Proteomic analysis of the pathophysiological process involved in the antisnake venom effect of Mucuna pruriens extract

Previously, we reported the antisnake venom properties of a Mucuna pruriens seed extract (MPE) and tested its in vivo efficacy against Echis carinatus venom (EV) in short- (1 injection) and long-term (three weekly injections) treatments. The aim of the present study was to investigate plasma proteome changes associated with MPE treatments and identify proteins responsible for survival of envenomated mice (CHALLENGED mice). Six treatment groups were studied. Three control groups: one saline, one short-term and one long-term MPE treatment. One group received EV alone. Two test groups received EV with either a short-term or long-term MPE treatment (CHALLENGED mice). The plasma from each group was analysed by 2-DE/MALDI-TOF MS. The most significant changes with treatment were: albumin, haptoglobin, fibrinogen, serum amyloid A and serum amyloid P. Most of these changes were explained by EV effects on coagulation, inflammation and haemolysis. However, MPE treatments prevented the EV-induced elevation in HPT. Consequently, HPT levels were similar to controls in the plasma of CHALLENGED mice. The plasma of CHALLENGED mice showed substantial proteomic modifications. This suggests the mechanism of MPE protection involves the activation of counterbalancing processes to compensate for the imbalances caused by EV.     

Study on the Chemical Constituens of Mucuna pruriens var. utilis (With Black seed Coat)

Five compounds were first isolated from the legumes of Mucuna pruriens (L.) DC. var. utilis (Wall. ex Wight) Baker ex Burck (M. atrocarpum Metcalf) with black seed coat. They were identified as L-dopa, stizolamine, D-erythro-neopterin, 6-hydroxymethylpterin and isoxanthopterin.     

Alkaloidal constituents of Mucuna pruriens seeds

The seeds of Mucuna pruriens (L.) DC. after chemical analysis afforded four tetrahydroisoquinoline alkaloids which have been isolated for the first time from M. pruriens. Out of them, two are new whose structures have been elucidated by spectroscopic methods.     

Mucuna pruriens seed powder feeding influences reproductive conditions and development in Japanese quail Coturnix coturnix japonica

This study was designed to test whether Mucuna pruriens, a natural source of l-dihydroxyphenylalanine (l-DOPA, a dopamine precursor) feeding, can influence development and reproductive conditions in the high food value bird, Japanese quail, Coturnix coturnix japonica. Experiments were performed in both male and female Japanese quail. One-week-old quail chicks were divided into three groups of 36 birds each. Group I was provided with normal diet and served as control. Group II was provided with food mixed with l-DOPA (50 mg/15 g food) and Group III was provided with food mixed with M. pruriens seed powder (480 g/kg food). At the age of 3 weeks (when birds were sexually distinguished) Group I was divided into two sub-groups IA (male) and IB (female) of six birds each. Similarly, Groups II and III were sub-divided into IIA (male), IIB (female) and IIIA (male), IIIB (female), respectively, of six birds each. Observations were made up to the age of 5 weeks. Male experimental groups (IIA and IIIA) showed significantly increased testicular activity, cloacal gland volume, body weight (BW), plasma testosterone and LH level in comparison to control (IA). Similarly female experimental groups (IIB and IIIB) showed significantly greater weight of reproductive organs (uterus, ovary, oviduct and ovarian follicle), BW, egg weight and size and number of follicles. On the other hand, plasma prolactin level was significantly low in comparison to control (IB). Results suggest that M. pruriens is a rich natural source of l-DOPA and the development and reproduction in Japanese quail might be associated with the dopaminergic system of the brain.     

Structural characterization of the N-glycans of gpMuc from Mucuna pruriens seeds

Mucuna pruriens seeds are used in some countries as a human prophylactic oral anti-snake remedy. Aqueous extracts of M. pruriens seeds possess in vivo activity against cobra and viper venoms, and protect mice against Echis carinatus venom. It was recently demonstrated that the seed immunogen generating the antibody that cross-reacts with the venom proteins is a multiform glycoprotein (gpMuc), and the immunogenic properties of gpMuc seemed to mainly reside in its glycan chains. In the present study, gpMuc was found to contain only N-glycans. Part of the N-glycans could be released with peptide-(N ⁴-(N-acetyl-β -glucosaminyl)asparagine amidase F (PNGase F-sensitive N-glycans); the PNGase F-resistant N-glycans were PNGase A-sensitive. The oligosaccharides released were analyzed by a combination of MALDI-TOF mass spectrometry, HPLC profiling of 2-aminobenzamide-labelled derivatives and ¹H NMR spectroscopy. The PNGase F-sensitive N-glycans comprised a mixture of oligomannose-type structures ranging from Man₅GlcNAc₂ to Man₉GlcNAc₂, and two xylosylated structures, Xyl₁Man₃GlcNAc₂ and Xyl₁Man₄GlcNAc₂. The PNGase A-sensitive N-glycans, containing (α 1-3)-linked fucose, were identified as Fuc₁Xyl₁Man₂GlcNAc₂ and Fuc₁Xyl₁Man₃GlcNAc₂. In view of the determined N-glycan ensemble, the immunoreactivity of gpMuc was ascribed to the presence of core (β 1-2)-linked xylose- and core α (1-3)-linked fucose-modified N-glycan chains.     

Effects of growth under elevated UV-B on photosynthesis and isoprene emission in Quercus gambelii and Mucuna pruriens

Increasing surface levels of UV-B resulting from stratospheric ozone reduction directly affect tropospheric photochemistry. There may also be indirect tropospheric effects due to changes in emission of organic compounds from vegetation. We treated woody and herbaceous isoprene-emitting species in the field with supplemental UV-B simulating 30% ozone depletion. For Quercus gambelii, photosynthesis and isoprene emission were significantly greater in elevated UV-B treatments when expressed on a leaf area basis, but not on a leaf mass basis. Leaves of Mucuna pruriens, however, showed no significant differences in photosynthesis or isoprene emission between treatments, nor when exposed for 45 min to acute high levels of UV-B. Elevated UV-B during growth did not elicit significant isoprene emission from Acer platanoides, a non-emitting species. Other potential UV-B effects, such as changes in leaf area or species composition, which may influence regional isoprene emissions, should be examined.     

Symbionts in Mucuna pruriens stimulate plant performance through nitrogen fixation and improved phosphorus acquisition

刺毛黎豆共生体通过固氮和提高磷吸收来提高植株的生长表现南非主要以热带稀树草原和草原生态系统为主,这些生态系统具有酸性和营养缺乏的属性,特别是磷和氮素的缺乏。刺毛黎豆(Mucuna pruriens)是在大多数非洲国家广泛存在的一种本土豆科植物,该 植物可以抵御这些不利的土壤条件。豆科植物在世界上许多国家具有药用价值,也被用于土壤施肥。尽管已有文献记载了在营养胁迫生态系统中刺毛黎豆的生长和建植,但尚未对其在磷缺乏条件下的共生互作、氮源偏好和与生长相关的碳成本进行研究。在本研究中,我们确定了微生物共生体对磷缺乏条件下的刺毛黎豆氮营养和生长相关的碳成本的影响。我们从4个不同的地理位置采集微生物接种土壤,然后将种子在这些天然土壤中发芽,在根瘤发育的早期,将幼苗转移到无菌石英砂中,并提供不同磷 浓度的营养培养基。16S RNA序列结果显示,在不考虑磷浓度的情况下,Burkholderia sp., Paenibacillus sp.和Bacillus 均有分枝。尽管磷缺乏导致总生物量/生长下降,但根系生物量、根瘤数量和碳成本增加。低磷供给的幼树丛枝菌根真菌根系定殖率最高。刺毛黎豆中,大气中的氮与磷水平呈正相关关系,幼树对大气氮素和土壤氮素具有双重依赖,低磷植株对土壤氮素的依赖程度增加。因此,在磷缺乏的情况下,刺毛黎豆表现出不同的形态和微生物共生关系。     

Carboxylesterases from the seeds of an underutilized legume, Mucuna pruriens; isolation, purification and characterization

Two carboxylesterases (ME-III and ME-IV) have been purified to apparent homogeneity from the seeds of Mucuna pruriens employing ammonium sulfate fractionation, cation exchange chromatography on CM-cellulose, gel-permeation chromatography on Sephadex G-100 and preparative PAGE. The homogeneity of the purified preparations was confirmed by polyacrylamide gel electrophoresis (PAGE), gel-electrofocussing and SDS–PAGE. The molecular weights determined by gel-permeation chromatography on Sephadex G-200 were 20.89 kDa (ME-III) and 31.62 kDa (ME-IV). The molecular weights determined by SDS–PAGE both in the presence and absence of 2-mercaptoethanol were 21 kDa (ME-III) and 30.2 kDa (ME-IV) respectively, suggesting a monomeric structure for both the enzymes. The enzymes were found to have Stokes radius of 2.4 nm (ME-III) and 2.7 nm (ME-IV). The isoelectric pH values of the enzymes, ME-III and ME-IV, were 6.8 and 7.4, respectively. ME-III and ME-IV were classified as carboxylesterases employing PAGE in conjunction with substrate and inhibitor specificity. The K_m of ME-III and ME-IV with 1-naphthyl acetate as substrate was 0.1 and 0.166 mM while with 1-naphthyl propionate as substrate the K_m was 0.052 and 0.0454 mM, respectively. As the carbon chain length of the acyl group increased, the affinity of the substrate to the enzyme increased indicating hydrophobic nature of the acyl group binding site. The enzymes exhibited an optimum temperature of 45 °C (ME-III) and 37 °C (ME-IV), an optimum pH of 7.0 (ME-III) and 7.5 (ME-IV) and both the enzymes (ME-III and ME-IV) were stable up to 120 min at 35 °C. Both the enzymes were inhibited by organophosphates (dichlorvos and phosphamidon), but resistant towards carbamates (carbaryl and eserine sulfate) and sulphydryl inhibitors (p-chloromercuricbenzoate, PCMB).     

In vitro rapid regeneration of plantlets from nodal explants of Mucuna pruriens– a valuable medicinal plant

A rapid and efficient protocol for the large‐scale propagation of a potential medicinal plant, Mucuna pruriens, through in vitro culture of nodal segment explants obtained from 15‐day‐old aseptic seedlings is described. Of the three different cytokinins, 6‐benzyladenine (BA), kinetin (Kin) and 2‐isopentenyl adenine (2‐iP) evaluated as supplements to Murashige and Skoog (MS) medium, BA at an optimal concentration of 5.0 μM was effective in inducing multiple shoots. Strength of the basal media also influenced the efficiency of shoot regeneration. The frequency of shoot regeneration tended to increase when the salt concentration in the basal media was reduced. Highest number of multiple shoots (23.3) and maximum average length (5.6 cm) were standardised on half‐strength MS medium supplemented with 5.0 μM BA along with 0.5 μM α‐naphthalene acetic acid (NAA) at pH 5.8. Rooting was best induced in shoots excised from proliferated shoot cultures on MS medium augmented with an optimal concentration of 1.0 μM indole‐3‐butyric acid (IBA). The in vitro‐raised plantlets with well‐developed shoots and roots were successfully established in earthen pots containing garden soil and were grown in greenhouse with 90% survival rate. The results of this study provide the first report on in vitro plant regeneration of M. pruriens.     

Continuous production of the pharmaceutical 7,8-dihydroxy N-di-n-propyl 2-aminotetralin using a phenoloxidase from cell cultures of Mucuna pruriens

Alginate-entrapped cells of Mucuna pruriens as well as the phenoloxidase isolated from the cell cultures, are able to ortho-hydroxylate several mono-, bi- and tri-cyclic monophenols. In this study, 7,8-dihydroxy N-di-n-propyl 2-aminotetralin, a catechol of pharmaceutical interest and difficult to prepare chemically, could be produced in considerable quantities by bioconversion of the precursor 7-hydroxy N-di-n-propyl 2-aminotetralin. A continuous flow system on a laboratory scale was used, which consisted of a phenoloxidase suspension in dialysis tubing as the biocatalysator in an airlift fermentor coupled with an aluminium oxide column for selective product isolation. Product formation continued for at least 50 h, resulting in ca. 130 mg product per liter, this being a bioconversion percentage of 25%. When the enzyme preparation was reused, 85% of the original activity was measured.Alan S. Horn deceased at January 2nd, 1990     

Estimation of genetic diversity in varieties of Mucuna pruriens using RAPD

Genetic diversity was estimated in 13 accessions of the otherwise self pollinated Mucuna pruriens (L.) DC. (velvetbean) comprising varieties pruriens and utilis collected from tropical humid forest using 15 RAPD primers. Similarity index value of 0.68 based on Nei and Li's similarity coefficient indicated high degree of genetic variability. Analysis of various genetic diversity indices like total heterozygosity, Nei's gene diversity, percentage of polymorphic loci, expected and observed number of alleles and Shannon index strongly suggests that variety pruriens is genetically more diverse than variety utilis. Chemical analysis with respect to 3,4-dihydroxy-L-phenylalanine (L-DOPA) content showed uniform distribution. Cluster analysis showed grouping of accessions into two major clusters and tendency of accessions of variety pruriens to group according to their geographical locations. Bootstrap analysis confirmed the robustness of the phenogram. The putative hybrid MMP6 with relatively low similarity value index and low L-DOPA content was promising as food or fodder.     

Roles of mouse sperm‐associated alpha‐L‐fucosidases in fertilization

Sperm‐associated α‐L ‐fucosidases have been implicated in fertilization in many species. Previously, we documented the existence of α‐L ‐fucosidase in mouse cauda epididymal contents, and showed that sperm‐associated α‐L ‐fucosidase is cryptically stored within the acrosome and reappears within the sperm equatorial segment after the acrosome reaction. The enrichment of sperm membrane‐associated α‐L ‐fucosidase within the equatorial segment of acrosome‐reacted cells implicates its roles during fertilization. Here, we document the absence of α‐L ‐fucosidase in mouse oocytes and early embryos, and define roles of sperm associated α‐L ‐fucosidase in fertilization using specific inhibitors and competitors. Mouse sperm were pretreated with deoxyfuconojirimycin (DFJ, an inhibitor of α‐L ‐fucosidase) or with anti‐fucosidase antibody; alternatively, mouse oocytes were pretreated with purified human liver α‐L ‐fucosidase. Five‐millimolar DFJ did not inhibit sperm–zona pellucida (ZP) binding, membrane binding, or fusion and penetration, but anti‐fucosidase antibody and purified human liver α‐L ‐fucosidase significantly decreased the frequency of these events. To evaluate sperm‐associated α‐L ‐fucosidase enzyme activity in post‐fusion events, DFJ‐pretreated sperm were microinjected into oocytes, and 2‐pronuclear (2‐PN) embryos were treated with 5 mM DFJ with no significant effects, suggesting that α‐L ‐fucosidase enzyme activity does not play a role in post‐fusion events and/or early embryo development in mice. The recognition and binding of mouse sperm to the ZP and oolemma involves the glycoprotein structure of α‐L ‐fucosidase, but not its catalytic action. These observations suggest that deficits in fucosidase protein and/or the presence of anti‐fucosidase antibody may be responsible for some types of infertility. Mol. Reprod. Dev. 80: 273–285, 2013. © 2013 Wiley Periodicals, Inc.     

Bioconversion of bi- and tri-cyclic monophenols by alginate-entrapped cells of Mucuna pruriens L. and by the partially purified Mucuna-phenoloxidase

Although alginate-entrapped cells of Mucuna pruriens L. possess a low substrate specificity, only para-substituted monocyclic phenols have been ortho-hydroxylated into catechols so far. In this study, compounds with more complex chemical structures were found to be substrates using entrapped cells of M. pruriens as well as the partially purified Mucuna-phenoloxidase. Thus, 5-, 6- and 7-hydroxylated 2-aminotetralins and a tricyclic compound, 9-hydroxy N-n-propyl hexahydronaphthoxazine, were converted into catechols. After isolation using preparative HPLC, the identity of the products was confirmed by MS. In general, for the entrapped cells and the enzyme preparation identical substrate specificities were found.This publication is dedicated to the memory of Prof. Alan S. Horn, Ph.D., who deceased at January 2, 1990     

Characterization of a group of pyrroloquinoline quinone‐dependent dehydrogenases that are involved in the conversion of L‐sorbose to 2‐Keto‐L‐gulonic acid in Ketogulonicigenium vulgare WSH‐001

Ketogulonicigenium vulgare WSH‐001 is an industrial strain used for vitamin C production. Based on genome sequencing and pathway analysis of the bacterium, some of its potential pyrroloquinoline quinone (PQQ)‐dependent dehydrogenases were predicted, including KVU_pmdA_0245, KVU_2142, KVU_2159, KVU_1366, KVU_0203, KVU_0095, and KVU_pmdB_0115. BLAST and function domain searches showed that enzymes encoded by these genes may act as putative PQQ‐dependent L ‐sorbose dehydrogenases (SDH) or L ‐sorbosone dehydrogenases (SNDH). To validate whether these dehydrogenases are PQQ‐dependent or not, these seven putative dehyrogenases were overexpressed in Escherichia coli BL21 (DE3) and purified for characterization. Biochemical and kinetic characterization of the purified proteins have led to the identification of seven enzymes that possess the ability to oxidize L ‐sorbose or L ‐sorbosone to varying degrees. In addition, the dehydrogenation of sorbose in K. vulgare is validated to be PQQ dependent, identification of these PQQ‐dependent dehydrogenases expanded the PQQ‐dependent dehydrogenase family. Besides, the optimal combination of enzymes that could more efficiently catalyze the conversion of sorbose to gulonic acid was proposed. These are important in supporting the development of metabolic engineering strategies and engineering of efficient strains for one‐step production of vitamin C in the future. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1398–1404, 2013     

Conformations and properties of the L‐tryptophyl‐containing peptides in solution,depending on the pH—Theoretical study vs. experiments

The conformational preference and electronic properties of three L ‐tryptophyl‐containing dipeptides, i.e., glycyl‐L ‐tryptophane (H‐Gly‐Trp‐OH), L ‐alanyl‐L ‐tryptophane (H‐Ala‐Trp‐OH), and L ‐methionyl‐L ‐tryptophane (L ‐Met‐Trp‐OH) in solution depending on the pH of the media are studied both theoretically and experimentally. The effect of the protonation of the COO^? and deprotonation of the NH as well as the alkaline hydrolysis of the amide fragment in a strong basic media on the electronic spectra are discussed. Ab initio and density functional theory (DFT) methods as well as the time‐dependent DFT (TD‐DFT) method as a function of the basis set are performed with a view to obtain the geometry and electronic properties of all of the species as well as the intermediate, obtained in the alkaline hydrolysis mechanism. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 727–734, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Channel‐like crystal structure of cinchoninium L‐O‐phosphoserine salt dihydrate

Studies on the interactions between L ‐O‐ phosphoserine, as one of the simplest fragments of membrane components, and the Cinchona alkaloid cinchonine, in the crystalline state were performed. Cinchoninium L ‐O‐phosposerine salt dihydrate (PhSerCin) crystallizes in a monoclinic crystal system, space group P2₁, with unit cell parameters: a = 8.45400(10) Å, b = 7.17100(10) Å, c = 20.7760(4) Å, α = 90°, β = 98.7830(10)°, γ = 90°, Z = 2. The asymmetric unit consists of the cinchoninium cation linked by hydrogen bonds to a phosphoserine anion and two water molecules. Intermolecular hydrogen bonds connecting phosphoserine anions via water molecules form chains extended along the b axis. Two such chains symmetrically related by twofold screw axis create a “channel.” On both sides of this channel cinchonine cations are attached by hydrogen bonds in which the atoms N1, O12, and water molecules participate. This arrangement mimics the system of bilayer biological membrane. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Roles of the three L‐domains in β‐retrovirus budding

Retroviral Gag protein plays a critical role during the late stage of virus budding and possesses a so‐called L‐domain containing PT/SAP, PPxY, YxxL or FPIV motifs that are critical for efficient budding. Mason–Pfizer monkey virus (M‐PMV) contains PSAP, PPPY, and YADL sequences in Gag. This study was performed to investigate the roles of these three L‐domain‐like sequences in virus replication in three different cell lines, 293T, COS‐7 and HeLa cells. It was found that the PPxY motif plays an essential role in progeny virus production as a major L‐domain in all three cell lines. The PSAP sequence was shown to function as an additional L‐domain in HeLa cells and to promote efficient release of M‐PMV; however, this sequence was dispensable for M‐PMV production in 293T and COS‐7 cells, suggesting that the role of the PSAP motif as an L‐domain in M‐PMV budding is cell type‐dependent. Viruses possessing multiple L‐domains appear to change the L‐domain usage to replicate in various cells. On the other hand, the YADL motif was required for M‐PMV production as a transport signal of Gag to the plasma membrane, but not as an L‐domain.