Secreted kinase phosphorylates extracellular proteins that regulate biomineralization

Protein phosphorylation is a fundamental mechanism regulating nearly every aspect of cellular life. Several secreted proteins are phosphorylated, but the kinases responsible are unknown. We identified a family of atypical protein kinases that localize within the Golgi apparatus and are secreted. Fam20C appears to be the Golgi casein kinase that phosphorylates secretory pathway proteins within S-x-E motifs. Fam20C phosphorylates the caseins and several secreted proteins implicated in biomineralization, including the small integrin-binding ligand, N-linked glycoproteins (SIBLINGs). Consequently, mutations in Fam20C cause an osteosclerotic bone dysplasia in humans known as Raine syndrome. Fam20C is thus a protein kinase dedicated to the phosphorylation of extracellular proteins.     

Metallothionein induction in cultured fibroblasts and liver of a marine flatfish,the turbot,Scophthalmus maximus

Intraperitoneal injection of turbot with Cd induced the synthesis of a low molecular weight hepatic Cd-binding protein and a 500bp mRNA, which hybridised to a plaice metallothionein (MT) cRNA probe. The Cd-binding protein displayed cross-reactivity in a competitive ELISA with antiserum raised against rainbow trout MT and had the characteristic amino acid composition, metal stoichiometry and spectral characteristics of a Class I MT. Only one isoform was apparent on ion exchange chromatography. Southern blot analysis of DNA cleaved with four restriction enzymes suggested that only a single MT gene is present in turbot.In an established turbot fibroblast cell line, Cd induced MT mRNA and MT levels in a dose and time-dependent manner. MT was also induced by Cu, Hg and Zn but not Pb exposure. Physiological concentrations of glucocorticoids and sex hormones did not induce MT synthesis, although at high concentrations a positive response to corticosterone, dexamethasone, hydrocortisone or progesterone was observedin vitro indicating the possible presence of a functional steroid regulatory element in the fish MT gene.Abbreviations used AMP adenosine monophosphate - CHSE chinook salmon embryo - DMSO dimethyl sulphoxide - HPLC high performance liquid chromatography - KB kenacid blue - MT metallothionein - Mr molecular weight (kDaltons) - NR neutral red - PEG polyethylene glycol - RTH rainbow trout hepatoma - SDS sodium dodecyl sulphate - SSC 0.15M NaCl, 0.015M sodium citrate - TF turbot fibroblast     

Application of sperm cryopreservation in selective breeding of the Pacific oyster,Crassostrea gigas (Thunberg)

The robustness of Pacific oyster, Crassostrea gigas (Thunberg), sperm cryopreservation in the context of selective breeding based on family lines was investigated. Irrespective of egg density, high fertilization success was achieved with cryopreserved sperm when sperm:egg ratios of 1000:1 to 10 000:1 were used. Variation among replicate runs on the same oyster batches was minimal, indicating that cryopreservation and larval rearing procedures were repeatable. Twenty independent single male–female crosses were made to assess the utility of cryopreserved sperm in selective breeding. The fertility of unfrozen sperm was generally a poor predictor of cryopreserved sperm fertility. Based on D‐larval yields, 17 of the 20 crosses were likely to yield adequate spat for selective breeding (>10⁵ D‐larvae from 1 million eggs), two were marginal (5 × 10⁴ D‐larvae) and one was inadequate (4 × 10³ D‐larvae). An alternative fertilization strategy to improve D‐yield from a given number of sperm was then tested. Fertilizing 10 million eggs at a sperm:egg ratio of 200:1 increased the total D‐yield when compared with fertilizing 1 million eggs at a sperm:egg ratio of 2000:1 for the same male–female pair. We conclude that, despite wide variation in fertility, cryopreserved sperm is useful for family production.     

Protein-DNA interactions in vivo upstream of a cell cycle-regulated human H4 histone gene

Cell cycle-dependent histone genes are transcribed at a basal level throughout the cell cycle, with a three- to fivefold increase during early S phase. Protein-DNA interactions in the 5' promoter region of a cell cycle-regulated human H4 histone gene have been analyzed at single-nucleotide resolution in vivo. This region contains two sites, with four potential protein-binding domains, at which the DNA is protected from reaction with dimethyl sulfate in cells and from digestion with deoxyribonuclease I in nuclei. These protein-DNA interactions persist during all phases of the cell cycle and dissociate with 0.16 to 0.2M sodium chloride.     

Physicochemical property guidelines for modern agrochemicals

The relentless need for the discovery and development of new agrochemicals continues as a result of driving forces such as loss of existing products through the development of resistance, the necessity for products with more favorable environmental and toxicological profiles, shifting pest spectra, and the changing agricultural needs and practices of the farming community. These new challenges underscore the demand for novel, high‐quality starting points to accelerate the discovery of new agrochemicals that address market challenges. This article discusses the efforts to identify the optimum ranges of physicochemical properties of agrochemicals through analysis of modern commercial products. Specifically, we reviewed literature studies examining physicochemical property effects and analyzed the properties typical of successful fungicides, herbicides, and insecticides (chewing and sap‐feeding pests). From the analysis, a new set of physicochemical property guidelines for each discipline, as well as building block class, are proposed. These new guidelines should significantly aid in the discovery of next‐generation agrochemicals. © 2018 Society of Chemical Industry     

Effect of xylem fluid from susceptible and resistant grapevines on developmental biology of Xylella fastidiosa

Xylella fastidiosa causes Pierce??s disease (PD), a serious disease in grapevines, and grapevine cultivars vary in susceptibility to X. fastidiosa in the field. The mechanism(s) by which this occurs has not been clearly elucidated. To explore possible mechanisms, X. fastidiosa cells from a PD strain were grown in pure xylem fluid of PD-susceptible grapevines, Vitis vinifera and V. labrusca, versus PD-resistant grapevines, V. champinii and V. smalliana. When grown in xylem fluid from the susceptible species, X. fastidiosa cells formed a heavier biofilm compared to those in xylem fluid from the resistant species. Differential expression of selected genes of X. fastidiosa cultured in the xylem fluids of V. vinifera and V. smalliana was analyzed using a DNA macroarray. Compared with xylem fluid of V. smalliana, xylem fluid of V. vinifera stimulated the expression of X. fastidiosa genes involved in virulence regulation, such as rpfC, gacA, xrvA, gcvR, and cysB, and genes involved in biogenesis of pili and twitching motility, such as pilI, pilU, pilE and pilG. Increased expression of virulence genes likely contributes to the expression of PD symptom in the susceptible grapevines, whereas reduced expression of these genes may lead to limitation of symptoms in resistant grapevines.     

Population variation of apple scab (Venturia inaequalis) within mixed orchards in the UK

Apple scab, caused by Venturia inaequalis, is one of the most important apple diseases worldwide. To investigate between- and within-orchard fungal variability, 212 isolates were sampled from two mixed orchards, one of 10?years of age and the other of 45?years of age, in the UK and genotyped with AFLP and SSR markers. Populations of isolates from the two orchards did not differ significantly in terms of allele frequencies at the screened AFLP and SSR loci. However, groups of isolates from individual cultivars differed significantly within each orchard and there were also significant differences between groups of isolates from individual trees of the same cultivar in the same orchard. These differences were less pronounced in the younger mixed orchard than in the older one. The existence of tree-to-tree fungal variability indicates a possible role for conidia as a source of primary inoculum. Non-random mating may be one of the factors causing the significant differences among fungal populations from different cultivars. These results suggest that apparently ??susceptible?? cultivars have different background genetic resistance factors, which can be exploited for disease management in mixtures.     

Spatial and temporal distribution of imidacloprid and thiamethoxam in citrus and impact on Homalodisca coagulata populations

Titers of two systemic neonicotinoid insecticides in citrus trees were measured in conjunction with conventional evaluations of their impact on glassy-winged sharpshooter (Homalodisca coagulata (Say); GWSS) populations. Xylem fluid samples were collected at regular intervals and from multiple locations within field-grown citrus trees to determine imidacloprid and thiamethoxam concentrations using commercial ELISA kits. Uptake profiles varied considerably with peak mean titers of imidacloprid occurring 6-8 weeks after application compared with 2 weeks for thiamethoxam. The persistence of each compound also varied as near-peak levels of imidacloprid were sustained for another 6-10 weeks before gradually declining. In contrast, thiamethoxam titers declined more rapidly after the initial peak, possibly reflecting an application rate only one-quarter of that used for imidacloprid. Within-tree distributions were more similar for the two compounds, with no significant effect due to height of the sample (upper or lower half) or to the quadrant location within the tree, with the exception of one quadrant in the thiamethoxam-treated trees. Substantial reductions in GWSS nymphs and adults were observed in imidacloprid-treated trees during the 2001 trial and were sustained for 4-5 months after treatment. Treatment effects on nymphs were not as well pronounced in the 2002 trial, when overall GWSS infestations were much reduced from the previous year. However, consistently lower adult infestations were still observed in 2002 for both treatments compared with untreated trees. Information on the spatial and temporal profiles in citrus trees was obtained for both compounds to complement field impact data and improve understanding of their pest management potential.