Nonstructural protein 5A of hepatitis C virus inhibits the function of karyopherin beta3

It has been suggested that nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) plays a role in the incapacitation of interferon by inactivation of RNA-dependent protein kinase PKR. In order to further investigate the role of NS5A, we tried to identify cellular proteins interacting with NS5A by using the yeast two-hybrid system. The karyopherin beta3 gene was isolated from a human liver cell library as a protein interacting with NS5A. The protein-protein interaction between NS5A and karyopherin beta3 was confirmed by in vitro binding assay and an in vivo coimmunoprecipitation method. The effect of NS5A on the karyopherin beta3 activity was investigated using a yeast cell line containing mutations in both PSE1 and KAP123, genes that are homologous to the human karyopherin beta3 gene. Human karyopherin beta3 complemented the loss of the PSE1 and KAP123 functions, supporting growth of the double mutant cells. However, expression of NS5A hampered the growth of the double mutant cells supplemented with human karyopherin beta3. On the other hand, expression of NS5A by itself had no effect on the growth of the double mutant expressing wild-type yeast PSE1. This indicates that NS5A may inhibit karyopherin beta3 function via protein-protein interaction. The role of NS5A in HCV replication is discussed.     

Phosphorylation of peptides derived from isolated soybean protein: effects on calcium binding, solubility and influx into Caco-2 cells

Calcium homeostasis in the human body is maintained primarily via the absorption of calcium through the intestine. In order to maintain an efficient absorption of calcium with minimal calcium loss due to the formation of calcium phosphate precipitates in the small intestinal lumen, we developed a calcium-binding mediator using peptides derived from isolated soybean protein (ISP). ISP was modified via tryptic digestion and chemical phosphorylation using sodium trimetaphosphate, thereby generating soybean phosphopeptides (SPP), and this was followed by conducting a binding reaction with calcium chloride. We have established an optimized procedure and reaction conditions for maximal phosphorylation and calcium binding. Consequently, the phosphorylation of soybean peptides resulted in considerable improvement in their calcium binding activities. Next, we demonstrated that SPP was able to render calcium ions resistant to precipitate formation with inorganic phosphates, which suggested the enhancement of calcium bioavailability. Finally, we noted that the addition of calcium-bound SPP induced an increase in cytosolic calcium concentration in the intestinal Caco-2 cells, due to an influx of calcium. These findings provide a new basis by which we may assess the possibility that SPP, as a potent calcium carrier, can be utilized in the prevention of poor absorption of dietary calcium in animals.     

Conversion of methylglyoxal to acetol by Escherichia coli aldo-keto reductases

Methylglyoxal (MG) is a toxic metabolite known to accumulate in various cell types. We detected in vivo conversion of MG to acetol in MG-accumulating Escherichia coli cells by use of (1)H nuclear magnetic resonance ((1)H-NMR) spectroscopy. A search for homologs of the mammalian aldo-keto reductases (AKRs), which are known to exhibit activity to MG, revealed nine open reading frames from the E. coli genome. Based on both sequence similarities and preliminary characterization with (1)H-NMR for crude extracts of the corresponding mutant strains, we chose five genes, yafB, yqhE, yeaE, yghZ, and yajO, for further study. Quantitative assessment of the metabolites produced in vitro from the crude extracts of these mutants and biochemical study with purified AKRs indicated that the yafB, yqhE, yeaE, and yghZ genes are involved in the conversion of MG to acetol in the presence of NADPH. When we assessed their in vivo catalytic activities by creating double mutants, all of these genes except for yqhE exhibited further sensitivities to MG in a glyoxalase-deficient strain. The results imply that the glutathione-independent detoxification of MG can occur through multiple pathways, consisting of yafB, yqhE, yeaE, and yghZ genes, leading to the generation of acetol.     

Antifungal mechanism of SMAP-29 (1-18) isolated from sheep myeloid mRNA against Trichosporon beigelii

The antifungal activity and mechanism of SMAP-29 (1-18) (SMAP-29), a cathelicidin-derived antimicrobial peptide deduced from N-terminal sequence of sheep myeloid mRNA, were investigated. SMAP-29 displayed a strong antifungal activity against various fungi. To understand the antifungal mechanism(s) of SMAP-29, we examined the interaction of SMAP-29 with the pathogenic fungus Trichosporon beigelii. Confocal microscopy showed that SMAP-29 was localized in the plasma membrane. The antifungal effects of SMAP-29 were further confirmed by using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a plasma membrane probe. Flow cytometric analysis revealed that SMAP-29 acted in an energy-dependent manner. This interaction is also dependent on the ionic environment. Furthermore, SMAP-29 caused significant morphological changes when testing the membrane disrupting activity using liposomes (phosphatidylcholine/cholesterol; 10:1, w/w), as shown by scanning electron microscopy. The results suggest that SMAP-29 may exert its antifungal activity by disrupting the structure of cell membranes, via direct interaction with the lipid bilayers and irregularly disrupted fungal membranes in an energy- and salt-dependent manner.     

Obesity Is Independently Associated with Spinal Anesthesia Outcomes: A Prospective Observational Study

The influence of body-mass index (BMI) on spinal anesthesia is still controversial, with discrepant results reported in previous studies. To compare spinal anesthesia in obese and non-obese subjects, the anesthesia profiles in patients who underwent spinal anesthesia using intrathecal hyperbaric bupivacaine were compared. A total of 209 patients undergoing elective total knee replacement arthroplasty (TKRA) surgery under spinal anesthesia were divided into an NO (non-obese) group (BMI < 30 kg/m², n = 141) and an O (obese) group (BMI ≥ 30 kg/m², n = 68). Anesthesia was deemed successful if a bilateral T12 sensory block occurred within 15 minutes of intrathecal drug administration, and if the level of sensory block was higher than T12 when the surgery ended. Logistic regression analysis with multiple variables known to influence spinal anesthesia was performed to identify which parameters independently determined the spinal anesthesia outcome. Similar doses of bupivacaine were administered to the NO and O groups. The incidence of anesthesia failure was significantly lower in the O group [n = 43 (30.5%) in the NO group vs. n = 10 (18.9%) in the O group, p = 0.014]. The independent predictors for successful anesthesia in all patients were dose of hyperbaric bupivacaine [odds ratio (OR) 2.12, 95% CI: 1.64–2.73] and obese status (BMI ≥ 30 kg/m², OR 2.86, 95% CI: 1.25–6.52). Time to first report of postoperative pain and time to first self-void were significantly longer in the O group. These results suggest that the duration of block with hyperbaric bupivacaine is prolonged in obese patients and obesity is independently associated with spinal anesthesia outcomes, as is bupivacaine dosage. A further study enrolling patients with morbid obesity and using a fixed bupivacaine dosage is required to confirm the effect of obesity on spinal anesthesia.     

Electromechanical Properties of Polymer Electrolyte‐Based Stretchable Supercapacitors

Aligned carbon nanotube (CNT) forests filled with a dehydrated polymer electrolyte are used to fabricate flexible solid state supercapacitors (SSCs) for multifunctional structural‐electronic applications. Local stiffness measurements on the composite electrodes determined through nano­indentation showed an 80% increase over the neat solid polymer electrolyte matrix. Electrochemical properties are monitored as a function of average tensile strain in the SSCs. Galvanostatic charge‐discharge tests with in situ microtensile testing on SSCs are used to show a 10% increase in the specific capacitance through the elastic region of the composite. The increase in capacitance is partly attributed to the enhanced double layer interaction that results from the partial alignment of the polymer electrolyte chains at the electrode‐electrolyte interface. When soaked in 1 m sulfuric acid, the specific capacitance of the CNT‐polymer electrolyte reached approximately 72 F g^–1 at 60 °C.     

Wild ginseng attenuates repeated morphine-induced behavioral sensitization in rats

Many studies have suggested that the behavioral and reinforcing effects of morphine are induced by hyperactivation of the mesolimbic dopaminergic system, which results in increases in locomotor activity, c-Fos expression in the nucleus accumbens (NAc), and tyrosine hydroxylase (TH) in the ventral tegmental area (VTA). In order to investigate the effect of wild ginseng (WG) on treating morphine addiction, we examined the behavioral sensitization of locomotor activity and c-Fos and TH expression in the rat brain using immunohistochemistry. Intraperitioneal injection of WG (100 and 200 mg/kg), 30 min before administration of a daily injection of morphine (40 mg/kg, s.c.), significantly inhibited morphine-induced increases in c-Fos expression in NAc and TH expression in VTA as well as in locomotor activity, as compared with Panax ginseng. It was demonstrated that the inhibitory effect of WG on the behavioral sensitization after repeated exposure to morphine was closely associated with the reduction of dopamine biosynthesis and postsynaptic neuronal activity. It suggests that WG extract may be effective for inhibiting the behavioral effects of morphine by possibly modulating the central dopaminergic system and that WG might be a useful resource to develop an agent for preventing and treating morphine addiction.     

GPA-9 is a novel regulator of innate immunity against Escherichia coli foods in adult Caenorhabditis elegans

Innate immune responses to pathogens are governed by the nervous system. Here, we investigated the molecular mechanism underlying innate immunity in Caenorhabditis elegans against Escherichia coli OP50, a standard laboratory C. elegans food. Longevity was compared in worms fed live or UV‐killed OP50 at low or high density food condition (HDF). Expression of the antimicrobial gene lys‐8 was approximately 5‐fold higher in worms fed live OP50, suggesting activation of innate immunity upon recognition of OP50 metabolites. Lifespan was extended and SOD‐3 mRNA levels were increased in gpa‐9‐overexpressing gpa‐9XS worms under HDF in association with robust induction of insulin/IGF‐1 signaling (IIS). Expression of ins‐7 and daf‐28 that control lys‐8 expression was reduced in gpa‐9XS, indicating that GPA‐9‐mediated immunity is due in part to ins‐7 and daf‐28 downregulation. Our results suggest that OP50 metabolites in amphid neurons elicit innate immunity through the IIS pathway, and identify GPA‐9 as a novel regulator of both the immune system and aging in C. elegans.