Auditory hair cell explant co-cultures promote the differentiation of stem cells into bipolar neurons

Auditory neurons, the target neurons of the cochlear implant, degenerate following a sensorineural hearing loss. The goal of this research is to direct the differentiation of embryonic stem cells (SCs) into bipolar auditory neurons that can be used to replace degenerating neurons in the deafened mammalian cochlea. Successful replacement of auditory neurons is likely to result in improved clinical outcomes for cochlear implant recipients. We examined two post-natal auditory co-culture models with and without neurotrophic support, for their potential to direct the differentiation of mouse embryonic SCs into characteristic, bipolar, auditory neurons. The differentiation of SCs into neuron-like cells was facilitated by co-culture with auditory neurons or hair cell explants, isolated from post-natal day five rats. The most successful combination was the co-culture of hair cell explants with whole embryoid bodies, which resulted in significantly greater numbers of neurofilament-positive, neuron-like cells. While further characterization of these differentiated cells will be essential before transplantation studies commence, these data illustrate the effectiveness of post-natal hair cell explant co-culture, at providing valuable molecular cues for directed differentiation of SCs towards an auditory neuron lineage.     

Yeast Coq5 C-methyltransferase is required for stability of other polypeptides involved in coenzyme Q biosynthesis

Coenzyme Q (Q) functions in the electron transport chain of both prokaryotes and eukaryotes. The biosynthesis of Q requires a number of steps involving at least eight Coq polypeptides. Coq5p is required for the C-methyltransferase step in Q biosynthesis. In this study we demonstrate that Coq5p is peripherally associated with the inner mitochondrial membrane on the matrix side. Phenotypic characterization of a collection of coq5 mutant yeast strains indicates that while each of the coq5 mutant strains are rescued by the Saccharomyces cerevisiae COQ5 gene, only the coq5-2 and coq5-5 mutants are rescued by expression of Escherichia coli ubiE, a homolog of COQ5. The coq5-2 and coq5-5 mutants contain mutations within or adjacent to conserved methyltransferase motifs that would be expected to disrupt the catalysis of C-methylation. The steady state levels of the Coq5-2 and Coq5-5 mutant polypeptides are not decreased relative to wild type Coq5p. Two other polypeptides required for Q biosynthesis, Coq3p and Coq4p, are detected in the wild type parent and in the coq5-2 and coq5-5 mutants, but are not detected in the coq5-null mutant, or in the coq5-4 or coq5-3 mutants. The effect of the coq5-4 mutation is similar to a null, since it results in a stop codon at position 93. However, the coq5-3 mutation (G304D) is located just four amino acids away from the C terminus. While C-methyltransferase activity is detectable in mitochondria isolated from this mutant, the steady state level of Coq5p is dramatically decreased. These studies show that at least two functions can be attributed to Coq5p; first, it is required to catalyze the C-methyltransferase step in Q biosynthesis and second, it is involved in stabilizing the Coq3 and Coq4 polypeptides required for Q biosynthesis.     

The purification and properties of yeast proteinase B from Candida albicans.

A serine proteinase (ycaB) from the yeast Candida albicans A.T.C.C. 10261 was purified to near homogeneity. The enzyme was almost indistinguishable from yeast proteinase B (EC 3.4.21.48), and an Mr of 30,000 for the proteinase was determined by SDS/polyacrylamide-gel electrophoresis. The initial site of hydrolysis of the oxidized B-chain of insulin, by the purified proteinase, was the Leu-Tyr peptide bond. The preferential degradation at this site, analysed further with N-blocked amino acid ester and amide substrates, demonstrated that the specificity of the proteinase is determined by an extended substrate-binding site, consisting of at least three subsites (S1, S2 and S'1). The best p-nitrophenyl ester substrates were benzyloxycarbonyl-Tyr p-nitrophenyl ester (kcat./Km 3,536,000 M-1 X S-1), benzyloxycarbonyl-Leu p-nitrophenyl ester (kcat./Km 2,250,000 M-1 X S-1) and benzyloxycarbonyl-Phe p-nitrophenyl ester (kcat./Km 1,000,000 M-1 X S-1) consistent with a preference for aliphatic or aromatic amino acids at subsite S1. The specificity for benzyloxycarbonyl-Tyr p-nitrophenyl ester probably reflects the binding of the p-nitrophenyl group in subsite S'1. The presence of S2 was demonstrated by comparison of the proteolytic coefficients (kcat./Km) for benzyloxycarbonyl-Ala p-nitrophenyl ester (825,000 M-1 X S-1) and t-butyloxycarbonyl-Ala p-nitrophenyl ester (333,000 M-1 X S-1). Cell-free extracts contain a heat-stable inhibitor of the proteinase.     

Diabetes decreases sensitivity of adipocyte lipolysis to inhibition by G-linked receptor agonists

(1) Streptozotocin-diabetes decreased the responsiveness of noradrenaline- or forskolin-stimulated lipolysis to inhibition by phenylisopropyladenosine (PIA), prostaglandin (E₁ (PGE₁) and nicotinate in rat adipocytes. (2) Diabetes had no effect on high affinity binding of [³H]PIA to adipocyte plasma membranes. (3) Plasma membranes from diabetic animals had increased abundance of -subunits of G_i1 and G_i2. The effect of pertussis toxin in overcoming inhibition of lipolysis by PIA was delayed in adipocytes from diabetic rats. (4) Diabetes decreased the GTP-dependent right-wards shift in the dose-curve for displacement of the antagonist [³H]DPCPX by PIA in adipocyte plasma membranes. (5) It is concluded that, despite increased abundance of G_i in diabetic adipocytes, less of this functional. This may contribute to reduced sensitivity to PIA, PGE₁ and nicotinate and explains some of the loss of control of lipolysis in insulin-dependent diabetes.     

Microscopy basics and the study of actin–actin-binding protein interactions

Actin is a multifunctional eukaryotic protein with a globular monomer form that polymerizes into a thin, linear microfilament in cells. Through interactions with various actin-binding proteins (ABPs), actin plays an active role in many cellular processes, such as cell motility and structure. Microscopy techniques are powerful tools for determining the role and mechanism of actin–ABP interactions in these processes. In this article, we describe the basic concepts of fluorescent speckle microscopy, total internal reflection fluorescence microscopy, atomic force microscopy, and cryoelectron microscopy and review recent studies that utilize these techniques to visualize the binding of actin with ABPs.     

Freshwater salinization syndrome: from emerging global problem to managing risks

Freshwater salinization is an emerging global problem impacting safe drinking water, ecosystem health and biodiversity, infrastructure corrosion, and food production. Freshwater salinization originates from diverse anthropogenic and geologic sources including road salts, human-accelerated weathering, sewage, urban construction, fertilizer, mine drainage, resource extraction, water softeners, saltwater intrusion, and evaporative concentration of ions due to hydrologic alterations and climate change. The complex interrelationships between salt ions and chemical, biological, and geologic parameters and consequences on the natural, social, and built environment are called Freshwater Salinization Syndrome (FSS). Here, we provide a comprehensive overview of salinization issues (past, present, and future), and we investigate drivers and solutions. We analyze the expanding global magnitude and scope of FSS including its discovery in humid regions, connections to human-accelerated weathering and mobilization of ‘chemical cocktails.’ We also present data illustrating: (1) increasing trends in salt ion concentrations in some of the world’s major freshwaters, including critical drinking water supplies; (2) decreasing trends in nutrient concentrations in rivers due to regulations but increasing trends in salinization, which have been due to lack of adequate management and regulations; (3) regional trends in atmospheric deposition of salt ions and storage of salt ions in soils and groundwater, and (4) applications of specific conductance as a proxy for tracking sources and concentrations of groups of elements in freshwaters. We prioritize FSS research needs related to better understanding: (1) effects of saltwater intrusion on ecosystem processes, (2) potential health risks from groundwater contamination of home wells, (3) potential risks to clean and safe drinking water sources, (4) economic and safety impacts of infrastructure corrosion, (5) alteration of biodiversity and ecosystem functions, and (6) application of high-frequency sensors in state-of-the art monitoring and management. We evaluate management solutions using a watershed approach spanning air, land, and water to explore variations in sources, fate and transport of different salt ions (e.g. monitoring of atmospheric deposition of ions, stormwater management, groundwater remediation, and managing road runoff). We also identify tradeoffs in management approaches such as unanticipated retention and release of chemical cocktails from urban stormwater management best management practices (BMPs) and unintended consequences of alternative deicers on water quality. Overall, we show that FSS has direct and indirect effects on mobilization of diverse chemical cocktails of ions, metals, nutrients, organics, and radionuclides in freshwaters with mounting impacts. Our comprehensive review suggests what could happen if FSS were not managed into the future and evaluates strategies for reducing increasing risks to clean and safe drinking water, human health, costly infrastructure, biodiversity, and critical ecosystem services.

     

Acute Simvastatin Inhibits KATP Channels of Porcine Coronary Artery Myocytes

Background

Statins (3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors) consumption provides beneficial effects on cardiovascular systems. However, effects of statins on vascular K_ATP channel gatings are unknown.

Methods

Pig left anterior descending coronary artery and human left internal mammary artery were isolated and endothelium-denuded for tension measurements and Western immunoblots. Enzymatically-dissociated/cultured arterial myocytes were used for patch-clamp electrophysiological studies and for [Ca²⁺]_i, [ATP]_i and [glucose]_o uptake measurements.

Results

The cromakalim (10 nM to 10 µM)- and pinacidil (10 nM to 10 µM)-induced concentration-dependent relaxation of porcine coronary artery was inhibited by simvastatin (3 and 10 µM). Simvastatin (1, 3 and 10 µM) suppressed (in okadaic acid (10 nM)-sensitive manner) cromakalim (10 µM)- and pinacidil (10 µM)-mediated opening of whole-cell K_ATP channels of arterial myocytes. Simvastatin (10 µM) and AICAR (1 mM) elicited a time-dependent, compound C (1 µM)-sensitive [³H]-2-deoxy-glucose uptake and an increase in [ATP]_i levels. A time (2–30 min)- and concentration (0.1–10 µM)-dependent increase by simvastatin of p-AMPKα-Thr¹⁷² and p-PP2A-Tyr³⁰⁷ expression was observed. The enhanced p-AMPKα-Thr¹⁷² expression was inhibited by compound C, ryanodine (100 µM) and KN93 (10 µM). Simvastatin-induced p-PP2A-Tyr³⁰⁷ expression was suppressed by okadaic acid, compound C, ryanodine, KN93, phloridzin (1 mM), ouabain (10 µM), and in [glucose]_o-free or [Na⁺]_o-free conditions.

Conclusions

Simvastatin causes ryanodine-sensitive Ca²⁺ release which is important for AMPKα-Thr¹⁷² phosphorylation via Ca²⁺/CaMK II. AMPKα-Thr¹⁷² phosphorylation causes [glucose]_o uptake (and an [ATP]_i increase), closure of K_ATP channels, and phosphorylation of AMPKα-Thr¹⁷² and PP2A-Tyr³⁰⁷ resulted. Phosphorylation of PP2A-Tyr³⁰⁷ occurs at a site downstream of AMPKα-Thr¹⁷² phosphorylation.