The cell density-dependent growth inhibition of human SK-N-MC neuroblastoma cells is initiated by increased ganglioside sialidase activity leading to elevated cell surface presentation of ganglioside GM1, a ligand of galectin-1. We herein show that the extent of the cell surface expression of the galectin coincides with marked increases of the sialidase activity. Reverse transcriptase-polymerase chain reaction analysis excludes a regulation at the transcriptional level. Exposure of cells to purified galectin-1 reveals its carbohydrate-dependent activity to reduce cell proliferation. Assays to detect DNA fragmentation biochemically and cytometrically and to block caspases render it unlikely that galectin-1 acts as a classical proapoptotic factor on these cells. Because the chimeric galectin-3 shares binding sites and binding parameters with galectin-1 for these cells, we tested whether this galectin will elicit the same response as the homodimeric cross-linking galectin-1. Evidently, galectin-3 fails to affect cell growth by itself but interferes with galectin-1 upon coincubation. Its proteolytically truncated variant, the C-terminal lectin domain with impaired capacity to form aggregates when surface bound, has only weak binding properties. Thus, the way in which the galectin-1 interacts topologically with an apparently common set of ligands relative to galectin-3 is crucial for eliciting post-binding events. We conclude that galectin-1 is a probable effector in the sialidase-dependent growth control in this system. Moreover, the experiments with galectin-3 reveal functional divergence, most probably based on different topologies of presentation of homologous carbohydratebinding sites.

5-lipoxygenase (5-LOThe enzyme 5-lipoxygenase (5-LO, arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) 1 catalyzes the conversion of arachidonic acid to (5S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HPETE) and further to leukotriene A 4 ((5S)-6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid) (1, 2).5-LO is expressed in a variety of immune competent cells including B-lymphocytes, granulocytes, monocytes, mast cells, and dendritic cells (3). Depending on the cell type, several cytokines have been shown to be inducers of the 5-LO pathway. In granulocytes 5-LO expression is stimulated by granulocytemacrophage colony-stimulating factor (GM-CSF) (4), whereas interleukin-3 regulates the development of the 5-LO pathway in mouse mast cells (5). In the human myeloid leukemic cell lines HL-60 and Mono Mac 6, cell differentiation by 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) and transforming growth factor-beta (TGF␤) leads to a strong induction of the 5-lipoxygenase pathway (6, 7). In Mono Mac 6 cells, the induction of 5-LO protein expression and activity by TGF␤ and 1,25(OH) 2 D 3 was accompanied by a 64-fold up-regulation of mature 5-LO mRNA and an up to 5-fold increase in 5-LO primary transcripts (7, 8), whereas no significant induction of 5-LO transcription was found in nuclear run-off assays (9). The human 5-LO gene promoter was first characterized by Hoshiko et al. (10). Several features of the putative 5-LO promoter region (such as the lack of TATAA or CCAAT boxes and repeated (GϩC)-rich elements) are characteristic for so-called housekeeping genes. Previous data suggest that the transcription factors Egr-1 and/or Sp1 are required for basal 5-LO transcription and that they functionally interact with the 5-LO promoter and activate it via repeated response elements located between positions Ϫ212 and Ϫ88 bp, relative to the translational start site (10, 11). Interestingly, naturally occurring mutations were found in the 5-LO promoter consisting of the deletion of one or two, or the addition of one Sp1 binding site (12). These mutations only slightly alter 5-LO promoter activity in reporter gene assays but have a significant impact on the response of asthma patients to 5-LO inhibitors (13).As yet, no data are available on the mechanisms involved in the cell type-specific activation of the 5-LO promoter in response to cell differentiation signals and inflammatory stimuli. Expression of several genes with (GϩC)-rich promoters has been shown to be regulated by DNA methylation (14). Whereas promoters of (GϩC)-rich housekeeping genes are usually unmethylated, methylation of promoters of tissue-specific genes is usually linked with silencing of the respective genes.Therefore, it was of interest to study the role of DNA methylation in the regulation of 5-lipoxygenase expression. The human myeloid cell lines Mono Mac 6 and HL-60 show prominent 5-LO gene expression and 5-LO activity after differentiation by TGF␤ and 1,25(OH) 2 D 3 (6, 7), whereas U937 cells and the HL-60TB cell line, a subline of the HL-60 ce...

We report a Turkish family with parental consanguinity and at risk for sialidosis type II, an inherited autosomal recessive disorder caused by lysosomal alpha-N-acetyl-neuraminidase (sialidase, NEU1) deficiency. The proband was a premature male infant that presented with hydrops, hepatomegaly, respiratory distress syndrome, and anemia and that died of respiratory insufficiency 2 months after birth despite intensive care. An abnormally increased [14C]methylamine incorporation and an isolated deficiency of lysosomal alpha-N-acetyl-neuraminidase were found in cultured skin fibroblasts. A previous pregnancy of the mother terminated in a spontaneous abortion in the 13th week of gestation. A successive pregnancy showed hydrops fetalis, and an enzymatic assay of cultured amniotic fluid cells indicated a deficiency of alpha-N-acetyl-neuraminidase. Following pregnancy termination at 20 weeks gestation, light microscopy of fetal tissues revealed classic vacuolation not only in liver, bone marrow, brain, and kidney, but also in endocrine organs such as the thyroid gland, adrenal gland, hypophysis, and testes, and in the thymus. DNA analysis of the family showed that both the proband and the third sibling had a novel homozygous nonsense point mutation at nucleotide 87 in exon 1 of the alpha-N-acetyl-neuraminidase (neu1) gene causing a substitution of tryptophan at codon 29 by a termination codon (W29X). DNA sequencing of polymerase chain reaction products identified the parents as heterozygous carriers. To detect neu1 mRNA expression, a real-time reverse transcription/polymerase chain reaction was performed, and similar rates of neu1 mRNA expression were found in the fibroblasts of the fetus, the 2nd sibling, and in controls. The very early termination codon with complete loss of neuraminidase activity is probably the molecular basis of the unusually severe vacuolation pattern in this form of congenital sialidosis.

The deficiency of the lysosomal neuraminidase (NEU1; sialidase) causes the storage disorder sialidosis with symptoms ranging from eye abnormalities and neurological disturbances to skeletal malformations, mental retardation and early death. Sialidosis patients encompassing a wide spectrum of clinical symptoms were screened for mutations in neu1. We identified the same homozygous interstitial deletion (11 kb) in two patients causing the fusion of exon 10 of CTL4 (New Gene 22; NG22) with the 3P P-UTR of neu1. In one patient we found the resulting CTL4/Neu1 fusion transcript, in the other we detected an alternatively spliced CTL4 transcript (retention of intron 9). ß

A subset of patients with type 2 Gaucher disease is characterized by intrauterine onset of rapidly progressive neuropathic disease, arthrogryposis, hydrops fetalis and in some cases restrictive dermopathy. beta-Glucocerebrosidase (beta-glucosidase) activity is usually low or undetectable. In most cases death ensues either in-utero or within hours or days after birth. We report on an infant born to non-consanguineous parents of Caucasian origin presenting at birth with hydrops, arthrogryposis, severe respiratory distress, hepatosplenomegaly, and liver failure. Death occurred within several hours after delivery and autopsy revealed typical Gaucher cells in multiple organs in combination with severe apoptotic neurodegeneration throughout the brain. beta-Glucocerebrosidase activity was 1% of the norm in fibroblasts and a novel heterozygous insertion c.1515_1516insAGTGAGGGCAAT was identified by genomic sequencing and an insertion-specific seminested PCR. In addition, molecular studies revealed a previously described in type 1 Gaucher disease missense mutation c.476G --> A which results in a heterozygous substitution of R120Q. Our observations confirm considerable genotypic heterogeneity in patients with type 2 Gaucher disease. The transheterozygous combination of a mutation, previously described in type 1 Gaucher disease, together with a newly identified insertion may result in this severe phenotype.