An aberrant retinal pathway and visual centers in Xenopus tadpoles share a common cell surface molecule, A5 antigen

A monoclonal antibody A5 (MAb-A5), which was raised against Xenopus tadpole tectal cells, recognizes a cell surface-related protein molecule (A5 antigen) expressed on the visual centers of Xenopus tadpoles (S. Takagi, T. Tsuji, T. Amagai, T. Takamatsu, and H. Fujisawa, 1987, Dev. Biol. 122, 90-100). The present immunohistochemistry using MAb-A5 indicated that, in addition to the visual centers, A5 antigen was expressed on the general somatic sensory tract in the medulla and spinal cord of Xenopus tadpoles. As the general somatic sensory tract has been shown to be a pathway for ectopically transplanted retinal axons (M. Constantine-Paton and R. R. Capranica, 1976, J. Comp. Neurol. 170, 17-32; M. J. Katz and R. J. Lasek, 1979, J. Comp. Neurol. 183, 817-832), we examined whether retinal axons transplanted close to the spinal cord or medulla preferentially grow into the A5 antigen-positive general somatic sensory tract. We performed eye transplantation at embryonic stages and detected precise locations and trajectories of transplanted retinal axons within the medulla and spinal cord in tadpoles after filling retinal axons with horseradish peroxidase (HRP). HRP histochemistry in combination with MAb-A5 immunohistochemistry indicated that almost all HRP-filled transplanted retinal axons joined the A5 antigen-positive general somatic sensory tract. These findings suggest the involvement of A5 antigen in specific cell-cell recognition between retinal axons and their targets.     

Amino acid sequence of the calcium-binding light chain of myosin from the lower eukaryote, Physarum polycephalum

We have established a new method for preparing Physarum myosin whose actin-activated ATPase activity is inhibited by micromolar levels of Ca2+. This Ca2+-inhibition is mediated by the Ca2+ binding to the myosin rather than by the Ca2+-dependent modification of the phosphorylated state of the myosin (Kohama, K., and Kendrick-Jones, J. (1986) J. Biochem. (Tokyo) 99, 1433-1446). Ca2+-binding light chain (CaLC) has been suggested to be primary importance in this Ca2+ inhibition (Kohama, K., Takano-Ohmuro, H., Tanaka, T., Yamaguchi, T., and Kohama, T. (1986) J. Biol. Chem. 261, 8022-8027). The amino acid sequence of CaLC was determined; it was composed of 147 amino acid residues and the N terminus was acetylated. The molecular weight was calculated to be 16,131. The homology of CaLC in the amino acid sequence with 5,5'-dithiobis-(2-nitrobenzoic acid) light chain and alkali light chain of skeletal muscle myosin were rather low, i.e., 25% and 30%, respectively. Interestingly, however, the CaLC sequence was 40% homologous with brain calmodulin. This amino acid sequence was confirmed by sequencing the cloned phage DNA accommodating cDNA coding CaLC. Northern and Southern blot analysis indicated that 0.8-kilobase pair mRNA was transcribed from a single CaLC gene. This is the first report on the amino acid sequence of myosin light chain of lower eukaryotes and nucleotide sequence of its mRNA.     

Ultrastructural visualization of selective peanut agglutinin binding sites in rat osteoclasts

We investigated the distribution of concanavalin A (ConA)-reactive alpha-D-mannosyl and alpha-D-glucosyl groups and peanut agglutinin (PNA)-reactive beta-D-galactose-(1----3)-N-acetyl-D-galactosamine residues on the surface of osteoclasts with pre-embedment ultrastructural lectin cytochemistry after aldehyde fixation of the metaphyses of the rat tibiae. By routine morphology, the plasma membrane of the ruffled border of the osteoclast was distinguished from the rest of the cell membrane, with the exception of the membrane of coated pits, by its characteristic thick coat at its cytoplasmic surface. Cytochemistry, using ConA in combination with horseradish peroxidase (ConA-HRP) and PNA conjugated to HRP, showed that binding of ConA was distributed over the entire cell surface of osteoclasts. In contrast, intense binding of PNA was limited to the membranes of the ruffled border and coated pits, whereas the remainder of the cell membrane stained weakly or not at all. These results demonstrate that preferential PNA binding sites of the cell surface correspond to coated membranes associated with osteoclastic endocytosis.     

The atypical M2 segment of the beta subunit confers picrotoxinin resistance to inhibitory glycine receptor channels.

Purified preparations of the inhibitory glycine receptor (GlyR) contain alpha and beta subunits, which share homologous primary structures and a common transmembrane topology with other members of the ligand-gated ion channel superfamily. Here, a beta subunit-specific antiserum was shown to precipitate the [3H]strychnine binding sites localized on alpha subunits from membrane extracts of both rat spinal cord and mammalian cells co-transfected with alpha and beta cDNAs. Further, inhibition of alpha homo-oligomeric GlyRs by picrotoxinin, a non-competitive blocker of ion flow, was reduced 50- to 200-fold for alpha/beta hetero-oligomeric receptors generated by cotransfection. Site-directed mutagenesis identified residues within the second predicted transmembrane segment (M2) of the beta subunit as major determinants of picrotoxinin resistance. These data implicate the M2 segment in blocker binding to and lining of the GlyR chloride channel.     

Incorporation of fatty acids by Streptococcus mutans

In a series of investigations into the cariogenicity of Streptococcus mutans, we studied the incorporation of exogenous fatty acids with reference to glucosyltransferase secretion and membrane fatty acid changes. When cells were grown with different fatty acids, both saturated and unsaturated fatty acids were readily incorporated into the membrane lipids and were biotransformed and elongated preferentially to the longer 16- and 18-carbon-chain fatty acids. This incorporation and chain-elongation led to significant changes in fatty acids composition. By adding fatty acids to the medium, it was possible to appropriately modify the degree of unsaturation and the relative ratio between specific fatty acids in the membrane lipids of S. mutans.     

Requirement of mitoses for the reversal of X-inactivation in cell hybrids between murine embryonal carcinoma cells and normal female thymocytes

By means of a 5-bromodeoxyuridine (BrdU) incorporation and acridine orange fluorescence staining method we studied reactivation of the inactivated X chromosome (Xi) in newly formed cell hybrids between the near-diploid HPRT-deficient OTF9-63 murine embryonal carcinoma cell (ECC) with an XO sex chromosome constitution and the normal female mouse thymocyte. As reported earlier, most near-tetraploid hybrid cells were ECC in morphology and retained all chromosomes from both parents including three X chromosomes. Synchronization of the late replicating X chromosome in such hybrid cells, indicative of reactivation, was found for the first time on Day 3, and the frequency of reactivation was attained 90% on Day 5. Inhibition of cell cycle progression either by methylglyoxal bis(guanylhydrazone) dihydrochloride, an inhibitor of polyamine metabolism, or by isoleucine-deficient medium after cell fusion delayed reactivation of the Xi, which implied that the number of cell division cycles traversed by individual cells rather than the length of time after cell fusion is critical for the reactivation. Double-labeling experiments using [3H]thymidine and BrdU indicated that hybrid cells had undergone three or four mitoses before reactivation of the Xi. Most probably reactivation of the Xi is consequent to reversion of the thymocyte genome to an undifferentiated state under the influence of OTF9 genome. DNA demethylation or dilution of Xi-specific factors by mitoses may be involved in this process.     

Endothelin: a new inhibitor of renin release

Endothelin is a recently-discovered vasoconstrictor peptide which is produced by endothelium and acts on vascular smooth muscle cells. At present its actions on other organs or cells are unknown. We studied the effect of endothelin on renin release in a dynamic superfusion system of dispersed rat juxtaglomerular (JG) cells. Endothelin in concentrations of 10(-11) M or more inhibited renin release dose-dependently and this inhibitory action vanished in the absence of extracellular Ca. It is suggested that endothelin is an inhibitory regulator of renin secretion from JG cells and its action is Ca-dependent.