Growth requirement for N as a criterion to assess the effects of physical manipulation on nitrate uptake fluxes in spinach

The effects of physical manipulation of hydroponically grown plants of spinach (Spinacia oleracea L., cvs Subito and Glares) on nitrate uptake fluxes were studied in a long-term experiment (3 days), and in short-term label experiments (2 h) with ¹³N-nitrate and ¹⁵N-nitrate. In the long-term experiment, net nitrate uptake rate (NNUR) was measured by following the nitrate depletion in the uptake solution, which was replaced at regular intervals. In the short-term experiments, NNUR and nitrate influx were measured by simultaneous application of ¹³N-nitrate and ¹⁵N-nitrate. Plants were gently transferred into the labelled uptake solution, as is usually done in nutrient uptake studies. In addition, a more severe physical manipulation was carried out, including blotting of the roots, to mimic pretreatments which involve more handling of the plants prior to uptake measurements. Nitrate influx was measured immediately after physical manipulation and after 2 h of recovery. To assess the impact of the physical manipulation the experimentally determined nitrate uptake fluxes were compared with the N demand for growth, defined as relative growth rate (RGR) times plant nitrogen concentration (PNC) of parallel plants, which were left undisturbed. Nitrate influx and efflux were both subject to changes after physical manipulation of the plants. Physical handling, however, did not always result in an alteration of NNUR, which complicates the determination of the length of the recovery period. The impact of the handling and the time course of the recovery depended on the severity of the disturbance and were independent of the light conditions during the experiments. Even after a gentle transfer of the plants, recovery, in most cases, was not complete within 2 h. The data emphasise the need for minimal disturbance of plants during the last hours prior to nutrient uptake measurements.     

Synthesis and SAR of 2,3-diarylpyrrole inhibitors of parasite cGMP-dependent protein kinase as novel anticoccidial agents

Several analogs of 2,3-diaryl pyrroles were synthesized and evaluated as inhibitors of Eimeria tenella cGMP-dependent protein kinase and in in vivo anticoccidial assays. A 4-fluorophenyl group enhances both in vitro and in vivo activities. The most potent analogs are the 5-(N-methyl, N-ethyl, and N-methylazetidine methyl) piperidyl derivatives 12, 23, and 34. These compounds have a broad spectrum of activity. Based on the in vivo efficacy and cost of synthesis, the N-ethyl analog 23 was chosen as a novel anticoccidial agent for a field trial.     

APC Inhibits Ligand-Independent Wnt Signaling by the Clathrin Endocytic Pathway

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Phosphorus effects on metabolic processes in monoxenic arbuscular mycorrhiza cultures

The influence of external phosphorus (P) on carbon (C) allocation and metabolism as well as processes related to P metabolism was studied in monoxenic arbuscular mycorrhiza cultures of carrot (Daucus carota). Fungal hyphae of Glomus intraradices proliferated from the solid minimal medium containing the colonized roots into C-free liquid minimal medium with different P treatments. The fungus formed around three times higher biomass in P-free liquid medium than in medium with 2.5 mM inorganic P (high-P). Mycelium in the second experiment was harvested at an earlier growth stage to study metabolic processes when the mycelium was actively growing. P treatment influenced the root P content and [(13)C]glucose administered to the roots 7 d before harvest gave a negative correlation between root P content and (13)C enrichment in arbuscular mycorrhiza fungal storage lipids in the extraradical hyphae. Eighteen percent of the enriched (13)C in extraradical hyphae was recovered in the fatty acid 16:1omega5 from neutral lipids. Polyphosphate accumulated in hyphae even in P-free medium. No influence of P treatment on fungal acid phosphatase activity was observed, whereas the proportion of alkaline-phosphatase-active hyphae was highest in high-P medium. We demonstrated the presence of a motile tubular vacuolar system in G. intraradices. This system was rarely seen in hyphae subjected to the highest P treatment. We concluded that the direct responses of the extraradical hyphae to the P concentration in the medium are limited. The effects found in hyphae seemed instead to be related to increased availability of P to the host root.     

Elevated p16ink4a Expression in Human Labial Salivary Glands as a Potential Correlate of Cognitive Aging in Late Midlife

Background

The cell-cycle inhibitor and tumor suppressor cyclin dependent kinase inhibitor, p16^ink4a, is one of the two gene products of the ink4a/ARF (cdkn2a) locus on chromosome 9q21. Up-regulation of p16^ink4a has been linked to cellular senescence, and findings from studies on different mammalian tissues suggest that p16^ink4a may be a biomarker of organismal versus chronological age.

Objective

The aim of this study was to examine the immunolocalization pattern of p16^ink4a in human labial salivary gland (LSG) tissue, and to analyze whether its expression level in LSGs is a peripheral correlate of cognitive decline in late midlife.

Methods

The present study was a part of a study of causes and predictors of cognitive decline in middle-aged men in a Danish birth cohort. It is based on data from 181 male participants from the Danish Metropolit birth cohort, born in 1953, who were examined for age-associated alterations in cognition, dental health, and morphological and autonomic innervation characteristics of the LSGs. The participants were allocated to two groups based on the relative change in cognitive performance from young adulthood to late midlife. LSG biopsies were analyzed by qRT-PCR for the expression level of p16^ink4a. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections of LSGs.

Results

p16^ink4a immunoreactivity was observed in LSG ductal, myoepithelial, and stromal cells, but not in acinar cells. The mean relative expression of p16^ink4a in LSGs was higher in the group of participants with decline in cognitive performance. A logistic regression analysis revealed that the relative p16 expression was predictive of the participant’s group assignment. A negative correlation was found between relative p16^ink4a expression and the participant’s standardized regression residuals from early adulthood to late midlife cognitive performance scores.

Conclusions

p16^ink4a expression in human LSGs may constitute a potential peripheral correlate of cognitive decline. Human labial salivary glands seem suitable for studies on organismal as opposed to chronological age.     

Fast identification of folded human protein domains expressed in <Emphasis Type="Italic">E. coli</Emphasis> suitable for structural analysis

Background  

High-throughput protein structure analysis of individual protein domains requires analysis of large numbers of expression clones to identify suitable constructs for structure determination. For this purpose, methods need to be implemented for fast and reliable screening of the expressed proteins as early as possible in the overall process from cloning to structure determination.     

Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites

O-GalNAc-glycosylation is one of the main types of glycosylation in mammalian cells. No consensus recognition sequence for the O-glycosyltransferases is known, making prediction methods necessary to bridge the gap between the large number of known protein sequences and the small number of proteins experimentally investigated with regard to glycosylation status. From O-GLYCBASE a total of 86 mammalian proteins experimentally investigated for in vivo O-GalNAc sites were extracted. Mammalian protein homolog comparisons showed that a glycosylated serine or threonine is less likely to be precisely conserved than a nonglycosylated one. The Protein Data Bank was analyzed for structural information, and 12 glycosylated structures were obtained. All positive sites were found in coil or turn regions. A method for predicting the location for mucin-type glycosylation sites was trained using a neural network approach. The best overall network used as input amino acid composition, averaged surface accessibility predictions together with substitution matrix profile encoding of the sequence. To improve prediction on isolated (single) sites, networks were trained on isolated sites only. The final method combines predictions from the best overall network and the best isolated site network; this prediction method correctly predicted 76% of the glycosylated residues and 93% of the nonglycosylated residues. NetOGlyc 3.1 can predict sites for completely new proteins without losing its performance. The fact that the sites could be predicted from averaged properties together with the fact that glycosylation sites are not precisely conserved indicates that mucin-type glycosylation in most cases is a bulk property and not a very site-specific one. NetOGlyc 3.1 is made available at www.cbs.dtu.dk/services/netoglyc.