Shunnosuke Natsuume-Sakai

Molecular cloning of mouse β2-glycoprotein I and mapping of the gene to chromosome 11

beta 2-Glycoprotein I (beta 2 GPI), a plasma protein that binds to anionic phospholipids, is composed of five repeating units called a short consensus repeat (SCR), which is found mostly in the regulatory proteins of the complement system. Recently the human beta 2 GPI gene has been assigned to chromosome 17, not to chromosome 1 where most of the genes of the SCR-containing proteins are clustered. In this report, we have isolated a full-length cDNA clone of mouse beta 2 GPI and determined the chromosomal localization of the gene. The amino acid sequence deduced from the nucleotide sequence of mouse beta 2 GPI revealed 76.1% identity with that of human beta 2 GPI. A genetic mapping by in situ hybridization and linkage analysis using 50 backcross mice has shown that the mouse beta 2 GPI gene (designated B2gp1) is located on the terminal portion of the D region of chromosome 11, closely linked to Gfap, and is 18 cM distal to Acrb, extending a conserved linkage group between mouse chromosome 11 and human chromosome 17. On the basis of these results, the evolutionary relationships among the SCR-containing proteins are discussed.

Different allotypes of C3 degrade at different rates

To determine whether different forms of C3 degrade at different rates, we compared two strains of mice with a B10 background. The only difference was that one is C3A, while the other is OR These strains allow comparison of C3A and C3B without the added complication of differing C3 convertases. Sera from the two strains were incubated with zymosan and the degradation products were detected by immunofixation following electrophoresis in agarose. The rate of degradation of mouse C3B was more rapid than that of C3A. Differences in the rates of degradation could not be explained by differing concentrations of C3. We suggest that the genetic differences in C3 determine the decay rate following activation via the alternate pathway.

Studies of the properties of a streptococcal preparation, OK-432 (NSC-B116209), as an immunopotentiator

SummaryThe present study was designed to examine the mechanism by which OK-432 triggers the cytotoxic activity of peritoneal exudate cells (PEC). When OK-432 was incubated with freshly harvested mouse serum, the formation of complexes of OK-432 with the third component of complement (C3) was demonstrated by using 131I-labeled mouse C3. The formation of C3-OK-432 complexes was totally abolished by a chelating compound, EDTA, which had been shown to inhibit the OK-432 induced activation of the alternative complement pathway. The C3-OK-432 complexes thus obtained bound to the resident PEC, which were subsequently shown to be activated. These activated PEC had augmented cytostatic activity against MM2 cells, a mouse mammary carcinoma.Further, the PEC from mice which had received an IP injection of OK-432 4–5 days previously were cytostatic against MM2 cells and also inhibited the growth of MM2 cells in culture. In contrast, resident PEC stimulated rather than inhibited the 3H-thymidine uptake by MM2 cells and the growth of MM2 cells. The mechanism of PEC (presumably macrophages) activation by OK-432 is discussed.

Quantitation of beta 1c/1A globulin (C3) in inbred mice: variation dependent upon strain, age, sex and environment.

The effect of age, sex, genetic backgrounds and environmental factors on mouse C3 level was studied. In general, C3 level increased as a mouse became older. Little difference in C3 level was observed between male and female of all ages. C3 level was found in a great variation from strain to strain, but we could not demonstrate any major role of H-2 in determining serum C3 level. Serum C3 level is not shown to be linked to the serum level of H-2 linked serum protein Ss (C4). On the other hand a close correlation between C3 level and C5 level was demonstrated. Serum C3 markedly increased, along with C5, when mice were injected with turpentine oil, suggesting both components are acute phase reactants. The degree of increase in C3 level in response to turpentine oil injection appeared to be controlled by a single or a very few genes probably not linked to H-2.

Structural polymorphism of murine factor B controlled by a locus closely linked to the H-2 complex and demonstration of multiple alleles

New alleles of murine factor B (Bf) protein were demonstrated. When ethylenediaminetetraacetic acid (EDTA)-plasmas from inbred and wild mice were analyzed by isoelectro-focusing (IEF) and immunofixation, murine Bf proteins were visualized as distinct protein bands in all mice tested. Four variants of murine Bf could be demonstrated in a large number of tested mice: Bf 1 (isoelectro-focusing point (P.I.) range of 5.8–6.1) exemplified by B10 and B10.BR, Bf 2 (P.I. range of 5.8–6.0) exemplified by B10.MOL (OHM), Bf 3 (P.I. range of 5.6–5.9) exemplified by B10.MOL (TEN2) and Mus musculus (Mus m.) subspecies Chc, Bf4 (P.I. range of 6.0–6.3) exemplified by Mus m. subspecies Shh. The genetic linkage between S locus and Bf locus was studied with two backcross progenies — [B 10.BR × (B10.BR × Mus m. subspecies Chc)F1] and [B 10.BR × (B10.BR × Mus m. subspecies Shh)F1]. Totally, 256 backcross progenies were typed for Bf type and for Ss type (plasma level of the fourth complement protein regulated by S locus). The results indicated that murine Bf was controlled by a single codominant locus located close to the H-2 complex because no mouse showing recombination between Bf locus and S locus was found.

Serological survey of complement factor H in common laboratory and wild mice: a new third allotype

Antigenic specificities of complement factor H from mice were studied serologically. In addition to previously reported allotypes, referred to as H.1 and H.2, a new allotype of complement factor H, H.3, was identified in the BFM/2Ms strain derived from European wild mice. Using three different alloantisera raised against the various mouse factor H allotype, a serological survey of the common laboratory strains and wild-derived strains of Mus musculus and its relatives, Mus spretus, Mus spretoides, and Mus spicilegus was carried out. All of the common laboratory strains examined in this survey had the H.1 allotype except for STR/N which had H.2. The geographical distributions of factor H allotypes in M. musculus were specific to the subspecies. Mice derived from Mus musculus domesticus and Mus musculus castaneus had the H.1 allotype. Mice derived from M. m. musculus, Mus musculus bactrianus, and Mus musculus molossinus had the H.2 allotype. Only BFM/2Ms and BFM/1Mpl strains derived from M. m. domesticus had the novel H.3 allotype. Sera of mice from strains derived from M. spretoides and M. spicilegus cross-reacted with H.2-specific antiserum, and those from M. spretus cross-reacted with H.3-specific antiserum.

Evaluation of basic procedures for adoptive immunotherapy for gastric cancer

Peripheral blood lymphocytes (PBL) of gastric cancer patients in advanced stages showed lymphokine activated killer (LAK) activities comparable to those of healthy donors, suggesting potential applicability of LAK cells induced from PBL stimulated with recombinant interleukin-2 (rIL-2) in adoptive immunotherapy (AIT) for gastric cancer. In order to generate a large number of LAK cells from PBL, lymphocytes were cultured with both rIL-2 and phytohemagglutinin (PHA). In this culture, the numbers of cells increased to a greater extent than those in culture with rIL-2 alone but cytotoxic activity did not augment, thus suggesting that this procedure would not afford sufficient clinical effects. On the other hand, a large number of LAK cells with high anti-tumor activities were efficiently induced from spleen cells of the patients by culture of rIL-2; hence clinical usefulness of these LAK cells is anticipated. In regional lymph node lymphocytes (RLNL) cultured with rIL-2, the cytotoxic activities were lower than in those induced in PBL, and a characteristic increase of CD8 + CD11 + suppressor T cells was observed after incubation with rIL-2. Nevertheless, an increase of CD4 + 4B4+ helper inducer T cells was also observed in RLNL after the culture with rIL-2. Furthermore, high cytotoxic activities were induced in RLNL in some cases in which metastasis to the regional lymph nodes was not detected. When gastric cancer patients were pretreated with biological response modifiers (BRM), especially with Lentinan, LAK cells from PBL showed higher NK and LAK activities as compared with those of patients without BRM pretreatment.

Structural polymorphism of mouse complement C2 detected by microscale peptide mapping: Linkage to H-2

Complement C2 was isolated from 17 mouse strains by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined for structural polymorphism by using micro-peptide mapping. By comparing the peptide maps of tryptic digests of C2 from various strains, two allotypic variations were detected. B 10 and 14 other mouse strains demonstrated C2.1 type, while a wild mouse line (M.Mol-Ohm) and one BIO congenic strain, B10.MOL.OHM, which carries the H-2 derived from M.Mol-Ohm, demonstrated C2.2 type. (B10 × Bl0.MOL.OHM)F1 demonstrated codominantly expressed C2 type (C2.1.2). Desialation of mouse C2 did not abolish the observed variation of mouse C2. It is concluded that an H-2-linked codominant locus controls the structure of mouse complement C2, further confirming the extensive homology of the major histocompatibility complex among higher vertebrate species.

Simplified method for purification of mouse beta 1H.

A simple three-step method was described for purification of murine beta 1H, one of the essential regulatory proteins of complement system. The method consists of heparin-Sepharose affinity chromatography; gel filtration on a Sepharose 6B column, and DNA-cellulose affinity chromatography. By this method over 10 mg of beta 1H can be purified by more than 200-fold from 100-ml of EDTA serum of various strains. Overall yield of beta 1H was about 45%. The purified beta 1H was homogeneous as judged by SDS-polyacrylamide gel electrophoresis and immunoelectrophoresis. The purified mouse beta 1H showed physicochemical properties very similar to those described for human beta 1H: mouse beta 1H is a beta-globulin consisting of a single polypeptide chain of molecular weight of 160,000. Purified mouse beta 1H retained its functional activity as the essential cofactor for the cleavage of fluid-phase human C3b by the human C3b inactivator. Immunization of rabbits with the purified mouse beta 1H resulted in the production of the potent and monospecific antibody.

THE MOUSE FACTOR H ALLOTYPES WITH MULTIPLE AMINO ACID REPLACEMENT, H.l AND H.2 SHOW INDISTINGUISHABLE CO‐FACTOR ACTIVITY FOR FACTOR I

The authors report the functional analysis of the purified mouse factor H allotypes H. 1 and H.2, which were clearly distinguished from each other by an immunodiffusion test. Both allotypes acted as a co‐factor for factor I in cleaving mouse C3b and we found no significant difference between their activities. The results strongly suggest that the function of mouse factor H for the co‐factor activity has been well conserved between two allotypes.