Effects of selective inhibition of nNOS and iNOS on neuropathic pain in rats.

Various animal models have been employed to understand the pathogenic mechanism of neuropathic pain. Nitric oxide (NO) is an important molecule in nociceptive transmission and is involved in neuropathic pain. However, its mechanistic actions remain unclear. The aim of this study was to better understand the involvement of neuronal and inducible isoforms of nitric oxide synthase (nNOS and iNOS) in neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve in rats. We evaluated pain sensitivity (mechanical withdrawal thresholds using Randall and Selitto, and von Frey tests, and thermal withdrawal thresholds using Hargreaves test) prior to CCI surgery, 14 days post CCI and after intrathecal injections of selective nNOS or iNOS inhibitors. We also evaluated the distribution of NOS isozymes in the spinal cord and dorsal root ganglia (DRG) by immunohistochemistry, synthesis of iNOS and nNOS by Western blot, and NO production using fluorescent probe DAF-2 DA (DA). Our results showed higher number of nNOS and iNOS-positive neurons in the spinal cord and DRG of CCI compared to sham rats, and their reduction in CCI rats after treatment with selective inhibitors compared to non-treated groups. Western blot results also indicated reduced expression of nNOS and iNOS after treatment with selective inhibitors. Furthermore, both inhibitors reduced CCI-evoked mechanical and thermal withdrawal thresholds but only nNOS inhibitor was able to efficiently lower mechanical withdrawal thresholds using von Frey test. In addition, we observed higher NO production in the spinal cord and DRG of injured rats compared to control group. Our study innovatively shows that nNOS may strongly modulate nociceptive transmission in rats with neuropathic pain, while iNOS may partially participate in the development of nociceptive responses. Thus, drugs targeting nNOS for neuropathic pain may represent a potential therapeutic strategy.

Intensive liquid feeding of dairy calves with a medium crude protein milk replacer: Effects on performance, rumen, and blood parameters.

The objective of this study was to evaluate the effects of different liquid-feeding systems using a medium crude protein milk replacer on performance, rumen, and blood parameters. Thirty newborn Holstein calves were blocked according to birth weight and date of birth, and randomly distributed to different liquid-feeding systems: conventional (4 L/d), intensive (8 L/d), or step-up/step-down (wk 1, 4 L/d; wk 2 to 6, 8 L/d; wk 7 and 8, 4 L/d). The commercial milk replacer (12.5% solids, 20.2% crude protein, 15.6% fat) was fed twice daily (0700 and 1700 h) until calves were weaned, at 8 wk of age. Calves were individually housed in wood hutches, with free access to water and starter concentrate, and to hay only after weaning. They were followed through 10 wk of age. Milk replacer and starter intake were inversely affected by feeding system. After weaning, starter intake and hay intake were similar among feeding systems. Total dry matter intake was higher during the liquid-feeding period for calves on the intensive system compared to calves on the conventional system, but conventional feeding resulted in the highest dry matter intake after weaning. Feed efficiency was similar among feeding systems before and after weaning. Average body weight and daily gain were not affected by feeding system before or after weaning. During liquid feeding, diarrhea occurrence was lower for calves on the conventional system; however, when calves on the step-up/step-down system were fed lower volumes of liquid feed, diarrhea occurrence was similar to that of calves on the conventional system. Plasma concentrations of ß-hydroxybutyrate were higher for calves on the conventional system, reflecting starter intake. Rumen pH, short-chain fatty acids, and N-NH3 were not affected by feeding system. Feeding higher volumes of milk replacer with a medium crude protein content had no beneficial effect on the performance of calves up to 10 wk of age.

Performance and Metabolism of Calves Fed Starter Feed Containing Sugarcane Molasses or Glucose Syrup as a Replacement for Corn.

The aim of this study was to evaluate the effect of replacing corn grain for sugar cane molasses (MO) or glucose syrup (GS) in the starter concentrate on performance and metabolism of dairy calves. Thirty-six individually housed Holstein male calves were blocked according to weight and date of birth and assigned to one of the starter feed treatments, during an 8 week study: i) starter containing 65% corn with no MO or GS (0MO); ii) starter containing 60% corn and 5% MO (5MO); iii) starter containing 55% corn and 10% MO (10MO); and iv) starter containing 60% corn and 5% GS (5GS). Animals received 4 L of milk replacer daily (20 crude protein, 16 ether extract, 12.5% solids), divided in two meals (0700 and 1700 h). Starter and water were provided ad libitum. Starter intake and fecal score were monitored daily until animals were eight weeks old. Body weight and measurements (withers height, hip width and heart girth) were measured weekly before the morning feeding. From the second week of age, blood samples were collected weekly, 2 h after the morning feeding, for glucose, ß-hydroxybutyrate and lactate determination. Ruminal fluid was collected at 4, 6, and 8 weeks of age using an oro-ruminal probe and a suction pump for determination of pH and short-chain fatty acids (SCFA). At the end of the eighth week, animals were harvested to evaluate development of the proximal digestive tract. The composition of the starter did not affect (p>0.05) concentrate intake, weight gain, fecal score, blood parameters, and rumen development. However, treatment 5MO showed higher (p<0.05) total concentration of SCFAs, acetate and propionate than 0MO, and these treatments did not differ from 10MO and 5GS (p>0.05). Thus, it can be concluded that the replacement of corn by 5% or 10% sugar cane molasses or 5% GS on starter concentrate did not impact performance, however it has some positive effects on rumen fermentation which may be beneficial for calves with a developing rumen.

Essential oils for dairy calves: effects on performance, scours, rumen fermentation and intestinal fauna.

UNLABELLED: The first cause of death of dairy calves is often diarrhea which is mainly caused by pathogenic bacteria, which can result in excessive use of antibiotics. However, facing the increase concern by the industry and consumers, the use of antibiotics not only to control pathogens, but also to manipulate growth, has become a challenge. Alternative additives, such essential oils, have the potential to decrease antibiotic use, without reducing performance or increasing mortality of dairy calves. The objective of this study was to evaluate the use of a commercial blend of essential oils, incorporated into the calf starter and/or milk replacer to monitor the effect on overall calf performance, fecal scores and rumen fermentation parameters. A total of 30 Holstein calves received 6 l/day of a liquid diet, consisting of a commercial milk replacer containing 20% CP : 15% fat (EE). Calves had free choice access to water and calf starter. Weaning occurred at week 8, and calves were followed until the 10th week of age. Calves were assigned to one of the three treatment groups in a randomized block design. TREATMENTS: (1) control without essential oils supplementation (C); (2) essential oils blend in the milk replacer at 400 mg/kg (MR) and (3) essential oils blend in the milk replacer (200 mg/kg) and starter feed (200 mg/kg) (MRS). From the 2nd week, calves were weighed and body measurements were taken, while concentrate intake and fecal scores were monitored daily. Blood samples were drawn weekly for determination of glucose and ß-hydroxybutyrate. Fecal samples were collected weekly and analyzed for lactic acid bacteria and Enterobacteria; and ruminal fluid for determination of pH, short chain fatty acids, ammonia-N and counts of amylolytic and cellulolytic bacteria, and protozoa. Performance, fecal scores and intestines microorganisms were not affected by the essential oils supplementation. Ruminal and blood parameters were also not affected, with the exception the rumen ammonia-N concentration, with higher values when essential oils were supplemented in a combination of milk replacer and starter feed. Most of the evaluated parameters were affected by age of calves, mainly as a response to the increase in concentrate intake as animals' aged. Essential oils are promising substitutes for antibiotics. However, the dose and routes of administration deserve further studies, allowing a better animal performance and health to be achieved.

Sensory analysis and species-specific PCR detect bovine milk adulteration of frescal (fresh) goat cheese.

The Brazilian market for dairy products made from goat milk is increasing despite the seasonality of production and naturally small milk production per animal, factors that result in high-priced products and encourage fraud. In Brazil, no official analytical method exists for detecting adulteration of goat dairy products with cow milk. The aim of this study was to design a strategy to investigate the adulteration of frescal (fresh) goat cheeses available in the Rio de Janeiro retail market, combining analysis of cheese composition and the perception of adulteration by consumers. Commercial goat cheeses were tested by using a duplex PCR assay previously designed to authenticate cheeses, by targeting the mitochondrial 12S ribosomal RNA genes of both species simultaneously. The PCR test was able to detect 0.5% (vol/vol) cow milk added during goat cheese formulation. The analysis of 20 locally produced goat cheeses (20 lots of 4 brands) showed that all were adulterated with cow milk, even though the labels did not indicate the addition of cow milk. To estimate the ability of consumers to perceive the fraudulent addition of cow milk, a triangle test was performed, in which cheeses formulated with several different proportions of goat and cow milk were offered to 102 regular consumers of cheese. Detection threshold analysis indicated that almost half of the consumers were able to perceive adulteration at 10% (vol/vol) cow milk. Effective actions must be implemented to regulate the market for goat dairy products in Brazil, considering the rights and choices of consumers with respect to their particular requirements for diet and health, preference, and cost.

Desempenho e parâmetros sanguíneos de bezerros leiteiros que receberam sucedâneo lácteo ou silagem de colostro / Performance and plasma metabolites of dairy calves fed a milk replacer or colostrum silage

O objetivo deste estudo foi avaliar o desempenho e os parâmetros sanguíneos de bezerros que consumiram colostro bovino fermentado sob condições anaeróbias. Após o nascimento, 18 bezerros da raça Holandês foram alojados em abrigos individuais e passaram a receber 4L da dieta líquida, sucedâneo lácteo ou silagem de colostro, divididos em duas refeições. O consumo de concentrado inicial e o escore fecal foram registrados diariamente, enquanto a pesagem e as colheitas de amostras de sangue para a determinação das concentrações plasmáticas de glicose, nitrogênio ureico, ácidos graxos livres, β-hidroxibutirato e proteínas totais séricas foram realizadas semanalmente. Os animais alimentados com silagem de colostro apresentaram menores consumo de concentrado, ganho de peso diário e peso vivo. Todos os parâmetros sanguíneos avaliados foram afetados pelos tratamentos, exceto a concentração plasmática de proteínas totais. O escore fecal foi afetado pelos tratamentos durante a segunda semana de vida, com animais alimentados com silagem de colostro apresentando fezes anormais e secas. O fornecimento de silagem de colostro como dieta líquida exclusiva não resultou em desempenho animal adequado, não sendo uma boa alternativa de substituto de leite.

The aim of this study was to evaluate the performance and plasma metabolites of calves fed colostrum fermented under anaerobic conditions as an exclusive liquid feed during the whole milk-feeding period. After birth, eighteen Holstein male calves were housed in individual hutches and fed four liters of liquid diet, milk replacer or colostrum silage, divided into two meals. The starter feed intake and fecal scores were recorded daily, and body weight and blood samples for the determination of plasma glucose, urea nitrogen, free fatty acids, β-hydroxybutyrate and serum total protein were taken weekly. Animals fed colostrum silage had lower intake of starter feed during the experimental period. Significant effects were also observed for average daily gain and body weight. All blood parameters measured were affected by the treatments, except the total protein plasma concentration. The fecal score was affected by treatments during the second week of life, with animals fed colostrum silage presenting abnormal and very dry feces. Feeding colostrum silage as exclusive liquid diet during the whole milk-feeding period resulted in inadequate animal performance, being considered a bad alternative as milk replacer.

Desempenho e parâmetros sanguíneos de bezerros leiteiros que receberam sucedâneo lácteo ou silagem de colostro / Performance and plasma metabolites of dairy calves fed a milk replacer or colostrum silage

O objetivo deste estudo foi avaliar o desempenho e os parâmetros sanguíneos de bezerros que consumiram colostro bovino fermentado sob condições anaeróbias. Após o nascimento, 18 bezerros da raça Holandês foram alojados em abrigos individuais e passaram a receber 4L da dieta líquida, sucedâneo lácteo ou silagem de colostro, divididos em duas refeições. O consumo de concentrado inicial e o escore fecal foram registrados diariamente, enquanto a pesagem e as colheitas de amostras de sangue para a determinação das concentrações plasmáticas de glicose, nitrogênio ureico, ácidos graxos livres, β-hidroxibutirato e proteínas totais séricas foram realizadas semanalmente. Os animais alimentados com silagem de colostro apresentaram menores consumo de concentrado, ganho de peso diário e peso vivo. Todos os parâmetros sanguíneos avaliados foram afetados pelos tratamentos, exceto a concentração plasmática de proteínas totais. O escore fecal foi afetado pelos tratamentos durante a segunda semana de vida, com animais alimentados com silagem de colostro apresentando fezes anormais e secas. O fornecimento de silagem de colostro como dieta líquida exclusiva não resultou em desempenho animal adequado, não sendo uma boa alternativa de substituto de leite.(AU)

The aim of this study was to evaluate the performance and plasma metabolites of calves fed colostrum fermented under anaerobic conditions as an exclusive liquid feed during the whole milk-feeding period. After birth, eighteen Holstein male calves were housed in individual hutches and fed four liters of liquid diet, milk replacer or colostrum silage, divided into two meals. The starter feed intake and fecal scores were recorded daily, and body weight and blood samples for the determination of plasma glucose, urea nitrogen, free fatty acids, β-hydroxybutyrate and serum total protein were taken weekly. Animals fed colostrum silage had lower intake of starter feed during the experimental period. Significant effects were also observed for average daily gain and body weight. All blood parameters measured were affected by the treatments, except the total protein plasma concentration. The fecal score was affected by treatments during the second week of life, with animals fed colostrum silage presenting abnormal and very dry feces. Feeding colostrum silage as exclusive liquid diet during the whole milk-feeding period resulted in inadequate animal performance, being considered a bad alternative as milk replacer.(AU)

Microbiological and chemical characteristics of Brazilian kefir during fermentation and storage processes.

The microbial community composition and chemical characteristics of a Brazilian milk kefir sample produced during its manufacturing and refrigerated storage were investigated by culture-dependent and -independent methods and HPLC. Lactococcus lactis ssp. cremoris and ssp. lactis, Leuconostoc mesenteroides, Acetobacter lovaniensis, and Saccharomyces cerevisiae were isolated, whereas the detected bands on denaturing gel gradient electrophoresis corresponded to Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus parakefiri, and S. cerevisiae. After fermentation, lactic acid bacteria were present at levels of 10 log units, whereas acetic acid bacteria and yeast were present at levels of 7.8 and 6 log units, respectively. The lactic acid bacteria and yeast counts remained constant, whereas acetic acid bacteria counts decreased to 7.2 log units during storage. From fermentation to final storage, the pH, lactose content and citric acid of the kefir beverage decreased, followed by an increase in the concentrations of glucose, galactose, ethanol, and lactic, acetic, butyric, and propionic acids. These microbiological and chemical characteristics contribute to the unique taste and aroma of kefir. This research may serve as a basis for the future industrial production of this beverage in Brazil.

Assessment of the microbial diversity of Brazilian kefir grains by PCR-DGGE and pyrosequencing analysis.

The microbial diversity and community structure of three different kefir grains from different parts of Brazil were examined via the combination of two culture-independent methods: PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing. PCR-DGGE showed Lactobacillus kefiranofaciens and Lactobacillus kefiri to be the major bacterial populations in all three grains. The yeast community was dominated by Saccharomyces cerevisiae. Pyrosequencing produced a total of 14,314 partial 16S rDNA sequence reads from the three grains. Sequence analysis grouped the reads into three phyla, of which Firmicutes was dominant. Members of the genus Lactobacillus were the most abundant operational taxonomic units (OTUs) in all samples, accounting for up to 96% of the sequences. OTUs belonging to other lactic and acetic acid bacteria genera, such as Lactococcus, Leuconostoc, Streptococcus and Acetobacter, were also identified at low levels. Two of the grains showed identical DGGE profiles and a similar number of OTUs, while the third sample showed the highest diversity by both techniques. Pyrosequencing allowed the identification of bacteria that were present in small numbers and rarely associated with the microbial community of this complex ecosystem.

Prevalência de tuberculose bovina em animais e rebanhos abatidos em 2009 no estado de Mato Grosso, Brasil / Prevalence of bovine tuberculosis in herds and animals slaughtered in 2009 in the State of Mato Grosso, Brazil

Estimou-se a prevalência de tuberculose em bovinos, e em seus respectivos rebanhos, abatidos em 2009 no estado de Mato Grosso, utilizando como diagnóstico confirmatório o exame bacteriológico e o molecular a partir de fragmentos de tecidos lesionados. Nos sete abatedouros selecionados, detentores de serviço de inspeção federal (SIF), foram inspecionados 41.193 bovinos, sadios ao exame ante mortem, procedentes de 492 rebanhos originários de 85 (60%) municípios mato-grossenses. Um total de 198 carcaças apresentaram lesões suspeitas. Apenas três carcaças (3/198) apresentaram lesões confirmadas como tuberculosas pelos diagnósticos laboratoriais. A prevalência aparente de tuberculose bovina em animais e rebanhos abatidos no estado de Mato Grosso foi de 0,007% [IC 95% = -0,001%; 0,016%] e 0,61% [IC 95% = -0,08%; 1,30%], respectivamente. O estado do Mato Grosso possui, naturalmente, um status sanitário considerado de baixa prevalência.

The prevalence of bovine tuberculosis in cattle, and its herds, slaughtered in 2009 in the state of Mato Grosso, Brazil, was estimated using bacteriological analysis and molecular test, from fragments of injured tissues as well as direct DNA templates. 41,193 cattle, which appeared healthy in the ante mortem examination, from seven selected slaughterhouses, under Brazilian federal inspection services (SIF), were inspected. The animals were from 492 herds located in 85 (60%) different cities of Mato Grosso. A total of the 198 carcasses had suspicious lesions. Three carcasses (3/198) had lesions that were found to be tuberculous in laboratory diagnosis. The apparent prevalence of bovine tuberculosis in the total number of animals and in herds slaughtered in Mato Grosso was 0.007% [IC 95% = -0.001%; 0.016%] and 0.61% [IC 95% = -0.08%; 1.30%], respectively. The sanitation status demonstrated in Mato Grosso indicates the progress in this state toward the eradication of bovine tuberculosis.

Prevalência de tuberculose bovina em animais e rebanhos abatidos em 2009 no estado de Mato Grosso, Brasil / Prevalence of bovine tuberculosis in herds and animals slaughtered in 2009 in the State of Mato Grosso, Brazil

Estimou-se a prevalência de tuberculose em bovinos, e em seus respectivos rebanhos, abatidos em 2009 no estado de Mato Grosso, utilizando como diagnóstico confirmatório o exame bacteriológico e o molecular a partir de fragmentos de tecidos lesionados. Nos sete abatedouros selecionados, detentores de serviço de inspeção federal (SIF), foram inspecionados 41.193 bovinos, sadios ao exame ante mortem, procedentes de 492 rebanhos originários de 85 (60%) municípios mato-grossenses. Um total de 198 carcaças apresentaram lesões suspeitas. Apenas três carcaças (3/198) apresentaram lesões confirmadas como tuberculosas pelos diagnósticos laboratoriais. A prevalência aparente de tuberculose bovina em animais e rebanhos abatidos no estado de Mato Grosso foi de 0,007% [IC 95% = -0,001%; 0,016%] e 0,61% [IC 95% = -0,08%; 1,30%], respectivamente. O estado do Mato Grosso possui, naturalmente, um status sanitário considerado de baixa prevalência.(AU)

The prevalence of bovine tuberculosis in cattle, and its herds, slaughtered in 2009 in the state of Mato Grosso, Brazil, was estimated using bacteriological analysis and molecular test, from fragments of injured tissues as well as direct DNA templates. 41,193 cattle, which appeared healthy in the ante mortem examination, from seven selected slaughterhouses, under Brazilian federal inspection services (SIF), were inspected. The animals were from 492 herds located in 85 (60%) different cities of Mato Grosso. A total of the 198 carcasses had suspicious lesions. Three carcasses (3/198) had lesions that were found to be tuberculous in laboratory diagnosis. The apparent prevalence of bovine tuberculosis in the total number of animals and in herds slaughtered in Mato Grosso was 0.007% [IC 95% = -0.001%; 0.016%] and 0.61% [IC 95% = -0.08%; 1.30%], respectively. The sanitation status demonstrated in Mato Grosso indicates the progress in this state toward the eradication of bovine tuberculosis.(AU)

Identification of the divergent calmodulin binding motif in yeast Ssb1/Hsp75 protein and in other HSP70 family members.

Yeast soluble proteins were fractionated by calmodulin-agarose affinity chromatography and the Ca2+/calmodulin-binding proteins were analyzed by SDS-PAGE. One prominent protein of 66 kDa was excised from the gel, digested with trypsin and the masses of the resultant fragments were determined by MALDI/MS. Twenty-one of 38 monoisotopic peptide masses obtained after tryptic digestion were matched to the heat shock protein Ssb1/Hsp75, covering 37% of its sequence. Computational analysis of the primary structure of Ssb1/Hsp75 identified a unique potential amphipathic alpha-helix in its N-terminal ATPase domain with features of target regions for Ca2+/calmodulin binding. This region, which shares 89% similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, is conserved in near half of the 113 members of the HSP70 family investigated, from yeast to plant and animals. Based on the sequence of this region, phylogenetic analysis grouped the HSP70s in three distinct branches. Two of them comprise the non-calmodulin binding Hsp70s BIP/GR78, a subfamily of eukaryotic HSP70 localized in the endoplasmic reticulum, and DnaK, a subfamily of prokaryotic HSP70. A third heterogeneous group is formed by eukaryotic cytosolic HSP70s containing the new calmodulin-binding motif and other cytosolic HSP70s whose sequences do not conform to those conserved motif, indicating that not all eukaryotic cytosolic Hsp70s are target for calmodulin regulation. Furthermore, the calmodulin-binding domain found in eukaryotic HSP70s is also the target for binding of Bag-1 - an enhancer of ADP/ATP exchange activity of Hsp70s. A model in which calmodulin displaces Bag-1 and modulates Ssb1/Hsp75 chaperone activity is discussed.

Identification of the divergent calmodulin binding motif in yeast Ssb1/Hsp75 protein and in other HSP70 family members

Yeast soluble proteins were fractionated by calmodulin-agarose affinity chromatography and the Ca2+/calmodulin-binding proteins were analyzed by SDS-PAGE. One prominent protein of 66 kDa was excised from the gel, digested with trypsin and the masses of the resultant fragments were determined by MALDI/MS. Twenty-one of 38 monoisotopic peptide masses obtained after tryptic digestion were matched to the heat shock protein Ssb1/Hsp75, covering 37 percent of its sequence. Computational analysis of the primary structure of Ssb1/Hsp75 identified a unique potential amphipathic alpha-helix in its N-terminal ATPase domain with features of target regions for Ca2+/calmodulin binding. This region, which shares 89 percent similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, is conserved in near half of the 113 members of the HSP70 family investigated, from yeast to plant and animals. Based on the sequence of this region, phylogenetic analysis grouped the HSP70s in three distinct branches. Two of them comprise the non-calmodulin binding Hsp70s BIP/GR78, a subfamily of eukaryotic HSP70 localized in the endoplasmic reticulum, and DnaK, a subfamily of prokaryotic HSP70. A third heterogeneous group is formed by eukaryotic cytosolic HSP70s containing the new calmodulin-binding motif and other cytosolic HSP70s whose sequences do not conform to those conserved motif, indicating that not all eukaryotic cytosolic Hsp70s are target for calmodulin regulation. Furthermore, the calmodulin-binding domain found in eukaryotic HSP70s is also the target for binding of Bag-1 - an enhancer of ADP/ATP exchange activity of Hsp70s. A model in which calmodulin displaces Bag-1 and modulates Ssb1/Hsp75 chaperone activity is discussed.

Shared control of maltose and trehalose utilization in Candida utilis.

Trehalose biosynthesis and its hydrolysis have been extensively studied in yeast, but few reports have addressed the catabolism of exogenously supplied trehalose. Here we report the catabolism of exogenous trehalose by Candida utilis. In contrast to the biphasic growth in glucose, the growth of C. utilis in a mineral medium with trehalose as the sole carbon and energy source is aerobic and exhibits the Kluyver effect. Trehalose is transported into the cell by an inducible trehalose transporter (K M of 8 mM and V MAX of 1.8 mol trehalose min-1 mg cell (dry weight)-1. The activity of the trehalose transporter is high in cells growing in media containing trehalose or maltose and very low or absent during the growth in glucose or glycerol. Similarly, total trehalase activity was increased from about 1.0 mU/mg protein in cells growing in glucose to 39.0 and 56.2 mU/mg protein in cells growing in maltose and trehalose, respectively. Acidic and neutral trehalase activities increased during the growth in trehalose, with neutral trehalase contributing to about 70% of the total activity. In addition to the increased activities of the trehalose transporter and trehalases, growth in trehalose promoted the increase in the activity of alpha-glucosidase and the maltose transporter. These results clearly indicate that maltose and trehalose promote the increase of the enzymatic activities necessary to their catabolism but are also able to stimulate each other's catabolism, as reported to occur in Escherichia coli. We show here for the first time that trehalose induces the catabolism of maltose in yeast.

Shared control of maltose and trehalose utilization in Candida utilis

Trehalose biosynthesis and its hydrolysis have been extensively studied in yeast, but few reports have addressed the catabolism of exogenously supplied trehalose. Here we report the catabolism of exogenous trehalose by Candida utilis. In contrast to the biphasic growth in glucose, the growth of C. utilis in a mineral medium with trehalose as the sole carbon and energy source is aerobic and exhibits the Kluyver effect. Trehalose is transported into the cell by an inducible trehalose transporter (K M of 8 mM and V MAX of 1.8 æmol trehalose min-1 mg cell (dry weight)-1. The activity of the trehalose transporter is high in cells growing in media containing trehalose or maltose and very low or absent during the growth in glucose or glycerol. Similarly, total trehalase activity was increased from about 1.0 mU/mg protein in cells growing in glucose to 39.0 and 56.2 mU/mg protein in cells growing in maltose and trehalose, respectively. Acidic and neutral trehalase activities increased during the growth in trehalose, with neutral trehalase contributing to about 70 percent of the total activity. In addition to the increased activities of the trehalose transporter and trehalases, growth in trehalose promoted the increase in the activity of alpha-glucosidase and the maltose transporter. These results clearly indicate that maltose and trehalose promote the increase of the enzymatic activities necessary to their catabolism but are also able to stimulate each other's catabolism, as reported to occur in Escherichia coli. We show here for the first time that trehalose induces the catabolism of maltose in yeast

Evidence for a modulation of neutral trehalase activity by Ca2+ and cAMP signaling pathways in Saccharomyces cerevisiae

Saccharomyces cerevisiae neutral trehalase (encoded by NTH1) is regulated by cAMP-dependent protein kinase (PKA) and by an endogenous modulator protein. A yeast strain with knockouts of CMK1 and CMK2 genes (cmk1cmk2) and its isogenic control (CMK1CMK2) were used to investigate the role of CaM kinase II in the in vitro activation of neutral trehalase during growth on glucose. In the exponential growth phase, cmk1cmk2 cells exhibited basal trehalase activity and an activation ratio by PKA very similar to that found in CMK1CMK2 cells. At diauxie, even though both cells presented comparable basal trehalase activities, cmk1cmk2 cells showed reduced activation by PKA and lower total trehalase activity when compared to CMK1CMK2 cells. To determine if CaM kinase II regulates NTH1 expression or is involved in post-translational modulation of neutral trehalase activity, NTH1 promoter activity was evaluated using an NTH1-lacZ reporter gene. Similar ß-galactosidase activities were found for CMK1CMK2 and cmk1cmk2 cells, ruling out the role of CaM kinase II in NTH1 expression. Thus, CaM kinase II should act in concert with PKA on the activation of the cryptic form of neutral trehalase. A model for trehalase regulation by CaM kinase II is proposed whereby the target protein for Ca2+/CaM-dependent kinase II phosphorylation is not the neutral trehalase itself. The possible identity of this target protein with the recently identified trehalase-associated protein YLR270Wp is discussed

Evidence for a modulation of neutral trehalase activity by Ca2+ and cAMP signaling pathways in Saccharomyces cerevisiae.

Saccharomyces cerevisiae neutral trehalase (encoded by NTH1) is regulated by cAMP-dependent protein kinase (PKA) and by an endogenous modulator protein. A yeast strain with knockouts of CMK1 and CMK2 genes (cmk1cmk2) and its isogenic control (CMK1CMK2) were used to investigate the role of CaM kinase II in the in vitro activation of neutral trehalase during growth on glucose. In the exponential growth phase, cmk1cmk2 cells exhibited basal trehalase activity and an activation ratio by PKA very similar to that found in CMK1CMK2 cells. At diauxie, even though both cells presented comparable basal trehalase activities, cmk1cmk2 cells showed reduced activation by PKA and lower total trehalase activity when compared to CMK1CMK2 cells. To determine if CaM kinase II regulates NTH1 expression or is involved in post-translational modulation of neutral trehalase activity, NTH1 promoter activity was evaluated using an NTH1-lacZ reporter gene. Similar beta-galactosidase activities were found for CMK1CMK2 and cmk1cmk2 cells, ruling out the role of CaM kinase II in NTH1 expression. Thus, CaM kinase II should act in concert with PKA on the activation of the cryptic form of neutral trehalase. A model for trehalase regulation by CaM kinase II is proposed whereby the target protein for Ca2+/CaM-dependent kinase II phosphorylation is not the neutral trehalase itself. The possible identity of this target protein with the recently identified trehalase-associated protein YLR270Wp is discussed.

Identification of an integral membrane 80 kDa protein of Saccharomyces cerevisiae induced in response to dehydration.

Using SDS-PAGE gels we observed the induced synthesis of a protein with a molecular mass of 80 kDa when cells of strains of Saccharomyces cerevisiae were subjected to dehydration. Physiological analysis showed that this protein is not present during growth on glucose but was found in derepressed cells from stationary phase. Furthermore, its synthesis was induced when cells were grown on medium containing alpha-methyl-glucoside as carbon source. However, the 80 kDa protein was not found in cells of mutants unable to transport trehalose. This protein was localized in the cytoplasmic membrane and showed trehalose-binding activity, determined by its partial purification on a trehalose-Sepharose 6B affinity column. The possible involvement of the 80 kDa protein with the trehalose transport system is discussed.

Regulation of UDPG-pyrophosphorylase isoforms in Saccharomyces cerevisiae and their roles in trehalose metabolism.

UDPG-pyrophosphorylase (EC 2.7.7.9) from Saccharomyces cerevisiae was studied and the presence of isoforms investigated. Its activity was monitored during growth of cultures in rich media containing glucose, galactose, sucrose, maltose or glycerol as carbon sources. The results suggest that UDPG-pyrophosphorylase is subject to both catabolite repression and catabolite inactivation. The inactivation process seems to be complex: in order to produce maximum inactivation, glucose and ammonium sulfate must be added together. Addition of glucose or ammonium sulfate separately produced little effect upon enzyme activity. Adsorption to and elution from a DEAE-Sephacel column of a crude protein extract prepared from yeast cells collected in stationary phase from a glucose medium showed three activity peaks, which we denominated isoform I, II, and III. Isoform I is constitutive, it was the only form present during exponential growth on glucose medium, and did not suffer any alteration after glucose exhaustion, heat shock or by growing cells on maltose. On the other hand, isoforms II and III were shown to be repressed by glucose, and induced by heat shock. Furthermore, isoform II of UDPG-pyrophosphorylase was present together with isoform I when yeast cells were grown on maltose. The presence of a MAL4C allele rendered isoform II constitutive. Interestingly, a gal3 mutant strain had low UDPG-pyrophosphorylase activity and isoforms I and II were not expressed. These results are discussed in relation to trehalose metabolism.

A regulatory role for TSL1 on trehalose synthase activity.

UDPG-dependent trehalose synthase activity was determined during growth on glucose medium in controls and yeast strains having deletions on components of the trehalose phosphate synthase complex. Deletion of TPS3 produced any alteration. In contrast, strains harboring deletions tsl1 delta or tsl1 delta/tps3 delta showed no activation of enzyme after glucose exhaustion. To evaluate the role played by TPS3 and TSL1 on trehalose synthase activity we have determined the effect of the addition of a cell free extract from a strain expressing only TPS3 and TSL1 to extracts of strains lacking TSL1, TPS3 or both. No effect was observed on trehalose synthase activity from tps3 delta mutant. The addition of the same extract to trehalose synthase from tsl1 delta or tsl1 delta/tps3 delta strains showed a two-fold activating effect, indicating that TPS3 and TSL1 regulated differently the UDPG-dependent trehalose synthase activity.