Production of recombinant beta-hexosaminidase A, a potential enzyme for replacement therapy for Tay-Sachs and Sandhoff diseases, in the methylotrophic yeast Ogataea minuta.

Human beta-hexosaminidase A (HexA) is a heterodimeric glycoprotein composed of alpha- and beta-subunits that degrades GM2 gangliosides in lysosomes. GM2 gangliosidosis is a lysosomal storage disease in which an inherited deficiency of HexA causes the accumulation of GM2 gangliosides. In order to prepare a large amount of HexA for a treatment based on enzyme replacement therapy (ERT), recombinant HexA was produced in the methylotrophic yeast Ogataea minuta instead of in mammalian cells, which are commonly used to produce recombinant enzymes for ERT.

1,25(OH)(2)D(3) blocks TNF-induced monocytic tissue factor expression by inhibition of transcription factors AP-1 and NF-kappaB.

An essential coagulation factor, tissue factor (TF), is rapidly expressed by human monocytes when exposed to a variety of agonists, such as lipopolysaccharide or tumor necrosis factor (TNF). We previously found that 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and its potent synthetic analogs downregulate TF and upregulate thrombomodulin expression on monocytic cells, counteracting the effects of TNF at the level of transcription. The human TF gene has characteristic binding sequences for activator protein-1 (AP-1) (c-Jun/c-Fos), nuclear factor-kappaB (NF-kappaB), Sp-1, and early growth response factor-1 (Egr-1).

Cytochemical analysis of storage materials in cultured skin fibroblasts from patients with I-cell disease.

Background: In cultured fibroblasts from I-cell disease patients the transport of many lysosomal enzymes is defective, and affected cells contain inclusion bodies filled with undegraded substrates. However, the contents of these inclusion bodies have not been well characterized yet. We attempted to identify accumulated substances in cultured I-cell disease fibroblasts cytochemically.

Significant decrease in tropoelastin gene expression in fibroblasts from a Japanese Costello syndrome patient with impaired elastogenesis and enhanced proliferation.

Costello syndrome is a connective tissue disorder associated with sparse, thin, and fragmented elastic fibers in tissues. In this study we demonstrated a significant decrease in the expression of tropoelastin mRNA in fibroblasts derived from a Japanese Costello syndrome patient with impaired elastogenesis and enhanced proliferation. In contrast, there were no changes in expression of the Harvey ras (HRAS), fibrillin-1, fibulin-5, microfibril-associated glycoprotein-1 (MAGP-1), lysyl oxidase (LOX), or 67-kDa non-integrin elastin-binding protein (EBP) gene.

Phospholipid storage in the myocardium of a unique Japanese case of idiopathic cardiomyopathy.

Background: A unique adult male patient who developed cardiomyopathy was first suspected to have cardiac Fabry disease based on the pathological findings in heart tissues obtained on biopsy, but the alpha-galactosidase activity in his leukocytes was normal and no mutation was detected in the coding region of the alpha-galactosidase gene. We identified accumulated materials in the myocardium of this patient.

Methods: Pathological and biochemical analyses were performed using the autopsied heart tissues as samples.

Corrective effect on Fabry mice of yeast recombinant human alpha-galactosidase with N-linked sugar chains suitable for lysosomal delivery.

We have previously reported the production of a recombinant alpha-galactosidase with engineered N-linked sugar chains facilitating uptake and transport to lysosomes in a Saccharomyces cerevisiae mutant. In this study, we improved the purification procedure, allowing us to obtain a large amount of highly purified enzyme protein with mannose-6-phosphate residues at the non-reducing ends of sugar chains. The products were incorporated into cultured fibroblasts derived from a patient with Fabry disease via mannose-6-phosphate receptors.

Establishment of immortalized Schwann cells from Sandhoff mice and corrective effect of recombinant human beta-hexosaminidase A on the accumulated GM2 ganglioside.

We have established spontaneously immortalized Schwann cell lines from dorsal root ganglia and peripheral nerves of Sandhoff mice. One of the cell lines exhibited genetically and biochemically distinct features of Sandhoff Schwann cells. The enzyme activities toward 4-methylumbelliferyl N-acetyl-beta-D-glucosamine (beta-hexosaminidases A, B, and S) and 4-methylumbelliferyl N-acetyl-beta-D-glucosamine-6-sulfate (beta-hexosaminidases A and S) were decreased, and GM2 ganglioside accumulated in lysosomes of the cells.

Induction of tissue factor expression in human monocytic cells by protease inhibitors through activating activator protein-1 (AP-1) with phosphorylation of Jun-N-terminal kinase and p38.

Tissue factor (TF) is expressed rapidly by human monocytes exposed to a variety of agonists such as lipopolysaccharide (LPS) or tumor necrosis factor-alpha. Activation of both activator protein-1 (AP-1; c-Jun/c-Fos) and nuclear factor-kappaB (NF-kappaB) pathways is necessary for maximal induction of the TF gene. It has been demonstrated that activation of both AP-1 and NF-kappaB is correlated with the degradation of both phosphorylated c-Jun and inhibitor kappaB (IkappaB) by proteasome.

A novel cell line derived from de novo acute myeloblastic leukaemia with trilineage myelodysplasia which proliferates in response to a Notch ligand, Delta-1 protein.

A novel human leukaemia cell line, designated TMD7, was established from blast cells of a patient with de novo acute myeloblastic leukaemia with trilineage myelodysplasia (AML/TLD). As seen in the original blast cells, TMD7 cells expressed CD7, CD13, CD33 and CD34 and showed an abnormal karyotype containing -5, -7, -8, der(16)t(10;16)(q22;q13). The cells proliferated without added growth factors.