Immunohistochemical detection of human gastrointestinal glutathione peroxidase in normal tissues and cultured cells with novel mouse monoclonal antibodies.

This is the first report to describe the successful detection of human gastrointestinal glutathione peroxidase in normal tissues by Western blotting and immunohistochemical staining techniques. Four hybridoma clones producing monoclonal antibodies (MAbs) against the human gastrointestinal glutathione peroxidase were established from mice immunized with a gastrointestinal glutathione peroxidase-derived peptide. The MAbs did not crossreact with other members of the glutathione peroxidase family, be it cellular glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, or extracellular glutathione peroxidase. Although the MAbs were found to react with a 24-kD protein in a Western blotting assay using gastric carcinoma cell extracts as antigen, they did not react with a B-lymphoblastoid cell extract. Immunohistochemical staining showed gastrointestinal glutathione peroxidase localized in the cytoplasm and in the nucleus of gastric carcinoma cells. Moreover, gastrointestinal glutathione peroxidase was detected in tissue extracts of human stomach, small intestine, large intestine, liver, and gallbladder by Western blotting, and its localization was immunohistochemically confirmed in the mucosal epithelia of the basal area of gastric pits and intestinal crypts.     

Human T-cell receptor TCRAV,TCRBV, and TCRAJ sequences newly found in T-cell clones reactive with allogeneic HLA class II antigens

Correspondence to: F. Obata.     

Isolation and characterization of mouse CD7 cDNA

The human CD7 antigen is a glycoprotein, M _r 40 000, expressed on the surface of peripheral blood T-lymphocytes and thymocytes, and is the earliest surface antigen to appear on T-cell lineage cells. In this study, putative mouse CD7 cDNA was identified based on its similarities with human CD7. Five independent clones originating from the same mRNA species were isolated (designated as mCD7) by screening a mouse thymocyte cDNA library with human CD7 cDNA, J61, under moderate stringency. The longest insert of a 995 base pair had an open reading frame of 210 amino acids. Northern blot analysis using the mouse CD7 cDNA probe demonstrated a single 1.2 kilobase mRNA ni the thymus, spleen, bone marrow, and small intestine. The protein deduced from mCD7 cDNA consisted of the leader, extracellular, transmembrane, and cytoplasmic domains of 24, 126, 21, and 39 amino acids, respectively, based on the hydrophobicity plot and the structure of human CD7. The extracellualr domain contained three potential N-glycosilation sites, while the cytoplasmic domain contained one potential protein kinase C phosphorylation site. The amino acid sequence had 45.5% similarity with human CD7, while the similarities for the individual domains ranged from 49.2% to 63.2%. The six highly conserved regions, which may possibly be involved with still unknown CD7-mediated functions, were located in the extracellular and cytoplasmic domains.The nucleotide sequence data reported in this paper have seen submitted to the GenBank, DDBJ, and EMBL nucleotide sequence database and have been assigned the accession number D10329.     

Cloning and complete nucleotide sequence of mouse immunoglobulin gamma 1 chain gene.

The 6.6 kb DNA fragment coding for the immunoglobulin γ1 chain was cloned from newborn mouse DNA using λgtWES·λB as the EK2 vector. The complete nucleotide sequence (1823 bases) of the γ1 chain gene was determined. The cloned gene contained the entire constant region gene sequence as well as the poly(A) addition site, but not the variable region gene. The results indicate that the variable and constant region genes of immunoglobulin heavy chain are separated in newborn mouse DNA. The constant region genes of other gamma chains (that is, γ2a, γ2b and γ3) are not present in the cloned DNA fragment. The sequence demonstrates that the γ1 chain gene is interrupted by three intervening sequences at the junction of the domains and the hinge region, as previously shown in the γ2b and α chain genes and in the γ1 chain gene cloned from myeloma. The results suggest that the intervening sequence was introduced into the heavy chain gene before divergence of the heavy chain classes, and also support the hypothesis that the splicing mechanism has facilitated the evolution of eucaryotic genes by linking duplicated domains or prototype peptides not directly adjacent to one another. Comparison of the nucleotide sequence of the γ1 chain gene around the boundaries of the coding and intervening sequences with those of other mouse genes revealed extensive divergence, although short prevalent sequences of AG-GTCAG at the 5′ border of the intervening sequence and TCTGCAG-GC at the 3′ border were deduced. A limited homology of nucleotide sequences was found among domains and between the hinge region and the 5′ portion of the CH2 domain. Comparison of 3′ untranslated sequences from the γ1 and γ2b chain genes and the mouse major β-globin gene shows significant homology and a palindrome sequence surrounding the poly(A) addition site.     

Steroidal control mechanism of cell proliferation in mouse uterine epithelium

The percentage of labeled cells in the uterine luminal epithelium of cycling mice showed the different zonal distributions at each stage of estrous cycle after cumulative labeling with 3H-thymidine for 36 hr. It was estimated that the proliferating fraction in the epithelium at proestrus, estrus, metestrus, and diestrus was 100%, 100%, 40% and 5%, respectively. The percentage of labeled cells in the uterine luminal epithelium of cycling mice treated with progesterone remained below 10% level for at least 20 hr after injections of progesterone. Total labeling was attained in the uterine epithelium of castrated mice by the administration of estradiol-17beta. On the other hand, the cell proliferation in the uterine epithelium of castrated mice treated with estradiol and progesterone was markedly suppressed and the percentage of labeled cells remained approximately at 35%. The remaining cell population, however, still showed the mitotic potency when mice received estradiol. It is suggested from this study that the effect of progesterone is to suppress the epithelial cell proliferation and transfer cells into resting cell fraction which is still evoked to proliferate as the effect of estradiol and that a key factor controlling epithelial proliferation in mouse uterus during the estrous cycle is proliferating fraction rather than cell cycle time.     

Correlative ontogenetic development of catecholamine- and LHRH-containing nerve endings in the median eminence of the rat

Summary The ontogenetic development of catecholamine (CA)-and LHRH-containing nerve endings in the median eminence of the rat was investigated by combining fluorescence histochemistry and immunohistochemistry in the same tissue section. LHRH-terminals appeared earlier than CA-terminals and were already detectable in the lateral part of the external layer of the central ME on the first day after birth. CA-nerve endings were first seen in a corresponding region of the ME on the seventh postnatal day. At this stage both types of terminals showed the earliest manifestation of a correlative pattern of their distribution. Subsequently the development of both types of nerve endings proceeded rapidly, and at 14 days their distribution pattern corresponded to that in adult animals. The authors conclude that at this stage the CA-neurons play a constant and significant role in the release of LHRH into the portal capillaries. The correlation between both types of nerve endings and the ontogenetic development of the capillary plexuses of the hypophysial portal system is discussed.This work was supported in part by a grant (No. 248093, 321426) from the Ministry of Education, Science and Culture, Japan     

Abrupt induction of GDP-fucose: Asialo GM1 fucosyltransferase in the small intestine after conventionalization of germ-free mice

We studied the time-course of the induction of GDP-fucose: asialo GM1 fucosyltransferase and its product, i.e. fucosyl asialo GM1, of the small intestine after introduction of microorganisms to germ-free mice (conventionalization). We found that the fucosyltransferase activity was abruptly induced and asialo GM1 was converted into fucosyl asialo GM1 within a few days after conventionalization. However, two weeks after conventionalization this enzyme activity dropped to approximately 10^?2 level of the maximum value and asialo GM1 appeared again as one of the major glycolipids. These results showed that the microbial colonization in the gut evoked a drastic change of the glycolipid pattern at the intestinal epithelial cell-surface via the induction of a fucosyltransferase.     

Single helical structure of curdlan triacetate

The molecular and crystal structure of curdlan triacetate, acetylated (1 → 3)β-D -glucan, was analyzed by means of an x-ray diffraction technique with the help of the linked-atom least-squares method. Unit cell dimensions are a = b = 11.00(1), c(fiber axis) = 22.91 (9) Å, and γ = 120°. The space group is P6₁. The unit cell contains six chemical repeating units related by 6/I-helical symmetry, which is essentially the same as the backbone conformation of one of the modifications (form I) of curdlan. During the refinement calculation, the terminal methyl in every acetyl moiety was elastically restrained to the trans conformation commonly observed in related oligosaccharide structures. The difference Fourier map, the observed and calculated densities, and the thermogravimetric measurement indicated one water molecule per glucose residue. The water oxygen is linked to two carbonyl oxygens in adjacent molecules by hydrogen bonds. The conformation of the primary acetyl moiety is a (skew, -gauche, trans). So far, no skew conformation was observed for the primary acetyl and hydroxyl moieties except in α, β-panose. In both cases, the unusual eclipsed orientation of the primary group is attributed to the hydrogen bond and this conformation is quite different from that of pachyman triacetate. © 1996 John Wiley & Sons, Inc.     

Guanidine hydrochloride-induced denaturation of Pseudomonas cepacia lipase.

The guanidine hydrochloride-induced denaturation of Pseudomonas cepacia lipase (PCL) was studied at pH 7 by monitoring the changes in the fluorescence and circular dichroism of the enzyme. The denaturation was irreversible as a whole, and the addition of Ca2+ ions decreased the velocity of the denaturation. The denaturation process was well explained consistently by a two-step mechanism, as follows: [see equation in text] where N is the native state of PCL, D(I) an intermediate denatured-state which can be refolded into the native state, and D(F) the final denatured-state that can not be renatured. Ethanol (10%) increased the denaturation velocity by decreasing the refolding step, D(I) + Ca2+ --> N x Ca2+, which would be caused by the stabilization of D(I) by ethanol.