Seiji Yoshizawa

Structure‐Function Relationships of Microcystins, Liver Tumor Promoters, in Interaction with Protein Phosphatase

Microcystins, isolated from toxic blue‐green algae, are potent inhibitors of protein phosphatases 1 and 2A. Recently, we have reported that mierocystin LR has a potent tumor‐promoting activity on rat liver initiated with diethylnitrosamine. The structure of microcystins is unique in having an unusual ammo acid, 3‐amino‐9‐methoxy‐10‐phenyl‐2,6,8‐trimethyl‐deca‐4(E),6(E)‐dienoic acid (Adda), which is thought to be significant for the activity. Geometrical isomers at C‐7 in the Adda portion of microcystins, 6(Z)‐Adda microcystins LR and RR, have been isolated from cyanohacteria. To estimate their tumor‐promoting activities and to understand the importance of the Adda portion for activity, the maternal microcystins LR and RR and their isomers were subjected to examination of their interaction with protein phosphatases 1 and 2A and the release of glutamic pyruvic transaminase from rat liver. 6(Z)‐Adda microcystins LR and RR bound to protein phosphatases 1 and 2A, inhibited their activities and released glutamic pyruvic transaminase from rat liver into serum, ten to one hundred times more weakly than the maternal microcystins LR and RR. These results indicated that the conjugated diene with 4(E),6(E) geometry in the Adda portion is important in the interaction with protein phosphatases.

Vimentin is hyperphosphorylated in primary human fibroblasts treated with okadaic acid

Okadaic acid and dinophysistoxin-1 (35-methylokadaic acid) induced hyperphosphorylation of a 58 kDa protein in primary human fibroblasts, due to inhibition of protein phosphatase 1 and 2A activities. The protein was present in the nuclear and cytosolic fractions. Its pI was 5.3. The hyperphosphorylated protein reacted with monoclonal and polyclonal anti-vimentin antibodies, but not with anti-nucleolin antibody. Phosphorylation of vimentin was stimulated in vitro by dinophysistoxin-1 dose-dependently in the presence of protein phosphatase 2A and protein kinases.

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.

Association of killer cell immunoglobulin-like receptor 2DL5 with systemic lupus erythematosus and accompanying infections

OBJECTIVE Identification of the association of killer cell immunoglobulin-like receptor (KIR) genes with SLE and accompanying infections. METHODS Presence or absence of all 14 KIR genes was studied for association with SLE by case-control studies. A total of 417 SLE cases, 72 RA cases and 256 controls, all of Japanese descent, were enrolled. RESULTS The carrier frequency of KIR2DL5 was significantly decreased in SLE patients compared with healthy controls [39.3 vs 50.4%; odds ratio (OR) = 0.64; 95% CI 0.36, 0.92; P = 0.005). When the prevalence of severe infections was analysed in 184 SLE patients, whose medical records were available, KIR2DL5 carriers were at an increased risk of overall infection and viral infection (crude OR = 2.66; 95% CI 1.43, 4.92; P = 0.017 and crude OR = 2.31; 95% CI 1.15, 4.62; P = 0.017, respectively). After adjusting for methylprednisolone pulse and/or cyclophosphamide pulse therapy, KIR2DL5 carriers were at significantly greater risk of infectious events overall (adjusted OR = 2.45; 95% CI 1.24, 4.81; P = 0.0095). However, KIR2DL5 carriers were marginally associated with an increased risk of viral infectious events (adjusted OR = 2.03; 95% CI 0.94, 4.41; P = 0.0718). CONCLUSION KIR2DL5 was significantly associated with a decreased risk of SLE as well as an increased risk of infectious events overall in SLE patients. Our data suggest a further role of KIRs in the pathogenesis of autoimmune diseases and infection.

Pendolmycin, a new tumor promoter of the teleocidin A class on skin of CD-1 mice.

Pendolmycin, isolated from Nocardiopsis, is a compound structurally similar to teleocidin A, one of the 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐type tumor promoters. Pendolmycin has a C5 dimethyl allyl group attached to C‐7 of (‐)‐indolactam‐V, whereas teleocidin A has a C10 linalyl group attached to the molecule. The structure‐activity relationships of a hydrophobic moiety attached to (‐)‐indolactam‐V were studied in four compounds, (‐)‐indolactam‐V, pendolmycin, teleocidin A and newly synthesized 7‐(nerolidyl)‐(‐)‐indolactam‐V in tests on inhibition of the specific [3H]TPA binding to a particulate fraction of mouse skin, activation of protein kinase C and induction of both adhesion of HL‐60 cells and ornithine decarboxylase in mouse skin. The potencies of the compounds for these activities increased mainly depending on the length of the hydrophobic group. Pendolmycin had a tumor‐promoting activity on mouse skin initiated with a single application of 7,12‐dimethyl‐benz[a]anthracene, and its potency was just between those of (‐)‐indolactam‐V and teleocidin A. The role of the hydrophobic moiety is discussed with particular emphasis on the results obtained with 7‐(nerolidyl)‐(‐)‐indolactam‐V.

Safety, pharmacokinetics, and pharmacodynamics of epratuzumab in Japanese patients with moderate-to-severe systemic lupus erythematosus: Results from a phase 1/2 randomized study

Abstract Objectives: This 12-week, randomized, double-blind, placebo-controlled, multicenter phase 1/2 study (NCT01449071) assessed the safety, pharmacokinetics, and pharmacodynamics of epratuzumab in Japanese patients with moderate-to-severe systemic lupus erythematosus despite standard of care. Methods: Twenty patients were randomized 1:1:1:1:1 to placebo or one of four epratuzumab dose regimens (100 mg every other week [Q2W], 400 mg Q2W, 600 mg every week [QW], or 1200 mg Q2W) administered during an initial 4-week dosing period. Adverse events (AEs), pharmacokinetics and pharmacodynamics were assessed. Results: Nineteen of 20 patients completed the study. All placebo patients and 13 of 16 epratuzumab patients reported ≥1 AE, 2 of 16 epratuzumab patients reported a serious AE. Cmax and AUCτ increased proportionally with dose after first and last infusion, t1/2 was similar across groups (∼13 days). Epratuzumab treatment was associated with decreased CD22 mean fluorescence intensity in total B cells (CD19+CD22+) and unswitched memory B cells (CD19+IgD+CD27+). Small-to-moderate decreases were observed in total B cell (CD20+) count. Conclusions: Epratuzumab was well-tolerated, with no new safety signals identified. The pharmacokinetics appeared linear after first and last infusions. Treatment with epratuzumab was associated with CD22 downregulation and with small-to-moderate decreases in total B cell count.

Impact of methotrexate dose on efficacy of adalimumab in Japanese patients with rheumatoid arthritis: Results from registered data analyses

Abstract Objective: Upper limit of methotrexate (MTX) for patients with rheumatoid arthritis (RA) was recently increased from 8 to 16 mg/week in Japan. We therefore examined the effect of concomitant MTX dose on the efficacy of adalimumab (ADA) in clinical practice. Method: Sixty-one consecutive RA patients treated with ADA were followed for minimum 52 weeks and retrospectively compared by MTX dose; patients receiving concomitant MTX of 10 mg/week or more (MTX ≥10 mg group) and <10 mg/week (MTX <10 mg group). Disease activity and remission were evaluated by the disease activity score 28 (DAS28) criteria. Results: The MTX ≥10 mg group consistently showed better improvement in DAS28 and resulted in more patients (52.8%) with DAS28-remission compared with the MTX <10 mg group (26.1%). Multivariate analysis showed that MTX ≥10 mg had a significant effect on DAS28 remission with odds ratio of 5.12. ADA retention rate was 72.2% in MTX ≥10 mg group compared with 52.0% in MTX <10 mg group. Discontinuation of ADA due to adverse events were comparable in the MTX ≥10 mg and MTX <10 mg groups (11.1% vs. 12.0%). Conclusions: These findings support the critical role of concomitant MTX in the efficacy of ADA, and recommend use of MTX ≥10 mg in Japanese RA patients.

Urinary C4 excretion in systemic lupus erythematosus

The fourth component of complement (C4) in urine was measured in 19 patients with systemic lupus erythematosus (SLE). Urinary C4 was detectable in all SLE patients using an enzyme linked immunosorbent assay. In 11 of 13 patients whose urinary C4 was serially measured, a decrease of urinary C4 was found in parallel with a decrease of disease activity of SLE. In the remaining 2 patients, the amount of C4 increased preceding a flare-up of the disease. The measurement of urinary C4 may be useful in evaluating the disease activity of SLE in individual patients and sometimes in predicting the flare-up of the disease. The specific hemolytic activity of excreted C4 and Western blotting analysis showed that urinary C4 consisted mainly of degraded fragments of C4. In two cases, camostat, a serine protease inhibitor, rapidly decreased the urinary C4 excretion, suggesting that the complement activation occurred in the glomerulus in lupus nephritis.

Tumor Antipromoters: Sarcophytols A and B, (–)–Epigallocatechin Gallate (EGCG), and Morusin

The Ministry of Health and Welfare of Japan supports the research on cancer chemoprevention within the framework of cancer research programs, because the increasing interest in cancer chemoprevention has been remarkable. However, the research on cancer chemoprevention is quite new in Japan. Therefore, we started to search for non-toxic inhibitory compounds against various cancers (anti-cancer agents as well as inhibitors of tumor promotion) from natural products; sarcophytols A and B, isolated from a soft coral, (–)–epigallocatechin gallate (EGCG), isolated from green tea infusion and morusin, isolated from the root bark of the mulberry tree. All these four compounds are inhibitors of tumor promotion induced by a tumor promoter, teleocidin, in two-stage carcinogenesis experiments on mouse skin. We would like to implement these non-toxic compounds in cancer chemoprevention in the near future.