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.

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.

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.

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.

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.

Dependence of apparent viscosity on mycelial morphology of Streptomyces fradiae culture in various nitrogen sources.

To examine what causes increased viscosity in culture broth in Streptomyces fradiae culture, various natural nitrogen sources were investigated. Extracellular protease activity increased with culture time and decomposed the natural nitrogen source into amino acids. In the case of gluten meal, after a culture time of 5 d, concentrations of glutamic acid and aspartic acid had increased to 600 and 200 mg/L, respectively, which were about 3- and 2-fold as high as levels in cultures under similar conditions using Pharmamedia. For various amino acids tested, the addition of glutamic acid or aspartic acid mixture to the culture medium raised the apparent viscosity to its highest demonstrated value, 260 mPa.s after 5 d of culture, which was 3-fold higher than without amino acids. Consumption of the decomposed glutamic acid and aspartic acid was dependent on the activities of glutamate dehydrogenase and aspartate aminotransferase, respectively. When ammonium ion was used as the nitrogen source, cell concentration reached 1.75 g/L measured as an intracellular nucleic acid concentration, which was about 2.3-fold higher than that with any other natural nitrogen source. However, apparent viscosity was only 75 mPa.s, a value one-third that of the amino acid mixture, and 70% of the pellets were bigger than 1.2 x 10(4) microm(2). In the case of gluten meal or the amino acid mixture, pellets bigger than 1.2 x 10(4) microm(2) comprised only 8%. This demonstrates that consumption of some amino acids affected the formation of filamentous morphology, which caused an increase in the apparent viscosity of the culture broth, and the apparent viscosity was not caused by the mycelial concentration but the mycelial morphology.

Efficient production of L-(+)-lactic acid using mycelial cotton-like flocs of Rhizopus oryzae in an air-lift bioreactor.

L-(+)-Lactic acid production was enhanced in a culture of Rhizopus oryzae by induction of a mycelial flocs morphology. By conventional culture the morphology of R. oryzae is that of a pellet-like cake; however, when mineral support and poly(ethylene oxide) are added to the culture, the morphology of R. oryzae takes on a cotton-like appearance. The formation of these cotton-like mycelial flocs was induced by the addition of 5 ppm poly(ethylene oxide) into a 12-14 h culture containing 3 g/L of the mineral support before the formation of the conventional pellet morphology. The cotton-like flocs were also formed in cultures grown in an air-lift bioreactor. This morphology allowed effective mass transfer inside the flocs and effective fluidity of culture broth in an air-lift bioreactor. L-(+)-Lactic acid concentration produced by mycelial flocs in an air-lift bioreactor, with the support and poly(ethylene oxide), was 104.6 g/L with a yield of 0.87 using 120 g/L of glucose as the substrate; for this culture without both, the concentration was 43.2 g/L. These results demonstrate that cotton-like mycelial flocs are the optimal morphology for use in the air-lift bioreactor culture of R. oryzae.

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.

Alterations in irregular and fractal heart rate behavior in growth restricted fetuses.

OBJECTIVES: To determine whether irregularity, and short- and long-term fractal correlation reflecting smoothness of heart rate behavior are changed in intrauterine growth restricted fetuses. STUDY DESIGN: Fetal heart rate data of 192 normal fetuses and 86 intrauterine growth restricted fetuses between 31 and 42 weeks of gestation were studied. Approximate entropy to quantify irregularity, and short-term (< or =80 beats, alpha1) and long-term (>80 beats, alpha2) fractal scaling exponents to quantify the short- and long-term fractal correlation were calculated. RESULTS: In the intrauterine growth restricted fetuses, the approximate entropy was significantly lower (P<0.001). The alpha2 value was significantly higher (P=0.0001) than in the normal fetuses, which performed better (F=34.2, P<0.0001) than other heart rate variability indexes in differentiating the intrauterine growth restricted fetuses from the normal fetuses in stepwise discriminant analysis. CONCLUSIONS: Compared to normal fetuses, intrauterine growth restricted fetuses show a difference in that the irregularity of the fetal heart is decreased. A more apparent difference is that the long-term fractal correlation of the fetal heart is increased and the fetal heart is smoother in the long-term scale.

Kinetics of soybean oil consumption and cephamycin C production in culture of Streptomyces sp. using mineral support.

A simple kinetics of soybean oil consumption and cephamycin C production in Streptomyces sp. culture using a mineral support is proposed in this study. The mineral support was used for both suspending the soybean oil as fine oil droplets and immobilizing mycelia. The optimum concentrations of oil and mineral support for obtaining the maximum cephamycin C production were determined to be 50 and 15 g/l, respectively, by the proposed kinetics. At the optimal concentrations, the concentration of cephamycin C estimated from the proposed model and from the experimental data was 2.82 and 2.80 g/l, respectively. The results of the simulation coincided well with the experimental data for various concentrations of the soybean oil and the support. This demonstrates that our model can explain the kinetics of a culture using vegetable oil as the carbon source and mineral support for both oil suspension and mycelial immobilization.

Image analysis of morphological change during arachidonic acid production by Mortierella alpina 1S-4.

The changes in mycelial morphology during arachidonic acid (AA) production by Mortierella alpina 1S-4 were investigated using an image analysis system. Cultivation was performed in a 10-kl fermentor, and the culture broth was separated into two fractions by sieving (0.5 mm aperture size): the filament fraction (F-fraction, <0.5 mm), and the pellet fraction (P-fraction, >0.5 mm). The effect of the mycelial morphology in each fraction on AA production was analyzed. As a result, a product distribution in the culture broth wherein the AA content in the mycelia of the P-fraction was observed to be higher than that in the mycelia of the F-fraction throughout the cultivation. Morphological analysis of the P-fraction revealed that the hairy pellets became smooth because the mycelia on the pellet surface were shaved off; some pellets were broken and reduced in size. The shaved-off mycelia from the hairy pellets surface moved into the F-fraction and aggregated there. From the above findings, it was likely that the low AA content in the F-fraction was due to mycelial damage during the cultivation. In addition, the morphology of the hairy pellets was found to contribute to an increase in the viscosity of culture broth.

Effect of nitrogen source on mycelial morphology and arachidonic acid production in cultures of Mortierella alpina.

The effects of nitrogen source on arachidonic acid (AA) production and morphological changes during the culture of Mortierella alpina were investigated using an image analysis system. When yeast extract, gluten meal, or corn steep liquor was used, a circular pellet morphology was obtained. However, when Pharmamedia, fish meal, or soybean meal was used, M. alpina formed radial filamentous mycelia. The radial filamentous area in the case of soybean meal was 75% of the total mycelial area. In a jar fermentor culture, M. alpina morphology varied with the cultivation period: (i) at 0-6 h culture, the inoculated pellet-like mycelia were adapted to the new environment, (ii) at 6 h-1 d culture, filamentous mycelia grew exponentially which yielded a feather-like morphology, (iii) at 1-2 d culture, the filamentous mycelia became disentangled as a result of the mechanical agitation; consequently, the proportion of filamentous mycelia was increased, (iv) at 2-4 d culture, mycelia showed stationary growth, but the AA concentration increased rapidly, and (v) at 4-6 d culture, hyphae grew thick radially with the AA concentration continuing to increase gradually. In the case of the cultures with feather-like morphology obtained using soybean meal, the AA yield was 0.14 g/g dry cell weight, which was two times higher than that in cultures grown using yeast extract. These results suggest that the feather-like morphology of culture of M. alpina is suitable for AA production.

Enhancement of epsilon-polylysine production by Streptomyces albulus strain 410 using pH control.

The enhancement of epsilon-poly-l-lysine (epsilon-PL) production by Streptomyces albulus strain no. 410 (S410) by means of a pH control strategy was investigated. S140 cells produce epsilon-PL at a high concentration if the culture pH remains at about 4.0; however, if it shifts to higher than 4.0, the accumulated epsilon-PL is depolymerized. We therefore suggest a pH control strategy for cell growth and epsilon-PL production aimed at increasing the amount of epsilon-PL produced. The cultivation was divided into two control phases. In phase I, cell growth was accelerated by maintaining the pH at higher than 5.0; in phase II, epsilon-PL production was increased by maintaining the pH at about 4.0. To avoid an increase in the pH during phase II as a result of glucose depletion, the glucose concentration was kept at around 10 g/l by glucose feeding. This control strategy enhanced the production of epsilon-PL to 48.3 g/l from 5.7 g/l in the case of batch culture.

Visualization of a recombinant gene protein in the baculovirus expression vector system using confocal scanning laser microscopy.

Expression of the recombinant protein beta-galactosidase in the Spodoptera frugiperda Sf-9 insect cell line infected by the Autographa californica nuclear polyhedrosis virus expressing beta-galactosidase (AcNPV-betagal) was visualized using confocal scanning laser microscopy with fluorescent staining of both the recombinant protein and the cell nucleus. The average size of the insect cells and the intracellular DNA concentration both increased markedly, respectively reading 3.8- and 2.3-fold the values before infection. The average beta-galactosidase activity began to increase at 20-24 h post infection and finally reached 1.9 x 10(4) units/ml. As the post infection time increased, the stained nucleus images expanded and spread broadly. Beta-galactosidase was first identified by fluorescent staining at 12 h post-infection, filled the cell at 27 h, began to be released at 36 h, and finally spread out of the cell. The locations of the nucleus and expressed beta-galactosidase were identified from computerized tomograms and 3-dimensional images.