Characterization of swine leukocyte antigen alleles and haplotypes on a novel miniature pig line, Microminipig.

Microminipigs are extremely small-sized, novel miniature pigs that were recently developed for medical research. The inbred Microminipigs with defined swine leukocyte antigen (SLA) haplotypes are expected to be useful for allo- and xenotransplantation studies and also for association analyses between SLA haplotypes and immunological traits. To establish SLA-defined Microminipig lines, we characterized the polymorphic SLA alleles for three class I (SLA-1, SLA-2 and SLA-3) and two class II (SLA-DRB1 and SLA-DQB1) genes of 14 parental Microminipigs using a high-resolution nucleotide sequence-based typing method. Eleven class I and II haplotypes, including three recombinant haplotypes, were found in the offspring of the parental Microminipigs. Two class I and class II haplotypes, Hp-31.0 (SLA-1*1502-SLA-3*070102-SLA-2*1601) and Hp-0.37 (SLA-DRB1*0701-SLA-DQB1*0502), are novel and have not so far been reported in other pig breeds. Crossover regions were defined by the analysis of 22 microsatellite markers within the SLA class III region of three recombinant haplotypes. The SLA allele and haplotype information of Microminipigs in this study will be useful to establish SLA homozygous lines including three recombinants for transplantation and immunological studies.

Effects of cortisol on muscle proteolysis and meat quality in piglets.

The present study was conducted to examine the effects of cortisol on muscle proteolysis and meat quality. Male piglets (n=14) were assigned to one of two treatment groups at 28 days of age. After 7 days adaptation period, each group was fed a commercial diet (86% total digestible nutrients, 21.5% crude protein) or the same commercial diet containing cortisol (120mg/kg diet) for 7 days from 35 days of age. All piglets were slaughtered at 42 days of age. The serum triiodothyronine (T3) concentration, µ- and m-calpain and proteasome activities and the content of easily releasable myofilament, which contains intermediates of the breakdown of myofibrils in the m. longissimus dorsi (LD) at slaughter were measured as parameters of muscle proteolysis. Serum T3 levels and µ-calpain activity were increased (P<0.01), as was the amount of easily releasable myofilament and m-calpain and proteasome activities were higher (P<0.05) in LD from cortisol-treated piglets than from non-treated controls. At 24 h postmortem, LD of cortisol-treated piglets showed higher (P<0.01) drip loss and lighter (P<0.05) color than those of the control. The results clearly show that the administration of cortisol increases serum T3 concentration and muscle proteolysis and reduces productivity and meat quality.

Relative validity of a semi-quantitative food frequency questionnaire versus 28 day weighed diet records in Japanese female dietitians.

OBJECTIVE: To assess the relative validity of a semi-quantitative food frequency questionnaire (SQFFQ) against 28 day weighed diet records (WDRs). SUBJECTS AND METHODS: The SQFFQ was administered to 106 (21 male and 85 female) Japanese dietitians in Aichi Prefecture in autumn, 1996 and four-season consecutive 7 day WDRs were carried out during 1996-1997. We evaluated validity of intakes of 15 foods and 31 macro- and micro-nutrients based on the SQFFQ against those according to 28 day WDRs among 79 Japanese female dietitians. RESULTS: Mean daily intakes of selected foods and nutrients determined by the SQFFQ were generally equivalent to those measured by 28 day WDRs. Pearson's de-attenuated correlation coefficients (CCs) with log-transformation and energy-adjustment between intakes of selected foods and nutrients quantified by the SQFFQ and 28 day WDRs (minimum-median-maximum) ranged from 0.17 (beverages)-0.52 to 0.74 (rice), and Spearman's rank CCs with energy-adjustment ranged from 0.28 (confectionery)-0.42 to 0.68 (rice). Respective Pearson's CCs for intakes of nutrients were 0.28 (PUFAs)-0.51 to 0.73 (magnesium), and Spearman's rank CCs ranged from 0.23 (n-3 PUFAs)-0.45 to 0.71 (magnesium). Favorably higher agreement for intakes of foods/nutrients was achieved along with lower disagreement. CONCLUSIONS: Satisfactorily higher relative validity was attained in Japanese female dietitians with the SQFFQ. This calibrated questionnaire seems therefore appropriate for administration to Japanese dietitians to clarify associations between diet and health/disease. SPONSORSHIP: A grant-in-aid from the Ministry of Education, Science, Sports and Culture (06454242).

Discrepancies in dietary intakes and plasma concentrations of fatty acids according to age among Japanese female dietitians.

OBJECTIVE: To clarify the influences of age on dietary intakes and plasma concentrations of fatty acids (FAs) in Japanese female dietitians. SUBJECTS AND METHODS: In autumn 1996, we estimated dietary FA intakes based on 7 day weighed diet records and analyzed plasma FA concentrations in 79 healthy Japanese female dietitians, and investigated their relationships with age, dividing into three age groups (young (32-42 y), middle-aged (43-50 y) and elderly (51-66 y)). RESULTS: Dietary intakes of total FA, saturated FAs, monounsaturated FAs, n-3 polyunsaturated FAs (PUFAs) and alpha-linolenic acid (18:3n-3) were significantly highest in the middle-aged group, and lowest in the elderly. Similar trends were observed for dietary intakes of n-6 PUFAs and linoleic acid (18:2n-6), but there were no differences with regard to eicosapentaenoic acid (EPA; 20:5n-3), docosahexaenoic acid (DHA; 22:6n-3) and n-3 highly unsaturated FAs (HUFAs=EPA+22:5n-3+DHA). On the other hand, plasma concentrations of all FAs except for arachidonic acid (20:4n-6) demonstrated positive correlations with age. Moreover, plasma concentrations of EPA in all age groups, DHA in the elderly and n-3 HUFAs in the middle-aged and the elderly were all positively correlated with dietary intakes. CONCLUSIONS: We should take into account the influence of age on dietary habit and lipid metabolism when interpreting associations between dietary FA intakes and plasma FA concentrations.

Age-related change in tissue-to-plasma partition coefficient of cefazolin for noneliminating organs in the rat.

Age-related changes in tissue distribution characteristics of cefazolin, a cephalosporin antibiotic, were examined for noneliminating organs of rats. The in vivo tissue-to-plasma partition coefficients (Kp,vivo) varied markedly among different ages and organs. In particular, muscle and skin acted as reservoirs for cefazolin distribution. There were also marked differences in interstitial fluid space (IS), determined using [14C]inulin, among different ages and organs. For muscle and bone, the magnitude of the age-related changes in Kp,vivo of cefazolin and IS was in the order of 1-week-old greater than 7-week-old = 100-week-old greater than 50-week-old rats. This is well correlated with the age-related changes in the volume of distribution at the steady state of cefazolin per body weight (Vdss/BW) and the extracellular fluid volume per body weight (Vecw/BW) determined previously using [14C]inulin. The predicted Kp value (Kp,pred) was estimated by incorporating the serum protein binding parameters of cefazolin, the IS values, and an interstitial-to-plasma albumin concentration ratio (AR) into equations derived from an extracellular fluid model. The Kp,pred values exhibited a fairly good correspondence with the Kp,vivo values determined for various organs, except gut, in rats of all four ages. These results suggest that the determinant of the age-related change in Vdss/BW is the difference in the IS value of muscle and bone, while the age-related change in serum protein binding plays only a modest role.

Prediction of steady-state serum theophylline concentration in children by first-order and zero-order absorption models.

We estimated the pharmacokinetic characteristics of theophylline in children with bronchial asthma after multiple oral dosing of Theo-Dur, in order to determine whether the absorption of the drug is best described by a zero-order absorption kinetic model (0-order model) or a first-order absorption kinetic model (1-order model). Thirteen children with bronchial asthma were evaluated in this study. Each patient received Theo-Dur twice daily at 12-hour intervals. Blood samples were taken at 0, 3, 6, 9 and 12 hours after the administration; plasma theophylline concentration was determined by the fluorescence polarization immuno assay method. Pharmacokinetic parameters for each patient were estimated by a one-compartment open model with 0-order or 1-order absorption and 1-order elimination rate constants. The regression curve predicted using the 0-order model fitted well to the observed value. In contrast, the use of the 1-order model was overestimated considerably in the absorption phase the precision of the prediction using the 0-order model was better than the use of the 1-order model. This study suggests that the 0-order model gave better estimated pharmacokinetic parameters when applied to Theo-Dur than did the 1-order model in children.

The design of oral sustained-release theophylline dosing after conversion from intravenous to oral therapy.

We studied the design of oral sustained-release theophylline dosing after conversion from constant aminophylline infusion. Twelve children with bronchial asthma (9 boys and 3 girls) were evaluated in this study. Each patient received a constant intravenous administration of aminophylline for 4-10 days. Three hours after conversion from constant aminophylline infusion, they received oral sustained-release theophylline twice daily at 12-hour intervals. Blood samples were obtained at least once during the aminophylline infusion, just before conversion from the aminophylline infusion, and 0, 3 and 6 hours, and 4-5 days after administering oral theophylline. Pharmacokinetic parameters were estimated using the serum theophylline concentrations that were obtained during constant aminophylline infusion. These estimates of pharmacokinetic parameters were used to predict the serum theophylline concentrations during oral theophylline therapy. Predicted serum theophylline concentrations using individual pharmacokinetic parameters were fitted with actual measured values in this study. When switching a patient from intravenous aminophylline to sustained-release oral theophylline, the use of Bayesian analysis of serum theophylline concentration values obtained during intravenous therapy works well in predicting serum theophylline concentrations and in determining oral dosages that maximize the drug's effectiveness.

Foods contributing to absolute intake and variance in intake of selected vitamins, minerals and dietary fiber in middle-aged Japanese.

Using 351 one-day weighted diet records, we selected foods providing vitamins, minerals and dietary fiber according to contribution analysis (CA) and multiple regression analysis (MRA). Vitamin C was supplied by various vegetables and fruits, and carotene was specifically derived from green-yellow vegetables based on MRA as well as CA. Vitamin A was provided by green-yellow vegetables, fruits, chicken egg and milk (whole) according to CA; whereas chicken liver and pork liver were major sources according to MRA. Vitamin E was mainly of vegetable origin as determined by CA, and largely of spinach, safflower oil and pumpkin as determined by MRA. Vitamin D was mainly derived from chicken egg, fish and mushroom based on CA, and particularly from fish based on MRA. Calcium was supplied by milk (whole), soy products and chicken egg as determined by CA; while milk, tofu and various small fishes were the main contributors to variance. Magnesium was provided by soy products, well-milled rice and spinach according to both analyses, and iron by chicken egg, spinach and soy products. Zinc was largely derived from well-milled rice, followed by chicken egg and milk (whole) as determined by CA, and copper was provided by well-milled rice, soy and wheat products. Dietary fiber was supplied by vegetable sources, whether water soluble or insoluble, based on both analytic methods.

Influence of the stocking density and rearing season on incidence of sudden death syndrome in broiler chickens.

Influences of the stocking density and rearing season on the incidence of sudden death syndrome (SDS) were investigated in commercial broiler chickens, which were reared until 63 d after birth. The SDS mortality and total mortality were measured at three different stocking densities of 12, 15, and 18 birds/m2 in rearing seasons of summer, autumn, and winter. At stocking densities of 15 and 12 birds/m2, neither SDS mortality nor total mortality was significantly different throughout the rearing season. However, at 18 birds/m2, SDS mortality significantly increased in summer and winter. The increased SDS mortality was accompanied by a significant increase in total mortality in summer but not in winter. Irrespective of the stocking density, body weight gain and feed intake were lower in summer with no significant change in feed efficiency. The data suggest that stocking density can act as a factor affecting the incidence of SDS in summer and winter, independent of feed efficiency.

Characterization of serum enzyme activities and electrolyte levels in broiler chickens after death from sudden death syndrome.

Serum activities of lactate dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT), and creatine phosphokinase (CPK), and serum levels of Na+ and K+ were measured in blood samples from healthy broilers and from broilers suffering from Sudden Death Syndrome (SDS) within 30 min after death. The LDH activity was higher in 4- to 9-wk-old SDS birds than in healthy birds (P < 0.05). The GOT activity was higher in SDS birds irrespective of age (P < 0.05). The CPK activity of SDS birds was not significantly different from that of healthy birds. The K+ level was about twofold higher in SDS birds of 4 to 6 and 7 to 9 wk of age than in healthy birds of the same age, whereas the Na+ level did not differ from that of healthy birds. The LDH, GOT, and CPK activities and Na+ level were unchanged for the post-mortem period of 30 min, but the K+ level increased. These results suggest that an elevations in serum LDH and GOT activities occur in association with SDS, and may be used as a characteristic sign of birds that are dying of SDS.

Characterization of lactic acid formation and adenosine triphosphate consumption in calcium-loaded erythrocytes of broiler chickens.

The formation of lactic acid and consumption of adenosine triphosphate (ATP) were measured in erythrocytes from broiler and White Leghorn chickens with the goal of providing further evidence that lactic acid, a metabolite from glycolysis, is an etiological factor for sudden death syndrome (SDS) in broiler chickens. When loaded with Ca2+, erythrocytes exhibited increased lactic acid concentrations. The effect of Ca2+ loading was significantly faster in broiler erythrocytes. In the absence of adenosine used as an energy-yielding substrate, Ca2+ loading was followed by a reduction in ATP concentrations. The effect was also significantly faster in broiler erythrocytes. Intravenous injection of a 20% lactate solution at 0.1 mL/kg produced SDS-like death of broilers but not White Leghorns. The results obtained in erythrocytes indicate that, in broilers, a greater amount of energy is required for regulation of intracellular Ca2+ concentrations, and, thereby, formation of lactic acid is more accelerated. In addition to the result that intravenous lactate injection caused death in broilers, an elevation of lactic acid concentrations in blood, arising from operation of Ca2+ regulation mechanisms, may predispose broilers to incidence of SDS.

Changes of mental stress biomarkers in ultramarathon.

We investigated the possible influence of an exhaustive physical exercise on mental stress biomarkers (serotonin, tryptophan, and beta-endorphin) along with dopamine, noradrenaline and free fatty acids in an ultramarathon race in which 45 km was run on the first day and 90 km on the second. We obtained serum samples at 6 different time points during and after the race from 18 Japanese male runners who completed the marathon. Overall changes of serum serotonin and tryptophan concentrations were statistically significant according to ANOVA for repeated measurements (p < 0.05). Serum serotonin levels elevated rapidly on the first day with the post hoc Tukey's test. Tryptophan concentrations inversely decreased during the race, possibly because of utilization for synthesis of serotonin. Levels of beta-endorphin appeared to increase on the first and second days, but were not statistically significant. In conclusion, serum serotonin, tryptophan and beta-endorphin appeared to be used for mental stress markers in physical exercise.

Soybean products and reduction of breast cancer risk: a case-control study in Japan.

Components of the Japanese diet, which might contribute to the relatively low breast cancer incidence rates in Japan, have not been clarified in detail. Since soybean products are widely consumed in Japan, a case-control study taking account of the menopausal status was conducted using data from the hospital-based epidemiologic research program at Aichi Cancer Center (HERPACC). In total, 167 breast cancer cases were included and 854 women confirmed as free of cancer were recruited as the control group. Odds ratios (OR) and 95% confidence intervals (95% CI) were determined by multiple logistic regression analysis. There were reductions in risk of breast cancer associated with high intake of soybean products among premenopausal women. Compared with women in the lowest tertile, the adjusted ORs for top tertile intake of tofu (soybean curd) was 0.49 (95% CI, 0.25-0.95). A significant decrease in premenopausal breast cancer risk was also observed for increasing consumption of isoflavones (OR=0.44; 95% CI, 0.22-0.89 for highest vs lowest tertile; P for trend=0.02). The present study found a statistically inverse association between tofu or isoflavone intake and risk of breast cancer in Japanese premenopausal women, while no statistically significant association was evident with the risk among postmenopausal women.

Age-related change of cefazolin binding to rat serum proteins and its relation to the molar ratio of free fatty acid to serum albumin.

The binding of cefazolin to rat sera has been studied as a function of age. A significant difference was observed in the cefazolin binding to serum protein among 1-, 2-, 3-, 5-, 7-, 50- and 100-week-old rats. There was a good correlation between the dissociation constants of cefazolin binding and the molar ratio of free fatty acid to albumin concentration in sera. This suggests that both changes of concentration of albumin and free fatty acid, which could be a major endogenous inhibitor of cefazolin binding, play an important role in the age-related changes of the serum protein binding. Removal of free fatty acid in 1- and 2-week-old rat sera showed marked increases of the cefazolin binding. On the contrary, addition of oleic acid to 7-week-old rat serum produced significant reduction of cefazolin binding to rat serum protein. Accordingly, free fatty acid could effectively inhibit the cefazolin binding in the physiological concentration range with increasing age, and the age-related changes of cefazolin binding to rat serum protein appear to be due to the fluctuation of the molar ratios of free fatty acid to albumin concentration in sera.

An analysis of the fatty acid composition of total lipids from mycoplasmas.

The fatty acid composition of the total lipids obtained from 9 species (22 strains) of Mycoplasma and 3 species (7 strains) of Acholeplasma was determined by gas-liquid chromatography (GLC). The major fatty acids of the Mycoplasma species were palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), and linoleic acid (C18:2). Lauric acid (C12:0) and myristic acid (C14:0) were present in small amounts in this genus. For the Mycoplasma species, the most prevalent fatty acid was C16:0 or C14:0, and other leading fatty acids were C12:0, C18:0, C18:1 and C18:2. A substantial amount of C12:0 and C14:0 was found in the Acholeplasma species. It was confirmed that C12:0 and C14:0 were synthesized de novo, based on the fact that 14C-acetate was incorporated into these acids. The total percentage of C16 fatty acid was less than that of C18 fatty acids in all the strains of Mycoplasma and Acholeplasma except 4 strains. This may be related to osmo-regulation of the L-forms of Streptococci as mentioned in other reports.

Preferential uptake of lactosylceramide-bearing dipalmitoylphosphatidylcholine-liposomes into liver: role of membrane fluidity.

The effect of the membrane fluidity of lactosylceramide (LacCer)-bearing liposomes on their liver uptake was investigated in rats. Liposomes consisting of phosphatidylcholine (PC): cholesterol:dicetylphosphate:LacCer (7:2:1:1, molar ratio) were prepared with various fluidities using dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC) and egg PC. These liposomes were all equally stable in serum and were small enough to pass freely through the fenestrae and be taken up easily by liver cells. The LacCer modification of DPPC-liposomes markedly facilitated blood clearance, whereas no enhancing effect of LacCer was observed with egg PC- and DMPC-liposomes. Tissue distribution studies showed the preferential liver uptake of LacCer-bearing DPPC-liposomes, which was largely compatible with the rapid clearance induced by the LacCer modification. In addition, electron spin resonance (ESR) spectroscopic analysis revealed that the LacCer modification of DPPC-liposomes significantly enhanced the order parameter S, indicating that LacCer-bearing DPPC-liposomes were the most rigid of those used in this study. These observations suggest that the membrane fluidity of liposomes in vivo is a crucial factor for their preferential liver uptake.