Allergenicity reduction of cow's milk proteins using latex peptidases.

The present study evaluated four laticifer fluids as a novel source of peptidases capable of hydrolyzing proteins in cow's milk. The latex peptidases from Calotropis procera (CpLP), Cryptostegia grandiflora (CgLP), and Carica papaya (CapLP) were able to perform total hydrolysis of caseins after 30 min at pH 6.5, as confirmed by a significant reduction in the residual antigenicity. Casein hydrolysis by Plumeria rubra latex peptidases (PrLP) was negligible. Moreover, whey proteins were more resistant to proteolysis by latex peptidases; however, heat pretreatment of the whey proteins enhanced the degree of hydrolysis and reduced the residual antigenicity of the hydrolysates. The in vivo assays show that the cow's milk proteins hydrolysed by CgLP and CapLP exhibited no immune reactions in mice allergic to cow's milk, similar to a commercial partially hydrolysed formula. Thus, these peptidases are promising enzymes for the development of novel hypoallergenic formulas for children with a milk allergy.

Lipid transfer protein isolated from noni seeds displays antibacterial activity in vitro and improves survival in lethal sepsis induced by CLP in mice.

In the present study, we aimed to evaluate the antibacterial activity of a lipid transfer protein isolated from Morinda citrifolia L. seeds, named McLTP1, and to investigate its effect in the cecal ligation and puncture (CLP) mouse sepsis model. Antimicrobial assays revealed that McLTP1 (12.5-800 µg/mL) significantly reduced Staphylococcus aureus (ATCC 6538P and ATCC 14458) and Staphylococcus epidermidis (ATCC 12228) planktonic growth, reaching maximal inhibition of approximately 50% and 98%, respectively. Furthermore, McLTP1 inhibited biofilm formation of both S. aureus strains, achieving percentages ranging from 39.1% to 69.1% (200-800 µg/mL) for ATCC 6538P and 34.4%-63% (12.5-800 µg/mL) for ATCC 14458. A synergistic interaction between McLTP1 and oxacillin against S. aureus and S. epidermidis was also observed, as determined by fractional inhibitory concentration indices of 0.18 and 0.38, respectively. McLTP1 showed no significant inhibitory effect against Gram-negative bacteria. In the in vivo experiments, sepsis was lethal to 83% of the animals, 72 h after CLP. In contrast, 100% of the animals treated with McLTP1 (8 mg/kg) before (intraperitoneal injection or oral dose) or after (oral dose) CLP were still alive 3 days later. In addition, oral or intraperitoneal administration of McLTP1 (8 mg/kg) significantly reduced the body weight loss, fever, leukocytosis, organ damage, and the level of inflammatory serum cytokines induced by sepsis. In conclusion, McLTP1 could be exploited for its antimicrobial properties, and can be considered a potential therapeutic candidate for the management of clinical sepsis.

Morinda citrifolia lipid transfer protein 1 exhibits anti-inflammatory activity by modulation of pro- and anti-inflammatory cytokines.

Previous reports have demonstrated that a thermostable lipid transfer protein isolated from noni seeds (McLTP1; 9.4kDa) displays anti-nociceptive and anti-inflammatory activities. This work aimed to investigate the underlying mechanisms of the anti-inflammatory activity of McLTP1 in mice. The protein was solubilised in sterile saline (0.9% NaCl) immediately before the treatment of mice by oral or intraperitoneal routes at doses of 8mg/kg. Given orally or intraperitoneally, McLTP1 significantly inhibited (p<0.05) cell migration in experimental models of carrageenan-induced peritonitis and the formation of paw oedema induced by carrageenan and dextran. Additionally, McLTP1 demonstrated the ability to significantly inhibit the production of the cytokines IL-1ß, IL-6, and TNF-α (p<0.05) and to promote an increase in the production of the anti-inflammatory cytokine IL-10. The treatment of mice with McLTP1 by the oral or i.p route reduced pancreatic injury and activities of amylase, lipase, and pancreatitis-associated lung injury. This study suggested that the observed anti-inflammatory effects of McLTP1 can be related to modulation of pro- and anti-inflammatory cytokine levels.

First insights into the diversity and functional properties of chitinases of the latex of Calotropis procera.

Chitinases (EC 3.2.1.14) found in the latex of Calotropis procera (Ait) R. Br. were studied. The proteins were homogeneously obtained after two ion exchange chromatography steps. Most proteins were identified individually in 15 spots on 2-D gel electrophoresis with isoelectric points ranging from 4.6 to 6.0 and molecular masses extending from 27 to 30 kDa. Additionally, 66 kDa proteins were identified as chitinases in SDS-PAGE. Their identities were further confirmed by mass spectrometry (MS) analysis of the tryptic digests of each spot and MS analysis of the non-digested proteins. Positive reaction for Schiff's reagent suggested the proteins are glycosylated. The chitinases exhibited high catalytic activity toward to colloidal chitin at pH 5.0, and this activity underwent decay in the presence of increasing amounts of reducing agent dithiothreitol. Spore germination and hyphae growth of two phytopathogenic fungi were inhibited only marginally by the chitinases but were affected differently. This suggested a complex relationship might exist between the specificity of the proteins toward the fungal species. The chitinases showed potent insecticidal activity against the Bruchidae Callosobruchus maculatus, drastically reducing survival, larval weight and adult emergence. It is concluded that closely related chitinases are present in the latex of C. procera, and the first experimental evidence suggests these proteins are involved more efficiently in defence strategies against insects rather than fungi.

First isolation and antinociceptive activity of a lipid transfer protein from noni (Morinda citrifolia) seeds.

In this study a novel heat-stable lipid transfer protein, designated McLTP1, was purified from noni (Morinda citrifolia L.) seeds, using four purification steps which resulted in a high-purified protein yield (72 mg McLTP1 from 100g of noni seeds). McLTP1 exhibited molecular masses of 9.450 and 9.466 kDa, determined by electrospray ionisation mass spectrometry. The N-terminal sequence of McLTP1 (AVPCGQVSSALSPCMSYLTGGGDDPEARCCAGV), as analysed by NCBI-BLAST database, revealed a high degree of identity with other reported plant lipid transfer proteins. In addition, this protein proved to be resistant to pepsin, trypsin and chymotrypsin digestion. McLTP1 given intraperitoneally (1, 2, 4 and 8 mg/kg) and orally (8 mg/kg) caused an inhibition of the writhing response induced by acetic acid in mice. This protein displayed thermostability, retaining 100% of its antinociceptive activity after 30 min incubation at 80 °C. Pretreatment of mice with McLTP1 (8 mg/kg, i.p. and p.o.) also decreased neurogenic and inflammatory phases of nociception in the formalin test. Naloxone (2 mg/kg, i.p.) antagonised the antinociceptive effect of McLTP1 suggesting that the opioid mechanisms mediate the analgesic properties of this protein.

Proteome of Soybean Seed Exudates Contains Plant Defense-Related Proteins Active against the Root-Knot Nematode Meloidogyne incognita.

Several studies have described the effects of seed exudates against microorganisms, but only few of them have investigated the proteins that have defensive activity particularly against nematode parasites. This study focused on the proteins released in the exudates of soybean seeds and evaluated their nematicidal properties against Meloidogyne incognita. A proteomic approach indicated the existence of 63 exuded proteins, including ß-1,3-glucanase, chitinase, lectin, trypsin inhibitor, and lipoxygenase, all of which are related to plant defense. The presence of some of these proteins was confirmed by their in vitro activity. The soybean exudates were able to reduce the hatching of nematode eggs and to cause 100% mortality of second-stage juveniles (J2). The pretreatment of J2 with these exudates resulted in a 90% reduction of the gall number in tobacco plants. These findings suggest that the exuded proteins are directly involved in plant defense against soil pathogens, including nematodes, during seed germination.

Soybean toxin (SBTX) impairs fungal growth by interfering with molecular transport, carbohydrate/amino acid metabolism and drug/stress responses.

Soybean toxin (SBTX) is an antifungal protein from soybeans with broad inhibitory activity against the growth and filamentation of many fungi, including human and plant pathogenic species such as Candida albicans, Candida parapsilosis, Aspergillus niger, Penicillium herquei, Cercospora sojina and Cercospora kikuchii. Understanding the mechanism by which SBTX acts on fungi and yeasts may contribute to the design of novel antifungal drugs and/or the development of transgenic plants resistant to pathogens. To this end, the polymorphic yeast C. albicans was chosen as a model organism and changes in the gene expression profile of strain SC5314 upon exposure to SBTX were examined. Genes that were differentially regulated in the presence of SBTX were involved in glucose transport and starvation-associated stress responses as well as in the control of both the induction and repression of C. albicans hyphal formation. Transmission electron microscopy showed that C. albicans cells exposed to SBTX displayed severe signs of starvation and were heavily granulated. Our data were indicative of C. albicans cell starvation despite sufficient nutrient availability in the medium; therefore, it can be speculated that SBTX blocks nutrient uptake systems. Because neither the starvation signal nor the alkaline response pathway lead to the induction of hyphae, we hypothesise that conflicting signals are transmitted to the complex regulatory network controlling morphogenesis, eventually preventing the filamentation signal from reaching a significant threshold.

Essential oil of Croton zehntneri and its major constituent anethole display gastroprotective effect by increasing the surface mucous layer.

Croton zehntneri, a plant native to northeastern Brazil, is widely used in folk medicine to treat gastrointestinal problems and has rich essential oil content. The effects of the essential oil of Croton zehntneri (EOCZ) and its main constituent anethole on several models of gastric lesions were studied in mice and rats. Oral treatment with EOCZ and anethole, both at doses of 30-300 mg/kg, caused similar and dose-dependent gastroprotection against ethanol- and indomethacin-induced gastric damage, but did not change cold-restraint stress-induced ulcers in rats. Furthermore, EOCZ and anethole (both at 30 and 300 mg/kg) similarly and significantly increased the mucus production by the gastric mucosa, measured by Alcian blue binding, in ethanol-induced ulcer model. However, at the same doses, neither EOCZ nor anethole promoted significant alteration in gastric production of non-protein sulfhydryl groups. In pylorus-ligated model, neither EOCZ nor anethole (both at 30 and 300 mg/kg) had a significant effect on the volume of gastric juice, pH, or total acidity. The results of this study show for the first time that EOCZ possesses a gastroprotective potential, an effect mostly attributed to the action of anethole. This activity is related predominantly to the ability of EOCZ and anethole to enhance the production of gastric wall mucus, an important gastroprotective factor. Furthermore, they suggest that EOCZ has potential therapeutic application for the treatment of gastric ulcers.

A novel chitin-binding protein from Moringa oleifera seed with potential for plant disease control.

A thermostable chitin-binding protein (14.3 kDa) with antifungal activity was isolated from Moringa oleifera seeds by affinity chromatography on chitin followed by ion exchange chromatography. NH(2-) CPAIQRCCQQLRNIQPPCRCCQ (Mo-CBP3) is a glycoprotein with 2.5% sugar, pI 10.8, without hemagglutination, chitinase or beta-glucanase activities. Mo-CBP3 possesses in vitro antifungal activity against the phytopathogenicfungi Fusarium solani, F. oxysporum, Colletotrichum musae and C. gloesporioides. Contrarily, Mo-CBP3 did not affect Pythium oligandrum, an oomycete. At 0.05 mg/ml, Mo-CBP3 showed to be fungistatic against F. solani, but at 0.1 mg/ml Mo-CBP3 behaved as a potent fungicidal agent as it inhibited both the spore germination and mycelial growth of F. solani. Surprisingly, the effect of Mo-CBP3 against spore germination was observed even when the protein was heated at 100 degrees C for 1 h or pretreated with 0.15M N-acetyl-D-glucosamine. Mo-CBP3 inhibited the glucose-stimulated acidification of the incubation medium by F. solani. This is apparently caused by structural plasma membrane disarrangement induced by Mo-CBP3. Altogether, these results suggest that Mo-CBP3 might be involved in plant defense mechanisms and could be used as potential antifungal agent for controlling fungal pathogens in plants.

Soybean toxin (SBTX), a protein from soybeans that inhibits the life cycle of plant and human pathogenic fungi.

Soybean toxin (SBTX) is a 44 kDa glycoprotein that is lethal to mice (LD(50) = 5.6 mg/kg). This study reports the toxicity of SBTX on pathogenic fungi and yeasts and the mechanism of its action. SBTX inhibited spore germination of Aspergillus niger and Penicillium herguei and was toxic to Candida albicans, Candida parapsilosis, Kluyveromyces marxiannus , Pichia membranifaciens, and Saccharomyces cerevisiae. In addition, SBTX hampered the growth of C. albicans and K. marxiannus and inhibited the glucose-stimulated acidification of the incubation medium by S. cerevisiae, suggesting that SBTX interferes with intracellular proton transport to the external medium. Moreover, SBTX caused cell-wall disruption, condensation/shrinkage of cytosol, pseudohyphae formation, and P. membranifaciens and C. parapsilosis cell death. SBTX is toxic to fungi at concentrations far below the dose lethal to mice and has potential in the design of new antifungal drugs or in the development of transgenic crops resistant to pathogens.