bluuebunny/crossref_metadata_2025 huggingface dataset:
Proliferating cell nuclear antigen (PCNA/cyclin) in plant proliferating cells: Immunohistochemical and quantitative analysis using autoantibody and murine monoclonal antibodies to PCNA
Authors: Daidoji Hideyuki , Takasaki Yoshinari , Nakane Paul K.
Published: May 2005

Proliferating‐cell nuclear antigen (PCNA), also known as cyclin, is synthesized in proliferative cells and recently was identified as DNA polymerase‐delta auxiliary protein. In this paper, the association of PCNA to the proliferative cells of plants was analysed using both autoantibodies to PCNA obtained from a patient with systemic lupus erythematosus (SLE) and murine monoclonal antibodies.

By immunohistochemical analysis, nuclei of cells around the growing point in soybean root tips reacted strongly with autoantibodies to PCNA in the serum from a patient with SLE. The plant PCNA in root tip cells was purified by ammonium sulfate fractionation, DEAE chromatography, and affinity chromatography. The partially purified plant PCNA was tested by immunoblotting and a 34 kD polypeptide reacted with both the human anti‐PCNA autoantibody and a mouse monoclonal antibody against human PCNA (TOB 7). In addition, the purified plant PCNA reacted with both antibodies in enzyme‐linked immunosorbent assay (ELISA). The binding of anti‐PCNA serum to the animal PCNA was blocked by the plant PCNA in this ELISA. The association of PCNA with growing cells in plants was further confirmed by quantitative sandwich type ELISA using two murine monoclonal antibodies to PCNA, TOB7 and TO17. Those results suggested that PCNA in both plant and animal cells had the same immunological and biochemical characteristics and the plant PCNA might play an important role in cell growth, existing as it does in proliferating plant cells.

The concentration of PCNA in soybean germ extract before germination was less than 5 ng ml−1 (protein concentration, 6·8 mg ml−1), but that of the root tip stem including the growing point increased to 887 ng ml−1 (protein concentration 3·8 mg ml−1) in the second day after germination.
Modifications of adhesion properties and proteoglycan structure in rat embryo fibroblast cultures with increasing passages
Authors: Pippia P. , Formato M. , Sciola L. , Tilloca G. , Bardoni A. , Pallavicini G. , Passi A. , Rindi S. , Salvini R. , De Luca G.
Published: May 2005

Adhesion properties of rat embryo fibroblast cultures and proteoglycans (PGs) produced both in the growth medium and in the cell layer were investigated with increasing passages. Both cell–cell and cell–substrate adhesion increased with increasing subculture number. Cell adhesion properties were improved by cell treatment with chondroitinase ABC. The increase in subculture number was coupled with a constant increase of PG molecular size, which was particularly evident in cell layer extracts. The ratio HS–PGs/DS–PGs increased with increasing passages. PG modifications are likely to represent evidence of changes in extracellular matrix organization and could play a role in the increase of cell adhesion properties.
Ornithine decarboxylase and ornithine decarboxylase‐inhibiting activity in rat thymocytes
Authors: Stefanelli Claudio , Rossoni Carmen , Ferrari Fabrizia , Flamigni Flavio , Caldarera Claudio M.
Published: May 2005

Isolation of thymocytes from rat thymus resulted in the disappearance of the high activity of ornithine decarboxylase (ODC) that characterizes the thymus of young rats, together with the appearance of an antizyme‐like ODC inhibiting activity, which showed a chromatographic profile that resembled that of dexamethasone‐treated rat thymus. Omission of serum or addition of dexamethasone or spermidine did not affect appreciably the extent of the antizyme‐like activity. On the other hand, a variety of hormonal effectors, i.e. insulin, glucagon, adrenalin and T3, as well as the phorbol ester, PMA or the mitogen, concanavalin A (Con A) induced ODC activity in cultured thymocytes together with the disappearance of the antizyme‐like activity. A paradoxical, transient induction of ODC was caused by the transcriptional inhibitor, actinomycin D. Complexed ODC was detected in rat thymus, but not in thymocytes, either quiescent or stimulated by mitogens. These results indicate that thymic lymphocytes can express either ODC activity or its inhibitor depending on the hormonal and proliferative status of the cells.
Phosphate efflux from jejunal enterocytes of the rat: Effect of phosphate concentration gradient and pH
Authors: Al‐Sawan S. , Skillen A. W.
Published: May 2005

The effect of variation in the intracellular and extracellular phosphate concentration on the Pi efflux across the basolateral membrane of pre‐loaded enterocytes has been examined. Efflux rate constants for Pi fell from 0·89 h−1 to 0·68 h−1 as the extracellular Pi concentration was increased from 0·5 mM to 5 mM. As the intracellular Pi concentration was raised from 0·5 to 3 mM the rate constant dropped from 0·95 h−1 to 0·77 h−1. The findings are indicative of the presence of Pi‐specific transporter at the basolateral membrane. The efflux rate constant of Pi at pH 7·1 was higher than that at pH 7·4 suggesting that the Pi flux across the basolateral membrane of enterocytes follows a similar pattern towards pH changes as do fluxes across the brush‐border membrane.
Normal and Ha‐ras‐1 oncogene transformed buffalo rat liver (BRL) cells show differential resistance to cytoskeletal protein inhibitors
Authors: Theodoropoulos P. A. , Gravanis A. , Saridakis I. , Stournaras C.
Published: May 2005

In the present study, using immunofluorescence microscopy, we have demonstrated that normal and Ha‐ras‐1 transformed Buffalo rat liver (BRL) cells which were exposed to cytoskeletal protein inhibitors, showed a differential resistance of their microfilament and microtubule networks. One hour exposure of normal BRL cells to 10−5 M cytochalasin B provoked a clear and already total breakdown of actin filaments. However, at this concentration of cytochalasin B, the microfilaments of transformed BRLHO6T1–1 cells were not seriously affected; a higher cytochalasin B concentration (≥ 2 × 10−5 M) was required to induce a significant breakdown of microfilaments in these transformed cells. The two cell lines also demonstrated differential microtubule stability when they were treated with either colchicine or triethyllead. Three hours exposure to 10−6 M of either antimicrotubule agents was sufficient to disrupt the microtubules of normal BRL cell, without affecting their counterparts in the transformed BRLHO6T1–1 cells. A 10‐fold higher drug concentration (10−5 M) was required to induce microtubular breakdown in the transformed BRL cells. The differential stability of microfilaments and microtubules in normal and transformed BRL cells that was observed could not be attributed to a differential internalization of the agents, as shown by experiments on the uptake of [3H]‐cytochalasin B and triethyllead. In addition, the transformed BRLHO6T1–1 cells did not express altered actin and tubulin isoforms, as demonstrated by isoelectric focusing followed by immunoblotting analysis. We conclude that the transformation of BRL cells with the Ha‐ras‐1 oncogene results in a greater stability of microfilaments and microtubules, leading to a structurally firmer cell shape. Transformed BRLHO6T1–1 cells offer an interesting model for the study of the molecular and cellular mechanisms by which oncogenic transformation alters cytoskeletal structures.
Ethanol‐induced inhibition of ventricular protein synthesis in vivo and the possible role of acetaldehyde
Authors: Siddiq Tahir , Richardson Peter J. , Mitchell William D. , Teare Julian , Preedy Victor R.
Published: May 2005

We have determined the extent to which acute ethanol administration perturbs the synthesis of ventricular contractile and non‐contractile proteins in vivo. Male Wistar rats were treated with a standard dose of ethanol (75 mmol kg−1 body weight; i.p.). Controls were treated with isovolumetric amounts of saline (0·15 mol 1−1 NaCl). Two metabolic inhibitors of ethanol metabolism were also used namely 4‐methylpyrazole (alcohol dehydrogenase inhibitor) and cyanamide (acetaldehyde dehydrogenase inhibitor) which in ethanol‐dosed rats have been shown to either decrease or increase acetaldehyde formation, respectively. After 2·5 h, fractional rates of protein synthesis (i.e. the percentage of tissue protein renewed each day) were measured with a large (i.e. ‘flooding’) dose of L‐[4‐3H]phenylalanine (150 μmol (100 g)−1 body weight into a lateral vein). This dose of phenylalanine effectively floods all endogenous free amino acid pools so that the specific radioactivity of the free amino acid at the site of protein synthesis (i.e. the amino acyl tRNA) is reflected by the specific radioactivity of the free amino acid in acid‐soluble portions of cardiac homogenates. The results showed that ethanol alone and ethanol plus 4‐methylpyrazole decreased the fractional rates of mixed, myofibrillar (contractile) and sarcoplasmic (non‐contractile) protein synthesis to the same extent (by approx. 25 per cent). Profound inhibition (i.e. 80 per cent) in the fractional rates of mixed, myofibrillar and sarcoplasmic protein synthesis occurred when cyanamide was used to increase acetaldehyde formation. There was also a significant decrease in cardiac DNA content. The results suggest that acute ethanol‐induced cardiac injury in the rat may be mediated by both acetaldehyde and ethanol.
Change of liver metabolism of 1,2‐dibromoethane during simultaneous treatment with carbon tetrachloride
Authors: Chiarpotto E. , Biasi F. , Aragno M. , Scavazza A. , Danni O. , Albano E. , Poli G.
Published: May 2005

The combination of carbon tetrachloride (CCl4) and 1,2‐dibromoethane (DBE) in isolated rat hepatocytes led to a significant potentiation of both lipid peroxidation and of plasma membrane damage observed after a single treatment with CCl4. Such a synergistic effect appeared to be related to the CCl4‐induced shift of DBE metabolism from the cytosolic conjugation with glutathione towards the microsomal transformation into toxic intermediates. In fact, CCl4 significantly inactivated hepatocyte total GSH‐transferase, i.e. the DBE detoxification pathway. Furthermore, while the microsomal metabolism of CCl4 was not affected by the simultaneous presence of DBE, the amount of DBE reactive metabolities covalently bound to hepatocyte protein was significantly enhanced in the presence of CCl4.
Ability of different hepatoma cells to metabolize 4‐hydroxynonenal
Authors: Canuto R. A. , Muzio G. , Maggiora M. , Poli G. , Biasi F. , Dianzani M. U. , Ferro M. , Bassi A. M. , Penco S. , Marinari U. M.
Published: May 2005

4‐Hydroxynonenal (4‐HNE), produced during the oxidative lipid breakdown of biological membranes, modulates various biochemical processes in normal liver and in hepatoma cells. It is very probable that the effects of 4‐HNE are related to the quantity formed in the cells and the cells' ability to metabolize it. Aldehyde catabolism takes place within the cells through oxidative and reductive enzymes, and through conjugation with intracellular glutathione. In this paper, the various enzymatic pathways involved in the metabolism of 4‐HNE were studied in normal hepatocytes and in hepatoma cells. The hepatocyte pathway undergoes a complex variety of change during neoplastic transformation.

In hepatoma cells, generally, 4‐HNE metabolism was due mainly to aldehyde dehydrogenases, whereas in normal hepatocytes 4‐HNE metabolism was mainly due to alcohol dehydrogenase and glutathione‐S‐transferase. The increase in oxidative enzymes compared to normal tissue was not the same in all types of hepatoma: in HTC hepatoma cells, the enzyme levels were considerably higher; in AH‐130 hepatoma cells of Yoshida, they were lower in subcellular particles and similar in the cytosol. Indeed, consumption of externally‐added 4‐HNE in hepatoma cells was proportional to their content of 4‐HNE metabolizing enzymes.
Propranolol has direct antithyroid activity: Inhibition of iodide transport in cultured thyroid follicles
Authors: Murakami Saburo , Nasu Michiyo , Fukayama Harushisa , Krishnan Lalita , Sugawara Masahiro
Published: May 2005

The effect of propranolol on the process of thyroid hormone formation was studied in a physiological culture system. Porcine thyroid follicles were preincubated with propranolol for 24 h. Iodide transport, iodine organification, and de novo thyroid hormone formation were measured by incubating these follicles with the mixture of carrier‐free 0·1 μCi Na 125I and 50 nM NaI for 2 to 6 h at 37°C. A concentration of propranolol greater than 100 μM inhibited iodide transport in a dose‐dependent manner; this inhibition was non‐competitive with iodide and independent of thyrotropin (TSH). Reduced iodine organification and thyroid hormone formation was seen with 150 μM propranolol or greater. The inhibitory action of propranolol was not caused by beta‐blocking activity, since D‐propranolol (devoid of beta‐blocking activity) inhibited iodide transport, and other beta‐blockers (metoprolol, atenolol, and labetalol) did not inhibit iodide transport. The inhibition of iodide transport was most likely caused by membrane stabilizing activity since quinidine, which possess the same membrane stabilizing activity as propranolol, also inhibited iodide transport. TSH‐mediated cAMP generation and Na +K+ ATPase activity, membrane functions for iodide transport, were unaffected by propranolol.

Our study has shown, for the first time, that propranolol has a direct antithyroid action, namely inhibition of iodide transport in the intact thyroid follicle.
Isolation and cloning by a polymerase chain reaction of a genomic DNA fragment of the human slow skeletal troponin (TNNT1) gene
Authors: Novelli G. , Gennarelli M. , Sangiuolo F. , D'Agruma L. , Cicero S. Lo , Melchionda S. , Dallapiccola B.
Published: May 2005

The genomic 3′ structure of the gene coding for the human slow skeletal troponin T (TNNT1) gene, is reported. An intron of 912 nucleotides containing an Alu‐element has been identified and characterized. The complexity of the sequenced region suggests an alternative exon use. The present results may be valuable for further studies on the gene structure of TNNT1 and the related troponin gene family.
Intracellular calcium mobilization is triggered by clustering of membrane glycoproteins in concanavalin A‐stimulated platelets
Authors: Ramaschi Giuseppe , Torti Mauro , Sinigaglia Fabiola , Balduini Cesare
Published: May 2005

Stimulation of human platelets with concanavalin A resulted in a significant increase in the concentration of cytoplasmic free Ca2+. This effect was due to two different processes: Ca2+ mobilization from internal stores and Ca2+ influx from the extracellular medium. Kinetic analysis revealed that the release of Ca2+ from internal storage sites occurred sooner than the opening of plasma membrane Ca2+ channels. The ability of concanavalin A to induce a sustained increase in cytoplasmic Ca2+ concentration was antagonized and reversed by methyl ∝‐D‐mannopyranoside, demonstrating that it was promoted by the interaction of the lectin with cell surface glycoproteins. Succinyl–concanavalin A, a dimeric derivative of the lectin, that does not promote patching/capping of the receptor, was able to bind to the platelet surface, and antagonized the effects of native concanavalin A. In addition, succinyl–concanavalin A, per se, was unable to induce Ca2+ mobilization in human platelets. Therefore, the action of the native concanavalin A was mediated by receptor clustering events. Concanavalin A mobilized Ca2+ from the same internal stores from which Ca2+ was mobilized in response to strong platelet agonists, such as thrombin and arachidonic acid. However, while thrombin was ineffective in inducing Ca2+ release after stimulation of platelets with Con A, Con A was able to cause a full discharge of Ca2+ from internal stores even in platelets previously stimulated with thrombin. These results demonstrate for the first time that the clustering of specific membrane glycoproteins can trigger platelet activation. The physiological implications during platelet aggregation are discussed.
PTH induces modification of transductive events in otosclerotic bone cell cultures
Authors: Fanó G. , Venti‐Donti G. , Belia S. , Paludetti G. , Antonica A. , Donti E. , Maurizi M.
Published: May 2005

We studied the effect of PTH (10–100 nM) on transductive mechanisms (adenylate cyclase activity, Ca2+ metabolism, IP3 levels) in cell cultures derived from normal and otosclerotic human bone fragments. The cultured cells were osteoblast‐like but with calcitonin‐receptors still present and with PTH receptors coupled with the adenylate cyclase system.

The results showed that PTH activated adenylate cyclase and increased the intracellular Ca2+ levels with qualitative and quantitative differences between the two cellular populations. In particular, otosclerotic cells responded less to hormone stimulation, which is in accord with the current hypothesis of a desensitization of the receptor/enzyme complex associated with the pathological status.
Estimation of metabolic flux rates in liver purine catabolism of tumour‐bearing mice by computer simulation of radioactive tracer experiments
Authors: Siems Werner G. , Schwendel Anke , Grune Tilman , Holzhütter Hermann‐Georg , Uhlig Rolf
Published: May 2005

Mouse hepatocytes from healthy control mice and from Ehrlich ascites tumour‐bearing mice were used for tracer‐kinetic studies of purine catabolism of liver cells during different periods of tumour growth. The dynamics of the radioactive tracers were modelled mathematically by a system of differential equations. Computer simulations, i.e. direct fitting of numerical solutions of these equations to the observed time‐courses of metabolites and specific radioactivites, enables one to estimate unknown kinetic parameters of a simplified model of pathways of hepatic purine catabolism in tumour‐bearing mice.

There occurred great differences of metabolic flux rates between control hepatocytes, hepatocytes of mice during the proliferating period of tumour growth (6th day after inoculation of the tumour) and hepatocytes of mice during the resting period of tumour growth (12th day after inoculation of the tumour). The final purine degradation of hepatocytes prepared during the proliferating period was lower in comparison with that of control hepatocytes, but it was markedly higher in hepatocytes prepared during the resting period of tumour growth. The changes in hepatocyte purine catabolism during the proliferating period of tumour growth argue for transitions which aim at the maintenance of high purine nucleotide levels in the liver itself rather than for an increased nucleoside and nucleobase supply for the tumour. This suggestion is in accordance with the increased ATP level of the liver during the proliferating phase of tumour growth. The drastic acceleration of the final steps of hepatic purine catabolism forming uric acid and allantoin during the resting period of tumour growth was predominantly due to increased flux rate from xanthosine and guanine in accordance with increased catabolism of monophosphorylated nucleotides.
The preparation and kinetic properties of multiple forms of chicken brain acetylcholinesterase
Authors: Al‐Jafari Abdulaziz A. , Kamal M. A.
Published: May 2005

A method for preparing various forms of acetylcholinesterase (A ChE) from chicken brain has been developed and they have been characterized in terms of kinetic parameters such as Km, rate constant (k), turnover number (kp), specificity constant (ksp), Vmax and half‐life (t1/2). The solubility experiments show that, there are two major forms of A ChE i.e. water‐soluble and membrane‐bound A ChE (MBA ChE). The MBA ChE shows several subforms, and on the basis of percentage activity only three MBA ChE forms have been selected for complete characterization by various kinetic parameters. It was found that these three forms of MBA ChE demonstrate significant differences in their kinetic properties.
No discrete complexes containing DNA polymerase α activity can be solubilized from the heat‐stabilized nuclear matrix prepared from HeLa S3 cells
Authors: Martelli Alberto M. , Cocco Lucio
Published: May 2005

Most of the DNA polymerase α activity, bound to the heat‐stabilized nuclear matrix prepared from HeLa S3 cells, was released as a matrix extract by sonication. When the extract was centrifuged in a 5–20 per cent linear sucrose gradient no definite peaks of activity could be identified. Most of the activity sedimented to the bottom of the tube under all the conditions tested, whilst the remaining activity was associated with matrix fragments of various and irregular size. No 10 S complexes, containing polymerase activity, were seen after incubation of the extract for 16 h before centrifugation. Other solubilization procedures (i.e. treatment of the matrix with chelating agents, high pH associated with reducing agents, ionic and nonionic detergents) failed to produce release of matrix‐bound DNA polymerase α activity. In contrast, we released 10 S complexes, containing polymerase activity, from the matrix prepared from nuclei not exposed to heat. We conclude that a 37°C incubation of isolated nuclei before extraction with 2 M NaCl and DNase I digestion causes DNA polymerase α to bind to the nuclear matrix in a form that cannot subsequently be released as discrete components, at variance with previous results obtained with the matrix prepared from regenerating rat liver.
Testing Drugs on Human Osteoarthritic Articular Cartilage
Authors: Chayen J. , Bitensky Lucille , Mehdizadeh S. , Dunham Jane , Older J.
Published: May 2005

New drugs are generally developed against animal models of the human disease. Before they are subjected to clinical trials it might be helpful to be able to test whether they are as effective against the disease in human tissue as they were in animals. It is proposed that this can be achieved by the use of organ maintenance culture of the human diseased tissue, the relevant biochemical parameters being measured by quantitative cytochemistry. In the present studies differences between the effect of indomethacin and of the ‘chondroprotective’ drug diclofenac sodium, on human osteoarthritic cartilage, have been measured.
Modulatory effect of dexamethasone on ornithine decarboxylase activity and gene expression: A possible post‐transcriptional regulation by a neutral metalloprotease
Authors: Nguyen‐Ba Giao , Robert Sophie , Dhalluin Stephane , Tapiero Haim , Hornebeck William
Published: May 2005

The intracellular effect of dexamethasone (DXME) on the activity and gene expression of ornithine decarboxylase (ODC) was studied in Syrian hamster embryo cells (SHE). The ODC activity (expressed as nmoles decarboxylated ornithine mg−1 protein h−1) was 4·61 ± 0·14 in untreated cells, whereas it increased to 14·38 ± 0·26 after 5h treatment with 1·6 × 10−7 M TPA. In contrast, DXME (2·5 × 10−5 M) reduced the ODC activity by 50 per cent to 2·35 ± 0·22. In cells co‐treated for 5 h with TPA and DXME, ODC acitivity decreased to the level of the untreated cells. However, when DXME was added 3 h after TPA treatment for 2 h, in the continuous presence of TPA, the ODC activity unexpectedly increased further to 16·44 ± 1·05. The modulation of ODC activity correlated partly with the level of ODC mRNA. Thus when cells were treated with TPA, and ODC mRNA increased threefold, whereas it decreased by 30 per cent when the cells were exposed to DXME. In TPA–DXME co‐treated cells, as in TPA pretreated cells followed by DXME for 2 h, a decrease (31·25 per cent and 12·5 per cent respectively) was observed in ODC mRNA. In turnover studies, DXME was found to increase the stability of ODC; the discrepancy between ODC activity and ODC mRNA levels could result from an inhibitory effect of the corticoid on proteolysis of ODC.

Studies of lysosomal protease showed that the activities of cathepsins L, B and H decreased following TPA treatment. DXME also inhibited cathepsin L and B activities, but stimulated cathepsin H. Analysis of neutral cytosolic protease activity by gelatin and casein zymograms showed that TPA strongly stimulated the activity of a 70 kDa gelatinase. DXME was able to inhibit the induction of such cytosolic proteases under all the treatment conditions. When the cytosolic protein was incubated in vitro, at 37°C, in the presence of 2 mM CaCl2, the ODC activity decreased by 50 per cent after 30 min incubation. Further decrease was achieved when p‐amino‐phenylmercuric acetate, a protease activator, was added. Proteolytic activity was not inhibited after the addition of 10 μg ml−1 of TIMP‐1. In contrast, the addition of EDTA restored the ODC activity completely.

We postulate that modification of the 70 kDa cytosolic metalloprotease activity could interact with the post‐transcriptional regulation of ODC.
Catalytic properties of DNA polymerase α activity associated with the heat‐stabilized nuclear matrix prepared from HeLa S3 cells
Authors: Martelli Alberto M. , Bareggi Renato , Narducci Paola
Published: May 2005

We have investigated whether or not ATP or other nucleoside di‐ and trisphosphates (including some nonhydrolysable ATP analogues) can stimulate the activity and/or the processivity of DNA polymerase α associated with the nuclear matrix obtained from HeLa S3 cell nuclei that had been stabilized at 37°C prior to subfractionation, as has been reported previously for DNA polymerase α bound to the nuclear matrix prepared from 22‐h regenerating rat liver. We have found that HeLa cell matrix‐associated DNA polymerase α activity could not be stimulated at all by ATP or other nucleotides, a behaviour which was shared also by DNA polymerase α activity that solubilizes from cells during the isolation of nuclei and that is thought to be a form of the enzyme not actively engaged in DNA replication. Moreover, the processivity of matrix‐bound DNA polymerase α activity was low (< 10 nucleotides). These results were obtained with the matrix prepared with either 2M NaCl or 0·25 M (NH4)2SO4 and led us to consider that a 37° incubation of isolated nuclei renders resistant to high‐salt extraction a form of DNA polymerase α which is unlikely to be involved in DNA replication in vivo.
Glycogen synthase, glycogen phosphorylase and α‐amylase activity in homogenates of islets of GK rats: Comparison with hepatic and pancreatic extracts
Authors: Zhang Tie‐Mei , Östenson Claes‐Göran , Malaisse Willy J.
Published: May 2005

Glycogen accumulation in pancreatic islet cells in situations of sustained hyperglycaemia may participate in the phenomenon of so‐called B‐cell glucotoxicity. Unexpectedly, however, previously little if any glycogen was found in islet cells of non‐insulin‐dependent diabetic Goto‐Kakizaki rats (GK rats). Therefore, the activities of glycogen synthase, glycogen phosphorylase and α‐amylase were measured in islets of control and GK rats. No significant difference in enzymatic activity was observed between the control and diabetic animals. In the liver, the activity of glycogen synthase appeared even somewhat higher in GK rats than in control animals. It is concluded that the diabetic syndrome in the GK rats does not involve any major anomaly of glycogen synthase and glycogen phosphorylase activity in the liver of these animals, as well as α‐amylase, in pancreatic islets.
Taxol causes rapid gross structural rearrangement of a native microtubule bundle
Authors: Hunt Cherryl , Stebbings Howard
Published: May 2005

Taxol is an anti‐mitotic agent now being used in the treatment of some cancers, although the manner of its interaction with the microtubular components of the cytoskeleton is still not fully characterized. Here we report the effects of taxol upon a huge, naturally occurring and experimentally amenable aggregate of parallel microtubules from the ovaries of hemipteran insects. Within seconds of exposure to taxol, the microtubule aggregate began to twist upon itself. After a few minutes this movement was complete, the drug having brought about a gross rearrangement of the microtubules, involving coiling on a massive scale. The final form assumed by the microtubule array was influenced by pH and by the presence of microtubule‐associated proteins, salt, cations, and both hydrolysable and non‐hydrolysable nucleotides. The possible mechanisms leading to this rapid structural change are considered.
Uric acid synthesis by rat liver supernatants from purine bases, nucleosides and nucleotides. Effect of allopurinol
Authors: Bleisch Siegfried , Sillero Maria A. Günther , Torrecilla Amparo , Sillero Antonio
Published: May 2005

The synthesis of uric acid from purine bases, nucleosides and nucleotides has been measured in reaction mixtures containing rat liver supernatant and each one of the following compounds at 1 mM concentration (except xanthine, 0·5 mM and guanosine and guanine, 0·1 mM). The rates of the reaction, expressed as nanomoles of uric acid synthesized g−1 of wet liver min−1 were: ATP, 10; ADP, 37; AMP, 62; adenosine, 108; adenine 6; adenylo‐succinate, 9; IMP 32; inosine, 112; hypoxanthine, 50; GTP, 19; GDP, 19; GMP, 27; guanosine, 34; guanine, 72; XMP, 10; xanthosine, 24; xanthine, 144. These figures divided by 55 correspond to nanomoles of uric acid synthesized min−1 per mg−1 of protein. The rate of synthesis of uric acid obtained with each one of those compounds at 0·1 and 0·05 mM concentrations was also determined. ATP (1 nM) strongly inhibited uric acid synthesis from 0·05 mM AMP (91 per cent) and from 0·05 mM ADP (88 per cent), but not from adenosine. CTP or UTP (1 mM) also inhibited (by more than 90 per cent) the synthesis of uric acid from 0·05 mM AMP. Xanthine oxidase was inhibited by concentrations of hypoxanthine higher than 0·012 mM. The results favour the view that the level of uric acid in plasma may be an index of the energetic state of the organism. Allopurinol, besides inhibiting uric acid synthesis, reduced the rate of degradation of AMP. The ability of crude extracts to catabolize purine nucleotides to uric acid is an important factor to be considered when some enzymes related to purine nucleotide metabolism, particularly CTP synthase, are measured in crude liver extracts.
Glucocorticoid‐induced changes in liver: Effect of dexamethasone administration on DNA topoisomerase I and II activities and distribution of histone H1 subtypes
Authors: Goodlad George A. J. , Clark Catherine M.
Published: May 2005

The activities of DNA topoisomerase I and II and the relative proportions of the histone H1 subtypes were investigated in rat liver which was undergoing hypertrophy and exhibiting increased transcriptional activity following the administration of dexamethasone. There was a rise in the level of activity of DNA topoisomerase I and a slight fall in that of DNA topoisomerase II. The relative proportions of the H1 subtypes were altered due to a preferential increase in H1.1. The results are discussed in relation to the effect of glucocorticoids on the transcription and replication of hepatic DNA.
Activation of phosphoinositide‐specific phospholipase C of rat neutrophils by the chemotactic aldehydes 4‐hydroxy‐2,3‐trans‐nonenal and 4‐hydroxy‐2,3‐trans‐octenal
Authors: Rossi Maria Armida , Di Mauro Clelia , Esterbauer Hermann , Fidale Fulvio , Dianzani Mario Umberto
Published: May 2005

A comparison has been made between the effects of 4‐hydroxy‐2,3‐trans‐nonenal (HNE) and 4‐hydroxy‐2,3‐trans‐octenal (HOE), two lipid peroxidation products, on the basal and GTPgammaS‐stimulated activities of phosphoinositide‐specific phospholipase C (PL‐C) of rat polymorphonuclear leukocytes. PL‐C activity was determined in vitro by measuring the hydrolysis of [3H] phosphatidylinositol‐4,5‐bis‐phosphate (PtdIns‐P2) added as exogenous substrate to neutrophil plasma membranes. PL‐C was activated by concentrations of HNE ranging from 10−8 to 10−6 M both in the presence and in the absence of 2 × 10−5 M GTPgammaS; HOE stimulated the enzymatic activity between 10−11 and 10−8 M; maximal stimulation was given by 10−11 M HOE plus GTPgammaS. The aldehyde concentrations able to accelerate PtdIns‐P2 breakdown displayed a good correspondence with those which have been reported to stimulate the oriented migration of rat neutrophils. Pretreatment of neutrophils with pertussis toxin prevented the stimulation of PL‐C by 10−11 M HOE and by HOE plus GTPgammaS. Our results suggest that the chemotactic action of HNE and HOE might depend on the activation of PL‐C; furthermore a regulatory G protein appears to be involved in the acceleration of PtdIns‐P2 turnover by HOE.
Release of arachidonic acid via Ca2+ increase stimulated by pyrophosphonucleotides and bradykinin in mammary tumour cells
Authors: Enomoto Koh‐ichi , Furuya Kishio , Yamagishi Shunichi , Oka Takami , Maeno Takashi
Published: May 2005

The relationship between the increase of intracellular Ca2+ and the release of arachidonic acid by bradykinin and pyrophosphonucleotides was studied in cultured mammary tumour cells, MMT060562. Bradykinin, ATP, UTP and UDP induced an increase of intracellular Ca2+ and the release of arachidonic acid from phospholipids into the extracellular fluid. Release of arachidonic acid was also induced by the application of the Ca2+ ionophore, A23187. Liberation of arachidonic acid by bradykinin and ATP was reduced by mepacrine, a blocker of phospholipase A2 and W‐7, a calmodulin antagonist.

It is suggested that the increase in cytosolic Ca2+‐induced release of arachidonic acid occurs through activation of calmodulin‐dependent phospholipase A2.
Increase of cyclin B by overexpression of cystatin α
Authors: Hiwasa Takaki , Ma Jun , Ike Yoshimasa , Katunuma Nobuhiko , Sakiyama Shigeru
Published: May 2005

Degradation of cyclin B was effectively suppressed when cells were treated with ALLN (N‐acetylleucylleucylnorleucinal) which inhibits proteasome, calpain and cysteine proteinase cathepsins. In order to examine which protease degrades cyclin B, the effect of a cathepsin inhibitor, cystatin α, was investigated. The cystatin α gene was inserted into an inducible expression vector, pMSG, and transfected into NIH3T3 mouse fibroblasts. The expression of cystatin α was induced effectively in the transfected cells after treatment with dexamethasone. Overexpression of cystatin α resulted in an increase of the amount of cyclin B, suggesting that cysteine proteinase cathepsins might be involved in the degradation of cyclin B.
Reduction of extracellular dehydroascorbic acid by K562 cells
Authors: Schweinzer Esther , Mao Yulu , Krajnik Paul , Getoff Nicola , Goldenberg Hans
Published: May 2005

K562 erythroleukaemic cells produced ascorbate when incubated with dehydroascorbic acid. The reduction depended on the number of cells and on the concentration of dehydroascorbic acid. The observed rate consists of a high affinity (apparent) Km 7 μM, Vmax 3·25 pmol min−1 (106 cells)−1 and a low affinity component, which was non‐saturable up to 1 mM of DHA (rate increase of 0·1 pmol min−1 (106 cells)−1 (1 μM of DHA−1). The rate was dependent on temperature and was stimulated by glucose and inhibited by phloretin, N‐ethylmaleimide, parachloro‐mercuribenzoate and thenoyltrifluoroacetone. Although uptake of DHA proceeded at a higher rate than its extracellular reduction, the generation of extracellular ascorbate from DHA cannot be accounted for by intracellular reduction and the release of ascorbate, since the latter was not linear with time and had an initial rate of approximately 3 pmol min−1 (106 cells−1). At a concentration of DHA of 100 μM this is 25 per cent of the observed reduction.
Interaction between Na ions and the Cl/HCO3 exchanger in basolateral membranes from rat jejunum
Authors: Orsenigo M. N. , Tosco M. , Faelli A.
Published: May 2005

The jejunal basolateral Cl/HCO3 exchanger is modulated by two Na‐dependent regulatory sites located on the inner and outer membrane surfaces. The aim of this work was to focus on the interaction between the anion exchanger and intracellular or extracellular sodium. Uptake studies, performed using basolateral membrane vesicles, provided kinetic parameters as a function of outside or inside Na concentration. The intracellular Na‐sensitive modifier site seems to be primarily involved in the modulation of the Cl/HCO3 exchanger.
Alteration of erythrocyte membrane Na, K‐ATPase in children with borderline or essential hypertension
Authors: Stojadinovic Nataša D. , Petronijević Milan R. , Pavićević Mirjana H. , Mršulja Bogomir B. , Kostić Milosav M.
Published: May 2005

The aim of this study was to evaluate the substrate (ATP) kinetics of erythrocyte membrane Na, K‐ATPase in children with borderline or essential hypertension. Although the activity of Na, K‐ATPase in the presence of in vivo concentrations of ATP was not significantly altered, kinetic studies showed an obvious inhibition of enzyme activity in the erythrocyte membrane of children with borderline or essential hypertension. Hanes plot analysis revealed a decrease of Vmax from 7·19 in erythrocytes from control subjects to 4·93 and 3·33 in those from children with borderline or essential hypertension, respectively. A mean value of the Km decreased from 0·10 in the control to 0·08 and 0·02 in children with borderline or essential hypertension, respectively. The energy status of erythrocytes, estimated by ATP, ADP and AMP levels, ATP/ADP ratio, and adenylate energy charge (AEC) was not significantly changed in the cells from hypertensive children. The use of a free radical‐generating system (FeSO4/ascorbate) in vitro significantly reduced enzyme activity in the control erythrocytes while in those from hypertensive children it was abolished completely. The level of lipid peroxides was considerably higher (+37 per cent) in the plasma, while that of reduced glutathione was significantly lower both in the erythrocytes and the plasma of children with essential hypertension than in healthy children. These results indicate significant alterations of the antioxidant status which could be the cause of the inhibited Na,K‐ATPase activity in erythrocyte membranes from hypertensive children.
Metabolism of glucose and glutamine in lymphocytes from graves' hyperthyroid patients: influence of methimazole treatment
Authors: Werner M. C. , Rosa L. F. B. P. Costa , Romaldini J. H. , Curi R.
Published: May 2005

Several studies have shown that thyroid hormones are able to influence selected immune responses such as cell mediated immunity, differentiation of B lymphocytes and the activity of NK cells. These hormones can also regulate the metabolism of glucose and glutamine in rat macrophages and their effects seem to occur mainly through the Krebs cycle. Alterations in the hexokinase, citrate synthase, glucose‐6‐phosphate dehydrogenase and glutaminase activities in lymphocytes from patients with Graves' disease, either untreated or on methimazole (MMI) therapy were investigated. Experiments were also done in vitro to determine the activities of these enzymes in normal lymphocytes cultured for 24 h in the presence of MMI, T3 and T4 using concentrations close to the physiological. Changes in the conversion of [U‐14C]‐glucose and [U‐14C]‐glutamine to 14CO2 as caused by the addition of MMI, T3 or T4 to the culture medium were also evaluated. The results indicate that high levels of thyroid hormones might stimulate the metabolism of glucose and glutamine for a short period of time but, if the stimulus is maintained, the utilization of glutamine by lymphocytes is then suppressed. Moreover, MMI does affect lymphocyte metabolism but the significance of this finding for its immunosuppressive effect remains to be examined.
Characterization of γ‐glutamyltranspeptidase in the liver of the frog: 3. Response to freezing and thawing in the freeze‐tolerant wood frogRana sylvatica
Authors: Hemmings Susan J. , Storey Kenneth B.
Published: May 2005

The freeze tolerant wood frogRana sylvaticawas studied to determine the impact of the freezing and thawing of this frog on the activity of γ‐glutamyltranspeptidase in the liver. On exposure to −2·5°C, for 1, 12 and 24 h, frogs were found to be cool, covered with ice crystals and frozen, respectively. Thawing for 24 h at 4°C recovered the frogs completely. A 45 per cent decrease in the liver weight: body weight ratio was notable after 1 h at −2·5°C, suggestive of an early hepatic capacitance response. A glycemic response to freezing was observed: blood glucose levels exhibited a 55 per cent decrease after 1 h at −2·5°C on cooling; a 10·5‐fold increase after 12 h at −2·5°C on the initiation of freezing; and a 22‐fold increase after 24 h at −2·5°C in the fully frozen state. Blood glucose levels remained elevated four‐fold in the thawed state. Plasma insulin levels were increased twofold in the frozen state and 1·8‐fold in the thawed state, while plasma ketone levels were increased 1·8‐fold in the frozen state and 1·5‐fold in the thawed state. Plasma total T3levels were decreased by 22 per cent in the frozen state and normalized on thawing.

In homogenates and plasma membranes isolated from the livers ofRana sylvatica, the activity of γ‐glutamyltranspeptidase was found to be elevated at all stages of the freeze–thaw process. After 1, 12 and 24 h at −2·5°C, activities were increased 2·5‐, 2·3‐, 2·4‐fold respectively in the homogenates and 2·5‐, 2·2‐, 2·4‐fold respectively in the plasma membranes. After thawing, activities were still increased 1·9‐fold in both homogenates and plasma membranes. In homogenates prepared from the kidneys ofRana sylvatica, the activity of γ‐glutamyltranspeptidase was increased 1·4‐fold after 1 h at −2·5°C after which it returned to normal. The role of thyroid hormone in producing the increase in γ‐glutamyltranspeptidase in the liver ofRana sylvaticain response to freezing is discussed as is the significance of the enzyme increase in terms of hepatic cytoprotection and freeze tolerance.
Hepatic fatty acid‐supported respiration in rats fed an energy‐dense diet
Authors: Liverini Giovanna , Iossa Susanna , Mollica Maria P. , Lionetti Lillà , Barletta Antonio
Published: May 2005

The energy balance and hepatic fatty acid‐supported respiration were studied in rats fed a control or an energy‐dense diet. In addition, state 3 and 4 respiratory rates as well as ketone body production with palmitoylcarnitine as substrate were determined in isolated mitochondria. Metabolizable energy intake and energy expenditure increased in rats fed an energy‐dense diet, but the gain in body weight and lipid content remained unchanged. No variation occurred in the mitochondrial palmitoylcarnitine utilization rate and ketone body production, but a significant increase in the mitochondrial content of ketone bodies and the serum levels was found in rats fed an energy‐dense diet. Furthermore, we have shown a significant increase in fatty acid‐stimulated respiration in hepatocytes from rats fed an energy‐dense diet. The enhanced hepatic fatty acid utilization as an energy substrate found in rats fed an energy‐dense diet may contribute to reduce the availability of lipids for storage, thus counteracting the development of obesity.
Influence of fibronectin on HIV‐1 infection and capability of binding to platelets
Authors: Pugliese A. , Savarino A. , Cantamessa C. , Torre D.
Published: May 2005

We have previously demonstrated that fibronectin (FN) can bind HIV‐1 envelope proteins, in particular gp120. The aim of the present study was to determine some biological effects of this phenomenon. Pretreating HIV‐1 with human FN increased the infectivity of HIV‐1, when a low concentration of the virus was used. In contrast, an RGD‐containing pentapeptide (Gly‐Arg‐Gly‐Asp‐Ser), which is a fundamental binding site of FN, reduced the infectivity of a suspension of HIV‐1 at high concentrations of the virus. It is likely that FN bridges the cell surface and the virions, while the RGD‐containing pentapeptide may saturate the HIV‐1 binding sites for cell surface receptors. Moreover, gp120 was bound to the FN present on the surface of platelets. The specifity of this binding was confirmed by the inhibition obtained by pretreating platelets with anti‐FN antibodies. The consequence of the surface modifications of the platelets could explain the thrombocytopenia that frequently occurs in patients infected with HIV and suggests also the possibility that platelets could be a vehicle for the virus in the circulation.
The design of calibration in near infra‐red reflectance analysis by clustering
Authors: Næs Tormod
Published: May 2005

A procedure for selecting samples to use in multivariate calibration, inspired by a paper by Zemroch, is proposed. The method developed is designed for multicollinear data and is based on cluster analysis. It selects among availabe spectra the samples to submit to chemical analysis and calibration. The method is tested on an example from NIR spectroscopy of biscuit dough pieces.
Estimation in compositional data analysis
Authors: Rayens William S. , Srinivasan Cidambi
Published: May 2005

Compositional data arise naturally in several branches of science, including chemistry, geology, biology, medicine, ecology and manufacturing design. In chemistry, these constrained data seem to occur typically when raw data are normalized or when output is obtained from a constrained estimation procedure, such as might be used in a source apportionment problem. It is important not only for chemists to be aware that the usual multivariate statistical techniques are not applicable to constrained data, but also to have access to appropriate techniques as they become available. The currently available methodology is due principally to Aitchison and is based on log–normal models. This paper suggests new parametric and non‐parametric approaches to significantly improve the existing methodology. In the parametric setting, some recent work of Rayens and Srinivasan is extended and a practical regression model is proposed. In the development of the non‐parametric approach, minimum distance methods coupled with multivariate bootstrap techniques are used to obtain point and region estimators.
Slowly converging parafac sequences: Swamps and two‐factor degeneracies
Authors: Mitchell Ben C. , Burdick Donald S.
Published: May 2005

The alternating least squares PARAFAC algorithm is a useful tool for resolving trilinear three‐way data arrays. Occasionally, however, it becomes bogged down for many iterations in the vicinity of a poor quality resolution before moving on to a much superior optimum fit. We investigate this behavior in a simulation study and suggest ways of overcoming the obstacles it presents.
The dorsal sack, the aulix and the diencephalic flexure
Authors: Wilder B. G.
Published: May 2005

In many of the lower vertebrates there is a pouch‐like evagination of the diatela (membranous roof of the diacele, or “third ventricle”) between the epiphysis and the paraphysis. In Ceratodus and in Polyodon it is anteverted and very large. In the green turtle it projects dorsad. Its mammalian representative is crowded caudad by the cerebrum and rests upon the retroverted epiphysis. It is commonly ignored in figures and descriptions, but was shown by Reichert. The speaker, without recognizing its homology, figured it in the New York Medical Journal, 1885, p. 320. It is large in the sheep and long in the cow and horse. The diaplexuses are continued into it.

Aulix is the mononym proposed by the speaker in 1882 for the “Sulcus Monroi” of Reichert—a sigmoid groove on the mesal surface of the thalamus just ventrad of the medicommissure and connecting the orifice of the mesocele with the porta (foramen of Monro) at either side. His and C. S. Minot regard it as the diencephalic representative of the “sulcus interzonalis,” the primary furrow between the dorsal and ventral zones of the nervous axis. But recent observations in the neurologic laboratory at Cornell upon embryo kittens render this interpretation somewhat doubtful, and the matter is under investigation.

In 1889, in the “Reference Hand‐Book of the Medical Sciences” the speaker called attention to the sharp angle between the prosencephalic and the diencephalic portions of the brain cavities. A recent review of the brains of all vertebrate classes warrants the generalization that a marked diencéphalic flexure occurs in all Reptiles, Birds and Mammals, while in Amphibians and “fishes” it is absent or insignificant.