bluuebunny/crossref_metadata_2025 huggingface dataset:
Distribution of tightly bound non‐histone proteins in chromatin fractions obtained by DNAase II digestion
Authors: Lonigro Renata I. , Altieri Fabio , Allegra Paola , Caiafa Paola
Published: May 2005

Digestion of pig liver chromatin with DNAse II afforded three different fractions which were characterized in terms of their DNA, RNA and tightly bound non‐histone protein content, their DNA fragment size and their template activity. Two of these fractions are soluble after digestion with DNAase II and have been separated on the basis of their different solubility in MgCl2. A third fraction is not solubilized even after extensive digestion, although the size of its DNA is comparable to that of the enzyme solubilized fractions. The three fractions show qualitatively and quantitatively different distribution of tightly bound non‐histone proteins, with specific protein components in each fraction; furthermore the non‐solubilized fraction is greatly enriched in proteins tightly bound to DNA. From all the data obtained it can be suggested that the tightly bound proteins of the insoluble fraction may play, directly or indirectly, a role in maintaining an organized chromatin structure.
Effect of tricyclohexylhydroxytin on synaptosomal Ca2+‐dependent ATP hydrolysis and rat brain subcellular calmodulin
Authors: Rao K. S. Prasada , Chetty C. S. , Trottman C. H. , Uzodinma J. E. , Desaiah D.
Published: May 2005

Effect of tricyclohexylhydroxytin (plictran) on Ca2+‐ATPase activity was studied in rat brain synaptosomes under in vitro and in vivo conditions. Plictran inhibited basal Ca2+‐ATPase activity with an IC50 value of 6 nM suggesting its interaction with calcium transport phenomenon. Plictran inhibited calmodulin (CaM) activated Ca2+‐ATPase in a concentration‐dependent manner. A complete reversal of calmodulin activation of Ca2+‐ATPase was observed with 2–3 nM plictran. A 50 per cent decrease of CaM activated Ca2+‐ATPase was observed with 0·5 nM plictran, a concentration at which no significant effect was observed on basel enzyme activity. Of all the brain fractions studied, calmodulin levels in P2 fractions alone were reduced significantly to about 75 per cent of control values in plictran treated rats. The synaptosomal Ca2+‐ATPase was also decreased by 35 per cent, 42 per cent and 65 per cent in 10, 20 and 40 mg plictran kg−1 day−1 treated rats for 3 days respectively. The activity levels of Ca2+‐ATPase in 10 and 20 mg plictran kg−1day−1 treated rats were restored to normal level by exogenously added calmodulin. These results suggest that plictran may disrupt synaptic function by altering calcium and calmodulin regulated processes in the central nervous system.
Studies on rat liver mitochondria. 6. The effect of contaminating particles in mitochondria stored at 0–4°C
Authors: Higuti Ilma Hiroko , Murata Lilia Yuko , Stencel Mario , Campello Annibal De Paiva , Nascimento Aguinaldo José Do
Published: May 2005

Rat liver mitochondria were stored at 0–4°C for several days using an appropriate medium and energy source. The elimination of the majority of microsomes and lysosomes, that normally contaminate isolated mitochondria, had a positive effect in preservation of respiratory control, P:O ratio, and monoamine oxidase activity during long term storage.
Is the saccharide component of the insulin receptor involved in hormonal imprinting?
Authors: Kovács P. , Csaba G.
Published: May 2005

Hormonal imprinting takes place at the first interaction of a given hormone with the cell (the Tetrahymena in the present case) and accounts for a greater responsiveness to the hormone on re‐exposure(s). The Tetrahymena is able to bind insulin and Concanavalin‐A (Con‐A) as well. Exposure to both ligands—simultaneously or in sequence—enhances the binding of both in the progeny generations. It follows that the lectin, which inhibits insulin binding by direct action, enhances rather than depresses the effect of insulin‐induced imprinting.
Biochemical significance of enhanced activity of fluorinated 1,25‐dihydroxyvitamin D3 in human cultured cell lines
Authors: Eisman John A. , Frampton Richard J. , McLean Fiona J.
Published: May 2005

Several human cancer cells possess receptors for 1,25‐dihydroxyvitamin D3[1,25‐(OH)2D3]. In these cells 1,25‐(OH)2D3 has a biphasic concentration‐dependent regulatory effect on cell replication and specifically induces its own metabolism. We have studied the effects on these parameters of the native hormone together with those of two analogues fluorinated at the 24‐carbon and of 1,24R,25‐trihydroxyvitamin D3[1,24R,25‐(OH)3D3]. The difluorinated analogue 24,24‐difluoro‐1,25‐(OH)2D3[24,24‐F2‐1,25‐(OH)2D3] is an approximately fivefold more potent inhibitor of cellular replication than the native hormone, while 1,24R,25‐(OH)3D3 is about fivefold less potent. This enhanced potency of the fluorinated analogue parallels its enhanced potency in in vivo studies of its effects on calcium and mineral metabolism. However, although the analogue retains replication stimulatory activity, it is clearly no more potent than the native hormone in this activity: 1,24R,25‐(OH)3D3 has no significant stimulatory activity. Exposure of the cells to 1,25‐(OH)2D3 at 0·05 nM for 6h increases the subsequent conversion of labelled hormone to aqueous phase soluble compounds by 6·7‐fold. None of the other compounds had a similar effect at this concentration. At 10nM all 1‐hydroxylated compounds increased aqueous phase radioactivity about equally (13 to 17‐fold); this effect is still specific since 25‐OH D3 had no such effect even at 10nM. Studies on the effects of the fluorinated analogues upon receptor binding of hormone in cell cytosols and uptake of hormone by intact cells clearly demonstrate that the enhanced activity of these analogues is not due to higher receptor affinity or more rapid access to intracellular receptor. These data suggest that this enhanced inhibitory potency, observed in these human cancer cells and by analogy their enhanced potency in vivo, is due to their resistance to metabolism by 24‐hydroxylation. In view of our previous observations that 24‐hydroxylation is part of the induced metabolic pathway, these data support the hypothesis that 24‐hydroxylation is part of an inactivation pathway in these human target cells.
Effect of Escherichia coli lipopolysaccharide on the microviscosity of liver plasma membranes and hepatocyte suspensions and monolayers
Authors: Portolés M. T. , Pagani R. , Díaz‐Laviada I. , Municio A. M.
Published: May 2005

The fluorescence probe 1,6‐diphenylhexa‐1,3,5‐triene (DPH) was used for monitoring structural perturbations induced by lipopolysaccharide (LPS) of Escherichia coli (0111 : B4) in plasma membranes of rat liver. Changes in microviscosity were observed in plasma membrane preparations from control rats after treatment with LPS and in plasma membrane preparations from liver perfused with LPS. In both systems fluorescence polarization was measured from which microviscosity was calculated. This parameter increases with LPS treatment. From temperature dependence studies was inferred that LPS interaction with plasma membrane preparations induces an increase of both the polarization term (r0/r–1)−1 and flow activation energy (ΔE). Addition of LPS to hepatocyte suspensions also induces an increase on microviscosity and a delay in the fall of microviscosity induced by a temperature rise in hepatocyte monolayers grown on microcover slides.

These data suggest that LPS interaction can be attributed to its binding to membrane hydrophobic regions in a non‐specific manner.
Inhibition by elastase inhibitors of the formyl met leu phe‐induced chemotaxis of rat polymorphonuclear leukocytes
Authors: Hornebeck W. , Soleilhac J. M. , Tixier J. M. , Moczar E. , Robert L.
Published: May 2005

Rat leukocyte elastase has been purified successively by AH‐Sepharose Kappa‐elastin affinity chromatography and by ion exchange chromatography on a carboxymethyl Sephadex resin. It has great similarities with human leukocyte elastase in its molecular weight, substrate specificity and inhibitory profile.

The effect of rat leukocyte elastase inhibitors in influencing the chemotactic response of rat PMN to fMetLeuPhe has been compared to that of other proteinase inhibitors. The results indicated that oleoyl (Ala)2ProValCH2Cl, a specific inhibitor of human and rat leukocyte elastases and Eglin C, which also inhibits human and rat cathepsin G, are among the powerful inhibitors of rat PMN chemotaxis induced by the formyl oligopeptide. This suggests that these neutral proteinases, in addition to their known participation in connective tissue catabolism, could play a role in PMN locomotion and chemotaxis.
Experimental conditions influence [3H]‐dihydroalprenolol binding characteristics to living HeLa cells due to morphological changes: A warning
Authors: Rademaker Ben , Stroes Jan W. , Bast Aalt , Timmerman Henk
Published: May 2005

Harvesting of plated growing HeLa cells, followed by incubation of these cells without any addition at 37°C was found to cause changes in the cell shape. This phenomenon is accompanied by a diminished binding of the beta‐adrenergic antagonist [3H]‐dihydroalprenolol and the alpha‐adrenergic antagonist phentolamine to a binding compartment not representing beta‐adrenergic receptors. These binding sites have a high affinity for hydrophobic agents and most probably represent lipophilic structures in the cellular membrane. Changes in the cell shape obviously cause alterations in the physical properties of the plasma membrane. This might lead to misinterpretations of the results from experiments in which the redistribution of beta‐adrenergic receptors is followed during incubation with agonists, as receptor occupation with subsequent receptor redistribution is possibly accompanied by effects on the membrane microviscosity.

It is concluded that investigations performed in order to follow physiological events like receptor redistribution and desensitization processes, may be obfuscated by changes in the normal physical state of the living cells.
Immunohistochemical localization of albumin and in situ hybridization of albumin mRNA
Authors: Shroyer Kenneth R. , Nakane Paul K.
Published: May 2005

Hepatocytes actively involved in albumin synthesis were identified by immunohistochemical method. In sections of perioidate–lysine–2 per cent (w/v) paraformaldehyde fixed normal rat liver, albumin was detected in all hepatocytes. At the untrastructural level, albumin was localized in the rough endoplasmic reticulum and in Golgi complexes located near the nucleus in only a small subpopulation of hepatocytes, while all other hepatocytes contained albumin only in Golgi complexes located near the bile canaliculi. Stimulation of albumin synthesis by puromycin aminonucleoside‐induced nephrosis resulted in an altered intracellular distribution of albumin at the light microscopic level. When examined at the ultrastructural level, albumin was localized in the rough endoplasmic reticulum as well as in Golgi complexes located near the nucleus in nearly all these hepatocytes.

Hepatocytes with the potential to synthesize albumin were identified by in situ hybridization of albumin mRNA. In sections of 0·1 per cent (v/v) glutaraldehyde perfusion fixed normal rat liver, albumin mRNA was detected in the cytoplasm of only a few hepatocytes scattered throughout the lobule. Following stimulation of albumin synthesis by the induction of nephrosis, albumin mRNA was detected in the cytoplasm of the hepatocytes.

The source of albumin in those hepatocytes which lacked albumin mRNA was identified in analbuminemic rats injected with rat albumin. At 6 h post injection, the light microscopic distribution of albumin in the liver of these animals was virtually indistinguishable from that in normal rat liver. At the ultrastructural level, injected albumin was localized in lysosomes and in Golgi complexes located near the bile canaliculi.
Differential time‐course of induction of rat liver gamma‐glutamyltransferase and drug‐metabolizing enzymes in the endoplasmic reticulum, golgi and plasma membranes after a single phenobarbital injection. Evaluation of protein variations by two‐dimensional electrophoresis
Authors: Antoine B. , Rahimi‐Pour A. , Siest G. , Magdalou J. , Galteau M. M.
Published: May 2005

This study was conducted to follow as a function of time the activity of gamma‐glutamyltransferase in the various membranes of rat liver cells after a single dose of phenobarbital (PB) (75 mg kg−1 body weight). Gamma‐glutamyltransferase induction was maximal 24 h after PB treatment in both the rough endoplasmic reticulum and the plasma membranes. This pattern of induction differed from that of some drug metabolizing enzymes. While total cytochrome P‐450 content was enhanced mainly in endoplasmic reticulum until 48 h after PB treatment, UDP‐glucuronosyltransferase activity was not greatly altered by PB under the same conditions.

The comparison of two‐dimensional electrophoretic polypeptide profiles of each subcellular membrane isolated from control and phenobarbital‐treated rats revealed important variations induced by PB. In plasma membranes, the heaviest subunit (apparent Mr = 60 × 103) of hepatic gamma‐glutamyltransferase was provisionally identified as a collection of polypeptides which differ only by their pI. The concentration of these polypeptides was smaller in the endoplasmic reticulum where they were of lower apparent molecular mass. This suggests that the gamma‐glutamyltransferase precursor is already processed at the level of the endoplasmic reticulum but it is still not completely mature or glycosylated. Five days of continuous PB treatment induced the appearance of new gamma‐glutamyltransferase isoforms in plasma membranes.

We demonstrate that after a single injection of PB, gamma‐glutamyltransferase activity increases simultaneously with some drug‐metabolizing enzymes, such as total cytochrome P‐450 but not with others, such as UDP‐glucuronosyltransferases.
Δ53β hydroxysteroid dehydrogenase activity of the rat corpus luteum exhibits positive substrate‐binding co‐operativity: A continuous monitoring microdensitometric study with unfixed ovarian tissue sections
Authors: Gordon M. , Robertson W. R.
Published: May 2005

Δ53β hydroxysteroid dehydrogenase activity transforms biologically inactive Δ53β hydroxy steroids into the active Δ43‐keto products (e.g. pregnenolone to progesterone). Using a cytochemical procedure which allows for the continuous microdensitometric monitoring of an enzyme reaction as it proceeds and a well described cytochemical assay for Δ53β HSD we have analysed the initial velocity rates (Vo) for dehydroepiandrosterone (DHEA) binding to this enzyme in regressing (i.e. 20α hydroxy steroid dehydrogenase positive) corpus luteum (CL) cells in unfixed tissue sections (5 μm) of the dioestrous and proestrous rat ovary. The results are mean ± S.E.M. The relationship between DHEA concentration (0 to 50 μM) and Δ53β HSD activity in the dioestrous corpora lutea was sigmoidal and had an atypical 1/Vo versus 1/S plot, the x intercept being positive. Using a 1/Vo versus 1/S2 plot the Vmax was determined to be 1·0 ± 0·08 μmol min−1 mg−1 CL (n = 6). The Hill constant was 2·7 ± 0·02 (n = 6) suggesting a high degree of positive co‐operativity for DHEA binding. The S concentration for half maximal activity was 17 ± 1 μmoles (n = 6). In the corpora lutea cells of the proestrous ovary, the Vmax for DHEA transformation was unchanged (0·95 ± 0·04 μmol min−1 mg−1, n = 3) whilst the S0·5 was significantly increased to 27 ± 0·1 (p < 0·01, n = 3). The Hill constant remained positive being 2·9 ± 0·2 (n = 3).

NAD+ binding to 3β HSD in regressing corpora lutea of the proestrous ovary has been demonstrated previously to be hyperbolic and fit the classical Michaelis‐Menten model.1 Extending the analysis of NAD+ binding to the regressing corpus luteum of the dioestrous rat ovary revealed similar kinetic characteristics to that seen with the proestrous enzyme, the apparent Vmax and Km being 0·84 ± 0·04 μmol min−1 mg−1 CL (n = 3) and 27 ± 7 μmol 1−1 (n = 3) respectively. The Hill constant was 1·1 ± 0·03 (n = 3), indicating no co‐operativity of co‐factor binding.
Cytophotometric analysis of alkaline phosphatase and 5′‐nucleotidase activity in regenerating rat liver after partial hepatectomy
Authors: Van Noorden Cornelis J. F. , Vogels Ilse M. C. , Houtkooper Joop M.
Published: May 2005

Alkaline phosphatase and 5′‐nucleotidase activities were analysed cytophotometrically in cryostat sections of female rat liver after partial hepatectomy. Alkaline phosphatase activity increased rapidly after operation up to a maximum seven‐fold rise at 24 h in comparison with sham operated or control rats. There was no indication of preferential localization of alkaline phosphatase activity in either periportal or pericentral areas at any time point in control rats, sham operated rats or hepatectomized rats. Microscopical observation revealed that (a) all alkaline phosphatase activity was present at the bile canalicular surface of hepatocytes and (b) hepatocytes in mitosis did not show any increase in activity. These findings indicate that the high alkaline phosphatase activity after partial hepatectomy is not involved primarily in proliferation processes because cell division mainly takes place periportally. It may be needed for enhanced bile secretion by conversion of intracellular phosphorylcholine into choline which can be transported into the bile. The intracellular phosphorylcholine level is high after operation due to changes in phospholipid metabolism. 5′‐Nucleotidase appeared to be three times higher pericentrally than periportally under normal conditions. Partial hepatectomy caused a 40 per cent decrease in activity in pericentral areas and only a small decrease periportally. It has been suggested that 5′‐nucleotidase plays a role in breakdown of messenger RNA and its activity in control liver could be considerably lower periportally because plasma protein synthesis mainly takes place in this area. 5′‐Nucleotidase activity may well be decreased after operation for accumulation of messenger RNA for enhanced plasma protein synthesis in order to compensate for 67 per cent of the liver cell functions lost during partial hepatectomy.
New synthesis of cyclic AMP‐dependent protein kinases during liver regeneration
Authors: Pujol Maria Jesùs , Soriano Montserrat , Coll Maria Josep , Domingo Jordi , Bachs Oriol
Published: May 2005

As has been previously reported one surge in cytosolic calmodulin is produced between 4 and 12 h after a partial hepatectomy. Moreoever, a surge in cytosolic cyclic AMP and another in cyclic AMP‐dependent protein kinase activity can be detected during the late period of the prereplicative phase of liver regeneration after a partial hepatectomy. It is known that these three surges are involved in triggering DNA synthesis.

By kinetic studies and by injecting transcription (actinomycin D) and translation (cycloheximide) inhibitors into hepatectomized rats we have demonstrated that the cyclic AMP‐dependent protein kinase surge is produced by new synthesis of the enzyme. In thyroparathyroidectomized rats subjected to a partial hepatectomy the calmodulin surge was similar to that observed in normal hepatectomized rats whereas the surge in cyclic AMP‐dependent protein kinase activity was strongly decreased suggesting that the cyclic AMP‐dependent protein kinase surge is not generated by the previous surge in calmodulin.
The effect of a native collagen gel substratum on the synthesis of collagen by bovine brain capillary endothelial cells
Authors: Scott D. M. , Kumar S. , Barnes M. J.
Published: May 2005

Cultured capillary endothelial cells, derived from bovine brain, and maintained on a plastic substratum synthesized predominantly interstitial collagens of which approximately 75 per cent were secreted into the medium. When grown on a native hydrated collagen type I gel, although no marked alteration in the ‘collagen synthetic pattern’ was observed, the overall level of collagen synthesis was increased by approximately 100 per cent. More dramatic, however, was the alteration in the distribution of these molecules between medium and cell layer. Interstitial collagens produced by cells grown on collagen gels were almost exclusively associated with the cell layer or collagenous gel. These studies, thus, demonstrate that an extracellular matrix may exert a considerable influence on the cellular synthetic activities and possibly cellular polarity of capillary endothelial cells.
Influence of long term treatment with 3‐methylcholanthrene or carbaryl on mixed‐function oxidase activity in 3T3 fibroblasts: Studies by microspectrofluorimetry on single cells
Authors: Lahmy Sylvie , Salmon Jean‐Marie , Viallet Pierre
Published: May 2005

Using benzo(a)pyrene (BaP) as a probe for aryl hydrocarbon hydroxylase (AHH) activity, differences in mixed‐function oxidase (MFO) activity were observed using microspectrofluorimetry in single living cells during long term treatment with 3‐methylcholanthrene (3‐MC) or carbaryl. Although these two compounds differ in chemical structure, similar effects were observed in 3T3 cell populations.

The results suggest that the two compounds activate the same enzymatic system and that individual cells of a supposed homogeneous cell population are not equally sensitive to xenobiotics, i.e. subpopulations were observed which have differences in AHH activity.
Intrarenal localization of angiotensin II specific binding in rat fetuses
Authors: Uva B. , Ghiani P. , Mandich A. , Masini M. A.
Published: May 2005

Specific binding sites for angiotensin II were localized in the developing rat kidney (18th day of pregnancy and immediately before birth) by autoradiography using [125I]‐ileu‐5‐angiotensin II either perfused in vivo through the fetal aorta or added in vitro to frozen sections in an incubation mixture. Specific binding was localized in the walls of the afferent and efferent arterioles, in the intraglomerular cells and in the peritubular arterioles of the subcapsular cortical zone. The immunohistochemical analysis, carried out on receptors saturated with unlabelled angiotensin II perfused through the mother's aorta, confirmed the autoradiographical localization. Antisera against ileu‐5‐angiotensin II were used in the indirect immunofluorescence technique and in the PAP method. Immunolocalization of angiotensin II was also found in the proximal tubule and in the thick ascending limb of Henle's loop.
Uptake of alpha 2‐macroglobulin–trypsin complex by human placenta is mediated by a microvillous membrane receptor
Authors: Eaton B. M. , Oakey M. P. , Sooranna S. R. , Contractor S. F.
Published: May 2005

The fate of native alpha 2‐macroglobulin (α2M) or its trypsin complex (α2M‐T) was studied in the isolated dually‐perfused lobule of term human placenta. [125I]‐α2M added to the maternal circuit was unchanged during the course of the perfusion with minimal activity becoming associated with the placental tissue. Transfer of radioactivity into the fetal circulation accounted for only 0·07 per cent of the initial dose after 2 h. In contrast, [125I]‐α2M‐T was rapidly taken up into the placental tissue (nearly 28 per cent of the initial dose during the 2‐h perfusion) and breakdown products were released into both maternal and fetal circulations. At the end of 2 h, radioactivity levels on the fetal side were 13 times higher than those found with the native protein. These indications of a classical receptor‐mediated uptake and breakdown pathway were confirmed in experiments in which the acidotrophic agent chloroquine was added to the maternal circuit prior to the α2M‐T. In the presence of chloroquine, tissue uptake was inhibited and the subsequent release of radioactive degradation products into the fetal circuit was similar to the levels seen with α2M. Incubation of term trophoblast cells at 37°C with [125I]‐α2M‐T revealed over three‐fold greater cell‐associated activity than was found with the native protein. In another series of experiments, a purified microvillous membrane fraction was prepared from term placentae using buffers containing 1 mM iodoacetate. In the presence of this proteolytic enzyme inhibitor, binding studies showed a single class of low affinity receptors for the α2M‐T complex capable of binding 4·8 ± 1·3 (SEM) μg of complex per mg of membrane protein. There was no binding of the native protein.
Inhibition of the glycolytic pathway by methylglyoxal in human platelets
Authors: Leoncini G. , Maresca M. , Buzzi E.
Published: May 2005

The incubation of human platelets with methylglyoxal and glucose produces a rapid transformation of the ketoaldehyde to D‐lactate by the glyoxalase system and a partial reduction in GSH. Glucose utilization is affected at the level of the glycolytic pathway. No effect of the ketoaldehyde on glycogenolysis and glucose oxidation through the hexose monophosphate shunt was demonstrated. Phosphofructokinase, fructose 1,6 diphosphate (F1, 6DP) aldolase, glyceraldehyde 3‐phosphate dehydrogenase and 3‐phosphoglycerate mutase were mostly inhibited by methylglyoxal. A decrease in lactate and pyruvate formation and an accumulation of some glycolytic intermediates (fructose 1,6 diphosphate, dihydroxyacetone phosphate, 3‐phosphoglycerate) was observed. Moreover methylglyoxal induced a fall in the metabolic ATP concentration. Since methylglyoxal is an intermediate of the glycolytic bypass system from dihydroxyacetone phosphate to D‐lactate, it may be assumed that ketoaldehyde exerts a regulating effect on triose metabolism.
Liposome–nucleus interactions. Flow cytometric study on the role of the nuclear surface
Authors: Caramelli E. , Papa S. , Billi A. M. , Vitale M. , Bartoletti A. , Manzoli L. , Capitani S.
Published: May 2005

The water‐soluble probe carboxyfluorescein (CF), contained in the internal aqueous phase of liposomes, was used to investigate the interaction of phospholipid vesicles with isolated nuclei. Ultrastructural analysis indicated that adherent liposomes coated the nuclear surface, and fluorescence microscopy showed that they contained quenching concentrations of the dye. Flow cytometry revealed that the transfer of the entrapped dye from the adhering liposomes to nuclei was blocked by chilling at 0°C. Chase experiments demonstrated that the most reliable mechanism of dye transfer involved fusion phenomena between the liposomal and the nuclear membranes. After the release of the fluorophore into the nucleus, empty liposomes could withdraw the intranuclear soluble fraction of the dye.
Cytochrome P450‐dependent alkoxyphenoxazone dealkylase activity in rat alveolar type II cells: Effect of pretreatment with β‐naphthoflavone
Authors: Rabovsky J. , Judy D. J. , Sapola N. A. , Pailes W. H. , McPeek M. , Castranova V.
Published: May 2005

Cytochrome P450‐dependent alkoxyphenoxazone dealkylase activity was measured in alveolar type II cells from control and β‐naphthoflavone (ip) treated‐rats. Type II cells were isolated from collagenase/elastase‐digested lung tissue and purified by centrifugal elutriation. The specificity of the cytochrome P450‐dependent activity towards four alkoxyphenoxazones (methoxy‐, ethoxy‐, pentoxy‐, and benzyloxyphenoxazone) was measured under conditions that minimized interference by cytosolic conjugating‐ and NADPH‐dependent quinone reductase activities. Ethoxyphenoxazone dealkylase activity was induced 17‐fold following β‐naphthoflavone treatment and was further characterized by its kinetic parameters and sensitivities toward in vitro inhibitors (Km(app)=0·20 μM, Vmax = 1·74 pmoles resorufin min−1 (106 cells)−1 106 cells; I50 (α‐naphthoflavone)=0·025 μM, and I50 (methyrapone) = 72 μM). β‐Naphthoflavone pretreatment of the rats did not result in statistically significant changes in methoxy‐, pentoxy‐, or benzyloxyphenoxazone dealkylase activity of alveolar type II cells, although, a trend towards decrease activity was observed for benzyloxyphenoxazone. β‐Naphthoflavone pretreatment had no effect on oxygen consumption of trypan blue exclusion in alveolar type II cells and macrophages, or on alveolar macrophage particle‐mediated superoxide release. Furthermore, alveolar macrophage ethoxyphenoxazone dealkylase and benzyloxphenoxazone dealkylase activities were not affected by the β‐naththoflavone pretreatment. The results show that exposure to β‐naphthoflavone resulted in an increase in type II cell cytochrome P450‐dependent ethoxyphenoxazone dealkylase activity but not in other alveolar type II cell or macrophage alkoxyphenoxazone dealkylase activities or in parameters that monitor viability and cell wall integrity.
Brush border membrane non‐esterified fatty acids. Physiological levels and significance for mucosal iron uptake in mouse proximal intestine
Authors: Simpson Robert J. , Venkatesan S. , Peters Timothy J.
Published: May 2005

Brush border membrane vesicles prepared using divalent cation precipitation methods can contain unphysiological levels of non‐esterified fatty acids. Fatty acid production from endogenous lipid during brush border membrane vesicle preparation is effectively prevented by the lipase inhibitor diethyl 4‐nitrophenylphosphate plus cooling. Vesicles prepared using this procedure have variable levels of non‐esterified fatty acids (range 22–193 nmol mg−1 protein). Changes in non‐esterified fatty acid levels in brush border membrane vesicles parallel Fe uptake by vesicles from Fe/ascorbate solutions. Brush border membrane vesicle fatty acids appear to be derived from the diet but hypoxic mice are able to maintain high brush border membrane non‐esterified fatty acid levels despite reduced dietary intake. Non‐esterified fatty acids in brush border membrane may thus provide a physiological mechanism of mucosal Fe uptake.
Effect of sodium arsentie on the induction and turnover of ornithine decarboxylase activity in erythroleukemia cells
Authors: Flamigni Flavio , Marmiroli Sandra , Caldarera Claudio M. , Guarnieri Carlo
Published: May 2005

Sodium arsenite proved effective in preventing the induction of ornithine decarboxylase (ODC) activity elicited by dilution of Friend erythroleukemia cells in fresh medium. A 50 per cent inhibition was produced at approximately 1 μM arsenite and complete inhibition was obtained at concentrations above 10 μM. However, addition of arsenite 5 h after cell dilution, i.e. when ODC was already induced, appeared to stabilize the enzyme. The half‐life of ODC activity, measured after cycloheximide treatment, increased almost six‐fold after addition of sodium arsenite. Agents known to provoke oxidative alteration of the thiol‐redox status in cells, also caused a similar effect on the induction and stability of ODC.
Blocking of human T lymphocyte activation by channel antagonists
Authors: Bono Maria Rosa , Simon Valeska , Rosemblatt Mario S.
Published: May 2005

It has been established that early events in lymphocyte activation involve a rise in intracellular Ca++ as well as changes in the flux of other ions. Although a Ca++ channel has been postulated to participate in the early Ca++ rise, its presence in lymphocytes remains controversial. Also although yet undetected, electrophysiological data suggest the presence of a Ca++ activated K+ channel on human peripheral blood lymphocytes (HPBL). Here we report on the effect of specific channel blockers as an approach to the identification of these channels on HPBL. At 40 nM nifedipine, an inhibitor of voltage‐gated Ca++ channels, fully inhibits the PHA‐promoted activation of HPBL. This effect is concentration dependant with a half maximum effect at approximately 10 nM and is demonstrable whether the drug is added at the same time as or up to 18 h after the addition of the mitogen. This inhibition of activation is not seen if the lymphocytes are activated using IL‐2 instead of PHA. Charybdotoxin a toxin which blocks a Ca++ activated K+ channel of muscle cells also blocks to almost 100 per cent the PHA‐induced activation of HPBL. This inhibition can be demonstrated regardless of whether the blocker is added together with or up to 4 h after PHA. As opposed to nifedipine charybdotoxin shows no effect if added 18 h after the initiation of the activation process. When nifedipine and charybdotoxin were tested on mice splenocytes we found that nifedipine fully inhibits the LPS‐promoted activation of these cells while charybdotoxin has no effect on their activation. Taken together these results strengthen the view regarding the participation of both a voltage‐gated Ca++ channel and a Ca++ activated K+ channel in human T lymphocyte activation.
The mechanism of placental alkaline phosphatase induction in vitro
Authors: Nozawa Shiro , Narisawa Sonoko , Iizuka Rihachi , Fukasawa Toshio , Kohji Takehiko , Nakane Paul K. , Hirano Kazuyuki , Millan Jose L.
Published: May 2005

The mechanism of placental alkaline phosphatase (PLAP) induction by prednisolone in a uterine cervical epidermoid cancer cell line SKG‐IIIa was investigated in vitro by enzyme‐cytochemistry, enzyme immunoassay, Northern and Southern blot analysis, and in situ hybridization. Enzyme‐cytochemical alkaline phosphatase (ALP) staining and immunoassay revealed increased levels of PLAP (heat‐stable ALP) in prednisolone‐treated cells. Northern blot analysis and in situ hybridization showed increased amounts of PLAP mRNA. Southern blot analysis indicated that PLAP was not a product of an amplified or rearranged gene.

These findings suggest that the induction of PLAP mRNA in SKG‐IIIa cells by prednisolone in turn increased the levels of PLAP.
4‐Hydroxynonenal interacts with tubulin by reacting with its functional ‐SH groups
Authors: Olivero Antonella , Miglietta Antonella , Gadoni Elena , Gabriel Ludovica
Published: May 2005

4‐Hydroxynonenal, which is one of the most important products of lipid peroxidation, alters microtubular organization and structure in 3T3 fibroblasts. Changes in cell shape and the disappearance of microtubules are observed by immunofluorescence after incubation with the aldehyde, and the colchicine binding activity of tubulin from 3T3 cells is modified. Moreover, the aldehyde determines a decrease in the ability of purified tubulin to polymerize and to bind colchicine. These effects may be related to the interaction of the aldehyde with functional ‐SH groups of tubulin which are necessary for protein integrity and functions. Indeed, the addition of cysteine protects against the damaging effects of the aldehyde.
ATP modifications in hexokinase deficient fibroblasts exposed to nutrient shifts
Authors: Chiarantini Laura , Vittoria Emanuela , Magnani Mauro
Published: May 2005

Human fibroblasts from a patient homozygous for hexokinase (HK) deficiency and normal controls were submitted to different nutrient shifts to investigate the role of glucose metabolism on ATP levels. HK‐deficient cells maintained in the presence of both glucose and L‐glutamine had 20 per cent less ATP than controls and an ATP/ADP ratio of 18 instead of 37–40. Glucose alone was not able to maintain normal ATP levels neither in the controls nor in the HK‐deficient cells. However while in control cells the ATP/ADP ratio was not modified, it was reduced to 10 in HK deficiency. The effect of several hexoses on ATP levels was also investigated. Mannose, which is metabolized by HK phosphorylation, was the least efficient for ATP maintenance in HK‐deficient cells. In contrast galactose, which is metabolized by a specific galactokinase, provided ATP values close to those observed in the presence of glutamine. These results suggest that glucose metabolism is an important determinant in the maintenance of ATP levels in cultured cells and that glutamine, although being a good metabolic substrate, is not sufficient to maintain normal ATP concentrations.
Effects of inositol 1,4,5‐trisphosphate on calcium release from the endoplasmic reticulum and golgi apparatus in mouse mammary epithelial cells: A comparison during pregnancy and lactation
Authors: Yoshimoto Akio , Nakanishi Keiko , Anzai Tadashi , Komine Senichi
Published: May 2005

It has been established that inositol 1,4,5‐trisphosphate(IP3) is responsible for the mobilization of calcium(Ca2+) from intracellular locations in a wide variety of tissues, and that this response triggers the stimulation of several hormones and neurotransmitters. However, these phenomena have yet to be examined in the mammary epithelium.

Ca2+ uptake from the medium into the endoplasmic reticulum(ER) and Golgi apparatus in vitro in both pregnant and lactating mouse mammary epithelial cells was studied and a strong Ca2+ release from these organelles into the medium with the use of IP3 was shown. The Ca2+ uptake and its release due to IP3 was also usually greater during pregnancy than lactation.
Impairment of ATP‐linked reactions in mitochondria isolated from skeletal muscle of halothane‐sensitive pigs
Authors: Ayoub S. , Monin G. , Rock E. , Younes A.
Published: May 2005

Oxygen consumption was depressed in mitochondria isolated from halothane sensitive pig (HP) muscle. The calculation of the respiratory control ratio (RCR) indicated that mitochondria were more affected at the site‐I level of the respiratory chain. Calcium accumulation in these mitochondria was not altered when driven by the oxidation of succinate. This process was abolished when linked to ATP as a source of energy. ATP transport was completely inhibited in (HP) mitochondria.
Decarboxylation of branched‐chain α‐ketoacids in hepatocytes from alloxan‐diabetic rats. The effect of insulin
Authors: Sterniczuk Anna , Wałajtys‐Rode Elżbieta I. , Wojtczak Anna B.
Published: May 2005

The flux through branched‐chain α‐ketoacid dehydrogenase and the activity of the branched‐chain α‐ketoacid dehydrogenase complex were measured in hepatocytes isolated from fed, starved and alloxan diabetic rats. The highest rate of branched‐chain α‐ketoacid oxidation was found in hepatocytes isolated from starved rats, slightly lower in those from fed rats, and significantly lower in diabetic hepatocytes. The amount of the active form of branched‐chain α‐ketoacid dehydrogenase was only slightly diminished in diabetic hepatocytes, whereas the flux through the dehydrogenase was inversely correlated with the rate of endogeneous ketogenesis. The same was observed in hepatocytes isolated from starved rats when branched‐chain α‐ketoacid oxidation was measured in the presence of added oleate. In both cases the diminshed flux through the dehydrogenase, restored by a short preincubation of hepatocytes with insulin, was parallelled by a decrease of fatty acid‐derived ketogenesis. The significance of these findings is discussed in relation to the role of insulin in branched‐chain α‐ketoacid oxidation in liver of diabetic rats.
Study of thiol proteases of normal human skin fibroblasts
Authors: Khalfan Hussain A.
Published: May 2005

The 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

The 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

The 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

The 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

We 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

The 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

The 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.