Embryonic Development and Postnatal Changes in Free d-Aspartate and d-Serine in the Human Prefrontal Cortex

Abstract: We have analyzed free chiral amino acids (aspartate and serine) in the human frontal cortex at different ontogenic stages (from 14 weeks of gestation to 101 years of age) by HPLC with fluorometric detection after derivatization with N-tert -butyl-oxycarbonyl- l -cysteine and o -phthaldialdehyde. Exceptionally high levels of free d -aspartate and d -serine were demonstrated in the fetal cortex at gestational week 14. The ratios of d -aspartate and of d -serine to the total corresponding amino acids were also high, at 0.63 and 0.27, respectively. The concentration of d -aspartate dramatically decreased to a trace level by gestational week 41 and then remained very low during all postnatal stages. In contrast, the frontal tip contained persistently high levels of d -serine throughout embryonic and postnatal life, whereas the d -amino acid content in adolescents and aged individuals was about half of that in the fetuses. Because d -aspartate and d -serine are known to have selective actions at the NMDA-type excitatory amino acid receptor, the present data suggest that these d -amino acids might play a pivotal role in cerebral development and functions that are related to the NMDA receptor.     

Recognition of the Qa-2k tumor antigen by T cell receptor γ/δ of an immunopotentiator-induced tumoricidal T cell of mice

Tumor-specific expression of Qa-2^k antigen coded by the Q5^k gene on various mouse tumor cells and immunological response of the host mice to the antigen have been demonstrated [Seo et al. (1992) J Exp Med 175: 547; Tanino et al. (1992) Cancer Immunol Immunother 35: 230]. The possibility was examined that Qa-2 antigen is one of the recognition target molecules of immunopotentiator-induced, H-2-nonrestricted tumoricidal lymphocytes of Qa-2^– mice. Lymphocytes stimulated in vivo withP. acnes or culture-induced anomalous killers of B6.K1 mice did not exhibit significant in vitro cytotoxicity against B6.K1 lymphoblasts but lysed their Qa-2,3-congenic counterpart B6 lymphoblasts. To demonstrate the Qa-2 specificity of such cytotoxic cells more precisely, an L cell transformant clone (L^Q7b/Kb), which expressed the 1 and 2 domains of the Qa-2 antigen (Q7^b gene product), was generated by transfecting a cloned plasmid DNA containing a hybrid gene constructed from the 5 half of the Q7^b gene and the 3 half of the H-2K^b gene (pQ7^b/K^b). Using L^Q7b/Kb cells as the target cells and the nylon-wool-nonadherent fraction of lymphocytes fromP. acnes-stimulated (C3H/He × B6.K1)F1 mice (H-2^k, Qa-2^–) as the effector cells of the in vitro cytotoxicity reaction, the presence of cytotoxic cells that recognize the 1/2 region of the Q7^b gene product was demonstrated. The cytotoxic activity was dependent on T cells bearing T cell receptors of the / type (TCR/). The (C3H/He × B6.K1)F1 effector cells, as well as the B6.K1 effector cells also lysed BW5147 lymphoma cells (Qa-2^k+) derived from AKR mice (Qa-2^–, H-2^k). By target-competition experiments it was shown that some of the effector cells lytic to BW5147 were identical to those that lysed L^Q7b/Kb. Therefore some of the tumoricidal cells induced by the immunopotentiator interact with the target tumor cells through recognition of the 1/2 region of the Qa-2^k tumor antigen by TCR/.     

Intraovarian thrombin and activated protein C signaling system regulates steroidogenesis during the periovulatory period

In addition to its role in blood coagulation, thrombin directly stimulates protease-activated receptors (PAR) or interacts with thrombomodulin (THBD) to activate membrane-bound protein C which stimulates PAR1 and PAR4 receptors to promote downstream pleiotropic effects. Our DNA microarray, RT-PCR, and immunostaining analyses demonstrated ovarian expression of THBD, activated protein C (APC) receptor [endothelial protein C receptor (EPCR)], as well as PAR1 and PAR4 receptors in mice. After treatment of gonadotropin-primed immature mice with an ovulatory dose of human chorionic gonadotropin (hCG) (a LH surrogate), major increases in the expression of THBD, EPCR, PAR1, and PAR4 were detected in granulosa and cumulus cells of preovulatory follicles. Immunoassay analyses demonstrated sustained increases in ovarian prothrombin and APC levels after hCG stimulation. We obtained luteinizing granulosa cells from mice treated sequentially with equine CG and hCG. Treatment of these cells with thrombin or agonists for PAR1 or PAR4 decreased basal and forskolin-induced cAMP biosynthesis and suppressed hCG-stimulated progesterone production. In cultured preovulatory follicles, treatment with hirudin (a thrombin antagonist) and SCH79797 (a PAR1 antagonist) augmented hCG-stimulated progesterone biosynthesis, suggesting a suppressive role of endogenous thrombin in steroidogenesis. Furthermore, intrabursal injection with hirudin or SCH79797 led to ipsilateral increases in ovarian progesterone content. Our findings demonstrated increased ovarian expression of key components of the thrombin-APC-PAR1/4 signaling system after LH/hCG stimulation, and this signaling pathway may allow optimal luteinization of preovulatory follicles. In addition to assessing the role of thrombin and associated genes in progesterone production by the periovulatory ovary, these findings provide a model with which to study molecular mechanisms underlying thrombin-APC-PAR1/4 signaling.     

Protection against metastasis by immunization with an allogeneic lymphocyte antigen

Q5 antigens are expressed on the surface of various experimental murine tumor cells. They share partially common antigenicity with Qa-2 alloantigens expressed on normal lymphocytes. For that reason we tested the immunoprotection by anti-Qa-2 immunization of mice against a Q5+ tumor. Nerve fibrosarcoma (NSFA) tumor, which specifically develops metastasis in the lung, has been reported to be poorly immunogenic. However, expression of the Q5 antigen was evident on the surface of NFSA cells. After immunizing (C3H/He x B6.K1)F1 (Qa-2-) mice with B6 (Qa-2+) lymphocytes, the protection against the proliferation of the semi-syngeneic NFSA tumor was examined First, immunization of normal mice induced resistance to NFSA cell transplants. Second, when the tumor cells were transplanted to the hind foot of a mouse and the resulting tumor was removed by amputating the leg, the mice were protected against the development of lung metastasis after immunization by intraperitoneal inoculation of B6 cells 3 days after tumor removal. Immunization with attenuated NFSA cells in this system failed to protect the mice from lung metastasis. On the other hand, inoculation of the mice with B6 cells without removal of the original tumor on the foot showed little effect on the progression of the tumor. Thus, cytotoxic T lymphocytes (CTL), which seemed to be present in an inactive form in the mice from which the tumor had not been removed, were induced in the mice after the removal of the major tumor followed by immunization with B6 lymphocytes. The induction of CTL by the immunization was suppressed in mice bearing large tumors. Cells stimulated by the tumor antigen seemed to be involved in the suppression. It was also shown that the Q5 antigen is the direct recognition target of the CTL since the activity of Q5-specific CTL clones in lysing tumor cells was inhibited by a monoclonal antibody specific for the Q5 antigen. In contrast to immunization with attenuated tumor cells, our novel allogeneic lymphocyte immunization procedure offers high CTL activation, by-passing the induction of T cell unresponsiveness.     

Mini-plasmin found in the epithelial cells of bronchioles triggers infection by broad-spectrum influenza A viruses and Sendai virus.

Extracellular cleavage of virus envelope fusion glycoproteins by host cellular proteases is a prerequisite for the infectivity of mammalian and nonpathogenic avian influenza viruses, and Sendai virus. Here we report a protease present in the airway that, like tryptase Clara, can process influenza A virus haemagglutinin and Sendai virus envelope fusion glycoprotein. This protease was extracted from the membrane fraction of rat lungs, purified and then identified as a mini-plasmin. Mini-plasmin was distributed predominantly in the epithelial cells of the upward divisions of bronchioles and potentiated the replication of broad-spectrum influenza A viruses and Sendai virus, even that of the plasmin-insensitive influenza A virus strain. In comparison with plasmin, its increased hydrophobicity, leading to its higher local concentrations on membranes, and decreased molecular mass may enable mini-plasmin to gain ready access to the cleavage sites of various haemagglutinins and fusion glycoproteins after expression of these viral proteins on the cell surface. These findings suggest that mini-plasmin in the airway may play a pivotal role in the spread of viruses and their pathogenicity.     

Studies on new intracellular proteases in various organs of rat. 3. Control of group-specific protease under physiological conditions.

1. To study the role of group-specific protease in enzyme degradation, alternation of its activity under various physiological conditions was examined. 2. Studies on the distribution of group-specific protease in various organs of rats showed high activity in skeletal muscle and the muscle layer of small intestine, and rather low activity in liver. The activity varied in different muscles, but red muscle tended to have higher activity than white muscle. Activity was much lower in the muscles of the stomach and colon than in those of the small intestine. 3. Group-specific protease in skeletal muscle increased under various dietary conditions (starvation, protein-free diet or high protein diet), but the activities in the muscle layer of the small intestine and liver were not greatly influenced by dietary conditions. None of the hormones tested (i.e. hydrocortisone, glucagon, insulin, growth hormone and estrogen) influenced the activity of group-specific protease in liver. 4. The level of group-specific protease in skeletal muscle was increased markedly fifteen days after denervation, with a reciprocal decrease in the level of muscle phosphorylase, which is a good substrate of the protease. 5. Liver protease activity appeared in the late suckling period. The activity in skeletal muscle was high at the time of birth and attained the adult level 3 weeks after birth. The activity in the muscle layer of the small intestine did not change after birth. Thus the mechanism for evoking these three specific proteases during development are apparently different. The activity of liver protease began to decrease approximately 12 h after partial hepatectomy and reached a minimum after about 72 h. Recovery of the protease activity was very slow and activity had not returned to the normal value 7 days after the operation. This observation seems to be consistent with the fact that there is little or no protease activity in liver in the neonatal period.     

Crystallization and properties of cathepsin B from rat liver.

Cathepsin B from rat liver was purified to apparent homogeneity by cell-fractionation, freezing and thawing, acetone treatment, gel filtration, DEAE-Sephadex and CM-Sephadex column chromatography, and was crystallized. The purified enzyme formed spindle-shaped crystals and its homogeneity was proved by disc gel electrophoresis in the presence of sodium dodecyl sulfate and by ultracentrifugal analysis. Its s20,w value was 2.8 S and its relative molecular mass was calculated to be 22,500 (+/- 900) by sedimentation equilibrium analysis. Crystalline cathepsin B was shown to consist of four isozymes with isoelectric points between pH 4.9 and 5.3, the main isozyme having an isoelectric point of pH 5.0. The enzyme was irreversibly inactivated by exposure to weak alkali. The pH optimum was 6.0 with alpha-N-benzoyl-DL-arginine-4-nitroanilide as substrate. Amino acid analysis showed that the enzyme contained hexosamine, glucosamine and galactosamine. Cathepsin B inactivated aldolase, glucokinase, apo-ornithine aminotransferase, and apo-cystathionase, but the rates of inactivation of glucokinase, apo-ornithine aminotransferase, and apocystathionase were lower than that of aldolase. Studies by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate showed that cathepsin B degraded apo-ornithine aminotransferase to two polypeptide chains differing in relative molecular mass and electrophoretic mobility.     

Catalytic Activities Of [GADV]-Peptides

We have previously postulated a novel hypothesis for the origin of life, assuming that life on the earth originated from “[GADV]-protein world”, not from the “RNA world” (see Ikehara's review, 2002). The [GADV]-protein world is constituted from peptides and proteins with random sequences of four amino acids (glycine [G], alanine [A], aspartic acid [D] and valine [V]), which accumulated by pseudo-replication of the [GADV]-proteins. To obtain evidence for the hypothesis, we produced [GADV]-peptides by repeated heat-drying of the amino acids for 30 cycles ([GADV]-P₃₀) and examined whether the peptides have some catalytic activities or not. From the results, it was found that the [GADV]-P₃₀ can hydrolyze several kinds of chemical bonds in molecules, such as umbelliferyl-β-D-galactoside, glycine-p-nitroanilide and bovine serum albumin. This suggests that [GADV]-P₃₀ could play an important role in the accumulation of [GADV]-proteins through pseudo-replication, leading to the emergence of life. We further show that [GADV]-octapaptides with random sequences, but containing no cyclic compounds as diketepiperazines, have catalytic activity, hydrolyzing peptide bonds in a natural protein, bovine serum albumin. The catalytic activity of the octapeptides was much higher than the [GADV]-P₃₀ produced through repeated heat-drying treatments. These results also support the [GADV]-protein-world hypothesis of the origin of life (see Ikehara's review, 2002). Possible steps for the emergence of life on the primitive earth are presented.