Phylogeny of seed plants based on evidence from eight genes

Relationships among the five groups of extant seed plants (cycads, Ginkgo, conifers, Gnetales, and angiosperms) remain uncertain. To explore relationships among groups of extant seed plants further and to attempt to explain the conflict among molecular data sets, we assembled a data set of four plastid (cpDNA) genes (rbcL, atpB, psaA, and psbB), three mitochondrial (mtDNA) genes (mtSSU, coxI, and atpA), and one nuclear gene (18S rDNA) for 19 exemplars representing the five groups of living seed plants. Analyses of the combined eight-gene data set (15?772 base pairs/taxon) with maximum parsimony (MP), maximum likelihood (ML), and Bayesian approaches reveal a gymnosperm clade that is sister to angiosperms. Within the gymnosperms, a conifer clade includes Gnetales as sister to Pinaceae. Cycads and Ginkgo are either successive sisters to this conifer clade (including Gnetales) or a clade that is sister to conifers and Gnetales. All analyses of the mtDNA partition and ML analyses of the nuclear partition yield very similar topologies. However, MP analyses of the combined cpDNA genes place Gnetales as sister to all other seed plants with strong bootstrap support, whereas ML and Bayesian analyses of the cpDNA data set place Gnetales as sister to Pinaceae. Maximum parsimony and ML analyses of first and second codon positions of the cpDNA partiation also place Gnetales as sister to Pinaceae. In contrast, MP analyses of third codon positions place Gnetales as sister to other seed plants, although ML analyses of third codon positions place Gnetales with Pinaceae. Thus, most of the discrepancies in seed plant topologies involve third codon positions of cpDNA genes. The likelihood ratio (LR) and Shimodaira-Hasegasa (SH) tests were applied to the cpDNA data. The preferred topology based on the LR test is that Gnetales are sister to Pseudotsuga. The SH test based on first and second codon and all three codon positions indicated that there is no significant difference between the best topology (Gnetales sister to Pseudotsuga) and Gnetales sister to a conifer clade. However, there is a significant difference between the best topology and topologies in which Gnetales are sister to the rest of the seed plants or Gnetales sister to angiosperms.     

Complementary expression of IGF-II and IGFBP-5 during anterior pituitary development

The specification of the five individual hormone-secreting cell types in the anterior pituitary requires a series of sequential cell fate decisions. We have immortalized cells at several stages along this pathway of pituitary differentiation. Here, we present analysis of differences in gene expression between an anterior pituitary precursor cell line, alphaT1-1, and an immature gonadotrope cell line, alphaT3-1, identified by using cDNA subtraction. Messenger RNA expression of members of the insulin-like growth factor signaling system, IGF-II and IGFBP-5, was found in the alphaT1-1 precursor cell line, but not in the more differentiated cell line, alphaT3-1. This inferred stage specificity was confirmed in the mouse embryo by using in situ hybridization on embryonic days e10.5 through e18.5. Expression of IGF-II and IGFBP-5 mRNAs was both temporally and spatially regulated during pituitary development. IGF-II was highly expressed in the epithelium surrounding Rathke's pouch at e10.5, while IGFBP-5 expression was restricted to the adjacent oral epithelium. At e11.5 (represented by alphaT1-1), IGF-II was expressed throughout the pouch, but was coexpressed with IGFBP-5 and alpha-subunit in the ventral portion of the pouch epithelium. On e12.5, the two mRNAs were expressed in opposing dorsoventral (IGF-II) and ventrodorsal (IGFBP-5) patterns, with IGF-II excluded from the rostral, alpha-subunit-expressing region. A decrease of both mRNAs was observed at e14.5 (equivalent to alphaT3-1), with IGF-II levels low and IGFBP-5 concentrated in the anterior pituitary rostral tip. These findings suggest that the timing of IGF-II expression and regulation of its accessibility by IGFBP-5 may play a role in anterior pituitary differentiation, survival, and/or proliferation.     

Selective inactivation of redox-sensitive mitochondrial enzymes during cardiac reperfusion

Reperfusion of ischemic myocardial tissue results in an increase in mitochondrial free radical production and declines in respiratory activity. The effects of ischemia and reperfusion on the activities of Krebs cycle enzymes, as well as enzymes involved in electron transport, were evaluated to provide insight into whether free radical events are likely to affect enzymatic and mitochondrial function(s). An in vivo rat model was utilized in which ischemia is induced by ligating the left anterior descending coronary artery. Reperfusion, initiated by release of the ligature, resulted in a significant decline in NADH-linked ADP-dependent mitochondrial respiration as assessed in isolated cardiac mitochondria. Assays of respiratory chain complexes revealed reduction in the activities of complex I and, to a lesser extent, complex IV exclusively during reperfusion, with no alterations in the activities of complexes II and III. Moreover, Krebs cycle enzymes alpha-ketoglutarate dehydrogenase and aconitase were susceptible to reperfusion-induced inactivation with no decline in the activities of other Krebs cycle enzymes. The decline in alpha-ketoglutarate dehydrogenase activity during reperfusion was associated with a loss in native lipoic acid on the E2 subunit, suggesting oxidative inactivation. Inhibition of complex I in vitro promotes free radical generation. alpha-Ketoglutarate dehydrogenase and aconitase are uniquely susceptible to in vitro oxidative inactivation. Thus, our results suggest a scenario in which inhibition of complex I promotes free radical production leading to oxidative inactivation of alpha-ketoglutarate dehydrogenase and aconitase.     

One-tube nested polymerase chain reaction for detection of Chlamydia trachomatis

Here we present a one-tube nested PCR test, which allows the detection of minimal quantities of Chlamydia trachomatis in human fluids. This assay includes the use of an internal control to avoid false negative results due to the presence of inhibitors. The results obtained show that this assay is robust enough to be used for clinical diagnosis.     

Infectious disease survey of lesser prairie chickens in north Texas

Lesser prairie chicken (Tympanuchus pallidicinctus) abundance, like that of most grassland birds, has declined rangewide for decades. Although habitat loss and degradation are likely ultimate causes for this decline, infectious agents, particularly microparasites, could be proximate contributors. No surveys of pathogenic bacteria or viruses have been published for this species. We surveyed 24 free-living lesser prairie chickens from Hemphill County, Texas (USA), for evidence of exposure to Salmonella typhimurium, S. pullorum, Mycoplasma gallisepticum, M. synoviae, Chlamydophila psittaci, and the avian influenza, Newcastle disease, infectious bronchitis, and reticuloendotheliosis viruses. Two of 18, and eight of 17 samples were seropositive for the Massachusetts and Arkansas serotypes of infectious bronchitis virus, respectively. Five of the eight positive individuals were juveniles, two of which were seropositive for both serotypes. All other serologic and genetic tests were negative. Because the ecological significance of these results is unknown, the pathogenesis, transmission, and/or population-level influences of infectious bronchitis and related avian coronaviruses for lesser prairie chickens deserves further study.     

Integrative nuclear FGFR1 signaling (INFS) pathway mediates activation of the tyrosine hydroxylase gene by angiotensin II,depolarization and protein kinase C

The integrative nuclear FGFR1 signaling (INFS) pathway functions in association with cellular growth, differentiation, and regulation of gene expression, and is activated by diverse extracellular signals. Here we show that stimulation of angiotensin II (AII) receptors, depolarization, or activation protein kinase C (PKC) or adenylate cyclase all lead to nuclear accumulation of fibroblast growth factor 2 (FGF-2) and FGFR1, association of FGFR1 with splicing factor-rich domains, and activation of the tyrosine hydroxylase (TH) gene promoter in bovine adrenal medullary cells (BAMC). The up-regulation of endogenous TH protein or a transfected TH promoter-luciferase construct by AII, veratridine, or PMA (but not by forskolin) is abolished by transfection with a dominant negative FGFR1TK-mutant which localizes to the nucleus and plasma membrane, but not by extracellularly acting FGFR1 antagonists suramin and inositolhexakisphosphate (IP6). Mechanism of TH gene activation by FGF-2 and FGFR1 was further investigated in BAMC and human TE671 cultures. TH promoter was activated by co-transfected HMW FGF-2 (which is exclusively nuclear) but not by cytoplasmic FGF-1 or extracellular FGFs. Promoter transactivation by HMWFGF-2 was accompanied by an up-regulation of FGFR1 specifically in the cell nucleus and was prevented FGFR1(TK-) but not by IP6 or suramin. The TH promoter was also transactivated by co-transfected wild-type FGFR1, which localizes to both to the nucleus and the plasma membrane, and by an exclusively nuclear, soluble FGFR1(SP-/NLS) mutant with an inserted nuclear localization signal. Activation of the TH promoter by nuclear FGFR1 and FGF-2 was mediated through the cAMP-responsive element (CRE) and was associated with induction of CREB- and CBP/P-300-containing CRE complexes. We propose a new model for gene regulation in which nuclear FGFR1 acts as a mediator of CRE transactivation by AII, cell depolarization, and PKC.     

5-HT7 receptors are involved in mediating 5-HT-induced activation of rat primary afferent neurons

The purpose of this study was to determine whether the 5-hydroxytryptamine7 (5-HT7) receptor is expressed by nociceptor-like neurons in the rat PNS and whether 5-HT activates these nociceptors via the 5-HT7 receptor subtype. Using a polyclonal antibody and the method of immunofluorescence staining, we demonstrated that the 5-HT7 receptor appears predominately on "nociceptor-like" neurons of the rat lumbar dorsal root ganglia. Using immunocytochemical methods, we showed that the immunoreactivity of the 5-HT7 receptor antibody complex is localized in the superficial layers of the spinal cord dorsal horn, which corresponds with laminae I, IIouter and IIinner. Furthermore, we demonstrated that noxious stimulation produced by knee injection of 5-HT or a 5-HT7 agonist dose-dependently increases c-Fos production of the rat spinal cord dorsal horn. This effect was significantly inhibited by the preinjection of a 5-HT7 antagonist. We conclude that the 5-HT7 receptor is expressed by rat primary afferent nociceptors which terminate in the superficial layers of the spinal cord dorsal horn and that the 5-HT7 receptor subtype is involved in nociceptor activation by 5-HT.     

Truncated, inactive N-acetylglucosaminyltransferase III (GlcNAc-TIII) induces neurological and other traits absent in mice that lack GlcNAc-TIII

N-Acetylglucosaminyltransferase III (GlcNAc-TIII), the product of the Mgat3 gene, transfers the bisecting GlcNAc to the core mannose of complex N-glycans. The addition of this residue is regulated during development and has functional consequences for receptor signaling, cell adhesion, and tumor progression. Mice homozygous for a null mutation at the Mgat3 locus (Mgat3(Delta)) or for a targeted mutation in the Mgat3 gene (previously called Mgat3(neo), but herein renamed Mgat3(T37) because the allele generates inactive GlcNAc-TIII of approximately 37 kDa) were found to exhibit retarded progression of liver tumors. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of neutral N-glycans from kidneys revealed no significant differences, and both mutants showed the expected lack of N-glycan species with an additional GlcNAc. However, the two mutants differed in several biological traits. Mgat3(T37/T37) homozygotes in a mixed or 129(SvJ) background were retarded in growth rate and exhibited an altered leg clasp reflex, an altered gait, and defective nursing behavior. Pups abandoned by Mgat3(T37/T37) mothers were rescued by wild-type foster mothers. None of these Mgat3(T37/T37) traits were exhibited by Mgat3(Delta/Delta) mice or by heterozygous mice carrying the Mgat3(T37) mutation. Similarly, no dominant-negative effect was observed in Chinese hamster ovary cells expressing truncated GlcNAc-TIII in the presence of wild-type GlcNAc-TIII. However, compound heterozygotes carrying both the Mgat3(T37) and Mgat3(Delta) mutations exhibited a marked leg clasp reflex, indicating that in the absence of wild-type GlcNAc-TIII, truncated GlcNAc-TIII causes this phenotype. The Mgat3 gene was expressed in brain at embryonic day 10.5 and thereafter and in neurons of adult cerebellum. The mutant Mgat3 gene was also highly expressed in Mgat3(T37/T37) brain. This may be the basis of the unexpected neurological phenotype induced by truncated, inactive GlcNAc-TIII in the mouse.     

Phosphatidylinositol 3-kinase activity negatively regulates stability of cyclooxygenase 2 mRNA

Human alveolar macrophages have both lipopolysaccharide (LPS)-induced and constitutive phosphatidylinositol 3-kinase (PI3K) activity. We observed that blocking PI3K activity increased release of prostaglandin E2 after LPS exposure, and increasing PI3K activity (interleukin-13) decreased release of prostaglandin E2 after LPS exposure. This was not because of an effect of PI3K on phospholipase 2 activity. PI3K inhibition resulted in an increase in cyclooxygenase 2 (COX2) protein, mRNA, and mRNA stability. PI3K negatively regulated activation of the p38 pathway (p38, MKK3/6, and MAPKAP2), and an active p38 was necessary for COX2 production. The data suggest that PI3K inhibition of p38 modulates COX2 expression via destabilization of LPS-induced COX2 mRNA.