperexcitability caused by reduced sarcolemmal chloride conductance due to mutations in CLCN1, the gene coding for the main skeletal muscle chloride channel ClC-1. The disorder may be transmitted as either an autosomal-dominant or recessive trait with close to 130 currently known mutations. Although this is a rare disorder, elucidation of the pathophysiology underlying myotonia congenita established the importance of sarcolemmal chloride conductance in the control of muscle excitability and demonstrated the first example of human disease associated with the ClC family of chloride transporting proteins.

BACKGROUND

Natural orifice transluminal endoscopic surgery (NOTES) is currently a very important topic for both gastroenterologists and surgeons. We have developed a technique of transvaginal hybrid NOTES cholecystectomy (TVC) that leaves no visible scar and is applicable to daily use. This technique is compared to the conventional laparoscopic cholecystectomy (CLC) in a matched-pair analysis.

METHODS

From June 2007 until February 2009, 108 NOTES cholecystectomies were performed. For a matched-pair analysis we first selected a group of 192 female patients who had undergone CLC and who were operated on by the same group of surgeons in the same time period. Then 108 pairs who had TVC were matched according to the degree of inflammation of the gallbladder and age. We were able to contact 208 patients at least 3 months after surgery. Hence, the study analysis was performed with 100 complete pairs.

RESULTS

All 200 cholecystectomies were performed successfully without conversion. The TVC procedure was significantly longer than CLC (52 vs. 35 min, p<0.001). There were no intraoperative complications in either group. There were no significant differences with respect to reoperations, wound infections, consumption of analgesic drugs, length of hospital stay, and sick leave. Seventy-five TVC and 73 CLC patients had sexual intercourse after the operation without any complaints.

CONCLUSIONS

We present here the largest series of NOTES for cholecystectomy published to date and the first comparative study with the gold standard. The TVC technique is as successful as the CLC, it causes no more complications than CLC, especially with respect to the vaginal approach, it is more time-consuming to perform, but has an ideal cosmetic result, i.e., no visible scar.

CLC proteins are found in cells from prokaryotes to mammals and perform functions in plasma membranes and intracellular vesicles. Several genetic human diseases and mouse models underscore their broad physiological functions in mammals. These functions range from the control of excitability to transepithelial transport, endocytotic trafficking and acidification of synaptic vesicles. The recent crystallization of bacterial CLC proteins gave surprising insights into CLC Cl(-)-channel permeation and gating and provides an excellent basis for structure-function studies. Surprisingly, the CLC from Escherichia coli functions as a Cl-/H+ exchanger, thus demonstrating the thin line separating transporters and channels.