Disulfide Biochemistry in 2-Cys Peroxiredoxin: Requirement of Glu50 and Arg146 for the Reduction of Yeast Tsa1 by Thioredoxin

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Application of methylotrophic yeast Pichia pastoris in the field of food industry - A review

Since ancient times, the utilization of yeasts by the man has a great impact on the socio-economic development. After the advent of the technology of recombinant DNA, great advances have occurred due to the acquisition of strains of mutant yeasts in the field of applied research, and Saccharomyces cerevisiae has soon been outstanding as an interesting candidate for the expression of heterologous proteins of biotechnological interest. As the time goes by other alternative systems of expression have been shown because they have advantages over Saccharomyces cerevisiae. Among those new systems, Pichia pastoris is outstanding as methylotrophic yeast capable of growing in a culture medium containing methanol as the only source of carbon and energy. The induction of production of glycerol-3-phosphate dehydrogenase (GPD, NAD(+): oxido-redutase EC 1.1. 1.8) by Pichia pastoris was accomplished in the medium containing methanol. One of the most important key parameters in Pichia pastoris expression system is the methanol concentration. Bibliographic reviews on the Pichia pastoris production system have shown that the best culture conditions vary according to the strain used and/or kind of heterologous protein desired to be expressed. Therefore, we have sought to develop a system, involving expression of glycerol-3-phosphate dehydrogenase in the yeast Pichia pastoris, for generating sufficient quantities of the enzyme in order to asses its potential value for use in various food bioanalytical determination. Dehydrogenases have been widely used in the enzymatic assays of diverse composites of industrial interest, being enclosed among them glycerol and a number of important bioanalytical applications.

PLANT-TO-SEED TRANSMISSION of CURTOBACTERIUM FLACCUMFACIENS pv. FLACCUMACIENS IN A DRY BEAN CULTIVAR

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Fluctuations in Richness and Abundance of Social Wasps During the Dry and Wet Seasons in Three Phyto-Physiognomies at the Tropical Dry Forest of Brazil

The social wasp nests were quantified in three different plant physiognomies (forested Caatinga, shrubby Caatinga, and agricultural systems) to analyze the effect of environmental seasonality and plant physiognomy on the richness, nest abundance, and species composition of social wasps in the region of tropical dry forest of Brazil. The forested Caatinga physiognomy had the greatest richness of species (S = 16), followed by shrubby Caatinga (S 13) and by agricultural system (S = 12). The first axis of detrended correspondence analysis (DCA) explained 67.8% of the variability and shows a gradient of the fauna from agricultural system and shrubby Caatinga to forested Caatinga. In the first axis, wet season scores were much higher than those for the dry season in forested Caatinga. The second axis explained 18.7% of the variability and shows a separation of samples collected during the wet or the dry periods in shrubby Caatinga. This separation was less evident in the agricultural system. Variations in nest abundance were more intense in arbustive caatinga (45% decrease in number of active nests in the dry period), moderate in forested Caatinga (24% decrease in number of active nests in the dry period), and low in agricultural systems (8% decrease in the dry period).

Time evolution of the structure function and dynamical scaling in porous SnO2 dry gels

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Distribution of exoantigens and a 43-kDa glycoprotein (gp43) in the yeast and mycelial forms of Paracoccidioides brasiliensis

Objective. Paracoccidioides brasiliensis antigens (strain 113) were located at ultrastructural level in both yeast and mycelial forms of the fungus. The reactivity of the sera employed was analysed. Materials and methods. Immunofluorescence and ultrastructural protein A-gold immunolabelling techniques were performed using two polyclonal antisera: one against P. brasiliensis exoantigens and the other against a 43-kDa glycoprotein (gp43). Immunoblotting assays were employed to define reactivity of these antisera with somatic and metabolic antigens of both forms of the fungus. Results. The techniques employed revealed in both yeast and mycelial forms of P. brasiliensis a similar antigenic distribution. The antigens deposits were seen within the cytoplasm, and over the cell wall of the fungus. The anti-exoantigen serum recognized several bands in both forms of the fungus. The anti-gp43 serum reacted strongly with the 43-kDa fraction and weakly with few other fractions. Conclusions. Immunocytochemical techniques suggest a protein synthesis within the cytoplasm followed by excretion through the cell wall. Similar results employing both polyclonal antisera were obtained.

Location of the β-galactosidase of the yeast Kluyveromyces marxianus var. marxianus ATCC 10022

During the growth of Kluyveromyces marxianus var. marxianus ATCC 10022 on lactose, peaks of glucose, but not β-galactosidase activity, were detected in culture medium. Harvested and washed whole cells produced glucose and galactose from lactose, or ortho-nitro-phenol from the chromogenic substrate ortho-nitro-phenyl-β-D-galactopyranoside (ONPG), indicating that β-galactosidase is physically associated with cells. ONPG hydrolysis by whole cells presented a monophasic kinetics (Km 36.6 mM) in lactose exponential growth phase cells, but a biphasic kinetics (Km 0.2 and 36.6 mM) in stationary growth phase cells. Permeabilization with digitonin or disruption of cells from both growth phases led to monosite ONPG hydrolysis (Km 2.2 to 2.5 mM), indicating that β-galactosidase is not located in the periplasm. In addition, the energy inhibitors fluoride or arsenate, as well as the uncouplercarbonyl cyanide m-chlorophenylhydrazone (CCCP) prevented ONPG hydrolysis by whole cells. These findings indicate that energy coupled transmembrane transport is the rate-limiting step for intracellular ONPG cleavage. The taxonomic and physiologic implications of the exclusive intracellular location of β-galactosidase of K. marxianus var. marxianus ATCC 10022 are discussed. © 1996 Kluwer Academic Publishers.

Growth-promoting factors for yeast cells of Paracoccidioides brasiliensis

Low-density seedings of yeast cells of Paracoccidioides brasiliensis give poor growth (as assessed by plating efficiency test) on conventional mycological agar media, and therefore growth-promoting factors for this fungus were sought. Water-extracts of yeast cells of six P. brasiliensis isolates were all considerably effective in promoting the growth of low-density seedings of P. brasiliensis isolates Pb-18 and Hachisuga, but had little effect on isolate Bt-4. Horse serum, at a concentration range of 2-4%, moderately or considerably promoted the growth of these P. brasiliensis isolates. Combinations of the fungus cell extracts with horse serum were highly effective in promoting the growth of all of the fungal isolates. The fungus cell extracts showed siderophore (microbial iron carrier) activity. An iron-chelator, ethylenediaminetetraacetic acid, at a concentration of 100 μM also highly promoted the growth of the fungal isolates in the presence of horse serum, and ferric ion added to culture medium was considerably effective in the growth promotion. These results suggest that deficient utilization of external iron by the fungus cell is one of the growth-limiting processes for low-density seedings of yeast cells of P. brasiliensis on conventional mycological agar media.

During the initiation of fermentation overexpression of hexokinase PII in yeast transiently causes a similar deregulation of glycolysis as deletion of Tps1

In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (=GGS1=FDP1=BYP1=CIF1=GLC6=TSS1)-encoded trehalose-6-phosphate synthase, is essential for restriction of glucose influx into glycolysis apparently by inhibiting hexokinase activity in vivo. We show that up to 50-fold overexpression of hexokinase does not noticeably affect growth on glucose or fructose in wild-type cells. However, it causes higher levels of glucose-6-phosphate, fructose-6-phosphate and also faster accumulation of fructose-1,6-bisphosphate during the initiation of fermentation. The levels of ATP and Pi correlated inversely with the higher sugar phosphate levels. In the first minutes after glucose addition, the metabolite pattern observed was intermediate between those of the tps1Δ mutant and tile wild-type strain. Apparently, during the start-up of fermentation hexokinase is more rate-limiting in the first section of glycolysis than phosphofructokinase. We have developed a method to measure the free intracellular glucose level which is based on the simultaneous addition of D-glucose and an equal concentration of radiolabelled L-glucose. Since the latter is not transported, the free intracellular glucose level can be calculated as the difference between the total B-glucose measured (intracellular + periplasmic/extracellular) and the total L-glucose measured (periplasmic/extracellular). The intracellular glucose level rose in 5 min after addition of 100 mM-glucose to 0.5-2 mM in the wild-type strain, ± 10 mm in a hxk1Δ hxk2Δ glk1Δ and 2-3 mM in a tps1Δ strain. In the strains overexpressing hexokinase PII the level of free intracellular glucose was not reduced. Overexpression of hexokinase PII never produced a strong effect on the rate of ethanol production and glucose consumption. Our results show that overexpression of hexokinase does not cause the same phenotype as deletion of Tps1. However, it mimics it transiently during the initiation of fermentation. Afterwards, the Tps1-dependent control system is apparently able to restrict Properly up to 50-fold higher hexokinase activity.

A demonstration of dry and viscous damping of an oscillating pendulum

Damped oscillatory motion is one of the most widely studied movements in physics courses. Despite this fact, dry damped oscillatory motion is not commonly discussed in physics textbooks. In this work, we discuss the dry and viscous dampec pendulum, in a teaching experiment that can easily be performed by physics or engineering students.

Influence of the diet on in situ disappearance of dry matter, organic matter, and neutral detergent fiber of hydrolysed sugar cane bagasse

The effects of two diets based on hydrolysed sugarcane bagasse (HSB) and whole cottonseed (WCS), with or without oat hay, were analyzed for the in situ disappearance of dry matter (DM), organic matter (OM) and neutral detergent fiber (NDF) of HSB. Six mature castrated rams with a permanent T ruminai cannula were used in a complete randomized split plot design. The incubation times were 3, 6, 9, 12, 24, 48 and 72h. The diet with oat hay showed higher disappearance indexes for the NDF fraction. Furthermore, the maximum degradation of HSB constituents was reached around 48h of incubation. The diets were T1=64% hydrolyzed sugarcane + 36% whole cottonseed and T2=14% hydrolyzed sugarcane bagasse + 36% cottonseed + 50% oat hay.

Hypogaeic anti epigaeic ant (Hymenoptera: Formicidae) assemblages of atlantic costal rainforest and dry mature and secondary amazon forest in Brazil: Continuums or communities

Meat, flour and sugar baits were used on the soil surface and buried to examine species composition of the ant fauna in three separate tropical forests in Brazil, and to control for the effect of the regional faunal pool. Compositional mosaic diversities were comparable among areas, bait types and foraging strata. Mosaic diversity was independent of mean assemblage size. The number of unique species per sampling unit was correlated with mean assemblage size. Canonical correspondence analysis ordered species first by foraging substrate, second by geographic location, and third by diet. The first axis was significantly correlated with mean similarity and affinity. Mean Mahanalobis distances between centroids of groups based upon foraging strata were significantly larger than between localities, indicating local ecological pressures stronger than regional species pool constraints. As most. species foraged in only one stratum in one geographical position and were not omnivorous, the response of species to environmental gradients (continuums) showed a lower coherency with these patterns than did communities, structured around guilds based upon foraging strata and diet.

Genetic interactions of yeast eukaryotic translation initiation factor 5a (eIF5A) reveal connections to poly(A)-binding protein and protein kinase C signaling

The highly conserved eukaryotic translation initiation factor eIF5A has been proposed to have various roles in the cell, from translation to mRNA decay to nuclear protein export. To further our understanding of this essential protein, three temperature-sensitive alleles of the yeast TIF51A gene have been characterized. Two mutant eIF5A proteins contain mutations in a proline residue at the junction between the two eIFSA domains and the third, strongest allele encodes a protein with a single mutation in each domain, both of which are required for the growth defect. The stronger tif51A alleles cause defects in degradation of short-lived mRNAs, supporting a role for this protein in mRNA decay. A multicopy suppressor screen revealed six genes, the overexpression of which allows growth of a tif51A-1 strain at high temperature; these genes include PAB1, PKC1, and PKC1 regulators WSC1, WSC2, and WSC3. Further results suggest that eIFSA may also be involved in ribosomal synthesis and the WSC/PKC1 signaling pathway for cell wall integrity or related processes.

Screening for yeast with antibacterial properties from an ethanol distillery

A general screening for the expression of antibacterial activity and non-flocculating type of yeast strains from must and fermented broth of alcohol distilleries was performed. From 60 strains only Saccharomyces sp. M26 presented a inhibitory halo in Lactobacillus fermentum culture and significant reduction in the culture turbidity (71%) and specific growth rate (56%) when compared to the control. Freezing did not affect the antibacterial activity of the Saccharomyces sp. M26 extract and heating at 90°C for 20 min completely destroyed this activity. It is expected the decrease of lactic acid bacteria growth in the S. cerevisiae alcoholic fermentation should allow for better control of these bacteria in the process. © 2003 Elsevier Ltd. All rights reserved.