The Ba2+ block of the ATP-sensitive K+ current of mouse pancreaticβ-cells

We studied the block of whole-cell ATP-sensitive K⁺ (K_ATP) currents in mouse pancreatic-cells produced by external Ba²⁺. Ba²⁺ produced a time- and voltage-dependent block of K_ATP currents, both the rate and extent of the block increasing with hyperpolarization. With 5.6 mM [K⁺]_o, the relationship between the steady-state KATP current and [Ba²⁺]_o, was fit by the Hill equation with aK _d of 12.5 ± 2.8 M at –123 mV and of 0.18 ± 0.02 mM at –62 mV The Hill coefficient (n) was close to 1 at all potentials indicating that binding of a single Ba²⁺ ion is sufficient to block the channel. When [K⁺]_o was raised to 28 mM the K_d was little changed (12.4 ± 4.1 gM at –123 mV 0.27 ± 0.05 mM at –62 mV) and n was unaffected, suggesting that K⁺ does not interact with the Ba²⁺ binding site. The kinetics of Ba²⁺ block were slow, 10 M Ba²⁺ blocking the K_ATP current with a time constant of 20 ms at –123 mV in 28 mM [K⁺]_o. The blocking rate constant was calculated as 1.7 mM^–1 ms^–1 and the unblocking rate as 0.02 ms^–1, at –123 mV The data are discussed in terms of a model in which Ba²⁺ binds to a site at the external mouth of the channel to inhibit the K_ATP channel.     

Regulation of the Na+2Cl–K+ cotransporter in isolated rat colon crypts

The Na(+2)Cl(-)K+ cotransporter accepts NH4+ at its K+-binding site. Therefore, the rate of cytosolic acidification after NH4+ addition to the bath (20 mmol/l) measured by BCECF fluorescence can be used to quantify the rate of this cotransporter. In isolated colon crypts of rat distal colon (RCC) addition of NH4+ led to an initial alkalinization, corresponding to NH3 uptake. This was followed by an acidification, corresponding to NH4+ uptake. The rate of this uptake was quantified by exponential curve fitting and is given in arbitrary units (delta fluorescence ratio units/1000 s). In pilot experiments it was shown that the pH signal caused by the Na(+)2Cl(-)K+ co-transporter could be amplified if the experiments were carried out in the presence of bath Ba2+ to inhibit NH4+ uptake via K+ channels. Therefore all subsequent experiments were performed in the presence of 1 mmol/l Ba2+. In the absence of any secretagogue, preincubation of RCC in a low-Cl- solution (4 mmol/l) for 10 min enhanced the uptake rate significantly from 1.70+/-0.11 to 2.54+/-0.27 U/1000 s (n=20). The addition of 100 mmol/l mannitol (hypertonic solution) enhanced the rate significantly from 1.93+/-0.17 to 2.84+/-0.43 U/1000 s (n=5). Stimulation of NaCl secretion by a solution containing 100 micromol/l carbachol (CCH) led to a small but significant increase in NH4+ uptake rate from 2.06+/-0.34 to 2.40+/-0.30 U/1000 s (n= 11). The increase in uptake rate observed with stimulation of the cAMP pathway by isobutylmethylxanthine (IBMX) and forskolin (100 micromol/l and 5 micromol/l, respectively) was from 2.39+/-0.24 to 3.06+/-0.36 U/1000 s (n=24). Whatever the mechanism used to increase the NH4+ uptake rate, azosemide (500 micromol/l) always reduced this rate to control values. Hence three manoeuvres enhanced loop-diuretic-inhibitable uptake rates of the Na(+)2Cl(-)K+ cotransporter: (1) lowering of cytosolic Cl- concentration; (2) cell shrinkage; (3) activation of NaCl secretion by carbachol and (4) activation of NaCl secretion by cAMP. The common denominator of all four activation pathways may be a transient fall in cell volume.     

On the mechanism of ionic regulation of apoptosis: would the Na+/K+-ATPase please stand up?

Apoptosis is an active process with distinct features including loss of cell volume, chromatin condensation, internucleosomal DNA fragmentation, and apoptotic body formation. Among the classical characteristics that define apoptosis, the loss of cell volume has become a very important component of the programmed cell death process. Changes in cell volume result from alterations in the homeostasis of ions and in particular the movement of Na+ and K+ ions. Most living cells have a high concentration of intracellular K+ and a low concentration of intracellular Na+. This is in contrast to the outside of the cell, where there is a high concentration of extracellular Na+ and a low concentration of extracellular K+. Thus a concentration gradient exists for the loss and gain of intracellular K+ and Na+, respectively. This gradient is maintained through the activity of various ionic channels and transporters, but predominantly the activity of the Na+/K+-ATPase. During apoptosis, there is compelling evidence indicating an early increase in intracellular Na+ followed by a decrease in both intracellular K+ and Na+ suggesting a regulatory role for these cations during both the initial signalling, and the execution phase of apoptosis. Recent studies have shown that the Na+/K+-ATPase is involved in controlling perturbations of Na+ and K+ homeostasis during apoptosis, and that anti-apoptotic Bcl-2 and Bcl-XL molecules influence these ionic fluxes. Finally, understanding the regulation or deregulation of ionic homeostasis during apoptosis is critical to facilitate the treatment of cardiovascular, neurological, and renal diseases where apoptosis is known to play a major role.     

Immunolocalisation of 11β-HSD-1 and -2, glucocorticoid receptor, mineralocorticoid receptor and Na+ K+-ATPase during the postnatal development of the rat epididymis

Glucocorticoids have been implicated in male reproductive function and 11β-HSD-1 and -2, the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), all of which are known to modulate glucocorticoid action, have been localised in the adult rat epididymis, but their developmental expression has not been investigated. Na(+)K(+)-ATPase activity, responsible for sodium transport, is induced by both mineralocorticoids and glucocorticoids in the kidney and colon, and has been localised in epididymal epithelium. This study examined the immunolocalisation of 11β-HSD-1 and -2, GR, MR and Na(+)K(+)-ATPase in rat epididymal epithelium (n = 5) at postnatal days (pnd) 1, 7, 15, 28, 40, 60, 75 and 104, and relative mRNA expression of 11β-HSD-1 and -2, and GR at pre-puberty (pnd 28) and post-puberty (pnd 75). 11β-HSD-1, GR and MR were localised in the epididymal epithelium from pnd 1, and 11β-HSD-2 and Na(+)K(+)-ATPase reactivity from pnd 15. At pnd 28 there was maximal immunoreactivity for both the GR and MR and 11β-HSD-1 and -2. 11β-HSD-1 mRNA expression in the caput increased from pre- to post-puberty, whereas 11β-HSD-2 mRNA expression fell over the same period (P < 0.01). GR mRNA expression was similar at pre- and post-puberty in both caput and cauda. Developmental changes in expression of 11β-HSD-1 and -2 suggest that overall exposure of the epididymis to glucocorticoids increases post-puberty, but cell-specific expression of the 11β-HSD enzymes still provides a capacity for intricate local control of glucocorticoid exposure.     

Differences in the protein-kinase-A-dependent regulation of CFTR Cl– channels and Na+–K+ pumps in guinea-pig ventricular myocytes

Protein-kinase-A- (PKA-) dependent regulation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- current (I(CFTR)) and Na+-K+ pump current (Ip) was studied in single guinea-pig ventricular myocytes. Both currents were measured simultaneously by means of whole-cell recording at 30 degrees C. The adenylyl cyclase activator forskolin was used to stimulate PKA activity. At -20 mV, forskolin (4 microM) induced a fast activation of I(CFTR) and a delayed stimulation of Ip. Despite the strikingly different time courses, however, the potency of the drug to regulate both currents was identical. Half-maximal activation of I(CFTR) and stimulation of Ip, respectively, were observed at 9.6 x 10(-8) M and 9.9 x 10(-8) M forskolin. Inclusion of a specific peptide inhibitor of PKA in the pipette solution (PKI, 20 microM) blocked forskolin's effect on Ip. However, regardless of the time allowed for cell dialysis, there still was a marked, transient activation of I(CFTR), which could be prevented by: (1) a short pre-activation of I(CFTR) with forskolin or (2) the additional inclusion in the pipette solution of a synthetic peptide (Ht31 peptide, 60 microM) that interferes with PKA binding to its anchoring proteins. Thus, there is a tight functional coupling between PKA and CFTR Cl- channels in guinea-pig ventricular myocytes. The coupling is probably due to the close physical proximity of channels and kinases mediated by PKA anchoring proteins. Na+-K+ pumps, on the other hand, though also regulated by PKA, appear to be loosely coupled to the kinases.     

The Na+2Cl–K+ cotransporter in the rectal gland of Squalus acanthias is activated by cell shrinkage

Effects of cAMP on Cl^– secretion, intracellular Cl^– activity and cell volume were studied in isolated perfused rectal gland tubules (RGT) of Squalus acanthias with electrophysiological and fluorescence methods. Recording of equivalent short-circuit current (I _sc) showed that cAMP stimulates Na⁺Cl^– secretion in a biphasic manner. The first and rapid phase corresponds to Cl^– exit via the respective protein-kinase-A- (PKA-) phosphorylated Cl^– conductance. The inhibitory effect of the loop diuretic furosemide (0.5 mmol/l, n=12) indicates that second phase reflects the delayed (1–2 min) activation of the Na⁺2Cl^–K⁺ cotransporter. During the first phase cytosolic Cl^– activity, as monitored by 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) fluorescence, fell to 78% (n=23) of the control value. Concomitantly, a transient fall in cell volume was recorded by calcein fluorescence to 92% (n=5) of the control value. Preincubation of the RGT with phalloidin (0.1 mmol/l, n=6) or cytochalasin D (0.1 mmol/l, n=4) almost completely prevented the development of the second phase of I _sc activation. When cytosolic Cl^– activity was increased by exposing the RGT to a high K⁺ concentration (25 mmol/l), in the presence of mannitol to prevent volume increases, stimulation was unaffected and biphasic. In contrast, when cell volume was clamped to an increased value (115%, n=8) by removing extracellular NaCl, the second phase was abolished completely (n=11). These data suggest that the primary and key process for triggering the Na⁺2Cl^–K⁺ cotransport is transient cell shrinkage. Received: 11 February 1999 / Accepted: 24 March 1999     

Blockade of Ca2+‐activated K+ channels in T cells: an option for the treatment of multiple sclerosis?

Voltage- and Ca(2+)-dependent K(+) channels in the membrane of both T and B lymphocytes are important for the cellular immune response. In the current issue of the European Journal of Immunology, Reich et al. demonstrate that selective blockade of the intermediate-conductance Ca(2+)-activated K(+) channel (the IK channel encoded by the KCNN4 gene) prevents cytokine production in the spinal chord and ameliorates the development of EAE caused by injection of myelin oligodendrocyte glycoprotein (MOG)(35-55) in mice. These data renew the focus on the IK channel as a potential target for the development of new immune-suppressant drugs for the treatment of autoimmune diseases.     

In skeletal muscle the relaxation of the resting membrane potential induced by K+ permeability changes depends on Cl− transport

In resting skeletal muscle the potassium permeability is determined by the permeability of the inwardly potassium rectifier. Continuous resting membrane potential measurements are done to follow the relaxation of the membrane potential upon changes in potassium permeability. Inhibition of the inwardly potassium rectifier, by extracellular application of 80 µM Ba²⁺, causes the cell to depolarize with mean time constants as follows: in control 127±7 s (n=23), in the presence of bumetanide, as an inhibitor of the Na⁺/K⁺/2Cl^– cotransporter, 182±23 s (n=7), in hypertonic media (340 mosmol/kg) 90.4±5 s (n=7) and in reduced chloride medium 64±8 s (n=5). The depolarizing relaxation of the membrane potential induced by reduction of extracellular potassium produces similar results. These time constants are at least three orders of magnitude slower than the time constants reported in the literature for the inhibition of the inwardly potassium rectifier. Chloride transport affects the relaxation of the membrane potential. A further characterization of chloride transport is done by following the relaxation of the membrane potential upon application of chloride transport modulators. It is argued that the electroneutral cotransporter, for which a flux was preliminarily estimated of 13.4 pmol cm^–2 s^–1, has a considerable role in the processes related to the resting membrane potential.     

A new class of inhibitors of cAMP-mediated Cl− secretion in rabbit colon,acting by the reduction of cAMP-activated K+ conductance

Previously we have shown that arylamino-benzoates like 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), which are very potent inhibitors of NaCl absorption in the thick ascending limb of the loop of Henle, are only poor inhibitors of the cAMP-mediated secretion of NaCl in rat colon. This has prompted our search for more potent inhibitors of NaCl secretion in the latter system. The chromanole compound 293 B inhibited the equivalent short-circuit current (I _sc) induced by prostaglandin E₂ (n=7), vasoactive intestinal polypeptide (VIP,n=5), adenosine (n=3), cholera toxin (n=4) and cAMP (n=6), but not by ionomycin (n=5) in distal rabbit colon half maximally (IC₅₀) at 2 mol/l from the mucosal and at 0.7 mol/l from the serosal side. The inhibition was reversible and paralleled by a significant increase in transepithelial membrane resistance [e.g. in the VIP series from 116±16 ·cm² to 136±21 ·cm² (n=5)]. A total of 25 derivatives of 293 B were examined and structure activity relations were obtained. It was shown that the racemate 293 B was the most potent compound with-in this group and that its effect was due to the enantiomer 434 B which acted half maximally at 0.25 mol/l. Further studies in isolated in vitro perfused colonic crypts revealed that 10 mol/l 293 B had no effect on the membrane voltage across the basolateral membrane (V _bl) in non-stimulated crypt cells: –69±3 mV versus –67±3 mV (n=10), whilst in the same cells 1 mmol/l Ba²⁺ depolarised (V _bl) significantly. However, 293 B depolarised (V _bl) significantly in the presence of 1 mol/l forskolin: –45±4mV versus –39±5 mV (n=7). Similar results were obtained with 0.1 mmol/l adenosine. 293 B depolarised (V _bl) from –40±5 mV to –30±4 mV (n=19). This was paralleled by an increase in the fractional resistance of the basolateral membrane. VIP had a comparable effect. The hyperpolarisation induced by 0.1 mmol ATP was not influenced by 10 mol/l 293 B: –75±6 mV versus –75±6 mV (n=6). Also 293 B had no effect on basal K⁺ conductance (n=4). Hence, we conclude that 293 B inhibits the K⁺ conductance induced by cAMP. This conductance is apparently relevant for Cl^– secretion and the basal K⁺ conductance is insufficient to support secretion.     

Activation of big conductance Ca2+-activated K+ channels (BK) protects the heart against ischemia–reperfusion injury

Activation of the large-conductance Ca²⁺-activated K⁺ channel (BK) in the cardiac inner mitochondrial membrane has been suggested to protect the heart against ischemic injury. However, these findings are limited by the low selectivity profile and potency of the BK channel activator (NS1619) used. In the present study, we address the cardioprotective role of BK channels using a novel, potent, selective, and chemically unrelated BK channel activator, NS11021. Using electrophysiological recordings of heterologously expressed channels, NS11021 was found to activate BK α + β1 channel complexes, while producing no effect on cardiac K_ATP channels. The cardioprotective effects of NS11021-induced BK channel activation were studied in isolated, perfused rat hearts subjected to 35 min of global ischemia followed by 120 min of reperfusion. 3 μM NS11021 applied prior to ischemia or at the onset of reperfusion significantly reduced the infarct size [control: 44.6 ± 2.0%; NS11021: 11.4 ± 2.0%; NS11021 at reperfusion: 19.8 ± 3.3% (p < 0.001 for both treatments compared to control)] and promoted recovery of myocardial performance. Co-administration of the BK-channel inhibitor paxilline (3 μM) antagonized the protective effect. These findings suggest that tissue damage induced by ischemia and reperfusion can be reduced by activation of cardiac BK channels.     

Distribution of metabotropic glutamate receptors in the parabrachial and Kölliker-Fuse nuclei of the rat

In the present study, we analysed the distribution and cellular localization of metabotropic glutamate receptors (1alpha, 2/3, 5) in parabrachial and K?lliker-Fuse nuclei using subtype-specific antisera. Immunolabelling revealed that different nuclei express different sets of metabotropic glutamate receptors. Metabotropic glutamate receptor la immunoreactivity was found in the K?lliker-Fuse nucleus and in several parabrachial nuclei, including the waist area, lateral crescent, medial, external medial and ventral lateral nuclei. The external lateral and internal lateral parabrachial nuclei were devoid of metabotropic glutamate receptor 1alpha immunoreactivity. Metabotropic glutamate receptor 5 immunoreactivity was observed in the K?lliker-Fuse and in the medial parabrachial nuclei, while in the remaining nuclei the staining was very weak. Again, the external lateral nucleus was devoid of metabotropic glutamate receptor 5 immunoreactivity. The metabotropic glutamate receptor 2/3 antisera stained all lateral parabrachial nuclei as well as the K?lliker-Fuse nucleus, while staining in the medial parabrachial nucleus was weak. Metabotropic glutamate receptor 1alpha immunoreactivity was observed on presumed dendritic profiles, while metabotropic glutamate receptor 5 immunoreactivity was found predominantly on neuronal cell bodies. Metabotropic glutamate receptor 2/3 immunoreactivity was present as a fine, punctate immunostaining in the neuropil. Our data suggest that glutamate release in the parabrachial and K?lliker-Fuse nuclei might induce a variety of second messenger cascades, as indicated by the presence or absence of certain types of metabotropic glutamate receptors.     

Large Na+ influx and high Na+, K+–ATPase activity in mitochondria-rich epithelial cells of the inner ear endolymphatic sac

Fluid in the mammalian endolymphatic sac (ES) is connected to the endolymph in the cochlea and the vestibule. Since the dominant ion in the ES is Na(+), it has been postulated that Na(+) transport is essential for regulating the endolymph pressure. This study focused on the cellular mechanism of Na(+) transport in ES epithelial cells. To evaluate the Na(+) transport capability of the ES epithelial cells, changes in intracellular Na(+) concentration ([Na(+)](i)) of individual ES cells were measured with sodium-binding benzofurzan isophthalate in a freshly dissected ES sheet and in dissociated ES cells in response to either the K(+)-free or ouabain-containing solution. Analysis of the [Na(+)](i) changes by the Na(+) load and mitochondrial staining with rhodamine 123 showed that the ES cells were classified into two groups; one exhibited an intensive [Na(+)](i) increase, higher Na(+), K(+)-ATPase activity, and intensive mitochondrial staining (mitochondria-rich cells), and the other exhibited a moderate [Na(+)](i) increase, lower Na(+), K(+)-ATPase activity, and moderate mitochondrial staining (filament-rich cells). These results suggest that mitochondria-rich ES epithelial cells (ca. 30% of ES cells) endowed with high Na(+) permeability and Na(+), K(+)-ATPase activity potentially contribute to the transport of Na(+) outside of the endolymphatic sac.     

Three distinct types of voltage-dependent K+ channels are expressed by Müller (glial) cells of the rabbit retina

There is ample evidence that retinal radial glial (Müller) cells play a crucial role in retinal ion homeostasis. Nevertheless, data on the particular types of ion channels mediating this function are very rare and incomplete; this holds especially for mammalian Müller cells. Thus, the whole-cell variation of the patch-clamp technique was used to study voltage-dependent currents in Müller cells from adult rabbit retinae. The membrane of Müller cells was almost exclusively permeable to K⁺ ions, as no significant currents could be evoked in K⁺-free internal and external solutions, external Ba²⁺ (1 mM) reversibly blocked most membrane currents, and external Cs⁺ ions (5 mM) blocked all inward currents. All cells expressed inwardly rectifying channels that showed inactivation at strong hyperpolarizing voltages ( –120 mV), and the conductance of which varied with the square root of extracellular K⁺ concentration ([K⁺]_e). Most cells responded to depolarizing voltages ( –30 mV) with slowly activating outward currents through delayed rectifier channels. These currents were reversibly blocked by external application of 4-aminopyridine (4-AP, 0.5 mM) or tetraethylammonium (TEA, > 20 mM). Additionally, almost all cells showed rapidly inactivating currents in response to depolarizing ( –60 mV) voltage steps. The currents were blocked by Ba²⁺ (1 mM), and their amplitude increased with the [K⁺]_e. Obviously, these currents belonged to the A-type family of K⁺ channels. Some of the observed types of K⁺ channels may contribute to retinal K⁺ clearance but at least some of them may also be involved in regulation of proliferative activity of the cells.     

Localization of Na+–K+-ATPase α/β, Na+–K+–2Cl-cotransporter 1 and aquaporin-5 in human eccrine sweat glands

In order to evaluate the function of the repaired or regenerated eccrine sweat glands, we must first localize the proteins involved in sweat secretion and absorption in normal human eccrine sweat glands. In our studies, the cellular localization of Na⁺–K⁺-ATPase α/β, Na⁺–K⁺–2Cl-cotransporter 1 (NKCC1) and aquaporin-5 (AQP5) in eccrine sweat glands were detected by immunoperoxidase labeling. The results showed that Na⁺–K⁺-ATPase α was immunolocalized in the cell membrane of the basal layer and suprabasal layer cells of the epidermis, the basolateral membrane of the secretory coils, and the cell membrane of the outer cells and the basolateral membrane of the luminal cells of the ducts. The localization of Na⁺–K⁺-ATPase β in the secretory coils was the same as Na⁺–K⁺-ATPase α, but Na⁺–K⁺-ATPase β labeling was absent in the straight ducts and epidermis. NKCC1 labeling was seen only in the basolateral membrane of the secretory coils. AQP5 was strongly localized in the apical membrane and weakly localized in the cytoplasm of secretory epithelial cells. The different distribution of these proteins in eccrine sweat glands was related to their functions in sweat secretion and absorption.     

Changes in the concentrations of Na+, K+ and Cl− in secretion from the skin during progressive increase in exercise intensity

Summary A simple method for sampling skin secretion in 1-min periods was developed for investigating the effects of progressive increases in exercise intensity on Na⁺, K⁺ and CI^– secretions from the skin of the forearm. Ten healthy male subjects performed exercise consisting of eight stepwise increases in intensity from 50 to 225 W, with a 25-W increase at each step. Exercise at each step was for 3 min followed by a 1-min recovery period. Samples of blood and skin secretion were taken during the recovery period. Significant positive correlations were found between the mean concentrations of Na⁺ and Cl^– and between those of K⁺ and Cl^– in the skin secretion. The concentrations of electrolytes in the skin secretion also showed significant correlations with the blood lactate concentrations. The inflection points for secretions of Na⁺, K⁺ and Cl^– were 4.04, 3.61 and 3.83 mmol · l^– of blood lactate; 64.42, 61.96 and 62.14% of maximal oxygen consumption ( ); and exercise intensities of 123.01, 117.65 and 125.07 W, respectively. No significant differences were observed between the value of 67.27% of or 134.00W at the onset of blood lactate accumulation (OBLA) and the inflection points. From these results we concluded that changes in electrolyte concentrations in skin secretion during incremental exercise according to this protocol were closely related with the change in the blood lactate concentration, and that the inflection points for electrolytes may have been near the exercise intensity at OBLA.     

Efficacy of BIO K+ CL1285? in the reduction of antibiotic-associated diarrhea – a placebo controlled double-blind randomized,multi-center study

Introduction

Antibiotic associated diarrhea (AAD) is a frequently encountered adverse event following antibiotic administration. Evidence suggests that probiotics may be beneficial in preventing and decreasing the severity of AAD.

Material and methods

Adult patients who were prescribed antibiotics for 3-14 days were enrolled from eight Canadian centers. Study treatment was randomized at a 1 : 1 ratio of BIO-K+CL1285^® or placebo and was administered within 24 h of initiation to 5 days after termination of antibiotherapy. Patients were followed for 21 days after last dose of study treatment. The primary outcome was severity and incidence of AAD. Severity was measured by the total number of days with diarrhea and incidence was defined as the number of patients with at least one day with diarrhea over the total number of patients enrolled in the study.

Results

216 patients were randomized to BIO-K+ and 221 to placebo. The mean (SD) number of days with diarrhea was 1.19. (3.20) days for the placebo and 0.67 (2.05) days for BIO-K+CL1285^® (p = 0.040). Adjusted multivariate linear regression results showed that the duration of diarrhea for BIO-K+CL1285 ^® vs. placebo was reduced by 51.5% (b[SE] = 0.515 [0.256], p = 0.045). The incidence of diarrhea was 21.8% for the BIO-K+ and 29.4% for the placebo group (OR = 0.667, p = 0.067). Multivariate logistic regression, showed that the adjusted odds ratio of AAD in patients receiving BIO-K+ vs. placebo was 0.627 (p = 0.037). Study treatment was well tolerated.

Conclusions

BIO-K+ is effective for preventing and reducing the severity of AAD in patients receiving antibiotic therapy in a hospital setting.     

Molecular properties of the lys1 + gene and the regulation of α-aminoadipate reductase in Schizosaccharomyces pombe

The -aminoadipate pathway for the biosynthesis of lysine is unique to fungi. Molecular properties of the cloned lys1 ⁺ gene and the regulation of the encoded -aminoadipate reductase (AAR) were investigated in the fission yeast Schizosaccharomyces pombe. A 5.2-kb HindIII-EcoRI fragment of S. pombe DNA, containing a functional lys1 ⁺ gene and a promoter, was subcloned to make the 10.7-kb plasmid pLYS1H. A nested 1.778-kb HindIII-EcoRI DNA fragment that complemented the lys1-131 mutant phenotype was sequenced from the plasmid pLYS1D, and shown to contain an open reading frame (ORF) of 470 amino acids, preceded by putative POLII promoter elements (TATA and CCAAT box elements, and two potential yeast GCN4-binding motifs) within 368 bp upstream of the start codon. This ORF shared with the corresponding region of the isofunctional AAR of Saccharomyces cerevisiae 49% amino-acid identity (62% similarity) overall, within which were smaller regions of marked sequence conservation. One such region coincided (95% identity) with a putative AMP-binding domain motif identified in the AAR of S. cerevisiae. In wild-type S. pombe, AAR activity from cells grown in lysine-supplemented minimal or YEPD media was less than the activity of cells grown in minimal mediu. The AAR of S. pombe was more sensitive to feedback inhibition by lysine in vitro than the AAR of S. cerevisiae. These results show the effects of extensive evolutionary divergence on the structure and expression of a pivotal enzyme in the -aminoadipate pathway. Presumably, delineated regions of strong sequence conservation correspond to discrete domains essential to AAR function.     

Analysis of deoxynucleosides in bacteriophages ?EF24C and K and the frequency of a specific restriction site in the genomes of members of the bacteriophage subfamily Spounavirinae

The genera SPO1-like and Twort-like viruses in the subfamily Spounavirinae of the family Myoviridae have been newly proposed, with the reorganization of the SPO1-related bacteriophages (phages). A criterion defining these viral genera is the presence/absence of DNA modifications. In this study, liquid chromatography/mass spectrometry showed that phages ?EF24C and K of the subfamily Spounavirinae have unmodified DNA, which classifies them as Twort-like viruses. Moreover, in the subfamily Spounavirinae, DNA modification and elimination of a particular DNA sequence were suggested to be the major antirestriction strategies of the SPO1-like and Twort-like viruses, respectively.