Shigeru Yoshizawa

Inhibition of protein phosphatases by microcystis and nodularin associated with hepatotoxicity

SummaryMicrocystins and nodularin, isolated from toxic blue-green algae, are hepatotoxic monocyclic polypeptides. Both microcystins and nodularin inhibited in vitro protein phosphatase activity present in a cytosolic fraction of mouse liver, bound to the okadaic acid receptors, protein phosphatases 1 and 2A, and thus resulted in the increase of phosphoproteins; this was referred to as the apparent “activation” of protein kinases. Their concentrations causing 50% of the maximal effects are comparable to that of okadaic acid, a potent protein phosphatase inhibitor and a potent tumor promoter, in the nanomolar range of concentration. The increase of phosphoproteins was observed in rat primary cultured hepatocytes and was subsequently associated with morphological changes, which appeared to be a step in the process of hepatotoxicity. The well-known hepatotoxic compounds,α-amanitin and phalloidin, did not show any effects similar to those of microcystins, nodularin and okadaic acid. It is suggested that the hepatotoxicity of microcystins and nodularin may result from inhibition of protein phosphatases and the increase of phosphoproteins.

In vitro and in vivo effects of protein phosphatase inhibitors, microcystins and nodularin, on mouse skin and fibroblasts

Three microcystins, YR, LR and RR and nodularin, all of which are hepatotoxic compounds, inhibited dose-dependently the activity of protein phosphatase 2A in and the specific [3H]okadaic acid binding to a cytosolic fraction of mouse skin, as strongly as okadaic acid. However, microcytins and nodularin did not induce any effects on mouse skin or primary human fibroblasts. Microinjection of microcystin YR into primary human fibroblasts induced morphological changes which were induced by incubation with okadaic acid. Microcystins and nodularin penetrate into the epithelial cells of mouse skin and human fibroblasts with difficulty, which reflects tissue specificity of the compounds.

Diarrhetic Shellfish Toxin, Dinophysistoxin‐1, Is a Potent Tumor Promoter on Mouse Skin

Dinophysistoxin‐1, 35‐methylokadaic acid, is a causative agent of diarrhetic shellfish poisoning. The biological activities and tumor‐promoting activity of dinophysistoxin‐1 were studied together with those of okadaic acid and 7‐O‐palmitoyl okadaic acid. Dinophysistoxin‐1 is a skin irritant and induces ornithine decarboxylase in mouse skin with the same potency as okadaic acid. 7‐O‐Palmitoyl okadaic acid induced a lower activity than the other compounds. Dinophysistoxin‐1 inhibited the specific [3H]okadaic acid binding to a participate fraction of mouse epidermis. The binding affinities of dinophysistoxin‐1 and okadaic acid to a particulate fraction were almost the same. Dinophysistoxin‐1 showed a tumor‐promoting activity as strong as that of okadaic acid in a two‐stage carcinogenesis experiment on mouse skin. The percentages of tumor‐bearing mice in the groups treated with 100 μg of 7,12‐dimethylbenz[α]anthracene (DMBA) followed by 5 μg of dinophysistoxin‐1, twice a week, and with DMBA followed by 5 μg of okadaic acid twice a week were 86.7% and 80.0% in week 30, respectively. The average number of tumors per mouse was 4.6 in the former group and 3.9 in the latter. Dinophysistoxin‐1 and okadaic acid act on cells through different pathways from the 12‐O‐tetradecanoylphorbol‐13‐acetate‐type tumor promoters.

Angina pectoris occurring during granulocyte colony‐stimulating factor‐combined preparatory regimen for autologous peripheral blood stem cell transplantation in a patient with acute myelogenous leukaemia

We describe a patient with acute myelogenous leukaemia who developed angina pectoris during pre‐transplant conditioning for autologous peripheral blood stem cell transplantation (PBSCT); the conditioning regimen consisted of cytotoxic drugs in combination with granulocyte colony‐stimulating factor (G‐CSF). Neutrophilia and hypercoagulability were observed at the time of angina pectoris. Recurrence of angina pectoris was not seen after nitrate and aspirin therapy. Exercise stress testing performed after PBSCT suggested the presence of myocardial ischaemia. Therefore cases at risk of vascular events should be carefully managed with prophylactic treatment during G‐CSF administration.

A functional M196R polymorphism of tumour necrosis factor receptor type 2 is associated with systemic lupus erythematosus: a case–control study and a meta-analysis

Objectives: To perform a case–control study of a functional M196R polymorphism of tumour necrosis factor receptor type 2 (TNF-RII) in a Japanese population and a meta-analysis of all published reports on the polymorphism to investigate the association of the M196R polymorphism of TNF-RII with systemic lupus erythematosus (SLE). Methods: The functional M196R polymorphism of TNF-RII was genotyped by using polymerase chain reaction combined with the subsequent single-strand conformation polymorphism (PCR—SSCP) analysis for screening, followed by nucleotide sequencing for confirmation. A total of 331 patients and 359 controls were subjected to a case–control study. A meta-analysis of the available case–control studies including all published data as well as our own data was performed to investigate the association of the functional M196R polymorphism of TNF-RII with SLE. Results: Our case–control study did not show any significant association of a functional M196R polymorphism of TNF-RII with SLE, although there was a trend towards association. A meta-analysis of seven case–control studies in eight different ethnic populations including our own showed that 196M/R and 196R/R genotypes combined was significantly associated with an increased risk of SLE (odds ratio (OR) 1.29, 95% confidence interval (CI) 1.04 to 1.60; p = 0.02). Stratification by ethnicity showed a more significant association in Asians, including Japanese, Korean and Vietnamese (OR 1.40, 95% CI 1.10 to 1.78; p = 0.006). The effect of the 196R allele on SLE was not clear in Caucasians. Conclusions: The 196R allele of the functional M196R polymorphism of TNF-RII is a risk factor for SLE, especially in the Asian population.

Anticentromere Antibody as a Risk Factor for Cancer in Patients with Systemic Sclerosis

Abstract: This study has estimated the cancer risk among patients with systemic sclerosis (SSc) using a population-based analysis. Using the inpatient and outpatient registries for patients at Kyushu University Hospital between 1982 and 1996, standardised incidence rates (SIRs) (ratio of observed-to-expected cancers) were calculated in 43 patients with SSc, 24 patients with polymyositis (PM) and 17 patients with dermatomyositis (DM). Risk factors predisposing to cancers were also investigated in the SSc patients. Compared with the Japanese general population, the SIR for developing cancer in SSc patients was 5.1 (95% confidence interval (CI), 1.7–10.8), while the SIRs for cancer in the PM and DM groups were 4.7 (95% CI, 1.5–10.3) and 61.2 (95% CI, 46.8–77.6), respectively. A statistically significant risk factor for cancers in the SSc patients was positivity for anticentromere antibody (ACA) (p<0.05), while the erythrocyte sedimentation rate, serum lactate dehydrogenase concentration, serum γ-globulin concentration, titre of antinuclear antibody and positivity for antitopoisomerase I antibody were not associated with cancer in SSc. Our population-based study confirms the increased risk of cancer among patients with SSc in Japan and provides new evidence that positivity for ACA should be considered as a risk factor for cancer in future monitoring of patients.

Prevention of steroid-induced osteonecrosis of femoral head in systemic lupus erythematosus by anti-coagulant.

Although osteonecrosis of femoral head (ONF) is one of the serious complications in systemic lupus erythematosus (SLE) associated with corticosteroid therapy, there has been few trials of prevention of ONF described. We aimed to prevent ONF in steroid-treated SLE patients using anticoagulant, warfarin, conducting a multicenter prospective study. Sixty newly diagnosed SLE patients requiring 40 mg/day or more prednisolone were alternately assigned to either of two groups; a warfarin group and a control one. Warfarin (1 ∼ 5 mg/day) was started together with the beginning of steroid therapy and continued at least for three months. Patients were observed for the development of silent ONF by magnetic resonance imaging (MRI) and symptomatic ONF by plain radiography for over five years. The warfarin group consisted of 31 patients (62 hips) and the control one 29 patients (58 hips). Silent ONF developed in 13 hips (21%) and 19 hips (33%) in the warfarin group and the control group, respectively (P = 0.13). On the other hand, warfarin tended to prevent symptomatic ONF; only three hips of 62 (4.8%) in the warfarin group and eight hips of 58 (14%) in the control group (P = 0.08) developed silent ONF. It was also found that silent ONF developed, if it did, very early; within three months in 16 of 18 patients (89%). Among risk factors for silent ONF, steroid pulse therapy was most outstanding and it seemed to overcome the effect of warfarin. Taken together, for the time being, anticoagulant therapy, if not significantly sufficient, may be of use for the prevention of steroid-induced ONF in SLE. We consider that this study added to important evidence for the pathogenesis and prevention of ONF.

Factors associated with anxiety, depression and suicide ideation in female outpatients with SLE in Japan.

Abstract: The subjects consisted of 84 female SLE outpatients who were all over 20 years of age. These patients were able to maintain relatively stable physical conditions and lead normal daily lives, and they were regularly treated at the outpatient clinic. All subjects were Japanese. Psychological features (trait anxiety, state anxiety, depression and suicide ideation) were evaluated using psychological tests, and the relationships between the respective psychological features and background factors were statistically evaluated using stepwise multiple logistic regression analyses. In this study, we found that ‘the self-evaluation of not having understood SLE at the time of starting SLE treatment’ was the background factor significantly affecting depression or trait anxiety. ‘No spouse’ had a statistically significant effect on depression, and ‘self-awareness as problems of side-effects due to steroids’ had a statistically significant effect on state anxiety. We also found ‘human relations among family members’ and ‘high daily steroid dosage’ to be significantly correlated with suicide ideation. However, there were no correlations between the psychological features and ‘disease activity at the time of investigation’ or ‘history of neuropsychiatric diseases’. In female SLE outpatients, performing psychological approaches focusing on ‘understanding SLE at the beginning of treatment’, ‘the human relationships among family members’, or ‘issues related to steroid therapy’ may be useful for the early treatment or prevention of various major mental problems.

Tumor-promoting activity of staurosporine, a protein kinase inhibitor on mouse skin

Staurosporine, which is a potent inhibitor of protein kinases, such as protein kinase C, inhibited both inductions of adhesion of human promyelocytic leukemia cells (50% effective dose = 9.0 nM) and Epstein-Barr virus early antigen in Raji cells (50% effective dose = 3.4 nM) by teleocidin. However, staurosporine induced irritation on mouse ear and histidine decarboxylase activity in mouse skin. It did not induce ornithine decarboxylase activity in mouse epidermis. The two-stage carcinogenesis experiments of staurosporine were carried out at two different doses. Experiment 1 revealed that the group treatment with a single application of 100 micrograms of 7,12-dimethylbenz(a)anthracene, followed by repeated applications of 50 micrograms of staurosporine, resulted in 85.7% of tumor-bearing mice at Wk 30, whereas group treatment with staurosporine alone or 7,12-dimethylbenz(a)anthracene alone gave 6.7% and 0%, respectively. Experiment 2 showed that group treatment with 7,12-dimethylbenz(a)anthracene followed by applications of 10 micrograms of staurosporine resulted in 33% of tumor-bearing mice at Wk 30. In addition, staurosporine treatment reduced the percentages of tumor-bearing mice treated with teleocidin from 100% to 67% in Wk 15. These results demonstrated that staurosporine is a weak tumor promoter of mouse skin compared with teleocidin, but staurosporine has some potency to inhibit tumor promotion by teleocidin.

Analysis of Fas ligand gene mutation in patients with systemic lupus erythematosus

OBJECTIVE To investigate the possible association of a Fas ligand (FasL) gene mutation(s) or polymorphism(s) with systemic lupus erythematosus (SLE). METHODS For amplification of the introns of the FasL gene, long polymerase chain reaction (PCR) using exon-based primers was utilized, followed by partial sequencing to construct exon-specific oligonucleotide primers for the analyses of FasL genomic DNA in SLE patients. Structural defects were studied by use of a composite analysis of reverse transcriptase-PCR/single-strand conformational polymorphism (SSCP) analysis of messenger RNA (mRNA) transcripts of the FasL gene in 35 SLE patients and PCR/SSCP analysis of FasL genomic DNA in 143 SLE patients. RESULTS The sizes of the introns were approximately 0.6 kb for intron 1, 4.3 kb for intron 2, and 1.3 kb for intron 3. By SSCP analysis, we did not identify any mutations or polymorphisms in the FasL mRNA transcripts or in any of the 4 exons or areas of the introns adjacent to the exons. CONCLUSION Using the same methods used in the present studies (PCR/SSCP), one group of investigators identified a structural defect of the FasL molecule in 1 of 75 SLE patients evaluated. Among the 143 SLE patients in the present study, however, we did not identify any mutations or polymorphisms of the FasL gene. Our results suggest that a FasL defect is not the major contributing factor in the pathogenesis of SLE.