Human cord blood mononuclear cells were cultured for 35 days in media containing recombinant human interleukin 5 (rhIL-5) supplemented with a fraction of the culture supernatant of phytohemagglutinin (PHA)-stimulated human T lymphocytes from which interleukin 2 (IL-2) was eliminated. Cultured cells were studied by electron microscopy and an immunogold procedure to detect subcellular site(s) of Charcot-Leyden crystal (CLC) protein. The majority of cells (greater than 70%) developing in this system were mature eosinophils, with descending frequency of other cells, including macrophages, mature basophils, eosinophilic myelocytes, and mature neutrophils. Mature eosinophils were characterized by increased numbers of primary granules, small granules, tubulovesicular structures, and decreased secondary granules. These eosinophils showed extensive piecemeal degranulation (PMD) characterized by partially empty and empty secondary granule chambers in the cytoplasm. Small, smooth vesicles were evident within empty granule chambers as well as adjacent to them. Eosinophils formed close associations with phagocytic macrophages that contained both standard-shaped and irregularly shaped CLC within phagolysosomes. Subcellular sites of CLC protein were demonstrated by immunogold in eosinophils and macrophages arising in these cultures. Charcot-Leyden crystal protein was present in the nuclear matrix and extraorganellar cytoplasm of eosinophils. Primary granules and some cytoplasmic vesicles were labeled for CLC protein, but full and empty secondary granules and the extensive network of tubulovesicles were not. Charcot-Leyden crystals were absent from eosinophils, nor were they present in the extracellular space. Charcot-Leyden crystals were absent from eosinophils, nor were they present in the extracellular space. Charcot-Leyden crystals within phagosomes of macrophages were labeled by the immunogold procedure for CLC protein. These results demonstrate that rhIL-5-supplemented, PHA-stimulated, T-cell-conditioned media induced the development of mature human eosinophils from cord blood cells. These eosinophils underwent PMD of secondary granule contents. Immunogold analysis showed eosinophil CLC protein in the cytoplasm, nucleus, and primary granules of eosinophils. Macrophages also were present in these cultures. They contained CLC protein-containing crystals in their phagosomes, suggesting active sequestration of eosinophil CLC protein by macrophages in vitro.

ClC-3 is a member of the ClC voltage-gated chloride (Cl(-)) channel superfamily. Recent studies have demonstrated the abundant expression and pleiotropy of ClC-3 in cardiac atrial and ventricular myocytes, vascular smooth muscle cells, and endothelial cells. ClC-3 Cl(-) channels can be activated by increase in cell volume, direct stretch of β1-integrin through focal adhesion kinase and many active molecules or growth factors including angiotensin II and endothelin-1-mediated signaling pathways, Ca(2+)/calmodulin-dependent protein kinase II and reactive oxygen species. ClC-3 may function as a key component of the volume-regulated Cl(-) channels, a superoxide anion transport and/or NADPH oxidase interaction partner, and a regulator of many other transporters. ClC-3 has been implicated in the regulation of electrical activity, cell volume, proliferation, differentiation, migration, apoptosis and intracellular pH. This review will highlight the major findings and recent advances in the study of ClC-3 Cl(-) channels in the cardiovascular system and discuss their important roles in cardiac and vascular remodeling during hypertension, myocardial hypertrophy, ischemia/reperfusion, and heart failure.

The osteopetroses are caused by reduced activity of osteoclasts which results in defective remodelling of bone and increased bone density. They range from a devastating neurometabolic disease, through severe malignant infantile osteopetrosis (OP) to two more benign conditions principally affecting adults [autosomal dominant OP (ADO I and II)]. In many patients the disease is caused by defects in either the proton pump [the a3 subunit of vacuolar-type H(+)-ATPase, encoded by the gene variously termed ATP6i or TCIRG1] or the ClC-7 chloride channel (ClCN7 gene). These pumps are responsible for acidifying the bone surface beneath the osteoclast. Although generally thought of as bone diseases, the most serious consequences of the osteopetroses are seen in the nervous system. Cranial nerves, blood vessels and the spinal cord are compressed by either gradual occlusion or lack of growth of skull foramina. Most patients with OP have some degree of optic atrophy and many children with severe forms of autosomal recessive OP are rendered blind; optic decompression is frequently attempted to prevent the latter. Auditory, facial and trigeminal nerves may also be affected, and hydrocephalus can develop. Stenosis of both arterial supply (internal carotid and vertebral arteries) and venous drainage may occur. The least understood form of the disease is neuronopathic OP [OP and infantile neuroaxonal dystrophy, MIM (Mendelian inheritance in man) 600329] which causes rapid neurodegeneration and death within the first year. Although characterized by the finding of widespread axonal spheroids and accumulation of ceroid lipofuscin, the biochemical basis of this disease remains unknown. The neurological complications of this disease and other variants are presented in the context of the latest classification of the disease.

Brain clearance of amyloid-β (Aβ42) by innate immune cells is necessary for maintenance of normal brain function. Phagocytosis of soluble Aβ42 by Alzheimer's disease (AD) macrophages is defective, recovered in all "Type I and Type II" AD patients by 1α,25(OH)2-vitamin D3 (1,25D3) and blocked by the nuclear vitamin D receptor (VDR) antagonist (23S)-25-dehydro-1α(OH)-vitamin D3-26,23-lactone (MK). Bisdemethoxycurcumin (BDC) is a VDR ligand and additive with 1,25D3 in promoting Aβ42 phagocytosis by Type I, but not by Type II macrophages. Here, we define the following intracellular mechanisms regulated by 1,25D3 that are associated with recovery of phagocytosis and consistent with the selectivity of BDC: 1) 1,25D3 potentiates a 4,4-diisothiocyanostilbene-2,2-disulfonic acid-sensitive chloride channel (i.e., ClC-3) currents in both Type I and II AD macrophages, but curcumin only potentiates the currents in Type I cells; 2) 1,25D3 is particularly effective in upregulating ClC-3 mRNA expression in Type II peripheral blood mononuclear cells (PBMCs) while both 1,25D3 and the BDC analog, C180, upregulate VDR mRNA, repressed by Aβ42 in Type II PBMCs; and 3) 1,25D3-induced Aβ42 phagocytosis is attenuated by the calcium-dependent ClC-3 blocker, inositol 3,4,5,6-tetraphosphate (IP4), in both AD Types and by the MEK1/2 inhibitor U0126 only in Type II macrophages. VDR hydrogen/deuterium exchange coupled mass spectrometry and computational results show differences between the abilities of 1,25D3 and curcuminoids to stabilize VDR helices associated with the regulation of gene transcription. The structure-function results provide evidence that 1,25D3 activation of VDR-dependent genomic and nongenomic signaling, work in concert to recover dysregulated innate immune function in AD.

ClC-2 is a ubiquitously expressed, two-pore homodimeric Cl- channel opened by hyperpolarisation. Little is known about its gating mechanisms. Crystallographic and functional studies in other ClC channels suggest that a conserved glutamate residue carboxylate side-chain can close protopores by interacting with a Cl--binding site in the pore. Competition for this site is thought to provide the molecular basis for gating by extracellular Cl-. We now show that ClC-2 gating depends upon intra- but not extracellular Cl- and that neutralisation of E217, the homologous pore glutamate, leads to loss of sensitivity to intracellular Cl- and voltage. Experiments testing for transient activation by extracellular protons demonstrate that E217 is not available for protonation in the closed channel state but becomes so after opening by hyperpolarisation. The results suggest that E217 is a hyperpolarisation-dependent protopore gate in ClC-2 and that access of intracellular Cl- to a site normally occupied by its side-chain in the pore stabilises the open state. A remaining hyperpolarisation-dependent gate might correspond to that closing both pores simultaneously in other ClC channels.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein kinase A and ATP-regulated Cl- channel that also controls the activity of other membrane transport proteins, such as the epithelial Na+ channel ENaC. Previous studies demonstrated that cytosolic domains of ENaC are critical for down-regulation of ENaC by CFTR, whereas others suggested a role of cytosolic Cl- ions. We therefore examined in detail the anion dependence of ENaC and the role of its cytosolic domains for the inhibition by CFTR and the Cl- channel CLC-0. Coexpression of rat ENaC with human CFTR or the human Cl- channel CLC-0 caused inhibition of amiloride-sensitive Na+ currents after cAMP-dependent stimulation and in the presence of a 100 mM bath Cl- concentration. After activation of CFTR by 3-isobutyl-1-methylxanthine and forskolin or expression of CLC-0, the intracellular Cl- concentration was increased in Xenopus oocytes in the presence of a high bath Cl- concentration, which inhibited ENaC without changing surface expression of alpha beta gammaENaC. In contrast, a 5 mM bath Cl- concentration reduced the cytosolic Cl- concentration and enhanced ENaC activity. ENaC was also inhibited by injection of Cl- into oocytes and in inside/out macropatches by exposure to high cytosolic Cl- concentrations. The effect of Cl- was mimicked by Br-, Br-, NO3(-), and I-. Inhibition by Cl- was reduced in trimeric channels with a truncated COOH terminus of betaENaC and gammaENaC, and it was no longer detected in dimeric alpha deltaCbeta ENaC channels. Deletion of the NH2 terminus of alpha-, beta-, or gammaENaC, mutations in the NH2-terminal phosphatidylinositol bisphosphate-binding domain of betaENaC and gammaEnaC, and activation of phospholipase C, all reduced ENaC activity but allowed for Cl(-)-dependent inhibition of the remaining ENaC current. The results confirm a role of the carboxyl terminus of betaENaC for Cl(-)-dependent inhibition of the Na+ channel, which, however, may only be part of a complex regulation of ENaC by CFTR.

The authors' goal was to provide a better understanding of the relationship between vocal production and perception in nonhuman primate communication. To this end, the authors examined the cotton-top tamarin's (Saguinus oedipus) combination long call (CLC). In Part 1 of this study, the authors carried out a series of acoustic analyses designed to determine the kind of information potentially encoded in the tamarin's CLC. Using factorial analyses of variance and multiple discriminant analyses, the authors explored whether the CLC encodes 3 types of identity information: individual, sex, and social group. Results revealed that exemplars could be reliably assigned to these 3 functional classes on the basis of a suite of spectrotemporal features. In Part 2 of this study, the authors used a series of habituation-dishabituation playback experiments to test whether tamarins attend to the encoded information about individual identity. The authors 1st tested for individual discrimination when tamarins were habituated to a series of calls from 1 tamarin and then played back a test call from a novel tamarin; both opposite- and same-sex pairings were tested. Results showed that tamarins dishabituated when caller identity changed but transferred habituation when caller identity was held constant and a new exemplar was played (control condition). Follow-up playback experiments revealed an asymmetry between the authors' acoustic analyses of individual identity and the tamarins' capacity to discriminate among vocal signatures; whereas all colony members have distinctive vocal signatures, we found that not all tamarins were equally discriminable based on the habituation-dishabituation paradigm.

Chloride fluxes are the main contributors to the resting conductance of mammalian skeletal muscle fibers. ClC-1, the most abundant chloride channel isoform in this preparation, is believed to be responsible for this conductance. However, the actual distribution of ClC-1 channels between the surface and transverse tubular system (TTS) membranes has not been assessed in intact muscle fibers. To investigate this issue, we voltageclamped enzymatically dissociated short fibers using a two-microelectrode configuration and simultaneously recorded chloride currents (I(Cl)) and di-8-ANEPPS fluorescence signals to assess membrane potential changes in the TTS. Experiments were conducted in conditions that blocked all but the chloride conductance. Fibers were equilibrated with 40 or 70 mM intracellular chloride to enhance the magnitude of inward I(Cl), and the specific ClC-1 blocker 9-ACA was used to eliminate these currents whenever necessary. Voltage-dependent di-8-ANEPPS signals and I(Cl) acquired before (control) and after the addition of 9-ACA were comparatively assessed. Early after the onset of stimulus pulses, di-8-ANEPPS signals under control conditions were smaller than those recorded in the presence of 9-ACA. We defined as attenuation the normalized time-dependent difference between these signals. Attenuation was discovered to be I(Cl) dependent since its magnitude varied in close correlation with the amplitude and time course of I(Cl). While the properties of I(Cl), and those of the attenuation seen in optical records, could be simultaneously predicted by model simulations when the chloride permeability (P(Cl)) at the surface and TTS membranes were approximately equal, the model failed to explain the optical data if P(Cl) was precluded from the TTS membranes. Since the ratio between the areas of TTS membranes and the sarcolemma is large in mammalian muscle fibers, our results demonstrate that a significant fraction of the experimentally recorded I(Cl) arises from TTS contributions.

The effect of intracellular adenosine triphosphate (ATP) on the "common gating" of the CLC-1 chloride channel has been studied by several laboratories with controversial results. Our previous study on the channel expressed in Xenopus oocytes using excised inside-out patch-clamp methods showed a robust effect of ATP in shifting the open probability curve of the common gate toward more depolarizing voltages (Tseng, P.Y., B. Bennetts, and T.Y. Chen. 2007. J. Gen. Physiol. 130:217-221). The results were consistent with those from studying the channel expressed in mammalian cells using whole cell recording methods (Bennetts, B., M.W. Parker, and B.A. Cromer. 2007. J. Biol. Chem. 282:32780-32791). However, a recent study using excised-patch recording methods for channels expressed in Xenopus oocytes reported that ATP had no direct effect on CLC-1 (Zifarelli, G., and M. Pusch. 2008. J. Gen. Physiol. 131:109-116). Here, we report that oxidation of CLC-1 may be the culprit underlying the controversy. When patches were excised from mammalian cells, the sensitivity to ATP was lost quickly--within 2-3 min. This loss of ATP sensitivity could be prevented or reversed by reducing agents. On the other hand, CLC-1 expressed in Xenopus oocytes lost the ATP sensitivity when patches were treated with oxidizing reagents. These results suggest a novel view in muscle physiology that the mechanisms controlling muscle fatigability may include the oxidation of CLC-1.

The possibilities for low-frequency horizontal transfer of the self-transmissible chlorocatechol degradative genes (clc) from Pseudomonas sp. strain B13 were investigated in activated-sludge microcosms. When the clc genes were transferred into an appropriate recipient bacterium such as Pseudomonas putida F1, a new metabolic pathway for chlorobenzene degradation was formed by complementation which could be selected for by the addition of mono- or 1, 4-dichlorobenzene (CB). Under optimized conditions with direct donor-recipient filter matings, very low transfer frequencies were observed (approximately 3.5 x 10(-8) per donor per 24 h). In contrast, in matings on agar plate surfaces, transconjugants started to appear after 8 to 10 days, and their numbers then increased during prolonged continuous incubation with CB. In activated-sludge microcosms, CB-degrading (CB+) transconjugants of strain F1 which had acquired the clc genes were detected but only when strain B13 cell densities of more than 10(5) CFU/ml could be maintained by the addition of its specific growth substrate, 3-chlorobenzoate (3CBA). The CB+ transconjugants reached final cell densities of between 10(2) and 10(3) CFU/ml. When strain B13 was inoculated separately (without the designated recipient strain F1) into an activated-sludge microcosm, CB+ transconjugants could not be detected. However, in this case a new 3CBA-degrading strain appeared which had acquired the clc genes from strain B13. The effects of selective substrates on the survival and growth of and gene transfer between bacteria degrading aromatic pollutants in a wastewater ecosystem are discussed.

We previously found that antisense oligonucleotide specific to ClC-3 (ClC-3 antisense) prevented rat aortic smooth muscle cell proliferation, which was related to cell volume regulation. In the present study, we further characterized the regulation of intracellular Cl(-) concentrations ([Cl(-)](i)) via volume-regulated ClC-3 Cl(-) channels in an embryo rat aortic vascular smooth muscle cell line (A10 cell) and ClC-3 cDNA-transfected A10 cells (ClC-3-A10) using multiple approaches including [Cl(-)](i) measurement, whole cell patch clamp, and application of ClC-3 antisense and intracellular dialysis of an anti-ClC-3 antibody. We found that hypotonic solution decreased [Cl(-)](i) and evoked a native I(Cl.vol) in A10 cells. The responses of [Cl(-)](i) and I(Cl.vol) to hypotonic challenge were enhanced by expression of ClC-3, and inhibited by ClC-3 antisense. The currents in A10 (I(Cl.vol)) and in ClC-3-A10 cells (I(Cl.ClC-3)) were remarkably inhibited by intracellular dialysis of anti-ClC-3 antibody. Reduction in [Cl(-)](i) and activation of I(Cl.vol) and I(Cl.ClC-3) in A10 and ClC-3-A10 cells, respectively, were significantly inhibited by activation of protein kinase C (PKC) by phorbol-12,13-dibutyrate (PDBu) and inhibition of tyrosine protein kinase by genistein. Sodium orthovanadate (vanadate), a protein-tyrosine phosphatase inhibitor, however, enhanced the cell swelling-induced reduction in [Cl(-)](i), accompanied by the activation of I(Cl.vol) and I(Cl.ClC-3) in a voltage-independent manner. Our results suggest that the volume-regulated ClC-3 Cl(-) channels play important role in the regulation of [Cl(-)](i) and cell proliferation of vascular smooth muscle cells.

Intracellular dialysis of NIH/3T3 cells with a commercially available anti-ClC-3 polyclonal antibody (Ab) for approximately 30 min completely inhibited expressed guinea-pig ClC-3 currents (IgpClC-3), while intracellular dialysis with antigen-preabsorbed anti-ClC-3 Ab failed to affect IgpClC-3. Anti-ClC-3 Ab was used as a selective probe to examine the relationship between endogenous ClC-3 expression and native volume-sensitive outwardly rectifying anion channels (VSOACs) in guinea-pig cardiac cells, canine pulmonary arterial smooth muscle cells (PASMCs) and Xenopus laevis oocytes. Intracellular dialysis or injection of anti-ClC-3 Ab abolished native VSOAC function in cardiac cells and PASMCs and significantly reduced VSOACs in oocytes. In contrast, native VSOAC function was unaltered by antigen-preabsorbed anti-ClC-3 Ab. It is suggested that endogenous ClC-3 represents a major molecular entity responsible for native VSOACs in cardiac and smooth muscle cells and Xenopus oocytes. Anti-ClC-3 Ab should be a useful experimental tool to directly test the relationship between endogenous ClC-3 expression and native VSOAC function, and help resolve existing controversies related to the regulation and physiological role of native VSOACs in a wide variety of different cells.

The divalent cation ionophore A23187 is frequently used for studies of eosinophil degranulation. Nonetheless, the mechanism whereby A23187 induces degranulation in human eosinophils is still unclear. In the present experiments, A23187 caused human eosinophils to release a granule protein, eosinophil-derived neurotoxin (EDN) and a membrane-associated protein, Charcot-Leyden crystal (CLC) protein in a calcium and a concentration-dependent manner. However, A23187 at a concentration (1 microgram/ml) that caused 15% EDN release and 30% CLC protein release also produced release of the cytoplasmic enzyme lactic dehydrogenase (LDH) and loss of cell viability, both of which were calcium dependent. CLC protein release preceded EDN release and was detectable even at 15 min after the addition of 1 microgram/ml A23187, whereas EDN release occurred after a lag period of 30 min, and coincided with LDH release. At 1 microgram/ml A23187, neither the release of LDH nor the loss of viability occurred with purified neutrophils obtained in the same blood sample as a by-product of eosinophil purification. Electron microscopic examination demonstrated that exposure to A23187 for 15 min resulted in an increase and elongation of microridges on the cell surface, and exposure for 45 min caused cell disruption followed by extrusion of membrane-bound granules through breaks in the plasma membrane. Only once was granule exocytosis observed. These results indicate that A23187 treatment of eosinophils causes an initial release of membrane-associated CLC protein by a noncytolytic mechanism, and causes degranulation as a result of eosinophil lysis.

ClC-3 is a Cl(-)/H(+) antiporter required for cytokine-induced intraendosomal reactive oxygen species (ROS) generation by Nox1. ClC-3 current is distinct from the swelling-activated chloride current (ICl(swell)), but overexpression of ClC-3 can activate currents that resemble ICl(swell). Because H(2)O(2) activates ICl(swell) directly, we hypothesized that ClC-3-dependent, endosomal ROS production activates ICl(swell). Whole-cell perforated patch clamp methods were used to record Cl(-) currents in cultured aortic vascular smooth muscle cells from wild type (WT) and ClC-3 null mice. Under isotonic conditions, tumor necrosis factor-alpha (TNF-alpha) (10 ng/ml) activated outwardly rectifying Cl(-) currents with time-dependent inactivation in WT but not ClC-3 null cells. Inhibition by tamoxifen (10 microm) and by hypertonicity (340 mosm) identified them as ICl(swell). ICl(swell) was also activated by H(2)O(2) (500 microm), and the effect of TNF-alpha was completely inhibited by polyethylene glycol-catalase. ClC-3 expression induced ICl(swell) in ClC-3 null cells in the absence of swelling or TNF-alpha, and this effect was also blocked by catalase. ICl(swell) activation by hypotonicity (240 mosm) was only partially inhibited by catalase, and the size of these currents did not differ between WT and ClC-3 null cells. Disruption of endosome trafficking with either mutant Rab5 (S34N) or Rab11 (S25N) inhibited TNF-alpha-mediated activation of ICl(swell). Thrombin also activates ROS production by Nox1 but not in endosomes. Thrombin caused H(2)O(2)-dependent activation of ICl(swell), but this effect was not ClC-3- or Rab5-dependent. Thus, activation of ICl(swell) by TNF-alpha requires ClC-3-dependent endosomal H(2)O(2) production. This demonstrates a functional link between two distinct anion currents, ClC-3 and ICl(swell).

OBJECTIVE

Gene silencing by hypermethylation plays an important role in proximal colon carcinogenesis. Conversely, DNA hypomethylation has been associated with distal colon cancer (CLC). Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of 5',10'-methylenetetrahydrofolate to 5'-methyl tetrahydrofolate, which serves as methyl donor in the remethylation of homocysteine to methionine. A common MTHFR 677 C->>T polymorphism is characterized by reduced catalytic activity, which affects methionine synthesis and DNA methylation. The aim of the study was to investigate the role of MTHFR 677 C->>T gene polymorphism in the tumorigenesis of proximal and distal CLC in a monoinstitutional group of patients in North Italy.

METHODS

One-hundred thirty four consecutive proximal and 142 consecutive distal CLC patients, and 279 control subjects without cancer were genotyped for MTHFR using PCR-restriction fragment-length polymorphism analysis.

RESULTS

The prevalence of the 677 TT genotype was significantly (P = 0.005) lower in patients with proximal tumors (10 of 134, 7%) than in subjects with distal tumors (28 of 142, 20%). Case/control approach indicated that individuals homozygous for the 677 TT allele had a significantly reduced risk (2.8-fold) (adjusted odds ratio, 0.36; 95% confidence intervals, 0.14-0.91) of developing proximal CLC compared with those harboring the wild-type or heterozygous genotype (677 CC or 677 CT). No significant association between CLC risk and TT genotype was observed in patients with distal tumors (odds ratio, 1.01; 95% confidence interval, 0.48-2.14).

CONCLUSIONS

Our findings support a role for MTHFR 677 TT genotype in reducing proximal CLC risk in North Italy.

Oxidative stress was proposed as a trigger of muscle impairment in various muscle diseases. The hindlimb-unloaded (HU) rodent is a model of disuse inducing atrophy and slow-to-fast transition of postural muscles. Here, mice unloaded for 14 days were chronically treated with the selective antioxidant trolox. After HU, atrophy was more pronounced in the slow-twitch soleus muscle (Sol) than in the fast-twitch gastrocnemius and tibialis anterior muscles, and was absent in extensor digitorum longus muscle. In accord with the phenotype transition, HU Sol showed a reduced expression of myosin heavy chain type 2A (MHC-2A) and increase in MHC-2X and MHC-2B isoforms. In parallel, HU Sol displayed an increased sarcolemma chloride conductance related to an increased expression of ClC-1 channels, changes in excitability parameters, a positive shift of the mechanical threshold, and a decrease of the resting cytosolic calcium concentration. Moreover, the level of lipoperoxidation increased proportionally to the degree of atrophy of each muscle type. As expected, trolox treatment fully prevented oxidative stress in HU mice. Atrophy was not prevented but the drug significantly attenuated Sol phenotypic transition and excitability changes. Trolox treatment had no effect on control mice. These results suggest possible benefits of antioxidants in protecting muscle against disuse.

The "Osteopetroses" are genetic diseases whose clinical picture is caused by a defect in bone resorption by osteoclasts. Three main forms can be distinguished on the basis of severity, age of onset and means of inheritance: the dominant benign, the intermediate and the recessive severe form. While several genes have been involved in the pathogenesis of the different types of osteopetroses, the CLCN7 gene has drawn the attention of many researchers, as mutations within this gene are associated with very different phenotypes. We report here the characterization of 25 unpublished patients which has resulted in the identification of 20 novel mutations, including 11 missense mutations, 6 causing premature termination, 1 small deletion and 2 putative splice site defects. Careful analysis of clinical and molecular data led us to several conclusions. First, intermediate osteopetrosis is not homogeneous, since it can comprise both severe dominant forms with an early onset and recessive ones without central nervous system involvement. Second, the appropriateness of haematopoietic stem cell transplantation in CLCN7-dependent ARO patients has to be carefully evaluated and exhaustive CNS examination is strongly suggested, as transplantation can almost completely cure the disease in situations where no primary neurological symptoms are present. Finally, the analysis of this largest cohort of CLCN7-dependent ARO patients together with some ADO II families allowed us to draw preliminary genotype-phenotype correlations suggesting that haploinsufficiency is not the mechanism causing ADO II. The availability of biochemical assays to characterize ClC-7 function will help to confirm this hypothesis.

BACKGROUND

Bartter syndrome (BS) is a genetic disorder accompanied by hypokalaemic metabolic alkalosis. BS with sensorineural deafness (SND, OMIM602522) is a newly identified phenotype caused by mutations in the BSND gene that encodes barttin, a beta-subunit for chloride channel ClC-Ka and ClC-Kb and classified as type IV BS. Type IV BS features the most severe phenotype entailing life-threatening neonatal volume depletion and chronic renal failure developing during infancy. A recent report described a case of BS with SND from a consanguineous family who showed homozygous mutations in the CLCNKA and CLCNKB genes. This case indicated the possibility of the occurrence of digenic inheritance in BS with SND resulting from double mutations in the CLCNKA and CLCNKB genes.

UNASSIGNED

The current report concerns a 2-year-old girl from a non-consanguineous family with BS accompanied by SND. In our case, four loss-of-function mutations, consisting of mutations in both parental alleles in both CLCNKA and CLCNKB, were identified. The paternal allele had a nonsense mutation (Q260X) in CLCNKA and a splicing site mutation (IVS17+1 g>a) in CLCNKB. The maternal allele had a large deletion mutation (about 12 kbp) extending from CLCNKA to CLCNKB. Our case provides clear evidence that loss-of-function alleles in both alleles of both CLCNKA and CLCNKB results in a phenotype indistinguishable from that of mutations in BSND (type IV BS).

CONCLUSIONS

Recent advances in genetics have resulted in a better understanding of many human inherited diseases, but most of them are monogenic disorders and more complex inheritance patterns remain unresolved. Our case provides clear evidence of digenic inheritance outside the scope of Mendelian inheritance disorders.

Genetic inactivation of ClC-5, a voltage-gated chloride channel prominently expressed in the kidney, leads to proteinuria because of defective apical endocytosis in proximal tubular cells. Because thyroid hormone secretion depends on apical endocytosis of thyroglobulin (Tg), we investigated whether ClC-5 is expressed in the thyroid and affects its function, using Clcn5-deficient knockout (KO) mice. We found that ClC-5 is highly expressed in wild-type mouse thyroid ( approximately 40% of mRNA kidney level). The protein was immunolocalized at the apical pole of thyrocytes. In Percoll gradients, ClC-5 overlapped with plasma membrane and early endosome markers, but best codistributed with the late endosomal marker, Rab7. ClC-5 KO mice were euthyroid (normal T4 and TSH serum levels) but developed a goiter with parallel iodine and Tg accumulation (i.e. normal Tg iodination level). When comparing ClC-5 KO with wild-type mice, thyroid 125I uptake after 1 h was doubled, incorporation into Tg was decreased by approximately 2-fold, so that trichloroacetic acid-soluble 125I increased approximately 4-fold. Enhanced 125I- efflux upon perchlorate and presence of 125I-Tg as autoradiographic rings at follicle periphery demonstrated delayed iodide organification. Endocytic trafficking of 125I-Tg toward lysosomes was not inhibited. Expression of pendrin, an I-/Cl- exchanger involved in apical iodide efflux, was selectively decreased by 60% in KO mice at mRNA and protein levels. Thus, ClC-5 is well expressed in the thyroid but is not critical for apical endocytosis, contrary to the kidney. Instead, the goiter associated with ClC-5 KO results from impaired rate of apical iodide efflux by down-regulation of pendrin expression.

We identified two ClC-2 clones in a guinea pig intestinal epithelial cDNA library, one of which carries a 30-bp deletion in the NH(2) terminus. PCR using primers encompassing the deletion gave two products that furthermore were amplified with specific primers confirming their authenticity. The corresponding genomic DNA sequence gave a structure of three exons and two introns. An internal donor site occurring within one of the exons accounts for the deletion, consistent with alternative splicing. Expression of the variants gpClC-2 and gpClC-2Delta77-86 in HEK-293 cells generated inwardly rectifying chloride currents with similar activation characteristics. Deactivation, however, occurred with faster kinetics in gpClC-2Delta77-86. Site-directed mutagenesis suggests that a protein kinase C-mediated phosphorylation consensus site lost in gpClC-2Delta77-86 is not responsible for the observed change. The deletion-carrying variant is found in most tissues examined, and it appears more abundant in proximal colon, kidney, and testis. The presence of a splice variant of ClC-2 modified in its NH(2)-terminal domain could have functional consequences in tissues where their relative expression levels are different.

Escherichia coli and other enterobacteria exploit the H+ -consuming reaction catalysed by glutamate decarboxylase to survive the stomach acidity before reaching the intestine. Here we show that chloride, extremely abundant in gastric secretions, is an allosteric activator producing a 10-fold increase in the decarboxylase activity at pH 5.6. Cooperativity and sensitivity to chloride were lost when the N-terminal 14 residues, involved in the formation of two triple-helix bundles, were deleted by mutagenesis. X-ray structures, obtained in the presence of the substrate analogue acetate, identified halide-binding sites at the base of each N-terminal helix, showed how halide binding is responsible for bundle stability and demonstrated that the interconversion between active and inactive forms of the enzyme is a stepwise process. We also discovered an entirely novel structure of the cofactor pyridoxal 5'-phosphate (aldamine) to be responsible for the reversibly inactivated enzyme. Our results link the entry of chloride ions, via the H+/Cl- exchange activities of ClC-ec1, to the trigger of the acid stress response in the cell when the intracellular proton concentration has not yet reached fatal values.

CLC-0, a member of the CLC family of Cl(-)-conducting ion channels, consists of an N-terminal hydrophobic core and a C-terminal region that is thought to be cytoplasmic. This study provides evidence that the C-terminal region is a mechanistically relevant cytoplasmic domain of the CLC-0 ion channel. Both a point mutation and a 37-residue deletion in this region cause drastic alterations in voltage-dependent gating of CLC-0 current expressed in Xenopus oocytes. CLC-0 current is not observed when the entire C-terminal region is deleted, but functional channels are efficiently reconstituted by co-injection of separate cRNA constructs encoding the N-terminal transmembrane and the C-terminal cytoplasmic domains. Moreover, reconstitution of CLC-0 can be achieved by co-injection of cRNA encoding the transmembrane domain along with Escherichia coli-expressed C-terminal domain polypeptide.

The absence of the chloride channel CLC-3 in Clcn3(-/-) mice results in hippocampal degeneration with a distinct temporal-spatial sequence that resembles neuronal loss in temporal lobe epilepsy. We examined how the loss of CLC-3 might affect GABAergic synaptic transmission in the hippocampus. An electrophysiological study of synaptic function in hippocampal slices taken from Clcn3(-/-) mice before the onset of neurodegeneration revealed a substantial decrease in the amplitude and frequency of miniature inhibitory postsynaptic currents compared with those in wild-type slices. We found that CLC-3 colocalized with the vesicular GABA transporter VGAT in the CA1 region of the hippocampus. Acidification of inhibitory synaptic vesicles induced by Cl(-) showed a marked dependence on CLC-3 expression. The decrease in inhibitory transmission in Clcn3(-/-) mice suggests that the neurotransmitter loading of synaptic vesicles was reduced, which we attribute to defective vesicular acidification. Our observations extend the role of Cl(-) in inhibitory transmission from that of a postsynaptic permeant species to a presynaptic regulatory element.

Acute respiratory distress syndrome (ARDS) is a severe form of acute lung injury. It is a response to various diseases of variable etiology, including SARS-CoV infection. To date, a comprehensive study of the genomic physiopathology of ARDS (and SARS) is lacking, primarily due to the difficulty of finding suitable materials to study the disease process at a tissue level (instead of blood, sputa or swaps). Hereby we attempt to provide such study by analyzing autopsy lung samples from patient who died of SARS and showed different degrees of severity of the pulmonary involvement. We performed real-time quantitative PCR analysis of 107 genes with functional roles in inflammation, coagulation, fibrosis and apoptosis; some key genes were confirmed at a protein expression level by immunohistochemistry and correlated to the degree of morphological severity present in the individual samples analyzed. Significant expression levels were identified for ANPEP (a receptor for CoV), as well as inhibition of the STAT1 pathway, IFNs production and CXCL10 (a T-cell recruiter). Other genes unassociated to date with ARDS/SARS include C1Qb, C5R1, CASP3, CASP9, CD14, CD68, FGF7, HLA-DRA, IGF1, IRF3, MALAT-1, MSR1, NFIL3, SLPI, USP33, CLC, GBP1 and TAC1. As a result, we proposed to therapeutically target some of these genes with compounds such as ANPEP inhibitors, SLPI and dexamethasone. Ultimately, this study may serve as a model for future, tissue-based analyses of fibroinflammatory conditions affecting the lung.