Indoxyl sulfate promotes vascular smooth muscle cell senescence with upregulation of p53, p21, and prelamin A through oxidative stress

We previously demonstrated that indoxyl sulfate (IS), a uremic toxin, induces aortic calcification in hypertensive rats and induces oxidative stress and the expression of osteoblast-specific proteins in vascular smooth muscle cells. This study aimed to clarify whether IS stimulates senescence of cultured human aortic smooth muscle cells (HASMCs) and aorta in Dahl salt-sensitive hypertensive rats and whether AST-120, an oral sorbent, prevents senescence of aorta in subtotally nephrectomized uremic rats. IS increased the mRNA expression of p53 and p21 in HASMCs, whereas it did not change that of p16 and retinoblastoma protein (pRb). The IS-induced expression of p53 and p21 was suppressed by N-acetylcysteine, an antioxidant. IS promoted protein expression of p53, p21, and senescence-associated β-galactosidase (SA-β-gal) activity in HASMCs, and N-acetylcysteine and pifithrin-α,p-nitro, a p53 inhibitor, blocked these effects. IS upregulated prelamin A, a hallmark of vascular smooth muscle cell senescence, and downregulated FACE1/Zempste24 protein expression in HASMCs, and N-acetylcysteine suppressed these effects. Administration of IS to hypertensive rats increased expression of SA-β-gal, p53, p21, prelamin A, and oxidative stress markers such as 8-hydroxyl-2'-deoxyguanosine (8-OHdG) and malondialdehyde (MDA) in the cells embedded in the calcification area of arcuate aorta. Further, the uremic rat model showed positive staining for SA-β-gal, p53, p21, prelamin A, 8-OHdG, and MDA in the cells embedded in the calcification area of arcuate aorta, whereas AST-120 reduced the expression of these biomarkers. Taken together, IS accelerates vascular smooth muscle cell senescence with upregulation of p53, p21, and prelamin A and downregulation of FACE1 through oxidative stress.     

The juvenile myoclonic epilepsy-related protein EFHC1 interacts with the redox-sensitive TRPM2 channel linked to cell death

The transient receptor potential M2 channel (TRPM2) is the Ca(2+)-permeable cation channel controlled by cellular redox status via β-NAD(+) and ADP-ribose (ADPR). TRPM2 activity has been reported to underlie susceptibility to cell death and biological processes such as inflammatory cell migration and insulin secretion. However, little is known about the intracellular mechanisms that regulate oxidative stress-induced cell death via TRPM2. We report here a molecular and functional interaction between the TRPM2 channel and EF-hand motif-containing protein EFHC1, whose mutation causes juvenile myoclonic epilepsy (JME) via mechanisms including neuronal apoptosis. In situ hybridization analysis demonstrates TRPM2 and EFHC1 are coexpressed in hippocampal neurons and ventricle cells, while immunoprecipitation analysis demonstrates physical interaction of the N- and C-terminal cytoplasmic regions of TRPM2 with the EFHC1 protein. Coexpression of EFHC1 significantly potentiates hydrogen peroxide (H(2)O(2))- and ADPR-induced Ca(2+) responses and cationic currents via recombinant TRPM2 in HEK293 cells. Furthermore, EFHC1 enhances TRPM2-conferred susceptibility of HEK293 cells to H(2)O(2)-induced cell death, which is reversed by JME mutations. These results reveal a positive regulatory action of EFHC1 on TRPM2 activity, suggesting that TRPM2 contributes to the expression of JME phenotypes by mediating disruptive effects of JME mutations of EFHC1 on biological processes including cell death.     

A role of synchronicity of neural activity based on dynamic plasticity of synapses in encoding spatiotemporal features of electrosensory stimuli

It is quite important for investigation of sensory mechanism to understand how dynamical property of neurons is used for encoding the feature of spatiotemporally varying stimuli. To consider concretely the problem, we focus our study on electrosensory system of a weakly electric fish. Weakly electric fish generate electric field around their body using electric organ discharge (EOD) and accurately detect the location of an object through the modulation of electric field induced by the object. We made a neural network model of electrosensory lateral-line lobe (ELL). Here we show that the features of EOD modulation depending specifically distance and size of an object are encoded into the timing of burst firing of ELL neurons. These features can be represented by the spatial area of synchronous burst firing and the interburst interval in the ELL network. We show that short-term changes of excitatory and inhibitory synapses, induced by efferent signals, regulate the ELL activity so as to effectively encode the features of EOD modulation.     

Dopaminergic modulation of salt sensitivity in patients with essential hypertension

The present study was designed to investigate the possible role of dopaminergic mechanisms in contributing to the pathogenesis of hypertension in salt sensitive patients. Eighteen patients with essential hypertension were studied while under a diet ranging from low salt to high salt, which enabled a classification in "salt-sensitive" (SS) and "nonsalt-sensitive" (NSS) groups based on a tentative criteria of a 10% increase of mean blood pressure with high salt diet. The SS patients showed reduced urinary excretion of sodium as compared with that from NSS patients. Urinary norepinephrine excretion in all patients with salt loading was suppressed, but urinary excretion of epinephrine showed a tendency to increase in SS patients after salt loading. Urinary excretion of dopamine increased in NSS patients with salt loading, but did not change in SS patients. To further evaluate the role of dopaminergic mechanisms in salt-sensitive hypertension, metoclopramide, a dopamine antagonist, was injected intravenously to all patients. With salt loading, plasma aldosterone levels increased after injection of metoclopramide in NSS patients, but did not change in SS patients. These results suggest that salt-sensitive hypertension is modulated by dopaminergic activity, which in turn is attenuated in SS patients. Decreased dopaminergic activity induced sodium retention both by a direct effect on the kidney as well as indirectly via relatively increased aldosterone secretion. Both mechanisms would help to increase intravascular volume and blood pressure in salt-sensitive hypertension.     

Liquid chromatographic-atmospheric pressure chemical ionization mass spectrometric analysis of glycine conjugates and urinary isovalerylglycine in isovaleric acidemia

n-Acetylglycine, n-propionylglycine, n-butyrylglycine, isobutyrylglycine, n-valerylglycine, isovalerylglycine, heptanoylglycine, phenylacetylglycine and isovalerylglucuronide were identified based on their liquid chromatographic-atmospheric pressure chemical ionization mass spectra (LC-APCI-MS). We were able to detect the presence of urinary isovalerylglycine in two cases of isovaleric acidemia using LC-APCI-MS. Membrane-filtered urine samples were injected into the LC-APCI-MS system in the negative-ion mode without any further pretreatment, and large amounts of isovalerylglycine were detected as the [M − H]⁻ ion. The urinary excretion of isovalerylglycine appeared to increase after -carnitine therapy. This analytical method is quick and easy and it may be a useful tool in understanding dysfunctional conditions in isovaleric acidemia.     

Nano‐sized bacterial magnetic particles displaying pyruvate phosphate dikinase for pyrosequencing

There is a high demand for inexpensive and high‐throughput DNA sequencing technologies in molecular biology and applied biosciences. In this study, novel nano‐sized magnetic particles displaying enzymes for pyrosequencing, a rather novel bioluminometric DNA sequencing method based on the sequencing‐by‐synthesis principle by employing a cascade of several enzymatic reactions, was developed. A highly thermostable enzyme, pyruvate phosphate dikinase (PPDK) which converts PPi to ATP was successfully expressed onto bacterial magnetic particles (BacMPs) using a novel protein display system of Magnetospirillum magneticum AMB‐1. The enzymatic stability of BacMPs displaying PPDK (PPDK‐BacMPs) to pH and temperature was evaluated and its broad range of properties was shown. Subsequently, PPDK‐BacMPs were applied in pyrosequencing and a target oligonucleotide was successfully sequenced. The PPDK enzyme displayed on BacMPs was shown to be recyclable in each sequence reaction as they can be manipulated by magnetic force. It was concluded that nano‐sized PPDK‐BacMPs are useful for the scale down of pyrosequencing reaction volumes, thus, permitting high‐throughput. The recycling of enzymes was also shown to be promising and applicable for the development of an inexpensive DNA sequencing at a low running cost. Biotechnol. Bioeng. 2009;103: 130–137. © 2008 Wiley Periodicals, Inc.     

Multiplex polymerase chain reaction (PCR) with color-tagged module-shuffling primers for comparing gene expression levels in various cells

A method based on the multiplex polymerase chain reaction (PCR) and gel electrophoresis for the comparative analysis of gene expression levels was developed. Using the method many cDNA fragments from different sources can be compared simultaneously. Competitive PCR amplification of expressed genes from different sources was performed by using ‘module-shuffling primers’ (MPSs). The MPSs (labeled with different fluorophores) consist of sequence modules of 3 or 4 nt. The modules are arranged in different orders in each primer; therefore, the base sequences of the primers are different but their melting temperatures are identical. The genes expressed in different sources are ligated with tags complementary with the MPSs. Tag-ligated fragments are mixed in one tube and amplified at the same amplification efficiency by the MPSs. Amplified fragments are detected separately by multiple-color gel electrophoresis. This method can detect different amounts of each expressed gene, up to a difference in amounts of 30%, and its detection limit is 0.1 amol per assay.