Characterization of a non-glycosylated fraction from honey proteins of Melipona beecheii with antimicrobial activity against Escherichia coli O157:H7.

AIMS: To analyse the non-glycosylated protein fraction from Melipona beecheii honey for antimicrobial activity against Escherichia coli O157:H7. METHODS AND RESULTS: The proteins from M. beecheii honey were separated according to their degree of glycosylation using Concanavalin A-affinity chromatography. The total protein extract and its fractions were analysed by 1D and 2D electrophoresis. We also determined the antimicrobial and antihaemolytic activities of the total protein extract and the non-glycosylated fraction. Furthermore, we evaluated the effect of this non-glycosylated fraction for the expression of the Stx1, Stx2, EAE and HlyA pathogen genes. Melipona beecheii honey contained at least 24 proteins with molecular weights ranging between 7·6 and 95 kDa and isoelectric points between 3 and 10, three proteins from the 24 are non-glycosylated. The non-glycosylated fraction had an MIC90 of 1·128 µg ml-1 , and this fraction inhibited the haemolytic activity of the pathogen, as well as reduced the expression of Stx1, Stx2 and HlyA. The MbF1-2 protein from the non-glycosylated fraction was sequenced and identified as a homologue of the royal jelly-like protein of Melipona quadrifasciata. CONCLUSIONS: The non-glycosylated protein fraction from M. beecheii honey greatly contributes to antibacterial activity and it is composed of at least three proteins, of which MbF1-2 provided over 50% of the antimicrobial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The study showed significant antimicrobial activity from several proteins present in the honey of M. beecheii. Interestingly, the non-glycosylated protein fraction demonstrated antihaemolytic activity and adversely affected the expression of virulence genes in Escherichia coli O157:H7; these proteins have the potential to be used in developing therapeutic agents against this bacterium.

The amoebicidal aqueous extract from Castela texana possesses antigenotoxic and antimutagenic properties.

Due to long-term treatment toxicity and clinical resistance to drugs commonly used against E. histolytica, new drugs against amoebiasis are urgently needed. Castela texana ("chaparro amargo") is a shrub taken traditionally in teas and capsules of dry plant to treat intestinal amoebic infections. An aqueous extract was prepared and its mutagenic, genotoxic and cytotoxicity properties were evaluated in prokaryotic and eukaryotic systems. This extract was neither mutagenic when evaluated with the Ames test in Salmonella typhimurium strains TA98, TA100 and TA102, nor genotoxic in unscheduled DNA synthesis in hepatocyte cultures, even at the highest concentrations tested. In fact, C. texana extract showed antimutagenic activity on S. typhimurium strains TA98 and TA100 in the Ames test. Furthermore, it was capable of protecting liver cell cultures against unscheduled DNA synthesis induced by 2-acetylaminofluorene at a concentration of 6.77 microg/ml. A free-radical scavenging test was used in order to explore the antioxidant capacity of C. texana extract with S. typhimurium strain TA102 pretreated with norfloxacin, a free radical producer. This extract showed a free radical withdrawal effect. The effective chemoprotective activity of this extract could be due to the antioxidant capacity of the C. texana extract components. In this paper it is shown that the antiamoebic natural product, C. texana, is also antimutagenic and protects against induction of preneoplastic lesions in rat liver. These results justify further studies to extend it use to human beings.

Survival of cultured plant cells grafted into the subcutaneous tissue of rats (preliminary report).

To evaluate the survival of plant tissue in an animal environment, cultured calli from a Mexican medicinal plant (Mimosa tenuiflora Poir.) were transplanted under sterile conditions into the subcutaneous tissue of rats. Microscopic studies of grafted areas were carried out at the 30th, 60th and 120th days after transplantation. Histological evidence of plant graft survival was found in specimens of all groups. during the first month of subcutaneous grafting a moderate inflammatory reaction around the callus was observed characterized by the presence of polymorphonuclear cells and some macrophages and the formation of a fibrous capsule. Nevertheless, the plant grafts remained viable and a decrease of the inflammatory reaction around the callus was observed in the specimens during the following months. In the fourth month specimens the formation of blood vessels inside the grafted plant tissue was observed. Once removed from rats, plant tissues showed high viability according to the fluorescein test. These calli were then transferred to the original in vitro medium showing growth capacity during the following weeks. These results demonstrate, for the first time, that cultivated cells of higher plants survive in an animal environment, suggesting the possibility to utilize pharmacologically active plant transplants in animals, a technique proposed here as inter-regni transplants. Further studies are required to explore this new field of research that opens numerous questions about plant-animal cellular interaction.

Sotolone production by hairy root cultures of Trigonella foenum-graecum in airlift with mesh bioreactors.

3-Hydroxy-4,5-dimethyl-2(5H)-furanone (sotolone) and 3-amino-4,5-dimethyl-2(5H)-furanone, the postulated precursor of sotolone, were detected in hairy root cultures of Trigonella foenum-graecum (fenugreek) by GC-MS. The hairy root cultures in both conical flasks and airlift with mesh bioreactors were achieved from hypocotyl of seedling by infection with Agrobacterium rhizogenes. In flasks, the mathematical relationship between hairy root growth and conductivity was established and afterward used to evaluate the biomass evolution in bioreactor cultures due to the difficulty of obtaining direct biomass samples from the bioreactor. The GC-MS analyses of ethanolic extracts from hairy roots revealed the presence of two important compounds: sotolone (1.2% of the volatile fraction) and 3-amino-4,5-dimethyl-2(5H)-furanone (17% of the volatile fraction). These results point out that biotechnological production of sotolone in bioreactors is possible. Additionally, these hairy root cultures offer, for the first time, an excellent biological model to study the biosynthetic pathway of sotolone in fenugreek.

Construction and characterization of a plant transformation-competent BIBAC library of the black Sigatoka-resistant banana Musa acuminata cv. Tuu Gia (AA).

A plant transformation-competent binary bacterial artificial chromosome (BIBAC) library was constructed from Musa acuminata cv. Tuu Gia (AA), a black Sigatoka-resistant diploid banana. After digestion of high-molecular-weight banana DNA by HindIII, several methods of DNA size selection were tested, followed by ligation, using a vector/insert molar ratio of 4:1. The library consists of 30,700 clones stored in 80 384-well microtiter plates. The mean insert size was estimated to be 100 kb, and the frequency of inserts with internal NotI sites was 61%. The majority of insert sizes fell into the range of 100+/-20 kb, making them suitable for Agrobacterium-mediated transformation. Only 1% and 0.9% of the clones contain chloroplast and mitochondrial DNA, respectively. This is the first BIBAC library for banana, estimated to represent five times its haploid genome (600 Mbp). It was demonstrated by hybridization that the library contains typical members of resistance gene and defense gene families that can be used for transformation of disease susceptible banana cultivars for banana genetic improvement.