Role of bradykinin in acid-induced mesenteric hyperemia and duodenal villous damage

Intestinal mucosal capsaicin-sensitive afferent nerves mediate, in part, the mesenteric hyperemia after intraduodenal acidification. The hyperemia plays a role in protecting the duodenal mucosa against acid damage. We tested the hypothesis that bradykinin contributes to this protective hyperemia. A specific antagonist of bradykinin will attenuate the hyperemia and exacerbate duodenal villous damage induced by acid. Study 1: Intravenous vehicle, or the specific bradykinin B2 receptor antagonist (HOE 140) was administered to anesthetized rats. This was followed by intraduodenal bolus administration of 160 microM capsaicin or 0.1 N HCl, and then intravenous bradykinin. Study 2: Intravenous administration of vehicle or HOE 140 was followed by duodenal perfusion with 0.1 N HCl. Superior mesenteric artery blood flow (pulsed Doppler flowmetry) (Study 1) and duodenal villous damage (histology) (Study 2) were recorded. HOE 140 significantly reduced the hyperemia induced by bradykinin and intraduodenal capsaicin or acid. Deep villous injury was significantly increased after treatment with HOE 140. These findings support the hypothesis that acid-induced and afferent nerve-mediated mesenteric hyperemia is modulated by a mechanism that involves bradykinin B2 receptor. Antagonism of bradykinin B2 receptor also increased acid-induced deep duodenal villous damage. Thus, maintenance of bradykinin-mediated mesenteric hyperemia, is a previous unrecognized mechanism associated with protection of the rat duodenal mucosa against acid-induced damage.     

An In Vitro ES Cell-Based Clock Recapitulation Assay Model Identifies CK2α as an Endogenous Clock Regulator

We previously reported emergence and disappearance of circadian molecular oscillations during differentiation of mouse embryonic stem (ES) cells and reprogramming of differentiated cells, respectively. Here we present a robust and stringent in vitro circadian clock formation assay that recapitulates in vivo circadian phenotypes. This assay system first confirmed that a mutant ES cell line lacking Casein Kinase I delta (CKIδ) induced ∼3 hours longer period-length of circadian rhythm than the wild type, which was compatible with recently reported results using CKIδ null mice. In addition, this assay system also revealed that a Casein Kinase 2 alpha subunit (CK2α) homozygous mutant ES cell line developed significantly longer (about 2.5 hours) periods of circadian clock oscillations after in vitro or in vivo differentiation. Moreover, revertant ES cell lines in which mutagenic vector sequences were deleted showed nearly wild type periods after differentiation, indicating that the abnormal circadian period of the mutant ES cell line originated from the mutation in the CK2α gene. Since CK2α deficient mice are embryonic lethal, this in vitro assay system represents the genetic evidence showing an essential role of CK2α in the mammalian circadian clock. This assay was successfully applied for the phenotype analysis of homozygous mutant ES cells, demonstrating that an ES cell-based in vitro assay is available for circadian genetic screening.     

An Anthelmintic Drug,Pyrvinium Pamoate,Thwarts Fibrosis and Ameliorates Myocardial Contractile Dysfunction in a Mouse Model of Myocardial Infarction

Metabolic adaptation to limited supplies of oxygen and nutrients plays a pivotal role in health and disease. Heart attack results from insufficient delivery of oxygen and nutrients to the heart, where cardiomyocytes die and cardiac fibroblasts proliferate – the latter causing scar formation, which impedes regeneration and impairs contractility of the heart. We postulated that cardiac fibroblasts survive metabolic stress by adapting their intracellular metabolism to low oxygen and nutrients, and impeding this metabolic adaptation would thwart their survival and facilitate the repair of scarred heart. Herein, we show that an anthelmintic drug, Pyrvinium pamoate, which has been previously shown to compromise cancer cell survival under glucose starvation condition, also disables cardiac fibroblast survival specifically under glucose deficient condition. Furthermore, Pyrvinium pamoate reduces scar formation and improves cardiac contractility in a mouse model of myocardial infarction. As Pyrvinium pamoate is an FDA-approved drug, our results suggest a therapeutic use of this or other related drugs to repair scarred heart and possibly other organs.     

Podophyllotoxin and nocodazole counter the effect of IKP104 on tubulin decay

Tubulin, the subunit protein of microtubules, undergoes a time-dependent loss of functional properties known as decay. We have previously shown that the drug 2-(4-fluorophenyl)-1-(2-chloro-3,5-dimethoxyphenyl)-3-methyl-6-phenyl-4(1H)-pyridinone (IKP104) accelerates decay, but that in the presence of colchicine, IKP104 becomes a stabilizer of tubulin. To see if this is due to conformational effects specific to colchicine or simply to occupancy at the colchicine site, we examined the effects of nocodazole and podophyllotoxin, two well-known competitive inhibitors of colchicine for binding to tubulin, on IKP104’s acceleration of decay. We found that podophyllotoxin abolished IKP104’s accelerating effect and, like colchicine, turned it into a stabilizer of tubulin. Nocodazole’s effects were similar to those of podophyllotoxin and colchicine, in that it abolished IKP104-induced enhancement of decay; however, in the presence of nocodazole, IKP104 caused little or no stabilization of tubulin. Since colchicine, nocodazole, and podophyllotoxin have very different interactions with tubulin, but all inhibit the IKP104-induced enhancement of decay, our findings suggest that this inhibition arises from occupancy of the colchicine site rather than from a direct conformational effect of these two drugs.     

Efficient biallelic mutagenesis with Cre/loxP-mediated inter-chromosomal recombination

The isolation of mutant cells with phenotypes caused by random mutagenesis has been hampered in mammalian cells because there are two alleles per gene and the disruption of both alleles is extremely rare. We describe a method for the efficient biallelic mutagenesis in embryonic stem cells. loxP sites were introduced near the centromeric regions of a pair of chromosome 1s. A mutant neo gene was inserted at the distal part of one of the loxP sites so that biallelic mutants would be selected by high-dose G418. Expression of Cre induced the recombination between homologous chromosomes and led to an elevation in the number of biallelic mutants. This system will facilitate phenotype-driven gene function study in the mammalian system.     

Study on placental transmission of Pneumocystis carinii in mice using immunodeficient SCID mice as a new animal model.

Placental transmission of Pneumocystis carinii in mice was examined in 39 animals obtained by caesarean section from 17 pregnant SCID females experimentally infected with P. carinii. When examined with toluidine blue O, DAPI and immunofluorescent antibody stains, P. carinii was detected in the lungs of infected mothers but not in the lungs of caesarean section-derived neonates even after the neonates were treated with dexamethasone for 8 weeks. However, 13 neonates born to five infected females developed P. carinii pneumonia. These results indicate that P. carinii cannot be transmitted transplacentally in mice.     

G alpha 12/13- and reactive oxygen species-dependent activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase by angiotensin receptor stimulation in rat neonatal cardiomyocytes

In the present study, we examined signal transduction mechanism of reactive oxygen species (ROS) production and the role of ROS in angiotensin II-induced activation of mitogen-activated protein kinases (MAPKs) in rat neonatal cardiomyocytes. Among three MAPKs, c-Jun NH(2)-terminal kinase (JNK) and p38 MAPK required ROS production for activation, as an NADPH oxidase inhibitor, diphenyleneiodonium, inhibited the activation. The angiotensin II-induced activation of JNK and p38 MAPK was also inhibited by the expression of the Galpha(12/13)-specific regulator of G protein signaling (RGS) domain, a specific inhibitor of Galpha(12/13), but not by an RGS domain specific for Galpha(q). Constitutively active Galpha(12)- or Galpha(13)-induced activation of JNK and p38 MAPK, but not extracellular signal-regulated kinase (ERK), was inhibited by diphenyleneiodonium. Angiotensin II receptor stimulation rapidly activated Galpha(13), which was completely inhibited by the Galpha(12/13)-specific RGS domain. Furthermore, the Galpha(12/13)-specific but not the Galpha(q)-specific RGS domain inhibited angiotensin II-induced ROS production. Dominant negative Rac inhibited angiotensin II-stimulated ROS production, JNK activation, and p38 MAPK activation but did not affect ERK activation. Rac activation was mediated by Rho and Rho kinase, because Rac activation was inhibited by C3 toxin and a Rho kinase inhibitor, Y27632. Furthermore, angiotensin II-induced Rho activation was inhibited by Galpha(12/13)-specific RGS domain but not dominant negative Rac. An inhibitor of epidermal growth factor receptor kinase AG1478 did not affect angiotensin II-induced JNK activation cascade. These results suggest that Galpha(12/13)-mediated ROS production through Rho and Rac is essential for JNK and p38 MAPK activation.     

Developmental and functional analyses of CD8(+) NK1.1(+) T cells in class-I-restricted TCR transgenic mice.

Using a class-I-restricted T cell receptor (TCR) transgenic mice (Tgm), 2C (Valpha3.1/Vbeta 8.2, specific for L(d) + LSPFPFDL), the development and cytokine production of tg-TCR(+) NKT cells were analyzed. We found that CD8(+) or double negative (DN) NKT cells constituted a major population of NKT cells in the H-2(b/b) 2C Tgm (positive selecting background) or the H-2(b/d) 2C Tgm (negative selecting background), respectively. Virtually no NKT cells were generated in the H-2(k/k) 2C Tgm (neutral selecting background). CD8(+) NKT cells in the H-2(b/b) 2C Tgm expressed CD8alphabeta heterodimers, whereas those in the H-2(b/d) 2C Tgm expressed CD8alphaalpha homodimers. These findings suggest that development of a subpopulation of NKT cells is influenced by the H-2 molecules. Upon stimulation with anti-CD3 mAb, tg-TCR(+) NKT cells generated in the H-2(b/b) and H-2(b/d) backgrounds produced IFN-gamma, but not IL-4.