Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The purpose of this study was to identify and characterize the functional activity of monocarboxylic acid transporter 1 (MCT1) on the human retinal pigmented epithelium (RPE) cell line, ARPE-19, and to evaluate whether the cell line can function as an in vitro screening tool for intravitreally administered drugs/prodrugs targeted to the MCT1 expressed in RPE. Uptake studies were carried out at 37 degrees C, for 30 s, with ARPE-19 cells. [(14)C]l-Lactic acid was selected as a substrate for this transporter. Uptake of [(14)C]L-lactic acid by ARPE-19 cells was found to exhibit saturable kinetics (K(m) = 3.1 +/- 0.6 mM and V(max) = 63.1 +/- 4.1 pmol/min/mg of protein). Monocarboxylic acids, such as benzoic acid, salicylic acid, and pyruvic acid, inhibited the uptake of [(14)C]L-lactic acid whereas di- and tricarboxylic acids, such as phthalic, succinic, and citric acids, did not demonstrate any inhibitory effect. Uptake was stereospecific where D-lactic acid was less effective in inhibiting [(14)C]L-lactic acid uptake than unlabeled L-lactic acid. ELISA indicated the expression of only MCT1, MCT4, and MCT8 isoforms by ARPE-19 cells. Increase in [(14)C]L-lactic acid uptake was observed as the uptake medium pH was lowered from 7.4 to 5.0. Moreover, inhibition of [(14)C]L-lactic acid uptake was observed in the presence of the protonophore 2,4-dinitrophenol. Uptake was significantly decreased in the presence of sodium azide, ouabain, p-chloromercuribenzoic acid (pCMBA), N-ethylmaleamide, dithiothreitol, and p-chloromercuribenzene sulfonate (pCMBS). However, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and L-thyroxine did not inhibit [(14)C]L-lactic acid. RT-PCR studies and sequence analysis of the PCR product confirmed the expression of MCT1 by ARPE-19 cells. Our results indicate that MCT1 is functionally active and is the only MCT isoform involved in the apical uptake of monocarboxylates by ARPE-19 cells. This cell line may thus be used as an effective screening tool for intravitreally administered drugs/prodrugs targeted toward MCT1 expressed on the RPE.
Mol Pharm
PMID:Functional activity of a monocarboxylate transporter, MCT1, in the human retinal pigmented epithelium cell line, ARPE-19. 1580 85

We propose a protocol for determination of lactate threshold (LT) and test the validity of one aerobic training based on LT in rats. In group I, V(LTi) (velocity at LT before training) was determined in all rats (n=10), each rat training at its own V(LTi) and in group II, animals (n=7) ran at 15 m min(-1), the mean V(LTi) of group I. The training consisted of daily runs at V(LTi) for 50 min, 5 days/week, for 4 weeks. In group I, this program increased V(LT) (V(LTi) 14.90+/-1.49 m min(-1) and V(LTf), after training, 22.60+/-1.17 m min(-1)) and the velocity at exhaustion (19.50+/-1.63 m min(-1) and 27.60+/-1.17 m min(-1)). [Lactate] at LT (2.62+/-0.43 mmol L(-1) versus 2.11+/-0.15 mmol L(-1)) and relative values of LT (76+/-3% versus 82+/-2%) stayed unaltered. In group II the V(LTf) was 20+/-1.8 m.mim(-1), the [lactate] at the LT, 2.02+/-0.17 mmol.L(-1); the exhaustion speed, 23.57+/-2.11 m.mim(-1) and relative value of LT, 82.71+/-2.29%. There were no significant differences in these parameters between groups I and II. Thus, this protocol based on LT is effective and the mean V(LT) determined in a small number of healthy untrained rats can be used for aerobic training in a larger group of healthy animals of same gender and age.
Comp Biochem Physiol A Mol Integr Physiol 2005 Apr
PMID:Method for diagnosis and control of aerobic training in rats based on lactate threshold. 1593 99

Human erythrocytes are highly specialized cells whose function is oxygen transport. These cells' sole metabolic source of energy is the fermentation of glucose via glycolysis. They contain an active insulin receptor and respond to insulin by increasing phosphorylation of tyrosine residues in several proteins. However, no metabolic effects have yet been associated with activation of this receptor in human erythrocytes. Here, we show that insulin increases the rate of glycolysis in human erythrocytes. Lactate production increased 56 and 173% in the presence of 10 and 100 nM insulin, respectively. A higher insulin concentration (1000 nM) partially reversed the stimulation of glycolysis. These effects occur through activation of the key glycolytic enzyme 6-phosphofructo-1-kinase, which exhibits the same pattern of modulation by insulin as seen for glycolytic flux. This modulation also occurs physiologically since ex vivo experiments revealed 50% stimulation of 6-phosphofructo-1-kinase (PFK) activity following a high carbohydrate meal. Insulin increases phosphorylation of PFK and redistributes the enzyme in red blood cells, causing it to detach from the erythrocyte membrane: upon insulin stimulation, the amount of enzyme associated with the plasma decreases by 86%. Detachment is a common mechanism of enzyme activation. As a consequence, insulin prevents up to 68% of red cells hemolysis. These results show that insulin regulates erythrocyte glycolysis and viability and suggest that this regulation is associated to other erythrocyte functions such as oxygen transport. Finally, we suggest that this regulatory mechanism might be compromised in patients with diabetes mellitus.
Mol Genet Metab 2005 Nov
PMID:Regulation of human erythrocyte metabolism by insulin: cellular distribution of 6-phosphofructo-1-kinase and its implication for red blood cell function. 1610 94

The object of the present study was to investigate the effect(s) of UV-B irradiation on the functional integrity, metabolic and detoxifying capacity of the isolated goat hepatocytes. Isolated goat hepatocytes were subjected to UV-B irradiation invitro for 0, 250, 500, 1250, 2500 and 7500 Joules/m2 which correspond to the irradiation time of 0, 1, 2, 5, 10 and 30 min. Cells were then analysed for Viability (Trypan blue exclusion test [TBE], 3-[4,5-dimethylthiozol-2yl]-2,5-diphenyltetrazolium bromide [MTT] assay, Membrane integrity (Lactate dehydrogenase [LDH] leakage, Lipid peroxidation) Detoxification (Ureagenesis, Cytochrome P450 activity [CYP450, Diazepam metabolism] and Glutathione-S-Transferase [GST] activity. The results show that there was no difference in functional, metabolic as well as detoxifying parameters of the hepatocytes when irradiated from 0-1250 Joules/m2, whereas a significant alteration was appreciable in the parameters such as LDH leakage, lipid peroxidation, and CYP450 activity when irradiated beyond 1250 Joules/m2. Our present findings suggest that the biologically compatible and feasible dose of UV-B irradiation for xenotransplantation appears to be 1250 Joules/m2.
Mol Cell Biochem 2005 Sep
PMID:Effect of ultraviolet B (302 nm) irradiation on viability, metabolic and detoxification functions of goat hepatocytes--in vitro study. 1613 14

The modified base queuine is a nutrient factor for lower and higher eukaryotes except yeast. It is synthesized in eubacteria and inserted into the wobble position of specific tRNAs (tRNA(GUN)) in exchange of guanine at position 34. The tRNAs of Q family are completely modified in terminally differentiated somatic cells. However, mainly free queuine is present in embryonic and fast proliferating cells, tRNA remains Q deficient. Lactate dehydrogenase (LDH) A mRNA and LDH A protein is known to increase when cells are grown in hypoxic conditions. In the present study, the level of LDH isozymes is analyzed in different tissues of normal and cancerous (DLA) mice and the effect of queuine treatment on LDH isozyme is observed. LDH A isozyme is shown to increase in serum and liver of DLA mice. The level and activity of LDH A decreases on queuine treatment. In skeletal muscle and heart, LDH A isozyme decreases while LDH B increases in DLA mice. Queuine administration leads to change back towards normal. In case of brain, LDH A increases but LDH B decreases in DLA mice. Queuine treatment leads to decrease in A4 anaerobic isozymes of LDH. The results suggest that queuine suppresses anaerobic glycolytic pathway, which leads to tumor suppression of DLA mice.
Mol Biol Rep 2005 Sep
PMID:Modulation of lactate dehydrogenase isozymes by modified base queuine. 1617 20

The metabolic acidosis resulting from an intense exercise bout is large in crocodilians. Here we studied recovery from this pH perturbation in the American alligator. Metabolic rate, minute ventilation, arterial pH and gases, and strong ion concentration were measured for 10 h after exhaustion to elucidate the mechanisms and time course of recovery. Exhaustion resulted in a significant increase in lactate, metabolic rate, and ventilation, and a decrease in arterial PCO2), pH and bicarbonate. By 15 min after exhaustion, oxygen consumption returned to rest though carbon dioxide excretion remained elevated for 30 min. Arterial PO2), [Na+], and [K+], increased following exhaustion and recovered by 30 min post-exercise. Minute ventilation, tidal volume, [Cl-], and respiratory exchange ratio returned to resting values by 1 h. The air convection requirement for oxygen was elevated between 15 and 60 min of recovery. Breathing frequency and pH returned to resting values by 2 h of recovery. Lactate levels remained elevated until 6 h post-exercise. Arterial PCO2) and [HCO3-] were depressed until 8 h post-exercise. Compensation during recovery of acid-base balance was achieved by altering ventilation: following the initial metabolic acidosis and titration of bicarbonate, a relative hyperventilation prevented a further decrease in pH.
Comp Biochem Physiol A Mol Integr Physiol 2006 Mar
PMID:Recovery from an activity-induced metabolic acidosis in the American alligator, Alligator mississippiensis. 1644 82

The influence of docosahexaenoic acid (DHA)- and eicosapentaenoic acid (EPA)-enriched phosphatidylcholine (PC) on the permeability, transport and uptake of phospholipids was evaluated in Caco-2 cells. The cells were grown on permeable polycarbonate transwell filters, thus allowing separate access to the apical and basolateral chambers. The monolayers of the cells were used to measure lucifer yellow permeability and transepithelial electrical resistance (TEER). Transcellular transportation of diphenylhexatriene (DPH) labeled-PC small unilamellar vesicles (SUV) from the apical to basolateral chamber, and uptake of the same SUV was monitored in the cell monolayers. Cell-membrane perturbation was evaluated to measure the release of lactate dehydrogenase and to determine the cell viability with sodium 2-(4-iodophenyl)-3-(4-nitrophenyl) -5-(2, 4-disulfophenyl)-2H-tetrazolium dye reduction assay. The lucifer yellow flux was 1.0 and 1.5 nmol/h/cm(2) with 50 microM PC, and 17.0 and 23.0 nmol/h/cm(2) with 100 microM PC when monolayers of Caco-2 cells were treated with DHA- and EPA-enriched PC, respectively. TEER decreased to 24 and 27% with 50 and 100 microM DHA-enriched PC, and to 25 and 30% with 50 and 100 microM EPA-enriched PC, respectively. Our results show that DHA- and EPA-enriched PC increases tight junction permeability across the Caco-2 cell monolayer whereas soy PC has no effect on tight junction permeability. Transportation and uptake of DHA- and EPA-enriched PC SUV differed significantly (P < 0.01) from those of soy PC SUV at all doses. We found that PC SUV transported across Caco-2 monolayer and was taken up by Caco-2 cells with very slight injury of the cell membrane up to 100 microM PC. Lactate dehydrogenase release and cell viability did not differ significantly between the treatment and control, emphasizing that injury was minimal. Our results suggest that DHA- and EPA-enriched PC enhance the permeability, transport and uptake of PC SUV across monolayers of Caco-2 cells.
Mol Cell Biochem 2006 Apr
PMID:Docosahexaenoic acid and eicosapentaenoic acid-enriched phosphatidylcholine liposomes enhance the permeability, transportation and uptake of phospholipids in Caco-2 cells. 1647 71

Lactic acid bacteria (LAB) employ sucrase-type enzymes to convert sucrose into homopolysaccharides consisting of either glucosyl units (glucans) or fructosyl units (fructans). The enzymes involved are labeled glucansucrases (GS) and fructansucrases (FS), respectively. The available molecular, biochemical, and structural information on sucrase genes and enzymes from various LAB and their fructan and alpha-glucan products is reviewed. The GS and FS enzymes are both glycoside hydrolase enzymes that act on the same substrate (sucrose) and catalyze (retaining) transglycosylation reactions that result in polysaccharide formation, but they possess completely different protein structures. GS enzymes (family GH70) are large multidomain proteins that occur exclusively in LAB. Their catalytic domain displays clear secondary-structure similarity with alpha-amylase enzymes (family GH13), with a predicted permuted (beta/alpha)(8) barrel structure for which detailed structural and mechanistic information is available. Emphasis now is on identification of residues and regions important for GS enzyme activity and product specificity (synthesis of alpha-glucans differing in glycosidic linkage type, degree and type of branching, glucan molecular mass, and solubility). FS enzymes (family GH68) occur in both gram-negative and gram-positive bacteria and synthesize beta-fructan polymers with either beta-(2-->6) (inulin) or beta-(2-->1) (levan) glycosidic bonds. Recently, the first high-resolution three-dimensional structures have become available for FS (levansucrase) proteins, revealing a rare five-bladed beta-propeller structure with a deep, negatively charged central pocket. Although these structures have provided detailed mechanistic insights, the structural features in FS enzymes dictating the synthesis of either beta-(2-->6) or beta-(2-->1) linkages, degree and type of branching, and fructan molecular mass remain to be identified.
Microbiol Mol Biol Rev 2006 Mar
PMID:Structure-function relationships of glucansucrase and fructansucrase enzymes from lactic acid bacteria. 1652 21

1. Glial cells are the most abundant cell population in the central nervous system. The aim of this study was to examine the effects of melatonin, 7-nitroindazole, and riluzole on glutamate toxicity in primary glial cell culture. 2. Glutamate toxicity was induced by addition of 100 microM glutamate to the cultures, and then 100 microM melatonin, 500 microM 7-nitroindazole, and 10 (M riluzole were administered in different groups. Lactate Dehydrogenase activity and nitrite levels were determined at the 1st, 6th, and 24th h. 3. Melatonin, 7-nitroindazole, and riluzole decrease Lactate Dehydrogenase activity at the 1st, 6th, and 24th h (at all hours, p<0.05). Nitrite levels were decreased by melatonin and riluzole at the 1st, 6th, and 24th h. 4. In this study, we observed that melatonin, 7-nitroindazole, and riluzole are effective as protective agents on glutamate toxicity in mixed glial cells.
Cell Mol Neurobiol 2007 Mar
PMID:Neuroprotective agents: is effective on toxicity in glial cells? 1675 18

Potential use of various nootropic drugs have been a burning area of research on account of various physical and chemical insult in brain under different toxicological conditions. One of the nootropic drug centrophenoxine, also known as an anti-aging drug has been exploited in the present experiment under aluminium toxic conditions. Aluminium was administered by oral gavage at a dose level of 100 mg/Kg x b x wt/day for a period of six weeks. To elucidate the region specific response, study was carried out in two different regions of brain namely cerebrum and cerebellum. Following aluminium exposure, a significant decrease in the activities of enzymes namely Hexokinase, Lactate dehydrogenase, Succinate dehydrogenase, Mg(2+) dependent ATPase was observed in both the regions. Moreover, the activity of acetylcholinesterase was also reported to be significantly decreased. Post-treatment with centrophenoxine was able to restore the altered enzyme activities and the effect was observed in both the regions of brain although the activity of lactate dehydrogenase and acetylcholinesterase did not register significant increase in the cerebellum region. Further, centrophenoxine was able to improve the altered short-term memory and cognitive performance resulted from aluminium exposure. From the present study, it can be concluded that centrophenoxine has a potential and can be exploited in other toxicological conditions also.
Mol Cell Biochem 2006 Oct
PMID:Evidence for centrophenoxine as a protective drug in aluminium induced behavioral and biochemical alteration in rat brain. 1696 89


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