Jasmonates Inhibit Flowering in Short-Day Plant Pharbitis nil

The role of jasmonates in the photoperiodic flower induction of short-day plant Pharbitis nil was investigated. The plants were grown in a special cycle: 72 h of darkness, 24 h of white light with lowered intensity, 24-h long inductive night, 14 days of continuous light. At 4 h of inductive night the cotyledons of non-induced plants contained about two times the amount of endogenous jasmonates (JA/JA-Me) compared to those induced. A 15-min long pulse of far red light (FR) applied at the end of a 24-h long white light phase inhibited flowering of P. nil. The concentration of jasmonates at 2 and 4 h of inductive night in the cotyledons of the plants treated with FR was similar. Red light (R) could reverse the effect of FR. R light applied after FR light decreased the content of jasmonates by about 50%. Methyl jasmonate (JA-Me) applied to cotyledons, shoot apices and cotyledon petioles of P. nil inhibited the formation of flower buds during the first half of a 24-h long inductive or 14-h long subinductive night. Application of JA-Me to the cotyledons was the most effective. None of the plants treated with JA-Me on the cotyledons in the middle of the inductive night formed terminal flower buds. The aspirin, ibuprofen and phenidone, jasmonates biosynthesis inhibitors partially reversed the effect of FR, stimulating the formation of axillary and terminal flower buds. Thus, the results obtained suggests that phytochrome system control both the photoperiodic flower induction and jasmonates metabolism. Jasmonates inhibit flowering in P. nil.     

The effect of calcium on the production of ethylene and abscisic acid by fungus Fusarium culmorum and by wheat seedlings infected with that pathogen

Wheat seedlings infected and non-infected with Fusarium culmorum were grown on mediums with different content of calcium (0, 2, 4, 8 mM). It was found that the higher the content of calcium in the medium, the greater the amounts of ethylene produced in both non-infected and infected wheat seedlings, whereas the level of ABA in their tissues was decreased. Taking into consideration the fact that ethylene inhibits, whereas ABA stimulates the growth and development of Fusarium culmorum, it may be assumed that the production of ethylene increased under the influence of calcium and the decreased level of ABA in wheat seedlings causes the reduction growth and development of pathogen and as a result it lowers the degree of infection of wheat seedlings by this fungus. Thus, on the base of the obtained results it may be concluded that the inhibiting influence of calcium on injurying the wheat seedlings by Fusarium culmorum may be caused by the influence of this cation on the hormone balance in the plant.     

Crystal structure and functional characterization of selenocysteine-containing glutathione peroxidase 4 suggests an alternative mechanism of peroxide reduction

Glutathione peroxidases (GPX) are anti-oxidative enzymes that reduce organic and inorganic hydroperoxides to the corresponding alcohols at the expense of reduced glutathione. The human genome involves eight GPX genes and five of them encode for selenocysteine-containing enzymes. Among the human GPX-isoforms, GPX4 is unique since it is capable of reducing complex hydroperoxy ester lipids such as hydroperoxy phospholipids and hydroperoxy cholesterolesters. Using a number of genetically modified mouse strains the biological role of GPX4 has comprehensively characterized but the molecular enzymology is less well explored. This lack of knowledge is partly related to the fact that mammalian selenoproteins are not high-level expressed in conventional overexpression systems. To explore the structural and functional properties of human GPX4 we expressed this selenoprotein in a cysteine-auxotrophic E. coli strain using a semi-chemical expression strategy. The recombinant enzyme was purified in mg amounts from the bacterial lysate to electrophoretic homogeneity and characterized with respect to its protein-chemical and enzymatic properties. Its crystal structure was solved at 1.3?Å resolution and the X-ray data indicated a monomeric protein, which contains the catalytic selenium at the redox level of the seleninic acid. These data suggest an alternative reaction mechanism involving three different redox states (selenol, selenenic acid, seleninic acid) of the catalytically active selenocysteine.     

The in vitro effect of progesterone on the orexin system in porcine uterine tissues during early pregnancy

Background

Orexin A (OXA) and orexin B (OXB) are hypothalamic-derived peptides that participate in the regulation of energy metabolism, food intake and reproductive function by influencing the hypothalamic-pituitary-ovarian axis. Orexins are also produced in the endometrium, myometrium and placenta, which suggests that they could act as a link between energy metabolism and the reproductive system. Changes in the expression of orexin and the orexin receptor genes and proteins during the oestrous cycle and early gestation in pigs imply that orexin activity may be regulated by local factors within the uterus. The aim of this study was to investigate the influence of progesterone (P₄) on the expression of orexin system genes, and proteins in the porcine uterus during early gestation. Gene expression was analyzed by real-time PCR. Adiponectin secretion was determined by ELISA, and the receptors proteins content was defined using western blot analysis.

Results

In the endometrium, P₄ enhanced OXA secretion on days 10 to 11 of gestation and OXB secretion on days 12 to 13. In the myometrium, P₄ inhibited the secretion of both orexins on days 15 to 16 and OXB secretion also on days 12 to 13. In the endometrium, P₄ inhibited the expression of orexin receptor 1 (OX1R) protein at nearly all times analyzed, whereas the expression of orexin receptor 2 (OX2R) protein was inhibited only on days 15 to 16 of gestation. In the myometrium, P₄ stimulated OX1R protein expression on days 12 to 13 and 15 to 16 of gestation and inhibited OX1R protein expression on days 27 to 28. The expression of OX2R protein in the myometrium increased on days 12 to 13 and decreased on days 10 to 11 and 15 to 16.

Conclusions

The results indicate that P₄ could regulate the expression of the orexin system in the porcine uterus during early pregnancy, which suggests the presence of a local feedback loop that could play an important role in the regulation of maternal metabolism during pregnancy. The findings may contribute to the existing knowledge of the mechanisms linking maternal energy metabolism with the regulation of the reproductive system during pregnancy.

     

Signaling Pathways in Murine Dendritic Cells That Regulate the Response to Vesicular Stomatitis Virus Vectors That Express Flagellin

Vesicular stomatitis virus (VSV) vectors that express heterologous antigens have shown promise as vaccines in preclinical studies. The efficacy of VSV-based vaccines can be improved by engineering vectors that enhance innate immune responses. We previously generated a VSV vaccine vector that incorporates two enhancing strategies: an M protein mutation (M51R) that prevents the virus from suppressing host antiviral responses and a gene encoding bacterial flagellin (M51R-F vector). The rationale was that intracellular expression of flagellin would activate innate immune pathways in addition to those activated by virus alone. This was tested with dendritic cells (DCs) from mice containing deletions in key signaling molecules. Infection of DC with either M51R or M51R-F vector induced the production of interleukin-12 (IL-12) and IL-6 and increased surface expression of T cell costimulatory molecules. These responses were dramatically reduced in DCs from IPS-1^−/− mice. Infection with M51R-F vector also induced the production of IL-1β. In addition, in approximately half of the DCs, M51R-F vector induced pyroptosis, a proinflammatory-type of cell death. These responses to flagellin were ablated in DCs from NLRC4^−/− mice but not Toll-like receptor 5-deficient (TLR5^−/−) mice, indicating that they resulted from inflammasome activation. These results demonstrate that flagellin induces additional innate immune mechanisms over those induced by VSV alone.     

Genetic diversity and horizontal transfer of genes involved in oxidation of reduced phosphorus compounds by Alcaligenes faecalis WM2072

Enrichment was performed to isolate organisms that could utilize reduced phosphorus compounds as their sole phosphorus sources. One isolate that grew well with either hypophosphite or phosphite was identified by 16S rRNA gene analysis as a strain of Alcaligenes faecalis. The genes required for oxidation of hypophosphite and phosphite by this organism were identified by using transposon mutagenesis and include homologs of the ptxD and htxA genes of Pseudomonas stutzeri WM88, which encode an NAD-dependent phosphite dehydrogenase (PtxD) and 2-oxoglutarate-dependent hypophosphite dioxygenase (HtxA). This organism also has the htxB, htxC, and htxD genes that comprise an ABC-type transporter, presumably for hypophosphite and phosphite transport. The role of these genes in reduced phosphorus metabolism was confirmed by analyzing the growth of mutants in which these genes were deleted. Sequencing data showed that htxA, htxB, htxC, and htxD are virtually identical to their homologs in P. stutzeri at the DNA level, indicating that horizontal gene transfer occurred. However, A. faecalis ptxD is very different from its P. stutzeri homolog and represents a new ptxD lineage. Therefore, this gene has ancient evolutionary roots in bacteria. These data suggest that there is strong evolutionary selection for the ability of microorganisms to oxidize hypophosphite and phosphite.     

Knockdown of Na,K-ATPase β-subunits in Oncopeltus fasciatus induces molting problems and alterations in tracheal morphology

The ubiquitously expressed transmembrane enzyme Na,K-ATPase (NKA) is vital in maintaining functionality of cells. The association of α- and β-subunits is believed to be essential for forming a functional enzyme. In the large milkweed bug Oncopeltus fasciatus four α1-paralogs and four β-subunits exist that can associate into NKA complexes. This diversity raises the question of possible tissue-specific distribution and function. While the α1-subunits are known to modulate cardenolide-resistance and ion-transport efficiency, the functional importance of the β-subunits needed further investigation. We here characterize all four different β-subunits at the cellular, tissue, and whole organismal scales. A knockdown of different β-subunits heavily interferes with molting success resulting in strongly hampered phenotypes. The failure of ecdysis might be related to disrupted septate junction (SJ) formation, also reflected in β2-suppression-induced alteration in tracheal morphology. Our data further suggest the existence of isolated β-subunits forming homomeric or β-heteromeric complexes. This possible standalone and structure-specific distribution of the β-subunits predicts further, yet unknown pump-independent functions. The different effects caused by β knockdowns highlight the importance of the various β-subunits to fulfill tissue-specific requirements.     

Microsatellite identification of ramet genotypes in a clonal plant with phalanx growth: The case of Cirsium rivulare (Asteraceae)

Cirsium rivulare is a perennial plant that forms patches consisting of ramets resulting from sexual reproduction by seeds and asexual propagation by rhizome fragmentation. We examined the relationship between the size of patches and genetic differentiation of ramets within and between patches. Ramet genotypes were identified using microsatellites. From among 216 ramets examined in the studied population, 123 had a unique genotype, while 93 were clonal, i.e., their genotype was present in at least two ramets. The frequency of ramets with clonal genotypes was 43% and the frequency of unique genotypes was 57%. Ramets with identical genotypes were dominant in small patches. Large patches consisted of ramets with both unique and clonal genotypes, usually with the predominance of the latter. A molecular variance analysis showed the highest level of variance between ramets and the lowest between patches. Additionally, 21.02% of the total variance was recorded between ramets and within patches. The size of patches was correlated with the number of clonal ramets and the number of unique ramets, but it was not correlated with the clonality index. This population of C. rivulare is currently in a phase of decline from 30 years of vegetation transformation, and there appears to have been an increase in sexual propagation based growth over clonal propagation based growth. Hence, a predominance of ramets with unique genotypes was observed. This can happen as a result of disintegration of large patches and formation of gaps between them. These gaps become convenient places for seed germination and the subsequent development of seedlings.     

Down-regulation by nuclear factor kappaB of human 25-hydroxyvitamin D3 1alpha-hydroxylase promoter

1,25-(OH)(2) vitamin D(3) is important for calcium homeostasis and cell differentiation. The key enzyme for the activation of liver-derived 25(OH) vitamin D(3) is 25-hydroxyvitamin D(3) 1alpha-hydroxylase. It is expressed mainly in the kidney but also in peripheral tissues. A 1413-bp fragment of the 1alpha-hydroxylase promoter was cloned into luciferase vectors pGL2basic and pGL3basic. Sequence analyses revealed four base exchanges and three base deletions compared with the published sequence which were identically found in five control persons. In silico promoter analyses revealed 17 putative nuclear factor (NF)kappaB sites, 10 of which were found to bind NFkappaB in EMSA experiments. Cotransfection of NFkappaB p50 and p65 subunits resulted in dramatic reduction of the promoter activity of the full-length construct as well as a series of 5'-deletion constructs. Deletion of the farmost 3'-situated NFkappaB-responsive element almost abolished NFkappaB responsiveness. Treatment of human embryonic kidney 293 cells with sulfasalazine, a NFkappaB inhibitor, resulted in enhanced 1alpha-hydroxylase mRNA production. Down-regulation of 1alpha-hydroxylase promoter through NFkappaB signaling may contribute to the pathogenesis of inflammation-associated osteopenia/osteoporosis.