Influence of genistein (4′,5,7-trihydroxyisoflavone) on the growth and proliferation of testicular cell lines

The effects of genistein on testicular cells, TM3, TM4, and GC-1 spg, were studied in vitro. First, each cell line was cultured with pre-determined concentrations of genistein for a maximum of 72 h to assess the effects of genistein on in vitro growth of the test cells. A second series of experiments were performed to determine the degree of genistein-induced apoptosis in these cells, using Apop-Tag^R kit reagents, to detect apoptotic cells in situ by specific end labeling, and detection of DNA fragments produced by the apoptotic process. The results obtained indicate that: i) genistein inhibits the growth and proliferation of testicular cells; ii) growth inhibition and proliferation is dose- and exposure-time dependent; iii) there is significant difference in sensitivity of the different testicular cells to genistein; iv) genistein induces apoptosis in testicular cells in a concentration-dependent manner. Genistein-induced apoptosis identifies genistein as a potential diagnostic and therapeutic tool in testicular pathophysiological research.     

Parallel airways inhomogeneity and lung tissue mechanics in transition to constricted state in rabbits

Toinvestigate whether changes of tissue resistance (Rti) duringmethacholine (MCh)-induced constriction correspond to an intrinsicmechanism or are an artifact of increased airways inhomogeneity, rabbits were studied after exposure to air(n = 7) or 1.5 parts/million O₃(n = 6). Animals were anesthetized andmechanically ventilated. Tracheal flow and pressure (Ptr) and fouralveolar capsule pressures (Pcap) were measured during 3 min afteradministration of an intrajugular bolus of 0.8 mg/ml MCh. By adjustmentof the equation of motion [P(t) = E · V(t) + R · dV(t)/dt + P₀] [whereP(t), V(t), and dV(t)/dt are pressure, volume, and flow as a function of time, respectively, Eis elastance, R is resistance, and P₀ is end-expiratorypressure] to Ptr, lung resistance(RL) and dynamic elastance(EL) were determined breath bybreath. Rti and airways resistance (Raw) were determined from Pcap in phase with rate of change of pulmonary expansion. Hysteresivity () was calculated. Parallel inhomogeneity wasestimated from the coefficients of variation (CV) of every Pcap at endinspiration and end expiration. Increase in CV significantly laggedRti, RL, and . A linearrelationship between EL and Rawwas observed. Our results suggest that changes in tissue mechanicsduring the transition to the constricted state are not artifactual.

     

Electrotransformation of Lactobacillus manihotivorans LMG 18010T and LMG 18011

An electroporation procedure was developed for the genetic transformation of intact cells of Lactobacillus manihotivorans , a new Lactobacillus species isolated from cassava sour starch fermentation in Colombia. Transformation efficiency of Lact. manihotivorans strains LMG 18010^T and LMG 18011 was measured and compared with electroporability of Lact. plantarum strains NCIMB 8299 and LMG 9211, by investigating the effect of changes in various parameters. For Lact. manihotivorans strain LMG 18010^T, the composition of the culture medium, such as the type of peptone and the presence of Tween-80, was found to be the most critical parameter, as well as the aeration conditions of the culture; better electroporation was obtained without air. The presence of MgCl₂ in the recovery medium was favourable to regeneration of electroporated cells. Plasmid-curing of the cells did not improve their electroporability. Transformants were obtained with Lact. manihotivorans strain LMG 18010^T and the plasmids pLZ12 and pGK13, whereas Lact. manihotivorans strain LMG 18011 was transformable with plasmids pLP825 and pLZ12, with different electroporation conditions.     

不同季节和冬眠时相中达乌尔黄鼠视前区温敏神经元特性和下丘脑内NA代谢的变化

比较了不同季节和冬眠时相中达乌尔黄鼠 (Citelleusdauricus)下丘脑内去甲肾上腺素 (noradrenaline ,NA)代谢和视前区 (POA)脑片中各类温敏神经元的比例、温度敏感性、放电活动的临界温度及下限温度 .结果表明 :与夏季动物相比 ,( 1)冬眠各时相中POA温敏神经元的比例和温敏性产生了与冬眠体温调节特性相关的适应性改变 ;( 2 )冬季和冬眠中POA神经元放电的下限温度和温敏神经元活动的临界温度均显著下移 ;( 3 )冬眠中POA神经元对NA反应的敏感性增高 ,冷敏神经元对NA的反应从夏季的抑制型转变为冬眠时的兴奋型 ;( 4)入眠和深冬眠时下丘脑内NA的含量和代谢水平下降 ,出眠时代谢水平升高 .这些变化可能解释动物入眠时主动降低体温和出眠时从深低体温中快速地升温的温度调节机理 .     

Sesquiterpene lactones from dicoria canescens

Dicoria canescens from Baja California, Mexico afforded the known monoterpene glucoside, (?)-cis-chrysanthenol O-β-d-glucopyranoside, ivalbatin, a xanthanolide and two new sesquiterpene lactones, 4-dihydroivalbatin (dicorin) and 11β, 13-tetrahydrovalbatin (dihydrocorin).     

Analysis of genetic diversity in red clover (Trifolium pratense L.) breeding populations as revealed by RAPD genetic markers.

Red clover is an important forage legume species for temperate regions and very little is known about the genetic organization of its breeding populations. We used random amplified polymorphic DNA (RAPD) genetic markers to address the genetic diversity and the distribution of variation in 20 breeding populations and cultivars from Chile, Argentina, Uruguay, and Switzerland. Genetic distances were calculated for all possible pairwise combinations. A high level of polymorphism was found and the proportion of polymorphic loci across populations was 74.2%. A population derived from a non-certified seedlot displayed a higher proportion of polymorphic loci than its respective certified seedlot. Gene diversity values and population genetics parameters suggest that the populations analyzed are diverse. An analysis of molecular variance (AMOVA) revealed that the largest proportion of variation (80.4%) resides at the within population level. RAPD markers are a useful tool for red clover breeding programs. A dendrogram based on genetic distances divided the breeding populations analyzed into three distinct groups. The amount and partition of diversity observed can be of value in identifying the populations that parents of synthetic cultivars are derived from and to exploit the variation available in the populations analyzed.     

Mapping oxidative DNA damage at nucleotide level

DNA damage induced by reactive oxygen species (ROS) is considered an important intermediate in the pathogenesis of human conditions such as cancer and aging. By developing an oxidative-induced DNA damage mapping version of the Ligation-mediated polymerase chain reaction (LMPCR) technique, we investigated the in vivo and in vitro frequencies of DNA base modifications caused by ROS in the human p53 and PGK1 gene. Intact human male fibroblasts were exposed to 50 mM H₂O₂, or purified genomic DNA was treated with 5 mM H₂O₂, 100 μM Ascorbate, and 50 μM, 100 μM, or 100 μM of Cu(II), Fe(III), or Cr(VI) respectively. The damage pattern generated in vivo was nearly identical to the in vitro Cu(II) or Fe(III) damage patterns; damage was non-random with guanine bases heavily damaged. Cr(VI) generated an in vitro damage pattern similar to the other metal ions, although several unique thymine positions were damaged. Also, extra nuclear sites are a major contributor of metal ions (or metal-like ligands). These data show that the local probability of H₂O₂-mediated DNA damage is determined by the primary DNA sequence, with chromatin structure having a limited effect. The data suggest a model in which DNA-metal ion binding domains can accommodate different metalions. LMPCR's unique aspect is a blunt-end ligation of an asymmetric double-stranded linker, permitting exponential PCR amplification. An important factor limiting the sensitivity of LMPCR is the representation of target gene DNA relative to non-targeted genes; therefore, we recently developed a method to eliminate excess non-targeted genomic DNA. Restriction enzyme-digested genomic DNA is size fractionated by Continuous Elution Electrophoresis (CEE), capturing the target sequence of interest. The amount of target DNA in the starting material for LMPCR is enriched, resulting in a stronger amplification signal. CEE provided a 24-fold increase in the signal strength attributable to strand breaks plus modified bases created by ROS in the human p53 and PGK1 genes, detected by LMPCR. We are currently taking advantage of the enhanced sensitivity of target gene-enriched LMPCR to map DNA damage induced in human breast epithelial cells exposed to non-cytotoxic concentrations of H₂O₂.     

Abundance and diversity of microfungi in three coastal beaches of Mexico

The abundance and diversity of species of microfungi was investigated on the beaches of Delfines, Km 24 Veracruz-Alvarado Highway, and El Coco, located on the coasts of the Caribbean Sea, Gulf of Mexico, and the Pacific Ocean, respectively. On each beach a sample composed of sand, subtidal wood or washed-up detritus with moist sand was collected. The samples were analyzed by three different methods, resulting in a total of 1,160 occurrences that fluctuated between 340 and 441 occurrences/sample. The number of species/sample fluctuated between 20 and 32. A total of 52 species was found, of which 12 were marine, and 40 nomarine, of terrestrial origin, and of these 15 were ascomycetes, 34 were hyphomycetes, 2 were blastomycetes and one was a coelomycete. The abundance distribution showed few species with high or low values, with the greates proportion having intermediate values. In order to compare species diversity among the samples frequency curves were utilized, based on the number of species expected from samples taken at random; the results showed that the beach at El Coco was richest in species.     

Substitution bias, rapid saturation, and the use of mtDNA for nematode systematics

Only relatively recently have researchers turned to molecular methods for nematode phylogeny reconstruction. Thus, we lack the extensive literature on evolutionary patterns and phylogenetic usefulness of different DNA regions for nematodes that exists for other taxa. Here, we examine the usefulness of mtDNA for nematode phylogeny reconstruction and provide data that can be used for a priori character weighting or for parameter specification in models of sequence evolution. We estimated the substitution pattern for the mitochondrial ND4 gene from intraspecific comparisons in four species of parasitic nematodes from the family Trichostrongylidae (38-50 sequences per species). The resulting pattern suggests a strong mutational bias toward A and T, and a lower transition/transversion ratio than is typically observed in other taxa. We also present information on the relative rates of substitution at first, second, and third codon positions and on relative rates of saturation of different types of substitutions in comparisons ranging from intraspecific to interordinal. Silent sites saturate extremely quickly, presumably owing to the substitution bias and, perhaps, to an accelerated mutation rate. Results emphasize the importance of using only the most closely related sequences in order to infer patterns of substitution accurately for nematodes or for other taxa having strongly composition-biased DNA. ND4 also shows high amino acid polymorphism at both the intra- and interspecific levels, and in higher level comparisons, there is evidence of saturation at variable amino acid sites. In general, we recommend using mtDNA coding genes only for phylogenetics of relatively closely related nematode species and, even then, using only nonsynonymous substitutions and the more conserved mitochondrial genes (e.g., cytochrome oxidases). On the other hand, the high substitution rate in genes such as ND4 should make them excellent for population genetics studies, identifying cryptic species, and resolving relationships among closely related congeners when other markers show insufficient variation.