The Role of the Formamide/Zirconia System in the Synthesis of Nucleobases and Biogenic Carboxylic Acid Derivatives

We describe the one-pot synthesis of a large variety of nucleic acid bases and related compounds from formamide in the presence of zirconium minerals as catalysts. The major products observed are: purine, 2-hydroxy pyrimidine, 5-hydroxy pyrimidine, isocytosine, adenine, urea, and carbodiimide. The synthesis of low molecular weight amides and carboxylic acid derivatives (intermediates of extant metabolism) was also observed: glyoxylamide, glycolic-, lactic-, succinic-, oxalic-, fumaric-, and maleic acids. As the major problem in the origin of informational polymers is the instability of their precursors, we also investigated the effects of zirconia minerals on the stability of ribooligonucleotides in formamide and in water. The relevance of these findings with respect to the origin of informational polymers and primordial metabolism is discussed.     

Heme-binding Protein HRG-1 Is Induced by Insulin-like Growth Factor I and Associates with the Vacuolar H+-ATPase to Control Endosomal pH and Receptor Trafficking

Endocytosis and trafficking of receptors and nutrient transporters are dependent on an acidic intra-endosomal pH that is maintained by the vacuolar H⁺-ATPase (V-ATPase) proton pump. V-ATPase activity has also been associated with cancer invasiveness. Here, we report on a new V-ATPase-associated protein, which we identified in insulin-like growth factor I (IGF-I) receptor-transformed cells, and which was separately identified in Caenorhabditis elegans as HRG-1, a member of a family of heme-regulated genes. We found that HRG-1 is present in endosomes but not in lysosomes, and it is trafficked to the plasma membrane upon nutrient withdrawal in mammalian cells. Suppression of HRG-1 with small interfering RNA causes impaired endocytosis of transferrin receptor, decreased cell motility, and decreased viability of HeLa cells. HRG-1 interacts with the c subunit of the V-ATPase and enhances V-ATPase activity in isolated yeast vacuoles. Endosomal acidity and V-ATPase assembly are decreased in cells with suppressed HRG-1, whereas transferrin receptor endocytosis is enhanced in cells that overexpress HRG-1. Cellular uptake of a fluorescent heme analogue is enhanced by HRG-1 in a V-ATPase-dependent manner. Our findings indicate that HRG-1 regulates V-ATPase activity, which is essential for endosomal acidification, heme binding, and receptor trafficking in mammalian cells. Thus, HRG-1 may facilitate tumor growth and cancer progression.     

Leptin modulates the survival of autoreactive CD4+ T cells through the nutrient/energy-sensing mammalian target of rapamycin signaling pathway

Chronic inflammation can associate with autoreactive immune responses, including CD4(+) T cell responses to self-Ags. In this paper, we show that the adipocyte-derived proinflammatory hormone leptin can affect the survival and proliferation of autoreactive CD4(+) T cells in experimental autoimmune encephalomyelitis, an animal model of human multiple sclerosis. We found that myelin olygodendrocyte glycoprotein peptide 35-55 (MOG(35-55))-specific CD4(+) T cells from C57BL/6J wild-type mice could not transfer experimental autoimmune encephalomyelitis into leptin-deficient ob/ob mice. Such a finding was associated with a reduced proliferation of the transferred MOG(35-55)-reactive CD4(+) T cells, which had a reduced degradation of the cyclin-dependent kinase inhibitor p27(kip1) and ERK1/2 phosphorylation. The transferred cells displayed reduced Th1/Th17 responses and reduced delayed-type hypersensitivity. Moreover, MOG(35-55)-reactive CD4(+) T cells in ob/ob mice underwent apoptosis that associated with a downmodulation of Bcl-2. Similar results were observed in transgenic AND-TCR- mice carrying a TCR specific for the pigeon cytochrome c 88-104 peptide. These molecular events reveal a reduced activity of the nutrient/energy-sensing AKT/mammalian target of rapamycin pathway, which can be restored in vivo by exogenous leptin replacement. These results may help to explain a link between chronic inflammation and autoimmune T cell reactivity.     

Left ventricular myofilament dysfunction in rat experimental hypertrophy and congestive heart failure

It is currently unclear whether left ventricular (LV) myofilament function is depressed in experimental LV hypertrophy (LVH) or congestive heart failure (CHF). To address this issue, we studied pressure overload-induced LV hypertrophy (POLVH) and myocardial infarction-elicited congestive heart failure (MICHF) in rats. LV myocytes were isolated from control, POLVH, and MICHF hearts by mechanical homogenization, skinned with Triton, and attached to micropipettes that projected from a sensitive force transducer and high-speed motor. A subset of cells was treated with either unphosphorylated, recombinant cardiac troponin (cTn) or cTn purified from either control or failing ventricles. LV myofilament function was characterized by the force-[Ca(2+)] relation yielding Ca(2+)-saturated maximal force (F(max)), myofilament Ca(2+) sensitivity (EC(50)), and cooperativity (Hill coefficient, n(H)) parameters. POLVH was associated with a 35% reduction in F(max) and 36% increase in EC(50). Similarly, MICHF resulted in a 42% reduction in F(max) and a 30% increase in EC(50). Incorporation of recombinant cTn or purified control cTn into failing cells restored myofilament Ca(2+) sensitivity toward levels observed in control cells. In contrast, integration of cTn purified from failing ventricles into control myocytes increased EC(50) to levels observed in failing myocytes. The F(max) parameter was not markedly affected by troponin exchange. cTnI phosphorylation was increased in both POLVH and MICHF left ventricles. We conclude that depressed myofilament Ca(2+) sensitivity in experimental LVH and CHF is due, in part, to a decreased functional role of cTn that likely involves augmented phosphorylation of cTnI.     

The liaison of sweet and savory

The sense of taste provides humans with necessary information about the composition and quality of food. For humans, five basic tastes are readily distinguishable and include sweet, bitter, salty, sour, and savory (or umami). Although each of these qualities has individualized transduction pathways, sweet and umami tastes are believed to share a common receptor element, the T1R3 receptor subunit. The two G-protein-coupled heteromer receptors that comprise an umami stimulus receptor (T1R1-T1R3) and a sweetener receptor (T1R2-T1R3) constitute a potential link between these two qualities of perception. While the role of the individual monomers in each human heteromer has been examined in vitro, very little is known of the implication of this research for human perception, or specifically, how sweet and savory taste perceptions may be connected. Using a psychophysical approach, we demonstrate that lactisole, a potent sweetness inhibitor that binds in vitro to hT1R3, also inhibits a significant portion of the perception of umami taste from monosodium glutamate. Following the molecular logic put forward by Xu et al. (2004, Proc. Natl Acad. Sci. USA, 101, 14258-14263), our psychophysical data support the in vitro hypothesis that the shared T1R3 monomer moderates the activation of both T1R2 and T1R1 in humans and impairs suprathreshold perception, respectively, of sweetness and, to a lesser degree, umaminess in the presence of lactisole.     

Light reaches the very heart of the zebrafish clock

Zebrafish are typically used as a model system to study various aspects of developmental biology, largely as a consequence of their ex vivo development, high degree of transparency, and, of course, ability to perform forward genetic mutant screens. More recently, zebrafish have been developed as a model system with which to study circadian clocks. Cell lines generated from early-stage zebrafish embryos contain clocks that are directly light-responsive. We describe recent experiments using single-cell luminescent imaging approaches to study clock function in this novel cell line system. Furthermore, studies examining the process of entrainment to light pulses within this cell population are described in this review, as are experiments examining light-responsiveness of early-stage zebrafish embryos.     

Ontogenic changes in lactoferrin receptor and DMT1 in mouse small intestine: implications for iron absorption during early life.

It has been proposed that lactoferrin receptor (LfR) may be involved in intestinal iron transport during early life. However, it is known that iron homeostasis is regulated by divalent metal transporter 1 (DMT1; Nramp2/DCT1) in the adult small intestine. To address the hypothesis that LfR may play a role as an alternative iron transport pathway during early life, we used immunohistochemistry (IHC) to examine the localization of mouse LfR (mLfR) and DMT1. In addition to studying the localization and abundance of LfR and DMT1 on the apical membrane, intestinal brush-border membrane vesicles (BBMV) were isolated during the first 3 postnatal weeks (postnatal day (PD) 0, 5, 10, and 20). We found that mLfR is expressed in fetal mice as early as gestational days (GD) 13.5, 15.5, and 18.5. A 34 kD band for mLfR was detected at PD 0 through PD 20 in total intestine homogenate. However, mLfR protein did not appear in the BBMV preparations until PD 5 and was highly expressed at PD 10. By IHC, DMT1 protein was minimally observed at PD 0 and PD 5, but by PD 10 DMT1 was predominantly localized in the apical membrane of the maturing intestine. BBMV fractionation revealed 50-120 kD protein bands for DMT1. In these BBMV preparations, the apical-membrane-associated 120 kD band for DMT1 increased in abundance with age. However, in the corresponding total homogenates, only the deglycosylated form of DMT1 (50 kD) was identified. These results indicate that DMT1 is mislocalized during late gestation, minimally expressed during early life, and predominantly expressed in its deglycosylated form until PD 20. The immunolocalization and abundant protein expression of mLfR suggest that accrual of iron from Lf may be the principal iron uptake pathway at this age. In conclusion, our findings support the notion that until the development-dependent expression of DMT1 in the intestine is induced, mLfR may serve as an alternative iron uptake pathway.     

Adrenomedullin immunoreactivity in the human carotid body

We studied by immunocytochemistry the expression of AM in human carotid bodies, sampled at autopsy from 16 adult subjects (mean age+/-S.D.: 44.3+/-3.4 years) and from six fetuses (mean gestational age+/-S.D.: 167+/-11 days). No AM immunoreactivity was visible in the type II cells of both series. The percentage of immunoreactive type I cells was higher in the adult subjects (32.3+/-7.7%) with respect to the fetuses (11.8+/-2.7%, P < 0.001). Dark cells showed a higher percentage of positive immunoreaction with respect to light cells, both in adult subjects (61.7+/-13.4% versus 19.2+/-5.2%) and in fetuses (25.3+/-4.4% versus 6.2+/-2.0%). AM may play a role in the regulation of chemoreceptor discharge through paracrine releasing action and/or vasodilator effect. The low expression of AM in fetuses may be ascribed to the absence of pulmonary respiration with lack of regulatory role of the carotid body during the prenatal period.