Use of trichostatin A alters the expression of HDAC3 and KAT2 and improves in vitro development of bovine embryos cloned using less methylated mesenchymal stem cells

The aim of this work was to investigate the methylation and hydroxymethylation status of mesenchymal stem cells (MSC) from amniotic fluid (MSC‐AF), adipose tissue (MSC‐AT) and fibroblasts (FIB‐control) and to verify the effect of trichostatin A (TSA) on gene expression and development of cloned bovine embryos produced using these cells. Characterization of MSC from two animals (BOV1 and BOV2) was performed by flow cytometry, immunophenotyping and analysis of cellular differentiation genes expression. The cells were used in the nuclear transfer in the absence or presence of 50 nM TSA for 20 hr in embryo culture. Expression of HDAC1, HDAC3 and KAT2A genes was measured in embryos by qRT‐PCR. Methylation results showed difference between animals, with MSC from BOV2 demonstrating lower methylation rate than BOV1. Meanwhile, MSC‐AF were less hydroxymethylated for both animals. MSC‐AF from BOV2 produced 44.92 ± 8.88% of blastocysts when embryos were exposed to TSA and similar to embryo rate of MSC‐AT also treated with TSA (37.96 ± 15.80%). However, when methylation was lower in FIB compared to MSC, as found in BOV1, the use of TSA was not sufficient to increase embryo production. MSC‐AF embryos expressed less HDAC3 when treated with TSA, and expression of KAT2A was higher in embryos produced with all MSC and treated with TSA than embryos produced with FIB. The use of MSC less methylated and more hydroxymethylated in combination with embryo incubation with TSA can induce lower expression of HDAC3 and higher expression of KAT2A in the embryos and consequently improve bovine embryo production.     

Sequential disruption of ALV host receptor genes reveals no sharing of receptors between ALV subgroups A,B, and J

Background: Previously, we showed that targeted disruption of viral receptor genes in avian leukosis virus(ALV)subgroups using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9))-based genome editing confers resistance to ALV subgroups B and J. Here, we used the same strategy to target the receptor expressed by ALV subgroup A(TVA) and generate chicken cells resistant to infection by this virus.Results: CRISPR/Cas9-based disruption of exon 2 within the tva gene of DF-1 fibroblasts conferred resistance to infection by ALV subgroup A regardless of whether frameshift mutations were introduced during editing. Conversely,overexpression of the wild-type TVA receptor(wtTVA) by tva-modified DF-1 clones restored susceptibility to ALV subgroup A. The results confirm that exon 2, which contains the low-density lipoprotein receptor class A domain of TVA, is critical for virus entry. Furthermore, we sequentially modified DF-1 cells by editing the tva, tvb, and Na~+/H~+ exchange 1(chNHE1) genes, which are the specific receptors for ALV subgroups A, B, and J, respectively.Conclusions: Simultaneous editing of multiple receptors to block infection by different subgroups of ALV confirmed that ALV subgroups A, B, and J do not share host receptors. This strategy could be used to generate cells resistant to multiple viral pathogens that use distinct receptors for cell entry.     

Hemolytic anemia,spherocytosis, and thrombocytopenia associated with honey bee envenomation in a dog

This case report describes a massive honey bee envenomation in a 14‐month‐old male Belgian Malinois dog from St. Kitts, West Indies. Acute and delayed onsets of hemolytic anemia, echinocytosis, spherocytosis, thrombocytopenia, hemoglobinemia, and hemoglobinuria developed following envenomation. The dog recovered after treatment with glucocorticoids and supportive therapy. Spherocytosis, hemolysis, and thrombocytopenia in patients with massive bee envenomation are likely due to the direct toxic effects of the primary components of bee venom, melittin and phospholipase A₂ (PLA₂). Mellitin causes hemolysis by forming large pores in erythrocytes resulting in leakage of hemoglobin and also causes spectrin stiffening and resultant echinocyte and spherocyte formation. Melittin also stimulates PLA₂, a hydrolase that causes echinocytosis and spherocytosis, in vivo and in vitro, and mitochondrial breakdown in platelets. However, delayed manifestations could be attributed to immune‐mediated mechanisms from the generation of antibodies against damaged erythrocytes and platelet membrane proteins.     

Potential for greenhouse gas emissions from soil carbon stock following biofuel cultivation on degraded lands

Consequent to the interest in converting degraded lands for cultivation of biofuel crops, concerns have been expressed about greenhouse gas (GHG) emissions resulting from changes in soil‐carbon (C) stock following land conversions. A literature‐based study was undertaken for estimating the magnitude of emission of GHGs, particularly carbon dioxide (CO₂), following an assessment of the extent and causes of land degradation and the nature of CO₂ emission from soils. The study estimated the potential for CO₂ emission resulting from changes in soil‐carbon stock following land conversions, using oil palm (Elaeis guineensis Jacq.) as a case study. The analysis indicated that, overall, the magnitude of CO₂ emission resulting from changes in soil C stock per se following opening up of degraded land would be low compared with other potential sources of CO₂ emission. However, lack of data on critical aspects such as baseline soil C status was a limitation of the study. Soil respiration is the single best measure of GHG emission from soils. Fixation of C in additional biomass will compensate, over time, for C loss through soil respiration following a change in land use or land management, unless such changes involve conversion of existing large C stocks. Therefore, any net CO₂ emission from soils resulting from changes in soil C stock following opening up of degraded land is likely to be a short‐term phenomenon. The estimations used in the study are based on various assumptions, which need to be validated by experimental field data. Copyright © 2010 John Wiley & Sons, Ltd.     

Production of ascorbyl palmitate by surfactant-coated lipase in organic media

The surface of a lipase from Burkholderia cepacia was coated with a nonionic surfactant, propylene glycol monostearate, and was used as a biocatalyst in the production of ascorbic acid in tert-butyl alcohol. The influence of various factors such as the type of surfactant, the pH of the buffer used for coating, the amount of surfactant in the coating, the organic solvent, and the temperature and molar ratio of the substrates used in the reaction on the conversion of ascorbyl palmitate were studied. After 24 h of reaction at 50 degrees C, a conversion of 47% was obtained using an ascorbic acid to palmitic acid molar ratio of 1:6. The native lipase showed only 6% conversion.     

Amelioration of obesity and glucose intolerance in high-fat-fed C57BL/6 mice by anthocyanins and ursolic acid in Cornelian cherry (Cornus mas)

Much attention has been focused on food that may be beneficial in preventing diet-induced body fat accumulation and possibly reduce the risk of diabetes and heart disease. Cornelian cherries (Cornus mas) are used in the preparation of beverages in Europe and also to treat diabetes-related disorders in Asia. In this study, the most abundant bioactive compounds in C. mas fruits, the anthocyanins and ursolic acid, were purified, and their ability to ameliorate obesity and insulin resistance in C57BL/6 mice fed a high-fat diet was evaluated. Mice were initially fed a high-fat diet for 4 weeks and then switched to a high-fat diet containing anthocyanins (1 g/kg of high-fat diet) and ursolic acid (500 mg/kg of high-fat diet) for an additional 8 weeks. The high-fat diet induced glucose intolerance, and this was prevented by anthocyanins and ursolic acid. The anthocyanin-treated mice showed a 24% decrease in weight gain. These mice also showed decreased lipid accumulation in the liver, including a significant decrease in liver triacylglycerol concentration. Anthocyanin and ursolic acid treated mice exhibited extremely elevated insulin levels. Both treatments, however, showed preserved islet architecture and insulin staining. Overall, these data suggest that anthocyanins and ursolic acid purified from C. mas fruits have biological activities that improve certain metabolic parameters associated with diets high in saturated fats and obesity.     

Impact of alkyl esters of caffeic and ferulic acids on tumor cell proliferation, cyclooxygenase enzyme, and lipid peroxidation

The antioxidant ferulic and caffeic acid phenolics are ubiquitous in plants and abundant in fruits and vegetables. We have synthesized a series of ferulic and caffeic acid esters and tested for tumor cell proliferation, cyclooxygenase enzymes (COX-1 and -2) and lipid peroxidation inhibitory activities in vitro. In the tumor cell proliferation assay, some of these esters showed excellent growth inhibition of colon cancer cells. Among the phenolics esters assayed, compounds 10 (C12-caffeate), 11 (C16-caffeate), 21 (C8-ferulate), and 23 (C12-ferulate) showed strong growth inhibition with IC50 values of 16.55, 13.46, 18.67, and 7.57 microg/mL in a breast cancer cell line; 9.65, 7.45, 17.05, and 4.35 microg/ mL in a lung cancer cell line; 5.78, 3.5, 4.29, and 2.46 microg/mL in a colon cancer cell line; 12.04, 12.21, 14.63, and 8.09 microg/ mL in a central nervous system cancer cell line; and 8.62, 7.76, 11.0, and 5.37 in a gastric cancer cell line. In COX enzyme inhibitory assays, ferulic and caffeic acid esters significantly inhibited both COX-1 and COX-2 enzymes. Caffeates 5-10 (C4-C12), inhibited COX-1 enzyme between 50% and 90% and COX-2 enzyme by about 70%, whereas ferulates 15-21 (C3-C8) inhibited COX-1 and COX-2 enzymes by 85-95% 25 microg/mL. Long-chain caffeates 11-14 (C16-C22) and short-chain ferulates 15-20 (C3-C5) were the most active in lipid peroxidation inhibition and showed 60-70% activity at 5 microg/mL concentration.     

Molecular characterization and identification of markers associated with yield traits in mulberry using ISSR markers

Mulberry (Morus indica L.) is an important tree crop being exploited for feeding the silk‐producing insect Bombyx mori L. In order to identify parents suitable for breeding to raise high‐yielding varieties for the non‐traditional areas of Kerala, India and also to identify markers associated with leaf yield attributing traits, the present study was undertaken with 44 mulberry genotypes. Variability on morpho‐biometric traits and molecular markers, generated with 12 selected ISSR primers, was estimated. Significant differences between genotypes were observed for all the traits. The dendrogram generated with morpho‐biometric characters clustered the genotypes into three distinct groups and one isolate, while the same using Inter simple sequence repeat (ISSR) markers clustered the genotypes into five groups and six isolates. The greater resolving power of the ISSR markers was evident form the dendrograms. Using step‐wise multiple regression analysis, a number of markers associated with number of branches, total shoot length, leaf weight, internodal distance, leaf chlorophyll, protein, leaf moisture percentage were identified. These markers could be of much use in Marker assisted selection (MAS) breeding programmes in mulberry, especially when no genetic information in terms of linkage maps and Quantitative Trait Locis (QTLs) is available a plant with high heterozygosity and a long juvenile period.     

Genetical and anatomical analyses of a leaf flecking mutant in Triticum aestivum L.

Flecking trait in the mutant C591 (M8) Triticum aestivum L. is a stable,developmentally programmed, dominant mutation under monogenic control resembling pathogenic attack and starts appearing only from boot leaf stage of the plant. Mutant plants differ significantly from normal plants in terms of total chlorphyll contents only at later stages of symptom spread when the flecks fully cover the leaf sheath and leaves. However, total grain weight per main spike of mutant did not differ significantly from the normal plants. Microscopic studies of the mutant leaves did not reveal any damaging effect of the mutation on leaf anatomy per se, even though differences were observed in chlorophyll filling in mesophyll cells. Considering the peculiar characteristics of the mutation, many of which resembling the disease lesion mimic mutations in other crops, this is suspected to be such a mutation in wheat. This revised version was published online in August 2006 with corrections to the Cover Date.     

Molecular characterization of mulberry genetic resources indigenous to India

Mulberry (Morus L.) is essential for the sericulture industry as the primary feed for the silkworm Bombyx mori L. in India, with its long tradition of practicing sericulture, has a large number of indigenous cultivars. Since knowledge on the genetic divergence of these cultivars/varieties is essential for proper conservation and utilization, Inter Simple Sequence Repeat (ISSR) profiling was employed to assess genetic relationships among 34 mulberry accessions, collected from different regions of India. By using 12 ISSR primers, which produced 72 markers displaying a high degree of polymorphism (94.4%), genetic dissimilarity coefficients were calculated for each pair of the accessions and clustering of the accessions with Unweighted Pair Group Method using Arithmetic average (UPGMA) analysis was done to unravel the genetic diversity among the accessions. The dissimilarity coefficients varied from 0.111 to 0.692. UPGMA analysis generated a dendrogram with six groups and five isolates. Clustering of the accessions did not correspond with the information on the geographic origin of many of the accessions. Two-dimensional representations of the relative positions of the accessions with regards to divergence using the first two canonical variates as co-ordinate axes revealed considerable variability among the cases in each group. Further, Discriminant Function Analysis (DFA) showed changes in the geographic origin of 11 accessions and species status of 20 accessions.