Maturational arrest from CD4+8+ to CD4+8- thymocytes in a mutant strain (LEC) of rat.

A mutant strain (LEC) of rats was found to have a novel defect in T cell maturation, that is, arrest of differentiation from CD4+8+ to CD4+8- but not to CD4-8+ thymocytes. FACS analyses demonstrated a deficiency in the CD4+8- T cell subset in the thymus and a marked decrease in CD4+ T cells in peripheral lymphoid organs. Expression of the T cell receptor (TCR)/CD3 complex in CD4+8+ and CD4-8+ thymocytes of LEC rats was normal. Expression of class II major histocompatibility complex (MHC) in the thymus of LEC rats was also the same as that of normal rats. These results indicate that maturational arrest occurs only in the transition pathway from CD4+8+ to CD4+8- thymocytes, and that this mutation can not be attributed to the default of expression of either TCR/CD3, CD4, or class II MHC antigen. Consequently, dysfunction of helper T cells was observed in LEC rats, while killer T cells and B cells functioned normally. Although the complete identification of the origin of this mutation requires further studies, it is hoped that such investigations will throw light on the mechanism of positive selection.

Elevation of metallothionein gene expression associated with hepatic copper accumulation in Long-Evans Cinnamon mutant rat.

The mechanism of the metallothionein (MT) gene expression was investigated in a mutant rat, LEC, which exhibits an abnormal accumulation of copper in hepatocytes. The levels of MT mRNA were extremely high and correlated with the hepatic copper concentrations in LEC rat liver. Gel retardation assays in nuclear extracts from LEC rat liver showed an increase in the copper-dependent binding proteins, which bind to the metal responsive element (MRE) of the MT gene. These results suggest that the high intracellular copper accumulation results in the elevation of the MT gene expression through increasing a putative trans-activating factor in LEC rat.

A missense mutation G2320R in the thyroglobulin gene causes non-goitrous congenital primary hypothyroidism in the WIC-rdw rat.

A convincing line of evidence is being developed that the congenital nongoitrous hypothyroidism and dwarfism observed in the WIC-rdw rat may indeed be caused by a primary defect in thyroid hormonogenesis. In support of this hypothesis, several recent reports have shown the presence of elevated molecular chaperone levels in the WIC-rdw thyrocytes, the endoplasmic reticulum of which was markedly dilated, suggesting a defect in intracellular protein transport. Here the studies were undertaken to identify the precise molecular defect in the WIC-rdw rat. First, the genetic linkage analysis revealed that the rdw locus was on rat chromosome 7 and was identical to the thyroglobulin (Tg) gene locus. Moreover, the Tg protein level was reduced in the WIC-rdw thyroid despite a similar level of the Tg gene transcripts that were indistinguishable in their size from the normal. Next, the complete sequencing of the rdw and the normal rat Tg cDNAs revealed a single nucleotide change, G6958C, resulting in a G2320R missense mutation in a highly conserved region of the Tg molecule. Finally, transient expression of the intact Tg cDNA containing the rdw mutation in the COS-7 cells showed no detectable Tg in the secreted media, indicating a severe defect in the export of the mutant Tg. Together, our observations suggest that a missense mutation, G2320R, in the Tg gene is responsible for the rdw mutation in the WIC-rdw rat.

Endothelin-induced interleukin-6 production by rat aortic endothelial cells.

Endothelin (ET) produced by endothelial cells has recently been found to be a potent vasoconstricting hormone. In this paper, ET isopeptides, both ET-1 and ET-3, were shown to be potent stimulators of interleukin (IL)-6 production by a rat aortic endothelial cell clone, WAE-1. Semi-quantitative polymerase chain reaction analysis indicated that augmentation of IL-6 production is due to an increase in IL-6 messenger RNA level. Ligand binding assay indicated that most of the [125I]ET-1 binding sites correspond to ET receptor type A (ETAR). However, ET receptor type B (ETBR) was shown to be also present on this cell line by reverse transcription polymerase chain reaction using ETBR-specific primers and by ligand binding assay using [125I]ET-3, although the protein receptor level is much lower than that of ETAR. ET-1, but not ET-3, induced inositol 1,4,5-triphosphate production and an increase in intracellular Ca2+ concentration with similar dose response. These data suggest that ET-1 stimulates IL-6 production through ETAR-phospholipase C activation axis, whereas ET-3 stimulates IL-6 production through different signaling pathway. The results shown in this paper raise the possibility that ET plays a role in inducing inflammation in endothelium.

Chronic estrogen treatment promotes a functional uncoupling of the D2 dopamine receptor in rat anterior pituitary gland.

The effect of chronic estrogen treatment on the anterior pituitary D2 dopamine receptor was studied by treating rats with diethylstilbestrol (DES) over a 6-week period. DES treatment resulted in an increase in anterior pituitary weight and PRL content and serum PRL levels compared to those in sham-treated controls. The status of the anterior pituitary D2 dopamine receptor was evaluated using both radioligand binding and adenylate cyclase assays. [125I]N-(p-aminophenethyl)spiroperidol [( 125I]NAPS), a derivative of the D2-selective antagonist spiperone, was used to quantitate D2 receptors. Saturation analysis of [125I]NAPS binding indicated that DES treatment had no effect on the affinity or maximum binding capacity of the radioligand for the D2 receptor. Competition analysis with unlabeled D2 antagonists for [125I]NAPS binding also indicated that DES treatment did not affect antagonist interactions with the receptor. In contrast, the interactions of agonists with the D2 receptors from DES-treated rats were modified, as assessed through [125I]NAPS competition analysis. Using control tissue, agonist competition curves revealed both high and low affinity agonist binding states of the receptor. In the presence of guanine nucleotides, the high affinity agonist binding state is abolished, reflecting coupling of the receptor with a guanine nucleotide regulatory (G) protein. In DES-treated tissue, agonist competition curves indicated the presence of only low affinity agonist binding, with minimal effects of guanine nucleotides, suggesting uncoupling of receptor-G-protein interactions. The functionality of the D2 receptor was further assessed by examining dopaminergic inhibition of vasoactive intestinal peptide-stimulated adenylate cyclase activity. Although DES treatment resulted in a reduction of vasoactive intestinal peptide-stimulated enzyme activity itself, the ability of dopaminergic agonists to inhibit this activity was reduced by about 50%. These results suggest that estrogen is capable of attenuating the functional coupling of the D2 receptor with its biochemical effector system in the anterior pituitary gland.

Expression of receptors for atrial natriuretic peptide on the murine bone marrow-derived stromal cells.

Atrial natriuretic peptide (ANP) receptors were identified on both murine bone marrow-derived stromal cell lines A-3 and ALC and primary cultured cells using [125I]ANP binding assays and Northern blot analyses. The binding of [125I] ANP to the stromal cells was rapid, saturable, and of high affinity. The dissociation constants between ANP and its receptors on these cells showed no difference among cell types, while maximal binding capacity values were different among cell types. Competitive inhibition of [125I]ANP binding with C-atrial natriuretic factor, specific for ANP clearance receptor (ANPR-C), revealed that most of [125I]ANP-binding sites corresponded to ANPR-C. Northern blotting data corroborated that bone marrow-derived stromal cells expressed ANPR-C. However, in ALC cells, ANP biological receptors (either ANPR-A or ANPR-B), the mol wt of which is approximately 130K, were detected, and cGMP was accumulated after stimulation with ANP. On the other hand, in another stromal cell clone, A-3 cells, the expression of biological receptor was not detected in the affinity cross-linking and competitive inhibition experiments using [125I]ANP. However, A-3 cells accumulated cGMP by responding to ANPR-B-specific ligand, C-type natriuretic peptide. These results suggest that ALC cells equally express ANPR-A and ANPR-B, while A-3 cells express ANPR-B dominantly. Although the physiological roles of these receptors in the bone marrow is still not resolved, ANP is expected to play a role in the regulation of stromal cell functions in bone marrow.

Detection and identification of proteins related to the hereditary dwarfism of the rdw rat.

Proteins having relations to hereditary dwarfism of the rdw rat (gene symbol: rdw) were searched for in various tissues of the rat with an improved two-dimensional gel electrophoresis technique followed by immunoblotting and microsequencing. Tissues inspected were cerebral cortex, cerebellum, brain trunk, hypothalamus, pituitary, thyroid gland, liver, testis, spleen, and thymus. Only pituitary and thyroid glands among those tissues showed abnormalities in protein contents. GH and PRL contents in the rdw pituitary were much less than in the normal one, which in the former were 1/15 and less than 1/30 times as much as in the latter, respectively, but the abnormalities in the rdw thyroid were far more serious than in the pituitary. At least 18 protein levels in the rdw thyroid were above, and 17 were below the normal. Those identified among the increased proteins were endoplasmin (GRP94), immunoglobulin heavy chain binding protein (BiP/GRP78), and heat shock protein 70 (hsp70), the contents of which respectively were 40 times, 10 times and more than 50 times as much in the rdw thyroid as in the normal tissue. Because BiP and endoplasmin are known to be ER resident proteins, and because all three belong to a chaperone protein family, accumulation of these proteins in the rdw thyroid suggests that protein folding and secreting disorders underlie the hypothyroidism of the rdw rat.

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) stimulate interleukin-6 production through the third subtype of PACAP/VIP receptor in rat bone marrow-derived stromal cells.

Regulation of Interleukin-6 (IL-6) production in bone marrow (BM)-derived stromal cells by neuropeptides, pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP), was examined. Both forms of PACAP, PACAP-27 and PACAP-38, as well as VIP significantly increased IL-6 production by rat BM-derived stromal cells at physiological concentrations ranging from 10(-10)-10(-8) M. The three related peptides (PACAP-27, -38, and VIP) stimulated the production of both cAMP and inositol 1,4,5-trisphosphate (IP3) in rat BM-derived stromal cells with similar 50% effective concentrations. The stimulatory potency of the three related peptides for the production of IL-6, cAMP, and IP3 was almost consistent, suggesting that the dual signaling transduction pathways may be involved in PACAP/VIP-induced IL-6 production in rat BM-derived stromal cells. The messenger RNA (mRNA) for the third subtype of PACAP receptor (PVR3) was found to be abundantly expressed in both BM-derived stromal cells and the BM tissue, whereas little of the mRNA for type 1 (PVR1) nor type 2 (PVR2) was detected. Furthermore, the mRNAs for PACAP and VIP were detected in the BM tissue, suggesting that both PACAP/VIP and PVR3 are synthesized in vivo in the BM. The results shown in this paper suggest that PACAP/VIP and their receptor play an important role in the IL-6 production and perhaps in the hematopoiesis in the BM.

Intestinal intra-epithelial lymphocytes in LEC mutant rats.

LEC rat is a novel strain showing a maturational arrest from CD4+8+ to CD4+8- cells but not to CD4-8+ cells in the thymus. In this study, we examined if this mutation affects the differentiation of intestinal intra-epithelial lymphocytes (IEL) in LEC rats. In normal rat IEL, all 4 subsets with respect to the CD4/CD8 expression were observed. The CD4-8+ population was dominant and a unique population, CD4+8+, was observed as already shown in previous papers. Both CD4+8- and CD4+8+ cells were CD3+, TCR-alpha/beta +, CD45RC-, and CD5+, whereas CD4-8+ cells consisted of a heterogeneous population, being CD3+, TCR-alpha/beta +/-, CD45RC+/-, and CD5-. In LEC rat IEL, CD4+8- and CD4+8+ cells existed normally and distribution of CD4/CD8 subsets was not different from that of normal rat IEL. Furthermore, the expression pattern of CD3, TCR-alpha/beta, CD45RC and CD5 was not different from that of normal rat IEL in each subset. These results suggest that maturational arrest of CD4+8- thymocytes does not affect IEL maturation, especially maturation of CD4+8- IEL, suggesting that the IEL maturation mechanism for CD4+8- cells is independent of that of thymocytes.

Maturational arrest from CD4+8+ to CD4+8- thymocytes is caused by the bone marrow-derived cells in LEC mutant rats.

LEC rats exhibit a congenital maturational arrest from CD4+8+ to CD4+8- but not to CD4-8+ cells in the thymus. To elucidate a cause of this mutation, bone marrow (BM) chimera rats were made between LEC and normal (WKAH) rats. In (WKAH-->LEC) BM chimera rats, donor-derived T cells matured normally, suggesting that LEC rat thymic stroma has a normal ability in supporting thymocyte differentiation. On the other hand in (LEC-->WKAH) BM chimera rats, LEC rat BM-derived T cells showed the arrest of maturation from CD4+8+ to CD4+8- cells in spite of having normal functions of WKAH rat-derived thymic stroma. In these chimeric rats, even though the maturational arrest from CD4+8+ to CD4+8- cells occurred in the thymus, CD4+ cells were found in peripheral lymph nodes (LNs), suggesting that these CD4+ cells differentiated extrathymically. These results suggest that the maturational arrest from CD4+8+ to CD+8- thymocytes is caused by BM-derived cells but not by thymic stroma.

The diversity of T-cell receptor repertoire of peripheral CD4+ lymphocytes in LEC mutant rats.

A novel mutant strain of rats, LEC, exhibits an arrest of T-cell maturation from CD4+CD8+ to CD4+CD8- but not the CD4-CD8+ cells in the thymus. Nevertheless, CD4+T cells arise in lymph nodes of LEC rats, implying extrathymic T-cell maturation. We analyzed the variable (V) and junctional region diversity of T-cell receptor (TCR) beta- chains in this population, since several reports have provided evidence that extrathymically matured T cells exhibit a biased expression of the TCR repertoire. CD4+ T cells in LEC rat lymph nodes exhibited a polymorphic V beta gene expression pattern. However, biased V beta gene expression was not identified in peripheral CD4+ T cells of LEC rats when compared with V beta gene expression in their thymocytes. Furthermore, junctional regions of TCR beta chains exhibited a vast diversity created by non-germline- encoded nucleotide (N nucleotide) additions. These results indicate that peripheral CD4+ T cells in LEC rats possess a vast diversity in the TCR repertoire as do thymocytes of the same rat, suggesting that they derive from the thymus.

The structural relation between the antigenic determinants to monoclonal antibodies and binding sites to rat brain synaptosomes and GT1b ganglioside in Clostridium botulinum type C neurotoxin.

The inhibition of the binding of 125I-labeled Clostridium botulinum type C neurotoxin to synaptosomes by unlabeled toxin indicated that there were two kinds of receptors on the synaptosomal membrane. The dissociation constants (Kd) were calculated as 79 pM and 35 nM from the concentration of unlabeled toxin that induced half-displacement of bound 125I-toxin. These values agree satisfactorily with the values obtained from direct binding experiments (Agui, T, Syuto, B., Oguma, K., Iida, H., & Kubo, S. (1983) J. Biochem. 94, 521-527). The inhibition of the binding of 125I-toxin to synaptosomes and N-acetylneuraminyl(alpha 2-3)galactosyl(beta 1-3)N-acetylgalactosaminyl(beta 1-4) [N-acetylneuraminyl(alpha 2-8) N-acetylneuraminyl(alpha 2-3)]galactosyl(beta 1-4)glucosyl(beta 1-1)ceramide (GT1b) by unlabeled heavy chain indicated that heavy chain facilitates the binding of toxin to synaptosomes and GT1b. The synaptosomal and heavy chain complex Kd values were estimated as 12 nM and 24 microM. Monoclonal antibodies C-9 and CA-12 recognized the binding sites to GT1b and synaptosomes, respectively. Antigenic determinants against the two antibodies are presumably partially overlapping, and the overlapping area seems to be essential to the reaction between toxin and C-9 antibody.

Agonist-induced desensitization of the D-2 dopamine receptor in the intermediate lobe of the rat pituitary gland.

The mechanism of agonist-induced desensitization of the D-2 dopamine receptor in the intermediate lobe (IL) of the rat pituitary gland was investigated. Exposure of neurointermediate lobe to 60 microM (-)apomorphine (APO) for 60 min altered the binding of [125I]-N-(p-aminophenethyl)spiperone (NAPS), a D-2 receptor-specific ligand. The capacity of the tissue to bind the ligand (Bmax) was not significantly altered by the exposure to (-)APO but the affinity for [125I]NAPS was decreased 3.6-fold in (-)APO-exposed tissue. The molar potency of YM-09151-2, a D-2 receptor-specific antagonist, showed a minimal difference between in control and (-)-APO-exposed tissue. However, the molar potency of (-)APO towards the D-2 receptor was diminished. The loss of [125I]NAPS binding in (-)APO-exposed tissue was reversed by the addition of guanyl nucleotide. These data suggest that exposure to agonist causes a persistent occupancy of the high affinity state of the receptor. Exposure to (-)APO had no effect on either basal or forskolin-activated adenylate cyclase activity of the intermediate lobe. However, the inhibitory effect of (-)APO upon adenylate cyclase activity of IL homogenates was diminished when the tissue was exposed to (-)APO before homogenization. Furthermore, the ability of GTP but not 5'-guanylyl imidodiphosphate [Gpp(NH)p] to inhibit enzyme activity diminished in the (-)APO-exposed tissue. These data suggest that an agonist-induced desensitization of D-2 receptor in rat IL is thought to occur by uncoupling the receptor from the inhibitory guanyl nucleotide binding protein (Gi) or potentiating the hydrolysis of GTP by Gi.

Binding of Clostridium botulinum type C neurotoxin to rat brain synaptosomes.

The binding of Clostridium botulinum type C neurotoxin to rat brain synaptosomes was determined by the use of 125I-neurotoxin. The binding was independent of the incubation temperature (0 degrees C and 37 degrees C) and was equilibrated in 10 min. The dose dependent of 125I-toxin binding to synaptosomes at 0 degrees C showed that there were two kinds of toxin receptors on the synaptosomal membrane; the association constants and maximum binding values were 1.05 x 10(10 M-1, 5.25 x 10(-13) mol/mg of synaptosomal protein and 5.00 x 10(6) M-1, 5.00 x 10(-12) mol/mg of synaptosomal protein, respectively. When the incubation of toxin with synaptosomes was continued at 37 degrees C after 125I-toxin had been pre-incubated with synaptosomes at 0 degrees C for 10 min, the displacement of labeled toxin by the addition of excess amounts of unlabeled toxin decreased slightly with increasing incubation time, and finally 0.4% of the bound 125I-toxin was not displaced from synaptosomes. The binding of 125I-toxin to synaptosomes was inhibited by anti-heavy chain IgG and a monoclonal antibody which neutralized toxin and recognized heavy chain. These results suggest that the binding sites of toxin to synaptosomes are localized on heavy chain and a small amount of the bound toxin is incorporated into the synaptosomal membrane or synaptosomes.

Opposite actions of transforming growth factor-beta 1 on the gene expression of atrial natriuretic peptide biological and clearance receptors in a murine thymic stromal cell line.

The regulation of the gene expression of the atrial natriuretic peptide receptor (ANPR) subtypes, ANPR-A, ANPR-B, and ANPR-C, was investigated in a murine thymic stromal cell line, MRL 104.8a. When MRL 104.8a cells were cultured with transforming growth factor (TGF)-beta1, [125I]ANP binding sites increased with increasing dose of TGF-beta1. These binding sites were identified as ANPR-C by a displacement experiment with ANPR-C-specific ligand, C-ANF, and by the affinity cross-linking of the [125I]ANP binding sites with a chemical cross-linker to determine the molecular weight of the ANPR. This augmentation of the ANPR-C expression was elucidated to occur at the transcriptional level by Northern blot experiment, comparison of the relative amounts of mRNA by reverse transcription (RT)-PCR, and in vitro nuclear transcription assay. Conversely, the expression of the ANP biological receptors, ANPR-A and ANPR-B, was shown to be down-regulated by TGF-beta1. These data suggest that TGF-beta1 regulates the gene expression of ANPRs in the thymic stromal cells and that ANP and TGF-beta1 might affect the thymic stromal cell functions.

Direct demonstration of guanine nucleotide sensitive receptors for vasoactive intestinal peptide in the anterior lobe of the rat pituitary gland.

The vasoactive intestinal peptide (VIP) receptors were identified on the membranes from the rat anterior pituitary gland with [125I]VIP. The dissociation constant (Kd) and the maximal binding capacity (Bmax) values were estimated from the competitive inhibition data. The Kd and Bmax values were 1.05 +/- 0.75 nM and 103 +/- 11 fmol/mg protein, respectively. The order of molar potency of related peptides to inhibit [125I]VIP binding was VIP greater than peptide histidine isoleucine (PHI) greater than secretin greater than glucagon. Glucagon was not effective to inhibit the binding. [125I]VIP binding was effectively inhibited by the addition of guanine nucleotides. The order of molar potency to inhibit the binding was Gpp(NH)p greater than GTP greater than GDP greater than GMP greater than ATP. These results directly suggest the coupling of VIP receptors with guanine nucleotide binding proteins in the anterior pituitary gland.

Identification of D1-dopamine receptor in chicken embryo retina with [125I]SCH 23982.

The D1-dopamine receptor in chicken embryo retina was identified with the D1-dopamine receptor specific ligand, [125I]SCH 23982. Binding of [125I]SCH 23982 to both pre-hatched and post-hatched chicken retina was rapid, saturable and of high affinity. The dissociation constant and maximal binding capacity were 795 +/- 25 pM (mean +/- S.E.M., n = 3) and 32.2 +/- 3.8 fmol/mg protein (mean +/- S.E.M., n = 3), respectively for 13-day-old chicken embryo retina, and 785 +/- 58 pM (mean +/- S.E.M., n = 3) and 96.9 +/- 4.1 fmol/mg protein (mean +/- S.E.M., n = 3), respectively for 1-day-old post-hatched chicken retina. The binding properties of the D1-dopamine receptor in chicken retina were similar to those in rat striatum. The maximal binding capacity of the D1-dopamine receptor for [125I]SCH 23982 was increased concomitant with embryonic development, but without any changes in either affinity or pharmacological properties. Dopamine-stimulated adenylate cyclase activity in the retinal homogenates increased concomitant with embryonic development, diminished in the presence of 1 microM SCH 23390 (a D1-dopaminergic antagonist) but remained unaffected by 1 microM YM-09151-2 (a D2-dopaminergic antagonist).

Multiple types of receptors for atrial natriuretic peptide.

The binding of atrial natriuretic peptide (ANP) to olfactory bulb, pituitary anterior lobe and thymus gland membranes was examined. [125I]ANP (rat, 99-126) bound specifically to the three types of membranes. However, the affinity for ANP receptor in olfactory bulb was much higher than those in either pituitary or thymus gland. Competitive inhibition of cold ANP (rat, 99-126) with [125I]ANP binding sites on olfactory bulb membranes gave a value of 796 +/- 80 pM (mean +/- S.E.M., n = 4) as a dissociation constant (Kd) of cold ANP (rat, 99-126), while on pituitary and thymus membranes, the competitive curve gave a value of 9.3 +/- 0.4 nM (mean +/- S.E.M., n = 3) and 25.5 +/- 2.2 nM (mean +/- S.E.M., n = 6) as a Kd of cold ANP (rat, 99-126), respectively. Furthermore, a truncated ANP fragment (rat, 111-126) did not inhibit the [125I]ANP binding in olfactory bulb, while this peptide fragment inhibited the [125I]ANP binding in either pituitary or thymus gland with affinities only 2- to 4-fold less potent than ANP (rat, 99-126). These data indicate the possibility of the existence of multiple types of ANP receptors. We propose alpha-receptor in olfactory bulb and beta-receptor in either pituitary anterior lobe or thymus gland.

A description method of handprinted chinese characters.

A description method of handprinted Chinese characters is presented. In the method, a Chinese character is composed of some partial patterns which are constructed using the concatenate relation, cross relation, and near relation. The relations of relative location among partial patterns are used for categorization of the partial pattems. A Chinese character is expressed from the results of categorization.

A coding method of chinese characters.

In this paper we propose a coding method of handprinted Chinese characters in which, defining a grammar, the process of block categorization is made to correspond to a process of sentential generation. We obtain two strings of production rules and block codes being equivalent to the descriptions of the structure and internal components of a character pattern, and a Chinese character is encoded effectively by the use of these two strings. It is shown that this coding method is available for the classification and discrimination of handprinted Chinese characters.