The disintegration of yam tuber gamma-glutamyl transpeptidase during tuber storage

As an attempt to understand some aspects of the protein catabolism in the resting yam (Dioscorea rotundata) tuber with respect to the storage problems of the tuber, gamma-glutamyl transpeptidase (gamma-GTP) was isolated from healthy tubers after aerobic storage for varying periods. The molecular weight was determined by SDS-gel electrophoresis and the activity by spectrophotometry using gamma-glutamyl alpha-naphthylamide as substrate. Molecular weight and activity were plotted versus duration of the tuber storage. The molecular weight of the gamma-GTP from newly harvested tuber was 2.2 . 10(5). Both the mol. wt and activity decreased linearly with tuber storage, the two processes having approximately equal first order rate constant, 1.2 . 10(-2)/week, with a half-life of 58 weeks. High molecular weight forms of tuber gamma-GTP when dissociated and gel electrophorized gave in each case a lower molecular weight form that also had enzymic activity. A plot of the mol. wt versus the activity gave a straight line which intersected the mol. wt axis at 200000, which is the mol. wt of the smaller subunit of gamma-GTP. These results show that yam tuber gamma-GTP undergoes progressive disintegration during tuber storage and in this process the components are lost probably from the larger subunit after proteolytic cleavage and subsequent dissociation of the cleaved components.     

Inhibition of intestinal peroxidase activity by nonsteroidal antiinflammatory drugs

The peroxidase activity of the mitochondrial fraction of rat intestine is inhibited in vitro by non-steroidal antiinflammatory drugs (NSAIDs), such as indomethacin (IMN) and acetylsalicylic acid (ASA), the former being more potent than the latter. The peroxidase was solubilised by cetab-NH₄Cl extraction and purified to apparent homogeneity by Sephadex G-150 gel filtration and affinity chromatography on Con-A Sepharose. The purified enzyme activity was 80% inhibited by 150 μM IMN and 50% by 2.67 mM ASA. IMN could also inhibit lactoperoxidase activity to the same extent but not the horseradish peroxidase activity. The inhibition of peroxidase-catalysed iodide oxidation by IMN and ASA was optimal at pH 5.5 and 4.5, respectively. Kinetic studies revealed that the inhibition by IMN was competitive with respect to iodide or guaiacol, while the inhibition by ASA was noncompetitive and reversible in nature. Studies of some structural analogues showed that indole-3-acetic acid was as effective as IMN, while salicylic acid was more potent than ASA. Spectral studies showed a small bathochromic shift of the Soret band of the enzyme by IMN, suggesting its possible interaction at or near the heme moiety. The competitive nature of IMN may be explained as due to its oxidation by the peroxidase to a product absorbing at 412 nm, the formation of which is inhibited by iodide. We suggest that IMN inhibits intestinal peroxidase activity by acting as a competitive substrate for the enzyme. As intestinal peroxidase is mainly contributed by the invading eosinophils, NSAIDs may affect the host defence mechanism by inhibiting the activity of the enzyme.     

Enhancement of DNA polymerase activity in potato tuber slices

DNA polymerase was extracted from potato (Solanum tuberosumL.) tuber discs and the temporal correlation of its activitychange to DNA synthesis in vivo was examined during aging ofthe discs. Most of the DNA polymerase was recovered as a boundform in the 18,000?g precipitate. Reaction with the bound-formenzyme was dependent on the presence of four deoxynucleosidetriphosphates, Mg²⁺, and a template. "Activated" DNA and heat-denaturedDNA, but not native DNA, were utilized as templates. The polymeraseactivity was sensitive to SH reagents. Fresh discs, which donot synthesize DNA in vivo, contained a significant amount ofDNA polymerase and its activity increased linearly with timeuntil 48 hr after slicing and became four times that of freshdiscs after 72 hr, whereas the activity of DNA synthesis invivo increased with time and decreased after reaching a maximumat 30 hr. Cycloheximide inhibited the enhancement of polymeraseactivity. DNA polymerase from aged and fresh discs had identicalrequirements for deoxynucleotides and a template in their reactions,sensitivity to SH reagent, and affinity to thymidine triphosphate. (Received February 18, 1977; )     

Antioxidant properties of some hydroalcoholic plant extracts with antiinflammatory activity

The hydroalcoholic extracts of Calendula officinalis, Hypericum perforatum, Plantago lanceolata and Glycyrrhiza glabra which exhibited different anti-inflammatory activities were evaluated for the possible mode of action by studying their antioxidant potential. In the present study we investigated if standardized hydroalcoholic extracts of plants such as Calendula officinalis, Hypericum perforatum, Plantago lanceolata and Glycyrrhiza glabra produced by Hofigal Stock Company could modulate the respiratory burst of human activated neutrophils, as a consequence of their antioxidant capacity. Their antioxidant properties were measured using a colorimetric assay (Total Antioxidant Status kit). We demonstrated that Hypericum perforatum and Calendula officinalis hydroalcoholic extracts possessed a significant antioxidant activity while Plantago lanceolata and Glycyrrhiza glabra hydroalcoholic extracts had a minor antioxidant status. Using reactive oxygen species-generating systems (OZ-activated human PMN neutrophils), Calendula officinalis and Hypericum perforatum extracts showed strong reactive oxygen species scavenging property, Hypericum perforatum extract exhibing the highest scavenging activity. These results confirm the potential of Calendula officinalis and Hypericum perforatum investigated hydroalcoholic extracts as medicinal remedies to be used in different inflammatory/allergic diseases. These extracts could be a useful tool for obtaining new antioxidant/anti-inflammatory agents.     

Wound-induced phenylalanine ammonia-lyase in potato tuber tissue. Development of enzyme activity and effects of antibiotics.

Phenylalanine ammonia-lyase [EC 4.3.1.5.] activity increased rapidly after a 3-hr lag period in potato tuber (Solanum tuberosum L. cv. May Queen) disks incubated in a suitable medium in the dark at 25 degrees. The activity reached a maxinum after incubation for about 40 hr. The effects of actinomycin D, 6-methylpurine, cycloheximide, chloramphenicol, and mitomycin C on the induction of phenylalanine ammonia-lyase were investigated during incubation of the disks. Actinomycin D, 6-methylpurine, and cycloheximide all inhibited the formation of phenylalanine ammonia-lyase, though cycloheximide was the most effective at low concentrations. Application of actinomycin D for the initial lag period (3 hr) caused strong inhibition; however, if it was supplied later it did not inhibit but actually increased phenylalanine ammonialyase formation. In contrast, cycloheximide was effective over most of the incubation period. Chloramphenicol and mitomycin C did not inhibit phenylalanine phenylalanine ammonialyase induction, but markedly stimulated it. Light was not an essential factor for phenylalanine ammonia-lyase induction in the wounded tissue.     

Cytotoxic (9βH)‐Pimarane and (9βH)‐17‐Norpimarane Diterpenes from the Tuber of Icacina trichantha

Three (9βH)‐pimaranes, 1, 2 , and 3 , and two (9βH)‐17‐norpimaranes, 4 and 5 , belonging to a rare compound class in nature, were obtained from the tubers of Icacina trichantha for the first time. Compound 1 is a new natural product, and 2 – 5 have been previously reported. The structures were elucidated based on NMR and MS data, and optical rotation values. The absolute configurations of (9βH)‐pimaranes were unambiguously established based on X‐ray crystallographic analysis. Full NMR signal assignments for the known compounds 2, 4 , and 5 , which were not available in previous publications, are also reported. All five isolates displayed cytotoxic activities on MDA‐MB‐435 cells (IC₅₀ 0.66–6.44 μM ), while 2, 3 , and 4 also exhibited cytotoxicities on HT‐29 cells (IC₅₀ 3.00–4.94 μM ).     

Regulation of uncoupling protein activity in phosphorylating potato tuber mitochondria

In isolated potato tuber mitochondria, palmitic acid (PA) can induce a H+ leak inhibited by GTP in the phosphorylating (state 3) respiration but not in the resting (state 4) respiration. The PA-induced H+ leak is constant when state 3 respiration is decreased by an inhibition of the succinate uptake with n-butyl malonate (nBM). We show that the efficiency of inhibition by GTP is decreased when state 3 respiration is progressively inhibited by antimycin A (AA) and is restored following subsequent addition of nBM. We propose that in phosphorylating potato tuber mitochondria, the redox state of ubiquinone, which can antagonistically be varied with AA and nBM, modulates inhibition of the PA-activated UCP-sustained H+ leak by GTP.     

The correlation of phenylalanine ammonia-lyase and cinnamic acid-hydroxylase activity changes in Jerusalem artichoke tuber tissues

Summary Phenylalanine ammonia-lyase (PAL) and cinnamic acid-hydroxylase (CAH) exhibit parallel activity changes in tissues of Jerusalem artichoke (Helianthus tuberosus L.) tubers during ageing and in vitro culture. The activity of both enzymes appears in the tissue only after slicing and can be stimulated by manganese. Inhibition of CAH, by the deprivation of the substrate O₂, leads to cinnamic acid accumulation and PAL inhibition, a process prevented by cycloheximide. It is propounded that the link between PAL and CAH activities is the result of changes of the cinnamic acid pool size produced by a change in the activity of CAH.     

Chemical properties and antiinflammatory activity of a galactomannoglucan from Arecastrum romanzoffianum

A galactomannoglucan (GMG) with an estimated weight-average molar mass of 1.5 × 10⁵ was obtained from an aqueous extract of the mesocarp of fruits of Arecastrum romanzoffianum (Cham.) Becc. by fractionation by Sephacryl S-300 HR and Sephadex G-25. Chemical and spectroscopic studies indicated that GMG has a chain of (1 → 4)-linked β-d-mannopyranosyl residues, a chain of (1 → 3)-linked β-d-galactopyranosyl residues, a chain of (1 → 4)-linked α-d-glucopyranosyl residues, repeating units of β-d-galactopyranosyl-(1 → 4)-β-d-mannopyranosyl and β-d-mannopyranosyl-(1 → 4)-α-d-glucopyranosyl and terminal residues of d-galactopyranosyl and d-glucopyranosyl which comprised galactose, mannose and glucose in the molar ratio of 10:37:53. The polysaccharide exhibited significant antiinflammatory activity against carrageenan-induced mouse paw oedema.     

Screening of antiradical and antioxidant activity of monodesmosides and crude extract from Leontice smirnowii tuber.

Leontice smirnowii is a member of the Berberidaceae family. In the current study we investigated the possible antiradical and antioxidant activity of the monodesmosides (MLS) and crude extract (CELS) of Leontice smirnowii using different antioxidant tests: 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging, scavenging of superoxide anion radical-generated non-enzymatic system, ferric thiocyanate (FTC) method, reducing power, hydrogen peroxide scavenging and metal chelating activities. Experiment revealed that MLS and CELS have an antioxidant effect concentration-dependently. Total antioxidant activity was performed according to FTC method. At the 30mug/ml concentration, the inhibition effects of MLS and CELS on peroxidation of linoleic acid emulsion were found to be 95.3% and 95.6%, respectively. On the other hand, percentage inhibition of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), alpha-tocopherol and trolox were found to be 98.2%, 98.5%, 84.0% and 87.9% inhibition of peroxidation of linoleic acid emulsion, respectively, at the same concentration. In addition, MLS and CELS had effective DPPH radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, reducing power and metal chelating activities. Also, these various antioxidant activities were compared with BHA, BHT, alpha-tocopherol and trolox which were accepted as references antioxidants.     

The amyloplast proteome of potato tuber

Potato (Solanum tuberosum) is the fourth largest crop worldwide in yield, and cv. Kuras is the major starch potato of northern Europe. Storage starch is packed densely in tuber amyloplasts, which become starch granules. Amyloplasts of soil-grown mini-tubers and agar-grown micro-tubers of cv. Kuras were purified. The mini-tuber amyloplast preparation was enriched 10-20-fold and the micro-tuber amyloplast approximately fivefold over comparative total protein extracts. Proteins separated by SDS-PAGE were digested with trypsin, analysed by mass spectrometry and identified by mascot software searches against an in-house potato protein database and the NCBI non-redundant plant database. The differential growth conditions for mini- and micro-tubers gave rise to rather different protein profiles, but the major starch granule-bound proteins were identical for both and dominated by granule-bound starch synthase I, starch synthase II and alpha-glucan water dikinase. Soluble proteins were dominated by starch phosphorylase L-1, other large proteins of the classes 'starch and sucrose metabolism', 'pentose phosphate pathway', 'glycolysis', 'amino acid metabolism', and other proteins such as plastid chaperonins. The majority of the identified proteins had a predicted plastid transit peptide, supporting their presence in the amyloplast. However, several highly expressed proteins had no transit peptide, such as starch phosphorylase H, or had a predicted mitochondrial location. Intriguingly, all polyphenol oxidases, a family of enolases, one transketolase, sulfite reductase, deoxynucleoside kinase-like and dihydroxy-acid dehydrase had twin-arginine translocation motifs, and a homologue to dihydrolipoamide dehydrogenase had a Sec (secretory) motif; these motifs usually target thylakoid-like structures.     

Nootropic activity of tuber extract of Pueraria tuberosa (Roxb)

Nootropic effect of alcoholic (ALE; 50, 75, 100 mg/kg) and aqueous (AQE; 100, 200, 400 mg/kg) extracts of P. tuberosa was evaluated by using Elevated Plus Maze (EPM), scopolamine-induced amnesia (SIA), diazepam-induced amnesia (DIA), clonidine-induced (NA-mediated) hypothermia (CIH), lithium-induced (5-HT mediated) head twitches (LIH) and haloperidol-induced (DA- mediated) catalepsy (HIC) models. Piracetam was used as the standard drug. A significant increase in inflexion ratio (IR) was recorded in EPM, SIA and DIA models. A significant reversal effect was observed on rectal temperature in CIH model, reduction of head twitches in LIH models. However no significant reduction in catalepsy scores in HIC models were observed with test extracts and standard piracetam. The results indicate that nootropic activity observed with ALE and AQE of tuber extracts of P. tuberosa could be through improved learning and memory either by augmenting the noradrenaline (NA) transmission or by interfering with 5-hydroxytryptamine (5-HT) release. Further, the extracts neither facilitated nor blocked release of the dopamine (DA). Thus ALE and AQE elicited significant nootropic effect in mice and rats by interacting with cholinergic, GABAnergic, adrenergic and serotonergic systems. Phytoconstituents like flavonoids have been reported for their nootropic effect and these are present in both ALE and AQE extracts of tubers of P. tuberosa (Roxb) and these active principles may be responsible for nootropic activity.     

Potato tuber cytokinin oxidase/dehydrogenase genes: Biochemical properties,activity, and expression during tuber dormancy progression

The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in lateral buds isolated from field-grown tubers. All five putative StCKX genes encoded proteins with in vitro CKX activity. All five enzymes were maximally active at neutral to slightly alkaline pH with 2,6-dichloro-indophenol as the electron acceptor. In silico analyses indicated that four proteins were likely secreted. Substrate dependence of two of the most active enzymes varied; one exhibiting greater activity with isopentenyl-type cytokinins while the other was maximally active with cis-zeatin as a substrate. [³H]-isopentenyl-adenosine was readily metabolized by excised tuber buds to adenine/adenosine demonstrating that CKX was active in planta. There was no change in apparent in planta CKX activity during either natural or chemically forced dormancy progression. Similarly although expression of individual StCKX genes varied modestly during tuber dormancy, there was no clear correlation between StCKX gene expression and tuber dormancy status. Thus although CKX gene expression and enzyme activity are present in potato tuber buds throughout dormancy, they do not appear to play a significant role in the regulation of cytokinin content during tuber dormancy progression.