A mobility shift detection method for DNA methylation analysis using phosphate affinity polyacrylamide gel electrophoresis

We describe a procedure for DNA methylation analysis using the bisulfite-mediated cytosine-to-uracil conversion of a target DNA followed by methylation-specific polymerase chain reaction (MSP) and phosphate affinity polyacrylamide gel electrophoresis (PAGE). The MSP was performed using a 1:1 mixture of 5′-phosphorylated methylation-specific and 5′-OH non-methylation-specific primers. The PAGE using an immobilized phosphate-binding tag molecule (i.e., a polyacrylamide-bound dizinc(II) complex, Zn²⁺-Phos-tag), which selectively captures the 5′-phosphorylated DNA fragment, enabled the mobility shift detection of the methylation-specific product as a slower migration band. Using this novel procedure, we demonstrated the detection of a methylated cytosine base in a pUC19 plasmid.     

Aminoguanidine pyridoxal adduct is superior to aminoguanidine for preventing diabetic nephropathy in mice.

Aminoguanidine inhibits the formation of advanced glycation end-products, and has been extensively examined in animals. However, administration of aminoguanidine decreases the hepatic content of pyridoxal phosphate. In order to avoid this problem, we developed an aminoguanidine pyridoxal Schiff base adduct and examined its efficacy in vitro as well as in a model of diabetic nephropathy. Mice with streptozotocin-induced diabetes were treated with aminoguanidine or aminoguanidine pyridoxal adduct for 9 weeks. An in vitro study was also performed to assess the antioxidant activity of aminoguanidine and its pyridoxal adduct. Neither drug altered glycemic control. Aminoguanidine pyridoxal adduct significantly improved urinary albumin excretion by 78.1 % compared with the diabetic control, and also had a better preventive effect on the progression of renal pathology than aminoguanidine did. Inhibition of glycation by both drugs was similar, but the antioxidant activity of the pyridoxal adduct was far superior. These findings suggest that aminoguanidine pyridoxal adduct may be superior to aminoguanidine, as it not only prevents vitamin B6 deficiency but is also better at controlling diabetic nephropathy, as this adduct inhibits oxidation as well as glycation.     

A physical map with yeast artificial chromosome (YAC) clones covering 63% of the 12 rice chromosomes.

A new YAC (yeast artificial chromosome) physical map of the 12 rice chromosomes was constructed utilizing the latest molecular linkage map. The 1439 DNA markers on the rice genetic map selected a total of 1892 YACs from a YAC library. A total of 675 distinct YACs were assigned to specific chromosomal locations. In all chromosomes, 297 YAC contigs and 142 YAC islands were formed. The total physical length of these contigs and islands was estimated to 270 Mb which corresponds to approximately 63% of the entire rice genome (430 Mb). Because the physical length of each YAC contig has been measured, we could then estimate the physical distance between genetic markers more precisely than previously. In the course of constructing the new physical map, the DNA markers mapped at 0.0-cM intervals were ordered accurately and the presence of potentially duplicated regions among the chromosomes was detected. The physical map combined with the genetic map will form the basis for elucidation of the rice genome structure, map-based cloning of agronomically important genes, and genome sequencing.     

Evidence for direct arrhythmogenic action of endothelin.

We studied electrophysiological effects of endothelin on canine cardiac tissues. Endothelin prolonged action potential duration and decreased spontaneous firing rate of the right bundle branch cells. At a concentration of 2 x 10(-7)M the plateau phase of action potentials was flattened, followed by the abrupt occurrence of early afterdepolarizations (EADs). ET, at a concentration as low as 2 x 10(-9)M, was capable of inducing EADs although their incidence was low. The EADs were initiated from the membrane potential less negative than -30mV and were suppressed by nicardipine, suggesting the involvement of dihydropyridine-sensitive Ca2+ channels in the induction of EADs. Because EADs are considered to underlie certain types of arrhythmias endothelin per se may have arrhythmogenic action.     

Expression and cloning of complementary DNA for a human enzyme that repairs O6-methylguanine in DNA

A cell line with an increased resistance to alkylating agents and an extremely high level of O6-methylguanine-DNA methyltransferase activity was isolated after transfection of methyltransferase-deficient Mer- cells with a cDNA library, prepared from methyltransferase-proficient human Mer+ (Raji) cells. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis revealed that a protein, with a molecular weight of approximately 25,000, accepted 3H label from DNA that had been treated with [3H]methylnitrosourea. Since the cDNA for methyltransferase was integrated into the chromosomal DNA, it was recovered by using the polymerase chain reaction. When the cDNA placed in an expression vector p500 was introduced into Mer- cells, the cells acquired an increased resistance to alkylating agents and exhibited a high level of O6-methylguanine-DNA methyltransferase activity. From the transformants the cDNA could be recovered as a part of the autonomously replicating plasmid. The nucleotide sequence of the cDNA was determined, and an open reading frame comprising 207 amino acid residues was found. The molecular weight of methyltransferase, calculated from the predicted amino acid sequence, was 21,700. The predicted amino acid sequence of the human methyltransferase exhibits an intensive homology with those of the bacterial counterparts, Ada and Ogt proteins of Escherichia coli and Dat protein of Bacillus subtilis, especially around possible methyl acceptor sites.     

Fibroblast growth factor stimulates colony formation of differentiated chondrocytes in soft agar

The effect of fibroblast growth factor (FGF) on the growth of chondrocytes in soft agar was examined. FGF induced colony formation by chick embryo and rabbit chondrocytes. The colony-forming efficiency of FGF-exposed chondrocytes was similar to that of Rous sarcoma virus-transformed chondrocytes (15-20%). Other mitogenic agents tested, such as epidermal growth factor, insulin, insulin-like growth factor-l, and platelet-derived growth factor, induced very low levels of colony formation. The induction of growth in soft agar of chondrocytes by FGF was not due to cells' phenotypic transformation, because chondrocytes grown in soft agar with FGF retained the ability to synthesize cartilage-characteristic proteoglycan. FGF did not induce growth in soft agar of chondrocytes whose phenotypic expression was suppressed by retinoic acid or 5-bromodeoxyuridine. In addition, FGF did not induce growth in soft agar of primary fibroblasts and normal rat kidney (NRK) cells. These results suggest that FGF selectively stimulates growth of differentiated chondrocytes in soft agar.     

Specific uptake of asialofetuin-tacked liposomes encapsulating interferon-gamma by human hepatoma cells and its inhibitory effect on hepatitis B virus replication.

Asialofetuin-tacked liposomes (AF-liposomes) encapsulating interferon (IFN)-gamma were bound and internalized into a human hepatoma cell line, HepG2 cells, selectively through asialoglycoprotein receptor, but not non-tacked liposomes (N-liposomes). AF-liposomal IFN-gamma was more effective for inhibition of viral DNA replication in hepatitis B virus (HBV)-producing clone from HepG2 cells transfected with HBV-DNA than N-liposomal IFN-gamma. AF-liposomes may increase the therapeutic potential of IFN-gamma through asialoglycoprotein receptor in treating HBV-infected hepatocytes.     

Adult onset globoid cell leukodystrophy (Krabbe disease): analysis of galactosylceramidase cDNA from four Japanese patients

We examined galactosylceramidase (GALC) cDNA in four Japanese patients with adult onset globoid cell leukodystrophy (Krabbe disease; AO-GLD) by polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) analysis, subsequent sequence determination, and restriction enzyme digestion of PCR products. Initial symptoms were the onset of slowly progressive spastic paraplegia from the middle of the second decade, and all patients had diminished GALC activity in their leukocytes. We identified three missense mutations (I66M, G270D, L618S) and one exon-6 skipping (535– 573del). Two of the patients had only the I66M mutant mRNA, and one only the G270D mutant mRNA. The fourth patient carried a compound heterozygous mutation of 535–573del and L618S. To determine the enzymatic activities produced by these mutations, we constructed mutated GALC cDNAs and expressed them in COS-1 cells. Three mutations, viz., G270D, L618S, and exon-6 skipping (535–573del), produced diminished GALC activity as expected. The I66M mutation in the wild-type GALC cDNA(I289) had normal activity, but when this mutation and the V289 polymorphism were introduced into the same allele, it had decreased activity. Thus, the combination of a unique mutation and polymorphism causes conformational change in the GALC enzyme, resulting in low enzymatic activity. AO-GLD mutations, including those found here, are located in the N-terminus (I66M, G270D, 535–573del) or C-terminus (L618S) of the GALC enzyme, whereas the reported mutations in the infantile form (IF-GLD) are in the central domain. This difference in mutation sites may affect the clinical features of GLD. Received: 4 February 1997 / Accepted: 28 April 1997     

Novel Defense by Metallothionein Induction Against Cognitive Decline: From Amyloid β<Subscript>1–42</Subscript>-Induced Excess Zn<Superscript>2+</Superscript> to Functional Zn<Superscript>2+</Superscript> Deficiency

The role of metallothioneins (MTs) in cognitive decline associated with intracellular Zn²⁺ dysregulation remains unclear. Here, we report that hippocampal MT induction defends cognitive decline, which was induced by amyloid β_1–42 (Aβ_1–42)-mediated excess Zn²⁺ and functional Zn²⁺ deficiency. Excess increase in intracellular Zn²⁺, which was induced by local injection of Aβ_1–42 into the dentate granule cell layer, attenuated in vivo perforant pathway LTP, while the attenuation was rescued by preinjection of MT inducers into the same region. Intraperitoneal injection of dexamethasone, which increased hippocampal MT proteins and blocked Aβ_1–42-mediated Zn²⁺ uptake, but not Aβ_1–42 uptake, into dentate granule cells, also rescued Aβ_1–42-induced impairment of memory via attenuated LTP. The present study indicates that hippocampal MT induction blocks rapid excess increase in intracellular Zn²⁺ in dentate granule cells, which originates in Zn²⁺ released from Aβ_1–42, followed by rescuing Aβ_1–42-induced cognitive decline. Furthermore, LTP was vulnerable to Aβ_1–42 in the aged dentate gyrus, consistent with enhanced Aβ_1–42-mediated Zn²⁺ uptake into aged dentate granule cells, suggesting that Aβ_1–42-induced cognitive decline, which is caused by excess intracellular Zn²⁺, can more frequently occur along with aging. On the other hand, attenuated LTP under functional Zn²⁺ deficiency in dentate granule cells was also rescued by MT induction. Hippocampal MT induction may rescue cognitive decline under lack of cellular transient changes in functional Zn²⁺ concentration, while its induction is an attractive defense strategy against Aβ_1–42-induced cognitive decline.     

Growth and photosynthetic response of two larches exposed to O<Subscript>3</Subscript> mixing ratios ranging from preindustrial to near future

In this study, we questioned whether ground-level ozone (O₃) induces hormesis in Japanese larch (Larix kaempferi) and its hybrid F₁ (L. gmelinii var. japonica × L. kaempferi). In order to answer the question, we exposed seedlings of both taxa to four O₃ treatments [ranging from ≈10 to 60 nmol(O₃) mol^–1] in open-top chambers for two consecutive growing seasons. We found a hormetic response in maximum photosynthetic rate (P_Nmax) at 1700 μmol(CO₂) mol^–1 and maximum rates of carboxylation (V_cmax) and electron transport (J_max) in both larches. Stimulation of P_Nmax, V_cmax, and J_max did not lead to suppressed plant productivity in Japanese larch, which followed a stress-tolerant strategy, but it did lead to suppressed plant productivity in hybrid larch which followed a competitive strategy. These findings are the first to suggest that stimulation of physiological functions by low O₃ exposures may have negative consequences for larch reproduction.