Isoelectric focusing and 2D electrophoresis of the human androgen receptor.

Nuclear androgen receptors from cultured genital skin fibroblasts were analyzed by non-denaturing isoelectric focusing (IEF) in ultrathin polyacrylamide gels before and after photoaffinity labeling with [3H]methyltrienolone. Both reversibly and covalently labeled receptors focused at pH 5.28 +/- 0.20 when extracted from nuclei with high salt. Lowering of the salt concentration yielded, in both cases, a second species which focused at pH 7.16. This species became predominant when nuclei were sonicated in IEF sample buffer containing no salt, even after extensive nucleic acid digestion. Low salt cytosols from both prostate and foreskin focused as a single peak of pI: 4.93 +/- 0.31 which remained unchanged when KCl was added to the cytosol up to a concentration of 0.6 M. SDS-polyacrylamide gel electrophoresis of photoaffinity labeled receptors revealed labeled proteins with Mw 90-95 kDa. Two-dimensional electrophoresis of photoaffinity labeled nuclear receptors, extracted in low or high salt, showed that the two isoforms (pI 5.28 and 7.16) contain the same steroid-binding subunit with Mw 90-95 kDa. Nuclear receptors from 4 patients with the receptor positive form of the Complete Androgen Insensitivity Syndrome (CAIS, Rc+) were analyzed by non-denaturing IEF: a single species was observed, focusing at pH 6.0 whether in high or low salt conditions. These results indicate that the nuclear androgen receptor is an acidic protein with pI 5.28 and Mw 90-95 kDa under maximum protein dissociation conditions. When extracted under low salt conditions, it can be isolated in a neutral form (pI 7.16) suggesting its association with a nuclear protein. Receptors of (CAIS, Rc+) patients have an abnormal charge and show no pI shift upon lowering of the salt concentration suggesting that this shift could be a significant step in the mechanism of action of androgens.     

Coprophilous mite communities as affected by concentration of plastic and glass particles

The community and trophic structure of mites was examined in cattle manure and their response to treatment of manure with solid-waste particles was investigated. Mites were collected from artificial dung pats exposed to natural colonization in the field for 16 days. The pats were composed of manure alone or manure separately mixed with waste-material particles of four types and concentrations.Mites were the numerically dominant taxon among arthropods in manure. The Prostigmata was the most abundant suborder, mainly represented by the families Pygmephoridae and Ereynetidae. The common families of Mesostigmata were all cosmopolitan. Correlations with other arthropods suggest that Mesostigmata are opportunistic predators, preying upon dipteran larvae, Collembola and other mites, and preyed upon by larger predatory insect larvae. Cryptostigmata and Astigmata were a numerically minor community component.The species composition and abundance upon treatment of manure with polyethylene, polystyrene and glass particles was similar to that of the treatment control, i.e. natural and inert sand. Relatively low concentrations, 5 and 30% (v/v), of particles in manure did not alter the mite communities despite important differences with untreated manure in final moisture content. These findings may be relevant to proposed methods of solid-waste disposal. High particle concentrations of 60 and 90% had detrimental effects to the abundance of mites in manure and were caused by the very low final moisture content and probably nutrient deficiency of these treatments. The persistence of members of the prostigmatid families Tydeidae, Nanorchestidae and Tarsonemidae in the moisture-deficient 90%-concentration treatments supports previous evidence of adaptations to low-water-content habitats.     

Essential Role of the EF-hand Domain in Targeting Sperm Phospholipase Cζ to Membrane Phosphatidylinositol 4,5-Bisphosphate (PIP2)

Sperm-specific phospholipase C-ζ (PLCζ) is widely considered to be the physiological stimulus that triggers intracellular Ca²⁺ oscillations and egg activation during mammalian fertilization. Although PLCζ is structurally similar to PLCδ1, it lacks a pleckstrin homology domain, and it remains unclear how PLCζ targets its phosphatidylinositol 4,5-bisphosphate (PIP₂) membrane substrate. Recently, the PLCδ1 EF-hand domain was shown to bind to anionic phospholipids through a number of cationic residues, suggesting a potential mechanism for how PLCs might interact with their target membranes. Those critical cationic EF-hand residues in PLCδ1 are notably conserved in PLCζ. We investigated the potential role of these conserved cationic residues in PLCζ by generating a series of mutants that sequentially neutralized three positively charged residues (Lys-49, Lys-53, and Arg-57) within the mouse PLCζ EF-hand domain. Microinjection of the PLCζ EF-hand mutants into mouse eggs enabled their Ca²⁺ oscillation inducing activities to be compared with wild-type PLCζ. Furthermore, the mutant proteins were purified, and the in vitro PIP₂ hydrolysis and binding properties were monitored. Our analysis suggests that PLCζ binds significantly to PIP₂, but not to phosphatidic acid or phosphatidylserine, and that sequential reduction of the net positive charge within the first EF-hand domain of PLCζ significantly alters in vivo Ca²⁺ oscillation inducing activity and in vitro interaction with PIP₂ without affecting its Ca²⁺ sensitivity. Our findings are consistent with theoretical predictions provided by a mathematical model that links oocyte Ca²⁺ frequency and the binding ability of different PLCζ mutants to PIP₂. Moreover, a PLCζ mutant with mutations in the cationic residues within the first EF-hand domain and the XY linker region dramatically reduces the binding of PLCζ to PIP₂, leading to complete abolishment of its Ca²⁺ oscillation inducing activity.     

Plasma adiponectin concentrations in patients with chronic renal failure: relationship with metabolic risk factors and ischemic heart disease.

AIMS: To compare plasma adiponectin levels between healthy controls and patients with chronic renal failure and to examine for a relationship between plasma adiponectin levels and ischemic heart disease as well as aortic distensibility which is an early marker of atherosclerosis. METHODS: We included 89 patients with CRF (45 on and 44 not on hemodialysis) and 70 controls in a cross-sectional study. Plasma adiponectin levels were measured by radioimmunoassay. Aortic distensibility was assessed by high-resolution ultrasonography. RESULTS: Plasma adiponectin levels were significantly almost twice as high in patients with renal failure compared to controls (9.7 +/- 1.1 vs. 5.4 +/- 0.6 microg/ml, p < 0.0001). No significant differences were found between renal patients on hemodialysis and not on hemodialysis (p = 0.71). Multivariate linear regression analysis in the renal patient group demonstrated a significant negative relationship between plasma adiponectin levels and ischemic heart disease (p = 0.02). The same analysis in the control subjects group showed a significant, negative relationship between plasma adiponectin levels and body mass index (p = 0.02) and a highly significant positive relationship with the high density lipoprotein cholesterol (p < 0.0001). In the total study population, glomerular filtration rate was the only independent predictor of plasma adiponectin concentrations. Aortic distensibility was lower in renal patients than in controls at a high level of significance (p < 0.0001). However, no significant relationship could be found between plasma adiponectin and aortic distensibility in either the controls or the renal patients. CONCLUSIONS: Plasma adiponectin levels are almost twice as high in patients with chronic renal failure in comparison with healthy controls, but not different between renal patients on and those not on hemodialysis. In addition, low plasma adiponectin levels are strongly associated with ischemic heart disease, but not with aortic distensibility in chronic renal failure.     

Short-term nitrous oxide emissions from pasture soil as influenced by urea level and soil nitrate

Nitrogen excreted by cattle during grazing is a significant source of atmospheric nitrous oxide (N₂O). The regulation of N₂O emissions is not well understood, but may vary with urine composition and soil conditions. This laboratory study was undertaken to describe short-term effects on N₂O emissions and soil conditions, including microbial dynamics, of urea amendment at two different rates (22 and 43 g N m⁻²). The lower urea concentration was also combined with an elevated soil NO ₃ ⁻ concentration. Urea solutions labelled with 25 atom%¹⁵N were added to the surface of repacked pasture soil cores and incubated for 1, 3, 6 or 9 days under constant conditions (60% WFPS, 14 °C). Soil inorganic N (NH ₄ ⁺ , NO ₂ ⁻ and NO ₃ ⁻ ), pH, electrical conductivity and dissolved organic C were quantified. Microbial dynamics were followed by measurements of CO₂ evolution, by analyses of membrane lipid (PLFA) composition, and by measurement of potential ammonium oxidation and denitrifying enzyme activity. The total recovery of¹⁵N averaged 84%. Conversion of urea-N to NO ₃ ⁻ was evident, but nitrification was delayed at the highest urea concentration and was accompanied by an accumulation of NO ₂ ⁻ . Nitrous oxide emissions were also delayed at the highest urea amendment level, but accelerated towards the end of the study. The pH interacted with NH ₄ ⁺ to produce inhibitory concentrations of NH₃(aq) at the highest urea concentration, and there was evidence for transient negative effects of urea amendment on both nitrifying and denitrifying bacteria in this treatment. However, PLFA dynamics indicated that initial inhibitory effects were replaced by increased microbial activity and net growth. It is concluded that urea-N level has qualitative, as well as quantitative effects on soil N transformations in urine patches.     

Targeting Ascophyllum nodosum (L.) Le Jol. extract application at five growth stages of winter wheat

Seaweed extracts are a category of biostimulant products that have the potential to promote plant growth and thus, have given a promise to develop more resilient systems of crop production that make efficient use of water and nutrients. This field study evaluated the effect of application timing of an Ascophyllum nodosum extract on nutrient uptake and grain yield of winter wheat when the extract was applied singly in five growth stages from 2017 to 2019. Analysis by orthogonal polynomial contrasts indicated that extract application significantly increased yield or biomass in all three site-years. Application at the tillering stage increased average yield by 17%, grain nutrient accumulation (N, P, K) by 21–24%, and N-use efficiency by 11% over the three site-year period, but extract effects were not consistent between site-years both in terms of optimal growth stages of application and magnitude of crop responses. While grain yield increased by 39% with extract application at tillering in 2017, yield gains ranging from 16 to 22% were obtained at the stem elongation and booting stages in 2018. These differences were likely the result of site-year variations in weather patterns and soil properties. Among macro-nutrients, only grain N accumulation was linearly correlated to grain yield across site-years indicating that increased soil N uptake and/or remobilization to the reproductive organs was a key process of the A. nodosum mode of action. An economic analysis indicated that the average return over extract cost was higher than that of the zero-extract control by €79-100 ha^-1 when the extract was applied at the tillering or stem elongation stage and assuming higher dilution rates recommended by the industry. Even greater economic benefits can result if N rates are substantially reduced without an adverse effect on yield gains.