Thyroid hormone induces sprouting angiogenesis in adult heart of hypothyroid mice through the PDGF‐Akt pathway

Study of physiological angiogenesis and associated signalling mechanisms in adult heart has been limited by the lack of a robust animal model. We investigated thyroid hormone‐induced sprouting angiogenesis and the underlying mechanism. Hypothyroidism was induced in C57BL/6J mice by feeding with propylthiouracil (PTU). One year of PTU treatment induced heart failure. Both 12 weeks‐ (young) and 1 year‐PTU (middle age) treatment caused a remarkable capillary rarefaction observed in capillary density. Three‐day Triiodothyronine (T3) treatment significantly induced cardiac capillary growth in hypothyroid mice. In cultured left ventricle (LV) tissues from PTU‐treated mice, T3 also induced robust sprouting angiogenesis where pericyte‐wrapped endothelial cells formed tubes. The in vitro T3 angiogenic response was similar in mice pre‐treated with PTU for periods ranging from 1.5 to 12 months. Besides bFGF and VEGF₁₆₄, PDGF‐BB was the most robust angiogenic growth factor, which stimulated notable sprouting angiogenesis in cultured hypothyroid LV tissues with increasing potency, but had little effect on tissues from euthyroid mice. T3 treatment significantly increased PDGF receptor beta (PDGFR‐β) protein levels in hypothyroid heart. PDGFR inhibitors blocked the action of T3 both on sprouting angiogenesis in cultured LV tissue and on capillary growth in vivo. In addition, activation of Akt signalling mediated in T3‐induced angiogenesis was blocked by PDGFR inhibitor and neutralizing antibody. Our results suggest that hypothyroidism leads to cardiac microvascular impairment and rarefaction with increased sensitivity to angiogenic growth factors. T3‐induced cardiac sprouting angiogenesis in adult hypothyroid mice was associated with PDGF‐BB, PDGFR‐β and downstream activation of Akt.     

The DNA deaminase activity of human APOBEC3G is required for Ty1, MusD, and human immunodeficiency virus type 1 restriction

Human APOBEC3G and several other APOBEC3 proteins have been shown to inhibit the replication of a variety of retrotransposons and retroviruses. All of these enzymes can deaminate cytosines within single-strand DNA, but the overall importance of this conserved activity in retroelement restriction has been questioned by reports of deaminase-independent mechanisms. Here, three distinct retroelements, a yeast retrotransposon, Ty1, a murine endogenous retrovirus, MusD, and a lentivirus, human immunodeficiency virus type 1 (HIV-1), were used to evaluate the relative contributions of deaminase-dependent and -independent mechanisms. Although human APOBEC3G can restrict the replication of all three of these retroelements, APOBEC3G lacking the catalytic glutamate (E259Q) was clearly defective. This phenotype was particularly clear in experiments with low levels of APOBEC3G expression. In contrast, purposeful overexpression of APOBEC3G-E259Q was able to cause modest to severe reductions in the replication of Ty1, MusD, and HIV-1(ΔVif). The importance of these observations was highlighted by data showing that CEM-SS T-cell lines expressing near-physiologic levels of APOBEC3G-E259Q failed to inhibit the replication of HIV-1(ΔVif), whereas similar levels of wild-type APOBEC3G fully suppressed virus infectivity. Despite the requirement for DNA deamination, uracil DNA glycosylase did not modulate APOBEC3G-dependent restriction of Ty1 or HIV-1(ΔVif), further supporting prior studies indicating that the major uracil excision repair system of cells is not involved. In conclusion, the absolute requirement for the catalytic glutamate of APOBEC3G in Ty1, MusD, and HIV-1 restriction strongly indicates that DNA cytosine deamination is an essential part of the mechanism.     

An upstream regulator and downstream target of phospholipase D1 activity during pheromone response in Saccharomyces cerevisiae

Phospholipase D1 (PLD1), which is the product of the SPO14 gene, has been shown to play a role in the process of polarized cell growth (PCG) during the pheromone response in Saccharomyces cerevisiae. PLD1 hydrolyzes phosphatidylcholine to produce phosphatidic acid (PA) and a free choline headgroup. This study investigated the interactions of PLD1 and PA with two proteins known to be involved in the cellular signaling leading to PCG in yeast, the small GTPase Cdc42p and the PAK family kinase Ste20p. Constitutively activated Cdc42p stimulates PLD1 activity. Protein-lipid binding blots confirmed the specific binding of Ste20p to the PLD1 product, PA. Finally, kinase activity assays provided evidence for the stimulation of Ste20p by PA. These findings highlight the important interactions among PLD1, Cdc42p and Ste20p during PCG in S. cerevisiae.     

The biflavonoid isoginkgetin is a general inhibitor of Pre-mRNA splicing

Membrane-permeable compounds that reversibly inhibit a particular step in gene expression are highly useful tools for cell biological and biochemical/structural studies. In comparison with other gene expression steps where multiple small molecule effectors are available, very few compounds have been described that act as general inhibitors of pre-mRNA splicing. Here we report construction and validation of a set of mammalian cell lines suitable for the identification of small molecule inhibitors of pre-mRNA splicing. Using these cell lines, we identified the natural product isoginkgetin as a general inhibitor of both the major and minor spliceosomes. Isoginkgetin inhibits splicing both in vivo and in vitro at similar micromolar concentrations. It appears to do so by preventing stable recruitment of the U4/U5/U6 tri-small nuclear ribonucleoprotein, resulting in accumulation of the prespliceosomal A complex. Like two other recently reported general pre-mRNA splicing inhibitors, isoginkgetin has been previously described as an anti-tumor agent. Our results suggest that splicing inhibition is the mechanistic basis of the anti-tumor activity of isoginkgetin. Thus, pre-mRNA splicing inhibitors may represent a novel avenue for development of new anti-cancer agents.     

Sexually different actions of leptin in proopiomelanocortin neurons to regulate glucose homeostasis

Leptin regulates energy balance and glucose homeostasis, at least in part, via activation of receptors in the arcuate nucleus of the hypothalamus located in proopiomelanocortin (POMC) neurons. Females have greater sensitivity to central leptin than males, suggested by a greater anorectic effect of central leptin administration in females. We hypothesized that the regulation of energy balance and peripheral glucose homeostasis of female rodents would be affected to a greater extent than in males if the action of leptin in POMC neurons were disturbed. Male and female mice lacking leptin receptors only in POMC neurons gained significantly more body weight and accumulated more body fat. However, female mice gained disproportionately more visceral adiposity than males, and this appeared to be largely the result of differences in energy expenditure. When maintained on a high-fat diet (HFD), both male and female mutants had higher levels of insulin following exogenous glucose challenges. Chow- and HFD-fed males but not females had abnormal glucose disappearance curves following insulin administrations. Collectively, these data indicate that the action of leptin in POMC neurons is sexually different to influence the regulation of energy balance, fat distribution, and glucose homeostasis.     

Genetic variation in endocannabinoid metabolism, gastrointestinal motility, and sensation

Cannabinoid agonist inhibits gastrointestinal motility. The endocannabinoid, anandamide, is inactivated by fatty acid amide hydrolase (FAAH). A single nucleotide polymorphism in the human FAAH gene (C385A) reduces FAAH expression. Our aim was to evaluate associations between FAAH genotype variation and symptom phenotype, gastric emptying and volume, colonic transit, and rectal sensation in patients with functional gastrointestinal disorders (FGID). 482 FGID patients [Rome II positive, 159 constipation disorders, 184 diarrhea disorders (D-IBS), 86 mixed bowel function (M-IBS), 20 chronic abdominal pain (CAP), 33 functional dyspepsia], and 252 healthy volunteers (HV) underwent questionnaires and studies of phenotype and genotype from 2000 to 2007: 250 gastric emptying, 210 fasting and postprandial gastric volume, 152 colonic transit, and 123 rectal sensation. All had FAAH genotype [CC vs. polymorphic (CA/AA)] determined by TaqMan. FAAH genotype distribution of FGID patients and HV did not deviate from Hardy-Weinberg equilibrium. There was a significant association of FAAH genotype with FGID phenotype (overall chi(2), P = 0.011) and with specific individual phenotypes (P = 0.048). Thus FAAH CA/AA increases the odds (relative to HV) for D-IBS (P = 0.008), M-IBS (P = 0.012), and, possibly, CAP (P = 0.055). There was a significant association of FAAH CA/AA genotype with accelerated colonic transit in D-IBS (P = 0.037). There was no association of FAAH genotype with rectal sensation thresholds or ratings. The association of genetic variation in metabolism of endocannabinoids with symptom phenotype in D-IBS and M-IBS and with faster colonic transit in D-IBS supports the hypothesis that cannabinoid mechanisms may play a role in the control of colonic motility in humans and deserve further study.     

Eye formation in the absence of retina

Eye development is a complex process that involves the formation of the retina and the lens, collectively called the eyeball, as well as the formation of auxiliary eye structures such as the eyelid, lacrimal gland, cornea and conjunctiva. The developmental requirements for the formation of each individual structure are only partially understood. We have shown previously that the homeobox-containing gene Rx is a key component in eye formation, as retinal structures do not develop and retina-specific gene expression is not observed in Rx-deficient mice. In addition, Rx−/− embryos do not develop any lens structure, despite the fact that Rx is not expressed in the lens. This demonstrates that during normal mammalian development, retina-specific gene expression is necessary for lens formation. In this paper we show that lens formation can be restored in Rx-deficient embryos experimentally, by the elimination of β-catenin expression in the head surface ectoderm. This suggests that β-catenin is involved in lens specification either through Wnt signaling or through its function in cell adhesion. In contrast to lens formation, we demonstrate that the development of auxiliary eye structures does not depend on retina-specific gene expression or retinal morphogenesis. These results point to the existence of two separate developmental processes involved in the formation of the eye and its associated structures. One involved in the formation of the eyeball and the second involved in the formation of the auxiliary eye structures.