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D J Tuma

Publications and source records attributed to D J Tuma.

At least 55 records · Page 3Linked to original sources
PubMed1994

Effects of ethanol feeding on the interaction of rat hepatocytes with laminin peptides.

Laminin, a complex glycoprotein of the extracellular matrix, contains a number of biologically active sites. These sites are involved in cell growth, attachment, differentiation, and gene expression. Our previous studies have shown that chronic ethanol consumption by rats impairs hepatocyte attachment to various components of the extracellular matrix including laminin. In this study, three synthetic peptides (PA22-2, YIGSR, and RGD) that correspond to three distinct functional sites on the laminin molecule were used to investigate the effect of ethanol consumption on their cognate receptors. Initially, varying concentrations of each peptide were incubated with isolated hepatocytes from ethanol-fed and pair-fed control rats. These hepatocytes were then assayed for the ability to attach to laminin. The results indicated that all three peptides effectively inhibited laminin-mediated cell adhesion: the degree of inhibition appeared similar between pair-fed controls and ethanol-fed animals. Of the three peptides, PA22-2 showed the most dramatic inhibition of attachment. Therefore, we investigated the ability of hepatocytes to attach directly to PA22-2 itself. Attachment of hepatocytes from ethanol-fed animals to PA22-2 was impaired by 30% after 4 days and 90% by 14 days. Conversely, no significant difference in attachment to the entire laminin molecule was observed in ethanol-fed animals at these early time points. These results indicated that the ethanol-induced impairment of hepatocyte attachment to laminin may be caused by the decreased interaction of hepatocytes with specific functional sites on the laminin molecule and that specific receptors on the hepatocyte may be affected differently.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcoholism↗
PubMed1994

Vapor phase exposure to acetaldehyde generated from ethanol inhibits bovine bronchial epithelial cell ciliary motility.

Acetaldehyde is a toxic product of the oxidation of ethanol and is known to induce slowing of ciliary motility in airway epithelium. Alcohol ingestion results in high exhaled breath concentrations of ethanol where lung microsomes and upper airway bacterial flora are capable of metabolizing it to acetaldehyde. Because acetaldehyde is very volatile, we hypothesized that the production and release of acetaldehyde vapor into the airway may result in ciliary slowing or ciliastasis. To test this hypothesis, ciliated bovine bronchial airway epithelial cells were maintained on collagen-coated dishes for 48 hr before coincubation with a separate dish containing control or test mixtures. In this arrangement, the ciliated cells were exposed only to the volatile components of the separate dish. The separate dish contained ethanol, acetaldehyde, or an acetaldehyde-generating system (AGS) that consisted of ethanol + glucose + glucose oxidase + catalase. This mixture was placed next to the ciliated cells within in a transparent sealed chamber. Ciliary motility was recorded at room temperature by video microscopy, and ciliary beat frequency was determined using computerized frequency spectrum analysis. Exposure to the AGS resulted in time- and concentration-dependent ciliary slowing with complete ciliastasis, with as low as 20 mM ethanol in the AGS. Direct vapor phase exposure to acetaldehyde alone served as a positive control and also resulted in time-dependent ciliary slowing with complete ciliastasis reached by 4 hr. The AGS-induced ciliastasis was reached 2 hr later than with acetaldehyde alone. When cells were pretreated with cyanamide, which is known to block acetaldehyde dehydrogenase, the time to ciliastasis was decreased by 10-30 min compared with untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaldehyde↗
PubMed1994

Detection of reduced acetaldehyde protein adducts using a unique monoclonal antibody.

Acetaldehyde (AA), the major product of alcohol metabolism, has been shown to bind to proteins in vivo and form chemical adducts. These AA-protein adducts have been shown to alter protein structure and function and may result in tissue damage. Recent reports have shown that polyclonal antibodies can be produced that recognize proteins modified in vitro with AA in the presence of sodium cyanoborohydride (NaCNBH3), a strong reducing (R) agent. Antibodies prepared in this way have been shown to recognize proteins in the livers of rats fed alcohol chronically. Because multiple AA-protein adducts can be recognized by polyclonal antisera, and a variety of adducts may be formed in vitro or in vivo, this study was designed to develop monoclonal antibodies specific for proteins modified by AA. In addition, adducts formed under R conditions are probably chemically different than those formed under nonreducing (NR) conditions, and monoclonal antibodies may provide the specificity required to distinguish these chemical differences. Balb/c mice were immunized with bovine brain tubulin that was modified by treatment with 5 mM AA for 7 days under NR conditions. Sera from immunized animals were tested for antibody activity to the immunogen (protein-NR) and for cross-reactivity to protein-R and unmodified protein. Although the highest serum antibody titers were seen toward the NR adduct, antibodies to the R adduct were also detected. This activity difference was independent of the carrier protein, because NR and R bovine serum albumin, keyhole limpet hemocyanin, and actin also gave similar results when used as the adducted protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaldehyde↗
PubMed1993

Chronic ethanol administration impairs ATP-dependent acidification of endosomes in rat liver.

Ethanol administration has been shown to alter various protein trafficking pathways in the liver, including protein secretion, plasma membrane assembly and receptor-mediated endocytosis. Molecules such as proteins are transported into and within the cells via endosomal vesicles, and acidification of these endosomes appears to be critical for efficient sorting and trafficking of proteins. In the present study we examined whether ethanol administration impaired the ATP-dependent acidification of endosomes. Liver endosomes were prepared from rats after intravenous labeling with fluorescently labeled ligands and the initial pH and extent of ATP-dependent acidification was determined. Our results showed that the intrinsic pH was significantly higher and that the ATP-dependent acidification of the interior of the endosomes was inhibited in ethanol-fed versus control animals. This impairment of endosomal acidification may play a role in the impaired protein trafficking which occurs as a result of ethanol administration.

Adenosine Triphosphate↗
PubMed1993

Time course of ethanol-induced impairment in fluid-phase endocytosis in isolated rat hepatocytes.

The time-course effects of long-term ethanol administration on fluid-phase endocytosis were studied in isolated rat hepatocytes. Rats were pair-fed an ethanol-supplemented liquid diet or an isocaloric control diet for 3 days, 1 wk, 2 wk or 5 wk. Hepatocytes were isolated and incubated at 37 degrees C with various concentrations of the fluid-phase marker Lucifer yellow. Net internalization of the marker dye was determined. After as little as 1 wk, ethanol-fed rats demonstrated marked decreases in the net internalization of dye compared with pair-fed controls; these changes persisted throughout 5 wk of feeding. Because net internalization is the balance between uptake into the cells vs. efflux from the cells, these components were examined individually. Early uptake was not significantly decreased by ethanol feeding; however, efflux of preloaded Lucifer yellow from cells from the ethanol-fed animals was markedly faster than efflux from pair-fed controls. This increased efflux was more prominent in the longer preload time (90 min) compared with a shorter preload time (15 min), indicating an alteration in dye distribution among various intracellular pools. These ethanol-induced changes in fluid-phase endocytosis were apparent for 1 wk through 5 wk of feeding and were similar for all Lucifer yellow concentrations examined. These results indicate that the decreased net internalization of Lucifer yellow through fluid-phase endocytosis is mainly a result of an ethanol-induced increase in efflux possibly caused by altered intracellular trafficking rather than by reduction in uptake.

Alcoholism↗
PubMed1993

Reaction of acetaldehyde with proteins: formation of stable fluorescent adducts.

The properties of stable acetaldehyde-protein adducts, using bovine serum albumin as a model protein, were investigated. Upon prolonged incubation at 37 degrees C and pH 7.4, the reaction of acetaldehyde and albumin yielded stable adducts that exhibited fluorescent properties. Reaction mixtures of acetaldehyde with polylysine or ethylamine also formed fluorescent products with similar fluorescent spectral properties like acetaldehyde-albumin adducts, indicating that the amino groups of protein alone can generate fluorescent products on reaction with acetaldehyde. When reactions of acetaldehyde with albumin or polylysine were conducted at 22 degrees C, stable binding reached a maximum after 24 hr of incubation and essentially remained at this level during the remaining 216 hr of incubation, and minimal-to-no fluorescence was associated with this binding. At 37 degrees C, stable binding was greater and increased continuously over the entire 216 hr of incubation. After an initial lag period of 24 to 48 hr, increases in fluorescence intensity paralleled the increases in stable binding. The presence of sodium cyanoborohydride, which reduces Schiff bases, in the reaction mixtures prevented fluorescence, indicating that Schiff bases are intermediates in the formation of fluorescent products. Both stable binding and fluorescence intensities were minimally affected by exhaustive dialysis (up to 144 hr), indicating that the fluorescent products were quite stable. These results suggest that an initial reaction of a Schiff base with another acetaldehyde molecule via an aldol condensation reaction gives rise to the formation of a crotonaldehyde Schiff base derivative. This reactive intermediate could then undergo further condensation reactions and form advanced conjugated products, some of which could be fluorescent.

Acetaldehyde↗
PubMed1993

Dietary betaine promotes generation of hepatic S-adenosylmethionine and protects the liver from ethanol-induced fatty infiltration.

Previous studies have shown that ethanol feeding to rats alters methionine metabolism by decreasing the activity of methionine synthetase. This is the enzyme that converts homocysteine in the presence of vitamin B12 and N5-methyltetrahydrofolate to methionine. The action of the ethanol results in an increase in the hepatic level of the substrate N5-methyltetrahydrofolate but as an adaptive mechanism, betaine homocysteine methyltransferase, is induced in order to maintain hepatic S-adenosylmethionine at normal levels. Continued ethanol feeding, beyond 2 months, however, produces depressed levels of hepatic S-adenosylmethionine. Because betaine homocysteine methyltransferase is induced in the livers of ethanol-fed rats, this study was conducted to determine what effect the feeding of betaine, a substrate of betaine homocysteine methyltransferase, has on methionine metabolism in control and ethanol-fed animals. Control and ethanol-fed rats were given both betaine-lacking and betaine-containing liquid diets for 4 weeks, and parameters of methionine metabolism were measured. These measurements demonstrated that betaine administration doubled the hepatic levels of S-adenosylmethionine in control animals and increased by 4-fold the levels of hepatic S-adenosylmethionine in the ethanol-fed rats. The ethanol-induced infiltration of triglycerides in the liver was also reduced by the feeding of betaine to the ethanol-fed animals. These results indicate that betaine administration has the capacity to elevate hepatic S-adenosylmethionine and to prevent the ethanol-induced fatty liver.

Animals↗
PubMed1993

Chronic ethanol administration impairs degradation of formaldehyde-treated albumin by the perfused rat liver.

Nonparenchymal cells of the liver appear to be important in the pathogenesis of various liver diseases, including that caused by ethanol. It is known that chronic ethanol administration impairs the process of receptor-mediated endocytosis in hepatocytes. Liver endothelial cells are also actively endocytic cells, playing a prominent role in the clearance from the circulation of a variety of macromolecules. In this study, we assessed the effect of ethanol administration on this "scavenger" function of liver endothelial cells by measuring the degradation of formaldehyde-treated albumin in isolated, perfused livers of ethanol-fed rats. Rats were pair-fed for 1 or 4 weeks with a liquid diet containing either ethanol as 36% of total calories or an isocaloric amount of carbohydrate. Chronic ethanol administration in this manner for 1 or 4 weeks significantly impaired the degradation of this endothelial cell ligand (by 60 +/- 9% and 37 +/- 9%, respectively). Liver perfusions were also performed on rats that had been administered ethanol acutely or in which ethanol was added to the perfusate. No acute effect of ethanol on the degradation of this ligand was seen. These results demonstrate that chronic ethanol ingestion impairs receptor-mediated endocytosis of formaldehyde-treated albumin by liver endothelial cells, indicating that the adverse effects of ethanol on protein trafficking within the liver are not limited to the hepatocytes.

Animals↗
PubMed1992

Substoichiometric inhibition of microtubule formation by acetaldehyde-tubulin adducts.

We have shown previously that acetaldehyde forms stable covalent adducts with tubulin, resulting in impaired microtubule formation. The present study explored the mechanism responsible for impaired microtubule formation caused by the substoichiometric stable binding of acetaldehyde to tubulin. The free tubulin dimer was much more reactive with acetaldehyde than microtubules, binding more than twice as much aldehyde. The dimer also formed nearly twice as many stable adducts on its alpha-chain as on its beta-chain, whereas microtubules exhibited an equal distribution of adducts between the two subunits. These data confirm that the alpha-chain of free tubulin, but not microtubules, has an accessible highly reactive lysine (HRL) residue that is a preferential target of acetaldehyde binding. Adduct formation with the HRL residue also correlated with impaired tubulin polymerization, and only 0.08 moles of acetaldehyde bound per mole of HRL was required for complete inhibition; however, adducts with other lysine residues (bulk adducts) did not affect assembly. Adducts to microtubule-associated proteins (MAPs) also impaired the assembly of tubulin, but were much less effective than HRL adducts. In a copolymerization assay, HRL-adducted tubulin, in addition to being itself assembly incompetent, also interfered with polymerization of normal (unadducted) tubulin. Bulk adducts did not alter assembly and were incorporated normally into the growing polymer. When tubulin was cleaved by the proteolytic enzyme, subtilisin, microtubule formation could readily take place in the absence of MAPs. In this polymerization system, HRL adducts, but not bulk adducts, still markedly inhibited assembly. When low concentrations of acetaldehyde (50 microM) were used to generate HRL adducts, an adduct on only 1 out of 20 tubulin molecules was sufficient to totally block polymerization. These findings indicate that substoichiometric amounts of acetaldehyde bound to HRL of tubulin can markedly inhibit microtubule formation via direct interference of dimer-dimer interactions, and further suggest that low concentrations of acetaldehyde could generate sufficient amounts of HRL adducts in cellular systems to alter microtubule formation and function.

Acetaldehyde↗
PubMed1992

The effects of chronic ethanol administration on the rates of internalization of various ligands during hepatic endocytosis.

The purpose of the present study was to further characterize the ethanol-induced impairments in hepatic endocytosis. Specifically, we examined the effects of ethanol treatment on receptor-ligand internalization via the coated and noncoated pit pathways. Insulin, epidermal growth factor (EGF) and asialoorosomucoid (ASOR) were used as model ligands to study internalization by isolated hepatocytes. ASOR and EGF are thought to be internalized strictly in coated pit regions of the cell membrane, while insulin may be internalized in both coated and uncoated membrane regions. Ethanol administration for 5-7 weeks decreased internalization of ASOR and EGF while internalization of insulin was unchanged during a single round of endocytosis of surface-bound ligand. Similarly, a more quantitative measure of endocytosis, the endocytic rate constant, was decreased for EGF and ASOR but not for insulin in livers of experimental rats. When endocytosis of Lucifer yellow, a fluorescent dye known to be internalized in the cell by fluid-phase endocytosis was examined, the initial rates of dye uptake were not significantly altered by alcohol administration. These results indicate that ethanol may selectively impair internalization occurring by coated pits while it has a minimal effect on initial uptake of molecules which are internalized by noncoated membrane regions.

Animals↗
PubMed1992

Impaired attachment of hepatocytes to extracellular matrix components after chronic ethanol administration.

BACKGROUND: Previous studies have shown that the assembly and properties of the hepatocyte plasma membrane are altered by ethanol administration, indicating possible changes in the receptor-mediated binding of the plasma membrane to extracellular matrix substrates. In the present study, the effects of chronic ethanol consumption on the ability of hepatocytes to attach to various components of the extracellular matrix were investigated. EXPERIMENTAL DESIGN: Rats were pair-fed for 5 weeks with a liquid diet containing either ethanol (as 36% of total calories) or isocaloric carbohydrate. The effects of ethanol treatment on hepatocyte-extracellular matrix interactions was ascertained by determining the ability of isolated hepatocytes to attach to various extracellular matrix substrates. RESULTS: The attachment of hepatocytes, isolated from the ethanol-fed rats, to laminin-coated plates was significantly decreased compared with hepatocytes from chow-fed or pair-fed controls. Greater decreases in attachment were seen when higher numbers of hepatocytes were seeded in the plates. Similar inhibitions of attachment were also observed when fibronectin or type I collagen were used as matrices. Time-course cell attachment assays indicated that the maximum extent of attachment rather than the rate of attachment was primarily altered by chronic ethanol feeding. Hepatocytes from the ethanol-fed rats also detached more readily from the matrix-coated plates than those from the controls. A reduced number of functional surface receptors for matrix components is likely the most important factor that accounts for the ethanol-induced impairment of hepatocyte attachment. CONCLUSIONS: These results indicate that chronic ethanol administration impairs the interactions of hepatocytes with their extracellular matrix and that this defect could lead to alterations of hepatocyte structure and function.

Animals↗
PubMed1992

A jejunoileal bypass rat model for rapid study of the effects of vitamin malabsorption.

Jejunoileal bypass operation was originally done to promote weight loss for treatment of morbid obesity. We used such a model to determine if dietary vitamin absorption is compromised by such an operation. Six rats were subjected to a jejunoileal bypass, 6 control rats were pair-fed to bypassed rats; and 6 were fed ad libitum. Vitamin content of folic, B6, riboflavin, nicotinate, pantothenate, thiamin, biotin, B12, vitamins A, E, and carotene in blood and liver was determined after 8 postoperative weeks. Aside from riboflavin, blood vitamin levels were significantly depressed in bypassed rats. The deepest depression was seen for B12, carotene and vitamin E. Liver vitamin stores of folate, riboflavin, thiamin, B12, clearly were significantly depressed in the bypassed animals compared to the pair-fed and ad libitum-fed controls. This model can serve for rapidly studying micronutrient depletion due to malabsorption without dietary manipulation or antibiotics for gut sterilization.

Animals↗
PubMed1991

Hepatic transmethylation and blood alcohol levels.

Golden Syrian hamsters that have elevated hepatic alcohol dehydrogenase activity were divided into four groups and group-fed on four different liquid diets for five weeks. Group I was fed a control diet formulated for hamsters. Group II was fed the control diet containing 20 micrograms of 4 methylpyrazole per litre. Group III was fed the hamster ethanol liquid diet (ethanol amounting to 36% of total calories). Group IV was fed the ethanol diet to which 4-methylpyrazole (20 micrograms/litre) was added. Groups I, II and III were group-fed the amount consumed by Group IV on a daily basis. Upon killing the animals, blood alcohol levels were found to be elevated in Group IV but not in Group III. Hepatic methionine synthetase (MS) was inhibited in Group IV. Betaine-homocysteine methyltransferase was induced in this group to compensate for the MS inhibition and liver betaine was lowered reflecting this induction. None of these changes were seen in Group III. Since none of the animals showed an aversion to their respective diets and gained weight normally, these data indicate that it was the elevated blood levels of ethanol rather than nutritional factors that were related to the changes in methionine metabolism.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1991

Chronic ethanol-induced impairments in receptor-mediated endocytosis of insulin in rat hepatocytes.

The effects of chronic ethanol administration on the receptor-mediated endocytosis of insulin were investigated in isolated hepatocytes. When hepatocytes were isolated from rats that were fed an ethanol liquid diet for 5-6 weeks, these cells bound 25% less insulin to their surface membrane than did cells from the chow-fed or pair-fed controls. This decreased binding was likely a result of reduced surface receptor number rather than changes in receptor affinity. Rates of insulin degradation were also reduced by 25-30% in hepatocytes from the ethanol-fed animals. In addition, chronic ethanol feeding induced a defect in the internalization of the receptor-insulin complex and altered the hepatocellular processing of the internalized insulin. These results indicate that chronic ethanol administration impairs both the surface binding and the endocytosis of insulin by the liver.

Alcoholism↗
PubMed1991

Acetaldehyde and microtubules.

Acetaldehyde covalently binds to tubulin to form stable and unstable adducts. Although tubulin has numerous lysine residues available to react with acetaldehyde, a key highly reactive lysine (HRL) on the alpha chain appears to be a preferential target for stable binding. The HRL residue is available for selective binding when tubulin is in the free (dimer) state but not when it is in the polymerized (microtubule) state. Stable binding of acetaldehyde to the HRL residue markedly inhibits tubulin assembly into microtubules, whereas stable binding to other residues (bulk adducts) has little influence on assembly. Substoichiometric stable binding of acetaldehyde to the HRL is sufficient to inhibit polymerization, via direct interference of tubulin dimer-dimer interactions, and an HRL adduct on only one out of 20 tubulin molecules can totally inhibit polymerization. These findings, along with our previous studies demonstrating impaired microtubule-dependent protein trafficking pathways in livers of ethanol-fed animals, indicate that low acetaldehyde concentrations, formed during ethanol oxidation in vivo, could generate sufficient amounts of HRL adducts on the alpha chain of tubulin in cellular systems to alter microtubule formation and function. In addition to alpha-tubulin, calmodulin and actin have also been found to have enhanced reactivity toward acetaldehyde. Thus, a general hypothesis to describe cellular injury induced by acetaldehyde adducts can be formulated: during ethanol oxidation, acetaldehyde forms stable adducts via binding to reactive lysine residues of preferential target proteins, resulting in selective functional impairment of these proteins and ultimately leading to cellular injury.

Acetaldehyde↗
PubMed1991

Acetaldehyde-mediated cilia dysfunction in bovine bronchial epithelial cells.

Acetaldehyde, which is present in significant concentrations in cigarette smoke and is elevated during alcohol ingestion, has been demonstrated to impair mucociliary clearance of the lung. Acetaldehyde is also known to impair protein function through the formation of acetaldehyde-protein adducts. We hypothesized that acetaldehyde impairs bronchial epithelial cilia motion by inhibiting cilia dynein adenosinetriphosphatase (ATPase) activity through the formation of acetaldehyde adducts with cilia proteins. Acetaldehyde induced concentration- and time-dependent slowing of cilia beating and cilia-derived dynein ATPase activity in primary cultures and isolated axonemes of bovine airway epithelial cells. Cilia slowing and ATPase inhibitory effects were also observed with related aldehydes but not with ethanol. Acetaldehyde binding, assessed by gel electrophoresis using [14C] acetaldehyde, was demonstrated to occur with the dynein heavy chains and with tubulin and closely paralleled ATPase inhibition. We conclude that acetaldehyde directly impairs bronchial cilia function causing slowing of cilia beating, inhibits cilia dynein ATPase activity, and binds to cilia proteins critical for motion including dynein and tubulin. These data suggest that acetaldehyde-induced cilia dysfunction may be related to direct cilia ATPase inactivation and adduct formation with cilia dynein and tubulin. This may be an important mechanism by which airway host defenses are impaired in clinical settings where acetaldehyde exposure occurs, e.g., with cigarette smoking and alcohol ingestion.

Acetaldehyde↗