A Positive Correlation between the Precursor Frequency of Cytotoxic Lymphocytes to Autologous Epstein-Barr Virus-Transformed B Cells and Antibody Titer Level against Epstein-Barr Virus-Associated Nuclear Antigen in Healthy Seropositive Individuals

A limiting dilution analysis was established to determine the precursor frequency (PF) of cytotoxic lymphocytes against autologous B cells transformed with the Epstein-Barr virus (EBV). This method was found to detect mainly self-restricted T-cell activity and little non-self-restricted cytotoxicity. The mean PF in 21 healthy EBV-seropositive persons was 1.4 × 10-³ (range: 0.03 × 10-³ to 8.7 × 10-³) for peripheral blood mononuclear cells, whereas 4 samples of mononuclear cells obtained from umbilical cord blood had PFs below 0.007 × 10-³. A positive correlation was observed between the PF and serum antibody titers against EBV-associated nuclear antigen among the seropositive persons.     

Effect of Substitution for Arginine Residues near Position 146 of the A Subunit of Escherichia coli Heat-Labile Enterotoxin on the Holotoxin Assembly

Escherichia coli heat-labile enterotoxin (LT) is a holotoxin which consists of one A and five B subunits. Although B subunit monomers released into periplasm can associate into pentameric structures in the absence of the A subunit, the A subunit accelerates the assembly. To express the function, A subunit constructs the proper spatial structure. However, the regions involved in the construction are unknown. To identify the regions, we substituted arginine residues near position 146 of the A subunit with glycine by oligonucleotide-directed site-specific mutagenesis and obtained the mutants expressing LT(R141G), LT(R143G), LT(R146G), LT(R143G, R146G), LT(R141G, R143G, R146G) and LT(R143G, R146G, R148G). We purified these mutant LTs by using an immobilized d -galactose column and analyzed the purified mutant LTs by SDS-PAGE to examine the amount of A subunit associated with B-subunit oligomer. The substitution of an arginine residue at any position did not induce a significant alteration in the amount of A subunit associated with B-subunit oligomer. However, the substitution of more than two arginine residues induced a significant decrease in the amount of A subunits associated with the B-subunit oligomer. Subsequently, we measured the level of the intracellular B-subunit oligomer of these mutant strains. The measurement revealed that the amount of B-subunit oligomer in cells decreased as the number of substituted arginine residues increased. These results show that all arginine residues near position 146 are important for the construction of the functional A subunit, and thus for holotoxin formation, although each individual arginine residue is not an absolute requirement.     

pag gene-like protein (ABP-25) of the Cynops embryo: regional distribution and gene expression during early embryogenesis

A maternal protein showing a unique distribution during early Cynops embryogenesis was screened by monoclonal antibody. The antigen protein, designated as ABP-25 (animal blastomere protein, molecular weight 25,000), was distributed uniformly in the uncleaved egg and concentrated into blastomeres of the animal half during cleavage. At the blastula stage, ABP-25 was definitely localized in cells of the animal half and a polarized distribution was observed within the cytoplasm. During gastrulation, immunohistochemical analysis indicated that the reactivity of the marginal zone (presumptive mesoderm) to the monoclonal antibody ABP-25 decreased after involution. At the end of gastrulation, a polarized distribution was still clearly observed in the ventral epidermis, but not in the neuroectoderm. Both Western and Northern blots indicated that the amount of antigen protein and the intensity of gene expresion were almost constant until the neurula stage. The deduced amino acid sequence of the ABP-25 cDNA showed a strong homology (84%) with that of the pag gene associated with cell proliferation.The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the accession number D37808     

Distribution of Endogenous Gibberellins in Vegetative Shoots of Rice

Levels of endogenous gibberellins in rice seedlings (Oryza sativaL., cv. Nipponbare) were compared between young and old leavesat the 4- and 5-leaf stages. The levels of GA₁, GA₁₉ and GA₅₃were higher in the youngest leaf than in older leaves at the5-leaf stage, but they did not differ significantly betweenthe leaf sheath and the leaf blade. At the 4-leaf stage, thelevel of GA₁, was highest in the third leaf sheaths which containedyoung elongating tissues. These results indicate that gibberellinsare synthesized in young vegetative tissues to promote theirelongation growth. The levels of GA₁ in the youngest leaf sheathsof two cultivars of dwarf rice, Tan-ginbozu and Waito-C, wereapproximately 10% of that in the normal rice at the 5-leaf stage.This result could explain the retardation of shoot elongationin these dwarf cultivars. (Received February 15, 1995; Accepted June 1, 1995)     

Isolation and Characterization of Two cDNAs Encoding 4-Coumarate:CoA Ligase in Lithospermum Cell Cultures

Two near full-length cDNAs (LE4CL-1, LE4CL-2), which encode4-coumarate:CoA ligase (4CL), were cloned from a library ofLithospermum erythrorhizon cell suspension cultures by the useof heterologous probe of potato 4CL. These cDNAs are 2.1 kband 2.2 kb in length, respectively. LE4CL-1 encodes 636 aminoacids, whose homologies to the 4CL protein sequences known topotato, parsley, pine and rice, were found to be 68%, 66%, 56%and 50% (identities on amino acid level), respectively, whereasthose of the predicted translation product of LE4CL-2 (594 aminoacids) to the above 4CL proteins were 49{small tilde}54%. Thesimilarity of the deduced amino acid sequences between the two4CLs from Lithospermum cell cultures was 49% in identity. Northernanalyses showed that the mRNA levels of both LE4CL-1 and LE4CL-2were much higher under illumination than in the dark, as reportedfor the 4CL genes of such plants as parsley. In comparison ofmRNA levels of LE4CL-1 and LE4CL-2, the former was demonstratedto be generally higher than the latter by means of an applicationof RT-PCR. The genomic southern blot experiments suggested thatthere are probably three copies of LE4CL-1 in the Lithospermumgenome DNA, whereas only one copy was detected for LE4CL-2. (Received May 26, 1995; Accepted August 16, 1995)     

Mval polymorphism in the proteolipid protein (PLP) gene

We report a rare polymorphism in the human proteolipid protein (PLP) gene. A synonymous mutation, 168 AG, was detected in exon 2 of the PLP gene. Mutations in this gene have been reported in some cases of Pelizaeus-Merzbacher disease.     

New monoclonal antibodies against gastric gland mucous cell-type mucins: a comparative immunohistochemical study

 The immunohistochemical reactivity of monoclonal antibodies raised against rat and pig gastric mucins (HIK1083, PGM36, and PGM37) was investigated in normal gastrointestinal tracts obtained from fish, amphibians, reptiles, birds, and mammals (including humans). These monoclonal antibodies exhibited highly selective reactivity with class III mucins, as identified by paradoxical concanavalin A stain, in the gastrointestinal tract of vertebrates. All three monoclonal antibodies reacted with the mucous neck cells and pyloric gland cells of amphibians, reptiles and mammals, the cardiac glands of reptiles and mammals, and Brunner’s glands of mammls. The deep crypt secretory cells of the rat colon and certain goblet-type cells deep in crypts in the pig colon differed from the above pattern only in that they did not show immunoreactivity with monoclonal antibody PGM36. These data suggest that the development of class III mucin is a fundamental evolutionary characteristic of vertebrate gastric mucins. These monoclonal antibodies should prove useful for the investigation of cell differentiation among gastrointestinal mucous cells and for the biochemical analysis of gastrointestinal mucins in different species. Accepted: 17 February 1998     

Effects of acute exercise on insulin binding to erythrocytes in normal men

Ten normal men were subjected to acute mild and moderate exercise tests, and the insulin binding to erythrocytes was determined before and immediately after exercise and also 10, 30 and 60 minutes after exercise in each test. The insulin binding significantly increased immediately after acute moderate exercise and did not increase immediately after acute mild exercise. In contrast, it decreased below the basal level at 30 and 60 minutes in each test. The changes in insulin receptor binding were due mainly to an alteration in insulin receptor affinity rather than a change in receptor number. These results suggest that isolated erythrocytes may be of some value for study of the effect of physical exercise on the insulin receptor.