Effects of thymulin and GnRH on the release of gonadotropins by in vitro pituitary cells obtained from rats in each day of estrous cycle

The effects of thymulin and GnRH on FSH and LH release were studied in suspension cultures of anterior pituitary cells from female adult rats sacrificed on each day of the estrous cycle. The spontaneous release of gonadotropins by pituitaries, as well as their response to GnRH or thymulin addition, fluctuated during the estrous cycle. Adding thymulin to pituitary cells from rats in diestrus 1 increased the concentration of FSH; while in cells from rats in estrus, FSH level decreased. Thymulin had a stimulatory effect on the basal concentration of LH during most days of the estrous cycle. Adding GnRH increased FSH release in cells from rats in diestrus 1, diestrus 2, or proestrus, and resulted in higher LH levels in cells obtained from rats in all days of the estrous cycle. Compared to the GnRH treatment, the simultaneous addition of thymulin and GnRH to cells from rats in diestrus 1, diestrus 2, or proestrus resulted in lower FSH concentrations. Similar results were observed in the LH release by cells from rats in diestrus 1, while in cells from rats in proestrus or estrus, LH concentrations increased. A directly proportional relation between progesterone serum levels and the effects of thymulin on FSH release was observed. These data suggest that thymulin plays a dual role in the release of gonadotropins, and that its effects depend on the hormonal status of the donor's pituitary.     

Purification and characterization of a thermostable α-amylase produced by the fungus Paecilomyces variotii

An α-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The α-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pI value of 4.5. Temperature and pH optima were 60 °C and 4.0, respectively. The enzyme was stable for 1 h at 55 °C, showing a t₅₀ of 53 min at 60 °C. Starch protected the enzyme against thermal inactivation. The α-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K_m of α-amylase on Reagen® and Sigma® starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to α-amylases from Bacillus sp. These results confirmed that the studied enzyme was an α-amylase ((1→4)-α-glucan glucanohydrolase).     

Reproductive performance of high growth rate gilts inseminated at an early age

The aim of this work was to determine if gilts, which have a high growth rate (GR) could be mated earlier without reducing the reproductive performance or increasing the culling rate up to the third parity. Gilts of Camborough 22 (C22, n=568) breeding were mated and allocated into three groups according to weight and age on the insemination day. G1 (n=164)-gilts with a GR>or=700 g/d and inseminated at <210 d. G2 (n=165)-gilts with a GR>or=700 g/d and inseminated at >or=210 d. G3 (n=239)-gilts with a GR<700 g/d and inseminated at >or=210 d. All females were fed ad libitum from 150 d on and were inseminated at their second estrus or later. The minimum weight at mating was 127 kg. Three parities were studied, with farrowing rate, litter size and culling rate being compared. At the first parity, G2 gilts produced, on average, one more piglet than the other groups (P<0.05). However, when analyzing three parities, there were no differences in total born (11.6 x 12.3 x 11.7), farrowing rate (87.1% x 88.7% x 89.8%) and culling rate (30.2% x 25.3% x 28.2%) among G1-G3 groups, respectively (P>0.05). In conclusion, gilts, which had a minimum weight of 127 kg can be inseminated at their second or greater estrus, between 185 and <210 d of age, without impairing their productive performance over three parities.     

Tracking down human contamination in ancient human teeth

DNA contamination arising from the manipulation of ancient calcified tissue samples is a poorly understood, yet fundamental, problem that affects the reliability of ancient DNA (aDNA) studies. We have typed the mitochondrial DNA hypervariable region I of the only 6 people involved in the excavation, washing, and subsequent anthropological and genetic study of 23 Neolithic remains excavated from Granollers (Barcelona, Spain) and searched for their presence among the 572 clones generated during the aDNA analyses of teeth from these samples. Of the cloned sequences, 17.13% could be unambiguously identified as contaminants, with those derived from the people involved in the retrieval and washing of the remains present in higher frequencies than those of the anthropologist and genetic researchers. This finding confirms, for the first time, previous hypotheses that teeth samples are most susceptible to contamination at their initial excavation. More worrying, the cloned contaminant sequences exhibit substitutions that can be attributed to DNA damage after the contamination event, and we demonstrate that the level of such damage increases with time: contaminants that are >10 years old have approximately 5 times more damage than those that are recent. Furthermore, we demonstrate that in this data set, the damage rate of the old contaminant sequences is indistinguishable from that of the endogenous DNA sequences. As such, the commonly used argument that miscoding lesions observed among cloned aDNA sequences can be used to support data authenticity is misleading in scenarios where the presence of old contaminant sequences is possible. We argue therefore that the typing of those involved in the manipulation of the ancient human specimens is critical in order to ensure that generated results are accurate.     

Oxidative DNA cleavage induced by an iron(III) flavonoid complex: synthesis, crystal structure and characterization of chlorobis(flavonolato)(methanol) iron(III) complex

A flavonol iron(III) complex, [Fe(flavonolato)(2)Cl(MeOH)], has been prepared. The compound has been characterized by X-ray crystallography, spectroscopy, magnetism and electronic paramagnetic resonance (EPR) at X- and Q-band. The geometrical environment around the metal is best described as rhombic distorted octahedral. This distortion has also been inferred from the magnetic measurements and from the EPR spectra at different temperatures, E/D(rhombicity parameter) approximately 0.06. The DNA cleavage activity of the iron(III) complex with and without ascorbate/hydrogen peroxide is reported. Mechanisms of the oxidative cleavage have been proposed when DNA strand scission is performed both with and without ascorbate/hydrogen peroxide activation.     

Agrobacterium-mediated transient transformation of marigold (Tagetes erecta)

Hypocotyls, roots, leaf sections and shoot tips from Tagetes erecta plantlets were inoculated with Agrobacterium tumefaciens, harboring the binary vector pCAMBIA2301, containing the β-glucuronidase gene. Histochemical GUS assays of infected tissues showed transient gus gene expression after 3 days.     

Age-growth models for tilapia Oreochromis aureus (Perciformes, Cichlidae) of the Infiernillo reservoir, Mexico and reproductive behaviour

In presence of a decrease in the catch from Infiernillo reservoir, Michoacán-Guerrero, Mexico, age and growth of tilapia Oreochromis aureus (Steindachner 1864) were estimated, as indispensable attributes for fishery management. Evaluations included scales and opercular bones readings and length frequency analysis. The population structure was determined too. Due to the great variation of tilapia individual growth and with the objective to have reliable growth estimations, linearized, non-parametric and non-linear methods were used and their applicability discussed. Comparison of ages determined from these methods revealed that opercular bones readings had advantages over scales. Reproduction and competition for space and food were considered as responsible factors for ring-formation on tilapia hard structures. The non-linear method was considered the best for modelling populations highly variable, which is characteristic of tilapia and tropical fish with multiple spawns. The growth parameters estimated were Linfinity = 479 mm, K = 0.46/yr, t0 = -0.055 yr. The tilapia population in Infiernillo reservoir is dominated by juveniles less than 1.5 years old. The fishery activity is affecting the tilapia population growth because are caught when they have not yet spawned. Therefore, this fishery is in urgent need of regulation. Increasing the mesh size of the gill nets might contribute to protect the reproductive potential of the stock.     

Characterization and comparison of digestive proteinases of the Cortez swimming crab, Callinectes bellicosus, and the arched swimming crab, Callinectes arcuatus

Abstract. Protease activity in the midgut gland, gastric chamber, and gastric juice from the crabs Callinectes bellicosus and Callinectes arcuatus was characterized by several methods, confirming that the composition of digestive proteases is the same in the gastric juice and the midgut gland. Gastric juice was suitable for the identification and characterization of the proteinases trypsin and chymotrypsin. Such enzymes were presented as isotrypsins and isochymotrypsins. Proteinase composition evaluated by SDS-PAGE and substrate-SDS-PAGE showed differences between species, but not between gender. Proteinases were thermostable at 40°–50°C for 1 h and showed maximum activity at pH 6–8, making the use of digestive proteinases for evaluations of protein digestibility by the pHstat method possible. We propose using gastric juice as a source of digestive enzymes for in vitro studies of enzymes in digestibility assays and characterization procedures.     

Clinical and immunological effects of Rituximab in patients with lupus nephritis refractory to conventional therapy: a pilot study

We studied the clinical and immunological effects of Rituximab (anti-CD20) therapy in patients with lupus nephritis. In an open clinical trial, 22 patients with active systemic lupus erythematosis and renal involvement (mainly class III and IV according to the WHO classification) that was refractory to conventional therapy were studied. In all these patients, Rituximab (0.5 to 1.0 g at days 1 and 15) was added to the immunosuppressive therapy and its therapeutic effect was evaluated. In addition, the levels and function of regulatory T lymphocytes and the apoptosis of immune cells were assessed. We found a significant reduction in disease activity (p < 0.05, MEX-SLEDAI index), and proteinuria (p < 0.05) at days 60 and 90 of Rituximab therapy. Although most patients showed improvement in creatinine clearance and erythrocyturia, no significant changes in these parameters were detected. In most patients (20/22), B cell depletion was observed, but no clear-cut effect of Rituximab on complement levels or auto-antibody titers was detected (p > 0.05 in all cases). One patient died at day 70 with invasive histoplasmosis. No important adverse effects of Rituximab therapy were registered in other patients. A significant enhancement in the levels of different CD4+ regulatory cells (T_REG, Th3, Tr1), but not CD8+ Ts lymphocytes, was observed at day 30. This increase was sustained for T_REG cells at day 90, and accompanied by an improvement in their regulatory function. In addition, we observed an unexpected increase in the apoptosis of T cells at day 30. Interestingly, the enhancement in the suppressive function of T_REG cells was not observed in the two patients that showed the poorest clinical response to Rituximab. We conclude that the data obtained in this open clinical trial suggest that Rituximab is a promising candidate for randomized controlled trials in patients with lupus nephritis refractory to the conventional immunosuppressive therapy. The effects of Rituximab on regulatory cells and apoptosis of T lymphocytes are interesting and its possible role in the putative effect of this biological agent in systemic lupus erythematosis deserves additional studies.