Evidence for involvement of clonal anergy in MHC class I and class II disparate skin allograft tolerance after the termination of intrathymic clonal deletion

Mechanisms of cyclophosphamide (CP)-induced tolerance to class I (D) and class II (IE) alloantigens were studied. Transplantation tolerance across H-2D plus IE Ag-barriers has been achieved when B10.Thy-1.1 (Kb,IAb,IE-,Db; Thy-1.1) mice were primed i.v. with 9 x 10(7) spleen cells plus 3 x 10(7) bone marrow cells from B10.A(5R) mice (5R; kb,IAb,IEb,Dd; Thy-1.2) and treated i.p. with 200 mg/kg of CP 2 days later. The tolerant state in the early and the late stage was confirmed by prolonged acceptance of donor-type skin grafts, and in vitro unresponsiveness to donor Ag. In the tolerant B10.Thy-1.1 mice treated with 5R cells 28 days earlier and followed by CP, intrathymic clonal deletion of V beta 11+ T cells reactive to IE-encoded antigens was observed in association with intrathymic mixed chimerism. 5R skin survived, however, even after the clonal deletion of V beta 11+ T cells terminated by 180 days after tolerance induction. V beta 11+ T cells, which reappeared in the periphery of the recipient B10.Thy-1.1 mice bearing 5R skin at this stage, were not capable of proliferating in response to receptor cross-linking with V beta 11-specific mAb. Furthermore, the CTL activity against class I (Dd) alloantigens of spleen cells from these tolerant mice was restored by the addition of IL-2 to MLC. Thus, our experiments provide direct evidence that tolerance to both class I (Dd) and class II (IEb) alloantigens by clonal allergy occurs during the termination of intrathymic clonal deletion. These results clearly show practical hierarchy of the mechanisms of transplantation tolerance.     

Generation of neutralization-resistant HIV-1 in vitro due to amino acid interchanges of third hypervariable env region.

Antigenic mutants of HIV-1 were isolated from three plaque-cloned viruses by the resistance of the virus to neutralizing mAb 0.5 beta against V3 domain of viral gp120, when the viruses were passaged in the presence of the antibody. However, when chronically infected MOLT-4 cells were treated with 0.5 beta mAb, recovered viruses from the 0.5 beta-treated cells showed no antigenic changes. The extent of genomic variation among antigenically distinct isolates was examined by nucleotide sequencing, which revealed a few base substitutions in 0.5 beta-binding site of all mutants isolated. The predicted amino acid replacements within 0.5 beta reacting epitope (V3 domain) causing the altered antigenicity were also identified for each of three isolates. Particularly, in one of the mutants, the most conserved Gly-Pro-Gly-Arg region located at the center of the V3 domain was changed to Gly-Gln-Gly-Arg. The radioimmunoprecipitation and synthetic peptide analyses revealed that this Pro320----Gln substitution reduced the binding affinity with 0.5 beta, although other mutations observed in the other mutants did not affect the binding affinity in radioimmunoprecipitation. We also observed that nucleic acid substitutions in the V3 domain occurred frequently in the absence of 0.5 beta mAb during our in vitro acute infection system using MT-4 cells.     

Role of carbohydrate moiety of IL-5. Effect of tunicamycin on the glycosylation of IL-5 and the biologic activity of deglycosylated IL-5

IL-5 is a T cell-derived lymphokine that induces B cell growth and differentiation in murine systems. In this study, we examined the role of carbohydrate moiety of IL-5 in the expression of biological function. IL-5 polypeptides translated in Xenopus oocytes were heterogeneous in terms of isoelectric point (pI 4.7 to 8.0) and m.w. (45,000 to 60,000 under nonreducing conditions) and yielded m.w. of 25,000 to 30,000 under reducing conditions. Treatment of rIL-5 with N-glycanase under reducing conditions yielded an IL-5 monomer of m.w. 12,000 to 14,000. Furthermore, deglycosylated rIL-5 that had been translated in the presence of tunicamycin showed very limited heterogeneity by two-dimensional gel electrophoresis (first dimension, nonequilibrium pH gradient electrophoresis; second dimension, SDS-PAGE). The m.w. was 27,000 to 28,000 under non-reducing conditions and migrated to m.w. 13,000 to 14,000 under reducing conditions. These results indicate that IL-5 is a glycoprotein carrying the N-glycosidically-linked carbohydrates. Treatment of IL-5 with sialidase caused the decrease in the heterogeneity in isoelectric point of IL-5. Deglycosylated rIL-5 that had been obtained from tunicamycin-treated oocytes could bind to IL-5-responding cells (T88-M), which express both high- and low-affinity IL-5 receptors, as efficient as intact rIL-5 under high-affinity conditions. Scatchard plot analysis of equilibrium binding of 35S-labeled rIL-5 to T88-M cells revealed that the dissociation constants (Kd) of glycosylated rIL-5 and deglycosylated rIL-5 were 127 pM and 110 pM, respectively. IL-5 activities determined by both B cell growth and differentiation assays were not affected by deglycosylation. These results indicate that N-linked glycoside moiety of IL-5 molecules may not play an essential role in the expression of its activity.     

Evolutionarily stable stalk to spore ratio in cellular slime molds and the law of equalization in net incomes

The evolutionarily stable stalk ratio (ESSR) in the cellular slime molds is studied when the fruiting body is formed by multiple clones of various size. The survival probability of a spore cell is assumed to depend on the stalk ratio and the fruiting body size. ESSR is obtained as the non-co-operative equilibrium (Nash solution) that maximizes the fitness of each clone. The following two predictions are obtained: (1) the number of spore cells produced by each clone forming a fruiting body tends to be equalized, even if a variation in clone size exists. As a result, the larger clones do not necessarily enjoy higher fitness than the smaller ones. (2) The stalk ratio and the overall fitness of the fruiting body decrease as the genetic diversity in the fruiting body increases. A condition for the stalk to spore ratio to be invariant of overall fruiting body size is also investigated. Finally, "the law of equalization in net incomes" is proposed, extending result (1) into the broader range of resource allocation problems.     

New human colorectal carcinoma cell lines that secrete proteinase inhibitors in vitro

Two new human cell lines, RCM-1 and CoCM-1, have been established from primary colorectal adenocarcinomas. Both cell lines were unique in that the cultures secreted trypsin inhibitors in vitro. The activities of these inhibitors were accumulated in serum-free media of both cell lines over a period of several days. Two inhibitors (PI-1 and PI-2) were isolated from serum-free conditioned medium in which RCM-1 was grown by anion-exchange and gel filtration high-performance liquid chromatography. PI-1 inhibited trypsin and chymotrypsin strongly, and pancreatic elastase weakly. Its molecular weight was about 57 kilodaltons (Kd) as determined by gel filtration chromatography. It cross-reacted with the antiserum elicited against human α₁-antitrypsin in double immunodiffusion. PI-1 corresponding to α₁ - antitrypsin was also demonstrated immunohistochemically in both cell lines. PI-2 inhibited trypsin strongly, and chymotrypsin, kallikrein and plasmin weakly. It had higher molecular weight (200–300 Kd) than that of PI-1, and did not crossreact with antisera against human α₁-antitrypsin, α₂-macroglobulin, α₁-antichymotrypsin, α₂-plasmin inhibitor, inter-α-trypsin inhibitor and urinary trypsin inhibitor. RCM-1 and CoCM-1 are the first colorectal adenocarcinoma cell lines that secrete functionally active trypsin inhibitors, including α₁-antitrypsin in vitro, and are useful for the study of tumor-cell derived proteinase inhibitors.     

Inheritance of mitochondrial aldehyde dehydrogenase: genotyping in Chinese,Japanese and South Korean families reveals dominance of the mutant allele

Summary Genotyping of mitochondrial aldehyde dehydrogenase (ALDH I) was performed in enzymatically amplified DNA of 20 Chinese, Japanese and South Korean families (85 individuals) and in 113 unrelated persons by employing allele-specific oligonucleotide probes and dot blot hybridization. Genotyping individuals with phenotypic deficiency of ALDH I activity always showed the presence of at least one mutant allele. The data are compatible with a model assuming dominant inheritance of the mutant allele, which we have previously suggested on the basis of a population study.     

Protection of OK-432, a Streptococcus pyogenes preparation, against lethal infection of mice with herpes simplex virus

We have studied the protective effect of OK-432, a biological response modifier (BRM) of Streptococcus pyogenes origin, on the lethal infection of mice with herpes simplex virus (HSV)-1. A single intraperitoneal (i.p.) injection of more than 10 micrograms of OK-432, when given at least two days before the infection, gave a marked effect yielding nearly 100% protection against ordinarily lethal infection. The protection was independent of the amount of infected virus inoculated. When given after the infection, the agent even at the maximal dose (100 micrograms), produced only a marginal effect. A single i.p. administration of OK-432 augmented the natural killer (NK) activity of peritoneal exudate cells and spleen mononuclear cells in mice 2 to 3 days after injection of OK-432, coinciding with the times when it induced a survival effect on HSV-infection. Treating OK-432-treated mice with a combination of an anti-macrophage agent, silica, and an anti-NK cell agent, anti-asialo GM1 serum, before infection diminished the antiviral effect of OK-432. The OK-432 protection against HSV infection was also markedly diminished in athymic nude mice. Thus, the protective effect of OK-432 on lethal HSV infection seems to be based on the activation of NK cells, macrophages, and T lymphocytes.     

Identification of Brassica oleracea monosomic alien chromosome addition lines with molecular markers reveals extensive gene duplication

Summary Chromosomes of Brassica oleracea (2n=18) were dissected from the resynthesized amphidiploid B. napus Hakuran by repeated backcrosses to B. campestris (2n=20), creating a series of monosomic alien chromosome addition line plants (2n=21). Using morphological, isozyme and restriction fragment length polymorphism markers (RFLPs), 81 putative loci were identified. Of nine possible synteny groups, seven were represented in the 25 monosomic addition plants tested. Sequences homologous to 26% of the 61 DNA clones utilized (80% were cDNA clones) were found on more than one synteny group, indicating a high level of gene duplication. Anomalous synteny associations were detected in four 2n=21 plants. One of these plants showed two markers from one B. oleracea chromosome associated with a second complete B. oleracea synteny group, suggesting translocation or recombination between non-homologous chromosomes in Hakuran or the backcross derivatives. The other three 2n=21 plants each contained two or more B. oleracea synteny groups, suggesting chromosome substitution.     

Expression and transport into mitochondria of bovine cytochrome P-450(SCC) in insect cells using the baculovirus expression system.

Bovine cytochrome P-450(SCC) introduced with the baculovirus host vector system was found to be expressed in Spodoptera frugiperda cells. Cell fractionation analysis indicated that the P-450(SCC) expressed as the precursor form was transported into mitochondria and converted to a mature form. However, this form did not exhibit definite activity for cholesterol side chain cleavage. These findings suggest that most of the P-450(SCC) expressed by this system is an inactive protein within mitochondria that is not folded to the conformation of the active enzyme and/or does not incorporate heme appropriately.     

Template-directed oligomerization of 5′-deoxy-5′-nucleosideacetic acid derivatives

5-Deoxy-5-nucleosideacetic acids II–V are isostructural analogues of nucleotides with a carboxylate group in the place of the 5-phosphate group. We have studied their oligomerization in aqueous solution using a water-soluble carbodiimide as the condensing agent in the presence or absence of an appropriate polynucleotide template. Condensation of adenylic acid analogues IIa, IIIa, and Va in the presence of polyuridylic acid were found to be the most efficient reactions. Cyclization of the activated monomers to lactones and the insolubility of the oligomers in aqueous solution were found to be obstacles to the efficient formation of long oligomers.