Methyl‐isobutyl amiloride reduces brain Lac/NAA,cell death and microglial activation in a perinatal asphyxia model

Na⁺/H⁺ exchanger (NHE) blockade attenuates the detrimental consequences of ischaemia and reperfusion in myocardium and brain in adult and neonatal animal studies. Our aim was to use magnetic resonance spectroscopy (MRS) biomarkers and immunohistochemistry to investigate the cerebral effects of the NHE inhibitor, methyl isobutyl amiloride (MIA) given after severe perinatal asphyxia in the piglet. Eighteen male piglets (aged < 24 h) underwent transient global cerebral hypoxia‐ischaemia and were randomized to (i) saline placebo; or (ii) 3 mg/kg intravenous MIA administered 10 min post‐insult and 8 hourly thereafter. Serial phosphorus‐31 (³¹P) and proton (¹H) MRS data were acquired before, during and up to 48 h after hypoxia‐ischaemia and metabolite‐ratio time‐series Area under the Curve (AUC) calculated. At 48 h, histological and immunohistochemical assessments quantified regional tissue injury. MIA decreased thalamic lactate/N‐acetylaspartate and lactate/creatine AUCs (both p < 0.05) compared with placebo. Correlating with improved cerebral energy metabolism, transferase mediated biotinylated d‐UTP nick end‐labelling (TUNEL) positive cell density was reduced in the MIA group in cerebral cortex, thalamus and white matter (all p < 0.05) and caspase 3 immunoreactive cells were reduced in pyriform cortex and caudate nucleus (both p < 0.05). Microglial activation was reduced in pyriform and midtemporal cortex (both p < 0.05). Treatment with MIA starting 10 min after hypoxia‐ischaemia was neuroprotective in this perinatal asphyxia model.     

Chirality Driven Twisting as a Driving Force of Primitive Folding in Binary Mixtures

Origins of Life and Evolution of Biospheres - The N-trifluoroacetylated α-aminoalcohols (TFAAAs) are able to form quasi-one-dimensional supramolecular fibers (strings) when chirally pure, and...     

The effect of folate-related SNPs on clinicopathological features,response to neoadjuvant treatment and survival in pre- and postmenopausal breast cancer patients

This study aimed to investigate the relationship of ten single nucleotide polymorphisms (SNPs) in the MTHFR, MTR, MTRR, DHFR, MTHFD1, TS, RFC1 and DNMT3b genes with cancer survival, therapeutic response to neoadjuvant chemotherapy and clinicopathological characteristics in 300 pre- and postmenopausal breast cancer patients of a Russian Western Siberian population. We found that the MTHFR 677CT genotype as well as combination of MTHFR 677CT and 677TT genotype was related to tumor size and estrogen-positive status in postmenopausal group. The RFC1 80А allele was associated with an increased risk of lymph node metastases among postmenopausal women. The MTHFR 677TT genotype was significantly correlated with a better progression-free survival in premenopausal patients. In contrast, a worse outcome was observed in this group patient with MTHFD1 1958AA genotype. In the multivariate analysis, the MTHFD1 1958AA genotype was identified as an independent prognostic factor for premenopausal breast cancer survival. Our findings provide evidence for associations of breast cancer survival with folate-related SNPs in a population of Western Siberian region of Russia and the MTHFD1 (1958G>A) may have additional prognostic value especially among premenopausal patients.     

Enhancement of flowering and branching phenotype in chrysanthemum by expression of <Emphasis Type="Italic">ipt</Emphasis> under the control of a 0.821 kb fragment of the <Emphasis Type="Italic">LEACO1</Emphasis> gene promoter

The cytokinin biosynthesis gene, isopentenyl transferase (ipt), under the control of an 821 bp fragment of the LEACO1 gene promoter (from Lycopersicon esculentum) was introduced into Dendranthema × grandiflorium ‘Iridon’ (chrysanthemum). LEACO1_0.821kb-ipt transgenic lines grown in the vegetative state, exhibited a range of phenotypic changes including increased branching and reduced internode lengths. LEACO1_0.821kb-ipt transgenic lines grown in the generative state, exhibited increased flower bud count that ranged from 3.8- to 6.7-times the number produced by wild-type plants. Dramatic increases in flower number were associated with a delay of flower bud development and a decrease in flower bud diameter. RT-PCR analysis indicated differences in ipt gene expression between individual transgenic lines that exhibited a range of phenotypes. Within an individual transgenic line, RT-PCR analysis revealed changes in ipt gene expression at different stages of generative shoot development. Expression of ipt in transgenic lines correlated well with high concentrations of the sum total to bioactive cytokinins plus the glucosides and phosphate derivatives of these species, under both vegetative and generative growth conditions. In general, transgenic lines accumulated higher concentrations of both storage-form cytokinins (O-glucosides) and deactivated-form cytokinins (N-glucosides) in generative shoots of than in vegetative shoots. Based on the range of phenotypes observed in various transgenic chrysanthemum lines, we conclude that the LEACO1 _0.821kb -ipt gene appears to have great potential for use in ornamental crop improvement. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Membrane property abnormalities in simulated cases of mild systematic and severe focal demyelinating neuropathies

The investigation of multiple nerve membrane properties by mathematical models has become a new tool to study peripheral neuropathies. In demyelinating neuropathies, the membrane properties such as potentials (intracellular, extracellular, electrotonic) and indices of axonal excitability (strength-duration time constants, rheobases and recovery cycles) can now be measured at the peripheral nerves. This study provides numerical simulations of the membrane properties of human motor nerve fibre in cases of internodal, paranodal and simultaneously of paranodal internodal demyelinations, each of them mild systematic or severe focal. The computations use our previous multi-layered model of the fibre. The results show that the abnormally greater increase of the hyperpolarizing electrotonus, shorter strength-duration time constants and greater axonal superexcitability in the recovery cycles are the characteristic features of the mildly systematically demyelinated cases. The small decrease of the polarizing electrotonic responses in the demyelinated zone in turn leads to a compensatory small increase of these responses outside the demyelinated zone of all severely focally demyelinated cases. The paper summarizes the insights gained from these modeling studies on the membrane property abnormalities underlying the variation in clinical symptoms of demyelination in Charcot-Marie-Tooth disease type 1A, chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome and multifocal motor neuropathy. The model used provides an objective study of the mechanisms of these diseases which up till now have not been sufficiently well understood, because quite different assumptions have been given in the literature for the interpretation of the membrane property abnormalities obtained in hereditary, chronic and acquired demyelinating neuropathies.     

The ATM cofactor ATMIN protects against oxidative stress and accumulation of DNA damage in the aging brain

Progressive accumulation of DNA damage is causally involved in cellular senescence and organismal aging. The DNA damage kinase ATM plays a central role in maintaining genomic stability. ATM mutations cause the genetic disorder ataxia telangiectasia, which is primarily characterized by progressive neurodegeneration and cancer susceptibility. Although the importance of ATM function to protect against oxidative DNA damage and during aging is well described, the mechanism of ATM activation by these stimuli is not known. Here we identify ATM interactor (ATMIN) as an essential component of the ATM signaling pathway in response to oxidative stress and aging. Embryos lacking ATMIN (atmin(Δ/Δ)) died in utero and showed increased numbers of cells positive for phosphorylated histone H2aX, indicative of increased DNA damage. atmin(Δ/Δ) mouse embryonic fibroblasts accumulated DNA damage and prematurely entered senescence when cultured at atmospheric oxygen levels (20%), but this defect was rescued by addition of an antioxidant and also by culturing cells at physiological oxygen levels (3%). In response to acute oxidative stress, atmin(Δ/Δ) mouse embryonic fibroblasts showed slightly lower levels of ATM phosphorylation and reduced ATM substrate phosphorylation. Conditional deletion of ATMIN in the murine nervous system (atmin(ΔN)) resulted in reduced numbers of dopaminergic neurons, as does ATM deficiency. ATM activity was observed in old, but not in young, control mice, but aging-induced ATM signaling was impaired by ATMIN deficiency. Consequently, old atmin(ΔN) mice showed accumulation of DNA damage in the cortex accompanied by gliosis, resulting in increased mortality of aging mutant mice. These results suggest that ATMIN mediates ATM activation by oxidative stress, and thereby ATMIN protects the aging brain by preventing accumulation of DNA damage.     

Mechanistic and structural analyses of the role of His67 in the yeast polyamine oxidase Fms1

The flavoprotein oxidase Fms1 from Saccharomyces cerevisiae catalyzes the oxidation of spermine and N(1)-acetylspermine to spermidine and 3-aminopropanal or N-acetyl-3-aminopropanal. Within the active site of Fms1, His67 is positioned to form hydrogen bonds with the polyamine substrate. This residue is also conserved in other polyamine oxidases. The catalytic properties of H67Q, H67N, and H67A Fms1 have been characterized to evaluate the role of this residue in catalysis. With both spermine and N(1)-acetylspermine as the amine substrate, the value of the first-order rate constant for flavin reduction decreases 2-3 orders of magnitude, with the H67Q mutation having the smallest effect and H67N the largest. The k(cat)/K(O2) value changes very little upon mutation with N(1)-acetylspermine as the amine substrate and decreases only an order of magnitude with spermine. The k(cat)/K(M)-pH profiles with N(1)-acetylspermine are bell-shaped for all the mutants; the similarity to the profile of the wild-type enzyme rules out His67 as being responsible for either of the pK(a) values. The pH profiles for the rate constant for flavin reduction for all the mutant enzymes similarly show the same pK(a) as wild-type Fms1, about ～7.4; this pK(a) is assigned to the substrate N4. The k(cat)/K(O2)-pH profiles for wild-type Fms1 and the H67A enzyme both show a pK(a) of about ～6.9; this suggests His67 is not responsible for this pH behavior. With the H67Q, H67N, and H67A enzymes the k(cat) value decreases when a single residue is protonated, as is the case with the wild-type enzyme. The structure of H67Q Fms1 has been determined at a resolution of 2.4 ?. The structure shows that the mutation disrupts a hydrogen bond network in the active site, suggesting that His67 is important both for direct interactions with the substrate and to maintain the overall active site structure.     

A putative Src homology 3 domain binding motif but not the C-terminal dystrophin WW domain binding motif is required for dystroglycan function in cellular polarity in Drosophila

The conserved dystroglycan-dystrophin (Dg.Dys) complex connects the extracellular matrix to the cytoskeleton. In humans as well as Drosophila, perturbation of this complex results in muscular dystrophies and brain malformations and in some cases cellular polarity defects. However, the regulation of the Dg.Dys complex is poorly understood in any cell type. We now find that in loss-of-function and overexpression studies more than half (34 residues) of the Dg proline-rich conserved C-terminal regions can be truncated without significantly compromising its function in regulating cellular polarity in Drosophila. Notably, the truncation eliminates the WW domain binding motif at the very C terminus of the protein thought to mediate interactions with dystrophin, suggesting that a second, internal WW binding motif can also mediate this interaction. We confirm this hypothesis by using a sensitive fluorescence polarization assay to show that both WW domain binding sites of Dg bind to Dys in humans (K(d) = 7.6 and 81 microM, respectively) and Drosophila (K(d) = 16 and 46 microM, respectively). In contrast to the large deletion mentioned above, a single proline to an alanine point mutation within a predicted Src homology 3 domain (SH3) binding site abolishes Dg function in cellular polarity. This suggests that an SH3-containing protein, which has yet to be identified, functionally interacts with Dg.