Effect of collidine (2,4,6-trimethylpyridine) on the osmiophilia and chromaffin reaction in the synaptic vesicles of rat pineal nerves

Summary Rat pineal nerve endings contain a population of small and of large synaptic vesicles that are either electron lucent or have electron-dense cores. It has been reported that their osmiophilia is elminated when collidine buffer is used in the fixation procedure. We investigated this effect and found that osmium tetroxide and potassium dichromate reactivity were abolished when excised pineal glands were briefly incubated with collidine buffer before glutaraldehyde-cacodylate fixation. Such an effect was not observed when collidine was applied after fixation. Glands that had been fixed in glutaraldehyde or osmium tetroxide buffered with collidine exhibited a peripheral zone containing reactive synaptic vesicles and a deeper, central zone where such reactivity was absent. These results indicate that the effect of collidine is due to depletion of monoamines rather than to chemical blockage of their reactivity, and further suggest that collidine has a higher rate of penetration into tissues than the tested fixatives.     

POCUS: mining genomic sequence annotation to predict disease genes

Here we present POCUS (prioritization of candidate genes using statistics), a novel computational approach to prioritize candidate disease genes that is based on over-representation of functional annotation between loci for the same disease. We show that POCUS can provide high (up to 81-fold) enrichment of real disease genes in the candidate-gene shortlists it produces compared with the original large sets of positional candidates. In contrast to existing methods, POCUS can also suggest counterintuitive candidates.     

Increased conformational rigidity of humic substances by oxidative biomimetic catalysis

A synthetic water-soluble meso-tetra(2,6-dichloro-3-sulfonatophenyl)porphyrinate of iron(III) chloride, Fe(TDCPPS)Cl, was employed as a biomimetic catalyst in the oxidative coupling of terrestrial humic materials. High-performance size-exclusion chromatography (HPSEC), solid-state nuclear magnetic resonance (CPMAS-(13)C NMR), electron paramagnetic resonance (EPR), and diffuse reflectance infrared spectroscopy (DRIFT) were used to follow conformational and structural changes brought about in different humic materials by the oxidative coupling. Increase in apparent weight-average molecular weight (Mw(a)) occurred invariably for all humic substances with the oxidative polymerization catalyzed by Fe(TDCPPS)Cl. HPSEC further showed that the polymerization reaction turned the loosely bound humic supramolecular structures into more stable conformations which could no longer be disrupted by the disaggregating effect of acetic acid. DRIFT spectroscopy suggested the formation of new alkyl and aromatic ethers following the oxidative coupling with the biomimetic catalyst. CPMAS-(13)C NMR and EPR spectra suggested a reduced molecular mobility of humic components and enhanced stabilization of free radicals in larger oxidized fragments. All findings concur in indicating that the biomimetic catalysis by Fe(TDCPPS)Cl increased the molecular mass and chemical rigidity of humic materials by formation of intermolecular covalent bonds via a free-radical mechanism. The development of a technology based on oxidative polymerization by biomimetic catalysis may be of importance in controlling the properties and reactivity of humic matter for industrial and environmental applications.     

Branch development controls leaf area dynamics in grapevine (Vitis vinifera) growing in drying soil

BACKGROUND AND AIMS: Soil water deficit is a major abiotic stress with severe consequences for the development, productivity and quality of crops. However, it is considered a positive factor in grapevine management (Vitis vinifera), as it has been shown to increase grape quality. The effects of soil water deficit on organogenesis, morphogenesis and gas exchange in the shoot were investigated. METHODS: Shoot organogenesis was analysed by distinguishing between the various steps in the development of the main axis and branches. Several experiments were carried out in pots, placed in a greenhouse or outside, in southern France. Soil water deficits of various intensities were imposed during vegetative development of the shoots of two cultivars ('Syrah' and 'Grenache N'). KEY RESULTS: All developmental processes were inhibited by soil water deficit, in an intensity-dependent manner, and sensitivity to water stress was process-dependent. Quantitative relationships with soil water were established for all processes. No difference was observed between the two cultivars for any criterion. The number of leaves on branches was particularly sensitive to soil water deficit, which rapidly and strongly reduced the rate of leaf appearance on developing branches. This response was not related to carbon availability, photosynthetic activity or the soluble sugar content of young expanding leaves. The potential number of branches was not a limiting factor for shoot development. CONCLUSIONS: The particularly high sensitivity to soil water deficit of leaf appearance on branches indicates that this process is a major determinant of the adaptation of plant leaf area to soil water deficit. The origin of this particular developmental response to soil water deficit is unclear, but it seems to be related to constitutive characteristics of branches rather than to competition for assimilates between axes differing in sink strength.     

Regulation of the mechanosensitive cation channels by ATP and cAMP in leech neurons

Single-channel recordings were used to study the modulation of stretch-activated channels (SACs) by intracellular adenosine nucleotides in identified leech neurons. These channels exhibited two activity modes, spike-like (SL) and multiconductance (MC), displaying different polymodal activation. In the absence of mechanical stimulation, internal perfusion of excised patches with ATP induced robust and reversible activation of the MC but not of the SL mode. The ATP effect on channel activity was dose-dependent within a range of 1 μM-1 mM and was induced at different values of intracellular pH and Ca²⁺. The non-hydrolyzable ATP analog AMP-PNP, ATP without Mg²⁺ or ADP also effectively enhanced MC activity. Adenosine mimicked the effect of its nucleotides. At negative membrane potentials, both ATP and adenosine activated the channel. Moreover, ATP but not adenosine induced a flickering block. Addition of cAMP during maximal ATP activation completely and reversibly inhibited the channel, with activation and deactivation times of minutes. However, cAMP alone only induced a weak and rapid channel activation, without inhibitory effects. The expression of these channels in the growth cones of leech neurons, their permeability to Ca²⁺ and their sensitivity to intracellular cAMP are consistent with a role in the Ca²⁺ oscillations associated with cell growth.     

Palmitate activates autophagy in INS-1E β-cells and in isolated rat and human pancreatic islets

We have investigated the in vitro effects of increased levels of glucose and free fatty acids on autophagy activation in pancreatic beta cells. INS-1E cells and isolated rat and human pancreatic islets were incubated for various times (from 2 to 24 h) at different concentrations of glucose and/or palmitic acid. Then, cell survival was evaluated and autophagy activation was explored by using various biochemical and morphological techniques. In INS-1E cells as well as in rat and human islets, 0.5 and 1.0 mM palmitate markedly increased autophagic vacuole formation, whereas high glucose was ineffective alone and caused little additional change when combined with palmitate. Furthermore, LC3-II immunofluorescence co-localized with that of cathepsin D, a lysosomal marker, showing that the autophagic flux was not hampered in PA-treated cells. These effects were maintained up to 18-24 h incubation and were associated with a significant decline of cell survival correlated with both palmitate concentration and incubation time. Ultrastructural analysis showed that autophagy activation, as evidenced by the occurrence of many autophagic vacuoles in the cytoplasm of beta cells, was associated with a diffuse and remarkable swelling of the endoplasmic reticulum. Our results indicate that among the metabolic alterations typically associated with type 2 diabetes, high free fatty acids levels could play a role in the activation of autophagy in beta cells, through a mechanism that might involve the induction of endoplasmic reticulum stress.     

A novel disorder caused by defective biosynthesis of N-linked oligosaccharides due to glucosidase I deficiency

Glucosidase I is an important enzyme in N-linked glycoprotein processing, removing specifically distal alpha-1,2-linked glucose from the Glc3Man9GlcNAc2 precursor after its en bloc transfer from dolichyl diphosphate to a nascent polypeptide chain in the endoplasmic reticulum. We have identified a glucosidase I defect in a neonate with severe generalized hypotonia and dysmorphic features. The clinical course was progressive and was characterized by the occurrence of hepatomegaly, hypoventilation, feeding problems, seizures, and fatal outcome at age 74 d. The accumulation of the tetrasaccharide Glc(alpha1-2)Glc(alpha1-3)Glc(alpha1-3)Man in the patient's urine indicated a glycosylation disorder. Enzymological studies on liver tissue and cultured skin fibroblasts revealed a severe glucosidase I deficiency. The residual activity was <3% of that of controls. Glucosidase I activities in cultured skin fibroblasts from both parents were found to be 50% of those of controls. Tissues from the patient subjected to SDS-PAGE followed by immunoblotting revealed strongly decreased amounts of glucosidase I protein in the homogenate of the liver, and a less-severe decrease in cultured skin fibroblasts. Molecular studies showed that the patient was a compound heterozygote for two missense mutations in the glucosidase I gene: (1) one allele harbored a G-->C transition at nucleotide (nt) 1587, resulting in the substitution of Arg at position 486 by Thr (R486T), and (2) on the other allele a T-->C transition at nt 2085 resulted in the substitution of Phe at position 652 by Leu (F652L). The mother was heterozygous for the G-->C transition, whereas the father was heterozygous for the T-->C transition. These base changes were not seen in 100 control DNA samples. A causal relationship between the alpha-glucosidase I deficiency and the disease is postulated.