Post-synthesis nanostructuration of BSA-Capsaicin nanoparticles generated by sucrose excipient.

In the pharmaceutical industry nano-hydrocolloid systems frequently coalesce or present nanoparticle aggregation after a long storage periods. Besides, the lyophilization process used to dry nanoparticles (NPs) produces loss of their original properties after dispersion. In this work we evaluated the effect on morphology and physicochemical properties of different protective excipients during drying of bovine serum albumin (BSA) NPs loaded with different concentrations of capsaicin. Capsaicin concentrations of 0, 812, 1625, 2437, and 3250 µg mL-1 were used; subsequently, NPs were dried with deionized water (DW), NaCl (DN), sucrose (DS), and not dried (ND). We found that ND, DW, and DN treatments showed a negative effect on the NPs properties; while, DS reduced the aggregation and produced the formation of isolated nanoparticles at higher concentrations of capsaicin (3250 µg mL-1), improving their circular shape, morphometrical parameters, and ζ-potential. The stability of the BSA-capsaicin NPs was associated to complex capsaicin/amino acid/water, in which GLY/GLN, ALA/HIS, ARG, THR, TYR, and Iso/CYS amino acids are involved in the restructuration of capsaicin molecules into the surface of nanoparticles during the drying process. The secondary nanostructuration in the post-synthesis stage can improve the molecular stability of the particles and the capacity of entrapping hydrophobic drugs, like capsaicin.

Generation of BSA-capsaicin Nanoparticles and Their Hormesis Effect on the Rhodotorula mucilaginosa Yeast.

Capsaicin is a chemical compound found in pungent chili peppers (Capsicum spp.). In biotechnology, capsaicin has been proposed as a pathogen control; however, its low solubility in water and high instability limits its uses. The aim of this work was to study the effect of high concentrations of capsaicin on the synthesis of nanoparticles and to evaluate their inhibitory effect on the growth of Rhodotorula mucilaginosa yeast. Bovine serum albumin (BSA)-capsaicin nanoparticles were formulated at 0, 16.2, 32.5, 48.7 and 65.0 µg of capsaicin per mg of BSA. Nanoparticle properties were evaluated and they were added to cultures of R. mucilaginosa to quantify their effect on cell viability. We found that increased capsaicin levels caused several changes to the physicochemical parameters, probably due to changes in the hydrophobicity sites of the albumin during the nanostructuration. The administration of nanoparticles to cultures of R. mucilaginosa produced a maximal viability with nanoparticles at 16.2 µg/mg; on the contrary, nanoparticles at 65.0 µg/mg caused maximal cell death. R. mucilaginosa cells displayed a hormesis effect in response to the nanoparticle dose concentration. The nanoparticles showed different responses during the uptake process, probably as a consequence of the nanostructural properties of capsaicin in the BSA molecules.

Heterologous expression of an active chitin synthase from Rhizopus oryzae.

Chitin synthases are highly important enzymes in nature, where they synthesize structural components in species belonging to different eukaryotic kingdoms, including kingdom Fungi. Unfortunately, their structure and the molecular mechanism of synthesis of their microfibrilar product remain largely unknown, probably because no fungal active chitin synthases have been isolated, possibly due to their extreme hydrophobicity. In this study we have turned to the heterologous expression of the transcript from a small chitin synthase of Rhizopus oryzae (RO3G_00942, Chs1) in Escherichia coli. The enzyme was active, but accumulated mostly in inclusion bodies. High concentrations of arginine or urea solubilized the enzyme, but their dilution led to its denaturation and precipitation. Nevertheless, use of urea permitted the purification of small amounts of the enzyme. The properties of Chs1 (Km, optimum temperature and pH, effect of GlcNAc) were abnormal, probably because it lacks the hydrophobic transmembrane regions characteristic of chitin synthases. The product of the enzyme showed that, contrasting with chitin made by membrane-bound Chs's and chitosomes, was only partially in the form of short microfibrils of low crystallinity. This approach may lead to future developments to obtain active chitin synthases that permit understanding their molecular mechanism of activity, and microfibril assembly.

A novel intracellular nitrogen-fixing symbiosis made by Ustilago maydis and Bacillus spp.

We observed that the maize pathogenic fungus Ustilago maydis grew in nitrogen (N)-free media at a rate similar to that observed in media containing ammonium nitrate, suggesting that it was able to fix atmospheric N2 . Because only prokaryotic organisms have the capacity to reduce N2 , we entertained the possibility that U. maydis was associated with an intracellular bacterium. The presence of nitrogenase in the fungus was analyzed by acetylene reduction, and capacity to fix N2 by use of (15) N2 . Presence of an intracellular N2 -fixing bacterium was analyzed by PCR amplification of bacterial 16S rRNA and nifH genes, and by microscopic observations. Nitrogenase activity and (15) N incorporation into the cells proved that U. maydis fixed N2 . Light and electron microscopy, and fluorescence in situ hybridization (FISH) experiments revealed the presence of intracellular bacteria related to Bacillus pumilus, as evidenced by sequencing of the PCR-amplified fragments. These observations reveal for the first time the existence of an endosymbiotic N2 -fixing association involving a fungus and a bacterium.

Proteomic analysis of Trichoderma atroviride reveals independent roles for transcription factors BLR-1 and BLR-2 in light and darkness.

The genus Trichoderma is one of the most widely used biological control agents of plant-pathogenic fungi. The main mechanism for survival and dispersal of Trichoderma is through the production of asexual spores (conidia). The transition from filamentous growth to conidiation can be triggered by light, nutrient deprivation, and mechanical damage of the mycelium. We conducted proteomic profiling analyses of Trichoderma atroviride after a blue light pulse. The use of two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) analysis allowed us to identify 72 proteins whose expression was affected by blue light. Functional category analysis showed that the various proteins are involved in metabolism, cell rescue, and protein synthesis. We determined the relationship between mRNA levels of selected genes 30 min after a light pulse and protein expression levels at different times after the pulse and found this correlation to be very weak. The correlation was highest when protein and mRNA levels were compared for the same time point. The transcription factors BLR-1 and BLR-2 are vital to the photoconidiation process; here we demonstrate that both BLR proteins are active in darkness and affect several elements at both the transcript and protein levels. Unexpectedly, in darkness, downregulation of proteins prevailed in the Δblr-1 mutant, while upregulation of proteins predominated in the Δblr-2 mutant. Our data demonstrate that the BLR proteins play roles individually and as a complex.