Trehalose synthesis inhibitor: A molecular in silico drug design.

Infectious diseases are serious public health problems, affecting a large portion of the world's population. A molecule that plays a key role in pathogenic organisms is trehalose and recently has been an interest in the metabolism of this molecule for drug development. The trehalose-6-phosphate synthase (TPS1) is an enzyme responsible for the biosynthesis of trehalose-6-phosphate (T6P) in the TPS1/TPS2 pathway, which results in the formation of trehalose. Studies carried out by our group demonstrated the inhibitory capacity of T6P in the TPS1 enzyme from Saccharomyces cerevisiae, preventing the synthesis of trehalose. By in silico techniques, we compiled sequences and experimentally determined structures of TPS1. Sequence alignments and molecular modeling were performed. The generated structures were submitted in validation of algorithms, aligned structurally and analyzed evolutionarily. Molecular docking methodology was applied to analyze the interaction between T6P and TPS1 and ADMET properties of T6P were analyzed. The results demonstrated the models created presented sequence and structural similarities with experimentally determined structures. With the molecular docking, a cavity in the protein surface was identified and the molecule T6P was interacting with the residues TYR-40, ALA-41, MET-42, and PHE-372, indicating the possible uncompetitive inhibition mechanism provided by this ligand, which can be useful in directing the molecular design of inhibitors. In ADMET analyses, T6P had acceptable risk values compared with other compounds from World Drug Index. Therefore, these results may present a promising strategy to explore to develop a broad-spectrum antibiotic of this specific target with selectivity, potency, and reduced side effects, leading to a new way to treat infectious diseases like tuberculosis and candidiasis.

Regulation of the yeast trehalose-synthase complex by cyclic AMP-dependent phosphorylation.

BACKGROUND: Trehalose is an important protectant in several microorganisms. In Saccharomyces cerevisiae, it is synthesized by a large complex comprising the enzymes Tps1 and Tps2 and the subunits Tps3 and Tsl1, showing an intricate metabolic control. METHODS: To investigate how the trehalose biosynthesis pathway is regulated, we analyzed Tps1 and Tps2 activities as well as trehalose and trehalose-6-phosphate (T6P) contents by mass spectrometry. RESULTS: Tsl1 deficiency totally abolished the increase in Tps1 activity and accumulation of trehalose in response to a heat stress, whereas absence of Tps3 only reduced Tps1 activity and trehalose synthesis. In extracts of heat stressed cells, Tps1 was inhibited by T6P and by ATP. Mg(2+) in the presence of cAMP. In contrast, cAMP-dependent phosphorylation did not inhibit Tps1 in tps3 cells, which accumulated a higher proportion of T6P after stress. Tps2 activity was not induced in a tps3 mutant. CONCLUSION: Taken together these results suggest that Tsl1 is a decisive subunit for activity of the TPS complex since in its absence no trehalose synthesis occurred. On the other hand, Tps3 seems to be an activator of Tps2. To perform this task, Tps3 must be non-phosphorylated. To readily stop trehalose synthesis during stress recovery, Tps3 must be phosphorylated by cAMP-dependent protein kinase, decreasing Tps2 activity and, consequently, increasing the concentration of T6P which would inhibit Tps1. GENERAL SIGNIFICANCE: A better understanding of TPS complex regulation is essential for understanding how yeast deals with stress situations and how it is able to recover when the stress is over.

Revisiting yeast trehalose metabolism.

Establishing the function of trehalose in yeast cells has led us, over the years, through a long path-from simple energy storage carbohydrate, then a stabilizer and protector of membranes and proteins, through a safety valve against damage caused by oxygen radicals, up to regulator of the glycolytic path. In addition, trehalose biosynthesis has been proposed as a target for novel drugs against several pathogens. Since this pathway is entirely absent in mammalian cells and makes use of highly specific enzymes, trehalose metabolism might be an interesting target for the development of novel therapies. In this review, we want to address some recent points investigated about trehalose metabolism in Saccharomyces cerevisiae, focusing mainly on the mechanism by which this simple disaccharide protects against stress and on the enzymes involved in its synthesis and breakdown. We believe that these concepts are of great importance for medical and biotechnological applications.

Ovarian morphology and follicular dynamics associated with ovarian aging in Bos indicus beef cows.

This study characterizes the ovarian volume, antral follicle count (AFC), and ovarian dynamics of cows at different ages. Nellore cows (Bos taurus indicus) were used in two experiments. In Experiment 1, 57 lactating cows ranging in age from 3 to 23 years had their estrous cycle synchronized based on progesterone/estradiol treatments. The ovaries were scanned by ultrasound to record ovarian volume and AFC prior to follicle aspiration (AFC1). A second AFC (AFC2) was performed 5 days later to count only the growing antral follicles. In Experiment 2, six long-lived (14- to 23-year-old) and three young (4- to 8-year-old) non-lactating cows were submitted to daily ovarian scanning ultrasound during an interovulatory interval. Blood samples were collected during the estrous cycle to assess serum progesterone concentration. Data were analyzed using Student's t-test, ANOVA, and regression analysis tests. In Experiment 1, there were more (P < 0.05) antral follicles in AFC1 (31.4 ± 3.5) than in AFC2 (22.6 ± 2.4). In AFC1, the volume of the right ovary (6.03 ± 0.5 cm3) was greater (P < 0.01) than that of the left one (4.53 ± 0.4 cm3), although the AFC did not differ between the two ovaries (15.3 ± 1.8 and 16.3 ± 1.8, respectively). In both AFC1 and AFC2, there was a decline in the number of antral follicles as the age of the cow increased (P < 0.01). Ovarian volume (average of both ovaries) was related (P < 0.01) with AFC (R² = 0.1499) and cow age (R² = 0.0911). In Experiment 2, young and old cows under the age of 20 had a pattern of follicular growth waves, while cows over 20 years old did not have waves of follicular growth. The progesterone profiles and corpus luteum size during the estrous cycle did not differ between the groups. In conclusion, although cows had increased ovarian volume and decreased follicular population as they age, the follicular growth pattern and corpus luteum functionality appear to be unaffected by age.

The effect of trehalose on the fermentation performance of aged cells of Saccharomyces cerevisiae.

The fermentation process offers a wide variety of stressors for yeast, such as temperature, aging, and ethanol. To evaluate a possible beneficial effect of trehalose on ethanol production, we used mutant strains of Saccharomyces cerevisiae possessing different deficiencies in the metabolism of this disaccharide: in synthesis, tps1; in transport, agt1; and in degradation, ath1 and nth1. According to our results, the tps1 mutant, the only strain tested unable to synthesize trehalose, showed the lowest fermentation yield, indicating that this sugar is important to improve ethanol production. At the end of the first fermentation cycle, only the strains deficient in transport and degradation maintained a significant level of the initial trehalose. The agt1, ath1, and nth1 strains showed the highest survival rates and the highest proportions of non-petites. Accumulation of petites during fermentation has been correlated to low ethanol production. When recycled back for a subsequent fermentation, those mutant strains produced the highest ethanol yields, suggesting that trehalose is required for improving fermentative capacity and longevity of yeasts, as well as their ability to withstand stressful industrial conditions. Finally, according to our results, the mechanism by which trehalose improves ethanol production seems to involve mainly protection against protein oxidation.

Effect of feeding rumen-protected methionine on productive and reproductive performance of dairy cows.

The objectives of this study were to evaluate the effects of daily top-dressing (individually feeding on the top of the total mixed ration) with rumen-protected methionine (RPM) from 30 ± 3 until 126 ± 3 Days in milk on productive and reproductive performance in lactating dairy cows. A total of 309 lactating dairy Holstein cows (138 primiparous and 171 multiparous) were randomly assigned to treatment diets containing either RPM (21.2 g of RPM + 38.8 g of dried distillers grain; 2.34% Methionine [Met] of metabolizable protein [MP]) or Control (CON; 60 g of dried distillers grain; 1.87% Met of MP). Plasma amino acids were evaluated at the time of artificial insemination (AI) and near pregnancy diagnosis. Milk production and milk composition were evaluated monthly. Pregnancy was diagnosed on Day 28 (by Pregnancy-specific protein B [PSPB]), 32, 47, and 61 (by ultrasound) and sizes of embryonic and amniotic vesicle were determined by ultrasound on Day 33 after AI. Feeding RPM increased plasma Met at 6, 9, 12, and 18 hours after top-dressing with a peak at 12 hours (52.4 vs 26.0 µM; P < 0.001) and returned to basal by 24 hours. Cows fed RPM had a small increase in milk protein percentage (3.08 vs 3.00%; P = 0.04) with no differences on milk yield and milk protein yield. Additionally, in multiparous cows, RPM feeding increased milk protein (3.03 vs 2.95%; P = 0.05) and fat (3.45 vs 3.14%; P = 0.01) percentages, although no effects were observed in primiparous cows. In multiparous cows fed RPM, pregnancy loss was lower between Days 28 to 61 (19.6 [10/51] vs. 6.1% [3/49]; P = 0.03) or between Days 32 to 61 (8.9 [4/45] vs. 0 [0/0] %; P = 0.03), although, there was no effect of treatment on pregnancy loss in primiparous cows. Consistent with data on pregnancy loss, RPM feeding increased embryonic abdominal diameter (P = 0.01) and volume (P = 0.009) and amniotic vesicle volume (P = 0.04) on Day 33 of pregnancy in multiparous cows but had no effect on embryonic size in primiparous cows. Thus, the increase in plasma Met concentrations after feeding RPM was sufficient to produce a small increase in milk protein percentage and to improve embryonic size and pregnancy maintenance in multiparous cows. Further studies are needed to confirm these responses and understand the biological mechanisms that underlie these responses as well as the timing and concentrations of circulating Met that are needed to produce this effect.

Proposal of a new model for CL regression or maintenance during pregnancy on the basis of timing of regression of contralateral, accessory CL in pregnant cows.

In bovine pregnancy, regression or maintenance of the corpus luteum (CL) is mediated through local communication pathways between embryo, uterus, and ovary with Days 16 to 25 of pregnancy generally recognized as the pivotal period determining either luteolysis or prevention of luteolysis. To evaluate this concept, accessory CL was generated by treating Holstein lactating dairy cows (n = 718) with GnRH on Day 5 of the first follicular wave to produce an accessory CL on the ovary either contralateral or ipsilateral to the gravid horn. In pregnant cows, 66.2% (86/130) of contralateral CL regressed by Day 75 of pregnancy, whereas few ipsilateral accessory CL regressed (11.9%; 8/67), on the basis of similar criteria (P < 0.0001). As hypothesized, some contralateral CL regressions (22/86 = 25.6%) happened on Days 19 to 25 of pregnancy. However, most contralateral CL regressions (64/86 = 74.4%) happened later than expected, from Days 33 to 60 of pregnancy. Later contralateral CL regression was more common in primiparous (84.3%) than multiparous (60.0%; P = 0.02) cows. Early accessory contralateral CL regression (Days 19-25) may be related to lack of exposure of the contralateral horn to interferon tau from the elongating embryo because pregnant cows without early accessory CL regression had a smaller uterine volume than nonpregnant cows or pregnant cows that had early accessory CL regression (128.4 ± 3.9 vs. 147.0 ± 3.8 vs. 143.6 ± 10.9 mm3, respectively; P = 0.003). These results indicate that there is a second distinct period for CL protection during bovine pregnancy from Days 30 to 60 and implicate local and not systemic pathways in occurrence or prevention of luteolysis during both the early (≤25 days) and later (≥33 days) critical periods since accessory contralateral CL regressed whereas the accessory ipsilateral CL of pregnancy remained.

Effect of deslorelin acetate treatment in oocyte recovery and in vitro embryo production in domestic cats.

Objectives The present study investigated the effect of contraceptive treatment with deslorelin acetate on in vitro embryo production and oocyte recovery in domestic queens. Methods Twenty-one mature domestic cats were used. Eleven queens (treated group) and one tom were kept in an experimental cattery, and 10 queens were privately owned (control group). When in interestrus or diestrus (day 0) a deslorelin acetate implant (Suprelorin, 4.7 mg/animal) was inserted into the subcutaneous tissue of the interscapular region in all queens in the treated group. After 6 months of treatment, all animals were ovariohysterectomized, and the ovaries were used for in vitro embryo production. Percentage of cleavage was determined 18 h after oocyte insemination and blastocyst formation was assessed on the eighth day of culture. The rate of cumulus-oocyte complexes (COCs) recovery was analyzed by an unpaired t-test. The cleavage and blastocyst rates were expressed as percentages and analyzed by Fisher's exact test. All analyses were performed using GraphPad Prism v5.0, with P <0.05 set as the level of significance. Results In the treated group, we recovered 8.3 ± 1.15 grade I COCs per queen; the cleavage rate was 60% and the blastocyst rate was 36%. In the control group, we recovered 18.4 ± 3.21 grade I COCs per queen; the cleavage rate was 55.97% and the blastocyst rate was 34%. Forty percent of treated females did not produce any blastocysts. In the treated group, we observed a significant decrease in COC recovery. Although there was no significant difference in cleavage and blastocyst rates between groups, 40% of treated females did not produce any blastocysts. Conclusions Recovery of grade I COCs is negatively affected by deslorelin treatment in domestic cats. Regarding embryo production, new studies are still necessary to evaluate the success of this technique owing to the individual effect caused by deslorelin acetate.

Non-Invasive Measurement of Adrenocortical Activity in Blue-Fronted Parrots (Amazona aestiva, Linnaeus, 1758).

Parrots kept in zoos and private households often develop psychological and behavioural disorders. Despite knowing that such disorders have a multifactorial aetiology and that chronic stress is involved, little is known about their development mainly due to a poor understanding of the parrots' physiology and the lack of validated methods to measure stress in these species. In birds, blood corticosterone concentrations provide information about adrenocortical activity. However, blood sampling techniques are difficult, highly invasive and inappropriate to investigate stressful situations and welfare conditions. Thus, a non-invasive method to measure steroid hormones is critically needed. Aiming to perform a physiological validation of a cortisone enzyme immunoassay (EIA) to measure glucocorticoid metabolites (GCM) in droppings of 24 Blue-fronted parrots (Amazona aestiva), two experiments were designed. During the experiments all droppings were collected at 3-h intervals. Initially, birds were sampled for 24 h (experiment 1) and one week later assigned to four different treatments (experiment 2): Control (undisturbed), Saline (0.2 mL of 0.9% NaCl IM), Dexamethasone (1 mg/kg IM) and Adrenocorticotropic hormone (ACTH; 25 IU IM). Treatments (always one week apart) were applied to all animals in a cross-over study design. A daily rhythm pattern in GCM excretion was detected but there were no sex differences (first experiment). Saline and dexamethasone treatments had no effect on GCM (not different from control concentrations). Following ACTH injection, GCM concentration increased about 13.1-fold (median) at the peak (after 3-9 h), and then dropped to pre-treatment concentrations. By a successful physiological validation, we demonstrated the suitability of the cortisone EIA to non-invasively monitor increased adrenocortical activity, and thus, stress in the Blue-fronted parrot. This method opens up new perspectives for investigating the connection between behavioural disorders and stress in this bird species, and could also help in their captive management.

Luteal changes after treatment with sub-luteolytic doses of prostaglandin (cloprostenol sodium) in cattle.

This study characterizes the physiological and morphological changes related to partial luteolysis in bovine corpus luteum (CL) after challenges with sub-doses of cloprostenol sodium on Day 6 (D6) of the estrous cycle. Cows (n = 12/treatment) were treated as follows: Control (2 mL, saline, i.m.); 2XPGF (two treatments i.m. 500 µg of cloprostenol sodium 2 h apart) and 1/6PGF (83.3 µg of cloprostenol sodium, i.m., once). Plasma progesterone (P4) concentration, CL volume and blood flow were measured immediately before the treatments, then every 8 h (h) for 48 h. In the Control, P4 concentrations were higher at 48 h than at 0 h. P4 decreased 8h after 2XPGF treatment (P < 0.05), and remained low until the end of the trial. P4 decreased in 1/6PGF between 8 and 16 h (P < 0.05), then began to rebound at 24 h. Luteal volume was higher in Controls at 48 h than at 0 h. Under 1/6PGF, luteal volume decreased at 24 h (P < 0.05) and began to rebound at 32 h. Luteal volume and blood flow were reduced starting at 24 and 32 h, respectively, after 2XPGF treatment (P < 0.05). In this study, we were able to describe the partial luteolysis phenomenon, induced by a treatment of a D6CL with cloprostenol sub-dose.

Role of interferon-tau in the maternal recognition of pregnancy / Atuação do Interferon-tau no reconhecimento materno da gestação / Papel del interferón tau en el reconocimiento materno de la gestación

Maternal recognition of pregnancy is a process by which the conceptus signals its presence to the mother in order to prolong the life of the corpus luteum (CL) thus maintaining the pregnancy. This process occurs between days 15 and 19 after fertilization and is the most important biological challenge for obtaining satisfactory reproductive indices in bovine. Interferon-tau (IFN-τ) glycoprotein -secreted in the uterus by the conceptus- has a paracrine action inhibiting the expression of estrogen receptors (ESR1) and oxytocin (OXTR) in the endometrium, thus preventing the release of luteolytic pulses of prostaglandin F2 alpha (PGF2α), hormone responsible for the onset of luteolysis. IFN-τ also increases the expression of several interferon-stimulated genes (ISGs) in the uterus, CL, and blood cells. Direct endocrine action of IFN-τ on extrauterine tissues stimulates ISGs expression, which in the corpus luteum seems to be involved with luteal resistance to luteolytic action of PGF2α. This review discusses recent findings on the luteolysis mechanism in the bovine and endocrine and paracrine mechanisms such as IFN-τ during the maternal recognition of pregnancy.

O reconhecimento materno da gestação é o processo pelo qual o concepto sinaliza sua presença à unidade materna, prolongando a vida do corpo lúteo (CL), determinando a manutenção da gestação. Esse processo que ocorre entre os dias 15 e 19 pós-fertilização, representa o desafio biológico mais importante para a obtenção de índices reprodutivos satisfatórios em bovinos. Nesta espécie, uma glicoproteína denominada Interferon-tau (IFN-τ), secretada pelo concepto no ambiente uterino, age de forma parácrina inibindo a expressão dos receptores de estrógenos (ESR1) e de ocitocina (OXTR) no endométrio, evitando a liberação de pulsos luteolíticos de prostaglandina F2 alfa (PGF2α), hormônio responsável pelo início da luteólise. O IFN-τ também aumenta a expressão de vários genes estimulados por interferons (ISGs) no útero, CL e em células sanguíneas. A ação endócrina direta do IFN-τ em tecidos extrauterinos estimula a expressão de ISGs, que no CL, parecem estar envolvidos com a resistência luteal à ação luteolítica da PGF2α. Portanto, esta revisão tem como objetivo discutir os achados recentes sobre o mecanismo de luteólise na espécie bovina, e a atuação parácrina e principalmente endócrina do IFN-τ, durante o período de reconhecimento materno da gestação.

El reconocimiento materno de la gestación es un proceso mediante el cual el concepto señaliza su presencia a la madre, prolongando la vida del cuerpo lúteo (CL), determinando el mantenimiento de la gestación. Este proceso que ocurre entre los días 15 y 19 después de la fertilización, representa el desafio biológico más importante para la obtención de índices reproductivos satisfactorios en bovinos. En esta especie, una glicoproteína denominada Interferon-tau (IFN-τ), es secretada por el concepto en el ambiente uterino, actúa de forma parácrina inhibiendo la expresión de los receptores de estrógenos (ESR1) y de oxitocina (OXTR) en el endometrio evitando la liberación de pulsos luteoliticos de prostaglandina F2 alfa (PGF2α), hormona responsable por el inicio de la luteólisis. El IFN-τ también aumenta la expresión de varios genes estimulados por interferon (ISGs) en el útero, CL y en las células sanguíneas. La acción endocrina directa del IFN-τ en tejidos extrauterinos estimula la expresión de ISGs, que en el cuerpo lúteo parece estar involucrados con la resistencia luteal a la acción luteolitica de la PGF2α. Por lo tanto esta revisión tiene como objetivo discutir los hallazgos recientes sobre el mecanismo de luteólisis en la especie bovina, así como el mecanismo paracrino y principalmente endocrino del IFN-τ durante el período de reconocimiento materno de la gestación.