Accumulation of Lysosphingolipids in Tissues from Patients with GM1 and GM2 Gangliosidoses

By using a sensitive method, we assayed lysocompounds of gangliosides and asialogangliosides in tissues from four patients with GM2 gangliosidosis (one with Sandhoff disease and three with Tay-Sachs disease) and from three patients with GM1 gangliosidosis [one with infantile type (fetus), one with late-infantile, and one with adult type]. In the brain and spinal cord of all the patients except for an adult GM1 gangliosidosis patient, abnormal accumulation of the lipids was observed, though the concentration in the fetal tissue was low. In GM2 gangliosidosis, the amounts of lyso GM2 ganglioside accumulated in the brain were similar among the patient with Sandhoff disease and the patients with Tay-Sachs disease, whereas the concentration of asialo lyso GM2 ganglioside in the brain was higher in the former patient than in the latter patients. By comparing the sphingoid bases of neutral sphingolipids, gangliosides, and lysosphingolipids, it was suggested that lysosphingolipids in the diseased tissue are synthesized by sequential glycosylation from free sphingoid bases, but not by deacylation of the sphingolipids. Because lysosphingolipids are known to be cytotoxic, the abnormally accumulated lysophingolipids may well be the pathogenetic agent for the neuronal degeneration in gangliosidoses.     

Hematopoietic, angiogenic and eye defects in Meis1 mutant animals

Meis1 and Hoxa9 expression is upregulated by retroviral integration in murine myeloid leukemias and in human leukemias carrying MLL translocations. Both genes also cooperate to induce leukemia in a mouse leukemia acceleration assay, which can be explained, in part, by their physical interaction with each other as well as the PBX family of homeodomain proteins. Here we show that Meis1-deficient embryos have partially duplicated retinas and smaller lenses than normal. They also fail to produce megakaryocytes, display extensive hemorrhaging, and die by embryonic day 14.5. In addition, Meis1-deficient embryos lack well-formed capillaries, although larger blood vessels are normal. Definitive myeloerythroid lineages are present in the mutant embryos, but the total numbers of colony-forming cells are dramatically reduced. Mutant fetal liver cells also fail to radioprotect lethally irradiated animals and they compete poorly in repopulation assays even though they can repopulate all hematopoietic lineages. These and other studies showing that Meis1 is expressed at high levels in hematopoietic stem cells (HSCs) suggest that Meis1 may also be required for the proliferation/self-renewal of the HSC.     

Spontaneous mutations in the ammonium transport gene AMT4 of Chlamydomonas reinhardtii

Evidence in several microorganisms indicates that Amt proteins are gas channels for NH(3) and CH(3)NH(2), and this has been confirmed structurally. Chlamydomonas reinhardtii has at least four AMT genes, the most reported for a microorganism. Under nitrogen-limiting conditions all AMT genes are transcribed and Chlamydomonas is sensitive to methylammonium toxicity. All 16 spontaneous methylammonium-resistant mutants that we analyzed had defects in accumulation of [(14)C]methylammonium. Genetic crosses indicated that 12 had lesions in a single locus, whereas two each had lesions in other loci. Lesions in different loci were correlated with different degrees of defect in [(14)C]methylammonium uptake. One mutant in the largest class had an insert in the AMT4 gene, and the insert cosegregated with methylammonium resistance in genetic crosses. The other 11 strains in this class also had amt4 lesions, which we characterized at the molecular level. Properties of the amt4 mutants were clearly different from those of rh1 RNAi lines. They indicated that the physiological substrates for Amt and Rh proteins, the only two members of their protein superfamily, are NH(3) and CO(2), respectively.     

Cytochrome aa3 in facultatively aerobic and hyperthermophilic archaeon Pyrobaculum oguniense

We partially purified and characterized the cytochrome aa3 from the facultatively aerobic and hyperthermophilic archaeon Pyrobaculum oguniense. This cytochrome aa3 showed oxygen consumption activity with N, N, N', N'-tetramethyl-1,4-phenylenediamine and ascorbate as substrates, and also displayed bovine cytochrome c oxidase activity. These enzymatic activities of cytochrome aa3 were inhibited by cyanide and azide. This cytochrome contained heme As, but not typical heme A. An analysis of trypsin-digested fragments indicated that 1 subunit of this cytochrome was identical to the gene product of subunit I of the SoxM-type heme--copper oxidase (poxC). This is the first report of a terminal oxidase in hyperthermophilic crenarchaeon belonging to the order Thermoproteales.     

Transcriptional regulation of cyclooxygenase-2 in the Human Microvascular Endothelial Cell Line,HMEC-1: Control by the combinatorial actions of AP2, NF-IL-6 and CRE elements

Interleukin-1beta (IL-1) is a potent inducer of cyclooxygenase-2 (COX-2) and prostaglandin biosynthesis in many types of cells, yet little is known about the molecular mechanisms regulating IL-1 mediated prostanoid biosynthesis in the endothelium of the microvasculature. Therefore, we examined the cis- and trans-acting factors regulating IL-1-induced COX-2 expression in the human microvascular endothelial cell line, HMEC-1. IL-1 enhanced steady state levels of COX-2 protein and mRNA synthesis by approximately 2-fold which preceded a 2-fold increase in PGF(alpha) biosynthesis. Expression of a series of COX-2 promoter-luciferase constructs in IL-1 treated HMEC-1 cells revealed that the 'full length' (-1432/+59 bp) promoter was 10 times more active than the SV-40 promoter/enhancer and that it could be further activated by IL-1. Surprisingly however, all except for the shortest COX-2 promoter construct retained the ability to respond to IL-1 and luciferase activity driven by -191/+59 bp COX-2 promoter was as responsive to IL-1 as the full-length promoter. Moreover, site-directed promoter mutagenesis and electophoretic mobility shift assays (EMSA) indicate that the combinatorial actions of AP2, NF-IL6, and CRE elements are critical for both constitutive and IL-1-inducible COX-2 promoter activity. Understanding the mechanism(s) regulating COX-2 gene expression and prostaglandin biosynthesis in the microvasculature has important implications with regard to inflammation and angiogenesis in vivo.     

Genomic organization of the AODEF gene in Asparagus officinalis L

The perianths of Liliaceae plants, such as lily and tulip, have two whorls of almost identical petaloid organs, which are called tepals. According to the modified ABC model proposed in tulip, the class B genes are expressed in whorl 1 as well as whorls 2 and 3, so that the organs of whorls 1 and 2 have the same petaloid structure. The floral structure of asparagus (Asparagus officinalis L.) is similar to that of Liliaceae plants, however, the expression of B-class genes (AODEF, AOGLOA, AOGLOB) was not found in whorl 1, but was confined to whorls 2 and 3. This result does not support the modified ABC model in asparagus. In order to gain a better understanding of asparagus flower development, we have characterized a genomic clone of the AODEF gene. We compared the genomic organization and promoter sequence of AODEF with three well-studied DEF-like genes, DEFICIENS (Antirrhinum), APETALA3 (Arabidopsis), and OSMADS16 (rice). Exon-intron structures of these genes are well-conserved except for the large fifth intron in the AODEF gene and the OSMADS16 gene. Putative cis-elements including CArG-boxes were found in the promoter region and forty-two microsatellites were found in the AODEF genomic sequence.     

Blood group A glycosphingolipid accumulation in the hair of patients with alpha-N-acetylgalactosaminidase deficiency

In the hair of individuals with blood group AB, the level of blood group A glycosphingolipids is much lower than that of blood group B. We hypothesized that in hair, blood group A determinants are converted by alpha-N-acetylgalactosaminidase (alpha-NAGA, E.C.3.2.1.49) to H determinants. To address our hypothesis, the relative amount of ABH glycosphingolipids in hairs and nails of normal subjects, patients with Kanzaki disease, and heterozygous carriers of alpha-NAGA deficiency were analyzed by dot-blotting and enzyme-linked immunosorbent assay. In hair from normal subjects with blood group B, ABH glycosphingolipids consisted of 88% blood group B- and 12% blood group H glycosphingolipids. In blood group A subjects, 14% were group A- and 86% were group H glycosphingolipids. In Kanzaki patients, 81% were blood group A- and 19% were blood group H glycosphingolipids. In 2 alpha-NAGA deficiency carriers, the ABH glycosphingolipids consisted of 67% blood group A- and 33% blood group H glycosphingolipids. These results indicate that blood group A glycosphingolipids are catabolized to H glycosphingolipids by alpha-NAGA, resulting in lower levels of blood group A glycosphingolipids in the hair of normal subjects, and alpha-NAGA deficiency causes accumulation of blood group A glycosphingolipids in the hair of Kanzaki patients. This finding is of clinical relevance because it suggests that hair may be used to diagnose and assess the alpha-NAGA status of individuals.     

Plant growth promotion abilities and microscale bacterial dynamics in the rhizosphere of Lupin analysed by phytate utilization ability

In the rhizosphere, phosphorus (P) levels are low because of P uptake into the roots. Rhizobacteria live on carbon (C) exuded from roots, and may contribute to plant nutrition by liberating P from organic compounds such as phytates. We isolated over 300 phytate (Na-inositol hexa-phosphate; Na-IHP)-utilizing bacterial strains from the rhizosheath and the rhizoplane of Lupinus albus (L.). Almost all of the isolates were classified as Burkholderia based on 16S rDNA sequence analysis. Rhizosheath isolates cultured with Na-IHP as the only source of C and P showed lower P uptake at the same extracellular phytase activity than rhizoplane strains, suggesting that bacteria from the rhizosheath utilized phytate as a C source. Many isolates also utilized insoluble phytate (Al-IHP and/or Fe-IHP). In co-culture with Lotus japonicus seedlings, some isolates promoted plant growth significantly.