Orosomucoid (ORM) typing by isoelectric focusing: evidence for gene duplication of ORM1 and genetic polymorphism of ORM2

Summary It has been demonstrated that the genetic polymorphism of human serum orosomucoid (ORM) is controlled by polymorphic ORM1 and monomorphic ORM2 loci. In this study a Japanese family was encountered in which several members had puzzling electrophoretic patterns consisting of four bands. The ORM patterns were due to the products of a duplicated ORM1 locus haplotype (ORM1 ^* 2·1) or the products of new variant alleles at the ORM2 locus. The ORM1 ^* 2·1 haplotype is very common in the Japanese population, occurring at an allele frequency of 0.16. The increased occurrence of ORM1 2-1 and the heterogeneity in band intensity among ORM1 2-1 phenotypes could be explained in terms of a duplicated gene ORM1 ^* 2·1. The ORM2 locus proved to be polymorphic, with six alleles in the Japanese population. Dedicated to Professor Dr. K. Nishigami on the occasion of his 60th birthday     

A re-evaluation of the cytology of cat Pacinian corpuscles

Summary The ultrastructure of cat mesenteric Pacinian corpuscles in cross and longitudinal sections has been examined. The terminal ends of lamellar cells of the inner core have been identified in longitudinal sections through the proximal portion of the inner core. These terminal bulbous expansions contain characteristic concentric membranes of rough endoplasmic reticulum and in some cases masses of oval membranous inclusions. The central axon as seen in cross section is oval in profile, having X-(short) and Y-(long) axes, and each axonal face is characterized by specializations of the axolemma. At the X-axis, the inner lamellae of the inner core tightly abut a smooth axolemma, with no intervening connective tissue matrix, in a manner reminiscent of a neuroepithelium. The axolemma of the Y-axis has numerous axonal spines (microspikes) that project into the cleft in the inner core. The extent of the axolemma having axonal spines can only be appreciated in longitudinal sections. The clefts contain a specialized connective tissue with elastic and collagen fibrils. The connective tissue compartment of fibers and matrix separating individual inner core lamellae is unique, in that it contains extremely thin collagen fibrils measuring approximately 15 nm in diameter. The diameter of collagen fibrils increases as the cleft is approached. Here the fibrils resemble typical endoneural collagen.     

Carbohydrate binding specificity of immobilized Allomyrina dichotoma lectin II

The carbohydrate binding specificity of Allomyrina dichotoma lectin II was investigated by analyzing the behavior of various complex type oligosaccharides and human milk oligosaccharides on an A. dichotoma lectin II-agarose column. Basically, the lectin interacts with the Gal beta 1----4GlcNAc group. Substitution of their terminal galactose residues by Neu5Ac alpha 2----6 will enhance their affinity to the lectin. By contraries, substitution at the C-2 or C-3 position of their terminal galactose with other sugars including sialic acid deprives their affinity to the lectin. With this characteristic, the immobilized lectin column can be used to separate complex type oligosaccharides with the Neu5Ac alpha 2----6Gal beta 1----4GlcNAc group from their isomeric oligosaccharides with the Neu5Ac alpha 2----3Gal beta 1----4GlcNAc group, where Neu5Ac is N-acetylneuraminic acid.     

Purification and characterization of a lectin from the beetle, Allomyrina dichotoma

A lectin was purified from the hemolymph of Allomyrina dichotoma larvae by affinity chromatography on acid-treated Sepharose 4B. The purified lectin showed two protein bands on polyacrylamide gel electrophoresis. These two lectin bands (allo A-I and -II) were separated by DEAE-Cellulofine column chromatography. By gel filtration on Sephadex G-100, the molecular weights of allo A-I and -II were estimated to be 65,000 and 66,500, respectively. On the other hand, by SDS-polyacrylamide gel electrophoresis after cross-linking of subunits with glutaraldehyde, they are estimated to be 38,000 and 39,000, respectively. On SDS-polyacrylamide gel electrophoresis, it was proved that both allo A-I and -II lectin consisted of two subunits, respectively. The molecular weights were 17,500 and 20,000 for allo A-I, and 19,000 and 20,000 for allo A-II. The isoelectric points of allo A-I and -II were estimated to be 6.4 and 5.9, respectively. On double immunodiffusion, allo A-I and -II gave single precipitin lines, which fused completely with each other, against the antibody to crude allo A. The hemagglutinating activity of allo A-I and -II was inhibited only by beta-linked D-galactose such as lactose and lactulose.     

Induction of proliferation of human follicular (B type) lymphoma cells by cognate interaction with CD4+ T cell clones

We examined stimuli which are required for the induction of in vitro proliferation of follicular lymphoma cells, a low grade non-Hodgkin's B cell lymphoma characterized by a specific chromosomal translocation, t(14;18)(q32;q21), and by in vivo growth of the lymphoma cells in germinal center-like follicles infiltrated with CD4+ T cells. The purified follicular lymphoma cells, which are morphologically uniform, small, and dense, did not respond to stimulation with soluble lymphokines in the absence of T cells. Vigorous in vitro proliferation of follicular lymphoma cells was induced, however, when the follicular lymphoma cells were cultured with a CD4+ T cell clone which recognized alloantigens expressed by the lymphoma cells. This response required B-T cell contact, and was inhibited by anti-class II but not by anti-class I MHC mAb, indicating that these neoplastic B cells behaved as normal B cells and responded to normal activation and differentiation signals from T cells. After the cognate B lymphoma-T cell interaction occurred in culture, addition of IL-2 or IL-4 enhanced the proliferation of the tumor cells. These results, with a monoclonal and homogeneous population of B cells, affirm the idea that cognate interaction between B cells and Th cells is required for the effective activation of resting B cells. Moreover, these results suggest that a critical host-tumor interaction occurs in vivo, and that the polyclonal CD4+ T cells that infiltrate follicular lymphomas play a role in sustaining rather than inhibiting tumor growth in vivo. If so, therapies directed not only against the neoplastic cell but also against specific T cells and their cognate interactions with tumor cells may have a rationale.     

A sensitive IL-1 thymocyte co-stimulator assay using a novel beta-D-galactoside specific lectin from the beetle, Allomyrina dichotoma

A sensitive thymocyte co-stimulator assay of IL-1 using a beta-D-galactoside specific lectin (allo A) obtained from the beetle (Allomyrina dichotoma) is reported here. Allo A stimulated [3H]thymidine uptake of mouse thymocytes in the presence of IL-1. The allo A assay was more sensitive than the PHA or PNA- thymocyte assay, especially at low doses of IL-1. Optimal conditions for the allo A assay were as follows: allo A, 2.5-5.0 micrograms/ml; whole thymocytes, 0.5-1.0 x 10(6) cells/well; incubation time, 72-96 hr. The assay is sensitive and convenient and can easily be performed in any laboratory.     

Correct splicing despite mutation of the invariant first nucleotide of a 5' splice site: a possible basis for disparate clinical phenotypes in siblings with adenosine deaminase deficiency.

Adenosine deaminase (ADA) deficiency usually causes severe combined immune deficiency in infancy. Milder phenotypes, with delayed or late onset and gradual decline in immune function, also occur and are associated with less severely impaired deoxyadenosine (dAdo) catabolism. We have characterized the mutations responsible for ADA deficiency in siblings with striking disparity in clinical phenotype. Erythrocyte dAdo nucleotide pool size, which reflects total residual ADA activity, was lower in the older, more mildly affected sib (RG) than in her younger, more severely affected sister (EG). Cultured T cells, fibroblasts, and B lymphoblasts of RG had detectable residual ADA activity, while cells of EG did not. ADA mRNA was undetectable by northern analysis in these cells of both patients. Both sibs were found to be compound heterozygotes for the following novel splicing defects: (1) a G+1-->A substitution at the 5' splice site of IVS 2 and (2) a complex 17-bp rearrangement of the 3' splice site of IVS 8, which inserted a run of seven purines into the polypyrimidine tract and altered the reading frame of exon 9. PCR-amplified ADA cDNA clones with premature translation stop codons arising from aberrant pre-mRNA splicing were identified, which were consistent with these mutations. However, some cDNA clones from T cells of both patients and from fibroblasts and Epstein-Barr virus (EBV)-transformed B cells of RG, were normally spliced at both the exon 2/3 and exon 8/9 junctions. A normal coding sequence was documented for clones from both sibs. The normal cDNA clones did not appear to arise from either contamination or PCR artifact, and mosaicism seems unlikely to have been involved. These findings suggest (1) that a low level of normal pre-mRNA splicing may occur despite mutation of the invariant first nucleotide of the 5' splice donor sequence and (2) that differences in efficiency of such splicing may account for the difference in residual ADA activity, immune dysfunction, and clinical severity in these siblings.     

Inhibition of the proliferation of cultured immortalized schwann cells by forskolin with a decreased basal level of diacylglycerol

The repetitive passages of a Schwann cell culture results in the appearance of immortalized cells. In order to investigate the direct effects of cyclic AMP (cAMP) on Schwann cell proliferation, we used the immortalized Schwann cells because the responses of a short-term Schwann cell culture to agents increasing the intracellular cAMP are more complicated and it does not seem that all of them are due to the direct effects of cAMP. By adding up to 200 M of forskolin, an adenylate cyclase activator, to the culture medium, Schwann cell proliferation was inhibited and the intracellular 1,2-diacylglycerol (DG) level was decreased in a dose-dependent manner to 44 and 53% of the control values, respectively. The protein phosphorylation activity in the cytosol from the cell treated with 100 M forskolin, assayed with myelin basic protein as the acceptor, decreased to 78% and this inhibition was then reversed by the addition of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeable DG, to the assay mixture. The cell proliferation inhibited by forskolin was also restored by the addition of OAG. These data suggest that cAMP inhibits both the activity of protein kinase C (PKC) and consequently cell proliferation through suppression of intracellular DG level, an activator of PKC. Since the inositol 1,4,5-triphosphate level and the hydrolysis of phosphatidylcholine to DG and phosphorylcholine were not affected, forskolin therefore appears to suppress the de novo synthesis of DG.     

IL-4-based helper activity of CD4 T cells is radiation sensitive

The capacity of CD4⁺ T cells to induce IgG synthesis in B cells has been known to be radioresistant for more than 20 years. However, the radiation sensitivity of helper T cells with regard to their ability to induce the synthesis of isotypes other than IgG has not been studied. We therefore irradiated KLH-primed lymph node T cells and examined their capacity to induce IgG, IgM, and IgE synthesis in hapten-primed B cells. We demonstrated that while the capacity of KLH-primed lymph node cells to induce IgG synthesis was not affected by irradiation, the capacity of such T cells to induce IgE synthesis was greatly reduced by γ-irradiation. This was consistent with our observations that IL-4 and IL-5 synthesis in such cells was greatly diminished by irradiation, whereas IL-2 synthesis was only minimally affected. A similar differential sensitivity to irradiation of the helper activity of Th1 and Th2 clones was observed with regard to their ability to induce IgE and IgG synthesis under cognate conditions. Irradiation greatly inhibited the capacity of Th2 clones to induce IgE synthesis, but only minimally affected the capacity of Th1 clones to induce IgG synthesis in primed B cells. The capacity of irradiated Th2 clones to induce IgE synthesis was restored by the addition of IL-4 and IL-5. These results taken together indicated that the sensitivity to irradiation of T helper cells with regard to the induction of IgE but not IgG synthesis was due to the sensitivity to irradiation of the production of IL-4 but not of IL-2. Thus, although some functions of CD4⁺ T cells are resistant to radiation, other functions, particularly those that depend on the production of IL-4 and IL-5, are greatly diminished by ionizing radiation.     

Genetic polymorphism of alpha-2-HS-glycoprotein: four new alleles and allele frequencies in Japanese

alpha 2-HS-glycoprotein (AHSG) phenotyping was done in 655 Japanese from the Goto Islands, western Japan, using isoelectric focusing followed by immunoblotting. Four new AHSG alleles were encountered, AHSG*G1-G4, whose genetic transmissions were established in family studies. The allele frequencies were: AHSG*1 = 0.7221; AHSG*2 = 0.2748, and AHSG*G1-G4 = 0.0008, respectively.