bluuebunny/crossref_metadata_2025 huggingface dataset:
Scanning‐integrating cytophotometric analyses of brain neuronal RNA and acetylcholinesterase in acute soman toxicated rats
Authors: Anthony A. , Doebler J. A. , Bocan T. M. A. , Zerweck C. , Shih T.‐M.
Published: May 2005

Cytophotometric analyses of RNA and acetylcholinesterase responses of caudate and cerebrocortical neurons of soman toxicated rats were conducted to characterize impairments in regulatory aspects of neuronal metabolism occurring in the acute phase of cholinesterase impairment. There was a severe and dose‐dependent suppression (20–60%) in neuronal acetylcholinesterase activity in both a.m. and p.m.‐treated rats; no diurnal differences were apparent in control acetylcholinesterase levels or neuronal acetylcholinesterase responsiveness to soman toxication. RNA levels, however, were markedly higher in p.m. than in a.m. saline‐treated controls. Soman depressed caudate neuron RNA contents in the afternoon, but not in the morning. Cerebrocortical neuron RNA levels were suppressed in both a.m. and p.m.‐toxicated rats, although this RNA depletion was more severe in the afternoon. These results indicate that soman can elicit marked alterations in neuronal transcriptional‐translational capabilities and that there are diurnal variations in cellular metabolic responsiveness to soman toxication. Although functional relationships between soman‐induced cholinesterase inhibition and RNA depletion remain to be elucidated, depressed RNA metabolism appears to be a maladaptive response preventing rapid regeneration of cholinesterase following poisoning.
The A12 acetylcholinesterase and polypeptide composition of electric organ basal lamina of electrophorus and some torpedinae fishes
Authors: Inestrosa Nibaldo C. , Méndez Bernardita
Published: May 2005

Basal lamina (BL) of Torpedo, Discopyge and Electrophorus electric organs was purified in order to establish polypeptide composition and association with acetylcholinesterase (AChE). Results indicate that BL presents a distinct peptide pattern and that the A12 form of AChE is directly attached to it. Comparison of the species studied demonstrated similarities both in polypeptide composition and AChE content of the purified BL. Extractions of BL with solutions of high ionic strength, guanidine–HCl and acetic acid indicated the differential solubilization of various domains of BL polypeptides.
Effects of mitogenic stimulation of lymphocytes on lysosomal enzyme activity
Authors: Olsen I. , Dean M. F. , Muir H. , Smith R. , Jenne B. M. , Hand C.
Published: May 2005

Changes in the activities of several lysosomal enzymes were studied during transformation of mouse spleen cells in vitro. The activity of β‐glucuronidase increased during culture in the presence of T or B‐cell mitogens, and lymphoblasts contained higher levels of activity than did small, non‐transformed lymphocytes. Moreover, lymphoblasts in well‐transformed cultures had higher activities than those in poorly‐transformed cultures. The activities of other lysosomal enzymes (N‐acetyl‐β‐glucosaminidase, α‐mannosidase, β‐glucosidase) also increased during mitogenic stimulation, but each at different rates, although aryl sulphatase was unaffected. Such differences may be of importance when lymphocytes are used for diagnosis of inherited lysosomal deficiency diseases.
Lysosomal membrane recovery following labilization by thyroid stimulators
Authors: Neylan D. , Smyth P. P. A.
Published: May 2005

Lysosomal membrane permeability was assessed by measuring freely available napthylamidase activity in intact preparations of guinea pig thyroid follicular cells following exposure of thyroid tissue to sequential stimulation by two thyroid stimulators, thyrotrophin (TSH) and thyroid stimulating immunoglobulins (TSI). These investigations showed that following labilization by TSH, the lysosomal membranes recovered and were capable of responding to a second thyroid stimulator (TSI). That such recovery represented restabilization of lysosomal membranes was confirmed by the finding that latent naphthylamidase activity was restored without a change in total activity of the enzyme.
Aerobic glycolysis of bone and cartilage: The possible involvement of fatty acid oxidation
Authors: Dunham Jane , Dodds R. A. , Nahir A. M. , Frost G. T. B. , Catterall A. , Bitensky Lucille , Chayen J.
Published: May 2005

The apparent paradox of aerobic glycolysis has been investigated in bone and in cartilage. A new cytochemical procedure for hydroxyacyl dehydrogenase (HOAD) activity showed that the maximal activity of this enzyme in both tissues was equivalent to the maximal activity of glyceraldehyde 3‐phosphate dehydrogenase (GAPD). The sum of these activities gave a measure of the maximum amount of acetyl‐coenzyme A that could be produced. In these tissues, but not in liver which does not exhibit aerobic glycolysis, this summed value exceeded the maximal activity of succinate dehydrogenase (SDH). Consequently, it suggested that where fatty acid oxidation is sufficient to supply all the acetyl‐coenzyme A required for the Krebs' cycle, that derived from fatty acid oxidation may inhibit pyruvate dehydrogenase causing accumulation of pyruvate which must be converted to lactate if pentose‐shunt activity is to be maintained.
Specific molybdenum binding to spectrin subunits
Authors: Mařík T. , Kselíková M. , Bíbr B. , Lener J.
Published: May 2005

Molybdenum in the form of its pentavalent complex binds primarily to spectrin when incubated with erythrocytes. Only the band 1 subunit is involved in this interaction thus indicating some structural differences between spectrin subunits.
In vitro cytotoxicity of a glass ionomer cement of a new generation
Authors: Meryon S. D. , Browne R. M.
Published: May 2005

The new glass ionomer dental cement, ChemFil, was found to be moderately cytotoxic when freshly mixed in vitro. This was assessed by a reduction in fibroblast and macrophage counts following 24 h exposure to the material, and by alterations in enzyme levels and enzyme staining kinetics relative to control cultures. Testing of set samples of the material showed that its toxicity decreased rapidly with setting. ChemFil behaved in a manner similar to two older glass ionomers, ASPA and ChemBond, although it was generally slightly less irritant.
Process of the combination of α‐lactalbumin with lysosomes in mammary epithelial cells
Authors: Komine Senichi , Nakanishi Keiko , Anzai Tadashi , Yoshimoto Akio
Published: May 2005

We previously reported that α‐lactalbumin combines with the lysosomal membrane of mammary epithelial cells and that it acts to release lysosomal enzymes. However, the details of this combination within the cells remained undetermined. We now report that 125I‐bovine‐α‐lactalbumin in the medium entered mouse mammary epithelial cells, and about 13 per cent of the α‐lactalbumin that entered the cell bound to lysosomes. About 75 per cent of the α‐lactalbumin that reached the lysosome was tightly bound to the lysosomal membrane. It appears that α‐lactalbumin in the secretory vesicles does not migrate out, because murine and bovine whey did not induce the release of Golgi enzymes in vitro.
Specificity of kirkman‐robbins hepatoma non‐histone chromatin proteins: Electrophoretic and immunological analyses
Authors: Krajewska Wanda M. , Kiliańska Zofia , Lipińska Anna , Klyszejko‐Stefanowicz Leokadia
Published: May 2005

The specificity of Kirkman‐Robbins hepatoma and hamster liver non‐histone chromatin proteins has been studied by comparing polypeptide patterns in polyacrylamide gel electrophoresis and by their immunological activity in the complement fixation test. Non‐histone proteins were separated from DNA with a polyethylene glycol‐dextran mixture and fractionated by hydroxylapatite chromatography into three classes named NHCP1, NHCP2, and NHCP3. Electrophoretic analysis indicated that among the non‐histone proteins of Kirkman‐Robbins hepatoma and hamster liver differences mainly of a quantitative nature can be observed. However, the polypeptides with molecular weight 25 000, 31 000, 36 000, 73 000 in NHCP1; 20 000, 40 000 in NHCP2 and 20 000, 32 000, 38 000, 44 000, 75 000, 80 000 in NHCP3 were found to be specific for hepatoma chromatin. Application of antibodies against NHCP1, NHCP2 and dehistonized chromatin of Kirkman‐Robbins hepatoma revealed that the highest specificity of NHCP2 eluted from hydroxylapatite with 100 mM phosphate buffer at pH 6·8. The NHCP1 of hepatoma shares some common antigenic determinants with analogous proteins of liver. On the other hand non‐histone proteins specific for heptoma dehistonized chromatin can be localized in the NHCP3 and partially in the NHCP1 fractions.
(Na‐K)ATPase activity along the nephrons in normal and adrenalectomized rats measured by quantitative cytochemistry
Authors: Snarski Joanna , Snarski Andrzej , Bachelet Marie , Bader Cyril , Ulmann André
Published: May 2005

A cytochemical method was used to measure total, ouabain insensitive and specific (Na‐K)ATPase activities along the rat nephron. Enzyme activity was expressed as per cent of mean integrated extinction with reference to a calibrated filter. The lowest mean values of total, ouabain‐insensitive, and (Na‐K)ATPase activities were found in the proximal convoluted tubule (PCT). In the distal convoluted tubule (DCT), total and ouabain‐insensitive activities (77·8 per cent and 45·8 per cent, respectively) were significantly higher than in the medullary thick ascending limb (MAL) (66·0 per cent and 24·6 per cent, respectively). Mean values of (Na‐K)ATPase activity were significantly lower in DCT than in MAL (32·0 per cent and 41·3 per cent, respectively). Using Lineweaver‐Burk plots, the KM ATP value for total ATPase activity was found to be 2·33, 1·79, and 3·63 mM in DCT, MAL, and PCT respectively. Maximal velocity was lower in PCT than in MAL and DCT. For (Na‐K)ATPase, the smallest KM value was found in MAL (0·95 mM) and was 2·73 and 5·71 mM in DCT and PCT respectively. Maximal velocity was the highest in MAL (49·3 per cent), lower in DCT (36·1 per cent) and least in PCT (22·5 per cent). ATPase was measured in the MAL and DCT from rats fed a normal (N‐Na+) or a high (Hi‐Na+) sodium diet, and from Hi‐Na+ rats one week after adrenalectomy (ADX). In the MAL, (Na‐K)ATPase tended to be higher in Hi‐Na+ than in N‐Na+ rats, but was significantly lower in ADX than in Hi‐Na+. A significant correlation was found between daily urinary Na excretion and (Na‐K)ATPase in N‐Na+ and Hi‐Na+ animals (UNa = 0·19 (Na‐K)ATPase − 3·10, n = 15, r = 0·53, P > 0·05). In the DCT, variations between N‐Na+ and ADX rats were minimal. ADX abolished the difference in (Na‐K)ATPase between MAL and DCT.

In summary, quantitative microdensitometry appears a suitable method for measuring (Na‐K)ATPase along the nephron, and this study indicates that the Na content of the diet and adrenal hormones modulate (Na‐K)ATPase in the MAL.
Improved rapid autoradiography with elimination of background
Authors: Spiridonidis Charalampos , Raza Azra , Zhao Shi‐Cheng , Preisler Harvey D.
Published: May 2005

A method has been developed to improve the quality of autoradiography. In addition to using high specific activity thymidine (3HTdr), low temperature exposure and scintillation fluid. FdUR was added to the cell suspension during incubation. It led to an increased amount of 3HTdr incorporation by the cells. By this method, the grain density over positive cells was increased and the background was minimal.
Utility and sensitivity of anti BrdU antibodies in assessing S‐phase cells compared to autoradiography
Authors: Raza Azra , Ucar Kalust , Bhayana Ranjan , Kempski Maureen , Preisler Harvey D.
Published: May 2005

A rapid and convenient method for estimating S‐phase cells in a population was developed which detects bromodeoxyuridine (BrdU) incorporation into DNA by means of monoclonal anti‐BrdU antibodies. This immunofluorescence technique (RPMB technique) was compared to autoradiographic (ARG) detection of tritiated thymidine (3HTdr) grains incorporated into the DNA. Using incubation periods for BrdU and 3HTdr ranging from one minute to one hour and detecting their incorporation by ARG and RPMB techniques, it became apparent that the RPMB technique was far more sensitive than ARG in addition to being extremely easy to perform. Some possible utilities of the RPMB technique are discussed.
Rat gastrointestinal transglutaminase: Demonstration of enzyme activity and cell and tissue distributions
Authors: Patel Ella K. , Bruce Sally E. , Bjarnason Ingvar , Peters Timothy J.
Published: May 2005

The properties, tissue and cellular distribution of intestinal transglutaminase have been investigated. Transglutaminase was assayed with dimethylcasein and [14C]putrescine as substrates. The enzyme has maximum activity at pH 10, although more reliable assays are made at pH 9. Transglutaminase showed an absolute requirement for Ca2+ and exhibited linear assay kinetics. The Km for putrescine was approx. 0·15 mmol/I.

Tissue distribution studies suggest transglutaminase is more active in the more muscular segments of the gut. The cellular localization in jejunum was investigated by sequential cell release techniques. Approximately 2 per cent of the total activity was found in the enterocytes and crypt cells. Most of the activity was in the submucosa and serosa suggesting an interstitial cell localization.

Acute hypoplastic enteropathy induced by methotrexate was accompanied by a striking decrease in mucosal transglutaminase but the activity returned to control values by 72 h. There was no significant increase in activity during the period of intense crypt cell hyperplasia and it is concluded that intestinal transglutaminase is not implicated in crypt cell proliferation.
Morphological changes in CHO and VERO cells treated with T‐2 mycotoxin. Correlation with inhibition of protein synthesis
Authors: Trusal Lynn R.
Published: May 2005

Exposure of Chinese hamster ovary and African green monkey kidney cells to T‐2 mycotoxin resulted in several morphological changes which were related to inhibition of protein synthesis, the basic in vitro mechanism of action of the toxin. These changes, which occurred in both cell types, included disassociation of polysomes and mitochondrial cristae alterations. In addition, CHO cells displayed membrane bleb formations similar to those found in CHO cells after exposure to established inhibitors of protein synthesis, puromycin and anisomycin. Blebs could be either a result of protein synthesis inhibition or a non‐specific early pathological response. Bleb formations were not observed in VERO cells under any experimental condition.
Heterogeneity of angiotensin II receptors in membranes of developing rat metanephros
Authors: Uva B. , Vallarino M. , Ghiani P.
Published: May 2005

Specific and high affinity binding sites for angiotensin II were demonstrated in the membranes of the developing rat metanephros during the second half of pregnancy and in the newborn by binding studies with 125I angiotensin II. Only one type of angiotesin receptor was found during intrauterine life while after birth two classes of angiotensin receptors were present in the membranes of the cortical renal tissue.
Effects of protease inhibitiors on adenylate cyclase activation and aldosterone production in rat adrenal zona glomerulosa cells
Authors: McAuley M. E. , Raven P. W. , Vinson G. P.
Published: May 2005

It has been shown that serine proteses are involved in aldosterone and 18‐hydroxycorticosterone production by the rat adrenal zona glomerulosa in response to a variety of stimulants. From evidence presented for various tissues, including the rat adrenal cortex, the observation that adenylate cyclase can be activated by proteolytic enzymes and inhibited by protease inhibitors has led to the suggestion that serine proteases may also be involved in the hormonal stimulation of adenylate cyclase. In studies designed to test this hypothesis using protease inhibitors, only high concentrations (>10−4M) of TAME (p‐tosyl‐L‐arginine methyl ester) inhibited ACTH stimulated steroid and cAMP production in rat adrenal glomerulosa cells. TPCK (tosyl‐L‐phenylalanine high concentrations) and TLCK (tosyl‐L‐lysine chloromethylketone) were found to have a similar effect at very high concentrations (10−2 M) but had no effect at the serine protease inhibitory concentration of 5 × 10−6 M. Other protease inhibitors tested had no effect on ACTH‐stimulated cAMP but the inhibitory effect of high concentrations of protease inhibitors on ACTH‐stimulated adenylate cyclase was duplicated by the polyanion dextran sulphate. The results suggest that the inhibitors act through non‐specific membrane effects and that proteases are not involved in the activation of zona glomerulosa adenylate cyclase by ACTH. In view of these findings it is concluded that a more rigorous approach should be applied to the use of protease inhibitors in whole cell systems, and that the concept of hormonal activation of adenylate cyclase via proteolytic events, which is based on studies with such inhibitors, should be reconsidered.
Differences among primates in defence against infection: Sensitivity of polymorphonuclear leukocytes to fMet‐Leu‐Phe
Authors: Suzuki Kazuo , Asaoka Kazuo , Takahashi Kenji , Fujikura Toshio
Published: May 2005

The sensitivity of polymorphonuclear leukocytes (PMN) to N‐formyl‐methionyl‐leucyl‐phenylalanine (fMet‐Leu‐Phe) for chemotaxis and for lysosomal enzyme release was examined using the PMN of four primate species, human (H. sapiens), chipanzee (P. troglodytes), rhesus monkey (M. mulatta), and cotton‐heded tamarin (S. (O) oedipus). The 50 per cent effective concentration (EC50) of fMet‐Leu‐Phe for chemoxaxis were 2·5 × 10−9 M in human, 10−9 M in chimpanzee, 8 × 10−8 M in rhesus moneky, and 3·3 × 10−6 M in tamarin. The EC50 values of fMet‐Leu‐Phe for myeloperoxides (MPO) release were 10−8 M in human, 4 × 10−8 M in chimpanzee, 4 × 10−8 M in rhesus monkey, and 10−6 M in tamarin and those for β‐glucuronidase release were 4 × 10−9 M, 6·4 × 10−8 M, 1·8 × 10−7 M, and 1·6 × 10−6 M, respectively. Thus, the sensitivity of fMet‐Leu‐Phe for chemotaxis was in the order: chimpanzee ≃ human > rhesus monkey > tamarin, and that for the release of lysosomal enzymes, MPO and β‐glucuronidase, was in the order: human > chimpanzee > rhesus monkey > tamarin.

These results appear to indicate that the sensitivity to fMet‐Leu‐Phe increases in the order of evolution of primates towards the human, and suggest that the sensitivity to fMet‐Leu‐Phe increases in the order of evolution of primates towards the human, and suggest that the sensitivity of PMN in the defence function against infection also increases in the same order.
Stimulation of tissue plasminogen activator production from epithelial cell lines
Authors: Electricwala Asgar , Griffiths Bryan
Published: May 2005

The aim of this study was to investigate the possibility of enhancing the yield of tissue plasminogen activator (tPA) from two epithelial cell lines of normal (non‐malignant) derivation grown in tissue culture. The three agents used in this investigation were chosen because of thier proven enhancing effect on analogous cells or products. The anabolic hormone stanozolol was found to have no significant stimulatory effect on these cell lines. A phorbol acetate (12‐O‐tetradecanoylphorbol 13‐acetate) caused a twofold enhancement in tPA yield but the most significant results were obtained with 5‐azacytidine. This agent increased the yield by up to fourfold in small stationary cultures and threefold in large‐scale microcarier cultures. A combination of azacytidine and phorbol acetate did not have an additive effect on total yield but did alter the kinetics of tPA expression with time. Indications were that the maximum yield with these types of potentiating agents was achieved as it could not be increased by using a combination of two different agents.
Fasting‐induced dissociation of cationic and secretory events in pancreatic islets
Authors: Carpinelli A. R. , Mathias P. C. F. , Leclercq‐Meyer V. , Malaisse W. J.
Published: May 2005

In pancreatic islets removed from 48 h‐fasted rats, as distinct from fed animals, the release of insulin evoked by D‐glucose is more severely impaired than that evoked by 2‐ketoisocaproate. This decreased secretory response to D‐glucose contrasts with an unimpaired cationic response to the sugar in terms of the glucose‐induced decrease in both 86Rb and 45Ca outflow from pre‐labelled islets. Likewise, fasting only causes a modest decrease of the secondary rise in 45Ca outflow evoked by D‐glucose in islets perifused at normal Ca2+ concentration. The latter decrease appears more marked, however, if the cationic response to glucose is expressed relative to that evoked by 2‐ketoisocaproate in islets removed from rats in the same nutritional state. It is concluded that, in the process of nutrient‐stimulated insulin release, neither the decrease in K+ conductance (inhibition of 86Rb outflow) nor the sequestration of Ca2+ by intracellular organelles and/or direct inhibition of Ca2+ outward transport (decrease in 45Ca outflow) represent the sole determinant(s) of the subsequent gating of Ca2+ channels (secondary rise in 45Ca efflux).
Effects of hydrocortisone on carbohydrase concentrations, de novo synthesis and turnover patterns in immature rat intestine
Authors: Tsuboi Kenneth K. , Kwong Linda K. , Fan Quing , Thompson Diana J. , D'Harlingue Arthur E. , Sunshine Philip
Published: May 2005

Hydrocortisone administration to infant rats enhanced cellobiase and maltase activities and induced precocious expression of sucrase and trehalase activities along the length of the small intestine. These activity changes reflected proportional concentration increases in the enzymes lactase (EC 3.2.1.23), maltase/glucoamylase (EC 3.2.1.20) and sucrase‐isomaltase (EC 3.2.1.48/10). Administration of an equivalent tracer dose of [3H]leucine (by body weight) to control and hydrocortisone‐treated infant rats resulted in greater accumulation of label in the carbohydrase pools of the treated rats, suggesting their increased de novo synthesis. The increased concentrations of lactase and maltase/glucoamylase induced by exogenous hydrocortisone were matched by the presence of corresponding greater amounts of label in their brush border pools. Accumulation of label in each of the lactase, maltase/glucoamylase and sucrase‐isomaltase pools was generally similar in the hydrocortisone‐treated rats, suggesting equivalent stimulation of their synthesis as a group by the humoral agent. The turnover rats of the carbohydrates as a group were found to be similar and did not appear to differ in control and hydrocortisone‐treated rats. Total protein synthesis rates were slightly greater in the intestine of the hydrocortisone‐treated group of rats.
Isolated adipocytes: An assessment of cell surface changes during their preparation
Authors: Al‐Jafari Abdulaziz A. , Lee Stephen R. , Tume Ronald K. , Cryer Anthony
Published: May 2005

A method is described, based on the detection of adipocyte‐specific cell surface antigens, which allows assessement of the relative surface damage incurred by the cells when they are prepared under a variety of conditions. Using the method it is possible to develop, for any set of reagents, a set of cell isolation conditions (collagenase concentration, time of incubation) which will produce minimally damaged cells which exhibit high levels of specific cell surface immunoreactivity. Under certain conditions a recovery from limited surface damage can be achieved, although, when cells are prepared under more extreme conditions irreversible surface damage occurs. The surface morphology of the cells as revealed by scanning electron microscopy, is also clearly affected by the conditions of cell isolation. The method has been used to define the conditions necessary for the isolation of cells to be used in the study of subtle biochemical responses.
Aggregation states of cyclic nucleotide phosphodiesterase of murine thymocytes
Authors: Sobolev A. S. , Rybalkin S. D.
Published: May 2005

In murine thymocytes cyclic nucleotide phosphodiesterase is represented by cAMP‐ and cGMP‐specific forms. cAMP and cGMP phosphodiesterase activities showed anomalous kinetic behaviour indicative of ‘low’ and ‘high’ affinity enzyme forms. Sucrose density gradient centrifugation resolved only ‘low’ affinity forms of cAMP and cGMP phosphodiesterases. Gel filtration on Ultragel Aca 34 column showed that cAMP and cGMP phosphodiesterases are probably oligomeric enzymes. Storage of enzyme preparation at 4°C for 24–48 h led to a decrease of higher molecular weight form and enhancement of cAMP and cGMP phosphodiesterase activities.
Synergism between cytosolic and mitochondrial oxidation: Its possible relevance to the metabolism of vitamin D3 in the kidney
Authors: Bachelet Maria , Lair Micheline , Thomas Michäel , Monet Dominique , Ulmann André , Bader Cyrille
Published: May 2005

Using frozen liver sections and quantitative cytochemistry it has been established that when cells are allowed to oxidize a cytoplasmic and a mitochondrial substrate simultaneously the resulting oxidative activity is markedly higher than the sum of the oxidation of each substrate measured separately. In the present study this type of synergistic interaction has been confirmed in the kidney, particularly in cells of the pars recta. Our results support the evidence of the influence of cytoplasmic NADPH on the intramitochondrial oxidative process and it is suggested that, in cells of the pars recta, cytosolic NADPH may be involved in intramitochondrial mixed function oxidases such as 1α‐hydroxylase: these results could further elucidate the mechanism responsible for the production of the hormonal form of vitamine D3.
Differentiation of myeloid cells in liquid culture. 1. Progenitor cells found in normal perpheral blood
Authors: Archimbaud Eric , Preisler Harvey D.
Published: May 2005

In order to establish a method for studying myeloid differentiation, light density, non‐adherent, T‐cell depleted mononuclear cells prepared from 26 normal peripheral blood buffy‐coats were cultured in McCoy' 5A medium supplemented with 15 per cent fetal calf serum (FCS) for three weeks at 37°C in a humidified 5 per cent CO2 atmosphere. The total number of viable cells in the cultures on weeks 1 and 2 represented 73 ± 10 per cent and 98 ± 41 per cent of the initial number of viable cells seeded. After one week, blasts represented 26 ± 10 per cent of the initial number of viable cells while all the initially contaminating mature granulocytes had disappeared. After two weeks, granulocytic differentiation was noted in most cultures and viable myelocytes and more mature cells represented 45 ± 26 per cent of the initial number of viable cells. The differentiation was independent on the lot of FCS used. The addition of PHA stimulated leukocyte conditioned medium to the cultures did not enhance granulocytic differentiation. The granulocytic differentiation observed in the absence of exogenous CSF persisted after removing the cells adhering to the bottom of the flasks on day 2 of the culture. An endogenous colony stimulating activity was detected in the cultures on week 3 but its intensity did not clearly correlate with the degree of granulocytic differentiation.
Biochemical aspects of the mechanism of action of antiarrhythmic drugs on mitochondria. VII. Effect on energy‐linked reactions and on membrane potential
Authors: Klüppel Maria Lúcia Wambier , Borba Hélcio Resende , Silveira Orieta , Lopes Luiz Carlos Vieira , De Paiva Campello Annibal
Published: May 2005

Effects of the antiarrhythmic drugs (propranolol, perhexiline maleate, lidoflazine and iproveratril) on energy‐linked reactions and on membrane potential were studied. Propranolol, perhexiline maleate and lidoflazine inhibit the ATPase activity of undamaged and broken mitochondria, and of submitochondrial particles. All drugs are inhibitors of either ATP‐driven or of succinate‐driven reduction of NADP+. The antiarrhythmics promote a decrease in the membrane potential upon energization of the mitochondrial membrane by α‐ketoglutarate, succinate, or ATP. It was suggested that these drugs have a primary action on the mitochondrial membrane, thus altering the activities of membrane proteins (channels and enzymes).
Differential hemoglobin synthesis in murino erythroleukemic cells: Hemin effects
Authors: Zucker R. M. , Umlas M. E. , Decal D. L. , Whittington K. B.
Published: May 2005

The addition of butyric acid to murine erythroleukemic cells (clone T3C12) induced the cells to differentiate, producing adult hemoglobin (A, α2,β2) and an embryonic hemoglobin (E2, α2Y2). The subsequent addition of hemin to the differentiating cells increased the synthesis of adult hemoglobin four‐fold and the synthesis of embryonic hemoglobin two‐fold; the relative synthesis of the α and β globins increased more than the Y globin. The embyronic hemoglobin was expressed prior to the adult hemoglobin in differentiating cells.
Isolation and characterization of parenchymal cells from normal and cirrhotic rat liver
Authors: Zimmermann Thomas , Franke Hans , Dargel Rolf
Published: May 2005

A technique is described for isolation of adult rat hepatocytes from micronodular cirrhotic livers based on a collagenase digestion procedure. Hepatocytes from normal livers and those chronically injured by thioacetamide did not differ with respect to the viability measured by the trypan blue exclusion test or to the cellular concentrations of protein and glycogen, but the triglycerde content of cells from cirrhotic livers was significantly reduced. Hepatocytes isolated from cirrhotic livers are ultrastructurally in a good state of preservation but they appear to be poorer than controls in RER membranes, although the well‐preserved mitochondria are somewhat richer in cristae. No differences were detected between the cell preparations in rates of gluconeogenesis and total de novo fatty acid synthesis, but the secreation of newly synthesized fatty acids was significantly reduced in cells from cirrhotic livers.

Thus adult rat hepatocytes can be isolated from thioacetamide‐induced micronodular cirrhotic livers with high yield and morphological integrity. Differentiated functions are maintained in suspension for at least 4 h.
Subcellular calmodulin distribution in rat liver after CCl4 poisoning
Authors: Barrera G. , Parola M. , Paradisi L. , Albin L. , Dianzani M. U.
Published: May 2005

Disturbed cellular calcium homeostasis has been observed during CCl4 poisoning, with an increase in calcium content 1 h after administration. Intracellular increase of calcium may be expected to alter membrane/cytosol distribution of calmodulin (CaM). This paper investigates changes in rat liver subcellular CaM distribution 30 min, 1 h and 2 h after CCl4 intoxication. The whole liver value remained unchanged, whereas the nuclear fraction increased and the microsomal and cytosolic fraction decreased. This may suggest that CaM is involved in the several liver cell alterations caused by CCl4 poisoning.
Biochemical and immunochemical determination of (Na+ + K+)‐ATPase content in subcellular fractions prepared from the avian salt gland: Evidence for enzyme heterogeneity
Authors: Addis John S. , Merrit William D. , Mazurkiewicz Joseph E. , Barrnett Russell J.
Published: May 2005

Subcellular membrane fractions were prepared from the salt glands of osmotically‐stressed ducklings. Two fractions were characterized biochemically with respect to (Na+ + K+)‐ATPase, alkaline phosphodiesterase I, succinate dehydrogenase, esterase, and galactosyltransferase activities and immunochemically with respect to (Na+ + K+)‐ATPase. The ratios of the estimates of the (Na+ + K+)‐ATPase contents obtained biochemically and immunochemically from the two fractions differed by more than 2 X. The results are consistent with the presence of at least two molecular species of (Na+ + K+)‐ATPase, unevenly distributed between the two fractions.
Inhibition of β‐adrenergic stimulated calcium pump of rat cardiac sarcoplasmic reticulum by tricyclohexyltin hydroxide
Authors: Sahib I. Kabeer Ahammed , Desaiah D.
Published: May 2005

The effects of tricyclohexyltin hydroxide (Plictran), an organotin acaricide, on 45Ca2+ uptake and Ca2+ ATPase were studied in vitro and in vivo in rat heart ventricular membrane vesicles, primarily sarcoplasmic reticulum. There was a concentration dependent inhibition of both 45Ca2+ uptake and Ca2+ ATPase in vivo as well as in vitro. Isoproterenol, a β‐adrenergic agonist, stimulated 45Ca2+ uptake and Ca2+ ATPase of sarcoplasmic reticulum and this was also inhibited by Plictran. Since cardiac relaxation is mediated by β‐adrenergic stimulation via Ca+ uptake by sarcoplasmic reticulum, the inhibition of calcium pump activity by Plictran may result in alterations in cardiac Ca2+ fluxes leading to cardiac dysfunction.
Differentiation of myeloid cells in liquid culture: 2. Acute myelocytic leukemia cells
Authors: Archimbaud Eric , Preisler Harvey D.
Published: May 2005

In a companion paper13 we demonstrated that normal peripheral blood granulocytic precursor cells differentiate after 2–3 weeks in suspension culture. In the studies described here leukemic blast cells obtained from 14 patients with acute myelocytic leukemia (AML) and two patients with chronic myelocytic leukemia in blastic crisis were cultured in McCoy's 5A medium containing 15 per cent fetal bovine serum for 2–3 weeks at 37°C in an atmosphere of 5 per cent CO2–95 per cent room air. ‘Spontaneous’ myeloid differentiation (20 × 104 viable mature myeloid cells ml−1) occurred in the cultures of cells obtained from 8 pts. The differentiation was granulocytic in three cases, monocytic in four cases and of mixed type in one case. Differentiation was independent of the growth of the cells in culture and occurred in four cases after the first week. Monocytic differentiation was seen only in AML of the FAB M4 type whereas granulocytic or mixed differentiation were seen only in AML of the FAB M1 or M2 types. When PHA leucocyte conditioned medium (PHA‐LCM) was added to the cultures monocytic/macrophage differentiation was favoured. Inducers of the differentiation of the HL‐60 cell line (N‐methylacetamide, cytosine arabinoside, or retinoic acid) had no consistent effect on the differentiation and were at times inhibitory. Three patients received therapy with low dose cytosine arabinoside and no correlation was observed between the outcome of the treatment and leukemic cell differentiation in culture in the presence of the drug.
Recovery of human erythrocytes from the echinocyte shape induced by added choline‐phospholipids is dependent on the acyl chain length
Authors: Tamura Akira , Sato Takashi , Fujii Tatsuzo
Published: May 2005

Addition of an amphiphilic lipid, such as phosphatidylcholine (PC) species with two identical saturated chains or lysophosphatidylcholine (lysoPC) species with one saturated acyl chain of various lengths, into a suspension of intact human erythrocytes resulted in lipid incorporation into the erythrocyte membrane to produce echinocytes (crenated cells). The altered shape gradually reverted on incubation at 37°C until the cells reassumed their normal disc shape. The rate of such recovery of shape increased with decreasing acyl chain length for both PC with C8–C12 acyl chains and lysoPC with a C14–C18 acyl chain, and was strongly influenced by incubation temperature. The identical rate of recovery of shape was observed for cells with normal, decreased or increased ATP content, implying that the metabolic state of the cell had no influence on the recovery process. Recovery of shape is therefore considered to be caused by translocation of the incorporated lipid molecules from the outer to the inner leaflet of the membrane lipid bilayer and the rate of recovery increases with decreasing hydrophobicity of the lipid.
Effect of cytochalasin b on glucose uptake, utilization, oxidation and insulinotropic action in tumoral insulin‐producing cells
Authors: Malaisse W. J. , Giroix M.‐H. , Sener A.
Published: May 2005

Cytochalasin B (17·3 μM) virtually abolished 3‐O‐methyl‐D‐[U‐14C]glucose uptake and D‐[5‐3H]glucose utilization in tumoral insulin‐producing cells of the RINm5F line. This coincided with a marked decrease in D‐[U‐14C] glucose oxidation and suppression of the stimulant action of D‐glucose upon insulin release. Cytochalasin B, however, augmented basal insulin release by the tumoral cells. The RINm5F cells appeared much more sensitive than normal islet cells to cytochalasin B, as judged by the relative magnitude of inhibition in either hexose uptake or utilization. In both cell types, the inhibitory action of cytochalasin B upon glucose metabolism seemed to be competitive, being more marked at low than high glucose concentration. These results are interpreted in support of the view that a decreased efficiency of hexose transport across the plasma membrane represents an essential deficiency of the RINm5F cells.
The preparative isolation of endosome fractions: A review
Authors: Mullock Barbara M. , Hinton Richard H. , Peppard Jane V. , Slot Jan W. , Luzio J. Paul
Published: May 2005

The endosome is an intracellular, acidic, membrane‐bound, subcellular compartment to which endocytosed ligands, receptors and plasma membrane proteins are conveyed before sorting and delivery to destinations elsewhere in the cell. The preparative isolation of elements of this compartment has been achieved successfully using various appropriate combinations of density gradient ultracentrifugation, electrophoretic, gel filtration and immunoaffinity techniques. These methods for isolating endosome fractions are reviewed together with the difficulties of establishing markers for such fractions. The isolation of an endosome fraction from the pathway of polymeric IgA transcytosis in rat liver is discussed to exemplify successful isolation procedures and appropriate subcellular markers.
A critical evaluation of the use of filipin‐permeabilized rat hepatocytes to study functions of the endoplasmic reticulum in situ
Authors: McEwen Bruce F. , Telford John N. , Handelman Caryl T. , Arion William J.
Published: May 2005

We have used transmission (TEM) and scanning electron microscopy (SEM) and leakage of lactate dehydrogenase (LDH; EC 1.1.1.27) to evaluate two published procedures which use filipin to render isolated rat hepatocytes permeable to ionic substrates. Cells treated by the procedure of Jorgenson and Nordlie6 retained less than 10 per cent of their LDH. TEM revealed severe damage to the internal structure of these cells, which included swelling, disintegration and extensive vesicularization of the endoplasmic reticulum (ER). Hepatocytes treated with filipin by the procedure of Gankema et al.13 retained 65–75 per cent of their LDH and displayed incomplete but highly variable permeability to Trypan blue. SEM revealed the loss of microvilli, other signs of swelling, and the presence of large lesions in the plasma membrane. TEM revealed signs of cell swelling, but the nuclei and the mitochondria were only moderately altered. The rough ER was not swollen, but significant fragmentation was evident and characteristic stacks of lamellar ER were never seen.

We conclude that useful information about the functions of the ER in situ cannot be obtained from studies of filipin‐treated cell. Our results indicate that retention of LDH is not a sufficient criterion of preservation of cell morphology and that staining with Trypan blue may significantly underestimate the permeability of cells to small ionic metabolites.
Age‐Dependent changes in rat liver lipid peroxidation and glutathione content induced by acute ethanol ingestion
Authors: Videla L. A. , Fernandez V. , Valenzuela A.
Published: May 2005

The study of the influence of the age of animals (13 to 53 weeks) on total liver thiobarbituric acid reactive substances (TBAR) content showed an increase which is maximal in rats of 39 weeks of age compared to young animals (13 weeks), followed by a dimunition in the 53 weeks old group. In this situation, the content of hepatic GSH and total GSH equivalents as well as the GSH/GSSG ratio were decreased with ageing, while GSSG levels were enhanced in the oldest group studied. Acute ethanol intoxication resulted in a marked increase in liver TBAR content in young animals, together with a decline in GSH, total GSH equivalents and GSH/GSSG ratio, and an enhancement in GSSG. These changes elicited by ethanol intake were reduced with ageing. It is concluded that ethanol‐induced oxidative stress in the liver is diminished during ageing, despite the progressive decrease in the glutathione content of the tissue observed in control animals.
Initial adhesion of murine fibroblasts to collagen and fibronectin occurs by two mechanisms
Authors: Bauvois Brigitte , Roth Stephen
Published: May 2005

The adhesion of Balb/c 3T12 cells to fibronectin (FN) and to denatured (DC) or native (NC) collagen is differentially sensitive to divalent cations and to sodium azide. Short‐time adhesion (10 min) to FN requires either Mg2+ or Mn2+, whereas only Mn2+ stimulates attachment to DC and NC. Azide treatment only slightly affects adhesion of cells to FN, but strongly inhibits cell attachment to DC and NC. Attachment to any of these substrata is unaffected by monensin and by treatment of the cells with an intracellular fraction, making unlikely the possibility that molecules released by secretion or cell lysis participate in the adhesive process. Soluble collagen inhibits the adhesion of cells to DC and NC, but does not affect adhesion to FN. Finally, rabbit antiserum against collagen binding proteins inhibits cell attachment to NC and DC; the cells, however, attach normally to FN in presence of this antiserum. Taken together, our results support the view that 3T12 cells attach directly to native or denatured collagens and that FN is not required for this process.
Inactivation of alpha‐1‐proteinase inhibitor in serum by stimulated human polymorphonuclear leucocytes. Evidence for a myeloperoxidase‐dependent mechanism
Authors: Shock Anthony , Baum Harold
Published: May 2005

Triggered polymorphonuclear leucocytes (PMNL) can decrease the elastase inhibitory capacity of serum by inactivating the main inhibitor of elastase alpha‐1‐proteinase inhibitor (alpha‐1‐PI). Maximal inactivation occurs with stimuli that release myeloperoxidase from PMNL along with hydrogen peroxide. Specific protection of alpha‐1‐PI function is obtained with antioxidants that interfere with this system. PMNL that are activated with phorbol myristate acetate release hydrogen peroxide but not myeloperoxidase, and only inactivate alpha‐1‐PI in the presence of exogenously‐added PMNL‐derived supernatants which contain this enzyme. Cell‐free inactivation requires both active enzyme and hydrogen peroxide, and is greatest at pH 6·2, the pH optimum for myeloperoxidase‐catalysed inactivation of alpha‐1‐PI. This data supports the notion that leucocyte myeloperoxidase may act to suppress the antiprotease screen afforded by alpha‐1‐PI by generating hypochlorous acid in the presence of chloride and respiratory burst‐derived hydrogen peroxide, and in the microenvironment of lowered pH associated with degranulation. Pulmonary emphysema seems to be associated with an imbalance between elastase and its inhibitors at the lung surface. PMNL are likely to play an important role in the pathogenesis of emphysema since they contain both elastase, which can solubilize connective tissue elastin, and the constituents of an oxidative system which can inactive the most important antielastase, alpha‐1‐PI.
Tightly‐bound non‐histone proteins in different nucleosome‐like subpopulations from pig kidney chromatin
Authors: Caiafa Paola , Tomassetti Antonella , Mastrantonio Stefania , Reale Anna , Spinelli Maurizio , Strom Roberto
Published: May 2005

By differential sucrose gradient centrifugation of pig kidney chromatin in the presence or absence of Na‐EDTA and under varying ionic strength conditions, three nucleosome‐like subpopulations with different buoyant densities can be obtained. These particles, on the basis of their histones and HMG protein pattern, of the 5‐methylcytosine level of their DNA and of the RNA polymerase activity associated with them, can be considered as originating from chromatin fractions differently involved in gene expression.

Two‐dimensional electrophoresis of the tightly‐bound non‐histone proteins shows a distinct pattern for each subpopulation, such protein components being notably present in restricted numbers but in high amounts in the subpopulation which was apparently derived from condensed heterochromatin.
Lipid peroxidation in purified plasma membrane fractions of rat liver in relation to the hepatoxicity of carbon tetrachloride
Authors: Le Page R. N. , Cheeseman K. H. , Osman N. , Slater T. F.
Published: May 2005

Preparations of rat liver sinusoidal plasma membrane have been tested for their ability to metabolize the hepatotoxin carbon tetrachloride (CCl4) to reactive free radicals in vitro and compared in this respect with standard preparations of rat liver microsomes. The sinusoidal plasma membranes were relatively free of endoplasmic reticulum‐associated activities such as the enzymes of the cytochrome P450 system and glucose‐6‐phosphatase. CCl4 metabolism was measured as (i) covalent binding of [14C]‐CCl4 to membrane protein, (ii) electron spin resonance spin‐trapping of CCl3· radicals and (iii) CCl4‐induced lipid peroxidation. By all of these tests, purified sinusoidal plasma membranes were found unable to metabolize CCl4.

The fatty acid composition of the plasma membranes was almost identical to that of the microsomal preparation and both membrane fractions exhibited similar rates of the lipid peroxidation that was stimulated non‐enzymically by γ‐radiation or incubation with ascorbate and iron. The absence of CCl4‐induced lipid peroxidation in the plasma membranes seems to be due, therefore, to an absence of CCl4 activation rather than an inherent resistance to lipid peroxidation.

We conclude that damage to the hepatocyte plasma membrane during CCl4 intoxication is not due to a significant local activation of CCl4 to CCl3· within that membrane.
Monoiodotyrosine formation during thyroglobulin processing in golgi vesicles
Authors: Bastiani P. , Rognoni J. B.
Published: May 2005

A golgi‐enriched subfraction was obtained from porcine throid glands by differential centrifugation. When incubated in a suitable medium, these vesicles were able to concentrate iodide from the medium and bind it to protein. The iodination process was inhibited by methylmercapto‐imidazole and was increased by the addition of an H2O2 generating system to the medium. Analysis of the protein content of the vesicles revealed the presence of 18 and 12–13 S thyroglobulin molecules, lacking mannose residues, and containing only monoiodotyrosine.

It is concluded that in vitro, iodination can begin before exocytosis, in the smooth‐surfaced vesicles derived from the golgi apparatus, as soon as N‐acetylglucosamine is incorporated onto the pre‐thyroglobulin molecule.
Ouabain‐insensitive active sodium transport in rat jejunum: Evidence from ATPase activities, Na uptake by basolateral membrane vesicles and in vitro transintestinal transport
Authors: Tosco M. , Orsenigo M. N. , Esposito G. , Faelli A.
Published: May 2005

The basolateral membrane of the jejunal enterocyte of the rat was separated by self‐orienting Percoll‐gradient centrifugation and further purified from brush border contamination. Pellets were analysed for Mg‐, Na‐ and (Na, K)‐ATPase activities. The uptake of 0·02 M NaCl was also followed by the rapid micro‐filtration technique. Transintestinal transport of fluid and electrolytes, and cell water, Na and K were determined in the in vitro everted and incubated jejunum.

There is ouabain‐insensitive Na‐ATPase in addition to the well‐known (Na, K)‐ATPase in the basolateral membrane. These are differently inhibited by furosemide and ethacrynate. Na uptake by osmotically active basolateral membrane vesicles is enhanced by ATP and a further enhancement is obtained if there is intravesicular K. The ATP effect is inhibited differently by strophanthidin, furosemide and ethacrynate. In the everted sac preparation, transintestinal transport of Na and fluid still occurs when the Na/K pump is totally inhibited by ouabain.

These experimental results suggest that there is also a ouabain‐insensitive Na pump, different from the Na/K pump, in the basolateral membrane.