bluuebunny/crossref_metadata_2025 huggingface dataset:
[Na‐K]ATPase activity in proximal and distal tubules of the rat kidney: Modification and application of a quantitative cytochemical technique
Authors: Hersey Roscoe M. , Gattone Vincent H. , Weisz Judith
Published: May 2005

A modified cytochemical assay for [Na‐K]ATPase in cryostat sections of kidney was further characterized and used to quantify activity in seven functionally distinct sites along the rat nephron. The activity of [Na‐K]ATPase was defined as the difference in ATPase activity in specifically identified tubules contained in serial sections incubated with and without ouabain. Preincubation of sections with ouabain was required for maximal inhibition of [Na‐K]ATPase activity in several distal sites. The concentration of oubain necessary for maximal inhibition of activity was 3·0 mM and half‐maximal inhibition was obtained in all regions with 30–100μM ouabain. In distal sites, [Na‐K]ATPase formed a higher proportion of total ATPase activity (60–80 per cent) than in proximal sites(20–40 per cent). Enzyme activity was quantified using two different methods. The first measured activity over the basal region of tubules and gave an index of the concentration of [Na‐K]ATPase over the basal lateral infoldings of cells composing the tubule. The second read activity over the entire cross section of tubules and provided an estimate of [Na‐K]ATPase per length of tubule. The highest activities over the basal region were obtained from tubules of the distal nephron including the inner (MALin) and outer (MALout) medullary ascending limb, distal convoluted tubule (DCT) and connecting segment (CS). Lower activities were obtained in proximal convoluted (PCT) tubules, proximal straight (PS) tubules and the papillary collecting duct (PD). Distal convoluted tubules contained the highest activity per length of tubule. Other sites contained lower levels of activity in the following order: MALin > MALout > PCT > PD > PS. The modifications introduced increase the sensitivity and precision of this assay and permit the application of this technique to studies of [Na‐K]ATPase activity in the major functional regions of the rat nephron.
Differential sensitivity to trypsin of human bone‐derived cells in culture: Surface changes detected by partitioning in aqueous two‐phase systems
Authors: Sharpe P. T. , Gallagher J. A. , Macdonald B. R. , Treffry T. E. , Russell R. G. G.
Published: May 2005

Human bone‐derived cells, grown in monolayer culture, were dissociated by incubations with trypsin/EDTA and subjected to thin‐layer counter‐current distribution in a ‘low potential’ aqueous two‐phase system. Two major populations of cells were detected. The number of cells in the second (more hydrophobic) population increased with length of trypsinization and time in culture. Cells allowed to ‘regain’ surface molecules lost by trypsinization did not produce the second population.

Cells occupying the second population after a short period of trypsinization had a lower rate of division than peak 1 cells but showed a higher rate of protein synthesis per rate of division than peak 1 cells.

These results show that the cells have markedly different sensitivities to trypsin digestion which may be related to cell division rate of growth. The possible relationship between this and osteoblast development are discussed.
The suppression of endogenous protein degradation by fractions of foetal calf serum: Dialysed serum is less able to suppress degradation in aged cells
Authors: Wharton S. A. , Riley P. A.
Published: May 2005

We have studied the effect of various fractions of foetal bovine serum upon the endogenous degradation of long labelled proteins in cultured MRC5 cells, and upon other cellular functions. Only heat‐inactivated serum was capable of suppressing protein degradation to a similar extent to complete serum. Acid‐treated and delipidized sera were moderately effective. Albumin on its own was able to replace 40 per cent of the effect of serum, indicating the exogenous protein might compete with endogenous protein for degradation in lysosomes. Albumin was not capable of supporting DNA synthesis. Dialysed serum showed an age‐related effect suppressing protein degradation to a lesser extent and being less effective in supporting DNA synthesis or cellular proliferation in aged cells. All the effects noted were related to lysosomal protein degradation. Serum diffusate did not suppress protein degradation.
Red blood cell phagocytosis and lysis following oxidative damage by phenylhydrazine
Authors: Magnani M. , Stocchi V. , Cucchiarini L. , Chiarantini L. , Fornaini G.
Published: May 2005

Red blood cells exposed in vitro to phenylhydrazine acquired Heinz bodies, bound autologous IgG and were then phagocytized when incubated with autologus mononuclear phagocytes. In vivo, phenylhdyrazine administered to rabbits, caused the appearance of high plasma hemoglobin levels and hemoglobinuria as well as Heinz body formations and IgG binding to erythrocytes. This suggests that while in vitro the main mechanism of red cell removal seems to be phagocytoses, in vivo both intravascular hemolysis and phagocytosis are active processes.

Preliminary biochemical studies on phenylhydrazine‐exposed erythrocytes showed that together with the well‐known appearance of Heinz bodies, methemoglobin and a drop in reduced glutathione, this drug also causes ATP depletion. This is initially concomitant with the appearance of ADP and AMP and subsequently hypoxantine. Thus, irreversible ATP depletion may contribute to the genesis of the hemolytic process observed in vivo.
The effects of acute phosphate depletion on isolated chick kidney tubule cells
Authors: Grahn Michael F. , Butterworth Peter J.
Published: May 2005

Isolated tubule cells from chick kidney respond to a short period of phosphate deprivation with increased phosphate uptake and a resistance to parathyroid hormone. During phosphate depletion a considerable amount of phosphate may be released from the cells, but intracellular inorganic phosphate levels are maintained by the hydrolysis of organic phosphate esters. It is suggested that the concomitant changes in metabolism might act as the signal causing the onset of the changes in phosphate handling associated with phosphate deprivation.
Phospholipids in chromatin: Incorporation of [32P]O in different subcellular fractions of hepatocytes
Authors: Viola‐Magni M. P. , Gahan P. B. , Albi E. , Iapoce R. , Gentilucci P. F.
Published: May 2005

Nuclear, chromatin and microsomal fractions were isolated from hepatocytes prepared from rats injected with [32P] O and killed subsequently at times between 1 and 48 h. Specific activities of the total phospholipids (PL) were determined for each subcellular fraction. The major points noted were (a) the initial specific activity of the chromatin PL was half that of both nuclear and microsomal PL at 1 h; (b) the first peak of labelling occurred at 6 h in both nuclear and microsomal PL, but was 3 h later (9h) in the chromatin PL; and (c) a second peak of labelling occurred in the chromatin and microsomal PL, but not in those of the nuclei.

On fractionation of the PL, the major and most metabolically active components were phosphatidylcholine + phosphatidylethanolamine, whilst sphingomyelin accounted for only about 8 per cent of the total PL. The chromatin and microsomal fractions were somewhat similar in their labelling patterns though with a delayed peaking of activity in the chromatin. This is indicative of a synthesis and transport of PL from the microsomes to the chromatin.
Aerobic glycolysis: A study of human articular cartilage
Authors: Nahir A. M.
Published: May 2005

Cartilage generally is one of those tissues that exhibit aerobic glycolysis. In a previous study on rat epiphyseal cartilage it had been suggested that this phenomenon is related to potentially excessive production of pyruvate and acetyl coenzyme A, the latter derived from fatty acid oxidation and inhibiting pyruvate dehydrogenase activity. The present study has shown that, in human articular cartilage, the contribution from fatty acid oxidation is too small to account for this phenomenon although the total potential production of pyruvate could still be in excess of the requirements for acetyl coenzyme A for the Krebs' cycle. Of greater relevance may be the apparent correlations that have been found between the activities of lactate and glyceraldehyde 3‐phosphate dehydrogenases (r = 0·82: 0·01 > p > 0·001) and between those of lactate and glucose 6‐phosphate dehydrogenases (r = 0·92; p < 0·001).
Relationship between melanin content and superoxide dismutase (SOD) activity in the liver of various species of animals
Authors: Schiel G. , Corsaro C. , Scalia M. , Sciuto S. , Geremia E.
Published: May 2005

The scavenger effect of melanin and of superoxide dismutase (SOD) activity on superoxide anion has been shown. In this work we show the relationship between melanin content and SOD activity in livers containing different quantities of melanin which were taken from various species of animals. The mitochondrial SOD activity disappears when the melanin content in the liver is very high; moreover it increases, in the liver of various species of animals examined, proportionally to the decrease of melanin content. No significant variation of the SOD activity localized in the soluble fraction has been detected when related to the melanin content. We think that in the pigmented liver the antioxidant activity of the melanin could mimic part of the function of SOD. The loss of Mn SOD activity could be mediated by a low intracellular level of superoxide anion due to the scavenger effect of melanin on superoxide anion; in fact, it is well known that the biosynthesis of Mn SOD is induced by intracellular levels of superoxide anion.
Avian thrombocyte thrombospondin
Authors: Wachowicz Barbara , Krajewski Tadeusz , Przybylska Maria
Published: May 2005

A high molecular weight glycoprotein (450 000) was obtained from thrombin‐treated duck thrombocytes by barium citrate adsorption technique followed by heparin‐agarose affinity chromatography. Amino acid composition (high number of acidic amino acids and cystine) as well as carbohydrate contents (1·3 per cent hexosamine, 0·9 per cent sialic acid and 1·5 per cent hexose) showed similarity to mammalian platelet thrombospondin.
Liver and biliary levels of glutathione and thiobarbituric acid reactants after acute lindane intoxication
Authors: Videla Luis A. , Barros Silvia B. De M. , Simizu Kiyoko , Junqueira Virgínia B. C.
Published: May 2005

The i.p. administration of 60 mg kg−1 body weight of lindane, the γ‐isomer of hexachlorocyclohexane, to fed rats led to an enhancement of hepatic lipid peroxidation after 24 h of treatment. This was evidenced by significant increases in the hepatic production and biliary release of thiobarbituric acid reactive substances, and in the biliary release of glutathione disulphide. Under these conditions, the content of cytochrome P450 was enhanced concomitantly with increases in the total microsomal oxygen uptake, superoxide radical generation and (+)‐catechin (cyanid‐3‐ol) sensitive respiration. The glutathione status of hepatocytes was altered by lindane as the content and biliary release of glutathione disulphide was drastically augmented, leading to a decrease in the cellular and biliary GSH/GSSG ratios. It is suggested that lindane treatment leads to an induced oxidative capacity, which, in turn, alters the glutathione status of the liver tissue.
Methotrexate: Studies on the cellular metabolism. I. Effect on mitochondrial oxygen uptake and oxidative phosphorylation
Authors: Yamamoto Nair , Oliveira Ma Benigna Martinelli , Campello Annibal De Paiva , Lopes Luiz Carlos Vieira , Klüppel Ma Lúcia Wambier
Published: May 2005

Effect of methotrexate (MTX) on mitochondrial oxygen uptake, oxidative phosphorylation and on the activity of several enzymes linked to respiratory chain was studied. MTX was able to inhibit state III respiration activated by ADP and to decrease the respiratory coefficient with the substrates α‐ketoglutarate and glutamate; these effects became pronounced when mitochondria were pre‐incubated with MTX for 10 min. No effect was observed on ATPase activity of undamaged or broken mitochondria; the same was true for NADH‐oxidase, NADH‐dehydrogenase, NADH‐cytochrome c reductase, succinate oxidase, and cytochrome c oxidase activity. The effect on the steady‐state of cytochrome b, as well as, the inhibitory effect on state III of respiration with NAD+‐linked substrates, offers a reasonable possibility to suggesting that the inhibition site of MTX could be in a place anterior to cytochrome b region, and not linked to respiratory chain.
Ornithine decarboxylase properties: Is there a role for a microsome‐bound inactivating activity?
Authors: Zuretti M. F. , Brossa O. , Gili P. , Gravela E.
Published: May 2005

Liver microsomes have a strong ornithine decarboxylase (ODC) inactivating capacity in vitro. The present results suggest that this may be involved in regulation of ODC activity in vivo: (1) the ODC inactivating capacity of microsomes appears susceptible to in vivo modulation: a single administration of thioacetamide, which induces ODC. also causes a significant increase in the inactivating capacity of the microsomes; (2) under conditions leading to increased microsome‐bound ODC‐inactivating capacity (e.g. liver from thioacetamide‐treated rates versus regenerating liver) ODC displays a greater thermal lability and inactivability in vitro.

A possible involvement of this microsomal activity in an autoregulatory pathway of ODC is suggested by the fact that it is induced by the administration of polyamines. However, inhibition of ODC activity by α‐difluoromethylornithine does not prevent the increase of the microsomal activity caused by thioacetamide. Thus, polyamine biosynthesis does not appear to be an absolute requirement for induction of the microsomal ODC‐inactivating capacity.

The apparent half‐life of ODC in vivo, as evaluated after cycloheximide administration, does not appear to correlate with the microsomal ODC‐inactivating capacity content and the stability properties of ODC in vitro.
Action of the antileukemic and anti‐HTLV‐III (anti‐HIV) agent avarol on the levels of superoxide dismutases and glutathione peroxidase activities in L5178y mouse lymphoma cells
Authors: Batke Erna , Ogura Ryohei , Vaupel Peter , Hummel Klaus , Kallinowski Friedrich , Gasić Miroslav J. , Schröder Heinz C. , Müllerm Werner E. G.
Published: May 2005

The antileukemic and anti‐HTLV‐III (anti‐HIV) agent avarol, a sesquiterpenoid hydroquinone, was determined to be converted into its corresponding quinone derivative avarone via the semiquinone free radical. Its g‐value was 2·0047; after hyperfine splitting the energy levels revealed 16 isotropic Hfs. The redox reaction products were identified at the pH values 4·0, 7·0 and 12·0 and the overall reaction pathways were formulated. In vivo experiments with L5178y mouse lymphoma cells in the ascites of mice revealed that the cytostatic potencies of avarol and avarone cannot be augmented by lowering the pH value. Incubation studies with L5178y cells in vitro showed that the intracellular levels of superoxide dismutases (SODases) and of glutathione (GSH) peroxidase activities significantly change after avarol administration. While both the Mn‐SODase and the Cu/Zn‐SODase activities dropped significantly, the GSH peroxidase activity increased inversely. From these experiments we assume that the anti‐tumour and the antiviral effects of avarol/avarone may be due to an increase, induced by the drug, of the intracellular concentrations of superoxide radicals.
Effect of phenylhydrazine on red blood cell metabolism
Authors: Magnani M. , Rossi L. , Cucchiarini L. , Stocchi V. , Fornaini G.
Published: May 2005

In addition to the well known effect of phenylhydrazine on red blood cells (methaemoglobin and Heinz body formation, autologous IgG binding, lipid peroxidation, etc.) an increased glucose utilization was observed. Measurement of 14CO2 formation from [1‐14C]‐glucose showed a maximum value at 2 mM phenylhydrazine followed by a progressive inhibition on increasing the drug concentration to 16 mM. Concomitantly we found a reduction in the reduced glutathione concentration but not a corresponding increase in the level of oxidized glutathione. Phenylhydrazine also causes ATP depletion. The ATP is in part dephosphorylated to ADP and AMP and in part converted to inosine monophosphate and hypoxanthine. Measurement of the cell content of reduced and oxidized pyridine nucleotides was also performed and showed a progressive increase in the reduced forms of these coenzymes. Thus phenylhydrazine promotes cellular ATP depletion followed by adenine nucleotide catabolism that is not efficiently counteracted by an increase in glucose utilization. The relevance of these data to the mechanism of phenylhydrazine‐induced anemia is discussed.
Effects of nonionic amphiphiles at sublytic concentrations on the erythrocyte membrane
Authors: Isomaa Boris , Hägerstrand Henry
Published: May 2005

The interactions of octaethyleneglycol alkylethers (C10–C16), pentaethyleneglycol dodecylether, and dodecyl D‐maltoside with the human erythrocyte membrane were studied. All the amphiphiles protected erythrocytes against hypotonic haemolysis. At concentrations where the amphipiles protected erythrocytes against hypotonic haemolysis they reduced phosphate efflux. The potency of the amphiphiles, at equiprotecting concentrations, was correlated negatively to the length of the alkyl chain. Pottasium fluxes were increased by all the amphiphiles at protective concentrations. The relative potency of the amphiphiles varied but it was not simply related to the length of the alkyl chain. The only amphiphile affecting active potassium influx was octaethyleneglycol decylether which induced a slight decrease. It is concluded that the increase in passive cation fluxes caused by the amphiphiles is due to an increased permeability of the lipid bilayer induced through a nonspecific interaction of the amphiphiles with the bilayer. The effect of the amphiphiles on ion transport mediated by membrane proteins is proposed to be due to an alteration of the state of the transporting protein.
Inhibition of monocyte luminol‐dependent chemiluminescence by tetrahydrobiopterin, and the free radical oxidation of tetrahydrobiopterin, dihydrobiopterin and dihydroneopterin
Authors: Heales Simon J. R. , Blair John A. , Meinschad Christian , Ziegler Irmgard
Published: May 2005

Luminol‐dependent chemiluminescence of normal human monocytes activated by zymosan is demonstrated to be inhibited by tetrahydrobiopterin in a concentration‐dependent manner. The reduced pterins tetrahydrobiopterin, dihydrobiopterin, and dihydroneopterin are all shown to be readily oxidized by the hydroxyl radical.

The susceptibility of reduced pterins to free radical attack may explain the inhibition of chemiluminescence observed and an additional role of reduced pterins as free radical scavengers in tissues is considered.
Isocitrate dehydrogenase activity and its regulation by estradiol in tissues of rats of various ages
Authors: Yadav R. N. Singh
Published: May 2005

The activity and hormonal regulation of NAD‐ and NADP‐linked isocitrate dehydrogenase (EC.1.1.1.41 and EC.1.1.1.42, respectively) in the brain, liver and kidney cortex of female rats of various ages was investigated. The activity of NAD‐ICDH of brain was greater than extramitochondrial (‐c) or intramitochondrial (‐m) NADP‐ICDH. In contrast, liver c‐NADP‐ICDH was much higher than NAD‐ or m‐NADP‐ICDH, whereas in kidney cortex the activity of m‐NADP‐ICDH is dominant over both NAD‐ and c‐NADP‐ICDH in all the age group of rats studied. The activity of the NAD‐ICDH of brain and all the enzymes of liver and kidney cortex increases until adulthood (33‐weeks) and decreases thereafter in old rats (85‐weeks). In brain c‐NADP‐ICDH was much higher in immature (6‐weeks) rats and decreases with increasing age of the animal, whereas m‐NADP‐ICDH showed no significant change with the age of the rats. Bilateral ovariectomy decreases the level of all the three forms of enzyme in all the tissues of 6‐, 13‐ and 33‐week rats but failed to show any significant effect in 85‐week old rats. Exogenous administration of estradiol induces all the three forms of enzyme in all the tissues of ovariectomized rats. The degree of response is tissue‐ and age‐specific.
Cell density dependent morphological changes in adult rat hepatocytes during primary culture
Authors: Koji T. , Nakane P. K. , Murakoshi M. , Watanabe K. , Terayama H.
Published: May 2005

In order to gain morphological insights about the cell density dependency, hepatocytes cultured at a low cell density (< about 0·1 × 105 nuclei (cm2)−1) and at a high cell density (> about 1 × 105 nuclei (cm2)−1) were examined ultrastructurally 24 h after plating (just prior to the beginning of DNA synthesis). The results were as follows: (i) glycogen rosettes disappeared completely in low density culture as compared with sections from an intact liver. In contrast, glycogen rosettes were still present in high density culture. (ii) Polysomes seemed increased in low density culture in comparison with those seen in sections from an intact liver and from the high density culture. (iii) In low density culture, the shape of mitochondria deviated from that of hepatocytes in an intact liver and the mitochondria often lost a characteristic close contact with rough endoplasmic reticulum (rough ER). (iv) In low density culture, bundles of filamentous structure were detected, which were not found in an intact liver or high density culture. The following features were found only in high density culture; (v) numberous villous cytoplasmic protrusions developed along the area facing adjacent cells, and seemed to intertwine with each other, and (vi) between the hepatocytes, only abortive junctions were found. These results indicate that the hepatocytes cultured at a low density express most of the characteristics of the hepatocytes in a regenerating liver and the features of the cells cultured at a high density are very similar to those of the hepatocytes in sections from an intact liver.
Metabolism of 4‐hydroxynonenal by the rat heptoma cell line MH1C1
Authors: Ferro Margherita , Marinari Umbertom M. , Poli Giuseppe , Dianzani Mario U. , Fauler Günter , Zollner Helmward , Esterbauer Hermann
Published: May 2005

The metabolism of the toxic lipid peroxidation product 4‐hydroxynonenal was investigated in the well‐differentiated rat heptoma cell line MH1C1.

When exposed to 0·1 mM 4‐hydroxynonenal (HNE), MH1C1 cells consumed it in a time‐dependent manner. There was a linear relationship between the amount of aldehyde consumed and cell number in the range 0·5–4 × 106 cells ml−1. This process was unaffected by pyrazole, suggesting that alcohol dehydrogenase is not involved.

The whole homogenate of MH1C1 cells consumed added HNE at a rate similar to that in intact cells. Fractionation of the homogenate showed that the highest HNE‐metabolizing activity is in the cytosol. The dialysed cytosol had almost no capacity to metabolize HNE, but this was restored by supplementation with NAD, NADH, NADP and NADPH.

The metabolism of HNE in MH1C1 cells is thus different from that in hepatocytes, which were shown to utilize cytosolic alcohol dehydrogenase for this process. Both reductive and oxidative pathways could be implicated in the metabolic activity of MH1C1 cells towards HNE as well as binding by glutathione.
Stimulatory effect of 1,25‐dihydroxyvitamin D3 on the glucose‐6‐phosphate dehydrogenase activity in the MCF‐7 human breast cancer cell line
Authors: Noun Aïcha , Garabedian Michèle , Monet Jean‐Dominique
Published: May 2005

Human breast cancer cell lines have been shown to possess high affinity receptors for 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) on the activity of glucose‐6‐phosphate dehydrogenase (G6PD) in cells of a human breast cancer cell line MCF‐7. G6PD, an enzyme which controls the hexose monophosphate shunt, is elevated and sensitive to 17β‐estradiol in breast tumors. G6PD activity was stimulated by 1,25(OH)2D3 in a dose‐dependent manner at very low concentrations of steroid (10−10–10−12 M). 1,25(OH)2D3 increased maximum velocity without modifying the affinity constant of the enzyme for glucose‐6‐phosphate.
Some effects of fructose‐1,6‐diphosphate on rat myocardial tissue related to a membrane‐stabilizing action
Authors: Galzigna L. , Rizzoli V. , Bianchi M. , Rigobello M. P. , Scuri R.
Published: May 2005

This study aims at elucidating the mechanism of action of extracellular fructose‐1,6‐diphosphate (FDP). FDP is able to inhibit Ca++ entry into the myocardial tissue with an IC50 value of 11·5 mM and in addition, it is bound by rat heart slices, the binding being activated by Zn and conditions of chemical hypoxia induced by KCN and iodoacetate. The overall effect of extracellular FDP includes an increase of frequency and amplitude of contraction of perfused heart at concentration below 1 mM, and, in general, a stimulation of the oxygen consumption of the tissue. The antihaemolytic effect of FDP suggests its action as a membrane stabilizer. The effects of extracellular FDP on the myocardial cell can be interpreted both on the basis of a limited permeability of the cell membrane to it and as a purely extracellular effect transduced through the cell membrane with a final response consisting of an increase in the intracellular FDP.
Prostaglandin derivatives inhibit the growth of malarial parasites in mice
Authors: Ohnishi S. Tsuyoshi , Ohnishi Noriko , Oda Yoshio , Katsuoka Masayuki
Published: May 2005

New prostaglandin oligomeric derivatives, termed MR‐256 and MR‐356, were found to inhibit the growth of murine malarial parasites, P. chabaudi and P. vinckei, within red blood cells in vivo. When mice were infected with P. chabaudi, both MR‐256 and MR‐356 suppressed the growth of parasites, but MR‐356 had a greater inhibitory effect than MR‐256. With P. vinckei, MR‐356 also inhibited the growth of parasites, and improved the survival rate. The effect of MR‐256 was much less. A possible inhibitory mechanism of action of these drugs is discussed.
Glucose 6‐phosphate plays a central role in the regulation of glycogen synthesis in a glycogen‐storing liver cell line
Authors: Mayer D. , Letsch I.
Published: May 2005

Two substrains of the epithelial liver cell line C1 I, one storing large amounts of glycogen, the other one being very poor in glycogen were used as a model for studying glycogen synthesis. The glycogen content of glycogen‐rich cells doubled during the proliferative phase and remained high in plateau phase although glycogen synthase I activity was not significantly altered during growth cycle and was too low to account for the increase in glycogen. However, the activity of the glucose 6‐phosphate (Glc6‐P)‐dependent synthase rose continuously during growth cycle, and intracellular Glc6‐P‐concentration increased about 10‐fold in log phase cells to 0·72 μmol g−1 wet weight. A0·5 of synthase for Glc6‐P was 0·79 mM. It was also found that in contrast to the enzyme from normal liver, glycogen phosphorylase a from C1 I cells was inhibited by Glc6‐P, the apparent Ki being 0·45 mM. It was concluded that glycogen accumulation in C1I cells was due to stimulation of synthase and inhibition of phosphorylase by Glc6‐P. Findings from the glycogen‐poor cell line which revealed similar specific activities of synthase and phosphorylase but only low Glc6‐P (0·056 μmol g−1 wet weight) supported this conclusion. Addition of glucose to starved cells resulted in a transient activation of synthase in both cell lines. Net glycogen synthesis, was, however, only observed in the cells with a high Glc6‐P‐content. Thus, modulation of synthase and phosphorylase by Glc6‐P and not activation/inactivation of the enzymes seems to play a predominant role in glycogen accumulation in this cell line.
Suppressive effect of adrenalectomy on growth of L1210 leukemic cells in ascites
Authors: Kimura Kotohiko , Sakamoto Yukiya
Published: May 2005

This study was designed to evaluate the effect of adrenalectomy on growth of L1210 leukemic cells in ascites of BDF1 mice. Varying doses of 1·5 × 104, 5·0 × 105, and 1·5 × 106 viable tumour cells were inoculated intraperitoneally into groups of either adrenalectomized or sham‐operated mice. At days 4 to 7 after the inoculation, adrenalectomized mice inoculated with 1·5 × 104 or 5·0 × 105 tumour cells had a smaller number of tumour cells in ascites than sham‐operated controls. However, after inoculation of 1·5 × 106 cells, no significant differences were found at days 2 to 4 between adrenalectomized and sham‐operated mice. The growth retardation by adrenalectomy was not observed in adrenalectomized mice supplemented with 4 or 6 μg dexamethasone per day per mouse. It suggested that the ablation of glucocorticoids was at least partially responsible for the growth retardation observed in adrenalectomized mice. Cell kinetic analysis revealed that the difference in a potential doubling time could not explain these results. Tumour retention in the peritoneal cavity was measured using [125I]‐iododeoxyuridine‐labelled tumour cells as a tracer. At days 4 to 6 after inoculation of 5·0 × 105 labelled cells, radioactivity in the peritoneal cavity in adrenalectomized mice was about 70 per cent of that in sham‐operated mice. This ratio was almost equivalent to the ratio of the number of cells in ascites of adrenalectomized mice to that of sham‐operated ones. Consequently, growth retardation observed in adrenalectomized mice resulted from an increase in tumour cell migration and/or in tumour cell death, but not from an increase in doubling time.
Subcellular localization of recently‐absorbed iron in mouse duodenal enterocytes: Identification of a basolateral membrane iron‐binding site
Authors: Snape Sue , Simpson Robert J. , Peters Timothy J.
Published: May 2005

The subcellular distribution of newly absorbed iron in isolated mouse duodenal enterocytes was investigated by analytical subcellular fractionation using sucrose density gradient centifugation. Two major peaks of mucosal 59Fe activity were observed: one soluble and one particulate (density 1·18–1·20 g ml−1). The latter was increased following prior exposure of animals to chronic hypoxia. The particulate 59Fe was localized to the basolateral membranes using the marker enzyme Na+, K+ activated, Mg2+ dependant, ATPase and by washing intact enterocytes with the selective plasma membrane perturbant digitonin.

The basolateral membrane can be selectively labelled by in vitro incubation of intact enterocytes at 0°C with 59Fe(III)‐nitrilotriacetate complex, confirming the presence of a 59Fe binding site on this membrane. No significant difference in in vitro iron binding to this site was observed between normal and chronically hypoxic animals. Iron binding to the basolateral membrane was significantly higher in disrupted, compared to intact enterocytes, indicating that this site is present on both sides of the basolateral membrane. It is therfore suggested that the increased labelling of this site in hypoxia, in vivo, is a consequence of an increase in a mucosal Fe pool which is available for binding to a membrane receptor.
Intracellular assembly of newly synthesized canine cardiac myosins
Authors: Seko Yoshinori , Naito Shigeto , Imataka Kouji , Fujii Jun , Nakane Paul K. , Takaku Fumimaro , Yazaki Yoshio
Published: May 2005

To investigate how newly synthesized cardiac myosins are assembled into myofilaments, we analysed the distribution of newly produced α‐myosin heavy chain isozyme in sarcomeres by immunoelectron microscopy using a monoclonal antibody (CMA19), which is specific for α‐myosin heavy chain. Isozymic changes in myosin heavy chains from β to α type were induced in canine ventricular muscles and cultured ventricular myocytes by administration of 1‐thyroxine. We incubated the glycerinated ventricular muscles or cultured ventricular myocytes with the enzyme (horseradish peroxidase) labelled Fab fragment of CMA19. After the reaction with 3, 3′‐diaminobenzidine and osmification, we prepared ultrathin sections of the ventricular muscles or cultured ventricular myocytes and analysed their staining patterns by electron microscopy.

There was apparent heterogeneity in the staining intensity of the myofilaments among different cells, among different myofibrils and even intramyofibrillarly. Higher magnification revealed that there were scattered foci of strong reaction which appeared to be foci of assembly of the newly synthesized α‐myosin heavy chain. Immunocytochemical study also showed heterogeneous reactions within myofilaments and that there were scattered foci of myofilament assembly, which were closely associated with polyribosomes producing newly induced α‐myosin heavy chain. These data suggest that newly synthesized cardiac myosins are assembled into myofilaments from the sites of synthesis, that is polyribosomes. This may explain the heterogeneity of the assembly pattern of newly synthesized cardiac myosins at the subcellular level.
Peculiar ultraviolet absorbing moieties of human osteoarthritic cartilage
Authors: Chayen J. , Bitensky Lucille , Nahir A. M.
Published: May 2005

The ultraviolet absorbing components of human cartilage have been measured by microspectrophotometry. The characteristics of the chondrocytes appeared to be identical, irrespective of the pathology. However the matrix of osteoarthritic cartilage contained components that absorbed maximally in the region of 270 to 250 nm; such components were not found in the matrix of cartilage of non‐arthritic joints. Substances that absorb maximally in this region of the ultraviolet could generate free radicals.
Modulation of membrane phenotype, matrix adhesion and microinvasiveness of metastatic tumour cells by HUdR
Authors: Timár J. , Pogány G. , Balázs M. , Szöllösi J. , Ladányi A. , Oláh J. , Timár F. , Lapis K. , Jeney A.
Published: May 2005

The effect of HUdR, proved to be anti‐metastatic in vivo, was studied in vitro on cell proliferation, nucleoside uptake, membrane fluidity, expression of galactosylated glycans and proteoglycans in metastatic HM tumour cells. The observed increase in membrane fluidity and the suppression of nucleoside transport were early events of the HUdR action followed by decrease of galactosylated glycan and HSPG expression. However, these changes did not influence the proliferation capacity of the cells at the concentrations studied. As a consequence of the membrane alterations a reduced adhesiveness and spreading on extracellular matrix components was detected. In addition, the HUdR treated HM cells showed reduced capacity to invade fibroblast monolayers in vitro. Based on these observations, HUdR could be the prototype of new anti‐metastatic agents acting at the level of tumour–host interaction.
Carboxylesterase activities during the establishment of the procambium and procortex in embryos of pisum sativum L.
Authors: Carmignac D. F. , Gahan P. B. , Loukopoulou Z.
Published: May 2005

A cytochemical and electrophoretic analysis of alpha‐naphthyl and naphthol AS‐D acetate esterases from the radicles of pea seeds germinated for 24, 36 and 48 h permitted the detection of the loss of activity from the procortex with time after germination, but a stability of this activity in the prostele. Inhibitor studies showed the procambial enzyme as a single band of carboxylesterase activity the remainder being acetylesterase activity. The procambial carboxylesterase which is present in the more mature root apex corresponds to that of the 48‐h embryo (separating at Rf 0·74).
Intracellular production of reactive oxygen species in human neutrophils following activation by the soluble stimuli FMLP, dioctanoylglycerol and ionomycin
Authors: Follin Per , Johansson Agneta , Dahlgren Claes
Published: May 2005

The stimuli, sn‐1, 2‐dioctanoylglycerol; (DG8) the calcium specific inophore, ionomycin, and the chemotactic peptide formylmethionyl‐leucyl‐phenylalanine (FMLP) can interact with normal human neutrophils and activate their superoxide/hydrogen peroxide generating NADPH‐oxidase. In response to the peptide as well as DG8, the neutrophils produced both superoxide (O2−) and hydrogen peroxide (H2O2). Since interaction between the cells and ionomycin was not associated with any notable superoxide production and hydrogen peroxide was induced only in the presence of azide, a potent inhibitor of the hydrogen peroxide‐consuming enzymes catalase and myeloperoxidase, we conclude that this stimulus can generate oxygen metabolites intracellularly. Since the DG8‐induced production of hydrogen peroxide was increased in the presence of azide, whereas the FMLP‐induced response was largely unaffected, we concluded that the three stimuli differ in their capacity to generate oxygen metabolites intracellularly.

The use of sn‐1,2‐didecanoylgylycerol (DG10) as stimulating agent did not result in any detectable activation of the NADPH‐oxidase. However, preincubation caused an increased (primed) response during stimulation with the chemotactic peptide FMLP. The response of primed neutrophils to FMLP proceeds with a time‐course different from that seen in normal cells. From the results presented on FMLP‐induced activity in the presence of azide, we conclude that FMLP causes normal cells to produce oxygen radicals which are released from the cells. However, the primed cells are also capable of generating oxygen metabolites that are retained inside the cells. In fact, measurement of the intracellularly generated metabolites discloses this to be the predominant part of the response.
Studies of the proliferation and differentiation of immature myeloid cells in vitro: 4: Preculture proto‐oncogene expression and the behaviour of myeloid leukemia cells in vitro
Authors: Yin Mo Ying , Gao Xue Zhi , Wang Zique , Preisler Harvey D.
Published: May 2005

Studies were conducted to determine the relationship between the pretherapy characteristics of leukemia cells and their behaviour during culture in vitro. Leukemia cells which proliferated well in vitro also proliferated well in vivo. Cells which manifested myeloid or monocytic differentiation in vivo tended to manifest differentiation along these lines in vitro. Cells which manifested high levels of expression of c‐fms, c‐fes, or triose phosphate isomerase prior to culture were likely to differentiate in vitro, with high levels of c‐fes expression being related to myeloid maturation. These observations suggest that differentiation at the molecular level prior to culture is a requisite for leukemia cell differentiation in vitro. The same may be true for differentiation in vivo under the influence of exogenously administered agents such as cytotoxic chemotherapy or recombinant growth factors.
Metabolic labelling and partial characterization of glycophospholipids in pancreatic islet cells
Authors: Albor Amador , Cáamara Javier , Mato José M. , Malaisse Willy J. , Verde Isabel Val
Published: May 2005

Insulin action is thought to be mediated by an inositol‐, glucosamine‐ and galactose‐containing oligosaccharide liberated by phosphodiesterase hydrolysis of a glycosyl‐phosphatidylinositol. This oligosaccharide inhibits insulin biosynthesis and secretion in pancreatic islets. In the present study, two main glycolipids (peak I and II) were resolved by sequential TLC of lipids extracted from islet cells labelled with tritiated glucosamine, galactose or myristate. The two glycolipids displayed comparable sensitivity to β‐galactosidase but differed from one another by their sensitivity to phosphatidylinositol‐specific phospholipase C. Moreover, structural heterogeneity within each peak was suggested by their partial resistance to nitrous acid deamination. These findings support the presence in islet cells of glycolipids similar to those currently considered as a possible postreceptor target for insulin in other cell types.
Effect of chronic ethanol consumption on postnatal development of renal (Na + K)‐ATPase in the rat
Authors: Rodrigo Ramón , Vergara Leoncio , Oberhauser Ernesto
Published: May 2005

Renal (Na + K)‐ATPase was studied to ascertain whether it follows the pattern of adaptation of membrane‐bound enzymes that are inhibited by acute ethanol exposure and develop greater activity after chronic ethanol treatment. A colony of rats was given 20 per cent (v/v) ethanol as sole drinking solution throughout gestation, lactation and following weaning. (Na + K)‐ATPase and ouabain‐insensitive Ca2+‐ATPase activities were determined; regional distribution of these enzymes was assessed in renal cortex and outer medulla. Control rats drank tap water.

(Na + K)‐ATPase in whole homogenate of kidney increased with age in controls and ethanol‐fed rats, but the latter showed higher values at every age studied. Between 15 and 60 days of age, the control group showed 2‐fold increases in cortex and 5‐fold in outer medulla, whereas ethanol‐fed rats reached a 3‐fold increase in the enzyme activity in both renal regions. Ca2+‐ATPase showed the same time course in developing kidney of both groups. Chronic ethanol treatment of adult rats resulted in an increase of (Na + K)‐ATPase activity in cortex and outer medulla, but no change in other ATPases.

Since an earlier maturational development of renal (Na + K)‐ATPase was displayed by ethanol‐fed rats, underlying mechanisms that may account for these results are discussed.
Simultaneous changes in various mechanisms that mediate the cell incorporation of folate compounds account for low levels of resistance to methotrexate
Authors: Cavalcanti F. , Calvo N. , Grau‐Oliete M. R. , Rivera‐Fillat M. P.
Published: May 2005

Changes in the mechanisms of folate incorporation were studied in cells treated with low concentrations of methotrexate in order to evaluate their contribution to the development of resistance to antifolate drugs. The uptake of methotrexate via reduced‐folate system, the membrane‐associated high‐affinity folate binding capacity and the activity, levels and affinity for methotrexate of dihydrofolate reductase were measured in L5178 murine leukemic lymphoblasts and in a subline, MTX/R16, 16 times more resistant to methotrexate which was isolated after a short exposure to the antifolate. Various simultaneous changes were characterized in MTX/R16 cells which co‐participated in the development of resistance: a decreased affinity of the carrier for methotrexate uptake via the reduced‐folate system of entry, the increase of dihydrofolate reductase activity and levels and a two‐fold increased expression of a membrane‐associated high‐affinity folate‐binding protein (mFBP). The increase of the mFBP expression, besides ensuring the growth of resistant cells by its contribution to the reduced folate intake, also participates in the methotrexate resistance by the internalization of folate cofactor which would compete with methotrexate hindering the effective inhibition of dihydrofolate reductase by the antifolate.
Identification of multi‐drug resistant cells in sensitive friend leukemia cells by quantitative videomicrofluorimetry
Authors: Lahmy S. , Salmon J. M. , Vigo J. , Viallet P.
Published: May 2005

The cellular resistance to cytotoxic drugs, particularly to anthracyclines, remains a major problem in cancer chemotherapy. A number of biochemical mechanisms have been described, one of them being a lower accumulation of drugs in resistant cells. The accumulation of Ho33342 in sensitive and resistant Friend leukemia cells was studied by quantitative fluorescence image analysis, a method which allows investigations to be made on living tissues and cells. The intensity of fluorescence is related to the amount of Ho33342 accumulated into the cells and has been found to be more intense in sensitive cells than in resistant ones. Moreover, the retention of this vital dye was inversely related to the degree of resistance in the three resistant cell lines. The addition of verapamil, which is known to reverse resistance to anthracyclines, resulted in an increase of the amount of Ho33342 accumulated in the resistant cells. Ho33342 presents a higher quantum yield than any other anthracyclines, such as adriamycin and can be used as a microfluorimetric probe to identify the resistant cells in a heterogeneous cell population.
The effects of monensin on blood‐borne arrest and glycosylation of BL/VL3 lymphoma cells
Authors: Di Virgilio Sergio , Rampelberg Murielle , Greimers Roland , Schnek Georges , Hooghe Robert
Published: May 2005

We have previously shown that inhibitors of N‐glycan processing alter both the cell surface carbohydrates and the homing properties in lymphoid cells. We have now studied the effects of the ionophore monensin (MON) on these parameters.

Arrest in the spleen of [111In]‐labelled BL/VL3 murine T lymphoma cells, injected intravenously was clearly reduced if the cells had been cultured for 24 h in the presence of monensin (0·1–1·0 μg ml−1).

We have characterized glycopeptides from BL/VL3 murine T lymphoma cells. Following labelling with tritiated precursors (fucose, mannose, galactose, glucosamine), surface glycopeptides from BL/VL3 murine T lymphoma cells, were released by trypsin and separated by gel filtration on Bio‐Gel P6 and by affinity chromatography on immobilized lectins.

After treatment with MON, a class of high molecular mass glycopeptides was no longer found. There were less complex and more high mannose glycans, as a consequence of a reduction of terminal glycosylation (sialylation, fucosylation or incorporation of N‐acetyl‐glucosamine). Similar findings were obtained with immunoprecipitated Thy‐1 antigen. However, as estimated by flow cytometry analysis, the cell surface expression of Thy‐1 was not reduced in MON‐treated cells. Taken together our results show that cell surface oligosaccharides are modified dramatically, but that at least, certain cell surface antigens are present in normal amounts. It is tempting to speculate that changes in glycosylation account for the abnormal homing properties of MON‐treated cells.
Stimulation of human platelets with concanavalin a involves phospholipase C activation
Authors: Torti Mauro , Balduini Cesare , Ramaschi Giuseppe , Sinigaglia Fabiola
Published: May 2005

In response to concanavalin A, cytoplasmic calcium movement was observed in human platelets, both in the presence of 1 mM Ca2+ or 1 mM EGTA in the medium. Concanavalin A also caused the activation of inositide turnover and the production of inositol phosphates, suggesting that activation of phospholipase C occurs. The mechanism by which concanavalin A stimulates phospholipase C does not depend on GTP‐binding transducers, because it was not inhibited by GDPβS, while experiments performed in the presence of cytochalasin B suggested a role for membrane glycoprotein IIb‐IIIa‐cytoskeleton interaction in this process. Ca2+‐proteases and Na +/H+ antiport also seemed to be related to concanavalin A‐induced phospholipase C activation, as suggested by experiments performed in the presence of leupeptin and amiloride.
Added ATP influences some responses of rat synaptosomes to glutamate
Authors: Galzigna L. , Bianchi M. , Rizzo V. , Battistin T. , Scarpa M. , Rigo A.
Published: May 2005

ATP added externally to rat synaptosomes activated uptake of both Ca2+ and glutamate which was partially accounted for by the uptake phenomena of synaptic vescles and mitochondria, as shown by using specific inhibitors of the latter. Increasing concentrations of glutamate stimulated Ca2+ entry linearly, as shown by using 45Ca or a Ca‐specific electrode. The processes of glutamate and Ca2+ uptake shared some common features and their ATP‐dependence may be correlated with an ouabain‐insensitive synaptosomal ectonucleotidase activity measured by a 31P‐NMR or a luminometric technique. The ATP hydrolysis catalysed by the synaptosomes was activated by both Ca2+ and glutamate. The present synaptosomal activities may represent a model for studying the modulatory effects of ATP on the glutamatergic neurotransmission.