Familial disorders of skeletal muscle excitability were initially described early in the last century and are now known to be caused by mutations of voltage-gated ion channels. The clinical manifestations are often striking, with an inability to relax after voluntary contraction (myotonia) or transient attacks of severe weakness (periodic paralysis). An essential feature of these disorders is fluctuation of symptoms that are strongly impacted by environmental triggers such as exercise, temperature, or serum K(+) levels. These phenomena have intrigued physiologists for decades, and in the past 25 years the molecular lesions underlying these disorders have been identified and mechanistic studies are providing insights for therapeutic strategies of disease modification. These familial disorders of muscle fiber excitability are "channelopathies" caused by mutations of a chloride channel (ClC-1), sodium channel (NaV1.4), calcium channel (CaV1.1), and several potassium channels (Kir2.1, Kir2.6, and Kir3.4). This review provides a synthesis of the mechanistic connections between functional defects of mutant ion channels, their impact on muscle excitability, how these changes cause clinical phenotypes, and approaches toward therapeutics.

The CLC protein family contains plasma membrane chloride channels and the intracellular chloride-proton exchangers ClC-3-7. The latter proteins mainly reside on the various compartments of the endosomal-lysosomal system where they are involved in the luminal acidification or chloride accumulation. Although their partially overlapping subcellular distribution has been studied extensively, little is known about their targeting mechanism. In a comprehensive study we now performed pulldown experiments to systematically map the differential binding of adaptor proteins of the endosomal sorting machinery (adaptor proteins and GGAs (Golgi-localized, γ-ear containing, Arf binding)) as well as clathrin to the cytosolic regions of the intracellular CLCs. The resulting interaction pattern fitted well to the known subcellular localizations of the CLCs. By mutating potential sorting motifs, we could locate almost all binding sites, including one already known for ClC-3 and several new motifs for ClC-5, -6, and -7. The impact of the identified binding sites on the subcellular localization of CLC transporters was determined by heterologous expression of mutants. Surprisingly, some vesicular CLCs retained their localization after disruption of interaction sites. However, ClC-7 could be partially shifted from lysosomes to the plasma membrane by combined mutation of N-terminal sorting motifs. The localization of its β-subunit, Ostm1, was determined by that of ClC-7. Ostm1 was not capable of redirecting ClC-7 to lysosomes.

Mutations in either ClC-7, a late endosomal/lysosomal member of the CLC family of chloride channels and transporters, or in its beta-subunit Ostm1 cause osteopetrosis and lysosomal storage disease in mice and humans. The severe phenotype of mice globally deleted for ClC-7 or Ostm1 and the absence of storage material in cultured cells hampered investigations of the mechanism leading to lysosomal pathology in the absence of functional ClC-7/Ostm1 transporters. Tissue-specific ClC-7-knockout mice now reveal that accumulation of storage material occurs cell-autonomously in neurons or renal proximal tubular cells lacking ClC-7. Almost all ClC-7-deficient neurons die. The activation of glia is restricted to brain regions where ClC-7 has been inactivated. The effect of ClC-7 disruption on lysosomal function was investigated in renal proximal tubular cells, which display high endocytotic activity. Pulse-chase endocytosis experiments in vivo with mice carrying chimeric deletion of ClC-7 in proximal tubules allowed a direct comparison of the handling of endocytosed protein between cells expressing or lacking ClC-7. Whereas protein was endocytosed similarly in cells of either genotype, its half-life increased significantly in ClC-7-deficient cells. These experiments demonstrate that lysosomal pathology is a cell-autonomous consequence of ClC-7 disruption and that ClC-7 is important for lysosomal protein degradation.

OBJECTIVE

In recent years, there have been significant advances in our understanding of the molecular mechanisms relating proximal tubule abnormalities to the pathogenesis of renal Fanconi syndrome. This review focuses on the role of intra-endosomal acidification-machinery proteins (V-ATPase, CLC-5, NHE-3), as well as apical receptors (megalin and cubilin), in the receptor-mediated endocytosis pathway and in the pathogenesis of proximal tubulopathies.

RESULTS

Animal models, including CLC-5 and megalin knockout mice, cubilin-deficient dogs and cadmium-toxicity studies in rats, have shed light on defects leading to low-molecular-weight proteinuria. In particular, the important contribution of defective endosomal acidification and membrane-protein recycling to the pathogenesis of the Fanconi syndrome has emerged from these studies. These observations, together with recent findings in patients with Dent's disease, Lowe's syndrome, autosomal-dominant idiopathic Fanconi syndrome and Imerslund-Grasbeck disease, show that the proteinuria of the Fanconi syndrome is more generalized than previously suspected. High concentrations of polypeptides, including hormones, vitamin-binding proteins and chemokines in urine from these patients and animals may play an important role in the progressive renal failure that is associated with the syndrome.

CONCLUSIONS

The molecular mechanism of proximal tubule protein reabsorption, which is defective in renal Fanconi syndrome, includes a crucial role for endosomal acidification-machinery proteins, in particular the V-ATPase and CLC-5 chloride channels, in the trafficking and acidification-dependent recycling of apical membrane proteins, including the endocytotic receptors megalin and cubilin. An increased understanding of the roles of V-ATPase and CLC-5 in proximal tubule endosomal acidification, in the regulation of the megalin/cubilin-mediated endocytosis pathway and finally in the pathogenesis of human Fanconi syndrome will help in the devising of appropriate strategies for therapeutic intervention for this disorder.

OBJECTIVE

Vascular endothelial growth factor (VEGF) is a primary driving force for both physiological and pathological angiogenesis, and its overexpression has been found in hepatocellular carcinoma (HCC). The aim of this study was to retrospectively clarify the usefulness of serum VEGF levels as a tumor marker in patients with hepatitis C virus (HCV)-related liver cirrhosis (CLC) and HCC.

METHODS

The patients with CLC were divided into three groups: 28 patients without HCC (CLC group), 11 patients with HCC (HCC group), and 48 patients with advanced HCC (aHCC group). The control group consisted of 37 patients with chronic HCV.

RESULTS

When the relation of serum VEGF to liver function was assessed, there was no significant difference of VEGF levels between the control group and the CLC group. When serum VEGF levels were assessed in relation to the presence of HCC, the VEGF levels of the HCC group and aHCC group were found to be significantly higher than that of the control group, while there was no significant difference between the control group and the CLC group. For the detection of cancer, serum VEGF had the largest area under the curve (AUC) and the highest accuracy when we employed the cut-off value obtained by receiver operating characteristic (ROC) analysis using the Youden index. Evaluation of various tumor markers in the aHCC group showed that the serum levels of α-fetoprotein (AFP) were higher in patients with infiltrating tumors than in patients with multiple discrete nodules or confluent multinodular tumors, while there were no significant differences in the serum levels of VEGF, Lens culinaris agglutinin-reactive fraction of AFP (AFP-L3), and des-γ-carboxy prothrombin. There were no significant differences on the serum levels of all four markers between tumor stages, but serum VEGF was higher in patients with vascular invasion than in those without vascular invasion.

CONCLUSIONS

The present findings suggest that the serum levels of VEGF might be a useful predictor of the presence of HCC in patients with CLC, while serum levels of AFP and VEGF can predict the tumor type and vascular invasion, respectively.

Adenovirus expressing <em>ClC</em>-3 (Ad-<em>ClC</em>-3) induces Cl(-)/H(+) antiport current (I(<em>ClC</em>-3)) in HEK293 cells. The outward rectification and time dependence of I(<em>ClC</em>-3) closely resemble an endogenous HEK293 cell acid-activated Cl(-) current (ICl(acid)) seen at extracellular pH <or= 5.5. ICl(acid) was present in smooth muscle cells from wild-type but not <em>ClC</em>-3 null mice. We therefore sought to determine whether these currents were related. ICl(acid) was larger in cells expressing Ad-<em>ClC</em>-3. Protons shifted the reversal potential (E(rev)) of I(<em>ClC</em>-3) between pH 8.2 and 6.2, but not pH 6.2 and 5.2, suggesting that Cl(-) and H(+) transport become uncoupled at low pH. At pH 4.0 E(rev) was completely Cl(-) dependent (55.8 +/- 2.3 mV/decade). Several findings linked <em>ClC</em>-3 with native ICl(acid); 1) RNA interference directed at <em>ClC</em>-3 message reduced native ICl(acid); 2) removal of the extracellular "fast gate" (E224A) produced large currents that were pH-insensitive; and 3) wild-type I(<em>ClC</em>-3) and ICl(acid) were both inhibited by (2-sulfonatoethyl)methanethiosulfonate (MTSES; 10-500 microm)-induced alkanethiolation at exposed cysteine residues. However, a <em>ClC</em>-3 mutant lacking four extracellular cysteine residues (C103_P130del) was completely resistant to MTSES. C103_P130del currents were still acid-activated, but could be distinguished from wild-type I(<em>ClC</em>-3) and from native ICl(acid) by a much slower response to low pH. Thus, <em>ClC</em>-3 currents are activated by protons and <em>ClC</em>-3 protein may account for native ICl(acid). Low pH uncouples Cl(-)/H(+) transport so that at pH 4.0 <em>ClC</em>-3 behaves as an anion-selective channel. These findings have important implications for the biology of Cl(-)/H(+) antiporters and perhaps for pH regulation in highly acidic intracellular compartments.

The function of voltage-gated chloride channels in neurons is essentially unknown. The voltage-gated chloride channel ClC-2 mediates a chloride current in pyramidal cells of the hippocampus. We directly show that ClC-2 assists chloride extrusion after high chloride load. Furthermore, the loss of this chloride channel leads to a dramatic increase of the input resistance of CA1 pyramidal cells, making these cells more excitable. Surprisingly, basal synaptic transmission, as judged from recordings of field EPSPs, was decreased. This difference was eliminated when GABAergic inhibition was blocked. Recordings from hippocampal interneurons revealed ClC-2-mediated currents in a subset of these cells. An observed increase in GABAergic inhibition could thus be explained by an increase in the excitability of interneurons, caused by the loss of ClC-2. Together, we suggest a dual role for ClC-2 in neurons, providing an additional efflux pathway for chloride and constituting a substantial part of the background conductance, which regulates excitability. In ClC-2 knock-out mice, an increased inhibition seemingly balances the hyperexcitability of the network and thereby prevents epilepsy.

Spleen dendritic cells (DC) and epidermal Langerhans cells (LC) belong to the same family of dendritic leukocytes and are considered to be prototypes of lymphoid DC and nonlymphoid DC, respectively. These cells are active APC in vitro and play a key role in the induction of primary T cell dependent immune responses in vivo. Two functional states of LC have been characterized in vitro, freshly isolated LC and cultured LC (cLC). That cLC closely resemble spleen DC in phenotype and function, has led to the hypothesis that LC undergo maturation toward DC while in culture, an event that has been correlated with the emigration of LC from skin into lymphoid organs. To date, however, DC have been studied only after overnight culture. To better understand the relationship between LC and DC, we examined DC shortly after their isolation from spleen, and after 24 h of culture. Freshly isolated DC (fDC) express high levels of MHC molecules and low levels of Fc gamma RII and C3biR; fDC also uniformly express the Ag recognized by the mAb 33D1, NLDC-145, and J11d. After culture, DC display a marked increase in the expression of MHC molecules, and they are induced to express the low affinity receptor for IL-2. By contrast, the expression of Fc gamma RII and F4/80 decreases with culture. With respect to function, fDC can efficiently present keyhole limpet hemocyanin to Ag-specific T cells, whereas cultured DC exhibit a marked reduction in this capacity. Finally, both fDC and cultured DC are capable of endocytosing surface Ia molecules, but only fDC are able to deliver them into acidic compartments. Our data indicate that fDC from spleen resemble freshly isolated LC from epidermis and that both cells undergo parallel changes during culture. These results suggest that LC and DC possess analogous attributes in vivo and respond similarly to external influences.

Charcot-Leyden crystals (CLC) are currently believed to be unique to the eosinophil and a hallmark of active eosinophilic inflammation or proliferation. The distinctiveness of the CLC to the eosinophil was questioned in 1965 by Archer and Blackwood (9), but their demonstration of CLC formation in basophils was ignored and later dismissed (1) as being the result of eosinophil contamination of basophil-enriched cell suspensions. We reexamined this question and showed that basophils obtained from the peripheral blood of normal individuals form CLC and that basophils contain a protein that is immunochemically indistinguishable from eosinophil CLC protein. These conclusions are based upon the findings that (a) crystal formation in basophils was demonstrated by specific histochemical staining of crystal-containing cells in highly enriched basophil suspensions prepared by fluorescence-activated cell sorter (FACS) purification of surface IgE-positive cells, (b) that enrichment for surface IgE-positive cells (primarily basophils) by the FACS also enriched for cells staining positively by immunofluorescence for eosinophil CLC protein, and (c) that CLC protein was measured by radioimmunoassay in cell extracts prepared from purified basophil suspensions containing 97-99% basophils and absolutely no contaminating eosinophils. These basophil extracts contained a protein immunochemically indistinguishable from eosinophil CLC protein. Based upon these findings, the CLC or the protein comprising the crystal (lysophospholipase) can no longer be considered as distinctive to the eosinophil. We must now consider the possibility that the presence of CLC in tissues, sputum, or stool may also represent basophil involvement in disease processes.

Cystic fibrosis is a fatal inherited disease that is caused by mutations in the gene encoding a cAMP-activated chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR). It has been suggested that the cystic fibrosis phenotype might be modulated by the presence of other Cl(-) channels that are coexpressed with CFTR in some epithelial cells. Because the broadly expressed plasma membrane Cl(-) channel, ClC-2, is present in the tissues whose function is compromised in cystic fibrosis, we generated mice with a disruption of both Cl(-) channel genes. No morphological changes in their intestine, lung, or pancreas, tissues affected by cystic fibrosis, were observed in these mice. The mortality was not increased over that observed with a complete lack of functional CFTR. Surprisingly, mice expressing mutant CFTR (deletion of phenylalanine 508), survived longer when ClC-2 was disrupted additionally. Currents across colonic epithelia were investigated in Ussing chamber experiments. The disruption of ClC-2, in addition to CFTR, did not decrease Cl(-) secretion. Colon expressing wild-type CFTR even secreted more Cl(-) when ClC-2 was disrupted, although CFTR transcript levels were unchanged. It is concluded that ClC-2 is unlikely to be a candidate rescue channel in cystic fibrosis. Our data are consistent with a model in which ClC-2 is located in the basolateral membrane.

Numerous Cl- channels have been identified in the kidney using physiological approaches and thus are thought to be involved in a range of physiological processes, including vectorial transepithelial Cl- transport, cell volume regulation, and vesicular acidification. In addition, expression of genes from several Cl- channel gene families has also been observed. However, the molecular characteristics of a number of Cl- channels within the kidney are still unknown, and the physiological roles of Cl- channels identified by molecular means remain to be determined. A gene knockout approach using mice might shed further light on the characteristics of these various Cl- channels. In addition, study of diseases involving Cl- channels (channelopathies) might clarify the physiological role of specific Cl- channels. To date, more is known about CLC Cl- channels than any other Cl- channels within the kidney. This review focuses on the physiological roles of CLC Cl- channels within the kidney, particularly kidney-specific ClC-K Cl- channels, as well as the recently identified maxi anion channel in macula densa, which is involved in tubulo-glomerular feedback.

The ClC proteins are members of a large family of chloride transport proteins, which are involved in a variety of physiological processes. All family members share a conserved molecular architecture consisting of a complex transmembrane transport domain and a soluble regulatory domain. To date, representative structures for the two parts are available, the transmembrane domain from the structure of a bacterial homologue, the soluble domain from a eukaryotic family member. Despite the strong conservation of the structural framework, the family members show an unusually broad variety of functional behaviors, as some members work as gated chloride channels and others as secondary chloride transporters. The conservation in the structure and the functional resemblance in gating and transport mechanism suggest a strong mechanistic relationship between seemingly contradictory transport modes.

1. The chloride channel from the Torpedo electric organ, ClC-0, is controlled by two distinct ('fast' and 'slow') voltage-dependent gates. Here we investigate the effects of mutations in a region after putative transmembrane domain D12. A mutation in this region has previously been shown to change fast gating and permeation. 2. We used a combination of site-directed mutagenesis with two-electrode voltage-clamp and patch-clamp measurements. 3. Most conservative substitutions have minor effects, while more drastic mutations change kinetics and voltage dependence of fast gating, as well as ion selectivity and rectification. 4. While ClC-0 wild-type (WT) channels deactivate fully in two-electrode voltage clamp at negative voltages, channels do not close completely in patch-clamp experiments. Open probability is increased by intracellular chloride in a concentration- but not voltage-dependent manner. 5. In several mutants, including K519R, the minimal macroscopic open probability of fast gating is larger than in WT. Mutant channels fluctuate at negative potentials between open and closed conformations. Open probability is much more effectively increased by intracellular chloride than in WT. The observations support the idea that permeating ions inside the pore stabilize the open state. 6. Besides effects on permeation and gating of single protopores, some mutations affect 'slow' gating. In summary, the region after D12 participates in fast as well as in slow gating; mutations additionally influence permeation properties.

1. A chloride current with mild outward rectification was induced in the native bovine non-pigmented ciliary epithelial (NPCE) cells by a 23 % hypotonic solution. The current showed no or little inactivation at depolarized steps. 2. ATP blocked 88 and 61 % of the outward and inward components of the volume-activated chloride current (ICl,vol) with an IC50 of 5.3 and 9.6 mM, respectively. 3. The volume-activated chloride current was decreased and the activation of the current was delayed by inhibiting endogenous ClC-3 expression using a ClC-3 antisense oligonucleotide. The inhibition of the current as a function of antisense concentration was asymptotic with a maximum about 60 %. The remaining current was probably not derived from ClC-3 and was inhibited by ATP. 4. ClC-3 expression in the bovine NPCE cells was verified by immunofluorescence studies. ClC-3 immunofluorescence was distributed throughout the cells but with the predominant location within the nucleus. The expression of ClC-3 protein was diminished by the ClC-3 antisense oligonucleotide with the greatest diminution occurring in the nuclear region. 5. The size of the volume-activated chloride current was positively correlated with the ClC-3 immunofluorescence level. 6. Regulatory volume decrease of the NPCE cells was reduced by ClC-3 antisense oligonucleotide. 7. We conclude that endogenous ClC-3 is associated with the volume-activated chloride current and is involved in cell volume regulation, but that it can only contribute towards a proportion of the current in NPCE cells. 8. The nuclear predominance of ClC-3 immunofluorescence in NPCE cells, the absence of basal activity of chloride current and the marked pharmacological differences between IClC-3 and ICl,vol argue against ClC-3 being the only, or even the main, volume-activated chloride channel in NPCE cells.

Neurons express adaptor (AP)-3 complexes assembled with either ubiquitous (beta3A) or neuronal-specific (beta3B) beta3 isoforms. However, it is unknown whether these complexes indeed perform distinct functions in neuronal tissue. Here, we explore this hypothesis by using genetically engineered mouse models lacking either beta3A- or beta3B-containing AP-3 complexes. Somatic and neurological phenotypes were specifically associated with the ubiquitous and neuronal adaptor deficiencies, respectively. At the cellular level, AP-3 isoforms were localized to distinct neuronal domains. beta3B-containing AP-3 complexes were preferentially targeted to neuronal processes. Consistently, beta3B deficiency compromised synaptic zinc stores assessed by Timm's staining and the synaptic vesicle targeting of membrane proteins involved in zinc uptake (ZnT3 and ClC-3). Surprisingly, despite the lack of neurological symptoms, beta3A-deficient mouse brain possessed significantly increased synaptic zinc stores and synaptic vesicle content of ZnT3 and ClC-3. These observations indicate that the functions of beta3A- and beta3B-containing complexes are distinct and divergent. Our results suggest that concerted nonredundant functions of neuronal and ubiquitous AP-3 provide a mechanism to control the levels of selected membrane proteins in synaptic vesicles.

CLC-K Cl(-) channels are selectively expressed in kidney and ear, where they are pivotal for salt homeostasis, and loss-of-function mutations of CLC-Kb produce Bartter's syndrome type III. The only ligand known for CLC-K channels is a derivative of the 2-p-chlorophenoxypropionic acid (CPP), 3-phenyl-CPP, which blocks CLC-Ka, but not CLC-Kb. Here we show that in addition to this blocking site, CLC-K channels bear an activating binding site that controls channel opening. Using the voltage-clamp technique on channels expressed in Xenopus laevis oocytes, we found that niflumic acid (NFA) increases CLC-Ka and CLC-Kb currents in the 10 to 1000 microM range. Flufenamic acid (FFA) derivatives or high doses of NFA produced instead an inhibitory effect on CLC-Ka, but not on CLC-Kb, and on blocker-insensitive CLC-Ka mutants, indicating that the activating binding site is distinct from the blocker site. Evaluation of the sensitivity of CLC-Ka to derivatives of NFA and FFA together with a modeling study of these ligands allow us to conclude that one major characteristic of activating compounds is the coplanarity of the two rings of the molecules, whereas block requires a noncoplanar configuration. These molecules provide a starting point for identification of diuretics or drugs useful in the treatment of Bartter's syndrome.

Salivary gland acinar cells shrink when Cl(-) currents are activated following cell swelling induced by exposure to a hypotonic solution or in response to calcium-mobilizing agonists. The molecular identity of the Cl(-) channel(s) in salivary cells involved in these processes is unknown, although ClC-3 has been implicated in several tissues as a cell-volume-sensitive Cl(-) channel. We found that cells isolated from mice with targeted disruption of the Clcn3 gene undergo regulatory volume decrease in a fashion similar to cells from wild-type littermates. Consistent with a normal regulatory volume decrease response, the magnitude and the kinetics of the swell-activated Cl(-) currents in cells from ClC-3-deficient mice were equivalent to those from wild-type mice. It has also been suggested that ClC-3 is activated by Ca(2+)-calmodulin-dependent protein kinase II; however, the magnitude of the Ca(2+)-dependent Cl(-) current was unchanged in the Clcn3(-/-) animals. In addition, we observed that ClC-3 appeared to be highly expressed in the smooth muscle cells of glandular blood vessels, suggesting a potential role for this channel in saliva production by regulating blood flow, yet the volume and ionic compositions of in vivo stimulated saliva from wild-type and null mutant animals were comparable. Finally, in some cells ClC-3 is an intracellular channel that is thought to be involved in vesicular acidification and secretion. Nevertheless, the protein content of saliva was unchanged in Clcn3(-/-) mice. Our results demonstrate that the ClC-3 Cl(-) channel is not a major regulator of acinar cell volume, nor is it essential for determining the secretion rate and composition of saliva.

1. Hyperpolarization-activated Cl- currents (ICl,hyp) were investigated in the T84 human adenocarcinoma cell line, using the patch-clamp whole-cell configuration. 2. During whole-cell recording with high-chloride and ATP-containing internal solutions, hyperpolarizing jumps from a holding potential of 0 mV elicited slow inward current relaxations, carried by Cl- and detected at membrane potentials more negative than -40 mV. Analysis of the relative permeabilities to monovalent anions gave the following sequence: Cl->> Br->> I->> glutamate. 3. ICl,hyp was partially inhibited by 1 mM diphenylamine-2-carboxylic acid or 0.1 mM 5-nitro-2-(3-phenylpropylamino)-benzoate, and was completely blocked by Cd2+ >> 300 microM). It was insensitive to 1 mM external 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid or 1 mM Ba2+. 4. ICl,hyp was inhibited by external application of 500 microM cptcAMP (8-(4-chlorophenylthio)-adenosine 3':5'-cyclic monophosphate) or 500 nM of the protein kinase C activator, phorbol 12-myristate, 13-acetate. 5. (i) Omission of ATP from the pipette solution, (ii) ATP replacement by the non-hydrolysable ATP analogue 5'-adenylylimidodiphosphate, and (iii) inhibition of protein kinase C by staurosporine or calphostin C accelerated the activation kinetics of the current and increased its amplitude, but did not alter its pharmacological properties. 6. We conclude that hyperpolarization-activated Cl- channels similar to those of ClC-2 channels (mammalian homologue of Torpedo chloride channel ClC-0) are present in T84 cells, and that their gating properties are modulated by phosphorylation.

Plant vacuoles play essential roles in many physiological processes, particularly in mineral nutrition, turgor provision and cellular signalling. The vacuolar membrane, the tonoplast, contains many membrane transporters that are critical in the execution of these processes. However, although increasing knowledge is available about the identity of proteins involved in these processes very little is known about the regulation of tonoplast transporters. By studying the phosphoproteome of tonoplast-enriched membranes, we identified 66 phosphorylation sites on 58 membrane proteins. Amongst these, 31 sites were identified in 28 membrane transporters of various families including tonoplast anion transporters of the CLC family, potassium transporters of the KUP family, tonoplast sugar transporters and ABC transporters. In a number of cases, the detected sites were well conserved across isoforms of one family pointing to common mechanisms of regulation. In other cases, isoform-unique sites were present, suggesting regulatory mechanisms tailored to the function of individual proteins. These results provide the basis for future studies to elucidate the mechanistic regulation of tonoplast membrane transporters.

BACKGROUND

Maternal employment has been one of the greatest barriers to breastfeeding. Women are increasingly solving this problem by expressing milk at work and taking it home to their infants.

OBJECTIVE

The objective was to determine duration of breast milk expression among working mothers enrolled in an employer-sponsored lactation program.

METHODS

Retrospective reviews were conducted on the lactation records of 462 women employed by 5 corporations in order to describe and characterize their experiences. The lactation program included the employees' choice of (a) a class on the benefits of breastfeeding; (b) services of a certified lactation consultant (CLC); and (c) private room in the workplace with equipment for pumping.

RESULTS

Breastfeeding was initiated by 97.5% of the participants, with 57.8% continuing for at least 6 months. Of the 435 (94.2%) who returned to work after giving birth, 343 (78.9%) attempted pumping milk at work, and 336 (98%) were successful. They expressed milk in the workplace for a mean of 6.3 months (SD = 3.9, range 2 weeks to 21 months). The mean age of infants when the mothers stopped pumping at work was 9.1 months (SD = 4.1, range 1.9 to 25 months). Most of the women who pumped their milk at work were working full time (84.2%). The mean postnatal maternity leave was 2.8 months. The proportion of women who chose to pump at work was higher among women who were salaried than among those who were paid hourly wages (p < 0.01).

CONCLUSIONS

Company-sponsored lactation programs can enable employed mothers to provide breast milk for their infants as long as they wish, thus helping the nation attain the Healthy People 2010 goals of 50% of mothers breastfeeding until their infants are 6-months-old.

The Lombard effect-an increase in vocalization amplitude in response to an increase in background noise-is observed in a wide variety of animals. We investigated this basic form of vocal control in the cotton-top tamarin (Saguinus oedipus) by measuring the amplitude of a contact call, the combination long call (CLC), while simultaneously varying the background noise level. All subjects showed a significant increase in call amplitude and syllable duration in response to an increase in background noise amplitude. Together with prior results, this study shows that tamarins have greater vocal control in the context of auditory feedback perturbation than previously suspected.

Probiotic properties are highly strain-dependent but rarely studied in enological lactic acid bacteria (LAB). In this study, the probiotic features of 11 strains of Lactobacillus spp., Pediococcus spp., and Oenococcus oeni, including saliva and acid resistance, bile tolerance and exopolysaccharides' production, were investigated. The assays included two probiotic reference strains (Lactobacillus plantarum CLC 17 and Lactobacillus fermentum CECT5716). The Lactobacillus and Pediococcus strains showed high resistance to lysozyme (>80% resistance to 100 mg/L of lysozyme under conditions simulating the in vivo dilution by saliva) and were capable of surviving at low pH values (pH 1.8) and bile salts, suggesting good adaptation of the wine strains to gastrointestinal conditions. The ability of the strains to adhere to the intestinal mucosa and the inhibition of the adhesion of Escherichia coli to human intestinal cells were also evaluated. Adhesion levels of enological LAB to Caco-2 cells varied from 0.37% to 12.2%, depending on the strain. In particular, Pediococcus pentosaceus CIAL-86 showed a high percentage of adhesion to intestinal cells (>12%), even higher than that shown by the probiotic reference strains, and a high anti-adhesion activity against E. coli CIAL-153 (>30%), all of which support this wine LAB strain as a potential probiotic.

The ClC transport protein family comprises both Cl(-) ion channel and H(+)/Cl(-) and H(+)/NO(3)(-) exchanger members. Structural studies on a bacterial ClC transporter reveal a pore obstructed at its external opening by a glutamate side-chain which acts as a gate for Cl(-) passage and in addition serves as a staging post for H(+) exchange. This same conserved glutamate acts as a gate to regulate Cl(-) flow in ClC channels. The activity of ClC-2, a genuine Cl(-) channel, has a biphasic response to extracellular pH with activation by moderate acidification followed by abrupt channel closure at pH values lower than approximately 7. We have now investigated the molecular basis of this complex gating behaviour. First, we identify a sensor that couples extracellular acidification to complete closure of the channel. This is extracellularly-facing histidine 532 at the N-terminus of transmembrane helix Q whose neutralisation leads to channel closure in a cooperative manner. We go on to show that acidification-dependent activation of ClC-2 is voltage dependent and probably mediated by protonation of pore gate glutamate 207. Intracellular Cl(-) acts as a voltage-independent modulator, as though regulating the pK(a) of the protonatable residue. Our results suggest that voltage dependence of ClC-2 is given by hyperpolarisation-dependent penetration of protons from the extracellular side to neutralise the glutamate gate deep within the channel, which allows Cl(-) efflux. This is reminiscent of a partial exchanger cycle, suggesting that the ClC-2 channel evolved from its transporter counterparts.

The ciliary neurotrophic factor alpha-receptor (CNTFRalpha) is required for motoneuron survival during development, but the relevant ligand(s) has not been determined. One candidate is the heterodimer formed by cardiotrophin-like cytokine (CLC) and cytokine-like factor 1 (CLF). CLC/CLF binds to CNTFRalpha and enhances the survival of developing motoneurons in vitro; whether this novel trophic factor plays a role in neural development in vivo has not been tested. We examined motor and sensory neurons in embryonic chicks treated with CLC and in mice with a targeted deletion of the clf gene. Treatment with CLC increased the number of lumbar spinal cord motoneurons that survived the cell death period in chicks. However, this effect was regionally specific, because brachial and thoracic motoneurons were unaffected. Similarly, newborn clf-/- mice exhibited a significant reduction in lumbar motoneurons, with no change in the brachial or thoracic cord. Clf deletion also affected brainstem motor nuclei in a regionally specific manner; the number of motoneurons in the facial but not hypoglossal nucleus was significantly reduced. Sensory neurons of the dorsal root ganglia were not affected by either CLC treatment or clf gene deletion. Finally, mRNA for both clc and clf was found in skeletal muscle fibers of embryonic mice during the motoneuron cell death period. These findings support the view that CLC/CLF is a target-derived factor required for the survival of specific pools of motoneurons. The in vivo actions of CLC and CLF can account for many of the effects of CNTFRalpha on developing motoneurons.