Microparticles obtained by complex coacervation: influence of the type of reticulation and the drying process on the release of the core material

Microparticles obtained by complex coacervation were crosslinked with glutaraldehyde or with transglutaminase and dried using freeze drying or spray drying. Moist samples presented Encapsulation Efficiency (%EE) higher than 96%. The mean diameters ranged from 43.7 ± 3.4 to 96.4 ± 10.3 µm for moist samples, from 38.1 ± 5.36 to 65.2 ± 16.1 µm for dried samples, and from 62.5 ± 7.5 to 106.9 ± 26.1 µm for rehydrated microparticles. The integrity of the particles without crosslinking was maintained when freeze drying was used. After spray drying, only crosslinked samples were able to maintain the wall integrity. Microparticles had a round shape and in the case of dried samples rugged walls apparently without cracks were observed. Core distribution inside the particles was multinuclear and homogeneous and core release was evaluated using anhydrous ethanol. Moist particles crosslinked with glutaraldehyde at the concentration of 1.0 mM.g-1 protein (ptn), were more efficient with respect to the core retention compared to 0.1 mM.g-1 ptn or those crosslinked with transglutaminase (10 U.g-1 ptn). The drying processes had a strong influence on the core release profile reducing the amount released to all dry samples

Fusarium graminearum growth inhibition mechanism using phenolic compounds from Spirulina sp

The application of natural antifungal substances is motivated by the need for alternatives to existing methods that are not always applicable, efficient, or that do not pose risk to consumers or the environment. Furthermore, studies on the behaviour of toxigenic species in the presence of natural fungicides have enabled their safe application in the food chain In this study, Spirulina LEB-18 phenolic extract was assessed for its antifungal activity on 12 toxigenic strains of Fusarium graminearum isolated from barley and wheat. The susceptible metabolic pathways were assessed through the determination of structural compounds (glucosamine and ergosterol) and enzyme activity of the microorganisms' primary metabolism. The results indicate that phenolic extracts reduced the growth rate of the toxigenic species investigated. The IC50 was obtained by applying 3 to 8% (p/p) of phenolic compounds in relation to the culture medium. The use of this natural fungicide proved promising for the inhibition of fungal multiplication, especially in terms of the inactivation of enzymatic systems (amylase and protease) of Fusarium graminearum.

Study of the enthalpy-entropy mechanism from water sorption of orange seeds (C. sinensis cv. Brazilian) for the use of agro-industrial residues as a possible source of vegetable oil production

Orange seeds are a promising agroindustry-waste which can be implemented in the extraction and production of vegetable oil. The relationship between moisture content and water activity provides useful information for the processing and storage of this waste item. The aim of this study was to determine the mechanism of water sorption enthalpy-entropy of orange seeds (C. sinensis cv. Brazilians) according to the moisture content. Therefore, desorption isotherms were determined at five different temperature (30, 40, 50, 60, and 70 ºC) under a wide range of moisture content (0.005-0.057 kg kg-1 d.b.) and water activity (0.02-0.756). Theoretical and empirical models were used for modeling the desorption isotherms. An analytical solution of the Clausius-Clapeyron equation was proposed to compute the isosteric heat of sorption, the differential entropy, and Gibbs free energy using the Oswin model when the effect of temperature on the hygroscopic equilibrium was considered.

Ultrastructural changes of Fusobacterium nucleatum as a defense mechanism against human neutrophil peptide-1 - A Transmission Electron Microscopy (TEM) study

Aims: The aim of this work was to assess the ultrastructural changes, cellular proliferation, and the biofilm formation ability of F. nucleatum as defense mechanisms against the effect of HNP-1. Materials and methods: The type strain of F. nucleatum (ssp. nucleatum ATCC 25586) and two clinical strains (ssp. polymorphum AHN 9910 and ssp. nucleatum AHN 9508) were cultured and incubated with four different test concentrations of recombinant HNP-1 (1, 5, 10 and 20 µg/ml) and one control group (0 µg/ml). Bacterial pellets from each concentration were processed for TEM imaging. Planktonic growth was assessed and colony forming units (CFU) were measured to determine the cellular proliferation. Scrambled HNP-1 was used for confirmation. Results: TEM analyses revealed a decrease in the outer membrane surface corrugations and roughness of the strain AHN 9508 with increasing HNP-1 concentrations. In higher concentrations of HNP-1, the strain AHN 9910 showed thicker outer membranes with a number of associated rough vesicles attached to the outer surface. For ATCC 25586, the treated bacterial cells contained higher numbers of intracellular granules with increasing the peptide concentration. Planktonic growth of the two clinical strains were significantly enhanced (P<0.001) with gradually increased concentrations of HNP-1. None of the planktonic growth results of the 3 strains incubated with the scrambled HNP-1 was statistically significant. HNP-1 decreased the biofilm formation of the two clinical strains, AHN 9910 and 9508, significantly (P<0.01 and P<0.001; respectively). Conclusions: The present in vitro study demonstrates that F. nucleatum has the ability to withstand the lethal effects of HNP-1 even at concentrations simulating the diseased periodontium in vivo. The increase in planktonic growth could act as defense mechanisms of F. nucleatum against HNP-1.

Sulphuric acid scarification effects on Brachiaria brizantha, B. humidicola and Panicum maximum seed dormancy release

Most tropical forage grass species have dormant seeds, which reduce percentages in germination tests. The objective of this study was to evaluate H2SO4 scarification effects on seed dormancy releasing, through germination time (T50) and variability among germination test replicates, in 630, 94 and 82 seed samples of B. brizantha, B. humidicola and P. maximum, respectively, tested at the Central Seed Testing Laboratory, Campinas, Brazil, from 1991 to 1999. Germination tests used two 4 x 100 replicates of intact and scarified seeds (15-, 10-, 5-minute treatments, respectively). Mean germination time (T50) and variability among germination replicates were also analysed. Statistical analysis was performed by t-test paired samples for means. Scarification promoted general decreases in T50, while variability among germination test replicates was reduced in B. brizantha. Scarification increase germination in B. brizantha and P. maximum, but is deleterious in B. humidicola.

Nanoclustering augmentation : a novel mechanism of Ras activation in cancer

Proteins of the Ras family are central regulators of crucial cellular processes, such as proliferation, differentiation and apoptosis. Their importance is emphasized in cancer, in which the isoforms H-ras, N-ras and K-ras are misregulated by mutations in approximately 20 – 30 % of cases. Thus, they represent major cancer oncogenes and one of the most important targets for cancer drug development. Ras proteins are small GTPases, which cycle between the GTP-bound active and GDP-bound inactive state. Despite the tremendous research conducted in the last three decades, many fundamental properties of Ras proteins remain poorly understood. For instance, although new concepts have recently emerged, the understanding of Ras behavior in its native environment, the membrane, is still largely missing. On the membrane Ras organizes into nanoscale clusters, also called nanoclusters. They differ between isoforms, but also between activation states of Ras. It is considered that nanoclusters represent the basic Ras signaling units. Recently, it was demonstrated that on the membrane Ras adopts distinct conformations, the so-called orientations, which are dependent on the Ras activations state. The membrane-orientation of H-ras is stabilized by the helix α4 and the C-terminal hypervariable region (hvr). The novel switch III region was proposed to be involved in mediating the change between different H-ras orientations. When the regions involved in this mechanism are mutated, H-ras activity is changed by an unknown mechanism. This thesis has explained the connection between the change of Ras orientation on the membrane and Ras activity. We demonstrated that H-ras orientation mutants exhibit altered diffusion properties on the membrane, which reflect the changes in their nanoclustering. The altered nanoclustering consequently rules the activity of the mutants. Moreover, we demonstrated that specific cancer-related mutations, affecting the switch III region of different Ras isoforms, exhibit increased nanoclustering, which consequently leads to stronger Ras signaling and tumorigenicity. Thus, we have discovered nanoclustering increase as a novel mechanism of Ras activity modulation in cancer. The molecular architecture of complexes formed on the membrane upon Ras activation is another poorly understood property of Ras. The following work has provided novel details on the regulation of Ras nanoclustering by a known H-ras-GTP nanoclustering stabilizer galectin-1 (Gal-1). Our study demonstrated that Gal-1 is not able to bind Ras directly, as it was previously proposed. Instead, its effect on H-ras-GTP nanoclustering is indirect, through binding of the effector proteins. Collectively, our findings represent valuable novel insights in the behavior of Ras, which will help the future research to eventually develop new strategies to successfully target Ras in cancer.

A note on the Pazos-Simonsen mechanism and Kaldor's early research on Latin American inflation

In contrast with the inflationary finance story, inflation acceleration in Latin America has been explained as the result of the interaction of inflation dynamics and the frequency of wage adjustments. Accordingly, small inflation disturbances are connected with a shift from moderate to high inflation (or beyond to hyperinflation) though a mechanism that makes adjustment intervals in wage contracts endogenous. Rudiger Dornbusch (1986) labeled this process the "Pazos-Simonsen mechanism". In this note we summarize the basic contribution of both Felipe Pazos (1978) and Mario Henrique Simonsen (1983) and find crucial differences between their views on wage dynamics, specifically regarding the endogeneity of the time interval between wage readjustments. A remarkable affinity with Pazos's view on wage dynamics and inflation is found in an early and almost unknown essay written by Nicholas Kaldor in 1957 (inspired in his brief experience in Latin America).

Coopetition as a lead generating mechanism: design, modelling and simulation

The aim of this research is to develop a tool that could allow to organize coopetitional relationships between organizations on the basis of two-sided Internet platform. The main result of current master thesis is a detailed description of the concept of the lead generating internet platform-based coopetition. With the tools of agent-based modelling and simulation, there were obtained results that could be used as a base for suggestion that the developed concept is able to cause a positive effect on some particular industries (e.g. web-design studios market) and potentially can bring some benefits and extra profitability for most companies that operate on this particular industry. Also on the basis of the results it can be assumed that the developed instrument is also able to increase the degree of transparency of the market to which it is applied.

Does Gamma-aminobutyric acid function as a plant resistance mechanism against phytophagous insect activity? /

Gamma-aminobutyric acid (GAB A) is a ubiquitous non-protein amino acid synthesized via the decarboxylation of L-glutamate in a reaction catalyzed by the cytosolic enzyme L-glutamate decarboxylase (GAD). In animals it functions as an inhibitory neurotransmitter. In plants it accumulates rapidly in response to various stresses, but its function remains unclear. The hypothesis that GABA accumulation in leaf tissue may function as a plant resistance mechanism against phytophagous insect activity was investigated. GABA accumulation in response to mechanical stimulation, mechanical damage and insect activity was demonstrated. In wt tobacco (Nicotiana tabacum cv Samsun), mechanical stimulation or damage caused GABA to accumulate within 2 min from mean levels of 14 to 37 and 1~9 nmol g-l fresh weight (FW), respectively. In the transgenic tobacco strain CaMVGAD27c overexpressing Petunia GAD, the same treatments caused GABA to accumulate from 12 to 59 and 279 nmol g-l FW, respectively. In the transgenic tobacco strain CaMVGADilC 11 overexpressing Petunia GAD lacking an autoinhibitory domain, mechanical stimulation or damage caused GABA to accumulate from 180 to 309 and 630 nmol g-l FW, respectively. Ambulatory activity by tobacco budworm (TBW) larvae (Heliothis virescens) on leaves of CaMVGAD27c tobacco caused GABA to accumulate from 28 to 80 nmol g-l FW within 5 min. Ambulatory and leaf-rolling activity by oblique banded leaf roller (OBLR) larvae (Choristoneura rosaceana cv Harris) on wt soybean leaves (Glycine max cv Harovinton) caused GABA to accumulate from 60 to 1123 nmol g-l FW within 20 min. Increased GABA levels in leaf tissue were shown to affect phytophagous preference in TBW larvae presented with wt and transgenic tobacco leaves. When presented with leaves of Samsun wt and CaMVGAD27c plants, TBW larvae consumed more wt leaf tissue (640 ± 501 S.D. mm2 ) than transgenic leaf tissue (278 ± 338 S.D. mm2 ) nine times out of ten. When presented with leaves of Samsun wt and CaMVGAD~C11 plants, TBW larvae consumed more transgenic leaf tissue (1219 ± 1009 S.D. mm2 ) than wt leaf tissue (28 ± 31 S.D. mm2 ) ten times out of ten. These results indicate that: (1) ambulatory activity of insect larvae on leaves results in increased GABA levels, (2) transgenic tobacco leaves with increased capacity for GABA synthesis deter feeding, and (3) transgenic tobacco leaves with constitutively higher GABA levels stimulate feeding.

The investigation of the reduction mechanism of the 5a-reductase enzyme of Penicillium decumbens /

The 5a-reductase of Penicillium decumbens ATCC 10436 was used as a model for the mammalian enzyme to investigate the mechanism of reduction of testosterone to 5adihydrotestosterone . The purpose of this study was to search for specific 5a-reductase inhibitors which antagonize prostate cancer . In a whole-cell biotransformation mode, this organism reduced testosterone (1) to 5a-dihydrosteroids (8) and 5aandrostane- 3, 17-dione (9) in yields of 28% and 37% respectively. Control experiments have shown that 5aandrostane- 3, 17-dione (9) can be produced from the corresponding alcohol (8) in a subsequent reaction separate from that catalysed by the 5a-reductase enzyme . Androst-4- ene-3, 17-dione (2) is reduced to give only (9) with a recovery of 80% The stereochemistry of the reduction was determined by 500 MHz ^H NMR analysis of the products resulting from the deuterium labelled substrates. The results were obtained by an analysis of the NOE difference spectra, double-quantum filtered phase sensitive COSY 2-D spectra, and ^^c-Ir 2-D shift correlation spectra of deuterium labelled products. According to the unambiguous assignment of the signals due to H-4a and H-4Ii in 5a-dihydro steroids, the NMR data show clearly that addition of hydrogen to the 4{5)K bond has occurred in a trans manner at positions 413 and 5a. To Study the reduction mechanism of this enzyme, several substrates were prepared as following; 3-methyleneandrost-4-en- 17fi-ol(3), androst-4-en-17i5-ol(5) , androst-4-en-3ii, 17fi-diol (6) and 4, 5ii-epoxyandrostane-3, 17-dione (7) . Results suggest that this enzyme system requires an oxygen atom at the 3-position of the steroid in order to bind the substrate. Furthermore, the mechanism of this 5a-reductase may proceed via direct addition of hydrogen at the 4,5 position without involvement of a carbonyl group as an intermediate.

The mechanism of the regulation of energy distribution between photosystems 1 and 2 in the cyanobacterium Synechococcus sp. strain PCC 7002 /

Cyanobacteria are able to regulate the distribution of absorbed light energy between photo systems 1 and 2 in response to light conditions. The mechanism of this regulation (the state transition) was investigated in the marine cyanobacterium Synechococcus sp. strain PCC 7002. Three cell types were used: the wild type, psaL mutant (deletion of a photo system 1 subunit thought to be involved in photo system 1 trimerization) and the apcD mutant (a deletion of a phycobilisome subunit thought to be responsible for energy transfer to photo system 1). Evidence from 77K fluorescence emission spectroscopy, room temperature fluorescence and absorption cross-section measurements were used to determine a model of energy distribution from the phycobilisome and chlorophyll antennas in state 1 and state 2. The data confirm that in state 1 the phycobilisome is primarily attached to PS2. In state 2, a portion of the phycobilisome absorbed light energy is redistributed to photo system 1. This energy is directly transferred to photo system 1 by one of the phycobilisome terminal emitters, the product of the apcD gene, rather than via the photo system 2 chlorophyll antenna by spillover (energy transfer between the photo system 2 and photo system 1 chlorophyll antenna). The data also show that energy absorbed by the photo system 2 chlorophyll antenna is redistributed to photo system 1 in state 2. This could occur in one of two ways; by spillover or in a way analogous to higher plants where a segment of the chlorophyll antenna is dissociated from photo system 2 and becomes part of the photo system 1 antenna. The presence of energy transfer between neighbouring photo system 2 antennae was determined at both the phycobilisome and chlorophyll level, in states 1 and 2. Increases in antenna absorption cross-section with increasing reaction center closure showed that there is energy transfer (connectivity) between photosystem 2 antennas. No significant difference was shown in the amount of connectivity under these four conditions.

Investigation of the mechanism of transfer of a-tocopherol by the human a-tocopherol transfer protein (H-a-TTP) /

The human a-tocopherol transfer protein (h-a-TTP) is understood to be the entity responsible for the specific retention of a-tocopherol (a-toc) in human tissues over all other forms of vitamin E obtained from the diet. a-Tocopherol is the most biologically active form of vitamin E, and to date has been studied extensively with regard to its antioxidant properties and its role of terminating membrane lipid peroxidation chain reactions. However, information surrounding the distribution of a-tocopherol, specifically its delivery to intracellular membranes by a-TTP, is still unclear and the molecular factors influencing transfer remain elusive. To investigate the mechanism of ligand transfer by the h-a-TTP, a fluorescent analogue of a-toc has been used in the development of a fluorescence resonance energy transfer (FRET) assay. (/?)-2,5,7,8-tetramethyl-2-[9-(7-nitro-benzo[l,2,5]oxdiazol-4-ylamino)-nonyl]- chroman-6-ol (NBD-toc) has allowed for the development of the FRET-based ligand transfer assay. This ligand has been utilized in a series of experiments where changes were made to acceptor lipid membrane concentration and composition, as well as to the ionic strength and viscosity of the buffer medium. Such changes have yielded evidence supporting a collisional mechanism of ligand transfer by a-TTP, and have brought to light a new line of inquiry pertaining to the nature of the forces governing the collisional transfer interaction. Through elucidation of the transfer mechanism type, a deeper understanding of the transfer event and the in vivo fate of a-tocopherol have been obtained. Furthermore, the results presented here allow for a deeper investigation of the forces controlling the collisional protein-membrane interaction and their effect on the transfer of a-toc to membranes. Future investigation in this direction will raise the possibility of a complete understanding of the molecular events surrounding the distribution of a-toc within the cell and to the body's tissues.

Localization of the mechanism of pH-dependent non- photochemical fluorescence quenching in thylakoid membranes

Higher plants have evolved a well-conserved set of photoprotective mechanisms, collectively designated Non-Photochemical Quenching of chlorophyll fluorescence (qN), to deal with the inhibitory absorption of excess light energy by the photosystems. Their main contribution originates from safe thermal deactivation of excited states promoted by a highly-energized thylakoid membrane, detected via lumen acidification. The precise origins of this energy- or LlpH-dependent quenching (qE), arising from either decreased energy transfer efficiency in PSII antennae (~ Young & Frank, 1996; Gilmore & Yamamoto, 1992; Ruban et aI., 1992), from alternative electron transfer pathways in PSII reaction centres (~ Schreiber & Neubauer, 1990; Thompson &Brudvig, 1988; Klimov et aI., 1977), or from both (Wagner et aI., 1996; Walters & Horton, 1993), are a source of considerable controversy. In this study, the origins of qE were investigated in spinach thylakoids using a combination of fluorescence spectroscopic techniques: Pulse Amplitude Modulated (PAM) fluorimetry, pump-probe fluorimetry for the measurement of PSII absorption crosssections, and picosecond fluorescence decay curves fit to a kinetic model for PSII. Quenching by qE (,..,600/0 of maximal fluorescence, Fm) was light-induced in circulating samples and the resulting pH gradient maintained during a dark delay by the lumenacidifying capabilities of thylakoid membrane H+ ATPases. Results for qE were compared to those for the addition of a known antenna quencher, 5-hydroxy-1,4naphthoquinone (5-0H-NQ), titrated to achieve the same degree of Fm quenching as for qE. Quenching of the minimal fluorescence yield, F0' was clear (8 to 130/0) during formation of qE, indicative of classical antenna quenching (Butler, 1984), although the degree was significantly less than that achieved by addition of 5-0H-NQ. Although qE induction resulted in an overall increase in absorption cross-section, unlike the decrease expected for antenna quenchers like the quinone, a larger increase in crosssection was observed when qE induction was attempted in thylakoids with collapsed pH gradients (uncoupled by nigericin), in the absence of xanthophyll cycle operation (inhibited by DTT), or in the absence of quenching (LlpH not maintained in the dark due to omission of ATP). Fluorescence decay curves exhibited a similar disparity between qE-quenched and 5-0H-NQ-quenched thylakoids, although both sets showed accelerated kinetics in the fastest decay components at both F0 and Fm. In addition, the kinetics of dark-adapted thylakoids were nearly identical to those in qEquenched samples at F0' both accelerated in comparison with thylakoids in which the redox poise of the Oxygen-Evolving Complex was randomized by exposure to low levels of background light (which allowed appropriate comparison with F0 yields from quenched samples). When modelled with the Reversible Radical Pair model for PSII (Schatz et aI., 1988), quinone quenching could be sufficiently described by increasing only the rate constant for decay in the antenna (as in Vasil'ev et aI., 1998), whereas modelling of data from qE-quenched thylakoids required changes in both the antenna rate constant and in rate constants for the reaction centre. The clear differences between qE and 5-0H-NQ quenching demonstrated that qE could not have its origins in the antenna alone, but is rather accompanied by reaction centre quenching. Defined mechanisms of reaction centre quenching are discussed, also in relation to the observed post-quenching depression in Fm associated with photoinhibition.

The 21-dehydroxylation of corticosteroids: evidence for an enol intermediate and the hydroxylation of steroids by fungal cyto-chrome P450: evidence for a stepwise mechanism

Two enzyme mechanisms were examined: the 21-dehydroxylation of corticosteroids by the anaerobe Eubacterium l en tum, and the hydroxylation of steroids by fungal cytochrome P450. Deuterium labelling techniques were used to study the enzymic dehydroxylation. Corticosteroids doubly labelled (2H) at the C-21 position were incubated with a culture of Eubacterium lentum. It was found that t he enzymic dehydroxylation proceeded with the loss of one 2H f rom C-21 per molecule of substrate. The kinetic isotope ef fect f or the reaction was found to be k~kD = 2. 28. These results suggest that enzyme/substr ate binding in this case may proceed via t he enol form of the substrate. Also , it appears that this binding is, at least in part, the rate determining step of t he reaction. The hydroxylation of steroids by fungal cytochrome P450 was examined by means of a product study. Steroids with a double bond at the A8 (9), ~( lO ), or ~ (ll) position were synthesized. These steroids were then incubated with fungal strains known to use a cytochrome P450 monooxygenase to hydroxylate at positions allylic to these doubl e bonds. The products formed in these incubations indicated that the double bonds had migrated during allylic hydroxylat ion. This suggests that a carbon centred radical or ion may be an intermediate i n the cytochrome P450 cat alytic cycle.

The kinetics and mechanism of the thermal decomposition of sec-Butyl peroxide in liquid phase /|nby Sandor Szilagyi. -- 260 St. Catharines, Ont. : [s. n.],

Re~tes artd pJ~oducts of tllerma]. d,ecom.position of sec-butyl peroxide at 110 - 150°C i.n four solvents h,ave been determined. The d,ecompos i tion vJas sb.o\'\Tn to be tlnlmolecl.llar wi tho energies of activation in toluene, benzene, and cyclohexane of 36 .7-+ 1.0, 33.2 +- 1..0, 33.t~) +.. 1.0 I'(:cal/mol respectively. The activation energy of thermal decomposition for the d,et.1terated peroxide was found to be 37.2 4:- 1.0 KC8:1/1TIol in toluene. A.bo1J.t 70 - 80/~ ol~ tJJ.e' pl~od.1..1CtS could, be explained by kn01rJ11 reactions of free allcoxy raclicals J and very littJ...e, i.f allY, disPl"Opox~tiol'lation of tll10 sec-butoxy radica.ls in t116 solvent cage could be detected. The oth,er 20 - 30% of the peroxide yielded H2 and metb.:'ll etb..yl 1{etol1e. Tl1.e yield. o:f H2 "'lIas unafJ:'ected by the nature or the viscosity of the solvent, but H2 was not formed when s-t1U202 lrJaS phctolyzed. in tolttene at 35°C nor 'tl!Jrl.en the peroxide 1;'JaS tl1.ermally o..ecoJnposed. in the gas p11ase. ~pC-Dideutero-~-butYlperoxide was prepared and decomposed in toluene at 110 - 150°C. The yield of D2 was about ·•e1ne same 248 the yield. of I{2 from s-Bu202, bU.t th.e rate of decomposition (at 135°C) 1iJas only 1/1.55 as fast. Ivlecl1.anisms fOl') J:1ydrogen produ.ction are discussed, but none satisfactorily explains all the evidence.