Masahiko Tsuda

Bone marrow transplantation for Niemann-Pick mice

The Niemann-Pick mice which received bone marrow transplants showed a decreased accumulation of sphingomyelin and cholesterol quantitatively in their spleen. The sphingomyelin deposit in the bone marrow was also reduced histochemically. However, the neurological manifestations were not improved by the bone marrow graft.

A lysosomal storage disorder in mice: a model of Niemann-Pick disease.

Recently a strain of C57 BL/KsJ mice showed a progressive degenerative neurologic manifestation with hepatosplenomegaly, similar to Niemann-Pick disease in humans (Miyawakiet al., 1982). Niemann-Pick disease (McKusick 25720), a rare genetic disorder, is characterized by sphingomyelin accumulation, parti cularly in the reticuloendothelial system, due to a genetic defect of sphingomyelin catabolism. Animal models as an analogue of human Niemann-Pick disease have been found in the Siamese cat (Chrispet al., 1970) and CBA mice (Lyonet al., 1965), but no other reports on these strains have been available. The ethical and scientific limitations of human experimentation have made necessary investigations with appropriate animal sys tems. We report here a new murine model to study a lysosomal storage disorder.

The primary structure of mouse saposin

The primary structure of mouse sphingolipid activator protein (saposin) was determined by cDNA sequencing. The amino acid sequence predicted by the cDNA sequence revealed that mouse saposin was highly homologous to human saposin and also to rat sertoli cell glycoprotein. Mouse saposin also has four functional domains, which are structurally similar to each other, and each domain has cysteines, prolines, and a potential glycosylation site at an almost identical position. An amino acid comparison between human and mouse saposins revealed that the similarity was approximately 70%, and human saposin lacks thirty-one amino acids between domains C and D. Heterogeneities of mRNA were found in both the coding and noncoding regions.

A novel mutation at a probable heme-binding ligand in neutrophil cytochrome b558 in atypical X-linked chronic granulomatous disease

A membrane-bound cytochrome b558, a heterodimer consisting of gp91-phox and p22-phox, is a critical component of the superoxide (O2-)-generating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes. Chronic granulomatous disease (CGD) is characterized by recurrent bacterial infection caused by a defect of the oxidase. Both subunits are absent from phagocytes in typical X-linked recessive CGD patients who are primarily defective in gp91-phox. We report here an atypical case of X-linked CGD in which neutrophils showed a complete absence of O2–-forming NADPH oxidase activity, but a small amount (about 10% of control) of both subunits was detected by immunoblot analysis. Spectrophotometric studies of the neutrophils with a recently developed sensitive method gave no evidence for the heme spectrum in the cytochrome b558 of this CGD. Reverse transcription/polymerase chain reaction and sequence analysis revealed a C to T transition replacing histidine at amino acid position 101 (His101) by tyrosine in gp91-phox. These results provide evidence that His101 of gp91-phox is the one of the heme-binding ligands of cytochrome b558.

No mutation in the SLC2A2 ( GLUT2) gene in a Turkish infant with Fanconi-Bickel syndrome.

Fanconi-Bickel syndrome (FBS), or glycogen storage disease type XI, is a rare, well-defined clinical entity. Recently, this disease was elucidated to link mutations in the SLC2A2 gene in many ethnic groups, indicating that FBS is a single gene disease. We report here an 8-month-old Turkish girl who developed characteristic findings of FBS. However, no mutation was detected in the protein-coding region of the SLC2A2 gene. Therefore, we propose that further molecular analysis is needed to determine whether other genes are involved in FBS.

16q12 microdeletion syndrome in two Japanese boys

Microdeletion of 16q12 is a rare chromosomal abnormality. We present the cases of two Japanese patients with developmental and renal symptoms of differing clinical severity. Both patients had 16q12 interstitial microdeletions that included the entire SALL1 gene. Patient 1 was a 15‐year‐old Japanese boy clinically diagnosed with branchio‐oto‐renal syndrome with mild developmental delay, but with no imperforate anus or polydactyly. Array comparative genome hybridization (aCGH) indicated a 5.2 Mb deletion in 16q12, which included SALL1. Patient 2 was a 13‐year‐old Japanese boy diagnosed with Townes–Brocks syndrome and severe developmental delay, epilepsy, and renal insufficiency requiring renal replacement therapy. Fluorescence in situ hybridization indicated deletion of the entire SALL1 gene. Subsequent aCGH showed a 6 Mb deletion in 16q12q13, which included SALL1. Precise analysis of the present two cases will give us some clues to elucidate the pathogenic mechanisms of 16q12 microdeletion syndrome.

Recombinant human granulocyte colony‐stimulating factor therapy for cyclic neutropenia associated with common variable immunodeficiency

A 14 year old boy with common variable immunodeficiency (CVID) had regularly recurring episodes of severe infections independently of the serum γ‐globulin level. Serial blood counts revealed that this patient also had cyclic neutropenia. Recently, recombinant human granulocyte colony‐stimulating factor (rhG‐CSF) was reported to be an effective treatment for this disease. We tried rhG‐CSF therapy for this patient and a prompt increase in the neutrophil count was noted. However, the cyclic alterations and duration of the nadir of the neutrophil count were not altered, which suggested that rhG‐CSF has a variable efficacy in at least some patients with cyclic neutropenia.

SPECTROPHOTOMETRIC DETERMINATION OF NEUTROPHIL CYTOCHROME B558 OF CHRONIC GRANULOMATOUS DISEASE

Abstract Background: Chronic granulomatous disease (CGD) is an inherited disease characterized clinically by severe recurrent bacterial infections from infancy. This disease is a disorder of the formation of superoxide (O2‐) by the neutrophil NADPH oxidase system, mostly due to defects in cytochrome b558 (cyt b558), which is one ol the oxidase components. Diagnosis of CGD has been performed by the assay of the O2‐ forming activity. immunological determination of defects in the oxidase components, and or spectrophotometry of cyt b558. However, spectrophotometric analysis of the b‐type heme is difficult with small amounts of blood from infant CGD patients, as the limited amounts of neutrophils are contaminated with a relatively high ratio of hemoglobin (Hb) that interferes with the heme spectrum of cyt b558. This report presents an accurate method foi the spectrophotometric analysis of cyt b558 in a small amount of CGD neutrophils that were treated with CO gas in a safe procedure instead of the previously reported CO‐bubbling method.

Lack of acid sphingomyelinase in the mitochondria-lysosome fraction of brain from Niemann―Pick mice

Niemann-Pick mice (spm/spm), a mutated strain of C57BL/KsJ with a marked accumulation of sphingomyelin and cholesterol in the visceral organs were established by Sakiyama et al. (1982) and Miyawaki et al. (1982). A significant decline of sphingomyelinase activity in the mouse liver and spleen was also reported. Although the accumulation of sphingomyelin and deficiency of sphingomyelinase activity were not obvious in the brain of affected mice, they showed severe neurological symptoms after 8 weeks of age, and died within 12-14 weeks of age. In order to elucidate the biochemical cause of their neurological manifestations, an additional enzymatic study on sphingomyelinase from brain and liver of Niemann-Pick mice was carried out. Spingomyelinases exist in various molecular forms in the liver and spleen of the control mice. In NiemannPick mice, a deficiency of acid sphingomyelinase in the liver and a specific lysosomal acid sphingomyelinase in the brain were noted. The lack of lysosomaI acid sphingomyelinase in the brain probably is responsible for the development of neurological manifestations in the affected mice. 16-18h with a 1.0 % (v/v) concentration of carrier ampholytes (pH 3.5-10.0) in a gradient of 0-92 % (w/v)

Metabolism: Therapeutic Bone Marrow Transplantation in Niemann‐Pick Mice

Mice with Niemann‐Pick disease were treated with bone marrow transplantation. The loss of Purkinje cells remained unchanged, but the accumulation of sphingomy elin and cholesterol was reduced in the visceral organs, especially in the spleen, three weeks after marrow graft. The activity of acid sphingomyelinase was increased in the grafted liver of mice, and in the spleen this activity was elevated to normal levels.