• Journal of Pharmaceutical Investigation
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• v.20 no.4
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• pp.223-228
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• 1990

The contribution of endogenous transport systems to the blood-brain barrier (BBB) transport of basic and acidic drugs was studied by using a carotid injection technique in rats and an isolated bovine cerebrovascular disease state were compared between the normotensive rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP) which have been well established as an animal model with pathogenic similarities to humans. Basic drugs such as eperisone, thiamine and scopolamine inhibited, in a concentration dependent manner the in vivo uptake of $[{^3}H]choline$ through BBB, whereas amino acids and acidic drugs such as salicylic acid and valproic acid did not inhibit the uptake. The uptake of $[^3H]choline$ by B-CAP increased with time and showed a remarkable temperature dependency. The uptake of $[^3H]choline$ by B-CAP showed the very similar inhibitory effects as observed in the in vivo brain uptake, and was competitively inhibited by a basic drug, eperisone. The in vivo BBB uptakes of $[^3H]acetic$ acid and $[^{14}C]salicylic$ acid were dependent on pH of the injectate and the concentration of drugs. Several acidic drugs such such as salicylic acid, benzoic acid and valproic acid inhibited the in vivo uptake of $[^3H]acetic$ acid, whereas amino acid, choline and a basic drug such as eperisone did not inhibit the uptake. The uptake of acetic acid by B-CAP was competitively inhibited by salicylic acid. The permeability surface area product (PS) through BBB for $[^3H]choline$ in SHRSP was significantly lower than that in WKY. The concentration of choline in the brain dialysate in SHRSP was about half of that in WKY, while no significant difference was observed in the plasma concentration of choline between SHRSP and WKY. No significant difference was observed in the transport of monocarboxylic acids, glucose and neutral amino acid through BBB between SHRSP and WKY. From these results, it was concluded that BBB transport system of choline contributes to the transport of basic drugs through BBB, that acidic drugs can be transported via a moncarboxylic acid BBB transport system and that the specific dysfuntion of the BBB choline transport in SHRSP was ascribed to the reduction of the maximum velocity of choline concentration in the brain interstitial fluids.

• Journal of Food Hygiene and Safety
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• v.25 no.4
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• pp.388-394
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• 2010

Metabolomics aims at the comprehensive, qualitative and quantitative analysis of wide arrays of endogenous metabolites in biological samples. It has shown particular promise in the area of toxicology and drug development, functional genomics, system biology and clinical diagnosis. In this study, analytical technique of MS instrument with high resolution mass measurement, such as time-of-flight (TOF) was validated for the purpose of investigation of amino acids, sugars and fatty acids. Rat urine and serum samples were extracted by selected each solvent (50% acetonitrile, 100% acetonitrile, acetone, methanol, water, ether) extraction method. We determined the optimized liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) system and selected appropriated columns, mobile phases, fragment energy and collision energy, which could search 17 metabolites. The spectral data collected from LC/TOFMS were tested by ANOVA. Obtained with the use of LC/TOFMS technique, our results indicated that (1) MS and MS/MS parameters were optimized and most abundant product ion of each metabolite were selected to be monitorized; (2) with design of experiment analysis, methanol yielded the optimal extraction efficiency. Therefore, the results of this study are expected to be useful in the endogenous metabolite fields according to validated SOP for endogenous amino acids, sugars and fatty acids.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.5
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• pp.420-424
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• 2006

A simple method for depleting E. coliS30 extracts of endogenous tRNA has been developed. An $ethanolamine-Sepharose^{(R)}$ column equilibrated with water selectively captured the tRNA molecules in E. coli S30 extracts. As a result, S30 extracts filtered through this column became completely dependent upon the addition of exogenous tRNA to mediate cell-free protein synthesis reactions. We anticipate that the procedures developed and described will be particularly useful for in vitro suppression reaction studies designed to introduce unnatural amino acids into protein molecules.

• BMB Reports
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• v.41 no.11
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• pp.790-795
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• 2008

An inducible lysine 6-dehydrogenase (Lys 6-DH), which catalyzes the oxidative deamination of the 6-amino group of L-lysine in the presence of $NAD^+$, was purified to homogeneity from Achromobacter denitrificans, yielding a homodimeric protein of 80 kDa. The enzyme was specific for the substrate L-lysine and $NAD^+$ served as a cofactor. The dimeric enzyme associated into a hexamer in the presence of 10 mM L-lysine. The $K_m$ values for L-lysine and $NAD^+$ were 5.0 and 0.09 mM, respectively. The lys 6-dh gene was cloned and overexpressed in E. coli. The open reading frame was 1,107 nucleotides long and encoded a peptide containing 368 amino acids with 39,355 Da. The recombinant enzyme was purified to homogeneity and characterized. Enzyme activities and kinetic properties of the recombinant enzyme were almost the same as those of the endogenous enzyme obtained from A. denitrificans. Crystals of the enzyme were obtained using the hanging drop method.

• Journal of the Korean Society of Food Science and Nutrition
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• v.20 no.2
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• pp.126-132
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• 1991

Nutritional factors affecting the urinary excretion of acid-soluble peptides(ASP) in rats were studied using protein-free diet, gluten diet, casein diet, and gluten supplemented with lysine and threonine(GLT) diet. The content of urinary ASP was lowest in protein-free diet group among the four kinds of diets above. But the amino acid pattern of urinary ASP in the four dietary groups were similar each other, suggesting that urinary ASP is mainly from endogenous origin under these nutritional conditions. The efficiency of dietary protein utilization was significantly lower in gluten diet than that of casein diet or GLT diet. Those findings suggest that the rate of urinary excretion of ASP-form amino acids can be employed as an index of protein metabolism, particularly as a simple index in the assessing the status of protein nutrition.

• BMB Reports
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• v.39 no.3
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• pp.335-338
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• 2006

Methylglyoxal (MG) is an endogenous physiological metabolite which is present in increased concentrations in diabetics. MG reacts with the amino acids of proteins to form advanced glycation end products. In this in vitro study, we investigated the effect of MG on the structure and function of ceruloplasmin (CP) a serum oxidase carrier of copper ions in the human. When CP was incubated with MG, the protein showed increased electrophoretic mobility which represented the aggregates at a high concentration of MG (100 mM). MG-mediated CP aggregation led to the loss of enzymatic activity and the release of copper ions from the protein. Radical scavengers and copper ion chelators significantly prevented CP aggregation. CP is an important protein that circulates in plasma as a major copper transport protein. It is suggested that oxidative damage of CP by MG may induce perturbations of the copper transport system and subsequently lead to harmful intracellular condition. The proposed mechanism, in part, may provide an explanation for the deterioration of organs in the diabetic patient.

• BMB Reports
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• v.46 no.4
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• pp.225-229
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• 2013

Methylglyoxal (MG) is an endogenous metabolite which is present in increased concentrations in diabetics and reacts with amino acids to form advanced glycation end products. In this study, we investigated whether ferritin enhances DNA cleavage by the reaction of MG with lysine. When plasmid DNA was incubated with MG and lysine in the presence of ferritin, DNA strand breakage was increased in a dose-dependent manner. The ferritin/MG/lysine system-mediated DNA cleavage was significantly inhibited by reactive oxygen species (ROS) scavengers. These results indicated that ROS might participate in the ferritin/MG/lysine system-mediated DNA cleavage. Incubation of ferritin with MG and lysine resulted in a time-dependent release of iron ions from the protein molecules. Our data suggest that DNA cleavage caused by the ferritin/MG/lysine system via the generation of ROS by the Fenton-like reaction of free iron ions released from oxidatively damaged ferritin.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.6
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• pp.873-882
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• 2008

Eight ileally cannulated pigs (BW $35.9{\pm}0.9kg$) were randomly allotted according to a $4{\times}3$ Latin square design to determine the effects of cellulose, pectin and starch on standardized ileal digestibility (SID) and apparent total tract digestibility (ATTD) of crude protein (CP) and amino acids (AA) as well as on the bacterial AA contribution in feces. The pigs were fed the control diet (20.2% CP, % dry matter (DM)) or one of the three experimental diets in which 25% of the control diet was substituted by cellulose, starch or pectin. Due to this substitution, dietary CP levels were lower in the cellulose (15.5% CP, % DM), pectin (15.4% CP, % DM) and starch diet (15.2% CP, % DM). Following a 15-d adaptation period, feces were collected for 5 d and ileal digesta for a total of 24 h. Starch increased SID of CP, while cellulose and pectin had no significant effect on the digestibility of CP. Overall, starch supplementation resulted in higher (p<0.05) SID values of histidine, isoleucine, threonine, alanine, aspartic acid, cysteine, glycine and serine compared with cellulose, while pectin decreased (p<0.05) SID of valine and proline compared with the starch and control diet. Both cellulose and pectin reduced (p<0.05) the ATTD of CP and AA, while starch decreased (p<0.05) ATTD of phenylalanine, alanine, proline and serine compared with the control. With regard to bacterial AA composition of the fecal mixed bacterial mass (MBM), cellulose supplementation increased (p<0.05) its content of N and almost all AA, except for valine, while pectin caused higher contents of arginine, histidine and proline compared with the control (p<0.05). The bacterial contribution of arginine in feces was higher (p<0.05) in the cellulose treatment, while pectin reduced (p<0.05) the bacterial contribution of leucine, alanine, glutamic acid and proline in feces compared with the control. In conclusion, the effects of cellulose, starch and pectin on SID were rather small. Bacterial activity in the large intestine can only explain the reduced ATTD values for arginine in the cellulose treatment, but not for the other AA in the cellulose and pectin treatments, suggesting higher endogenous losses of these AA in the large intestine.

• Korean journal of applied entomology
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• v.15 no.4 s.29
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• pp.169-178
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• 1976

The concentration of amino acids, total nitrogen, trehalose, lipids and the activities of respiratory, acid$\cdot$alkaline phosphatase, glutamic oxalozcetic transaminase and glutamic pyruvic transaminase during larval stage in Pine leaf gall midge, Thecodiplosis janensis Uchi. et Inouye were measured using Paper chromatographic method, micro-Kjeldahl method, Thin layer chromatographic method, Warburg's manometric method, Bessey-Lowry method and Reitman-Frankel method, respectively. Healthy specimens )yore chosen as samples of each larval stages; alrva in gall and larva in soil. Amino acids present in the alcoholic extracts were alanine, glutamic acid, glycine, histidine, methionine, proline, threonine, tryptophan and valine. The total nitrogen concentration reached to 31.348mg/g during the larva in gall and the larval stage in soil of the value was decreased to 29.027mg/g. The hemolymph sugar, trehalose value for larva in soil was about two times of the value for larva in gall. Total lipid, phospholipid,monoacylglycerol, triacylglycerol, sterol, free fatty acid and ester cholesterol were identified at larval stages in gall and soil. Triacylglycerol concentration reached high level in contrast with other lipid contents during larvae in gall and larva in soil. Free fatty acid, sterol except decreased lipids during larval stage in soil. Endogenous respiration, succinate of respiratory activities decreased at larval stage in soil compare with larva in gall. The activities of acid phosphatase decreased larval stage in soil but the activities of alkaline phosphatase increased remarkably. The activities of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase reached high level of the larva in gall.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.6
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• pp.856-865
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• 2007

Caveolin-1 of the caveolin family of proteins regulates mammary gland development and has been shown to play a contradictory role in breast tumor progression. A specific anti-Caveolin-1 antibody will be useful for functional study of Caveolin-1 in different tissues. In this study, we generated a rabbit polyclonal antibody that specifically recognizes the N-terminal amino acids 50-65 of Caveolin-1. This polyclonal antibody specifically reacted with Caveolin-1 extracted from cells of different species, including human epithelial A431 cells, goat primary mammary epithelial cells and mice fibroblast NIH 3T3 cells, by Western blotting. Endogenous Caveolin-1 protein expressing in cells and normal human tissues was detected by this polyclonal antibody using immunocytofluorescent and immunohistochemical staining, respectively. Furthermore, an apparent decrease in Caveolin-1 expression in tumorous breast and colon tissues was detected by this polyclonal antibody. In conclusion, we have identified amino acids 50-65 of Caveolin-1, which contains an epitope that is specific to Caveolin-1 and is conserved in the human, goat and mouse. In future, this anti-Caveolin-1 antibody can be used to examine the progression of breast and colon cancers and to study functions of Caveolin-1 in human, goat and mouse cells.