The presence of multiple NS5A RASs is associated with the outcome of sofosbuvir and ledipasvir therapy in NS5A inhibitor-naïve patients with chronic HCV genotype 1b infection in a real-world cohort.

It is unclear whether multiple nonstructural (NS) 5A resistance-associated substitutions (RASs) correlate with the outcome of sofosbuvir (SOF) and ledipasvir (LDV) therapy. We investigated the effects of multiple NS5A RASs in NS5A inhibitor-naïve patients with chronic hepatitis C virus genotype 1b infection treated with SOF/LDV. In 313 patients treated with SOF/LDV, we assessed the effects of multiple NS5A RASs on the sustained virological response (SVR). RASs at L28, R30, L31, Q54, P58, Q62, A92, and Y93 in the NS5A region were examined by direct sequencing. The prevalence of RASs was as follows: 2.6% at L28, 8.7% at R30, 6.1% at L31, 48.7% at Q54, 9.9% at P58, 9.9% at Q62, 5.1% at A92, 13.8% at Y93, and 19.2% at L31 or Y93. A total of 133 patients had no RASs. SVR was achieved in 98.7% of the patients. SVR rates significantly differed between patients with and without the L31 or Y93 RAS (93.0% [53/57] vs 100% [250/250], P = .0011). In addition, among patients with the L31 or Y93 RAS, 29.8%, 45.6% and 24.6% had one, two and three or more NS5A RASs, respectively. The SVR rate was significantly lower in patients with the L31 or Y93 RAS with more than three NS5A RASs compared to those with fewer than three NS5A RASs (71.4% [10/14] vs 100% [43/43], P = .0025). Although the prevalence of multiple NS5A RASs at baseline was low in NS5A inhibitor-naïve patients, the presence of multiple NS5A RASs was associated with the effectiveness of SOF/LDV therapy.

L-type Ca²âº channel blockade with antihypertensive medication disrupts VTA synaptic plasticity and drug-associated contextual memory.

Drug addiction is driven, in part, by powerful and enduring memories of sensory cues associated with drug intake. As such, relapse to drug use during abstinence is frequently triggered by an encounter with drug-associated cues, including the drug itself. L-type Ca(2+) channels (LTCCs) are known to regulate different forms of synaptic plasticity, the major neural substrate for learning and memory, in various brain areas. Long-term potentiation (LTP) of NMDA receptor (NMDAR)-mediated glutamatergic transmission in the ventral tegmental area (VTA) may contribute to the increased motivational valence of drug-associated cues triggering relapse. In this study, using rat brain slices, we found that isradipine, a general LTCC antagonist used as antihypertensive medication, not only blocks the induction of NMDAR LTP but also promotes the reversal of previously induced LTP in the VTA. In behaving rats, isradipine injected into the VTA suppressed the acquisition of cocaine-paired contextual cue memory assessed using a conditioned place preference (CPP) paradigm. Furthermore, administration of isradipine or a CaV1.3 subtype-selective LTCC antagonist (systemic or intra-VTA) before a single extinction or reinstatement session, while having no immediate effect at the time of administration, abolished previously acquired cocaine and alcohol (ethanol) CPP on subsequent days. Notably, CPP thus extinguished cannot be reinstated by drug re-exposure, even after 2 weeks of withdrawal. These results suggest that LTCC blockade during exposure to drug-associated cues may cause unlearning of the increased valence of those cues, presumably via reversal of glutamatergic synaptic plasticity in the VTA.

Induction of elastin expression in vascular endothelial cells relates to hepatoportal sclerosis in idiopathic portal hypertension: possible link to serum anti-endothelial cell antibodies.

Hepatoportal sclerosis accompanied by dense elastic fibre deposition is generally regarded as the primary lesion in the development of idiopathic portal hypertension (IPH). This study was performed to clarify the mechanism of elastic fibre deposition in the peripheral portal tracts of IPH liver in relation to serum anti-endothelial cell antibodies (AECA). In-vitro experiments were performed using human dermal microvascular endothelial cells (HMVEC) and patients' sera. The presence of serum AECA was assayed by a cell-based enzyme-linked immunosorbent assay (ELISA) using HMVEC. Immunohistochemical analysis of elastin was performed using liver tissue sections of IPH patients. IPH sera contained one or more AECA that could bind to the vascular endothelial cells of the peripheral portal tracts of the liver. When the value of AECA greater than the mean ± 2 standard deviations of healthy controls was regarded as positive, the positive detection rate of either immunoglobulin (Ig)G, IgA or IgM AECA in IPH sera was 30% (10 of 33 cases). IPH sera induced the expression of elastin in HMVEC, which appeared to be associated with the presence of AECA. Apoptosis was also induced in HMVEC by the stimulation with IPH sera. In vivo, elastin expression was observed in the endothelial cells of the peripheral portal tracts of IPH livers in a proportion of cases. The disease pathogenesis of IPH seems to be heterogeneous, and this study elucidated a possible contribution of the induction of elastin expression in the portal vessels to hepatoportal sclerosis of IPH, which might be linked to serum AECA as a causative factor.

Activation of inflammatory signaling by lipopolysaccharide produces a prolonged increase of voluntary alcohol intake in mice.

Previous studies showed that mice with genetic predisposition for high alcohol consumption as well as human alcoholics show changes in brain expression of genes related to immune signaling. In addition, mutant mice lacking genes related to immune function show decreased alcohol consumption (Blednov et al., 2011), suggesting that immune signaling promotes alcohol consumption. To test the possibility that activation of immune signaling will increase alcohol consumption, we treated mice with lipopolysaccaride (LPS; 1mg/kg, i.p.) and tested alcohol consumption in the continuous two-bottle choice test. To take advantage of the long-lasting activation of brain immune signaling by LPS, we measured drinking beginning one week or one month after LPS treatment and continued the studies for several months. LPS produced persistent increases in alcohol consumption in C57BL/6J (B6) inbred mice, FVBxB6F1 and B6xNZBF1 hybrid mice, but not in FVB inbred mice. To determine if this effect of LPS is mediated through binding to TLR4, we tested mice lacking CD14, a key component of TLR4 signaling. These null mutants showed no increase of alcohol intake after treatment with LPS. LPS treatment decreased ethanol-conditioned taste aversion but did not alter ethanol-conditioned place preference (B6xNZBF1 mice). Electrophysiological studies of dopamine neurons in the ventral tegmental area showed that pretreatment of mice with LPS decreased the neuronal firing rate. These results suggest that activation of immune signaling promotes alcohol consumption and alters certain aspects of alcohol reward/aversion.

Add-on combination therapy with adefovir dipivoxil induces renal impairment in patients with lamivudine-refractory hepatitis B virus.

Combination therapy with adefovir dipivoxil (ADV) and lamivudine (LAM) is recommended for patients infected with LAM-refractory hepatitis B virus (HBV). However, the effects of such therapy on renal function and serum phosphorus levels have not been fully evaluated. Combination therapy with ADV and LAM was given to 37 patients infected with LAM-refractory HBV, including 17 with hepatic cirrhosis. Serum HBV DNA levels decreased to below 2.6 log(10) copies/mL in 23 (62%) of 37 patients at 12 months, 25 (78%) of 32 patients at 24 months, and 16 (84%) of 19 patients at 36 months. Except for one cirrhotic patient, serum alanine aminotransferase levels were below 50 IU/L in all patients during combination therapy. Serum creatinine levels increased in 14 (38%) of 37 patients, and serum phosphate levels decreased to below 2.5 mg/mL in 6 (16%) of 37 patients during combination therapy. Patients who received combination therapy for 36 months or longer had a significantly incidence of elevated serum creatinine levels. Fanconi syndrome occurred in a 57-year-old woman with cirrhosis after ADV was added to LAM. Combination therapy with ADV and LAM can maintain biochemical remission in patients with LAM-refractory HBV. However, the dosing interval of ADV should be adjusted according to renal function and serum phosphate levels in patients receiving long-term treatment.

Amphetamine selectively blocks inhibitory glutamate transmission in dopamine neurons.

Amphetamine is a highly addictive psychostimulant that promotes the release of the catecholamines dopamine and norepinephrine. Amphetamine-induced release of dopamine in the midbrain inhibits the activity of dopamine neurons through activation of D2 dopamine autoreceptors. Here we show that amphetamine may also excite dopamine neurons through modulation of glutamate neurotransmission. Amphetamine potently inhibits metabotropic glutamate receptor (mGluR)-mediated IPSPs in dopamine neurons, but has no effect on ionotropic glutamate receptor-mediated EPSCs. Amphetamine desensitizes the mGluR-mediated hyperpolarization through release of dopamine, activation of postsynaptic alpha1 adrenergic receptors, and suppression of InsP3-induced calcium release from internal stores. By selectively suppressing the inhibitory component of glutamate-mediated transmission, amphetamine may promote burst firing of dopamine neurons. Through this mechanism, amphetamine may enhance phasic release of dopamine, which is important in the neural processing of reward.

The rck/p54 candidate proto-oncogene product is a 54-kilodalton D-E-A-D box protein differentially expressed in human and mouse tissues.

Expression of the RCK gene, which is a target gene on 11q23 of the t(11;14) (q23;q32) translocation in the B-cell lymphoma cell line RC-K8, was studied by Northern and Western blot analyses. The RCK gene product is a member of the D-E-A-D box protein/RNA helicase family. With the use of Northern blot analysis, a 7.5-kb transcript of the RCK gene was shown to be expressed ubiquitously in human and mouse tissues. Polyclonal antibodies against the RCK gene product were raised, and the RCK gene expression pattern was examined in human and mouse tissues. Two different polyclonal anti-rck antibodies detected a specific 54-kilodalton product named rck/p54 in the majority of human and mouse tissues tested by Western blot analysis. However, rck/p54 was shown to be very low in the human brain and was not detectable in lumbar muscle and lung tissues, although RCK mRNA is abundantly present in these tissues. It is of interest that malignant transformed human cells arising from tissues with low or no expression of rck/p54, such as neuroblastoma, glioblastoma, rhabdomyosarcoma, and lung cancer cell lines, produced a moderate amount of rck/p54 protein, suggesting that rck/p54 plays a role in tumorigenesis. In addition, the rck/p54 protein was localized to cytoplasm by immunostaining with the use of laser microscopy and by subcellular fractionation.

Development of non-invasive method for assessment of hepatic steatosis.

Steatosis is a critical feature of liver disease and is considered to play a pivotal role in the progression of nonalcoholic fatty liver disease, as well as being a surrogate marker of metabolic syndrome. The purpose of this study was to develop a non-invasive diagnostic method for assessment of liver steatosis. It is well known that ultrasonic velocity depends on materials and temperature. For example, the ultrasonic velocity in water is 1530m/s at 37°C and 1534m/s at 39°C, while that in fat is 1412m/s at 37°C and 1402m/s at 39°C. On this basis, we thought that the percentage of fat in hepatic steatosis could be assessed by detecting changes of ultrasonic in the liver, caused by warming. In order to confirm the effectiveness of this method, we obtained the ultrasonic velocity changes of tissue phantom including lard oil and the liver of living rabbit by ultrasonic warming, and then succeeded in 2-D imaging of ultrasonic velocity changes of the phantom and the liver of living rabbit. We named this the ultrasonic velocity-change method. The experimental results show the possibility that hepatic steatosis could be characterized using our novel, non-invasive method.

Inositol 1,4,5-triphosphate-evoked responses in midbrain dopamine neurons.

Synaptically released glutamate evokes slow IPSPs mediated by metabotropic glutamate receptors (mGluRs) in midbrain dopamine neurons. These mGluR IPSPs are caused by release of Ca(2+) from intracellular stores and subsequent activation of small-conductance Ca(2+)-activated K(+) channels (SK channels). To further investigate the intracellular mechanisms involved, the effect of photolyzing intracellular caged inositol 1,4,5-triphosphate (InsP(3)) on membrane conductance and intracellular Ca(2+) concentration ([Ca(2+)](i)) was examined in rat midbrain slices. Photolytic release of InsP(3) elicited a transient outward current and a sharp rise in [Ca(2+)](i) that lasted for approximately 5 sec. Apamin, a blocker of SK channels, abolished the InsP(3)-induced outward current without affecting the rise in [Ca(2+)](i). Depleting intracellular Ca(2+) stores with cyclopiazonic acid completely blocked both the outward current and the Ca(2+) transient elicited by InsP(3). InsP(3)-evoked Ca(2+) mobilization was not affected by blockade of ryanodine receptors with ruthenium red, whereas depleting ryanodine-sensitive Ca(2+) stores with ryanodine almost eliminated InsP(3)-induced Ca(2+) release. Increasing the size of intracellular Ca(2+) stores by means of prolonged depolarization added a late component to the outward current and a slow component to the rising phase of [Ca(2+)](i). These effects of depolarization were blocked by ruthenium red. These results show that InsP(3) activates SK channels by releasing Ca(2+) from InsP(3)-sensitive stores that also contain ryanodine receptors. Increasing intracellular Ca(2+) stores boosts InsP(3)-evoked responses by invoking Ca(2+)-induced Ca(2+) release through ryanodine receptors. This intracellular signaling pathway may play a significant role in regulating the excitability of midbrain dopamine neurons.

Short communication: emission of nitrous oxide (N2O) from transgenic tobacco expressing antisense NiR mRNA

The emission of N2 and N2O from intact transgenic tobacco (clone 271) expressing antisense nitrite reductase (NiR) mRNA, and wild-type plants grown aseptically, on NO3-, NO2- or NH4+ -containing medium was investigated. 15N contents of gas sampled from gas-sealed pots, in which the plants were grown on 15N-containing medium, were analyzed by gas chromato- graphy and mass spectrometry (GC-MS). No emission of N2 was detected in either of the gas samples from plant clone 271 or the wild-type grown on NO3--containing medium. N2O emission from clone 271 grown on NO3--containing medium was detected, but not from the wild-type plants. The N2O emission rate of clone 271 was 106 ng N2O mg-1 incorporated N week-1 and the N2O emission was inhibited by tungstate (a nitrate reductase inhibitor). No emission of N2O was found from clone 271 or wild-type plants grown on medium containing NH4+. Emission of N2O also was detected from clone 271 grown on NO2--containing medium and its emission rate increased with increasing NO2- levels in plants. We speculate that NO3- is reduced to NO2- and that a part of NO2- is metabolized to N2O in clone 271.

Expression of the [beta]-Glucuronidase Gene in Pollen of Lily (Lilium longiflorum), Tobacco (Nicotiana tabacum), Nicotiana rustica, and Peony (Paeonia lactiflora) by Particle Bombardment.

A [beta]-glucuronidase (GUS) gene that is under the control of the anther-specific LAT52 promoter of tomato (Lycopersicon esculentum) and the nopaline synthetase polyadenylation terminator was successfully expressed in pollen of Lilium longiflorum, Nicotiana tabacum, Nicotiana rustica, and Paeonia lactiflora using a pneumatic particle gun. The GUS gene in plasmid pBI221 was also expressed, to a lesser extent, in pollen of all of these species. The presence of methanol in the substrate solution for histochemical GUS assay and the incubation time in this solution influenced successful detection of GUS expression in bombarded pollen. Cytological analysis of GUS-expressing pollen of lily showed that introduced gold particles were seen in intracellular compartments of pollen, including the vegetative cytoplasm, vegetative nucleus, and generative cytoplasm.

Growth hormone, prolactin and chorionic somatomammotropin in normal and molar pregnancy.

Twenty patients with molar pregnancy, ten normal pregnant women and ten healthy non-pregnant women were given 30 g of arginine intravenously. The serum concentration of growth hormone, prolactin and chorionic somatomammotropin (CS) was determined by radioimmunoassay. In addition, serum 17beta-estradiol, estriol and progesterone were also measured. Arginine infusion induced a sharp rise of GH in patients with molar pregnancy and in nonpregnant subjects, but the response in normal pregnancy was blunted. The response of PRL was high in patients with molar pregnancy, blunted in normal pregnancy and very small in nonpregnant subjects. CS did not respond at all to arginine infusion both in normal pregnancy and molar pregnancy. The high response to argine of PRL, normal response of GH and low baseline secretion and no response of CS may be characteristic of molar pregnancy.

Structures of transgene loci in transgenic Arabidopsis plants obtained by particle bombardment: junction regions can bind to nuclear matrices.

To clarify the molecular structure of the integration sites of transgenes, we used particle bombardment to examine the DNA sequences of transgene loci. Three transgenic Arabidopsis lines gave a single Southern hybridization band with a selectable gene as the probe. Junction regions flanked by the transgenes were cloned by the inverse polymerase chain reaction method, and the characteristics of the DNA sequences of the 10 junction regions were investigated. All but two of these were AT-rich sequences bearing motifs characteristic of a scaffold/matrix-attachment region (S/MAR). Calculations showed that seven of them should have a propensity for curvature. An assay of in-vitro binding to tobacco nuclear matrices showed that all the junction regions bound to nuclear matrices and that the two input DNAs did not bind. The 12 chromosome/transgene (CT) junctions in these three transgene loci were investigated. Cleavage sites for topoisomerase I were found at 10 of the 12, near the junction point. The other two junctions had sites within 6bp of the junction point. The sequence near one terminal of the transgene in the transgene loci was compared with that near the other terminal. Short, direct repeats consisting of 4-6bp were present within 10bp of the junction points in the sequence. We speculate that the transgene introduced by particle bombardment is delivered on AT-rich S/MAR that has a propensity for curvature, and then a nucleotide near the short, direct repeat on the transgene is joined near the cleavage sites on the genome for topoisomerase I.

Gene transfer into intact plant cells by electroinjection through cell walls and membranes.

Tobacco mosaic virus (TMV) RNA was introduced directly into mesophyll cells of Nicotiana tabacum var. Samsun using electric-field pulses (electroinjection). The injected gene was successfully expressed in the recipient cells as judged by the assay for the virus coat protein using immunofluorescence and by the virus infectivity assay of the homogenate of the electroinjected cells for local lesions on tobacco leaves. As much as 50% of the cells that survived 24 days after electroinjection showed immunofluorescent specks.

Activation of mitogen-activated protein kinase by the nociceptin receptor expressed in Chinese hamster ovary cells.

Activation of the nociceptin receptor stably expressed in Chinese hamster ovary cells induced a transient mitogen-activated protein kinase (MAPK) activation, via pertussis toxin-sensitive G-proteins. The nociceptin receptor-mediated MAPK activation was partially blocked by down-regulation or inhibition of protein kinase C, and suppressed by pretreatment with a phosphatidylcholine-specific phospholipase C inhibitor, D609. Furthermore, a tyrosine protein kinase inhibitor, genistein, and phosphatidylinositol 3-kinase inhibitors, wortmannin and LY294002, affected the nociceptin-induced MAPK activity. The nociceptin-induced MAPK activation may lead to activation of phospholipase A2 and induce changes in gene expression.

Desensitization and resensitization of delta-opioid receptor-mediated Ca2+ channel inhibition in NG108-15 cells.

1. To approach the mechanisms underlying desensitization of the opioid receptor-mediated Ca2+ channel inhibition, the effects of prolonged application of [D-Ala2, D-Leu5]enkephalin (DADLE) on Ba2+ currents (I(Ba)) through Ca2+ channels were analysed in NG108-15 neuroblastoma x glioma hybrid cells. 2. Inhibition of I(Ba) by 100 nM DADLE desensitized by 57% with a time constant of 4.4 min. 3. Maximal desensitization of the delta-opioid receptor-Ca2+ channel coupling was attained by 1 microM DADLE. The EC50 value for desensitization was estimated to be 78 nM. 4. RNA blot hybridization analysis and immunoblot analysis revealed the expression of beta-adrenoceptor kinase-1 (betaARK1) in NG108-15 cells. 5. Heparin, an inhibitor of betaARK, significantly reduced the magnitude and rate of desensitization, whereas Rp-cyclic AMPS and PKI (14-24)amide, inhibitors of cyclic AMP-dependent protein kinase (PKA), or long-term treatment with phorbol 12-myristate 13-acetate to induce down-regulation of protein kinase C (PKC) had no significant effect. 6. Recovery from desensitization (resensitization) proceeded with a time constant of 6.7 min. Okadaic acid, an inhibitor of serine/threonine phosphatases 1 and 2A, significantly attenuated the degree of resensitization. 7. In summary, we have characterized the time course and concentration-dependence of the desensitization of DADLE-induced I(Ba) inhibition in NG108-15 cells. This desensitization was reversible after removal of DADLE. It is suggested that betaARK, but neither PKA nor PKC, is involved in desensitization, while serine/threonine phosphatases mediate resensitization.

Stimulation of DNA synthesis in trophoblasts and human umbilical vein endothelial cells by hepatocyte growth factor bound to extracellular matrix.

Hepatocyte growth factor (HGF) promotes the growth not only of hepatocytes but also of several other types of cells such as cytotrophoblasts and endothelial cells. Recent studies have revealed that HGF is trapped in the extracellular (ECM) matrix through heparan sulphate in vivo, thereby acting as a mitogen for hepatocytes in cooperation with heparan sulphate. In this study, we detected HGF protein in chorionic tissue and placental tissue extracts, and found that HGF and heparan sulphate were co-distributed in the endothelial basement membrane and trophoblast basement membrane on immunohistochemical examination. The rates of DNA synthesis in primary cultured cytotrophoblasts and human umbilical vein endothelial cells (HUVEC) cultured on HGF-bound Matrigeltrade mark were 6-8 times those of control cytotrophoblasts and HUVEC. When Matrigeltrade mark dishes were pretreated with heparinase and heparitinase prior to binding of HGF, stimulation of DNA synthesis was markedly decreased. A considerable decrease in stimulation of DNA synthesis was observed following washing of HGF-bound Matrigeltrade mark with 1 m acetic acid, 1 m NaCl and 0.1 per cent trypsin, but not following treatment with chondroitinase ABC. These observations suggest that HGF can be trapped in ECM in vivo, thereby acting as a mitogen for cytotrophoblasts and placental vein endothelial cells in cooperation with heparan sulphate.

Recurrent cerebral infarctions and del(10)(p14p15.1) de novo in HDR (hypoparathyroidism, sensorineural deafness, renal dysplasia) syndrome.

We report on a Japanese boy with HDR syndrome (hypoparathyroidism, sensorineural deafness, renal dysplasia) and recurrent cerebral infarctions in the basal ganglia. The patient experienced cerebral infarctions four times between age 7 months and age 20 months. Chromosome analysis of the patient demonstrated a 46,XY, del(10)(p14p15.1) de novo. This suggests that the putative gene responsible for HDR syndrome is located at 10p14-p15.1.

Enhancement by stem cell factor of interleukin-2 (IL-2)-induced DNA synthesis in human decidual CD16- CD56bright natural killer cells mediated by increased expression of the IL-2 receptor alpha chain.

Extracts of chorionic villous and decidual tissue specimens from women in the early stages of pregnancy contained stem cell factor (SCF), the amount in the latter tissue (246.6+/-119.7 pg/mg protein) being approximately three times that in the former. Immunohistochemical analysis revealed the presence of SCF in the mesenchymal cells of the chorion, the trophoblast, and decidual stromal cells, whereas the SCF receptor, c-kit, was detected in the trophoblast and decidual mononuclear leukocytes but not in decidual stromal cells. Reverse transcription and polymerase chain reaction analysis detected transcripts corresponding to both secretory and membrane-bound types of SCF in chorionic tissue, but only those encoding the secretory type in decidual tissue. Flow cytometric analysis showed that c-kit was expressed on decidual CD16- CD56bright natural killer (NK) cells, CD14+ macrophages, and CD34+ hematopoietic progenitor cells, but not on CD3+ T cells or CD16+ NK cells. Although SCF alone had no effect on DNA synthesis in decidual CD16- CD56bright NK cells, it enhanced the proliferative effect of interleukin-2 (IL-2) at IL-2 concentrations that selectively saturate the high-affinity IL-2 receptor (IL-2R). Flow cytometry of decidual mononuclear leukocytes cultured in the presence of SCF demonstrated that this factor increased the expression of the IL-2Ralpha chain, but not IL-2Rbeta and gamma chain expression on CD16- CD56bright NK cells. Results suggest that SCF produced in the decidua increases the expression of the IL-2Ralpha which is usually present in smaller amounts than other two IL-2R chains on decidual CD16- CD56bright NK cells, and thereby promotes the proliferation of these cells in response to low concentrations of IL-2, resulting in an increase of the high affinity IL-2Rs.