In-vitro anti-inflammatory activity of serine protease inhibitor from Cassia siamea and Dolichos biflorus: A comparative study.

Cassia siamea is a nonedible legume belonging to Fabaceae. The seed of C. siamea contains ~16% of protein. The study reports the biochemical characterization of purified novel serine protease inhibitor from seeds of C. siamea, aimed with assessing the anti-inflammatory activity. The seed extract was subjected to ammonium sulfate precipitation followed by fast protein liquid chromatography (FPLC)-anion exchange chromatography and affinity-chromatography to obtain a relative pure protease inhibitor. Thirty-fivefold purification with the specific activity of 250 U/mg of trypsin inhibitory unit was obtained. The characterization of protease inhibitor for optimum temperature, pH, and metal ions were measured using N-α-benzoyl-DL-arginine-p-nitroanilide (BAPNA) assay and casein zymogram. The C. siamea trypsin inhibitor (CsTI) has a relative molecular mass of 25.540 kDa. Purified CsTI and Dolichos biflorus were tested for anti-inflammatory efficacy against A549 and RAW264.7 cell lines. The inhibitory activity of both purified inhibitors are comparable and are potent toward anti-inflammatory activity. The purified inhibitor shows to be a promising candidate as anti-inflammatory agent by targeting the serine proteases.

Structural characteristic of pectin-glucuronoxylan complex from Dolichos lablab L. hull.

Polysaccharides are important constituents in Dolichos lablab hull. Herein, pectin-glucuronoxylan complex from D. lablab hull designated as DLHP-3 (D. lablab hull polysaccharide,) was prepared by ion exchange and gel permeation chromatography, and further characterized by acid degradation and enzymatic hydrolysis, methylation combined with GC-MS, NMR and MALDI-TOF-MS analysis. Both of pectin and glucuronoxylan regions were found in DLHP-3. The glucuronoxylan region consisted of a →4)-ß-Xylp-(1→ backbone with branches of α-GlcpA-(1→ substituted at O-2 site, and the ratio of xylose to glucuronic acid was about 5:1. Acetyl groups were mainly attached to O-3 site of →2,4)-ß-Xylp-(1→ residues. The main chain of pectin region could be represented by →4)-α-GalpA-(1→4)-α-GalpA-(1→ and →2)-α-Rhap-(1→4)-α-GalpA-(1→ with partial methyl-esterification. The side chains were deduced to embrace arabinan and arabinogalactan linked to rhamnogalacturonan-I region. Pectin was probably covalently bound to glucuronoxylan. Our findings uncovered the molecular structure of pectin-glucuronoxylan complex from D. lablab hull.

High resolution structural and functional analysis of a hemopexin motif protein from Dolichos.

It is increasingly evident that seed proteins exhibit specific functions in plant physiology. However, many proteins remain yet to be functionally characterized. We have screened the seed proteome of Dolichos which lead to identification and purification of a protein, DC25. The protein was monomeric and highly thermostable in extreme conditions of pH and salt. It was crystallized and structure determined at 1.28 Å resolution using x-ray crystallography. The high-resolution structure of the protein revealed a four-bladed ß-propeller hemopexin-type fold containing pseudo four-fold molecular symmetry at the central channel. While the structure exhibited homology with 2S albumins, variations in the loops connecting the outermost strands and the differences in surface-charge distribution may be relevant for distinct functions. Comparative study of the protein with other seed hemopexins revealed the presence of four conserved water molecules in between the blades which cross-link them and maintain the tertiary structure. The protein exhibited intrinsic peroxidase activity, which could be inhibited by binding of a heme analog. The identification of redox-sensitive cysteine and inhibition of peroxidase activity by iodoacetamide facilitated characterization of the possible active site. The determined peroxidase activity of DC25 may be responsible for rescuing germinating seeds from oxidative stress.

Triterpene Glycosides from the Seeds of Dolichos lablab.

Two new triterpene glycosides (1 and 2), together with nine known triterpene glycosides (3-11), were isolated from the seeds of Dolichos lablab (Leguminosae). The structures of the new compounds were determined by spectroscopic analysis, including two-dimensional NMR spectroscopy, and chromatographic analysis of the hydrolyzed products. The isolated compounds did not show cytotoxicity against HL-60 human leukemia cells and HepG2 human hepatoma cells at sample concentrations of 20 µM.

Expression profiling of the Dolichos lablab lectin during germination and development of the seed.

The temporal expression of the field bean (Dolichos lablab) galactose specific lectin, DLL-II, during germination, post-germination and seed development was evaluated using Native-PAGE followed by activity staining, immunodetection and quantitative Real Time PCR (qPCR). A rapid and steep decline in the polyphenol oxidase (PPO) and hemagglutinating activity during the initial stages of germination, which did not correlate with the slow decline in total protein was observed. During post germination period, PPO and hemagglutination activities were negligible, whereas a rapid resorption of the protein was evident. These results suggest that DLL-II is not a storage protein. The presence of mRNA in the quiescent seed and initial stages of germination are indicative of a very stable mRNA. DLL-II was expressed in high copies during seed development and increased dramatically between 10 and 20 days after flowering (DAF), suggesting a switch over stage in DLL-II expression. Transcript levels reached a maximum at the mature stage of seed development. Among the non-seed tissues examined, root showed the highest level. The high affinity binding to kinetin and indole acetic acid, the key hormones that regulate root development and its vascular differentiation add a new dimension to the physiological role of DLL-II in the seed. This finding, coupled with the PPO and hemagglutinating activity makes DLL-II, truly a multifunctional protein.

Supplemental UV-B radiation induced changes in growth, pigments and antioxidant pool of bean (Dolichos lablab) under field conditions.

Present study is conducted to evaluate the response of bean (Dolichos lablabcv . pusa early prolific) plants to supplemental UV-B (sUV-B: 280-315 nm: 7.2 kJ m(-2) d(-1)) radiation. UV-B caused alteration in biomass translocation pattern with more retention of biomass in below ground parts leading to an increment in root shoot ratio. Specific leaf area (SLA) which is the measure of leaf thinness, increased in plants under sUV-B exposure by 95.7 and 82.3% after 15 and 30 days after germination. Photosynthetic machinery of bean plants was the potential target of UV-B as photosynthetic rate was decreased by 88.6% at 30 days after germination. sUV-B lead to the formation of reactive oxygen species thus generating oxidative stress. Stimulation of antioxidant defense system (enzymatic and non-enzymatic) was observed due to sUV-B radiation. Phenolic content decreased (34.7 and 18.6%) but protein showed varied response, increased initially (34%) thereafter declined (10.2%) under sUV-B radiation.

Complete primary structure of a newly characterized galactose-specific lectin from the seeds of Dolichos lablab.

A new unique lectin (galactose-specific) purified from the seeds of Dolichos lablab, designated as DLL-II is a heterodimer composed of closely related subunits alpha and beta. These were separated by SDS-PAGE and isolated by electroelution. By ESI-MS analysis their molecular masses were found to be 30.746 kDa (alpha) and 28.815 kDa (beta) respectively. Both subunits were glycosylated and displayed similar amino acid composition. Using advanced mass spectrometry in combination with de novo sequencing and database searches for the peptides derived by enzymatic and chemical cleavage of these subunits, the primary sequence was deduced. This revealed DLL-II to be made of two polypeptide chains of 281(alpha) and 263(beta) amino acids respectively. The beta subunit differed from the alpha subunit by the absence of some amino acids at the carboxy terminal end. This structural difference suggests that possibly, the beta subunit is derived from the alpha subunit by posttranslational proteolytic modification at the COOH-terminus. Comparison of the DLL-II sequence to other leguminous seed lectins indicates a high degree of structural conservation.

Cloning and functional expression of the gene encoding an inhibitor against Aspergillus flavus alpha-amylase, a novel seed lectin from Lablab purpureus (Dolichos lablab).

Maize is one of the more important agricultural crops in the world and, under certain conditions, prone to attack from pathogenic fungi. One of these, Aspergillus flavus, produces toxic and carcinogenic metabolites, called aflatoxins, as byproducts of its infection of maize kernels. The alpha-amylase of A. flavus is known to promote aflatoxin production in the endosperm of these infected kernels, and a 36-kDa protein from the Lablab purpureus, denoted AILP, has been shown to inhibit alpha-amylase production and the growth of A. flavus. Here, we report the isolation of six full-length labAI genes encoding AILP and a detailed analysis of the activities of the encoded proteins. Each of the six labAI genes encoded sequences of 274 amino acids, with the deduced amino acid sequences showing approximately 95-99% identity. The sequences are similar to those of lectin members of a legume lectin-arcelin-alpha-amylase inhibitor family reported to function in plant resistance to insect pests. The labAI genes did not show any of the structures characteristic of conserved structures identified in alpha-amylase inhibitors to date. The recombinant proteins of labAI-1 and labAI-2 agglutinated human red blood cells and inhibited A. flavus alpha-amylase in a manner similar to that shown by AILP. These data indicate that labAI genes are a new class of lectin members in legume seeds and that their proteins have both lectin and alpha-amylase inhibitor activity. These results are a valuable contribution to our knowledge of plant-pathogen interactions and will be applicable for developing protocols aimed at controlling A. flavus infection.

Tryptophan environment, secondary structure and thermal unfolding of the galactose-specific seed lectin from Dolichos lablab: fluorescence and circular dichroism spectroscopic studies.

Fluorescence and circular dichroism spectroscopic studies were carried out on the galactose-specific lectin from Dolichos lablab seeds (DLL-II). The microenvironment of the tryptophan residues in the lectin under native and denaturing conditions were investigated by quenching of the intrinsic fluorescence of the protein by a neutral quencher (acrylamide), an anionic quencher (iodide ion) and a cationic quencher (cesium ion). The results obtained indicate that the tryptophan residues of DLL-II are largely buried in the hydrophobic core of the protein matrix, with positively charged side chains residing close to at least some of the tryptophan residues under the experimental conditions. Analysis of the far UV CD spectrum of DLL-II revealed that the secondary structure of the lectin consists of 57% alpha-helix, 21% beta-sheet, 7% beta-turns and 15% unordered structures. Carbohydrate binding did not significantly alter the secondary and tertiary structures of the lectin. Thermal unfolding of DLL-II, investigated by monitoring CD signals, showed a sharp transition around 75 degrees C both in the far UV region (205 nm) and the near UV region (289 nm), which shifted to ca. 77-78 degrees C in the presence of 0.1 M methyl-beta-D-galactopyranoside, indicating that ligand binding leads to a moderate stabilization of the lectin structure.

Crystallization and preliminary X-ray crystallographic analysis of a galactose-specific lectin from Dolichos lablab.

The galactose-specific lectin from the seeds of Dolichos lablab has been crystallized using the hanging-drop vapour-diffusion technique. The crystals belong to space group P1, with unit-cell parameters a = 73.99, b = 84.13, c = 93.15 A, alpha = 89.92, beta = 76.01, gamma = 76.99 degrees. X-ray diffraction data to a resolution of 3.0 A have been collected under cryoconditions (100 K) using a MAR imaging-plate detector system mounted on a rotating-anode X-ray generator. Molecular-replacement calculations carried out using the available structures of legume lectins as search models revealed that the galactose-specific lectin from D. lablab forms a tetramer similar to soybean agglutinin; two such tetramers are present in the asymmetric unit.

Hypolipidemic effect of methanolic extract of Dolichos biflorus Linn. in high fat diet fed rats.

High fat diet fed rats showed significant increased levels of plasma and tissue total cholesterol, triglycerides, free fatty acids, phospholipids, plasma LDL cholesterol and decreased level of plasma HDL cholesterol. Methanolic extract of D. biflorus administration to high fat diet fed rats showed near to normal levels of the above lipids in plasma and tissues. Higher dose of the extract (400 mg/kg body weight) showed comparable results with standard drug atorvastatin. It is concluded that the methanolic extract of D. biflorus possesses hypolipidemic activity in high fat diet fed rats.

BMPs and BMP receptors in mouse metanephric development: in vivo and in vitro studies.

BMPs have recently emerged as likely regulators of development of the permanent kidney (metanephros). Transcripts for BMPs and their receptors have been localised in the developing metanephros. In vitro, BMPs 2, 4 and 7 have direct or indirect roles in regulation of ureteric branching morphogenesis and branch formation. In vivo, renal phenotypes have been reported in BMP7 homozygous null mutant mice and BMP4 heterozygous null mutant mice. In the present study, in vivo and in vitro roles of BMPs and BMP receptors in metanephric development were further analysed. Stereology and histology were used to analyse kidneys from mice heterozygous for mutations in either BMP2, BMPR-IA or ActR-IA. Roles of BMPs 2 and 4 in mouse metanephric development in vitro were analysed by culturing whole metanephroi in the presence of BMP2, BMP4, the BMP inhibitor noggin, and BMP4 plus noggin. Ureteric branching morphogenesis and nephrogenesis were analysed. By qualitative histology, kidneys from BMP2, BMPR-IA and ActR-IA heterozygous null mutant mice were found to be the same as those from wild type mice. The kidneys of the heterozygous mice contained the normal complement of nephrons. In vitro, high concentrations of BMP4 inhibited branching of the ureteric epithelium and changed its morphology, while nephrogenesis was inhibited by 50%. A range of concentrations of BMP2 did not alter ureteric or mesenchyme morphology, or the number of glomeruli formed. Noggin did not alter metanephric development in vitro, but did block the effect of BMP4. The experiments described in this study have shown that BMP4 has distinct roles from BMP2 in metanephric development.