Hyperforin inhibits cell proliferation and differentiation in mouse embryonic stem cells.

OBJECTIVES: Hyperforin, a phloroglucinol derivative of St. John's Wort, has been identified as the major molecule responsible for this plant's products anti-depressant effects. It can be expected that exposure to St. John's Wort during pregnancy occurs with some frequency although embryotoxic or teratogenic effects of St. John's Wort and hyperforin have not yet been experimentally examined in detail. In this study, to determine any embryotoxic effects of hyperforin, we have attempted to determine whether hyperforin affects growth and survival processes of employing mouse embryonic stem (mES) cells (representing embryonic tissue) and fibroblasts (representing adult tissues). MATERIALS AND METHODS: We used a modified embryonic stem cell test, which has been validated as an in vitro developmental toxicity protocol, mES cells, to assess embryotoxic potential of chemicals under investigation. RESULTS: We have identified that high concentrations of hyperforin inhibited mouse ES cell population growth and induced apoptosis in fibroblasts. Under our cell culture conditions, ES cells mainly differentiated into cardiomyocytes, although various other cell types were also produced. In this condition, hyperforin affected ES cell differentiation into cardiomyocytes in a dose-dependent manner. Analysis of tissue-specific marker expression also revealed that hyperforin at high concentrations partially inhibited ES cell differentiation into mesodermal and endodermal lineages. CONCLUSIONS: Hyperforin is currently used in the clinic as a safe and effective antidepressant. Our data indicate that at typical dosages it has only a low risk of embryotoxicity; ingestion of large amounts of hyperforin by pregnant women, however, may pose embryotoxic and teratogenic risks.

α(1D)-Adrenoceptor regulates the vasopressor action of α(1A)-adrenoceptor in mesenteric vascular bed of α(1D)-adrenoceptor knockout mice.

1 The pressor action of the α(1A)-adrenoceptor (α(1A)-AR) agonist A61603 (N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl] methanesulfonamide) and the α(1)-ARs agonist phenylephrine and their blockade by selective α(1)-ARs antagonists in the isolated mesenteric vascular bed of wild-type (WT) mice and α(1D)-AR knockout (KO α(1D)-AR) mice were evaluated. 2 The apparent potency of A61603 to increase the perfusion pressure in the mesenteric vascular bed of WT and KO α(1D)-AR mice is 86 and 138 times the affinity of phenylephrine, respectively. 3 A61603 also enhanced the perfusion pressure by ≈1.7 fold in the mesenteric vascular bed of WT mice compared with KO α(1D)-AR mice. 4 Because of its high affinity, low concentrations of the α(1A)-AR selective antagonist RS100329 (5-methyl-3-[3-[4-[2-(2,2,2,-trifluoroethoxy) phenyl]-1-piperazinyl] propyl]-2,4-(1H)-pyrimidinedione) shifted the agonist concentration-response curves to the right in the mesenteric vascular bed of WT and KO α(1D)-AR mice. 5 The α(1D)-AR selective antagonist BMY7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5] decane-7,9-dione) did not modify the A61603 or the phenylephrine-induced pressor effect. 6 The α(1B/D)-ARs alkylating antagonist chloroethylclonidine (CEC) shifted the agonist concentration-response curves to the right and decreased the maximum phenylephrine-induced vascular contraction in KO α(1D)-AR mice when compared to WT mice; however, CEC only slightly modified the contraction induced by A61603. 7 The results indicate that the isolated mesenteric vascular bed of WT and KO α(1D)-AR mice expresses α(1A)-AR, that the pressor action of α(1A)-AR is up-regulated for α(1D)-AR in WT mice and suggest an important role of α(1B)-AR in the vascular pressure evoked by phenylephrine in KO α(1D)-AR mice.

Angiotensin II modifies the expression of α(1)-adrenoceptors in aorta smooth muscle cells of α(1D)-adrenoceptor knockout mice.

1 The effect of angiotensin II (Ang II) on α(1A)-, α(1B)-, and a(1D)-adrenoceptors (α(1)-AR) expression was analyzed in aorta smooth muscle cells obtained from wild-type (WT) and knock out of α(1D)-AR (α(1D)-AR KO) mice. 2 The relative abundance of mRNA for the three α(1)-ARs was determined in WT and α(1D)-AR KO aortic smooth muscle cells. There were no significant differences between WT and α(1D)-AR KO cells. 3 As early as 1 h Ang II increased α(1B)-AR mRNA in WT cells ≈ 2 fold compared with control; in contrast, in α(1D)-AR KO cells the α(1B)-AR transcript was ≈ 50% of control. 4 Western blot assays showed that Ang II incremented protein content for α(1A)-AR, 86% and 107% in WT and α(1D)-AR KO cells, respectively. 5 Protein for α(1B)- and α(1D)-ARs did not change significantly with Ang II in both WT and a(1D)-AR KO cells. 6 The effect of Ang II on α(1B)-AR mRNA seems to be influenced by the absence of α(1D)-AR in aortic smooth muscle cells, which might be important to understand the interactions among α(1)-ARs.

Alpha(1D)-adrenoceptors mediate nerve and agonist-evoked contractions in mouse vas deferens: evidence obtained from knockout technology.

1 It has been demonstrated that nerve-evoked contractions of the rat vas deferens involve alpha(1D)-adrenoceptors. Definitive evidence for a similar alpha(1D)-adrenoceptor-mediated response in mouse vas deferens has been more difficult to obtain. In this study, we have used alpha(1D)-adrenoceptor knockout (alpha(1D)-KO) mice to aid in the pharmacological characterization. 2 Mouse whole vas deferens was stimulated with a single pulse every 5 min. Once a stable response had been obtained, vehicle or antagonist was administered cumulatively at 5-min intervals and a response to stimulation obtained 5 min later. Cumulative concentration-response curves were also obtained for noradrenaline. 3 In vas deferens from alpha(1D)-KO mice, the contractile response to low concentrations of noradrenaline and the contractile response to a single stimulus were significantly reduced as compared to wild type (WT). 4 The alpha(1D)-adrenoceptor selective antagonist, BMY 7378, produced a concentration-dependent inhibition of single pulse-evoked contractions of vas deferens from WT and alpha(1D)-KO mice. BMY 7378 was significantly less potent in inhibiting stimulation-evoked contractions in vas deferens from alpha(1D)-KO mice. 5 It is concluded that alpha(1D)-adrenoceptors mediate a component of nerve- and agonist-evoked contractions of the vas deferens of WT mice.

Study of antibody titres after measles vaccination: fever within 7 days of vaccination and efficacy of booster doses.

We investigated seroconversion rates in febrile children after measles vaccination. Among 6364 vaccinees, 501 children had a temperature of 37.5 degrees C or higher within 7 days of vaccination. The seroconversion rate assessed by a haemagglutination-inhibition assay was 76.6% in 501 febrile children but 95.2% in 84 afebrile controls. Measles vaccination has been reported to provide immunity in at least 95% of cases. The number of patients infected with measles has dramatically decreased since the introduction of measles vaccination. However, problems remain, including primary vaccine failure (PVF), failure to develop immunity after vaccination, and secondary vaccine failure (SVF), that is, the development of infection because of waning antibodies after vaccination. In this study, we investigated the effect of febrile upper respiratory tract infection (URTI) after vaccination and found a lower rate of seroconversion to measles and a lower mean antibody titre in those who developed a fever within 7 days of measles vaccination.

Alpha1-adrenoceptors are required for normal male sexual function.

BACKGROUND AND PURPOSE: Alpha(1)-adrenoceptor antagonists are extensively used in the treatment of hypertension and lower urinary tract symptoms associated with benign prostatic hyperplasia. Among the side effects, ejaculatory dysfunction occurs more frequently with drugs that are relatively selective for alpha(1A)-adrenoceptors compared with other drugs of this class. This suggests that alpha(1A)-adrenoceptors may contribute to ejaculation. However, this has not been studied at the molecular level. EXPERIMENTAL APPROACH: The physiological contribution of each alpha(1)-adrenoceptor subtype was characterized using alpha(1)-adrenoceptor subtype-selective knockout (KO) mice (alpha(1A)-, alpha(1B)- and alpha(1D)-AR KO mice) since the subtype-specific drugs available are only moderately selective. We analysed the role of alpha(1)-adrenoceptors in the blood pressure and vascular response as well as ejaculation by determining these variables in alpha(1)-adrenoceptor subtype-selective KO mice and in mice with all their alpha(1)-adrenoceptor subtypes deleted (alpha(1)-AR triple-KO mice). KEY RESULTS: The pregnancy rate was reduced by 50% in alpha(1A)-adrenoceptor KO mice, and this reduction was dramatically enhanced in alpha(1)-adrenoceptor triple-KO mice. Contractile tension of the vas deferens in response to noradrenaline was markedly decreased in alpha(1A)-adrenoceptor KO mice, and this contraction was completely abolished in alpha(1)-adrenoceptor triple-KO mice. This attenuation of contractility was also observed in the electrically stimulated vas deferens. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that alpha(1)-adrenoceptors, particularly alpha(1A)-adrenoceptors, are required for normal contractility of the vas deferens and consequent sperm ejaculation as well as having a function in fertility.

Body water balance and body temperature in vasopressin V1b receptor knockout mice.

In an attempt to determine whether there is a specific vasopressin receptor (V(1b)) subtype involved in the regulation of body water balance and temperature, vasopressin V(1b) receptor knockout mice were used. Daily drinking behavior and renal excretory function were examined in V(1b)-deficient (V(1b)(-/-)) and control (V(1b)(+/+)) mice under the basal and stress-induced condition. In addition, body temperature and locomotor activity were measured with a biotelemetry system. The baseline daily water intake and urine volume were larger in V(1b)(-/-) mice than in V(1b)(+/+) mice. V(1b)(-/-) mice (V(1b)(-/-)) had significantly higher locomotor activity than wild-type, whereas the body temperature and oxygen consumption were lower in V(1b)(-/-) than in the V(1b)(+/+) mice. Next, the V(1b)(-/-) and V(1b)(+/+) mice were subjected to water deprivation for 48 hr. Under this condition, their body temperature decreased with the time course, which was significantly larger for V(1b)(-/-) than for V(1b)(+/+) mice. Central vasopressin has been reported to elicit drinking behavior and antipyretic action, and the V(1b) receptor has been reported to be located in the kidney. Thus, the findings suggest that the V(1b) receptor may be, at least in part, involved in body water balance and body temperature regulation.

Chloroethylclonidine reveals that alpha (1 A)-adrenoceptors mediate contraction in aorta of alpha (1 D)-adrenoceptor knockout mice.

1 We have characterized the alpha(1)-adrenoceptor subtypes present in isolated aorta of the alpha(1D)-adrenoceptor knockout (KO) mice, by chloroethylclonidine (CEC)-induced alkylation and their protection by selective alpha(1)-adrenoceptor antagonists. 2 The alpha(1D)-adrenoceptor is involved in the contractile response to noradrenaline in wild type (WT) mouse aorta. 3 In WT mice 5-methylurapidil (5-MU, an alpha(1A)-adrenoceptor antagonist) or BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl] ethyl]-8-azaspiro[4.5] decane-7,9 dione, a selective alpha(1D)-adrenoceptor antagonist), protected the receptors from CEC-induced (alpha(1B/D)-adrenoceptor) alkylation, the combination of both antagonists resulted in complete protection, while AH11110A (1-[biphenyl-2-yloxy]-4-imino-4-piperidin-1-yl-butan-2-ol, an alpha(1B)-adrenoceptor antagonist) did not protect. 4 In aorta of KO mice there was a 19-fold rightward shift in noradrenaline effective concentration (EC(50)) compared with WT; while 5-MU alone or in combination with AH11110A protected alpha(1)-adrenoceptors to the same extent. 5 The data indicate that alpha(1A)-adrenoceptors mediate contraction and suggest their role in maintaining homeostasis in the alpha(1D)-adrenoceptors KO mice.

MCM3AP, a novel acetyltransferase that acetylates replication protein MCM3.

The MCM proteins are essential for the initiation of DNA replication. We have isolated an MCM3-associated protein (MCM3AP) in a two-hybrid screen using MCM3. Here we demonstrate that MCM3AP is an acetyltransferase which acetylates MCM3 and that chromatin-bound MCM3 is acetylated in vivo. The MCM3 acetylase, MCM3AP, is also chromatin-bound. This study also indicates that MCM3AP contains putative acetyl CoA binding motifs conserved within the GCN5-related N-acetyltransferase superfamily. Mutation of those motifs significantly inhibits the MCM3 acetylase activity. Over-expression of MCM3AP inhibits DNA replication, whereas mutation of the acetylase motifs abolishes this effect, suggesting that acetylation plays a role in DNA replication. Taken together, we suggest that MCM3 acetylation is a novel pathway which might regulate DNA replication.

Functional genomic research of alpha 1-adrenoceptors.

The Human Genome Project is now almost completed, and we are about to move into the post-genome sequence era of functional genomics. The advent of genome science has markedly changed the way life science research including pharmacological study is conducted; thus, systematic and integrated 'genome-wide' survey is feasible. The stream of 'Genome-->Transcriptome--> Proteomics' is logical and, in each aspect, approaches for functional genomics are now pursued at a high pace. We have recently developed a standardized technical platform (in various levels, such as transcription, cell and whole animal levels, etc.), and applied these techniques to the study of functional genomics of G-protein-coupled receptors, particularly alpha1-adrenoceptors as a model. Combining the genome information and technology, future pharmacological studies would become the genome-based search and research.

[High-throughput techniques for analyzing SNPs and application of SNPs to pharmacogenomics].

Progress in the human genome project can contribute to molecular diagnosis of various diseases and development of novel treatments. The next logical step for human genetics is the exploration and elucidation of genes involved in differential pharmacological response. An understanding of the role that genes have in pharmacological response is the cornerstone of personalized medicine. The pharmacogenomics approach is necessary and useful for this purpose. This article introduces these concepts to provide a context for the use of SNPs in pharmacogenomics and high-throughput techniques to analyze them.

Characterization of calcium signaling by purinergic receptor-channels expressed in excitable cells.

ATP-gated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct Ca(2+) and facilitate voltage-sensitive Ca(2+) entry in excitable cells. To study Ca(2+) signaling by P2XRs and its dependence on voltage-sensitive Ca(2+) influx, we expressed eight cloned P2XR subtypes individually in gonadotropin-releasing hormone-secreting neurons. In all cases, ATP evoked an inward current and a rise in [Ca(2+)](i). P2XR subtypes differed in the peak amplitude of [Ca(2+)](i) response independently of the level of receptor expression, with the following order: P2X(1)R < P2X(3)R < P2X(4)R < P2X(2b)R < P2X(2a)R < P2X(7)R. During prolonged agonist stimulation, Ca(2+) signals desensitized with different rates: P2X(3)R > P2X(1)R > P2X(2b)R > P2X(4)R >> P2X(2a)R >> P2X(7)R. The pattern of [Ca(2+)](i) response for each P2XR subtype was highly comparable with that of the depolarizing current, but the activation and desensitization rates were faster for the current than for [Ca(2+)](i). The P2X(1)R, P2X(3)R, and P2X(4)R-derived [Ca(2+)](i) signals were predominantly dependent on activation of voltage-sensitive Ca(2+) influx, both voltage-sensitive and -insensitive Ca(2+) entry pathways equally contributed to [Ca(2+)](i) responses in P2X(2a)R- and P2X(2b)R-expressing cells, and P2X(7)R operated as a nonselective pore capable of conducting larger amounts of Ca(2+) independently on the status of voltage-gated Ca(2+) channels. Thus, Ca(2+) signaling by homomeric P2XRs expressed in an excitable cell is subtype-specific, which provides an effective mechanism for generating variable [Ca(2+)](i) patterns in response to a common agonist.

Cloning and characterization of the promoter of murine cytohesin-1 gene.

Mouse cytohesin-1 (CTH-1) is a guanine nucleotide exchange factor, specific for ADP ribosylation factors. The CTH-1 gene promoter was characterized by deletion mapping and mutational analysis. The region between -101 and -38 relative to the transcription start site showed the essential promoter activity when transfected into both NIH3T3 and COS7 cells. The nucleotides of the core promoter region contain three tandem GC boxes, which can offer potential binding sites for the ubiquitous transcription factor Sp-1 family. Mutational analysis revealed that these tandem GC boxes are indispensable for activation of the gene transcription.

A novel nonsense mutation of the PEPD gene in a Japanese patient with prolidase deficiency.

A nonsense mutation at amino acid residue 184 in the human peptidase D (PEPD) gene caused the production of a truncated polypeptide. Characterizing molecular defects in patients provides clues to elucidate the relationship between the phenotype and the genotype.

Characterization of the mouse alpha1D-adrenergic receptor gene.

Alpha1-adrenergic receptors (alpha1-ARs) play critical roles in the regulation of a variety of physiological processes. Increasing evidence suggests that multiple receptor subtypes of alpha1-ARs regulate these physiological processes. Molecular cloning has identified three distinct cDNAs encoding alpha1-AR subtypes (alpha1A, alpha1B and alpha1D) that are structurally homologous. Among the alpha1-AR subtypes, the function of the alpha1D-AR remains unclear. In order to examine the physiological role of alpha1D-AR, we cloned and characterized a gene for the mouse alpha1D-AR. Using a mouse alpha1D-AR cDNA as a probe, we isolated the gene for the mouse alpha1D-AR from a mouse genomic library. The alpha1D-AR consists of two exons and an intron that interrupts the coding region of the putative sixth transmembrane domain. The 5'-flanking region of exon 1 contains neither a TATA box nor a CAAT box but is high in GC content and contains several Sp1 binding sites (GC boxes). This pattern is similar to promoters described for other members of alpha1-ARs. The untranslated region also contains putative cyclic AMP response elements. Isolation of this gene will allow further investigation, via gene knock-outs and deletion mutants, of the mechanisms of transcriptional regulation and a greater understanding of the physiological role of alpha1D-AR.

Structure and sequence of the mouse V1a and V1b vasopressin receptor genes.

The structural organization and 5'-flanking region of the mouse V1a and V1b vasopressin receptor genes were determined. The mouse V1a receptor gene was located within an 8-kb XbaI fragment, and the mouse V1b receptor gene was located within a 14-kb EcoRV fragment. Both genes were comprised of two coding exons that were separated by a 2.3-kb and a 8.0-kb intron, respectively, located before the respective seventh transmembrane domain of the receptor sequence. The availability of these genes would allow us to study the functional role of V1a and V1b receptors by disrupting the gene in mice.

Excess copper and ceruloplasmin biosynthesis in long-term cultured hepatocytes from Long-Evans Cinnamon (LEC) rats, a model of Wilson disease.

Immortalized hepatic cell lines obtained from laboratory animals or patients with defects in copper metabolism in the liver provide new approaches to examine related metabolism and toxicity. We established a series of hepatic cell lines from the liver of Long-Evans Cinnamon (LEC) rats, using recombinant adenovirus which expresses SV40 large T. Cells from the LEC rats were cultured and accumulated larger amounts of copper than did control cells, when the concentrations of copper in the culture medium exceeded 5 microM. The secretion of ceruloplasmin (CP) from the cultured cells was not reduced in hepatocytes from LEC cells, as compared with the control cells. As accumulation of copper did not affect CP secretion, CP production was not likely to be affected by the accumulation of copper in LEC rat hepatocytes. The production of holo-CP was further investigated by transfection of human CP cDNA and detection of human holo-CP by immunological procedures and use of a monoclonal antibody (mAb CP60) which recognizes human holo-CP but not human apo-CP and rat CP. Hepatocytes from the LEC rats processed and secreted holo-CP into the medium, even with excess copper present in the medium. These observations suggest that the genetic defect in LEC rats did not alter biosynthetic and secretory pathways of CP and that the intracellular copper concentration did not regulate the synthesis and processing of CP in the cultured hepatocytes. Low ceruloplasmin levels are observed in most, but not all, patients with Wilson disease, as well as in LEC rats. Our results do suggest that the copper transporting ATPase encoded in the Wilson disease gene is not a integral part of the biochemical mechanism of copper incorporation into apoprotein. The cell lines and immunological procedures we used are expected to add to information on biologically important process related to copper metabolism and to CP biosynthesis.

Structural organization and analysis of the human fumarylacetoacetate hydrolase gene in tyrosinemia type I.

Fumarylacetoacetate hydrolase (FAH) is a metabolic enzyme functioning at the last step of tyrosine catabolism. Deficiency in this enzyme activity is associated with tyrosinemia type I, characterized by hypertyrosinemia, liver dysfunction, renal tubular dysfunction, liver cirrhosis, and hepatic tumors. We isolated from a human gene library a chromosomal gene related to FAH. The human FAH gene is 30 kilobases long and is split into 14 exons. All of the splice donor and acceptor sites conform to the GT/AG rule. We also analyzed findings in a patient with tyrosinemia type I with respect to the mutation responsible for defects in the enzyme. A nucleotide change from T to G was found in the exon 2 of the gene and this change was accompanied by an amino acid substitution (Phe62Cys). Transfection and expression analysis of the cDNA in cultured BMT-10 cells with the nucleotide substitution demonstrated that the substitution was indeed responsible for the decreased activity of the enzyme in the patient. These results confirmed that the T to G mutation was one of the causes of tyrosinemia type I. Structure of the FAH gene and tests for expression of the mutant FAH will facilitate further understanding of various aspects of FAH.