bluuebunny/crossref_metadata_2025 huggingface dataset:
Inositol lipids in friend erythroleukemia cells: Evidence for changes in nuclear metabolism after differntiation
Authors: Capitani S. , Billi A. M. , Bertagnolo V. , Previati M. , Mazzoni M. , Neri L. M. , Manzoli F. A.
Published: May 2005

The incorporation of 32Pi into phospholipids was studied in Friend erythroleukemia cells either induced or not to erythroid differentiation with 4 mM hexamethylenebisacetamide (HMBA). The effect of the differentiating agent on the recovery of radiolabelled phospholipids was compared in whole cells, isolated nuclei and nuclear matrix after in vivo labelling for 1 hr. The procedure employed for the isolation of nuclei was demonstrated to allow only negligible lipid redistribution caused by cell manipulations. Among the lipids extractable from nuclei, acidic phospholipids, and particularly polyphosphoinositides, were more represented than in whole cells, while small differences were found in the other phospholipid classes examined. The comparison between the uninduced and induced condition showed that the relative amounts of nuclear inositol lipids were modified by HMBA treatment of the cells, with a decreased recovery of phosphatidylinositol 4,5 bisphosphate.

These results indicate that phosphatidylinositol and its phosphorylation products synthesized in vivo show a different metabolism in nuclei and whole cells. They appear to be tightly bound nuclear components, also present in membrane‐deprived nuclei and nuclear matrix, and are probably related to the nuclear events involved in erythroid differentiation.
Effects of rGM‐CSF on leukemia cell proliferation and on the incorporation of cytosine arabinoside into DNA
Authors: Gao Xue Zhi , Yin Moying , Wang Zique , Raza Azra , Preisler Harvey D.
Published: May 2005

In vitro studies of the effects of recombinant granulocyte macrophage‐colony stimulating factor (rGM‐CSF) on freshly obtained human leukemia cells were conducted to determine if there is a relationship between the effects of this growth factor on the proliferative characteristics of leukemia cells and on their incorporation of cytosine arabinoside (araC) into DNA. While rGM‐CSF was found to be able to stimulate both leukemia cell proliferation and araC incorporation, for individual leukemia specimens there was no consistent relationship among these effects. In some specimens proliferation was stimulated without an increase in araC incorporation. The reverse was also observed. These studies demonstrate the difficulty in identifying assays capable of predicting the clinical effects of growth factors on leukemia cells in patients since the effect in vitro vary with the assay.
Experimental studies on the mechanism of action of 4‐Hydroxy‐2,3‐trans‐nonenal, a lipid peroxidation product displaying chemotactic activity toward rat neutrophils
Authors: Rossi Maria Armida , Curzio Marina , Mauro Clelia Di , Fidale Fulvio , Garramone Andrea , Esterbauer Hermann , Torrielli Mario , Dianzani Mario Umberto
Published: May 2005

The effects of 4‐hydroxy‐2,3‐trans‐nonenal (HNE) and nonanal on the activity of phosphoinositide‐specific phospholipase C of rat neutrophils have been studied in parallel with their action on neutrophil oriented migration. Concentrations of HNE ranging from 10−7 to 10−5 M significantly stimulated the oriented migration of rat polymorphonuclear leukocytes. HNE stimulated both the basal and GTP gamma S‐induced phospholipase C activity when used at concentrations between 10−8 and 10−6 M. Nonanal was devoid both of chemotactic activity and of any action on phospholipase C activity. The effect of GTP gamma S on the stimulation of phospholipase C induced by HNE was higher when the lowest dose of the aldehyde was used; the finding of an additive effect between 10−8 M HNE and 2 × 10−5 M GTP gamma S suggests that the two compounds may share a final common pathway of action.

These results suggest that the chemotactic activity of HNE might be mediated, like that of other more well‐known chemoattractants, by the stimulation of phosphoinositide‐specific phospholipase C.
Tellurite effect on ATPase activity of contractile membrane protein
Authors: Mirčevová Libuše , Kodíček Milan
Published: May 2005

The effect of tellurite on ATPase activity of the contractile membrane protein in human erythrocytes was studied. Tellurite, even at a concentration of 0·01 mM, inhibited 25 per cent of the saponin‐stimulated ATPase activity of the contractile membrane protein; the inhibition increased with increasing tellurite concentration, and was reversible. On the other hand, in erythrocytes preincubated with tellurite, the ATPase activity of the membrane contractile protein, non‐stimulated by saponin, increased, and the increase was further enhanced by washing the erythrocytes. The behaviour is analogous to the tellurite effect on erythrocyte morphology: incubation of erythrocytes with tellurite caused morphological changes which were more pronounced after removing the tellurite by washing. The complex effect of tellurite is discussed.
The effect of ligands on the uptake of iron by cells in culture
Authors: Jonas S. K. , Riley P. A.
Published: May 2005

Uptake of iron by a mammalian epithelial cell line (CNCM I‐221) was shown to be dependent on the nature of the iron complex. Iron uptake was demonstrated by cytochemical staining and determination of redox‐reactive iron in cell lysates. Three classes of ligands were investigated: (i) low molecular weight hydrophilic compounds, represented by ethylenediamine‐tetraacetic acid (EDTA) and other charged ligands such as adenosine phosphates (ATP, ADP, AMP) and diethylenetriaminepentaacetic acid (DTPA), (2) low‐molecular weight lipophilic ligands such as 8‐hydroxyquinoline (8‐HQ) and (3) a high molecular mass ligand, dextran. Iron complexed to 8‐HQ accumulated intracellularly, the uptake rate of iron being 4·16 fmoles cell−1 h−1 of exposure at 37°C or 3·86 fmoles cell−1 h−1 at 4°C. Iron‐dextran was endocytosed and retained in phagosomes. The uptake rate of iron following exposure to iron dextrans was found to be 5°6 fmoles cell−1 h−1 of exposure at 37°C. In contrast to iron/8‐HQ, uptake of iron dextran by cells was inhibited at 4°C. Iron complexed to low molecular weight hydrophilic ligands was not taken up by cells.

Cytotoxicity was measured by reduction of plating efficiency or tritiated thymidine incorporation. These tests showed that toxic effects of added iron were demonstrable only in cells exposed to the complex with 8‐HQ.
Phorbol myristate acetate inhibits phosphoinositol lipid‐specific phospholipase C activity via protien kinase C activation in conditions inducing differentiation in HL‐60 cells
Authors: Cost Hélène , Barreau Patricia , Basset Michèle , Le Peuch Christian , Geny Blandine
Published: May 2005

We have studied, in streptolysin O‐permeabilized HL‐60 cell membrane preparations, the effects of phorbol 12‐myristate 13‐acetate (PMA) on polyphosphoinositide‐specific phospholipase C (PLC) activity and on terminal differentiation towards macrophagic‐like cells. We showed that terminal differentiation was induced when differentiating concentrations of the drug were present for only 1–2 h in the culture medium. Conditions inducing differentiation also inhibited PLC activity for a long lasting period (at least 5 h). When terminal differentiation affected only part of the cell population, inhibition of phospholipase C activity was found to be less marked and reversible over the period studied. Moreover in experiments done in an HL‐60 clone resistant to PMA, no inhibition of PLC activity was provoked by this tumour promotor.

In order to study the involvement of protein kinase C in this process, we measured modifications of PLC activity by PMA in the presence of two different protein kinase C inhibitors, staurosporine and H‐7. They both prevented the inhibition of PLC activity by PMA indicating that this inhibition is likely to be related to the effect of PMA on protein kinase C activity. This was also confirmed by the fact that active protein kinase C, by itself, was able to decrease PLC activity when added to membrane preparations or to streptolysin O‐permeabilized control HL‐60 cells.

These results indicate that PMA acts in inhibiting phospholipase C activity through its effect on protein kinase C activation and/or on protein kinase C translocation to the plasma membrane and that terminal differentiation, might be related to changes in both protein kinase C and PLC activities.
Inhibitory action of melatonin and structurally related compounds on testosterone production by mouse leydig cells in vitro
Authors: Persengiev S. , Kehajova J.
Published: May 2005

The possible effect of melatinin, 5‐methoxytryptamine, 5‐methoxytryptophol, 6‐chloromelatonin and 2‐iodomelatonin on testosterone production by Leydig cells in vitro was investigated. The ability of individual indoles to inhibit testosterone production was found to depend on the concentration used. The relative inhibitory potency of the compounds tested was: 6‐chloromelationin > 2‐iodomelatonin > melatonin > 5‐methoxytryptamine > 5‐methoxytryptophol. The results revealed that natural indoles which are synthesized in the pineal gland and their halogenized derivatives are capable of influencing directly testosterone production by Leydig cells. Also, these results demonstrated that melatonin exerts its remarkable antigonadotrophic effects, at least in part, through the direct decrease of testosterone production. Moreover, 6‐chloromelationin and 2‐iodomelatonin, which are reported to inhibit melatonin binding to target tissues, possess properties of biological melatonin analogues under the conditions of the model system used.
Protamine‐induced permeability changes in the neutrophil plasma membrane as the basis of activation of exocytosis
Authors: Elferink J. G. R.
Published: May 2005

Protamine induces a gradual change in plasma membrane permeability in rabbit neutrophils, which is evident from the increase of indol fluorescence, and the leakage of quin2 from quin2‐loaded neutrophils. The influx of extracellular Ca2+ into the neutrophil provides an explanation for exocytosis which occurs in the presence of Ca2+ and protamine. The dependence of exocytosis on Ca2+ concentration follows the same pattern as is observed in neutrophils permeabilized by other means. In the absence of Ca2+, and in the presence of protamine, La3+ has an activating effect on exocytosis. At higher concentrations La3+ inhibits exocytosis that occurs in the presence of Ca2+ and protamine, as do some other metal ions. The resemblance between the membrane effects of a number of toxins, as reported in literature, and protamine‐induced membrane damage suggests that they occur via the same mechanism.
Phospholipid N‐methylation in diabetic erythrocytes: Effects on membrane Na+, K+ ATPase activity
Authors: Kowluru Anjaneyulu , Kowluru Renu A.
Published: May 2005

Phospholipid methylation was quantified in non‐diabetic and streptozotocin diabetic rat erythrocytes. While the total mass of methylated lipids remained the same in both groups, the relative abundance of individual methylated lipid species differed significantly in diabetic erythrocytes. Moreover, incubation of erythrocytes membranes with S‐adenosyl methionine, a substrate for methyl transferases, not only increased membrane lipid methylation but also decreased Na+, K+ ATPase activity significantly. These results suggest that phospholipid methylation may cause the observed depression of erythrocyte Na+, K+ ATPase activity in diabetes and could contribute to the altered rheology of erythrocytes in diabetes.
Cloturin: Effect on energy‐producing processes in ehrlich ascites and P388 murine leukaemia cells
Authors: Miko Milan , Krepelka Jiri , Melka Milan
Published: May 2005

The main purpose of the present investigation was to study the effect of cloturin on aerobic glycolysis, endogenous and exogenous respiration and the level of ATP in both Ehrlich ascites carcinoma (EAC) and P388 murine leukaemia cells incubated in vitro. Also its effect on the level of total (T‐SH) and non‐protein (NP‐SH) thiol groups was investigated. A significant inhibition of aerobic glycolysis was found only in P388 cells after 60 min of cloturin action. Cloturin inhibited both endogenous and exogenous respiration of EAC with succinate as substrate. Cloturin decreased the level of ATP after 2 h incubation in both types of tumour cell. The level of NP–SH was decreased more than that of T‐SH in both types of cell.
Effects of hyperthermia on intracellular calcium concentration and responses of cancerous mammary cells in culture
Authors: Furukawa Masahiko , Enomoto Koh‐Ichi , Kato Hirokazu , Ishida Tetsuya , Maeno Takashi
Published: May 2005

Effects of hyperthermia on the intracellular calcium concentration (Cai) of an established mouse breast cancer cell line, MMT060562, were studied using fura‐2 fluorescence microscopy and the whole‐cell clamp technique. A sudden change of temperature from 37 to 45°C induced a transient increase in the fluorescence ratio permeability of the cell membrane and inward current. Deletion of extracellular calcium abolished the fluorescence ratio response to the rise in temperture. Cai of some cells increased after hyperthermia treatment at 44–48°C for 20 min, but the average increase of Cai was negligible. After hyperthermia treatment, spontaneous oscillation of Cai, chemical responses to ATP and bradykinin and the mechanically‐induced spreading reponse diminished. However, the mechanically induced increase of Cai within the stimulated cell remained even after hyperthermia treatment. Suppression of the ATP‐induced Cai response recovered to about half the original level within 12 h. Blockage of protein synthesis with cycloheximide (100 μM) had no effect on the recovery. The D‐myo‐inositol 1,4,5‐triphosphate (IP3)‐dependent increase of Cai remained intact even after hyperthermia treatment. It is concluded that hyperthermia treatment increases both the permeability of the cell membrane and Cai, but decreases the sensitivity of cells to ATP and bradykinin, presumably due to modification of the signal transduction mechanism.
Studies on the ATPase of Bacillus cereus
Authors: Higuti I. H. , Stencel M. , Nascimento K. H. , Nascimento A. J.
Published: May 2005

The membrane ATPase (EC 3.6.1.3) of Bacillus cereus was solubilized by a ‘shock‐wash’ process and purified. The non‐specific phosphatase contaminant was separated by glycerol density gradient centrifugation. The optimum temperature was 39·5°C and the pH optimum at 7·5. On SDS‐polyacrylamide gel electrophoresis two classes of subunits were observed in equal proportions with molecular weights of 70 K and 83 K. The effect of various compounds on the enzymatic activity was studied. The enzyme was insensitive to NaN3, oligomycin and to divalent cations, but was inhibited by citrate and oxalate.
Effect of inhibitors and activators of tyrosine kinase on insulin imprinting in Tetrahymena
Authors: Kovács P. , Csaba G.
Published: May 2005

Primary exposure of Tetrahymena cells to insulin gave rise to hormonal (insulin) imprinting in the offspring generations, as judged from the increase in binding upon reexposure to insulin. Vanadate mimicked the action of insulin, inasmuch as it also induced imprinting for insulin, whereas the other tyrosine kinase activator tested, namely H2O2, had no such effect. However, combined treatment with vanadate + H2O2 + insulin induced a more pronounced imprinting for insulin than either insulin or vanadate on their own. The tyrosine kinase inhibitor genistein, a plant flavonoid, did not change the value for insulin binding significantly relative to the control immediately after exposure, but increased it slightly in the offspring generations after 24 h at high dilution. Upon combination with insulin, 10−4 M genistein inhibited imprinting by insulin. These experimental observations suggest that there may be a key role for tyrosine kinase activity in the mechanism (development) of imprinting.
An in situ study of beta‐glucosidase activity in normal and gaucher fibroblasts with fluorogenic probes
Authors: Kohen Elli , Kohen Cahide , Hirschberg Joseph G. , Santus Rene , Grabowski Gregory , Mangel Walter , Gatt Shimon , Prince Jeffrey
Published: May 2005

Beta‐glucosidase activity was evaluated in situ by means of fluorogenic probes in normal human fibroblasts and fibroblasts from homozygous carriers of the Gaucher trait. Probe internalization, targeting to lysosomes and post‐cleavage probe retention were the primary concerns. Internalization and targeting were attempted by in situ photosensitized labilization of lysosomal membranes, lysosomotropic detergents and the use of low density lipid (LDL) or the receptor ligand apolipoprotein E (ApoE). Post‐cleavage increase of fluorescence with fluoresceinyl (bis) betaglucopyranoside was appreciably above the rather large pre‐cleavage emission. In cells incubated overnight with nonylumbelliferylbetaglucoside (UG9) in the presence of bovine serum albumin and in the absence of ApoE, the probe was dealt with as a cytotoxic agent, accumulating in a paranuclear cap, most likely comprising elements of the endoplasmic reticulum (ER) and Golgi apparatus. Targeting of UG9 to lysosomes occurred within 1 to 3 h of preincubation in the presence of ApoE. There was some evidence of specificity, as Gaucher fibroblasts exhibited weaker cleavage of UG9 (by 50 per cent or more) compared to normal fibroblasts, but in the Gaucher cells there was some residual beta‐glucosidase activity. Cleavage of UG9 was nearly totally suppressed in Gaucher cells treated with the beta‐glucosidase inhibitor, conduritol B epoxide, for 24 h to 7 days. Suppression in the control fibroblasts was evident but to a lesser degree. The in situ method of fluorogenic assay established for beta‐glucosidase deficiency, is in principle applicable to enzyme deficiencies in other lysosomal storage diseases, or to evaluate enhanced enzyme activity following gene therapy.
Dual effects of formylpeptides on the adhesion of endotoxin‐primed human neutrophils
Authors: Bellavite P. , Chirumbolo S. , Lippi G. , Andrioli G. , Bonazzi L. , Ferro I.
Published: May 2005

Neutrophils, treated with sequential additions of bacterial products such as endotoxin (E. Coli lipopolysaccharide, LPS) and the chemotactic peptide N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP), undergo to metabolic activation and express membrane‐anchoring proteins that promote adhesion to serum‐coated culture wells. By investigating the dose–response relationships of these phenomena, we have found that: (a) resting neutrophils do not produce a significant amount of superoxide (O) and show only minimal adhesion to serum‐coated plastic surfaces; (b) fully activatory doeses (> 5 × 10−8 M) of fMLP induce the release of O and a significant increase of the cell adhesion; (c) pretreatment of the cells for 1 h with LPS augments cell adhesion to serum‐coated culture wells in the absence of further stimulation and primes the neutrophils to enhanced fMLP‐dependent O release; (d) addition of low, substimulatory doses of fMLP (from 10−10 M to 5 × 10−9 M) inhibits and reverses the adhesion of LPS‐treated cells, (e) high fMLP doses (> 10−7 M) are additive to LPS in promoting adhesion. Phorbol‐myristate acetate (> 10−9M) increased adhesion in both normal and LPS‐treated neutrophils, but low doses of this stimulant did not inhibit adhesion. Low doses (10−9 M) of fMLP increased intracellular cyclic AMP in both normal and LPS‐treated neutrophils, suggesting that stimulus‐induced rises in cAMP may be the negative signal responsible for down‐modulation of adhesion. Low (5 × 10−9 M) and high (5 × 10−7 M) fMLP doses induced the same increase of expression of CD11/CD18 integrins, indicating that the inhibition of adhesion caused by low doses is not due to quantitative down‐regulation of integrins. These findings may provide an in vitro model of the complex biological events involved in the regulation of neutrophil adhesion.
Characterization of over‐expressed alkaline phosphodiesterase I in tumour‐derived fibroblasts from patients with neurofibromatosis
Authors: Maruyama Etsuko , Takashima Sachio
Published: May 2005

Alkaline phosphodiesterase I from cultured fibroblasts from patients with neurofibromatosis was partially purified and characterized following extraction with Triton X‐100, and fractionation with high‐performance liquid chromatography. Some properties were compared with the enzyme extracted from normal‐appearing fibroblasts. The isoelectric points of both the tumour and normal‐appearing cell enzymes were 6·0. The enzyme required Zn2+ for its activity, was heat labile, and nicked superhelical covalently closed circular ϕX174 DNA. The activity was inhibited by GTP, DTT and EDTA. The native molecular weight of alkaline phosphodiesterase I was determined to be 430 000. No differences were found in properties of the tumour‐derived and normal cell enzymes. On purification it was observed that the peak pattern of enzyme activity corresponded to that of 125 kDa protein, which was more abundant upon SDS‐PAGE analysis in tumour cells than in normal cells. The most active fraction of isoelectric focusing, which was performed using disulfide cross‐linked polyacrylamide gel, was used to produce an antibody. The bands of 125, 60 and 40 kDa were immuno‐stained in tumour cell preparation. These results indicate that alkaline phosphodiesterase I, of which the molecular weight is probably 125 kDa, is over‐expressed in tumour‐derived fibroblasts from neurofibromatosis patients.
Stimulation of HeLa cell growth by physiological concentrations of 4‐hydroxynonenal
Authors: Zarkovic Neven , Ilic Zoran , Jurin Mislav , Schaur R. Jörg , Puhl Herbert , Esterbauer Hermann
Published: May 2005

The aim of this study was to analyze the growth response of HeLa cells over a prolonged period of time to a single exposure of physiological and supraphysiological concentrations of 4‐hydroxynonenal (HNE), a peroxidation product of omega‐6‐polyunsaturated fatty acids. Furthermore, the growth modulating effect of serum factors, particularly albumin, on the growth pattern was examined. The effects of HNE on the growth rate and viability of the cells, as well as on the incorporation of labelled amino acids were monitored daily over a period of four days. Fetal calf serum not only had a growth stimualting effect but also modulated the action of HNE. In neither respect was albumin able to substitute for serum indicating that the influence of serum was not exerted via an albumin–HNE conjugate.

HNE had a clear dose‐dependent effect and a distinction could be made between a supraphysiological concentration (100 μM), which was primarily cytotoxic and a physiological range (below 10 μM) which showed growth modulatory effects. These effects consisted of a transient inhibition in the initial phase of the cell growth, which under optimal conditions (in presence of serum) was followed by a period of increased proliferation, compared to untreated control cultures, until confluence was attained. It is suggested that HNE is not only a toxic product of lipid peroxidation, but a physiological growth regulating factor as well.
On the association of DNA primase activity with the nuclear matrix in HeLa S3 cells
Authors: Martelli Alberto M.
Published: May 2005

We have reinvestigated the association of DNA primase activity with the nuclear matrix prepared from exponentially growing HeLa S3 cells. We have found that 25–30 per cent of the nuclear primase activity resists extraction with 2 M NaCl and digestion with Dnase I. Unlike previous investigations, done with the same cell line, the results showed that nuclear matrix‐bound DNA primase activity represented less than 10 per cent of the total cell activity. Association of high levels of primase activity with the nuclear matrix was strictly dependent on a 37°C incubation of isolated nuclei prior to subfractionation. Evidence was obtained that the method used for preparing nuclei can have a dramatic effect on the amount of primase activity which is recovered both in the postnuclear supernatant and in isolated nuclei, thus seriously affecting the interpretation of the results about the quantity of DNA primase activity bound to the nuclear matrix.
Enalapril maleate affects 2‐oxoglutarate metabolism in mitochondria from the rat kidney cortex
Authors: Merlin Ma. Eliane , Campello Annibal P. , Klüppel Ma. Lúcia W.
Published: May 2005

Enalapril maleate (EM) is the salt of N‐{(S)‐1‐(ethoxycarbonyl)‐3‐phenylpropyl}‐L‐alanyl‐L‐proline, used therapeutically as an anti‐hypertensive agent. The effects of EM on some aspects of the energy metabolism and membrane properties of mitochondria from rat liver and kidney cortex were studied, but only the latter were significantly affected. With 0·8 mM of EM and 2‐oxoglutarate as oxidizable substrate for isolated mitochondria from rat kidney cortex, the findings were: (a) inhibition of the respiratory rate in state III (37 per cent) and decrease (45 per cent) in respiratory control ratio (RCR), with only one addition of ADP; (b) reinforcement of the inhibition when a second addition of ADP was made; (c) no significant effect either on the rate of respiration in state IV or on the ADP/O ratio; (d) no effect on the ATPase activity of mitochondria from liver or kidney cortex; (e) inhibition of the transmembrane potential (Δψ) after a second addition of ADP; (f) inhibition of the 2‐oxoglutarate dehydrogenase complex. It is suggested that in kidney mitochondria, EM interferes in the gluconeogenesis dependence of at least five substrates: 2‐oxoglutarate, glutamine, glutamate, lactate, and pyruvate. Also EM may inhibit Na+/H+ exchange causing natriuresis.
Mechanism by which ethanol inhibits phosphatidylcholine biosynthesis in human leukemic monocyte‐like U937 cells
Authors: Chu Arthur J.
Published: May 2005

A previous study showing that ethanol (ETOH) blocked [3H]choline incorporation into phosphatidylcholine (PC) suggested an inhibition of PC biosynthesis in human leukemic monocyte‐like U937 cells. The mechanism of the inhibitory action of ETOH was investigated. Cells were pulsed with [3H]choline for 30 min and chased in the presence or absence of ETOH for up to 6 h. PC biosynthesis was inhibited drastically within 1 h after exposure to ETOH which increased intracellular cAMP appreciably. After a 3‐h treatment, ETOH significantly inhibited both choline kinase (CK) and the cytosolic CTP: cholinephosphate cytidylyltransferase (CT). The inactivated CT was no longer stimulated by exogenous phosphatidylglycerol (PG). There was no evidence for redistribution of CT activity between cytosol and microsomes. When cells were exposed to 8‐Bromo‐cAMP ranging from 100 to 300 μM, PC biosynthesis remained unaffected despite the drastically elevated cAMP. These results seem to suggest that the raised cAMP is not a prerequisite for the inhibition of PC biosynthesis in U937 cells. Following pretreatment with protein kinase inhibitors (H‐89 and K‐252a), PC biosynthesis was decreased significantly and the inhibitory effect of ETOH was potentiated. Taken together, our results suggest that the inhibition of PC biosynthesis and the inhibitory effect of ETOH are independent of the activation of cAMP‐dependent protein kinase. Unlike protein kinase inhibitors, pretreatment with tyrosine kinase inhibitors (erbstatin, genistein and tyrphostin 25) resulted in differential effects on PC biosynthesis and on the inhibitory action of ETOH. Genistein stimulated PC biosynthesis by 30 per cent as well as partially preventing /reversing the ETOH action, while tyrphostin 25 produced a synergistic inhibition. The relevance of tyrosine phosphorylation/dephosphorylation to the regulation of PC biosynthesis and ETOH action remains to be established.
Lidocaine inhibits choline uptake and phosphatidylcholine biosynthesis in human leukemic monocyte‐like U937 cells
Authors: Chu Arthur J. , Lee Jack M.
Published: May 2005

The effect of lidocaine on [3H]choline uptake and the incorporation of label into phosphatidylcholine (PC) in human monocyte‐like U937 cells was investigated. Lidocaine inhibited the rate of choline uptake in a dose‐dependent manner; at 3·2 mM it resulted in a drastic reduction, by as much as 65 per cent (n = 10; p < 0·0005) or 55 per cent (n = 10; p < 0·0006) in a 3‐ or 6‐h incubation, respectively. Lidocaine also decreased the rate of choline incorporation into PC in a dose‐dependent manner. At the highest dose, nearly 70 per cent or 45 per cent reduction was seen in a 3‐ or 6‐h incubation, respectively. Analysis of choline‐containing metabolites showed that the major label association with phosphocholine and PC was reduced to a similar extent which was also parallel to the inhibition of choline uptake. At 3·2 mM lidocaine, the reduction of choline uptake was shown to follow a competitive inhibition. In the case of [3H] choline incorporation into PC, the inhibitory pattern was shown to be of a mixed type. The pulse‐chase study dissecting the effect on choline metabolism from that on total choline uptake indicated that lidocaine exerted an additionally inhibitory effect on intracellular choline metabolism into PC. In a separate protocol in which the labelled cells were first allowed to be chased until 3H‐incorporation into PC reached a steady state, lidocaine no longer showed any effect. These results seem to exclude the possibility of enhanced PC breakdown and further suggest that the main inhibitory effect is on the CDP‐choline pathway for PC biosynthesis. After a 3‐h treatment, CTP: cholinephosphate cytidylyltransferase (CYT) in both the cytosolic and microsomal fractions was inhibited by approximately 20 per cent, while choline kinase (CK) and choline phosphotransferase (CPT) remain relatively unchanged. There was no evidence for translocation of CYT between cytosol and microsomes. Taken together, we have demonstrated a dual inhibitory function of lidocaine which inhibits PC biosynthesis in addition to its ability to block choline uptake profoundly in U937 cells.
Energy metabolism of reticulocytes: Two different sources of energy for Na+K+‐ATPase activity
Authors: Kostić Milosav. M. , Živković R. V.
Published: May 2005

Total energy production in rabbit reticulocytes amounted to 136·52 ± 6·50μmol ATP h−1ml−1 of reticulocytes: 88·3 per cent was provided by oxidative phosphorylation, whereas only 11·7 per cent by aerobic glycolysis. Na+K+‐ATPase accounted for 23 per cent, i.e. 27·65 ± 2·55μmol ATP h−1ml−1 of reticulocytes, in the overall energy consumption in reticulocytes of rabbits. Under basal conditions ATP for Na+K+‐ATPase activity was derived exclusively from oxidative phosphorylation. However, when the activity of Na+K+‐ATPase was increased due to the stimulation of adenylate cyclase by (−)‐isoprenaline, the additional energy required was provided by aerobic glycolysis. These results indicate that two different compartments, one cytosolic and the other mitochondrial, provide energy for Na+K+‐ATPase activity in reticulocytes.
Effect of omeprazole on secretion, synthesis and the gene expression of pepsinogen in the guinea pig stomach mucosa
Authors: Tsukada Shinko , Ichinose Masao , Kakei Nobuyuki , Tatematsu Masae , Tezuka Noriaki , Matsushima Masashi , Miki Kazumasa , Kurokawa Kiyoshi , Nozawa Masayuki , Kageyama Takashi , Takahashi Kenji , Fukamachi Hiroshi , Nakane Paul K.
Published: May 2005

The effects of omeprazole, a proton pump inhibitor, on gene expression, protein synthesis, intracellular storage and secretion of pepsinogen in guinea pig stomach were investigated.

After treatment with omeprazole for five days, acid and pepsinogen secretion into the gastric lumen was significantly reduced. Concomitant with this, there was an increase in intracellular pepsinogen as demonstrated by increased pepsin activity in the gastric mucosa, more intense immunohistochemical staining by antibodies specific of pepsinogen and accumulation of secretory granules in the cells producing pepsinogen. In these cells, the amount for pepsinogen mRNA was reduced as revealed by Northern blotting and in situ hybridization. Ultrastructurally the endoplasmic reticulum of these cells was poorly developed, the findings being consistent with a reduction in protein synthesis. It appears that omeprazole inhibits the secretion of pepsinogen, increasing the intracellular store and leading to the reduction in gene expression probably by a feedback mechanism and consequent reduction in pepsinogen synthesis. Since these changes were most evident in the acid‐secreting fundic gland mucosa, as compared with other mucosae secreting only pepsinogen, namely pyloric and duodenal mucosa, it appears probable that these changes are linked with omeprazole‐induced reduction in the acid secretion.
N‐3 but not N‐6 fatty acids reduce the expression of the combined adhesion and scavenger receptor CD36 in human monocytic cells
Authors: Pietsch A. , Weber C. , Goretzki M. , Weber P. C. , Lorenz R. L.
Published: May 2005

CD36, a multifunctional adhesion receptor e.g. for thrombospondin and collagen, as well as a scavenger receptor for oxidized low density lipoprotein, is expressed e.g. on platelets and monocytes. By this dual role it might be involved in early steps of atherosclerosis like the recruitment of monocytes and formation of foam cells. We therefore studied the effects of n‐3 fatty acids on CD36 expression in human monocytic cells. Incorporation of eicosapentaenoic acid (EPA, C20:5n‐3) and docosahexaenoic acid (DHA, C22:6n‐3) into cellular phospholipids resulted in a significant reduction of CD36 expression at the mRNA and protein level, whereas arachidonic acid (AA, C20: 4n‐6) and linoleic acid (LA, C18:2n‐6) tended to increase CD36 expression compared to the control. This specific down‐regulation of CD36 by n‐3 fatty acids in cells involved in the initiation and progression of atherogenesis and inflammation, represents a further mechanism that may contribute to the beneficial effects of n‐3 polyunsaturated fatty acids (PUFA) in these disorders.
Antimicrotubular effect of calvatic acid and of some related compounds
Authors: Gadoni Elena , Gabriel Ludovica , Olivero Antonella , Bocca Claudia , Miglietta Antonella
Published: May 2005

A structure–activity relationship has been established between calvatic acid and some related synthetic compounds, and their ability to inhibit GTP‐induced microtubular protein polymerization in vitro. These compounds were effective in a dose‐ and a time‐dependent manner. The most active drug was the p‐chloro substituted compound, which showed its inhibitory activity without any preincubation period, which the others needed. Since if cysteine was present, polymerization was no longer affected, an involvement of titratable –SH groups of tubulin could be suggested. In contrast, taxol‐induced polymerization was only slightly inhibited by these compounds, and colchicine‐binding activity was not generally impaired.
The effect of insulin on macrophage metabolism and function
Authors: Rosa Luís F. B. P. Costa , Safi Danilo A. , Cury Yara , Curi Rui
Published: May 2005

This study examined the effect of insulin on rat macrophage metabolism and function. The following parameters were studied: cell migration in response to thioglycollate and BCG stimuli, macrophage phagocytic capacity, H2O2 production, glucose and glutamine metabolism as indicated by the measurement of enzyme activities, the utilization of metabolites and production and oxidation of substrates. The results indicate that insulin: (1) did not affect cell migration in response to thioglycollate and BCG; (2) enhanced the phagocytic capacity of macrophages and the production of H2O2 by macrophages; (3) increased the metabolism of glucose and reduced that of glutaminase.
Inositolphosphoglycans and diacylglycerol are possible mediators in the glycogenic effect of GLP‐1(7‐36)amide in BC3H‐1 myocytes
Authors: Galera C. , Clemente F. , Alcántara A. , Trapote M. A. , Perea A. , López‐Delgado M. I. , Villanueva‐Peñacarrillo M. L. , Valverde I.
Published: May 2005

A potent glycogenic effect of GLP‐1(7‐36)amide has been found in rat hepatocytes and skeletal muscle, and specific receptors for this peptide, which do not seem to be associated with the adenylate cyclase—cAMP system, have been detected in these tissue membranes. On the other hand, inositolphosphoglycan molecules (IPGs) have been implicated as second messengers of the action of insulin. In this work, we have found, in differentiated BC3H‐1 myocytes, specific binding of [125I]GLP‐1(7‐36)amide, and a stimulatory effect of the peptide on glycogen synthesis, confirming the findings in rat skeletal muscle. Also, GLP‐1(7‐36)amide modulates the cell content of radiolabelled glycosylphosphatidylinositols (GPIs) and increases the production of diacylglycerol (DAG), in the same manner as insulin acts, indicating hydrolysis of GPIs and an immediate and short‐lived generation of IPGs. Thus, IPGs and DAG could be mediators in the glycogenic action of GLP‐1(7‐36)amide in skeletal muscle.
Reversal of doxorubicin resistance by the amiloride analogue EIPA in multidrug resistant human colon carcinoma cells
Authors: Pannocchia Antonella , Revelli Silvia , Tamponi Giacomo , Giorgianni Angelica , Todde Roberta , Bosia Amalia , Ghigo Dario
Published: May 2005

Although multidrug resistance (mdr) may arise through a variety of mechanisms, the most widely studied and accepted form is associated with an increased concentration of P‐glycoprotein (P‐gp), a 170kd protein found in the membrane fraction of a number of mammalian cells. Since mdr seems to be related to the ability of resistant cells to extrude drugs and the circumvention of mdr is supposed to be due to the restored ability to accumulate drugs, membrane has been regarded as the crucial site for such a regulation and an important role for membrane ion exchangers has been suggested. The aim of this work was to elucidate whether the Na+/H+ antiporter is involved in the mechanism of regulation and circumvention of mdr and if 5‐(N‐ethyl‐N‐isopropyl) amiloride (EIPA), a selective inhibitor of the Na+/H+ exchanger, can modulate the functional expression of the mdr phenotype. The effect of EIPA on doxorubicin (DX) resistant cells (LoVo/DX) obtained from a human colon adenocarcinoma cell line (LoVo) was studied. EIPA at concentrations ranging from 10 to 50 μM was able to increase the antibiotic cytotoxicity in the resistant Lovo/DX cells. The reversal of DX resistance paralleled an increase of the ability of the cells to accumulate the drug. Both drug loading and sensitivity to the inhibitory effect of DX on cell proliferation were restored by EIPA in a dose‐dependent way. These results suggest a new mechanism of mdr reversal and indicate that amiloride and its derivatives may be useful in reversing DX resistance and in enhancing the clinical effectiveness of chemotherapeutics.
Comparison of different In Vitro tests for evaluating immune reactivity
Authors: Pugliese A. , Comito G. , Cantamessa C. , Pollono A. M. , Savarino A.
Published: May 2005

We performed some in vitro tests for the detection of the immune state and compared the results. In particular we studied the production of various cytokines obtained by stimulating peripheral blood mononucleated cells (PBMC) with different inducers, using optimal and suboptimal doses. This was compared with the results of blastic transformation of lymphocytes, and with the evaluation of the capping effect of macrophages, and of the Multitest Mérieux. The correlation between the different investigations was generally good. This permits a simplification of the study of immune reactivity, selecting some of the tests proposed. The use of suboptimal doses of inducers improves the evaluation of very moderate deficits and supplies an in vitro model for the study of immunomodulant drugs.
Influence of culture conditions on monoamine oxidase A and B activity in rat astrocytes
Authors: Carlo Pia , del Rio Meris , Violani Elisabetta , Sciabà Luigi , Picotti Giovanni B.
Published: May 2005

Astroglial cells dispersed from newborn rat hemispheres were established in medium supplemented with 20 per cent fetal calf serum (FBS) and then grown to a confluent monolayer in the presence of 10 per cent FBS or charcoal‐stripped FBS (CS). Type 1 astrocytes were subcultured and either maintained under the same conditions of the primary cultures or converted to serum‐free chemically defined medium (CDM). No differences were found in either MAO A or MAO B activity of astrocytes grown in the presence of FBS or CS after 15 and 21 days in vitro (day 1 and 6 of subculture). In contrast, on day 21 both MAO A and MAO B activities were markedly higher in astrocytes subcultured in CDM compared with cells maintained in serum‐supplemented medium. This difference appeared to be due to increased number of enzyme molecules, since kinetic analysis showed an increase in Vmax of both MAO isoenzymes in serum‐free medium, but no change in Km. Consistently, the recovery of MAO A and MAO B activity after irreversible enzyme inhibition by clorgyline and deprenyl was faster in CDM than in FBS‐supplemented medium, indicating enhanced enzyme synthesis under serum‐free condition. Estimates of half‐lives for the recovery of MAO A and MAO B activity indicated that, under both culture conditions, type A activity had a higher turnover rate than type B. The effect of CDM on astrocyte MAO does not appear to be due to selection of a subpopulation of cells, but rather linked to a morphological change (differentiation) with increased synthesis of both MAO isoenzymes.
Effects of methylglyoxal on platelet hydrogen peroxide accumulation, aggregation and release reaction
Authors: Leoncini Giuliana , Poggi Massimiliano
Published: May 2005

Methylglyoxal generates a slight increase in the basal level of hydrogen peroxide in platelets. The oxidation effect of methylglyoxal significantly potentiated by thrombin, depends on both the ketoaldehyde and the agonist concentrations. A further significant increase in hydrogen peroxide accumulation was obtained in platelets pretreated with the alkylating agent N‐ethylmaleimide which depletes GSH and blocks glutathione peroxidase. Resting platelets completely transform the ketoaldehyde into D(−)lactate, whereas stimulated platelets transform about 10–15 per cent of the metabolized methylglyoxal into D(−)lactate. The metabolic modifications generated by methylglyoxal such as the GSH depletion and hydrogen peroxide accumulation induce modifications in platelet function. Methylglyoxal inhibits platelet aggregation induced by several agonists and ATP release induced by thrombin.
Hepatic and pancreatic effects of polyenoylphosphatidylcholine in rats with alloxan‐induced diabetes
Authors: Buko Vyacheslav , Lukivskaya Oxana , Nikitin Vasily , Tarasov Yury , Zavodnik Lev , Borodinsky Alexandr , Gorenshtein Bronislava , Janz Barbara , Gundermann Karl‐Josef , Schumacher Rainer
Published: May 2005

Polyenoylphosphatidylcholine (PPC: 100 or 300 mg kg−1 b.w., by gastric intubation for 30 days) produced a clearcut protection of the liver of rats treated with alloxan (150 mg kg−1 b.w., i.p.). The liver of rats treated with alloxan was characterized by hydropic dystrophy and lymphocytic infiltrations. Treatment with alloxan increased serum γ‐GT and ALAT activities. The liver structure of rats treated with PPC did not differ from the liver of control animals. PPC normalized the biochemical abnormalities caused by the diabetes. The number of pancreatic islets and β/α; cell ratio decreased in the diabetic rats. A number of β‐cells in this group did not contain granules. PPC prevented the decrease in the number of islets and the β/α; cell ratio in the pancreas of the diabetic rats. The intensity of staining of β‐cell granules in the pancreas of PPC‐treated rats had a position intermediate between the control and diabetic groups. Alloxan increased the blood glucose content where treatment with PPC decreased this. The results suggest that PPC acts as a cytoprotector in the liver and pancreas of rats with experimental diabetes induced by alloxan.
Cytotoxicity of two new ribosome‐inactivating proteins, cinnamomin and camphorin, to carcinoma cells
Authors: Ling Jun , Liu Wang‐Yi
Published: May 2005

Cinnamomin (two‐chain) and camphorin (single‐chain), two novel ribosome‐inactivating proteins (RIPs) purified from the seeds of Cinnamomum camphora, produced inhibitory effects in cultured carcinoma cells. The IC50 of cinnamomin to the human hepatocarcinoma cell‐line 7721 and the melanoma cell‐line M21 were 18·8 nmol and 11·7 nmol respectively. The IC50 of camphorin to the human hepatocarcinoma cell‐line 7721 was 59 nmol, whereas the melanoma cell‐line M21 was not susceptible to camphorin. Furthermore, cinnamomin exhibited a remarkable inhibitory effect on the growth of solid melanoma in the skin of the nude mouse. An R‐fragment could be isolated from ribosomes of cinnamomin‐ or camphorin‐treated carcinoma cells after incubation with acidic aniline, indicating that the cytotoxicity of these two new RIPs to carcinoma cells might result from modification to the ribosomes.
Effect of 4‐hydroxynonenal on superoxide anion production from primed human neutrophils
Authors: Dianzani Chiara , Parrini Marinella , Ferrara Cinzia , Fantozzi Roberto
Published: May 2005

HNE (4‐hydroxy‐2,3‐trans‐nonenal), an aldehydic product of lipid peroxidation, has been reported to modulate different functional parameters of human and rat neutrophils (PMNs), such as chemiluminescence, migration and some enzymatic activities, thus exerting effects that varied according to the concentration tested. Experiments were done to evaluate the effects of HNE on superoxide anion (O2−.) production from human PMNs, isolated from healthy volunteers. After having tested that HNE by itself was not able to activate the cells, comparisons were made between its effects on PMNs, stimulated by either a single stimulus, N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP), or a combination of stimuli, such as FMLP and the neuropeptide substance P (SP; primed PMNs). In the concentration range tested (10−12–10−4 M), HNE inhibited FMLP‐evoked O2−. production with an IC50 of 11·6 ± 1·5 × 10−6 M; at concentrations ≤10−6 M, HNE enhanced O2−. production elicited by FMLP + SP, while higher concentrations were inhibitory. There was a bell‐shaped dose–response curve to the enhancing effects of HNE, depending on the incubation time being recorded after only short periods (≤5 min) of the exposure of the cells to HNE; this was not shown by structurally‐related aldehydes, such as 2‐nonenal and nonanal. These results suggest that low concentrations of HNE may participate in the evolution of the inflammatory process, by contributing to the activation of PMNs. The effects of high concentrations of the aldehyde may represent a mechanism which contributes to the regulation of the extent of the inflammatory response.
The expression of the human chorionic gonadotropin beta subunit gene depends on negative control
Authors: Chen Yen‐Hui , Chen Tzer‐Ming , Hsieh Chang‐Yao
Published: May 2005

Transcription of the human chorionic gonadotropin (hCG) genes begins in the very early embryo stage and decreases or even disappears in nonplacental tissues. We have studied the regulation of hCG‐beta genes by cell fusion and by the reverse transcription‐polymerase chain reaction (RT‐PCR). The choriocarcinoma cell lines, JAR and JEG‐3, express high levels of the hCG‐beta subunit while HeLa cells express extremely low levels of it. Most HeLa × JAR and HeLa × JEG‐3 fusion clones expressed only a trace of the hCG‐beta subunit mRNA, while JAR × JEG‐3 fusion clones still expressed high levels of the hCG‐beta subunit. Most transcripts of the hCG‐beta subunit genes in JAR and JEG‐3 came from the hCG‐beta 5 subunit. Even the trace amount of hCG‐beta transcripts from fusion clones came mainly from the beta‐5 gene. The results suggest that the expression of the hCG‐beta subunit genes depends on negative control. Probably when embryonic cells differentiate to form nonplacental tissues, specific inhibitors may appear and inhibit the expression of the hCG‐beta subunit genes.