The role of tropomyosin in the interactions of F-actin with caldesmon and actin-binding protein (or filamin)

The interactions of actin filaments with actin-binding protein (filamin) and caldesmon under the influence of tropomyosin were studied in detail using falling-ball viscometry, binding assay and electron microscopy. Caldesmon decreased the binding constant of filamin with F-actin. In contrast, the maximum binding ability of filamin to F-actin was decreased by tropomyosin. The filamin-induced gelation of actin filaments was inhibited by caldesmon. Tropomyosin also inhibited this gelation. The effect of caldesmon became stronger under the influence of tropomyosin. Furthermore, both caldesmon and tropomyosin additionally decreased the filamin binding to F-actin. From these results, caldesmon and tropomyosin appeared to influence filamin binding to F-actin with different modes of actin. In addition, there was no sign of direct interactions between filamin, caldesmon and tropomyosin as judged from gel filtration. Under the influence of caldesmon and tropomyosin, calmodulin conferred Ca2+ sensitivity on the filamin-induced gelation of actin filaments.     

Paraquat Tolerance of Transgenic Nicotiana tabacum with Enhanced Activities of Glutathione Reductase and Superoxide Dismutase

Transgenic tobacco with enhanced cytosolic activities of glutathionereductase and superoxide dismutase were generated by cross-fertilization.Leaves of the hybrids exhibited further increased toleranceto a O₂^-generating herbicide paraquat than those of their parents.This result indicates the efficiency of manipulating more thanone gene in improving resistance of plants to photooxidativestress. ⁴Present address: Department of Regulation Biology, NationalInstitute for Basic Biology, Myodaiji, Okazaki, 444 Japan     

A new highly sensitive chemiluminescent assay of alkaline phosphatase using lucigenin and its application to enzyme immunoassay

The chemiluminescent reaction of lucigenin with various reducing sugars and reducing compounds has been studied. It was found that dihydroxyacetone gave the most intense chemiluminescence (CL). We have developed highly sensitive chemiluminescent methods for alkaline phosphatase (ALP) based on the production of dihydroxyacetone using NADP⁺ or glycerol-3-phosphate as substrate. The detection limits for ALP using each substrate were 1.25 × 10^?19 mol/assay and 2.5 × 10^?19 mol/assay, and the coefficient of variation (n = 7) was 2.8% and 3.7%, respectively. We have also applied the method using NADP⁺ as substrate in enzyme immunoassays (EIA) for cholecystokinin (CCK) and human chorionic gonadotropin (hCG). CCK-8 (octapeptide sulphated form of a carboxy terminal fragment of CCK) concentrations released from alimentary canal of rat were assayed using the chemiluminescent EIA (CLEIA) and a fluorimtric EIA (ALP label). The correlation between CCK-8 values obtained by these methods was y = 1.04x + 18.21, r = 0.946, n = 28. hCG values in serum and in urine were measured. The correlation between hCG values in serum samples obtained using the CLEIA and a time-resolved fluoroimmunoassay (TR-FIA), and in urine samples obtained using the CLEIA and the fluorimetric EIA using ALP were satisfactory. The correlations were y = 1.00x ? 0.04, r = 0.997 (n = 51) and y = 1.00x ? 0.03, r = 0.999 (n = 10), respectively.     

Localization of synapsin I in normal fibers and regenerating axonal sprouts of the rat sciatic nerve

The localization of synapsin I, a synaptic vesicle-associated protein, was investigated immunocytochemically in normal nerve fibers and regenerating axonal sprouts following crush-injuries to the rat sciatic nerve. In normal myelinated axons, weak synapsin I immunoreactivity was found in the axoplasmic/smooth endoplasmic domains, but not in the cytoskeletal domains comprising neurofilaments and microtubules. In non-myelinated axons without dense cytoskeletal structures, moderate immunoreactivity was distributed diffusely throughout the axoplasm. In the crush-injured nerves, intense synapsin I immunoreactivity was demonstrated by light microscopy in early regenerating sprouts emerging from nodes of Ranvier. These nodal sprouts subsequently elongated as regenerating axons through the space between the basal lamina and the myelin sheath (or Schwann cell plasma membrane). Intense synapsin I immunoreactivity was also found in the growth cones of such long regenerating axons. Electron microscopy revealed that synapsin I immunoreactivity was associated mainly with vesicular organelles in the nodal sprouts and growth cones of regenerating axons. Long regenerating axons exhibited no synapsin I immunoreactivity in the shaft, which contained an abundance of neurofilaments. However, vesicle accumulations remaining in the periphery of the shaft still exhibited intense synapsin I immunoreactivity. Thus, it can be concluded that synapsin I is localized at especially high density in the domains comprising vesicular organelles, which are characteristic of early nodal sprouts, as well as in growth cones of regenerating axons. These findings, together with the proposed functions of synapsin I investigated in other studies, suggest that synapsin I may play important roles in vesicular dynamics including the translocation of vesicles to the plasma membrane in sprouts and growth cones of regenerating axons.     

Metachronous serous endometrial intraepithelial carcinoma and serous peritoneal carcinoma: analysis of probable independent lesions

Background

Uterine serous endometrial intraepithelial carcinoma (SEIC) is an immediate precursor of invasive carcinoma. The majority of stage IA SEICs are curable, but those with latent peritoneal metastasis and/or capillary lymphatics invasion may have poor prognoses Careful pathologic staging is thus needed to predict the risk of recurrence and to determine postoperative therapeutic strategies.

Case Presentation

A 71-year-old woman was hospitalized for the treatment of peritoneal carcinoma. She had undergone total hysterectomy and bilateral salpingo-oophorectomy due to SEIC (stage IA) at age 63 years, and had received medical check-ups every year since. Elevated serum CA125 (184 U/mL) was detected for the first time 8 years after surgery. A thorough workup revealed no potential primary lesion other than that in the peritoneum. Tumor reduction surgery was performed. Histologic analysis of the peritoneal lesion was high-grade serous carcinoma. The peritoneal carcinoma was diffusely immunostained for p53; thus, possible recurrence of SEIC was suspected. Tumor DNAs were microdissected from the uterine and peritoneal lesions and p53 mutation analysis was done. SEIC and peritoneal carcinomas had distinct p53 mutations that were mutually exclusive.

Conclusions

The present case raised a concern about the difficulty of histologic staging for SEICs. Although SEICs confined to the uterine endometrium in most cases predict a good prognosis, microscopic metastasis to the peritoneum may not be detectable at hysterectomy. If secondary malignancies of a serous phenotype develop years later, comprehensive reexamination of SEIC is mandated, with the help of DNA analysis.

     

Immunohistochemical Techniques for Detection of Dermatan Sulfate Proteoglycan in Tissue Sections

Dermatan sulfate proteoglycan chains were detected in tissue sections treated with chondroitin Blyase (0.01 units/ml) in 20 mM Tris-HC1 (pH 8.0) for i hr, followed by staining with antibody 9A2 specific for unsaturated uronic acid coupled to N-acetylgdactosaa- m i n d sulfate. In contrast, after treatment with chondroitin B-lyase, no positive stpining was observed with antibodies 3B3 and 1B5 which react to the unsaturated uronic acid coupled to N-acetylgalactosamine 6-sulfate and unsaturated uronic acid coupled to N-acetylgalactospmine, respectively. The distribution of dermatan sulfate thus revealed was mnfirmed by comparison with that found by monoclonal antibody 6B6 which reacts with small pmteoglycans carrying dermatan sulfate side chains. The localization of positive staining in fib- connective tissues was almost identical with these two procedures.     

Establishment of a cell line producing basement membrane components from an adenoid cystic carcinoma of the human salivary gland

A new cell line has been established from an adenoid cystic carcinoma arising in the submandibular gland of a 63-year-old woman. The cultured epithelial-like cells grew vigorously and adhered together to form a sheet. Immunohistochemical stainings for type IV collagen, laminin and fibronectin were clearly positive in the intercellular matrix and on the surface of the culture cells. Chondroitin 6-sulfate proteoglycan and heparan sulfate were also detected. Ultrastructural studies showed that the cells had abundant rough endoplasmic reticulum and a well-developed Golgi apparatus. Rough endoplasmic reticulum near the cell surface was markedly dilated, and contained material of low electron density. This cell line would be useful for biological and biochemical studies on the mechanisms by which the stromal component is formed.     

Caldesmon: a common actin-linked regulatory protein in the smooth muscle and nonmuscle contractile system

Caldesmon was originally purified from gizzard smooth muscle as a major calmodulin-binding protein which also interacts with actin filaments. It has an alternative binding ability to either calmodulin or actin filaments depending upon the concentration of Ca2+ ("flip-flop binding"). Two forms of caldesmon (Mr's in the range of 120-150 kDa and 70-80 kDa) have been demonstrated in a wide variety of smooth muscles and nonmuscle cells. Immunohistochemical studies suggest that caldesmon is colocalized with actin filaments in vivo. Considering its abundance, the Ca2+-dependent flip-flop binding ability to either calmodulin or actin filaments, and its intracellular localization, caldesmon is expected to be involved in contractile events. Recent results from our laboratory have led to the conclusion that caldesmon regulates the smooth muscle and nonmuscle actin-myosin interaction and the smooth muscle actin-high Mr actin-binding protein (ABP or filamin) interactin in a flip-flop manner. It might function in cell motility by regulating the contractile system.