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Biomedical subjects

D J Tuma

Publications and source records attributed to D J Tuma.

At least 109 records · Page 6Linked to original sources
PubMed1985

Measurement of protein synthetic activity by determination of peptidyl[3H]puromycin formation in liver slices after ethanol administration.

We investigated the utility of [3H]puromycin as an alternate and adjunct precursor to amino acids for measuring protein synthetic activity in rat liver slices. Slices were incubated in the presence of either [3H]puromycin or radiolabeled valine to compare the incorporation of these isotopic precursors into nascent hepatocellular proteins. Compared to liver slices from controls, comparable decreases in the incorporation of both [3H]puromycin and labeled valine were observed in experiments using slices from fasted rats and in slices preincubated with 25 mM ethanol. Radiolabeling of nascent polypeptides with either [3H]puromycin or labeled valine in liver slices from rats fed a liquid diet containing ethanol was also decreased compared to slices from pair-fed control and chow-fed animals. Our results demonstrated the validity of using [3H]puromycin to detect changes in protein synthetic activity under these conditions. The potential advantage of using [3H]puromycin for in vivo studies is discussed.

Alcoholism↗
PubMed1985

Covalent binding of acetaldehyde to hepatic proteins during ethanol oxidation.

Acetaldehyde production and the radiolabeling of hepatic proteins were determined in rat liver slices incubated with 14C-ethanol (10 mmol/L). Significant labeling of hepatic proteins occurred in the presence of protein synthesis inhibitors, indicating that, under these conditions, the radiolabeling of protein did not occur via de novo protein synthesis. Additional experiments indicated that the major source of protein-bound radioactivity derived from 14C-ethanol oxidation was the formation of 14C-acetaldehyde adducts with proteins. This conclusion was made from observations that pyrazole, an inhibitor of ethanol oxidation and, therefore, acetaldehyde formation, decreased radiolabeling of protein, whereas cyanamide, which elevated hepatic acetaldehyde levels, markedly increased the labeling of protein. Furthermore, L-cysteine, which can bind acetaldehyde and, therefore, act as an acetaldehyde trap, substantially reduced protein-bound radioactivity. It was also demonstrated that acetaldehyde formed both stable and unstable adducts with hepatic proteins and that unstable adducts may undergo conversion to form stable adducts during incubation.

Acetaldehyde↗
PubMed1985

Colchicine-binding properties of hepatic tubulin. The role of "time-decay".

Determination of total and polymerized tubulin in the liver by the [3H]colchicine-binding assay is hampered by rapid loss of binding sites. One solution, which has been proposed to overcome this problem, is the use of a "time-decay" corrected assay that extrapolates to an initial binding capacity based on the results of testing multiple aliquots of liver supernatants over time. If such an approach is valid, it follows that the extrapolated initial value should be independent of the rate of time-decay. To test this relationship, we modulated the half-life (T1/2) for time-decay of a fixed amount of hepatic microtubule-derived tubulin using various concentrations of organic acids, sodium glutamate and glucose-1-phosphate. We found that the initial binding capacity based on simple first order decay did vary with the T1/2. Similar results were also obtained with bovine brain tubulin added to hepatic supernatants. These results indicate that the quantification of hepatic tubulin derived by extrapolated time-decay values is inaccurate and may overestimate the actual amount of tubulin present, especially when time-decay is rapid.

Animals↗
PubMed1984

Enhancement of acetaldehyde-protein adduct formation by L-ascorbate.

The effect of L-ascorbate on the binding of [14C]acetaldehyde to bovine serum albumin was examined. In the absence of ascorbate, acetaldehyde reacted with albumin to form both unstable (Schiff bases) and stable adducts. Ascorbate (5 mM) caused a time-dependent increase in the formation of total acetaldehyde-albumin adducts, which were comprised mainly of stable adducts. Significant enhancement of adduct formation by ascorbate was observed at acetaldehyde concentrations as low as 5 microM. An ascorbate concentration as low as 0.5 mM was still effective in stimulating stable adduct formation. The electron acceptor, 2,6 dichlorophenolindophenol, prevented the ascorbate-induced increase in albumin-adduct formation. Ascorbate also caused enhanced acetaldehyde adduct formation with other purified proteins, including cytochrome c and histones, as well as the polyamino acid, poly-L-lysine. These results indicate that ascorbate, acting as a reducing agent, can convert unstable acetaldehyde adducts to stable adducts, and can thereby increase and stabilize the binding of acetaldehyde to proteins.

2,6-Dichloroindophenol↗
PubMed1984

Impaired plasma membrane glycoprotein assembly in the liver following acute ethanol administration.

The in vivo effects of acute ethanol administration on hepatic plasma membrane assembly were studied in the rat. When [14C]fucose and [3H]N-acetylmannosamine, a sialic acid precursor, were injected following an acute dose of ethanol, minimal effects on fucose and a slight reduction of sialic acid incorporation into the total pool of hepatic membrane glycoproteins were observed. However, the assembly of labeled fucoproteins and sialoproteins into the plasma membrane was markedly inhibited in the ethanol-treated animals. These results indicate that ethanol administration impairs the late stages of membrane assembly which include the transport of glycoproteins from the Golgi complex to the plasma membrane and/or the insertion of glycoproteins into the membrane.

Animals↗
PubMed1984

Effect of ethanol on hepatic secretory proteins.

Both acute and chronic ethanol administration inhibit the secretion of albumin and glycoproteins from the liver. Impairment of posttranslational steps of the secretory process are mainly involved in this secretory defect, although in some instances altered synthesis of the protein moiety may be a factor. Decreased secretion following ethanol administration results in the intrahepatic retention of export proteins. The secretory defect is a consequence of the metabolism of ethanol and is likely mediated via acetaldehyde, although more conclusive proof is still required. The manner by which acetaldehyde impairs the secretory process is unknown, but may be related to its high reactivity with hepatocellular proteins. The specific posttranslational steps or processes involved in the secretory defect are still unclear; however, it appears that the final steps of secretion (post-Golgi events) may be the primary site of impairment. Impaired secretion of proteins from the liver could contribute to altered levels of plasma proteins and hepatomegaly as well as to the liver injury observed in the alcoholic.

Acetaldehyde↗
PubMed1984

Methotrexate hepatotoxicity.

An inhibitor of folate metabolism, amethopterin (methotrexate) has been successfully used in the treatment of psoriasis and neoplastic disease. This drug produces several dangerous side effects of both an acute and chronic nature which have widely curtailed its use. A serious chronic side effect of the drug is its hepatotoxicity, which may culminate in hepatic cirrhosis and death. To date the underlying mechanism of methotrexate in producing liver damage is unknown. Results of three studies conducted in this laboratory on the nutritional effects of methotrexate offer some evidence that the hepatotoxicity may possibly be incurred through the effect of the drug on methionine biosynthesis and methylation processes. This thesis is discussed in the light of methylating agents vital to the synthesis of methionine.

Animals↗
PubMed1984

Increasing inhibition of hepatic protein secretion by colchicine during development.

Colchicine and other antimicrotubular drugs have been shown to impair plasma protein secretion by fetal rat liver at late gestation (day 21) markedly less than by the mature organ. In the present study, the effect of colchicine on plasma protein secretion by liver slices was determined at several times during rat development. Secretion of [14C]leucine-labeled albumin and [3H]glucosamine-labeled glycoproteins was only minimally impaired (20%) by colchicine (50 microM) on gestation day 19. Inhibition of protein secretion increased to about 40% just before birth and remained unchanged throughout the perinatal period. Inhibition increased thereafter until the maximum effect (75%) was first observed 28 days after birth. Studies in which [3H]-colchicine was used indicated that neither reduced colchicine levels in tissue nor reduced affinity of tubulin for colchicine caused the diminished impairment of secretion in immature liver. These findings suggest that participation of microtubules in liver plasma protein secretion is reduced during development and that full participation is delayed beyond the perinatal period in this species.

Animals↗
PubMed1984

Acute effects of ethanol on hepatic glycoprotein secretion in the rat in vivo.

The in vivo effects of acute ethanol administration on the secretion of plasma glycoproteins and albumin were studied. Ethanol (6 g/kg body wt) was administered intragastrically to nonfasted rats, while control animals received isocaloric glucose. Hepatic protein and glycoprotein secretion in these animals was assessed by determining the rate of appearance of [3H]leucine- and [14C]fucose-labeled proteins into the plasma. Ethanol treatment decreased the secretion of both leucine- and fucose-labeled proteins into the plasma. This inhibition of secretion was accompanied by a corresponding increase in the hepatic retention of both leucine- and fucose-labeled immunoprecipitable secretory proteins. Pretreatment of the ethanol-treated animals with pyrazole, an inhibitor of ethanol metabolism, prevented the inhibition of hepatic protein secretion. These in vivo studies confirm our earlier in vitro findings that acute ethanol administration inhibits the secretion of plasma proteins, resulting in their hepatocellular retention.

Animals↗
PubMed1984

Reduced effect of microtubule inhibitors on hepatic protein secretion in the fetal rat.

Drugs that block the assembly of microtubules inhibit protein secretion by mature hepatocytes. In this study, we ascertained the effects of these drugs on hepatic protein secretion during development. Slices prepared from adult and late-gestation (day 21) fetal rat livers were incubated with colchicine, vinblastine, or podophyllotoxin, and the secretion of [14C]leucine-labeled proteins was then determined. Colchicine (10 microM) decreased trichloroacetic acid-precipitable protein secretion by 65% in adult liver, but only by 31% in fetal liver. A higher (50 microM) concentration of colchicine caused minimal further impairment of secretion in both age groups. Colchicine (10 microM) impaired the secretion of immunoprecipitable albumin to about the same degree as trichloroacetic acid-precipitable proteins in both adult and fetus. Determinations of [3H]colchicine uptake into slices indicated that reduced hepatocellular levels of the drug were not the cause of the diminished effect on protein secretion observed in the fetus. Like colchicine, various concentrations of vinblastine and podophyllotoxin inhibited trichloroacetic acid-precipitable protein and albumin secretion consistently less from the fetal liver (30%-50%) than from the adult liver (50%-75%). These results indicate a reduced dependence on normal microtubule function for protein secretion in the developing liver.

Albumins↗
PubMed1983

Betaine, metabolic by-product or vital methylating agent?

The possible physiological role of betaine, the oxidative product of choline, is considered. It is proposed that betaine, instead of merely being a metabolic by-product of choline oxidation, may serve as an important methylating agent when normal methylating pathways are impaired by ethanol ingestion, drugs or nutritional imbalances. Furthermore, betaine may prove to have therapeutic application in cases of altered folate, vitamin B12 or methionine metabolism.

Alcohol Oxidoreductases↗
PubMed1983

Hair and plasma zinc levels following exclusion of biliopancreatic secretions from functioning gastrointestinal tract in humans.

Pancreatic secretions have been shown to be important in zinc absorption in experimental animals. Recently, complete surgical diversion of biliary and pancreatic secretions from the functioning small intestine has been utilized in Europe to treat morbid obesity. To determine the importance of pancreatic secretions in zinc absorption in the human, we measured hair and serum zinc levels in 14 patients having undergone complete biliopancreatic bypass 12-56 months earlier and compared these values with those obtained from 11 patients subjected to only partial biliopancreatic bypass as well as to six morbidly obese controls. No differences were observed in either hair or serum zinc levels between any of these groups of patients. Pancreatic secretions do not appear to be necessary for adequate zinc absorption in humans.

Adult↗
PubMed1983

Acetaldehyde adducts with proteins: binding of [14C]acetaldehyde to serum albumin.

Acetaldehyde, the immediate oxidation product of ethanol metabolism, was assessed for its ability to bind covalently to a purified protein in solution. Bovine serum albumin (BSA) was used as the model protein incubated in the presence of 0.2 mM [14C]acetaldehyde at pH 7.4 and at 37 degrees C. Acetaldehyde formed both stable and unstable adducts with serum albumin. Unstable adducts were identified following stabilization with the reducing agent sodium borohydride. We examined both types of binding using trichloroacetic acid precipitation, gel filtration, and dialysis as means to separate bound from free acetaldehyde. All three methods of analysis gave comparable results except that the number of stable acetaldehyde adducts with albumin were significantly lower following separation by dialysis. The effects of L-cysteine, L-lysine, and reduced glutathione were assessed for their abilities as competitive reagents to decrease binding of [14C]acetaldehyde to BSA. Addition of cysteine caused a rather dramatic concentration-dependent reduction in [14C]acetaldehyde binding to BSA when compared to that caused by lysine which displayed a relatively mild effect on covalent binding. The presence of glutathione caused a concentration-dependent decrease in protein-bound radioactivity that was stronger than that by lysine but not as effective as cysteine. The ability of each reagent to reverse the formation of preformed acetaldehyde adducts with BSA was also examined. Only L-cysteine effectively decreased the number of unstable acetaldehyde adducts with BSA while stable binding of acetaldehyde remained essentially unaffected by any of the three reagents. These results indicate that acetaldehyde can covalently bind to protein and form unstable as well as stable adducts.

Acetaldehyde↗
PubMed1983

Hepatic protein synthesis and secretion after jejunoileal bypass in the rat.

The rat was used as an animal model to explore the mechanism responsible for the development of hepatomegaly and hypoproteinemia which commonly occur after jejunoileal bypass. Sprague-Dawley rats. 300 to 350 g, were divided into three groups of 12 animals. Six of the 12 rats per group served as study animals and six as controls. The first six were subjected to 90% jejunoileal bypass and the six controls were sham-operated and pair-fed. In the second group, six animals were subjected to 90% jejunoileal resection and six controls were sham-operated and pair-fed. Six animals in the third group were underfed so that their weights mimicked that of the bypassed animals and six controls were fed ad libitum. After 8 wk the animals were killed. Liver weights, hepatic protein content, and serum protein and triglycerides were determined. Synthesis and secretion of proteins and glycoproteins were measured using incorporation of 14C-leucine and 14C-glucosamine, respectively, into hepatic and medium proteins by liver slices. Bypassed animals demonstrated hepatomegaly, decreased serum proteins and triglycerides, and increased hepatic protein content. While both protein and glycoprotein synthesis remained normal, the secretion of these proteins into the medium appeared to be impared. Comparable changes did not occur after jejunoileal resection or after underfeeding. This study suggests that the impairment of glycoprotein and protein secretion may be a contributing factor in the increased liver weight and protein content in conjunction with decreased serum protein observed in the bypassed rat.

Animals↗
PubMed1983

Effects of chronic ethanol administration on hepatic glycoprotein secretion in the rat.

The effects of chronic ethanol feeding on protein and glycoprotein synthesis and secretion were studied in rat liver slices. Liver slices from rats fed ethanol for 4-5 wk showed a decreased ability to incorporate [14C]glucosamine into medium trichloracetic acid-precipitable proteins when compared to the pair-fed controls; however, the labeling of hepatocellular glycoproteins was unaffected by chronic ethanol treatment. Immunoprecipitation of radiolabeled secretory (serum) glycoproteins with antiserum against rat serum proteins showed a similar marked inhibition in the appearance of glucosamine-labeled proteins in the medium of slices from ethanol-fed rats. Minimal effects, however, were noted in the labeling of intracellular secretory glycoproteins. Protein synthesis, as determined by measuring [14C]leucine incorporation into medium and liver proteins, was decreased in liver slices from ethanol-fed rats as compared to the pair-fed controls. This was the case for both total proteins as well as immunoprecipitable secretory proteins, although the labeling of secretory proteins retained in the liver slices was reduced to a lesser extent than total radiolabeled hepatic proteins. When the terminal sugar, [14C]fucose, was employed as a precursor in order to more closely focus on the final steps of hepatic glycoprotein secretion, liver slices obtained from chronic ethanol-fed rats exhibited impaired secretion of fucose-labeled proteins into the medium. When ethanol (5 or 10 mM) was added to the incubation medium containing liver slices from the ethanol-fed rats, the alterations in protein and glycoprotein synthesis and secretion caused by the chronic ethanol treatment were further potentiated. The results of this study indicate that liver slices prepared from chronic ethanol-fed rats exhibit both impaired synthesis and secretion of proteins and glycoproteins, and these defects are further potentiated by acute ethanol administration.

Animals↗
PubMed1983

Binding of metabolically derived acetaldehyde to hepatic proteins in vitro.

Homogenates of rat liver incubated with 10 mM [14C]ethanol were analyzed for acetaldehyde production and for both stable and unstable radiolabeled acetaldehyde adducts with proteins. During incubation, formation of acetaldehyde and of 14C-labeled proteins both increased with time in a parallel manner. Acetaldehyde generation and subsequent formation of radiolabeled proteins were potentiated by supplementation of the cell-free system with NAD+ (1 mM). Cycloheximide (0.1 mM) caused no significant reduction in protein-bound radioactivity, whereas the addition of strong nucleophiles L-cysteine (5 mM) and reduced glutathione (5 mM) each decreased radiolabeling by 50 to 60%. Preheating of crude homogenates at 90 degrees C, prior to incubation with [14C]ethanol profoundly decreased subsequent production of acetaldehyde and formation of 14C-labeled proteins. The results indicate that the major source of protein-bound radioactivity derived from [14C]ethanol oxidation in this system is due to binding of enzymatically derived [14C]acetaldehyde to hepatic proteins.

Acetaldehyde↗