Increase in reelin-positive cells in the marginal zone of Pax6 mutant mouse cortex.

The extracellular matrix molecule reelin is a crucial molecule in CNS development, in particular in the cerebellum and cerebral cortex. In the cerebral cortex, reelin is provided by a small number of neurons located in the marginal zone (MZ). These neurons belong to the earliest neurons generated, but little is known about the molecular mechanisms of their specification. Here we describe that reelin-positive cells are strongly increased in the developing cortex of the Pax6 mutant mice Small eye. Shortly after the onset of reelin expression, the number of reelin- and calretinin-positive cells is doubled in the cortex of Pax6 mutants and this increase is further enhanced during development. In contrast, calbindin-positive cells in the MZ do not co-express reelin and are not altered in the Pax6 mutant cortex. The split of the preplate cells was also defective in the Pax6 mutant cortex, suggesting that the amount of reelin is crucial for positioning of the cortical plate between the MZ and subplate. We further show that Pax6 mutant cortical cells isolated in vitro do not develop an increase in reelin-positive cells, while cells isolated from the entire telencephalon do. Consistent with non-cell-autonomous mechanisms contributing to the increase in reelin-positive cells in the Pax6-deficient cortex, tangential migration of diverse cell types from the ventral telencephalon into the cortex is enhanced in the Pax6 mutant mice. Taken together, these experiments further elucidate how patterning of the forebrain by the transcription factor Pax6 regulates the specification of distinct neuronal subtypes in the cortical MZ.

Emx2 directs the development of diencephalon in cooperation with Otx2.

The vertebrate brain is among the most complex biological structures of which the organization remains unclear. Increasing numbers of studies have accumulated on the molecular basis of midbrain/hindbrain development, yet relatively little is known about forebrain organization. Nested expression among Otx and Emx genes has implicated their roles in rostral brain regionalization, but single mutant phenotypes of these genes have not provided sufficient information. In order to genetically determine the interaction between Emx and Otx genes in forebrain development, we have examined Emx2(-/-)Otx2(+/-) double mutants and Emx2 knock-in mutants into the Otx2 locus (Otx2(+/Emx2)). Emx2(-/-)Otx2(+/-) double mutants did not develop diencephalic structures such as ventral thalamus, dorsal thalamus/epithalamus and anterior pretectum. The defects were attributed to the loss of the Emx2-positive region at the three- to four-somite stage, when its expression occurs in the laterocaudal forebrain primordia. Ventral structures such as the hypothalamus, mammillary region and tegmentum developed normally. Moreover, dorsally the posterior pretectum and posterior commissure were also present in the double mutants. In contrast, Otx2(+/Emx2) knock-in mutants displayed the majority of these diencephalic structures; however, the posterior pretectum and posterior commissure were specifically absent. Consequently, development of the dorsal and ventral thalamus and anterior pretectum requires cooperation between Emx2 and Otx2, whereas Emx2 expression is incompatible with development of the commissural region of the pretectum.

Characterization of a proximal element in the rat preadipocyte factor-1 (Pref-1) gene promoter.

Preadipocyte factor-1 (Pref-1) was shown to negatively regulate adipocyte differentiation. We recently reported that ZOG, a rat homolog of Pref-1, was specifically expressed in the adrenal zona glomerulosa. Results of the investigation of Pref-1 expression in preadipocyte and in undifferentiated adrenal cortex suggested that down-regulation of Pref-1 gene was closely correlated with the differentiation process. In this study we demonstrate that an upstream region (from -76 to -47) of the rat Pref-1 gene was essential for its expression in adrenocortical carcinoma-derived H295R cells. A nucleotide sequence found in this region, GCGTGGGCGTGGGCGGGGG (Egr/GC-box), seemed to contain three elements, two early growth response (Egr) elements and one GC-box, overlapping each other. Mutations of four or five nucleotides in a 7-nucleotides-stretch in the midst of the Egr/GC-box eliminated the binding of Sp1/3, abolished the activation by Egr-factor(s) and diminished the Pref-1 promoter activity. When mutations were introduced into the outside of the middle portion, the binding of Sp1/3 to the Egr/GC-box was abolished similarly. However, the decrease in the promoter activity was less than that found with the construct mutated at the middle. These results indicated that an element present at the 7-nucleotides-stretch in the midst of the Egr/GC-box might be important for the Pref-1 promoter activity, and this proximal element was possibly activated by a still-unidentified nuclear factor(s). This element would function as the promoter of the Pref-1 gene in H295R cells, but not in HeLa cells.

Evidence for the involvement of a Src-related tyrosine kinase in Xenopus egg activation.

Recently, we have purified a Src-related tyrosine kinase, named Xenopus tyrosine kinase (Xyk), from oocytes of Xenopus laevis and found that the enzyme is activated within 1 min following fertilization [Sato et al. (1996) J. Biol. Chem. 271, 13250-13257]. A concomitant translocation of a part of the activated enzyme from the membrane fraction to the cytosolic fraction was also observed. In the present study, we show that parthenogenetic egg activation by a synthetic RGDS peptide [Y. Iwao and T. Fujimura, T. (1996) Dev. Biol. 177, 558-567], an integrin-interacting peptide, but not by electrical shock or the calcium ionophore A23187 causes the kinase activation, tyrosine phosphorylation, and translocation of Xyk. A synthetic tyrosine kinase-specific inhibitor peptide was employed to analyze the importance of the Xyk activity in egg activation. We found that the peptide inhibits the kinase activity of purified Xyk at IC50 of 8 microM. Further, egg activation induced by sperm or RGDS peptide but not by A23187 was inhibited by microinjection of the peptide. In the peptide-microinjected eggs, penetration of the sperm nucleus into the egg cytoplasm and meiotic resumption in the egg were blocked. Indirect immunofluorescence study demonstrates that Xyk is exclusively localized to the cortex of Xenopus eggs, indicating that Xyk can function in close proximity to the sperm-egg or RGDS peptide-egg interaction site. Taken together, these data suggest that the tyrosine kinase Xyk plays an important role in the early events of Xenopus egg activation in a manner independent or upstream of calcium signaling.

Aromatase inhibitors and enzyme stability.

The effects of two steroidal (4-hydroxyandrostenedione and atamestane) and three non-steroidal (fadrozole, vorozole, and pentrozole) aromatase inhibitors on the levels of aromatase mRNA and protein were examined in vitro and in vivo. Immunocytochemistry revealed increased quantities of immunoreactive aromatase in human choriocarcinoma-derived JEG-3 cells in response to pretreatment with the non-steroidal inhibitors. To elucidate this effect in detail, aromatase protein in JEG-3 cells after treatment with various inhibitors was quantified using an enzyme-linked immunosorbent assay (ELISA). A time-dependent increase in aromatase protein in the cells was observed with all the aromatase inhibitors except 4-hydroxyandrostenedione, whereas aromatase mRNA levels in the cells remained unchanged during the inhibitor treatment. The three non-steroidal agents caused an approximately fourfold increase in aromatase protein in the cells 24 h after the treatment, as compared with untreated controls. The increase in aromatase protein in the cells was not blocked by treatment with cycloheximide, an inhibitor of protein synthesis. The inhibitors also appeared to block the rapid degradation observed in JEG-3 cells after induction by forskolin. In vivo, daily injection of the inhibitors into adult female mice caused increases in levels of both aromatase mRNA and protein in the ovary. The increase in aromatase mRNA in this in vivo study could be explained by an increase in gonadotropin concentrations in response to decreased plasma concentrations of estrogens. In conclusion, we suggest that aromatase inhibitors increase aromatase protein through stabilization and reduced protein turnover.

Cloning of a membrane-spanning protein with epidermal growth factor-like repeat motifs from adrenal glomerulosa cells.

The three zones of adrenal cortex are thought to arise from a single multipotential stem cell, but the mechanisms underlying the zonal differentiation during embryonic development of adrenal cortex are poorly understood. Employing subtraction cloning strategy, we isolated three distinct clones that were specifically expressed in the rat glomerulosa zone. One clone, named zona glomerulosa specific clone, encoded a membrane-spanning protein with a signal peptide at the N-terminus, six epidermal growth factor-like repeat motifs, and a transmembrane domain near the C-terminus. It was identified as a rat homolog of preadipocyte factor-1 (Pref-1), a factor involved in maintaining the undifferentiated status of preadipocyte. Immunohistochemical studies confirmed the presence of Pref-1 protein in the glomerulosa zone. Detailed examination revealed that the zone is divided into two layers; the first is a few-cells-thick layer present underneath the capsule (expressing both Pref-1 protein and aldosterone synthase cytochrome P450), and the second layer is beneath the first (containing Pref-1 protein but not aldosterone synthase). Moreover, another cell layer was found beneath the second layer and above the fasciculata zone, whose cells contained no Pref-1 protein, aldosterone synthase, or 11beta-hydroxylase. These findings suggest that a recently reported aldosterone synthase- and 11beta-hydroxylase-less cell layer between the two zones is composed of two kinds of cell: Pref-1 protein-positive and -negative cells. The level of Pref-1 message in the adrenal glands of animals having various pituitary-adrenal axis activities, as well as various plasma salt concentrations, correlated with the total number of glomerulosa cells. However, the specific content of Pref-1 message in a cell was fairly constant. When the adrenal gland was surgically enucleated and the remaining capsule regenerated, the level of Pref-1 transcript was significantly suppressed at the early phase. At this phase, only a minor population of the cortical cells expressed Pref-1 protein, most of these cells already expressing a fasciculata/reticularis-specific marker, inner zone antigen. These findings suggest that the capsular cells, mostly composed of the glomerulosa cells, may have potential for differentiating into other zones' cells, and the down-regulation of Pref-1 expression may be an important step in the adrenal zonal differentiation.

Adrenocortical and gonadal expression of the mammalian Ftz-F1 gene encoding Ad4BP/SF-1 is independent of pituitary control.

Ad4BP/SF-1 is a transcription factor essential for the development of the adrenal gland and the gonads as well as for the maintenance of their functions through regulating tissue-specific gene transcription. In the whole body, hypothalamo-pituitary-gonadal and -adrenal axes are known to play prominent roles in mediating the function of the gonads and adrenal. In this study, the effects of the tropic peptide hormones secreted by the pituitary on the regulation of the rat Ftz-F1 (rFtz-F1) gene encoding Ad4BP/SF-1 were investigated. Immunochemical studies revealed that Ad4BP/SF-1 was expressed even in the adrenal cortex of hypophysectomized rats. Such persistent expression of Ad4BP/SF-1 was also observed in the testes and ovaries of the hypophysectomized animals. In contrast to Ad4BP/SF-1, the expressions of steroidogenic P450s were reduced significantly. The transcriptional activities of the endogenous and transfected rFtz-F1 genes were examined with Y-1 and I-10 cells derived from mouse adrenocortical and testicular Leydig cell tumors, respectively. Neither gene appeared to be activated significantly by cAMP, whereas both endogenous and exogenous CYP11A genes encoding P450(SCC) were activated. Taken together, these observations indicate that the expression of the rFtz-F1 gene is mainly regulated by a mechanism independent of the neuroendocrine axes.

Inhibitors of aromatase prevent degradation of the enzyme in cultured human tumour cells.

The effects of two steroidal (4-hydroxyandrostenedione and atamestane) and three non-steroidal (fadrozole, vorozole and pentrozole) aromatase inhibitors on the levels of aromatase mRNA and protein were examined using cultured JEG-3 and HepG2 cells. Immunocytochemical studies demonstrated increased quantities of immunoreactive aromatase in both cell types as a result of these treatments. To clarify this effect in detail, quantitation of aromatase protein in JEG-3 cells was performed after various treatments using an enzyme-linked immunosorbent assay. Time-dependent increase was observed with all the aromatase inhibitors except 4-hydroxyandrostenedione. The three non-steroidal agents caused an approximately fourfold elevation in the cells 24 h after the treatment compared with untreated controls. The inhibitors also appeared to block the rapid degradation observed in JEG-3 cells after induction with forskolin. However, aromatase mRNA levels in JEG-3 cells remained unchanged. Furthermore, the increase in aromatase protein in JEG-3 cells due to the inhibitor action was not blocked by treatment with cycloheximide, an inhibitor of protein synthesis. These results thus suggest that aromatase inhibitors increase aromatase protein through stabilization and reduced protein turnover as a side-effect of their binding.

Identification of functional domains of rat intestinal phospholipase B/lipase. Its cDNA cloning, expression, and tissue distribution.

A cDNA encoding a rat intestinal Ca(2+)-independent phospholipase B/lipase (PLB/LIP) was cloned from an ileac mucosa cDNA library using a probe amplified by polymerase chain reaction based on the purified enzyme's sequence. PLB/LIP consists of an NH2-terminal signal peptide, four tandem repeats of about 350 amino acids each, and a hydrophobic domain near the COOH terminus. The enzyme purified previously was found to be derived from the second repeat part. To examine the function of each domain, the full-length PLB/LIP, individual repeats, and a protein lacking the COOH-terminal hydrophobic stretch were expressed in COS-7 cells. The results showed that the second repeat, but not the other repeats, had all the activities (phospholipase A2, lysophospholipase, and lipase) found in the purified natural and expressed full-length enzymes, suggesting repeat 2 is a catalytic domain. The full-length enzyme was mainly present in membrane fractions and efficiently solubilized by treatment with 1% Triton X-100, but not with phosphatidylinositol-specific phospholipase C. Deletion of the COOH-terminal hydrophobic stretch caused the secretion of > 90% of synthesized PLB/LIP into culture media. These results suggest the hydrophobic domain is not replaced by a glycosylphosphatidylinositol anchor but serves as a membrane anchor directly. A message of the full-length PLB/LIP was abundantly expressed in the ileum and also, in a smaller, but significant amount, in the esophagus and testis. Immunohistochemistry showed that PLB/LIP is localized in brush border membranes of the absorptive cells, Paneth cells, and acrosomes of spermatid, suggesting its roles related and unrelated to intestinal digestion.

Implication of ZOG protein (zona glomerulosa-specific protein) in zone development of the adrenal cortex.

The three zones of adrenal cortex are thought to arise from a single multipotential stem cell. Immunohistochemical studies of fetal and adult adrenals using an antibody against a previously-cloned ZOG protein, a rat homolog of Pref-1, were conducted to explore its roles in the differentiation of cortical tissues. At the early embryonic stage, ZOG was already expressed in adrenogonadal primordial cells. The ZOG-positive cells gradually formed the adrenal primordium by E14.5. By E17.5 the expression was repressed in the inner part of the aggregate and these cells began to express CYP11B1. The ZOG-positive cells at this stage existed at the periphery of the aggregate but they did not express CYP11B2 yet. Not until E20.5 did the aldosteronogenic cells appear among the ZOG-positive cells at the outermost part of the gland. Based on these and the other findings the zonal development of the adrenal cortex is discussed.

Zona glomerulosa-specific factor: cloning and function.

The rat adrenal cortex is composed of three zones: the zona glomerulosa, the zona fasciculata, and the zona reticularis. Several investigators have claimed the presence of a zona intermedia between the zonae glomerulosa and fasciculata. The cells of zona glomerulosa, a few layers of cells just beneath the adrenal capsule, synthesize and secrete aldosterone, whereas those of zonae fasciculata and reticularis secrete glucocorticoids and androgens, respectively. The function of the cells in zona intermedia is unclear, because they express neither aldosterone synthase nor 11 beta-hydroxylase. To investigate the mechanism underlying the zonal differentiation of adrenocortical steroidogenesis, attempts have been made to isolate and characterize zone-specifically expressed proteins such as steroidogenic enzymes and putative regulatory factors. Having subtracted the mRNAs present in the decapsulated adrenal gland from those in the adrenal capsule, we successfully isolated three distinct clones, each specifically expressed in the zona glomerulosa. One clone encoded a protein named zona glomerulosa-specific factor (ZOG), which had a putative signal peptide at the N-terminus, six tandem epidermal growth factor (EGF)-like repeats, and a transmembrane domain in the central portion and a short cytosolic stretch at the C-terminus. Immunohistochemical studies using the antibody raised against ZOG confirmed the presence of the protein in all layers of cells in the zona glomerulosa. In contrast, cells possessing aldosterone synthase were present only in the periphery of zona glomerulosa, just beneath the capsule. These findings suggest that there are at least two kinds of zona glomerulosa cells in the rat adrenal cortex, one expressing aldosterone synthase as well as ZOG, and another expressing only ZOG. The cells in the zona intermedia did not express ZOG, aldosterone synthase, or 11 beta-hydroxylase, but did express Ad4BP. ZOG was not detected in zonae fasciculata and reticularis where 11 beta-hydroxylase was present.

Transplantation of genetically marked cardiac muscle cells.

We examined the possibility that cardiomyocytes could be genetically marked or modified before being grafted to the heart under conditions applicable to the clinical setting. We used a replication-defective recombinant adenovirus carrying the beta-galactosidase reporter gene, and delivered it to cultured murine fetal cardiac myocytes. Virtually all fetal cardiomyocytes in a primary culture expressed beta-galactosidase 24 hours after recombinant adenovirus infection. These cells were transplanted to the hearts of syngenic adult recipient mice. Expression of the beta-galactosidase gene in the grafted cells was demonstrated by staining with 5-bromo-4-chloro-3-indoyl-beta-D-galactosidase, resulting in a blue color at the histochemical level and an electron-dense deposit on transmission electron microscopic analysis. Gene expression was recognized from 7 days to 12 weeks after transplantation. Implanted cardiomyocytes aligned themselves along the layers of the host myocardium. Formation of gap junctions was demonstrated by transmission electron microscopy. Neither inflammation nor fibrous scar tissue was detectable by histologic analysis. This study demonstrates that ex vivo gene transfer to the heart by means of the adenoviral vector is possible.

Polyadenylation-mediated translational regulation of maternal P450(11beta) mRNA in frog oocytes.

Northern blot analysis of bullfrog tissues using a cDNA probe of cytochrome P450(11beta) showed that a large amount of message was present in the ovary as well as in the adrenal tissue. Two kinds of mRNA of different sizes were found in the ovary. Sequence determination of the two cDNAs and analysis by reverse-transcription polymerase chain reaction indicated that the protein encoded by the larger mRNA was identical to the adrenal enzyme, while the protein encoded by the smaller had a truncated sequence lacking an extension peptide necessary for the protein transport to the mitochondria. The mRNAs were present in the oocytes but not in the follicular cells, and their content in an oocyte varied little during its maturation. Immunoblot analyses of the mitochondrial fraction of oocytes failed to demonstrate the presence of P450(11beta) protein. In contrast the eggs were found to contain a large amount of enzymatically active protein. Interestingly the mRNA has a cis-element called cytoplasmic polyadenylation element at its 3' untranslated region. When poly(A) tails of the message prepared from eggs and oocytes were examined by RNase H digestion or reverse-transcription polymerase chain reaction, those of eggs were about 150 nucleotides longer than those of oocytes. These results suggest that translation of the message is stimulated during the oocyte maturation as a result of enhanced polyadenylation at its 3'-end. Finally a finding is presented that progesterone was converted to 11beta-hydroxyprogesterone by the frog P450(11beta), implying that the enzyme expressed in eggs may control a level of progesterone which is needed to initiate the oocyte maturation.

Identical origin of adrenal cortex and gonad revealed by expression profiles of Ad4BP/SF-1.

BACKGROUND: Ad4BP/SF-1 was originally identified as a steroidogenic tissue-specific transcription factor. Recent gene disruption studies with the mammalian Ftz-F1 gene encoding Ad4BP/SF-1 clearly revealed the essential function of the factor for adrenal and gonadal differentiation. RESULTS: In this study, we examined the early development of these tissues using Ad4BP/SF-1 as the marker. In rat foetuses of 11.5 days post-coitum (d.p.c.), a cell population designated adrenogenital primordium was firstly observed on symmetrical lines extending from the dorsal aorta to the dorsal coelomic epithelia of the primitive urogenital ridges. From 12.5 d.p.c., the rostral half of the adreno-genital primordium started to separate into two distinct cell populations. Judging from the distribution of primordial germ cells, the cell population on the dorsal aortal side is a primordium for the adrenal cortex whereas that on the coelomic epithelial side is for the gonads. At 13.5 d.p.c., these two primordia have separated completely. CONCLUSION: These observations clearly identified a novel adreno-genital primordium from which both the adrenal cortex and the gonads originate. An RT-PCR study conducted to detect adrenal- and gonad-specific mRNAs supported the above observations.

Identification of a minimum enhancer sequence for the type II collagen gene reveals several core sequence motifs in common with the link protein gene.

The type II collagen gene (Col2a1) is expressed primarily in chondrocytes. Transcription of Col2a1 is mediated by cell-specific regulatory elements located within the promoter and first intron. Here, we map a minimal enhancer and identify elements that determine cartilage-specific Col2a1 expression by analyzing the activity of a series of chimeric genes consisting of rat Col2a1 first intron deletion mutants ligated to the chloramphenicol acetyltransferase reporter gene. We show that a 100-base pair (bp) segment within the first intron is the minimum size necessary for high level, cell type-specific expression of Col2a1. Sequence analysis of this 100-bp Col2a1 enhancer revealed several sequence motifs similar to motifs present within the regulatory region of the link protein gene, another cartilage gene. These motifs include an AT-rich element, a C1 motif and a C3 motif. Deletion of any of these elements reduced Col2a1 enhancer activity in chick embryo chondrocytes. We also tested enhancer-mediated activity in CFK2 cells which differentiate to a chondrogenic phenotype and begin to express type II collagen mRNA after extended culture. In stably transfected CFK2 cells, constructs containing the 100-bp enhancer were activated during the transition from prechondrogenic to chondrogenic cell populations and deletions within the enhancer strongly down-regulated activity. Chondrocyte-specific DNA-protein complexes were identified using nuclear extracts prepared from chick embryo chondrocytes and 32P-labeled oligonucleotides from these regions of the first intron. These results suggest that interaction of chondrocyte specific nuclear factors with multiple core elements from a small region within the first intron are important for cell-type specific Col2a1 enhancer activity.

Function and distribution of a steroidogenic cell-specific transcription factor, Ad4BP.

Ad4BP was identified as an essential transcription factor regulating steroidogenic cell-specific and cAMP-dependent transcription of the genes of steroidogenic P450s. The Ad4BP transcript was detected in steroidogenic tissues such as adrenal gland, testis, ovary, placenta and brain by RT-PCR, and showed good correlation with the expression of steroidogenic P450s. The genes of steroidogenic P450s, which are transcribed only in steroidogenic cells, were transcribed in non-steroidogenic cells when an Ad4BP expression vector was introduced into the cells. To study the function of Ad4BP in the differentiation of the steroidogenic tissues, immunochemical and immunohistochemical studies were performed with the tissues prepared from various developmental stages of rats. Adrenal cortex expressed Ad4BP since the tissue was detected in the dorsal wall of the fetus. Gonadal tissues expressed Ad4BP in a sex-dependent manner. High levels of Ad4BP expression were detected in fetal and prepubertal testes and in prepubertal and adult ovaries, whereas low level expressions were observed in the adult testes and in the fetal ovaries. The expression of Ad4BP in the gonads correlates well with the expression of the Müllerian inhibiting substance gene as well as the steroidogenic P450 gene for both sexes. These observations indicate that Ad4BP plays an important role in the development and differentiation of the steroidogenic tissues including sexual differentiation of the gonadal tissues through activation of the transcription of its target genes.

Cytochrome P450(11 beta): structure-function relationship of the enzyme and its involvement in blood pressure regulation.

Cytochrome P450(11 beta) is deeply involved in the final steps of biosynthesis of mineralocorticoids. This paper deals with following issues about this enzyme. (1) The structure and function of the enzymes of various animal species are discussed. By making alignment of amino acid sequences of the enzymes, we identified peptide domains essential for the enzyme actions such as a putative steroid binding domain and a heme binding region. Estimates of molecular similarity among the P450(11 beta) family enzymes suggested that the enzymes having both 11 beta-hydroxylation activity and aldosterone (ALDO) synthetic activity of certain animals such as frog, cattle and pig are more similar to the ALDO synthases of the other animals, such as rat, mouse and human, than the 11 beta-hydroxylases of these animals. (2) The molecular nature of the P450(11 beta) family enzymes of genetically hypertensive rats as well as adrenal regeneration hypertension (ARH) rats is examined. (i) Mutation was found in the P450(11 beta) gene of Dahl's salt-resistant normotensive rat. Steroidogenic activity expressed by the mutated gene accounted well for abnormal plasma levels of steroid hormones in this rat. (ii) 11 beta-, 18- and 19-Hydroxylation activities of adrenal mitochondrial prepared from spontaneously hypertensive rat (SHR), Wistar-Kyoto rat (WKY), and stroke-prone (SP)-SHR were not significantly different from each other. Levels of mRNA of ALDO synthase in adrenal glands of 50-week-old SHR was significantly lower than those of 10-week-old SHR, WKY and SHR-SP. (iii) No significant difference in 19-hydroxylation activity was found between adrenal mitochondria prepared from ARH rat and those from control rat. The level of message of ALDO synthase was lower in adrenal glands of ARH rat.

[99mTc-MIBI scintigraphy for the detection of parathyroid lesions in patients with hyperparathyroidism].

The usefulness of 99mTc-MIBI scintigraphy for the detection of parathyroid lesions was evaluated in 17 patients with hyperparathyroidism. Delayed image was used to evaluate the lesions. Detectability of MIBI for parathyroid lesions was 86% (18/21). The smallest lesion detected was parathyroid hyperplasia weighted 270 mg. Ectopic parathyroid adenoma and bone metastases of parathyroid carcinoma were clearly demonstrated. Detectability of MIBI scintigraphy for the lesions including ectopic and metastatic lesions was the highest among those of ultrasonography, CT and MRI methods. MIBI scintigraphy was thought to be useful for the detection of parathyroid lesions, especially for ectopic and metastatic lesions.

Collagen II promoter and enhancer interact synergistically through Sp1 and distinct nuclear factors.

The collagen II gene is expressed primarily in chondrocytes. Its transcription is activated through the interaction of cell type-specific regulatory elements located in the promoter region and in the first intron. In this study, we found that a short promoter sequence including two GC boxes was required for efficient enhancer-mediated transcription. Gel-shift analysis, site mutations, and footprint analysis showed that one of the GC boxes bound functionally to an Sp1-related factor and that an oligonucleotide containing this GC box did interact with an enhancer-nuclear factor complex. Additionally, an enhancer-derived oligonucleotide was found to complex CIIZFP, a zinc-finger protein that binds to the enhancer within the first intron and Sp1, but only in presence of CIIZFP. Antibodies against Sp1 specifically inhibited the formation of this complex. Western/Southwestern analysis also showed that a protein complex including Sp1 was able to bind the enhancer and the promoter regions in non-denaturing conditions. This complex was dissociated by denaturation. These results suggest that the formation of a nuclear protein-mediated loop structure between the promoter and enhancer may regulate transcription of the collagen II gene transcription.