Anaid Antaramian

Induction of peroxisomal proliferator-activated receptor γ and peroxisomal proliferator-activated receptor γ coactivator 1 by unsaturated fatty acids, retinoic acid, and carotenoids in preadipocytes obtained from bovine white adipose tissue

The importance of dietary fat components, such as fatty acids, in the expression of multiple genes is clear. In the case of beef cattle, fat in the form of fatty acids (saturated or unsaturated), vitamin A (mainly retinoic acid), or carotenoids (beta-carotene and lutein) is obtained from dietary feed or pasture. The aim of this work was to study the effect of fatty acids (phytanic and pristanic acids), vitamin A (all-trans and 9-cis retinoic acid), and carotenoids (beta-carotene and lutein) on the expression of PPARgamma and its coactivator PGC-1alpha during differentiation of bovine white adipose tissue. Samples were collected at slaughter from subcutaneous adipose tissue and processed in a solution containing type II collagenase for 2 h at 37 degrees C. Cells were resuspended in basal medium, Dulbeccos modified Eagles medium containing 5% fetal bovine serum, plated on 24-well culture plates at a density of 1 x 10(4) cells/cm(2), and incubated at 37 degrees C in a 5% CO(2) atmosphere. Preadipocyte differentiation after reaching confluence was induced by various treatments: rosiglitazone (20 microM); unsaturated fatty acids: phytanic acid (25, 50, 100 microM) and pristanic acid (25, 50, 100 microM); retinoids: 9-cis retinoic acid (0.5, 0.75, 1 microM) and all-trans retinoic acid (0.5, 0.75, 1 microM); and carotenoids: beta-carotene (10, 20, 30 microM) and lutein (10, 20, 30 microM). Expression of PPARgamma and PGC-1alpha was measured in differentiated cells. Phytanic acid, all-trans retinoic acid, and 9-cis retinoic acid were the best activators of PPARgamma expression, and the combination of 9-cis and all-trans retinoic acid was the best activator of PGC-1alpha expression (P < 0.05). Therefore, these are powerful agents for the promotion of bovine adipogenesis and constitute promising compounds to be used in bovine fattening.

Bovine sirtuins: initial characterization and expression of sirtuins 1 and 3 in liver, muscle, and adipose tissue.

Sirtuins, the mammalian homologs of the silent information regulator 2 gene of Saccharomyces cerevisiae, are members of the NAD(+)-dependent family of histone deacetylases. In vertebrates, 7 sirtuins have been described, with different cellular localizations and target proteins. Glucose and lipid metabolism are among the processes regulated by these enzymes. In ruminants, gluconeogenesis is the main biochemical pathway by which glucose is obtained. Because sirtuins in bovines have not been studied, the aim of this work was to obtain sequences coding for the 7 sirtuins and determine the expression patterns of sirtuin1 (Sirt1) and sirtuin3 (Sirt3) in the liver, muscle, and adipose tissue of calves and bulls. Using PCR amplification, we obtained sirtuin gene sequences and reported them to the National Center for Biotechnology Information GenBank. Characteristic sequence motifs corresponding to the sirtuin catalytic core domain were found, including the active and zinc-binding sites. Relative expression patterns of Sirt1 and Sirt3 in liver, muscle, and adipose tissue were quantified by real-time PCR, normalizing to the geometric mean of the housekeeping genes cyclophilin A and β-actin. Expression of Sirt1 was less in liver and muscle, whereas it was greater in adipose tissue of adult animals, with statistical differences (P=0.0071) only in the latter. In the case of Sirt3, expression was greater in all 3 adult tissues, but statistical differences were found only in liver (P=0.0141) and muscle (P=0.0017). The greatest expression was observed in liver for Sirt1 and in muscle for Sirt3, whereas the least expression was in muscle for Sirt1 and in adipose tissue for Sirt3. In other species, sirtuin expression (both Sirt1 and Sirt3) in liver is reported to be the greatest among these 3 tissues, a pattern different from what we measured. These differences in expression can be associated with metabolic differences between nonruminant and ruminant species. However, further research on the relationship between bovine sirtuins and ruminant metabolism is required for a better understanding of these fields.

Enhanced inhibitory avoidance learning prevents the long-term memory-impairing effects of cycloheximide, a protein synthesis inhibitor

Interference with activity of numerous cerebral structures produces memory deficiencies; in many instances, however, when animals are over-trained such interference becomes innocuous. Systemic administration of protein synthesis inhibitors impairs long-term retention; this effect has been interpreted to mean that protein synthesis is required for memory consolidation, though little is known about the effect of protein synthesis inhibitors on memory of enhanced learning in the rat. To further analyze the protective effect of enhanced learning against amnesic treatments, groups of Wistar rats were trained in a one-trial step-through inhibitory avoidance task, using different intensities of foot-shock during training. Cycloheximide (CXM; 2.8 mg/kg), an inhibitor of protein synthesis, was injected either 30 min before training or immediately after training. Twenty-four hours after training retention latencies were recorded. Our data showed that both pre- and post-training administration of CXM produced amnesia in those groups that had been trained with relatively low foot-shock intensities, but no impairment in retention was observed when relatively high intensities of foot-shock were administered. These and similar results lead us to conclude that protein synthesis inhibitors may interfere with memory consolidation, but their effect disappears when animals are submitted to an enhanced learning experience, calling into question the idea that protein synthesis is required for memory consolidation.

The deduced primary structure of subunit I from cytochrome c oxidase suggests that the genus Polytomella shares a common mitochondrial origin with Chlamydomonas

We cloned and sequenced the mitochondrial gene encoding subunit I of cytochrome c oxidase (coxI) of Polytomella spp., a colorless alga related to Chlamydomonas. The purpose was to explore whether homology between the two species also exists at the level of a mitochondrial enzyme. The gene is 1512 bp long and contains no introns. The translated protein sequence exhibits 73.8% identity with its Chlamydomonas reinhardtii counterpart. The data obtained support the hypothesis that the separation of the colorless alga from the Chlamydomonas lineage was a late event in evolution, that occurred after the endosymbiotic process that gave rise to mitochondria.

Subunit structures of purified beef mitochondrial cytochrome bc1 complex from liver and heart.

The existence of tissue-specific isozymes of cytochromec oxidase has been widely documented. We have now studied if there are differences between subunits of mitochondrialbc1 complexes isolated from liver and heart. For this purpose, we have developed a method for the purification of an active ubiquinol-cytochromec oxidoreductase from adult bovine liver that includes solubilization of submitochondrial particles with deoxycholate, ammonium acetate fractionation, resolubilization with dodecyl maltoside, and ion exchange chromatography. The electrophoretic pattern of the liver preparation showed the presence of 11 subunits, with apparent molecular weights identical to the ones reported for the heart complex. Western blot analysis and isoelectric focusing followed by two-dimensional gels ofbc1 complexes from liver and heart were compared, and no qualitative differences were observed. In addition, the high-molecular-weight subunits of the purified complexes from both tissues, subunits I, II, V, and VI, were isolated by PAGE in the presence of Coomasie Blue and subjected to limited proteolysis and to chemical digestion with cyanogen bromide and BNPS-skatol, and the peptide patterns were compared. Finally, two of the small-molecular-weight subunits from the liver complex were isolated (subunits VII and X), partially analyzed by amino terminal sequencing, and found to be identical with the reported sequence of their heart counterparts. The data suggest that, in contrast to the case of cytochromec oxidase,bc1 complexes from liver and heart do not exhibit tissue-specific differences.

Ghrelin stimulates myogenic differentiation in a mouse muscle satellite cell line and in primary cultures of bovine myoblasts

Ghrelin is an acylated hormone that influences food intake, energy metabolism and reproduction, among others. Ghrelin may also stimulate proliferating myoblast cell differentiation and multinucleated myotube fusion. The aim of this work was to assess the effect of human ghrelin (hGHRL) and human ghrelin fragment 1-18 (hGHRL1-18) on myoblast differentiation by means of mRNA expression and protein level. Two types of cells were tested, the cell line i28 obtained from mouse skeletal muscle and primary cultures of bovine myoblasts. Both ghrelin and its N-terminal fragment hGHRL1-18 were used at concentrations of 0, 0.01, 0.1, 1, 10 and 100 nm. Treatments were applied to pre-confluent cultures and were maintained for 4 days. We determined that between 0.1 and 100 nm, hGHRL and hGRHL1-18 had similar effects on myogenic differentiation of i28 cells (p < 0.01). On the other hand, only the higher concentrations (10 and 100 nm) of hGHRL stimulated bovine myoblast differentiation. These results could be attributed to the presence, in both i28 cells and in bovine myoblasts, of the mRNA for GHS-R1a and CD36 receptors. The use of ghrelin in livestock production is still questionable because of the limited effects shown in this study, and additional research is needed in this field.

Potential role of sirtuins in livestock production.

Sirtuins are NAD(+)-dependent histone and protein deacetylases, which have been studied during the last decade with a focus on their role in lifespan extension and age-related diseases under normal and calorie-restricted or pathological conditions. However, sirtuins also have the ability to regulate energy homeostasis as they can sense the metabolic state of the cell through the NAD(+)/NADH ratio; hence, changes in the diet can modify the expression of these enzymes. Dietary manipulations are a common practice currently being used in livestock production with favorable results, probably due in part to the enhanced activity of sirtuins. Nevertheless, sirtuin expression in livestock species has not been a research target. For these reasons, the goal of this review is to awaken interest in these enzymes for future detailed characterization in livestock species by presenting a general introduction to what sirtuins are, how they work and what is known about their role in livestock.

Steroid Receptors and Aromatase Gene Expression in Different Brain Areas of Copulating and Sexually Sluggish Male Rats

INTRODUCTION Sexually sluggish (SS) males have been identified in several species of mammals including rats. These animals take more than 30 minutes to ejaculate; they do not ejaculate or do so inconsistently despite being tested repeatedly with sexually receptive females. Different brain areas and hormones play an important role in the control of male sexual behavior. AIMS Determine gene expression for the androgen receptor (AR), the estrogen receptor alpha (ERα), the progesterone receptor (PR), and the aromatase enzyme (ARO), in brain regions important in the control of male sexual behavior including the medial preoptic area (MPOA), the amygdala (AMG), the olfactory bulb (OB), and, as a control, the cortex (CTX) of copulating (C) and SS male rats. METHODS Males that ejaculated within 30 minutes in three tests with receptive females were included in the C group, while those males that ejaculated in one or none of the four tests were included in the SS group. RNA was isolated 1 week after the last test of sexual behavior, and cDNA was synthesized from the brain areas listed above. MAIN OUTCOMES MEASURES Expression of the AR, ERα, PR, and ARO genes was determined by quantitative polymerase chain reaction (qPCR). Cyclophilin A (CycA) and tyrosine 3-monooxygenase-tryptophan activation protein zeta (Ywhaz) were housekeeping genes used to determine relative gene expression with the 2(-ΔΔCt) method. RESULTS The expression of mRNA for AR and ARO increased in the MPOA of SS males. ARO mRNA was increased in the AMG of SS males. In the OB, ERα mRNA was increased and AR mRNA reduced in SS males. CONCLUSION These results indicate SS and C males show differences in gene expression within brain regions controlling sexual behavior.

Time course of retinal degeneration associated with the absence of 1, 4, 5-inositol trisphosphate receptor in Drosophila melanogaster

The absence of the inositol trisphosphate receptor is associated with a gradual retinal degeneration in Drosophila melanogaster. To characterize the time-course profile of this process, mosaic flies expressing a null allele of the itp gene in the eye were studied by electroretinograms and electronic microscopy. Membrane contour alterations, disrupted mitochondria, altered morphology and even loss of photoreceptors were increased progressively starting 5 d after hatching, were more evident during days 10–15 and promoted highly disorganized structures thereafter. The synaptic transmission and membrane potential of retinal cells were also significantly distorted, showing reduced ON and OFF transients as well as membrane potential from day 10 of hatching, and the functional defects became progressively more severe. Unexpectedly, these alterations were detected not only in the non-pigmented mutant ommatidia, but also in the pigmented ommatidia, including heterozygous and twin clones expressing 1, 4, 5-inositol trisphosphate receptor (IP3R). To explore the mechanism underlying this degenerative process, the progression of pro-oxidant and apoptotic reactions was characterized by immunohistochemical techniques. Mutant ommatidia showed intermittent episodes of increased pro-oxidant reactions (detected as adducts of 4-hydroxy-nonenal) throughout the flys life. Similarly, several episodes of active caspase 3, an apoptotic effector, were evident with the same time pattern. Episodes of enhanced lipid peroxidation and apoptosis were also observed in the pigmented ommatidia of the mosaic eyes. The results indicate that photoreceptors lacking IP3R suffer episodes of increased lipid peroxidation, which eventually perturb the retinal subcellular organization and disrupt the phototransduction process and cell viability. Pigmented ommatidia also showed a similar pattern of damage, indicating that the degenerative process is non-autonomous and is so intense that it propagated to the non-mutant retinal cells in the mosaic eyes. In conclusion, ommatidia with a null mutation of IP3R degenerate by a process associated with intermittent lipid peroxidation and apoptotic activities.

Basal activity of GIRK5 isoforms

G protein-coupled inwardly rectifying K(+) channels (GIRK or Kir3) form functional heterotetramers gated by Gbetagamma subunits. GIRK channels are critical for functions as diverse as heart rate modulation and neuronal post-synaptic inhibition. GIRK5 (Kir3.5) is the oocyte homologue of the mammalian GIRK subunits that conform the K(ACh) channel. It has been claimed that even when the oocytes express GIRK5 proteins they do not form functional channels. However, the GIRK5 gene shows three initiation sites that suggest the existence of three isoforms. In a previous work we demonstrated the functionality of homomultimers of the shortest isoform overexpressed in the own oocytes. Remarkably, the basal GIRK5-Delta25 inward currents were not coupled to the activation of a G-protein receptor in the oocytes. These results encouraged us to study this channel in another expression system. In this work we show that Sf21 insect cells can be successfully transfected with this channel. GIRK5-Delta25 homomultimers produce time-dependent inward currents only with GTPgammaS in the recording pipette. Therefore, alternative modes of stimulus input to heterotrimeric G-proteins should be present in the oocytes to account for these results.