Identification of C4 responsive genes in the facultative C4 plant Hydrilla verticillata

The aquatic monocot Hydrilla verticillata (L.f.) Royle is a well-documented facultative C₄ NADP-malic enzyme species in which the C₄ and Calvin cycles operate in the same cell with the specific carboxylases confined to the cytosol and chloroplast, respectively. Several key components had already been characterized at the molecular level, thus the purpose of this study was to begin to identify other, less obvious, elements that may be necessary for a functional single-cell C₄ system. Using differential display, mRNA populations from C₃ and C₄ H. verticillata leaves were screened and expression profiles compared. From this study, 65 clones were isolated and subjected to a customized macroarray analysis; 25 clones were found to be upregulated in C₄ leaves. Northern and semi-quantitative RT-PCR analyses were used for confirmation. From these screenings, 13 C₄ upregulated genes were identified. Among these one encoded a previously recognized C₄ phosphoenolpyruvate carboxylase, and two encoded distinct pyruvate orthophosphate dikinase isoforms, new findings for H. verticillata. Genes that encode a transporter, an aminotransferase and two chaperonins were also upregulated. Twelve false positives, mostly housekeeping genes, were determined from the Northern/semi-quantitative RT-PCR analyses. Sequence data obtained in this study are listed in the dbEST database (DV216698 to DV216767). As a single-cell C₄ system that lacks Kranz anatomy, a better understanding of how H. verticillata operates may facilitate the design of a transgenic C₄ system in a C₃ crop species.Srinath K. Rao and Hiroshi Fukayama contributed equally to this study.     

Urotensin-II receptor blockade with SB-611812 attenuates cardiac remodeling in experimental ischemic heart disease

It is now well established that urotensin-II (UII) levels are increased in several cardiovascular diseases. We previously demonstrated that UII and the UII receptor (UT) protein levels are significantly increased in the hearts of both humans and rats with congestive heart failure (CHF). We have also recently demonstrated that UII blockade, with a selective UII antagonist, improves heart function in a rat model of ischemic CHF. Here, we evaluated the attenuation of cardiac remodeling associated with UII antagonism in the same rat model of ischemic CHF. Animals were administered a specific UT receptor antagonist, SB-611812 (30 mg/kg/day, gavage), or vehicle 30 min prior to coronary artery ligation followed by daily treatment for 8 weeks. Myocardial interstitial fibrosis was analyzed by Masson's trichrome and picrosirius red staining. RT-PCR analysis was utilized for mRNA expression studies. We used Western blotting to assess levels of collagen types I and III. Mitogenic activity of UII on cultured neonatal cardiac fibroblasts was also evaluated. Following coronary ligation, SB-611812 significantly attenuated both myocardial and endocardial interstitial fibrosis, and reduced collagen type I:III ratio (P<0.01). UII induced proliferation of cardiac fibroblasts and this mitogenic effect was significantly inhibited with 1 microM of SB-611218 (P<0.05). We demonstrate here that selective blockade of UT reduces diastolic dysfunction by decreasing myocardial fibrosis post-coronary ligation in vivo, and inhibits UII-mediated fibroblast proliferation in vitro.     

Adrenal expression of orexin receptor subtypes is differentially regulated in experimental streptozotocin induced type-1 diabetes

Orexins (hypocretins) are involved in the regulation of energy homeostasis and sleeping behavior. Orexins were also implicated in the regulation of neuroendocrine and autonomic functions. Recent data show the expression of orexin receptors within the hypothalamic-pituitary-adrenal (HPA) axis and suggest specific actions of orexins at the pituitary and adrenal glands. To further evaluate the role of orexin in the HPA axis, we investigated the mRNA expression of prepro-orexin (PPO) and orexin receptors within the HPA axis of streptozotocin-injected (STZ) rats showing type-1 like diabetes. PPO, as well as OX(1) and OX(2) receptor levels were analyzed by quantitative real-time PCR (qPCR). STZ rats were characterized by decreased body weight, plasma insulin, and leptin levels and by increased plasma glucose. Hypothalamic PPO mRNA levels were significantly reduced in STZ compared to non-diabetic control rats. No differences were found in the mRNA levels of hypothalamic or pituitary OX(1) and OX(2) receptors between control and STZ rats. In adrenals, OX(1) receptor mRNA levels were significantly elevated in STZ rats while OX(2) receptors were significantly reduced. Our results imply distinct functions of adrenal orexin receptor subtypes during type-1 like diabetes.     

Coordinated action of NSF and PKC regulates GABAB receptor signaling efficacy

The obligatory heterodimerization of the GABAB receptor (GBR) raises fundamental questions about molecular mechanisms controlling its signaling efficacy. Here, we show that NEM sensitive fusion (NSF) protein interacts directly with the GBR heterodimer both in rat brain synaptosomes and in CHO cells, forming a ternary complex that can be regulated by agonist stimulation. Inhibition of NSF binding with a peptide derived from GBR2 (TAT-Pep-27) did not affect basal signaling activity but almost completely abolished agonist-promoted GBR desensitization in both CHO cells and hippocampal slices. Taken with the role of PKC in the desensitization process, our observation that TAT-Pep-27 prevented both agonist-promoted recruitment of PKC and receptor phosphorylation suggests that NSF is a priming factor required for GBR desensitization. Given that GBR desensitization does not involve receptor internalization, the NSF/PKC coordinated action revealed herein suggests that NSF can regulate GPCR signalling efficacy independently of its role in membrane trafficking. The functional interaction between three bona fide regulators of neurotransmitter release, such as GBR, NSF and PKC, could shed new light on the modulation of presynaptic GBR action.     

Thermodynamic characterization of an engineered tetracycline-binding riboswitch

Riboswitches reflect a novel concept in gene regulation that is particularly suited for technological adaptation. Therefore, we characterized thermodynamically the ligand binding properties of a synthetic, tetracycline (tc)-binding RNA aptamer, which regulates gene expression in a dose-dependent manner when inserted into the untranslated region of an mRNA. In vitro, one molecule of tc is bound by one molecule of partially pre-structured and conformationally homogeneous apo-RNA. The dissociation constant of 770 pM, as determined by fluorimetry, is the lowest reported so far for a small molecule-binding RNA aptamer. Additional calorimetric analysis of RNA point mutants and tc derivatives identifies functional groups crucial for the interaction and including their respective enthalpic and entropic contributions we can propose detailed structural and functional roles for certain groups. The conclusions are consistent with mutational analyses in vivo and support the hypothesis that tc-binding reinforces the structure of the RNA aptamer, preventing the scanning ribosome from melting it efficiently.