Nutrient ileal digestibility evaluation of dried mealworm (Tenebrio molitor) larvae compared to three animal protein by-products in growing pigs.

OBJECTIVE: This study was to investigate the nutrient ileal digestibility of dried mealworm (Tenebrio molitor) larvae and compare with those of three animal protein by-products in growing pigs. METHODS: A total of 12 crossbred ([Landrace×Yorkshire]×Duroc) growing pigs with average body weights of 24.12±0.68 kg were surgically equipped with simple T-cannulas after being deprived of feed for 24 h according to published surgical procedures. These pigs had a recovery period of two weeks. A total of 12 pigs were assigned to individual metabolic crates and allotted to one of four treatments with 3 replicates in a fully randomized design. Dietary treatments included the following: i) Fish meal, corn-vegetable by-product basal diet+9.95% fish meal; ii) Meat meal, corn-vegetable by-product basal diet+9.95% meat meal; iii) Poultry meal, corn-vegetable by-product basal diet+9.95% poultry meal; iv) Tenebrio molitor, corn-vegetable by-product basal diet+9.95% dried Tenebrio molitor larvae. RESULTS: Results showed that the apparent ileal digestibility (AID) of Lys was higher (p<0.05) in pigs fed Tenebrio molitor diet than that in pigs fed fish meal diet. Pigs fed Tenebrio molitor diet showed increased (p<0.05) AID of His and Arg compared to pigs fed Fish meal or Meat meal diet. The AID of Cys was increased (p<0.05) in pigs fed poultry meal and Tenebrio molitor diets compared to that in pigs fish meal diet. Pigs fed meat meal, poultry meal, and Tenebrio molitor diets showed higher (p<0.05) standardized ileal digestibility (SID) of total energy compared to pigs fed fish meal diet. The SID of Arg was higher (p<0.05) in pigs fed Tenebrio molitor diet than that in pigs fed fish meal or meat meal diet. Furthermore, pigs fed poultry meal or Tenebrio molitor diets showed increased (p<0.05) SID of Cys compared to pigs fed fish meal diet. CONCLUSION: In conclusion, providing pigs with diets that contained Tenebrio molitor larvae meal improved AID and SID of nutrients as well as essential and non-essential amino acids. The digestibility of dried mealworm larvae protein and its utilization in vivo are also good. Therefore, dried mealworm larvae protein can be used as protein source at 10% level in growing pigs.

Variations in fat mass contribution to bone mineral density by gender, age, and body mass index: the Korea National Health and Nutrition Examination Survey (KNHANES) 2008-2011.

UNLABELLED: The relationship of body composition and bone mineral density is complex and controversial. When classifying Korean population based on gender, age, and body mass index, fat mass had varying contributions to bone mineral density. INTRODUCTION: The relationship between body composition and bone mineral density (BMD) is complex, and it is uncertain how components of body mass variably affect BMD. METHODS: This cross-sectional observational study was performed in subjects ≥20 years based on the Korea National Health and Nutrition Examination Survey (KNHANES) 2008 to 2011. Among 17,583 subjects, the mean ages were 49.1 ± 16.0 years (M, n = 7495) and 49.3 ± 16.3 years (F, n = 10,088). Subjects were divided into age groups, either <50 or ≥50 years for males, or menopausal state, either premenopausal or postmenopausal, for females. A further classification used BMI, either <25 or ≥25 kg/m(2). Anthropometric and body composition parameters were compared and evaluated to look for correlations with BMD. Further, appendicular lean mass (ALM), fat mass (FM), fat percentage (FP), and waist circumference (WC) were included for multivariate analysis with BMD, controlling for covariates in each age group and BMI subgroup. RESULTS: Anthropometric and body composition parameters significantly correlated with BMD in all age groups for both genders. After adjusting for covariates, ALM strongly affected BMD in all age groups for both genders. FM, FP, and WC significantly affected BMD in both age groups of women and in older men, but they did not affect BMD in younger men. Fat indices positively affected BMD of all sites in all non-obese women and in non-obese older men. However, little contribution was found in obese subgroups of both genders and in non-obese younger men. CONCLUSION: Considering different weights of covariates, ALM strongly contributed to BMD in all gender, age, and BMI groups. On the other hand, fat indices positively affected BMD of both age groups in women and older men with normal BMI, but they showed little contribution to BMD within the same age groups with high BMI or any BMI subgroups of younger men.

Diversity of Tn1546 in vanA-positive Enterococcus faecium clinical isolates with VanA, VanB, and VanD phenotypes and susceptibility to vancomycin.

AIM: To investigate diversity in the vanA cluster in Enterococcus faecium isolates from nontertiary hospitals. METHODS AND RESULTS: We identified 43 vanA-positive Ent. faecium isolates, including two vancomycin-susceptible isolates, from hospitals between 2003 and 2006. Of these isolates, >85% were resistant to ampicillin, erythromycin and ciprofloxacin. The vanA cluster was classified into six types using overlapping PCR, but the prototype transposon Tn1546 was not found. Most vanA-positive vancomycin-resistant Enterococcus (VRE) carried IS1216V and belonged to Type III (58·1%) or Type II (20·9%). vanY, vanZ and IS1216V were observed in the left and right ends of Type III with long-range PCR. IS1216V was also observed within vanS and vanX in the two vancomycin-susceptible isolates and in two vancomycin-resistant isolates. No VRE isolates with VanB and VanD phenotypes contained point mutations in vanS, unlike in previous reports. Sequence types (STs) of all isolates belonged to clonal complex 17, and ST78 was predominant. CONCLUSIONS: Insertion sequences, especially IS1216V, cause structural variation in the vanA cluster. We report the first observation of vanY and vanZ at the left end of Tn1546 in clinical isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the frequency of vancomycin resistance and diversity of Tn1546 in vanA-positive Ent. faecium isolates from nontertiary hospitals.

The effect of rosiglitazone on insulin sensitivity and mid-thigh low-density muscle in patients with Type 2 diabetes.

AIMS: We examined the effect of rosiglitazone on insulin sensitivity, abdominal fat and mid-thigh intramuscular fat distribution, and plasma concentrations of adipocytokines in patients with Type 2 diabetes. METHODS: Rosiglitazone was administered at a daily dose of 4 mg to 42 Type 2 diabetes patients [age 32-70 years, body mass index (BMI) 17.5-32.6 kg/m(2), 15 women, 27 men] for 12 weeks. Various anthropometric and metabolic profiles, plasma adiponectin, leptin, and resistin levels were measured, and insulin resistance was calculated from the short insulin tolerance test. Body fat composition was assessed by computed tomography. RESULTS: Twelve weeks' rosiglitazone treatment resulted in improved insulin resistance despite increases in body weight and BMI. There was a significant decrease in abdominal visceral adipose tissue area (145 +/- 65.6 vs. 129 +/- 73.1 cm(2), P = 0.049). Mid-thigh low-density muscle area (TLDMA) increased from 23 +/- 9.6 to 26 +/- 8.2 cm(2) (P = 0.009). There were significant changes in plasma adipocytokines, but they were not significantly correlated with changes in insulin resistance. CONCLUSIONS: Rosiglitazone treatment resulted in an improvement of insulin responsiveness in Type 2 diabetic subjects, which was associated with the redistribution of visceral and subcutaneous adipose tissue, an increase in TLDMA, and changes in serum adipocytokine levels. Further studies are needed to elucidate the insulin sensitizing mechanism of rosiglitazone on peripheral skeletal muscles.

Effects of phenyllactic acid on growth performance, intestinal microbiota, relative organ weight, blood characteristics, and meat quality of broiler chicks.

This study was conducted to determine the effects of dietary supplementation with phenyllactic acid (PLA) on growth performance, intestinal microbiota, relative organ weight, blood characteristics, and meat quality in broilers. A total of 500 male broilers (BW = 46.3 g) were randomly allotted into 1 of the following 5 dietary treatments: 1) basal diet (CON), 2) basal diet + 44 mg/kg of avilamycin (ANT), 3) basal diet + 0.2% PLA (PLA0.2), 4) basal diet + 0.4% PLA (PLA0.4), 5) basal diet + 0.2% PLA + 44 mg/kg of avilamycin (PA). Chicks fed PLA had lower feed intake (FI) from d 0 to 7 (P < 0.05) than those fed CON and ANT. From d 21 to 35, BW gain was greater in ANT, PLA0.4, and PA diets than CON and PLA0.2 diets (P < 0.05), whereas the FI was lowest in the PLA0.4 diet. Feed efficiency was depressed (P < 0.05) by the antibiotics and PLA supplementation during d 0 to 7, whereas it was improved (P < 0.05) in the PLA and ANT diets during d 21 to 35, with the best value in PLA0.4.The population of Escherichia coli in the large intestine was lower in the ANT, PLA0.4, and PA groups than the CON and PLA0.2 groups (P < 0.05). The relative weights of gizzard, liver, spleen, bursa of Fabricius, breast, and abdominal fat were unaffected by any of the dietary supplementations. Treatment of PLA led to an increase (P < 0.05) in the concentrations of white blood cells and lymphocyte percentage. The yellowness of breast muscle decreased by ANT, PLA0.4, and PA treatment. In conclusion, PLA can improve growth performance when it is supplemented in finisher diet (d 21 to 35), whereas it can depress BW gain and FI in earlier days (d 0 to 7). In addition, PLA can also decrease the number of E. coli in the large intestine and improve the number of immune-related blood cells.

Role of three rab5-like GTPases, Ypt51p, Ypt52p, and Ypt53p, in the endocytic and vacuolar protein sorting pathways of yeast.

The small GTPase rab5 has been shown to represent a key regulator in the endocytic pathway of mammalian cells. Using a PCR approach to identify rab5 homologs in Saccharomyces cerevisiae, two genes encoding proteins with 54 and 52% identity to rab5, YPT51 and YPT53 have been identified. Sequencing of the yeast chromosome XI has revealed a third rab5-like gene, YPT52, whose protein product exhibits a similar identity to rab5 and the other two YPT gene products. In addition to the high degree of identity/homology shared between rab5 and Ypt51p, Ypt52p, and Ypt53p, evidence for functional homology between the mammalian and yeast proteins is provided by phenotypic characterization of single, double, and triple deletion mutants. Endocytic delivery to the vacuole of two markers, lucifer yellow CH (LY) and alpha-factor, was inhibited in delta ypt51 mutants and aggravated in the double ypt51ypt52 and triple ypt51ypt52ypt53 mutants, suggesting a requirement for these small GTPases in endocytic membrane traffic. In addition to these defects, the here described ypt mutants displayed a number of other phenotypes reminiscent of some vacuolar protein sorting (vps) mutants, including a differential delay in growth and vacuolar protein maturation, partial missorting of a soluble vacuolar hydrolase, and alterations in vacuole acidification and morphology. In fact, vps21 represents a mutant allele of YPT51 (Emr, S., personal communication). Altogether, these data suggest that Ypt51p, Ypt52p, and Ypt53p are required for transport in the endocytic pathway and for correct sorting of vacuolar hydrolases suggesting a possible intersection of the endocytic with the vacuolar sorting pathway.

Effects of chitooligosaccharide supplementation on performance, blood characteristics, relative organ weight, and meat quality in broiler chickens.

This study was conducted to evaluate the effects of dietary supplementation with chitooligosaccharide (COS) on growth performance, blood characteristics, relative organ weight, and meat quality in broilers. A total of 480 broilers with an average initial BW of 45.04 g per chick were randomly allocated into 1 of the following 4 dietary treatments (20 broilers per pen with 6 pens per treatment): 1) CON (basal diet), 2) ANT (basal diet + 44 mg/kg of avilamycin), 3) COS0.2 (basal diet + 14 g/kg of COS), 4) COS0.4 (basal diet + 28 g/kg of COS). The experiment lasted for 5 wk and avilamycin was administered from d 0 to 21. Growth performance was measured on d 0, 21, and 35, and all other response criteria were measured on d 35. No change in feed conversion (G:F) was observed in response to any of the treatments throughout the experimental period (P > 0.05). However, BW gain and feed intake were greater (P < 0.05) in broilers provided with feed supplemented with COS than in those in the control group. In addition, broilers had significantly greater (P < 0.05) red blood cell and high-density lipoprotein cholesterol concentrations when they were provided with the COS0.4 diet, whereas the triglyceride concentration was lower (P < 0.05) in broilers in the COS0.2 treatment group. No other blood characteristics were affected by the treatments. Additionally, as the dietary COS concentration increased, the liver weight increased (P < 0.05). Conversely, as the concentrations of dietary COS increased, abdominal fat decreased (P < 0.05). Furthermore, meat yellowness decreased (P < 0.05) as the concentration of COS increased. Finally, the breast meat and abdominal fat of birds provided with feed supplemented with COS had a lower (P < 0.05) saturated fatty acid concentration but a greater concentration of total monounsaturated fatty acids (P < 0.05) than that of birds in the control. In conclusion, COS can improve the performance and breast meat quality of broilers while increasing the red blood cell and high-density lipoprotein cholesterol concentrations in blood. In addition, COS can induce a decrease in abdominal fat and improve meat quality.

The anti-diabetic effects of ethanol extract from two variants of Artemisia princeps Pampanini in C57BL/KsJ-db/db mice.

The anti-diabetic effects of two variants of Artemisia princeps Pampanini, sajabalssuk (SB) and sajuarissuk (SS), were investigated in type 2 diabetic animal using their ethanol extracts. Male C57BL/KsJ-db/db (db/db) mice were divided into control, SB ethanol extract (SBE), SS ethanol extract (SSE), or rosiglitazone (RG) groups and their age-matched littermates (db/+) were used. Supplementation of the SBE (0.171 g/100g diet), SSE (0.154 g/100g diet), and RG (0.005 g/100g diet) improved glucose and insulin tolerance and significantly lowered blood glycosylated hemoglobin levels, as compared to the control group. Plasma insulin, C-peptide and glucagon levels in db/db mice were higher in the db/+ mice, however these values were significantly lowered by SBE, SSE or RG-supplement. Hepatic GK activity was significantly lower in the db/db mice than in the db/+ mice, while hepatic G6Pase activity was vice versa. Supplementation of SBE, SSE and RG reversed these hepatic glucose-regulating enzyme activities. In addition, SBE and SSE markedly increased the hepatic glycogen content and muscle ratio as compared to the control group, but they did not alter the food intake, body weight and plasma leptin level. The RG group, however, showed a significant increase in the food intake, body weight and plasma leptin. These results suggest that SBE and SSE exert an anti-diabetic effect in type 2 diabetic mice.

Vesicle-associated membrane protein 4 is implicated in trans-Golgi network vesicle trafficking.

The trans-Golgi network (TGN) plays a pivotal role in directing proteins in the secretory pathway to the appropriate cellular destination. VAMP4, a recently discovered member of the vesicle-associated membrane protein (VAMP) family of trafficking proteins, has been suggested to play a role in mediating TGN trafficking. To better understand the function of VAMP4, we examined its precise subcellular distribution. Indirect immunofluorescence and electron microscopy revealed that the majority of VAMP4 localized to tubular and vesicular membranes of the TGN, which were in part coated with clathrin. In these compartments, VAMP4 was found to colocalize with the putative TGN-trafficking protein syntaxin 6. Additional labeling was also present on clathrin-coated and noncoated vesicles, on endosomes and the medial and trans side of the Golgi complex, as well as on immature secretory granules in PC12 cells. Immunoprecipitation of VAMP4 from rat brain detergent extracts revealed that VAMP4 exists in a complex containing syntaxin 6. Converging lines of evidence implicate a role for VAMP4 in TGN-to-endosome transport.

Biophoton emission of MDCK cell with hydrogen peroxide and 60 Hz AC magnetic field.

We studied biophoton characteristics of Madin-Darby canine kidney (MDCK) cells under the influence of H2O2 by employing a photomultiplier tube (PMT) and a fluorescence microscope. H2O2 was used for producing reactive oxygen species (ROS) in the measurement. Images from a fluorescence microscope show an increase of photon intensity emitted from the sample due to H2O2. By using a PMT we measured quantitative change in biophoton emission with application of H2O2 to the MDCK cell culture, found that the increase of the biophoton is dependent upon the amount of H2O2. The agreement between the results of the PMT and the fluorescence microscope suggests the possibility of quantitative measurement of the influence of ROS on living tissue or cell. In addition we applied a 60 HzAC magnetic field on the cells to investigate the change in reaction between MDCK cell and ROS. It showed that a decay of chemiluminescence intensity has taken a different path following exposure to the magnetic field. As a result, the PMT measurement might be considered as a useful tool for studying biochemical characteristics in relation to ROS.

Enzyme specificity in the metabolic activation of N-nitrosodimethylamine to a mutagen for Chinese hamster V79 cells.

In order to elucidate the mechanism of the metabolic activation of N-nitrosodimethylamine (NDMA), the relationship between NDMA demethylase (NDMAd) and NDMA mutagenicity in Chinese hamster V79 cells was investigated. The microsome-mediated activation system produced NDMA mutagenicity similar to the S9-mediated activation system, suggesting that microsomes are solely responsible for the activation process. Pretreatment of rats with ethanol- or acetone-induced microsomal NDMAd activity, and such treatment also enhanced microsome-mediated NDMA mutagenicity 6-7-fold. The patterns of NDMA activation by ethanol- and acetone-induced microsomes differed distinctly from that by phenobarbital-induced microsomes for both NDMAd and the mutagenicity. The former type of microsomes had a low Km for NDMA, but the latter appeared to have very high Km values, and NDMAd was highly positively related to NDMA mutagenicity. Purified cytochrome P-450 isozymes from acetone- and phenobarbital-induced microsomes, P-450ac and P-450b, respectively, were effective for the activation of NDMA to a mutagen in a reconstituted monooxygenase system. In parallel fashion to NDMAd activity, P-450ac was effective at low substrate concentrations, whereas P-450b exhibited appreciable activity only at high NDMA concentrations. The results demonstrate clearly that NDMAd, which is effectively catalyzed by a specific P-450 isozyme inducible by compounds such as ethanol, acetone, and isopropanol, is primarily responsible for the activation of NDMA to a mutagen.

Metabolism of N-nitrosodialkylamines by human liver microsomes.

The metabolism of N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine, N-nitrosobenzylmethylamine, and N-nitrosobutylmethylamine was investigated in incubations with human liver microsomes. All of the 16 microsomal samples studied were able to oxidize NDMA to both formaldehyde and nitrite at NDMA concentrations as low as 0.2 mM; the rates of product formation of the samples ranged from 0.18 to 2.99 nmol formaldehyde/min/mg microsomal protein (median, 0.53 nmol). At a concentration of 0.2 mM NDMA, the rates of denitrosation (nitrite formation) were 5 to 10% (median, 6.3%) those of demethylation (formaldehyde formation); the ratio of denitrosation to demethylation increased with increases in NDMA concentration, in a similar manner to rat liver microsomes. Immunoblot analysis with antibodies prepared against rat P-450ac (an acetone-inducible form of cytochrome P-450) indicated that the P-450ac [P-450j (isoniazid-inducible form)] orthologue in human liver microsomes had a slightly higher molecular weight than rat P-450ac and the amounts of P-450ac orthologue in human liver microsomes were highly correlated with NDMA demethylase activities (r = 0.971; P less than 0.001). Analysis of four selected microsomal samples showed that human liver microsomes exhibited at least three apparent Km and corresponding Vmax values for NDMA demethylase. This result, suggesting the metabolism of NDMA by different P-450 enzymes, is similar to that obtained with rat liver microsomes, even though most of the human samples had lower activities than did the rat liver microsomes. The high affinity Km values of the four human samples ranged from 27 to 48 microM (median, 35 microM), which were similar to or slightly lower than those observed in rat liver microsomes, indicating that human liver microsomes are as efficient as rat liver microsomes in the metabolism of NDMA. The human liver microsomes also catalyzed the dealkylation and denitrosation of other nitrosamines examined. The rates of product formation and the ratios of denitrosation to dealkylation varied with the structures and concentrations of the substrates as well as with the microsomal samples tested. The results indicate that human liver microsomes are capable of metabolizing N-nitrosodialkylamines via the pathways that have been established with rat liver microsomes.

Metabolism and activation of N-nitrosodimethylamine by hamster and rat microsomes: comparative study with weanling and adult animals.

It has been reported that hamster liver preparations are more effective for the metabolic activation of N-nitrosodimethylamine (NDMA) to a mutagen than rat liver preparations. The enzymatic basis for this phenomenon, however, has not been clearly elucidated. The present study was undertaken to examine the enzymology of NDMA metabolism by different hepatic subcellular fractions prepared from hamsters and rats of two different ages, and to investigate the correlation between the metabolism and the activation of NDMA to a mutagen for Chinese hamster V79 cells. The content of cytochrome P-450 was approximately 1.5-fold higher in hamster microsomes than in rat microsomes from both ages (1.19-1.38 versus 0.73-0.83 nmol P-450/mg protein). Weanling hamster microsomes exhibited multiple apparent Km values for NDMA metabolism as did weanling rat microsomes. The apparent Km I value of NDMA demethylase (NDMAd) in hamster microsomes was about one-half that in rat microsomes (36 versus 83 microM) with corresponding Vmax values of 2.09 and 2.57 nmol/min/nmol P-450. The Km I values for denitrosation did not differ from the corresponding values for NDMAd with Vmax values of 0.17 and 0.22 nmol/min/nmol P-450 for hamster and rat microsomes, respectively. These apparent Km values were affected neither by sonication nor by the presence of cytosolic proteins in S9 fractions. Adult rat liver microsomes showed less than one-half the NDMAd activity in weanling rat liver microsomes, whereas such age difference was not observed in hamster liver microsomes. This result was confirmed by Western blotting showing the levels of P-450ac (an acetone-inducible form of P-450) of these microsomes at comparable levels to their NDMAd activities. NDMAd was highly correlated to the metabolic activation of NDMA to a mutagen for V79 cells in an activation system mediated by microsomes prepared from hamsters and rats of different ages. The results from this study clearly demonstrate the enzymatic basis for the more effective metabolism of NDMA in adult hamsters than in adult rats.

Nature of N-nitrosodimethylamine demethylase and its inhibitors.

The present study was undertaken to examine the nature of the low Km (KmI) form of rat liver microsomal N-nitrosodimethylamine demethylase (NDMAd) and its inhibition by organic compounds which are commonly present in the assay mixture. Using radiometric and colorimetric assay methods with an NADPH-generating system consisting of 0.4 mM NADP, 10 mM glucose-6-phosphate, and glucose-6-phosphate dehydrogenase (0.4 units/ml), Km values of 40-50 microM were obtained. These Km values were lower than the values of 60-80 microM reported previously. This decrease was due to the elimination of inhibitors such as glycerol in the assay mixture. Glycerol was a competitive inhibitor, and this observation explained in part why purified P-450ac (acetone-inducible form of P-450), displayed a higher Km value in a reconstituted NDMAd system, which contained glycerol, than in microsomes. Semi-carbazide which had been used in many previous assays of NDMAd was also found to be a competitive inhibitor of this enzyme. Other inhibitors studied include the commonly used solvents dimethylsulfoxide, acetone, ethylene glycol, dimethylformamide, ethyl acetate, benzene, and hexane as well as thiol compounds dithiothreitol and mercaptoethanol. Although very low Km values (10-20 microM) for N-nitrosodimethylamine metabolism were reported in studies with perfused liver, liver slices, and isolated liver cells, we believe that the KmI form of liver NDMAd is responsible for the metabolism and activation of N-nitrosodimethylamine in the rat liver.

cis-Hydroxyproline inhibits proliferation, collagen synthesis, attachment, and migration of cultured bovine retinal pigment epithelial cells.

PURPOSE: Proliferative vitreoretinopathy (PVR) is characterized by the proliferation and migration of retinal pigment epithelial (RPE) and other cells into the vitreous cavity. The PVR membrane formation also is associated with collagen production by RPE. The authors examined the effect of a proline analog, cis-hydroxyproline (CHP), on proliferation, collagen synthesis, attachment, and migration of bovine RPE in vitro. METHODS: The effect of CHP on cell proliferation was determined as a function of dosage and days in culture by counting the cell numbers on days 3, 6, and 9. Collagen synthesis was determined by trichloroacetic acid precipitation of the radiolabeled samples before and after bacterial collagenase digestion. The attachment assay involved type I collagen or fibronectin substrates or both (2.5 micrograms/well). For migration experiments, RPE cells were removed from a defined area of a confluent culture, and migration was quantitated by counting the number of cells migrating into the denuded area over 30 hours. RESULTS: The addition of CHP inhibited RPE proliferation in both a dose- and a time-dependent manner; collagen synthesis, attachment, and migration also were inhibited by CHP in a dose-dependent manner. When the culture plates were coated with collagen, < 100 micrograms/ml of CHP had no effect on cell attachment. Higher doses of CHP resulted in mild inhibition of attachment on collagen-coated plates. Simultaneous addition of L-proline to the cultures resulted in blockade of these inhibitory effects on proliferation, collagen synthesis, attachment, and migration. CONCLUSIONS: The results show that RPE functions critical to the development of PVR are inhibited by CHP, suggesting the possibility that this drug may have potential clinical application.

Effects of thiamine deficiency on hepatic cytochromes P450 and drug-metabolizing enzyme activities.

To elucidate the mechanisms by which thiamine deficiency affects hepatic microsomal monooxygenase activities, the effect of thiamine deficiency on two constitutive cytochrome P450 isozymes, P450IIE1 and P450IIC11, was investigated, using weanling male Sprague-Dawley rats. The clinical signs of thiamine deficiency were apparent after feeding a thiamine-deficient diet for 3 weeks. Thiamine deficiency caused an increase in P450IIE1, which was determined by N-nitrosodimethylamine demethylase assay and immunoquantitation of P450IIE1. This increase in the P450IIE1 level was mainly attributed to thiamine deficiency per se but not to dietary restriction. Ketone bodies were not elevated in thiamine-deficient rats, whereas ketone bodies were elevated and may have served as inducing factors in calorically restricted pair-fed animals. Injections of pyruvate or pyrithiamine in addition to thiamine deficiency did not potentiate the induction effect. On the other hand, thiamine deficiency did not affect the level of P450IIC11 during the 3 weeks of feeding the thiamine-deficient diet. In addition, thiamine deficiency increased cytosolic glutathione S-transferase activity but not steroid isomerase activity. The present study demonstrates the specificity of thiamine deficiency per se in the induction of P450IIE1 which does not involve an increase in the ketone body level.

Modulation of the levels of cytochromes P450 in rat liver and lung by dietary lipid.

This study was undertaken to investigate the effect of dietary lipid on the regulation of several constitutive P450 isozymes. Male Sprague-Dawley rats with body weights of 130-140 g were fed either a 20% corn oil (CO) diet or a fat-free (FF) diet for 4 days following 2 days of fasting. Using liver microsomes, the catalytic activities and immunochemically detectable protein levels of P450s 1A1 and 2, 2A1, 2B1 and 2, 2C11, 2E1, and 3A were determined. The microsomes from rats fed the 20% CO diet exhibited 2-fold higher levels in N-nitrosodimethylamine demethylase activity and P450 2E1 protein than those from rats fed the FF diet. The CO group also showed 2.5-fold higher levels in 6 beta-hydroxylation of testosterone and P450 3A protein than the FF group. In contrast, the CO diet did not affect the immunodetectable level of P450 2C11 protein and its catalytic activities such as benzphetamine demethylase activity and 2 alpha-hydroxylation of testosterone. P450 1A1 was not detectable in either group, but 1A2 was 2.5-fold higher in the CO group than in the FF group. In the liver, the P450 2B1 level was very low in both groups as measured by pentoxyresorufin dealkylase activity and the protein level, whereas 2B2 was 2.5-fold higher in the CO diet group. In lung microsomes from rats fed different amounts of CO, an inverse relationship was observed between the P450 2B1 level and the dietary CO level. The results suggest that the constitutive levels of P450 isozymes are modulated by dietary lipid in a selective manner; the levels of hepatic P450s 1A2, 2B2, 2E1, and 3A were regulated positively but the level of pulmonary P450 2B1 was suppressed by dietary lipid.

Application of fast atom bombardment combined with tandem mass spectrometry to the structural elucidation of O-demethylabierixin and related polyether antibiotics.

O-Demethylabierixin, a new polyether antibiotic, was isolated from a Streptomyces spp. Its structure elucidation was carried out with fast atom bombardment mass spectrometry (FAB-MS) and fast atom bombardment mass spectrometry-mass spectrometry (FAB-MS/MS) by comparing spectral patterns with those of structurally similar polyether compounds, nigericin and abierixin. The collision-induced dissociation (CID) of sodium-adduct and deprotonated molecular ions produced many cyclic ether rings-opened product ions via a series of the dissociative processes. Because of the negative charge of the carboxylate group at the end of the molecule, the charge-remote fragmentation pattern in the negative CID spectra of deprotonated molecular ions was very helpful for complete identification of product ions which are characteristic for cyclic ether structures. From this study, the new polyether antibiotic was identified to be O-demethylabierixin in which the methoxy group of six-membered ether ring in abierixin was replaced by the hydroxy group.

Non-NMDA receptor antagonist attenuates antinociception induced by morphine but not beta-endorphin, D-Pen2-D-Pen5-enkephalin, and U50, 488H administered intracerebroventricularly in mice.

The antinociception induced by morphine but not beta-endorphin, D-Pen2-D-Pen5-enkephalin (DPDPE), or U50, 488H (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl] benzeocetamide) administered intracerebroventricularly (i.c.v.) has been previously demonstrated to be mediated by the N-Methyl-D-Aspartic Acid (NMDA) receptor. The present study was designed to determine if non-NMDA receptors are involved in opioid-induced antinociception. Antinociception was assessed by the tail-flick test in male ICR mice. Various doses of CNQX (6-Cyano-7-nitroquinoxaline-2,3-dione), a competitive non-NMDA receptor antagonist, (0.001 to 0.5 microgram) injected intracerebroventricularly (i.c.v.) alone did not show any antinociceptive effect. CNQX (0.01 to 1 micrograms, i.c.v.) dose-dependently attenuated the inhibition of the tail-flick response induced by morphine (1 microgram). However, inhibition of the tail-flick response induced by i.c.v. administered beta-endorphin (1 microgram), DPDPE (10 micrograms), or U50, 488H was not affected by i.c.v. administered CNQX. It is concluded that non-NMDA receptors are involved in i.c.v. morphine-induced antinociception. However, non-NMDA receptors are not involved in i.c.v. administered beta-endorphin-, DPDPE-, and U50, 488H-induced antinociception at the supraspinal level.

Proteome analysis of light-induced proteins in Synechocystis sp. PCC 6803: identification of proteins separated by 2D-PAGE using N-terminal sequencing and MALDI-TOF MS.

The cyanobacterium Synechocystis sp. PCC 6803 is an ideal model organism for the proteome study of light-induced gene expression because the whole genomic sequence has been determined. The soluble proteins extracted from light- and dark-cultured cells were separated by two-dimensional polyacrylamide gel electrophoresis. Light-induced protein spots electroblotted on a polyvinyldiene difluoride membrane were analyzed by N-terminal Edman sequence determination and followed by CyanoBase. The tryptic digests of some proteins were also confirmed by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) and MS-Fit search. Interestingly, eight proteins were related to photosynthesis and respiration (RbcS/L, CbbA, Gap2, AtpB, CpcB, PsbO, and PsbU). Four proteins (SodB, DnaK, GroEL2, and Tig) were involved in cellular processes and the functions of another two proteins (rehydrin and membrane protein) were unknown. The proteome analysis by N-terminal Edman sequencing and MALDI-TOF enabled us to characterize one-shot protein profiles expressed under different physiological conditions.