Detection of a novel HLA-DQ specificity: serological and immunochemical analyses by a monoclonal antibody

A monoclonal antibody (mAb) with a novel human B-cell allospecificity was produced by immunizing a C3H/He mouse with the human B lymphoblastoid cell line EBV-Wa (HLA-DR4/Dw15/DQblank homozygous). The mAb, termed HU-46, reacted with B cells from not only DR4/Dw15-positive individuals but also certain DRw8/Dw8-positive ones whose DQ phenotypes had not yet been defined. Two-dimensional gel analyses indicated that the mAb recognized class II antigens which were encoded by the HLA-DQ locus. Furthermore, in genetic analysis, the gene encoding the class II antigen detected by HU-46 met the Hardy-Weinberg condition as a fourth allele of the DQ locus. We provisionally labeled this novel DQ specificity DQWa.     

Formation of mast cell colonies in methylcellulose by mouse peritoneal cells and differentiation of these cloned cells in both the skin and the gastric mucosa of W/Wv mice: evidence that a common precursor can give rise to both "connective tissue-type" and "mucosal" mast cells

We investigated the issue of mast cell heterogeneity by cloning mast cell colonies from peritoneal cells in methylcellulose, injecting the cloned cells into the skin and stomach of mast cell-deficient (WB X C57BL/6)F1-W/Wv (WBB6F1-W/Wv) mice, and staining the mast cells that developed in these sites with Berberine sulfate, a fluorescent dye that identifies heparin-containing mast cells. When peritoneal cells of nontreated WBB6F1-+/+ mice were plated in methylcellulose containing pokeweed mitogen-stimulated spleen cell conditioned medium, pure mast cell colonies developed. In contrast, the peritoneal cavity of genetically mast cell-deficient WBB6F1-W/Wv mice lacked the progenitor cells that made mast-cell colonies. The clonal nature of the mast cell colonies was determined by using the giant granules of C57BL/6-bgJ/bgJ mice as a marker: even when mixture of peritoneal cells of C57BL/6-bgJ/bgJ mice and C57BL/6-+/+ mice were plated, all of the resulting colonies consisted of either bgJ/bgJ-type mast cells alone or +/+-type mast cells alone. Individual mast c 11 colonies of WBB6F1-+/+ mouse origin were divided into two parts; one part was directly injected into the wall of the glandular stomach of a WBB6F1-W/Wv mouse, and another part was injected into the skin of the same W/Wv mouse. Injections of 14 of 46 such colonies resulted in development of mast cells in both the "connective tissues" (skin or stomach muscle or both) and the stomach mucosa. Mast cells in the connective tissues were stained with Berberine-sulfate, indicating that they contained heparin, whereas mast cells in the stomach mucosa were not. These results suggest that a single precursor cell can give rise to both "connective tissue-type" and "mucosal" mast cells.     

Study of the shape of the binding site of bovine opsin using 10-substituted retinal isomers

The 9-cis, 11-cis, 13-cis, and all-trans isomers of 10-fluoro-, 10-chloro-, 10-methyl-, and 10-ethylretinals have been prepared and characterized. Results of their interaction with bovine opsin are reported. The data have been analyzed in terms of the conformational properties of the retinal isomers and their steric compatibility with the binding site as defined by the two-dimensional map disclosed earlier. The need to expand the active zone and previously undetected restrictions in the third dimension are noted.     

Phospholipid modulates in vitro replication of autonomous replicating sequence from human cells

A cloned plasmid, pmyc(H-K), containing sequences derived from human c-myc gene replicated in vitro in Raji nuclear extract in a semiconservative manner. Using this system, it was found that phosphatidylinositol and cardiolipin strongly inhibited the replication of pmyc(H-K) in vitro, whereas other phospholipids, i.e., phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, and sphingomyelin, had no appreciable effect. The concentrations of phosphatidylinositol and cardiolipin producing 50% inhibition of the replication were 4.6 and 5.4 microM, respectively. Phosphatidylinositol and cardiolipin inhibited the relaxation of pmyc(H-K) supercoiled DNA, but showed little or weaker effects on DNA polymerase alpha and topoisomerase II in Raji nuclear extract. These results suggest that phosphatidylinositol and cardiolipin antagonize the replication of pmyc(H-K) in vitro, through, at least in part, the interaction with topoisomerase I.     

Mesocestoides lineatus: trypsin induced development to adult mediated by Ca2+ and protein kinase C

A mode of action of the inducible treatment with trypsin for the development of Mesocestoides lineatus tetrathyridium to adult was analyzed by administering various agents effective on Ca2+-dependent metabolic pathways in the cells: protein kinase C activators such as a synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol, and a tumor promoting phorbol, 12-O-tetra-decanoyl-phorbol-13-acetate, enhanced the trypsin induced developmental processes. On the contrary, a calmodulin inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide, cyclic adenosine 3',5'-monophosphate, and adenylate cyclase activators such as forskolin and cholera toxin, inhibited the triggering action of trypsin. Furthermore, a combined administration of Ca2+ ionophore (A23187) and the phorbol showed a similar effect with trypsin treatment, and sodium taurocholate acted as a potent enhancer like the activators of protein kinase C. These results strongly suggest that the initiation of development to adult in this cestode may be regulated synergistically by Ca2+ and protein kinase C, and that a bile acid may be involved in an activation mechanism of protein kinase C.     

Toxicity of Microcystis aeruginosa K-139 strain

Toxicity of the cells of a newly established axenic Microcystis aeruginosa K-139 strain to mice was studied. LD50 of the cells harvested in the mid-log phase was 7.3 mg/kg. The organs of acute dead mice were examined histopathologically. The blood congestion and necrosis of the parenchymal cells around the central veins in the liver were observed, but other organs seemed to be normal. The liver damage was confirmed by the tests of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) activities in the sera of the mice after the injection with the K-139 cells. Furthermore, the K-139 cells were capable of inducing interleukin 1 (IL-1) production by peritoneal macrophages in vitro.     

Stimulation of insulin secretion by transforming growth factor-beta

Effects of transforming growth factor-beta (TGF-beta) on insulin secretion were studied in rat pancreatic islets. When islets were incubated in a batch incubation system with various concentrations of TGF-beta in the presence of 2.8 mM glucose, TGF- beta increased insulin release in a concentration-dependent manner. Both TGF- beta 1 and TGF- beta 2 were equally effective. The stimulatory action of TGF- beta was greater in the presence of stimulatory concentration of glucose. In perifusion system, TGF- beta induced an immediate monotonic increase in insulin secretion. These results indicate that TGF- beta is a stimulator of insulin secretion.     

Isolation and long-term cultivation of human tonsil follicular dendritic cells

Highly purified follicular dendritic cells (FDC) were isolated from human tonsils and cultivated for up to 150 days. The cell separation method employed produced pure aggregates (FDC-clusters) composed of FDC and germinal center lymphoid cells, useful for the analysis of the relationship between these two cell types and of the behavior of FDC in culture. During the first few days of culture, lymphoid cells located between FDC extensions survived better than those which were free or partly covered by FDC. After 6 days, the lymphoid population degenerated and only the FDC survived. The unique antigenic pattern of FDC (positive for HLA-DR. DRC-1, CD14b, CD21, CD23, CD35) disappeared within a few days of culture. Recombinant interferon-gamma exerted a positive effect either on retaining HLA-DR expression or on the reexpression of these antigens by FDC. HLA-ABC antigens were traced until the 10th day and desmosomal junctions until the 14th day. Subsequently, FDC presented peculiar features, including oval and rhomboid shapes, one to ten nuclei, fine amoeboid extensions, stress fibers and a radical dense zone in their cytoplasm. FDC possessed actin, tubulin and vimentin, but neither desmin nor cytokeratin. After 40 days of culture, FDC enlarged and were covered with abundant membrane extensions. Even when kept as long as 150 days in vitro. FDC did not proliferate in any of the culture conditions employed.     

In vitro fertilization and development of frozen-thawed bovine oocytes.

Bovine oocytes were vitrified (V-oocytes) or frozen slowly (S-oocytes) at the germinal vesicle (GV) stage or after maturation in vitro (IVM) and their survival assessed morphologically and also by in vitro fertilization (IVF) and culture. The morphological survival of S-oocytes was 30.7% after freezing at the GV stage and 53.3% after IVM. The corresponding survival rates of V-oocytes were significantly lower, viz. 14.6 and 14.0%, respectively. The fertilization rate of S-oocytes frozen after IVM (51.0%) was lower than that of unfrozen controls (75.8%), but higher than after other treatments. Development continued in 16.0% of the fertilized S-oocytes, compared to 39.4% of control IVF zygotes and 1.6% developed into morulae or blastocysts (4.5% in controls). Only 0.8% of frozen-thawed GV stage oocytes and 4.6% of post-IVM V-oocytes cleaved after IVF and none formed morulae or blastocysts. Transfer of four embryos (two morulae and two blastocysts) derived from post-IVM S-oocytes into a recipient heifer resulted in pregnancy and the birth of twin calves.     

Improvement in the heterogeneous N-termini and the defective N-glycosylation of human interleukin-6 by genetic engineering.

Recombinant human interleukin-6 (IL-6), expressed in Chinese hamster ovary cells, has heterogeneous N-termini of Ala1 and Val3, as does naturally occurring IL-6. This heterogeneity is thought to be caused by difficulty in cleavage of the signal sequence. To obtain homogeneous IL-6, Pro at -1 was exchanged for Ala by site-directed mutagenesis. Alternatively, the signal sequence was replaced with that of human granulocyte-colony-stimulating factor. In both cases, the IL-6 designed to start with Ala1 was still heterogeneous, while the IL-6 designed to start with Val3 showed a homogeneous N-terminus. It is suggested that the heterogeneity of the N-terminus is caused not only by the signal sequence, but also by the succeeding sequences of the mature protein. Only a portion of recombinant human IL-6 is N-glycosylated. Asn46, being exchanged for Gln by site-directed mutagenesis, was confirmed to be partially N-glycosylated. The defective N-glycosylation was assumed to be caused by interference or tension from a disulfide bond near the N-glycosylation site. To verify this hypothesis, the Cys45 and Cys51 forming the disulfide bond were exchanged for Ser. The N-glycosylated species became predominant upon this substitution, suggesting that formation of the disulfide bond is a cause of the defective N-glycosylation.