LH surge in Nelore cows (Bos indicus), after induced estrus or after ovarian superestimulation

Considering that there is limited information about the preovulatory LH surge in Zebu cattle (Bos indicus), the purpose of the present work was to assess the LH surge in Nelore cows during the estrous cycle and after ovarian superestimulation of ovarian follicular development with FSH. This information is particularly important to improve superovulatory protocols associated with fixed-time artificial insemination. Nelore cows (n=12) had their estrus synchronized with an intravaginal device containing progesterone (CIDR-B) associated with estradiol benzoate administration (EB, 2.5 mg, i.m., Day 0). Eight days later all animals were treated with PGF2alpha (Day 8) in the morning (8:00 h) and at night, when CIDR devices were removed (20:00 h). Starting 38h after the first PGF2alpha injection, blood sampling and ovarian ultrasonography took place every 4h, during 37 consecutive hours. Frequent handling may have resulted in a stress-induced suppression of LH secretion resulting in only 3 of 12 cows having ovulations at 46.7+/-4.9 and 72.3+/-3.8 h, respectively, after removal of CIDR-B. Thirty days later, the same animals received the described hormonal treatment associated with FSH (Folltropin), total dose=200 mg) administered twice a day, during 4 consecutive days, starting on Day 5. Thirty-six hours after the first injection of PGF2alpha, to minimize stress, only seven blood samples were collected at 4h interval each, and ultrasonography was performed every 12 h until ovulation. In 11 of 12 cows (92%) the LH surge and ovulation were observed 34.6+/-1.6 and 59.5+/-1.9 h, respectively, after removal of progesterone source. The maximum values for LH in those animals were 19.0+/-2.6 ng/ml (mean+/-S.E.M.). It is concluded that, in Nelore cows submitted to a ovarian superstimulation protocol, the LH surge occurs approximately 35 h after removal of intravaginal device containing progesterone, and approximately 12h before the LH surge observed after an induced estrus without ovarian superstimulation.     

Desarrollo de un bioadsorbente laminar con Phanerochaete chrysosporium hipertolerante al cadmio,al níquel y al plomo para el tratamiento de aguas

BackgroundThe use of basidiomycetes for metal removal is an alternative to traditional methods. In this, the biomass acts as a natural ionic exchanger removing metals from solution.ObjectiveTo develop a laminar biosorbent using a basidiomycete fungus resistant to high Cd, Ni and Pb concentrations.MethodsThe tolerance of Trametes versicolor, Pleurotus ostreatus and Phanerochaete chrysosporium was evaluated using increasing concentrations of the heavy metal salts, cadmium sulphate, lead acetate and nickel chloride. A biosorbent system was developed based on polyethylene sheets with a fungal biomass. It was evaluated in bubble columns using synthetic wastewater with the 3 metal salts at a rate of 300 mg/l. Finally, in a complementary experiment using shake flasks, the effect of a higher amount of biomass related to the metal removal efficiency was evaluated.ResultsP. chrysosporium strain was more tolerant to C₄H₆O₄Pb (10,000 mg/l), Cl₂Ni (300 mg/l) and CdSO₄·8H₂O (1,500 mg/l). In a reactor, under non-ligninolytic conditions, the fungus removed 69% of the chemical oxygen demand and produced enzymes such as LiP (0.01 U/l) and MnP (0.6 U/l.). An accumulation of metals in the wall was observed. By increasing the biomass to 1.6 (w/v), the metal biosorption was favored in the mixture (57% Pb, 74% Cd, and 98% Ni) and separately (95% Pb, 60% Cd, and 56% Ni). Competition between Ni and Pb by ligands of the wall was observed.ConclusionA novel laminar system based on P. chrysosporium viable biomass was developed. It has a large surface area and tolerance to high concentrations of Cd, Ni and Pb. It seems to be an alternative for the removal of metals from water.     

Microvesicles Derived from Adult Human Bone Marrow and Tissue Specific Mesenchymal Stem Cells Shuttle Selected Pattern of miRNAs

Background

Cell-derived microvesicles (MVs) have been described as a new mechanism of cell-to-cell communication. MVs after internalization within target cells may deliver genetic information. Human bone marrow derived mesenchymal stem cells (MSCs) and liver resident stem cells (HLSCs) were shown to release MVs shuttling functional mRNAs. The aim of the present study was to evaluate whether MVs derived from MSCs and HLSCs contained selected micro-RNAs (miRNAs).

Methodology/Principal Findings

MVs were isolated from MSCs and HLSCs. The presence in MVs of selected ribonucleoproteins involved in the traffic and stabilization of RNA was evaluated. We observed that MVs contained TIA, TIAR and HuR multifunctional proteins expressed in nuclei and stress granules, Stau1 and 2 implicated in the transport and stability of mRNA and Ago2 involved in miRNA transport and processing. RNA extracted from MVs and cells of origin was profiled for 365 known human mature miRNAs by real time PCR. Hierarchical clustering and similarity analysis of miRNAs showed 41 co-expressed miRNAs in MVs and cells. Some miRNAs were accumulated within MVs and absent in the cells after MV release; others were retained within the cells and not secreted in MVs. Gene ontology analysis of predicted and validated targets showed that the high expressed miRNAs in cells and MVs could be involved in multi-organ development, cell survival and differentiation. Few selected miRNAs shuttled by MVs were also associated with the immune system regulation. The highly expressed miRNAs in MVs were transferred to target cells after MV incorporation.

Conclusions

This study demonstrated that MVs contained ribonucleoproteins involved in the intracellular traffic of RNA and selected pattern of miRNAs, suggesting a dynamic regulation of RNA compartmentalization in MVs. The observation that MV-highly expressed miRNAs were transferred to target cells, rises the possibility that the biological effect of stem cells may, at least in part, depend on MV-shuttled miRNAs. Data generated from this study, stimulate further functional investigations on the predicted target genes and pathways involved in the biological effect of human adult stem cells.     

An engineered hyaluronan synthase: characterization for recombinant human hyaluronan synthase 2 Escherichia coli

The Class I hyaluronan synthase (HAS) is a unique glycosyltransferase synthesizing hyaluronan (HA), a polysaccharide composed of GlcUA and GlcNAc, by using one catalytic domain that elongates two different monosaccharides. As for the synthetic mechanism, there are two alternative manners for the sugar elongation process. Some bacterial HASs add new sugars to the non-reducing end of the acceptor to grow polymers. On the other hand, some vertebrate enzymes seem to transfer sugars to the reducing end. Expression of vertebrate HASs as active and soluble proteins will accelerate further precise insight into mechanisms of sugar elongation reactions by natural HASs. Since large scale production of HA polymers and oligomers would become powerful tools both for basic studies and new biotechnology to create functional carbohydrates in medicinal purposes, advent of an efficient method for the expression of HASs in Escherichia coli is strongly expected. Here we communicate the first success of the production of recombinant human HAS2 proteins composed of only the catalytic region in E. coli as the active form. It was demonstrated that an engineered HAS2 expressed in E. coli exhibited significant activity to synthesize a mixture of HAS oligomers from 8-mer (HA8) to 16-mer (HA16). Engineered HAS2 prepared herein elongated sugars from exogenous tetrasaccharide to form polymers with a direction to the non-reducing end. According to the present results, large scale production of engineered recombinant HASs is to be performed using E. coli that will provide practical and economic advantages in manufacturing enzymes for use in the synthesis of various oligomeric HA molecules and their industrial applications.     

Increase in secretion of glial cell line-derived neurotrophic factor from glial cell lines by inhibitors of vacuolar ATPase

Glial cell line-derived neurotrophic factor (GDNF) was reported to be effective for treating subjects with neurodegenerative diseases such as Parkinson's disease. In search of finding a compound which promotes GDNF secretion, we found that concanamycin A (ConA), a vacuolar ATPase (V-type ATPase) inhibitor purified from Streptomyces diastatochromogens, enhanced GDNF secretion from glioma cells. The rat glioma cell line, C6, and the human glioma cell lines, U87MG and T98G, abundantly expressed GDNF mRNA, and secreted GDNF into culture media, and this event was potently enhanced by a Ca(2+) ionophore and by phorbol ester, as noted in other cells. ConA concentration dependently and potently increased GDNF release from C6, U87MG and T98G cells into culture media. In addition, ConA enhanced GDNF secretion from astrocyte primary cultures prepared from the human fetus with the same potency seen in glioma cell lines. Likewise, another V-type ATPase inhibitor, bafilomycinA1 facilitated GDNF release from C6, U87MG and T98G glioma cells, in a concentration-dependent manner. The potencies of these V-type ATPase inhibitors in enhancing GDNF secretion were consistent with those which inhibited V-type ATPase activity. These results suggest that blockade of V-type ATPase potently stimulates the secretion of GDNF from glial cells. The V-type ATPase inhibitors may be beneficial to use for the treatment of diseases in which increase in GDNF could be effective.     

Biological evaluation of sphingomyelin analogues as inhibitors of sphingomyelinase

Seeking neutral sphingomyelinase inhibitors, we designed and synthesized hydrolytically stable analogues of sphingomyelin. These novel analogues replace the phosphodiester moiety of sphingomyelin with carbamate and urea moiety, resulting in inhibition of neutral sphingomyelinase. Compound 1 prevented ceramide generation and apoptotic neuronal cell death in a model of ischemia based on organotypic hippocampal slice cultures.     

Genetic mosaic in a marine species flock

We used molecular approaches to study the status of speciation in coral reef fishes known as hamlets (Serranidae: Hypoplectrus). Several hamlet morphospecies coexist on Caribbean reefs, and mate assortatively with respect to their strikingly distinct colour patterns. We provide evidence that, genetically, the hamlets display characteristics common in species flocks on land and in freshwaters. Substitutions within two mitochondrial DNA (mtDNA) protein-coding genes place hamlets within a monophyletic group relative to members of two related genera (Serranus and Diplectrum), and establish that the hamlet radiation must have been very recent. mtDNA distances separating hamlet morphospecies were slight (0.6 +/- 0.04%), yielding a coalescent estimate for the age of the hamlet flock of approximately 430 000 years. Morphospecies did not sort into distinct mtDNA haplotype phylogroups, and alleles at five hypervariable microsatellite loci were shared broadly across species boundaries. None the less, molecular variation was not distributed at random. Analyses of mtDNA haplotype frequencies and nested clades in haplotype networks revealed significant genetic differences between geographical regions and among colour morphospecies. We also observed significant microsatellite differentiation between geographical regions and in Puerto Rico, among colour morphospecies; the latter providing evidence for reproductive isolation between colour morphospecies at this locale. In our Panama collection, however, colour morphospecies were mostly genetically indistinguishable. This mosaic pattern of DNA differentiation implies a complex interaction between population history, mating behaviour and geography and suggests that porous boundaries separate species in this flock of brilliantly coloured coral reef fishes.     

Decreased Histamine-Stimulated Phosphoinositide Hydrolysis in the Cerebral Cortex of a Rat Line Selectively Bred for High Alcohol Preference

This study sheds light on the comparative analysis of agonist-stimulated phosphoinositide (PI) hydrolysis in the cerebral cortex of alcohol-naive rats from established lines selectively bred for low alcohol preference (LAP) and high alcohol preference (HAP). The effect of histamine (1.0 mM), but neither norepinephrine (0.1 mM) nor carbachol (0.5 mM), on PI hydrolysis was significantly reduced in HAP rats (0.4 +/- 5.0 fmol/mg protein [3H]inositol phosphates formed over basal) compared with LAP rats (25.5 +/- 10.0 fmol/mg protein). The contents of monoamines (dopamine, norepinephrine, and serotonin) and histamine in the cerebral cortex did not significantly differ between LAP and HAP rats, nor did the contents of their metabolites, except 3-methoxy-4-hydroxyphenylglycol (one of the metabolites of norepinephrine) and N(tau)-methylhistamine, which was not detected in our system. The histamine stimulatory effect was unchanged in the cerebral cortex of an intact Wistar rat that was treated with intraperitoneal injection of alcohol (1.0 g/kg once per day for 14 days). The results of the current study indicate that the decrease in the histamine effect on PI hydrolysis in HAP rats might be attributed to that particular rat line.     

Do high progesterone concentrations decrease pregnancy rates in embryo recipients synchronized with PGF2alpha and eCG?

The objective of this study was to evaluate the effects of equine chorionic gonadotropin (eCG) treatment on the number of induced accessory corpora lutea (CL), plasma progesterone concentrations and pregnancy rate in cross-bred heifers after transfer of frozen-thawed (1.5M ethylene glycol) embryos. All recipients received 500 microg PGF2alpha (dl-cloprostenol, i.m.) at random stages of the estrous cycle (Day 0) and were observed for estrus for 7 days. On Day 14, heifers detected in estrus between 2 and 7 days after PGF2alpha treatment were randomly allocated to four groups ( n=83 per group) and given 0 (control), 200, 400, or 600 IU of eCG. Two days later (Day 16), these recipients were given PGF2alpha and observed for estrus. Six to eight days after detection of estrus, plasma samples were collected to determine progesterone concentration and ultrasonography was performed to observe ovarian structures. Heifers with multiple CL or a single CL >15 mm in diameter received an embryo by direct transfer. Embryos of excellent and good quality were thawed and transferred to the recipients by the same veterinarian. Pregnancy was diagnosed by ultrasonography and confirmed by transrectal palpation 21 and 83 days after embryo transfer (ET), respectively. Plasma progesterone concentrations on the day of transfer (Day 7 of the estrous cycle) were 3.9+/-0.7, 4.2+/-0.4,6.0+/-0.4 and 7.8+/-0.6 ng/ml for groups Control, 200, 400, and 600, respectively (Control versus treated groups P=0.009; 200 versus 400 and 600 groups P=0.0001; and 400 versus 600 P=0.012 ). Conception rates 83 days after ET were 41.9, 50.0, 25.0, and 20.9% for groups Control, 200, 400, and 600, respectively (200 versus 400 and 600 groups P=0.0036 ). In conclusion, an increase in progesterone concentration, induced by eCG treatment, did not improve pregnancy rates in ET recipients. Conversely, there was a decline in conception rates in the animals with the highest plasma progesterone concentrations.