Expression and efficient secretion of a functional chitinase from Chromobacterium violaceum in Escherichia coli.

BACKGROUND: Chromobacterium violaceum is a free-living ß-proteobacterium found in tropical and subtropical regions. The genomic sequencing of C. violaceum ATCC 12472 has revealed many genes that underpin its adaptability to diverse ecosystems. Moreover, C. violaceum genes with potential applications in industry, medicine and agriculture have also been identified, such as those encoding chitinases. However, none of the chitinase genes of the ATCC 12472 strain have been subjected to experimental validation. Chitinases (EC 3.2.1.14) hydrolyze the ß-(1,4) linkages in chitin, an abundant biopolymer found in arthropods, mollusks and fungi. These enzymes are of great biotechnological interest as potential biocontrol agents against pests and pathogens. This work aimed to experimentally validate one of the chitinases from C. violaceum. RESULTS: The open reading frame (ORF) CV2935 of C. violaceum ATCC 12472 encodes a protein (439 residues) that is composed of a signal peptide, a chitin-binding domain, a linker region, and a C-terminal catalytic domain belonging to family 18 of the glycoside hydrolases. The ORF was amplified by PCR and cloned into the expression vector pET303/CT-His. High levels of chitinolytic activity were detected in the cell-free culture supernatant of E. coli BL21(DE3) cells harboring the recombinant plasmid and induced with IPTG. The secreted recombinant protein was purified by affinity chromatography on a chitin matrix and showed an apparent molecular mass of 43.8 kDa, as estimated by denaturing polyacrylamide gel electrophoresis. N-terminal sequencing confirmed the proper removal of the native signal peptide during the secretion of the recombinant product. The enzyme was able to hydrolyze colloidal chitin and the synthetic substrates p-nitrophenyl-ß-D-N,N'-diacetylchitobiose and p-nitrophenyl-ß-D-N,N',N"-triacetylchitotriose. The optimum pH for its activity was 5.0, and the enzyme retained ~32% of its activity when heated to 60°C for 30 min. CONCLUSIONS: A C. violaceum chitinase was expressed in E. coli and purified by affinity chromatography on a chitin matrix. The secretion of the recombinant protein into the culture medium was directed by its native signal peptide. The mature enzyme was able to hydrolyze colloidal chitin and synthetic substrates. This newly identified signal peptide is a promising secretion factor that should be further investigated in future studies, aiming to demonstrate its usefulness as an alternative tool for the extracellular production of recombinant proteins in E. coli.

Osmotin from Calotropis procera latex: new insights into structure and antifungal properties.

This study aimed at investigating the structural properties and mechanisms of the antifungal action of CpOsm, a purified osmotin from Calotropis procera latex. Fluorescence and CD assays revealed that the CpOsm structure is highly stable, regardless of pH levels. Accordingly, CpOsm inhibited the spore germination of Fusarium solani in all pH ranges tested. The content of the secondary structure of CpOsm was estimated as follows: α-helix (20%), ß-sheet (33%), turned (19%) and unordered (28%), RMSD 1%. CpOsm was stable at up to 75°C, and thermal denaturation (T(m)) was calculated to be 77.8°C. This osmotin interacted with the negatively charged large unilamellar vesicles (LUVs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-1-glycerol (POPG), inducing vesicle permeabilization by the leakage of calcein. CpOsm induced the membrane permeabilization of spores and hyphae from Fusarium solani, allowing for propidium iodide uptake. These results show that CpOsm is a stable protein, and its antifungal activity involves membrane permeabilization, as property reported earlier for other osmotins and thaumatin-like proteins.

In vitro toxicological characterisation of the antifungal compound soybean toxin (SBTX).

Soybean toxin (SBTX) is a protein isolated from soybean seeds and composed of two polypeptide subunits (17 and 27 kDa). SBTX has in vitro activity against phytopathogenic fungi such as Cercospora sojina, Aspergillus niger, and Penicillium herguei, and yeasts like Candida albicans, C. parapsilosis, Kluyveromyces marxiannus, and Pichia membranifaciens. The present study aimed to analyze in vitro whether SBTX causes any side effects on non-target bacterial and mammalian cells that could impede its potential use as a novel antifungal agent. SBTX at 100 µg/mL and 200 µg/mL did not hinder the growth of the bacteria Salmonella enterica (subspecies enterica serovar choleraesuis), Bacillus subtilis (subspecies spizizenii) and Staphylococcus aureus. Moreover, SBTX at concentrations up to 500 µg/mL did not significantly affect the viability of erythrocytes, neutrophils, and human intestinal Caco-2 cells. To study whether SBTX could induce relevant alterations in gene expression, in vitro DNA microarray experiments were conducted in which differentiated Caco-2 cells were exposed for 24 h to 100 µg/mL or 200 µg/mL SBTX. SBTX up-regulated genes involved in cell cycle and immune response pathways, but down-regulated genes that play a role in cholesterol biosynthesis and platelet degranulation pathways. Thus, although SBTX did not affect bacteria, nor induced cytotoxity in mammalian cells, it affected some biological pathways in the human Caco-2 cell line that warrants further investigation.

Mo-CBP4, a purified chitin-binding protein from Moringa oleifera seeds, is a potent antidermatophytic protein: In vitro mechanisms of action, in vivo effect against infection, and clinical application as a hydrogel for skin infection.

Dermatophytes belonging to Trichophyton ssp. are important anthropophilic and zoophilic pathogens, which developed resistance to griseofulvin, the common antifungal drug used to treat dermatophytosis. In this context, Moringa oleifera seed proteins have been described as antifungal agents with potential applications. Thus, this work aimed to evaluate the antidermatophytic in vitro, focusing on mechanisms, and in vivo potential of Mo-CBP4, purified from M. oleifera seeds. Mo-CBP4was purified after protein extraction with 50 mM Tris-HCl buffer, pH 8.0, and chromatography on chitin and CM Sepharose™ columns and antidermatophytic potential of Mo-CBP4 evaluated in vitro and in vivo. In vitro, Mo-CBP4 reduced in 50% the germination of microconidia of Trichophyton mentagrophytes at 45 µM; but did not show inhibition of mycelial growth. Mo-CBP4 (45 µM) presents the inhibitory activity even when incubated with N-acetyl-d-glucosamine (NAG). Analysis of the mechanisms of Mo-CBP4 revealed an increase in membrane permeability, ROS overproduction and damage to cell wall leading to microconidia death. Furthermore, using in vivo models, Mo-CBP4 (5, 10 and 20 mg g-1) reduced the severity and time of dermatophytosis. Altogether, these findings indicate that Mo-CBP4 has great potential for the development of novel antifungal drugs for the clinical treatment of dermatophytosis.

Orally hypoglycemic activity of an insulin mimetic glycoprotein isolated from Cnidoscolus quercifolius Pohl. (Euphorbiaceae) seeds, Cq-IMP.

The genus Cnidoscolus (Euphorbiaceae) is widely distributed in tropical areas. In the Northeast of Brazil, the species C. quercifolius is endemic and has been used in traditional medicine. In this study, a novel protein was isolated from C. quercifolius seeds and characterized by its molecular weight, primary structure, isoelectric point (pI), and carbohydrate content. The hypoglycemic activity of this protein was investigated by in vitro assay with the RIN-5F glucose-responsive cell line and in vivo test using alloxan-induced diabetic mice models. In addition, safe use of the protein was also investigated by cytotoxicity, hemagglutinating, and immunogenicity assays. The protein which was named Cq-IMP (Cnidoscolus quercifolius - Insulin Mimetic Protein) showed a single 11.18 KDa glycopolypeptide chain (16.4% of carbohydrates, m/m), pI of 8.0 and N-terminal sequence (TKDPELKQcKKQQKKqQQYDDDDKK) with similarity around 46-62% to sucrose binding protein-like and vicilin-like protein that was confirmed by mass spectrometry tryptic peptides analysis. Besides that, Cq-IMP presented anti-insulin antibody cross-reactivity as hypoglycemic activity in both in vitro and in vivo models. Additionally, it did not present any toxicity by methods tested. In conclusion, Cq-IMP is an insulin-mimetic protein, with a potent hypoglycemic activity and no toxicity showing great potential for therapeutic applications and drug development.

Role of membrane sterol and redox system in the anti-candida activity reported for Mo-CBP2, a protein from Moringa oleifera seeds.

Plant proteins are emerging as an alternative to conventional treatments against candidiasis. The aim of this study was to better understand the mechanism of action of Mo-CBP2 against Candida spp, evaluating redox system activity, lipid peroxidation, DNA degradation, cytochrome c release, medium acidification, and membrane interaction. Anti-candida activity of Mo-CBP2 decreased in the presence of ergosterol, which was not observed with antioxidant agents. C. albicans treated with Mo-CBP2 also had catalase and peroxidase activities inhibited, while superoxide dismutase was increased. Mo-CBP2 increased the lipid peroxidation, but it did not alter the ergosterol profile in live cells. External medium acidification was strongly inhibited, and cytochrome c release and DNA degradation were detected. Mo-CBP2 interacts with cell membrane constituents, changes redox system enzymes in C. albicans and causes lipid peroxidation by ROS overproduction. DNA degradation and cytochrome c release suggest apoptotic or DNAse activity. Lipid peroxidation and H+-ATPases inhibition may induce the process of apoptosis. Finally, Mo-CBP2 did not have a cytotoxic effect in mammalian Vero cells. This study highlights the biotechnological potential of Mo-CBP2 as a promising molecule with low toxicity and potent activity. Further studies should be performed to better understand its mode of action and toxicity.

A resistant cowpea (Vigna unguiculata [L.] Walp.) genotype became susceptible to cowpea severe mosaic virus (CPSMV) after exposure to salt stress.

In nature, plants are simultaneously challenged by biotic and abiotic stresses. However, little is known about the effects of these combined stresses for most crops. This work aimed to evaluate the responsed of the virus-resistant cowpea genotype BRS-Marataoã to the exposure of salt stress combined with CPSMV infection. Cowpea plants were exposed to 200 mM NaCl either simultaneously (SV plant group) or 24 h prior to the CPSMV infection [S(24 h)V plant group]. Physiological, biochemical, and proteomic analyses at 2 and 6 days post salt stress (DPS) revealed that cowpea significantly reprogrammed its cellular metabolism. Indeed, plant size, photosynthetic parameters (net photosynthesis, transpiration rate, stomatal conductance, and internal CO2 partial pressure) and chlorophyll and carotenoid contents were reduced in S(24 h)V compared to SV. Moreover, accumulation of viral particles at 6 DPS in S(24 h)V was observed indicating that the salt stress imposed prior to virus infection favors viral particle proliferation. Proteomic analysis showed differential contents of 403 and 330 proteins at 2 DPS and 6 DPS, respectively, out of 733 differentially abundant proteins between the two plant groups. The altered leaf proteins are involved in energy and metabolism, photosynthesis, stress response, and oxidative burst. BIOLOGICAL SIGNIFICANCE: This is an original study in which a virus-resistant cowpea genotype (BRS-Marataoã) was (i) exposed simultaneously to 200 mM NaCl and inoculation with CPSMV (SV plant group) or (ii) exposed to 200 mM NaCl stress 24 h prior to inoculation with CPSMV [S(24 h)V plant group]. The purpose was to shed light on how this CPSMV resistant cowpea responded to the combined stresses. Numerous key proteins and associated pathways were altered in the cowpea plants challenged with both stresses, but unexpectedly, the salt stress imposed 24 h prior to CPSMV inoculation allowed viral proliferation, turning the cowpea genotype from resistant to susceptible.

A cysteine protease from the latex of Ficus benjamina has in vitro anthelmintic activity against Haemonchus contortus.

Haemonchus contortus is a gastrointestinal nematode that is responsible for high mortality rates in ruminant herds. The resistance of nematodes to synthetic anthelmintics is widespread and requires a continuous search for new bioactive molecules, such as proteins. The objective of this study was to evaluate the anthelmintic potential of a protease purified from the latex of Ficus benjamina against H. contortus . Fresh latex was collected from plants via small incisions in the green stems, the rubber was removed by centrifugation, and the latex protein extract (LPE) was obtained. After LPE fractionation with ammonium sulfate and chromatography of the fraction containing the highest proteolytic activity on CM-cellulose, a cysteine protease (FbP) was purified. FbP has a molecular mass of approximately 23.97 kDa, and its proteolytic activity was stable between pH 6.0 and pH 10 and over a broad temperature range, with optimum activity at 60 °C. FbP inhibited both the development and exsheathment of H. contortus larvae, with 50% effective concentrations of 0.26 and 0.79 mg/mL, respectively. We conclude that this cysteine protease from F. benjamina latex with anthelmintic activity against H. contortus could be a promising alternative for the development of products for use in parasite control programmes.

Expression of the Canavalia brasiliensis lectin (ConBr) in tobacco plants.

Tobacco plants were transformed with gene constructs encoding prepro-ConBr (Canavalia brasiliensis lectin). Transgenic plants confirmed by PCR expressed the recombinant protein as revealed by Western blot. However, the apparent molecular mass of the recombinant polypeptide (ca. 34 kDa) was higher than the native lectin (about 30 kDa), showing that further proteolytic processing of pro-ConBr was not detected.

Superoxide dismutase, catalase and peroxidase activities do not confer protection against oxidative damage in salt-stressed cowpea leaves.

• The aim of this study was to determine whether guaiacol peroxidase (POX), superoxide dismutase (SOD) and catalase (CAT) activities are effective in the protection and recovery of cowpea (Vigna unguiculata (L.) Walp.) leaves exposed to a salt-induced oxidative stress. The salt treatment (200 mm NaCl) was imposed during six consecutive days and the salt withdrawal after 3 d (recovery treatment). Control plants received no NaCl treatment. • The salt treatment caused almost complete cessation of leaf relative growth rate in parallel with the transpiration rate. The restriction in leaf growth was associated with a progressive increase in membrane damage, lipid peroxidation and proline content. Salt withdrawal induced a significant recovery in both leaf growth rate and transpiration. Surprisingly, these prestressed/recovered plants showed only a slight recovery in leaf lipid peroxidation and membrane damage. • Leaf CAT activity experienced a twofold decrease only after 1 d NaCl treatment, and salt withdrawal had no effect on its recovery. SOD activity did not change compared with control plants. By contrast, POX activity significantly increased after 1 d NaCl treatment and showed a significant recovery to levels near to those of control. • In conclusion, it appears that the ability of cowpea plants to survive under high levels of salinity is not caused by an operating antioxidant system involving SOD, POX and CAT activities in mature leaves.

Proline accumulation and glutamine synthetase activity are increased by salt-induced proteolysis in cashew leaves.

In this study cashew (Anacardium occidentale) plants were exposed to a short- and long-term exposure to NaCl in order to establish the importance of the salt-induced proteolysis and the glutamine synthetase activity on the proline accumulation. The cashew leaf showed a prominent proline accumulation in response to salt stress. In contrast, the root tissue had no significant changes in proline content even after the drastic injury caused by salinity on the whole plant. The leaf proline accumulation was correlated to protease activity, accumulation of free amino acid and ammonia, and decrease of both total protein and chlorophyll contents. The leaf GS activity was increased by the salt stress whereas in the roots it was slightly lowered. Although the several amino acids in the soluble pool of leaf tissue have showed an intense increment in its concentrations in the salt-treated plants, proline was the unique to show a proportional increment from 50 to 100 mol m-3 NaCl exposure (16.37 to 34.35 mmol kg-1 DM, respectively). Although the leaf glutamate concentration increased in the leaves of the salt-stressed cashew plants, as compared to control, its relative contribution to the total amino acid decreased significantly in stressed leaves when compared to other amino acids. In addition, when the leaf discs were incubated with NaCl in the presence of exogenous precursors (Glu, Gln, Orn or Arg) involved in the proline synthesis pathways, the glutamate was unique in inducing a significant enhancement of the proline accumulation compared to those discs with precursor in the absence of NaCl. These results, together with the salt-induced increase in the GS activity, suggest an increase in the de novo synthesis of proline probably associated with the increase of the concentration of glutamate. Moreover, the prominent salt-induced proline accumulation in the leaves was associated with the higher salt-sensitivity in terms of proteolysis and salt-induced senescence as compared to the roots. In conclusion, the leaf-proline accumulation was due, at least in part, to the increase in the salt-induced proteolysis associated with the increments in the GS activity and hence the increase in the concentration of glutamate precursor in the soluble amino acid pool.

Identification of a novel antimicrobial peptide from Brazilian coast coral Phyllogorgia dilatata.

The marine ecosystem is able to provide enormous biomolecule diversity that could be used for treatment of various diseases. In this highly competitive environment, organisms need chemical barriers to reduce or avoid microorganism contamination. Among the molecules that protect these animals the antimicrobial peptides (AMPs) are included. In the present study, crude extracts of coral coral specimens Carijoa riisei, Muriceopsis sulphurea, Neospongodes atlantica, Palythoa caribeorum, Phyllogorgia dilatata and Plexaurella grandiflora were challenged against multiple Grampositive and -negative bacteria showing different activities. P. dilatata crude extract showed the antibacterial activity, and was ammonium-sulfate (0-40%) fractionated, being able to control the growth of K. pneumoniae, S. flexineri and S. aureus. Rich-fraction was further purified by using Amicon® Ultra Centrifugal 10 kDa associated with reversed-phase HPLC chromatography (C18), producing the peptide named Pd-AMP1. Pd-AMP1 was able to inhibit S. aureus development. Mass spectrometry analyses showed a monoisotopic mass of 5372.66 Da and N-terminal sequence showed no significant match with databank. In this view, the prospecting of protein biomolecules and biotechnological potential from marine animals is still little explored and may serve as an alternative to common antibiotics.

Chemical composition, nutritive value, and toxicological evaluation of Bauhinia cheilantha seeds: a legume from semiarid regions widely used in folk medicine.

Among the Bauhinia species, B. cheilantha stands out for its seed protein content. However, there is no record of its nutritional value, being used in a nonsustainable way in the folk medicine and for large-scale extraction of timber. The aim of this study was to investigate the food potential of B. cheilantha seeds with emphasis on its protein quality to provide support for flora conservation and use as raw material or as prototype for the development of bioproducts with high added socioeconomic value. B. cheilantha seeds show high protein content (35.9%), reasonable essential amino acids profile, low levels of antinutritional compounds, and nutritional parameters comparable to those of legumes widely used such as soybean and cowpea. The heat treatment of the seeds as well as the protein extraction process (to obtain the protein concentrate) increased the acceptance of diets by about 100% when compared to that of raw Bc diet. These wild legume seeds can be promising alternative source of food to overcome the malnutrition problem faced by low income people adding socioeconomic value to the species.

A cysteine protease from the latex of Ficus benjamina has in vitro anthelmintic activity against Haemonchus contortus

Haemonchus contortus é um nematoide gastrintestinal, responsável por altas taxas de mortalidade em rebanhos de pequenos ruminantes. A resistência dos nematoides aos anti-helmínticos sintéticos está generalizada e requer uma busca contínua por novos compostos bioativos, como as proteínas. O objetivo deste trabalho foi avaliar o potencial anti-helmíntico da protease purificada do látex de Ficus benjamina contra H. contortus. O látex fresco foi coletado das plantas por pequenas incisões nas hastes verdes e o extrato proteico de látex (EPL) foi obtido. Após o fracionamento do EPL com sulfato de amônio e cromatografia da fração contendo a maior atividade proteolítica da CM-Celulose, uma protease cisteínica (FbP) foi purificada. A FbP tem massa molecular de cerca de 23,97 kDa, a atividade proteolítica foi estável entre pH 6,0 e pH 10 e ao longo de uma ampla faixa de temperatura, com atividade ótima a 60 °C. A FbP inibiu tanto o desenvolvimento quanto o desembainhamento das larvas de H. contortus, com 50% de inibição nas concentrações de 0,26 e 0,79 mg/mL, respectivamente. Concluímos que esta protease cisteínica do látex de F. benjamina, com ação anti-helmíntica contra H. contortus, pode ser uma alternativa promissora para o desenvolvimento de produtos a serem utilizados em programas de controle de parasitos.

A cysteine protease from the latex of Ficus benjamina has in vitro anthelmintic activity against Haemonchus contortus / Atividade anti-helmíntica in vitro de uma protease cisteínica do látex de Ficus benjamina contra Haemonchus contortus

Abstract Haemonchus contortus is a gastrointestinal nematode that is responsible for high mortality rates in ruminant herds. The resistance of nematodes to synthetic anthelmintics is widespread and requires a continuous search for new bioactive molecules, such as proteins. The objective of this study was to evaluate the anthelmintic potential of a protease purified from the latex of Ficus benjamina against H. contortus . Fresh latex was collected from plants via small incisions in the green stems, the rubber was removed by centrifugation, and the latex protein extract (LPE) was obtained. After LPE fractionation with ammonium sulfate and chromatography of the fraction containing the highest proteolytic activity on CM-cellulose, a cysteine protease (FbP) was purified. FbP has a molecular mass of approximately 23.97 kDa, and its proteolytic activity was stable between pH 6.0 and pH 10 and over a broad temperature range, with optimum activity at 60 °C. FbP inhibited both the development and exsheathment of H. contortus larvae, with 50% effective concentrations of 0.26 and 0.79 mg/mL, respectively. We conclude that this cysteine protease from F. benjamina latex with anthelmintic activity against H. contortus could be a promising alternative for the development of products for use in parasite control programmes.

Resumo Haemonchus contortus é um nematoide gastrintestinal, responsável por altas taxas de mortalidade em rebanhos de pequenos ruminantes. A resistência dos nematoides aos anti-helmínticos sintéticos está generalizada e requer uma busca contínua por novos compostos bioativos, como as proteínas. O objetivo deste trabalho foi avaliar o potencial anti-helmíntico da protease purificada do látex de Ficus benjamina contra H. contortus . O látex fresco foi coletado das plantas por pequenas incisões nas hastes verdes e o extrato proteico de látex (EPL) foi obtido. Após o fracionamento do EPL com sulfato de amônio e cromatografia da fração contendo a maior atividade proteolítica da CM-Celulose, uma protease cisteínica (FbP) foi purificada. A FbP tem massa molecular de cerca de 23,97 kDa, a atividade proteolítica foi estável entre pH 6,0 e pH 10 e ao longo de uma ampla faixa de temperatura, com atividade ótima a 60 °C. A FbP inibiu tanto o desenvolvimento quanto o desembainhamento das larvas de H. contortus, com 50% de inibição nas concentrações de 0,26 e 0,79 mg/mL, respectivamente. Concluímos que esta protease cisteínica do látex de F. benjamina, com ação anti-helmíntica contra H. contortus, pode ser uma alternativa promissora para o desenvolvimento de produtos a serem utilizados em programas de controle de parasitos.

Osmotin purified from the latex of Calotropis procera: biochemical characterization, biological activity and role in plant defense.

A protein, similar to osmotin- and thaumatin-like proteins, was purified from Calotropis procera (Ait.) R.Br latex. The isolation procedure required two cation exchange chromatography steps on 50mM Na-acetate buffer (pH 5.0) CM-Sepharose Fast Flow and 25 mM Na-phosphate buffer (pH 6.0) Resource-S, respectively. The protein purity was confirmed by an unique N-terminal sequence [ATFTIRNNCPYTIWAAAVPGGGRRLNSGGTWTINVAPGTA]. The osmotin (CpOsm) appeared as a single band (20,100 Da) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as two spots in two-dimensional electrophoresis (pI 8.9 and 9.1). Both polypeptides were further identified by mass spectrometry as two osmotin isoforms with molecular masses of 22,340 and 22,536 Da. The CpOsm exerted antifungal activity against Fusarium solani (IC50=67.0 µg mL⁻¹), Neurospora sp. (IC50=57.5 µg mL⁻¹) and Colletotrichum gloeosporioides (IC50=32.1 µg mL⁻¹). However, this activity was lost when the protein was previously treated with a reducing agent (DTT, Dithiothreitol) suggesting the presence of disulfide bounds stabilizing the protein. The occurrence of osmotin in latex substantiates the defensive role of these fluids.

Purification of a chitin-binding protein from Moringa oleifera seeds with potential to relieve pain and inflammation.

Moringa oleifera Lam. is a perennial multipurpose tree that has been successfully used in folk medicine to cure several inflammatory processes. The aim of this study was to purify and characterize a chitin-binding protein from Moringa oleifera seeds, named Mo-CBP4, and evaluate its antinociceptive and anti-inflammatory effects in vivo. The protein was purified by affinity chromatography on chitin followed by ion exchange chromatography. Acetic acid-induced abdominal constrictions assay was used for the antinociceptive and anti-inflammatory activity assessments. Mo-CBP4 is a glycoprotein (2.9% neutral carbohydrate) composed of two protein subunits with apparent molecular masses of 28 and 18 kDa (9 kDa in the presence of reducing agent). The intraperitoneal injection of Mo-CBP4 (3.5 and 10 mg/kg) into mice 30 min before acetic acid administration potently and significantly reduced the occurrence of abdominal writhing in a dose dependent manner by 44.7% and 100%, respectively. In addition, the oral administration of the protein (10 mg/kg) resulted in 18% and 52.8% reductions in abdominal writhing when given 30 and 60 min prior to acetic acid administration, respectively. Mo-CBP4, when administered by intraperitoneal route, also caused a significant and dose-dependent inhibition of peritoneal capillary permeability induced by acid acetic and significantly inhibited leukocyte accumulation in the peritoneal cavity. In conclusion, this pioneering study describes that the chitin-binding protein Mo-CBP4, from M. oleifera seeds, exhibits anti-inflammatory and antinociceptive properties and scientifically supports the use of this multipurpose tree in folk medicine.

Effects of a novel pathogenesis-related class 10 (PR-10) protein from Crotalaria pallida Roots with papain inhibitory activity against root-knot nematode Meloidogyne incognita.

A novel pathogenesis-related class 10 (PR-10) protein with papain inhibitory activity, named CpPRI, was purified from Crotalaria pallida roots by ammonium sulfate precipitation followed by three reverse-phase high-performance liquid chromatographies (HPLCs). CpPRI is made up of a single polypeptide chain with a M(r) of 15 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This protein exhibited a K(i) value of 1.8 x 10(-9) M and operates via a noncompetitive inhibition mechanism. The alignment of the N-terminal amino acid sequence of CpPRI with other proteins revealed its identity with PR-10 proteins. CpPRI acts against digestive proteinase from root-knot nematode Meloidogyne incognita and demonstrated nematostatic and nematicide effects on this parasite in bioassays. In a localization study, fluorescein-5-isothiocyanate (FITC)-CpPRI was observed to internalize and diffuse over the entire J2 body after 6 h of incubation. This fact could explain the natural tolerance of this plant species to nematodes.

The carbohydrate-binding specificity and molecular modelling of Canavalia maritima and Dioclea grandiflora lectins

The carbohydrate-binding specificity of lectins from the seeds of Canavalia maritima and Dioclea grandiflora was studied by hapten-inhibition of haemagglutination using various sugars and sugar derivatives as inhibitors, including N-acetylneuraminic acid and N-acetylneuraminic acid and N-acetylmuramic acid. Despite some discrepancies, both lectins exhibited a very similiar carbohydrate-binding specificity as previously reported for other lectins from Diocleinae (tribe Phaseoleae, sub-tribe Diocleinae). Accordingly, both lectins exhibited almost identical hydropathic profiles and their three-dimensional models built up from the atomic coordinates of ConsA looked very similar. However, doking experiments of glucose and mannose in their monosaccharide-binding sites, by comparison with the ConA-mannose complex used as a model, reveled conformational changes in side chains of the animo acid residues invlved in the binding of monosaccharides. These results fully agree with crystallographic data showing that binding of specific ligands to ConsA requires conformational chances of this monosaccharide-binding site.

Plant lectins, chemical and biological aspects

Lectins, carbohydrate-binding proteins of non-immune origin, that agglutinate cells or precipitate polysaccharides and glycoconjugates, are well distributed in nature, mainly in the Plant Kingdom. The great majority of the plante lectins are present in seed cotyledons where they are found in the cytoplasm or int he protein bodies, although they have also been found in roots, stems and leaves. Due to their peculiar properties, the lectins are used as a tool both for analytical and preparative purposes in biochemistry, cellular biology, immunology and related areas. In agriculture and medicine the use of lectins greatly improved in the last few years. The lextins, with few exceptions, are glycoproteins, need divalent cations to display full activity and are, in general, oligomers with variable molecular weight. Although the studies on lectins have completed a century, their role in nature is yet ynknown . Several hypotheses on their physiological functions have been suggested. Thus, lectins could play important roles in defense against pathogens, plant-microorganism symbiosis, cell organization, embryo morphogenesis, phagocytosis, cell wall elongation, pollen recognition and as reserve proteins. A brief review on the general properties and roles of the lectins is given.