Relationship between Exchangeable Acidity and Persistence of Orchardgrass (\u3ci\u3eDactylis glomerata\u3c/i\u3e) in Temperate Pastures under Different Management

Orchardgrass is a high productive, highly nutritive grass, but its persistence is low under acid soil condition. Therefore, to obtain the information on the effect of soil acidity on the persistence of orchardgrass in acid soil grasslands, we investigated the relationship between exchangeable acidity (y1) and orchardgrass in sown pastures. Eight temperate pastures (four cutting meadows [CMs] and four cattle grazing pastures [GPs]) were used for vegetation survey and soil samplings. Six or three line transects (50 m each) were fixed in each meadow or pasture, and measurement location (three quadrats 20 cm×20 cm in size and 50 cm apart from each other) was set along the transect at a 10 m interval. The most dominant plant species was recorded after first cut of the meadows in 2018. Soil samples were collected in the area around quadrats, at depth of 0–5 cm for measurement of y1 and other chemical properties. Orchardgrass was dominated in 78% and 37% of CM and GP, respectively. Tall fescue (Festuca arundinacea) was also dominated in 2% and 22% of CM and GP, respectively. In CM, the locations with higher y1 showed a tendency of higher frequency of orchardgrass (P\u3c 0.1) and significantly low frequency of tall fescue (P\u3c 0.05). In contrast in GP, soil y1 did not show significant relationship to the frequency of orchardgrass and tall fescue. Soil chemical properties such as pH, NO3-N, K2O, CaO and MgO show negative relationship to y1 (P\u3c 0.05) in CM or GP, although they did not show significant relationship to the frequency of orchardgrass. These results suggest that survival rate of orchardgrass increases with increase of y1, through preventing establishment of other plant species such as tall fescue under cutting condition, while this trend is not clear under grazing

Polyphenol Concentration of Native Plant Species, and Its Effect on Blood Antioxidant Capacity in Grazing Cattle in a Species-Rich Vegetation in Japan

It is known that polyphenols in plants have a high antioxidant capacity. However, there is scarce information on its concentration in native plants and the effect of antioxidant capacity to grazing animals. In this study, polyphenol concentration of available plants was investigated in a species-rich grazing area in north-eastern district of Japan. In addition, polyphenol concentration in blood serum of grazing cattle was also measured. Eight beef cows grazed in a grazing area (hill pasture 3.1 ha; forest 16.9 ha) from late spring to mid-autumn (142 days), and four cows among the eight grazed at an orchardgrass pasture in mid-summer (10 days). During the grazing period, foraging behaviour was directly observed, and plant species proportion in ingesta and residence time in the hill pasture and the forest were measured. Based on these results, the top 10–13 species were hand-clipped by mimicking foraging manner of cows, and total polyphenol and catechin concentration were measured. Blood samples of cows were also collected during the grazing period, and potential antioxidant [PAO] and serum total antioxidant status [STAS] were analysed. The cows ingested 17–32 plant species in the hill pasture, and 53–73 species in the forest. In contrast, the cows ingested mainly orchardgrass in the sown pasture in mid-summer. Total polyphenol and catechin concentration were higher in tree leaves (112.8–209.3 g/kg DM, 0.081–6.250 g/kg DM) than monocots (11.9–34.0 g/kg DM, 0–0.159g/kg DM). However, those concentration in ingesta of the cows were low throughout the seasons (35.0–56.9 g/kg DM, 0.108–0.467 g/kg DM), as in the sown pasture (26.2 g/kg DM, 0.158 g/kg DM), due to high proportion of monocots in ingesta at the hill pasture (67–75%). PAO (373.4–455.8 μ mol/L) and STAS (769.8–910.0 μ mol/L) of the cows were almost constant throughout the seasons

Tah1 helix-swap dimerization prevents mixed Hsp90 co-chaperone complexes

Specific co-chaperone adaptors facilitate the recruitment of client proteins to the Hsp90 system. Tah1 binds the C-terminal conserved MEEVD motif of Hsp90, thus linking an eclectic set of client proteins to the R2TP complex for their assembly and regulation by Hsp90. Rather than the normal complement of seven α-helices seen in other tetratricopeptide repeat (TPR) domains, Tah1 unusually consists of the first five only. Consequently, the methionine of the MEEVD peptide remains exposed to solvent when bound by Tah1. In solution Tah1 appears to be predominantly monomeric, and recent structures have failed to explain how Tah1 appears to prevent the formation of mixed TPR domain-containing complexes such as Cpr6-(Hsp90)2-Tah1. To understand this further, the crystal structure of Tah1 in complex with the MEEVD peptide of Hsp90 was determined, which shows a helix swap involving the fifth α-helix between two adjacently bound Tah1 molecules. Dimerization of Tah1 restores the normal binding environment of the bound Hsp90 methionine residue by reconstituting a TPR binding site similar to that in seven-helix-containing TPR domain proteins. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes

Multiplex SNP typing by bioluminometric assay coupled with terminator incorporation (BATI)

A multiplex single-nucleotide polymorphism (SNP) typing platform using ‘bioluminometric assay coupled with terminator [2′,3′-dideoxynucleoside triphosphates (ddNTPs)] incorporation’ (named ‘BATI’ for short) was developed. All of the reactions are carried out in a single reaction chamber containing target DNAs, DNA polymerase, reagents necessary for converting PPi into ATP and reagents for luciferase reaction. Each of the four ddNTPs is dispensed into the reaction chamber in turn. PPi is released by a nucleotide incorporation reaction and is used to produce ATP when the ddNTP dispensed is complementary to the base in a template. The ATP is used in a luciferase reaction to release visible light. Only 1 nt is incorporated into a template at a time because ddNTPs do not have a 3′ hydroxyl group. This feature greatly simplifies a sequencing spectrum. The luminescence is proportional to the amount of template incorporated. Only one peak appears in the spectrum of a homozygote sample, and two peaks at the same intensity appear for a heterozygote sample. In comparison with pyrosequencing using dNTP, the spectrum obtained by BATI is very simple, and it is very easy to determine SNPs accurately from it. As only one base is extended at a time and the extension signals are quantitative, the observed spectrum pattern is uniquely determined even for a sample containing multiplex SNPs. We have successfully used BATI to type various samples containing plural target sequence areas. The measurements can be carried out with an inexpensive and small luminometer using a photodiode array as the detector. It takes only a few minutes to determine multiplex SNPs. These results indicate that this novel multiplexed approach can significantly decrease the cost of SNP typing and increase the typing throughput with an inexpensive and small luminometer

Structural basis for phosphorylation-dependent recruitment of tel2 to hsp90 by pih1

Client protein recruitment to the Hsp90 system depends on cochaperones that bind the client and Hsp90 simultaneously and facilitate their interaction. Hsp90 involvement in the assembly of snoRNPs, RNA polymerases, PI3-kinase-like kinases, and chromatin remodeling complexes depends on the TTT (Tel2-Tti1-Tti2), and R2TP complexes-consisting of the AAA-ATPases Rvb1 and Rvb2, Tah1 (Spagh/RPAP3 in metazoa), and Pih1 (Pih1D1 in humans)-that together provide the connection to Hsp90. The biochemistry underlying R2TP function is still poorly understood. Pih1 in particular, at the heart of the complex, has not been described at a structural level, nor have the multiple protein-protein interactions it mediates been characterized. Here we present a structural and biochemical analysis of Hsp90-Tah1-Pih1, Hsp90-Spagh, and Pih1D1-Tel2 complexes that reveal a domain in Pih1D1 specific for binding CK2 phosphorylation sites, and together define the structural basis by which the R2TP complex connects the Hsp90 chaperone system to the TTT complex

Rtp1p Is a Karyopherin-Like Protein Required for RNA Polymerase II Biogenesis

The assembly and nuclear transport of RNA polymerase II (RNA pol II) are processes that require the participation of many auxiliary factors. In a yeast genetic screen, we identified a previously uncharacterized gene, YMR185w (renamed RTP1), which encodes a protein required for the nuclear import of RNA pol II. Using protein affinity purification coupled to mass spectrometry, we identified interactions between Rtp1p and members of the R2TP complex. Rtp1p also interacts, to a different extent, with several RNA pol II subunits. The pattern of interactions is compatible with a role for Rtp1p as an assembly factor that participates in the formation of the Rpb2/Rpb3 subassembly complex and its binding to the Rpb1p-containing subcomplex. Besides, Rtp1p has a molecular architecture characteristic of karyopherins, composed of HEAT repeats, and is able to interact with phenylalanine-glycine-containing nucleoporins. Our results define Rtp1p as a new component of the RNA pol II biogenesis machinery that plays roles in subunit assembly and likely in transport through the nuclear pore complex

γCOP Is Required for Apical Protein Secretion and Epithelial Morphogenesis in Drosophila melanogaster

Background: There is increasing evidence that tissue-specific modifications of basic cellular functions play an important role in development and disease. To identify the functions of COPI coatomer-mediated membrane trafficking in Drosophila development, we were aiming to create loss-of-function mutations in the γCOP gene, which encodes a subunit of the COPI coatomer complex. Principal Findings: We found that γCOP is essential for the viability of the Drosophila embryo. In the absence of zygotic γCOP activity, embryos die late in embryogenesis and display pronounced defects in morphogenesis of the embryonic epidermis and of tracheal tubes. The coordinated cell rearrangements and cell shape changes during tracheal tube morphogenesis critically depend on apical secretion of certain proteins. Investigation of tracheal morphogenesis in γCOP loss-of-function mutants revealed that several key proteins required for tracheal morphogenesis are not properly secreted into the apical lumen. As a consequence, γCOP mutants show defects in cell rearrangements during branch elongation, in tube dilation, as well as in tube fusion. We present genetic evidence that a specific subset of the tracheal defects in γCOP mutants is due to the reduced secretion of the Zona Pellucida protein Piopio. Thus, we identified a critical target protein of COPI-dependent secretion in epithelial tube morphogenesis. Conclusions/Significance: These studies highlight the role of COPI coatomer-mediated vesicle trafficking in both general and tissue-specific secretion in a multicellular organism. Although COPI coatomer is generally required for protein secretion, we show that the phenotypic effect of γCOP mutations is surprisingly specific. Importantly, we attribute a distinct aspect of the γCOP phenotype to the effect on a specific key target protein

DNAAF1 links heart laterality with the AAA+ ATPase RUVBL1 and ciliary intraflagellar transport

DNAAF1 (LRRC50) is a cytoplasmic protein required for dynein heavy chain assembly and cilia motility, and DNAAF1 mutations cause primary ciliary dyskinesia (PCD; MIM 613193). We describe four families with DNAAF1 mutations and complex congenital heart disease (CHD). In three families, all affected individuals have typical PCD phenotypes. However, an additional family demonstrates isolated CHD (heterotaxy) in two affected siblings, but no clinical evidence of PCD. We identified a homozygous DNAAF1 missense mutation, p.Leu191Phe, as causative for heterotaxy in this family. Genetic complementation in dnaaf1-null zebrafish embryos demonstrated the rescue of normal heart looping with wild-type human DNAAF1, but not the p.Leu191Phe variant, supporting the conserved pathogenicity of this DNAAF1 missense mutation. This observation points to a phenotypic continuum between CHD and PCD, providing new insights into the pathogenesis of isolated CHD. In further investigations of the function of DNAAF1 in dynein arm assembly, we identified interactions with members of a putative dynein arm assembly complex. These include the ciliary intraflagellar transport protein IFT88 and the AAA+ (ATPases Associated with various cellular Activities) family proteins RUVBL1 (Pontin) and RUVBL2 (Reptin). Co-localization studies support these findings, with the loss of RUVBL1 perturbing the co-localization of DNAAF1 with IFT88. We show that RUVBL1 orthologues have an asymmetric left-sided distribution at both the mouse embryonic node and the Kupffer’s vesicle in zebrafish embryos, with the latter asymmetry dependent on DNAAF1. These results suggest that DNAAF1-RUVBL1 biochemical and genetic interactions have a novel functional role in symmetry breaking and cardiac development

An invasive podosome-like structure promotes fusion pore formation during myoblast fusion

An F-actin–enriched protrusion resembling an invasive podosome promotes fusion pore formation between muscle founder cells and fusion-competent myoblasts