Electronic excitations of a magnetic impurity state in the diluted magnetic semiconductor (Ga,Mn)As.

The electronic structure of doped Mn in (Ga,Mn)As is studied by resonant inelastic x-ray scattering. From configuration-interaction cluster-model calculations, the line shapes of the Mn L3 resonant inelastic x-ray scattering spectra can be explained by d-d excitations from the Mn ground state dominated by charge-transferred states, in which hole carriers are bound to the Mn impurities, rather than a pure acceptor Mn2+ ground state. Unlike archetypical d-d excitation, the peak widths are broader than the experimental energy resolution. We attribute the broadening to a finite lifetime of the d-d excitations, which decay rapidly to electron-hole pairs in the host valence and conduction bands through the hybridization of the Mn 3d orbital with the ligand band.

Hypothalamic ATF3 is involved in regulating glucose and energy metabolism in mice.

AIMS/HYPOTHESIS: The pancreas and hypothalamus are critical for maintaining nutrient and energy homeostasis, and combined disorders in these organs account for the onset of the metabolic syndrome. Activating transcription factor 3 (ATF3) is an adaptive response transcription factor. The physiological role of ATF3 in the pancreas has been controversial, and its role in the hypothalamus remains unknown. To elucidate the roles of ATF3 in these organs, we generated pancreas- and hypothalamus-specific Atf3 knockout (PHT-Atf3-KO) mice in this study. METHODS: We crossed mice bearing floxed Atf3 alleles with Pdx1-cre mice, in which cre is specifically expressed in the pancreas and hypothalamus, and analysed metabolic variables, pancreatic morphology, food intake, energy expenditure and sympathetic activity in adipose tissue. We also used a hypothalamic cell line to investigate the molecular mechanism by which ATF3 regulates transcription of the gene encoding agouti-related protein (Agrp). RESULTS: Although PHT-Atf3-KO mice displayed better glucose tolerance, neither plasma glucagon nor insulin level was altered in these mice. However, these mice exhibited higher insulin sensitivity, which was accompanied by a leaner phenotype due to decreased food intake and increased energy expenditure. We also observed decreased hypothalamic Agrp expression in PHT-Atf3-KO mice. Importantly, an increase in ATF3 levels is induced by fasting or low glucose in the hypothalamus. We also showed that ATF3 interacts with forkhead box-containing protein, O subfamily 1 (FoxO1) on the Agrp promoter and activates Agrp transcription. CONCLUSIONS/INTERPRETATION: Our results suggest that ATF3 plays an important role in the control of glucose and energy metabolism by regulating Agrp.

Contribution of Kv2.1 channels to the delayed rectifier current in freshly dispersed smooth muscle cells from rabbit urethra.

We have characterized the native voltage-dependent K(+) (K(v)) current in rabbit urethral smooth muscle cells (RUSMC) and compared its pharmacological and biophysical properties with K(v)2.1 and K(v)2.2 channels cloned from the rabbit urethra and stably expressed in human embryonic kidney (HEK)-293 cells (HEK(Kv2.1) and HEK(Kv2.2)). RUSMC were perfused with Hanks' solution at 37°C and studied using the patch-clamp technique with K(+)-rich pipette solutions. Cells were bathed in 100 nM Penitrem A (Pen A) to block large-conductance Ca(2+)-activated K(+) (BK) currents and depolarized to +40 mV for 500 ms to evoke K(v) currents. These were unaffected by margatoxin, κ-dendrotoxin, or α-dendrotoxin (100 nM, n = 3-5) but were blocked by stromatoxin-1 (ScTx, IC(50) ∼130 nM), consistent with the idea that the currents were carried through K(v)2 channels. RNA was detected for K(v)2.1, K(v)2.2, and the silent subunit K(v)9.3 in urethral smooth muscle. Immunocytochemistry showed membrane staining for both K(v)2 subtypes and K(v)9.3 in isolated RUSMC. HEK(Kv2.1) and HEK(Kv2.2) currents were blocked in a concentration-dependent manner by ScTx, with estimated IC(50) values of ∼150 nM (K(v)2.1, n = 5) and 70 nM (K(v)2.2, n = 6). The mean half-maximal voltage (V(1/2)) of inactivation of the USMC K(v) current was -56 ± 3 mV (n = 9). This was similar to the HEK(Kv2.1) current (-55 ± 3 mV, n = 13) but significantly different from the HEK(Kv2.2) currents (-30 ± 3 mV, n = 11). Action potentials (AP) evoked from RUSMC studied under current-clamp mode were unaffected by ScTx. However, when ScTx was applied in the presence of Pen A, the AP duration was significantly prolonged. Similarly, ScTx increased the amplitude of spontaneous contractions threefold, but only after Pen A application. These data suggest that K(v)2.1 channels contribute significantly to the K(v) current in RUSMC.

Loss of functional K+ channels encoded by ether-à-go-go-related genes in mouse myometrium prior to labour onset.

There is a growing appreciation that ion channels encoded by the ether-à-go-go-related gene family have a functional impact in smooth muscle in addition to their accepted role in cardiac myocytes and neurones. This study aimed to assess the expression of ERG1-3 (KCNH1-3) genes in the murine myometrium (smooth muscle layer of the uterus) and determine the functional impact of the ion channels encoded by these genes in pregnant and non-pregnant animals. Quantitative RT-PCR did not detect message for ERG2 and 3 in whole myometrial tissue extracts. In contrast, message for two isoforms of mERG1 were readily detected with mERG1a more abundant than mERG1b. In isometric tension studies of non-pregnant myometrium, the ERG channel blockers dofetilide (1 microM), E4031 (1 microM) and Be-KM1 (100 nM) increased spontaneous contractility and ERG activators (PD118057 and NS1643) inhibited spontaneous contractility. In contrast, neither ERG blockade nor activation had any effect on the inherent contractility in myometrium from late pregnant (19 days gestation) animals. Moreover, dofetilide-sensitive K(+) currents with distinctive 'hooked' kinetics were considerably smaller in uterine myocytes from late pregnant compared to non-pregnant animals. Expression of mERG1 isoforms did not alter throughout gestation or upon delivery, but the expression of genes encoding auxillary subunits (KCNE) were up-regulated considerably. This study provides the first evidence for a regulation of ERG-encoded K(+) channels as a precursor to late pregnancy physiological activity.

Nature of magnetic coupling between Mn ions in As-grown Ga1-xMnxAs studied by X-ray magnetic circular dichroism.

The magnetic properties of as-grown Ga1-xMnxAs have been investigated by the systematic measurements of temperature and magnetic field dependent soft x-ray magnetic circular dichroism (XMCD). The intrinsic XMCD intensity at high temperatures obeys the Curie-Weiss law, but a residual spin magnetic moment appears already around 100 K, significantly above the Curie temperature (T_{C}), suggesting that short-range ferromagnetic correlations are developed above T_{C}. The present results also suggest that the antiferromagnetic interaction between the substitutional and interstitial Mn (Mn_{int}) ions exists and that the amount of the Mn_{int} affects T_{C}.

Molecular expression and pharmacological identification of a role for K(v)7 channels in murine vascular reactivity.

BACKGROUND AND PURPOSE: This study represents a novel characterisation of KCNQ-encoded potassium channels in the vasculature using a variety of pharmacological and molecular tools to determine their role in contractility. EXPERIMENTAL APPROACH: Reverse transcriptase polymerase chain reaction (RT-PCR) experiments were undertaken on RNA isolated from mouse aorta, carotid artery, femoral artery and mesenteric artery using primers specific for all known KCNQ genes. RNA isolated from mouse heart and brain were used as positive controls. Pharmacological experiments were undertaken on segments from the same blood vessels to determine channel functionality. Immunocytochemical experiments were performed on isolated myocytes from thoracic aorta. KEY RESULTS: All blood vessels expressed KCNQ1, 4 and 5 with hitherto 'neuronal' KCNQ4 being, surprisingly, the most abundant. The correlated proteins K(v)7.1, K(v)7.4 and K(v)7.5 were identified in the cell membranes of aortic myocytes by immunocytochemistry. Application of three compounds known to activate K(v)7 channels, retigabine (2 -20 microM), flupirtine (20 microM) and meclofenamic acid (20 microM), relaxed vessels precontracted by phenylephrine or 1 mM 4-aminopyridine but had no effect on contractions produced by 60 mM KCl or the K(v)7 channel blocker XE991 (10 microM). All vessels tested contracted upon application of the K(v)7 channel blockers XE991 and linopirdine (0.1-10 microM). CONCLUSIONS AND IMPLICATIONS: Murine blood vessels exhibit a distinctive KCNQ expression profile with 'neuronal' KCNQ4 dominating. The ion channels encoded by KCNQ genes have a crucial role in defining vascular reactivity as K(v)7 channel blockers produced marked contractions whereas K(v)7 channel activators were effective vasorelaxants.

C-type natriuretic peptide activates a non-selective cation current in acutely isolated rat cardiac fibroblasts via natriuretic peptide C receptor-mediated signalling.

In the heart, fibroblasts play an essential role in the deposition of the extracellular matrix and they also secrete a number of hormonal factors. Although natriuretic peptides, including C-type natriuretic peptide (CNP) and brain natriuretic peptide, have antifibrotic effects on cardiac fibroblasts, the effects of CNP on fibroblast electrophysiology have not been examined. In this study, acutely isolated ventricular fibroblasts from the adult rat were used to measure the effects of CNP (2 x 10(-8) M) under whole-cell voltage-clamp conditions. CNP, as well as the natriuretic peptide C receptor (NPR-C) agonist cANF (2 x 10(-8) M), significantly increased an outwardly rectifying non-selective cation current (NSCC). This current has a reversal potential near 0 mV. Activation of this NSCC by cANF was abolished by pre-treating fibroblasts with pertussis toxin, indicating the involvement of G(i) proteins. The cANF-activated NSCC was inhibited by the compounds Gd(3+), SKF 96365 and 2-aminoethoxydiphenyl borate. Quantitative RT-PCR analysis of mRNA from rat ventricular fibroblasts revealed the expression of several transient receptor potential (TRP) channel transcripts. Additional electrophysiological analysis showed that U73122, a phospholipase C antagonist, inhibited the cANF-activated NSCC. Furthermore, the effects of CNP and cANF were mimicked by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG), independently of protein kinase C activity. These are defining characteristics of specific TRPC channels. More detailed molecular analysis confirmed the expression of full-length TRPC2, TRPC3 and TRPC5 transcripts. These data indicate that CNP, acting via the NPR-C receptor, activates a NSCC that is at least partially carried by TRPC channels in cardiac fibroblasts.

Pharmacological and molecular evidence for the involvement of Kv4.3 in ultra-fast activating K+ currents in murine portal vein myocytes.

BACKGROUND AND PURPOSE: The aim of this study was to determine the molecular identity of a transient K+ current (termed IUF) in mouse portal vein myocytes using pharmacological and molecular tools. EXPERIMENTAL APPROACH: Whole cell currents were recorded using the ruptured patch con from either acutely dispersed single smooth muscle cells from the murine portal vein or human embryonic kidney cells. Reverse transcriptase polymerase reaction (RT-PCR) experiments were undertaken on RNA isolated from mouse portal vein using primers specific for various voltage-dependent K+ channels, auxillary subunits and calcium-binding proteins. Immunocytochemistry was undertaken using an antibody specific for Kv4.3. KEY RESULTS: IUF had a mean amplitude at +40 mV of 558 +/- 50 pA (n = 32) with a mean time to peak at +40 mV of approximately 4 ms. IUF activated and inactivated with a half maximal voltage of -12 +/- 2 mV and -85 +/- 2 mV, respectively. IUF was relatively resistant to 4-aminopyridine (5 mM produced 30 +/- 6 % block at +20 mV) but was inhibited effectively by flecainide (IC50 value was 100 nM) and phrixotoxin II. This pharmacological profile is consistent with IUF being comprised of Kv4.x proteins and this is supported by the results from the quantitative PCR and immunocytochemical experiments. CONCLUSIONS AND IMPLICATIONS: These data represent a rigorous investigation of the molecular basis of vascular transient K+ currents and implicates Kv4.3 as a major component of the channel complex.

K+ currents regulate the resting membrane potential, proliferation, and contractile responses in ventricular fibroblasts and myofibroblasts.

Despite the important roles played by ventricular fibroblasts and myofibroblasts in the formation and maintenance of the extracellular matrix, neither the ionic basis for membrane potential nor the effect of modulating membrane potential on function has been analyzed in detail. In this study, whole cell patch-clamp experiments were done using ventricular fibroblasts and myofibroblasts. Time- and voltage-dependent outward K(+) currents were recorded at depolarized potentials, and an inwardly rectifying K(+) (Kir) current was recorded near the resting membrane potential (RMP) and at more hyperpolarized potentials. The apparent reversal potential of Kir currents shifted to more positive potentials as the external K(+) concentration ([K(+)](o)) was raised, and this Kir current was blocked by 100-300 muM Ba(2+). RT-PCR measurements showed that mRNA for Kir2.1 was expressed. Accordingly, we conclude that Kir current is a primary determinant of RMP in both fibroblasts and myofibroblasts. Changes in [K(+)](o) influenced fibroblast membrane potential as well as proliferation and contractile functions. Recordings made with a voltage-sensitive dye, DiBAC(3)(4), showed that 1.5 mM [K(+)](o) resulted in a hyperpolarization, whereas 20 mM [K(+)](o) produced a depolarization. Low [K(+)](o) (1.5 mM) enhanced myofibroblast number relative to control (5.4 mM [K(+)](o)). In contrast, 20 mM [K(+)](o) resulted in a significant reduction in myofibroblast number. In separate assays, 20 mM [K(+)](o) significantly enhanced contraction of collagen I gels seeded with myofibroblasts compared with control mechanical activity in 5.4 mM [K(+)](o). In combination, these results show that ventricular fibroblasts and myofibroblasts express a variety of K(+) channel alpha-subunits and demonstrate that Kir current can modulate RMP and alter essential physiological functions.

Thermoresponsive artificial extracellular matrix for tissue engineering: hyaluronic acid bioconjugated with poly(N-isopropylacrylamide) grafts.

Thermoresponsive hyaluronans (HAs) were prepared by graft polymerization of N-isopropylacrylamide (NIPAM) on HA (number-averaged molecular weight, Ma, ca. 1.5 x 10(5) and 5.0 x 10(5)) using dithiocarbamate which is a kind of iniferter (initiator, transfer agent and terminator). The degree of dithiocarbamylation (DD) as an iniferter ranged from 0.4 to 11.4% per disaccharide unit of HA. The estimated Mn of the grafted polyNIPAM (PNIPAM) ranged from approximately 5.0 x 10(3) to 8.4 x 10(4). The PNIPAM-grafted HAs (PNIPAM-HAs) were water-soluble at room temperature, while they precipitated at temperatures above approximately 34 degrees C in water. The temperature at the onset of precipitation (lower critical solution temperature: LCST) was independent of parameters of molecular architecture such as Mn of HA, degree of grafting of PNIPAM, and Mn of PNIPAM. Equilibrium transmittance of the aqueous solution above LCST decreased with an increase in both degree of grafting and Mn of PNIPAM. At physiological temperature, the PNIPAM-HA film cast from a cold solution was very wettable with water. A markedly reduced adhesion of endothelial cells to the film was observed, indicating that the PNIPAM-HA film may serve as a non-cell-adhesive matrix. Scanning electron microscopic observation appeared to differentiate supramolecular structures between rapidly freeze-dried PNIPAM-HA and nongrafted HA:PNIPAM-HA exhibited a nonuniform fibrous network, whereas the morphology of which is markedly different from that of a nongrafted HA gel exhibited a mixture of sharp needle- and platelike structures.

[In vitro and in vivo activities of panipenem against penicillin-resistant Streptococcus pneumoniae].

Efficacy of panipenem/betamipron (PAPM/BP) against experimental pneumonia caused by penicillin-resistant Streptococcus pneumoniae (PRSP: MIC of benzylpenicillin, > or = 1.56 micrograms/ml) in mice was compared with those of imipenem/cilastatin (IPM/CS), meropenem (MEPM), cefozopran (CZOP), ceftriaxone (CTRX), ampicillin (ABPC), and vancomycin (VCM). The infection was induced by inoculating a PRSP clinical isolate, 9601 (serotype 6) or 10,693 (serotype 19), into ddY male mice intranasally. Drugs were administered subcutaneously at doses of 0.4, 2, and 10 mg/kg, 18, 26, 42, and 50 hours post-infection. Viable cell counts in the lungs were determined 66 hours post-infection. PAPM/BP showed the greatest efficacy against the infections among tested drugs. MICs of PAPM against PRSP 9601 and 10,693 were both 0.125 microgram/ml, which were superior to those of IPM (0.25 and 0.5 microgram/ml, respectively), MEPM (0.5 and 1 microgram/ml, respectively), CZOP (2 and 1 microgram/ml, respectively), CTRX (both 1 microgram/ml), ABPC (both 4 micrograms/ml), and VCM (0.5 and 0.25 microgram/ml, respectively). These results suggest that the potent in vivo activity of PAPM/BP reflects the potent in vitro activity of PAPM. MICs of PAPM, IPM, MEPM, and CZOP against clinical isolates, penicillin-susceptible S. pneumoniae (PSSP: MIC of benzylpenicillin, < or = 0.05 microgram/ml), penicillin-intermediate S. pneumoniae (PISP: MIC of benzylpenicillin, 0.1-0.78 microgram/ml), and PRSP, were tested by an agar dilution method. MIC90s of the drugs against the PSSP, PISP, and PRSP were as follows: PAPM, 0.012, 0.05, and 0.39 microgram/ml; IPM, < or = 0.006, 0.1, and 0.78 microgram/ml; MEPM, 0.05, 0.39, and 1.56 micrograms/ml; and CZOP, 0.2, 0.78, and 6.25 micrograms/ml, respectively. Thus, PAPM showed the most potent activity among tested drugs against clinical isolates of PISP and PRSP.

Usefulness and limitation of DiBAC4(3), a voltage-sensitive fluorescent dye, for the measurement of membrane potentials regulated by recombinant large conductance Ca2+-activated K+ channels in HEK293 cells.

The usefulness of bis-(1,3-dibutylbarbituric acid)-trimethine oxonol (DiBAC4(3)), a voltage-sensitive fluorescent dye, for the measurement of membrane potentials (MPs) was evaluated in HEK293 cells, where alpha or alpha plus beta1 subunits of large conductance Ca2+-activated K+ (BK) channels were expressed (HEKBK alpha and HEKBK alphabeta). The fluorescent intensity of DiBAC4(3) was measured at various potentials under voltage-clamp for calibration to estimate the absolute MP semi-quantitatively. The resting MPs measured with DiBAC4(3) were roughly comparable to those recorded with a microelectrode; the MP in HEKBK alphabeta was 10-20 mV more negative than that in native HEK. In HEKBK alpha, the membrane hyperpolarization induced by 10 microM Evans blue, a BK channel opener, was detected with DiBAC4(3). NS-1619, another BK channel opener, induced gradual but substantial change in F/F(K) even in native HEK, while the BK channel opening effect was detected. Oscillatory membrane hyperpolarization was induced in HEKBK alphabeta by application of 10 microM acetylcholine via increase in intracellular Ca2+ concentration. The oscillatory hyperpolarization was, however, detected only as a slow hyperpolarization with DiBAC4(3). It can be concluded that relatively slow effects of BK channel modulators can be semi-quantitatively measured by use of DiBAC4(3) in HEKBK, while the limited temporal resolution and possible artifacts should be taken into account.

Local Ca(2+) transients and distribution of BK channels and ryanodine receptors in smooth muscle cells of guinea-pig vas deferens and urinary bladder.

1. The relationship between Ca(2+) sparks spontaneously occurring at rest and local Ca(2+) transients elicited by depolarization was analysed using two-dimensional confocal Ca(2+) images of single smooth muscle cells isolated from guinea-pig vas deferens and urinary bladder. The current activation by these Ca(2+) events was also recorded simultaneously under whole-cell voltage clamp. 2. Spontaneous transient outward currents (STOCs) and Ca(2+) sparks were simultaneously detected at -40 mV in approximately 50 % of myocytes of either type. Ca(2+) sparks and corresponding STOCs occurred repetitively in several discrete sites in the subplasmalemmal area. Large conductance Ca(2+)-dependent K(+) (BK) channel density in the plasmalemma near the Ca(2+) spark sites generating STOCs was calculated to be 21 channels microm(-2). 3. When myocytes were depolarized from -60 to 0 mV, several local Ca(2+) transients were elicited within 20 ms in exactly the same peripheral sites where sparks occurred at rest. The local Ca(2+) transients often lasted over 300 ms and spread into other areas. The appearance of local Ca(2+) transients occurred synchronously with the activation of Ca(2+)-dependent K(+) current (I(K,Ca)). 4. Immunofluorescence staining of the BK channel alpha-subunit (BKalpha) revealed a spot-like pattern on the plasmalemma, in contrast to the uniform staining of voltage-dependent Ca(2+) channel alpha1C subunits along the plasmalemma. Ryanodine receptor (RyR) immunostaining also suggested punctate localization predominantly in the periphery. Double staining of BKalpha and RyRs revealed spot-like co-localization on/beneath the plasmalemma. 5. Using pipettes of relatively low resistance, inside-out patches that included both clustered BK channels at a density of over 20 channels microm(-2) and functional Ca(2+) storage sites were obtained at a low probability of approximately 5%. The averaged BK channel density was 3-4 channels microm(-2) in both types of myocyte. 6. These results support the idea that a limited number of discrete sarcoplasmic reticulum (SR) fragments in the subplasmalemmal area play key roles in the control of BK channel activity in two ways: (i) by generating Ca(2+) sparks at rest to activate STOCs and (ii) by generating Ca(2+) transients presumably triggered by sparks during an action potential to activate a large I(K,Ca) and also induce a contraction. BK channels and RyRs may co-localize densely at the junctional areas of plasmalemma and SR fragments, where Ca(2+) sparks occur to elicit STOCs.

Comparative study of the molecular and functional expression of L-type Ca2+ channels and large-conductance, Ca2+-activated K+ channels in rabbit aorta and vas deferens smooth muscle.

The relationship between the density of ionic currents through major two channels, voltage-dependent L-type Ca2+ channels (L-type VDCC) and large-conductance, Ca2+-activated K+ channels (BKC), and the mRNA expression levels of alpha1C subunit of L-type VDCC (alpha1C) and alpha/beta subunits of BKC (alphaBK/betaBK) were compared in smooth muscle cells (SMC) of rabbit aorta and vas deferens using whole cell-voltage clamp and reverse transcription-polymerase chain reaction (RT-PCR) analyses. The density of the currents through VDCC (ICa) and BKC (IK,Ca) at +10 mV in aortic SMC was approximately one-seventh and one-sixth respectively of that in vas deferens. Whilst application of the Ca2+ channel agonist Bay K 8644 increased ICa by 75-90% in these SMC, the increase in IK,Ca was far greater in aorta than in vas deferens. The expression of the alpha1C transcript in vas deferens was approximately 3.5 times higher than that in aorta. In contrast, expression of alphaBK/betaBK was almost identical in both tissues, indicating the dissociation of IK,Ca density from the expression levels of BKC transcripts in aorta. The results were supported by Western blot and immunocytochemical analyses using subunit-specific antibodies. The lower Ca2+ influx through VDCC in aorta activates only a very limited fraction of BKC compared with that in vas deferens. The greater expression of BKC than of VDCC in aortic SMC contributes to a strong negative feed-back mechanism that minimizes membrane depolarization and acts as a safety margin to maintain low membrane excitability.

Molecular cloning and expression of the novel splice variants of K(+) channel-interacting protein 2.

Two cDNAs encoding the splice variants of K(+) channel-interacting protein 2 (KChIP2) recently reported as human KChIP2 have been identified from rat, mouse, and human heart by RT-PCR. A longer variant, KChIP2L encodes a protein of 270 amino acids, which has a 50-amino-acid insertion in N-terminus in comparison with a shorter one, KChIP2S. Interestingly, both KChIP2S and KChIP2L (KChIP2S/L) but not the original KChIP2 were expressed in human heart and umbilical vein endothelial cells (HUVECs). KChIP2S transcripts but not KChIP2L were predominantly expressed in rat, mouse, and human heart and HUVECs, whereas both transcripts were expressed at low levels in other tissues such as brain, aorta, and kidney. Using chimeric proteins of green fluorescence protein (GFP) fused to the N-terminus of KChIP2S/L, the interactions between Kv4.3 and KChIP2S/L were analyzed in native and Kv4.3-expressed HEK293 cells. Specific localization of GFP-fused KChIP2S/L proteins on or near cell membrane was observed only in Kv4.3-expressed HEK293 cells.

Regional expression of the splice variants of Kv4.3 in rat brain and effects of C-terminus deletion on expressed K+ currents.

In situ hybridization and RT-PCR analyses have revealed that, among three Kv4.3 splice variants (a, b, and c) with distinct C-terminal cytoplasmic domains, the mRNA for Kv4.3a is abundant in cerebral cortex, cerebellum, olfactory bulb, and medulla oblongata, whereas the mRNA for Kv4.3c is localized mainly to hippocampus. Three new distinct splice variants of Kv4.3 (Kv4.3d, e and f), which consist of 601, 635, and 628 amino acids, respectively, and have distinct C-terminal cytoplasmic domains, were isolated from rat brain by RT-PCR. Kv4.3b, d, e and f were expressed at much lower levels in brain. Mutagenesis which removed 149 amino acids in C-terminal domain of Kv4.3a significantly slowed its rate of recovery from inactivation as measured in heterologous expression in HEK293 cells. Surprisingly, however, neither the rate of inactivation nor voltage dependence of the activation and inactivation were changed.

Hypersusceptibility of the Pseudomonas aeruginosa nfxB mutant to beta-lactams due to reduced expression of the ampC beta-lactamase.

The Pseudomonas aeruginosa nfxB mutant lacking mexAB-oprM showed hypersusceptibility to 9 out of 24 beta-lactams tested. This hypersusceptibility was found for the nfxB mutant lacking mexAB-oprM-mexXY (N108) but not for the nfxB mutant lacking both mexAB-oprM-mexXY and ampC. The level of the AmpC beta-lactamase induction was reduced in N108. Thus, the reduced AmpC induction must be the cause of the hypersusceptibility.

Principles and application of in-vitro drug pharmacokinetic simulation systems controlled by stepwise method.

The principles and application of an in-vitro pharmacokinetic simulation system controlled by a stepwise method are described. In this system, drug concentrations in a culture medium are regulated by a periodic stepwise-adjustment method. The drug concentration is increased by adding a portion of concentrated drug solution, or decreased by draining a portion of the culture medium and the subsequent addition of drug-free medium. The drug concentration is adjusted each minute with peristaltic pumps controlled by a microcomputer. Concentrations of two drugs with independent pharmacokinetics can be simulated in the same medium. An algorithm for controlling drug concentrations by a microcomputer is presented in this report. This pharmacokinetic system was successfully applied to a urinary tract infection system, in which three infection models with different severity were reproduced in vitro. The in-vitro pharmacokinetic simulation system described here could be applicable not only for evaluating the bactericidal activities of antibacterial agents but also for evaluating other categories of drugs, such as antitumor agents, based on their pharmacokinetics.

Synthesis and in vitro antifungal activities of novel triazole antifungal agent CS-758.

Synthesis and in vitro antifungal activities of a novel triazole antifungal agent CS-758 (former name, R-120758) are described. The minimum inhibitory concentrations (MICs) of a series of dioxane-triazole compounds related to R-102557 were examined. Variation of the length of the chain between the dioxane ring and the phenyl ring revealed that the linkage with two double bonds is the most preferable. When a cyano group was introduced to the C4 position on the benzene ring, MICs improved further. A fluorine atom was introduced to obtain CS-758. The MICs of CS-758 surpassed those of fluconazole and itraconazole against Candida, Aspergillus and Cryptococcus species. The precursor (E,E)-aldehyde was synthesized stereoselectively from 3-fluoro-4-methylbenzonitrile using the Horner-Wadsworth-Emmons reaction.

Synthesis and antibacterial activity of novel 1beta-methyl carbapenems with cycloalkylamine moiety at the C-2 position.

Novel 1beta-methyl carbapenems with a cycloalkylamine moiety as a side chain were synthesized and their structure-activity relationships were studied. These carbapenems showed potent antibacterial activities against a wide range of Gram-positive and Gram-negative bacteria, and moderate urinary recovery when administered intraperitoneally in mice.