Volume 55, Issue 1-2

A new oxyprenyl coumarin was isolated from the lipophilic exudate of Ozothamnus lycopodioides. Its structure was established as 7-(3,3′-dim ethylallyloxy)-5-hydroxy-6-methoxycoumarin from its UV, MS and NMR spectral data, especially two dimensional experiments. In addition to six earlier reported flavonols, we found four highly substituted flavones, including two rare methylenedioxyflavones.

Twenty-two species of Scophulariaceae have been found to accumulate flavonoid aglycones externally on their leaves and stems. They belong to the genera Anarrhinum , Antirrhinum , Asarina, Calceolaria, Mimulus, and Odontites. Most of the flavonoids are methylated flavones and flavonols, some with 6-O and/or 8-O -substitution. One of them is the natural isobutyryl ester of a rare flavone.

Xylitol, a sweetener comparable to sucrose, is anticariogenic and can be consumed by diabetics. This sugar has been employed sucessfully in many foods and pharmaceutical products. The discovery of microorganisms capable of converting xylose present in lignocellulosic biomass into xylitol offers the opportunity of producing this poliol in a simple way. Xylitol production by biotechnological means using sugar cane bagasse is under study in our laboratories, and fermentation parameters have already been established. However, the downstream processing for xylitol recovery is still a bottleneck on which there is only a few data available in the literature. The present study deals with xylitol recovery from fermented sugar cane bagasse hydrolysate using 5.2 g/l of aluminium polychloride associated with activated charcoal. The experiments were performed at pH 9, 50 °C for 50 min. The results showed that aluminium polychloride and activated charcoal promoted a 93.5% reduction in phenolic compounds and a 9.7% loss of xylitol from the fermented medium , which became more discoloured, facilitating the xylitol separation.

Spirulina platensis trichomes grown in Zarrouks medium having total Na + concentration as 0.14 ᴍ when transfered to fresh Zarrouks medium containing enhanced level of Na + ions equal to 0.86 ᴍ showed 30% more accumulation of Na + intracellularly as compared to the control. An inhibition of photosystem II activity to almost 66% was observed. Also due to this exposure to high Na + , the room temperature absorption characteristics of Spirulina trichomes and the thylakoid membrane preparations w ere altered indicating changes in the chromophore protein interactions and alterations in the phycocyanin/allophycocyanin ratio; there by affecting the energy harvest and energy transfer processes. An increase in the carotenoid absorption was two fold over the control in the treated sample. Similarly, room temperature and low temperature (77 K) fluorescence emission spectra collectively suggested alterations in the chlorophyll a emissions, F 726 of photosystem I reflecting changes in the lipid protein environment of the thylakoid. Our results indicate that in Spirulina the enhanced Na + level alters the energy harvest and transfer processes. It also affected the emission characteristics of chlorophyll a of photosystem I.

Research was done on dormant and non-dorm ant barley cv. Ars caryopses and triticale cv. Grado caryopses treated and non-treated with abscisic acid (ABA). During germination higher participation of populations of so-called tightly-bound polysom es (TBP) in embryos of dormant barley caryopses was observed, as well as their high metabolic activity. In embryos of triticale caryopses of which dormancy was imposed in an artificial way by ABA (100 μM), the strongest incorporation of 14 C -amino acids into nascent polypeptide chains in vivo was found in population of TBP, as well as the highest participation among three of the studied fractions (free polysomes, membrane-bound polysom es and tightly-bound polysomes). These results may indicate the significant role of TBP (putative cytoskeleton-bound polysomes - CBP) in maintaining dormancy during imbibition of cereal caryopses.

Oat phytoalexins, avenanthramides, occur as constitutive components in seeds. The amounts of each avenanthram ide were analyzed. The com position of avenanthram ides in dry seeds was different from that in elicitor-treated leaves. In seeds, avenanthram ide C was most abundant with an amount two times larger than that of avenanthramide A or B. On the other hand, avenanthramide A was the major component in elicitor-treated leaves. The total amount of avenanthram ides in seeds increased 2.5 times during imbibition for 48 h although the composition did not change. The hydroxycinnam oyl-CoA :hydroxyanthranilate N-hydroxycinnam oyltransferase (HHT, EC 2.3.1.-) activity, which is responsible for the final condensation step in the avenanthramide biosynthesis, was detected in dry seeds. The activity was localized in endosperm and scutellum , and slightly increased during 48-h imbibition. The enzyme was partially purified by anion exchange chromatography from both dry seeds and elicitor-treated leaves. The activity was separated into two peaks by chromatography, indicating that HHT consists of at least two isoforms. The substrate specificities of HHT isoforms from seeds were different from each other.

Exposure to the herbicide acifluorfen resulted in marked increase of glutathione S-transferase (GST) enzyme activity in wheat seedlings, primarily in shoot tissues. From the six major, constitutively expressed GST subunits found in un treated w heat shoots subunits 2 and 3 were selectively induced by acifluorfen. No new subunit could be detected. The induced subunits belong to those GST isoenzymes, which metabolize diphenyl ether herbicides.

Seven flavonoids isolated from chloroform , ethyl acetate and butanol extracts, obtained from the aerial parts of Retama sphaerocarpa, have been assessed for cytotoxic activity against three human cancer cell lines: TK-10 (renal adenocarcinom a), MCF -7 (breast adenocarcinom a) and UACC-62 (m elanom a), using the SRB assay. All of them , extracts and flavonoids, were actives in, at least, one of the three cell lines at the recom m ended N ational C ancer Institute doses. They produce a d ose-dependent inhibition of cell grow th at concentrations in the 10 -6 -10 -4 ᴍ and 25 -250 μg/ml range for the flavonoids and extracts respectively, being the flavonol rhamnazin the most cytotoxic.

In response to the exogenous application of elicitors and attempted invasion by pathogens, plants exhibit a wide range of defense reactions. To understand the defense mechanism s at the level of gene activation and deactivation, differential screenings were perform ed to isolate cDNA clones which are differentially expressed in pathogen-inoculated resistant chickpea plants and elicitor-treated cell cultures. A plenty of genes were isolated and arranged in 5 groups, namely defense-related pathways, signal transduction pathways, regulation of gene expression, catabolic pathways and primary metabolism . Most of these genes were activated although several genes were also found to be suppressed. We discuss the plausible functions of cDNA products in plant defense responses. The cDNA s provide a variety of tools to investigate m olecular mechanisms of defense responses and clearly reflect the massive genomic and metabolic changes which occur during manifestation of antimicrobial defense.

Catalytic activities of catalase (CAT) immobilized on graphite - GMZ™ , soot - “NORIT ” and “PM -100” to mediate decomposition of 3-Cl-C 6 H 4 COOOH (3-CPBA)in acetonitrile have been investigated. Under these conditions, the kinetic parameters K m , k, E a , V max , and Z o were calculated. Conclusions on a probable mechanism of the catalytic process observed were drawn from the calculated values of ΔG*, ΔH*, and ΔS*. A quantitative UV-spectrophotometrical approach was used as the basic analytical tool. The electrochemical reduction of oxygen generated in enzyme catalysed 3-CPBA decomposition was examined with polarization curves method.

Xanthine oxidase (E . C. 1.2.3.2) was immobilized by adsorption on electrochemically modified graphite plate to obtain an enzyme electrode. The current of the enzyme electrode in substrate (xanthine) solutions was found to be a result of the electrooxidation of H 2 O 2 generated in the enzyme layer. The linearity of the amperometric signal was up to a substrate concentration of 65 μm at 0.6 V (vs. Ag/AgCl). The response time was 2 minutes. The enzyme electrode preserves 80% of its initial activity activity after a three-week storage in air at room temperature.

Xylanase production of newly isolated thermophilic alkali-tolerant Bacillus sp. strain SP and strain BC was investigated in batch and continuous cultures. Enzyme synthesis was inducible with both strains and was observed only in xylan-containing media. Xylan from oat spelt is a better inducer than xylan from birch for strain Bacillus sp. BC while such difference was not observed for strain SP Compared with batch cultures xylanase production of both strains increased about two times and its rate became more than four times faster in continuous cultures at a dilution rate of 0.2 h -1 .

Three propolis samples from Austria, Germany and France were investigated by GC/MS, where eleven compounds were being new for propolis. The samples showed some similarities in their qualitative composition. Phenylethyl-trans-caffeate, benzyl ferulate and galangin were predominant in German propolis. Benzyl caffeate was predominant in French sample. Pinocembrin was predominant in French and Austrian propolis and trans-p-coumaric acid was predominant in all samples. The antimicrobial activity against Staphylococcus aureus; Escherichia coli, and Candida albicans was evaluated. German propolis showed the highest antimicrobial activity against Staphylococcus aureus and Escherichia coli. While Austrian propolis has the highest activity against Candida albicans. French propolis was effective against all pathogens but less than German and Austrian propolis.

Chemometrics has been shown quite efficient to uncover relationships between chemical composition of a sample and its geographical origin. Forty propolis samples originated from the the South and South East of Brazil were analyzed by HPLC and 18 compounds of interest were studied which included: caffeic, p-coumaric and ferulic acids, and some of their derivatives, pinobanksin, a derivative of kaempferol and five phenolic compounds (assigned as 3-prenyl-4-hydroxycinnamic acid (PHCA ); 2,2-dimethyl-6-carboxyethnyl-2H-l-benzopyran (DCBE); 3,5-diprenyl-4-hydroxycinnamic acid (DHCA ); compound E (still unknown) and 6-propenoic-2,2-dimethyl-8-prenyl-2H-l-benzopyran acid (DPB). Principal Component Analysis (PCA) indicated three different groups of propolis samples, having the same typical chromatogram, evaluated by HPLC. Samples from the South East group were rich in derivatives of kaempferol. Samples from the South group I had a high content of DPB compound, but a low concentration of kaempferol derivatives and of DCBEN compound. Samples from the South group II were characterized by a high concentration of DCBEN , DHCA , p-coum-aric and DPB compounds. Therefore, the identification of new compounds in Brazilian propolis can give useful information about the plant sources of a given geographic region.

A marine soft coral species of the genus Heteroxenia collected from Mindoro Island, Philippines yielded two cadinene sesquiterpenes, (+)-α-m uurolene (1) and a novel derivative (+)- 6 -hydroxy-α-muurolene (2 ), as well as the biologically active polyhydroxysterol, sarcoaldosterol A (3). The structure of the novel compound was unambiguously established on the basis of NMR spectroscopic ( 1 H, 13 C, COSY, 1 H-detected direct and long range 13 C - 1 H correlations) and mass spectrometric (EIMS) data. All compounds were active against the phytopathogenic fungus Cladosporium cucumerinum. The isolated terpenes were also active in the brine shrimp lethality test.

Sixty products, derived from marine organisms, typical of the Bulgarian Black Sea coast, were examined for inhibitory activity on the reproduction of influenza viruses in tissue cultures. The antiviral effect was investigated by the reduction of virus infectivity. Using representative strains of influenza virus it was shown that apparently the inhibitory effect was strain-specific. The most effective products were further studied in fertile hen’s eggs and in experimental influenza infection in white mice.

Toxicity of the extracellular products (ECP) and the lethal attribute of serine protease secreted by five pathogenic Vibrio alginolyticus strains from various sources in kuruma prawn Penaeus japonicus were studied. The ECPs of organisms originally isolated from diseased kuruma prawn or small abalone Haliotis diversicolor supertexta were more lethal (LD 50 value of 0.48 or 0.41 μg protein/g prawn) than those from diseased tiger prawn P.monodon, yellowfin porgy Acanthopagrus latus or horse mackerel (LD 50 value of 0.98 -1.17 μg protein/g prawn). All the ECPs manifested strong, weak and no activities against gelatin, sheep erythrocytes and chitin, respectively. In immunodiffusion tests using rabbit antiserum to a purified 33 kDa serine protease of strain Swy against ECP of each tested strain produced one single precipitation band in each treatment. Furtherm ore, the serine protease was suggested to be the dominant protease secreted by V. alginolyticus strains tested since the majority of enzymatic activity of the respective ECP was inhibited by phenylmethanesulfonyl fluoride (PMSF). A higher inhibition of serine protease activity by PMSF resulted in lower mortality rate of the ECPs injected into the prawns suggesting that the protease is one of the major lethal factor(s) secreted by Valginolyticus.

The substrate saturation and temperature-dependent kinetic properties of soluble and membrane-bound forms of acetylcholinestarase (AChE) from brain and butyrylcholinesterase (BChE) from heart and liver were examined. In simultaneous studies these parameters were also measured for AChE in erythrocyte membranes and for BChE in the serum from rat and humans. For both soluble and membrane-bound forms of the enzyme from the three tissues, two components were discernible. In the brain, K m of component I (high affinity) and component II (low affinity) was somewhat higher in membrane-bound form than that of the soluble form components, while the V max values were significantly higher by about five fold. In the heart, K m of component II was lower in membrane-bound form than in the soluble form, while V max for both the components was about four to six fold higher in the membrane-bound form. In the liver, V max was marginally higher for the two components of the membrane-bound enzyme; the K m only of component I was higher by a factor of 2. In the rat erythrocyte membranes three components of AChE were present showing increasing values of K m and V max . In contrast, in the human erythrocyte membranes only two components could be detected; the one corresponding to component II of rat erythrocyte membranes was absent. In the rat serum two components of BChE were present while the human serum was found to possess three components. Component I of the human serum was missing in the rat serum. Temperature kinetics studies revealed that the Arrhenius plots were biphasic for most of the systems except for human serum. Membrane binding of the enzyme resulted in decreased energy of activation with shift in phase transition temperature (T t ) to near physiological temperature.

The binding affinity of synthetic nonapeptides to human leucocyte antigens (HLA) molecules of the A0201 allotype, the most common in Caucasian, is enhanced or reduced by suitable amino acid substitutions at position 4, as a result of increased or decreased chain flexibility. A higher flexibility of the bond at this position correlates with an easier accomodation of the fragment into the HLA groove, while rigidity of the peptide chain appears to interfere. These data are based on two lines of evidence: a) most natural high affnity ligands for HLA-A0201 possess, at position 4, flexible residues b) substitutions of such residues by rigid amino acids results in a decrease of binding affinity.

Model derivatives of plasmalogens and chemically synthesized oxidative degradation products as found e.g. during oxidation of low density lipoproteins show strong effects on phagocytosis induced secretion of reactive oxygen species of macrophages which was measured by luminol-enhanced chemiluminescence. Whereas a plasmalogen epoxide showed enhancing effects in submicromolar range, inhibition was found with higher concentrations as well as with a-hydroxyaldehydes. The substances showed only little effects on the non-cellular ROSdependent chemiluminescence of the reaction between hydrogen peroxide and opsonized zymosan and no cytotoxic effects under the assay conditions used. These results show that oxidative modification and degradation of plasmalogens occuring also under pathophysiological situations in vivo produces effective modulators of macrophage function which could be important; e.g. during inflammation or atherogenesis.

Simvastatin is an inhibitor of HMG -CoA reductase used in the treatment of hypercholesterolemia. In the present study simvastatin-induced contraction was observed in rat aortic thoracic rings, this effect increased when the endothelium was removed and when NO synthase was blocked by L-NOARG (3 x 10 -5 ᴍ) . The contractile effect of simvastatin on intact aortic rings diminished when cyclo-oxygenase was inhibited with indomethacin (10 -5 ᴍ). Also in the presence of endothelium, pretreatment with mevalonate (1 mM), the product of HMGCoA reductase activity, significantly inhibited the contraction. In other experiments carried out on endothelium-removed preparations and in medium containing the calcium antagonist, diltiazem (10 -5 and 10 -6 ᴍ) , the contraction dose-response curves were significantly reduced and the same happened in the presence of the inhibitor of sarcoplasmic reticulum Ca- 2+ - ATPase, cyclopiazonic acid (CPA) (3 x 10 -6 ᴍ) . The results suggest that simvastatin might increase intracellular calcium concentration. This effect could lead to an activation of NO synthase and cyclooxygenase pathways in endothelial cells and to contraction in vascular smooth muscle cells. This rise in Ca 2+ concentration could be due to an inhibition of isoprenoid synthesis prevented by mevalonate.

Lom-AKH-I is a member of the adipokinetic hormone/ red pigment concentrating hormone (AKH / RPCH) family of peptides found in flying insects. A molecular dynamics simulation at room temperature (293 K) in water has been performed to survey the folding path of the Lom-AKH-I peptide in water and to establish the secondary structure of Lom-AKH-I. The obtained results indicate the presence of an undefined extended conformation.