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The effects of high saline concentrations on the plasma osmolytes of the four populations of the Pacific Treefrog, Pseudacris regilla, were investigated. Two of the populations, Chris Wicht and Trabuco Canyon, were in fresh water environments and two populations, Newport Back Bay and Alkaline Marsh, were in brackish water environments. The Newport Backbay treefrogs were most tolerant of increased sodium chloride solutions with 11 of 15 individuals surviving at 240 mM sodium chloride at 72 hrs. The Trabuco Canyon population was the least tolerant with 10 of 15 specimens surviving 120 mM sodium chloride at 72 hrs but none at the 180 mM concentration at 72 hrs. The Chris Wicht and Alkaline Marsh treefrogs were intermediate in their survival times with frogs surviving in 180 mM saline solutions at 72 hrs. The Newport treefrogs appeared to be the most efficient in osmoregulating by mainstreaming the plasma sodium and increasing plasma urea to compensate for the osmotic gradient produced by the increased external saline concentrations. The Trabuco Canyon and the Chris Wicht treefrogs appeared to be intermediate in their osmoregulatory abilities with the Alkaline Marsh treefrogs being least efficient and more likely an osmoconformer. The difference in osmoregulatory responses appears to be a population difference and a function of habitat. Sodium chloride was the major plasma osmolyte in the four populations of treefrogs. Urea increased in importance as the external concentration of the saline media increased, thus the role of urea was significant in maintaining the internal osmoconcentration. These simple physiological mechanisms allow this widespread treefrog to occur in so many different habitats., The Pacific Treefrog, or Pacific Chorus Frog, Pseudacris regilla, formerly Hyla regilla, is the most wide spread Western United States' amphibian. It occurs in a variety of habitats (grasslands, woodlands, [...]

The purpose of this article is to provide recommendations on the choice of peritoneal dialysis (PD) fluids in children by the European Pediatric Dialysis Working Group. The literature on experimental and clinical studies with PD solutions in children and adults was analyzed together with consensus discussions within the group. A grading was performed based on the international KDIGO nomenclature and methods. The lowest glucose concentration possible should be used. Icodextrin may be applied once daily during the long dwell, in particular in children with insufficient ultrafiltration. Infants on PD are at risk of ultrafiltration-associated sodium depletion, while anuric adolescents may have water and salt overload. Hence, the sodium chloride balance needs to be closely monitored. In growing children, the calcium balance should be positive On behalf of the European Pediatric Dialysis Working Group and dialysate calcium adapted according to individual needs. Limited clinical experience with amino acid-based PD fluids in children suggests good tolerability. The anabolic effect, however, is small; adequate enteral nutrition is preferred. CPD fluids with reduced glucose degradation products (GDP) content reduce local and systemic toxicity and should be preferred whenever possible. Correction of metabolic acidosis is superior with pH neutral bicarbonate-based fluids compared with single-chamber, acidic, lactate-based solutions. Prospective comparisons of low GDP solutions with different buffer compositions are still few, and firm recommendations cannot yet be given, except when hepatic lactate metabolism is severely compromised. Keywords Peritoneal dialysis fluids * Pediatrics * Biocompatible * Icodextrin * Amino acid * Consensus * Glucose degradation products, Introduction Peritoneal dialysis (PD) is the preferred renal replacement therapy in children until renal transplantation can be realized. The choice of PD solution has gained particular importance, in the light [...]

The purpose of this study was to investigate whether luminal leptin alters ion transport properties of the intestinal epithelium under acute inflammatory conditions. Monolayers of human intestinal T84 epithelial cells and a rat model of chemotherapy-induced enterocolitis were used. Cells were treated with leptin and mounted in Ussing chambers to measure basal and secretagogue-induced changes in transepithelial short-circuit current (ISC). Furthermore, the role of MAPK and phosphatidylinositol 3-kinase (PI3K) signaling pathways in mediating responses to leptin was investigated. Acute colitis in Sprague-Dawley rats was induced by intraperitoneal injection of 40 mg/kg methotrexate. Leptin (100 ng/ml) induced a time-dependent increase in basal [I.sub.SC] in [T.sub.84] intestinal epithelial cells (P < 0.01). Moreover, pretreatment of [T.sub.84] cells with leptin for up to 1 h significantly potentiated carbachol- and forskolin-induced increases in [I.sub.SC]. Pretreatment with an inhibitor of MAPK abolished the effect of leptin on basal, carbachol- and forskolin-induced chloride secretion (P < 0.05). However, the PI3K inhibitor, wortmannin, only blunted the effect of leptin on forskolin-induced increases in [I.sub.SC]. Furthermore, leptin treatment evoked both ERK1/2 and Akt1 phosphorylation in T84 cells. In the rat model, luminal leptin induced significant increases in [I.sub.SC] across segments of proximal and, to a lesser extent, distal colon (P < 0.05). We conclude that luminal leptin is likely an intestinal chloride secretagogue, particularly when present at elevated concentrations and/or in the setting of inflammation. Our findings may provide a mechanistic explanation, at least in part, for the clinical condition of secretory diarrhea both in hyperleptinemic obese patients and in patients with chemotherapy-induced intestinal inflammation. intestinal ion transport doi: 10.1152/ajpgi.00320.2009.

Am J Physiol Gastrointest Liver Physiol 298: G37-G44, 2010. First published October 29, 2009; doi: 10.1152/ajpgi.00202.2009.--Neuroimmune agonists induce epithelial [Cl.sup.-] secretion through elevations in intracellular [Ca.sup.2+] or cAMP. Previously, we demonstrated that epidermal growth factor receptor (EGFR) transactivation and subsequent ERK MAPK activation limits secretory responses to [Ca.sup.2+] -dependent, but not cAMP-dependent, agonists. Although JNK MAPKs are also expressed in epithelial cells, their role in regulating transport function is unknown. Here, we investigated the potential role for JNK in regulating [Cl.sup.-] secretion in [T.sub.84] colonic epithelial cells. Western blot analysis revealed that a prototypical [Ca.sup.2+]-dependent secretagogue, carbachol (CCh; 100 [micro]M), induced phosphorylation of both the 46-kDa and 54-kDa isoforms of JNK. This effect was mimicked by thapsigargin (TG), which specifically elevates intracellular [Ca.sup.2+], but not by forskolin (FSK; 10 [micro]M), which elevates cAMP. CCh-induced JNK phosphorylation was attenuated by the EGFR inhibitor, tyrphostin-AG1478 (1 [micro]M). Pretreatment of voltage-clamped [T.sup.84] cells with SP600125 (2 [micro]M), a specific JNK inhibitor, potentiated secretory responses to both CCh and TG but not to FSK. The effects of SP600125 on CCh-induced secretion were not additive with those of the ERK inhibitor, PD98059. Finally, in apically permeabilized [T.sub.84] cell monolayers, SP600125 potentiated CCh-induced [K.sup.+] conductances but not [Na.sup.+]/[K.sup.+] ATPase activity. These data demonstrate a novel role for JNK MAPK in regulating [Ca.sup.2+] but not cAMP-dependent epithelial [Cl.sup.-] secretion. JNK activation is mediated by EGFR transactivation and exerts its antisecretory effects through inhibition of basolateral [K.sup.+] channels. These data further our understanding of mechanisms regulating epithelial secretion and underscore the potential for exploitation of MAPK-dependent signaling in treatment of intestinal transport disorders. ion transport; colon; epithelium

This study investigates the link between adaptation to biocides and antibiotics in Pseudomonas aeruginosa. An enrichment continuous culture of P. aeruginosa NCIMB 10421 (MIC 25 mg BKC [l.sup.-1]) was operated (D=0.04 [h.sup.-1], 792 h) with added benzalkonium chloride (BKC). A derivative, PA-29 (696 h), demonstrated a >12-fold decrease in sensitivity to the biocide (MIC >350 mg BKC [l.sup.-1]). The variant demonstrated a 256-fold increase in resistance to ciprofloxacin, with a mutation in the gyrA gene (Thr-83[right arrow]lle). Similarly, culturing of the original strain in a continuous-culture system with ciprofloxacin selection pressure led to the evolution of BKC-adapted populations (MIC 100 mg BKC [l.sup.-1]). Efflux pump activity predominantly contributed to the developed phenotype of PA-29. An amino acid substitution (Val-51[right arrow]Ala) in nfxB, the Mex efflux system regulator gene, was observed for PA-29. Overexpression of both MexAB-OprM and MexCD-OprJ was recorded for PA-29. Similarly, mexR, a repressor of the Mex system, was downregulated. Competition studies were carried out in continuous culture between PA-29 and the original strain (in the presence of subinhibitory concentrations of RKC). The outcome of competition was influenced by the concentration of biocide used and the nature of limiting nutrient. The inclusion of 1 mg BKC [l.sup.-1] in the medium feed was sufficient to select (S=0.011) for the BKC-adapted strain in magnesium-limited culture. Conversely, the presence of 10 mg BKC [l.sup.-1] in the medium supply was insufficient to select for the same organism (S=-0.017) in the glucose-limited culture. These results indicate the importance of environmental conditions on selection and maintenance of biocide adaptation. DOI 10.1099/mic.0.029751-0

Although [Cl.sup.-] transport in fetal lung is important for fluid secretion and normal lung development, the role of [Cl.sup.-] transport in adult lung is not well understood. In physiological studies, the cystic fibrosis transmembrane regulator (CFTR) plays a role in fluid absorption in the distal air spaces of adult lung, and alveolar type II cells cultured for 5 days have the capacity to transport [Cl.sup.-]. Although both alveolar type I and type II cells express CFTR, it has previously not been known whether type I cells transport [Cl.sup.-]. We studied [Cl.sup.-] uptake in isolated type I cells directly, using either radioisotopic tracers or halidesensitive fluorescent indicators. By both methods, type I cells take up [Cl.sup.-]. In the presence of [beta]-adrenergic agonist stimulation, [Cl.sup.-] uptake can be inhibited by CFTR antagonists. Type I cells express both the [Cl.sup.-]/HC[O.sub.3] anion exchanger AE2 and the voltage-gated [Cl.sup.-] channels CLC5 and CLC2. Inhibitors of AE2 also block [Cl.sup.-] uptake in type I cells. Together, these results demonstrate that type I cells are capable of [Cl.sup.-] uptake and suggest that the effects seen in whole lung studies establishing the importance of [Cl.sup.-] movement in alveolar fluid clearance may be, in part, the result of [Cl.sup.-] transport across type I cells. chloride transport; cystic fibrosis transmembrane regulator; CLC5 doi: 10.1152/ajplung.90466.2008.

Experiments were performed to test the hypothesis that three members of the SLC26 anion transporter gene family (SLC26a3, A4, and A6; hereafter termed za3, za4, and za6) mediate branchial [Cl.sup.-]/[HCO.sup.-.sub.3] exchange in adult zebrafish (Danio rerio). Real-time RT-PCR demonstrated that the gill expressed relatively high levels of za6 mRNA; za3 and za4 mRNA, while present, were less abundant. Also, za4 and za6 were expressed at relatively high levels in the kidney. The results of in situ hybridization or immunocytochemistry (za3 only) experiments performed on gill sections revealed that the SLC26 transporters were predominantly expressed on the filament epithelium (especially within the interlamellar regions) and to a lesser extent on the lamellar epithelium at the base of lamellae. This distribution pattern suggests that the SLC26 anion transporters are localized to mitochondrion-rich cells (ionocytes). Transferring fish to water containing low [[Cl.sup.-]] (0.02 mmol/l) resulted in significant increases in branchial SLC26 mRNA expression after 5-10 days of exposure relative to fish raised in normal water [[Cl.sup.-]] (0.4 mmol/l); transferring fish to [Cl.sup.-]-enriched water (2.0 mmol/l) was without effect on mRNA levels. Transferring fish to water containing elevated levels of NaHC[O.sub.3] (10-12.5 mmol/l) caused marked increases in branchial SLC26 mRNA expression between 3 and l0 days of transfer that was associated with a significant 40% increase in [Cl.sup.-]-uptake (as measured upon return to normal water after 7 days). A decrease in whole body net acid excretion (equivalent to an increase in net base excretion) in fish previously maintained in high [NaHC[O.sub.3]] water, concurrent with increases in [Cl.sup.-] uptake and SLC26 mRNA levels, suggests a role for these anion transporters in [Cl.sup.-] uptake and acid-base regulation owing to their [Cl.sup.-]/HC[O.sup.-.sub.3] exchange activities. SLC26a3; SLC26a4; SLC26a6; pendrin; ionic regulation; acid-base balance; mitochondrion rich cell; gill doi: 10.1152/ajpregu.00327.2009.

T-wave alternans, characterized by a beat-to-beat change in T-wave morphology, amplitude, and/or polarity on the ECG, often heralds the development of lethal ventricular arrhythmias in patients with left ventricular hypertrophy (LVH). The aim of our study was to examine the ionic basis for a beat-to-beat change in ventricular repolarization in the setting of LVH. Transmembrahe action potentials (APs) from epicardium and endocardinm were recorded simultaneously, together with transmural ECG and contraction force, in arterially perfused rabbit left ventricular wedge preparation. APs and [Ca.sup.2+]-activated chloride current ([I.sub.Cl,Ca]) were recorded from left ventricular myocytes isolated from normal rabbits and those with renovascular LVH using the standard microelectrode and whole cell patch-clamping techniques, respectively. In the LVH rabbits, a significant beat-to-beat change in endocardial AP duration (APD) created beat-to-beat alteration in transmural voltage gradient that manifested as T-wave alternans on the ECG. Interestingly, contraction force alternated in an opposite phase ('out of phase') with APD. In the single myocytes of LVH rabbits, a significant beat-to-beat change in APD was also observed in both left ventricular endocardial and epicardial myocytes at various pacing rates. APD alternans was suppressed by adding 1 [micro]M ryanodine, 100 [micro]M 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and 100 [micro]M 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). The density of the [Ca.sup.2+]-activated chloride currents ([I.sub.Cl,Ca]) in left ventricular myocytes was significantly greater in the LVH rabbits than in the normal group. Our data indicate that abnormal intracellular [Ca.sup.2+] fluctuation may exert a strong feedback on the membrane [I.sub.Cl,Ca], leading to a beat-to-beat change in the net repolarizing current that manifests as T-wave alternans on the ECG. T-wave alternans

Neurotransmitter:sodium symporters (NSS) have a critical role in regulating neurotransmission and are targets for psychostimulants, anti-depressants and other drugs (1,2). Whereas the non-homologous glutamate transporters mediate chloride conductance (3), in [...]

In airway epithelia, purinergic receptor (P2[Y.sub.2]-R) stimulation of intracellular calcium ([Ca.sup.2+.sub.i])-regulated ion transport is restricted to the membrane domain ipsilateral to receptor activation, implying compartmentalization of [Ca.sup.2+.sub.i] signaling. Because mitochondria can spatially restrict cellular [Ca.sup.2+.sub.i] signals, immunocytochemical, electron microscopic, and fluorescent studies of mitochondria localization were performed in human airway epithelia. Although concentrated at the apical domain, mitochondria were found distributed at both the apical and the basolateral poles and in close association with the endoplasmic reticulum. The role of mitochondria in locally restricting P2[Y.sub.2]-R-induced [Ca.sup.2+.sub.i] signals was investigated by measuring changes in mitochondrial [Ca.sup.2+] ([Ca.sup.2+.sub.m]) in human airway epithelial monolayers. P2[Y.sub.2]-R activation induced [Ca.sup.2+.sub.m] accumulation in mitochondria confined to the domain ipsilateral to P2[Y.sub.2]-R stimulation, which was blocked by mitochondrial uncoupling with 1 [micro]M CCCP and 2.5 [micro] g/ml oligomycin. The role of mitochondria in restricting the cellular cross-talk between basolateral P2[Y.sub.2]-R-dependent [Ca.sup.2+.sub.i] mobilization and apical membrane [Ca.sup.2+]-activated [Cl.sup.-] secretion was investigated in studies simultaneously measuring [Ca.sup.2+.sub.i] and [Cl.sup.-] secretion in cystic fibrosis human airway epithelial monolayers. Activation of basolateral P2[Y.sub.2]-Rs produced similar increases in [Ca.sup.2+.sub.i] in monolayers without and with pretreatment with uncouplers, whereas [Ca.sup.2+.sub.i]-activated [Cl.sup.-] secretion was only efficiently triggered in mitochondria-uncoupled conditions. We conclude that (a) mitochondria function as a [Ca.sup.2+.sub.i]-buffering system in airway epithelia, compartmentalizing [Ca.sup.2+.sub.i]-dependent functions to the membrane ipsilateral to receptor stimulation; and (b) the mitochondria provide structural barriers that protect the airway epithelia against nonspecific activation of [Ca.sup.2+.sub.i]-modulated functions associated with [Ca.sup.2+.sub.i] signals emanating from the apical or the basolateral membrane domains. KEY WORDS: calcium signaling * mitochondria * endoplasmic reticulum * chloride secretion * purinergic receptors

Chloride concentration ([[Cl.sup.-]]) was measured in defined organellar compartments using fluorescently labeled transferrin, [[alpha].sub.2]-macroglobulin, and cholera toxin B-subunit conjugated with [Cl.sup.-]-sensitive and -insensitive dyes. In pulse-chase experiments, [[Cl.sup.-]] in Tf-labeled early/ recycling endosomes in J774 cells was 20 mM just after internalization, increasing to 41 mM over ~10 min in parallel to a drop in pH from 6.91 to 6.05. The low [[Cl.sup.-]] just after internalization (compared with 137 mM solution [[Cl.sup.-]]) was prevented by reducing the interior-negative Donnan potential. [[Cl.sup.-]] in [[alpha].sub.2]-macroglobulin-labeled endosomes, which enter a late compartment, increased from 28 to 58 mM at 1-45 min after internalization, whereas pH decreased from 6.85 to 5.20. [Cl.sup.-] accumulation was prevented by bafilomycin but restored by valinomycin. A [Cl.sup.-] channel inhibitor slowed endosomal acidification and [Cl.sup.-] accumulation by ~2.5-fold. [[Cl.sup.-]] was 49 mM and pH was 6.42 in cholera toxin B subunit-labeled Golgi complex in Vero cells; Golgi compartment [Cl.sup.-] accumulation and acidification were reversed by bafilomycin. Our experiments provide evidence that [[Cl.sup.-] is the principal counter ion accompanying endosomal and Golgi compartment acidification, and that an interior-negative Donnan potential is responsible for low endosomal [[Cl.sup.-]] early after internalization. We propose that reduced [[Cl.sup.-]] and volume in early endosomes permits endosomal acidification and [[Cl.sup.-]] accumulation without lysis.

Indicator molecules for caspase-3 activation have been reported that use fluorescence resonance energy transfer (FRET) between an enhanced cyan fluorescent protein (the donor) and enhanced yellow fluorescent protein (EYFP; the acceptor). Because EYFP is highly sensitive to proton ([H.sup.+]) and chloride ion ([Cl.sup.-]) levels, which can change during apoptosis, this indicator's ability to trace the precise dynamics of caspase activation is limited, especially in vivo. Here, we generated an [H.sup.+]- and [Cl.sup.-]--insensitive indicator for caspase activation, SCAT, in which EYFP was replaced with Venus, and monitored the spatio-temporal activation of caspases in living cells. Caspase-3 activation was initiated first in the cytosol and then in the nucleus, and rapidly reached maximum activation in 10 min or less. Furthermore, the nuclear activation of caspase-3 preceded the nuclear apoptotic morphological changes. In contrast, the completion of caspase-9 activation took much longer and its activation was attenuated in the nucleus. However, the time between the initiation of caspase-9 activation and the morphological changes was quite similar to that seen for caspase-3, indicating the activation of both caspases occurred essentially simultaneously during the initiation of apoptosis.

Research has been conducted on Photosystem II component, protein CP43. Results demonstrate that (super)305 Arg (approaches) (super)305 Ser mutation in this protein produces mutant R305S which is exacerbates when the strain grows under chloride-limiting conditions.

Research has been conducted on urea and guanidine hydrochloride. Results indicate that these compounds stabilize ferrocytochrome c at subdenaturing concentrations of denaturants and reveal the nature of protein-denaturant interactions.

Research has been conducted on ability of bactria to adapt to disinfectants. The morphological and chemical exposure effects to sublethal levels of benzalkonium chloride on Listeria monocytogenes have been investigated, and the details are reported.

Research has been conducted on gram-positive moderately halophilic bacterium Halobacillus halophilus. The biological functions of chloride in this bacterium have been investigated, and the results suggest that chloride can function as a signal molecule in salt adaptation in moderately halophilic bacterium H. halophilus which may sense salinity via chloride concentration of its environment.

Research has been conducted on cytochrome c oxidase. The role of bound Cl (super)- in cytochrome c oxidase's redox-linked protonation kinetics and stoichiometry has been investigated via the use of multiwavelength stopped-flow spectroscopy and the results demonstrate that H (super)+ promotes this protonation and Cl (super)- dissociation.

We report the molecular and functional characterization of murine Slc26a6, the putative apical chloride-formate exchanger of the proximal tubule. The Slc26a6 transcript is expressed in several tissues, including kidney. Alternative splicing of the second exon generates two distinct isoforms, denoted Slc26a6a and Slc26a6b, which differ in the inclusion of a 23-residue N[H.sub.2]-terminal extension. Functional comparison with murine Slc26a1, the basolateral oxalate exchanger of the proximal tubule, reveals a number of intriguing differences. Whereas Slc26a6 is capable of [Cl.sup.-], S[O.sup.2-.sub.4], formate, and oxalate uptake when expressed in Xenopus laevis oocytes, Slc26a1 transports only S[O.sup.2-.sub.4] and oxalate. Measurement ofintracellular pH during the removal of extracellular [Cl.sup.-] in the presence and absence of HC[O.sup.-.sub.3] indicates that Slc26a6 functions as both a [Cl.sup.-]/HC[O.sup.-.sub.3] and a [Cl.sup.-]/O[H.sup.-] exchanger; simultaneous membrane hyperpolarization during these experimental maneuvers reveals that HC[O.sup.-.sub.3] and O[H.sup.-] transport mediated by Slc26a6 is electrogenic. Cis-inhibition and efflux experiments indicate that Slc26a6 can mediate the exchange of both [Cl.sup.-] and S[O.sup.2-.sub.4] with a number of substrates, including formate and oxalate. In contrast, S[O.sup.2-.sub.4] and oxalate transport by Slc26a1 are mutually cis-inhibited but activated significantly by extracellular halides, lactate, and formate. The data indicate that Slc26a6 encodes an apical [Cl.sup.-]/formate/oxalate and [Cl.sup.-]/base exchanger and reveal significant mechanistic differences between apical and basolateral oxalate exchangers of the proximal tubule. oxalate; proximal tubule; formate; anion exchange; chloride

Research has been conducted on the octahedral hexanuclear cluster halides of the transition metals. The low temperature solid-state approaches to these compounds carried out via the reduction of TaCl (sub)5 with gallium and gallium dichloride have been investigated and the results are reported.

Research has been conducted on the aryl halides. Results demonstrate that the amination of these compounds carried out in the presence of air-stable alkali metal hydroxide bases and a palladium catalyst has produced arylamines.

The early proximal tubule preferentially reabsorbs organic solutes and bicarbonate over chloride ions, resulting in a luminal fluid with a higher chloride concentration than that in blood. From this late proximal tubular fluid, one-half of NaCl reabsorption by the adult proximal tubule is active and transcellular and one-half is passive and paracellular. The purpose of the present in vitro microperfusion study was to determine the characteristics of passive chloride transport and permeability properties of the adult and neonatal proximal straight tubules (PST). In tubules perfused with a late proximal tubular fluid, net passive chloride flux was 131.7 [+ or-] 37.7 pmol*[mm.sup.-1]*[min.sup.-1] in adult tubules and -17.1 [+ or-] 23.3 pmol*[mm.sup.-1]*[min.sup.-1] in neonatal proximal tubules (P < 0.01). Chloride permeability was 10.94 [+ or -] 5.21 x [10.sup.-5] cm/s in adult proximal tubules and -1.26 [+ or-] 1.84 x [10.sup.-5] cm/s in neonatal proximal tubules (P < 0.05). Thus neonatal PST have a chloride permeability not different from zero and have no net passive chloride transport. Bicarbonate permeability is also less in neonates than adults in this segment (-0.07 [+ or-] 0.03 x [10.sup.-5] vs. 0.93 [+ or -] 0.27 x [10.sup.-5] cm/s, P < 0.01). Neonatal PST have higher sodium-to chloride and bicarbonate-to-chloride permeability ratios than adult PST. However, mannitol and sucrose permeabilities were not different in adult proximal tubules and neonatal PST. Transepithelial resistance was measured using current injection and cable analysis. The resistance was 6.7 [+ or-] 0.7 [OMEGA]*[cm.sup.2] in adult tubules and 11.3 [+ or-] 1.4 [OMEGA]*[cm.sup.2] in neonatal PST (P < 0.01). In conclusion, there are significant maturational changes in the characteristics of the PST paracellular pathway affecting transport in this nephron segment. chloride transport; renal development; passive transport; tight junction; chloride permeability; bicarbonate permeability

The present studies were carried out with the aims to determine the cDNA sequence for cyclooxygenase (COX) in an elasmobranch species and to study its role in regulation of chloride secretion in the perfused shark rectal gland (SRG). With the use of long primers (43 bp) derived from regions of homology between zebrafish and rainbow trout COX-2 genes, a 600-bp product was amplified from SRG and was found to be almost equally homologous to mammalian COX-1 and COX-2 (65%). The full-length cDNA sequence was obtained by 5'-RACE and by analyzing an EST clone generated by the EST Project of the Mt. Desert Island Biological Laboratory Marine DNA Sequencing Center. The longest open reading frame encodes a 593-amino acid protein that has 68 and 64% homology to mammalian COX-1 and COX-2, respectively. The gene and its protein product is designated as shark COX (sCOX). The key residues in the active site ([Try.sup.385], [His.sup.388], and [Ser.sup.530]) are conserved between the shark and mammalian COX. sCOX contains [Val.sup.523] that has been shown to be a key residue determining the sensitivity to COX-2-specific inhibitors including NS-398. The mRNA of sCOX, detected by RT-PCR, was found in all tissues tested, including rectal gland, kidney, spleen, gill, liver, brain, and heart, but not in fin. In the perfused SRG, vasoactive intestinal peptide (VIP) at 5 nM induced rapid and marked C1- secretion (basal: vasoactive intestinal peptide; prostaglandins; shark rectal gland; NS-398; C-type natriuretic peptide; 5'-rapid amplification of cDNA ends

The mechanisms of proguanylin synthesis and secretion in the intestine are incompletely understood. We designed an in vitro model to study proguanylin secretion in a model of intestinal villous epithelial cells. The C2/bbe1 cell line, a differentiated subclone of Caco-2 cells, was used to examine the direction of proguanylin secretion and the potential for feedback regulation via activators of the guanylyl cyclase C signal transduction pathway. When cells were grown on Transwell inserts, proguanylin was secreted into the apical and basolateral media, consistent with other models of intestinal guanylin secretion. Proguanylin synthesis and secretion were not decreased on activation of guanylyl cyclase C-mediated chloride secretion, implying a regulatory system other than negative-feedback inhibition. These data describe the use of C2/bbe1 cells as a model for proguanylin secretion in villous epithelial cells and demonstrate their potential use for the study of the regulatory mechanisms governing proguanylin synthesis and secretion. guanylin; chloride secretion; Transwell inserts

The present studies were undertaken to determine the direct effects of nitric oxide (NO) released from an exogenous donor, S-nitroso-N-acetyl pencillamine (SNAP) on [Cl.sup.-]/O[H.sup.-] exchange activity in human Caco-2 cells. Our results demonstrate that NO inhibits [Cl.sup.-]/O[H.sup.-] exchange activity in Caco-2 cells via cGMP-dependent protein kinases G (PKG) and C (PKC) signal-transduction pathways. Our data in support of this conclusion can be outlined as follows: 1) incubation of Caco-2 cells with SNAP (500 [micro]M) for 30 min resulted in ~50% inhibition of DIDS-sensitive [sup.36]Cl uptake; 2) soluble guanylate cyclase inhibitors Ly-83583 and (1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one significantly blocked the inhibition of [Cl.sup.-]/O[H.sup.-] exchange activity by SNAP; 3) addition of 8-bromo-cGMP (8-BrcGMP) mimicked the effects of SNAP; 4) specific PKG inhibitor KT-5823 significantly inhibited the decrease in [Cl.sup.-]/O[H.sup.-] exchange activity in response to either SNAP or 8-BrcGMP; 5) [Cl.sup.-]/O[H.sup.-] exchange activity in Caco-2 cells in response to SNAP was not altered in the presence of protein kinase A (PKA) inhibitor (Rp-cAMPS), demonstrating that the PKA pathway was not involved; 6) the effect of NO on [Cl.sup.-]/O[H.sup.-]-exchange activity was mediated by PKC, because each of the two PKC inhibitors chelerythrine chloride and calphostin C blocked the SNAP-mediated inhibition of [Cl.sup.-]/O[H.sup.-]-exchange activity; 7) S[O.sup.2-.sub.4]/O[H.sup.-] exchange in Caco-2 cells was unaffected by SNAP. Our results suggest that NO-induced inhibition of [Cl.sup.-]/O[H.sup.-] exchange may play an important role in the pathophysiology of diarrhea associated with inflammatory bowel diseases. chloride absorption; human intestine; guanylate cyclase; protein kinase C regulation

Research has been conducted on the s-triazine herbicides. Results indicate that these herbicides hydrolytically displace chloride and amino substituents of 2-chloro-4,6-diamino-s-triazine.

Research has been conducted on Staphylococcus aureus cells in biofilms. The disinfectant test developed for these cells and the membrane-active compound benzalkonium chloride and oxidizing agent hypochlorite used in this test are discussed.

Research has been conducted on the alpha-sulfinylamides. The preparation of these compounds has been carried out via condensation of the primary amine with ketone and via the reaction of the resulting imine with ethylsulfenylacetyl chloride followed by the oxidation with sodium periodate.

Research has been conducted on the oxime-derived chloro-bridged palladacycles. The effect of bases, additives and solvents on the containing phenylboronic acids aryl bromide and chloride Suzuki couplings with oxime-derived palladacycles as catalysts has been investigated and the details are reported.

The effect of acidosis on the electrical activity of isolated rat atrial myocytes was investigated using the patch-clamp technique. Reducing the pH of the bathing solution from 7.4 to 6.5 shortened the action potential. Acidosis had no significant effect on transient outward or inward rectifier currents but increased steady-state outward current. This increase was still present, although reduced, when intracellular [Ca.sup.2+] was buffered by 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA); BAPTA also inhibited acidosis-induced shortening of the action potential. [Ni.sup.2+] (5 mM) had no significant effect on the acidosis-induced shortening of the action potential. Acidosis also increased inward current at -80 mV and depolarized the resting membrane potential Acidosis activated an inwardly rectifying [Cl.sup.-] current that was blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which also inhibited the acidosis-induced depolarization of the resting membrane potential. It is concluded that an acidosis-induced increase in steady-state outward [K.sup.+] current underlies the shortening of the action potential and that an acidosis-induced increase in inwardly rectifying [Cl.sup.-] current underlies the depolarization of the resting membrane potential during acidosis. action potential; potassium current; chloride current; perforated patch

Research has been conducted on the ruthenium(II)- and ruthenium(III)-chloride-dimethyl sulfoxide precursors and the antimetasatic drug [ImH][trans-RuCl (sub)4 (dmso-S)] (Im= imidazole, dmso= dimethyl sulfoxide). The reactivity of these compounds toward nitric oxide has been studied and the details are reported.

Research has been conducted on the allylstannanes and allylsilanes. The results of the intramolecular reaction of these compounds with alkynes carried out in the presence of Pt(II), Ru(II), Pd(II) and Au(II) chlorides are discussed.

Research has been conducted on the alcohol conversion into formate esters. Results indicate that this conversion can be carried out in methyl chloride at room temperature via the use of 2,4,6-trichloro-1,3,5-triazine and N,N-dimethylformamide in the presence of lithium fluoride.

Research has been conducted on fluorocarbonyl sulfenyl chloride, methylfluorocarbonyl disulfide and bis(fluorocarbonyl) trisulfide. The photoelectron spectroscopy study of these compounds and the results of the molecular orbital analysis and ionization energy calculations are presented.

Research has been conducted on the chiral nonracemic and racemic alpha-unsubstituted and alpha-substituted beta-fluoroalkyl beta amino acid derivatives. The synthesis of these acid derivatives has been carried our via fluorinated imidoyl chlorides and ester enolates and the results indicate that the process takes place with good diastereoselectivity.

Research has been conducted on the gamma-irradiated CCl (sub)3 F/n-alkane solutions. The studies of these solutions carried out via the use of the electron paramagnetic resonance spectroscopy and gas chromatographic analysis after melting are discussed.

Research has been conducted on the hydrogen chloride. The computational results of the hydrogen chloride elimination from Cl (sub)3 GaNH (sub)3 and intermediates' gas-phase polymerization are presented.

Research has been conducted on the peptide-based channel replacement therapy used in cystic fibrosis treatment. The hypothesis that the chloride conductance can be restored via the anion selective pore introduction produced by the channel-forming peptide has been tested and the results are reported.

Research has been conducted on the sterically bulky, dimeric diethylamido-bridged gallium hydride. The synthesis of this compound and related chloride derivatives and the studies of the preliminary chemical vapor deposition have been carried out and are discussed.

Research has been conducted on the functional vinylstannane. The palladium-catalyzed stereoselective annulation of this compound carried out by acyl chlorides has been explored and the results indicate that this annulation proceeds via the Stille reaction/cyclization sequence.

Research has been conducted on the range of pyrazole 1-oxides. The preparation of these oxides has been carried out via the regioselective deprotonation or bromine-magnesium exchange and transmetalation with ZnCl (sub)2 and palladium(0)-catalyzed cross-coupling.

Research has been conducted on the acyl chlorides and nitriles. The insertion reaction of these compounds carried out in producing 3-oxo-1-alkenylphosphonates in high yields is described.

Research has been conducted on the air-stable palladium complexes. Results indicate that these complexes serve as catalysts for the vinyl and aryl chloride cross-coupling reactions with thiols, arylzinc reagents and arylboronic acids.

Research has been conducted on the tridentate thioether pincer ligands. The ligation of these ligands to rhodium(II) has been carried out and the details are presented.

Research has been conducted on the substituted epoxy ethers. The results of the intramolecular radical cyclization of these ethers carried out via the use of bis(cyclopentadienyl)titanium(III) chloride as the radical source are presented.

Research has been conducted on the aryl chlolides and amines. The study of the nickel-catalyzed aryl amination processes carried out without NaH has demonstrated that the reaction scope could be extended to the primary and aromatic alkylamines.

Research has been conducted on the second indole sulfenylation mechanism. Results indicate that the second sulfenylation occurs via the indolenium 3,3-bis-sulfide intermediate formation followed by the sulfide group migration to the 2-position.

Sodium/proton exchangers [[Na.sup.+]/[H.sup.+] (NHEs)] play an important role in salt and water absorption from the intestinal tract. To investigate the contribution of the apical membrane NHEs, NHE2 and NHE3, to electroneutral NaC1 absorption, we measured radioisotopic [Na.sup.+] and [Cl.sup.-] flux across isolated jejuna from wild-type [NHE(+)], NHE2 knockout [NHE2(-)], and NHE3 knockout [NHE3(-)] mice. Under basal conditions, NHE(+) and NHE2(-) jejuna had similar rates of net [Na.sup.+] (~6 [micro]eq/[cm.sup.2] * h) and [Cl.sup.-] (~3 [micro]eq/[cm.sup.2] * h) absorption. In contrast, NHE3(-) jejuna had reduced net [Na.sup.+] absorption (~2 [micro]eq/ cm.sup.2] * h) but absorbed [Cl.sup.-] at rates similar to NHE(+) and NHE2(-) jejuna. Treatment with 100 [micro]M 5-(N-ethyl-N-isopropyl) amiloride (EIPA) completely inhibited net [Na.sup.+] and [Cl.sup.-] absorption in all genotypes. Studies of the [Na.sup.+] absorptive flux [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] indicated that [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] in NHE(+) jejunum was not sensitive to 1 [micro]M EIPA, whereas [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] in NHE3(-)jejunum was equally sensitive to 1 and 100 [micro]M EIPA. Treatment with forskolin/ IBMX to increase intracellular cAMP ([cAMP.sub.i]) abolished net NaCl absorption and stimulated electrogenic [Cl.sup.-] secretion in all three genotypes. Quantitative RT-PCR of epithelia from NHE2(-) and NHE3(-) jejuna did not reveal differences in mRNA expression of NHE3 and NHE2, respectively, when compared with jejunal epithelia from NHE(+) siblings. We conclude that 1) NHE3 is the dominant NHE involved in small intestinal [Na.sup.+] absorption; 2) an amiloride-sensitive [Na.sup.+] transporter partially compensates for [Na.sup.+] absorption in NHE3(-) jejunum; 3) [cAMP.sub.i] stimulation abolishes net [Na.sup.+] absorption in NHE(+), NHE2(-), and NHE3(-)jejunum; and 4) electroneutral [Cl.sup.-] absorption is not directly dependent on either NHE2 or NHE3. sodium; chloride; cystic fibrosis transmembrane conductance regulator; adenosine 3',5'-cyclic monophosphate; chloridebicarbonate exchanger; sodium-hydrogen exchange; jejunum; small intestine; cystic fibrosis

Research has been conducted on the full-length colicin B and its isolated C-terminal domain. The conformational stabilities of the colicin B C-terminal domain have been investigated via unfolding induced by guanidine hydrochloride and the results indicate that this domain consists of two subdomains.