Estudio de la actividad antibacteriana y la toxicidad en gambas en salmuera de dos especies de Polygonum / Assessment of anti-bacterial activity and brine shrimp toxicity of two Polygonum species

Se estudi¨® la actividad antibacteriana de las partes a¨¦reas de Polygonum barbatum var. barbata y Polygonum stagninum(Familia: Polygonaceae) frente a diversas cepas bacterianas mediante el ensayo de difusi¨®n en disco, as¨ª comola toxicidad en gambas en salmuera mediante el ensayo de letalidad de gambas en salmuera. Todos los extractos/fracciones, a excepci¨®n del extracto/fracci¨®n de MeOH, presentaron niveles de actividad antibacteriana de bajosa moderados frente a la mayor¨ªa de las cepas de la prueba (zona de inhibici¨®n = 7-21 mm). Todos los extractos yfracciones presentaron considerable toxicidad general hacia las gambas en salmuera. Los valores de LD50 de losextractos/fracciones de la prueba se encontraron en el rango de 2,19 a 114,81 ¦Ìg/mL, mientras que la del controlpositivo (sulfato de vincristina) fue de 0,61 ¦Ìg/mL

The extracts of the aerial parts of Polygonum barbatum var. barbata and Polygonum stagninum (Family: Polygonaceae)were assessed for anti-bacterial activity against a number of bacterial strains using the disc diffusion assay, andbrine shrimp toxicity using the brine shrimp lethality assay. All extracts/fractions, except the MeOH extract/fraction,exhibited low to moderate levels of anti-bacterial activity against most of the test strains (zone of inhibition = 7-21mm). All extracts and fractions displayed considerable general toxicity towards brine shrimps. The LD50 values of thetest extracts/fractions were within the range of 2.19 to 114.81 mg/mL, whereas that of the positive control (vincristinesulphate) was 0.61 mg/mL

Antibacterial activity of Clausena heptaphylla.

The antibacterial activity of different crude extracts of Clausena heptaphylla leaves as well as three purified coumarins, obtained from the cold methanol extract, is reported.

[Ag(I)(Et(2)PCH(2)CH(2)PPh(2))(2)]NO(3): An Antimitochondrial Silver Complex.

The silver(I) complex [Ag(eppe)(2)]NO(3) (eppe = Et(2)PCH(2)CH(2)PPh(2)) is shown by X-ray crystallography to be tetrahedral with Ag - PEt(2) and Ag - P Ph(2) bond lengths of 2.482 and 2.518 A, respectively. The complex is selectively antimitochondrial and inhibits the growth of a number of yeast strains in non-fermentable media at concentrations as low as 2.5 muMu and induces the mitochondrial mutation petite The effect is largely reversed by the presence of aspirin. The complex is shown to be stable in the cell culture media and in the presence of glutathione, but readily reacts with disulfides of oxidized glutathione and serum albumin. Surprisingly, neither [Au(eppe)(2)]Cl nor [Au(eppe)(2)]Cl (dppe = Ph(2)PCH(2)CH(2)PPh(2)) showed any mitochondrial selectivity in the same screening protocol.

Rapid production of a panel of blood group A-active oligosaccharides using chemically synthesized di- and tri-saccharide primers and an easily prepared porcine (1-->3)-alpha-N-acetyl-D-galactosaminyltransferase.

A porcine (1-->3)-alpha-N-acetyl-D-galactosaminyltransferase was obtained in a state suitable for preparative-scale (mg-scale) synthesis using simple procedures requiring only three days of effort. The enzyme thus prepared transferred GalNAc efficiently from UDP-GalNAc to six different chemically synthesized di- and tri-saccharide H-active structures to yield blood-group A-active oligosaccharides that were characterized by 1H NMR spectroscopy and mass spectrometry. This work further demonstrates the efficiency and attractiveness of using glycosyltransferases in a combined chemoenzymatic approach for the rapid production of biologically active oligosaccharides.

Artificial antigens. Synthetic carbohydrate haptens immobilized on crystalline bacterial surface layer glycoproteins.

The crystalline surface-layer glycoproteins of Clostridium thermohydrosulfuricum L111-69, Bacillus stearothermophilus NRS 2004/3a and Bacillus alvei CCM 2051 were used for immobilization of spacer-linked blood group A-trisaccharide (alpha GalNAc(1----3)[alpha Fuc(1----2)]beta Gal) and of the spacer-linked, tumor-associated T-disaccharide [beta Gal(1----3)alpha GalNAc]. The immobilization involved the glycan portions of surface-layer glycoproteins. Different activation methods were used, namely, periodate oxidation, or treatment with epichlorohydrin or divinyl sulfone, followed by coupling of the hapten under appropriate conditions. The resulting conjugates are useful for assessing the application potential of haptenated surface layer preparations as carrier/adjuvants for the induction of immunity to poorly immunogenic molecules.

An improved affinity support and immunoadsorbent with a synthetic blood group oligosaccharide and polymer coating for hemoperfusion.

An improved affinity support and an immunoadsorbent suitable for extracorporeal perfusion of whole blood (or plasma) are reported. The affinity support consists of calcined diatomite-type silica particles to which a synthetic oligosaccharide hapten, viz. A-trisaccharide representing human blood group A, with a linking spacer-arm is chemically attached. The immunoadsorbent thus obtained is surface-modified with a polymer coating. The modified immunoadsorbent is not hemolytic and shows no loss of biological activity in reducing antibody titers in vitro. An important feature of the improved immunoadsorbent is that the polymer coating provides a better surface resistance and therefore stability to the affinity support to prevent the release of potentially harmful fines. The usefulness of a physically stable support as an affinity adsorbent for the selective removal of specific antibodies or unwanted substances directly from the blood circulation by extracorporeal immunoadsorption has profound medical significance because this would provide an efficient but safe and practical alternative to therapeutic intervention using plasma exchange or plasma perfusion, both of which require plasmapheresis.

Synthesis and bioactivity of copolymers with fragments of heparin.

A new type of biocompatible copolymer comprising small fragments of heparin, (octa- to dodecasaccharides) copolymerized with a synthetic monomeric component, viz. acrylamide, has been prepared. The heparin fragments are produced by enzymatic or chemical means and are copolymerized, directly or after suitable derivatization, with acrylamide as the major polymerizable component. The polymeric material incorporates the heparin segments as pendant moieties such that their essential functional groups and structural features for specific binding with the selective serine protease coagulation factor inhibitor antithrombin III are preserved. An important feature of this copolymer is its biocompatibility which relates specifically to its antithrombotic and antithrombogenic activity derived from those of heparin fragments. The biological activity of heparin fragments and copolymers thereof are determined in terms of APTT and anti-Xa activity, their antithrombotic potential being expressed as a ratio of anti-Xa activity to APTT. The copolymers reported have biological activities similar to equivalent amounts of respective heparin fragments, and show higher antithrombotic activity compared to intact heparin or commercially available low-molecular-weight heparin (4,000-6,000 Da).

Immunoadsorbents with synthetic oligosaccharide hapten representing blood group A substances.

Immunoadsorbents with a synthetic oligosaccharide hapten representing human blood group A specific substances are prepared. The synthetic hapten, known as A-trisaccharide, which carries a space arm, is chemically attached to various solid supports, either directly through a suitable functional group at the end of the spacer arm or indirectly via a protein conjugated to the hapten. The preparation involves simple and mild procedures for the activation and/or derivatization of the supports. The latter includes naturally occurring polyhydroxy materials such as agarose, cellulose, or cellulose derivatives, and other particulate materials such as inorganic diatomites and a synthetic organic copolymer. The methods used for the coupling concern specifically the preparation of controlled-capacity and high-efficiency immunoadsorbents, with limited incorporations, which may be prepared easily and used for the selective removal, or affinity chromatographic separation, of specific antibodies from plasma environment or blood. It has been found that while hapten incorporation to the support may be varied rather easily, the physical nature of the support as well as the form of the hapten is important in determining the efficiency of an immunoadsorbent.

Flow rate dependence of in vitro removal of anti-A and anti-B antibodies by immunoadsorbents with synthetic oligosaccharides representing blood group substances.

In vitro removal of anti-A and anti-B antibodies by immunoadsorbents with synthetic oligosaccharide structures representing blood group substances have been studied under controlled and simulated extracorporeal perfusion conditions at different flow rates ranging from 20 to 160 mL/min. There is a gradual decline in antibody titers in the plasma, as it relates to the antibody binding capacity and efficiency of the immunoadsorbents, both as a function of time and plasma volume at any of the flow rates employed. Approximately the same maximal binding capacity is achieved in about 4 hours of perfusion regardless of the flow rate, whereas increased number of plasma volumes are required at higher flow rates to compensate for the appropriate residence times needed in the column to utilize the same capacity. The perfusion time alone appears to be a critical factor which via the flow rate determines the residence time between the plasma and the immobilized hapten in the column. Also, the optimal flow rate at which maximum binding capacity of the immunoadsorbent may be utilized efficiently appears to fall within a range of the flow rate. The factors determining this range and the efficiency of the immunoadsorbents under dynamic conditions are discussed. The results obtained under the controlled conditions and the discussions that follow are expected to be generally useful, at least, as approximate clinical guidelines, for selective removal of specific substances by extracorporeal perfusion techniques utilizing therapeutic intervention with immunoadsorbents in this important area of medical therapy.

Kinetics of yeast alcohol dehydrogenase and its coenzyme coimmobilized in a tubular flow reactor.

Yeast alcohol dehydrogenase and nicotinamide adenine dinucleotide (NAD) were coimmobilized, with covalent attachment, to the interior surface of a nylon tube. The NAD was attahed at the N(6) group of the adenine moiety; an NAD derivative was prepared and attached to free carboxyl groups at a partially hydrolyzed nylon surface. The enzyme was attached, through glutaraldehyde residues, to free amino groups on the surface. Kinetic studies were carried out in which the reduced NAD was recycled by means of phenazine ethosulfate and 2,6-dichlorophenol indophenol. The reaction was studied over a range of flow rates and ethanol concentrations. The variation of rate with flow rate suggested that there was little diffusion control with respect to ethanol and that there was no observable inhibition by the reaction products. These conclusions were confirmed by evidence based on dimensionless parameters for the reaction and by direct inhibition experiments. The apparent Michaelis constant was lower than when only the enzyme was immobilized, suggesting that the immobilized enzyme-coenzyme system is of high efficiency. Overall rates of reaction were lower than when there was saturation with NAD. The tube showed no measurable loss of catalytic activity over a period of one month.

pH dependence of free and immobilized yeast alcohol dehydrogenase kinetics.

A study was made of the influence of pH on the reaction between NAD and ethanol, catalyzed by yeast alcohol dehydrogenase, both in free solution and attached to the inner surface of a nylon tube. A new least-squares analysis of the results has been devised; it is simpler to apply and is more realistic than those previously employed. Analysis of the results for the free enzyme indicated that the free enzyme has two active ionizing groups having pK values of about 6.6 and 8.8. These pK values undergo only small changes when the enzyme is bound to NAD and when it is bound to both NAD and ethanol. With the immobilized enzyme and saturating concentrations of ethanol the rates went through a maximum as the pH was varied from 6.5 to 10.0. With saturating concentrations of NAD there was a steady increase in rate, with no falling off at pH 10. Immobilization generally brought about an increase in the pK values. These increases are attributed partly to a residual negative surface charge which attracts the leaving H+ ions. They are also attributed partly to the formation in the reaction of H+ ions, which cause the local pH to be lower than that in the bulk solution. This effect is more important with saturating NAD ions, since the buffer anions will then be less mobile and less able to mediate the movement of protons.

Flow kinetics of yeast alcohol dehydrogenase attached to nylon tubing.

Yeast alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) was attached covalently to the inner surface of nylon tubing, and the immobilized enzyme retained its activity over a period of months. A study was made of the flow kinetics for the reaction between ethanol and NAD. With the ethanol held at saturating concentrations there was partial diffusion control, the extent decreasing with increasing flow rate and increasing NAD concentration. With the NAD at saturating concentrations there was no appreciable diffusion control. The apparent Michaelis constants varied with flow rate vf, being linear in vf-1/3, and extrapolation to infinite flow rate (vf-1/3 = 0) gave the intrinsic Michaelis constants. The inhibition by products was also studied. The results for both NADH and acetaldehyde showed mixed competitive and non-competitive inhibition, with a preponderance of the former. Acetaldehyde is the stronger inhibitor, and this is consistent with the lack of dissusion control with variable ethanol. Inhibition by acetaldehyde is not affected by flow rate, but inhibition by NADH is affected, presumably because of the greater degree of diffusion control with variable NAD.