Effects of auxin and gibberellin on conidial germination and elongation of young hyphae in a cyclic 3′:5′ adenosine monophosphate-dependent protein kinase mutant of Neurospora crassa

The possibility that plant growth regulators may relate to a cyclic 3:5 adenosine monophosphate (cAMP)-dependent protein kinase through the control of cAMP level in the conidial germination process of Neurospora crassa was examined using a cAPM-dependent protein kinase mutant (cpk mutant) which is thought to be cAMP-independent because of defect in the regulatory subunit of cAMP-dependent protein kinase. IAA, 2,4-D and GA₃ promoted conidial germination and elongation of young hyphae in the mutant as well as in the wild-type. The result suggests that the effects of auxin and gibberellin on germination and hyphal elongation are not mediated by cAMP.     

Cardiac myocyte-specific ablation of follistatin-like 3 attenuates stress-induced myocardial hypertrophy

Transforming growth factor-β family cytokines have diverse actions in the maintenance of cardiac homeostasis. Follistatin-like 3 (Fstl3) is an extracellular regulator of certain TGF-β family members, including activin A. The aim of this study was to examine the role of Fstl3 in cardiac hypertrophy. Cardiac myocyte-specific Fstl3 knock-out (KO) mice and control mice were subjected to pressure overload induced by transverse aortic constriction (TAC). Cardiac hypertrophy was assessed by echocardiography and histological and biochemical methods. KO mice showed reduced cardiac hypertrophy, pulmonary congestion, concentric LV wall thickness, LV dilatation, and LV systolic dysfunction after TAC compared with control mice. KO mice displayed attenuated increases in cardiomyocyte cell surface area and interstitial fibrosis following pressure overload. Although activin A was similarly up-regulated in KO and control mice after TAC, a significant increase in Smad2 phosphorylation only occurred in KO mice. Knockdown of Fstl3 in cultured cardiomyocytes inhibited PE-induced cardiac hypertrophy. Conversely, adenovirus-mediated Fstl3 overexpression blocked the inhibitory action of activin A on hypertrophy and Smad2 activation. Transduction with Smad7, a negative regulator of Smad2 signaling, blocked the antihypertrophic actions of activin A stimulation or Fstl3 ablation. These findings identify Fstl3 as a stress-induced regulator of hypertrophy that controls myocyte size via regulation of Smad signaling.     

Calreticulin Induces Dilated Cardiomyopathy

Background

Calreticulin, a Ca²⁺-buffering chaperone of the endoplasmic reticulum, is highly expressed in the embryonic heart and is essential for cardiac development. After birth, the calreticulin gene is sharply down regulated in the heart, and thus, adult hearts have negligible levels of calreticulin. In this study we tested the role of calreticulin in the adult heart.

Methodology/Principal Findings

We generated an inducible transgenic mouse in which calreticulin is targeted to the cardiac tissue using a Cre/loxP system and can be up-regulated in adult hearts. Echocardiography analysis of hearts from transgenic mice expressing calreticulin revealed impaired left ventricular systolic and diastolic function and impaired mitral valve function. There was altered expression of Ca²⁺ signaling molecules and the gap junction proteins, Connexin 43 and 45. Sarcoplasmic reticulum associated Ca²⁺-handling proteins (including the cardiac ryanodine receptor, sarco/endoplasmic reticulum Ca²⁺-ATPase, and cardiac calsequestrin) were down-regulated in the transgenic hearts with increased expression of calreticulin.

Conclusions/Significance

We show that in adult heart, up-regulated expression of calreticulin induces cardiomyopathy in vivo leading to heart failure. This is due to an alternation in changes in a subset of Ca²⁺ handling genes, gap junction components and left ventricle remodeling.     

Monitoring of Ralstonia eutropha KT1 in Groundwater in an Experimental Bioaugmentation Field by In Situ PCR

Ralstonia eutropha KT1, which degrades trichloroethylene, was injected into the aquifer after activation with toluene, and then the number of bacteria was monitored by in situ PCR targeting the phenol hydroxylase gene and by fluorescent in situ hybridization (FISH) targeting 16S rRNA. Before injection of the bacterial suspension, the total concentration of bacteria in the groundwater was approximately 3 × 10⁵ cells/ml and the amount of Ralstonia and bacteria carrying the phenol hydroxylase gene as a percentage of total bacterial cells was less than 0.1%. The concentration of bacteria carrying the phenol hydroxylase gene detected by in situ PCR was approximately 3 × 10⁷ cells/ml 1 h after injection, and the concentration of Ralstonia detected by FISH was similar. The number of bacteria detected by in situ PCR was similar to that detected by FISH 4 days after the start of the extraction of groundwater. On and after day 7, however, the number of bacterial cells detected by FISH was less than that detected by in situ PCR.     

Mutations that alter the allosteric nature of cAMP receptor protein of Escherichia coli.

Mutations which permit cAMP binding protein (CRP) to act in the absence of cAMP have been isolated by in vitro mutagenesis of a plasmid containing the cloned crp gene. Adenylate cyclase deficient cells harbouring the mutant (crp*) plasmids exhibited a variety of fermentation profiles on MacConkey indicator plates containing various sugars. beta-galactosidase synthesis in cells carrying the crp* plasmids was activated most by the addition of cGMP as well as cAMP. The sites of mutations which are responsible for the cAMP independent phenotype were determined by in vitro recombination and DNA sequencing. The amino acid substitutions in the mutant proteins were found in two specific regions of the crp gene encoding residues 53-62 and 141-148 of CRP polypeptide. The first region may participate in cAMP binding, while the second appears to be the inter-domain region of the N-terminal cAMP-binding and C-terminal DNA-binding domains.     

Establishment of a monoclonal antibody PMab-233 for immunohistochemical analysis against Tasmanian devil podoplanin

Monoclonal antibodies (mAbs) against not only human, mouse, and rat but also rabbit, dog, cat, bovine, pig, and horse podoplanins (PDPNs) have been established in our previous studies. PDPN is used as a lymphatic endothelial cell marker in pathological diagnoses. However, mAbs against Tasmanian devil PDPN (tasPDPN), which are useful for immunohistochemical analysis, remain to be developed. Herein, mice were immunized with tasPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/tasPDPN) cells, and hybridomas producing mAbs against tasPDPN were screened using flow cytometry. One of the mAbs, PMab-233 (IgG₁, kappa), specifically detected CHO/tasPDPN cells by flow cytometry and recognized tasPDPN protein by western blotting. Furthermore, PMab-233 strongly detected CHO/tasPDPN cells by immunohistochemistry. These findings suggest that PMab-233 may be useful as a lymphatic endothelial cell marker of the Tasmanian devil.     

Excitation and deexcitation processes of Eu(III) benzo-15-crown-5 complex studied on comparing with its first coordination sphere

The coordination sphere and the deexcitation mechanism of the Eu(III) benzo-15-crown-5 complex, Eu(B15C5), were studied with references of the Eu(III) complexes with a similar coordination sphere; the dibenzo-18-crown-6 complex, Eu₃(B₂18C6)₂, and the cryptand[2.2.1] complex, Eu([2.2.1]). NMR spectroscopy reveals that the Eu(B15C5) complex is quite stable in acetonitrile solution whereas only 40% of the Eu(III) ion forms the complex in the equimolar Eu(NO₃)₃ and B₂18C6 acetonitrile solution. The coordination sphere of the Eu(III) complexes in acetonitrile solutions were also discussed by the degenerate ⁷F₀→⁵D₀ transition energy levels. The Eu(B15C5) have a negative shift compared with the europium(III) nitrate in acetonitrile and it is explained by the coordination of both nitrate ions and the crown ether ligand. Energy transfer from the n–π* excited state located in the catechol structure to the central europium ion was first observed as the sensitized luminescence of ⁵D₀→⁷F_J. The excited state lifetime of the Eu(B15C5) complex was first determined as 201 μs in the present study.