We have devised an analytical method for the determination of fatty acid composition of erythrocyte membrane sphingomyelin by chemical ionization mass spectrometry combined with capillary column gas-liquid chromatography. Fatty acid composition of erythrocyte membrane sphingomyelin from 8 patients with adrenoleukodystrophy (ALD) and 16 healthy controls were examined by this method. The ratio of hexacosanoic acid (C26:0) to docosanoic acid (C22:0) in erythrocyte membrane sphingomyelin from ALD patients was 2.6-fold higher than that of the controls. This result suggests that biochemical diagnosis of ALD is possible by the analysis of fatty acid composition of erythrocyte membrane sphingomyelin. Furthermore, it demonstrates that biochemical abnormality in ALD is the generalized abnormal metabolism of very long-chain saturated fatty acids.

Morphological and biochemical studies were performed on the CNS of neurologically affected NCTR-Balb/C mouse. Histological and electron microscopic techniques demonstrated severe myelin deficiency in the affected brains. Neither the presence of lipid-containing macrophages nor reactive gliosis was apparent. Analysis of myelin-associated lipids and proteins revealed prominent depletion of galactocerebroside, sulfatide, and proteolipid proteins. In contrast to the scarcity of myelin-specific constituents a marked accumulation of GM2 and GM3 gangliosides and several neutral glycolipids, i.e., glucocerebroside, lactosylceramide, gangliotriaosylceramide, and gangliotetraosylceramide were found in affected CNS. These abnormalities were already apparent in 12-day-old pups as well as in 65-day-old mice. A significant deficit in the proportion of long-chain fatty acids (C24), notable in both normal and alpha-hydroxy acids of cerebrosides from affected white matter, was measured. The lack of reactive gliosis, the observed depletion of galactocerebroside and sulfatide at the early age of 12 days, and the relative decrease in long-chain fatty acids in affected CNS strongly suggest a defect in myelinogenesis in this mutant rather than a secondary process of myelin breakdown.

Mouse kidney ␤-1,6-GlcNAc-transferase (GNT) is the key enzyme for the synthesis of a glycosphingolipid (Gal␤1-4(Fuc␣1-3)GlcNAc␤1-6(Gal␤1-3)GalNAc␤1-3Gal␣1-4Gal␤1-4Glc␤1-ceramide) that contains the Le X trisaccharide epitope at its nonreducing terminus. The expression of this glycolipid in the kidney is polymorphic; it is expressed in BALB/c but not DBA/2 mice; and a single autosomal gene (Gsl5) is responsible for this polymorphism. We report here the cDNA sequence that encodes the kidney GNT of BALB/c mice, which possess a wild-type Gsl5 gene. The deduced amino acid sequence exhibits 84% identity to that of human core 2 ␤-1,6-GlcNAc-transferase, which suggests that kidney GNT is a mouse homologue of human core 2 ␤-1,6-GlcNAc-transferase. The GNT mRNA is expressed abundantly in the kidney, but was not detected in other BALB/c organs or in the kidneys of DBA/2 mice by Northern blot analysis. In addition, we were able to clone and sequence another homologous cDNA from the submandibular gland. The two sequences differ only in their 5-untranslated region. The submandibular gland type of cDNA was detected in various organs of DBA/2 mice by reverse transcription-polymerase chain reaction, which indicates that the submandibular gland type is ubiquitous and that its expression is not regulated by the Gsl5 gene. Results obtained using the long accurate polymerase chain reaction method indicate that the GNT gene is ϳ45 kilobases long, and the order of the exons from the 5-end is exon 1 of the kidney type, exon 1 of the ubiquitous type, exon 2, and exon 3. Exons 2 and 3 are present in both transcripts, and the translated region is in exon 3. These data suggest that the expression of GNT is regulated by an alternative splicing mechanism and also probably by tissue-specific enhancers and that Gsl5 regulates the expression of GNT only in the kidney.Carbohydrate chains of cell-surface glycoconjugates play important roles in cell-cell and cell-matrix communication (1). The diversity of the carbohydrate structures provides a basis for cell-specific recognition. The expression of carbohydrate chains is highly regulated and changes during embryogenesis, differentiation, and oncogenic transformation (2). The regulatory process may be mediated by many different gene products, including glycosyltransferases (3, 4), transcription factors (5-10), nucleotide sugar transporters (11-14), kinases and phosphatases that act on transferases (15,16), and other genes. The objective of our studies is to understand the genetic basis and mechanisms that regulate the expression of carbohydrates.We have identified an autosomal mouse gene (Gsl5) that controls the expression of GlcNAc␤1-6(Gal␤1-3)GalNAc␤1-3Gb 3 Cer and its elongated glycolipids by regulating ␤-1,6-GlcNAc-transferase (GNT) 1 activity in the kidney (17). DBA/2 mice are not able to express detectable levels of GNT activity or the glycolipids containing GlcNAc␤1-6(Gal␤1-3)GalNAc␤1-3Gb 3 Cer as a core structure because of a defect of the Gsl5 gene. To elucidate the role of Gsl5, the mouse k...

The proximal straight tubule (S3 segment) of the kidney is highly susceptible to ischemia and toxic insults but has a remarkable capacity to repair its structure and function. In response to such injuries, complex processes take place to regenerate the epithelial cells of the S3 segment; however, the precise molecular mechanisms of this regeneration are still being investigated. By applying the “toxin receptor mediated cell knockout” method under the control of the S3 segment-specific promoter/enhancer, Gsl5, which drives core 2 β-1,6-N-acetylglucosaminyltransferase gene expression, we established a transgenic mouse line expressing the human diphtheria toxin (DT) receptor only in the S3 segment. The administration of DT to these transgenic mice caused the selective ablation of S3 segment cells in a dose-dependent manner, and transgenic mice exhibited polyuria containing serum albumin and subsequently developed oliguria. An increase in the concentration of blood urea nitrogen was also observed, and the peak BUN levels occurred 3–7 days after DT administration. Histological analysis revealed that the most severe injury occurred in the S3 segments of the proximal tubule, in which tubular cells were exfoliated into the tubular lumen. In addition, aquaporin 7, which is localized exclusively to the S3 segment, was diminished. These results indicate that this transgenic mouse can suffer acute kidney injury (AKI) caused by S3 segment-specific damage after DT administration. This transgenic line offers an excellent model to uncover the mechanisms of AKI and its rapid recovery.

In this study, we developed a rapid, simple and reliable method for the simultaneous analysis of HLA and HPA antibodies. ICFA can also be used as an alternative to the lymphocyte cytotoxicity test for HLA cross-matching.