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.     

Effects of pancreastatin on insulin and pancreatic polypeptide secretion in the dog

Porcine pancreastatin (1.19 nmol) was administered into the peripheral vein (i.v.) or the third cerebral ventricle (i.t.v.) of dogs and its effect on the secretion of insulin and pancreatic polypeptide (PP) studied. Neither means of administration had any effect on basal and glucose-induced insulin or PP secretion. However, i.v. pancreastatin did inhibit the i.v. CCK-8-induced insulin but not PP release. Pancreastatin may thus play a role in the regulation of insulin secretion in the canine pancreas.     

Altered localization of antigen recognized by proteinuriainducing monoclonal antibody in experimental nephrosis

Change in the localization of the antigen recognized by the proteinuria-inducing monoclonal antibody (MA) 5-1-6 in experimental nephrosis was studied by indirect and biotin-avidin immunofluorescence, and immunoperoxidase at light and electron microscopical levels. The proteinuric state was induced by the administration of the aminonucleoside of puromycin (PAN) or adriamycin. The antigen decreased in quantity and/or its distribution changed with an increase in the amount of protein excreted in both experimental models. Recovery from the alterations observed during the development and proteinuria appeared to occur when PAN-induced proteinuria subsided. This antigenic molecule may thus be essential for maintaining the normal permselectivity of glomerular capillary walls.     

Modification of the amino acid acceptor stem of E. coli tRNAMetf by ligation of chemically synthesized ribooligonucleotides

The single-stranded region of the amino acid acceptor stem corresponding to the 3'-end of E. coli tRNAMetf was replaced by ligation of chemically synthesized ribooligonucleotides, in order to change the length of the single-stranded CCA terminus. The chemically synthesized ribooligomers, CCA, ACCA, AACCA and CAACCA, were ligated to nuclease-treated E. coli tRNAMetf, which lacked the ACCA sequence at the 3'-end. The methionine acceptor activities of these modified tRNAs were examined using E. coli methionyl-tRNA synthetase. Ligation of the chemically synthesized pentamer (AACCA) to the acceptor terminus restored the methionine acceptor activity, whereas ligation of the hexamer (CAACCA) or trimer (CCA) to the acceptor terminus did not Modification of the acceptor terminus had no effect on the formylation of accepted methionine.     

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.     

Remodeling of Human Sperm Chromatin Mediated by Nucleoplasmin from Amphibian Eggs

The molecular events associated with decondensation of human sperm nuclei were analyzed by incubating sperm with egg extracts from an amphibian, Bufo japonicus . Acid-urea-Triton polyacrylamide gel electrophoresis (AUT-PAGE) showed that the nuclear basic proteins of human sperm consist mainly of protamines (HPI, HPII) with minor amounts of nucleosomal histones. On incubation of lysolecithin (LC)- and dithiothreitol (DTT)-treated human sperm with the egg extract, the nuclei lost HPI and HPII within 15 min in association with extensive nuclear decondensation, and the acquirement of a whole set of nucleosomal histones. Incubation of LC-DTT-sperm with nucleoplasmin purified from Bufo eggs also induced nuclear decondensation and loss of protamines within 30 min. Native-PAGE and Western blot analyses of incubation medium indicated tight association of the released protamines to nucleoplasmin, strongly suggesting that protamines are removed from sperm nuclei not enzymatically but by their specific binding to nucleoplasmin. On incubation of LC-DTT-sperm with nucleoplasmin and exogenous nucleosomal core histones, micrococcal nuclease-protected DNA fragments were released, although their unit repeat length was slightly less than that of somatic nucleosomes. Thus remodeling of human sperm during fertilization can be mimicked under defined conditions with nucleoplasmin and exogenous histones.     

Mutualism based on stress: selective synthesis and phosphorylation of a stress protein by an intracellular symbiont.

The effects of a temperature shift-up and various metabolic inhibitors on the protein synthesis of an endosymbiont isolated from the pea aphid were studied. The syntheses of at least three major polypeptides were stimulated transiently immediately after a temperature shift-up, and treatment with ethanol and heavy metals (Cd2+ and As2+). One of these proteins, the 63 kDa heat-shock protein (63-kDa HSP), was immunoprecipitated with antiserum raised against symbionin, which is selectively synthesized by the endosymbiont harbored by the aphid bacteriocytes. The 63 kDa heat-shock protein has a molecular mass of 800 kDa and is more acidic than symbionin. It was also shown that symbionin is subject to phosphorylation in vivo and in vitro after a temperature shift-up. It was thought likely that forms of environmental stress such as heat shock and metabolic inhibitors stimulate the synthesis of a phosphorylated form of symbionin. It was also suggested that the in vitro phosphorylation of symbionin is due to its own catalytic activity. Since symbionin is a homolog of the Escherichia coli groEL protein, a stress protein, it is likely that the endosymbiont suffers stress when harbored by the bacteriocytes and responds in a similar manner to environmental stress when outside these cells.     

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.     

Expression of a synthetic gene for human cap binding protein (human IF-4E) in Escherichia coli and fluorescence studies on interaction with mRNA cap structure analogues

An artificial gene coding for the human cap binding protein (hCBP: human IF-4E) was chemically synthesized and expressed in Escherichia coli under the control of a trp promoter. The DNA duplex of 662 bp was designed and constructed from 44 oligodeoxynucleotide fragments of typically 30 nucleotides in length. Although the hCBP gene was not directly expressed in E. coli HB101, we succeeded in its high-level expression as a fusion protein connected with a portion of human growth hormone through a tetradecapeptide (Asp-Asp-Pro-Pro-Thr-Val-Glu-Leu-Gln-Gly-Leu-Val-Pro-Arg) that contains the recognition sequence for a site-specific protease alpha-thrombin. Upon induction with 3-indoleacrylic acid, the fusion protein accumulated with a yield of about 20% of the total proteins of the host cell. Upon the treatment of the fusion protein with alpha-thrombin, which recognizes the sequence "Val-Pro-Arg," specific proteolysis at the fused junction occurred efficiently. In this system, nonspecific digestion by alpha-thrombin was not marked. About 15 mg of recombinant hCBP was obtained from a 1-liter culture. Association constants between the recombinant hCBP and mRNA cap structure analogues were determined by fluorescence spectroscopy. The values obtained for the m7GpppA, m7GTP, and m7GMP were almost the same as those reported for the IF-4E isolated from human erythrocyte cells.(ABSTRACT TRUNCATED AT 250 WORDS)