Control of glyceroneogenic activity in rat brown adipose tissue

Brown adipose tissue (BAT) glyceroneogenesis was evaluated in rats either fasted for 48 h or with streptozotocin-diabetes induced 3 days previously or adapted for 20 days to a high-protein, carbohydrate-free (HP) diet, conditions in which BAT glucose utilization is reduced. The three treatments induced an increase in BAT glyceroneogenic activity, evidenced by increased rates of incorporation of [1-14C]pyruvate into triacylglycerol (TAG)-glycerol in vitro and a marked, threefold increase in the activity of BAT phosphoenolpyruvate carboxykinase (PEPCK). BAT glycerokinase activity was not significantly affected by fasting or diabetes. After unilateral BAT denervation of rats fed either the HP or a balanced diet, glyceroneogenesis activity increased in denervated pads, evidenced by increased rates of nonglucose carbon incorporation into TAG-glycerol in vivo (difference between 3H2O and [14C]glucose incorporations) and of [1-14C]pyruvate in vitro. PEPCK activity was not significantly affected by denervation. The data suggest that BAT glyceroneogenesis is not under sympathetic control but is sensitive to hormonal/metabolic factors. In situations of reduced glucose use there is an increase in BAT glyceroneogenesis that may compensate the decreased generation of glycerol-3-phosphate from the hexose.     

Antimicrobial activity of essential oils isolated from Portuguese endemic species of Thymus

AIMS: Thymus species are wild species mostly found in the arid lands of Portugal. Possible antimicrobial properties of Thymus essential oils have been investigated. The chemical composition of the essential oils and the antimicrobial activity of Thymus mastichina (L) L. subsp. mastichina, T. camphoratus and T. lotocephalus from different regions of Portugal were analysed. METHODS AND RESULTS: Hydrodistillation was used to isolate the essential oils and the chemical analyses were performed by gas chromatography (GC) and GC coupled to mass spectrometry. The antimicrobial activity was tested by the disc agar diffusion technique against Candida albicans, Escherichia coli, Listeria monocytogenes, Proteus mirabilis, Salmonella spp. and Staphylococcus aureus. Pure linalool, 1,8-cineole and a mixture (1 : 1) of these compounds were included. Linalool, 1,8-cineole or linalool/1,8-cineole and linalool/1,8-cineole/linalyl acetate were the major components of the essential oils, depending on the species or sampling place. The essential oils isolated from the Thymus species studied demonstrated antimicrobial activity but the micro-organisms tested had significantly different sensitivities. CONCLUSIONS: The antimicrobial activity of essential oils may be related to more than one component. SIGNIFICANCE AND IMPACT OF THE STUDY: Portuguese endemic species of Thymus can be used for essential oil production for food spoilage control, cosmetics and pharmaceutical use. Further studies will be required to elucidate the cell targets of the essential oil components.     

The interaction of resveratrol with ferrylmyoglobin and peroxynitrite; protection against LDL oxidation

Resveratrol (3,4',5-trihydroxystilbene) is a natural phytoalexin synthesized in response to injury or fungal attack, found in the grape skin and wine, specially red wine. A large number of studies have demonstrated that resveratrol regulates many biological activities, namely protection against atherosclerosis by a set of pharmacological properties, including the antioxidant activity. In this study, we explored the capacity of resveratrol in protecting low density lipoproteins (LDL) against either ferrylmyoglobin- or peroxynitrite-mediated oxidation and the underlying mechanisms of its antioxidant potential. Resveratrol efficiently decreases the accumulation of hydroperoxides in LDL promoted by ferrylmyoglobin, a potent oxidant formed by the reaction of metmyoglobin with hydrogen peroxide, in a concentration-dependent manner, promptly reducing the oxoferryl complex to metmyoglobin. Simultaneously, resveratrol is consumed as detected by the rapid decrease in the characteristic peak at 310 nm, in a similar way to that observed upon its reaction with peroxidase/H 2 O 2 , pointing to a mechanism of one-electron oxidation and subsequent resveratrol dimer formation. On the other hand, resveratrol inhibits LDL apoprotein modifications induced by peroxynitrite, another potent oxidant formed by the reaction between superoxide and nitric oxide, as assessed by the decrease in apo-B net charge alterations and in carbonyl groups formation mediated by that oxidant. Resveratrol also interacts with peroxynitrite in a similar way to that observed with laccases, suggesting a mechanism of resveratrol oxidation rather than a nitration one. These mechanisms are discussed. Considering that either ferrylmyoglobin or peroxynitrite are physiologically relevant oxidants implicated in several pathologies, including atherosclerosis, our results certainly contribute to the understanding of the antioxidant action of resveratrol and consequently provide a new approach for the cardiovascular benefits associated with moderate consumption of red wine.     

Modulation of gastrin and epidermal growth factor by pyrrolizidine alkaloids obtained from Senecio brasiliensis in acute and chronic induced gastric ulcers

We investigated the antiulcerogenic activity of pyrrolizidine alkaloids (PAs) integerrimine, retrorsine, senecionine, usaramine and seneciplhylline, an alkaloidal extract obtained from Senecio brasiliensis. The PA extract demonstrated significantly activity in both, acute and chronic gastric ulcers on rats. The effects of PA extract were dose dependent. The mechanisms implicated on this activity were evaluated by determination of gastrin plasma levels in rats subjected to the acute treatment with PA extract and by expression of mRNA of Epidermal Growth Factor (EGF) after chronic treatment with this extract. The results showed that the PA extract increased both the levels of gastrin and the expression of EGF on these animals. Moreover, the histological examinations showed a reduction of exfoliation of superficial cells, hemorrhages and blood cell infiltration. We concluded that the PAs showed an important and qualitative antiulcerogenic activity mediated by increase in gastrin secretion and mRNA expression of EGF.     

Human immunodeficiency virus type 1 Brazilian subtype B variant showed an increasing avidity of the anti-V3 antibodies over time compared to the subtype B US/European strain in São Paulo, Brazil

The Brazilian variant of human immunodeficiency virus type 1 (HIV-1) subtype B, (serotype B"-GWGR), has a tryptophan replacing the proline in position 328 the HIV-1 envelope. A longer median time period from infection to acquired immunodeficiency syndrome (AIDS) for serotype B (B"-GWGR) infected subjects compared to the B-GPGR US/European strain was reported. In a cohort study, in S?o Paulo city, 10 B"-GWGR patients had a statistically significant increased avidity of the anti-V3 antibodies, from 79% +/- 33% to 85% +/- 75%, versus from 48% +/- 59% to 32% +/- 17% for the 10 B-GPGR subjects (p = 0.02). The T CD4+ cells showed a mean increase of + 0.45 cells/month for the B-GPGR subjects and for B"-GWGR the slope was + 1.24 cells/month (p = 0.06), for 62 and 55 months of follow up, respectively. RNA plasma viral load decreased from 3.98 +/- 1.75 to 2.16 +/- 1.54 log10 in the B"-GWGR group while B-GPGR patients showed one log10 reduction in viral load from 4.09 +/- 0.38 to 3.17 +/- 1.47 log10 over time (p = 0.23), with a decreasing slope of 0.0042 +/- log10,/month and 0.0080 +/- log10/month, for B-GPGR and B"-GWGR patients, respectively (p = 0.53). Neither group presented any AIDS defining events during the study, according to Center for Diseases Control criteria. Although the sample size is small, these results may indicate that differences in the pathogenicity of the 2 HIV-1 B serotypes which co-circulate in Brazil may be correlated to the avidity of anti-V3 antibodies.     

Use of internal artificial vaginas for breeding soundness evaluation in range bulls: an alternative for electroejaculation allowing observation of sex drive and mating ability

The objective of this study was to test the efficacy of an inexpensive, reusable internal artificial vagina (IAV) developed for breeding soundness evaluation of range beef bulls. In addition, sexual behavior during semen collection by IAV was compared to behavior during pasture breeding. Breeding soundness exams (BSEs) were conducted on 165 bulls in two consecutive years (96 in Year 1 and 69 in Year 2). In Year 1, BSEs were done twice in all bulls, once by a conventional protocol using electroejaculation (EEJ), followed by the IAV method, one week later. In Year 2, all BSEs were done on one day; 69 bulls by the IAV method followed by EEJ in 21 bulls that failed to serve the IAV. For semen collection using an IAV, mount cows were restrained in breeding crates and an IAV was inserted into the vagina just beyond the depth of the vestibular sphincter. After each copulation, the IAV was replaced for the next bull to be tested. Semen collection by IAV was successful for all bulls that mounted and penetrated cows during the testing period (54.3 and 69.6% of the bulls served the cows with IAVs in Year 1 and 2, respectively). Semen was collected successfully by EEJ from all bulls in both years. Differences were observed between semen collection methods in semen volume and percentage of sperm staining alive; however, the differences were opposite in Year 1 and 2 and, therefore, were probably due to natural variations in time and within bull rather than the method of semen collection. Semen collection by IAV allowed the detection of problems that prevented copulation in 8 bulls (4.8%) that were determined to be satisfactory potential breeders when semen was collected by EEJ. In Year 1, breeding observations were made at pasture for 15 bulls that served, and 15 bulls that did not serve cows with an IAV. Bulls that did not serve the IAV during the test period had fewer mounts, attempts to mount, and completed services at pasture than bulls that had served the IAV, indicating that such bulls are likely to be less efficient breeders. In conclusion, IAVs might be used as an alternative to EEJ for breeding soundness evaluation in about 50-70% of range bulls. Semen collection by IAV also allows observation of sex drive and mating ability thus improves the ability to select sound breeding bulls.     

Molecular and functional characterization of the Azorhizobium caulinodans ORS571 hydrogenase gene cluster

In this work, we report the cloning and sequencing of the Azorhizobium caulinodans ORS571 hydrogenase gene cluster. Sequence analysis revealed the presence of 20 open reading frames hupTUVhypFhupSLCDFGHJK hypABhupRhypCDEhupE. The physical and genetic organization of A. caulinodans ORS571 hydrogenase system suggests a close relatedness to that of Rhodobacter capsulatus. In contrast to the latter species, a gene homologous to Rhizobium leguminosarum hupE was identified downstream of the hyp operon. A hupSL mutation drastically reduced the high levels of hydrogenase activity induced by the A. caulinodans ORS571 wild-type strain in symbiosis with Sesbania rostrata plants. However, no significant effects on dry weight and nitrogen content of S. rostrata plants inoculated with the hupSL mutant were observed in plant growth experiments.     

The saposin-like domain of the plant aspartic proteinase precursor is a potent inducer of vesicle leakage

A unique feature of plant aspartic proteinase precursors is the presence of an internal domain, known as plant-specific insert, whose function is not completely understood. The three-dimensional structure of the plant-specific insert resembles that of saposin-like proteins, a group of lipid-binding proteins involved in a variety of physiological processes. Here we show that recombinant plant-specific insert is able to interact with phospholipid vesicles and to induce leakage of their contents in a pH- and lipid-dependent manner. The leakage activity is higher at pH 4.5 and requires the presence of acidic phospholipids such as phosphatidylserine. To determine whether the same effect could be observed when the plant-specific insert is part of the precursor form, procardosin A and a mutant form lacking this specific domain were produced and characterized. Procardosin A displays a similar activity profile, whereas the mutant without the plant-specific insert shows only residual activity. These findings indicate that the plant-specific insert domain of plant aspartic proteinases mediates an interaction of their precursors with phospholipid membranes and induces membrane permeabilization. It is therefore possible that the plant-specific insert, alone or in conjunction with the proteolytic activity of plant aspartic proteinases, may function either as a defensive weapon against pathogens or in late autolysis of plant cells.     

Interaction of cellular proteins with the 5' end of Norwalk virus genomic RNA

The lack of a susceptible cell line and an animal model for Norwalk virus (NV) infection has prompted the development of alternative strategies to generate in vitro RNAs that approximate the authentic viral genome. This approach has allowed the study of viral RNA replication and gene expression. In this study, using mobility shift and cross-linking assays, we detected several cellular proteins from HeLa and CaCo-2 cell extracts that bind to, and form stable complexes with, the first 110 nucleotides of the 5' end of NV genomic RNA, a region previously predicted to form a double stem-loop structure. These proteins had molecular weights similar to those of the HeLa cellular proteins that bind to the internal ribosomal entry site of poliovirus RNA. HeLa proteins La, PCBP-2, and PTB, which are important for poliovirus translation, and hnRNP L, which is possibly implicated in hepatitis C virus translation, interact with NV RNA. These protein-RNA interactions are likely to play a role in NV translation and/or replication.