bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo and in vitro evidence concerning the role of lipid peroxidation in the mechanism of hepatocyte death due to carbon tetrachloride
Authors: Biasi Fiorella , Albano Emanuele , Chiarpotto Elena , Corongiu Francesco P. , Pronzato Maria A. , Marinari Umberto M. , Parola Maurizio , Dianzani Mario U. , Poli Giuseppe
Published: May 2005

Isolated rat hepatocytes exposed to CCl4 showed a stimulated formation of malonaldehyde after only 30–60 min incubation. Conversely, the onset of hepatocyte death was a relatively late event, being significant only after 2–3 h of treatment. A cause–effect relationship between the two phenomena has been demonstrated by using hepatocytes isolated from rats pretreated with alpha‐tocopherol. Comparable results were obtained in vivo where supplementation with alpha‐tocopherol 15 h before CCl4 dosing induced a partial or complete protection against the drug's necrogenic effect, depending on the concentration of the haloalkane used. Moreover, the vitamin supplementation prevented the CCl4‐induced increase of liver total calcium content, probably by blocking alterations in the liver cell plasma membranes due to lipid peroxidation.
Chromatin phospholipid changes during rat liver development
Authors: Albi Elisabetta , Magni Mariapia Viola , Lazzarini Remo , Gahan Peter B.
Published: May 2005

The chromatin extracted from rat hepatocytes of different ages has been shown to contain a phospholipid fraction representing 0·47–0·59 per cent of total chromatin in newborn animals and 0·22 per cent in 45‐day‐old animals. No such age‐related differences are observed in the nuclei.

The phospholipid composition of the nuclei at different ages shows a higher level of sphingomyelin and a lower level of phosphatidylserine in newborn than in adult animals. Chromatin phospholipids have a completely different composition from that of nuclei with respect to age, particularly in newborn rats, where there is a decrease in phosphatidylcholine and an increase in phosphatidylserine.
Comparative subcellular distribution of benzaldehyde and acetaldehyde dehydrogenase activities in two hepatoma cell lines and in normal hepatocytes
Authors: Ferro Margherita , Muzio Giuliana , Bassi Anna M. , Biocca Maria E. , Canuto Rosa A.
Published: May 2005

The NAD‐ and NADP‐dependent aldehyde dehydrogenase (ALDH) activities were evaluated in two rat hepatoma cell lines, namely the well‐differentiated MH1C1 line and the less differentiated HTC line. Each activity was determined in parallel in isolated rat hepatocytes, for comparison. The aliphatic aldehyde acetaldehyde (ACA) and the aromatic aldehyde benzaldehyde (BA) were used as substrates. With the first substrate the ALDH activities found in the crude cytoplasmic extracts were lower in hepatoma cells than in normal hepatocytes, especially when measured with NADP as coenzyme (ACA/NADP). Otherwise, with benzaldehyde as substrate the NAD‐dependent enzyme activity (BA/NAD) was increased about 9‐fold in HTC cells over hepatocytes and decreased in MH1C1 cells, while the NADP‐dependent (BA/NADP) activity was increased 38‐ and 2·5‐fold in HTC and MH1C1 cell lines, respectively. Studies on the subcellular distribution of these enzyme activities showed that the activity measured with acetaldehyde and NAD (ACA/NAD) was almost equally distributed between the cytosol and the subcellular particles in the three cell populations, but the ACA/NADP activity was shifted towards the cytosolic compartment in hepatomas, especially in HTC cells. The BA/NAD and BA/NADP ALDH activities found in the organelles of hepatoma cells were markedly reduced in comparison with hepatocytes, in favour of the cytosol. The most striking difference between the normal and the transformed cells was the 94‐fold increase over hepatocytes of the BA/NADP activity, found in the cytosolic fractions of HTC cells. MH1C1 cells showed a less pronounced (7·5‐fold) enhancement of this tumour‐associated specific activity. These results confirm that the cytosolic NADP‐dependent benzaldehyde dehydrogenase activity can be considered as a tumour marker, the activity being inversely correlated with the degree of differentiation of the two hepatoma cell lines.
Mechanisms of interaction between human skin fibroblasts and elastin: Differences between elastin fibres and derived peptides
Authors: Groult V. , Hornebeck W. , Ferrari P. , Tixier J. M. , Robert L. , Jacob M. P.
Published: May 2005

3H‐Labelled kappa‐elastin peptides (kE:75 kDa molecular weight) were shown to bind to confluent human skin fibroblast (HSF) cultures in a time‐dependent and saturable manner. Scatchard analysis indicated the presence of high affinity binding sites with kD = 2·7 × 10−10 M and 19 000 sites per cell. Binding of kE to its receptor on HSF accelerates and intensifies the adhesion of insoluble elastin fibres (iE) to confluent HSF. Optimal effect was attained for a kE concentration of 0·3 × 10−9 M close to kD. This stimulatory effect of kE on the binding of iE to HSF could be inhibited by neomycin, retinal and pertussis toxin, substances which act at different levels of the transduction mechanism following the activation of the receptor and the subsequent triggering of cell biological events (chemotaxis, modification of calcium fluxes). The stimulation of iE adhesion to HSF induced by kE as well as kE binding to the cells could by inhibited by lactose and laminin but not by Arg‐Gly‐Asp‐Ser (RGDS) peptides. This indicates that the elastin peptide receptor on HSF possesses lectin‐like properties and shares homology with the laminin receptor as also shown for other cell types. None of the substances tested, that is inhibitors of the transduction mechanism, lactose, laminin and Arg‐Gly‐Asp‐Ser (RGDS) peptides were shown to interfere significantly with the binding of iE (in the absence of added kE) to confluent HSF. The proteins adhering strongly to elastin fibres were isolated by a sequential extraction procedure and the final hydrochloride guanidinium‐DTT extract was analysed by SDS–PAGE under reducing conditions, Western blots using specific antibodies against several connective tissue proteins and affinity for [3H]‐kE following nitrocellulose electro‐transfer of proteins. Fibronectin, vitronectin, tropoelastin(s), and a 120 kDa cysteine rich glycoprotein previously designated as elastonectin were identified. Among these proteins, [3H]‐kE was found to bind exclusively to a 65 kDa protein that could be eluted selectively from elastin fibres with a neutral buffer containing 100 mM lactose. Therefore the elastin peptide receptor on human skin fibroblasts shares properties with the elastin receptor characterized from other cell types. Conformational differences between elastin peptides and elastin fibres could explain the differences in the mechanisms of interactions between elastin fibres and elastin peptides with HSF in culture. The stimulatory effect of elastin‐derived peptides on the adhesion of elastin fibres to HSF could have implications in the oriented biosynthesis of elastin fibres.
Mitochondrial nucleotide translocase from skeletal muscle of halothane sensitive pigs: An electrophoretic study
Authors: Ayoub S. , Berson G. , Rock E. , Younes A.
Published: May 2005

ATP translocation into mitochondria isolated from halothane‐sensitive pig (HP) muscle was dramatically reduced compared with normal pigs (NP). To determine if this was due to a decreased amount of ATP translocase in the mitochondrial membranes, or a structural modification of this protein, an electrophoretic study was undertaken. Total proteins and purified translocase preparations from (NP) and (HP) mitochondria were analysed by SDS gel electrophoresis. In the two type of mitochondria no significant differences were observed either in the amount of ATP translocase or in the molecular weight. Also, neither nonequilibrium pH gradient gel electrophoresis nor the analysis of peptides produced by limited proteolysis revealed any structural difference between the two types of protein. On the basis of these results, the depressed translocase activity observed in (HP) mitochondria cannot be explained by a reduced amount of the nucleotide translocase, nor a structural alteration of this protein. Possible inhibition of (HP) translocase activity by Ca2+ accumulation or by other mechanisms is discussed.
A comparative study of the effects of the diamine oxidase inhibitor aminoguanidine, with or without dietary restriction, on the nucleic acid and protein composition of cardiac and type I and type II skeletal muscles of the rat
Authors: Siddiq Tahir , Preedy Victor R
Published: May 2005

It has been proposed that the diamine oxidase inhibitor aminoguanidine may be a potential therapeutically important anabolic agent. An investigation was therefore made into the effects of aminoguanidine treatment with or without nutritional restriction, on cardiac and skeletal muscles containing mainly of either Type I (i.e. soleus) or Type II fibres (i.e. plantaris) or a mixture of Type I and II fibres (i.e. gastrocnemius).

After 3 weeks, dietary restrictions reduced cardiac weight, protein, RNA and DNA contents by between 31 per cent and 36 per cent. Similar, but smaller, reductions were observed in the soleus (18–31 per cent), plantaris (22–34 per cent) and gastrocnemius (22–34 per cent).

Aminoguanidine had no effect on the heart of the rats fed ad libitum, nor did it alter the response to dietary restriction. Treatment with aminoguanidine had no overt anabolic effect on skeletal muscle, but a reduction in DNA content was observed.

It was concluded that cardiac protein and nucleic acid contents are more sensitive to dietary deprivation than either anaerobic or aerobic skeletal muscles. Furthermore, aminoguanidine does not appear to promote growth or reduce catabolism as previous studies have suggested.
Reduced neutrophil superoxide generation and plasma lipoperoxide levels in pigs fed with cod liver oil
Authors: Guarnieri Carlo , You‐Zhou Li , Roth Elizabeth , Biagi Pierluigi , Ussia Giovanni , Baccarini Elisabetta , Farruggia Francesco , Anderson Gary L. , Barker John H. , Galletti Giovanni
Published: May 2005

Circulating neutrophils, isolated from pigs fed for 8 weeks with a diet supplemented with CLO, had an accentuated n‐3 fatty acid incorporation into the plasma membrane, as evidenced by an approximately four‐fold greater n‐3/n‐6 ratio as compared with the control diet group. Moreover, the neutrophils of the CLO fed pigs produced less superoxide when stimulated with PMA or f‐MLP, as well as showing a more prolonged latency period before O2−‐generation. In the plasma of pigs fed with CLO there were higher levels of thiobarbituric reactive material and lipofuscin, while the content of GSSG was similar in both dietary groups.

The results of this study indicate that dietary supplementation with CLO reduces the activation of circulating neutrophils and favours the presence in the plasma of lipoperoxides.
Relation between hyaluronan and sulphated glycosaminoglycan synthesis and degradation in cultured embryonic fibroblasts. Effect of concanavalin a and ammonium chloride administration
Authors: Carinci P. , Locci P. , Evangelisti R. , Marinucci L. , Rossi L. , Becchetti E.
Published: May 2005

In order to evaluate the relationship between glycosaminoglycan (GAG) synthesis and degradation, the effect of NH4Cl, which inhibits lysosomal degradation, on GAG production was analysed in vitro in concanavalin A (Con A) stimulated fibroblasts from 7 and 14‐day‐old chick embryos. 35SO4 incorporation into total proteoglycan (PG), 3H incorporation into individual GAG classes, β‐N‐acetyl‐D‐glucosaminidase and β‐D‐glucuronidase activity were determined. The results indicate a correlation between Con A and NH4Cl effects: NH4Cl induced a reduction principally in the GAG classes most stimulated by Con A. Thus HA and DS are much more stimulated by Con A and inhibited by NH4Cl than are CS and HS.
Hepatic selective adjustments in short‐term cold exposed rats
Authors: Iossa S. , Barletta A. , Liverini G.
Published: May 2005

The metabolic adjustments which occur in rat liver cells during the first days of cold exposure have been investigated. We have measured both mitochondrial mass and oxidative capacities as well as ATP production after five and 10 days of cold exposure. In addition, we have measured plasma membrane Na+/K+‐ATPase activity. Cold exposure elicited a significant increase in mitochondrial mass as well as in state 3 oxidative rates and ATP production in isolated mitochondria using various substrates. Moreover, our results show that Na+‐pumping activity significantly increased after both five and 10 days of cold exposure.

Our results suggest that during the first days of cold exposure liver cells undergo alterations which are useful for survival in the cold.
Metabolic activity in isolated hepatocytes from cold exposed rats subjected to 24‐hour fasting
Authors: Iossa S. , Barletta A. , Liverini G.
Published: May 2005

The aim of this work was to study the metabolic activity in isolated hepatocytes from control rats and rats exposed for 15 or 30 days to cold, all subjected to 24‐h fasting. Hepatocyte oxygen consumption was used as an index of metabolic activity. The results show that 24‐h fasting induces a decrease in energy expenditure at the level of the liver in cold‐exposed rats but not in control animals.
Balancing of energy‐consuming processes of K 562 cells
Authors: Siems Werner G. , Schmidt Heike , Gruner Stefan , Jakstadt Manuela
Published: May 2005

A balance of ATP‐consuming processes in human erythroleukemia (K 562) cells by use of the decreased 14CO2 formation from [1‐14C]‐glutamate following inhibition of energy‐requiring processes is presented. This method was tested on Ehrlich mouse ascites tumour cells and was used in suspensions of K 562 cells with a low cell content. More than 90 per cent of the ATP produced by oxidative phosphorylation could be accounted for in K 562 cells. Protein synthesis consumed about 35 per cent, Na+/K+‐ATPase about 20 per cent and transcription processes 5–10 per cent of the total ATP. The share of the Ca2+‐dependent reactions was notably high at 25 per cent in comparison with Ehrlich mouse ascites tumour cells, reticulocytes or hepatocytes. ATP consumption by DNA synthesis was assessed at 5–10 per cent. Only less than 10 per cent of the consumption of ATP produced oxidatively remained for other cellular reactions. The degree of coupling of K 562 cells was high in comparison with that of other eukaryotic cell types.
In situ localization of male germ cell‐associated kinase (mak) mRNA in adult mouse testis: Specific expression in germ cells at stages around meiotic cell division
Authors: Koji Takehiko , Jinno Atsushi , Matsushime Hitoshi , Shibuya Masabumi , Nakane Paul K.
Published: May 2005

Biochemical analysis of the male germ cell‐associated kinase (mak) gene, which was isolated recently by using weak cross‐hybridization with the v‐ros tyrosine kinase gene, revealed that the gene was highly expressed in mammalian testicular germ cells, but not in ovarian cells. In order to identify the cells which express the mak gene in more detail, we localized mak mRNA in frozen sections of mouse testis by non‐radioactive in situ hybridization. In this study, we utilized thymine–thymine (T‐T) dimerized mak cDNA as a haptenic, non‐radioactive probe, and the signal was detected enzyme‐immunohistochemically by using an anti‐T‐T antibody. As a result, mak mRNA was localized intensely in late pachytene (stage X) and diplotene (stage XI) spermatocytes, and faintly in dividing spermatocytes (stage XII) and early round spermatids (stage I–II), suggesting that the gene may play an important role in the phase around meiotic cell division, but not throughout the entire meiosis.
Kinetics of expression of prion protein in uninfected and scrapie‐infected N2a mouse neuroblastoma cells
Authors: Pfeifer Karin , Bachmann Michael , Schröder Heinz C. , Forrest Jock , Müller Werner E. G.
Published: May 2005

The scrapie prion protein, PrPSc, is formed from its isoform, the cellular PrPc. There is evidence available indicating that PrPSc is necessary component of the infectious prion particle to cause a series of transmissible spongiform encephalopathies. We have used immunocytochemistry and RNA blotting techniques to investigate if infection with prions results in an increased PrP gene expression. For the experiments we used N2a cells which had been infected with prions (ScN2a cells). We demonstrated by confocal laser scanning microscopy that PrP‐protein was present in the nucleus (predominantly in the nucleoli) of ScN2a cells. Analysis of the PrP‐mRNA levels both in N2a‐ and in ScN2a cells using cDNA encoding PrPc revealed no marked alteration of the mRNA steady state level between the two cell strains. Likewise, in run‐off experiments no changes in either PrP‐specific transcription or in general transcriptional activity were found. The half‐life of PrP‐mRNA was found to be identical in both cell strains (7 h). Taken together, these results show that PrPSc and /or PrPc is present in the nucleus (nucleoli) of ScN2a cells but does not display and effect on the expression of the PrP gene.
Proliferation‐associated increase in sensitivity of mammary epithelial cells to inositol‐1,4,5‐trisphosphate
Authors: Enomoto Koh‐ichi , Furuya Kishio , Yamagishi Shunichi , Maeno Takashi
Published: May 2005

Injection of D‐myo‐inositol‐1,4,5‐trisphosphate (IP3) was found to induce a transient increase of intracellular Ca2+ concentration in cancerous mammary cells (MMT060562) and in normal mammary cells treated with epidermal growth factor. Responses to injection of either D‐myo‐inositol‐1,4‐bisphosphate (IP2) or D‐myo‐inositol‐1,3,4,5‐tetrakisphosphate (IP4) were small or absent. Furthermore, normal mammary cells cultivated with low‐protein serum replacement alone or in the presence of differentiation‐inducing hormones (insulin + cortisol + prolactin) were less sensitive to IP3.

Thapsigargin induced a transient increase of Ca2+ due to the release of Ca2+ from an intracellular pool. There was no difference in the peak heights of the thapsigargin‐induced Ca2+ increase when mammary cells were cultivated in the presence or absence of epidermal growth factor or insulin + cortisol + prolactin. These findings suggest that the releasable intracellular Ca2+ pool remained unchanged whereas sensitivity to IP3 increases during the proliferation stage.

Mechanical stimulus of a mammary cell induces an increase of intracellular Ca2+ in the stimulated cell. A certain stimulating factor is released from the mechanically stimulated cell into the extracellular space, and it induces an increase of Ca2+ in surrounding cells.18 In contrast, the IP3‐induced Ca2+ increase in both cancerous and epidermal growth factor‐treated normal mammary cells did not spread to adjacent cells. Therefore, increase of Ca2+ is not sufficient to account for the release of stimulating substances from mammary cells in the mechanically‐induced spreading response.
9‐Hydroxybenfluron induced inhibition of proliferation and metabolism in hela cells
Authors: Jantovà S. , Horàkovà K.
Published: May 2005

The highest concentration of 9‐hydroxybenfluron (HBF) tested, namely 4·05 μmol1−1, induced immediate cytotoxic effects which were manifested by total inhibition of cell proliferation after only 24 h of culture. In a certain proportion of the cells cytolytic effects were observed at longer times of culture. Lower concentrations of HBF induced toxicity that was concentration‐ and time‐dependent.

The toxic effect appeared to occur in two phases. Cells, which had lost their ability to divide, did not stop their metabolism in which glutamine was the main source of energy. The results suggest that HBF primarily interferes with one of the phases of the cell cycle and only secondarily influences energy processes. Because benfluron (BF) and HBF have similar effects, it is suggested that the cytotoxicity of BF can be ascribed to the influence of its metabolite, HBF.
Hexose metabolism in pancreatic islets: Succinate dehydrogenase activity in islet homogenates
Authors: Rasschaert Joanne , Malaisse Willy J.
Published: May 2005

Succinate dehydrogenase activity was measured in rat pancreatic islet homogenates incubated in the presence of [1,4‐14C]succinate, the reaction velocity being judged through the generation of 14CO2 in the auxiliary reactions catalysed by pig heart fumarase and chicken liver NADP‐malate dehydrogenase. In the presence of 1·0 mM succinate, the reaction velocity averaged 5·53 ± 0·44 pmol min−1 μg−1 islet protein. The Km for succinate was close to 0·4 mM and the enzymic activity was restricted to mitochondria. These kinetic results indicate that, under the present experimental conditions, the activity of succinate dehydrogenase does not vastly exceed that of either NAD‐isocitrate dehydrogenase or the 2‐ketoglutarate dehydrogenase complex, at least when the latter enzymes are activated by ADP and/or Ca2+. Nevertheless, the activity of succinate dehydrogenase is sufficient to account for the increase in O2 uptake evoked in intact islets by the monomethyl ester of succinic acid. It could become a rate‐limiting step of the Krebs cycle in models of B‐cell dysfunction.
Effect of diets containing adenosine, guanosine, inosine or xanthosine on the nucleotide content of Artemia. Influence of mycophenolic acid
Authors: Sillero Antonio , Sillero María A. Günther , Hernandorena Arantxa
Published: May 2005

Artemia uses the stored diguanosine tetraphosphate as a source of adenine and guanine nucleotides during development from the encysted gastrula to the free swimming larva. Further development of the larvae depends on a dietary source of purine rings. We have investigated the growth of Artemia in axenic cultures supplemented with 0·6 mg ml−1 of adenosine, guanosine, inosine or xanthosine. The total protein and soluble nucleotide content of Artemia grown in the presence of adenosine, guanosine or inosine was very similar, around (2 A260 units and 500 mg protein) and (4 A260 units and 1000 mg protein) after 4 and 6 days of postlarval development, respectively. The nucleotide pattern of those extracts subjected to HPLC were almost identical, the major peaks corresponding to ATP, ADP and AMP. Other nucleotides, not well characterized, were also present in those extracts. Mycophenolic acid (10 μg ml−1) inhibited the growth of Artemia (as measured by their protein and soluble nucleotide content) in the presence of adenosine and inosine as the purine source, and had no appreciable effect in the presence of guanosine. A quantitative analysis of the chromatographic peaks obtained from Artemia grown in the presence of any of the three nucleosides ± mycophenolic acid showed that the effect of the antibiotic on each one of the chromatographic peaks was very similar, suggesting that Artemia, and probably other organisms as well, tend to maintain a balance between all nucleotides and to adjust the overall level to the limiting step(s) in their rates of synthesis/interconversion. Xanthosine was not able to support the development of Artemia.
Lectin binding and uptake and glycoprotein characterization of isolated porcine aortic endothelial and smooth muscle cells
Authors: Schumacher Udo , Vischer Peter , Völker Wolfgang , Engelmann Bernd
Published: May 2005

Endothelial and smooth muscle cells were isolated from porcine aorta and kept in short‐term culture. To determine the terminal carbohydate composition of the plasma membranes from both cell populations, the cells were incubated with a panel of fluorescein‐labelled lectins. Both cell populations shared a number of terminal carbohydrates, but the N‐galactosamine specific lectin Wistaria floribunda agglutinin labelled only the endothelial cells. A lectin which selectively labelled smooth muscle cells was not found. Western blot analysis of isolated endothelial cell membrane glycoproteins indicated that most membrane glycoproteins are labelled by Wistaria floribunda agglutinin.
Modifications of proteoglycans produced by human skin fibroblast cultures during replicative senescence
Authors: Passi A. , Albertini R. , Bardoni A. , Rindi S. , Salvini R. , Pallavicini G. , De Luca G.
Published: May 2005

The properties of proteoglycans (PGs) produced by normal human skin fibroblast were investigated with increasing passage. The increase of subculture number was associated with a constant increase in PG molecular size, which was particularly evident in cell layer extracts. In the cell layer, the ratio of DS‐PGs/HS‐PGs was markedly higher in early passage cultures. Moreover, the cell layer from young cells contained lower amounts of radioactivity incorporated into the most hydrophobic PG populations, suggesting that the PG core protein might also undergo significant modification with increasing subcultures. There was no significant difference in energy charge value between early and late passage cultures, whereas the NAD/NADH ratio was found to decrease markedly in senescent cells.
Stimulation of phosphatidylcholine biosynthesis by hemicholinium‐3, a potent inhibitor of choline uptake in human leukemic monocyte‐like U937 cells
Authors: Chu Arthur J.
Published: May 2005

The effect of hemicholinium‐3 (HC‐3) on choline uptake and phosphatidylcholine (PC) biosynthesis was examined in human leukemic monocyte‐like U937 cells. HC‐3 inhibited [3H]choline uptake in a dose‐ and time‐dependent manner. After a 3 h treatment, HC‐3 (100 μM) decreased choline uptake by as much as 80 per cent (p < 0·0001; n = 4). Reduction of incorporation of label into PC was also detected in a dose‐dependent manner; the extent of inhibition, however, was always 10–20 per cent less than that observed in the total uptake. At 3 h HC‐3 decreased the incorporation into PC by 65 per cent (p < 0·0001; n = 5). Kinetic studies in vivo showed that HC‐3 inhibited total uptake and incorporation into PC differently, suggesting that the labelling of PC is not simply dictated by [3H]choline uptake. In separate experiments, cells were pretreated with 100 μM HC‐3 for 3 h. After washing, the inhibitory effect on total uptake was no longer observed, while a 20 per cent stimulation of the incorporation into PC was obtained in these pretreated cells. In pulse‐chase studies, the cells were prelabelled with [3H]choline for 30 min and chased with HC‐3 for up to 3 h; the results showed a significant stimulation of incorporation into PC in a longer chase with 100 μM HC‐3. After a 3 h treatment, the cytosolic CTP:cholinephosphate cytidylytransferase (CT) was activated by 56 per cent, while choline kinase (CK) was inhibited slightly. The stimulation of CT was not simply due to the intact HC‐3 molecule, and there was no redistribution of CT between cytosol and microsomes. Taken together, the results suggest that HC‐3 activates PC biosynthesis apart from the inhibitory effect on choline uptake.
Regulation of intracellular creatine in erythrocytes and myoblasts: Influence of uraemia and inhibition of Na, K‐ATPase
Authors: Bennett Susan E. , Bevington Alan , Walls John
Published: May 2005

The regulation of intracellular creatine concentration in mammalian cells is poorly understood, but is thought to depend upon active sodium‐linked uptake of creatine from extracellular fluid. In normal human erythrocytes, creatine influx into washed cells was inhibited by 40 per cent in the absence of extracellular sodium. In washed cells from uraemic patients, sodium‐independent creatine influx was normal, whereas the sodium‐dependent component of creatine influx was 3·3 times higher than normal, possibly relecting the reduced mean age of uraemic erythrocytes. In spite of this, the intracellular creatine concentration was no higher than normal in uraemic erythrocytes, implying that some factor in uraemic plasma in vivo inhibits sodium‐dependent creatine influx. Both in normal and uraemic erythrocytes, the creatine concentration was 10 times that in plasma, and the concentration in the cells showed no detectable dependence on that in plasma, suggesting that the intracellular creatine concentration is controlled by an active saturable process. Active sodium‐dependent accumulation of creatine was also demonstrated in L6 rat myoblasts and was inhibited when transport was measured in the presence of 10−4M ouabain or digoxin, implying that uptake was driven by the transmembrane sodium gradient. However, when creatine influx was measured immediately after ouabain or digoxin had been washed away, it was higher than in control cells, suggesting that Na,K‐ATPase and/or sodium‐linked creatine transport are up‐regulated when treated with inhibitors of Na,K‐ATPase.
Mitochondrial pyruvate metabolism in liver and kidney during acidosis
Authors: Oliver F. Javier , Salto Rafael , Sola María M. , Vargas Alberto M.
Published: May 2005

Pyruvate transport and carboxylation have been determined in mitochondria from liver and kidney cortex isolated from Wistar rats with acidosis produced by three different treatments: fasting, exercise and ingestion of ammonium chloride. Fasting for 48 h or swimming for 2 h resulted in an increased rate of CO2 fixation by mitochondria from both organs incubated with pyruvate. This increase was accompanied by a rise in the rate of pyruvate transport in all cases except in mitochondria derived from the kidney of the fasted animals. Acute acidosis produced by the ingestion of ammonium chloride resulted in increases in pyruvate transport and carboxylation in kidney mitochondria, but a drop in pyruvate carboxylation was observed in mitochondria from the liver. The results are discussed in terms of the differential regulation of the mitochondria steps for gluconeogenesis from three carbon precursors in liver and kidney, taking into consideration the hormonal status of the animals and the prevailing available substrates in each condition.
Characterization of γ‐glutamyltranspeptidase in the liver of the frog: 2. Response to season, temperature and thyroid hormone in Rana pipiens
Authors: Sulakhe‐Hemmings Susan J. , Xing Hongmei
Published: May 2005

The impact of season and temperature on frog liver γ‐glutamyltranspeptidase was assessed by measuring the activity of this enzyme in plasma membranes isolated from the livers of Rana pipiens obtained as summer and winter frogs; subjected to short‐term (3 weeks) temperature acclimation; and subjected to multiple‐temperature shifts. Plasma levels of T3 were determined. γ‐Glutamyltranspeptidase was found to be 2·2‐fold higher in the summer frog relative to the winter frog; decreased by 44 percent in the summer frog by cold acclimation and increased by 1·7‐fold in the winter frog by warm acclimation; and increased by 1·9‐fold in the summer frog and 2·8‐fold in the winter frog subjected to multiple‐temperature shifts. Plasma T3 levels were found to be 42‐fold higher in the summer frog relative to the winter frog; decreased by 42 percent by cold acclimation and increased by 2·9‐fold by warm acclimation; and decreased by 39 percent and 38 percent in the summer and winter frogs subjected to multiple temperature shifts. T3 replacement during the last phase of the multiple‐temperature shift protocol, restored the plasma T3 levels to 75 percent of the control levels and prevented the increase evoked by the multiple‐temperature shifts in γ‐glutamyl‐transpeptidase activity. Indeed, enzyme activity in the T3 replaced state was 19 percent lower than in the control state. The involvement of thyroid hormone as a negative regulator of enzyme activity is discussed.
Differences in the G/total actin ratio and microfilament stability between normal and malignant human keratinocytes
Authors: Katsantonis J. , Tosca A. , Koukouritaki S. B. , Theodoropoulos P. A. , Gravanis A. , Stournaras Christos
Published: May 2005

The state of polymerization of actin and the organization of actin filaments is widely believed to be related to cellular transformation. Since the intracellular monomer (G) and filamentous (F) actin content reflects the state of microfilament polymerization, we measured the G/total actin ratio in primary cultures of normal and malignant human keratinocytes. In normal keratinocytes the mean value of this ratio was 0·30 ± 0·03 (mean ± SE, n = 15), while in basal cell carcinoma (BCC) keratinocytes it was 0·49 ± 0·03 (n = 8) and in squamous cell carcinoma keratinocytes (SCC) 0·5 ± 0·07 (n = 4), indicating a 1·7‐fold increase of the G/total actin ratio in malignant cells. These results imply that the proportion of polymerized actin is decreased markedly in malignant keratinocytes, suggesting alterations of microfilament structures which probably occur during the transformation process. This was supported by the morphological changes of microfilament structures as assessed by fluorescence microscopy. A different distribution of actin filaments in normal and malignant cells became evident; stress‐fibres were converging in patches at several points in SCC cells, when compared to normal keratinocytes. Furthermore, incubation of normal and malignant keratinocytes with cytochalasin B indicated differences in the resistance of their microfilament networks. After 1 h exposure to 10−6 and 10−5 M cytochalasin B, microfilaments in normal cells appeared to be less affected than their counterparts in neoplastic cells. Even in a high excess of cytochalasin B (10−4 M), normal keratinocytes preserved their shape, while both basal cell and SCC were totally disrupted. We concluded that the G/total actin ratio was significantly increased in malignant keratinocytes. This seems to be correlated with altered microfilament morphology and resistance to cytochalasin B treatment. Our results suggest that the process of malignant transformation may be characterized by changes in the state of the polymerization of actin and in the stability of the microfilament network indicating that both features could potentially serve as markers determine the transformed state of keratinocytes.
Effect of phorbol 12‐myristate 13‐acetate (PMA) on the phosphoinositol (PI) system in Tetrahymena. Study of the 32P incorporation and breakdown of phospholipids
Authors: Kovács P. , Csaba G.
Published: May 2005

Phorbol 12‐myristate 13‐acetate (PMA) treatment elicited an increased 32P incorporation into phospholipids namely phosphatadyl‐inositol (PI); phosphatidyl‐inositol‐4‐phosphate (PIP); phosphatidyl‐inositol‐4,5‐bis‐phosphate (PIP2); phosphatidyl‐acid (PA); phosphatidyl‐choline (PC) and phosphatidyl‐ethanolamine (PE) particularly at the 20–30th min after treatment. The ratio of members of the phosphoinositol system, especially PIP and PI, related to the total phospholipid content was increased. PMA (2 × 10−7 M) was the most effective of the three concentrations tested. The results call attention to the presence of a working phosphoinositol system in Protozoa.
Assessment of growth inhibition by aldehydic lipid peroxidation products and related aldehydes by Saccharomyces cerevisiae
Authors: Wonisch Willibald , Schaur R. Jörg , Bilinski Tomasz , Esterbauer Hermann
Published: May 2005

The effect of pretreatment with aldehydes on the subsequent colony forming efficiency (CFE) of Saccharomyces cerevisiae was investigated. All 21 aldehydes tested inhibited CFE in a dose‐dependent manner. The effective doses, however, differed markedly from 300 mM to 0·07 mM depending on the functional groups and chain length of the aldehydes. Amongst the nine representatives of n‐alkanals, formaldehyde was the most potent inhibitor, reducing CFE to 50 per cent at a dose of 0·3 mM (IC50). In the series of 2‐trans‐alkenals, acrolein was most effective with an IC50 of 0·08 mM and amongst the 4‐hydroxy 2‐trans‐alkenals, 4‐hydroxynonenal was most effective with IC50 of 0·07 mM. In general, effectiveness decreased in the order: 4‐hydroxyalkenals > 2‐alkenals ≫ n‐alkenals. It is proposed that S. cerevisiae is a promising target cell to elucidate further the molecular mechanisms by which aldehydes, particularly the lipid peroxidation product 4‐hydroxynonenal, inhibits cell proliferation.
Modification of frequency augmentation‐potentiation by GTPγS in the frog neuromuscular junction
Authors: Enomoto Koh‐Ichi , Maeno Takashi
Published: May 2005

The effect of modifiers of guanine nucleotide‐binding proteins (G proteins) on the frequency augmentation‐potentiation of transmitter release were studied in the frog neuromuscular junction. Using GenetransferR as a carrier the mean quantal content of the endplate potential increased by penetration of GTPγS into the presynaptic nerve terminal. Neither GTPγS alone nor carrier alone had any effect. The relationship of log (mean quantal content) versus stimulation frequency changed from a single linear to a dual linear function, suggesting that the immediately releasable pool was modified. GDPβS + carrier also had similar effects, but was less potent. Aluminium fluoride was without effect. Extracellularly recorded presynaptic nerve action potentials remained unchanged with GTPγS + carrier. Also, GTPγS + carrier did not affect the action potential nor the cytosolic Ca2+ concentration in differentiated NG108–15 hybrid cells. It is suggested that some smg‐type G protein‐dependent processes are involved in determining frequency augmentation‐potentiation.
The secretion of urokinase‐like plasminogen activator is inhibited by microtubule‐interacting drugs
Authors: Santibañez Juan F. , Maccioni Ricardo B. , Martínez Jorge
Published: May 2005

The secretion of proteinases into the extracellular matrix is one of the main features of tumour cells, as related to their invasive behaviour. Considering the role of the microtubule cytoskeleton, and particularly the action of microtubule‐associated protein (MAPs) in mediating protein secretion, the effects of the anti‐microtubule drugs estramustine and taxol, on the secretion of urokinase‐type plasminogen activator (u‐PA) and the 72 kDa gelatinase were investigated. Treatment of 5637 bladder carcinoma cells with estramustine and taxol inhibited u‐PA secretion into the conditioned medium in a drug concentration‐dependent fashion. This inhibition was confirmed by determinations of u‐PA enzymatic activities and by measurements of the levels of immunoreactive activator. Studies using gelatin zymograms also showed an inhibition of another tumoural proteinase namely the 72 kDa gelatinase. Time‐course uptake experiments showed that estramustine was incorporated into the cells, a process which depended on temperature. On the other hand, immunofluorescence studies indicated that the microtubule network was affected by taxol with the formation of bundles of microtubules at different cell domains. Minor effects were visualized after treatment of the cells with estramustine‐phosphate, a drug that blocks primarily the action of microtubule‐associated proteins. The studies provide a way to analyse the relationships between u‐PA secretion and the integrity of the cytoskeletal network.
Cation‐induced efflux of calcium from the mitochondria of Hymenolepis diminuta
Authors: Wani Javaid H. , Siddiqui Abrar , Srivastava V. M. L.
Published: May 2005

We have previously reported that Ca2+ influx into the mitochondria of Hymenolepis diminuta, a rat intestinal eestode, takes place through an electrophoretic uniport system. Sodium and lithium were found to induce efflux of 45Ca2+ from the mitochondria of H. diminuta. The two cations induced the efflux in a hyperbolic and linear fashion, respectively. The efflux as well as an exchange of external Ca2+ with internal 45Ca2+ was inhibited by lanthanum. The type of Ca2+ transport system in the cestode organelle has been discussed and compared with that of the host (mammalian) counterpart.
Enzymatic, biophysical and ultrastructural changes of plasma membranes in chemical‐induced rat hepatoma
Authors: Paradisi Luciana , Losa Gabriele A. , Dianzani Mario U.
Published: May 2005

Plasma membranes from liver of control rats or from chemical‐induced hepatoma were prepared. The basal activity of adenylate cyclase was increased significantly in the rat plasma membranes of DEN‐induced hepatoma compared to normal tissue. The glucagon‐induced response on the cellular effector systems via guanine nucleotide‐binding regulatory proteins (G proteins) was inhibited in hepatoma plasma membranes. These findings suggest that in hepatoma membranes, unlike normal hepatic membranes, the response to hormonal stimuli through regulatory G proteins results in a loss of response to glucagon, as well as to GTP plus glucagon or to GTPγS. However, the activating effects of forskolin, which catalyses the formation of cyclic AMP from ATP acting on the catalytic subunit, were to some extent retained. The methyltransferase‐I behaved in the opposite direction to the adenylate cyclase, showing a decreased activity in hepatoma plasma membranes compared to control membranes. In contrast, the activity of the ecto‐5′‐nucleotidase was significantly increased in hepatoma. These enzymatic changes have been found to influence the membrane fluidity and to be responsible for the ultrastructural modifications of hepatoma plasma membranes which are induced by chemical carcinogens.
Lovastatin induces differentiation of Mono Mac 6 cells
Authors: Weber C. , Erl W. , Weber P. C.
Published: May 2005

The proliferation of human monocytic Mono Mac 6 cells was significantly retarded by treatment with lovastatin (LOV, 10 μM) for 72 h. Treatment of Mono Mac 6 cells with LOV increased surface protein expression of monocyte‐associated CD14 and the integrin‐chain CD11b towards levels found in isolated human blood monocytes. These effects were dose‐dependent and completely reversed by the isoprenoid precursor mevalonate (MVA). LOV failed to induce growth retardation and upregulation of CD11b or CD14 in the less mature premonocytic U937 cell line. While CD11b expression was comparable in Mono Mac 6 cells treated with LOV (10 μM), TNF (100 U ml−1) or LPS (10 ng ml−1), upregulation of CD14 by LOV was less pronounced. Basal CD23 expression was unaffected by LOV but markedly reduced by treatment with TNF or LPS. Moreover, LOV enhanced Mono Mac 6 adhesiveness to human umbilical vein endothelial cells to levels found in isolated human blood monocytes, probably due to the increased CD11b and CD14 expression. In conclusion, LOV can induce differentiation of monocytic cells which is reflected by the retardation of growth, expression of CD14 and CD11b, and enhanced adhesiveness.
Microdensitometric measures of cytoplasmic RNA and total protein in pyramidal neurons of the insular cortex and midfrontal gyrus in patients with Alzheimer's disease
Authors: Colurso Gloria J. , Kan Robert K. , Anthony Adam
Published: May 2005

Scanning and integrating microdensitometry of azure B‐ and Coomassie brilliant blue G‐stained tissue sections was used to measure the levels of RNA and protein, respectively, in pyramidal neurons of the insular cortex (INS) and midfrontal gyrus (MFG) in patients with Alzheimer's disease (AD) and age‐matched, nondemented control subjects. AD was associated with a decreased neuronal RNA (by 7·4 per cent) and protein (by 28·7 per cent) content in INS. Although the neuronal RNA content was maintained at the control amounts in MFG, the average protein level was lower (14·7 per cent) in AD patients. These results demonstrate a disease‐related impairment in metabolic function in two brain regions connected via discrete corticocortical pathways. Such findings support the hypothesis that a primary site of pathology occurs in AD, and specific neural deficits occur secondarily in certain connected brain regions.
Proteoglycan alterations in skin fibroblast cultures from patients affected with pseudoxanthoma elasticum
Authors: Passi A. , Albertini R. , Contri M. Baccarani , de Luca G. , de Paepe A. , Pallavicini G. , Ronchetti I. Pasquali , Tiozzo R.
Published: May 2005

Proteoglycans (PGs) were investigated in fibroblast cultures from both apparently normal and involved areas of skin from two patients affected with Pseudoxanthoma elasticum (PXE) and compared to control normal cells. Biochemical analysis showed that cells from the PXE‐affected patients produced a PG population with stronger polyanion properties, as well as a markedly increased amount of high hydrodynamic‐size PGs. Moreover, PGs from PXE‐affected cells showed abnormal hydrophobic interaction properties when examined under associative conditions and included heparan sulphate (HS)‐containing populations with anomalous electrophoretic mobility. These phenomena were particularly evident in the case of PGs secreted into the growth medium. In agreement with these findings immunohistochemical study showed alterations affecting decorin and biglycan, as well as a different content and distribution of HS‐PGs in PXE‐affected cells. The same biochemical and morphological alterations were confirmed for both patients on different cell cultures and were present in cells from both apparently normal and affected skin areas, being more pronounced in the latter. Our results indicate that PXE‐affected fibroblasts in culture exhibit an abnormal PG metabolism, which could affect the normal assembly of extracellular matrix.
Long‐term treatment of swiss 3T3 fibroblasts with dexamethasone attenuates MAP kinase activation induced by insulin‐like growth factor‐I (IGF‐I)
Authors: Hansson Anders , Hehenberger Karin , Thorén Marja
Published: May 2005

Bone formation is reduced in hyperglucocorticoid states, e.g. Cushing's syndrome or long‐term treatment with synthetic glucocorticoids during rheumatic diseases. Possibly related to decreased sensitivity of the target to insulin‐like growth factor‐I (IGF‐I). In this study, we have sought to identify postreceptor‐mechanisms for glucocorticoid‐induced resistance to insulin‐like peptides in a model system. Treatment of Swiss 3T3 fibroblasts with 100 nM dexamethasone for 48 h reduced IGF‐I‐induced activation of mitogen‐activated protein kinase (MAP kinase). The level of insulin receptor substrate‐1 (IRS‐1) was reduced in dexamethasone‐treated cells, as measured by Western blot; however, the pattern of tyrosine‐phosphorylated protein subsequent to stimulation with IGF‐I (1 min) was not altered. No inhibitory effect of dexamethasone was observed on the level of phosphotyrosine in IRS‐1 in extracts from IGF‐I‐treated cells. The amount of IGF‐I‐induced association of insulin receptor substrate‐1 and phosphatidylinositol 3‐kinase was increased in steroid treated cells. Addition of IGF‐I increased the synthesis of lipid, glycogen and protein, and the reduction of a tetrazolium dye, MTS, in untreated cells. The response to IGF‐I in terms of glycogen synthesis was blunted, whereas the effect of IGF‐I was unaffected for the other three parameters in cells pretreated with dexamethasone. These findings indicate that the activation of MAP kinase may be dissociated from IGF‐I‐induced anabolic pathways and tyrosine phosphorylationof IRS‐1. The results agree with the previously proposed role for the activation of MAP kinase in the regulation of glycogen synthesis. Furthermore, they suggest that dexamethasone‐induced reduction of IRS‐1 expression may be important for the impaired activation of MAP kinase by insulin‐like peptides in steroid‐treated cells.