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Candida species were tested for susceptibility to caspofungin, anidulafungin, and micafungin in order to evaluate the roles of Etest and Sensititre YeastOne in antifungal susceptibility testing for daily routines and to survey resistance. A total of 104 Candida species isolates detected from blood cultures were investigated. With EUCAST broth microdilution as the reference method, essential agreement (EA), categorical agreement (CA), very major errors (VME), major errors (ME), and minor (MIN) errors were assessed by reading MICs at 18, 24, and 48 h. By use of EUCAST broth microdilution and species-specific clinical breakpoints (CBPs), echinocandin resistance was not detected during the study period. Using EUCAST CBPs, MIC readings at 24 h for the Etest and Sensititre YeastOne resulted in CA levels of 99% and 93% for anidulafungin and 99% and 97% for micafungin. Using revised CLSI CBPs for caspofungin, CA levels were 92% and 99% for Etest and Sensititre YeastOne. The Etest proved an excellent, easy-to-handle alternative method for testing susceptibility to anidulafungin and micafungin. Due to misclassifications, the Etest is less suitable for testing susceptibility to caspofungin (8% of isolates falsely tested resistant). The CA levels of Sensititre YeastOne were 93% and 97% for anidulafungin and micafungin (24 h) by use of EUCAST CBPs and increased to 100% for both antifungals if CLSI CBPs were applied and to 100% and 99% if Sensititre YeastOne epidemiological cutoff values (ECOFFs) were applied. No one echinocandin could be demonstrated to be superior to another in vitro Since resistance was lacking among our Candida isolates, we cannot derive any recommendation from accurate resistance detection by the Etest and Sensititre YeastOne., (Copyright © 2017 American Society for Microbiology.)

Background: Osteoporotic fractures of the proximal humerus show an increasing incidence. Osteoporosis not only influences the fracture risk after low-energy trauma, but also affects the mechanical stability of internal fixation. Preoperative assessment of the local bone quality may be useful in the surgical treatment of patients sustaining these injuries. The aim of the present study was to present a method for the preoperative assessment of the local cancellous bone mineral density (BMD) of the proximal humerus using CT data., Methods: In the first part of the study, CT scans of 30 patients with unilateral fractures of the proximal humerus after low-energy trauma were used. The local BMD was assessed on the contralateral uninjured side. All 30 patients additionally underwent dual-emission X-ray absorptiometry (DXA) of the lumbar spine, proximal femur, and forearm of the side of the uninjured proximal humerus within 6 weeks after trauma. Three independent trauma surgeons performed measurements on the uninjured proximal humerus twice with a time interval of 4 weeks in order to assess the inter- and intraobserver reliability of the method. In the second part of the study, the local BMD of 507 patients with either proximal humerus fractures or chronic shoulder instability was assessed by a single trauma surgeon. In both parts, the average HU values in standardized ROIs of the humeral head were automatically calculated after correcting for HU values below the water equivalent. A linear calibration equation was computed for the calculation from HU to BMD using a calibration device (EFP)., Results: The intra- and interobserver reliability was high (ICC > 0.95). Correlation coefficients between the local BMD of the proximal humerus and other anatomical sites were between 0.35 (lumbar spine) and 0.64 (forearm). We found a high correlation between the local BMD and age. The BMD in the fracture group was significantly lower than in the instability group. These patients were significantly older and more likely to be female., Conclusion: Our method may provide a preoperative tool for the assessment of the local bone quality of the proximal humerus using CT data. Therapeutic adjustments such as augmentation or primary arthroplasty may be considered in patients with very low local BMD.

Introduction: Distal forearm fractures in younger adults are rare injuries resulting from high energy trauma. Treatment options vary from cast fixation, external fixator, percutaneus pinning and open reduction and internal fixation., Method: We retrospectively reviewed 13 patients aged 18-59 from 1996 to 2005 with a distal unstable forearm fracture. All were treated with open reduction and internal fixation of the radius. The ulna was stabilized either by an open reduction and internal fixation or by a closed reduction with or without pin fixation and cast fixation in all cases. At follow-up, we evaluated the radiologic results in terms of forearm fracture retention and functional outcome according to the wrist score by Krimmer., Result: Radial inclination amounted to 24 degrees at the injured side when compared to 27 degrees at the non-injured side, palmar tilt was 3 degrees versus 7 degrees and ulna variance was -2 versus -1 mm. According to the modified wrist score by Krimmer, seven excellent, two good and four fair results were achieved. The range of motion of the injured wrist joint was 149 degrees of rotation, in the sagittal plane 106 degrees , frontal plane 61 degrees and on the non-injured side rotation was 171 degrees , and movement in the sagittal plane was 146 degrees and 79 degrees in the frontal plane. Decreased forearm rotation (107 degrees vs. 162 degrees ) and decreased range of motion in the sagittal plane (77 degrees vs. 114 degrees ) were measured in patient following open reduction and internal fixation of radius and ulna compared to the outcome in patients with open reduction and internal fixation of the radius and closed reduction of the ulna. Grip strength of the injured side averaged 350 N versus 440 N which is 76% of that of the opposite side. All patients stated no pain at rest and some experienced slight pain at work. Three patients had an excellent performance at daily activities, nine patients presented problems with certain activities, and one patient showed severe limitations., Conclusions: Open reduction and internal fixation of the radius is the keystone in treating distal forearm fracture. In case of stable retention of the ulnar head after closed reduction, cast fixation with or without percutaneus pin fixation is a sufficient method to treat unstable distal forearm fractures. In patients with remaining instability of the distal ulna fracture, ORIF is indicated.

Purpose: To evaluate the sequelae of distal intraarticular radius fracture with regard to the development of arthritis and clinical symptoms., Patients and Method: In a retrospective follow-up examination, 72 patients with a distal intraarticular radius fracture could be included for clinical and radiological investigation 9 years following the trauma. All fractures were treated by ORIF and cortico-cancellous bone grafting., Results: Radiological evaluation revealed 5.1 degrees palmar tilt, 19.1 degrees radial tilt and the ulnar variance amounted to -0.5 mm. The articular cavity depth in the sagittal plane measured with 4.6 mm, 1.2 mm more than the non-involved side. Articular step-off was noticed in 6 patients. According to the Knirk and Jupiter classification system, two patients healed without arthritis, 43 patients presented arthritis stage 1, and 27 stage 2. Evaluation of the data showed a significant correlation between arthritis and articular cavity depth. But arthritis had neither influence on the DASH, nor the pain level. On the other hand, arthritis led to decreased sagittal wrist motion., Conclusion: ORIF of distal intraarticular radius fractures led to predictable results concerning restoration of length and form of the distal radius. Arthritis had a minor influence on the clinical end result.

Introduction: The study was made to evaluate the role of MR imaging in pediatric distal forearm fractures by comparison with the findings of plain radiographs and MRI., Material and Method: 38 patients (27 boys and 11 girls, mean age of 12 years, range 7 to 15 years) with radiographically open distal radius and ulna growth plates requiring first aid for a fracture of the distal third of the forearm, were included in this study. The fractures were diagnosed on plain radiographs and conservative treatment was performed. In 35 patients MR imaging was performed within 3 weeks after the accident and in 3 patients MRI was performed after 6 to 9 weeks because of persistent wrist pain., Results: Fifteen Salter/Harris II injuries of the radius and 1 of the ulna, 1 torus fracture of the radius and 2 of the ulna, 12 greenstick fractures of the radius and 3 of the ulna, 10 complete displaced radius fractures and 15 ulnar styloid fractures were found on plain radiographs. Twelve patients had evidence of associated triangular fibrocartilage complex (TFCC) lesions in MRI, there was no statistical correlation between TFCC lesions and fracture types, fracture dislocations or patients age (p > 0.5). One patient had an avulsion of the radioscaphocapitate ligament from the radius accompanying a greenstick fracture of the distal radius. 19 bone bruises and two radiographically occult fractures were identified. In 2 patients, a bone marrow oedema was seen in the radial epiphysis immediately adjacent to the germinal zone of the growth plate. In these patients premature physeal arrest occurred., Conclusion: MRI plays an important role in the evaluation of acute pediatric wrist injuries. It allows a better evaluation of osseous lesions than plain radiographs. In our study, a tear of the triangular fibrocartilage complex accompanied distal radius fractures in 32 % of patients. Simultaneous rupture of the TFCC insertion in the fovea ulnaris and ulnar styloid fracture lead to destabilisation of the distal radioulnar joint (DRUJ).

ICln is a multifunctional protein involved in regulatory mechanisms as different as membrane ion transport and RNA splicing. The protein is water-soluble, and during regulatory volume decrease after cell swelling, it is able to migrate from the cytosol to the cell membrane. Purified, water-soluble ICln is able to insert into lipid bilayers to form ion channels. Here, we show that ICln159, a truncated ICln mutant, which is also able to form ion channels in lipid bilayers, belongs to the pleckstrin homology (PH) domain superfold family of proteins. The ICln PH domain shows unusual properties as it lacks the electrostatic surface polarization seen in classical PH domains. However, similar to many classical PH domain-containing proteins, ICln interacts with protein kinase C, and in addition, interacts with cAMP-dependent protein kinase and cGMP-dependent protein kinase type II but not cGMP-dependent protein kinase type Ibeta. A major phosphorylation site for all three kinases is Ser-45 within the ICln PH domain. Furthermore, ICln159 interacts with LSm4, a protein involved in splicing and mRNA degradation, suggesting that the ICln159 PH domain may serve as a protein-protein interaction platform.

Introduction: The purpose of this retrospective study was to investigate the frequency and extent of clinical and radiological late sequelae and to identify predicting factors., Materials and Methods: A total of 220 patients of growing age with 232 closed, conservatively treated fractures were re-examined clinically and radiologically at a median follow-up time of 10 years (range 5-16 years). Clinical and radiological findings were summarised as an overall result., Results: Of the total of patients, 19% reported pain in the injured wrist, and wrist mobility was limited in 5% of patients. Forearm rotation was decreased in 16%, primarily in epiphyseal separation of the ulna ( p=0.0033). Radial inclination was different in 6% of patients, palmar tilt in 2%, and ulnar variance in 37%, compared with the contralateral side. Ulnocarpal impaction syndrome was present in 75% of the patients with positive ulnar variance. Overall outcome was excellent in 72%, good in 19%, moderate in 6%, and poor in 3% of patients. The younger the children were at the time of injury, the more favourable the results were ( p=0.009). Children who were older than 10 years when they suffered a severe fracture dislocation had the poorest results ( p=0.008). Further factors having a negative influence on outcome were repeated reduction maneuvers and an additional fracture of the ulna., Conclusion: Our follow-up examinations showed that the majority of patients achieved good results, especially in children under 10 years old. Large dislocations at the time of fracture healing do not influence long-term results in this age group and thus can be tolerated. Patients over 10 years old, whose fractures healed with an angular deformity of more than 20 degrees and/or fragment dislocation over half the breadth of the shaft showed the poorest results. Thus, such dislocations should not be tolerated, and reduction should be attempted in this age group by only one reduction maneuver.

Introduction: The purpose of this retrospective study was to compare the remodeling capacity and functional outcome of palmarly and dorsally displaced pediatric radius fractures in the distal one-third., Materials and Methods: Fifty-three children with a residual dorsal angulation of 15 degrees (range 10 degrees -28 degrees, +/-SD 5.32) and 31 children with a residual palmar angulation of 15 degrees (range 10 degrees -30 degrees, +/-SD 4.88) at fracture healing were re-examined clinically and radiologically with a median follow-up time of 10 years (range 5-15 years)., Results: There was no difference in the remodeling capacity, palmar tilt, radial inclination, and ulnar variance between both groups at follow-up. Pain as well as flexion/extension of the wrist and pronation showed no difference in both groups. Palmarly displaced fractures showed a significantly higher restriction of supination ( p=0.01)., Conclusion: We conclude that remodeling of residual palmar angulation occurs to the same extent as it does in dorsal angulation. Functional outcome differs in forearm supination.

ICln is a multifunctional protein that is essential for cell volume regulation. It can be found in the cytosol and is associated with the cell membrane. Besides its role in the splicing process, ICln is critically involved in the generation of ion currents activated during regulatory volume decrease after cell swelling (RVDC). If reconstituted in artificial bilayers, ICln can form ion channels with biophysical properties related to RVDC. We investigated (i) the cytosol versus cell membrane distribution of ICln in rat kidney tubules, NIH 3T3 fibroblasts, Madin-Darby canine kidney (MDCK) cells, and LLC-PK1 epithelial cells, (ii) fluorescence resonance energy transfer (FRET) in living fibroblasts between fluorescently tagged ICln and fluorochromes in the cell membrane, and (iii) possible functional consequences of an enhanced ICln presence at the cell membrane. We demonstrate that ICln distribution in rat kidneys depends on the parenchymal localization and functional state of the tubules and that cell swelling causes ICln redistribution from the cytosol to the cell membrane in NIH 3T3 fibroblasts and LLC-PK1 cells. The addition of purified ICln protein to the extracellular solution or overexpression of farnesylated ICln leads to an increased anion permeability in NIH 3T3 fibroblasts. The swelling-induced redistribution of ICln correlates to altered kinetics of RVDC in NIH 3T3 fibroblasts, LLC-PK1 cells, and MDCK cells. In these cells, RVDC develops more rapidly, and in MDCK cells the rate of swelling-induced depolarization is accelerated if cells are swollen for a second time. This coincides with an enhanced ICln association with the cell membrane.

Introduction: An intact distal radioulnar joint (DRUJ) is essential for normal functioning of the upper limb. Osteoarthritis of the DRUJ often leads to ulnar wrist pain, limitation of forearm rotation and reduced grip strength, all of which limit activities of daily living. Once the joint is damaged, salvage procedures are recommended., Materials and Methods: Between 1986 and 1996 a modified Sauvé-Kapandji procedure was performed in 117 patients with painfully limited forearm rotation and osteoarthritis of the distal radioulnar joint (DRUJ). Of the 117 patients, 73 women and 32 men, whose ages at operation ranged from 22 to 74 years (average 58 years), were retrospectively reviewed clinically and radiologically 8 years (range 5-12 years) after the operation. The DASH questionnaire was used with 53 patients, 43 patients were accepted for the study, and 10 were excluded., Results: Forearm rotation improved in all patients, ulnar wrist pain was reduced in 97% of the patients, and 9% had mild pain at the proximal ulnar stump. Grip strength improved from a preoperative mean of 38% to a postoperative mean of 55% compared with the contralateral side. The mean DASH score was 28 points (range 0-53 points). In all cases the arthrodesis fused within 8 weeks. The radiographs showed approximation between the proximal ulna stump and the radius compared with the preoperative situation in 74% of the patients., Conclusion: Our clinical and radiological findings suggest that the Sauvé-Kapandji procedure is indicated in symptomatic, non-reconstructable disorders of the DRUJ. The DASH questionnaire provides a general view of the functional outcome after the Sauvé-Kapandji procedure. The DASH questionnaire is very helpful in evaluating the effect of the Sauvé-Kapandji procedure on the entire upper limb.

Purpose: The present study was designed to evaluate long-term outcome of upper extremities and subjective self-assessment of patient disability after a Kapandji-Sauvé procedure by means of the DASH score., Patients and Method: Between 1986 and 1996, a modified Kapandji-Sauvé procedure was performed in 117 patients with painfully limited forearm rotation and arthrosis of the distal radioulnar joint (DRUJ). Of the 117 patients, 73 women and 32 men, whose ages at operation ranged from 22 to 74 years (average, 58 years) were retrospectively reviewed clinically and radiologically eight years (range, five to twelve years) after the operation. The DASH questionnaire was used in 43 patients., Results: The mean DASH score was 28 points (range, 0 to 53 points). The mean score in part A was 1.9 points, in part B 1.8 points. Worst outcomes were noted for activities requiring the exertion of force. Pain was reduced in 97 % of the patients. Forearm rotation and grip strength improved in all patients., Conclusion: Our clinical findings suggest that the Kapandji-Sauvé procedure is indicated in symptomatic, non-reconstructable disorders of the DRU-joint with or without ulnocarpal impaction syndrome. The DASH questionnaire provides a general view of functional outcome after the Kapandji-Sauvé procedure, though rotation is absolutely necessary to evaluate the success of the operation.

Background: Distal radius fracture often presents a metaphyseal void which is more extended in elderly, osteoporotic patients. Bone graft and bone substitutes are reported to be beneficial in maintaining metaphyseal reduction., Methods: We performed a prospective study on 52 menopausal, osteoporotic women with unstable intra-articular distal radius fractures to compare the outcome of percutaneous pinning and immobilisation in a cast for 6 weeks with that using injectable calcium phosphate bone cement (Norian Skeletal Repair System, SRS) to supplement pin and screw fixation and immobilisation in a cast for 3 weeks. All patients were reviewed 2 years (range 21-29 months) after surgery., Results: Patients treated with SRS had better functional outcome, restoration of movement and grip strength ( p<0.001). In this group there was 1 mm loss of radial length, 3 degrees loss of radial inclination and 7 degrees loss of palmar tilt. In the control group radial length decreased 3 mm, radial inclination decreased 11 degrees and palmar tilt 12 degrees. Loss of reduction was significantly higher in the control group ( p<0.001)., Conclusion: We conclude that the use of Norian SRS to supplement pin and screw fixation is effective in maintaining the reduction of unstable intra-articular distal radius fractures in osteoporotic patients and provides a better clinical outcome than percutaneous pinning.

ICln is an ion channel cloned from renal epithelial cells. The reconstitution of the protein in 1,2-diphytanoyl- sn-glycero-3-phosphocholine (Diph-PC) bilayer membranes reveals potassium-selective channels, which become more chloride selective in the presence of calcium. Here we show that the ion selectivity of ICln also depends on the lipid environment in which the channels are reconstituted. Diph-PC is a synthetic lipid commonly used for reconstituting ion channels. However, since this lipid is not found in native membranes, we reconstituted the ICln ion channels in a polar heart-lipid extract. Using this lipid mixture the reconstituted ICln ion channels are chloride selective in the presence of calcium and an acidic pH. The relative ion selectivity of ICln under these conditions is similar to the cation versus anion selectivity of native ion channels activated by cell swelling.

ICln is an ion channel identified by expression cloning using a cDNA library from Madin-Darby canine kidney cells. In all organisms tested so far, only one transcript for the ICln protein could be identified. Here we show that two splice variants of the ICln ion channel can be found in Caenorhabditis elegans. Moreover, we show that these two splice variants of the ICln channel protein, which we termed IClnN1 and IClnN2, can be functionally reconstituted and tested in an artificial lipid bilayer. In these experiments, the IClnN1-induced currents showed no voltage-dependent inactivation, whereas the IClnN2-induced currents fully inactivated at positive potentials. The molecular entity responsible for the voltage-dependent inactivation of IClnN2 is a cluster of positively charged amino acids encoded by exon 2a, which is absent in IClnN1. Our experiments suggest a mechanism of channel inactivation that is similar to the "ball and chain" model proposed for the Shaker potassium channel, i.e. a cluster of positively charged amino acids hinders ion permeation through the channel by a molecular and voltage-dependent interaction at the inner vestibulum of the pore. This hypothesis is supported by the finding that synthetic peptides with the same amino acid sequence as the positive cluster can transform the IClnN1-induced current to the current observed after reconstitution of IClnN2. Furthermore, we show that the nematode ICln gene is embedded in an operon harboring two additional genes, which we termed Nx and Ny. Co-reconstitution of Nx and IClnN2 and functional analysis of the related currents revealed a functional interaction between the two proteins, as evidenced by the fact that the IClnN2-induced current in the presence of Nx was no longer voltage-sensitive. The experiments described indicate that the genome organization in nematodes allows an effective approach for the identification of functional partner proteins of ion channels.

In previous studies we have shown that the expression of the transforming Ha-ras oncogene in NIH 3T3 fibroblasts stimulates cellular calcium entry, which triggers oscillatory calcium induced calcium release from internal stores. The intracellular calcium oscillations lead to cytoskeletal remodeling by actin stress fiber depolymerization and activation of the Na(+)/H(+) exchanger thus mediating cell swelling and intracellular alkalosis, both important mitogenic signals. This is evidenced by abrogation of Ha-ras induced growth factor independent cell proliferation by interference with any of these events, i.e. by inhibition of cellular calcium entry or inhibition of the Na(+)/H(+) exchanger. As shown in this study, simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key enzyme for cholesterol biosynthesis, is able to prevent these events following the expression of the transforming Ha-ras oncogene. We show, that simvastatin inhibits farnesylation dependent membrane translocation of a CAAX motive bearing yellow fluorescent protein and suppresses Ha-ras stimulated cellular calcium influx, which can be identified as capacitative calcium entry. In addition simvastatin is able to block regulatory volume decrease channels and to suppress the cytoskeletal remodeling, intracellular alkalinization, increase in cell volume and growth factor independent cell proliferation induced by the oncogene. Thus simvastatin is able to prevent crucial cellular events following expression of the transforming Ha-ras oncogene., (Copyright 2002 S. Karger AG, Basel)

Cell volume alterations are involved in numerous cellular events like epithelial transport, metabolic processes, hormone secretion, cell migration, proliferation and apoptosis. Above all it is a need for every cell to counteract osmotic cell swelling in order to avoid cell damage. The defence against excess cell swelling is accomplished by a reduction of the intracellular osmolarity by release of organic- or inorganic osmolytes from the cell or by synthesis of osmotically less active macromolecules from their specific subunits. De-spite the large amount of experimental data that has accumulated, the intracellular mechanisms underlying the sensing of cell volume perturbations and the activation of volume compensatory processes, commonly summarized as regulatory volume decrease (RVD), are still only partly revealed. Moving into this field opens a complex scenario of molecular rearrangements and interactions involving intracellular messengers such as calcium, phosphoinositides and inositolphosphates as well as phosphoryla-tion/dephosphorylation processes and cytoskeletal reorganization with marked cell type- and tissue specific variations. Even in one and the same cell type significant differences regarding the activated pathways during RVD may be evident. This makes it virtually im-possible to unambigously define common sensing- and sinaling pathways used by differ-ent cells to readjust their celll volume, even if all these pathways converge to the activa-tion of comparatively few sets of effectors serving for osmolyte extrusion, including ion channels and transporters. This review is aimed at providing an insight into the manifold cellular mechanisms and alterations occuring during cell swelling and RVD., (Copyright 2002 S. Karger AG, Basel)

ICln is a ubiquitously expressed eukaryotic protein. Expression of the protein in Xenopus laevis oocytes, the knocking-down of the protein in fibroblasts, or the reconstitution of the protein in lipid bilayer led to the assumption that this protein is an ionic channel or a significant part thereof. However, other possible roles for ICln in potential regulatory mechanisms have been postulated, as diverse as regulator of cell morphology by interacting with the Skb1 protein and/or interaction with core spliceosomal proteins. Here we show that ICln is able to interact with SnRNP core proteins SmD1, SmD2, SmD3, SmX5 and SmB/B'.

The Na(+)/H(+) exchangers (NHEs) are among the major ion transporters involved in cell volume regulation. NHE activation leads to a cellular influx of Na(+) ions and extrusion of H(+) ions, which are readily replenished from intracellular buffers. This will result in a net import of Na(+). In many systems NHE operates in parallel to Cl(-)/ HCO3(-) exchange, resulting in cellular uptake of NaCl. The influx of osmotically obliged water will consequently lead to cell swelling. This makes NHEs suitable to serve as powerful mechanisms for increasing cell volume (CV). The low volume threshold for NHE activation enables the cells to respond to very minute reductions of the CV. By the coupling to the export of H(+) ions cell volume regulatory NHE activation may lead to changes in intracellular pH. On the other hand NHEs are activated by a broad variety of ligands and by intracellular acidosis, which, in turn, may consequently lead to cell swelling. In addition, NHEs are linked to other intracellular proteins and structures, like e.g. the cytoskeleton, which themelves are involved in the regulation of numerous cellular processes. Therefore NHEs link CV regulation to a diversity of cellular functions, both in physiological and pathophysiological conditions. Six isoforms of the Na(+)/H(+) exchanger, termed NHE1--6, have been cloned so far. NHE 1--5 are located in the plasma membrane, whereas NHE6 is sorted to the mitochondrial membrane. NHE1 and NHE6 are the ubiquitously expressed isoforms. The expression of the isoforms NHE2 to NHE5 is restricted to specific tissues and the pattern of their expression, as well as their subcellular localization indicate that they fulfill specialized functions. Cell shrinkage induced activation has been shown for NHE1,2 and 4. In contrast, NHE3 is inhibited by cell shrinkage. In many cells several isoforms are present and assigned to specific membrane domains where they may serve a functional crosstalk between the different ion transporters.

Reconstitution of purified ICln in lipid bilayer leads to functional ion channels showing varying rectification. The reconstituted single channels have a conductance of approximately equal to 3 pS and their open probability is sensitive to nucleoside analogues. Mutation of a putative nucleotide binding site identified at the predicted extracellular mouth of the ICln channel protein leads to the reduction of the nucleoside-analogue sensitivity. Reconstituted ICln channels can be permeated both by cations and anions. The relative permeability of cations over anions depends on the presence of calcium. In the presence of calcium reconstituted ICln channels are more permeable to bromide than chloride, and more permeable to potassium than sodium. Similarly in NIH3T3 fibroblasts, the relative permeability of cations over anions of swelling-dependent chloride channels depends on extracellular calcium. Site-directed mutagenesis revealed the calcium-binding site responsible for the shift of the selectivity from cations towards anions of reconstituted ICln channels. Additional indirect structural information has been obtained by mutating a histidine in the predicted pore region of ICln. This histidine seems to have access to the ion-conducting tunnel of the pore. Our experiments show that ICln can act as an ionic channel, which does not exclude additional functions of the protein in regulatory mechanisms of the cell. Since knocking down the ICln protein in fibroblasts and epithelial cells leads to an impaired regulatory volume decrease (RVD) after cytoplasmic swelling and reconstituted ICln channels show several biophysical features of ion channels activated after swelling, ICln is a molecular candidate for these channels.

1. It was postulated that swelling dependent chloride channels are involved in the proton secretion of parietal cells. Since omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 are structurally related to phenol derivatives known to block swelling dependent chloride channels, we set out to test, whether these substances--which are known to block the H,K-ATPase--could also lead to an inhibition of swelling-dependent chloride channels. Swelling-dependent chloride channels--characterized in many different cell types--show highly conserved biophysical and pharmacological features, therefore we investigated the effect of omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 on swelling-dependent chloride channels elicited in fibroblasts, after the reduction of the extracellular osmolarity. 2. Omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 are able to block swelling-dependent chloride channels (IClswell). 3. Lansoprazole and its protonated metabolite AG2000 act on at least two different sites of the IClswell protein: on an extracellular site which seems to be in a functional proximity to the nucleotide binding site, and on an intracellular site which allows the formation of disulfide-bridges. 4. The inhibition of the proton pump and the simultaneous blocking of chloride channels by omeprazole, lansoprazole and its acid activated sulphenamide form AG2000, as described here could be an effective mode to restrict proton secretion in parietal cells.

Normal function of organs and cells is tightly linked to the cytoarchitecture. Control of the cell volume is therefore vital for the organism. A widely established strategy of cells to counteract swelling is the activation of chloride and potassium channels, which leads to a net efflux of salt followed by water - a process termed regulatory volume decrease. Since there is evidence for swelling-dependent chloride channels (IClswell) being activated also during pathological processes, the identification of the molecular entity underlying IClswell is of utmost importance. Several proteins are discussed as the channel forming IClswell, i.e. phospholemman, p-glycoprotein, CLC-3 and ICln. In this review we would like to focus on the properties of ICln, a protein cloned from a Madin Darby canine kidney (MDCK) cell library whose expression in Xenopus laevis oocytes resulted in a nucleotide sensitive outwardly rectifying chloride current closely resembling the biophysical properties of IClswell., (Copyright 2000 S. Karger AG, Basel.)

ICln is a cloned chloride channel paramount for regulatory volume decrease. Two different loci that carry the coding region for ICln were identified in the human genome. By PCR strategies an intronless copy of the gene was located on chromosome 6 at position 6p12.1-6q13 (CLNS1B). By fluorescence in situ hybridization a copy carrying introns with a putative length of 19 kb was located at chromosome 11 on position 11q13.5-q14.1 (CLNS1A). The characterization and chromosomal localization of the ICln gene offer the opportunity to study the regulatory sites of this gene in greater detail and could be helpful in establishing linkages between ICln and potential human diseases.

Fluorescence-optical measurements of the intracellular chloride concentration facilitate identification of chloride movements across the cell membrane of living cells. The two main dyes used for this purpose are 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) and 6-methoxy-quinolyl acetoethyl ester (MQAE). The use of both substances is impaired by their poor membrane permeability and therefore limited loading of the cells to be studied. Here we report the use of 6-methoxy-N-ethylquinolinium iodide (MEQ), a chloride-sensitive dye for which a membrane-permeable form is easily prepared. This makes the loading procedure as easy as with the acetoxymethyl (AM) forms of other dyes for sensing intracellular ions. In addition, the original method, which described absolute concentration measurements of chloride in the cytosol, was modified in so far as only relative measurements were made. This avoids the known limitations of single wavelength excitation and emission dyes with respect to exact concentration measurements. Moreover, to enhance the signal-to-noise ratio the driving force for chloride was considerably increased by changing the original direction of the anion flux in the cells under investigation. We verified the method by using fibroblasts and activating ICln, a putative chloride channel cloned from epithelial cells and of paramount importance in the regulatory volume decrease in these cells. In the presence of SCN- the MEQ quench measured in NIH 3T3 fibroblasts is dramatically enhanced in hypotonically challenged cells compared with cells under isotonic conditions. Antisense oligodeoxynucleotides sensing ICln considerably impeded the swelling-induced chloride current (ICl) in NIH 3T3 fibroblasts. Accordingly, the chloride movement measured by the SCN- quench of the MEQ signal was significantly reduced. Similar results can be obtained in the presence of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) or 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), two known blockers of chloride transport in the plasma membrane of a variety of cells. In conclusion, fluroscence-optical measurements using MEQ as the chloride-sensitive dye provide a reliable and easy-to-use method for measuring changes of the chloride flux across the cell membrane of living cells.

1. In NIH3T3 fibroblasts, the chloride channel involved in regulatory volume decrease (RVD) was identified as ICln, a protein isolated from a cDNA library derived from Madin Darby canine Kidney (MDCK) cells. ICln expressed in Xenopus laevis oocytes gives rise to an outwardly rectifying chloride current, sensitive to the extracellular addition of nucleotides and the known chloride channel blockers, DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)-benzoic acid). We set out to study whether substances structurally similar to NPPB are able to interfere with RVD. 2. RVD in NIH3T3 fibroblasts and MDCK cells is temperature-dependent. 3. RVD, the swelling-dependent chloride current and the depolarization seen after reducing extracellular osmolarity can be blocked by gossypol and NDGA (nordihydroguaiaretic acid), both structurally related to NPPB. 4. The cyclic AMP-dependent chloride current elicited in CaCo cells is less sensitive to the two substances tested while the calcium-activated chloride current in fibroblasts is insensitive. 5. The binding site for the two phenol derivatives onto ICln seems to be distinct but closely related to the nucleotide binding site identified as G x G x G, a glycine repeat located at the predicted outer mouth of the ICln channel protein.

Cell volume regulation is an essential feature of most cells. After swelling in hypotonic media, the simultaneous activation of potassium and chloride channels is believed to be the initial, time-determining step in cell volume regulation. The activation of both pathways is functionally linked and enables the cells to lose ions and water, subsequently leading to cell shrinkage and readjustment of the initial volume. NIH 3T3 fibroblasts efficiently regulate their volume after swelling and bear chloride channels that are activated by decreasing extracellular osmolarity. The chloride current elicited in these cells after swelling is reminiscent of the current found in oocytes expressing an outwardly rectifying chloride current termed ICln. Introduction of antisense oligodeoxynucleotides complementary to the first 30 nucleotides of the coding region of the ICln channel into NIH 3T3 fibroblasts suppresses the activation of the swelling-induced chloride current. The experiments directly demonstrate an unambiguous link between a volume-activated chloride current and a cloned protein involved in chloride transport.

NIH 3T3 fibroblasts expressing the ras oncogene (+ras cells) respond to bradykinin, bombesin or serum with sustained oscillations of cell membrane potential reflecting oscillations of intracellular calcium activity and subsequent activation of calcium-sensitive K+ channels. In contrast, identical cells not expressing the oncogene (-ras cells) respond to bradykinin with a single, transient hyperpolarization of the cell membrane. Furthermore, +ras cells are characterized by a serum-independent proliferation, an increase in cell volume and a marked reorganization of the cytoskeleton. It has been shown previously that the calcium channel blocker nifedipine, but not verapamil and diltiazem, inhibits oscillations of cell membrane potential as well as proliferation. In this study, we have examined the effect of several calcium channel blockers (bepridil, nifedipine, verapamil, diltiazem) on the proliferation, volume and cytoskeletal reorganization of +ras cells. Bepridil (10 mumol/l), which is also shown here to inhibit oscillations of cell membrane potential, and nifedipine (10 mumol/l) caused a decrease in cell number, whereas verapamil and diltiazem (10 mumol/l each) resulted in growth rates which did not differ from untreated +ras cells. The increase in cell volume as observed in untreated +ras cells was also observed for cells treated with verapamil and diltiazem, whereas cell volumes of +ras cells treated with bepridil and nifedipine were markedly reduced and similar to the values obtained for -ras cells. In addition, bepridil and nifedipine markedly inhibited cytoskeletal rearrangement, i.e depolymerization of actin-containing stress fibers. This inhibitory effect was not observed for verapamil and diltiazem.(ABSTRACT TRUNCATED AT 250 WORDS)

Background: The antiviral drugs AZT and acyclovir are generally used in the treatment of infections with human immunodeficiency virus (HIV) and herpes simplex virus (HSV). These substances are known to impede virus replication by premature nucleic acid chain termination. It is not yet clear, however, if this is the sole mechanism responsible for the antiviral and/or the numerous side effects observed in patients treated with these agents. We investigated the swelling-induced chloride current in fibroblasts, which we demonstrated is closely related or identical to a cloned epithelial chloride channel, ICln: This chloride channel can be blocked by nucleotides., Materials and Methods: Electrophysiological, fluorescence optical, and volume measurements were made to determine the effect of nucleoside analogs on the swelling-dependent chloride current (ICl) in NIH 3T3 fibroblasts and in human T cell lymphoma (H9) cells and the cAMP-dependent chloride current in CaCo cells., Results: AZT and acyclovir block the swelling-dependent chloride current and the chloride flux in fibroblasts, and the regulatory volume decrease (RVD) and ICl in H9 cells. This immediate effect can be substantially reduced by the simultaneous incubation of the cells with thymidine-5'-diphosphate (TDP) or uridine, both of which are by themselves unable to affect ICl., Conclusions: We show here a novel molecular mechanism by which antiviral drugs of the nucleoside analog family could lead to impairments of the kidney, bone marrow, gastrointestinal, and neuronal functions, and how these side effects could possibly be restricted by the presence of TDP or uridine.