Citrate diminishes hypothalamic acetyl-CoA carboxylase phosphorylation and modulates satiety signals and hepatic mechanisms involved in glucose homeostasis in rats

The hypothalamic AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) pathway is known to play an important role in the control of food intake and energy expenditure. Here, we hypothesize that citrate, an intermediate metabolite, activates hypothalamic ACC and is involved in the control of energy mobilization. Initially, we showed that ICV citrate injection decreased food intake and diminished weight gain significantly when compared to control and pair-fed group results. In addition, we showed that intracerebroventricular (ICV) injection of citrate diminished (80% of control) the phosphorylation of ACC, an important AMPK substrate. Furthermore, citrate treatment inhibited (75% of control) hypothalamic AMPK phosphorylation during fasting. In addition to its central effect, ICV citrate injection led to low blood glucose levels during glucose tolerance test (GTT) and high glucose uptake during hyperglycemic-euglycemic clamp. Accordingly, liver glycogen content was higher in animals given citrate (ICV) than in the control group (23.3+/-2.5 vs. 2.7+/-0.5 microg mL(-1) mg(-1), respectively). Interestingly, liver AMPK phosphorylation was reduced (80%) by the citrate treatment. The pharmacological blockade of beta3-adrenergic receptor (SR 59230A) blocked the effect of ICV citrate and citrate plus insulin on liver AMPK phosphorylation. Consistently with these results, rats treated with citrate (ICV) presented improved insulin signal transduction in liver, skeletal muscle, and epididymal fat pad. Similar results were obtained by hypothalamic administration of ARA-A, a competitive inhibitor of AMPK. Our results suggest that the citrate produced by mitochondria may modulate ACC phosphorylation in the hypothalamus, controlling food intake and coordinating a multiorgan network that controls glucose homeostasis and energy uptake through the adrenergic system.     

Analysis of the activity patterns of two sympatric sandfly siblings of the Lutzomyia longipalpis species complex from Brazil

Abstract.  Lutzomyia longipalpis s.l. (Lutz & Neiva) (Diptera: Psychodidae) is the main vector of visceral leishmaniasis in Latin America. Differences in copulation songs, pheromones and molecular markers show that L.  longipalpis is a species complex in Brazil. The patterns of activity of insect vectors are important in disease transmission. In addition, differences in activity rhythms have a potential role as a temporal reproductive isolation mechanism in closely related species. We compared the activity patterns of males and females of two sympatric species of the Longipalpis complex from Sobral (Ceará State, Brazil) in controlled laboratory conditions. We observed small but significant differences between the two species in the activity phase in both males and females.     

Protein phosphatase 2A subunit PR70 interacts with pRb and mediates its dephosphorylation

The retinoblastoma tumor suppressor protein (pRb) regulates cell proliferation and differentiation via phosphorylation-sensitive interactions with specific targets. While the role of cyclin/cyclin-dependent kinase complexes in the modulation of pRb phosphorylation has been extensively studied, relatively little is known about the molecular mechanisms regulating phosphate removal by phosphatases. Protein phosphatase 2A (PP2A) is constituted by a core dimer bearing catalytic activity and one variable B regulatory subunit conferring target specificity and subcellular localization. We previously demonstrated that PP2A core dimer binds pRb and dephosphorylates pRb upon oxidative stress. In the present study, we identified a specific PP2A-B subunit, PR70, that was associated with pRb both in vitro and in vivo. PR70 overexpression caused pRb dephosphorylation; conversely, PR70 knockdown prevented both pRb dephosphorylation and DNA synthesis inhibition induced by oxidative stress. Moreover, we found that intracellular Ca²⁺ mobilization was necessary and sufficient to trigger pRb dephosphorylation and PP2A phosphatase activity of PR70 was Ca²⁺ induced. These data underline the importance of PR70-Ca²⁺ interaction in the signal transduction mechanisms triggered by redox imbalance and leading to pRb dephosphorylation.     

Population dynamics of Triatoma vitticeps (Stål, 1859) in Itanhomi, Minas Gerais, Brazil

Taxonomic markers (head structure morphometry, isoenzymes and randon amplified polymorphism of DNA - RAPD) were used to understand the population dynamics of Triatoma vitticeps, predominant triatomine species in Itanhomi district, using samples obtained from domestic, peridomiciliary and sylvatic habitats. Morphometric analysis revealed sexual dimorphism within the three samples although specimens could not be separated according to the habitat in which they were captured. Forty-two bands were analyzed from RAPD profiles generated using four primers. A dendrogram constructed from Dice's similarity coefficient values showed that migration of the insects between the habitats has occurred, without structuring of populations. Moreover, the dendrogram obtained from the genetic distance values showed an important gene flow between the sylvatic and domestic habitats. No polymorphism was found in the electrophoretic mobility of proteins for the ten enzymes studied. Our results revealed movement of triatomines between the three habitats, suggesting that the presence of T. vitticeps in houses should not be ignored. As invasion of houses by sylvatic insects is frequent and the natural infection indices of this species are among the highest known, epidemiological vigilance studies may reveal possible changes in T. vitticeps behaviour which could present future risks to public health.     

Molecular mapping of soybean rust (Phakopsora pachyrhizi) resistance genes: discovery of a novel locus and alleles

Soybean production in South and North America has recently been threatened by the widespread dissemination of soybean rust (SBR) caused by the fungus Phakopsora pachyrhizi. Currently, chemical spray containing fungicides is the only effective method to control the disease. This strategy increases production costs and exposes the environment to higher levels of fungicides. As a first step towards the development of SBR resistant cultivars, we studied the genetic basis of SBR resistance in five F(2) populations derived from crossing the Brazilian-adapted susceptible cultivar CD 208 to each of five different plant introductions (PI 200487, PI 200526, PI 230970, PI 459025, PI 471904) carrying SBR-resistant genes (Rpp). Molecular mapping of SBR-resistance genes was performed in three of these PIs (PI 459025, PI 200526, PI 471904), and also in two other PIs (PI 200456 and 224270). The strategy mapped two genes present in PI 230970 and PI 459025, the original sources of Rpp2 and Rpp4, to linkage groups (LG) J and G, respectively. A new SBR resistance locus, rpp5 was mapped in the LG-N. Together, the genetic and molecular analysis suggested multiple alleles or closely linked genes that govern SBR resistance in soybean.     

Effect of early luteolysis in progesterone-based timed AI protocols in Bos indicus, Bos indicus x Bos taurus, and Bos taurus heifers

The objective of this study was to evaluate the effects of treatment with an intravaginal progesterone-releasing device (CIDR) and estradiol benzoate (EB) on follicular dynamics in Bos indicus (n=23), Bos taurus (n=25), and cross-bred (n=23) heifers. To assess the influence of reduced serum progesterone concentrations during 8 days of treatment with a progesterone-releasing device on follicular dynamics, half of the heifers received PGF at CIDR insertion (Day 0; 3 x 2 factorial design). Mean (+/-S.E.M.) serum progesterone concentrations during CIDR treatment varied (P<0.05) among genetic groups: B. indicus (5.4+/-0.1 ng/mL), B. taurus (3.3+/-0.0 ng/mL), and cross-bred (4.3+/-0.1 ng/mL). Maximum diameter of the dominant follicle (DF) was smaller (P<0.01) in B. indicus heifers (9.5+/-0.5 mm) than in cross-bred (12.3+/-0.4 mm) or B. taurus heifers (11.6+/-0.5 mm). B. indicus experienced lower (P<0.01) ovulation rate (39.1%) than did B. taurus (72.7%) and cross-bred (84.0%). Heifers treated with PGF on Day 0 had lower (P<0.05) serum progesterone concentrations during progesterone treatment. The PGF treatment on Day 0 increased (P<0.01) the diameter of the DF (11.9+/-0.4 mm vs. 10.5+/-0.4 mm). Moreover, greater (P=0.02) ovulation rates (78.8 vs. 54.0%) occurred in heifers treated with PGF on Day 0. In summary, B. indicus heifers had greater serum progesterone concentrations, smaller DF diameter, and a lower ovulation rate compared to B. taurus heifers. Prostaglandin treatment on the day of CIDR insertion reduced serum progesterone during treatment, and resulted in increased maximum DF diameter and ovulation rate.     

Changes in calcium-dependent membrane permeability properties in mitochondria of livers from arthritic rats

The involvement of the mitochondrial permeability transition pore (PTP) in the responses of mitochondria from adjuvant-induced arthritic rats to Ca(2+) addition was investigated. The respiratory activity, the Ca(2+)-induced osmotic swelling and the electrophoretic (45)Ca(2+) uptake were evaluated in the absence and in the presence of cyclosporin A (CsA), a well-known inhibitor of the mitochondrial PTP. The Ca(2+)-induced mitochondrial permeability transition (MPT) process occurred in mitochondria from arthritic rats even in the presence of a low Ca(2+) concentration. Whereas in the normal condition, the Ca(2+)-induced uncoupling of oxidative phosphorylation and osmotic swelling was observed in the presence of 10 or 20 microM Ca(2+) concentration, in the arthritic condition, these events occurred at 1.0 microM concentration. In addition, mitochondria from arthritic rats presented an impaired ability to accumulate (45)Ca(2+). All these effects were completely prevented by the administration of CsA. The results of the present study suggest that the higher sensitivity of mitochondria from arthritic rats to Ca(2+)-induced MPT may be an important factor in the pathogenesis of the arthritis disease.     

Effects of phosphatidylethanolamine glycation on lipid-protein interactions and membrane protein thermal stability

Non-enzymatic glycation of biomolecules has been implicated in the pathophysiology of aging and diabetes. Among the potential targets for glycation are biological membranes, characterized by a complex organization of lipids and proteins interacting and forming domains of different size and stability. In the present study, we analyse the effects of glycation on the interactions between membrane proteins and lipids. The phospholipid affinity for the transmembrane surface of the PMCA (plasma-membrane Ca(2+)-ATPase) was determined after incubating the protein or the phospholipids with glucose. Results show that the affinity between PMCA and the surrounding phospholipids decreases significantly after phosphospholipid glycation, but remains unmodified after glycation of the protein. Furthermore, phosphatidylethanolamine glycation decreases by approximately 30% the stability of PMCA against thermal denaturation, suggesting that glycated aminophospholipids induce a structural rearrangement in the protein that makes it more sensitive to thermal unfolding. We also verified that lipid glycation decreases the affinity of lipids for two other membrane proteins, suggesting that this effect might be common to membrane proteins. Extending these results to the in vivo situation, we can hypothesize that, under hyperglycaemic conditions, glycation of membrane lipids may cause a significant change in the structure and stability of membrane proteins, which may affect the normal functioning of membranes and therefore of cells.     

Pattern of silver nitrate-staining during meiosis and spermiogenesis in testicular lobes of Antiteuchus tripterus (Heteroptera: Pentatomidae)

The pattern of silver nitrate (Ag)-staining differed among testicular lobes of Antiteuchus tripterus. In general, these differences are in regard to the number, size, shape, coloring intensity, and location of the stained bodies or masses, observed during meiosis and spermiogenesis. These characteristics were similar in lobes 1-3. Lobes 4-6, however, differed from each other and from lobes 1-3 as well. Because the Ag-staining method is specific for nucleolar organizing regions and nucleolar material, the observations in meiosis of lobes 1-3 suggested the presence of a single pair of nucleolar organizing region-bearing chromosomes in A. tripterus, as previously found in other Pentatomidae species. In general, the amount of Ag-stained material seen in meiosis of the testicular lobes 1-3 of A. tripterus is smaller than in the other lobes. The differences among lobes observed during spermiogenesis included a striking variation in morphology of the Ag-stained material found in the head and tail of the spermatids. Given that the key role of the nucleolar material is to participate in protein synthesis, interlobular variations seem to be related to the different functions attributed to each lobe (reproduction to lobes 1-3 and basically nutrition to lobes 4-6). To our knowledge, this is the first time that the nucleolar material was studied in each testicular lobe during spermatogenesis. The present observations encourage further studies since, in addition to being of basic biological interest, several Pentatomidae species are agricultural pests and added knowledge of their biology, mainly in reproduction, may be important for the development of control strategies.