Biochemical characterization of Bombyx mori α-N-acetylgalactosaminidase belonging to the glycoside hydrolase family 31.

Horizontal gene transfer is an important evolutionary mechanism not only for bacteria but also for eukaryotes. In the domestic silkworm Bombyx mori, a model species of lepidopteran insects, some enzymes are known to have been acquired by horizontal transfer; however, the enzymatic features of protein BmNag31, belonging to glycoside hydrolase family 31 (GH31) and whose gene was predicted to be transferred from Enterococcus sp. are unknown. In this study, we reveal that the transcription of BmNag31 increases significantly during the prepupal to pupal stage, and decreases in the adult stage. The full-length BmNag31 and its truncated mutants were heterologously expressed in Escherichia coli and characterized. Its catalytic domain exhibits α-N-acetylgalactosaminidase activity and the carbohydrate-binding module family 32 domain shows binding activity towards N-acetylgalactosamine, similar to the Enterococcus faecalis homolog, EfNag31A. Gel filtration chromatography and blue native polyacrylamide gel electrophoresis analyses indicate that BmNag31 forms a hexamer whereas EfNag31A is monomeric. These results provide insights into the function of lepidopteran GH31 α-N-acetylgalactosaminidase.

The combined effect of socioeconomic status and metabolic syndrome on depression: the Korean National Health and Nutrition Examination Survey (KNHANES).

BACKGROUND: Depression shows different patterns depending on socioeconomic status (SES) and metabolic syndrome (MS). However, the nature of this association remains poorly understood. The aim of this study was to examine whether the combination of MS and lower SES was associated with the prevalence of depression, based on data from the Korea National Health and Nutrition Examination Survey (KNHANES). METHODS: Data were obtained from a cross-sectional study of 24,102 adults (> 19 years of age) who participated in the KNHANES during 2008-2013 and for whom MS and depression data were available. MS was defined using the diagnostic criteria of the modified National Cholesterol Education Program Adult Treatment Panel III. Measure of depression was ascertained from self-reports of physician diagnosis. Multiple logistic regression analysis was used to evaluate the association between depression and MS as well as SES (alone and in combination). RESULTS: Overall, 622 of the 24,102 subjects (2.6%) met the criteria for depression. The prevalence of depression was associated with MS, a lower high-density lipoprotein cholesterol level, an elevated triglyceride level, a lower education level, and a lower household income. Participants with MS and a low SES had a higher likelihood of depression than those without MS and a high SES (odds ratio [OR] = 4.180 for low education level and OR = 3.994 for low household income level). CONCLUSIONS: This study suggests that the combination of SES and MS may play an important role in depression, which has implications for healthcare policy and depression management.

Evidence for the role of INSL3 on sperm production in boars by passive immunisation.

Insulin-like factor 3 (INSL3), previously called relaxin-like factor, is essential for foetal testis descent and has been implicated in sperm production in adult males. This study investigated the role of INSL3 in sperm production by examining the effect of neutralising INSL3 by passive immunisation on testicular function and sperm output in boars. Six male Duroc boars were randomly assigned to passive immunisation and control groups (n = 3 each). The immunisation group was intravenously injected with an IgG fraction of anti-INSL3 antibody developed against the B domain of INSL3 at 2-week intervals from 21-40 weeks of age. The control group was treated with normal IgG in the same manner. Antibody administration reduced testis weight and caused a fourfold increase in the frequency of apoptotic germ cells, which was associated with upregulation of the pro-apoptotic caspase 3 and BAX, and downregulation of the anti-apoptotic XIAP and BCL2, and a substantial marked reduction in sperm concentration. Neutralising INSL3 delivered by passive immunisation reduced testis weight and sperm concentration by inducing germ cell apoptosis, suggesting that INSL3 acts as a germ cell survival/anti-apoptotic factor in the maintenance of sperm production.

Proposed mechanism in the change of cellular composition in the outer medullary collecting duct during potassium homeostasis.

Potassium depletion (K⁺-D) induces hypertrophy and hyperplasia of collecting duct cells, and potassium repletion (K⁺-R) induces regression of these changes. The purpose of this study was to examine the time courses of the changes in cellular composition, the origin of intercalated cells (ICs) and the mechanism responsible for these changes. SD rats received K⁺-depleted diets for 1, 7, or 14 days. After K⁺-D for 14 days some of the rats received normal diets for 1, 3, 5, or 7 days. In the inner stripe of the outer medulla, K⁺-D increased significantly the number and proportion of H⁺-ATPase-positive ICs, but decreased the proportion of H⁺-ATPase-negative principal cells (PCs). However, proliferation was limited to H⁺-ATPase-negative PCs. During K⁺-R, the cellular composition was recovered to control level. Apoptosis increased during K⁺-R and exclusively limited in H⁺-ATPase-negative PCs. Double immunolabeling with antibodies to PC and IC markers identified both cells negative or positive for all markers during both K⁺-D and K⁺-R. Electron microscopic observation showed that ultrastructure of AE1-positive some cells were similar to AE1-negative some cells during K⁺-R. LC3 protein expression increased significantly and autophagic vacuoles appeared particularly in PCs on days 14 of K⁺-D and in ICs on days 3 of K⁺-R. These results suggest that PCs and ICs may interconvert in response to changes in dietary K+ availability and that autophagic pathways may be involved in the interconversion.

The arcuate NPY, POMC, and CART expressions responding to food deprivation are exaggerated in young female rats that experienced neonatal maternal separation.

This study was conducted to examine the effect of neonatal maternal separation on the hypothalamic feeding peptides expression in young female offspring. Sprague-Dawley pups were separated from dam for 3h daily during PND 1-14 (MS), or left undisturbed except routine cage cleaning (NH). Weanling female pups were housed in group and the arcuate mRNA levels of neuropeptide Y (NPY), proopiomelanocortin (POMC), and cocaine-amphetamine regulated transcript (CART) were examined at two months of age with or without food deprivation. The basal arcuate expression levels of these peptides did not differ between NH and MS group. However, a 48 h of food deprivation significantly increased NPY mRNA level, and decreased POMC and CART, in the arcuate nucleus of MS females, but not in NH females. Fasting-induced elevation of the plasma corticosterone tended to be greater in MS group than in NH, but the basal levels did not differ between the groups. Plasma leptin levels were decreased in MS females compared with NH, and food deprivation significantly suppressed the leptin levels both in NH and MS groups. Results suggest that MS experience may increase stress vulnerability in female rats and exaggerate the feeding peptides expression in the arcuate nucleus responding to metabolic stress food deprivation.

The effect of combining dexamethasone with ondansetron for nausea and vomiting associated with fentanyl-based intravenous patient-controlled analgesia.

We investigated whether combined dexamethasone and ondansetron is more effective than ondansetron alone in preventing postoperative nausea and vomiting in patients with fentanyl-based intravenous patient-controlled analgesia. One hundred and thirty patients undergoing video-assisted thoracoscopic surgery were assigned to either an ondansetron group or a dexamethasone and ondansetron group. In all patients, ondansetron 4 mg was administered at the end of surgery and 12 mg was added to the patient-controlled analgesia solution. The dexamethasone and ondansetron group received dexamethasone 8 mg at the induction of anaesthesia. The overall incidence of nausea and vomiting during the first 48 h postoperatively did not differ between groups (34/61 (56%) vs 28/62 (45%) in the ondansetron group and dexamethasone and ondansetron groups, respectively). The incidence of severe nausea and vomiting (≥ 7 nausea on an 11-point verbal numerical rating scale, retching or vomiting) was higher in the ondansetron group than in the dexamethasone and ondansetron group (15/61 (25%) vs 6/62 (10%, respectively, p=0.028). Combined dexamethasone and ondansetron is more effective in reducing severe nausea and vomiting than ondansetron alone in patients receiving fentanyl-based intravenous patient-controlled analgesia.

Tracheal intubation using the Airtraq: a comparison with the lightwand.

SUMMARY: The Airtraq laryngoscope is a new tracheal intubation device that has been developed for the management of normal and difficult airways. As with the lightwand, the Airtraq can be used without placing the patient in the 'sniffing position' for direct laryngoscopy. The purpose of this study was to compare the efficacy and usability of the Airtraq with that of the lightwand during routine airway management. One hundred ASA 1-2 patients scheduled for elective surgery under general anaesthesia were randomly assigned to either the Airtraq (n = 50) or lightwand (n = 50). Intubation was performed by one of two anaesthetists experienced in the use of both devices. There was no difference in success rate, intubation time, and haemodynamic response between the two groups. In conclusion, the Airtraq and lightwand have similar efficacy in patients without risk factors for difficult intubation.

The effect of pneumoperitoneum in the steep Trendelenburg position on cerebral oxygenation.

BACKGROUND: daVinci robot-assisted laparoscopic radical prostatectomy (RALP) requires pneumoperitoneum in the steep Trendelenburg position, which results in increased intracranial pressure and cerebral blood flow. The aim of this study was to evaluate the effect of pneumoperitoneum in a 30 degrees Trendelenburg position on cerebral oxygenation using regional cerebral oxygen saturation (rSO2). METHODS: Thirty-two male patients of ASA I and II physical status without previous episodes of cerebral ischemia or hemorrhage undergoing daVinci RALP were enrolled. The rSO2 was continuously monitored with near-infrared spectroscopy (INVOS) 5100) during the study period. Measurements were obtained immediately after anesthesia induction (T0; baseline), 5 min after a 30 degrees Trendelenburg position (T1), 5 min after 15 mmHg pneumoperitoneum in a supine position (T2), 30, 60 and 120 min after the pneumoperitoneum in a Trendelenburg position (T3, T4 and T5, respectively) and after desufflation in a supine position (T6). RESULTS: The change in the left and right rSO2 was statistically significant (Left P=0.004 and Right P=0.023). Both the right and the left rSO2 increased significantly during pneumoperitoneum in a Trendelenburg position (from T3 to T5) and at T6 compared with the baseline value at T0. The partial pressure of carbon dioxide (PaCO2) was increased significantly at T2, T3, T5 and T6 compared with the baseline value at T0. CONCLUSIONS: During daVinci RALP, cerebral oxygenation, as assessed by rSO2, increased slightly, which suggests that the procedure did not induce cerebral ischemia. The PaCO2 should be maintained within the normal limit during pneumoperitoneum in a Trendelenburg position in patients undergoing daVinci RALP because the rSO2 increased in conjunctions with the increase in PaCO2.

Effect of epidural saline washout on regression of sensory and motor block after epidural anaesthesia with 2% lidocaine and fentanyl in elderly patients.

Seventy elderly males received lumbar epidural anaesthesia with 12 ml of 2% lidocaine containing fentanyl 50 mug. At the end of transurethral surgery, the washout group (n = 33) received an epidural bolus of 30 ml saline while the control group (n = 34) did not. Mean (SD) times to 1-grade (17.2 (11.9) vs 32.7 (11.3) min) and 2-grade regression (23.8 (12.2) vs 56.0 (23.9) min) of motor block, 3-dermatomal sensory regression (31.4 (11.6) vs 42.2 (14.4) min for cold and 30.8 (15.6) vs 40.6 (14.2) min for pinprick), and regression to S1 (57.7 (16.1) vs 76.2 (20.2) min for cold and 56.8 (17.3) vs 69.2 (16.2) min for pinprick) were significantly shorter in the washout group than the control group. There were no differences in postoperative pain scores and side effects between the two groups. We concluded that epidural washout facilitates regression of both motor and sensory block following epidural anaesthesia without reducing the postoperative analgesic benefit.

Caudal midazolam does not affect sevoflurane requirements and recovery in pediatric day-case hernioplasty.

BACKGROUND: This prospective, randomized, double-blind study aimed to determine whether caudal midazolam combined with ropivacaine affects anesthetic requirements, recovery profiles, and post-operative analgesia compared with ropivacaine alone in pediatric day-case hernioplasty. METHODS: Sixty boys (2-5 years old) received caudal injections of 0.2% ropivacaine 1 ml/kg and epinephrine 1 : 200,000 with (RM group) or without (R group) 50 microg/kg of midazolam under sevoflurane anesthesia. The sevoflurane requirement was determined by adjusting to a bispectral index score=50. RESULTS: Concentrations of end-tidal sevoflurane (ETsevo%) after induction were similar in both groups. After caudal block, ETsevo% before and after surgical stimuli did not show significant intra- or intergroup differences. Recovery characteristics, including post-operative sedations, were similar in both groups. Post-operative pain scores were significantly lower in the RM group than the R group. CONCLUSIONS: Caudal midazolam (50 microg/kg) added to 2% ropivacaine did not influence sevoflurane requirement or recovery but improved post-operative analgesia compared with ropivacaine alone in pediatric day-case hernioplasty.

Diffusive coupling can induce synchronized periodic activity in neural networks.

Diffusive coupling also known as nearest-neighbor coupling is a common form of coupling but its role in the behavior of neural circuits is unclear. Previous experimental and theoretical studies have shown that potassium lateral diffusion coupling (i.e., diffusive coupling) was responsible for synchronization of neuronal activity. We tested the hypothesis that potassium lateral diffusion coupling is required to generate periodic epileptiform activity in a zero-Ca(2+) CA1 pyramidal neuron network model. The simulation results show that potassium lateral diffusion coupling is crucial for establishing a periodic synchronized epileptiform activity similar to that observed in experimental preparations. This results suggest that potassium lateral diffusion coupling - a physiological realization of the concept of diffusive coupling - can play a role in network behavior.

Isolation of Ashbya gossypii mutant for an improved riboflavin production targeting for biorefinery technology.

AIMS: To isolate a strain overproducing riboflavin and to improve riboflavin production for practical use in a biorefinery technology. METHODS AND RESULTS: Ashbya gossypii spores were mutagenized by exposure to UV light and mutant ZP4 strain, producing riboflavin threefold the riboflavin that of the wild-type strain, was isolated by the first and second screenings. Proteomic analysis of ZP4 strain showed the expression patterns of eight types of genes related to riboflavin biosynthesis different from those of the wild-type strain and those enzyme activities were investigated. When activated bleaching earth (ABE) containing 75 g l(-1) rapeseed oil was added in the culture of the ZP4 strain with oxygen-enriched air supplied, riboflavin concentration increased to 8.7 g l(-1) at 5 days of culture. Riboflavin production yield was 0.17 g g(-1) of consumed oil, which was eightfold higher than that of the wild-type strain. CONCLUSIONS: The results show that the mutant ZP4 strain shows potential for improving riboflavin production for practical utilization using vegetable oil as the sole carbon source. SIGNIFICANCE AND IMPACT OF STUDY: Our results indicate that the mutant ZP4 strain shows potential for producing riboflavin from vegetable oil, and therefore will be contributed to biorefinery technology.

Effects of amino acid on morphological development and nucleus formation of arachidonic acid-producing filamentous micro-organism, Mortierella alpina.

AIMS: Effects of amino acid on morphological development and nucleus formation of arachidonic acid-producing filamentous micro-organism, Mortierella alpina were investigated using flow-through chamber. METHODS AND RESULTS: Mortierella alpina CBS 754.68 was cultivated in flow through chamber using nutrient-rich, minimal and specific amino acid-containing minimal media. To investigate the effect of amino acid on morphological parameters either 0.28 g l(-1) alanine, 0.53 g l(-1) sodium glutamate one hydrate or 0.42 g l(-1) valine was added to the minimal medium. In a flow-through chamber, the growth of hyphal elements and nucleus formation of arachidonic acid-producing fungus M. alpina were studied on-line, using image analysis techniques. When the Ala- and Val-containing media were used, the hyphal growth units (HGUs) were 90.2 and 86.7 microm per tip, respectively, which were 2.4-fold higher than that in the nutrient-rich medium, indicating that Ala and Val stimulate the elongation of hyphae. The specific nucleus formation rates were Glu->Val-containing media>minimal and nutrient-rich media>Ala-containing medium. The nucleus doubling times in Glu- and Val-containing media were 1.9 and 2 h, respectively, which were not significant different. CONCLUSIONS: Ala and Val stimulate the elongation of M. alpina hyphae, and nucleus formation rates were Glu->Val->Ala-containing media. SIGNIFICANCE AND IMPACT OF THE STUDY: Formation of fungal morphology and nucleus were shown using the flow-through chamber coupled with image analysis, which making possible to discuss the relationship between mycelial morphology and nucleus formation of M. alpina.

Monitoring of morphological development of the arachidonic-acid-producing filamentous microorganism Mortierella alpina.

Morphological parameters, such as hyphal growth rate, tip formation rate, tip extension rate and branch formation rate, of Mortierella alpina have been measured using a flow-through chamber under 25 different combinations of carbon and nitrogen concentrations. Morphological parameters were influenced not by C/N ratio but by carbon concentration in the medium. Specific rates of hyphal growth and tip formation both remained constant at a low carbon concentration of 5 g/l. Tip extension rate from one tip was 60 microm tip(-1) h(-1) at a carbon concentration below 15 g/l, and the branching formation rate was independent of carbon concentration. Tip extension rate was a function of specific hyphal growth rate, which in turn was linearly proportional to the specific tip formation rate, demonstrating that tip extension rate was exponentially proportional to the specific tip formation rate. Branch formation rate per hyphal element remained unchanged even at tip extension rates lower than 60 microm tip(-1) h(-1) and at specific hyphal growth rates lower than 0.83 h(-1), but decreased drastically at higher rates of tip extension and hyphal growth.

Nociceptin/orphanin FQ increases ANP secretion in neonatal cardiac myocytes.

Nociceptin (N/OFQ) is a novel heptadecapeptide with an amino acid sequence similar to that of endogenous opioid peptide dynorphin A. Dynorphin have been reported to increase the secretion of atrial natriuretic peptide (ANP) via selective activation of kappa-opioid receptor in cultured atrial cardiocytes. The present study was designed to investigate the direct effect of N/OFQ on the ANP secretion in cultured neonatal rat cardiac myocytes via N/OFQ receptor (NOP) activation. The secretion of ANP from cultured neonatal cardiac myocytes was increased in terms of incubation time. N/OFQ, at a dose of 0.3, 1, 3, and 10 microM, caused increases in ANP secretion in a dose-dependent manner. The N/OFQ-induced ANP secretion was completely antagonized by antagonists of NOP, 1 microM each of [Phe1 (CH2-NH) Gly2] nociceptin (1-13)-NH2 ([FG]N/OFQ(1-13)NH2) or naloxone benzoylhydrazone. In contrast, naloxone (1 microM), the non-selective opioid receptor antagonist, did not alter ANP response to N/OFQ. N/OFQ at 3 microM inhibited basal and forskolin-stimulated cAMP production, which was partially antagonized with the pretreatment of [FG]N/OFQ(1-13)NH2. An increase in ANP secretion by N/OFQ was also partially blocked by the pretreatment of forskolin. Homologous competition studies in neonatal cardiomyocyte membranes revealed the presence of two distinct sites. The high affinity site (10.9 +/- 1.6 nM) was far less abundant than the low affinity site. Therefore, these results suggest that N/OFQ causes an increase in ANP secretion in cultured neonatal cardiac myocytes by decreasing cAMP through its binding sites.

4-Hydroxy-6-oxo-6,7-dihydro-thieno[2,3-b] pyrimidine derivatives: synthesis and their biological evaluation for the glycine site acting on the N-methyl-D-aspartate (NMDA) receptor.

Bioisostere approach has been shown to be useful to augment potency or to modify certain physiological properties of a lead compound. Based upon well documented bioisosterism, an isosteric replacement of benzene ring of 4-hydroxy-2-quinolone compound (L-695902) with a thiophene moiety was carried out to prepare the title compounds, 4-hydroxy-6-oxo-6,7-dihydro-thieno[2,3-b] pyrimidines 15. The resulting bioisosteric compounds 15 were evaluated for their antagonistic activity (binding assay) for NMDA receptor glycine site.

Mycelial pellet intrastructure and visualization of mycelia and intracellular lipid in a culture of Mortierella alpina.

The intrastructure of mycelial pellets of Mortierella alpina, which accumulate fatty acids in mycelia, was visualized following labeling with fluorescein isothiocyanate (FITC) and Nile red using fluorescence microscopy. The pellet was an ellipse shape, but its intrastructure was shaped as a doughnut with a cave inside. Using three-dimensional image analysis, it was shown that the lipid was produced on the edge of the pellet, which corresponded to the area where the mycelial density was high. The cavity ratio of the pellet section was determined on the basis of the FITC fluorescence intensity, and in the early culture stage remained at 0.2 in a 10-kl fermentor culture, but finally increased to 0.35. Mycelial pellet volume paralleled the cavity ratio. Application of the technique used here allows analysis of the intrastructure of fungal pellets and new types of fungal biological study.

Interleukin 12 gene therapy of cancer by peritumoral injection of transduced autologous fibroblasts: outcome of a phase I study.

A phase I dose-escalation clinical trial of peritumoral injections of interleukin 12 (IL-12)-transduced autologous fibroblasts was performed in patients with disseminated cancer for whom effective treatment does not exist. The goals of this study were to assess the safety and toxicities as well as the efficacy, and ancillarily the immunomodulatory effects, of peritumoral IL-12 gene transfer. Primary dermal fibroblasts cultured from the patients were transduced with retroviral vector carrying human IL-12 genes (p35 and p40) as well as the neomycin phosphotransferase gene (TFG-hIL-12-Neo). Patients received four injections at intervals of 7 days. Nine patients were enrolled in this dose-escalation study, with secreted IL-12 doses ranging from 300 ng/24 hr for the first three patients to 1000, 3000, and 5000 ng/24 hr for two patients in each subsequent dosage level. Although a definite statement cannot be made, there appears to be perturbation of systemic immunity. Also, the locoregional effects mediated by tumor necrosis factor alpha (TNF-alpha) and CD8+ T cells were observed with tumor regression. Treatment-related adverse events were limited to mild to moderate pain at the injection site; clinically significant toxicities were not encountered. Transient but clear reductions of tumor sizes were observed at the injected sites in four of nine cases, and at noninjected distant sites in one melanoma patient. Hemorrhagic necrosis of tumors was observed in two melanoma patients. These data indicate that gene therapy by peritumoral injection of IL-12-producing autologous fibroblasts is feasible, and promising in patients with advanced cancer.

Enzymatic hydrolysis of waste office paper using viscosity as operating parameter.

Enzymatic hydrolysis of waste office (WO) paper with feeding WO paper in a reactor was investigated using apparent viscosity as operating parameter. Since the apparent viscosity was correlated with the concentration of pulping WO paper, the amount of hydrolyzed WO paper was assumed by measuring the decrease in the apparent viscosity. Then the amount of hydrolysis WO paper and the amount of enzyme corresponding to the desired ratio were fed into the reactor. When the WO paper and 1% (to the amount of WO paper) enzyme were fed to the hydrolytic reaction, 87 g/L of reducing sugar (RS) with a hydrolytic yield of 42.2% was obtained for a 24-h hydrolysis. However, when nonpulping WO paper and 5% (to the amount of WO paper) enzyme were fed to the hydrolytic reaction, 120 g/L of RS with a hydrolytic yield of 40% was obtained for a 24-h hydrolysis. Therefore, the RS concentration from this hydrolysis process feeding WO paper using apparent viscosity as operating parameter may be of sufficient concentration to serve as a carbon source in microorganism culture or chemical feedstock.

Influence of ligand binding to human cytochrome P-450 1A2: conformational activation and stabilization by alpha-naphthoflavone.

Human cytochrome P-450 (P-450) 1A2 expressed in Escherichia coli is readily converted into non-native cytochrome P-420 (P-420) in the presence of detergents. alpha-Naphthoflavone (ANF) has been used to prevent P-450 1A2 inactivation to P-420 during purification. However, the mechanism by which ANF modulates P-450 1A2 is not clearly understood. We observed that recombinant human P-450 1A2 prepared in the absence of ANF has an approx. 5 times higher maximum catalytic activity in the O-deethylation of 7-ethoxycoumarin than that in the presence of ANF, with the same K(m) values. The results revealed that the enzyme purified with ANF is not catalytically fully active, indicating that ANF tightly binds to the enzyme, only to be dissociated by heat denaturation. Furthermore, the inactive P-420 form of the enzyme could be reconverted to P-450 by ANF in high concentrations of detergents. The reconversion was concentration-dependent, confirming ANF-induced regeneration of active P-450 1A2. The reconversion coincided with the conformational change of the enzyme including increased alpha-helix content. The conformation of P-450 1A2 was also stabilized by ANF, resulting in an approx. 5 degrees C increase in thermal stability.