Characterization and engineering of glycosyltransferases responsible for steroid saponin biosynthesis in Solanaceous plants

Solanaceous plants contain steroid saponins that have diverse biological and pharmacological activities. The structures of their sugar chains play an important role in their activities. A functional glucosyltransferase SaGT4A from Solanum aculeatissimum glucosylates both steroidal sapogenins and steroidal alkaloids. A potato (S. tuberosum) glycosyltransferase StSGT, which has a high degree of sequence homology with SaGT4A, exhibits the same substrate specificity toward steroidal compounds as SaGT4A. To identify the residues or domain structures responsible for these enzymatic activities, we determined the residues that are essential for SaGT4A activity, compared the specific activities of SaGT4A and StSGT, and constructed several SaGT4A/StSGT chimeric proteins, focusing on the donor-sugar recognition domain. These proteins were heterogeneously expressed in E. coli and purified, and their glycosyltransferase activities were evaluated using a coupled assay. His369 and Glu377, located in the consensus motif for plant glycosyltransferases, and Cys121, Cys247, and Cys370 were shown to be important for SaGT4A activity. StSGT exhibited more activity with UDP-galactose as a sugar donor than with UDP-glucose, whereas SaGT4A exhibited glucosyltransferase activity exclusively. The sugar selectivities of SaGT4A and StSGT were not altered by exchanging their domains, and some of the chimeric proteins showed no activity. These results suggest that the differences in the SaGT4A and StSGT amino acid sequences do not simply reflect their distinct sugar-donor specificities. We also successfully converted the non-functional SaGT4A homolog, SaGT4R, into an active glucosyltransferase.     

Ligninolytic enzymes activities of Oyster mushrooms cultivated on OMW (olive mill waste) supplemented media, spawn and substrates

Ligninolytic enzymes activities (laccases, peroxidases (total, MnP and MiP) and aryl-alcohol oxidase (AAO)) were measured during the cultivation of six commercial Pleurotus sp. strains on MMP media, on cereal grains (spawn) and on straw substrates (the three commonly utilized cultivation steps to obtain fruiting bodies) supplemented with several concentrations of autoclaved (OMW) or gamma-irradiated (iOMW) olive mill waste. Results indicated that all the strains were able to grow on MMP media and spawn containing up to 30% OMW and iOMW and on straw substrates mixed with 50% OMW. None of the strains showed AAO activity and there was not a single strain which showed the highest laccases and peroxidases activities, independently of the utilized substrate. Pleurotus mycelia adjusted their enzymatic mechanisms depending on their variety, type of substrate, concentration of OMW or iOMW added. OMW was a better supplement to use than iOMW because OMW induced higher exo-enzymes activities.     

Four new immunomodulating steroidal glycosides from the stems of Stephanotis mucronata

Four new C-21 steroidal glycosides, mucronatosides E (1), F (2), G (3), and H (4), were isolated from the stems of Stephanotis mucronata. Two of them had the rare aglycone with a double bond between C-6 and C-7. Their structures were elucidated on the basis of spectroscopic data and chemical evidence. These isolated compounds were assayed for their immunological activities in vitro against concanavalin A (Con A)- and lipopolysaccharide (LPS)-induced proliferation of mice splenocytes. Compounds 2 and 4 showed immunosuppressive activities in a dose-dependent manner, while compounds 1 and 3 possessed immunoenhancing activities.     

Evidence that displacement activities facilitate behavioural transitions in ring-tailed lemurs

Displacement activities are behavioural patterns defined by their apparent irrelevance to an animal's ongoing actions. Despite being identified in diverse taxa, their function remains poorly understood. One hypothesis posits that displacement activities facilitate transitions between different behaviours by mediating changes in animals' motivational state. Under this hypothesis, it is predicted that displacement activities will occur more frequently around changes in behaviour than at other times, and also that rates of displacement activities will be higher before than after such behavioural transitions. We tested these two predictions in wild ring-tailed lemurs (Lemur catta). During focal observations, animals' behavioural state was continuously recorded, as were all occurrences of self-scratching, a common displacement activity in this species. Self-scratching rates were found to be significantly elevated both before and after behavioural transitions. Furthermore, self-scratching rates were significantly higher before behavioural transitions occurred than after. These results, therefore, provide support for the hypothesis that displacement activities facilitate behavioural transitions in L. catta.     

Two Kazal-type protease inhibitors from Macrobrachium nipponense and Eriocheir sinensis: comparative analysis of structure and activities

Kazal-type inhibitors (KPIs) play important roles in many biological and physiological processes, such as blood clotting, the immune response and reproduction. In the present study, two male reproductive tract KPIs, termed Man-KPI and Ers-KPI, were identified in Macrobrachium nipponense and Eriocheir sinensis, respectively. The inhibitory activities of recombinant Man-KPI and Ers-KPI against chymotrypsin, elastase, trypsin and thrombin were determined. The results showed that both of them strongly inhibit chymotrypsin and elastase. Kinetic studies were performed to elucidate their inhibition mechanism. Furthermore, individual domains were also expressed to learn further which domain contributes to the inhibitory activities of intact KPIs. Only Man-KPI_domain3 is active in the inhibition of chymotrypsin and elastase. Meanwhile, Ers-KPI_domain2 and 3 are responsible for inhibition of chymotrypsin, and Ers-KPI_domains2, 3 and 4 are responsible for the inhibition of elastase. Meanwhile, the inhibitory activities of these two KPIs toward Macrobrachium rosenbergii, M. nipponense and E. sinensis sperm were compared with that of the Kazal-type peptidase inhibitor (MRPINK) characterized from the M. rosenbergii reproductive tract in a previous study. The results demonstrated that KPIs can completely inhibit the gelatinolytic activities of sperm proteases from their own species, while different levels of cross-inhibition were observed between KPI and proteases from different species. These results may provide new perspective to further clarify the mechanism of KPI-proteases interaction in the male reproductive system.     

Desiccation tolerance of the resurrection plant Ramonda serbica is associated with dehydration-dependent changes in levels of proteolytic activities

The unique response of desiccation-tolerant, or resurrection plants, to extreme drought is accompanied by major changes in the protein pool, raising the possibility of the involvement of proteases. We detected and characterized proteases present in their active state in leaf extracts of desiccated Ramonda serbica Pan?., a resurrection plant from the Balkan Peninsula. Plants desiccated under laboratory conditions and maintained in anhydrobiosis for 4 and 14 months revived upon rehydration. Protease activities were determined spectrophotometrically in solution and by zymography on gels. Several endo- and aminopeptidases were detected and characterized by their pH profiles. Their enzyme class was determined using specific inhibitors. Those with higher activities were a serine endopeptidase active against Bz-Arg-pNA with a pH optimum around 9, and aminopeptidases optimally active at pHs from 7 to 9 against Leu-pNA, Met-pNA, Phe-pNA, Pro-pNA and Ala-pNA. The levels of their activities in leaf extracts from desiccated plants were significantly higher than those from rehydrated plants and from regularly watered plants, implying their involvement in the recovery of vegetative tissues from desiccation.     

O-Glucosyltransferase activities toward phenolic natural products and xenobiotics in wheat and herbicide-resistant and herbicide-susceptible black-grass (Alopecurus myosuroides).

Herbicide safeners manipulate herbicide selectivity by enhancing the activities of detoxifying enzymes, such as glutathione transferases (GSTs) and cytochrome P450 mono-oxygenases (CYPs) in cereal crops. As part of a study examining the importance of O-glucosyltransferases (OGTs) in pesticide metabolism in hexaploid bread wheat (Triticum aestivum L.), seedlings were grown in the presence of dichlormid, a safener used in maize and cloquintocet mexyl, a wheat safener. The efficacy of the treatments was confirmed by monitoring changes in the abundance of phi and tau class GSTs. OGT activities in the root and shoot tissue were assayed using phenolics of natural and xenobiotic origin to determine if they were enhanced by safeners. Cloquintocet mexyl selectively increased OGT activities toward xenobiotics (4-nitrophenol and 2,4,5-trichlorophenol) and flavonoids, (quercetin, luteolin, genistein and coumestrol) in both the roots and shoots. However, OGT activity towards simple phenols and phenylpropanoids was not enhanced by cloquintocet mexyl. Dichlormid was a much weaker enhancer of OGT activity, with the same subset of OGT activities increased as determined with cloquintocet mexyl, but with the effect being largely restricted to the roots. OGT activities were also determined in black-grass (Alopecurus myosuroides L.), an agronomically important weed in wheat. Two populations of black-grass differing in their sensitivity to herbicides were analysed. The population Peldon, which is resistant to multiple classes of herbicides due in part to the elevated expression of CYPs and GSTs active in herbicide detoxification, contained higher OGT activities than herbicide sensitive black-grass. Unlike wheat, treatment with cloquintocet mexyl or dichlormid, had no effect on OGT activities in either black-grass population.     

Phenolic compounds from Gastrodia rhizome and relaxant effects of related compounds on isolated smooth muscle preparation

Gastrol (1), together with 10 known phenolic compounds, has been isolated from the MeOH extract of the rhizomes of Gastrodia elata Blume (Orchidaceae), and their structures were elucidated by detailed spectral analyses including by 2D NMR spectroscopic analyses. The relaxant effects of these constituents on smooth muscle preparations isolated from guinea-pig ileum were also studied in order to reveal their characteristic pharmacological activities.     

Effects of salicylic acid and cold treatments on protein levels and on the activities of antioxidant enzymes in the apoplast of winter wheat leaves

The effects of salicylic acid (SA) and cold on apoplastic protein levels and activities of apoplastic catalase (CAT), peroxidase (POX) and polyphenol oxidase (PPO) were investigated in winter wheat (Triticum aestivum cv. Dogu-88) leaves. The plants were grown with and without 10 microM SA treatment at both control (20/18 degrees C for 30 and 45-day) and cold (10/5 degrees C for 30-day and 5/3 degrees C for 45-day) acclimatisations. Molecular masses of the apoplastic polypeptides were shown ranging in size from 20 to 66 kDa on SDS-PAGE. Accumulation and pattern of the polypeptides were changed by both SA and cold. It is observed that CAT, POX and PPO activities at 45-day control leaves were higher than at 30-day. When the activities with SA and cold treatments are compared to their controls, CAT activities were decreased while POX and PPO activities were increased by both the treatments. When the activities with cold+SA treatment are compared to their cold treatments, CAT and POX activities were decreased while PPO activity was increased by SA. It is concluded that exogenous SA can be involved in cold tolerance by regulating apoplastic proteins and antioxidant enzyme activities.     

Xanthones from Garcinia cowa Roxb. latex

Five xanthones named cowagarcinone A-E and six previously reported xanthones were isolated from the latex of Garcinia cowa Roxb. Their structures were determined on the basis of spectroscopic analysis. The crude latex and the isolated compounds were investigated for their radical scavenging activities.     

Acanthamoeba castellanii Neff: In vitro activity against the trophozoite stage of a natural sesquiterpene and a synthetic cobalt(II)-lapachol complex

In this study, the in vitro activities of a natural sesquiterpene, α-cyperotundone, isolated from the root bark of Maytenus retusa and a cobalt(II)-complex of a natural occurring prenyl hydroxynaphthoquinone (lapachol) were evaluated against the trophozoite stage of Acanthamoeba castellanii Neff using a previously developed colorimetric 96-well microtiter plate assay, based on the oxido-reduction of Alamar Blue®. The obtained activities showed that these two compounds were able to inhibit the in vitro growth of the amoebae at relatively low concentrations. Further identification of the molecular targets of these products and their effects on acanthamoebae should be determined to evaluate their possible therapeutic use.     

Molluscicidal activity of various solvent extracts from Solanum nigrum var. villosum L. aerial parts against Galba truncatula

Molluscicidal activity of Solanum nigrum var. villosum (morelle velue) extracts and their fractions were tested against the mollusca gastropoda Galba truncatula intermediate host of Fasciola hepatica. The results indicated that the hydro-methanol (MeOH-H2O) immature fruit extract possess the highest molluscicidal activity (LC50 = 3.96 mg/L) against Galba truncatula compared with other tested compounds. After acido-basic treatment, the methanolic extract fraction isolated from the immature fruits and the richest in alkaloids was the most toxic (LC50 = 1.65 mg/L). The fractions richest in saponosides obtained from the hydromethanolic and methanolic extracts of immature fruits showed interesting molluscicidal activities (LC50 = 6.15 mg/L and LC50 = 7.91 mg/L, respectively). The observed molluscicide activity could be attributed to the presence of alkaloids or saponosides. So, the immature fruits of Solanum nigrum var. villosum could be substrates of choice for molluscicide activity. In addition, total alkaloids and saponosides present in this plant deserve further investigations in order to identify the active principles and demonstrate their activities on mollusks in their natural habitat. According to the World Health Organization's guidelines on screening for plant molluscicides, use of these fractions may add to the arsenal of methods to control snail transmitting fasciolosis in tropical and Third World countries where fasciolosis is a common disease.     

Stilbenes from the roots of Pleuropterus ciliinervis and their antioxidant activities

Two stilbene glycosides, pieceid-2"-O-gallate and pieceid-2"-O-coumarate, were isolated from the MeOH extract of the roots of Pleuropterus ciliinervis Nakai (Polygonaceae), together with two known compounds, resveratrol and pieceid. Their structures were determined spectroscopically, particularly by 2D NMR spectroscopic analysis. The antioxidant activities of stilbenes isolated were determined in vitro against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, superoxide radicals and by determining their lipid peroxidation inhibitory activities. Among the compounds isolated, pieceid-2"-O-gallate had the most potent inhibitory scavenging effect on DPPH, superoxide radicals and upon lipid peroxidation inhibition with IC50 values of 16.5, 23.9 and 5.1 microM, respectively.     

Biochemical activities of Iranian Mentha piperita L. and Myrtus communis L. essential oils

GC-MS analysis of essential oils of Iranian Mentha piperita and Myrtus communis extracted by hydrodistillation lead to identification of 26 and 32 compounds, respectively. The oils had good to excellent antimicrobial activities against Escherichia coli, Staphylococcus aureus and Candida albicans with the oil of M. piperita being more active. The findings suggest feasibility of application of M. piperita oil in treatment of the infections caused by C. albicans and E. coli. D-values on exposure to M. piperita and Myrtus communis oils were (2.14 and 2.8min), (1.4 and 12.8min) and (4.3 and 8.6min) for E. coli, S. aureus and C. albicans , respectively. The oils were screened for their possible antioxidant activities by two complementary test systems, namely DPPH free radical scavenging and beta-carotene/linoleic acid systems. M. piperirta oil exerted greater antioxidant activity than that of M. communis. Phytochemical and phytobiological characteristics of these oils may lead to extraction and production of active compounds in single or combined forms with useful applications.     

Association of thymidine and uridine kinase activities with changes in nucleic acid levels during peanut fruit ontogeny

Various stages of pegs, cotyledons and embryonic axes from maturing peanut fruits were examined for their ability to phosphorylate thymidine and uridine. Highest specific activities during peg elongation were found just prior to increases in endosperm nuclei and embryo cell numbers. In the developing cotyledons and axes, the net kinase activities of crude extracts reached a maximum 1–2 weeks before maximal RNA and DNA contents were attained. An exception was the apparent lack of any relationship between uridine kinase activities and RNA levels in developing embryonic axes. The present results support the observation that peanut axes are devoid of thymidine and uridine kinases during the first 24 hr of germination, as fully developed fruits had very low specific activities for both of these phosphate transferases.     

Changes in glycosidase activities during galactoglucomannan oligosaccharide inhibition of auxin induced growth

The inhibition of 2,4-D-induced elongation growth by galactoglucomannan oligosaccharides (GGMOs) in pea stem segments (Pisum sativum L. cv. Tyrkys) after 18 h of incubation results in changes of extracellular, intracellular and cell wall glycosidase activities (beta-D-glucosidase, beta-D-mannosidase, beta-D-galactosidase, beta-D-xylosidase, alpha-D-galactosidase, and alpha-L-arabinosidase). GGMOs lowered the glycosidase activities in the extracellular fraction, while in the cell wall fractions their activities were markedly increased. The intracellular enzyme alpha-d-galactosidase increased while the beta-d-galactosidase decreased in activity in response to the GGMO treatment. Extracellular enzymes showed low values of activities in comparison with intracellular and cell wall glycosidases. It is evident that GGMOs can alter auxin induced elongation and glycosidase activities in different compartments of the cell, however, the mode and site of their action remains unclear.     

Differences in enzyme activities between two species of Hematodinium, parasitic dinoflagellates of crustaceans

Parasitic dinoflagellates of the genus Hematodinium infect several commercially important decapod crustaceans. Different species of Hematodinium have different levels of virulence in their respective hosts. Enzyme activities were studied from two species of Hematodinium, one isolated from the Norway lobster (Nephrops norvegicus) and the other from the American blue crab (Callinectes sapidus). We report the identification of differences in secretion of acid phosphatase (AP) and leucine arylamidase from two parasite species. Leucine arylamidase was only contained and secreted by the species infecting the blue crab. Both parasite species contained AP, but only the species infecting the Norway lobster secreted this enzyme. In this species, AP activity was predominantly in the soluble fraction (69.5%). AP activity was localized to cytoplasmic granules and on the membranes surrounding the cell nucleus. In addition to providing information on the cellular metabolism of the parasite, the pattern of activities of these enzymes may also be useful in distinguishing among different species of Hematodinium.     

Hypoglycemic activities of A- and B-type procyanidin oligomer-rich extracts from different Cinnamon barks

Procyanidin oligomers in Cinnamon are thought to be responsible for the biological activity in the treatment of diabetes mellitus (DM). To clarify types of procyanidin oligomers in different Cinnamon species and investigate their different effects, the present study investigated procyanidin oligomers in polyphenolic oligomer-rich extracts of three Cinnamon samples by LC-MS methods, and their hypoglycemic activities were detected in vivo and in vitro. The results showed that two of the three samples from Cinnamomum cassia were rich in B-type procyanidin oligomers, and the other sample was rich in A-type procyanidin oligomers. The Cinnamon extracts were administered at doses of 200 and 300 mg/kg body wt. in high-fat diet-fed and low-dose streptozotocin (STZ)-induced diabetic mice for 14 days. The results showed that blood glucose concentrations were significantly decreased in all Cinnamon extract groups compared with the control group (p < 0.05). Administration of the Cinnamon extracts significantly increased the consumption of extracellular glucose in insulin-resistant HepG2 cells and normal HepG2 cells compared with the control group. These results suggest that both A- and B-type procyanidin oligomers in different Cinnamon species have hypoglycemic activities and may improve insulin sensitivity in type 2 DM.     

2-β-Benzothiazolyl-6-iminopyridylmetal dichlorides and the catalytic behavior towards ethylene oligomerization and polymerization

A series of 2-(2-benzothiazolyl)-6-(1-(arylimino)ethyl)pyridines and their metal (Fe or Co) complexes were prepared. All organic compounds were fully characterized by NMR, FT-IR spectra and elemental analysis, and all metal complexes were identified by FT-IR spectroscopic and elemental analysis. The molecular structures of representative metal complexes were confirmed by single-crystal X-ray diffraction and displayed the distorted trigonal bipyramid geometry. Upon activation with modified methylaluminoxane (MMAO), the iron pro-catalysts showed good catalytic activities up to the range of 10⁷ g mol⁻¹(Fe) h⁻¹ in ethylene reactivity with the high selectivity for the vinyl-type products of both oligomers and polyethylene waxes; whereas the cobalt pro-catalysts showed moderate activities towards ethylene oligomerization. The correlations between metal complexes and their catalytic activities and products were investigated in detail under various reaction parameters and discussed.