Blockade of sphingosine 1-phosphate receptor 2 signaling attenuates streptozotocin-induced apoptosis of pancreatic β-cells

Sphingosine 1-phosphate (S1P) is a potent sphingolipid mediator that acts through five cognate G protein-coupled receptors (S1P₁-S1P₅) and regulates many critical biological processes. Recent studies indicated that S1P at nanomolar concentrations significantly reduces cytokine-induced apoptosis of pancreatic β-cells in which genes for S1P₁-S1P₄ are co-expressed. However, the S1P receptor subtype(s) involved in this effect remains to be clarified. In this study, we investigated the potential role of S1P₂ in streptozotocin (STZ)-induced apoptosis of pancreatic β-cells and progression of diabetes. S1P₂-deficient (S1P₂^-/-) mice displayed a greater survive ability, lower blood glucose levels, and smaller numbers of TUNEL-positive apoptotic β-cells to administration of a high dose of STZ than wild-type (WT) mice. S1P₂^-/- mice showed higher insulin/glucose ratios (an index of relative insulin deficiency) and larger insulin-positive islet areas to administration of a low dose of STZ than WT mice. Moreover, administration of JTE-013, a S1P₂-specific antagonist, to WT mice ameliorated STZ-induced blood glucose elevation and reduced the incidence of diabetes. Our findings indicate that blockade of S1P₂ signaling attenuates STZ-induced apoptosis of pancreatic β-cells and decreases the incidence of diabetes.     

Protective effect of nicotinamide against poly(ADP-ribose) polymerase-1-mediated astrocyte death depends on its transporter-mediated uptake

AimPoly(ADP-ribose) polymerase-1 (PARP-1) is a DNA repair enzyme, and its excessive activation, following ischemia, trauma, etc., depletes cellular nicotinamide adenine dinucleotide (NAD⁺) as a substrate and eventually leads to brain cell death. Nicotinamide, an NAD⁺ precursor and a PARP-1 inhibitor, is known to prevent PARP-1-triggered cell death, but there is no available information on the mechanisms involved in its transport. Here we clarified the transport characteristics of nicotinamide in primary cultured mouse astrocytes.Main methodsUptake characteristics of [¹⁴C]nicotinamide were assessed by a conventional method with primary cultured mouse astrocytes. Cell viability and PARP-1 activity were determined with intracellular LDH activity and immunocytochemical detection of PAR accumulation, respectively.Key findingsPARP-1 activation was induced by treatment of astrocytes with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), an alkylating agent. MNNG-triggered astrocyte death and PAR accumulation were completely inhibited by treatment with nicotinamide as with DPQ (3,4-dihydro-5-(4-(1-piperidinyl)butoxy)-1(2H)-isoquinolinone), a second generation PARP inhibitor. The uptake of [¹⁴C]nicotinamide was time-, temperature-, concentration- and pH-dependent, and was inhibited and stimulated by co- and pre-treatment with N-methylnicotinamide, a representative substrate of an organic cation transport system, respectively. Co-treatment of astrocytes with nicotinamide and N-methylnicotinamide resulted in a decrease in PAR accumulation and absolute prevention of cell death.SignificanceThese findings suggest that nicotinamide has a protective effect against PARP-1-induced astrocyte death and that its transporter-mediated uptake, which is extracellular pH-sensitive and common to N-methylnicotinamide, is critical for prevention of PARP-1-triggered cell death.     

Zierane sesquiterpene lactone,cembrane and fusicoccane diterpenoids,from the Tahitian liverwort Chandonanthus hirtellus

Cembrane-type diterpenoids, 13,18,20-epi-iso-chandonanthone (1) and (8E)-4α-acetoxy-12α,13α-epoxycembra-1(15),8-diene (2), two fusicoccane-type diterpenoids, fusicoauritone 6α-methyl ether (3) and 6β,10β-epoxy-5β-hydroxyfusicocc-2-ene (4) and a zierane sesquiterpene γ-lactone, chandolide (5) were isolated from the Tahitian liverwort Chandonanthus hirtellus (Web.) Mitt., together with eight known diterpenoids, chandonanthine (6), fusicogigantone A (7), fusicogigantone B (8), fusicogigantepoxide (9), anadensin (10), fusicoauritone (11), ent-verticillol (12) and ent-epi-verticillol (13). Their structures were established by a combination of extensive NMR spectroscopy and/or X-ray crystallographic analyses. Compounds 1, 5 and 10 showed weak cytotoxic activity against HL-60. Compound 3 also indicated weak cytotoxic activity against KB cell lines.     

Short-limbed dwarfism: slw is a new allele of Npr2 causing chondrodysplasia

Short-limbed dwarfism (SLW) is a new mutant mouse characterized by a dwarf phenotype with markedly short body, limbs, and tail. In the present study, we investigated the skeletal phenotypes of the SLW mouse and determined the chromosomal localization to identify the gene responsible for the phenotypes (slw). Skeletal preparations stained with alcian blue and alizarin red revealed that longitudinal growth of the extremities of the affected (slw/slw) mice was significantly reduced in comparison with that of normal mice, whereas the positions and numbers of skeletal elements were normal. Histological examination of tibial growth plates of the affected mice showed that the numbers of proliferating and hypertrophic chondrocytes were obviously diminished. These phenotypes resembled to those of human chondrodysplasias caused by defective chondrocyte proliferation and differentiation. We mapped the slw locus on an 11.7-cM interval of the proximal region of mouse chromosome 4 by linkage analysis. Furthermore, allelism test using Npr2(cn) locus, a mutant allele of Npr2 gene encoding a natriuretic peptide receptor B, revealed that slw locus is an allele of the Npr2 gene. These results suggest that the dwarf phenotype of the SLW mouse is caused by the disturbed endochondral ossification, and a mutation in the Npr2 gene is expected to be responsible for the phenotypes of the SLW mouse.     

Effect of pre-treatment with St John's Wort on nephrotoxicity of cisplatin in rats

An herbal health care supplement, St John's Wort (SJW, Hypericum perforatum) has become widely used in the treatment of depression, and is known to interact with therapeutic drugs. Here we report a preventive effect of SJW on cisplatin nephrotoxicity in rats. Rats were given SJW (400 mg/kg/day, p.o.) for 10 consecutive days, and were injected with cisplatin (5 mg/kg, i.v.) on the day after the final SJW treatment. Cisplatin treatment increased the serum creatinine level, which is an index of nephrotoxicity, to 1.51+/-0.22 mg/dl (mean+/-SE) from 0.28+/-0.05 mg/dl (control) on day 5 after the cisplatin injection. This increase fell significantly to 0.86+/-0.13 mg/dl by pre-treatment with SJW. Cisplatin-induced histological abnormality of the kidney was blocked by pre-treatment with SJW. When SJW was administered for 10 days, the amounts of renal metallothionein (MT) and hepatic multidrug resistance protein 2 (Mrp2) were increased to 164.8+/-13.0% and 220.8+/-39.3% (mean+/-SE) of controls, respectively. GSH levels in the kidney and liver were not changed. Total and free cisplatin concentration in serum was not influenced by SJW treatment. In conclusion, the results suggest that pre-treatment with SJW may diminish cisplatin nephrotoxicity.     

In vitro and in silico analysis reveals an efficient algorithm to predict the splicing consequences of mutations at the 5' splice sites

We have found that two previously reported exonic mutations in the PINK1 and PARK7 genes affect pre-mRNA splicing. To develop an algorithm to predict underestimated splicing consequences of exonic mutations at the 5′ splice site, we constructed and analyzed 31 minigenes carrying exonic splicing mutations and their derivatives. We also examined 189 249 U2-dependent 5′ splice sites of the entire human genome and found that a new variable, the SD-Score, which represents a common logarithm of the frequency of a specific 5′ splice site, efficiently predicts the splicing consequences of these minigenes. We also employed the information contents (R_i) to improve the prediction accuracy. We validated our algorithm by analyzing 32 additional minigenes as well as 179 previously reported splicing mutations. The SD-Score algorithm predicted aberrant splicings in 198 of 204 sites (sensitivity = 97.1%) and normal splicings in 36 of 38 sites (specificity = 94.7%). Simulation of all possible exonic mutations at positions −3, −2 and −1 of the 189 249 sites predicts that 37.8, 88.8 and 96.8% of these mutations would affect pre-mRNA splicing, respectively. We propose that the SD-Score algorithm is a practical tool to predict splicing consequences of mutations affecting the 5′ splice site.     

Transforming activity of purinergic receptor P2Y, G-protein coupled, 2 revealed by retroviral expression screening

Colorectal cancer (CRC) is one of the leading causes of cancer death in humans. In order to identify novel cancer-promoting genes in CRC, we here constructed a retroviral cDNA expression library from a CRC cell line RKO, and used it for a focus formation assay with mouse 3T3 fibroblasts, leading to the identification of 42 independent cDNAs. One of such cDNAs turned out to encode purinergic receptor P2Y, G-protein coupled, 2 (P2RY2). The oncogenic potential of P2RY2 was confirmed in vitro with the focus formation assay as well as soft agar-growth assay, and also in vivo with a tumorigenicity assay in nude mice. While our P2RY2 cDNA encodes a protein with two amino-acid substitutions compared to the reported one, we have confirmed that the wild-type P2RY2 has a strong transforming potential as well. These results indicate an unexpected role of P2RY2 in the carcinogenesis of human cancers.     

A strong association between human earwax-type and apocrine colostrum secretion from the mammary gland

Here we provided the first genetic evidence for an association between the degree of apocrine colostrum secretion and human earwax type. Genotyping at the earwax-type locus, rs17822931 within the ABCC11 gene, revealed that 155 of 225 Japanese women were dry-type and 70 wet-type. Frequency of women without colostrum among dry-type women was significantly higher than that among wet-type women (P < 0.0002), and the measurable colostrum volume in dry-type women was significantly smaller than in wet-type women (P = 0.0341).