Interference of L-α-aminoocy-β-phenylpropionic acid with cold-induced sphagnorubin synthesis in Sphagnum magellanicum BRID

The ability of the phenylalanine ammonia-lyase (PAL)-inhibitor L--aminooxy--phenyl-propionic acid (AOPP) to suppress the synthesis of the main reddish-violet wall pigment of Sphagnum magellanicum (sphagnorubin) was investigated. Fifty percent inhibition is achieved with 14 M AOPP in mosses stimulated to intensive coloring by sugar feeding. AOPP does not affect the content of free amino acids, except for phenylalanine, during cold-induced sphagnorubin synthesis. AOPP dramatically amplifies the increase in extractable PAL activity in response to cold treatment. Phenylalanine applied in vivo causes an eminent increase in PAL activity, above the level of the cold-treated mosses. The results from the feeding experiments are discussed in connection with a possible end-product repression in PAL activity with sphagnorubin-synthesizing mosses. These results are correspond best to the theory that the enzyme level is regulated independently from a mechanism of feedback repression.Abbreviations L-AOPP L--aminooxy--phenylpropionic acid - DCMU 3-(3,4-dichlorophenyl)-1.1-dimethyl urea - PAL L-phenylalanine ammonia-lyase (EC. 4.3.1.5.) - TAL L-tyrosine ammonia-lyase     

Inhibition of anthocyanin formation in seedlings and flowers by the enantiomers of α-aminooxy-β-phenylpropionic acid and their N-benzyloxycarbonyl derivatives

Both enantiomers of -aminooxy--phenylpropionic acid (AOPP), potent inhibitors of L-phenylalanine ammonia-lyase, and their N-benzyloxycarbonyl (N-BOC) derivatives inhibit anthocyanin formation in developing flowers of Ipomoea tricolor Cav. and Catharanthus roseus Don. as well as in seedlings of Brassica oleracea var. caulo-rapa DC (kohlrabi) and B. oleracea var. capitata L. (red cabbage) with little interference with their normal development. Kohlrabi seedlings tolerate up to 0.3 mM L-AOPP and N-BOC-L-AOPP without a reduction of fresh weight or chlorophyll content, while anthocyanin is reduced to less than 20%.Abbreviations AOPP -aminooxy--phenylpropionic acid - N-BOC N-benzyloxycarbonyl - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5)     

The influence of lignification on the development of vascular tissue inVigna radiata L.

Summary During the early development of mungbean seedlings, treatment with L--aminooxy--phenylpropionic acid (AOPP), a potent specific inhibitor of phenylalanine ammonia-lyase, results in an inhibition of anthocyanin and lignin synthesis. The xylem vessels of the hypocotyl and root of AOPP treated seedlings collapse, and the cellulose microfibrils of the unlignified secondary wall are separated from one another and lie disorganized in the lumen of the mature xylem cell. The differentiation of the secondary cell wall appears unaffected by AOPP treatment, as does the ultrastructure of the wall of the mature phloem fibers of the root which is also lignified in untreated tissue. The results are discussed in the light of current thinking on the role and development of lignification in the xylem vessel.Abbreviation AOPP L-aminooxy--phenylpropionic acid     

Extracellular peroxidases of suspension culture cells of spruce (Picea abies): fungal elicitor-induced inactivation

Extracellular peroxidases of suspension cultures of spruce (Picea abies) (L.) (Karst) become inactivated when the cell suspension is elicited with a cell wall preparation of the spruce pathogenic fungus Rhizosphaera kalkhoffii. In contrast, cellular peroxidases are induced under these conditions. Both changes of activity are reflected in the isoenzyme profiles.Inactivation of the extracellular peroxidases is caused by an effector, arising from the cells after contact with the elicitor. Formation of the effector is limited to the beginning of elicitation, showing maximal activity at this period of time. Subsequently it becomes increasingly ineffective, probably due to inactivation. The effector is able to also inactivate commercial (horseradish) peroxidase. Inactivation was not the result of the action of a protease present in the medium.The elicitor exerts two different effects on the spruce cell suspension culture. It induces synthesis of enzymes correlated with lignin synthesis and an accumulation of lignin-like material. It also induces secretion of the negative effector which inactivates extracellular peroxidases.The elicitor-induced inactivation is not specific for peroxidases. Other extracellular enzymes, -glucosidase and acid phosphatase (secreted by the cells into the medium) and -amylase and pectinase (from Aspergillus strains) are also inactivated.     

Peroxidase from Catharanthus roseus (L.) G. Don and the biosynthesis of α-3′,4′-anhydrovinblastine: a specific role for a multifunctional enzyme

Summary. We have characterized a basic peroxidase with -3,4-anhydrovinblastine (AVLB) synthase activity, which was purified from Catharanthus roseus leaves. This enzyme was the single peroxidase isoenzyme detected in C. roseus leaves, and the single AVLB synthase activity detected in C. roseus extracts. It was observed that the monomeric substrates of AVLB, vindoline and catharanthine, are both suitable electron donors for the oxidizing intermediates of the basic peroxidase, compounds I and II. Results also showed that the reaction proceeds by a radical-propagated mechanism. Substrate specificity studies of the enzyme revealed that it was also able to oxidize several common peroxidase substrates, indicating a broad range of substrate specificity that is characteristic of class III plant peroxidases. Cytochemical studies showed that the enzyme is localized in C. roseus mesophyll vacuoles, in individual spots at the inner surface of the tonoplast. This particular location suggests a meaningful spatial organization that led to the proposal of a metabolic channeling model for the peroxidase-mediated synthesis of AVLB. The importance of this type of mechanism in the regulation of peroxidase isoenzyme functions in vivo is discussed. In view of the results obtained it is concluded that the basic peroxidase present in C. roseus leaves fulfills all the requirements to be considered as an AVLB synthase, and it is proposed that this specific function of this multifunctional enzyme is determined by metabolic channeling resulting from specific protein–protein interactions.Correspondence and reprints: Institute for Molecular and Cell Biology, University of Porto, rua Campo Alegre 823, 4150-180 Porto, Portugal.Received March 15, 2002; accepted February 4, 2003; published online August 26, 2003     

Modulation of the serotonin-activated K+ channel by G protein subunits and nucleotides in rat hippocampal neurons

In hippocampal neurons, 5-hydroxytryptamine (5-HT) activates an inwardly rectifying K⁺ current via G protein. We identified the K⁺ channel activated by 5-HT (K_5-HT channel) and studied the effects of G protein subunits and nucleotides on the K⁺ channel kinetics in adult rat hippocampal neurons. In inside-out patches with 10 m 5-HT in the pipette, application of GTP (100 m) to the cytoplasmic side of the membrane activated an inwardly rectifying K⁺ channel with a slope conductance of 36±1 pS (symmetrical 140 mm K⁺) at –60 mV and a mean open time of 1.1±0.1 msec (n=5). Transducin activated the (K_5-HT) channels and this was reversed by -GDP. Whether the K_5-HT channel was activated endogenously (GTP, GTPS) or exogenously (), the presence of 1 mm ATP resulted in a 4-fold increase in channel activity due in large part to the prolongation of the open time duration. These effects of ATP were irreversible and not mimicked by AMPPMP, suggesting that phosphorylation might be involved. However, inhibitors of protein kinases A and C (H-7, staurosporine) and tyrosine kinase (tyrphostin 25) failed to block the effect of ATP. These results show that G activates the G protein-gated K⁺ channel in hippocampal neurons, and that ATP modifies the gating kinetics of the channel, resulting in increased open probability via as yet unknown pathways.     

The feeding ecology of a carnivorous plant (Pinguicula nevadense): prey analysis and capture constraints

Summary The taxonomic composition and size of arthropods captured by Pinguicula nevadense, an endemic carnivorous plant of the high-mountain zone of the Sierra Nevada (southern Spain), are analysed. The actual prey of P. nevadense and the available arthropods trapped by mimic-traps are compared, in order to identify the capture constraints of the plant. The results show that P. nevadense captures various arthropod taxa. Winged insects, especially Nematocera, make up the main component of the diet. The range of prey sizes in all P. nevadense populations studied is similar. The taxonomic composition of arthropods trapped by the mimic-traps is similar to that of the actual prey of P. nevadense. However, the plant captures prey only below a specific size threshold. These size constraints appear to be the principal factor determining the actual prey of this carnivorous plant.     

Antimutagenic Role of Autonomous 3′ → 5′-Exonucleases

Original and published data on the antimutagenic role of autonomous 3 5-exonucleases (AE) are analyzed. AE are not bound covalently to DNA polymerases but are often involved in replicative complexes. AE overproduction in bacterial cells is accompanied by a sharp suppression of mutagenesis, whereas AE inactivation in bacteria and higher fungi results in the increase in mutation rates by two to three orders of magnitude. The combined action of AE and DNA polymerases substantially improves the fidelity of their functioning in vitro. The fidelity of nuclease-free DNA polymerases and increases by two to three orders of magnitude in the presence of AE. The fidelity of moderately processive DNA polymerase I increases by two orders of magnitude, and that of highly processive DNA polymerase increases by a factor of 5–10, although both these polymerases possess their own 3 5-exonucleolytic activity. In biochemical experiments, AE was shown to participate directly in the correction of errors made by DNA polymerase I. The presence of AE in multienzyme DNA polymerase complexes increases their fidelity by a factor of 5–10. A model of extrinsic proofreading by AE in DNA biosynthesis is proposed. An investigation of thirty objects from all three kingdoms of life (from archaea and bacteria to mammals, including humans) has shown that AE account for 30–90% of the total cellular 3 5-exonucleolytic activity. Therefore, AE increase significantly the intracellular ratio of 3 5-exonuclease to DNA polymerase activities in a wide phylogenetic variety of species, which always leads to the increasing fidelity of DNA biosynthesis.     

Gynoecium development inRubiaceae-Vanguerieae,with particular reference to the “stylar head”-complex and secondary pollen presentation

Gynoecium development in taxa of the tribeVanguerieae (Rubiaceae, subfam.Antirheoideae) was studied, using primarily a 2- and a 5-carpellate genus (Keetia andVangueria) as examples. All investigated taxa, characterized by ovaries with a solitary, ± apically inserted, pendulous anatropous ovule per locule/carpel, showed a very similar gynoecium development. Comparisons with other uniovulateRubiaceae (taxa of subfam.Rubioideae) revealed that notwithstanding the place of insertion and orientation of the solitary ovules (apically vs. basally inserted, pendulous vs. erect, anatropous ovules) the gynoecium development follows the same pattern; differing ovule insertion and orientation can easily be explained by the principle of variable proportions. — Since secondary pollen presentation is characteristic for the tribe, particular attention was paid to the development of the conspicuous “stylar head”-complex, defined here as structural unit comprised of pollen presenting organ, “receptaculum pollinis”, plus stigmatic (i.e., receptive) surfaces. It was found that their morphological differentiation, following the same pattern in all investigated taxa, starts at very early stages of floral development. They already had their final shape at a stage when the ovule development in the ovary had just barely started. The fully developed, actual “receptaculum pollinis” is made up of enormously enlarged, ± cylindrical epidermis cells which have peculiar ring-like thickenings in vicinity of and parallel to the outer tangential walls (a “mechanical barrier” preventing pollen tubes from entering?) and a much smaller-celled subepidermal tissue. With regard to shape, size, and exposure of the stigmatic surfaces, the investigated taxa exhibited certain morphological differences. These were found to be correlated with differences in androecium structure (and, ultimately, pollen presentation).     

Induction of cellulose- and xylan-degrading enzyme complex in the yeast Trichosporon cutaneum

The specificity of induction of cellulose- and xylan-degrading enzymes was investigated on the yeast strain Trichosporon cutaneum CCY 30-5-4 using series of compounds structurally related to cellulose and xylan, including monosaccharides, glycosides, glucooligosaccharides and xylooligosaccharides. Determination of activities of secreted cellulase and -xylanase, intracellular, cell wall bound and extracellular -glucosidase and -xylosidase revealed that: (1) The synthesis of xylan-degrading enzymes is induced in the cell only by xylosaccharides, 1,3--xylobiose, 1,2--xylobiose, 1,4--xylosyl-L-arabinose, 1,4--xylobiose and thioxylobiose being the best inducers. The xylan-degrading enzymes show different pattern of development in time and discrete cellular localization, i.e. intracellular -xylosidase precedes extracellular -xylanase. (2) A true cellulase is not inducible by glucosaccharides and cellulose. Negligible constitutive cellulase activity was detected which was about two orders lower than an induced cellulase in the typical cellulolytic fungus Trichoderma reesei QM 9414. (3) The best inducer of intracellular -glucosidase splitting cellobiose was thiocellobiose in a wide range of concentration (0.1–10 mM), whereas xylosaccharides at high concentrations induced -xylosidase of xylobiose type and a non-specific aryl -D-glucosidase.The results were confirmed by growing cells on cellulose and xylan. T. cutaneum was found to be a xylan-voracious yeast, unable to grow on cellulose.     

Effects of D‐mannoheptulose upon D‐glucose metabolism in tumoral pancreatic islet cells

D[³H]mannoheptulose was recently reported to be poorly taken up by tumoral pancreatic islet cells of the RINm5F and INS1 lines. We have now investigated the effects of Dmannoheptulose upon Dglucose metabolism in these two cell lines. Dmannoheptulose (1.0–10.0 mM) only caused a minor decrease of Dglucose metabolism in RINm5F cells, whether at low (1.1 mM) or higher (8.3 mM) Dglucose concentration. A comparable situation was found in INS1 cells examined after more than 20 passages. In both cases, however, the hexaacetate ester of Dmannoheptulose (5.0 mM) efficiently inhibited Dglucose metabolism. In the INS1 cells, the relative extent of the inhibitory action of Dmannoheptulose upon Dglucose metabolism increased from 12.4 ± 2.6 to 38.3 ± 3.8% as the number of passages was decreased from more than 20 to 13–15 passages, the latter percentage remaining lower, however, than that recorded in INS1 cells also examined after 13–15 passages but exposed to Dmannoheptulose hexaacetate (66.9 ± 2.2%). These findings when compared to our recent measurements of D[³H]mannoheptulose uptake, reinforce the view that the entry of the heptose into cells and, hence, its inhibitory action on Dglucose metabolism are dictated by expression of the GLUT2 gene.     

Isolation and characterization of the ecto-5′-nucleotidase from a rat glioblastoma cell line

Summary 5-Nucleotidase has been purified from rat glioblastoma cells (Rugli cells). The enzyme has been solubilized from plasma membranes by using Triton X-100 and CHAPS. Two affinity chromatographies on concanavalin A and 5-AMP-Sepharose render the purified enzyme with a high specific activity (76.36 mol AMP-min^–1-mg^–1). The purified enzyme gives a single polypeptide band on SDS-PAGE with an apparent molecular mass of 74 kDa. Active forms with an apparent molecular mass of 135 kDa and 268 kDa are observed when the purified enzyme is analyzed by gel filtration in the presence of either 0.6% sodium deoxycholate or 0.1% Triton X-100, respectively. The purified 5-nucleotidase presents optimum activity at pH 7.8–8.1 either in the presence or in the absence of Me²⁺. A linear Arrhenius plot is observed in the 25–46° C temperature range and an activation energy of 33.7 KJ/mol is calculated. The enzyme is inhibited by EDTA; the activity is partially restored by different divalent cations as Zn²⁺, Mn²⁺, and Co²⁺. The hydrolysis of nucleosides 5-monophosphate shows Michaelis kinetic. The enzyme is inhibited by nucleosides di- and triphosphate. 5-Nucleotidase is a glycoprotein, being its activity inhibited at different extent by various lectins.     

Transglycosylation reactions by exoglycosidases from the termite Macrotermes subhyalinus

The ability of four exoglycosidases (-galactosidase, -glucosidase, -glucosidase and invertase) from the termite Macrotermes subhyalinus to catalyse tranglycosylation reactions was tested using lactose, cellobiose, maltose and sucrose as glycosyl donors and 2-phenylethanol as glycosyl acceptor. The experimental conditions were optimized in relation to the time course of the reaction, pH and concentrations of glycosyl donor and acceptor. Whereas the hydrolytic activity was largely predominant over the transferase activity with -galactosidase and -glucosidase, the transglycosylation activity represented 68% with -glucosidase. In addition, as demonstrated by the transglycosylation product formed, the hydrolysis of sucrose was catalysed by -glucosidase and not by invertase. On the basis of this work, -glucosidase from M. subhyalinus appears to be a valuable tool for the preparation of neoglycoconjugates.     

Occurrence of phytoalexins and other putative defense-related substances in uninfected parsley plants

Considerable amounts of the following substances were found in uninfected parsley (Petroselinum crispum) cotyledons: furanocoumarins, the putative phytoalexins of this and some related plant species, two enzymes of the furanocoumarin pathway (S-adenosyl-L-methionine: xanthotoxol and S-adenosyl-L-methionine: bergaptol O-methyltransferases), two hydrolytic enzymes (1,3--glucanase, EC 3.2.1.39, and chitinase, EC 3.2.1.14), and pathogenesis-related proteins. The furanocoumarins and the methyltransferase activities reached their highest levels at the onset of cotyledon senescence as the hydrolytic enzymes increased from low to relatively high activity values. The relative amounts of pathogenesis-related proteins 1 and 2, as well as the corresponding mRNAs, also increased markedly. Two enzymes of general phenylpropanoid metabolism, L-phenylalanine ammonia-lyase and 4-coumarate: CoA ligase, decreased in activity in a biphasic fashion during cotyledon development. At all developmental stages, the levels of these putative defense-related agents in total cotyledon extracts were too high to enable detection of, possibly, additional changes upon infection with zoospores of Phytophthora megasperma f. sp. glycinea, a fungal pathogen to which parsley shows a non-host, hypersensitive resistance response.Abbreviations BMT S-adenosyl-L-methionine: bergaptol O-methyltransferase (EC 2.1.1.-) - 4CL 4-coumarate: CoA ligase (EC 6.1.1.12) - CMT S-adenosyl-L-methionine: caffeate O-methyltransferase (EC 2.1.1.-) - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5) - PR pathogenesis-related - XMT S-adenosyl-L-methionine: xanthotoxin O-methyltransferase (EC 2.1.1.-)     

Influence of several nucleotides on the competence development of Bacillus subtilis

The influence of adenosine-3,5-cyclic monophosphate (cAMP) and other nucleotides on the competence development of Bacillus subtilis was studied. The stimulation of competence which can be achieved by exposing physiologically low-competent cells to supernatants from highly competent cultures can be inhibited with different cAMP doses. When the same cells were suspended in a minimal medium with cAMP, varying degrees of stimulation of competence were observed depending on the time of addition of the drug. This effect is not specific for cAMP. It appears to be correlated to an increase of the amount of DNA bound to the competent cells. cAMP activities were antagonized by equimolar doses of adenosine-triphosphate (ATP) and guanosine-triphosphate (GTP).List of Abbreviations ATP adenosine triphosphate - AMP adenosine monophosphoric acid - GTP guanosine-triphosphate - cGMP guanosine 3,5-cyclic-monophosphoric acid - PLC physiologically low-competent cells - TY triptone yeast - CSA competence-stimulating activity - SF filtered supernatants - NCS non-competent supernatants - MBW minimal Bott and Wilson     

Δ′-Dehydrogenation of steroids byArthrobacter simplex immobilized in calcium polygalacturonate beads

Summary Arthrobacter simplex ATCC 6946 (viable cells) was immobilized in a calcium polygalacturonate gel. The trapped cells were used for repeated batchwise bioconversion of steroids. Reichstein's compound S and hydrocortisone were dehydrogenated introducing a double bond between C₁ and C₂ of ring A. The products 1-dehydro S and prednisolone, respectively, were identified by high pressure liquid chromatography. Steroid dehydrogenase activity increased in the system when an artificial electron acceptor, such as menadione (vitamin K₃) was present in the reaction mixture. An airlift-type reactor was used to bioconvert up to 90% of substrate in 15 min, under optimal conditions. The gel entrapped cell preparations were used for repeated batch bioconversion during 30 days; 69 batch bioconversions for Reichstein's compound S were performed during 15 days of operation of the reactor. The operational stability of the process and the feasibility of repeated batch bioconversions was shown to be comparable to similar processes.     

Different evolution rates within the lens-specificβ-crystallin gene family

Summary We have determined the sequence of a rat A3/A1-crystallin complementary DNA (cDNA) clone and the (partial) sequence of the human B3-crystallin gene. Calculation of the ratio of silent to nonsynonymous substitution between orthologous A3/A1-, B3-, and other - and -crystallin sequences revealed that the region encoding the two globular domains of the A3/A1-crystallin sequence is the best conserved during evolution, much better than the corresponding region of the B1-, B3-, or the -crystallin sequences, and even better (at least in the rodent/frog comparison) that the well-conserved A-crystallin sequence. Remarkably, the rate of change of the A3/A1-crystallin coding sequence does not differ in the rodent and primate lineages, in contrast with previous findings concerning the evolution rates of the A- or -crystallin sequences in these two lineages. Comparison of the regions that encode the four motifs of the -crystallin between orthologous mammalian sequences showed that the extent of nonsynonymous substitution in each of these four homologous motif regions is the same. However, when the orthologous -crystallin genes of more distantly related species (mammals vs chicken or frog) are compared, the extent of nonsynonymous substitution is higher in the regions encoding the external motifs I and III than in the regions encoding the internal motifs II and IV. This phenomenon is also observed when paralogous members of the /-crystallin supergene family are compared.     

Peptide modification by incorporation ofα-trifluoromethyl substituted amino acids

Summary Metabolic stabilization of pharmacologically active peptides can be achieved by incorporation of sterically hindered non-natural amino acids, e.g. C ^, -disubstituted amino acids.-Trifluoromethyl substituted amino acids, a subclass of C ^, -disubstituted amino acids, also fulfil this requirement while featuring additional properties based on the electronic influence of the fluorine substituents.This review summarizes the results concerning the stability of peptides containing-TFM amino acids towards proteolysis by-chymotrypsin. Furthermore, configurational effects of-TFMAla on the proteolytic stability of peptides are explained using empirical force field calculations. The influence of-TFMAla incorporation on the secondary structure of selected tripeptide amides is compared to the effects exerted by its fluorine-free analogue, aminoisobutyric acid.Finally, results on metabolic stabilization and biological activity of modified thyrotropin releasing hormone are interpreted.