The benzodiazepine receptor (BZDR) of the embryonic chick brain contained three subunit proteins with molecular weights of 48-kilodalton (KD), 50-KD and 51-KD at a pI of 5.6, as demonstrated by two-dimensional gel electrophoresis and fluorography of the 3H-flunitrazepam (FNZ)-photolabeled receptor. Monoclonal antibodies (mAB) against the receptor were produced by using the spleen cells of one mouse immunized with the three subunit proteins extracted from SDS-PAGE gels. When the radioligand-labeled membranes were subjected to two-dimensional gel electrophoresis followed by immunoblotting using the mAB 2C3, both 50-KD and 51-KD bands with a pI of 5.6 were immunoreactive and radioactive. Thus, the mAB 2C3 recognized a common epitope on the 50-KD and 51-KD subunits of the BZDR. In addition, the mAB 2C3 was used with immunocytochemistry to determine the distribution of the receptor in the chick embryo brain. The BZDR immunoreactivity was observed among various brain areas, including hippocampus, optic tectum and cerebellum. The reaction product was localized in the neuronal membranes and cytoplasm. Certain neurons in the culture derived from embryonic chick brains were also immunoreactive as detected by immunocytochemical staining.
1. The endocytic pathway of horseradish peroxidase (HRP) was investigated in the perikarya of cultured neurons by electron microscopy and enzyme cytochemistry. The tracer was observed in endocytic pits and vesicles, endosomes, multivesicular bodies, and lysosomes. It took approximate 15 min for the transfer of HRP from the exterior of the cell to the lysosomes. 2. Monensin induced distension of the Golgi apparatus and formation of intracellular vacuoles. When neurons were incubated with both monensin and HRP for 30 to 120 min, the number of HRP-labeled endosomes was greater than that in the monensin-free group, whereas the reverse was seen for HRP-positive lysosomes. The formation of HRP-positive lysosomes in monensin-treated cells was blocked by 47 to 79%. 3. These results indicate that the intracellular transport of the endocytosed macromolecule is pH dependent. It is also possible that the export of lysosomal enzymes is inhibited by monensin, resulting in an accumulation of the endosomes and a reduction of the lysosomes.
The newly developed Ti-15Mo-1Bi alloy not only possesses all the desirable mechanical properties inherent to beta-Ti Mo alloys, but may even enjoy better clinical applicability with the addition of bismuth element, which has long been administered as antibacterial and antitumor medicines. A significantly higher viability of 3T3 cells was demonstrated when they were grown on Ti-15Mo-1Bi alloy than on Ti-15Mo and Ti-6Al-4V. Cells incubated in the medium conditioned by Bi powder at 37 degrees C for 96 h exhibited viability similar to that in the blank group and higher than that in the Ni conditioned group. In vivo experiments using 6 mm x 2 mm metal pin implanted into the epicondyle of rabbit femur revealed superior potential of new bone growth and better persistence of the deposited bony tissue with the Ti-15Mo-1Bi alloy in contrast to that with Ti-6Al-4V. The difference is evident at 12th week and become even more prominent after 26 weeks, with the new bone area measuring 249% of that around Ti-6Al-4V alloy. In summary, Ti-15Mo-1Bi alloys show no cytotoxicity in the in-vitro test and demonstrates superior ability to retain bone in the in-vivo implantation experiment as compared with Ti-6Al-4V alloys.
Effects of monensin were examined on the intracellular processing of the GABAA/benzodiazepine receptor (GABAA/BZDR) in neuron cultures derived from embryonic chicken brain, using 3H-flunitrazepam as the probe for the benzodiazepine modulator site on the receptor. Incubation of cultures with 0.1 or 1 microM monensin for 3 h blocked the binding of 3H-flunitrazepam by about 18%. Loss of ligand binding was due to a reduction in the number of binding sites, with no significant changes in receptor affinity. The general cellular protein synthesis and glycosylation in the cells were inhibited by 26% and 56%, respectively, in the presence of 1 microM monensin, as detected by assaying the incorporation of 3H-leucine and 3H-galactose. In contrast, an increase was observed for mannose incorporation by the cultures in the presence of the drug. Moreover, the results from in situ trypsinization of the cultures following monensin treatment showed that monensin did not alter the distribution of intracellular and surface receptors. The data suggest that monensin induces the down-regulation of GABAA/BZDR by generating abnormal glycosylation of the receptor and interrupting its transport within the Golgi apparatus, as well as from the Golgi apparatus to the intracellular pool and cell membrane. The galactosylation of receptor proteins may be important for the maturation of the receptor.
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