Ca2+ and calmodulin regulate microtubule-associated protein-actin filament interaction in a flip-flop switch.

MAP2 (microtubule-associated protein 2) and tau factor are calmodulin-binding and actin filament-interacting proteins, respectively. We have examined the effect of Ca2+ and calmodulin on MAP-induced actin gelation by the low-shear falling-ball method, the high-speed centrifugation method, and electron microscopy using negative staining. Each MAP crosslinks actin filaments to increase the apparent viscosities and finally to form gels. Calmodulin inhibited MAP2- and tau factor-induced actin gelation (MAP2- and tau factor-actin interaction) only in the presence of Ca2+, but not in its absence. There were no differences in actin filament crosslinking activity of respective MAPs with or without Ca2+. MAP2 was not coprecipitated with F-actin only in the presence of Ca2+ and calmodulin determined by the high-speed centrifugation method. But MAP2 was found to bind to F-actin under any other conditions examined. In contrast, the tau factor-actin filament interaction could only be detected by the low-shear viscosity, but not by the high-speed centrifugation method. MAP2 and tau factor aggregated to form actin bundles as shown by electron microscopy. MAP2- or tau factor-induced bundle formation of actin filaments was inhibited only in the presence of Ca2+ and calmodulin, but not in the presence or absence of Ca2+. In conclusion, the interaction of MAP2- and tau factor-actin filaments is regulated by Ca2+ and calmodulin in a flip-flop switch.

Crosslinking of actin filaments is caused by caldesmon aggregates, but not by its dimers.

A recent report by Bretscher [(1984) J. Biol. Chem. 259, 12873-12880] showed that caldesmon prepared by his method crosslinks actin filaments to form thick bundles. This is in contrast to the results of previous work that caldesmon binds to F-actin but does not cause any gelation [(1981) Proc. Natl. Acad. Sci. USA 78, 5652-5655]. The present work clearly showed that caldesmon purified according to Bretscher does not cause any gelation of F-actin. However, caldesmon aggregates formed by concentration or by freeze-thawing gelated F-actin to form bundles.

Purification of an 80 kDa Ca2+-dependent actin-modulating protein, which severs actin filaments, from bovine adrenal medulla.

A protein with a molecular weight of 80 kDa, which binds Ca2+-dependently to actin, was purified chromatographically from bovine adrenal medulla by using Sephacryl S-300, DEAE-Sepharose, actin-DNase I Sepharose, and Sephacryl S-200. This protein was retained on an actin-DNase I affinity column only in the presence of Ca2+, and could be eluted from this column by EGTA. The 80 kDa protein is a monomer and binds to G-actin in a Ca2+-dependent manner at an equimolar ratio. It caused fragmentation of actin filaments at more than 4 X 10(-7) M free Ca2+ concentration, as determined by low-shear viscometry and electron microscopy. Saturating amounts of tropomyosin showed a slight protective effect on the fragmentation of actin filaments by the 80 kDa protein. Considering the mode of action on actin filaments, the 80 kDa protein reported here seems to be a gelsolin-like protein. Gel electrophoresis of this protein revealed changes in mobility depending upon the concentration of Ca2+. This result also indicates that the 80 kDa protein itself is a Ca2+-binding protein.

[Therapeutic effect of cefotaxime in the field of pediatrics].

Cefotaxime (CTX) was administered to 130 children with various bacterial infections of 41 to 400 mg/kg/day for 2 to 21 days. The clinical effect of CTX was very satisfactory in respiratory tract infection, urinary tract infection and meningitis. The overall clinical effect was excellent in 59, good in 39, fair in 17 and failure in 8 with effective rate of 79.7%. During this therapy, side effects were seen in 3 cases, diarrhea in 1 and rash in 2. Abnormal laboratory findings were seen in 5 cases, elevation of GOT in 1, GOT, GPT and A1-P in 1, GOT, GPT and T. Bil. in 1, elevation of BUN, increase of number of basophils and albuminuria in 1 and observation of albuminuria in 1. The above results demonstrate that CTX is a clinically useful antibiotic for the therapy of pediatric infections.

[Combination chemotherapy with cis-diamminedichloroplatinum, vinblastine and bleomycin for a rhabdomyosarcoma of the prostate in a child: report of a case].

A case of prostatic rhabdomyosarcoma in a 5-year-old boy is reported. He was brought to our clinic on Apr. 1, 1982 with complaints of pollakisuria and urethral pain. X-ray examinations revealed a huge intrapelvic tumor, and it was histopathologically diagnosed as embryonal rhabdomyosarcoma with a specimen of transrectal needle biopsy. Since the tumor was too huge to resect completely, he was initially treated with combination chemotherapy regimen of vincristine, actinomycin D and cyclophosphamide (VAC therapy), and resulted in failure. Then another combination chemotherapy consisting of cis-diamminedichloroplatinum, vinblastine and bleomycin (PVB therapy) was tried, and the tumor showed reduction in size. On Oct. 15, 1982, total cystectomy with ileal conduit urinary diversion was performed. Histopathologically, degenerative change and partial necrosis of the tumor cell were recognized. After the operation, he was treated with radiation therapy and prophylactic VAC therapy. But six months later, multiple pulmonary metastases occurred and gradually increased in size and number. They did not respond to any other chemotherapy. He died on July 13, 1983. We discussed the chemotherapy for rhabdomyosarcoma, and emphasized that the PVB therapy should be tried on rhabdomyosarcoma as an initial chemotherapy.

Infantile granulomatous urachal abscess with acute localized peritonitis and appendicitis.

Infected urachal remnants in early childhood are occasionally seen. However, intraperitoneal penetration of an infected urachal remnant, resulting in acute peritonitis, is rare. We report a case of infantile granulomatous urachal abscess extending into the peritoneum and appendix. The diagnosis and treatment of urachal abscess in childhood are discussed.

Purification and characterization of caldesmon77: a calmodulin-binding protein that interacts with actin filaments from bovine adrenal medulla.

Caldesmon150, a protein composed of the Mr 150,000/147,000 doublet, alternately binds to calmodulin and actin filaments in a Ca2+-dependent "flip-flop" fashion. In all fibroblast cell lines examined, we also found a Mr 77,000 protein that crossreacts with anti-caldesmon150 antibody by using an immunoprecipitation technique [Owada, M.K., Hakura, A., Iida, K., Yahara, I., Sobue, K. & Kakiuchi, S. (1984) Proc. Natl. Acad. Sci. USA 81, 3133-3137]. In this report, we examine the tissue distribution of caldesmon by the method of immunoblotting, using caldesmon-specific antibody. Both caldesmon150 and caldesmon77 show widespread distribution in the tissues examined. Caldesmon77 is more widely distributed than caldesmon150, and we have purified caldesmon77 from bovine adrenal medulla. Its molecular weight estimated by NaDodSO4/polyacrylamide gel electrophoresis was 77,000, and a tetramer of this polypeptide may constitute the native molecule (Mr, 300,000). Caldesmon77 possesses a number of features in common with caldesmon150, including flip-flop binding to calmodulin and actin filaments depending on the concentration of Ca2+ and crossreactivity with caldesmon150-specific antibody. Analysis of caldesmon77-F actin interaction by sedimentation and electrophoresis revealed that 0.5 mg of caldesmon77 bound to 1 mg of F actin. This indicated that the molar ratio between caldesmon77 (tetramer) and actin monomer was calculated to be 1:12-14. In addition, caldesmon77 regulated the actin-myosin interaction in Ca2+-sensitive actomyosin obtained from adrenal medulla. These results suggest that caldesmon77 might be a ubiquitous actin-linked regulator of nonmuscle contractile processes, including those in adrenal medulla.