Susceptibility to exogenously added interferon-beta protein depends on intracellular interferon-beta mRNA level in human glioma cells

Exogenously added human interferon-beta (HuIFN-beta) protein possesses a remarkable antiproliferative activity in human glioma and melanoma. Endogenous HuIFN-beta protein, which is produced by its gene transfer using cationic liposomes, has much more effective antiproliferative activity against these tumors, even in cells resistant to exogenously added HuIFN-beta protein. As the first step to elucidate the possible difference in antiproliferative mechanisms between exogenous and endogenous HuIFN-beta protein, we here investigated the relationship between the intracellular level of its mRNA and susceptibility to exogenously added HuIFN-beta protein. In this study, we used seven human glioma cell lines (SK-MG-1, SK-MG-4, SK-AO2, U87MG, U251SP, U251MG and T98) and one human melanoma cell line (MMAN). At first, we examined the relationship between spontaneous expression of HuIFN-beta mRNA and susceptibility to exogenously added HuIFN-beta protein (50 IU/ml) in human glioma cells and then confirmed a significant correlation between them. Next, we confirmed that administration of 0-100 IU/ml exogenously added HuIFN-beta protein upregulated the HuIFN-beta mRNA in a dose-dependent manner using the RT-PCR technique and that the HuIFN-beta mRNA was suppressed by siRNA for HuIFN-beta in SK-MG-1 and MMAN cells. Furthermore, we confirmed that the siRNA for HuIFN-beta significantly suppressed the antiproliferative effect of SK-MG-1 cells treated with 10-100 IU/ml HuIFN-beta protein and MMAN cells with 25 and 50 IU/ml HuIFN-beta protein. We found this phenomenon in another human glioma cell line, U87MG cells, as well. This finding would suggest that susceptibility to exogenously added HuIFN-beta protein is related to the amount of intracellular HuIFN-beta mRNA in human glioma and melanoma cells.     

Effects of bacteria-produced human alpha, beta, and gamma interferons on in vitro immune functions

The effects of bacteria-produced human interferons (HuIFN) alpha, beta, and gamma on in vitro immune functions of human peripheral blood mononuclear cells (PBMC) were studied. Proliferative response to phytohemagglutinin was significantly inhibited by the addition of HuIFN-alpha 2 or HuIFN-beta at 10, 100, or 1000 U/ml. In contrast, HuIFN-gamma showed suppressive activities only when added at 1000 U/ml. HuIFN-alpha 2 or HuIFN-beta caused significant inhibition of human mixed-lymphocyte reaction (MLR) as measured by [3H]thymidine incorporation. Similar inhibition was caused by HuIFN-gamma when it was added only at very low concentrations (1 U/ml); 10, 100, or 1000 U/ml resulted in no or only a modest increase in MLR. All three interferons exhibited dose-related effects on PWM-induced immunoglobulin synthesis in cultures of PBMC. These data demonstrate that purified interferons produced by recombinant DNA technology can significantly alter in vitro immune functions and that HuIFN-gamma has properties which are different from those of HuIFN-alpha 2 or HuIFN-beta.     

Acute oral toxicity and anti-inflammatory activity of hydroalcoholic extract from Lampaya medicinalis Phil in rats

Background

Algesia and inflammation are related with several pathological conditions. It is known that many drugs available for the treatment of these problems cause unwanted side effects. This study was aimed at evaluating acute toxicity and anti-inflammatory activity of Lampaya medicinalis Phil. (Verbenaceae) widely used in the folk medicine of Northern Chile against rheumatism, arthritis and body joints pain.

Results

Oral administration of hydroalcoholic extract (HAE) at the highest dose of 3000 mg/ Kg body weight resulted in no mortalities or evidence of significant behavioral changes. Histological examination revealed normal architecture and no significant adverse effects were observed on the liver, kidney, heart, lung or ovaries and testicles. The results suggest that the oral administration of hydroalcoholic extract (HAE) from Lampaya medicinalis did not produce any toxic effect in rats. Hydroalcoholic extract (HAE) significantly inhibited the carrageenan-induced rat paw edema in dose – response relationship, at test doses of 37.5, 75, 150 and 300 mg/Kg body weight. Maximum inhibition (61.98 ± 2.69%) was noted at 300 mg/Kg after 2 h of drug treatment carrageenan induced paw edema, whereas indomethacin produced 47.90 ± 1.16% of inhibition. The inhibitory values of edema at 3 h postcarrageenan were 31.04±0.75%, 40.51 ± 2.36%, 48.97 ± 1.14% and 56.87 ± 0.41% for 37.5, 75, 150, and 300 mg/kg of extract respectively. Indomethacin (10 mg/Kg) gave a percentage inhibition of 49.44 ± 1.44. HAE (300 and 150 mg/kg) induced an anti-inflammatory effect greater than (or comparable) with the effect of indomethacin from 2nd to 4th hours of the experiment.

Conclusions

Our results reveal for first time that compounds contained in the hydroalcoholic extract of Lampaya medicinalis Phil exert anti-inflammatory effect and the oral administration is safe and non toxic up to dose level 3000 mg/kg body weight. The anti-inflammatory activity may be associated with the presence of flavonoids. These findings also justify the traditional use of the plant for treating pain.     

Mutagenicity and genotoxicity of dicapthon insecticide

Mutagenic and genotoxic effects of dicapthon were investigated by using the bacterial reverse mutation assay in Salmonella typhimurium TA97, TA98, TA100 and TA102 strains with or without metabolic activation system (S9 mix), and chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and micronucleus (MN) tests in human peripheral blood lymphocytes in vitro. Dicapthon was dissolved in dimethyl sulfoxide for all test systems. 0.1, 1, 10 and 100 μg/plate doses of dicapthon were found to be weakly mutagenic on S. typhimurium TA 98 without S9 mix. The human peripheral lymphocytes were treated with four experimental concentrations of dicapthon (25, 50, 100, and 200 μg/mL) for 24 and 48 h. Dicapthon increased the frequency of SCE only at the 100 μg/mL concentration for the 24 and 48 h applications. Dicapthon also induced abnormal cell frequency, CA/cell ratio and frequency of MN dose dependently for 24 and 48 h. Dicapthon showed a statistically significant cytotoxic effect by decreasing the mitotic index in all concentrations and a cytostatic effect by decreasing nuclear division index in 100 and 200 μg/mL concentrations for both treatment periods when compared with both untreated and solvent controls. These values decreased also in a dose dependent manner.     

Effect of bovine pituitary hormone preparations on newt lens regeneration in vitro: stimulation by thyrotropin

The anterior lobe of the pituitary gland can stimulate lens regeneration from the dorsal iris in the newt Notophthalmus viridescens. We have studied the effect of pituitary hormone preparations on this process. Dorsal irises were cultured for 20 days in diluted Medium 199 supplemented with 10% fetal calf serum. Bovine thyrotropin TSH-B8 at concentrations of 30 to 3000 μg/ml significantly stimulated lens regeneration in these dorsal irises. Well-developed lenses, up to stage 9, were formed, in which γ-crystallin, a protein specific for lens fibers of young lenses, was detected by immunofluorescence. Additionally, the mitotic index was 5.5 times elevated in these explants when compared to their controls. Lutropin LH-B10 at concentrations of 30 to 3000 μg/ml, prolactin PRL-B4 at concentrations of 23 to 1600 μg/ml, and porcine adrenocorticotropin ACTH-6002 at concentrations of 3 to 300 μg/ml did not stimulate lens regeneration. A weak stimulation of lens formation was observed in iris cultures with 2700 μg/ml of follitropin FSH-B1 or 3000 μg/ml somatotropin GH-B18, but not at concentrations of 30 μg/ml. Our results suggest that the inherent ability of the dorsal iris to form lens can be activated by the bovine thyrotropin preparation TSH-B8.     

Inhibition of Bacillus licheniformis spore growth in milk by nisin, monolaurin, and pH combinations

The effects of nisin and monolaurin, alone and in combination, were investigated on Bacillus licheniformis spores in milk at 37 degrees C. In the absence of inhibitors, germinated spores developed into growing vegetative cells and started sporulation at the end of the exponential phase. In the presence of nisin (25 IU ml-1), spore outgrowth was inhibited (4 log10 reduction at 10 h). Regrowth appeared between 10 and 24 h and reached a high population level (1.25 x 10(8) cfu ml-1) after 7 d. Monolaurin (250 micrograms ml-1) had a bacteriostatic effect during the first 10 h but thereafter, regrowth occurred slowly with a population level after 7 d (4 x 10(5) cfu ml-1) lower than that of nisin. Different combined effects of nisin (between 0 and 42 IU ml-1), monolaurin (ranging from 0 to 300 micrograms ml-1), pH values (between 5.0 and 7.0) and spore loads (10(3), 10(4), 10(5) spores ml-1) were investigated using a Doehlert matrix in order to study the main effects of these factors and the different interactions. Results were analysed using the Response Surface Methodology (RSM) and indicated that nisin and monolaurin had no action on spores before germination; only pH values had a significant effect (P < or = 0.001), i.e. spore count decreased as the pH value increased in relation to germination. Sublethal concentrations of nisin (30 IU ml-1) and monolaurin (100 micrograms ml-1) in combination acted synergistically on outgrown spores and vegetative cells, showing total inhibition at pH 6.0, without regrowth, within 7 d at 37 degrees C.     

间歇性低压低氧方式对发育大鼠心脏缺血/再灌注损伤的影响

本研究旨在探讨两种不同形式的间歇性低压低氧（intermittent hypobaric hypoxia,IHH）对发育大鼠心脏缺血,再灌注损伤的影响。雄性Sprague-Dawley（SD）新生大鼠72只,随机分为三组：对照组、IHH3000in组（IHH3000）、IHH5000m组（IHH5000）。低氧组大鼠出生后立即于低压氧舱分别接受28d、42d和56d（海拔5000m、每天6h：海拔3000m、每天5h）的低压低氧处理。应用Langendorff离体心脏灌流技术,给予心脏缺血（停灌30min）／再灌注（复灌60min）处理,分别在缺血前5min及复灌后l、5、10、20、30、60min记录心功能和冠状动脉流量变化,并测定乳酸脱氢酶（1actate dehydrogenase,LDH）活性。实验结束时测定心脏重量。结果显示：（1）IHH3000组大鼠体重增长与对照组无明显差异;IHH5000组大鼠体重增长明显慢于对照组及IHH3000组大鼠（P〈0．01）。（2）IHH3000组人鼠表现明显的心脏保护效应。与对照组相比较,在心脏停灌,再灌注60min时,心功能（LVDP、±LVdp／drmax）恢复增强（P〈0．05）、LDH活性降低（P〈0．05）、冠状动脉流量增多（P〈0．05）;心脏重量与对照组大鼠无差异;IHH42d处理的大鼠心功能恢复明显好于IHH28d处理的大鼠（P〈0．05）。（3）IHH5000组大鼠表现出明显的心脏损伤效应,各项心功能指标（LVDP、±LVdp／dtmax）的恢复均低于对照组（P〈0．05）,复灌过程中LDH活性明显高于相应对照组（P〈0．05）,右心室重量明显高于对照组大鼠（P〈0．05）。结果表明,适当的IHH增强发育大鼠心脏对缺血,再灌注损伤的抵抗能力;间歇性低氧方式是影响其心脏保护作用的重要因素。     

Alpha-fetoprotein concentrations measured by radioimmunoassay in diagnosing and excluding hepatocellular carcinoma.

Serum alpha-fetoprotein (AFP) concentrations were estimated by sensitive radioimmunoassay in 30 patients with cirrhosis complicated by hepatocellular carcinoma and in 100 patients with cirrhosis in whom malignancy was excluded. Twenty-nine of the 30 patients with hepatocellular carcinoma had concentrations above 10 IU/ml (10.5 ng/ml) (median 3500 IU/ml (3675 ng/ml)), whereas only one of the 100 patients with cirrhosis and no tumour development had a raised concentration. Eleven out of 20 patients in whom hepatocellular carcinoma had developed in an apparently normal liver had raised AFP concentrations. In this group the differential diagnosis is usually secondary carcinoma, and three of 50 such patients had AFP concentrations above 10 IU/ml. Noting raised AFP concentrations is thus of considerable value both in detecting and in excluding hepatocellular carcinoma in cirrhosis, for in this case such concentrations gave only 1% false-positive and 3% false-negative results. They are less useful, however, in distinguishing between primary tumours arising in patients without cirrhosis and secondary hepatic deposits, giving 6% false-positive and 45% false-negative results.     

Synergistic effects of lectins in the interaction of thrombin with human platelets

Several lectins have been studied for their effects on the interaction of thrombin with human platelets. Wheat germ agglutinin, concanavalin A and Ricinus communis lectin increased the number of high affinity sites for diisopropylphosphothrombin on washed platelets from 3000 to about 12 000 but the binding affinities were unchanged (Kd approx 4 nM). Two other lectins, Lens culinaris and Bandieria simplicifolia, were without effect. (2) Using formalinized platelets to avoid possible complications of the platelet release reaction, wheat germ agglutinin showed a marked increase (5-fold) in the binding of active thrombin, peanut agglutinin had no effect while Ricinus communis and :Bandieria simplicifolia showed marginal increases (2-fold). Thrombin binding was decreased to about one quarter with Lens culinaris, Phaseolus vulgaris and concanavalin A. (3) Wheat germ agglutinin caused a synergistic increase of platelet aggregation at low concentrations of thrombin (12.5 mU/ml) and ADP (1 microM), both in the absence and presence of added fibrinogen, but had no effect on ristocetin-induced aggregation.     

High dietary vitamin A interferes with tissue α-tocopherol concentrations in fattening pigs: a study that examines administration and withdrawal times

This study aimed to assess the interaction between different dietary vitamin A (dVitA) levels and the same concentration of vitamin E (100 IU all-rac-α-tocopheryl acetate/kg feed) in growing-finishing pigs. In the first experiment, two fat sources × two dVitA levels (0 v. 100 000 IU) were used. The supplementation of 100 000 IU dVitA induced a range of 5.13 to 30.03 μg retinol/g liver, 62.78 to 426.88 μg retinol palmitate/g liver, and 0.60 to 1.96 μg retinol/g fat. Dietary fat did not affect retinol or retinyl palmitate deposition in pigs. The high concentration of dVitA produced lower fat and liver α-tocopherol concentrations, and increased susceptibility of muscle tissue to oxidation. A second experiment was carried out to study the retinol and α-tocopherol retention at different withdrawal times prior to slaughter (two dVitA levels; 0 v. 100 000 IU). A high dose of 100 000 IU vitamin A during a short 2-week period was enough to induce α-tocopherol depletion in liver and fat to a similar extent as when 100 000 IU were administered during the whole fattening. Muscle, fat and liver α-tocopherol concentrations were not affected by dVitA in the 1300-13 000 IU/kg range, but liver α-tocopherol concentration was higher when vitamin A was removed from the vitamin mix 5 weeks prior to slaughter (experiment 3).     

Direct effects of heparin on basal and stimulated aldosterone production in rat adrenal glomerulosa cells

Direct effects of heparin (0.1-10 IU/ml) on basal and stimulated aldosterone production have been studied using intact rat adrenal glomerulosa cells. Heparin at any dose did not affect basal aldosterone production when added to the incubation medium. Heparin at a 0.01 IU/ml dose had no effect on aldosterone production maximally stimulated by angiotensin II (AII, 4.8 X 10(-8) M), ACTH (4.3 X 10(-9) M) or potassium (8.0 mM). However, heparin at 0.1 and 0.3 IU/ml doses selectively blocked aldosterone production maximally stimulated by AII but not by ACTH or potassium, while the compound at 1 and 10 IU/ml doses inhibited aldosterone production maximally stimulated by these three stimuli. In addition, the inhibitory effect of 0.3 IU/ml heparin occurred as early as 30 min after incubation with heparin. These data suggest that heparin at 0.1 and 0.3 IU/ml doses acts directly on adrenal zona glomerulosa to selectively block the stimulatory action of AII, while the compound at 1 and 10 IU/ml doses inhibits all the stimulatory actions of AII, ACTH and potassium.     

Influence of tannic acid application on alfalfa hay: in vitro rumen fermentation, serum metabolites and nitrogen balance in sheep

Alfalfa protein is poorly utilised by ruminants due to its rapid degradation in rumen. The objective of the study was to assess the influence of spraying tannic acid (TA) on chopped alfalfa hay on in vitro rumen fermentation and nitrogen (N) retention by sheep. Alfalfa hay with and without TA was fed to sheep to determine nutrient digestibility and N balance. TA was sprayed on chopped alfalfa at three concentrations to determine its effect on in vitro fermentation of dry matter (DM) and N balance in sheep. Final TA concentrations were 0, 30, 60 and 90 g TA per kg DM. The control was sprayed with the same amount of water but without TA. In vitro DM degradation and the production of gas, ammonium-N (NH4-N) and short-chain fatty acid (SCFA) were measured. TA-sprayed alfalfa and the control were fed to sheep to determine nutrient digestibility and N retention. Addition of TA had no influence on the extent and rate of gas production but significantly decreased NH4-N concentration at 30 (P < 0.05), 60 and 90 (P < 0.0001) g/kg DM. Addition of polyethylene glycol (PEG) to TA-sprayed alfalfa increased NH4-N to a level comparable to non-TA-sprayed alfalfa. Spraying of alfalfa with TA significantly decreased (P < 0.05) isovalerate but did not affect the total and individual SCFA acid production. Tannic acid significantly (P < 0.05) reduced in vitro true degradability of DM (IVTD) after 24 h incubation at levels of 60 and 90 g TA per kg DM. Neutral-detergent fibre digestibility (dNDF) after 24 h (P < 0.01), 60 and 90 (P < 0.0001) g TA per kg DM. The effect of TA on either IVTD or dNDF was not significant (P > 0.05) after 48 h of incubation. There was a strong linear relationship between percentage increase in gas production due to PEG and protein precipitation capacity (R2 = 0.94). N digestibility was significantly reduced with all three levels of TA additions. However, the proportion of urine-N to total N output was reduced by adding 60 g (P < 0.05) and 90 g (P < 0.01) TA per kg DM. Serum metabolites and liver enzymes were not affected by TA (P > 0.05). Higher faecal N as the TA level increased indicates incomplete dissociation of tannin-protein complexes post ruminally. Factors affecting dissociation of tannin-protein complexes need further study.     

白藜芦醇对哇巴因所致豚鼠乳头状肌迟后去极化及触发活动的效应

研究旨在应用标准玻璃微电极技术,观察白藜芦醇对哇巴因所引起的离体豚鼠乳头状肌迟后去极化(delayed after depolarization,DAD)及触发活动(triggered activity,TA)的效应。结果显示：(1)预先给予白藜芦醇(30、60、120μmol／L)可剂量依赖性地抑制哇巴因所引起的乳头状肌DAD及TA;(2)预先应用L型钙通道开放剂Bay K8644(0．25μmol／L),可取消白藜芦醇的上述效应;(3)预先应用一氧化氮合酶抑制剂L-NAME(1mmol／L),对白藜芦醇的上述效应无影响;(4)单独应用17β-雌二醇(E2,5μmol／1．0或白藜芦醇(30μmol／L)对DAD及TA无明显影响,而联合应用相同剂量的E2和白藜芦醇则对DAD及TA产生明显的抑制效应;(5)预先应用雌激素受体拮抗剂他莫昔芬(10μmol／L)不能取消白藜芦醇对DAD及TA的抑制作用。以上结果表明,白藜芦醇具有抑制乳头状肌DAD及TA的作用,这一效应可能与其抑制钙离子内流有关,但此作用机制中NO和雌激素受体的作用并不显著。白藜芦醇这种抗心律失常作用对于心血管系统具有一定的保护意义。     

Study on mutagenicity and antimutagenicity of BHT and its derivatives in a bacterial assay

The mutagenicity of butylated hydroxytoluene (BHT) and its derivatives was investigated by the Ames method using Salmonella typhimurium TA98 and TA100 with or without S9 mix. The compounds were not mutagenic in either indicator strain at concentrations ranging from 50 to 330 micrograms/plate (SQ: 3,5,3',5'-tetra-tert-butylstilbenequinone; VI-III: unidentified), 500 micrograms/plate (BE: 3,5,3',5'-tetra-tert-4,4'-dihydroxy-1,2-diphenylethylene; VI: 2,6-di-tert-butyl-4-methyl-4-tert-butylperoxy-2,5-cyclohexadienone ; VI-I: unidentified; VI-II: 3-acetyl-2,5-di-tert-cyclopenta-2,4-dienone) and 1000 micrograms/plate (BHT). The antimutagenic effects of BHT and its derivatives on mutagenesis by chemical agents were investigated in Salmonella typhimurium TA98 and TA100 and Escherichia coli WP-2 hcr-. VI-II suppressed the mutagenesis induced in TA98 and TA100 by 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) and that induced in WP-2 hcr- by 4-nitroquinoline-1-oxide (4NQO) without decreasing cell viability. In WP-2 hcr-, the mutagenesis induced by AF-2 and ethyl methanesulfonate was also suppressed significantly. Mutations induced by methyl methanesulfonate were slightly inhibited. However, VI-II had no effect on the mutagenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine.     

Reversible effects of triamcinolone and lack of effects with aspirin or L-656,224 on external genitalia of male Sprague-Dawley rats exposed in utero.

Inhibitors of the arachidonic acid cascade were given to pregnant rats during the critical period for morphogenesis of the external genitalia. Groups treated subcutaneously (s.c.) with 0.1 or 0.25 mg/kg/day of triamcinolone acetonide (TA) on gestational days (GD) 14-19 had male fetuses on GD 20 with moderate decreases in absolute anogenital distance (AGD), but gross and histological examinations revealed no alterations to the genital tubercle (i.e., no hypospadias). The s.c. coadministration of arachidonic acid at 100 mg/kg/day had minimal to no effect on AGD in the TA-exposed groups. No effect on AGD was observed in male fetuses from groups administered aspirin orally at 150 mg/kg/day, and only a 6% decrease was observed in the 300-mg/kg/day group. Neither TA nor aspirin adversely affected AGD of female fetuses. In another study, TA was administered on GD 11-19 at dose levels of 0.05 and 0.1 mg/kg/day, and dams were allowed to deliver. High-dose male offspring examined on postcoitum day (PCD) 23, had moderate decreases in AGD. In both studies with TA, there were also significant decreases in offspring weights. The contribution of the decreased weight to the decrease in absolute AGD was examined by a variety of methods (ratio of AGD to cube root of weight or biparietal distance, comparison to weight-matched controls, and covariance analysis). We conclude that TA caused a specific decrease in AGD on GD 20 that was largely reversed by PCD 23. When examined as adults (8 weeks old), the external genitalia of TA-exposed offspring were normal. Thus, the TA-induced decreases in AGD on GD 20 did not predict irreversible malformation. TA also caused other effects, which included a somewhat flattened genital tubercle and apparently thinned and glossy skin between the tubercle and the anus in both sexes on GD 20 and PCD 23, but not as adults. In addition, there were high pup mortality and high incidences of micrognathia and omphalocele (in the 0.25-mg/kg/day group only). Aspirin at 75 or 150 mg/kg/day and a specific lipoxygenase inhibitor (L-656,224) at 1,000 or 2,000 mg/kg/day were also administered from GD 14 to 19, and no offspring effects were observed. Thus, of the three agents that potentially inhibit the arachidonic acid cascade, only triamcinolone produced moderate effects on rat external genitalia that were largely reversible.(ABSTRACT TRUNCATED AT 400 WORDS)     

Limited Utility of Plasma M30 in Discriminating Non-Alcoholic Steatohepatitis from Steatosis – A Comparison with Routine Biochemical Markers

Introduction

The utility of Cytokeratin-18 fragment, namely CK18Asp396 (M30), for the diagnosis of non-alcoholic steatohepatitis (NASH) is currently uncertain. We aimed to provide further data in this area among multi-ethnic Asian subjects with NAFLD.

Materials and Methods

The accuracy of M30 for detecting NASH was compared with serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma glutamyl transpeptidase (GGT) levels in consecutive adult subjects with biopsy-proven non-alcoholic fatty liver disease (NAFLD).

Results

Data for 93 NAFLD subjects (mean age 51.0±11.1 years old and 51.6% males) and 20 healthy controls (mean age 50.2±16.4 years old and 33.3% males) were analyzed. There were 39 NASH subjects (41.9%) and 54 non-NASH subjects (58.1%) among the NAFLD subjects. Plasma M30 (349 U/L vs. 162 U/L), and serum ALT (70 IU/L vs. 26 IU/L), AST (41 IU/L vs. 20 IU/L) and GGT (75 IU/L vs. 33 IU/L) were significantly higher in NAFLD subjects than in healthy controls. Serum ALT (86 IU/L vs. 61 IU/L), AST (58 IU/L vs. 34 IU/L) and GGT (97 IU/L vs. 56 IU/L) were significantly higher in NASH subjects compared to non-NASH subjects, but no significant difference was observed with plasma M30 (435 U/L vs. 331 U/L). The accuracy of plasma M30, and serum ALT, AST and GGT was good for predicting NAFLD (AUROC 0.91, 0.95, 0.87 and 0.85, respectively) but less so for NASH (AUROC 0.59, 0.64, 0.75 and 0.68, respectively). Serum ALT and AST, but not plasma M30 showed a significant trend with increasing grades of ballooning and lobular inflammation.

Conclusion

The utility of M30 in the detection of NASH in clinical practice appears limited, in comparison to routine biochemical markers.     

Enhanced secretion of glucagon-like peptide 1 by biguanide compounds

Metformin was reported to increase plasma active glucagon-like peptide-1 (GLP-1) in humans. There are two possible mechanisms for this effect: (1) metformin inhibits dipeptidyl peptidase IV (DPPIV), an enzyme degrading GLP-1, and (2) metformin enhances GLP-1 secretion. To elucidate the mechanism(s), we examined (1) IC(50) of metformin for DPPIV inhibition, (2) plasma active GLP-1 changes after oral biguanide (metformin, phenformin, and buformin) treatment in fasting DPPIV-deficient F344/DuCrj rats, and (3) plasma intact GLP-1 excursions after oral administration of metformin and/or valine-pyrrolidide, a DPPIV inhibitor, in fasting DPPIV-positive F344/Jcl rats. Our in vitro assay showed that metformin at up to 30mM has no inhibitory activity towards porcine or rat DPPIV. Metformin treatment (30, 100, and 300mg/kg) increased plasma active GLP-1 levels dose-dependently in DPPIV-deficient F344/DuCrj rats (approximately 1.6-fold at 3 and 5h after administration of 300mg/kg). This treatment had no effect on blood glucose levels. Similarly, phenformin and buformin (30 and 100mg/kg) elevated plasma intact GLP-1 levels in F344/DuCrj rats. In DPPIV-positive F344/Jcl rats, coadministration of metformin (300mg/kg) and valine-pyrrolidide (30mg/kg) resulted in elevation of plasma active GLP-1, but neither metformin nor valine-pyrrolidide treatment alone had any effect. These findings suggest that metformin has no direct inhibitory effect on DPPIV activity and that metformin and the other biguanides enhance GLP-1 secretion, without altering glucose metabolism. Combination therapy with metformin and a DPPIV inhibitor should be useful for the treatment of diabetes.     

Mutagenicity studies with N6, O2'-dibutyryl cyclic adenosine 3',5'-monophosphate (DBcAMP)

N6,O2'-Dibutyryl cyclic adenosine 3,5-monophosphate (DBcAMP) was studied for mutagenicity using the rec assay, the Ames method, and in vitro cytogenetics. DBcAMP had no mutagenic effect on B. subtilis in the rec assay, or on S. typhimurium (TA1535, TA1537, TA1538, TA98 and TA100) or E. coli (WP2 uvrA). In the cytogenetic study, a significant increase in chromosomal aberrations was observed at a concentration of 50,000 micrograms/ml, but it was considered that this effect could be attributed to the secondary effect of the high osmotic pressure in the culture medium. These results suggest that DBcAMP has no mutagenic potential.     

Influence of vitamins A, D3 and E status on post-mortem meat quality in steers under winter housing or pasture finishing systems

We investigated the influence of Swedish recommended vitamins A, D3 and E supplementation levels on muscle tenderness and fatty acid (FA) composition under indoor or outdoor finishing programmes. Swedish Red breed steer calves were divided into vitamin supplemented (n = 12) and non-supplemented (n = 15) groups while on pasture prior to the finishing period. This trial began at the beginning of the winter housing period during which the steers were fed a 55 : 45 dry matter barley : grass silage diet indoors. The indoor finished group was comprised of vitamin supplemented (n = 6) and non-supplemented (n = 8) steers slaughtered after about 155 days on feed. Vitamin supplemented steers were provided with 100 g mineral supplement providing 400 000 IU vitamin A, 100 000 IU D3 and 3000 IU E daily as recommended for Swedish production practices. In spring, outdoor finished vitamin supplemented (n = 6) and non-supplemented (n = 7) steers grazed semi-natural grassland for an additional 120 days before slaughter. During pasture, vitamin supplemented steers had free-choice access to a mineral supplement containing vitamins A, D3 and E. The mineral supplement for the non-supplemented steers did not contain vitamins A, D3 and E and was provided at the same amount as the vitamin supplemented steers. Shear force values were similar between vitamin supplemented and non-supplemented steers after ageing 2, 7 and 14 days within indoor and outdoor finishing programmes. The shear force values had decreased by 14 days of ageing within all programmes. The μ- and m-calpain activity did not differ between vitamin supplemented and non-supplemented steers for either the indoor or outdoor finishing programmes. The calpastatin activity was higher for the indoor, vitamin supplemented steers. Indoor finished vitamin supplemented steers had a greater proportion of C18:1c-9 and total monounsaturated fatty acids, whereas the non-supplemented steers had a greater proportion of total saturated fatty acids. We concluded that the meat quality from steers not receiving vitamin supplementation was similar to that of steers receiving vitamins A, D3 and E supplementation at Swedish recommended levels under indoor and outdoor finishing programmes.     

Subdose of human chorionic gonadotropin applied at the Hou Hai acupoint on follicular dynamics and luteal development in donkeys

The objective of this study was to evaluate the effects of an hCG subdose applied at the Hou Hai acupoint as an ovulation inducer in donkeys. Eleven donkeys were distributed in randomized blocks in T1 = application of 1,500 IU of hCG intravenous (IV); T2 = 450 IU of hCG applied at the false acupoint (IV), and T3 = 450 IU of hCG applied at the Hou Hai acupoint. There was no difference (P > 0.05) between the treatments regarding the mean diameter of the pre-ovulatory follicle (34.5 ± 1.3 mm), the ovulation rate (96.97%), the interval between induction and ovulation (58.07 ± 16.82 h), the mean diameter of the CL (D0 = 23.0 ± 0.6; D2 = 27.7 ± 1.9 and D8 = 28.2 ± 0.8mm), and serum P₄ concentrations (10.50 ± 2.99 ng.mL^-1). The application of 450 IU of hCG at the Hou Hai acupoint increased ovulation rate (72.73%) more than 48 h after induction (P = 0.03) and a larger diameter of the CL on D4 (30.7 ± 5.1 mm) (P = 0.04). The vascularization area of the CL on D8, obtained by minimum number of colored pixel (NCP), was greater (P < 0.05) in the donkeys that received 1,500 IU of IV hCG (T1, 41.91 ± 1.17), and we found a positive correlation (P < 0.05) between mean NCP and P₄ concentration in the donkeys that received 450 IU of hCG IV at the false acupoint (T2) or at the Hou Hai acupoint (T3). The application of 450 IU of hCG by IV route at the false acupoint or the Hou Hai acupoint was sufficient to induce ovulation in donkeys, demonstrating that the average dosage commonly used for this species is too high.