bluuebunny/crossref_metadata_2025_split huggingface dataset:
Study of thiol proteases of normal human skin fibroblasts
Authors: Khalfan Hussain A. 
Published: May 2005

AbstractThe protease activity of cultured normal human skin fibroblasts was studied using the synthetic fluorigenic peptides, the modified protein 4‐methylumbelliferyl‐casein, the thiol inhibitors and the affinity for concanavalin A‐Sepharose. The majority of the activity to N‐benzyloxycarbonyl‐L‐phenylalanyl‐L‐arginyl‐7‐amido‐4‐methyl‐coumarin and N‐a‐benzyloxycarbonyl‐L‐arginyl‐arginyl‐7‐amido‐4‐methylcoumarin had a pH optimum of 6·0, and was thiol‐dependent and inhibited by leupeptin and antipain. The activity toward N‐benzyloxycarbonyl‐L‐phenylalanyl‐L‐arginyl‐7‐amido‐4‐methylcoumarin represents both cathepsin B and cathepsin L, whereas the activity towards 4‐methylumbelliferyl‐casein represent only cathepsin L. Cathepsin H could not be detected when assayed with L‐arginine‐7‐amido‐4‐methylcoumarin substrate. Cathepsin D was present in comparatively small amounts when assayed with 4‐methylumbelliferyl‐casein. Activity towards 4‐methylumbelliferyl‐casein had pH optima at 3 and 6 and was stimulated by dithiothreitol. A proportion of the activity at pH 6·0 was not dependent on thiols and not inhibited by leupeptin, and had the general characteristics of a carboxyl proteinase. Over 70 per cent of the activity was in the lysosomal fraction and showed structure‐linked latency. All the detectable protein emerged from the immobilized concanavalin A column and the fractions eluted by α‐methyl‐D‐mannoside were significantly hydrolysed the synthetic peptides. Only that fraction which bound to concanavalin A was active towards 4‐methylumbelliferyl‐casein. Cathepsin B had no affinity for concanavalin A‐Sepharose due to the absence of glycoprotein content, unlike cathepsin L which showed a strong affinity for concanavalin A‐Sepharose.
Interstitial collagen synthesis by somatic testicular cells in culture
Authors: Rodriguez J. Pablo , Fernández Mireya , Minguell Jośe J. 
Published: May 2005

AbstractThe synthesis, distribution and types of collagen produced by somatic testicular cells in culture was studied. To investigate whether changes in collagen synthesis correlate with the age of the animal, cultures derived from immature and pubertal rats were established. Immature rats synthesize 40 per cent more collagen than pubertal rats. Both groups of animals synthesize procollagen types I and III. Pro‐collagen type I is present in the culture medium as well as in the cell fraction, while type III is only detected in the culture medium. In the transition from immature to pubertal rat, the ratio of procollagen type III to procollagen type I diminishes from 5·7 to 1·7. These results indicate that the synthesis, distribution and molecular characteristics of interstitial collagens changes with the age of the animal. Since, the content of other extracellular matrix components such as proteoglycans and collagen type IV also varies with age, we postulate that the composition of the extracellular matrix in the testes is not constant but changes with sexual development.
Mode of action of 2‐(diethylamino)‐7‐ethoxychromone on human platelets
Authors: Leoncini Giuliana , Maresca Mitzi , Colao Clorinda , Buzzi Enrica , Mazzei Mauro 
Published: May 2005

AbstractThe in vitro effect of 2‐(diethylamino)‐7‐ethoxychromone (RC39XVIII) on human platelet aggregation induced by several agonists and on thromoboxane B2 formation, granule release and intracellular cAMP elevation has been studied. The chromone‐derivative exerts a dose‐dependent inhibitory effect on aggregation producted by U46619, arachidonic acid, thrombin, collagen and ADP. RC39XVIII inhibits aggregation, TxB2 formation and granule release in parallel. Moreover the drug potentiates cAMP accumulation induced by iloprost and forskolin. The drug also inhibits soluble cAMP phosphodiesterase in dose‐dependent manner. No effect on adenylate cyclase activity measured in platelet membranes was evident.
Phorbol ester inhibits 13‐cis‐retinoic acid‐induced hydrolysis of phosphatidylinositol 4,5‐bisphosphate in cultured murine keratinocytes: A possible negative feedback via protein kinase C‐activation
Authors: Tang Wilson , Ziboh Vincent A. 
Published: May 2005

AbstractThe incubation of double‐labelled ([14C]‐glycerol and [3H]‐myoinositol) keratinocytes with 13‐cis retinoic acid induced the transient and simulataneous release of [3H]‐inositol trisphosphate ([3H]‐InsP3) and [14C]‐diacylglycerol ([14C]‐DAG) indicating that a possible mode of action of this retinoid on murine keratinocytes may be at least in part the early trasient release of the two putative messengers (InsP3 and DAG) from phosphatidylinositol‐4,5 bisphosphate (PtdIns4, 5P2). In contrast, the preincubation of the keratinocytes with 12‐O‐tetradecanolyphorbol‐13‐acetate (TPA) prior to incubation with 13‐cis‐RA suppressed the 13‐cis‐RA‐induced release of [3H]‐InsP3 and [14C]‐DAG. The specificity of the TPA effect was established by the lack of effect of the biologically inactive 4α‐phorbol 12, 13‐didecanoate. Furthermore, the incubation of the TPA‐primed keratinocytes with 13‐cis‐RA caused a delayed and sustained accumulation of [14C]‐DAG. An exploration of the source of this late relase of [14C]‐DAG revealed that this [14C]‐DAG was released from non‐inositol containing phospholipids, particularly, phosphatidylcholine. This latter DAG released in the TPA‐primed cells correlated with the translocation of the cytoplasmic protein kinase C (PKC) activity to the membrane associated PKC activity. Taken together, these results suggest that alteration of PKC activity, presumably induced by DAG released from non‐inositol phospholipids, may play a major role in the TPA‐induced negative feedback inhibition of 13‐cis RA‐induced hydrolysis of keratinocyte PtdIns4, 5P2.
Differential effects of unsaturated fatty acids on modulation of endotoxin‐induced tissue factor activation in cultured human leukemia U937 cells
Authors: Chu Arthur J. , Moore John 
Published: May 2005

AbstractWe examined the direct effects of unsaturated fatty acids, oleic (18:1 n‐9), linoleic (18:2 n‐6), eicosapentaenoic (20:5 n‐3) and docosahexaenoic (22:6 n‐3) on tissue factor (TF) activity in the human leukemia monocytic U937 cell line. After exposing cells to fatty acids for 16 h, there were no significant effects on either TF activity or its activation induced by bacterial endotoxin (LPS). When the cells were primed with fatty acids for 24 h, 48 h or 72 h, the TF activity remained essentially unchanged. However, the extent of TF‐activation induced by LPS depended on the length of priming, and the dose and the degree of unsaturation of the fatty acids to which cells were exposed. After a 72‐h priming, 18:1 produced 40–60 per cent elevation in LPS‐challenge. In contrast, approximately 20–50 per cent reduction in LPS‐challenge was achieved by 18:2, 20:5 and 22:6 at high concentrations. The results suggest that chronic exposure of U937 cells to unsaturated fatty acids leads to modulation of the TF‐activation in response to LPS.
Aldehyde‐induced modifications of the microtubular system in 3T3 fibroblasts
Authors: Olivero Antonella , Miglietta Antonella , Gadoni Elena , Gabriel Ludovica 
Published: May 2005

AbstractThe molecular structure of aldehydes is closely related to their antimicrotubular effect. Morphological modifications of the microtubular system in living cells after incubation with certain aldehydes are consistent with biochemical alterations detected in previous research. The microtubular arrangement was visualized by an immunofluorescence technique with antitubulin antibodies, while the content of tubulin in the cells was evaluated by a colchicine binding assay. 2‐Nonenal behaved similarly to 4‐hydroxynonenal, a lipid peroxidation product, disorganizing microtubular network in 3T3 fibroblasts and decreasing the amounts of tubulin able to bind labelled colchicine. Nonanal did not significantly impair the tubulin characteristics in the cells, despite the fact that it has been shown to be active on the purified microtubular system; benzaldehyde was ineffective. This would appear to explain the mechanisms of interaction of aliphatic aldehydes which might be suitable for use as antimicrotubular drugs.
Generation of hydrogen peroxide in resting and activated platelets
Authors: Maresca M. , Colao C. , Leoncini G. 
Published: May 2005

AbstractThe production of hydrogen peroxide was measured by following the oxidation of dichlorofluorescein (DCFH) entrapped into platelets. Resting platelets produced nanomolar quantities of DCF, which was proportional to the concentration of platelets and was steady during 1 h of incubation. A significant increase of basal DCF fluorescence was induced by stimuli namely thrombin, arachidonic acid, the Ca2+ ionophore A23187 and PMA. The effect of agonists has been also measured in the presence of 3‐amino‐1,2,4‐triazole (AT) or N‐ethylmaleimide (NEM), inhibitors of catalase and glutathione peroxidase, respectively. A further significant enhancement of DCF produced in stimulated platelets was detected only in the presence of NEM. A correlation was found between the increase in DCF and externally added hydrogen peroxide or the oxidizing species formed by xanthine oxidase plus acetaldehyde. The yield was not affected by superoxide dismutase and was higher in the presence of AT or NEM. A cooperative effect in the presence of both inhibitors was shown. Glutathione peroxidase plus glutathione diminished the level of DCF to basal levels.
Mechanism of citrinin‐induced dysfunction of mitochondria. II. Effect on respiration, enzyme activities, and membrane potential of liver mitochondria
Authors: Chagas Generoso M. , Oliveira Ma. Bengna M. , Campello Annibal P. , Klüppel Ma. Lúcia W. 
Published: May 2005

AbstractThe mycotoxin citrinin, depressed the phosphorylation efficiency of liver mitochondria as deduced from a decrease of respiratory coefficent and of the ADP/O ratio. Citrinin (1·0 mM) inhibited some enzymes linked to the respiratory chain, namely NADH oxidase and NADH cytochrome c reductase involved with complex I. The activities of enzymes related with other enzymatic complexes of the respiratory chain were either unaffected or enhanced. ATPase activity was inhibited by the mycotoxin. Malate, glutamate, and 2‐oxoglutarate dehydrogenases were also inhibited. The transmembrane potential (Δψ), developed by energized mitochondria and depolarization on the addition of ADP, was decreased. The results suggest that citrinin promotes a partial dissipation of the transmembrane potential, different from that resulting from a classical uncoupler such as 2,4‐dinitrophenol.
Regulation of the phosphate (Pi) concentration in UMR 106 osteoblast‐like cells: Effect of Pi, Na+ and K+
Authors: Kemp Graham J. , Khouja Hamed I. , Ahmado Abdulla , Russell R. Graham G. , Bevington Alan 
Published: May 2005

AbstractOsteoblast‐like cells possess Na‐dependent transporters which accumulate orthophosphate (Pi) from the extracellular medium. This may be important in bone formation. Here we describe parallel measurements of Pi uptake and cellular [Pi] in such cells from the rat (UMR 106–01 and UMR 106–06) and human (OB), and in non‐osteoblastic human fibroblasts (Detroit 532 (DET)). In UMR 106–01, cellular [Pi] was weakly dependent on extracellular [Pi] and higher than expected from passive transport alone. [32Pi]‐uptake was inhibited by Na deprivation, but paradoxically increased on K deprivation. With Na, 87 per cent of cellular 32P was found in organic phosphorus pools after only 5 min. Na deprivation also decreased cellular [Pi], in both UMR 106–01 and DET, but the decrease was smaller than that in [32Pi]‐uptake. Ouabain decreased [32Pi]‐uptake and cellular [Pi] in DET, but not in UMR 106–01. Regulation of cellular [Pi] is therefore at least partly dependent on Na/Pi co‐transport, but this does not seem to be an exclusive property of osteoblasts.
Calciotropic hormones raise the chemically detectable [Pi] in UMR 106–06 osteoblast‐like cells
Authors: Ahmado Abdulla , Khouja Hamed I. , Kemp Graham J. , Guilland‐Cumming Diane F. , Russell R. Graham G. , Bevington Alan 
Published: May 2005

AbstractUptake of orthophosphate (Pi) by osteoblast‐like cells is known to be stimulated by parathyroid hormone (PTH), but effects on intracellular [Pi] have not been investigated. Here we show in rat osteoblast‐like cells (UMR 106‐06) that PTH (10−11 to 10−7 M) increases both 32Pi uptake and cellular [Pi] by up to 50 per cent. 1,25 Dihydroxyvitamin D3 (1,25D) (10−12 to 10−6 M) and salmon calcitonin (CT) (10−12 to 10−6 g ml−1) also increased cellular [Pi] (by up to 60 per cent), but the percentage increases in total cellular 32Pi uptake were smaller. The effects of 1,25D were transient (observable at 80 min and 6 h but not 24 h), and were also observed with 24,25 dihydroxy‐ and 25 hydroxyvitamin D3. Transient degradation of organic phosphorus pools to Pi might contribute to this increased [Pi]. These pools remain to be identified but were not shown to be phospholipids. Foetal bovine serum also affected cellular [Pi]. Care is therefore needed in distinguishing direct hormonal effects on cellular [Pi] from indirect effects arising from changes in the rate of cell growth.
The effects of tunicamycin on the metabolism of acetylated low density lipoproteins
Authors: Armstrong David P. , White David A. 
Published: May 2005

AbstractThe effect of tunicamycin (TM) on the metabolism of acetylated low‐density lipoprotein (AcLDL) was examined to determine whether N‐linked glycosylation is required for the proper function of the AcLDL pathway. Proteolytic degradation of [125I]‐AcLDL was increased twofold in the presence of TM. This did not occur via an increase in total lysosomal enzyme activity or extracellular proteolysis; rather, the rate of uptake of [125I]‐AcLDL was increased. The enhanced degradation of AcLDL did not lead to a commensurate increase in the rate of synthesis of cholesteryl oleate. Conversely, the rate of cholesterol esterification was reduced in the presence of TM. The uptake of [125I]‐AcLDL was more sensitive to inhibition by chloroquine in TM‐treated cells. However, the presence of TM did not affect the ability of chloroquine to inhibit constitutive recycling of AcLDL binding sites. These results suggest that N‐linked glycosylation may be involved in the regulation of AcLDL metabolism in J774 cells.
Extracellular calcium modulates proliferation of factor dependent hemopoietic cells
Authors: Martínez Jorge , Santibañez Juan F. 
Published: May 2005

AbstractBlockade of the calcium channel inhibited, in a dose‐dependent manner, the proliferation of the IL‐3 dependent FDCP‐1 cell line and normal murine bone marrow cells. Similar results were obtained by lowering the amount of extracellular calcium using specific chelators or a calcium‐free medium. These results suggest that factor‐dependent cell proliferation is highly sensitive to fluctuations in the concentration of extracellular calcium.
Adaptation to phosphate deprivation in osteoblast‐like cells
Authors: Ha Richard , Steenbergen Dawn K. , Kempson Stephen A. 
Published: May 2005

AbstractThe rat osteosarcoma cell line UMR‐106–01 has an osteoblast‐like phenotype. When grown in monolyer culture these cells transport inroganic phosphate and L‐alanine via Na+‐dependent transport systems. Exposure of these cells to a low phosphate medium for 4 h produced a 60–70 per cent increase in Na+‐dependent phosphate uptake compared to control cells maintained in medium with a normal phosphate concentration. In contrast, Na+‐dependent alanine uptake and Na+‐independent phosphate uptake were not changed during phosphate deprivation. The increased phosphate uptake was due, in part, to an increased Vmax and was blocked completely by pretreatment with cycloheximide (70 μM). In these cells recovery of intracellular pH after acidification with NH4Cl is due primarily to the Na+/H+ exchange system. The rate of this recovery process, monitored with a pH sensitive indicator (BCECF), was decreased by more than 50 per cent in phosphate‐deprived cells compared to controls indicating that Na+/H+ exchange was inhibited during phosphate deprivation.
Enzymatic, metabolic and secretory perturbations in pancreatic islets of thyroidectomized rats
Authors: Sener Abdullah , Malaisse‐Lagae Francine , Gervy‐Decoster Christine , Malaisse Willy J. 
Published: May 2005

AbstractIn thyroidectomized rats, the activity of FAD‐linked glycerophosphate dehydrogenase was severely diminished in liver homogenates but not affected significantly in pancreatic islet homogenates, whilst the activity of 2‐ketoglutarate dehydrogenase was decreased modestly in both liver and islet homogenates. Likewise, in intact islets of thyroidectomized rats, the generation of3HOH from [2‐3H]glycerol was not decreased, and the ratio between oxidative and total glycolysis not significantly lower than in islets from sham‐operated rats, at least in the presence of a high concentration of D‐glucose. Nevertheless impaired oxidation of both D‐[3,4‐14C]glucose and D‐[6‐14C]glucose was observed in islets of thyroidectomized rats, the relative magnitude of such a decrease being more pronounced at a low than at a high D‐glucose concentration. Such metabolic anomalies coincided with a lower level of plasma insulin and a decreased output of insulin by islets incubated at low (2·8 mM), but not higher, concentrations of D‐glucose. It is concluded that hypothyroidism does not mimic the deficiency in islet FAD‐linked glycerophosphate dehydrogenase activity found in rats with inherited or acquired non‐insulin‐dependent diabetes.
Stability and optimization of canalicular function in hepatocyte couplets
Authors: Wilton Joanne C. , Coleman Roger , Lankester David J. , Chipman J. Kevin 
Published: May 2005

AbstractAn enriched preparation of rat hepatocyte couplets was obtaied by collagense perfusion and subsequent elutriation (> 85 per cent couplets and triplets; viability of over 95 per cent).Canalicular secretory activity (the ability to accumulate cholyl‐lysyl‐fluorescein, CLF) was first apparent after 2 h of culture at 37°C and was present in over 80 per cent of the total population after 5–6 h. This remained almost constant for at least 4 h in both elutriated and directly plated cells. Initial storage of freshly prepared couplets at 4°C for up to 6 h prior to incubation had no adverse effect upon secretory function.Reduction of canalicular secretory activity occurred at a concentration of the hepatotoxic agent menadione (IC50 17 μM) that was lower than that required to induce mild plasma‐membrane blebbing (IC50 43 μM).This study has optimized and characterized the canalicular secretory effectiveness and stability of an enriched preparation of hepatocyte couplets, and established the feasibility of studies of toxic agents on hepatobiliary function in a heterogeneous population of hepatocytes. In this preparation other biochemical parameters can be assessed, thus complementing previous techniques using individual couplets.
Differential effects of unsaturated fatty acids on phospholipid synthesis in human leukemia monocytic U937 cells
Authors: Chu Arthur J. , Nguyen Camtu T. , Moore John 
Published: May 2005

AbstractThe biosynthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in monocyte‐like leukemia U937 cells was monitored by adding [3H]choline, [14C]ethanolamine or [14C]glycerol to the culture media; incorporation into phospholipid (PL) increased with time. The effect of unsaturated fatty acids (UFA) on PC and PE synthesis was investigated by pretreating U937 cells for 72h with 10 μM 18:1 (n –9), 18:2 (n –6), 18:3 (n –3), 20:4 (n –6) and 20:5 (n –3). The UFA caused no alteration in cell growth, as evidenced by light microscopy and the incorporation of [3H]thymidine and [3H]leucine. Total cellular uptake of radioactive precursors remained unaffected by all the treatments. Pretreatment with 20:5 resulted in approximately 25 per cent reduction in the incorporation of [3H]choline into PL, while no significant effect was detected with the other UFAs. 18:3, 20:4 and 20:5 depressed the incorporation of [14C]ethanolamine into PL by 34 per cent, 28 per cent and 49 per cent respectively. However, there was no redistribution of label with any of the treatments. 18:3, 20:4 and 20:5 also antagonized the stimulatory effect of endotoxin (LPS) on PC and PE synthesis. In addition, the incorporation from [14C]glycerol into PC and PE was reduced by 18:3, 20:4 and 20:5.Although the PL composition of the cells remained essentially unaffected, our study shows that chronic treatment of U937 cells with n –3 PUFA (20:5) depressed PC and PE synthesis, and 18:3 and 20:4 also caused inhibition of PE synthesis.
Continuous column culture system for adherent cells
Authors: Ishihara Yoshimi , Ohkubo T. , Shimazaki H. , El Borai Nadia , Takano J. , Yamamura M. 
Published: May 2005

AbstractA new continuous column culture system for adherent cells was developed using beads. The beads were packed in a column and an appropriate medium was continuously passed through. The whole system was kept under closed conditions.L cells and C6 cells were cultured by this new system. The number of cells increased linearly up to 16 days and reached a maximum at around 18 days. As the heat production remained constant for 16 days, it can be concluded that cells grown in this system had identical characteristics. The final concentration of cells reached was 1.0 × 108ml−1. The cells could grow both in the upward and the downward direction.Advantages of this system are: (1) Cells can be recovered in their adherent form on the beads; (2) cells can easily be collected from the column by trypsinization, and (3) cells remaining in the column after trypsinization can grow again.
Characterization of γ‐glutamyltranspeptidase in the liver of the frog: 1. Comparison to the rat liver enzyme
Authors: Sulakhe‐Hemmings Susan J. , Xing Hongmei 
Published: May 2005

AbstractThe characteristics of the enzyme γ‐glutamyltranspeptidase were determined in frog liver and compared to those of the rat. In Rana pipiens, tissue distribution studies indicated the order of activity to be: kidney >>> liver >> nerve > egg > lung > heart > skeletal muscle in homogenates. In the Rana pipiens relative to the Fischer 344 rat, the activity of the liver enzyme was somewhat greater (1·8‐fold) and the kidney enzyme substantially less (25‐fold). Frog liver γ‐glutamyltranspeptidase displayed strain‐dependent differences in activity with Rana pipiens and Rana sylvatica exhibiting comparable activities and Xenopus laevis exhibiting 20‐fold lower activities. No influence of sex was apparent in Rana pipiens in contrast to the sex dependent differences observed in the Fischer 344 rat: ♀ : ♂ = 7:1. In homogenates and plasma membrane fractions of Rana pipiens, Xenopus laevis and the Fischer 344 rat, high, and comparable relative specific activities, were observed, 8–11, coupled with protein yields of 2·2–2·5 per cent indicating the enzyme to be plasma membrane bound and associated with the sinusoidal surface of the liver cell. Both the frog Rana pipiens and Xenopus laevis and Fischer 344 rat liver plasma membrane enzymes displayed comparable temperature‐induced activation (1·51–1·74‐fold) but with a peak for the frogs at 60°C and for the rat at 50°C. Both Acivicin and maleate inhibited the liver plasma membrane γ‐glutamyltranspeptidase of both Rana pipiens and the Fischer 344 rat, but the frog enzyme was less sensitive (89 per cent decrease versus 97 per cent decrease) to 150 μM Acivicin and more sensitive (65 per cent decrease versus 35 per cent decrease at 150 mM maleate) to maleate. Kinetic studies indicated that the liver plasma membrane enzyme from Rana pipiens had a Km of 0·61 mM and Vmax of 55·6 nmol mg−1 min−1 and that from the Fischer 344 rat had a Km of 3·57 mM and Vmax of 71·4 nmol mg−1 min−1.