Susumu Tomonaga

Two populations of immunoglobulin-forming cells in the skate, Raja kenojei: Their distribution and characterization

We recently reported the presence in the skate, Raja kenojei, of two immunoglobulins, a high molecular weight immunoglobulin analogous to mammalian IgM, and a low molecular weight immunoglobulin belonging to a hitherto undescribed class. In view of these findings, we studied lymphoid organs of the skate using morphological and immunocytochemical means. The spleen and the intestinal mucosa had numerous plasma cells and lymphocytes, while the Leydig organ of the esophagus, the epigonal organ and the liver contained these cells in a much lesser frequency. Immunocytochemical studies proved these plasma cells and some of the lymphocytes to be immunoglobulin-forming cells. Double immunofluorescence staining of the spleen and other lymphoid tissues demonstrated the occurrence at a 1: 1 ratio of two distinct populations of cells, one forming the high molecular weight immunoglobulin and the other producing the low molecular weight immunoglobulin.

Isolation and characterization of immunoglobulin of hagfish, Eptatretus burgeri, a primitive vertebrate

The immunoglobulin of the hagfish, Eptatretus burgeri, one of the most primitive vertebrates extant, was isolated from the serum of non-immune normal adult hagfish in a pure form. Analysis of the immunoglobulin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing condition indicated that the immunoglobulin was composed of heavy (H) and light (L) chains. The mol. wt of the H-chain was 68,000, slightly smaller than that of the human mu-chain. The L-chain of the immunoglobulin appeared as 2 bands on SDS-PAGE, with mol. wts of 25,000 and 22,000. These findings were confirmed by gel filtration of reduced-alkylated immunoglobulin in 5 M guanidine-HCl. The H:L molar ratio of the immunoglobulin was roughly 1:1. Gel filtration of the immunoglobulin in non-dissociating buffer indicated that the mol. wt of the intact immunoglobulin was 150,000-160,000. Thus, the subunit chain composition of the immunoglobulin was assumed to be H2L2, identical with the fundamental structure of immunoglobulins. The instability of the hagfish immunoglobulin was ascertained by the fact that it dissociated into heterogeneous mol. wt components ranging from approx. 90,000 to 160,000 upon SDS-PAGE under non-reducing conditions. However, almost no free or monomeric H- or L-chains were dissociated from the immunoglobulin by this procedure and also by gel filtration in 5 M guanidine-HCl. Theses results indicated that the hagfish immunoglobulin is unusually labile in its tertiary structure but has disulfide binding between at least more than 2 subunit chains.

Component C3 of hagfish complement has a unique structure: identification of native C3 and its degradation products.

A protein from hagfish serum that cross-reacted with the third component of hagfish complement (C3) was purified to homogeneity and its structural properties were compared with those of C3 which has a two-subunit chain structure (115 and 72 kDa). This protein (designated C3b), when purified from plasma, consisted of three disulfide-linked polypeptide chains (77, 72 and 30 kDa). On immunoelectrophoresis, purified C3b migrated more rapidly towards the anode than the beta mobility of C3. However, immunochemical analysis revealed that C3b, after the first step in its purification, consisted of two disulfide-linked polypeptide chains (105 and 72 kDa). Treatment of C3b with methylamine, prior to spectrophotometric titration of the free sulfhydryl groups, did not significantly affect the end-point of the titration, suggesting the absence of a thioester bond in this molecule. Analysis of the amino acid sequences of the amino-termini of the subunits of C3b revealed that 77 amino acid residues at the amino-terminus of the native alpha chain were missing from both the 77-kDa and the 105-kDa polypeptides from C3b. These results indicate that the C3b in this study was analogous to mammalian C3b. Furthermore, amino acid sequencing data indicated that most of the native C3 from hagfish serum has an irregular two-subunit (alpha+gamma and beta)-linked structure, as a result of one-sided processing of putative hagfish pro-C3 at the beta-alpha processing site exclusively. Moreover, it appears that only the molecular features of degenerated hagfish C3 (C3b) are altered during its purification to generate a three-chain structure.

Studies on immunoglobulin and immunoglobulin-forming cells in Heterodontusjaponicus, a cartilaginous fish

Immunoglobulin (Ig), lymphoid tissues and Ig-forming cells of the Japanese bullhead shark, Heterodontus japonicus were analyzed biochemically, histologically and immunocytochemically. The serum of Heterodontus contains two Igs with different molecular weights one with 900 K and the other with 180 K daltons. Heavy chains of the two Igs showed an identical molecular weight of 68 K and the same antigenicity, indicating that the two Igs belong to the same class with different molecular structure. Light chains of Heterodontus Igs showed two distinct bands using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one with the molecular weight of 25 K and the other with 22 K daltons. The latter finding indicates the possible existence of two light chain types in the Heterodontus Igs. White pulp of the spleen appeared as a well-developed lymphoid tissue accompanied large number of Ig-forming cells especially around blood vessels. Massive lymphocytic aggregations were found in the central area of the intestinal valves and certain lymphoid cells were demonstrated to be Ig-forming cells. Ig-forming cells were also observed in the epigonal organ, although the frequency was much less than in the former two tissues. Although the spleen is the major Ig-forming organ in Heterodontus japonicus, the valvular intestine and the epigonal organ also appear to share the function of Ig production.

Primordial germ cells and lymphomyeloid system in the embryos of the Aleutian skate,Bathyraja aleutica

Organogenesis in embryos (1 and 5 mm-disk widths) of the Aleutian skate,Bathyraja aleutica was described histologicaily in complete serial paraffin sections. Special attention was paid to localization of primordial germ cells (PGC) and development of lymphoid tissues. The embryo of 1 mm-disk width was at the somite stage with no evidence of organogenesis. PGC, gathered in the genital ridge, were seen in the somatic mesodermal layer as well as in the mesenchyme between the endoderm and splanchnic mesoderm of the 1 mm-embryo. Most of the visceral organs were developing in the embryo of 5 mm-disk width. With respect to the development of immune system, two pairs of thymus anlagen, filled with numerous thymic lymphocytes, were recognized in the pharyngeal region. A small focus of numerous immature blood cells appeared to be an anlage of the spleen was found between the stomach and the liver.

Characterization of the Effects of Opsonins in Normal Hagfish Serum on the Ingestion of Rabbit Erythrocytes by Hagfish Macrophages

Abstract Normal serum from the hagfish Eptatretus burgeri strongly enhanced the phagocytic activity of homologous peritoneal macrophages against rabbit erythrocytes (RRBC). Such phagocytosis was effectively but incompletely inhibited by rabbit antibodies against hagfish C3 (HC3). However, when RRBC were pretreated with purified HC3 or normal hagfish serum in the presence of EDTA, little enhancement of ingestion was observed. The results indicate that HC3 binds to RRBC, in collaboration with other serum factor(s), and that bound HC3 functions as an opsonin. Additionally, we obtained evidence for the possible presence of another opsonin (HOP) that was resistant to EDTA in C3-depleted hagfish serum. HOP prepared from plasma had a molecular mass of approximately 1,000 kDa, consisting of various small polypeptides that were able to form aggregates via disulfide bonds and/or noncovalent interactions. The functions of HC3 and HOP might be essential to immunity in hagfish because no clear evidence for the production of antibodies in cyclostomes has been reported to date.

Immunohistochemical localization and biological significance of the phylogenically conserved thymus-brain antigen (UB-13 antigen) in skate, rat and human

A monoclonal antibody (UB-13) originally raised against the brain of the skate (Raja kenojei, a cartilaginous-fish) was found to react with lymphoid and brain tissues from many species when examined immunohistochemically. In rat and human thymus, UB-13 antigen was observed to be closely associated with reticular tissue in the medulla and cortex. Interestingly, a few or several thymocytes were encircled by the UB-13-reactive reticular tissue. At 14 days gestation, rat thymus consisted mainly of reticular epithelial tissue, after which strong thymocyte production started. At this stage, some of the reticular tissue was heavily stained with UB-13. In the thymus tissues of the irradiated and recovering rats, where reduction and massive reproduction of thymocytes were observed, extensive UB-13 antigen expression localized on the reticular epithelial tissue, an observation which may support the thymocyte re-population. These findings suggest that the antigen recognized by UB-13 may be important for thymocyte proliferation and maturation. UB-13 antigen was found in the fibrous structure of the molecular and granular layer of the human cerebellum. Some glial cells were also stained strongly with UB-13 in the human cerebellar or cerebral grey and white matter. In rat, glial cells, especially astroglias, and the endothelial structure of blood vessels were stained strongly with UB-13. These findings suggest that UB-13 may be a useful monoclonal antibody for analysis of brain-lymphoid antigen in many species.

Structure of the urinary bladder in the Pacific cod

The urinary bladder of the Pacific cod,Gadus macrocephalus was investigated. A pair of ureters was found to unite and form a sac-like protrusion on the posteroventral aspect of the gonad. This protrusion was confirmed as the urinary bladder based on its histology and the presence of ammonia and urea in the fluid within this structure. The urinary bladder consisted of a central lumen and two lateral expansions found on the right and left sections. The luminal epithelial cells of the Pacific cod urinary bladder, unlike those of other animals, were characterized by microvilli on the free surface, and the presence of a number of vesicles and mitochondria across the apical portion on the cells. This suggests that the luminal epithelium of the urinary bladder might be actively engaged in transportation of water or other materials from the urine. The Pacific cod urinary bladder may therefore be closely associated with osmoregulation by reabsorbing water from the urine. Ammonia-N and urea-N levels of 65–213 μg/dl and 1.5–3.5 mg/dl were measured in the fluid which filled the urinary bladder.