Sachiko Kamei

1α,25-Dihydroxyvitamin D3 and Its Potent Synthetic Analogs Downregulate Tissue Factor and Upregulate Thrombomodulin Expression in Monocytic Cells, Counteracting the Effects of Tumor Necrosis Factor and Oxidized LDL

BackgroundWe have recently found that a hormonally active form of vitamin D, 1&agr;,25-dihydroxyvitamin D3 [1,25(OH)2D3], exerts anticoagulant effects by upregulating the expression of an anticoagulant glycoprotein, thrombomodulin (TM), and downregulating the expression of a critical coagulation factor, tissue factor (TF), in monocytic cells including human peripheral monocytes. In this study, we investigated the counteracting effects of 1,25(OH)2D3 and its potent analogs on TF induction and TM downregulation by tumor necrosis factor and oxidized LDL in monocytic cells and the modulatory effects of potent analogs on TF and TM expression. Methods and ResultsEffects of 1,25(OH)2D3 and its potent synthetic analogs (22R)-22-methyl-20-epi-1,25(OH)2D3 (KY3) and 22-oxacalcitriol on TF and TM antigen levels, cell surface activities, and mRNA levels in monocytic cells were examined. 1,25(OH)2D3 and its potent analogs showed anticoagulant effects in monocytic cells by downregulating TF and upregulating TM expression, counteracting the effects of tumor necrosis factor and oxidized LDL. KY3 was most potent in its regulatory effect on TF and TM expression. ConclusionsBecause KY3 has the highest affinity for vitamin D receptor, our findings suggest that TF and TM regulation by 1,25(OH)2D3 analogs is also mediated by vitamin D receptor. The 1,25(OH)2D3 analogs KY3 and 22-oxacalcitriol may have the potential to serve as an agent for preventing and treating atherosclerotic and other cytokine-mediated thrombotic diseases and as a tool for studying the molecular mechanisms of TF and TM regulation.

α-Galactosidase gene mutations in Fabry disease: heterogeneous expressions of mutant enzyme proteins

Five point mutations were identified in unrelated Japanese Fabry disease hemizygotes: three new missense mutations, C142Y (425 G → A), A156V (467 C → T), and L166V (496 C → G) in exon 3; one new splice site mutation at the 3′ end of the consensus sequence in exon 4; one previously reported nonsense mutation, W44X (131 G → A). C142Y expressed 50% of the normal enzyme protein in COS-1 cells, but catalytic activity was not detected. Both A156V and L166V expressed significant amounts of residual enzyme activity (6.7% and 9.8%) and enzyme proteins (10% each), the latter were more thermolabile at neutral pH than at acid pH, in vitro.

Molecular and structural studies of Japanese patients with sialidosis type 1

AbstractTo gain insight into the pathogenesis of sialidosis type 1, we performed molecular investigations of two unrelated Japanese patients. Both of them are compound heterozygotes for base substitutions of 649G-to-A and 727G-to-A, which result in amino acid alterations V217M and G243R, respectively. Using homology modeling, the structure of human lysosomal neuraminidase was constructed and the structural changes caused by these missense mutations were deduced. The predicted change due to V217M was smaller than that caused by G243R, the latter resulting in a drastic, widespread alteration. The overexpressed gene products containing these mutations had the same molecular weight as that of the wild type, although the amounts of the products were moderately decreased. A biochemical study demonstrated that the expressed neuraminidase containing a V217M mutation was partly transported to lysosomes and showed residual enzyme activity, although a G243R mutant was retained in the endoplasmic reticulum/Golgi area and had completely lost the enzyme activity. Considering the data, we surmise that the V217M substitution may be closely associated with the phenotype of sialidosis type 1 with a late onset and moderate clinical course.

Diagnostic and prognostic usefulness of N1,N8-diacetylspermidine and N1,N12-diacetylspermine in urine as novel markers of malignancy

Abstract Recently, we found N1,N8-diacetylspermidine (Ac2Spd) and N1,N12-diacetylspermine (Ac2Spm) in human urine, and noted that their amount increased significantly in patients with urogenital malignancies. Previous findings that simultaneous reference to these diacetylpolyamines is useful in distinguishing cancer patients from healthy persons were confirmed by more recent analytical data on urine samples from several cancer patients. Further examination revealed that urinary Ac2Spm and Ac2Spd tended to decrease when cancer patients were treated and entered partial remission. In cases where the Ac2Spm and Ac2Spd levels were normal or near-normal after treatments, the prognosis of the patients was generally good. In contrast, when their level remained far above the normal limits after apparently effective treatment, the prognosis of the patients was poor. When a patient is in remission for more than 3 years, urinary levels of both Ac2Spm and Ac2Spd are stabilized and stay below the normal limits, with rare exceptions. The recurrence of a cancer as well as the complication of a second one during the period of follow-up examination was accompanied by elevation of urinary diacetylpolyamines. These observations indicate that urinary Ac2Spm and Ac2Spd are useful as prognostic indicators after treatment and during follow-up examination of cancer patients.

Significance of urinary N1,N8-diacetylspermidine and N1,N12-diacetylspermine as indicators of neoplastic diseases

N1,N8-Diacetylspermidine (Ac2Spd) andN1,N12 diacetylspermine (Ac2Spm), the occurrence of which in healthy human urine was demonstrated recently, increased much more frequently and markedly than total polyamines, acetylputrescine,N1-acetylspermidine andN8-acetylspermidine in patients with urogenital malignancies. Ac2Spd was hardly elevated in cases of benign disease, while Ac2Spm only infrequently stayed within normal limits in patients with malignant disorders. Urine samples from more than 90% of healthy persons, but fewer than 10% of patients with malignancies, gave values within normal limits for both Ac2Spd and Ac2Spm. Simultaneous reference to these diacetylpolyamines is therefore useful in distinguishing patients with malignancies from healthy persons.

Screening and detection of gene mutations in Japanese patients with Fabry disease by non-radioactive single-stranded conformation polymorphism analysis

We have applied non-radioactive polymerase chain reaction (PCR)-single-stranded conformation polymorphism (SSCP) to the detection of gene mutations causing Fabry disease. Nineteen of 22 known mutations were detected as electrophoretic mobility shifts on PCR-SSCP analysis. Then, DNA from newly diagnosed Japanese patients with the classical form of Fabry disease was subjected to PCR-SSCP analysis, and 4 novel mutations (1 small deletion, 1 nonsense mutation and 2 missense mutations) and 1 neutral polymorphism were identified. Furthermore, identification of an asymptomatic heterozygote and a hemizygote with moderate clinical manifestations was successfully achieved by application of this method to a family with the variant form of Fabry disease. PCR-SSCP is useful for the gene diagnosis of etiologically heterogeneous Fabry disease.

Two novel mutations in the α-galactosidase gene in Japanese classical hemizygotes with Fabry disease

SummaryFour α-galactosidase gene mutations were identified in Japanese male patients with Fabry disease who had no detectable α-galactosidase activity. Two of them were novel mutations, an 11-bp deletion in exon 2 and a g−1 to t substitution at the 3′ end of the splice acceptor site in intron 1. The former caused a frameshift and led to the creation of a new stop codon at codon 118. The latter was predicted to provoke aberrant mRNA splicing followed by accelerated degradation of the mRNA. A nonsense mutation, R301X, and a 2-bp deletion starting at nucleotide position 718, which were reported previously, were also identified in unrelated patients.

Arsenic trioxide (As2O3) gradually downregulates tissue factor expression without affecting thrombomodulin expression in acute promyelocytic leukemia cells.

Arsenic trioxide (As 2 O 3 ) gradually downregulates tissue factor expression without affecting thrombomodulin expression in acute promyelocytic leukemia cells

Occurrence of serum M-protein species in Japanese patients older than 50 years based on relative mobility in cellulose acetate membrane electrophoresis.

We investigated the occurrence of serum M‐protein species in 2,007 Japanese patients older than 50 years of age. All sera samples were analyzed by cellulose acetate membrane electrophoresis. The relative mobility of an M‐protein band was calculated by dividing the migration distance of M protein by that of albumin. M proteins were found to be present in 71 of 2,007 cases (3.5%). Men 80–89 years old showed the highest occurrence of M proteins, 11.0%. The relative mobility of M‐protein bands, especially the band of the IgA‐type M protein, increased as the patients age advanced. The patients had higher levels of the IgG‐type M protein than healthy Japanese subjects. We found that the occurrence of M‐protein species in Japanese patients increases with their age. The IgG‐type M protein was most frequently expressed among other types. The mobility of the M protein was greater in older patients probably because of aging‐related changes in the carbohydrate chain of immunoglobulins composing an M‐protein molecule. J. Clin. Lab. Anal. 14:64–69, 2000.

Simultaneous analysis of serum immunoglobulins in patients with M protein using cellulose acetate membrane isoelectric focusing.

We developed a method for the simultaneous analysis of microheterogeneity of human serum IgG, IgA, IgM, IgD, and IgE, and serum protein pattern using cellulose acetate membrane isoelectric focusing, and analyzed in 11 healthy subjects and 67 patients with M protein (17 cases of multiple myeloma [MM] and 50 cases of monoclonal gammopathy of undetermined significance [MGUS]). Using this method, bands indicating the microheterogeneity of each immunoglobulin could clearly be detected. Among healthy subjects, the detected IgG, IgA, and IgM bands did not vary, but the detected IgE and IgD bands did vary. Therefore, IgA, IgM, and IgG were selected for comparison of serum immunoglobulins in MM and in MGUS. In the IgA‐type M protein group, normal IgM and IgG bands were decreased in MM patients compared to MGUS patients, while the M band and other bands were increased in MM patients compared to MGUS patients, but the differences between the two groups were not significant. In the IgG‐type M protein group, normal IgM, IgA, and IgG were significantly decreased in MM patients compared to MGUS patients. We examined the changes in electrophoretic pattern in six MM patients and eight MGUS patients with IgA‐type M protein after neuraminidase treatment. The width of the M band in MM patients with IgA‐type M protein decreased with neuraminidase treatment. On the other hand, the width of the M band in MGUS patients with IgA‐type M protein increased with neuraminidase treatment. We concluded that the decrease of the normal immunoglobulins in MM patients with IgG type M protein could be detected by this method, and IgA type of M protein binding sugar chain were different between MM and MGUS patients. J. Clin. Lab. Anal. 13:145–150, 1999.