CD9-positive cells in the intermediate lobe migrate into the anterior lobe to supply endocrine cells.

The adenohypophysis is composed of the anterior and intermediate lobes (AL and IL), and secretes important hormones for growth, sexual development, metabolism, and reproduction. In the marginal cell layer (MCL) facing Rathke's cleft between the IL and AL, cluster of differentiation (CD) 9-, CD81-, S100ß-, and SOX2-quadruple positive (CD9/CD81/S100ß/SOX2-positive) cells in the adult IL are settled as tissue-resident stem/progenitor cells supplying hormone-producing cells to the AL. However, it is unclear how CD9/CD81/S100ß/SOX2-positive cells in the IL-side MCL migrate into the AL across Rathke's cleft. In the present study, we performed chimeric pituitary tissue culture using S100ß/GFP-transgenic rats and Wistar rats, and traced the footprint of S100ß/GFP-expressing cells. We detected IL-side S100ß/GFP-expressing cells in the AL tissue, demonstrating that these cells migrate from the IL to the AL. However, the cells failed to migrate in the opposite direction. Consistently, scanning electron microscopic analysis revealed well-developed cytoplasmic protrusions in the IL-side MCL, but not in the AL-side MCL, suggesting that IL-side CD9/CD81/S100ß/SOX2-positive cells had higher migratory activity. We also searched for a specific marker for IL-side CD9/CD81/S100ß/SOX2-positive cells and identified tetraspanin 1 (TSPAN1) from microarray analysis. Downregulation of Tspan1 by specific siRNA impaired cell migration and significantly reduced expression of snail family transcriptional repressor 2 (Slug), a marker of epithelial-mesenchymal transition (EMT). Therefore, CD9/CD81/S100ß/SOX2-positive cells in the IL-side MCL can be stem/progenitor cells that provide stem/progenitor cells to the AL-side MCL via SLUG-mediated EMT and cell migration.

The WRPW motif of the hairy-related basic helix-loop-helix repressor proteins acts as a 4-amino-acid transcription repression and protein-protein interaction domain.

Hairy-related proteins include the Drosophila Hairy and Enhancer of Split proteins and mammalian Hes proteins. These proteins are basic helix-loop-helix (bHLH) transcriptional repressors that control cell fate decisions such as neurogenesis or myogenesis in both Drosophila melanogaster and mammals. Hairy-related proteins are site-specific DNA-binding proteins defined by the presence of both a repressor-specific bHLH DNA binding domain and a carboxyl-terminal WRPW (Trp-Arg-Pro-Trp) motif. These proteins act as repressors by binding to DNA sites in target gene promoters and not by interfering with activator proteins, indicating that these proteins are active repressors which should therefore have specific repression domains. Here we show the WRPW motif to be a functional transcriptional repression domain sufficient to confer active repression to Hairy-related proteins or a heterologous DNA-binding protein, Ga14. This motif was previously shown to be necessary for interactions with Groucho, a genetically defined corepressor for Drosophila Hairy-related proteins. Here we show that the WRPW motif is sufficient to recruit Groucho or the TLE mammalian homologs to target gene promoters. We also show that Groucho and TLE proteins actively repress transcription when directly bound to a target gene promoter and identify a novel, highly conserved transcriptional repression domain in these proteins. These results directly demonstrate that Groucho family proteins are active transcriptional corepressors for Hairy-related proteins and are recruited by the 4-amino acid protein-protein interaction domain, WRPW.

Identification of an alternative form of the Drosophila Ca2+/calmodulin-dependent protein kinase II that is maternally derived.

Four forms of the Drosophila Ca2+/calmodulin-dependent protein kinase II are generated from a single gene by alternative splicing (Ohsako et al. (1993) J. Biol. Chem. 268, 2052-2062). We identified a fifth form of the cDNA encoding the enzyme expressed in the ovary, unfertilized egg and early embryos by reverse transcription-polymerase chain reaction, which suggests that it is maternally derived. The fifth form was also generated from the gene by alternative splicing and was identical to the cDNA encoding the 530-amino-acid polypeptide, the longest of the four forms previously identified, except that it lacked exon 11. Three splicing derivatives which lost one amino acid from the 509- and 530-amino-acid polypeptides were also found in 4 to 10 h embryos.

Apoptosis in the effector phase of autoimmune diabetes, multiple sclerosis and thyroiditis.

The immune system is unusual in two respects. It produces billions of new cells daily that traffic throughout the body and cells within the system proliferate rapidly following exposure to an infectious agent. Both of these attributes require that cell production be regulated by cell death. Human diseases characterized by accelerated cell death leading to immunodeficiency disorders or by reduced cell death leading to systemic autoimmune diseases have been identified. In certain autoimmune diseases, the immune system directs its powerful cytotoxic effector mechanisms against specialized cells such as oligodendrocytes in multiple sclerosis, the beta cells of the pancreas in diabetes mellitus and thyrocytes in Hashimoto's thyroiditis. In this review, we examine the cytotoxic effector pathways implicated in cell death in organ specific autoimmune disorders.

Phosphatidic acid mimics the muscarinic action of acetylcholine in cultured bovine chromaffin cells.

In cultured bovine chromaffin cells, acetylcholine as well as muscarine stimulated the 32Pi incorporation into phosphatidic acid, induced the efflux of 45Ca2+ from prelabelled cells, and, in parallel, elevated intracellular cyclic GMP content. Phosphatidic acid added to the medium also stimulated the efflux of 45Ca2+ and the synthesis of cyclic GMP in the cells in the same fashion as muscarinic agents, whereas it did not induce the secretion of catecholamines indicating that the effect of phosphatidic acid is specific to muscarinic action. The result supports the hypothesis that phosphatidic acid produced during phosphatidylinositol turnover is linked to the regulation mechanism of Ca2+ mobilization and cyclic GMP synthesis by muscarinic stimulation.

Subcellular distribution of alpha and beta subunit proteins of Ca2+/calmodulin-dependent protein kinase II expressed in Chinese hamster ovary cells.

When two cDNAs respectively encoding the entire coding regions of alpha and beta subunits of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) were introduced into Chinese hamster ovary cells, the expressed alpha and beta subunits were differently associated with subcellular structure. Although alpha subunit was loosely associated with subcellular structure, about 80% of CaM kinase II activity of alpha subunit was found in soluble fraction. More than 50% of the beta subunit bound to the membrane, and the remainder was soluble but was loosely associated with subcellular structure. The relative rate of phosphorylation for substrate proteins of the beta subunit bound to membrane was significantly different from that of the soluble form.

Comparative uterine gene expression analysis after dioxin and estradiol administration.

The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) adversely affects many organisms. TCDD exposure is known to be associated with abnormal development, hepatotoxicity and endocrine effects. It has also been reported to have antiestrogenic activity in addition to estrogenic activity. In order to clarify the effects of TCDD in the uterus, we evaluated the patterns of gene expression after TCDD and estradiol administration. Of the 10 000 arrayed genes, only a few were affected by both estradiol and TCDD. Although the subset of genes that responded to estrogen was also activated by TCDD, the response to TCDD was more limited than that observed in response to estradiol. Therefore, according to our analysis of gene expression patterns, TCDD had partial and weak estrogenic activity in the uterus.

Immunocytochemical observation of the 90 KD heat shock protein (HSP90): high expression in primordial and pre-meiotic germ cells of male and female rat gonads.

We used immunocytochemistry to detect the 90 KD major heat shock protein (HSP90), a potential regulator of gene expression, during male and female rat gonad development. In the Day 13.0 post-coital (dpc) fetal gonad, strong immunoreactivity to anti-HSP90 antibody was shown in the cytoplasm of primordial germ cells (PGCs). Other somatic cells in the gonad showed only faint reactivity. During testicular development, strong immunostaining was observed in the cytoplasm of embryonic germ cells and in spermatogonia and spermatocytes of the pre-pubertal testis. In adult testis reactivity of spermatogonia and pachytene spermatocytes was strong but reactivity of post-meiotic spermatogenic cells, i.e., secondary spermatocytes and spermatids, was extremely reduced. During ovarian development, immunostaining was also observed in the oogonia and the oocytes of pre-pubertal ovary. However, the staining of oocytes was reduced with the development of primordial follicles during the first week after birth. This study revealed that HSP90 is highly expressed in PGCs and continues to be expressed in both male and female pre-meiotic germ cells. The HSP90 accumulation may be essential for both male and female mammalian pre-meiotic germ cells.

Ca2+/calmodulin-dependent protein kinase II in the rat cerebellum: an immunohistochemical study with monoclonal antibodies specific to either alpha or beta subunit.

Monoclonal antibodies specific to either alpha or beta subunit of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) of the rat brain were produced and the distribution of each subunit in the rat cerebellum was examined immunohistochemically. Each antibody detected solely the corresponding subunit in immunoblot analysis of crude homogenates of the rat forebrain and cerebellum, and purified CaM kinase II from the rat forebrain. Immunoreactivity for alpha subunit was present selectively in Purkinje cells: perikarya, dendrites with their spines, axons and their terminal-like structures in the cerebellar cortex, cerebellar nuclei and lateral vestibular nucleus. Many of these alpha subunit-immunoreactive axons from the cerebellum were traced only through the inferior cerebellar peduncle. beta Subunit was detected in perikarya and dendrites of a limited number of Purkinje cells, many granule cells and neurons in the cerebellar nuclei. Thus, different distributions of alpha and beta subunits of CaM kinase II in the cerebellum were demonstrated.

Identification and characterization of a Drosophila homologue of the yeast UBC9 and hus5 genes.

The yeast UBC9 and hus5 gene products have been identified as putative E2 members of the ubiquitin-conjugating enzyme (UBC) family and have been shown to play an essential role in cell cycle progression. We have identified a Drosophila Ubc9/Hus5 homologue (termed dUBC9) in an attempt to identify proteins that interact with the amino-terminal transcriptional repression domain of the Groucho corepressor by use of the yeast two-hybrid system. The predicted dUBC9 protein consists of 159 amino acids and shows 85, 68, and 54% amino acid sequence identities with human UBC9 homologue, Schizosaccharomyces pombe Hus5, and Saccharomyces cerevisiae Ubc9 proteins, respectively. Expression of dUBC9 cDNA complements a temperature-sensitive ubc9-1 mutation of S. cerevisiae to fully restore normal growth, indicating that the dUBC9 protein can act as a substitute for the yeast Ubc9 protein. The dUBC9 transcripts were about 1.2 kb and were detected at all stages of Drosophila development and in ovaries and Schneider cells. However, an increased level was observed in early embryos and ovaries. The dUBC9 gene is present as a single copy in the genome and localized in segment 21C-D on the left arm of the second chromosome.

Role of threonine-286 as autophosphorylation site for appearance of Ca2(+)-independent activity of calmodulin-dependent protein kinase II alpha subunit.

To confirm directly the role of Thr-286 as the autophosphorylation site responsible for the appearance of Ca2(+)-independent activity of Ca2+/calmodulin-dependent protein kinase II alpha subunit, we constructed two mutated cDNAs of Thr-286 to Pro or Ala using site-directed mutagenesis and introduced into Chinese hamster ovary cells. The mutant enzymes expressed in stable cell lines were partially purified and their catalytic properties were confirmed to be similar to those of wild-type kinase, except that the mutant kinase which were deprived of Thr-286 as an autophosphorylation site could not be converted to Ca2(+)-independent forms upon autophosphorylation. Other autophosphorylation sites of the mutants were essentially unchanged from those of the wild-type kinase and phosphorylation of such sites did not convert them to Ca2(+)-independent forms. The results indicate that Thr-286 is the only indispensable autophosphorylation site for the appearance of Ca2(+)-independent activity of calmodulin-dependent protein kinase II alpha subunit.

Maternal exposure to a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppressed the development of reproductive organs of male rats: dose-dependent increase of mRNA levels of 5alpha-reductase type 2 in contrast to decrease of androgen receptor in the pubertal ventral prostate.

To assess the health risks associated with exposure to 2,3,7,8-tetrachlorodebenzo-p-dioxin (TCDD), we studied the effects of a relatively low dose of TCDD on the male reproductive system of rats, using the experimental protocol of T. A. Mably et al. (1992, Toxicol. Appl. Pharmacol. 114, 97-107, 108-117, 118-126), and searched for the most sensitive and reliable among several indices of TCDD toxicity. Pregnant Holtzman rats were given a single oral dose of 0, 12.5, 50, 200, or 800 ng TCDD/kg body weight on gestational day (GD) 15, and male offspring were sacrificed on postnatal day (PND) 49 or 120. GC-MS analysis of the abdominal fat tissue and testis clearly showed increased amounts of TCDD in these offspring. However, there was no TCDD effect on body weight of offspring. There were no changes on testicular or epididymal weights by TCDD administration, even at the 800-ng/kg dose in rats sacrificed on either PND 49 or 120. In addition, TCDD administration resulted in no changes in daily sperm production or sperm reserve at any of the doses used. However, the weight of the urogenital complex, including the ventral prostate, was significantly reduced at doses of 200 and 800 ng TCDD/kg in rats sacrificed on PND 120. Moreover, the anogenital distance (AGD) of male rats sacrificed on PND 120 showed a significant decrease in the groups receiving doses greater than 50 ng TCDD/kg. TCDD administration resulted in no apparent dose-dependent changes in levels of either serum testosterone or luteinizing hormone. Interestingly, reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that, in the ventral prostates of the PND 49 group, TCDD administration resulted in both a dose-dependent increase in 5alpha-reductase type 2 (5alphaR-II) mRNA level and a dose-dependent decrease in androgen receptor (AR) mRNA level. These results suggest that low-dose TCDD administration had a greater effect on the development of the external genital organs and ventral prostate than on development of the testis and other internal genital organs. Moreover, it is highly suggested that the decrease in the size of the ventral prostate by maternal TCDD exposure might be due to decreased responsiveness of the prostate to androgen due to an insufficient expression level of androgen receptor during puberty.

Effect of interferon therapy in a patient with chronic active hepatitis type C associated with interstitial pneumonia and rheumatoid arthritis: a case report.

Interferon (IFN) is widely used to treat patients with chronic hepatitis types B and C but has been found to occasionally aggravate rheumatoid arthritis (RA) or cause interstitial pneumonia. We administered 6 MIU/d IFN-beta by intravenous injection for 6 weeks to a 55-year-old man with chronic active hepatitis type C associated with RA and interstitial pneumonia. Transaminase levels rapidly returned to normal after treatment and hepatitis C virus-RNA (nested RT-PCR method) was negative on completion of treatment. No significant adverse reactions or aggravation of RA or interstitial pneumonia occurred. These findings suggest that use of IFN-beta in the treatment of patients with chronic hepatitis type C associated with RA and/or interstitial pneumonia presents no problem if appropriate precautions are taken.

Characterization of calcium/calmodulin-dependent protein kinase II isoforms from forebrain and cerebellum.

Calcium/calmodulin-dependent protein kinase II (CaM kinase II) is composed of two distinct but related subunits, alpha and beta, in various ratios. To investigate the physiological significance of this variation, we have studied the effect of autophosphorylation of CaM kinase II isoforms purified from forebrain and cerebellum on the activity, and analyzed their endogenous protein substrates. Autophosphorylation of two kinases resulted in the appearance of Ca2(+)-independent activity and the substrate specificity of the Ca2(+)-independent form differed from that of the Ca2(+)-dependent, non-phosphorylated form of the enzyme. Increased phosphorylation of two kinases resulted in a decrease in the enzyme activity. The decrease in the enzyme activity of forebrain CaM kinase II was larger than that of cerebellar kinase. Phosphorylated forms of two kinases were less stable than the non-phosphorylated forms, and the phosphorylated form of forebrain kinase was less stable than that of cerebellar kinase. Many endogenous protein substrates of respective CaM kinase II were found in both soluble and particulate fractions of forebrain and cerebellum using gel electrophoresis. Although the major protein substrates of CaM kinase II were almost the same in forebrain and cerebellum, some of the endogenous protein substrates of respective CaM kinase II were found to be different in both soluble and particulate fractions of forebrain and cerebellum.

Expression and characterization of hydroxyindole O-methyltransferase from a cloned cDNA in Chinese hamster ovary cells.

We have used two kinds of expression systems to test whether the cloned cDNA encoding hydroxyindole O-methyltransferase (HIOMT) of the bovine pineal gland was functional or not. First, when mRNA was synthesized in vitro by the SP6 system and injected into Xenopus oocytes, the enzymatic activity was expressed in the oocytes. Second, the cloned cDNA was recombined to a vector under the control of the simian virus 40 early promoter and transfected to Chinese hamster ovary (CHO) cells. The enzymatic activity of the crude supernatant of transfected cells (CHO-HT2) reached to 400 pmol melatonin formed per min per mg of protein, which value was approximately 9% of that of bovine pineal supernatant. The amounts of enzyme protein estimated by immunoblotting were proportional to the enzymatic activity in both CHO and pineal gland. The content of HIOMT protein was 8- to 30-fold larger in pineal gland compared to CHO cells. On the other hand, the content of mRNA encoding the enzyme measured by dot hybridization with [32P]cDNA, was in the same range in both CHO cells and pineal glands. These data suggest that the 11-fold higher enzymatic activity in pineal gland is due to an accumulation of the enzyme protein, not to a high level of the mRNA and also indicate that the cloned cDNA can express an intact hydroxyindole O-methyltransferase enzyme in CHO cells.

Glutamate receptors expressed in Xenopus oocyte by messenger RNA from invertebrate muscle.

Glutamate induced responses in the surface membrane of Xenopus oocyte by injection with mRNA from crustacea and insect muscle. Glutamate (10(-5) to 10(-4) M) elicited smooth current responses which resembled those induced by kainate. This smooth current reversed direction at about 0 mV and was blocked by a spider toxin (JSTX). Higher concentrations (more than 1 x 10(-3) M) of glutamate induced JSTX-insensitive oscillatory current responses which may be caused by Cl- activation.

Production of an antiserum using a fusion protein produced by a cDNA for rat choline acetyltransferase.

A fusion protein produced by a cDNA for rat choline acetyltransferase (ChAT) inserted into a translation vector was used for immunization of rabbits. An antiserum was obtained that could recognize a single protein band in immunoblot analysis of a partially purified enzyme preparation of the rat brain. The antiserum revealed ChAT immunoreactivity in the motoneurons and terminal-like structures in the neuropil of the ventral horn in cryostat sections of the cervical spinal cord of the rat. This antiserum may be of particular use to study the development of the cholinergic neuron.

Differential expression of glutamate receptors in Xenopus oocytes injected with messenger RNA from lobster muscle.

In the neuromuscular synapse of lobster walking leg, application of glutamate and its agonists generated transient depolarization of the postsynaptic membrane. The order of potency was quisqualate greater than glutamate greater than kainate. NMDA was inactive on lobster muscle. However, the membrane of Xenopus oocytes injected with mRNA from lobster muscle acquired sensitivity to NMDA. The order of potency for inducing current responses in the oocyte membrane was kainate greater than glutamate = NMDA much greater than quisqualate. The results suggest that mRNAs coding for glutamate receptors in the lobster muscle are expressed differently in Xenopus oocyte membrane.

Cellular mechanism involved in the synthesis of cyclic GMP in nervous tissues.

Intracellular cyclic GMP content responds to the stimulation of muscarinic receptor in a variety of tissues. Several aspects of the cellular mechanism involved in the synthesis of cyclic GMP were investigated. 1. In cultured bovine chromaffin cells, acetylcholine as well as muscarine stimulated the 32Pi incorporation into phosphatidic acid, induced Ca2+ mobilization across the cells, and, in parallel, elevated intracellular cyclic GMP content. Phosphatidic acid added to culture medium also stimulated the efflux and influx of Ca2+ and the synthesis of cyclic GMP in bovine chromaffin cells and in neuroblastoma cells in the same fashion as acetylcholine. 2. We have succeeded in a purification of an endogenous activator for guanylate cyclase from rat brain and identified it as L-arginine. L-Arginine, but not D-arginine, activated soluble guanylate cyclase 10- to 20-fold at a low concentration (1-2 X 10(-5) M). The activation of the enzyme by L-arginine seemed to require Ca2+. Calcium accumulated in cells in response to muscarinic stimulation would activate guanylate cyclase in collaboration with L-arginine. 3. Using a specific monoclonal antibody, we demonstrated the cellular and subcellular localizations of guanylate cyclase in rat brain. An intense reaction was observed in the brain regions which were rich in muscarinic receptor. Electron microscopic examination revealed that guanylate cyclase was concentrated in the postsynaptic perikaryon and dendrites of some type of neurons indicating its involvement in neural transmission.

Effects of 3,3',4,4',5-pentachlorobiphenyl, a coplanar polychlorinated biphenyl congener, on cultured neonatal mouse testis.

3,3',4,4',5-Pentachlorobiphenyl (PCB126), a congener with a planar configuration, has been established to have relatively strong toxicities similar to those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via aryl hydrocarbon receptors. We investigated the effects of this coplanar PCB on mammalian early spermatogenesis and steroidogenesis in a mouse neonatal testicular organ culture system. Testes collected from newborn mice were subjected to organ culture in medium containing 0, 10, 100 or 1000 nM PCB126. Histochemical analysis revealed that the BrdU-labeling indices of both spermatogenic cells and Sertoli cells were unchanged in all testis specimens exposed to the coplanar PCB. CYP1A1 and steroidogenic enzymes (P450scc, P450c17, 3beta-HSD and 17beta-HSD) mRNA levels were determined by semiquantitative RT-PCR. The CYP1A1 mRNA level in cultured testis was significantly increased by PCB126 in a dose-dependent manner. Although mRNA levels of 3beta-HSD and 17beta-HSD were unchanged, the P450scc mRNA level was significantly down-regulated by PCB126 in a dose-dependent manner. In contrast, the P450c17 mRNA level was significantly higher in 1000 nM PCB126-exposed testis than in control testis. These results suggest that the coplanar PCB does not alter the proliferative activity of spermatogenic cells and Sertoli cells in neonatal testis, but that it directly affects the expression of steroidogenic enzyme genes.