Cloning of the rat angiotensin II type 2 receptor gene and identification of its functional promoter region.

We have cloned the rat angiotensin II receptor type 2 (AT2) gene, whose physiological function remains unclear. Sequence analysis indicated that exons 1 and 2 exist in the 5'-untranslated region and the initiation codon ATG is located in exon 3. The 1.6-kb genomic fragment at positions -1567 to +26 relative to the putative transcription start site was found to contain a functional promoter region using transient chloramphenicol acetyltransferase assay. This is the first report demonstrating the nucleotide sequence of the promoter region of this gene.

Isolation and characterization of two alternatively spliced complementary DNAs encoding a Xenopus laevis angiotensin II receptor.

We have isolated two cDNAs of 1.7 and 3.0 kb, produced by alternative splicing, that encode a angiotensin II (AII) receptor from a Xenopus laevis heart cDNA library. The two clones had identical coding regions with each other and were found to belong to the G protein-coupled receptor superfamily like the mammalian type 1 AII receptors (AT1); their amino acid sequence was 68.7% homologous with the human AT1 receptor sequence. However, there was a 1.3 kb insertion at the 3'-untranslated region of the longer clone. The insertion contained 9 repeats of an ATTTA motif, suggesting that the two mRNAs undergo distinct post-transcriptional regulation by virtue of a difference in their stability. Although the Xenopus receptor exhibited distinct specificities for AII receptor antagonists compared with mammalian AII receptors, several common characteristics, including the effect of dithiothreitol and guanosine 5'-O-(3-thiotriphosphate), demonstrated that the cloned receptor is a counterpart of the mammalian AT1 receptor. Moreover, the cloned receptor was expressed most abundantly in the Xenopus heart, which is inconsistent with the tissue distribution of mammalian AII receptors. This indicated that the Xenopus heart, unlike that of mammals, plays a major role in the AII-dependent regulation of blood pressure and extracellular fluid volume.

Ventral mesoderm induction and patterning by bone morphogenetic protein heterodimers in Xenopus embryos.

Bone morphogenetic proteins (BMPs) perform diverse functions in vertebrate development. Here we demonstrate that the heterodimeric BMP-4/7 protein directly induces ventral mesoderm and blood in Xenopus animal caps, and BMP-2/7 heterodimers may function similarly. We also provide indirect evidence that BMP heterodimers function in embryos, using assays with dominant-negative BMP ligands. Homodimeric BMP-2 and BMP-4 proteins do not induce mesoderm, but they ventralize mesoderm induction by activin. In contrast, BMP-7 protein interferes with mesoderm induction by activin, but BMP-7 stimulates ventral mesoderm induction by the heterodimer, BMP-4/7. This novel property of BMP-7 distinguishes it from other BMPs. BMP-7 may therefore function in early embryogenesis to antagonize activin signals and potentiate BMP signals. We propose that BMP heterodimers convey signals for ventral mesoderm induction and patterning in Xenopus development.

Identification of chick frizzled-10 expressed in the developing limb and the central nervous system.

We have identified chick frizzled (Fz)-10, encoding a Wnt receptor, and examined the expression pattern during embryogenesis. Fz-10 is expressed in the region posterior to the Hensen's node at stage 6. Fz-10 expression is detected in the dorsal domain of the neural tube and the central nervous system of the developing embryo. In the developing limb, Fz-10 expression starts at stage 18 in the posterior-dorsal region of the distal mesenchyme, and gradually expands to the anterior-distal region. Fz-10 is also expressed in the feather bud and branchial arch. Implantation of Sonic hedgehog (Shh)-expressing cells into the anterior margin of the limb bud resulted in the induction of Fz-10 expression in anterior-dorsal mesenchyme.

Diurnal regulation of per repeat family in the suprachiasmatic nucleus of rat brain.

We have recently reported fluctuations in the expression of the period repeat sequence, pp2.5, during light-dark cycles in the suprachiasmatic nucleus (SCN) of rat. Presently, we performed in situ hybridization which shows that the fluctuation of pp2.5 expression continues during constant darkness conditions in the SCN of rat. The light exposure during subjective night but not subjective day triggered its elevated expression in a time-dependent manner which is parallel to that of c-fos expression. In this review, the cloning and characterization of multiple per repeat sequences from mouse genom and rat brain mRNA were summarized. The abundance of a novel per repeat mRNA (designated as RB15) fluctuates during a light-dark cycle in the SCN. These findings suggest that per repeat sequence may have a role for the mammalian circadian rhythms. The evolutionary relationship between the mammarian per repeat sequence and the Drosophila period gene is also discussed.

Human renin gene of renin-secreting tumor.

A large amount of renin mRNA was found to be expressed in the juxtaglomerular cell (JGC) tumor, as determined by Northern analysis. We have isolated the long 5'-flanking region of the human renin gene from the tumor, and characterized the promoter region with respect to nucleotide (nt) sequence and mRNA transcription start point. Of two sets of CAAT and TATA box at 29 bp upstream from the capping site is demonstrated to be a functional promoter by the primer extension. The 1:6-kb sequence, containing the 5'-flanking region, exon 1, and part of the first intron, obtained from the tumor was in complete agreement with that of the clone from fetal liver, which does not produce renin. This indicates that abnormal expression of the human renin gene in the JGC tumor involves no major alteration in the primary structure within 1.2 kb of the 5'-flanking region. Within 1.2 kb of the 5'-flanking region, there are several nt segments exhibiting homology with the glucocorticoid, estrogen, and progesterone receptor-binding sites and enhancers. These structures may be related to the tissue-specific expression of the renin gene.

Multiple genes for Xenopus activin receptor expressed during early embryogenesis.

Four distinct cDNAs for activin receptor designated as XSTK2, 3, 8 and 9 have been cloned from a Xenopus laevis cDNA library. The protein structures deduced from the cDNAs have shown that they all have a putative extracellular ligand-binding domain, a single transmembrane domain and cytoplasmic Ser/Thr kinase domain, except that XSTK2 is extremely similar to the XSTK3 gene but lacks a carboxyl-terminal part of the kinase motif. Northern blot analysis showed that all transcripts are maternally inherited. The levels of transcript for XSTK2, 3 and 8 appeared to fluctuate during early development while those for XSTK9 maintain constant.

A carboxyl-terminal truncated version of the activin receptor mediates activin signals in early Xenopus embryos.

The function of a carboxyl-terminal truncated version of the Xenopus activin receptor, encoded by a previously isolated gene XSTK2, was investigated in early embryos. The transcript corresponding to the truncated receptor gene was detected throughout embryonic development although the temporal expression pattern was different from that of an intact receptor. Injection of XSTK2 mRNA into early embryos resulted in the formation of a duplicated body axis. Mesoderm induction as evaluated by the activation of the alpha-actin gene in presumptive ectoderm (animal cap) treated with exogenous activin was significantly enhanced by the injection of XSTK2 mRNA. These results suggest that the truncated receptor is capable of transmitting the activin signal to the same extent as the native receptor.

Molecular cloning of a gene under control of the circadian clock and light in the rodent SCN.

We recently found a mouse unusual per repeat genomic gene showing circadian expression in the suprachiasmatic nucleus (SCN) of rat brain. As an initial step to the better understanding of biological functions of mammalian per repeat family, we isolated a new cDNA clone that encodes for the putative open reading frame of 133 amino acids, designating as mp41, having a per repeat sequence of (ACAGC)32 which lacks one base pair from a mouse unusual per repeat sequence (ACAGGC)n. In situ hybridization showed that the mRNA of mp41 gene expression is detected in the rat pancreas, uterus, ovary, liver, adrenal glands, kidney, intestine, spleen and brain. In brain, daily fluctuations of mp41 mRNA levels were found in the SCN under light and dark cycles--high during the day time and lower during the night time, even in constant darkness for 15 days. After exposing rats to light, mp41 mRNA increased only during the subjective night of the circadian cycle when light also induced the c-fos mRNA expression in the SCN. These results suggest that the transcriptional control of mp41 gene is regulated by light and a circadian clock and indicate that mp41 is a new marker gene for a cycling transcript in the SCN.

Serum amyloid A, C-reactive protein and remnant-like lipoprotein particle cholesterol in type 2 diabetic patients with coronary heart disease.

BACKGROUND: Serum amyloid A (SAA) and C-reactive protein (CRP) have been suggested to be involved in the process of coronary heart disease (CHD) and to be potential markers and/or predictors of CHD. Remnant-like lipoprotein particles (RLPs), which are regarded as atherogenic remnant lipoprotein, are reported to be increased in type 2 diabetic patients. We assessed the association of CHD with SAA, CRP and RLP-cholesterol in type 2 diabetic patients. METHODS: One hundred and twenty-six diabetic patients without CHD and 41 patients with CHD were recruited from our hospital. Plasma SAA was measured by the latex agglutination nephelometric immunoassay. Plasma high-sensitivity CRP was measured by a latex immunoturbidity method. Plasma RLP-cholesterol was measured by an immunoabsorption enzyme method. RESULTS: The mean standard deviation values of RLP-cholesterol in patients with and without CHD were 0.22 (0.26) mmol/L and 0.15 (0.10) mmol/L, respectively (P <0.05). Median (interquartile ranges) for SAA in patients with and without CHD were 7.4 (4.2-11.2) mg/L and 3.9 (2.2-5.9) mg/L, respectively (P <0.001). Median (interquartile ranges) for CRP in patients with and without CHD was 1.14 (0.45-2.08) mg/L and 0.43 (0.19-1.25) mg/L, respectively (P <0.001). For all patients, the Spearman rank correlation statistics for RLP-cholesterol compared with SAA and with CRP were 0.213 (P <0.05) and 0.301 (P <0.01), respectively. CONCLUSION: These data suggest that SAA, CRP and RLP-cholesterol are increased in type 2 diabetic patients with CHD, and that the inflammatory proteins correlate with remnant lipoprotein.

Differential expression of Xenopus BMPs in early embryos and tissues.

Expression levels of mRNA for Xenopus bone morphogenetic proteins (BMPs) in early embryos and adult organs were examined. Reverse transcription-polymerase chain reaction (RT-PCR) that used specific primers for each BMP subtype revealed that mRNAs for BMP-2 and BMP-4 are distributed in a wide variety of adult tissues including lung, heart, and kidney. Unexpectedly, distribution of mRNA for BMP-7 was found to be limited to ovary and early embryos. The result suggests that Xenopus BMP-7 may have a specific role in ovary and early embryos.

Effects of nucleosides and a nucleotide on DNA and RNA syntheses by the salvage and de novo pathway in primary monolayer cultures of hepatocytes and hepatoma cells.

Studies were made on the effects of inosine, guanosine 5'-monophosphate (GMP), cytidine, uridine, thymidine, and their mixture (4:4:4:3:1, OG-VI) on DNA and RNA syntheses in primary monolayer cultures of normal hepatocytes and cultures of hepatoma cells, AH130, to use these compounds for total parenteral nutrition. Addition of an appropriate amount of inosine, GMP, uridine, or thymidine to primary cultures of hepatocytes enhanced both DNA and RNA syntheses by the salvage and de novo pathways. Cytidine appeared to have lower optimal concentration for enhancing these pathways. The OG-VI mixture also enhanced the syntheses of DNA and RNA, but the composition of the mixture was not optimal. Additions of inosine, GMP, uridine, and thymidine to cultured hepatoma cells also enhanced their DNA and RNA syntheses, but the cells consumed more of the added nucleic acid compounds than hepatocytes did. Addition of cytidine had no effect on proliferation of the cells. The OG-VI mixture at relatively higher concentration inhibited the syntheses of DNA and RNA by hepatoma cells. Addition of high concentrations of nucleic acid compounds was found to suppress the proliferation of both hepatocytes and hepatoma cells. These results suggest that addition of optimal amounts of nucleic acid compounds such as nucleosides and nucleotides would enhance growth of hepatocytes, particularly during liver regeneration, but that they may also enhance proliferation of tumor cells in the liver.

Effect of methionine-deprived nutrition on cell growth and cell kinetics in cell cultures and experimental tumors.

The effect of methionine-deprived nutrition on cell growth and cell kinetics was investigated in cell cultures and in tumor-bearing rats using the total parenteral nutrition (TPN) technique. A simultaneous flow cytometric measurement of the cellular DNA content and the amount of 5-bromodeoxyuridine incorporated into cellular DNA was performed for analysis of cell kinetics. The methionine-free medium demonstrated a cytocidal effect on the growth of SLC cells after 6 hours of culturing. It decreased viability from 80% in the control medium to 23%, and it decreased the S phase and increased the G0/G1 phase of the cell cycles. The methionine-deprived medium showed a concentration-dependent inhibition in cellular growth. Methionine-deprived TPN was seen to inhibit AH109A and SLC tumor growth compared with conventional TPN and decreased the S phase and increased the G0/G1 phase of cell cycles. These results confirm that methionine deprivation blocks cells from processing into the G1 phase and recycling, and that it is effective in inhibiting tumor growth in cultures and in vivo.

A Novel Therapeutic and Prophylactic Vaccine (HVJ-Envelope / Hsp65 DNA + IL-12 DNA) against Tuberculosis Using the Cynomolgus Monkey Model.

We have developed a novel tuberculosis (TB) vaccine; a combination of the DNA vaccines expressing mycobacterial heat shock protein 65 (HSP65) and interleukin 12 (IL-12) delivered by the hemagglutinating virus of Japan (HVJ)-envelope and -liposome (HSP65 + IL-12/HVJ). An IL-12 expression vector (IL-12DNA) encoding single-chain IL-12 proteins comprised of p40 and p35 subunits were constructed. This vaccine provided remarkable protective efficacy in mouse and guinea pig models compared to the BCG vaccine on the basis of C.F.U of number of TB, survival, an induction of the CD8 positive CTL activity and improvement of the histopathological tuberculosis lesions. This vaccine also provided therapeutic efficacy against multi-drug resistant TB (MDR-TB) and extremely drug resistant TB (XDR-TB) (prolongation of survival time and the decrease in the number of TB in the lung) in murine models. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. This novel vaccine provided a higher level of the protective efficacy than BCG based upon the assessment of mortality, the ESR, body weight, chest X-ray findings and immune responses. All monkeys in the control group (saline) died within 8 months, while 50% of monkeys in the HSP65+hIL-12/HVJ group survived more than 14 months post-infection (the termination period of the experiment). Furthermore, the BCG priming and HSP65 + IL-12/HVJ vaccine (booster) by the priming-booster method showed a synergistic effect in the TB-infected cynomolgus monkey (100% survival). In contrast, 33% of monkeys from BCG Tokyo alone group were alive (33% survival). Furthermore, this vaccine exerted therapeutic efficacy (100% survival) and augmentation of immune responses in the TB-infected monkeys. These data indicate that our novel DNA vaccine might be useful against Mycobacterium tuberculosis including XDR-TB and MDR-TB for human therapeutic clinical trials.

Activin as a cell differentiation factor.

Activin, originally discovered as a polypeptide hormone that is capable of stimulating follicle-stimulating hormone secretion from pituitary cells in vitro, has recently been found to have a much wider range of biological activities. There are a number of reports of activin action as a cell differentiation factor on various types of cells rather than as a modulator of hormone secretion, as predicted initially, based on its structural similarity to transforming growth factor-beta. Studies of the distribution of activin and its receptor in a variety of tissues and its wide-ranging actions clearly illustrates its multifunctional properties. In particular, activin has been shown to be a potential regulator of early development of Xenopus laevis. Observation of activin effect in embryogenesis is of general importance to our understanding of the role of the family of growth factors in developmental processes.

Molecular cloning and functional analysis of a new activin beta subunit: a dorsal mesoderm-inducing activity in Xenopus.

A cDNA that encodes a new member of the TGF-beta superfamily most similar to activin beta A, beta B, and recently reported beta C has been isolated from Xenopus laevis. Expression of the gene in early embryos suggested its biological importance in Xenopus embryogenesis. Microinjection of a synthetic mRNA transcribed from the cDNA into ventral blastomeres of early Xenopus embryo led to the formation of secondary body axis. Mesodermal marker genes were induced in isolated animal cap by a similar mRNA injection. These results demonstrate that the gene encodes a new member of activin subfamily whose function is closely related to mesoderm induction.

Involvement of frizzled-10 in Wnt-7a signaling during chick limb development.

The dorsal ectoderm of the limb bud is known to regulate anterior-posterior patterning as well as dorsal-ventral patterning during vertebrate limb morphogenesis. Wnt-7a, expressed in the dorsal ectoderm, encodes a key molecule implicated in these events. In the present study, chicken frizzled-10 (Fz-10) encoding a Wnt receptor was used to study mechanisms of Wnt-7a signaling during chick limb patterning, because its expression is restricted to the posterior-distal region of the dorsal limb bud. Fz-10 transcripts colocalize with Sonic hedgehog (Shh) in the dorsal side of stages 18-23 chick limb buds. It was demonstrated that Fz-10 interacts with Wnt-7a to induce synergistically the expression of Wnt-responsive genes, such as Siamois and Xnr3, in Xenopus animal cap assays. In the chick limb bud, Fz-10 expression is regulated by Shh and a signal from the dorsal ectoderm, presumably Wnt-7a, but not by signals from the apical ectodermal ridge. These results suggest that Fz-10 acts as a receptor for Wnt-7a and has a positive effect on Shh expression in the chick limb bud.

Immunodetection of Xenopus bone morphogenetic protein-4 in early embryos.

Specific antibodies to Xenopus laevis bone morphogenetic protein-4 (xBMP-4) were raised by immunizing rabbits with a fusion protein of bacterial beta-galactosidase and xBMP-4. The antibodies were used to detect xBMPs expressed in mammalian cells by Western blotting. The antibodies were found to recognize xBMP-4 specifically and not to cross-react with either xBMP-2 or xBMP-7 which are similar to xBMP-4. In addition, the antibodies recognized dimeric xBMP-4 whereas our previous antibodies recognized the reduced form only. The present antibodies detected an immunoreactive 27 kDa protein in extracts of developing Xenopus embryos from oocyte to tailbud embryo. The xBMP-4 peptide appeared to be monomeric in structure because the molecular weight did not shift upon reduction of disulfide bond(s).