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.     

Deoxyribonucleoside triphosphate imbalance. 5-Fluorodeoxyuridine-induced DNA double strand breaks in mouse FM3A cells and the mechanism of cell death

The mechanism of cytotoxic action of 5-fluorodeoxyuridine (FdUrd) in mouse FM3A cells was investigated. We observed the FdUrd-induced imbalance of intracellular deoxyribonucleoside triphosphate (dNTP) pools and subsequent double strand breaks in mature DNA, accompanied by cell death. The imbalance of dNTP pools was maximal at 8 h after 1 microM FdUrd treatment; a depletion of dTTP and dGTP pools and an increase in the dATP pool were observed. The addition of FdUrd in culture medium induced strand breaks in DNA, giving rise to a 90 S peak by alkaline sucrose gradient sedimentation. The loss of cell viability and colony-forming ability occurred at about 10 h. DNA double strand breaks as measured by the neutral elution method were also observed in FdUrd-treated cells about 10 h after the addition. These results lead us to propose that DNA double strand breaks play an important role in the mechanism of FdUrd-mediated cell death. A comparison of the ratio of single and double strand breaks induced by FdUrd to that observed following radiation suggested that FdUrd produced double strand breaks exclusively. Cycloheximide inhibited both the production of DNA double strand breaks and the FdUrd-induced cell death. An activity that can induce DNA double strand breaks was detected in the lysate of FdUrd-treated FM3A cells but not in the untreated cells. This suggests that FdUrd induces the cellular DNA double strand breaking activity. The FdUrd-induced DNA strand breaks and cell death appear to occur in the S phase. Our results indicate that imbalance of the dNTP pools is a trigger for double strand DNA break and cell death.     

A transgenic class I antigen is recognized as self and functions as a restriction element

The function of a transgenic Dd class I molecule in the induction of immunologic tolerance to major histocompatibility complex antigens and in directing major histocompatibility complex restriction in C57BL/6 mice were investigated. All of the transgenic Dd mouse strains were found to be tolerant for the Dd antigen. Spleen cells from transgenic mice were immunocompetent but consistently failed to generate an anti-Dd cytotoxic T lymphocyte response in vitro, and skin grafts between transgenic Dd mice were not rejected. These data suggests that the Dd antigen was recognized as a self molecule. In addition, the transgenic Dd mice generated antigen-specific Dd-restricted cytotoxic T lymphocyte, indicating that the Dd antigen also functioned as a restriction element for antigen recognition. These observations demonstrate the usefulness of the transgenic mouse system for studying class I antigen expression and function.     

Existence of a polar digitalis-like factor in mammalian hypothalamus

We were able to partially purify a polar digitalis-like factor from rat and bovine hypothalami based on the capacity to inhibit [3H]ouabain binding to intact human erythrocytes. This factor was characterized in reference to the digitalis-like factor that we have isolated and reported on. Hypothalamic factor shared digitalis-like activities and physicochemical properties with the one derived from human urine and mammalian plasma. These findings strongly suggest that a polar digitalis-like factor identical to the circulatory factor does exist in mammalian hypothalamus.     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A previous paper has demonstrated that enhanced tumor-specific immunity could be induced by priming mice with Bacillus Calmette Guerin (BCG) and subsequently immunizing them with syngeneic tumor cells modified with BCG-cross-reactive muramyl dipeptide (MDP) hapten [15]. The present study establishes a tumorspecific immunotherapy protocol for a murine chronic leukemia based on the above T-T cell collaboration between antitumor effector T cells and anti-MDP hapten helper T cells induced by BCG priming. BALB/c mice which had been primed to BCG were injected intravenously (i.v.) with viable, syngeneic BCL1 leukemia cells. One week later, these mice were immunized intraperitoneally (i.p.) with unmodified or MDP hapten-modified, 10,000 R X-irradiated BCL1 cells, followed by 4 booster immunizations at 5-day intervals. The administration of unmodified BCL1 tumor cells into BCG-primed mice failed to prevent them from tumor death due to the persistent growth of preinjected BCL1 cells. In contrast, the immunization of BCG-primed, BCL1 leukemia-cell-bearing mice with MDP-modified BCL1 cells resulted in a high growth inhibition of leukemia cells and protection of these mice from death by leukemia. It was also revealed that potent tumorspecific, T-cell-mediated immunity was generated in mice which survived in this immunotherapy model. Thus, these results indicate that administration of MDP hapten-modified, syngeneic leukemia cells into leukemia-bearing mice which have been primed with BCG results in potent tumor-specific, T-cell-mediated immunity attributable to preventing the growth of disseminated leukemic cells.This work was supported by a Grant-in-Aid for the Special Project Cancer-Bioscience from the Ministry of Education, Science, and Culture, Japan Abbreviations used: TATA, tumor-associated transplantation antigens; MDP, muramyl dipeptide; MTP, muramyl tripeptide; BCG, Bacillus Calmette Guerin     

Covalent binding of 4-nitrobenzyl mercaptan S-sulfate to the sulfhydryl groups of hepatic cytosolic proteins and bovine serum albumin with mixed disulfide bond formation

4-Nitrobenzyl [35S]mercaptan S-sulfonic acid ([35S]NBM S-sulfate), a new type of reactive metabolite of the thiol [35S]NBM in rat liver cytosol fortified with 3'-phosphoadenosine 5'-phosphosulfate, bound rapidly and covalently at pH 7.4 and 37 degrees C to the sulfhydryl groups of rat liver cytosolic proteins with formation of disulfide bonds. From the radioactive proteins was isolated and identified the sole amino acid adduct, S-([35S]NBM)cysteine, after their acid hydrolysis under the anaerobic conditions. Bovine serum albumin (BSA), a model protein with a single SH group, also reacted readily with radioactive NBM S-sulfate to form a disulfide bond in stoichiometric manner. S-([35S]NBM)-cysteine was also isolated and identified as the sole amino acid adduct from the well-washed, radioactive BSA after the same anaerobic acid hydrolysis. A normal hepatic level of GSH not only retarded the BSA-NBM adduct formation completely, but also detached the radioactivity from BSA by the reduction of the disulfide bond with formation of [35S]NBM and its disulfide. Of twenty-one amino acids examined at pH 7.4 and 37 degrees C, only cysteine reacted with NBM S-sulfate and afforded S-(NBM)cysteine with concomitant formations of S-sulfocysteine, cystine, NBM, and its disulfide.     

The complete amino acid sequence of the A-chain of human plasma alpha 2HS-glycoprotein

Normal human plasma alpha 2HS-glycoprotein has earlier been shown to be comprised of two polypeptide chains. Recently, the amino acid and carbohydrate sequences of the short chain were elucidated (Gejyo, F., Chang, J.-L., Bürgi, W., Schmid, K., Offner, G. D., Troxler, R.F., van Halbeck, H., Dorland, L., Gerwig, G. J., and Vliegenthart, J.F.G. (1983) J. Biol. Chem. 258, 4966-4971). In the present study, the amino acid sequence of the long chain of this protein, designated A-chain, was determined and found to consist of 282 amino acid residues. Twenty-four amino acid doublets were found; the most abundant of these are Pro-Pro and Ala-Ala which each occur five times. Of particular interest is the presence of three Gly-X-Pro and one Gly-Pro-X sequences that are characteristic of the repeating sequences of collagens. Chou-Fasman evaluation of the secondary structure suggested that the A-chain contains 29% alpha-helix, 24% beta-pleated sheet, and 26% reverse turns and, thus, approximately 80% of the polypeptide chain may display ordered structure. Four glycosylation sites were identified. The two N-glycosidic oligosaccharides were found in the center region (residues 138 and 158), whereas the two O-glycosidic heterosaccharides, both linked to threonine (residues 238 and 252), occur within the carboxyl-terminal region. The N-glycans are linked to Asn residues in beta-turns, while the O-glycans are located in short random segments. Comparison of the sequence of the amino- and carboxyl-terminal 30 residues with protein sequences in a data bank demonstrated that the A-chain is not significantly related to any known proteins. However, the proline-rich carboxyl-terminal region of the A-chain displays some sequence similarity to collagens and the collagen-like domains of complement subcomponent C1q.     

Analysis of nephritogenic antigens in human glomerular basement membrane by two-dimensional gel electrophoresis

Collagenase digests of GBM were partially purified by column chromatography and analyzed by 2-D gel electrophoresis. Silver staining of 2-D gels showed charge- and size-related heterogeneity of proteins in the 45 to 50 kDa and 25 to 27 kDa regions. These components were transferred to nitrocellulose sheets and reacted with 10 human anti-GBM autoantibodies. Detection of bound anti-GBM autoantibodies to blotted proteins was carried out with peroxidase-labeled goat anti-human IgG and revealed binding predominantly to the cationic (pI 8 to 9.0) 45 to 50 kDa and 25 to 27 kDa components. Positive-staining patterns of blotted proteins were similar with all anti-GBM autoantibodies except that three sera additionally identified neutral (pI 5.5 to 6.5) protein components. One anti-GBM autoantibody, which developed following renal transplantation, lacked reactivity with the most cationic components in the 25 to 27 kDa region. These findings suggest heterogeneity of nephritogenic GBM antigens. The cationic 45 to 50 kDa components were sensitive to reduction, while one neutral 45 to 50 kDa component was resistant; a complex array of 25 to 30 kDa proteins (pI 5.5 to 7.5) were observed by silver staining postreduction. None of the reduced protein components reacted with anti-GBM antibodies, suggesting that epitopes on nephritogenic GBM antigens may be related to disulfide-bonded regions. Although there is variable immunohistochemical reactivity of anti-GBM autoantibodies with the GBM of infant kidneys, 2-D gels of collagenase-digested human infant GBM blotted and reacted with anti-GBM autoantibodies and showed staining patterns similar to that of adult GBM. These studies demonstrate the presence of nephritogenic antigens in the GBM of immature human kidney which are not detectable by immunohistochemical analysis.     

Spider toxin (JSTX) on the glutamate synapse

A new neurotoxin (JSTX) was separated from spider (Nephila clavata, Joro spider) venom. JSTX irreversibly suppressed the excitatory postsynaptic potential (EPSP) and the glutamate potential in the lobster neuromuscular junction with high degree of specificity. The threshold concentration for suppressing EPSPs corresponds to a small fraction of the toxin in a venom gland, roughly estimated as low as 10(-10) M/l. 10(-10) M/l. In the giant synapse of squid stellate ganglion JSTX suppressed EPSPs without affecting the antidromic response. Glutamate-induced membrane depolarization was blocked by JSTX. In mammalian brain slice preparation, JSTX suppressed the orthodromic spike response but failed to affect on the antidromic spike in the hippocampal pyramidal neuron of CA1 and CA3 region. The above results strongly support the view that the squid giant synapse and synapses in the hippocampal pyramidal neuron are mediated by glutamate.