We investigated the effects of a high-protein diet and resveratrol supplementation on immune cells changes induced by abdominal irradiation in rats. Female Wistar rats were divided into 5 groups: 1) control diet, 2) control diet with irradiation 3) 30% high-protein diet with irradiation, 4) normal diet with resveratrol supplementation and irradiation, and 5) 30% high-protein diet with resveratrol supplementation and irradiation. We measured blood protein and albumin concentrations, lipid profiles, white blood cell (WBC) counts, proinflammatory cytokine production, and splenocyte proliferation in rats that had been treated with a 17.5 Gy dose of radiation 30 days prior. A high-protein diet affected plasma total cholesterol and very low density lipoprotein-cholesterol levels, which were increased by the radiation treatment. In addition, the lymphocyte percentage and immunoglobulin M (IgM) concentration were increased, and the neutrophil percentage was decreased in rats fed a high-protein diet. Resveratrol supplementation decreased the triglyceride (TG) level, but increased the IgM concentration and splenocyte proliferation. Proinflammatory cytokine production was lower in rats fed a high-protein diet supplemented with resveratrol than in rats fed a control diet. The results of the present study indicate that high-protein diets, with or without resveratrol supplementation, might assist with recovery from radiation-induced inflammation by modulating immune cell percentages and cytokine production.
This study was performed to investigate effect of dietary protein and calcium levels on cad-mium intoxication in rats. Adult Sprague-Dawley male rate(245$\pm$21g) were blocked into 18 groups of 7 animals according to body weight Nine experimental diets different with protein(40%, 15%, 7%) and calcium (1.3%, 0.6%, 0.1%) levels were prepared. Nine groups of animals were fed each diet with 50ppm cadmium in drinking water and the other 9 groups without cadmium for 30days. Results were summarized as follows: 1) Body weight gain F. E. R(Food Efficiency Ratio) and weights of liver kidney and femur were higher in high protein groups among cadmium exposed groups. 2) Cadmium contents in liver and intestine were higher in rats fed high protein diet or low calcium diet among cadmium exposed groups. Fecal cadmium excretion was highest in high protein-high calcium diet group among cadmium exposed animals. Metallothionein contents in liver kidney and intestine were higher in animals exposed to cadmium and fed high protein diets. 3) Gel filtration chromatography of cytosolic solution showed that the higher dietary protein and calcium levels were the more cadmium was found in metallothionein fractions. 4) No gross histopathological change was seen in liver kidney and intestine of cadmium exposed rats. However a significant increase of smooth endoplasmic reticulum which was alleveated by high protein-high calcium diet was observed. Results obtained indicated that not only high protein diet but also high calcium diet showed preventive effect on cadmium intoxication by increasing the induction of metallothionein syn-thesis and decreasing the cadmium absorption.
This study was performed to investigate the effects of dietary protein and calcium levels on calcium metabolism in eight healthy Korean adult females. The 2-day metabolic study consisted of a 2 day adaptation period and three 6-day experimental periods. Three experimental diets were low protein low calcium(LPLCa : protein 44g, Ca 422mg), higher protein low calcium(HPLCa : protein 85g, Ca 365mg), and high protein high calcium (HPHCa : protein 84g, Ca 727mg). The apparent calcium absorption was likely to be affected by the calcium intake rather than by the protein intake. Average calcium absorption rate was about 23-29% of calcium intake. The calcium balance was -21.44mg for LPCa, -25.02mg for HPLCa, and -3.22mg for HPHCa. Avergae urinary calcium excretion was 127.7mg for LPLCa, 108.6mg for HPLCa, and 215.4mg for HPHCa. Urinary calcium excretion was more closely related to the changes of calcium intake rather than of protein intake. These results seemed to be due to the interactions between the high phosphours contained in the high protein diet and the little discrepancy of protein intake levels.
To study the effect of menopause and dietary protein level on Ca metabolism, ovariectomy (OVAX) and sham operations were performed in 16-week old female rats. Each treatment group was fed for 16 weeks either 5%(L) or 50%(H) casein diets, forming SH, SL, OH, OL groups. High protein groups(SH, OH) showed higher Ca and hydroxyproline excretion in urine. Urinay hydroxyproline was also higher in OVAX, which tells the possibility of increased bone resorption by OVAX and by high dietary protein. At 16th wee, however, urinary Ca and hydroxyproline of SH caught up with OH group, whereas those of OL remained higher than SL. Therefore it seems that high dietary protein overrides the effect of OVAX with time. Urnary protein measured at 8th week was higher in high protein groups, especially in OH. GFR was not differ significantly among groups at 8th week. At 16th week, however, high protein groups showed twice the GFR value of low protein groups. Therefore the increase of urinary Ca and hydroxyproline in SH and OH groups can be explained partly by the increased GFR. The tendency of increased GFR and urinary excretion of protein, Ca, and hydroxyproline was most obvious in OH group. It seems that the effect of high protein diet is likely to accelerated by ovariectomy. The effect of Ovax and dietary protein on the composition of femur, scapular, and lumbar bones, was not pronounced. However, when only the high protein groups were compared, OVAX resulted in the reduction of bone weight, ash and Ca contents, especially in femur. The reason that was no significant effect on bone might be due to the short experimental period to induce that was no significant effect on bone might be due to the short experimental period to induce the changes on bone composition and dietary Ca content used in this experiment may have been high enough to prevent bone loss.
We assessed heterologous protein expression in 64 strains obtained from the Escherichia coli Reference (ECOR) collection, a collection representing diverse natural E. coli populations. A plasmid generating a glutathione S-transferase and plant carbonic anhydrase fusion protein (GST-CA) under the control of the tac promoter was introduced into the ECOR strains, and the quantity of the fusion protein was determined by SDS-PAGE. The foreign protein was generated at various levels, from very high (40 strains, high producers) to very low (six strains, low producers). Immunoblotting showed that the high producers expressed approximately 250-500 times more GST-CA protein than the low producers. The results of semi-quantitative RT-PCR showed that the low producers generated mRNA levels comparable to those of the high producers, thereby suggesting that, at least in this case, inefficient translation is a major cause of the low production. We introduced a different plasmid, which expressed a maltose binding protein and plant guanylate kinase fusion protein (MBP-GK) into the six low producers. Interestingly, five of these expressed MBP-GK at very high levels. Thus, we conclude that the production of a particular protein from an expression vector can vary considerably, depending on the host strain. Strains in the ECOR collection could function as useful alternative hosts when a desired level of protein expression is not obtained from commonly used strains, such as E. coli K12 or B derivatives.
Based on plasmid display technology by the complexes of fusion protein and the encoding plasmid DNA, an in vitro selection method for high affinity DNA-binding protein was developed and experimentally demonstrated. The GAL4 DNA-binding domain (GAL4 DBD) was selected as a model DNA-binding protein, and enhanced green fluorescent protein (EGFP) was used as an expression reporter for the selection of target proteins. Error prone PCR was conducted to construct a mutant library of the model. Based on the affinity decrease with increased salt concentration, mutants of GAL4 DBD having high affinity were selected from the mutant protein library of protein-encoding plasmid complex by this method. Two mutants of (Lys33Glu, Arg123Lys, Ile127Lys) and (Ser47Pro, Ser85Pro) having high affinity were obtained from the first generation mutants. This method can be used for rapid in vitro selection of high affinity DNA-binding proteins, and has high potential for the screening of high affinity DNA-binding proteins in a sequence-specific manner.
To evaluate the differences of the levels and sources of protein intake human protein metabolism, an 26-day metabolic balance study was conducted in 10 healthy Korean adult females. In the pre-study, the subjects recorded their own diets for 3 days. The metabolic balance study consisted of 6-day adaptation period, 10-day moderate protein period(60-65g/d) and 10-day high protein period(90-95g/d). During the moderate and high protein period, 5 subjects were fed the higher animal protein meals and the other 5 subjects were fed the high plant protein meals. Body weight, nitrogen balance and blood chemistries were monitored through out the study. The urine volume were sighificantly larger in the animal protein group and, the dietary fiber and fecal weights were significantly heavier in the plant protein diet group. But no statistically significant differences were found between the two dietary groups in apparent nitrogen digestability, urinary nitrogen excretion and nitrogen balance. Body weight, serum protein, albumin and HDL-cholesterol levels were not changed, but serum total cholesterol level in the animal protein diet group was elevated significantly from 143.8mg/dl on moderate potein diet to 173.0mg/dl on high proetin diet. In conclusion, from the observation of this short-term N balance study, plant diet on the adequate level of calorie and protein intake had almost the same effect of animal protein diet for protein maintenace in adults.
Albio rats right after weaning, weighing $50{\sim}55g$, were divided into the control, high-carbohydrate-, high-lipid-, and high-protein-fed groups. and were fed for 12 weeks with the respective diets to observe the increase in body weights as well as changes in the chemical constituentes and enzyme activities in the liver tissue, with the following results. (1) There was little difference in the rate of increase in the body weights among the groups, showing normal growth, except the high-protein-fed group which showed decrease in rate of body weight increase from the 7th week after feeding. (2) The liver weight was either increased after 12 weeks of feeding with the high-carbohydrate and high-Lipid diets, or showed no difference with the high-protein diet, as compared to the control weight. (3) The liver cytosol protein content was increased when fed with the high-protein diet, but decreased when fed with the high-carbohydrate and high lipid diets, as compared to the control content. (4) The triglyceride and cholesterol contents in the liver were decreased in the high-protein-fed group, but increased markedly in the high-carbohydrate- and high-lipid-fed groups as compared to the control values. (5) The hepatic glucokinase, G6PD, LDH, and fatty acid synthetase activities were increased in the high-carbohydrate and high-lipid-fed groups, and GOT and CPT activities were increased in the high-protein-fed group. From the above results. it was known that the high-carbohydrate and high-lipid diets stimulated the hepatic lipid metabolism, giving rise to lipogenesis, but the high- protein diet could prevent the lipogeuesis leading to the body weight increase.
To investigate the effect of estrogen and dietary protein level on Ca metabolism, female rats were undergone ovariectomy or sham-operation. Ovariectomized rate were divided into either estrogen-or vehicle-treated groups. Each treatment group was again divided into 40%-casein(H) or 10%-casein(L) diet groups. All experimental diets contained 0.2% Ca, 0.4% P and fed to rats for 8 weeks. Apparant Ca absorption and Ca balance were not affected by dietary protein level and ovariectomy, however they were increased by estrogen injection and this effect was even higher in low protein groups. Urinary Ca excretion were higher in high protein groups. GFR was not affected by dietary protein level, ovariectomy, or by estrogen injection. Urinary protein excretion was higher in high protein groups, which implies that the kidney funtion was deteriorated by high protein diet, and this may account partly for the higher urinary Ca in high protein groups. Ovariectomy or estrogen treatment had no effect on urinary protein excretion. Urinary hydroxyproline was higher in ovariectomized rats and increased in high protein grous. Elevated value of ovarictomized rats was lowered by estrogen injection, especially in low protein group. Alkaline phosphatase tended to increase in ovariectomized groups and lowered with estrogen treatment, but this difference was not statistically significant. Serum PTH was not affected by ovariectomy and dietary protein level. Therefore the increased hydroxproline excretion does not seem to be attributed to PTH. Dietary protein level, ovariectomy and estrogen treatment did not affect the weights and components of femur, scapular, and 4th vertebra. Ash/wt ratio of femur was, however, lower in ovariectomized rats and increased with estrogen treatment. Therefore, among the bones studied, femur seemed to be the most vulnerable. The results of this study shows that estrogen treatment may alleviate or reduce bone loss in postmenopausal women somewhat, especially for those people with low protein diet.
In recent years, the importance of proteomic works, such as protein expression, detection and identification, has grown in the fields of proteomic and diagnostic research. This is because complete genome sequences of humans, and other organisms, progress as cellular processing and controlling are performed by proteins as well as DNA or RNA. However, conventional I protein analyses are time-consuming; therefore, high throughput protein analysis methods, which allow fast, direct and quantitative detection, are needed. These are so-called protein microarrays or protein chips, which have been developed to fulfill the need for high-throughput protein analyses. Although protein arrays are still in their infancy, technical development in immobilizing proteins in their native conformation on arrays, and the development of more sensitive detection methods, will facilitate the rapid deployment of protein arrays as high-throughput protein assay tools in proteomics and diagnostics. This review summarizes the basic technologies that are needed in the fabrication of protein arrays and their recent applications.
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