Immunocytochemical detection of triphosphoinositide in vestibular hair cells

Triphosphoinositide (TPI), an aminoglycoside receptor and a possible regulator of cationic permeation through its ability to bind with Ca++, was localized by the protein-A gold technique in vestibular sensory epithelia using an antibody highly specific to TPI. TPI was detected on the stereocilia, kinocilia, and cuticular plate of hair cells, and in the reticular membrane of supporting cells. The cilia of hair cells are damaged by aminoglycosides at a relatively early stage of toxicity. Ca++-regulated bioactivity in this area is probably involved.     

The immunohistolocalization of carbonic anhydrase III in the submandibular gland of rats and hamsters

Summary Carbonic anhydrase III has been localized using the avidin-biotin-glucose oxidase complex (ABC) method in the submandibular gland of the rat and hamster. This isozyme, which is predominant in skeletal muscle, was observed in intercalated duct, striated duct and excretory duct cells in the rat submandibular glands. In contrast, only some striated duct cells in hamster submandibular glands were stained.     

Analysis of urinary aldosterone metabolites in the rabbit by gas chromatography-mass spectrometry

After a large amount of aldosterone was injected into a male rabbit, urine was collected for 48 h. Separation of urinary aldosterone metabolites into monoglucosiduronate fraction and monosulphate fraction was carried out by a combination of countercurrent distribution and DEAE-Sephadex A-25 column chromatography. Each fraction was hydrolyzed with enzyme and free steroids released were separated by Sephadex LH-20 column chromatography. The free steroid was then identified by gas chromatography-mass spectrometry. In monoglucosiduronate fraction, 3 alpha, 5 beta-tetrahydroaldosterone and 3 beta, 5 alpha-tetrahydroaldosterone were found. On the other hand, 3 alpha, 5 beta-tetrahydroaldosterone was the only aglycone detected in monosulphate fraction. These findings comfirmed results in the preceding paper, where the free steroid was characterized on the basis of the mobility of the steroid and its derivatives on paper chromatography.     

Immunocytochemical detection of triphosphoinositide in vestibular hair cells

Summary Triphosphoinositide (TPI), an aminoglycoside receptor and a possible regulator of cationic permeation through its ability to bind with Ca⁺⁺, was localized by the protein-A gold technique in vestibular sensory epithelia using an antibody highly specific to TPI. TPI was detected on the stereocilia, kinocilia, and cuticular plate of hair cells, and in the reticular membrane of supporting cells. The cilia of hair cells are damaged by aminoglycosides at a relatively early stage of toxicity. Ca⁺⁺-regulated bioactivity in this area is probably involved.     

Aminoglycoside binding sites in the cochlea as revealed by neomycin-gold labelling

Summary Neomycin/bovine serum albumin/gold was used as a probe to detect the binding sites of aminoglycosides on the thin sections of the cochlea embedded in Spurr. The binding sites were mainly located on the stereocilia, the cuticular plate of hair cells, the head plates of Deiters' cells, fibrous structures in pillar cells, in the spiral limbus and tectorial membrane and basilar membrane, plasma membranes, mitochondria and the chromatin of various kinds of cells. Triphosphoinositide, acidic glycosaminoglycans, and RNA were considered to be responsible for the binding activity.     

A tobacco protein kinase, NPK2, has a domain homologous to a domain found in activators of mitogen-activated protein kinases (MAPKKs)

A cDNA (cNPK2) that encodes a protein of 518 amino acids was isolated from a library prepared from poly(A)⁺ RNAs of tobacco cells in suspension culture. The N-terminal half of the predicted NPK2 protein is similar in amino acid sequence to the catalytic domains of kinases that activate mitogen-activated protein kinases (designated here MAPKKs) from various animals and to those of yeast homologs of MAPKKs. The N-terminal domain of NPK2 was produced as a fusion protein in Escherichia coli, and the purified fusion protein was found to be capable of autophosphorylation of threonine and serine residues. These results indicate that the N-terminal domain of NPK2 has activity of a serine/threonine protein kinase. Southern blot analysis showed that genomic DNAs from various plant species, including Arabidopsis thaliana and sweet potato, hybridized strongly with cNPK2, indicating that these plants also have genes that are closely related to the gene for NPK2. The structural similarity between the catalytic domain of NPK2 and those of MAPKKs and their homologs suggests that tobacco NPK2 corresponds to MAPKKs of other organisms. Given the existence of plant homologs of an MAP kinase and tobacco NPK1, which is structurally and functionally homologous to one of the activator kinases of yeast homologs of MAPKK (MAPKKKs), it seems likely that a signal transduction pathway mediated by a protein kinase cascade that is analogous to the MAP kinase cascades proposed in yeasts and animals, is also conserved in plants.     

Localization of triphosphoinositide in the cochlea

Summary Triphosphoinositide (TPI) has been demonstrated to be a receptor for aminoglycosides in the cochlea and may regulate ionic permeability by its binding with Ca⁺⁺. This phospholipid was localized by a protein A-gold technique in the cochlea at the electronmicroscopic level. TPI was prepared by a neomycin column and antibodies to it were raised in rabbits. The antibody used in this study reacted virtually only to TPI among the tested lipids. TPI was localized mainly at stereocilia, cuticular plates, head plates of Deiter's cells, plasma membrane, and mitochondria of various cells in the organ of Corti. In the vascular stria, TPI was found mainly at the plasma membrane of basal infoldings of the marginal cells. Possible physiological and pathophysiological roles of TPI in the cochlea are briefly discussed.     

Binding sites of an aminoglycoside in the cochlea examined by immunocytochemistry

Summary To localize the binding sites of aminoglycosides in the cochlea, immunocytochemistry was used with the antibody to gentamicin and the protein-A/gold complex. We found that the main binding sites were the stereocilia, the cuticular plates of hair cells, the head plates of Deiters' cells, cell filaments and the cones of pillar cells, tectorial membranes, basilar membranes, the matrix of the spiral limbus, plasma membranes, mitochondria, and the chromatin of various kinds of cells. Triphosphoinositide and acidic glycosaminoglycans are the two most likely candidates for the cause of binding activity.     

Mutagenic and nonmutagenic bypass of DNA lesions by Drosophila DNA polymerases dpoleta and dpoliota

cDNA sequences were identified and isolated that encode Drosophila homologues of human Rad30A and Rad30B called drad30A and drad30B. Here we show that the C-terminal-truncated forms of the drad30A and drad30B gene products, designated dpoletaDeltaC and dpoliotaDeltaC, respectively, exhibit DNA polymerase activity. dpoletaDeltaC and dpoliotaDeltaC efficiently bypass a cis-syn-cyclobutane thymine-thymine (TT) dimer in a mostly error-free manner. dpoletaDeltaC shows limited ability to bypass a 6-4-photoproduct ((6-4)PP) at thymine-thymine (TT-(6-4)PP) or at thymine-cytosine (TC-(6-4)PP) in an error-prone manner. dpoliotaDeltaC scarcely bypasses these lesions. Thus, the fidelity of translesion synthesis depends on the identity of the lesion and on the polymerase. The human XPV gene product, hpoleta, bypasses cis-syn-cyclobutane thymine-thymine dimer efficiently in a mostly error-free manner but does not bypass TT-(6-4)PP, whereas Escherichia coli DNA polymerase V (UmuD'(2)C complex) bypasses both lesions, especially TT-(6-4)PP, in an error-prone manner (Tang, M., Pham, P., Shen, X., Taylor, J. S., O'Donnell, M., Woodgate, R., and Goodman, M. F. (2000) Nature 404, 1014-1018). Both dpoletaDeltaC and DNA polymerase V preferentially incorporate GA opposite TT-(6-4)PP. The chemical structure of the lesions and the similarity in the nucleotides incorporated suggest that structural information in the altered bases contribute to nucleotide selection during incorporation opposite these lesions by these polymerases.     

Degradation of estrogens by Rhodococcus zopfii and Rhodococcus equi isolates from activated sludge in wastewater treatment plants

We have isolated four strains of Rhodococcus which specifically degrade estrogens by using enrichment culture of activated sludge from wastewater treatment plants. Strain Y 50158, identified as Rhodococcus zopfii, completely and rapidly degraded 100 mg of 17beta-estradiol, estrone, estriol, and ethinyl estradiol/liter, as demonstrated by thin-layer chromatography and gas chromatography-mass spectrometry analyses. Strains Y 50155, Y 50156, and Y 50157, identified as Rhodococcus equi, showed degradation activities comparable with that of Y 50158. Using the random amplified polymorphism DNA fingerprinting test, these three strains were confirmed to have been derived from different sources. R. zopfii Y 50158, which showed the highest activity among these four strains, revealed that the strain selectively degraded 17beta-estradiol during jar fermentation, even when glucose was used as a readily utilizable carbon source in the culture medium. Measurement of estrogenic activities with human breast cancer-derived MVLN cells showed that these four strains each degraded 100 mg of 17beta-estradiol/liter to 1/100 of the specific activity level after 24 h. It is thus suggested that these strains degrade 17beta-estradiol into substances without estrogenic activity.