No abstract

Mutant mouse models are indispensable tools for clarifying the functions of genes and for elucidating the underlying pathogenic mechanisms of human diseases. Currently, several large-scale mutagenesis projects that employ the chemical mutagen N-ethyl-N-nitrosourea (ENU) are underway worldwide. One specific aim of our ENU mutagenesis project is to generate diabetic mouse models. We screened 9375 animals for dominant traits using a clinical biochemical test and thereby identified 11 mutations in the glucokinase (Gk) gene that were associated with hyperglycemia. GK is a key regulator of insulin secretion in the pancreatic beta-cell. Approximately 190 heterozygous mutations in the human GK gene have been reported to cause maturity onset diabetes of the young, type 2 (MODY2). In addition, five mutations have been reported to cause permanent neonatal diabetes mellitus (PNDM) when present on both alleles. The mutations in our 11 hyperglycemic mutants are located at different positions in Gk. Four have also been found in human MODY2 patients, and another mutant bears its mutation at the same location that is mutated in a PNDM patient. Thus, ENU mutagenesis is effective for developing mouse models for various human genetic diseases, including diabetes mellitus. Some of our Gk mutant lines displayed impaired glucose-responsive insulin secretion and the mutations had different effects on Gk mRNA levels and/or the stability of the GK protein. This collection of Gk mutants will be valuable for understanding GK gene function, for dissecting the function of the enzyme and as models of human MODY2 and PNDM.

Modified galactosylceramide with a long-chain cyclic acetal at the sugar moiety, plasmalogalactosylceramide, was isolated from equine brain. To identify the isomeric stereostructure of the natural product, the plasmalo derivative was chemically synthesized from galactosylceramide through acetalization. The presence of cyclic acetal linkage, the linked position and length of the acetal chain of the synthesized and natural products were determined by proton nuclear magnetic resonance spectroscopy and fast-atom bombardment-mass spectrometry, as well as gas chromatography-mass spectrometry and gas-liquid chromatography. The orientation of the acetal chain linked to galactoside was characterized by connectivity between the cyclic acetal proton and ring proton(s) on the sugar moiety using the homonuclear Overhauser effect. This revealed that, of the two positional isomers of the acetal linkage with 4,6-Oacetal and 3,4-O -acetal derivatives obtained from the acetalization reaction, the former positional isomer, separated into two spots, was identified to 'endo'-and 'exo'-type acetal chains. In comparison to the NMR data of the synthesized derivative, equine brain acetalized lipid was found to be an 'endo'-type 4,6-O -acetal derivative.-

Equine brain gangliosides were isolated and their structures were characterized, to examine whether equine brain has N-glycolyl neuraminic acid in gangliosides, since other mammals predominantly possess N-acetyl neuraminic acid in brain gangliosides, and equine erythrocytes and organs except the brain have gangliosides exclusively containing N-glycolyl neuraminic acid. The gangliosides purified from the brain were identified by proton NMR spectroscopy and mass spectrometry, as well as GLC, resulting in their identification as GM4, GM3, GM2, GM1, GD1a, GD1b, and GT1b. Of these gangliosides, GM3 possessed N-glycolyl neuraminic acid as a minor component (18% of the total GM3), whereas other gangliosides exclusively contained N-acetyl neuraminic acid. The N-glycolyl neuraminic acid residue of the GM3 was confirmed by TLC immunostaining. The possibility of contamination of the GM3 by erythrocytes was eliminated based on the finding that the lipid compositions were characteristic of brain gangliosides. The presence, even as a minor component, of the N-glycolyl neuraminic acid in equine brain gangliosides is exceptional among the sialic acid species in mammalian central nervous system.

Dihydroceramide desaturase, which catalyzes the introduction of a double bond at the 4,5-position of the sphingosine base in a dihydroceramide, was assayed in vitro using radiolabeled D-erythro-C18-dihydroceramide (N-stearoyl sphinganine) and homogenates of fetal rat skin, and some enzymatic properties, including substrate specificity, were determined. The ceramide structure, as the enzymatic product, was confirmed by (i) oxidation of the product with 2,3-dicyano-5,6-dichlorobenzoquinone, which revealed the conversion to 3-ketoceramide (3,3'-didehydroceramide), indicating that a double bond was introduced at the adjacent to the C-3 hydroxyl residue of sphinganine, and (ii) mass spectrometry of a long chain base released from the enzymatic product, which revealed a spectrum identical to that of authentic sphingenine. A short chain dihydroceramide, which was radiolabeled at sphinganine through a newly established method, having a C2- or C6-fatty acid was not desaturated by the skin enzyme, whereas that having a C10-, C14-, or C18-acid was desaturated, maximal reactivity being observed for the C14-dihydroceramide. Other enzymatic properties were confirmed: NAD(H) or NADP(H) and a detergent were required for elevation of the activity; the optimum pH was approximately 6.7; and metal cations were not essential, but Zn2+, Cu2+, and Fe2+ were rather inhibitory. These properties of rat skin desaturase were partly similar to those of rat liver microsomes, as reported recently, however, their substrate specificities were different.