Negative control of p53 by Sir2alpha promotes cell survival under stress.

The NAD-dependent histone deacetylation of Sir2 connects cellular metabolism with gene silencing as well as aging in yeast. Here, we show that mammalian Sir2alpha physically interacts with p53 and attenuates p53-mediated functions. Nicotinamide (Vitamin B3) inhibits an NAD-dependent p53 deacetylation induced by Sir2alpha, and also enhances the p53 acetylation levels in vivo. Furthermore, Sir2alpha represses p53-dependent apoptosis in response to DNA damage and oxidative stress, whereas expression of a Sir2alpha point mutant increases the sensitivity of cells in the stress response. Thus, our findings implicate a p53 regulatory pathway mediated by mammalian Sir2alpha. These results have significant implications regarding an important role for Sir2alpha in modulating the sensitivity of cells in p53-dependent apoptotic response and the possible effect in cancer therapy.     

Excitability of oxytocin neurons in paraventricular nucleus is regulated by voltage-gated potassium channels Kv4.2 and Kv4.3

Oxytocin is produced by neurons in the paraventricular nucleus (PVN) and the supraoptic nucleus in the hypothalamus. Various ion channels are considered to regulate the excitability of oxytocin neurons and its secretion. A-type currents of voltage-gated potassium channels (Kv channels), generated by Kv4.2/4.3 channels, are known to be involved in the regulation of neuron excitability. However, it is unclear whether the Kv4.2/4.3 channels participate in the regulation of excitability in PVN oxytocin neurons. Here, we investigated the contribution of the Kv4.2/4.3 channels to PVN oxytocin neuron excitability. By using transgenic rat brain slices with the oxytocin-monomeric red fluorescent protein 1 fusion transgene, we examined the excitability of oxytocin neurons by electrophysiological technique. In some oxytocin neurons, the application of Kv4.2/4.3 channel blocker increased firing frequency and membrane potential with extended action potential half-width. Our present study indicates the contribution of Kv4.2/4.3 channels to PVN oxytocin neuron excitability regulation.

Abbreviation: PVN, paraventricular nucleus; Oxt-mRFP1, Oxt-monometric red fluorescent protein 1; PaTx-1, Phrixotoxin-1; TEA, Tetraethylammonium Chloride; TTX, tetrodotoxin; aCSF, artificial cerebrospinal fluid;PBS, phosphate buffered saline 3v, third ventricle.     

Parkin suppresses dopaminergic neuron-selective neurotoxicity induced by Pael-R in Drosophila

Parkin, an E3 ubiquitin ligase that degrades proteins with aberrant conformations, is associated with autosomal recessive juvenile Parkinsonism (AR-JP). The molecular basis of selective neuronal death in AR-JP is unknown. Here we show in an organismal system that panneuronal expression of Parkin substrate Pael-R causes age-dependent selective degeneration of Drosophila dopaminergic (DA) neurons. Coexpression of Parkin degrades Pael-R and suppresses its toxicity, whereas interfering with endogenous Drosophila Parkin function promotes Pael-R accumulation and augments its toxicity. Furthermore, overexpression of Parkin can mitigate alpha-Synuclein-induced neuritic pathology and suppress its toxicity. Our study implicates Parkin as a central player in the molecular pathway of Parkinson's disease (PD) and suggests that manipulating Parkin expression may provide a novel avenue of PD therapy.     

Expression of an mNSC1 in mammalian cells.

We have cloned a cDNA inducing a cation-permeable current (mNSC1) from pancreatic beta-cells, which shows niflumate-sensitive current in Xenopus oocytes. To elucidate the expression in mammalian cells, mNSC1 was expressed in CHO cells. The reversal potential by mNSC1 was shifted toward positive which was significantly reversed by flufenamic acid. Single-channel analysis showed a characteristic of a Ca-activated nonselective cation channel. Therefore, we may conclude that mNSC1 expresses a fenamates-sensitive cation channel, inducing membrane depolarization in a mammalian cell.     

Nampt/PBEF/Visfatin regulates insulin secretion in beta cells as a systemic NAD biosynthetic enzyme

Intracellular nicotinamide phosphoribosyltransferase (iNampt) is an essential enzyme in the NAD biosynthetic pathway. An extracellular form of this protein (eNampt) has been reported to act as a cytokine named PBEF or an insulin-mimetic hormone named visfatin, but its physiological relevance remains controversial. Here we show that eNampt does not exert insulin-mimetic effects in vitro or in vivo but rather exhibits robust NAD biosynthetic activity. Haplodeficiency and chemical inhibition of Nampt cause defects in NAD biosynthesis and glucose-stimulated insulin secretion in pancreatic islets in vivo and in vitro. These defects are corrected by administration of nicotinamide mononucleotide (NMN), a product of the Nampt reaction. A high concentration of NMN is present in mouse plasma, and plasma eNampt and NMN levels are reduced in Nampt heterozygous females. Our results demonstrate that Nampt-mediated systemic NAD biosynthesis is critical for beta cell function, suggesting a vital framework for the regulation of glucose homeostasis.     

Non-invasive diagnosis of cutaneous leishmaniasis by the direct boil loop-mediated isothermal amplification method and MinION™ nanopore sequencing

Cutaneous leishmaniasis (CL) is gaining attention as a public health problem. We present two cases of CL imported from Syria and Venezuela in Japan. We diagnosed them as CL non-invasively by the direct boil loop-mediated isothermal amplification method and an innovative sequencing method using the MinION? sequencer. This report demonstrates that our procedure could be useful for the diagnosis of CL in both clinical and epidemiological settings.     

Site-directed mutagenesis of hepatitis B surface antigen sequence at codon 160 from arginine to lysine for conversion of subtypic determinant from r to w

Site-directed mutagenesis from G to A was induced at nucleotide 479 in the S gene of hepatitis B virus DNA, cloned from an individual carrying the surface antigen of subtype ayr. HepG2 cells were transfected with the plasmid DNA containing the mutant. They produced surface antigen of subtype ayw, unlike HepG2 cells harboring the parent viral DNA that produced surface antigen of subtype ayr. These results indicate that a point mutation from G to A at nucleotide 479 in the S gene, changing codon 160 for arginine to that for lysine, can convert the subtypic determinant of hepatitis B surface antigen from r to its allelic determinant w.     

Specific inhibitors of tyrosine-specific protein kinase, synthetic 4-hydroxycinnamamide derivatives

Several newly synthesized 4-hydroxycinnamamide derivatives such as 3-(3',5'-di-isopropyl-4'-hydroxybenzylidene)-2-oxindol (ST 280), 3-(3',5'-di-methylthiomethyl-4'-hydroxybenzylidene)-2-oxindole (ST 458), alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide (ST 638) and 3-(3'-ethoxy-4'-hydroxy-5'-phenylthiomethylbenzylidene)-2-pyrol idinone (ST 642) were found to inhibit tyrosine-specific protein kinase activity of the epidermal growth factor (EGF) receptor with IC50 values of 0.44 microM, 0.44 microM, 0.37 microM and 0.85 microM, respectively. None of them showed inhibitory effect on the enzyme activities of serine- and/or threonine-specific protein kinases such as cAMP-dependent protein kinase, Ca2+/phospholipid-dependent protein kinase C, casein kinase I and casein kinase II. In addition, none of them had effect on Na+/K+-ATPase or 5'-nucleotidase. The results suggest that the compound ST 280, ST 458, ST 638 and ST 642 are potent and specific inhibitors of tyrosine-specific protein kinase.     

Inhibition of pentagastrin-stimulated gastric acid secretion by intraileal administration of bile and elevation of plasma concentrations of gut glucagon-like immunoreactivity in anesthetized dogs

The effects of intraileal administration of bile on gastric acid secretion stimulated by a submaximal dose of intravenous pentagastrin infusion and on plasma concentrations of gut glucagon-like immunoreactivity (gut GLI) were studied in anesthetized dogs. Gastric acid secretion was measured for a 2-h period at 15-min intervals before and after intraluminal instillation of test solutions. 100 ml of canine bladder bile diluted to 10% in saline evoked a significant inhibition (20%) of gastric acid secretion. The inhibition of gastric acid secretion was accompanied by an elevation of plasma concentration of gut GLI, whereas saline instillation (in controls) caused no responses. Although the inhibition of gastric acid secretion and the elevation of plasma gut GLI are parallel phenomena, gut GLI can be reasonably postulated as one of the candidate mediators of bile-induced inhibition of gastric acid secretion, since its structurally related peptides, pancreatic glucagon, glicentin and oxyntomodulin have been reported as inhibitors of gastric acid secretion.