Green tea phenolics inhibit butyrate-induced differentiation of colon cancer cells by interacting with monocarboxylate transporter 1.

Diet has a significant impact on colorectal cancer and both dietary fiber and plant-derived compounds have been independently shown to be inversely related to colon cancer risk. Butyrate (NaB), one of the principal products of dietary fiber fermentation, induces differentiation of colon cancer cell lines by inhibiting histone deacetylases (HDACs). On the other hand, (-)-epicatechin (EC) and (-)-epigallocatechin gallate (EGCG), two abundant phenolic compounds of green tea, have been shown to exhibit antitumoral properties. In this study we used colon cancer cell lines to study the cellular and molecular events that take place during co-treatment with NaB, EC and EGCG. We found that (i) polyphenols EC and EGCG fail to induce differentiation of colon adenocarcinoma cell lines; (ii) polyphenols EC and EGCG reduce NaB-induced differentiation; (iii) the effect of the polyphenols is specific for NaB, since differentiation induced by other agents, such as trichostatin A (TSA), was unaltered upon EC and EGCG treatment, and (iv) is independent of the HDAC inhibitory activity of NaB. Also, (v) polyphenols partially reduce cellular NaB; and (vi) on a molecular level, reduction of cellular NaB uptake by polyphenols is achieved by impairing the capacity of NaB to relocalize its own transporter (monocarboxylate transporter 1, MCT1) in the plasma membrane. Our findings suggest that beneficial effects of NaB on colorectal cancer may be reduced by green tea phenolic supplementation. This valuable information should be of assistance in choosing a rational design for more effective diet-driven therapeutic interventions in the prevention or treatment of colorectal cancer.

The changes in the energy metabolism of human muscle induced by training.

The biochemical effects of training programmes have been studied with a kinetic model of central metabolism, using enzyme activities and metabolite concentrations measured at rest and after 30 s maximum-intensity exercise, collected before and after long and short periods of training, which differed only by the duration of the rest intervals. After short periods of training the glycolytic flux at rest was three times higher than it had been before training, whereas during exercise the flux and energy consumption remained the same as before training. Long periods of training had less effect on the glycolytic flux at rest, but increased it in response to exercise, increasing the contribution of oxidative phosphorylation.

The importance of polymerization and galloylation for the antiproliferative properties of procyanidin-rich natural extracts.

Grape (Vitis vinifera) and pine (Pinus pinaster) bark extracts are widely used as nutritional supplements. Procyanidin-rich fractions from grape and pine bark extract showing different mean degrees of polymerization, percentage of galloylation (percentage of gallate esters) and reactive oxygen species-scavenging capacity were tested on HT29 human colon cancer cells. We observed that the most efficient fractions in inhibiting cell proliferation, arresting the cell cycle in G(2) phase and inducing apoptosis were the grape fractions with the highest percentage of galloylation and mean degree of polymerization. Additionally, the antiproliferative effects of grape fractions were consistent with their oxygen radical-scavenging capacity and their ability to trigger DNA condensation-fragmentation.

Anticancer effect of a new benzophenanthridine isolated from Zanthoxylum madagascariense (Rutaceline).

Fractionation of the cyclohexane extract from the stem bark powder of Zanthoxylum madagascariense led to the isolation of a new benzophenanthridine-type alkaloid, hydrochloride of 2,3-methylendioxy-8-hydroxy- 7-methoxy-benzo[C]phenanthridine (Rutaceline), characterized on the basis of its spectral data. Rutaceline was evaluated for its antiproliferative capacity on the human colorectal adenocarcinoma (Caco-2) and the African green monkey kidney (Vero) cell lines. The 50% inhibition of cell growth (IC50) obtained after 24 h incubation was similar for both cells lines (110-115 microg/ml, i.e. 269-281 microM), but at 48 h the IC50 value for the Caco-2 cells was lower than for the Vero cells (20 microg/lml, i.e. 49 microM versus 90 microg/ml, i.e. 220 microM) indicating a higher cell growth inhibitory effect on the colon adenocarcinoma cells. At the respective IC50 concentrations, Rutaceline did not significantly induce apoptosis but induced cell cycle arrest in the GO/G1 phase, as well as a decrease of cells in S phase. Rutaceline also induced DNA fragmentation in both cell lines, as revealed by agarose gel electrophoresis, and a dose-dependent clastogenic effect in both cell lines as revealed by the Comet assay.

Energetic coupling of Na-glucose cotransport.

(1) Energetic coupling in Na-linked glucose transport in renal brush border membrane vesicles has been studied in terms of various carrier models differing with respect to reaction order (random vs. ordered), and to rate limitation of steps within the routes of carrier-mediated solute transfer (translation across the membrane barrier vs. binding/release between carrier and bulk solution). (2) By computer simulation it was found that effective energetic coupling requires the leakage routes to be significantly, if not predominantly, rate-limited by their (barrier-crossing) translatory steps. This does not apply to the transfer route of the ternary complex, as coupling is possible whether or not this route is rate-limited by the translatory step. (3) The system transports glucose in the absence of Na+ (uniport) and the unidirectional flux is stimulated by unlabeled glucose on the trans side (negative tracer coupling). It is concluded that glucose binds to the carrier on either side without Na, as would be consistent with either a random system or one mode of ordered system with mirror symmetry (glucose binds before Na) but inconsistent with either mode of glide symmetry. The tracer coupling appears to indicate that the rate coefficient of carrier-mediated glucose transfer exceeds that of the empty carrier. (4) The Na-linked zero-trans flow of glucose in either direction is strongly trans-inhibited by Na. This consistent with a random system in which Na blocks or retards the translocation of the glucose-free carrier, thereby reducing 'slipping' through an internal leakage route. It is also consistent with the above mentioned ordered system, (i.e., in the absence of Na-transport without D-glucose) if it is assumed that trans Na interferes with the dissociation of the ternary complex, thereby slowing the release of glucose. (5) Minimum equilibrium exchange of glucose is stimulated in the presence of Na. This appears to indicate that Na expands the flow density of carrier-mediated glucose transfer. This expansion does not result from a 'velocity effect' (the ternary complex moving faster than the binary glucose carrier complex), as Na fails to stimulate maximum equilibrium exchange. It can instead be accounted for by an 'affinity effect' (the affinity of the carrier for glucose being increased by Na) as Na depresses the Michaelis constant of equilibrium exchange.(ABSTRACT TRUNCATED AT 400 WORDS)

Further characterization of adenosine transport in renal brush-border membranes.

Adenosine transport has been further characterized in rat renal brush-border membranes (BBM). The uptake shows two components, one sodium-independent and one sodium-dependent. Both components reflect, at least partly, translocation via a carrier mechanism, since the presence of adenosine inside the vesicles stimulates adenosine uptake in the presence as well as in the absence of sodium outside the vesicles. The sodium-dependent component is saturable (Km adenosine = 2.9 microM, Vmax = 142 pmol/min per mg protein) and is abolished at low temperatures. The sodium-independent uptake has apparently two components: one saturable (Km = 4-10 microM, Vmax = 174 pmol/min per mg protein) and one non-saturable (Vmax = 3.4 pmol/min per mg protein, Km greater than 2000 microM). Inosine, guanosine, 2-chloroadenosine and 2'-deoxyadenosine inhibit the sodium-dependent and -independent transport, as shown by trans-stimulation experiments, probably because of translocation via the respective transporter. Uridine and dipyridamole inhibited only the sodium-dependent uptake. Other analogs of adenosine showed no inhibition. The kinetic parameters of the inhibitors of the sodium-dependent component were further investigated. Inosine was the most potent inhibitor with a Ki (1.9 microM) less than the Km of adenosine. This suggests a physiological role for the BBM ecto-adenosine deaminase (enzyme which extracellularly converts adenosine to inosine), balancing the amount of nucleoside taken up as adenosine or inosine by the renal proximal tubule cell.

In vivo measurements of control coefficients for hexokinase and glucose-6-phosphate dehydrogenase in Xenopus laevis oocytes.

Hexokinase and glucose-6-phosphate dehydrogenase activities were increased in Xenopus laevis oocytes by microinjection of commercial pure enzymes. The effect of increased fractional activities on glycogen synthesis or on the production of 14CO(2) (the oxidative portion of the pentose phosphate pathway) was investigated by microinjection of [1-(14)C]glucose and measurements of the radioactivity in glycogen and CO(2). Control coefficients calculated from the data show that hexokinase plays an important role in the control of glycogen synthesis (control coefficient=0.7) but its influence on the control of the pentose phosphate pathway is almost nil (control coefficient=-0.01). Glucose-6-phosphate dehydrogenase injections did not affect the production of 14CO(2) by the pentose phosphate pathway, indicating that other factors control the operation of this pathway. In addition, an almost null control of this enzyme on glycogen synthesis flux was observed.

Excitatory amino acid neurotransmission. Pathways for metabolism, storage and reuptake of glutamate in brain.

In the nervous system, glutamate is an excitatory aminoacid which at higher concentrations has been implicated in a number of disorders. Glutamate is stored in presynaptic vesicles and is released by calcium-dependent exocytosis. After its action on ionotropic receptors (iGluR, related to ionic channels) or metabotropic receptors (mGluR, related to metabolic formation of second messengers), glutamate can be removed from the synaptic cleft through two processes: re-uptake back into pre-synaptic terminals or diffusion out of synaptic cleft for uptake by glial cells. This is achieved by glutamate transporters. In pre-synaptic terminals, glutamate is packed into the specialized secretory vesicles by means of a specific vesicular transporter. The level of glutamate available for neurosecretion is regulated by the vesicular transport activity. In order to achieve a proper concentration of the neurotransmitter in synaptic vesicles, glutamate must be synthesized. Glutamine is obtained in astroglial cells from the glutamate reuptaken, and as it has no neurotransmitter activity, it is the metabolite which regenerates glutamate in neurones (glutamate-glutamine cycle). Moreover, glutamate is also obtained from glucose by an intermediate of TCA cycle. In this paper we want to introduce some aspects of glutamate biosynthesis and release: glutamate receptors, neurotransmitter uptake by the glutamate transporters and neurotransmitter inactivation and new formation by metabolism.

Surface adenosine deaminase. A novel B-cell marker in chronic lymphocytic leukemia.

Previous studies found that ADA is present on the surface of mononuclear blood cells from healthy patients. Because the expression of this surface antigen depends upon the cell type, the presence of ADA on the plasma membrane of cells from patients with malignant hematologic diseases was studied by flow cytometry. The highest percentage of expression was found in CLL, whereas the lowest was found in T-cell-derived malignancies. The enzyme expression in immortalized cell lines showed a similar pattern, with the highest expression (95% +/- 5%) in the SKW64 B-derived cell line, the lowest (15% +/- 5%) in Jurkat T-lymphoma derived cells, and the intermediate (32% +/- 8%) in K562 cells derived from a chronic myelogenous leukemia. Double labeling ADA/CD5 and ADA/CD19, as well as the correlation of ADA expression with the expression of other surface markers, indicate that surface ADA might be considered a novel marker for CLL.

Structural requirements of benzodiazepines for the inhibition of pig brain nitric oxide synthase.

Nitric oxide synthases (NOS) are heme-containing enzymes which catalyse the oxidation of L-arginine to nitric oxide and L-citrulline. Some nitrogenated compounds have been reported to coordinate with the iron atom from the heme group, thus inhibiting NOS. 1,4-Benzodiazepines are nitrogenated compounds which have many physiological effects such as antianxiety, antiepileptic, hypnotic, and muscle relaxation properties. The aim of this paper was to measure the effect of different benzodiazepines on NOS activity in pig brain extracts. Medazepam, pinazepam, diazepam, oxazepam and alprazolam competitively inhibited NOS with IC(50) in the micromolar range. Other benzodiazepines showed no effect at concentrations as high as 200 microM. Due to the structural similarity of the benzodiazepine ring nucleus with L-arginine, we propose a benzodiazepine-enzyme interaction to explain the competitive inhibitions. By comparing benzodiazepine effects and their structures, the inhibitory effect of benzodiazepines on NOS is related to the absence of substituents on N4 and to the absence of a halogen substituent on C5 phenyl group. Although benzodiazepine's inhibitions observed in this study are not in the physiological range in normal cases, these inhibitions could be significant in drug abuse situations and should be taken into account for the rational design of drugs which specifically inhibit NOS.

Sources of interference in the use of 2,3-diaminonaphthalene for the fluorimetric determination of nitric oxide synthase activity in biological samples.

The use of 2,3-diaminonaphthalene (DAN) for the fluorimetric determination of nitric oxide synthase (NOS) activity in rat brain extracts has been re-examined. Two types of interference were observed, due either to components of the reaction mixture or to the enzymatic sample itself. One of the substrates (NADPH) and some cofactors (FADH(2), FMNH(2)) required for the enzyme activity interfere in the assay by quenching the fluorescence produced. Interference was minimized by using lower FADH(2), FMNH(2) and NADPH concentrations (1 micromol/l) and a NADPH recycling system in the reaction mixture. The addition of bovine serum albumin or hemoglobin to the sample quenched fluorescence intensity, but these protein interferences could be reduced by filtering the samples after reaction. We conclude that the DAN fluorimetric assay as originally described is not suitable for the determination of NOS activity in crude extracts such as rat brain cytosolic fraction, due to the presence of interfering substances. Nevertheless, DAN could be used for the determination of enzyme activity after reducing protein interference by filtering, or in less complex samples such as cell cultures (e.g. activated macrophages), or in chromatographic fractions obtained during the purification of the enzyme. A careful use of the commercial kits based on the use of DAN for the determination of NOS activity is recommended.

Heterogeneity of the gradients performed by the freeze-thaw method.

Gradients produced by the freeze-thaw method were analyzed at various rates of freezing. The shape of the gradient depended on the rate of freezing. At high rates the pattern did not change but at low rates the steepness of the gradient increased with time. Solutes concentrated at the bottom in a fashion which depended on the density of the solvent and on the rate of freezing. It should also be noted that the gradient was not uniform over the entire surface as the concentration of solute increased near the wall of the test tube.

Is adenosine deaminase involved in adenosine transport?

The hypothesis that adenosine metabolizing enzymes may have a key role in the transport of adenosine is discussed. The enhancement of adenosine transport by inhibitors of adenosine deaminase (the enzyme which deaminates adenosine to inosine) and the ecto-localization of adenosine deaminase suggest a contribution of the enzyme in taking up nucleosides. Two possible mechanisms are suggested: 1) transport and deamination of adenosine as a coupled process, or 2) uptake of inosine after cleavage of adenosine by ecto-adenosine deaminase. In both cases, the so-called adenosine deaminase binding protein which is a membrane protein could be the real nucleoside transporter. This behaviour of adenosine deaminase as an ectoenzyme anchored to a membrane protein remembers the behaviour of periplasmic binding proteins of bacteria. Thus, adenosine deaminase as well as, for instance, adenosine kinase would be a kind of 'periplasmic proteins' of eukaryotic cells. The function of adenosine deaminase and adenosine kinase would then be to take adenosine and give it to the true transporters.

Theoretical study about the variability of the genome of foot-and-mouth disease viruses.

1. The possibilities of change in amino acids of a protein are discussed in terms of a point mutation. 2. Whereas Met and Trp are forced to change due to a point mutation, other amino acids (Ala, Arg, Gly, Leu, Pro and Val) have a probability of 1/3 to survive in the sequence. 3. On basis of these considerations, the genome from 5 strains (CSP, C3Ind, O1K, A10 and A12) of the foot-and-mouth disease virus was studied. 4. A hypothetical genealogic tree for these strains is suggested, where CSP and C3Ind are close and also A10 and A12. O1K is closer to A10 and A12 than to CSP or C3Ind.

Slight differences between adenosine deaminases from different species an immunochemical study.

IgGs against adenosine deaminase from rat brain, rat liver, mouse duodenum and human erythrocyte were purified from rabbit antisera with yields of 82-87%. The inhibition of adenosine deaminase by the antienzyme is studied, and it is demonstrated that rat and mouse antibodies are tight-binding inhibitors. These antibodies inhibit either the rat or the mouse enzymes and do not inhibit the human erythrocytes enzyme. The human antibody does not inhibit either the human or the rat or mouse enzyme. These results indicate that some differences in antigenic behaviour near the active site must be encountered among species. Comparing the sequenced of the two products corresponding to two adenosine deaminase genes recently sequenced (human and murine) a hypothesis concerning the localization of the adenosine deaminase active site is proposed.

Use of alpha-toxin from Staphylococcus aureus to test for channelling of intermediates of glycolysis between glucokinase and aldolase in hepatocytes.

We investigated whether hepatocytes permeabilized with alpha-toxin from Staphylococcus aureus are a valid model for studying the channelling of intermediates of glycolysis between glucokinase and triosephosphate isomerase. These cells are permeable to 2-aminoisobutyrate, ATP, glucose 6-phosphate (Glc6P) and fructose 2, 6-bisphosphate [Fru(2,6)P(2)], but maintain cell integrity in the presence of ATP as judged by the retention of cytoplasmic enzymes. During incubation with 25 mM glucose, an ATP-generating system and saturating concentrations of Fru(2,6)P(2), rates of detritiation of [2-(3)H]glucose and [3-(3)H]glucose were similar. Exogenous Glc6P (1 mM) and to a lesser extent fructose 6-phosphate, but not Fru(1, 6)P(2), decreased the rate of detritiation of [3-(3)H]glucose. During incubation with 25 mM glucose and Glc6P (0.2-1 mM), with either [3-(3)H]glucose or [3-(3)H]Glc6P as labelled substrate, there was dilution of metabolism of [3-(3)H]glucose with increasing Glc6P but no overall increase in glycolytic flux from glucose and Glc6P, indicating that glycolysis is apparently saturated with Glc6P despite the permeability of the cells to this metabolite. These findings could be explained by partial channelling of Glc6P between glucokinase and glycolysis in the presence of saturating concentrations of Fru(2,6)P(2). They provide an alternative explanation for the concept that there is more than one Glc6P pool.

Distribution of adenosine deaminase in some rat tissues. Inhibition by ethanol and dimethyl sulfoxide.

The level of adenosine deaminase in various rat tissues has been tested. The enzyme activity of cytosolic fractions decreased in the following order: lung greater than spleen greater than small intestine greater than stomach greater than kidney greater than heart greater than liver greater than skeletal muscle greater than forebrain greater than cerebellum. The enzyme had identical patterns from tissue to tissue with respect to Km, V, and Ki values for ethanol and for dimethyl sulfoxide, with respect to electrophoretic behaviour and to inhibition by antibodies anti-rat brain adenosine deaminase.

Determination of the characteristics, properties and homogeneity of rat brain microsomes. Binding of lactate dehydrogenase, malate dehydrogenase and 5' nucleotidase to microsomal membranes.

Quantitatively, the amount of microsomes obtained using dimethyl sulfoxide is larger than that obtained from sucrose solutions (Centelles, Franco & Bozal (1986) Biol. Chem. Hoppe Seyler 367, 461-475). In this paper it is demonstrated that from a qualitative point of view they appeared to be indistinguishable with respect to molecular characteristics. Thus, both types of microsomes had the same behaviour in experiments of isopicnic ultracentrifugation with Percoll, isoelectric focusing and gel permeation. In these experiments, the 5'-nucleotidase, lactate dehydrogenase and malate dehydrogenase activities bound to the microsomal fraction were also studied. Lactate and malate dehydrogenase activities were always found in free and membrane-bound form. In contrast, 5'-nucleotidase activity was always encountered bound to microsomal membranes.

Purification and partial characterization of brain adenosine deaminase: inhibition by purine compounds and by drugs.

Rat brain adenosine deaminase (E.C. 3.5.4.4.) was purified 667-fold from the supernatant fraction by the following techniques: heat treatment (60 degrees C), fractionation with ammonium sulfate, column chromatography on DEAE-Sepharose, and preparative gel electrophoresis. The purified enzyme was homogeneous by the criterion of polyacrylamide disc gel electrophoresis and isoelectric focusing. Amino acid composition is given. The isoelectric point of the enzyme (5.2) was determined by isoelectric focusing on agarose. The apparent molecular weight was estimated to be 39,000 (Stokes Radius [Rs] = 27.3 A) using a calibrated Sephacryl S-300 column. The study of the influence of the temperature on the initial reaction rates allowed calculation of Ea (8.9 Kcal/mole) and delta H (5.0 Kcal/mole) values. The variation of V and Km with pH suggests the existence of a sulfhydryl group and an imidazole group in the enzyme-substrate complex. The enzyme had a Km (adenosine) of 4.5 X 10(-5) M and was inhibited by inosine, guanosine, adenine, and hypoxanthine but not by other intermediates of purine metabolism. None of the inhibitors were active as substrates. The enzyme was also inhibited by dimethyl sulfoxide and ethanol. Inhibition by ethanol can account partially for the CNS depressant effects of levels 3 and 4 of alcohol intoxication. A number of drugs having therapeutic uses such as sedative, anxiolytic, analgesic, and relaxant are modulators of the enzyme. Among these, lidoflazine, phenylbutazone, and chlordiazepoxide are the most potent as inhibitors (Ki 30, 54, and 83 microM, respectively), whereas medazepam is the most potent as activator (Ka 0.32 mM). Thus, it is concluded that some drugs that inhibit adenosine uptake also modulate adenosine deaminase activity. Besides, since the enzyme is located extracellularly [Franco et al, 1986], these drugs can modulate the physiological effects exerted by extracellular adenosine.