Treatment of dermal ulcer with autologous fibrin glue: Two case reports of an exploratory prospective pilot study.

INTRODUCTION: The healing of recurrent and refractory skin ulcers requires a long time, during which there is risk of infection, and hospital admission is occasionally required for surgical or daily conservative treatment. Therefore, the development of promising treatments that promote faster, uneventful healing is a must. Composed of cryoprecipitate and thrombin, fibrin glue has a history of surgical use for preventing bleeding and spinal fluid leakage. Moreover, in-house cryoprecipitates contain higher concentrations of coagulation factors and cytokines that may enhance wound healing than commercially available products. However, the efficacy of completely autologous fibrin glue (AFG) in tissue repair has not yet been fully demonstrated. PATIENT CONCERNS: This study aimed to evaluate the efficacy of AFG in the treatment of refractory skin ulcers in comparison with the conventional treatment. DIAGNOSIS: Two patients with skin ulcer on their lower extremities due to trauma or scleroderma who showed resistance to conventional treatment were included in the study. Both study participants were diagnosed with refractory skin ulcer and were ineligible for autologous skin transplantation. INTERVENTIONS: AFG was prepared following autologous blood donation using a Cryoseal® system. Subsequently, AFG was administered to 50% of the area of each ulcer and observed for 4 weeks in comparison with recombinant basic fibroblast growth factor with bucladesine sodium treatment that was administered to the rest of the ulcer. OUTCOMES: The skin ulcer after trauma in participant 1 showed better improvement in the AFG-treated area. Although AFG did not show superiority regarding the ulcer area of a patient with scleroderma, it guarded the continuous exudation from the edge of the swollen skin surrounding the ulcer. CONCLUSION: AFG showed effective and beneficial results for wound healing of refractory skin ulcer and prevented exudation without any severe adverse events.

Efficacy of optical frequency domain imaging in detecting peripheral artery disease: the result of a multi-center, open-label, single-arm study.

Optical frequency domain imaging (OFDI) is a high-resolution intracoronary imaging modality with fast automated longitudinal pullback. We aimed to evaluate the ability of performing OFDI from the superficial femoral artery (SFA) to the below-knee (BK) artery. This clinical trial was a multi-center, single-arm, open-label study. The primary endpoint was to obtain a clear image of the intra-vascular lumen from the SFA to the BK artery, specifically > 270° visualization of the blood vessel lumen with > 16/21 cross sections. The proportion of the clear image (≥ 85%) was regarded as confirmatory of the ability of OFDI to visualize the vessel lumen. Overall, 20 patients were enrolled. The proportion of the primary endpoint was 90% (18/20), and the pre-specified criterion was successfully attained. The proportion of the clear image assessed by the operator was 100% (20/20), and an additional statistical analysis for the proportion of the visualization, > 270°, of the blood vessel lumen revealed a significantly higher cut-off value than that for the pre-specified criterion, 85% (p = 0.0315). There were three adverse events not related to OFDI. OFDI achieved acceptable visualization of the vessel lumen without any adverse event related to it. After regulatory approval based on the present study, OFDI will be available as a new option of endovascular imaging for peripheral artery diseases in daily practiceTrial registration: This study was registered in the Japanese Registry of Clinical Trials (jRCT 2052190025, https://jrct.niph.go.jp/latest-detail/jRCT2052190025 ).

Amelioration of Radiation Enteropathy by Dietary Supplementation With Reduced Coenzyme Q10.

PURPOSE: Effective methods to ameliorate radiation enteropathy have not been developed. To address this issue, we investigated the reduced form of coenzyme Q10 (rCoQ10) as a potential radioprotector in a mouse model. METHODS AND MATERIALS: rCoQ10 was added to a standard laboratory mouse diet at a final concentration of 1.0% 9 days before irradiation and 30 days thereafter or dissolved in corn oil and administered transorally. Accumulated amounts of coenzyme Q10 (CoQ10) or coenzyme Q9 in the intestine were measured by high-performance liquid chromatography. Reactive oxygen species (ROS), apoptosis, and morphologic changes in the intestine were assessed by immunohistochemistry after administration of 13 Gy of x-ray to the mouse abdomen. Body weight and survival were monitored for 30 days after irradiation. Cytotoxicity using 3 human cancer cell lines and the tumor growth-inhibiting effect in a xenograft were investigated to determine whether rCoQ10 interferes with radiation-specific cytotoxic effects on tumor growth. RESULTS: CoQ10 was greatly accumulated in all sections of the intestine after both massive transoral dosing and dietary administration, whereas coenzyme Q9 was not. Administration of rCoQ10 suppressed ROS production and inhibited apoptosis in the crypts, resulting in preservation of villi structures after irradiation. Notably, 92% of mice fed the rCoQ10-supplemented diet were healthy and alive 30 days after irradiation, whereas 50% of control mice died (P < .05). Moreover, rCoQ10 did not interfere with radiation-specific cytotoxic effects on tumors either in vitro or in vivo. CONCLUSIONS: Administration of rCoQ10 led to its accumulation in the intestine and induced radioprotective effects by inhibiting ROS-mediated apoptosis, thereby preserving intestinal structures. Our results indicated that rCoQ10 supplementation effectively ameliorated radiation enteropathy.

Low amounts of dietary fibre increase in vitro production of short-chain fatty acids without changing human colonic microbiota structure.

This study investigated the effect of various prebiotics (indigestible dextrin, α-cyclodextrin, and dextran) on human colonic microbiota at a dosage corresponding to a daily intake of 6 g of prebiotics per person (0.2% of dietary intake). We used an in vitro human colonic microbiota model based on batch fermentation starting from a faecal inoculum. Bacterial 16S rRNA gene sequence analysis showed that addition of 0.2% prebiotics did not change the diversity and composition of colonic microbiota. This finding coincided with results from a clinical study showing that the microbiota composition of human faecal samples remained unchanged following administration of 6 g of prebiotics over seven days. However, compared to absence of prebiotics, their addition reduced the pH and increased the generation of acetate and propionate in the in vitro system. Thus, even at such relatively low amounts, prebiotics appear capable of activating the metabolism of colonic microbiota.

Kv3.1 channels stimulate adult neural precursor cell proliferation and neuronal differentiation.

Adult neural stem/precursor cells (NPCs) play a pivotal role in neuronal plasticity throughout life. Among ion channels identified in adult NPCs, voltage-gated delayed rectifier K(+) (KDR) channels are dominantly expressed. However, the KDR channel subtype and its physiological role are still undefined. We used real-time quantitative RT-PCR and gene knockdown techniques to identify a major functional KDR channel subtype in adult NPCs. Dominant mRNA expression of Kv3.1, a high voltage-gated KDR channel, was quantitatively confirmed. Kv3.1 gene knockdown with specific small interfering RNAs (siRNA) for Kv3.1 significantly inhibited Kv3.1 mRNA expression by 63.9% (P < 0.001) and KDR channel currents by 52.2% (P < 0.001). This indicates that Kv3.1 is the subtype responsible for producing KDR channel outward currents. Resting membrane properties, such as resting membrane potential, of NPCs were not affected by Kv3.1 expression. Kv3.1 knockdown with 300 nm siRNA inhibited NPC growth (increase in cell numbers) by 52.9% (P < 0.01). This inhibition was attributed to decreased cell proliferation, not increased cell apoptosis. We also established a convenient in vitro imaging assay system to evaluate NPC differentiation using NPCs from doublecortin-green fluorescent protein transgenic mice. Kv3.1 knockdown also significantly reduced neuronal differentiation by 31.4% (P < 0.01). We have demonstrated that Kv3.1 is a dominant functional KDR channel subtype expressed in adult NPCs and plays key roles in NPC proliferation and neuronal lineage commitment during differentiation.

γ-Aminobutyric acid type B (GABAB) receptor expression is needed for inhibition of N-type (Cav2.2) calcium channels by analgesic α-conotoxins.

α-Conotoxins Vc1.1 and RgIA are small peptides isolated from the venom of marine cone snails. They have effective anti-nociceptive actions in rat models of neuropathic pain. Pharmacological studies in rodent dorsal root ganglion (DRG) show their analgesic effect is mediated by inhibition of N-type (Ca(v)2.2) calcium channels via a pathway involving γ-aminobutyric acid type B (GABA(B)) receptor. However, there is no direct demonstration that functional GABA(B) receptors are needed for inhibition of the Ca(v)2.2 channel by analgesic α-conotoxins. This study examined the effect of the GABA(B) agonist baclofen and α-conotoxins Vc1.1 and RgIA on calcium channel currents after transient knockdown of the GABA(B) receptor using RNA interference. Isolated rat DRG neurons were transfected with small interfering RNAs (siRNA) targeting GABA(B) subunits R1 and R2. Efficient knockdown of GABA(B) receptor expression at mRNA and protein levels was confirmed by quantitative real time PCR (qRT-PCR) and immunocytochemical analysis, respectively. Whole-cell patch clamp recordings conducted 2-4 days after transfection showed that inhibition of N-type calcium channels in response to baclofen, Vc1.1 and RgIA was significantly reduced in GABA(B) receptor knockdown DRG neurons. In contrast, neurons transfected with a scrambled nontargeting siRNA were indistinguishable from untransfected neurons. In the HEK 293 cell heterologous expression system, Vc1.1 and RgIA inhibition of Ca(v)2.2 channels needed functional expression of both human GABA(B) receptor subunits. Together, these results confirm that GABA(B) receptors must be activated for the modulation of N-type (Ca(v)2.2) calcium channels by analgesic α-conotoxins Vc1.1 and RgIA.

Purification of immature neuronal cells from neural stem cell progeny.

Large-scale proliferation and multi-lineage differentiation capabilities make neural stem cells (NSCs) a promising renewable source of cells for therapeutic applications. However, the practical application for neuronal cell replacement is limited by heterogeneity of NSC progeny, relatively low yield of neurons, predominance of astrocytes, poor survival of donor cells following transplantation and the potential for uncontrolled proliferation of precursor cells. To address these impediments, we have developed a method for the generation of highly enriched immature neurons from murine NSC progeny. Adaptation of the standard differentiation procedure in concert with flow cytometry selection, using scattered light and positive fluorescent light selection based on cell surface antibody binding, provided a near pure (97%) immature neuron population. Using the purified neurons, we screened a panel of growth factors and found that bone morphogenetic protein-4 (BMP-4) demonstrated a strong survival effect on the cells in vitro, and enhanced their functional maturity. This effect was maintained following transplantation into the adult mouse striatum where we observed a 2-fold increase in the survival of the implanted cells and a 3-fold increase in NeuN expression. Additionally, based on the neural-colony forming cell assay (N-CFCA), we noted a 64 fold reduction of the bona fide NSC frequency in neuronal cell population and that implanted donor cells showed no signs of excessive or uncontrolled proliferation. The ability to provide defined neural cell populations from renewable sources such as NSC may find application for cell replacement therapies in the central nervous system.

Physiological roles of ion channels in adult neural stem cells and their progeny.

Elucidation of the machinery of adult neurogenesis is indispensable for the treatment of neurodegenerative diseases by therapeutic drugs and/or by neural stem cell (NSC) transplantation. It is well known that membrane ion channels play a critical role in cell function, including proliferation, apoptosis and migration in a wide range of cells. In NSC research, interdisciplinary collaboration between cell biologists and membrane physiologists has been pursued principally to monitor ion channel and synaptic currents as a hallmark of neuronal differentiation and maturation of NSC progeny. Nevertheless, less attention had been paid to a functional role of ion channels in NSCs or their immature progeny. Recently, however, evidence regarding their functional relevance has started to accumulate. In focusing on the early stages of the neurogenic process during which NSCs give rise to neuroblasts, this review highlights the latent ability of ion channels to act as functional regulators of adult neurogenesis.

[Drug-induced liver injury caused by an herbal medicine, bofu-tsu-sho-san].

A 37-year-old woman was admitted to a hospital with jaundice. Within a couple of weeks, her liver function improved with only symptomatic therapy. About 30 to 60 days before admission, she had taken a herbal medicine, bofu-tsu-sho-san. A diagnosis of drug-induced liver injury was made according to the diagnostic scale proposed at the Digestive Disease Week-Japan 2004. A drug-lymphocyte stimulation test for each ingredient of bofu-tsu-sho-san; the results were positive for Cnidii Rhizoma, Angelicae Radix and Menthae Herba. The liver biopsy specimen revealed features of acute hepatitis. Physicians should be aware that bofu-tsu-sho-san can cause liver injury, as this drug is commonly used as an over-the-counter medicine.

K(ir) and K(v) channels regulate electrical properties and proliferation of adult neural precursor cells.

The functional significance of the electrophysiological properties of neural precursor cells (NPCs) was investigated using dissociated neurosphere-derived NPCs from the forebrain subventricular zone (SVZ) of adult mice. NPCs exhibited hyperpolarized resting membrane potentials, which were depolarized by the K(+) channel inhibitor, Ba(2+). Pharmacological analysis revealed two distinct K(+) channel families: Ba(2+)-sensitive K(ir) channels and tetraethylammonium (TEA)-sensitive K(v) (primarily K(DR)) channels. Ba(2+) promoted mitogen-stimulated NPC proliferation, which was mimicked by high extracellular K(+), whereas TEA inhibited proliferation. Based on gene and protein levels in vitro, we identified K(ir)4.1, K(ir)5.1 and K(v)3.1 channels as the functional K(+) channel candidates. Expression of these K(+) channels was immunohistochemically found in NPCs of the adult mouse SVZ, but was negligible in neuroblasts. It therefore appears that expression of K(ir) and K(v) (K(DR)) channels in NPCs and related changes in the resting membrane potential could contribute to NPC proliferation and neuronal lineage commitment in the neurogenic microenvironment.

The doublecortin-expressing population in the developing and adult brain contains multipotential precursors in addition to neuronal-lineage cells.

Doublecortin (DCX) has recently been promulgated as a selective marker of cells committed to the neuronal lineage in both the developing and the adult brain. To explore the potential of DCX-positive (DCX+) cells more stringently, these cells were isolated by flow cytometry from the brains of transgenic mice expressing green fluorescent protein under the control of the DCX promoter in embryonic, early postnatal, and adult animals. It was found that virtually all of the cells (99.9%) expressing high levels of DCX (DCX(high)) in the embryonic brain coexpressed the neuronal marker betaIII-tubulin and that this population contained no stem-like cells as demonstrated by lack of neurosphere formation in vitro. However, the DCX+ population from the early postnatal brain and the adult subventricular zone and hippocampus, which expressed low levels of DCX (DCX(low)), was enriched for neurosphere-forming cells, with only a small subpopulation of these cells coexpressing the neuronal markers betaIII-tubulin or microtubule-associated protein 2. Similarly, the DCX(low) population from embryonic day 14 (E14) brain contained neurosphere-forming cells. Only the postnatal cerebellum and adult olfactory bulb contained some DCX(high) cells, which were shown to be similar to the E14 DCX(high) cells in that they had no stem cell activity. Electrophysiological studies confirmed the heterogeneous nature of DCX+ cells, with some cells displaying characteristics of immature or mature neurons, whereas others showed no neuronal characteristics whatsoever. These results indicate that DCX(high) cells, regardless of location, are restricted to the neuronal lineage or are bone fide neurons, whereas some DCX(low) cells retain their multipotentiality.

Omega-conotoxin CVIB differentially inhibits native and recombinant N- and P/Q-type calcium channels.

Omega-conotoxins are routinely used as selective inhibitors of different classes of voltage-gated calcium channels (VGCCs) in excitable cells. In the present study, we examined the potent N-type VGCC antagonist omega-conotoxin CVID and non-selective N- and P/Q-type antagonist CVIB for their ability to block native VGCCs in rat dorsal root ganglion (DRG) neurons and recombinant VGCCs expressed in Xenopus oocytes. Omega-conotoxins CVID and CVIB inhibited depolarization-activated whole-cell VGCC currents in DRG neurons with pIC50 values of 8.12 +/- 0.05 and 7.64 +/- 0.08, respectively. Inhibition of Ba2+ currents in DRG neurons by CVID (approximately 66% of total) appeared to be irreversible for > 30 min washout, whereas Ba2+ currents exhibited rapid recovery from block by CVIB (> or = 80% within 3 min). The recoverable component of the Ba2+ current inhibited by CVIB was mediated by the N-type VGCC, whereas the irreversibly blocked current (approximately 22% of total) was attributable to P/Q-type VGCCs. Omega-conotoxin CVIB reversibly inhibited Ba2+ currents mediated by N- (Ca(V)2.2) and P/Q- (Ca(V)2.1), but not R- (Ca(V)2.3) type VGCCs expressed in Xenopus oocytes. The alpha2delta1 auxiliary subunit co-expressed with Ca(V)2.2 and Ca(V)2.1 reduced the sensitivity of VGCCs to CVIB but had no effect on reversibility of block. Determination of the NMR structure of CVIB identified structural differences to CVID that may underlie differences in selectivity of these closely related conotoxins. Omega-conotoxins CVIB and CVID may be useful as antagonists of N- and P/Q-type VGCCs, particularly in sensory neurons involved in processing primary nociceptive information.

Usefulness of noninvasive transient elastography for assessment of liver fibrosis stage in chronic hepatitis C.

AIM: To evaluate the method of noninvasive transient elastography for assessment of histological stage of liver fibrosis in patients with chronic hepatitis C (CHC). METHODS: Two hundred and thirty-seven patients with CHC were included in this study. Liver biopsy was performed under ultrasonography on 217 of the patients, excluding twenty with clear clinical evidence of liver cirrhosis. Fifty subjects without liver disease were enrolled as a control group (stage 0). Twenty-five patients with sustained virological response (SVR) to interferon (IFN) therapy were also enrolled. These patients underwent liver biopsy before IFN therapy. Examination of liver stiffness (LS) was performed by elastography. RESULTS: Medians (50% levels) of LS were 4.1 (3.5-4.9), 6.3 (4.8-8.5), 8.8 (6.8-12.0), 14.6 (10.5-18.6), and 22.2 (15.4-28.0), respectively, in the fibrosis stages 0-4 (P < 0.001). LS was significantly correlated with four serum fibrosis markers. LS values in patients with SVR were 3.8 (3.5-5.6), 5.2 (4.4-6.8), 6.8 (6.1-7.6), and 6.1 (3.6-7.9), respectively, in the fibrosis stages 1-4. In all stages, LS for patients with SVR was significantly lower than that for patients who did not undergo IFN therapy. LS was significantly correlated with serum concentrations of hyaluronic acid, type IV collagen, type IV collagen 7S, and type III procollagen N peptide. CONCLUSION: LS correlated well with the histological stage of fibrosis. Changes in liver fibrosis stage may thus be estimated noninvasively using transient elastography.

Mode of HCV infection examined by polymorphism of hypervariable region-1 in cases of acute hepatitis C after accidental exposure to blood-borne pathogens.

Acute hepatitis C is known to respond better to interferon therapy than chronic hepatitis C. The reason for this difference remains unclear. The present study was undertaken to examine HCV quasispecies in blood from patients with acute hepatitis C caused by accidental exposure to blood-borne pathogens and in blood from the source patients. Three patients who developed hepatitis C (recipient patients; R-Pt.) and two patients who served as a source of HCV infection (source patients; S-Pt.) were the subjects of this study. The number of quasispecies and the genetic diversity in hypervariable region-1 (HVR-1) were examined on the basis of fluorescence single-strand conformation polymorphism and sequence analysis (FSSA). On the day of the accident, the number of quasispecies and genetic diversity were 13 and 36 in S-Pt.1 and 6 and 20 in S-Pt.3, respectively. At the time of diagnosis of acute hepatitis, the number of quasispecies and nucleotide diversity were 2 and 2 in R-Pt.1, 2 and 0 in R-Pt.2, and 4 and 0 in R-Pt.3, respectively. Immediately before the start of treatment, the number of quasispecies and genetic diversity were 4 and 4 in R-Pt.1, 2 and 0 in R-Pt.2, and 3 and 0 in R-Pt.3., respectively. In three R-Pts, interferon therapy resulted in eradication of HCV. These findings indicate that in the early stage of HCV infection, only a portion of HCV transmitted from S-Pts to R-Pts can proliferate. The low number of quasispecies of HCV appears to be one of the reasons why acute hepatitis responds well to interferon therapy.

Auxiliary subunit regulation of high-voltage activated calcium channels expressed in mammalian cells.

The effects of auxiliary calcium channel subunits on the expression and functional properties of high-voltage activated (HVA) calcium channels have been studied extensively in the Xenopus oocyte expression system, but are less completely characterized in a mammalian cellular environment. Here, we provide the first systematic analysis of the effects of calcium channel beta and alpha(2)-delta subunits on expression levels and biophysical properties of three different types (Ca(v)1.2, Ca(v)2.1 and Ca(v)2.3) of HVA calcium channels expressed in tsA-201 cells. Our data show that Ca(v)1.2 and Ca(v)2.3 channels yield significant barium current in the absence of any auxiliary subunits. Although calcium channel beta subunits were in principle capable of increasing whole cell conductance, this effect was dependent on the type of calcium channel alpha(1) subunit, and beta(3) subunits altogether failed to enhance current amplitude irrespective of channel subtype. Moreover, the alpha(2)-delta subunit alone is capable of increasing current amplitude of each channel type examined, and at least for members of the Ca(v)2 channel family, appears to act synergistically with beta subunits. In general agreement with previous studies, channel activation and inactivation gating was regulated both by beta and by alpha(2)-delta subunits. However, whereas pronounced regulation of inactivation characteristics was seen with the majority of the auxiliary subunits, effects on voltage dependence of activation were only small (< 5 mV). Overall, through a systematic approach, we have elucidated a previously underestimated role of the alpha(2)-delta(1) subunit with regard to current enhancement and kinetics. Moreover, the effects of each auxiliary subunit on whole cell conductance and channel gating appear to be specifically tailored to subsets of calcium channel subtypes.

The alpha2delta auxiliary subunit reduces affinity of omega-conotoxins for recombinant N-type (Cav2.2) calcium channels.

The omega-conotoxins from fish-hunting cone snails are potent inhibitors of voltage-gated calcium channels. The omega-conotoxins MVIIA and CVID are selective N-type calcium channel inhibitors with potential in the treatment of chronic pain. The beta and alpha(2)delta-1 auxiliary subunits influence the expression and characteristics of the alpha(1B) subunit of N-type channels and are differentially regulated in disease states, including pain. In this study, we examined the influence of these auxiliary subunits on the ability of the omega-conotoxins GVIA, MVIIA, CVID and analogues to inhibit peripheral and central forms of the rat N-type channels. Although the beta3 subunit had little influence on the on- and off-rates of omega-conotoxins, coexpression of alpha(2)delta with alpha(1B) significantly reduced on-rates and equilibrium inhibition at both the central and peripheral isoforms of the N-type channels. The alpha(2)delta also enhanced the selectivity of MVIIA, but not CVID, for the central isoform. Similar but less pronounced trends were also observed for N-type channels expressed in human embryonic kidney cells. The influence of alpha(2)delta was not affected by oocyte deglycosylation. The extent of recovery from the omega-conotoxin block was least for GVIA, intermediate for MVIIA, and almost complete for CVID. Application of a hyperpolarizing holding potential (-120 mV) did not significantly enhance the extent of CVID recovery. Interestingly, [R10K]MVIIA and [O10K]GVIA had greater recovery from the block, whereas [K10R]CVID had reduced recovery from the block, indicating that position 10 had an important influence on the extent of omega-conotoxin reversibility. Recovery from CVID block was reduced in the presence of alpha(2)delta in human embryonic kidney cells and in oocytes expressing alpha(1B-b). These results may have implications for the antinociceptive properties of omega-conotoxins, given that the alpha(2)delta subunit is up-regulated in certain pain states.

Overexpressed Ca(v)beta3 inhibits N-type (Cav2.2) calcium channel currents through a hyperpolarizing shift of ultra-slow and closed-state inactivation.

It has been shown that beta auxiliary subunits increase current amplitude in voltage-dependent calcium channels. In this study, however, we found a novel inhibitory effect of beta3 subunit on macroscopic Ba(2+) currents through recombinant N- and R-type calcium channels expressed in Xenopus oocytes. Overexpressed beta3 (12.5 ng/cell cRNA) significantly suppressed N- and R-type, but not L-type, calcium channel currents at "physiological" holding potentials (HPs) of -60 and -80 mV. At a HP of -80 mV, coinjection of various concentrations (0-12.5 ng) of the beta3 with Ca(v)2.2alpha(1) and alpha(2)delta enhanced the maximum conductance of expressed channels at lower beta3 concentrations but at higher concentrations (>2.5 ng/cell) caused a marked inhibition. The beta3-induced current suppression was reversed at a HP of -120 mV, suggesting that the inhibition was voltage dependent. A high concentration of Ba(2+) (40 mM) as a charge carrier also largely diminished the effect of beta3 at -80 mV. Therefore, experimental conditions (HP, divalent cation concentration, and beta3 subunit concentration) approaching normal physiological conditions were critical to elucidate the full extent of this novel beta3 effect. Steady-state inactivation curves revealed that N-type channels exhibited "closed-state" inactivation without beta3, and that beta3 caused an approximately 40-mV negative shift of the inactivation, producing a second component with an inactivation midpoint of approximately -85 mV. The inactivation of N-type channels in the presence of a high concentration (12.5 ng/cell) of beta3 developed slowly and the time-dependent inactivation curve was best fit by the sum of two exponential functions with time constants of 14 s and 8.8 min at -80 mV. Similar "ultra-slow" inactivation was observed for N-type channels without beta3. Thus, beta3 can have a profound negative regulatory effect on N-type (and also R-type) calcium channels by causing a hyperpolarizing shift of the inactivation without affecting "ultra-slow" and "closed-state" inactivation properties.

Development of a high-throughput fluoroimmunoassay for Syk kinase and Syk kinase inhibitors.

Syk is a tyrosine kinase which is indispensable in immunoglobulin Fc receptor- and B cell receptor-mediated signal transduction in various immune cells. This pathway is important in the pathophysiology of allergy. In this study we established a quantitative nonradioactive kinase assay to identify inhibitors of Syk. We used recombinant GST-tagged Syk purified from baculovirus-infected insect cells. As a substrate, biotinylated peptide corresponding to the activation loop domain of Syk, whose tyrosine residues are autophosphorylated upon activation, was employed to screen both ATP- and substrate-competitive inhibitors. After the kinase reaction in solution phase, substrate was trapped on a streptavidin-coated plate, followed by detection of the phosphorylated tyrosine with europium-labeled anti-phosphotyrosine antibody. The kinase reaction in solution phase greatly enhanced phosphorylation of substrate compared to that of plate-coated substrate. High signal-to-background ratio and low data scattering were obtained in the optimized high-throughput screening (HTS) format. Further, several kinase inhibitors showed concentration-dependent inhibition of recombinant Syk kinase activity with almost the same efficacy for immunoprecipitated Syk from a human cell line. These data suggest that this assay is useful to screen Syk kinase inhibitors in HTS.

Omega-conotoxin CVID inhibits a pharmacologically distinct voltage-sensitive calcium channel associated with transmitter release from preganglionic nerve terminals.

Neurotransmitter release from preganglionic parasympathetic neurons is resistant to inhibition by selective antagonists of L-, N-, P/Q-, R-, and T-type calcium channels. In this study, the effects of different omega-conotoxins from genus Conus were investigated on current flow-through cloned voltage-sensitive calcium channels expressed in Xenopus oocytes and nerve-evoked transmitter release from the intact preganglionic cholinergic nerves innervating the rat submandibular ganglia. Our results indicate that omega-conotoxin CVID from Conus catus inhibits a pharmacologically distinct voltage-sensitive calcium channel involved in neurotransmitter release, whereas omega-conotoxin MVIIA had no effect. omega-Conotoxin CVID and MVIIA inhibited depolarization-activated Ba(2+) currents recorded from oocytes expressing N-type but not L- or R-type calcium channels. High affinity inhibition of the CVID-sensitive calcium channel was enhanced when position 10 of the omega-conotoxin was occupied by the smaller residue lysine as found in CVID instead of an arginine as found in MVIIA. Given that relatively small differences in the sequence of the N-type calcium channel alpha(1B) subunit can influence omega-conotoxin access (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 15728-15735), it is likely that the calcium channel in preganglionic nerve terminals targeted by CVID is a N-type (Ca(v)2.2) calcium channel variant.