Retinol-binding protein (RBP) was expressed in Escherichia coil using the cDNA for rat RBP, and characterized. The expressed RBP was fused to maltose-binding protein (MBP) at the N-terminal end (MBP-RBP), and MBP was enzymatically removed from the MBP-RBP with proteinase factor Xa. The binding of retinol and transthyretin (TTR) to the recombinant RBP was monitored by means of gel filtration. The recombinant RBP specifically bound to retinol with an affinity similar to that of purified RBP from rat serum. Furthermore, the retinol-bound recombinant RBP formed hetero-complexes with TTR similar to RBP. Thus, the results showed that the recombinant RBP expressed in E. codi is as functional as serum RBP in terms of retinol and TTR bindings.
Our previous study revealed that no retinyl esters were detectable in chick and hen lungs, suggesting that the retinol esterification system may be absent in these tissues. This possibility encouraged us to investigate whether chick lungs exhibit the activity of a retinol esterifying enzyme, i.e., lecithin : retinol acyltransferase (LRAT). The LRAT activ ity was assayed with dilauroyl phosphatidylcholine and either complex of retinol-cellular retinol-binding protein, type two or retinol-cellular retinol binding protein in microsomal preparations of lung, duodenum and liver of 7-day-old chicks. Relatively high levels of LRAT activity were present in the duodenum and the liver of chicks as well as in the rat lung. However, the chick lung exhibited no LRAT activity. The lungs of both rat and chick showed similar and low levels of acyl-CoA : retinol acyl transferase (ARAT) activity, but only rat lung, but not chick lung, contained a detectable amount of retinyl esters. Thus, the retinyl ester storage in the lung seems to depend on the presence of LRAT activity in the lung, but it is independent of the presence of ARAT activity in the lung. The absence of LRAT activity and retinyl esters in the chick lung suggests that the retinol in the chick lung may not be provided from retinyl ester storage, and the retinol transferred directly from serum should be utilized to generate retinoic acid.
Thirty-four foods were analyzed in order to determine the content of water-soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) Using the results with the standard table for 227 foods, the intake ratio of IDF/SDF of an average Japanese was calculated for the period 1946-1990. The ratio was 3.22 in 1990 as calculated on the food intakes shown in the national nutrition survey, and the secular change was not detected since 1946 when the ratio was 3.30. The ratio was also shown to be well preserved between types of households including the age of the head. Using dietary records of 60 healthy city workers (average 42.8 years) for 4 weeks, however, the weekly average ratio for an individual was found to vary in the range of 2.25-5.13 although the total average for 60 individuals was 3.33. Thus, the well preserved IDF/SDF intake ratio for an average Japanese showed, on the contrary, a wide variation of food selection between each person.
The hyperlipidemia and atherosclerosis-prone (HAP) Japanese quail is a strain developed for the study of atherosclerosis by genetic selection from the commercially available (CA) Japanese quail. To delineate the characteristics of cholesterol metabolism in this strain, concentrations of serum lipids as well as hepatic enzyme activities were compared between HAP and CA quail. The hepatic enzymes studied are involved in the key step reaction in cholesterol metabolism: HMG-CoA reductase, ACAT, and cholesterol 7α-hydroxylase. The animals were fed ad libitum with either 1% cholesterol or cholesterol-free semipurified diet for 28 days. Although a significant increase (p<0.01) in serum cholesterol was observed in both strains on elapse of cholesterol feeding, formation of atheroma was seen exclusively in HAP quail of the cholesterol-fed group. The serum and liver cholesterol levels of HAP quail fed the cholesterol diet were significantly higher (p<0.01) than those of CA quail. No significant differences were seen in the rate of cholesterol biosynthesis (HMG-CoA reductase activity), cholesterol ester formation (ACAT activity) and cholesterol catabolism (7α-hydroxylase activity) between CA and HAP quail. Furthermore, the fecal excretions of acidic and neutral sterol showed no significant difference between strains. Although the formation of atheroma in HAP quail may be presumably due to the contribution of the marked increase in serum cholesterol level, the rate of cholesterol catabolism and synthesis in HAP quail compared well with those of CA quail. These observations suggest that the retarded rate of cholesterol biosynthesis or catabolism is not responsible for hyperchol-esterolemia in HAP quail.
Effects of fatty acids on accumulation and secretion of histamine in rat basophilic leukemia RBL-2H3 cells and leukotriene release from peritoneal exudate cells isolated from Wistar rats were examined in relation to the manifestation of type I allergic reactions. When RBL-2H3 cells were cultured for 24h in the presence of 1 mM short chain fatty acids, a marked increase in histamine accumulation was observed, especially with butyric acid. In addition, Ca-ionophore A23187-stimulated histamine release was enhanced in the cells treated with 0.1mM mono to hexa unsaturated fatty acids with 18 to 22 carbonchains. On the other hand, LTB4 release from rat peritoneal exudate cells was inhibited in the presence of polyunsaturated fatty acids, both n-6 and n-3, having more than 3 double bonds. Inhibitory activity was enhanced by an increase in the number of double bonds, and docosahexaenoic acid (DHA) exerted the highest activity with complete inhibition at 0.1mM and 3 5.5% inhibition even at 10μM. A hydrophobic radical scavenger (9, 10-diphenylanthracene) and two antioxidants (butyrated hydroxytolu-ene and α-tocopherol) inhibited the production of LTB4, but hydrophilic counterparts (mannitol and ascorbic acid) did not. These results suggest that lipophilic anti-oxidative agents, as well as PUFA, inhibit the production of LTB4.
The effects of calcium gluconate on the utilization of mag nesium and nephrocalcinosis in male Wistar rats made magnesium deficient by adding excess dietary phosphorus (1.195g of phosphorus/100g of diet) and calcium (1.04g of calcium/100g of diet) were compared with the effects of calcium carbonate. The effects of dietary magnesium concentration on the magnesium status and nephrocalcinosis were also examined. Adding excess dietary phosphorus and calcium decreased the apparent magnesium absorption ratios and the concentrations of magnesium in the serum and femur and increased the deposition of calcium in the kidney, and the low magnesium condition (0.024g of magnesium/ 100g of diet) aggravated the deposition of calcium and the low magnesium status. The apparent magnesium absorption ratios and femur magnesium concentration in the rats fed a calcium gluconate diet (an equimolar mixture of calcium gluconate and calcium carbonate was used as a source of calcium) were significantly higher than in the rats fed a calcium carbonate diet (only calcium carbonate was used as a source of calcium), irrespective of dietary magnesium concentration. Dietary calcium gluconate lessened the accumulation of calcium in the kidney and increased the serum magnesium concentration compared with dietary calcium carbonate, when the rats were fed the normal magnesium diet (0.049g of magnesium/100g of diet) but not the low magnesium diet. We speculate that the increased utilization of magnesium by feeding the calcium gluconate diet to a limited extent prevented the low magnesium status and the severity of nephrocalcinosis caused by adding excess dietary phosphorus and calcium.
The effects of a rice protein isolate (RPI) on 7, 12-dimeth-ylbenz[α] anthracene (DMBA)-induced mammary tumor progression were investigated in female Sprague-Dawley rats. At 6 weeks of age, rats were fed a casein, RPI or soybean protein isolate (SPI) diet, respectively. After 1 week, DMBA was administered orally at the dose of 30mg/kg body weight. The mean tumor number per tumor-bearing rat at autopsy was significantly lower only in rats fed RPI than in those fed casein. Palpable tumors at the mid point of the experiment were significantly lower in rats fed RPI and SPI than in those fed casein. Serum estradiol-17β concentrations were lower in rats fed the SPI (but not in those fed RPI) than in those fed casein. In a further experiment, no differences were found in hepatic microsomal DMBA-arylhydrocarbon hydroxylase activity after 7 days of feeding the respective diets. These results suggest that RPI exerts its inhibitory effect on DMBA-induced mammary tumor-igenesis irrespective of changes in circulating estrogens or modulation of hepatic DMBA metabolism.
A low casein diet supplemented with 0.2-0.3% L-methionine promotes growth in rats, although it causes liver fat accumulation. Oligo-L-methionine on the other hand, prepared by papain-catalyzed oligomerization of L-methionine ethylester, stimulates growth in rats without causing liver fat accumulation, when added to a 8% casein diet. Oligo-L-methionine is a mixture of oligomers, with a oligomerization degree of 5 to 12, and is hydrolyzed very slowly by pancreatic juice and everted gut rings of rats. A methionine derivative, cyclo (LL-dimethionine), was examined in this study as it is hydrolyzed and/or absorbed very slowly from the intestinal tract and it was expected to be utilized as a methionine source without causing liver fat accumulation, when added to a low casein diet. It consists of two methionine residues linked together by two cis-peptide bonds and, therefore, is expected to be hydrolyzed and/or absorbed very slowly from intestinal tract. However, although cyclo (LL-dimethionine) was absorbed slowly from the intestinal tract, it was not utilized by rats as a methionine source, when added to 8% casein.
The effects of protein nutrition on insulin-like growth factor-I (IGF-I) receptor in various tissues of rats were investigated. Northern blot and dot blot hybridization analyses were performed using RNA from testis, heart, lung, intestine, stomach, kidney, and brain of rats fed on a 12% casein diet, on a 12% gluten diet which was marginally deficient in lysine and threonine, or on a protein-free diet. The mRNA content of IGF-I receptor in the testis and heart of the rats fed on the 12% gluten and protein-free diets was significantly larger than those of the rats fed on the 12% casein diet. Whereas in other tissues examined, IGF-I receptor mRNA content did not change significantly under the different nutritional conditions. The amount of IGF-I receptor in these tissues and the affinity to IGF-I were determined by measuring the amount of 125I-labelled-IGF-I bound to solubilized IGF-I receptor. The affinity of IGF-I receptor to IGF-I in each tissue under the various nutritional conditions did not show any marked differences. The number of receptors did not change in the testis, lung, intestine, brain or kidney; possibly increased in stomach of the rats fed on the 12% gluten or protein-free diet; and slightly decreased in heart of the rats fed on the 12% gluten diet compared with that of the rats fed on the 12% casein diet. These results indicate that the synthesis of IGF-I receptor is regulated in a distinctive way in each tissue in response to protein nutrition, and suggest that the regulation may have some physiological meaning in signal transmission of IGF-I. The amount of IGF-I receptor, however, was relatively constant in most tissues. Because the plasma IGF-I and IGF-binding protein concentrations change dramatically under different nutritional conditions, we conclude that concentrations of plasma IGF-I and IGF-binding proteins may mainly regulate the IGF-I action in tissues in response to protein nutrition.
The feasibility of near-infrared (NIR) diffuse reflectance spectroscopy scanning from 1100 to 2500 nm in the analyses of the amounts of moisture, protein, starch, amylose, and tannin in buckwheat flours was examined. Fifty ground samples comprised of 27 different cultivars harvested in 12 different countries were divided into two sets: 35 samples as a calibration set and 15 samples as a prediction set. The multiple regression equations (MREs) established between the second derivative NIR spectra data and the reference data, which were obtained by chemical analyses of the calibration set, gave multiple correlation coefficients of higher than 0.93 for moisture, protein, and starch, and workable standard error of predictions (SEPs) in relation to the reference standard deviation data. In contrast, the MREs for amylose and tannin were judged as unstable because the SEPs had no difference with their reference standard deviation data. The influential wavelengths were 1925 nm for water (assigned to the combination of the stretching and bending vibrations of hydroxyl [OH]), 2057nm for protein (assigned to the combination of NH and amide II or III), and 2100nm for starch (assigned to the combination of OH and CO). The obtained results indicated that the NIR procedure can be used as a nondestructive analysis method for rapid and simple measurement of the amounts of moisture, protein, and starch in buckwheat flours.
Rice bran trypsin inhibitor (RBTI) was digested by pepsin alone or by pepsin and pancreatin with or without bovine serum albumin (BSA) to clarify the changes in trypsin inhibitory activity, apparent antigenicity, and molecular size of RBTI. In vitro pepsin digestion of RBTI in the absence of BSA caused the gradual loss of the trypsin inhibitory activity and antigenicity. This was mostly due to a progressive degradation of the native 14.5-kDa RBTI molecule to small molecular mass products. The presence of BSA in the digestion mixture prevented the RBTI degradation and was accompanied with a considerable protection of the activity and antigenicity. Similar results were also given by in vitro pepsin-pancreatin digestion. These findings suggest that RBTI may be present in its active form in the gastrointestinal tract when fed to animals, especially with a dietary protein.