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.

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.

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.

Preferential inhibition of nucleosome assembly by ultraviolet-induced (6-4)photoproducts.

We reconstituted nucleosomes in vitro using two kinds of damaged pBR322 plasmid DNA carrying cyclobutane pyrimidine dimers (CPD) or (6-4)photoproducts. The results indicate that nucleosome assembly is inhibited preferentially by (6-4)photoproducts compared with CPD, suggesting that the regions carrying (6-4)photoproducts retain their nucleosome-free form, i.e. linker-like conformation until completion of the repair processes.

Frog cytochrome P-450 (11 beta,aldo), a single enzyme involved in the final steps of glucocorticoid and mineralocorticoid biosynthesis.

A cDNA for cytochrome P-450(11 beta,aldo) was cloned from a library of bullfrog interrenal tissue (tissue corresponding to the mammalian adrenal gland). The 1919-bp cDNA encoded a protein of 517 amino acids. Its amino acid sequence was highly similar to the sequences of bovine P-450(11 beta) and rat P-450(11 beta,aldo) when P-450(11 beta) family enzymes reported to date were examined. The enzyme expressed in COS7 cells had the 11 beta-hydroxylation, 18-hydroxylation activities and aldosterone synthetic activity. Northern-blot and immunoblot analyses suggested that a single P-450(11 beta) enzyme was expressed in bullfrog interrenal tissue. These results suggest that a single enzyme catalyzes the final steps of glucocorticoid and mineralocorticoid biosynthesis in bullfrog interrenal tissue as in bovine adrenal gland. A phylogenetic tree of CYP11B genes suggests that the frog enzyme diverged at an earlier evolutionary time from other vertebrate enzymes. Immunohistochemical and in situ hybridization studies indicated that steroidogenic cells existed in the outer region of interrenal tissue more densely than in the inner region, whereas some medullary cells made clusters like islets. Most of the cells were diffusely distributed in the tissue.

Sex-dependent expression of a transcription factor, Ad4BP, regulating steroidogenic P-450 genes in the gonads during prenatal and postnatal rat development.

We investigated the expression of Ad4BP (also known as SF-1), a transcription factor regulating steroidogenic P-450 genes, in the steroidogenic tissues such as adrenal glands, testes and ovaries through the prenatal and postnatal life of rats. Ad4BP was detected in the primordial adrenal glands and gonads of the 13.5 day postcoitum (d.p.c.) fetus. After the appearance of Ad4BP, a steroidogenic P-450 (P-450(SCC)) was also detected in the adrenal glands and its amount increased gradually. In the fetal gonads of 14.5 d.p.c., a significant amount of Ad4BP was detected in the somatic cells of the testes, whereas only a trace amount was present in the ovaries. The sexually dimorphic expression of Ad4BP continued throughout the neonatal age. Drastic alterations occurred during the first to third week of postnatal age accompanied by functional and structural changes of the gonads. The expression of Ad4BP in the testes attained a maximal level one week after birth and decreased markedly thereafter. By contrast, increase of Ad4BP in the ovary was detected after the first postnatal week. Expression of P-450c17 showed a good correlation with the proliferation of Leydig cells in the testes and theca cells in the ovaries. Immunohistochemical studies revealed the presence of Ad4BP in Sertoli cells as well as Leydig cells up to the pubertal age. In the adult rat testis, however, staining of Sertoli cells decreased significantly. Ad4BP was detected in granulosa, theca, corpus luteum and interstitial gland cells in the ovary although the expression levels in granulosa cells varied among follicles. It is suggested that the Müllerian inhibitory substance gene may be a target of Ad4BP since this gene has a conserved Ad4-binding site within the promoter, which is recognized by Ad4BP expressed in the fetal testes.

The effects of ultraviolet C on in vitro nucleosome assembly and stability.

The effects of ultraviolet C (UVC) irradiation on nucleosome assembly and its stability were investigated quantitatively using an in vitro nucleosome assembly system comprising a plasmid DNA of pBR322 and core histones isolated from rat ascites hepatoma cells. Nucleosomal formation was estimated by analyzing the resulting DNA supercoils. When UVC-irradiated (3000 J/m2) DNA was used as a substrate for the nucleosome assembly system, the nucleosomal formation efficiency was reduced by half compared with nonirradiated DNA. On the other hand, when the reconstituted nucleosomes (minichromosomes) on the nonirradiated DNA were irradiated with UVC (3000 J/m2), about half each were disrupted and retained. These results indicate that it is difficult for UV-damaged DNA regions to supercoil around the histone octamers to form nucleosomes and that the histone octamers in the UV-damaged nucleosomes tend to be dissociated from DNA.

Functional difference between Ad4BP and ELP, and their distributions in steroidogenic tissues.

Ad4BP, a zinc finger DNA-binding protein, was identified as a transcription factor regulating steroidogenic P-450 genes in a cAMP-dependent manner. Immunochemical and immunohistochemical studies with steroidogenic tissues, adrenal, ovary, and testis, were performed using the antiserum to Ad4BP. Ad4BP was expressed to the same extent in the three zones of the adrenal cortex. Immunohistochemical examination of ovarian follicle and corpus luteum showed the expression of Ad4BP. The granulosa and thecal cells, the two distinct types of the steroidogenic cells in the follicle, gave Ad4BP signals, which were stronger than in the latter cells than in the former. Immunoblot analyses of mature and regressed corpora lutea indicated a parallel expression of Ad4BP and side-chain cleavage P-450, and both proteins significantly decreased in the regressed tissues. Leydig cells surrounding seminiferous tubules gave clear immunostaining signals for Ad4BP. ELP, a mammalian counterpart of Drosophila FTZ-F1 detected in EC cells, and are isoforms transcribed from the same gene. The Ad4BP and ELP forms recognize same nucleotide sequences. Reverse transcriptase-polymerase chain reaction with specific primers for ELP revealed that steroidogenic tissues contained ELP as well as Ad4BP. The effects of the two proteins on the transcription of the CYP11B gene were compared using the expression vectors of Ad4BP and ELP. ELP did not activate transcription and showed a weak inhibitor effect on the Ad4BP-dependent transactivation of the CYP11B gene promoter when transfected simultaneously. A gel shift analysis using in vitro synthesized Ad4BP and ELP revealed that the binding activity of ELP is significantly weaker than that of Ad4BP.

Panning and immune electron-microscopic study of mucinous ovarian-cancer antigen-specific cells derived from malignantly transformed bloom-syndrome B-lymphoblastoid cells.

We separated characteristic mucinous ovarian cancer (OVC) antigen cells from malignantly transformed Bloom syndrome (BS) cell line (BS-SHI-4M) with the panning procedure using OVC patients sera. We undertook an immune electron-microscopic and scanning electron-microscopic study to acquire information regarding the antigenic determinant of the membrane using pre-embedding method, as well as immunofluorescence (IF) study. The distribution of Protein A colloidal gold (PAG) grains on the cell membrane of mucinous OVC antigen cells paralleled that of fluorescein-conjugated anti-human IgG observed in the IF study. The three patterns of PAG labeling of uniform labeling, uniform partial labeling, and partial labeling of one side of the cell paralleled the three patterns of IF labeling observed under IF. These findings strongly suggest the immunological reaction of BS-SHI-4M OVC-MU antigen cells with the antibody of mucinous OVC patient serum. Western blot analysis demonstrated that the antigen which characterizes mucinous OVC has a band at 84000 MW.

A new approach to collagen fiber orientation in cow skin by the microwave method.

The fiber orientation of cow leather sheets corresponding to the corium of the middle layer in the cow skin was studied by measuring the angular dependence of transmitted microwave intensity and the complex dielectric constant at a microwave frequency of 3.9 GHz. The direction of the minimum transmitted microwave intensity and the maximum dielectric loss agrees with that of the maximum mechanical breaking strength, suggesting that the collagen fibers constituting the cow leather should be preferably oriented in the direction of minimum transmitted microwaves and the maximum dielectric loss. The electron microphotograph supported this suggestion. The main chain direction of collagen fibers and the degree of orientation varied from position to position in the sheet. The present microwave method is convenient for quick determination of the orientation of collagen fibers in the cow skin. It can be applied non-destructively and contact-freely.

Presence of sex difference of cytochrome P-450 in the rat preoptic area and hypothalamus with reference to coexistence with oxytocin.

Localization of female type cytochrome P-450 (F1) in the preoptic area and hypothalamus of the rat was examined immunocytochemically using antiserum against purified hepatic P-450 (F1). This antiserum recognizes both P-450 (F1) and P-450 (M3). Western immunoblotting using the antiserum demonstrated that female rat brain contains P-450 (F1) but not P-450 (M3), since microsomes from the brain and liver displayed only one immunoreactive band at 50 kD, coinciding with that of P-450 (F1) purified from female rat liver. On the other hand, the male brain has P-450 (M3) but not P-450 (F1), as liver- and brain-derived microsomes produced single band at 49 kD, which represents a mol. wt. identical to that of P-450 (M3) extracted from male rat liver. These results indicate that P-450 (F1)-like immunoreactivity (LI) occurs in the female rat brain, while P-450 (M3)-LI takes place in the male rat brain. Immunocytochemical analysis further demonstrated the detailed cellular localization of these two P-450-LIs in the preoptic area and hypothalamus of female and male rats. Localization of P-450 (F1)-LI in the female rat hypothalamus resembled that of P-450 (M3)-LI in the male rat hypothalamus. Magnocellular neurosecretory neurons in the paraventricular nucleus and supraoptic nucleus were labeled and were found to contain oxytocin but lack vasopressin when serial sections of these areas were analyzed. In addition, groups of immunoreactive cells were seen in the median preoptic nucleus, medial and lateral preoptic area, caudal portion of the bed nucleus of the stria terminalis, lateral hypothalamus at the level of the paraventricular nucleus, periventricular zone from the preoptic area to the paraventricular nucleus, and parvocellular portion of the paraventricular nucleus.

Enzymatic activities of P-450(11 beta)s expressed by two cDNAs in COS-7 cells.

Expression plasmids were constructed using two cDNA clones of P-450(11 beta), pcP-450-(11 beta)-2, and pcP-450(11 beta)-3 (Morohashi et al. (1987) J. Biochem. 102, 559-568 and Kirita et al. (1988) J. Biochem. 104, 683-686), and introduced into COS-7 cells by electroporation. The expression of P-450(11 beta) proteins and their localization in the mitochondria were demonstrated by immunoblotting, immunofluorescence microscopy, and immunoelectron microscopy. The enzymatic activities of the expressed P-450(11 beta)s were determined using deoxycorticosterone (DOC), deoxycortisol, and corticosterone as substrates. Though the activities of the two P-450(11 beta)s for 11-, 18-, and 19-hydroxylation of DOC were almost equal, the production of 18-hydroxycorticosterone and aldosterone from corticosterone by P-450(11 beta)-3 was greater than that by P-450(11 beta)-2.

Immunocytochemical localization of adrenodoxin in bovine adrenal cortex by protein A-gold technique.

To investigate the intracellular localization of the enzymes that are involved in steroid hormone synthesis, an immunocytochemical study of the distribution of adrenodoxin in cells of the bovine adrenal cortex was carried out by the post-embedding immunostaining method and by immuno-cryoultramicrotomy in combination with the protein A-gold technique. Gold particles were seen on the matrix and the inner membrane of all the mitochondria examined, which have typical vesicular or tubulo-vesicular cristae, in parenchymal cells of the zona fasciculata and the zona reticularis. Gold particles were distributed homogeneously among the mitochondria. The density of gold particles on mitochondria in the parenchymal cells of the zona glomerulosa was lower than that of the zona fasciculata, which was similar to that of the zona reticularis. Gold particles were also seen on round, electron-dense intramitochondrial bodies in the parenchymal cells. The intramitochondrial bodies were abundant in the zona glomerulosa and the outer region of the zona fasciculata.