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The Wilms' tumor gene WT1 is overexpressed in various types of solid tumors, including lung and breast cancer and WT1 protein is a tumor antigen for these malignancies. In phase I clinical trials of WT1 peptide-based cancer immunotherapy, two patients with advanced lung cancer were intradermally injected with 0.3 mg of an HLA-A*2402-restricted, 9-mer WT1 peptide emulsified with Montanide ISA51 adjuvant. Consecutive WT1 vaccination at 2-week intervals resulted in a reduction in tumor markers such as chorio-embryonic antigen (CEA) and sialyl Lewis (x) (SLX) and by a transient decrease in tumor size. No adverse effects except for local erythema at the injection sites of WT1 vaccine were observed. These results provided us with the first clinical evidence demonstrating that WT1 peptide-based immunotherapy should be a promising treatment for patients with lung cancer.

The Wilms tumor gene, WT1, is overexpressed not only in leukemias and myelodysplastic syndrome (MDS) but also in various types of solid tumors, including lung and breast cancer, and the WT1 protein is a tumor antigen for these malignancies. In clinical trials of WT1 peptide-based cancer immunotherapy, patients with overt leukemia from MDS or MDS with myelofibrosis were injected intradermally with 0.3 mg of an HLA-A*2402-restricted, 9-mer WT1 peptide emulsified with Montanide ISA51 adjuvant. Only a single dose of WT1 vaccination resulted in an increase in WT1-specific cytotoxic T-lymphocytes, which was followed by a rapid reduction in leukemic blast cells. Severe leukopenia and local erythema at the injection sites of WT1 peptide were observed as adverse effects. These results have provided us with the first clinical evidence suggesting that WT1 peptide-based immunotherapy is an attractive treatment for patients with leukemias or MDS.

Interconnection between surface microdomains and the actin cytoskeleton is vital to various cellular activities. We studied the responses of okadaic acid (OKA)-treated K562 leukemia cells to crosslinking of membrane microdomains. Although OKA alone induced clustering of surface-bound F-actin, addition of a biotinylated poly(ethylene glycol) derivative of cholesterol (bPEG-Chol) and subsequent binding of streptavidin (SA) further induced accumulation of the clusters, resulting in the formation of a spherical cell extrusion. This extrusion was also induced by direct crosslinking of a raft marker, CD59, and ganglioside GM1. In addition, we found that knockout of the gene encoding Fyn kinase inhibited formation of the spherical extrusion in murine T-cells. In bPEG-Chol/SA-treated cells, CD59, ganglioside GM1, and clathrin/AP-2 were all accumulated on the surface of the actin-rich extrusion, whereas dynamin and transferrin receptors were unaffected. Intermediate filaments, mitochondria, and other vesicles also accumulated. These results suggest that crosslinking of membrane domains exaggerates the linkage between actin and a defined set of membrane proteins in OKA-treated cells.

The Wilms' tumor gene WT1 is overexpressed in most types of leukemias and various kinds of solid tumors, including lung and breast cancer, and participates in leukemogenesis and tumorigenesis. WT1 protein has been reported to be a promising tumor antigen in mouse and human. In the present study, a single amino-acid substitution, M-->Y, was introduced into the first anchor motif at position 2 of the natural immunogenic HLA-A*2402-restricted 9-mer WT1 peptide (CMTWNQMNL; a.a. 235-243). This substitution increased the binding affinity of the 9-mer WT1 peptide to HLA-A*2402 molecules from 1.82 x 10(-5) to 6.40 x 10(-7) M. As expected from the increased binding affinity, the modified 9-mer WT1 peptide (CYTWNQMNL) elicited WT1-specific cytotoxic T lymphocytes (CTL) more effectively than the natural 9-mer WT1 peptide from peripheral blood mononuclear cells (PBMC) of HLA-A*2402-positive healthy volunteers. CTL induced by the modified 9-mer WT1 peptide killed the natural 9-mer WT1 peptide-pulsed CIR-A*2402 cells, primary leukemia cells with endogenous WT1 expression and lung cancer cell lines in a WT1-specific HLA-A*2402-restricted manner. These results showed that this modified 9-mer WT1 peptide was more immunogenic for the induction of WT1-specific CTL than the natural 9-mer WT1 peptide, and that CTL induced by the modified 9-mer WT1 peptide could effectively recognize and kill tumor cells with endogenous WT1 expression. Therefore, cancer immunotherapy using this modified 9-mer WT1 peptide should provide efficacious treatment for HLA-A*2402-positive patients with leukemias and solid tumors.

The ability to predict MHC-binding peptides remains limited despite ever expanding demands for specific immunotherapy against cancers, infectious diseases, and autoimmune disorders. Previous analyses revealed position-specific preference of amino acids but failed to detect sequence patterns. Efforts to use computational analysis to identify sequence patterns have been hampered by the insufficiency of the number/quality of the peptide binding data. We propose here a dynamic experiment design to search for sequence patterns that are common to the MHC class I-binding peptides. The method is based on a committee-based framework of query learning using hidden Markov models as its component algorithm. It enables a comprehensive search of a large variety (20(9)) of peptides with a small number of experiments. The learning was conducted in seven rounds of feedback loops, in which our computational method was used to determine the next set of peptides to be analyzed based on the results of the earlier iterations. After these training cycles, the algorithm enabled a real number prediction of MHC binding peptides with an accuracy surpassing that of the hitherto best performing positional scanning method.

Molecular characterization of plant group II chaperonin (CCT, c-cpn, or TriC) still remains elusive. By PCR-based cloning techniques using soybeans, we have made a successful attempt to clone a delta-subunit homologue of CCT (CCTdelta). This subunit is responsible for the binding of an in vivo substrate, alpha-actin, by assisting the correct folding of the cytoskeletal protein in mouse, and the occurrence of the subunit homologue in plant CCT was unclear. As the cloning strategy, a putative amino acid segment, NH(2)-Gly-Gly-Gly-Ala-Pro-Glu-COOH, which is tightly conserved in all known animal and yeast CCTdelta subunits, was chosen for designing a degenerate primer of the PCR-cloning. The resultant 1881-bp cDNA was found to have an open-reading frame of 533 amino acids with a calculated molecular mass of 57,677 Da and to share about 58-65% identity overall at the amino acid level with the corresponding subunits known to date. Using antibodies raised against Escherichia coli-produced soybean insoluble CCTdelta as a monitoring tool, we purified soybean CCT from the extract of its immature seeds. STEM images demonstrated that the molecular shape of soybean CCT is a double eight-membered ring, which resembles the known group II chaperonins. The CCT also reactivated a denatured firefly luciferase with a significant, but limited level of the native enzymic activity in an in vitro system. Northern blot analysis showed that soybean CCTdelta gene, which is intronless and composed of a small family, was only expressed at a very early stage of seed development of soybean.

Wilms tumor gene WT1 is expressed at high levels in hematopoietic malignancies, such as leukemias and myelodysplastic syndromes (MDS), and in various kinds of solid tumors, including lung cancer, and it exerts an oncogenic function in these malignancies. IgM and IgG WT1 antibodies were measured by means of dot blot assay in 73 patients with hematopoietic malignancies (16 acute myeloid leukemia [AML], 11 acute lymphoid leukemia [ALL], 13 chronic myeloid leukemia [CML], and 33 MDS) and 43 healthy volunteers. Immunoglobulin IgM, IgG, and IgM+IgG WT1 antibodies were detected in 40 (54.8%), 40 (54.8%), and 24 (32.8%), respectively, of the 73 patients with hematopoietic malignancies, whereas 7 (16.2%), 2 (4.7%), and none of the 43 healthy volunteers had IgM, IgG, or IgM+IgG WT1 antibodies, respectively. Furthermore, immunoglobulin isotype class switching of WT1 antibodies from IgM to IgG occurred in conjunction with disease progression from refractory anemia (RA) to RA with excess of blasts (RAEB), and further to RAEB in transformation (RAEB-t) in MDS patients. These results showed that humoral immune responses against the WT1 protein could be elicited in patients with WT1-expressing hematopoietic malignancies, and they suggested that the helper T-cell responses needed to induce humoral immune responses and immunoglobulin isotype class switching from IgM to IgG were also generated in these patients. Our findings may provide new insight into the rationale for elicitation of cytotoxic T-cell responses against the WT1 protein in cancer immunotherapy using the WT1 vaccine.

A major histcompatibility complex (MHC) class I protein H-2K(b) was expressed in a large scale as a fusion protein with thioredoxin and hexahistidine at the N-terminus to analyze the interaction with the antigen peptide SIYRYYGL. NMR spectra of the peptide in the mixture solution with the protein showed very broad signals, indicating the obviously clear existence of the dynamic interaction between the class I protein and the antigen peptide. The interaction of the protein and peptide was discussed as well as the surrounding atmosphere of the peptide in the complex., (Copyright 2000 Academic Press.)

Specificities of three mouse major histocompatibility complex (MHC) class I molecules, Kb, Db, and Ld, were analyzed by positional scanning using combinatorial peptide libraries. The result of the analysis was used to create a scoring program to predict MHC-binding peptides in proteins. The capacity of the scoring was then challenged with a number of peptides by comparing the prediction with the experimental binding. The score and the experimental binding exhibited a linear correlation but with substantial deviations of data points. Statistically, for approximately 80% of randomly chosen peptides, MHC-binding capacity could be predicted within one log concentration of peptides for a half-maximal binding. Known cytotoxic T-lymphocyte epitope peptides could be predicted, with a few exceptions. In addition, frequent findings of MHC-binding peptides with incomplete or no anchor amino acid(s) suggested a substantial bias introduced by natural antigen processing in peptide selection by MHC class I molecules.

A soybean protein was purified from mature dry seeds. Amino-acid sequencing of the nine internal peptides derived from this N-terminally blocked protein showed that it has a significant similarity to the soluble epoxide hydrolases known to date. A degenerate series of 23-mer oligonucleotides with sequences corresponding to an internal region of eight amino-acid residues was synthesized as a probe mixture for detection of a putative epoxide hydrolase cDNA in a developing cotyledon cDNA library. The 1332-bp cDNA obtained was found to have an open-reading frame encoding the seed epoxide hydrolase-like precursor consisting of 341 amino-acid residues, suggesting that 25 amino-acid residues upstream from the second methionine correspond to a transit peptide. Employing an Escherichia coli expression system, the putative mature epoxide hydrolase-like protein was overexpressed and purified to homogeneity. This recombinant protein was confirmed to exhibit its epoxide-diol converting activity using styrene oxide as substrate. The Vmax and Km values for styrene oxide are 1.36 micromol x min-1 x mg-1 and 1500 microM, respectively. Sedimentation equilibrium experiments showed that the active form of this epoxide hydrolase is monomeric in solution. Using the above cDNA as a probe, a 12-kb genomic clone was selected and the sequence of a 1933-bp fragment from this clone was found to cover the entire coding region together with 5'- and 3'-flanking regions of the soybean epoxide hydrolase gene. The coding region of the gene, interrupted by two short introns, was identical to the corresponding regions of the cDNA. Northern blot analyses showed that this epoxide hydrolase gene was expressed strongly at a very early stage (13 days after flowering) and then the level of expression gradually decreased and almost ceased at a very late stage (58 days after flowering) of seed development, whereas its expression was markedly up-regulated by ethylene treatment. In stems (hypocotyl portion), the epoxide hydrolase transcript was detected at significant levels and was also up-regulated in response to ethylene. On the other hand, it is hardly expressed in leaves, even though they were treated with the phytohormone. Overall, the results obtained may indicate that soluble-type epoxide hydrolase mRNA is expressed at the maximum level in an early stage of seed development. Later, oil bodies are formed and subsequently epoxy fatty acids, naturally occurring metabolites, accumulate within those bodies. The temporal induction of this epoxide hydrolase transcript in some tissues in response to ethylene also indicates that this epoxide hydrolase may play a crucial role in self-defense systems of plant.

We recently have reported that Wilms' tumor gene WT1 is highly expressed not only in leukemias but also in various types of solid tumors and that WT1 protein is a novel tumor antigen against which cytotoxic T lymphocytes (CTLs) can be elicited by immunization with 9-mer WT1 peptides capable of binding to major histocompatibility complex (MHC) class I molecules. In the present study, plasmid DNA encoding murine full-length WT1 protein was injected intramuscularly into C57BL/6 mice. The mice vaccinated with the WT1 plasmid DNA elicited CTLs against the WT1 protein, and the CTLs specifically killed WT1-expressing tumor cells in a MHC class I-restricted manner. Furthermore, the vaccinated mice rejected the challenges of WT1-expressing tumor cells and survived with no signs of autoimmunity caused by the CTLs. These results demonstrated that vaccination with the WT1 plasmid DNA can elicit CTL responses specific for the WT1 protein, resulting in the acquisition of rejection activity against challenges of WT1-expressing tumor cells. This WT1 DNA vaccination may find clinical application for various types of solid tumors as well as leukemias.

The Wilms' tumor gene WT1 is expressed at high levels not only in acute myelocytic and lymphocytic leukemia and in chronic myelocytic leukemia but also in various types of solid tumors including lung cancers. To determine whether the WT1 protein can serve as a target Ag for tumor-specific immunity, three 9-mer WT1 peptides (Db126, Db221, and Db235), which contain H-2Db-binding anchor motifs and have a comparatively higher binding affinity for H-2Db molecules, were tested in mice (C57BL/6, H-2Db) for in vivo induction of CTLs directed against these WT1 peptides. Only one peptide, Db126, with the highest binding affinity for H-2Db molecules induced vigorous CTL responses. The CTLs specifically lysed not only Db126-pulsed target cells dependently upon Db126 concentrations but also WT1-expressing tumor cells in an H-2Db-restricted manner. The sensitizing activity to the Db126-specific CTLs was recovered from the cell extract of WT1-expressing tumor cells targeted by the CTLs in the same retention time as that needed for the synthetic Db126 peptide in RP-HPLC, indicating that the Db126-specific CTLs recognize the Db126 peptide to kill WT1-expressing target cells. Furthermore, mice immunized with the Db126 peptide rejected challenges by WT1-expressing tumor cells and survived for a long time with no signs of autoaggression by the CTLs. Thus, the WT1 protein was identified as a novel tumor Ag. Immunotherapy targeting the WT1 protein should find clinical application for various types of human cancers.

Lipopolysaccharides of gram-negative bacteria are potent activators of B cells, dendritic cells and monocytes/macrophages. We have investigated the use of LPS-activated spleen cells as antigen-presenting cells to induce CD8+ cytotoxic T lymphocytes in vivo that are reactive to MHC class I binding peptides. Compared with resting spleen cells, CTL induction was more efficient and less variable for different peptides with LPS-activated spleen cells. Cytotoxic responses were specific for the immunized peptides and contained high affinity CD8+ T cells. The removal of dendritic cells and monocytes/macrophages by Sephadex G10 column did not show profound effects on CTL induction, indicating that B-cell blasts were largely responsible. This easily accessible method should facilitate the screening of MHC class I binding peptides to determine whether or not the host's T-cell repertoire contains reactive T cells.

A soybean seed-specific PR-8 chitinase, named Chib2, has a markedly extended C-terminal segment compared to other plant Chib1 homologues of the PR-8 chitinase family known to date. To further characterize the molecular structure and the expression pattern of this chitinase family, we cloned two typical Chib1-similar cDNAs (Chib1-1 and Chib1-2) from soybeans by PCR-cloning techniques. The deduced primary sequence of Chib1-1 chitinase is composed of a signal peptide segment (26 amino acid residues) and a mature 273 amino acid sequence (calculated molecular mass 28,794, calculated pI 3.7). This Chib1-1 enzyme is more than 90% identical to Chib1-2 chitinase but is below 50% identical to Chib2 enzyme. Thus, we confirmed the occurrence of two distinct classes, Chib1 and Chib2 in the plant PR-8 chitinase family. The Chib1 genes, interrupted by one intron, were found to be up-regulated in response to ethylene in stems and leaves, but scarcely expressed in developing soybean seeds. Chib1 chitinases may be responsible for protecting the plant body from various pathogenic attacks.

Duchenne muscular dystrophy is a lethal and common X-linked recessive disease caused by a defect in dystrophin. Normal myoblast transplantation and dystrophin gene transfer have been expected to correct the deficiency in the muscles, but their clinical application has been hampered by the limited preservation of dystrophin-positive myofibers. In this study we investigated the mechanism for immunologic rejection of normal C57BL/10 (B10) myoblasts transplanted into dystrophin-deficient mdx mice, an animal model of Duchenne muscular dystrophy. We found that mdx mice develop CTL specific for dystrophin itself, which were CD8 dominant and restricted by H-2Kb. We identified several antigenic peptides derived from dystrophin that bind to H-2Kb and are recognized by the mdx anti-B10 CTL. Immunologic tolerance against dystrophin was successfully induced by i.v. injection of these peptides before B10 myoblast transplantation, which resulted in sustained preservation of dystrophin-expressing myofibers in mdx mice. These results demonstrate that dystrophin is antigenic in dystrophin-deficient mice and that immunologic regimen would be necessary to achieve the persistent expression of introduced dystrophin in the muscles of dystrophin-deficient individuals.

Assuming that the amount of superoxide radicals generated in vivo correlates with the production of ergastic substances such as storage proteins, the coordinated response of detoxication enzymes such as superoxide dismutases is largely exploited to understand the self-defense systems of plant. Here we examined expression of the genes for superoxide dismutases during seed development of soybean. The cDNAs encoding a cytosolic copper/zinc form and an iron form of the above enzyme have been cloned and then employed as probes, separately. Northern blotting results suggested that both superoxide dismutase mRNAs are expressed at the maximum level, preceding a developmental stage when mRNA encoding glycinin, soybean 11S-storage protein, at the maximum.

An epitope that acted as a weak agonist in the cytotoxicity assay was identified as part of the capsid protein of a hepatitis C virus (HCV) variant. In a low concentration, the variant epitope also had a weak antagonistic effect. When a minute amount of this variant epitope was added to the culture for induction, it selectively attenuated the expansion of major cytotoxic T cell populations and drastically reduced the cytotoxic responses against the wild-type epitope. Thus, antagonism to induction suppressed immune responses against both the wild type and the variant, thereby helping the persistence of not only variant itself but also the wild-type HCV. Because this variant was a weak agonist, most cytotoxic T cells induced with the wild-type epitope were cross-reactive with the variant and susceptible to the antagonism to induction. Only the T cells which were not cross-reactive with the variant and not susceptible to the antagonism survived the antagonism in induction. This implied that the specificity of the remaining immune response, if any, was directed exclusively to the wild-type epitope after the emergence of the variant. For viruses like HCV, being heterogeneous itself may contribute significantly toward persistent infection through antagonism to induction.

A protein fraction was isolated from defatted soybean flour by extraction at acid pH, 40% ammonium sulfate precipitation, hydrophobic interaction chromatography, and gel filtration chromatography. SDS-PAGE, under reducing conditions, confirmed it as a homogeneous preparation. This conclusion was consistent with N-terminal amino acid sequence data (20 cycles) which showed a major sequence identical to those reported for soybean Bowman-Birk-type protease inhibitor (BBI), and also indicated a minimum 95% purity based on recoveries of PTH-amino acid residues. The purified fraction inhibited both trypsin and chymotrypsin with average specific activities of 350 and 672 units mg(-1), respectively. Compared with classical BBI purification, this procedure is very rapid requiring only 72-96 h to achieve a yield of 37 mg purified BBI per 200 g starting material.

Multiple peptides have been used as molecular tools to probe the selective, yet degenerative, recognition of peptides by class I major histocompatibility complex (MHC) molecules and T-cell receptors. The additive contributions from individual amino acids that have been identified experimentally can often be used to predict the binding of any peptide to class I MHC molecules, although other contributing factors will often result in this being an oversimplification.

Targeted disruption of beta 2-microglobulin gene results in deficient major histocompatibility complex class I expression and failure to develop CD4-8+ T cells. Despite this, beta 2 M-/- mice reject skin grafts and cope with most viral infections tested. We asked whether CD4+8- cytotoxic T cells would play a role in compensating for the defect in CD4-8+ cytotoxic T cell function. We found that the cytotoxic activity against class II+ targets is significantly higher among CD4+8- T cells of beta 2M-/- than among those of beta 2M+/+ mice. In the limiting dilution experiment, we showed that the precursor frequency for the cytotoxic, CD4+8-, class II-specific T cells is at least fivefold higher in beta 2M-/- than in beta 2M+/+ mice. These results suggest that CD4+8- cytotoxic T cells could play a major role in carrying out cytotoxic function in beta 2M-/- mice.

A 6 month old boy was diagnosed as a case of combined immunodeficiency (with predominant T cell defect by previous classification). His T cell count was decreased, his B cell count in peripheral blood was increased, his serum IgG level was decreased, his serum IgM level was normal and the thymus was not evident on CT scans and magnetic resonance imaging. Administration of the thymus hormone, thymosin, led to a partial recovery of T cell function without normalization of the T cell count. At age 26 months the patient received an irradiated thymus transplantation from a 16 week old female fetus. After the transplantation, the T cell count (mainly CD4+ cells) increased by 50-70%. A mild graft-versus-host reaction (GVHR) occurred and several immunosuppressants were prescribed. Chromosome analysis showed that the T cells have both 46 XY and 46 XX karyotypes while the B cells have the 46 XY karyotype alone. His cellular immunity (skin tests, DNA synthesis, mixed lymphocyte reaction, cytotoxic activity and natural killer cell function) and his serum IgG level remained low. However, being on regular r-globulin therapy and oral anti-fungal drugs, he is now living normally with almost no trouble at age 6 years and 3 months. This case showed that irradiated thymus transplantation might be a useful method when an adequate donor for bone marrow transplantation is not available. The unexpected observation that the increased T cells were mainly CD4 may be related to the mild GVHR and the clinical improvement.

A bacterial expression system in Escherichia coli has been developed that produces as much as 10 mg/l of culture of the VH protein associated with monoclonal antibodies specific for the 5-dimethylaminonaphthalene-2-sulfonyl (Dns) group. This system has been applied to the expression of the VH genes derived from a low-affinity, IgM-producing hybridoma and from a high-affinity, IgG-producing cell line. The plasmid vectors (contributed by Dr William F. Studier) utilize a T7 expression cassette whose activity is initiated by infection with a lambda phage derivative carrying the T7 RNA polymerase gene. The VH proteins were extracted from the bacterial pellet in 8 M urea and purified by chromatography in 8 M urea. Recombinants with the homologous light (L) chains were prepared to yield VHL molecules. These were used to measure intrinsic affinity for Dns-lysine by resonance energy transfer. The association constants were 7 x 10(6) M-1 and 7 x 10(9) M-1 for the low- and high-affinity systems, respectively. These values are not significantly different from those observed with monoclonal antibodies secreted from the corresponding cell lines. This system lends itself to the quantitative evaluation of the binding properties of the VH protein itself as well as the modulation of affinity by site-directed mutagenesis.

By following dissociation kinetics of radiolabelled C1q from rabbit IgG antibody-sensitized sheep red blood cells (SRBC) before and after its incorporation in the C1 complex, it was demonstrated that the binding stability is markedly enhanced by the presence of the C1r2-C1s2 subunit of C1 which by itself exhibits no significant binding capacity to immune complexes. The dissociation of C1q was decreased by up to 95%, the extent of decrease being pronounced as the cell surface IgG antibody density increased. However, such a stabilizing effect of C1r2-C1s2 was largely abolished when SRBC sensitized with the dimeric fragment F(acb)2 lacking C gamma 3 was used as the C1 binder, whereas the dissociation rate of uncomplexed C1q from F(acb)2-sensitized cells was similar to that from whole IgG-sensitized cells. It was also shown that, although the C1r2-C1s2 subunit is dissociated selectively from C1 bound to either IgG- or F(acb)2-sensitized cells in the presence of EDTA, it is held on much longer by the former cells than the latter cells. These results were taken to indicate that, although the C1 fixation by immune complexes of IgG is undertaken primarily by the interaction between C1q and the C gamma 2 domain, it is also strengthened by the secondary interaction between the C1r2-C1s2 subunit of C1 and the C gamma 3 domain or a structure which is dependent on the pair of C gamma 3 domains.

A number of fragments derived from acid-treated rabbit IgG by digestion with plasmin have been separated by high-resolution gel filtration. Fragments isolated included a dimer and monomer Facb, named F(acb)2 and Facb, respectively and a heterodimer composed of Facb and Fab subunits, named F(acb)(ab). A C gamma 2 fragment was obtained by papain digestion of Facb. A heterodimer composed of Facb and Fab', named F(acb)(ab'), was also prepared by oxidizing a reduced mixture of these fragments. Fragments thus obtained are classified into two groups--those carrying paired C gamma 2 domains, i.e. F(acb)2, and the disulfide-linked dimeric C gamma 2 fragment; and those having a single C gamma 2 domain, i.e. reduced, alkylated Facb and C gamma 2 fragment, F(acb)(ab) and F(acb)(ab'). These fragments exhibited marked differences in their capacity to activate complement in assay systems of hemolysis and complement consumption by immune complexes or aggregates on polystyrene latex. Fragments of the former group could activate complement but with a definitely reduced efficiency (50%) compared to intact IgG, whereas fragments of the latter group were practically inactive. Although it was not determined whether the C1-binding capacity itself is changed by monomerization of the C gamma 2 domain, the results suggested that the intact paired C gamma 2 module is required at least for the activation process of complement.

As the cDNAs encoding A1aB1b and A2B1a subunit precursors of the glycinin A2 subfamily contain a unique NcoI site sequence, (A)CCATGG, occurring at their translation initiation sites, plasmids were constructed to direct the synthesis of those precursor proteins by inserting NcoI/PstI fragments derived from those cDNA clones into the NcoI/PstI-pKK233-2 expression vector in Escherichia coli MV1190, respectively. The resultant plasmids directed the expression of 57-kDa protein components that have molecular masses in agreement with those of the in vitro translation products directed by glycinin A2 subfamily mRNAs, by the addition of isopropyl beta-D-thiogalactoside. These proteins, which comprised as much as 1% of the total bacterial protein, are immunoprecipitable with rabbit antibodies specific for glycinin subunits. This procedure makes glycinin subunits available as a model for studying structure-function relationships in seed proteins using site-directed mutagenesis. This is the first expression of glycinin-like storage protein in E. coli.

The single site binding constants of rabbit IgG and its plasmin-derived fragments F(acb)2, Facb and F(ab)2 for human C1q were measured by the sedimentation velocity method. The intact IgG and F(acb)2 having the paired C gamma 2 domains gave an identical association constant at 20 degrees C (Ka) of 3.02 X 10(4) M-1 in the presence of a physiological concn of salt and on the basis of six sites per C1q. The C1q-binding affinity was found to be decreased to 1.04 X 10(4) M-1 in the reduced, monomerized fragment Facb. Under the same conditions F(ab)2, which is completely unable to activate the classical complement cascade, gave an apparent C1q-affinity of 0.36 X 10(4) M-1. The results, together with previous observations, led us to the conclusion that the C1q-binding site of rabbit IgG is constituted associatively by the pair of C gamma 2 domains, each of which providing a limited, complementary part of the binding free energy between IgG and C1q.

The complete nucleotide sequence of a cloned cDNA, designated pGA5A4B3822, corresponding to glycinin A5A4B3 mRNA was determined. Analysis of the cDNA insert revealed that it contained 1899 nucleotides of mRNA sequence with a 5'-terminal non-translated region of 31 nucleotides, a signal peptide region corresponding to 23 amino acids, an acidic subunit region (A5) corresponding to 97 amino acids, an acidic subunit region (A4) corresponding to 257 amino acids followed by a basic subunit region (B3) corresponding to 185 amino acids, and a 3'-terminal non-translated region of 182 nucleotides. These results show that the glycinin A4 subunit, which is not found to be linked to a basic subunit via a disulfide bond, is synthesized as a full-sized precursor, i.e. the A5A4B3 subunit complex, from a single mRNA, followed by post-translational processing to generate an intermediary subunit complex (A5-B3), covalently linked by a disulfide bond, and the mature A4 subunit, which may associate with the above subunit complex by non-covalent interactions. From the results obtained by the Chou-Fasman rules we speculated that the two post-translational cleavage sites of this subunit precursor might be processed by the same proteolytic enzyme.

Age-related alterations in bone marrow cellularity were examined on SD and Wistar SPF rats. Six rats/strain/sex were sacrificed at 58, 83, 109 and 123 or 135 weeks of age and quantitative assessment and categorization of the bone marrow cells were performed. One male and two female SD rats suffering from leukemia were excluded from statistics. Age-related increases of myeloblasts and plasma cells were observed in rats of either strain and either sex. Age-related decreases in percentages of lymphocytes were noticed in SD, but not in Wistar rats. Age-related alterations in absolute numbers of marrow lymphocytes were equivocal in either strain.

The correlation between morphological and biochemical changes produced by the antigen-antibody reaction was studied in cultures of tissue monocytes taken from sensitized animals. The cells were grown under conditions which allowed collection of samples from the culture fluid as well as microscopic observation. Introduction of the antigen into the culture medium causes rapid release of a protease characterized by its susceptibility to sulfhydryl block and its optimum pH in the neutral range. Protease activation occurs simultaneously with morphological changes in the cytoplasm of the cultured cells. Delayed changes affecting the mitochondria and Golgi bodies appear after the peak of the proteolytic reaction and may be secondary to it. The gradual inactivation of the protease observed in the course of the antigen-antibody reaction will be discussed in a separate paper.