Diets enriched in unsaturated fatty acids enhance early embryonic development in lactating Holstein cows

We hypothesized that a diet enriched in alpha-linolenic acid would enhance embryonic development relative to diets enriched in linoleic or saturated fatty acids. Twenty-four lactating Holstein cows (86+/-22 d postpartum) were assigned to one of three diets containing saturated fatty acids (SAT; high in palmitic and stearic acids), whole flaxseed (FLX; high in alpha-linolenic acid) or sunflower seed (SUN; high in linoleic acid). Rations were formulated to provide 750 g supplemental fat/cow/d in all dietary groups. Ovulation (Day 0) was synchronized approximately 20 d after diets began. Ultrasound-guided follicular ablation of all follicles >8 mm was performed 5 d after ovulation; super stimulatory treatments began 2 d after follicular ablation, and embryos were collected non-surgically 7 d after AI. Fertilization rate, numbers of follicles and ovulations, and total and transferable embryos did not differ (P>0.05) among dietary groups. Sixty-one transferable embryos were stained and total blastomere number determined. Blastomere number was affected by diet (P<0.01); without regard to stage of development, embryos collected from cows fed SAT had lower (P<0.01) blastomere numbers (mean+/-S.E.M.; 77.1+/-3.9) than those from cows fed FLX (93.4+/-3.3) or SUN (97.2+/-3.5). Differences were most evident in the expanded blastocyst stage; at this stage, embryos of cows fed FLX and SUN diets had more blastomeres (P<0.02) than those of cows fed SAT (115.4+/-6.3, 132.3+/-8.3, and 89.3+/-9.6 cells, respectively). Although our hypothesis was only partially supported, embryonic development was enhanced in Holstein cows fed unsaturated fatty acids compared to those fed saturated fatty acids.     

Phase I trial of a recombinant yeast-CEA vaccine (GI-6207) in adults with metastatic CEA-expressing carcinoma

Yeast-CEA (GI-6207) is a therapeutic cancer vaccine genetically modified to express recombinant carcinoembryonic antigen (CEA) protein, using heat-killed yeast (Saccharomyces cerevisiae) as a vector. In preclinical studies, yeast-CEA induced a strong immune response to CEA and antitumor responses. Patients received subcutaneous vaccines every 2 weeks for 3 months and then monthly. Patients were enrolled at 3 sequential dose levels: 4, 16, and 40 yeast units (10⁷ yeast particles/unit). Eligible patients were required to have serum CEA > 5 ng/mL or > 20 % CEA⁺ tumor block, ECOG PS 0–2, and no history of autoimmunity. Restaging scans were performed at 3 months and then bimonthly. Peripheral blood was collected for the analysis of immune response (e.g., by ELISPOT assay). Twenty-five patients with metastatic CEA-expressing carcinomas were enrolled. Median patient age was 52 (range 39–81). A total of 135 vaccines were administered. The vaccine was well tolerated, and the most common adverse event was grade 1/2 injection-site reaction. Five patients had stable disease beyond 3 months (range 3.5–18 months), and each had CEA stabilization while on-study. Some patients showed evidence post-vaccination of increases in antigen-specific CD8⁺ T cells and CD4⁺ T lymphocytes and decreases in regulatory T cells. Of note, a patient with medullary thyroid cancer had substantial T cell responses and a vigorous inflammatory reaction at sites of metastatic disease. Yeast-CEA vaccination had minimal toxicity and induced some antigen-specific T cell responses and CEA stabilization in a heterogeneous, heavily pre-treated patient population. Further studies are required to determine the clinical benefit of yeast-CEA vaccination.     

Analyses of the Xylem Sap Proteomes Identified Candidate Fusarium virguliforme Proteinacious Toxins

Background

Sudden death syndrome (SDS) caused by the ascomycete fungus, Fusarium virguliforme, exhibits root necrosis and leaf scorch or foliar SDS. The pathogen has never been identified from the above ground diseased foliar tissues. Foliar SDS is believed to be caused by host selective toxins, including FvTox1, secreted by the fungus. This study investigated if the xylem sap of F. virguliforme-infected soybean plants contains secreted F. virguliforme-proteins, some of which could cause foliar SDS development.

Results

Xylem sap samples were collected from five biological replications of F. virguliforme-infected and uninfected soybean plants under controlled conditions. We identified five F. virguliforme proteins from the xylem sap of the F. virguliforme-infected soybean plants by conducting LC-ESI-MS/MS analysis. These five proteins were also present in the excreted proteome of the pathogen in culture filtrates. One of these proteins showed high sequence identity to cerato-platanin, a phytotoxin produced by Ceratocystis fimbriata f. sp. platani to cause canker stain disease in the plane tree. Of over 500 soybean proteins identified in this study, 112 were present in at least 80% of the sap samples collected from F. virguliforme-infected and -uninfected control plants. We have identified four soybean defense proteins from the xylem sap of F. virguliforme-infected soybean plants. The data have been deposited to the ProteomeXchange with identifier PXD000873.

Conclusion

This study confirms that a few F. virguliforme proteins travel through the xylem, some of which could be involved in foliar SDS development. We have identified five candidate proteinaceous toxins, one of which showed high similarity to a previously characterized phytotoxin. We have also shown the presence of four soybean defense proteins in the xylem sap of F. virguliforme-infected soybean plants. This study laid the foundation for studying the molecular basis of foliar SDS development in soybean and possible defense mechanisms that may be involved in conferring immunity against F. virguliforme and other soybean pathogens.     

The role of abscisic acid (ABA) in ethylene insensitive Gladiolus (Gladiolus grandiflora Hort.) flower senescence

Gladiolus flowers are ethylene insensitive and the signals that start catabolic changes during senescence of gladiolus flower are largely not known. Therefore, experiments were performed to understand the role of abscisic acid (ABA) in ethylene insensitive floral senescence in gladiolus (Gladiolus grandiflora Hort.). It was observed that ABA accumulation increased in attached petals of gladiolus flowers as they senesced. Exogenous application of ABA in vase solution accelerated senescence process in the flowers due to change in various senescence indicators such as enhanced membrane leakage, reduced water uptake, reduced fresh weight and ultimately vase life. Enhancement of in vivo ABA level in petals by creating osmotic stress also upregulates the same parameters of flower senescence as those occurring during natural senescence and also akin to exogenous application of ABA. Attempts to increase vase life of flowers by application of putative ABA biosynthesis inhibitor fluridone in vase solution to counteract ABA effect were unsuccessful. In contrast, ABA action was mitigated by application of GA₃ in holding solution along with ABA which is basically an antagonist of ABA action. The present study provides valuable insights into the role of ABA as a hormonal trigger in ethylene insensitive senescence process and therefore would be helpful for dissecting the complex mechanism underlying ABA-regulated senescence process in gladiolus.     

PeptideMine - A webserver for the design of peptides for protein-peptide binding studies derived from protein-protein interactomes

Background  

Signal transduction events often involve transient, yet specific, interactions between structurally conserved protein domains and polypeptide sequences in target proteins. The identification and validation of these associating domains is crucial to understand signal transduction pathways that modulate different cellular or developmental processes. Bioinformatics strategies to extract and integrate information from diverse sources have been shown to facilitate the experimental design to understand complex biological events. These methods, primarily based on information from high-throughput experiments, have also led to the identification of new connections thus providing hypothetical models for cellular events. Such models, in turn, provide a framework for directing experimental efforts for validating the predicted molecular rationale for complex cellular processes. In this context, it is envisaged that the rational design of peptides for protein-peptide binding studies could substantially facilitate the experimental strategies to evaluate a predicted interaction. This rational design procedure involves the integration of protein-protein interaction data, gene ontology, physico-chemical calculations, domain-domain interaction data and information on functional sites or critical residues.     

A major determinant of cyclophilin dependence and cyclosporine susceptibility of hepatitis C virus identified by a genetic approach

Since the advent of genome-wide small interfering RNA screening, large numbers of cellular cofactors important for viral infection have been discovered at a rapid pace, but the viral targets and the mechanism of action for many of these cofactors remain undefined. One such cofactor is cyclophilin A (CyPA), upon which hepatitis C virus (HCV) replication critically depends. Here we report a new genetic selection scheme that identified a major viral determinant of HCV's dependence on CyPA and susceptibility to cyclosporine A. We selected mutant viruses that were able to infect CyPA-knockdown cells which were refractory to infection by wild-type HCV produced in cell culture. Five independent selections revealed related mutations in a single dipeptide motif (D316 and Y317) located in a proline-rich region of NS5A domain II, which has been implicated in CyPA binding. Engineering the mutations into wild-type HCV fully recapitulated the CyPA-independent and CsA-resistant phenotype and four putative proline substrates of CyPA were mapped to the vicinity of the DY motif. Circular dichroism analysis of wild-type and mutant NS5A peptides indicated that the D316E/Y317N mutations (DEYN) induced a conformational change at a major CyPA-binding site. Furthermore, nuclear magnetic resonance experiments suggested that NS5A with DEYN mutations adopts a more extended, functional conformation in the putative CyPA substrate site in domain II. Finally, the importance of this major CsA-sensitivity determinant was confirmed in additional genotypes (GT) other than GT 2a. This study describes a new genetic approach to identifying viral targets of cellular cofactors and identifies a major regulator of HCV's susceptibility to CsA and its derivatives that are currently in clinical trials.     

Distinctive repertoire of contingency genes conferring mutation- based phase variation and combinatorial expression of surface lipoproteins in Mycoplasma capricolum subsp. capricolum of the Mycoplasma mycoides phylogenetic cluster

The generation of surface variation among many divergent species of Mollicutes (mycoplasmas) occurs through stochastic expression patterns of diverse lipoprotein genes. The size and wide distribution of such variable gene sets in minimal (approximately 0.6- to 1.4-Mb) mycoplasmal genomes suggest their key role in the adaptation and survival of these wall-less monoderms. Diversity through variable genes is less clearly established among phylogenetically similar mycoplasmas, such as the Mycoplasma mycoides cluster of ruminant pathogens, which vary widely in host range and pathobiology. Using (i) genome sequences from two members of this clade, Mycoplasma capricolum subsp. capricolum and M. mycoides subsp. mycoides small colony biotype (SC), (ii) antibodies to specific peptide determinants of predicted M. capricolum subsp. capricolum gene products, and (iii) analysis of the membrane-associated proteome of M. capricolum subsp. capricolum, a novel set of six genes (vmcA to vmcF) expressing distinct Vmc (variable M. capricolum subsp. capricolum) lipoproteins is demonstrated. These occur at two separate loci in the M. capricolum subsp. capricolum genome, which shares striking overall similarity and gene synteny with the M. mycoides subsp. mycoides SC genome. Collectively, Vmc expression is noncoordinate and combinatorial, subject to a single-unit insertion/deletion in a 5' flanking dinucleotide repeat that governs expression of each vmc gene. All vmc genes share modular regions affecting expression and membrane translocation. In contrast, vmcA to vmcD genes at one locus express surface proteins with highly structured size-variable repeating domains, whereas vmcE to vmcF genes express products with short repeats devoid of predicted structure. These genes confer a distinctive, dynamic surface architecture that may represent adaptive differences within this important group of pathogens as well as exploitable diagnostic targets.     

Antioxidant defense mechanism under salt stress in wheat seedlings

The present study was carried out to study the effect of salt stress on cell membrane damage, ion content and antioxidant enzymes in wheat (Triticum aestivum) seedlings of two cultivars salt-tolerant KRL-19 and salt-sensitive WH-542. Seedlings (4-d-old) were irrigated with 0, 50 and 100 mM NaCl. Observations were recorded on the 3^rd and 6^th day after salt treatment and 2^nd day after salt removal. The relative water content declined with induction of salt stress, more in WH-542 than in cv. KRL-19. K⁺/Na⁺ ratio in KRL-19 was higher than in WH-542. WH-542 suffered greater damage to cellular membranes due to lipid peroxidation as indicated by higher accumulation of H₂O₂, MDA and greater leakage of electrolytes than KRL-19. The activities of catalase, peroxidase and ascorbate peroxidase and glutathione reductase increased with increase in salt stress in both the cultivars, however, superoxide dismutase activity declined. Upon desalanization, partial recovery in the activities of these enzymes was observed in KRL-19 and very slow recovery in WH-542.     

Water status of isolated Negev desert populations of Acacia raddiana with different mortality levels

Many populations of native Acacia raddiana trees in ephemeral riverbeds in the Negev desert are suffering from high mortality. Road-building techniques that cut off water to trees in downstream populations and the pumping of aquifers for agriculture have been mooted as causes of this mortality. We studied the water relations of nine isolated populations of these trees using a pressure chamber and stable carbon isotopes in order to determine whether current water stress could be the cause of this mortality. High mortality populations had more negative water potentials, lower relative water contents, and lower cell water volumes at full turgor than low mortality populations. Thus, mortality is correlated with current inter-population differences in water stress. However, there was no difference in water potentials between trees upstream and downstream of roads. Furthermore, greater water stress in trees after a dry winter than in the previous summer indicates that these trees are largely drawing on surface floods for water. Thus, road-building practices and aquifer pumping are unlikely causes of current water stress. Hence, water stress is linked to mortality in these trees, although our results indicate that irregular water stress (perhaps in drought years only) is probably the cause of this mortality.