Hormone physiology of pea mutants prevented from flowering by mutations gi or veg1

The veg1 (vegetative) mutant in pea (Pisum sativum L.) does not flower under any circumstances and gi (gigas) mutants remain vegetative under certain conditions. gi plants are deficient in production of floral stimulus, whereas veg1 plants lack a response to floral stimulus. During long days in particular, these non-flowering mutant plants eventually enter a stable compact phase characterised by a large reduction in internode length, small leaves and growth of lateral shoots from the upper-stem (aerial) nodes. The first-order laterals in turn produce second-order laterals and so on in a reiterative pattern. The apical bud is reduced in size but continues active growth. Endogenous hormone measurements and gibberellin application studies with gi-1, gi-2 and veg1 plants indicate that a reduction in gibberellin and perhaps indole-3-acetic acid level may account, at least partially, for the compact aerial shoot phenotype. In the gi-1 mutant, the compact phenotype is rescued by transfer from a 24- to an 8-h photoperiod. We propose that in plants where flowering is prevented by a lack of floral stimulus or an inability to respond, the large reduction in photoperiod gene activity during long days may lead to a reduction in apical sink strength that is manifest in an altered hormone profile and weak apical dominance.

Regulation of male sexual development by Sry and Sox9

Sry, a gene from the Y chromosome, is known to initiate testis formation and subsequent male differentiation in mammals. A related gene, Sox9, also plays a critical role in testis determination, possibly in all vertebrates. A number of models have been presented regarding the molecular modes of action of these two genes. However, details regarding their regulation, regulatory target genes, and interacting protein factors and co-factors have not been established with any certainty. In this review, we examine new evidence and re-examine existing evidence bearing on these issues, in an effort to build up an integrative model of the network of gene activity centred around Sry and Sox9. J. Exp. Zool. 290:463-474, 2001. (C) 2001 Wiley-Liss, Inc.

Chromatin dynamics in Arbidopsis development: a live cell imaging approach

Dissertation presented to obtain the Ph.D degree in Biology

Fitness correlates with the extent of cheating in a bacterium.

There is growing awareness of the importance of cooperative behaviours in microbial communities. Empirical support for this insight comes from experiments using mutant strains, termed 'cheats', which exploit the cooperative behaviour of wild-type strains. However, little detailed work has gone into characterising the competitive dynamics of cooperative and cheating strains. We test three specific predictions about the fitness consequences of cheating to different extents by examining the production of the iron-scavenging siderophore molecule, pyoverdin, in the bacterium Pseudomonas aeruginosa. We create a collection of mutants that differ in the amount of pyoverdin that they produce (from 1% to 96% of the production of paired wild types) and demonstrate that these production levels correlate with both gene activity and the ability to bind iron. Across these mutants, we found that (1) when grown in a mixed culture with a cooperative wild-type strain, the relative fitness of a mutant is negatively correlated with the amount of pyoverdin that it produces; (2) the absolute and relative fitness of the wild-type strain in the mixed culture is positively correlated with the amount of pyoverdin that the mutant produces; and (3) when grown in a monoculture, the absolute fitness of the mutant is positively correlated with the amount of pyoverdin that it produces. Overall, we demonstrate that cooperative pyoverdin production is exploitable and illustrate how variation in a social behaviour determines fitness differently, depending on the social environment.

Molecular evidence for a functional ecdysone signaling system in Brugia malayi.

BACKGROUND: Filarial nematodes, including Brugia malayi, the causative agent of lymphatic filariasis, undergo molting in both arthropod and mammalian hosts to complete their life cycles. An understanding of how these parasites cross developmental checkpoints may reveal potential targets for intervention. Pharmacological evidence suggests that ecdysteroids play a role in parasitic nematode molting and fertility although their specific function remains unknown. In insects, ecdysone triggers molting through the activation of the ecdysone receptor: a heterodimer of EcR (ecdysone receptor) and USP (Ultraspiracle). METHODS AND FINDINGS: We report the cloning and characterization of a B. malayi EcR homologue (Bma-EcR). Bma-EcR dimerizes with insect and nematode USP/RXRs and binds to DNA encoding a canonical ecdysone response element (EcRE). In support of the existence of an active ecdysone receptor in Brugia we also cloned a Brugia rxr (retinoid X receptor) homolog (Bma-RXR) and demonstrate that Bma-EcR and Bma-RXR interact to form an active heterodimer using a mammalian two-hybrid activation assay. The Bma-EcR ligand-binding domain (LBD) exhibits ligand-dependent transactivation via a GAL4 fusion protein combined with a chimeric RXR in mammalian cells treated with Ponasterone-A or a synthetic ecdysone agonist. Furthermore, we demonstrate specific up-regulation of reporter gene activity in transgenic B. malayi embryos transfected with a luciferase construct controlled by an EcRE engineered in a B. malayi promoter, in the presence of 20-hydroxy-ecdysone. CONCLUSIONS: Our study identifies and characterizes the two components (Bma-EcR and Bma-RXR) necessary for constituting a functional ecdysteroid receptor in B. malayi. Importantly, the ligand binding domain of BmaEcR is shown to be capable of responding to ecdysteroid ligands, and conversely, ecdysteroids can activate transcription of genes downstream of an EcRE in live B. malayi embryos. These results together confirm that an ecdysone signaling system operates in B. malayi and strongly suggest that Bma-EcR plays a central role in it. Furthermore, our study proposes that existing compounds targeting the insect ecdysone signaling pathway should be considered as potential pharmacological agents against filarial parasites.

Identification of reference genes for quantitative RT-PCR in ascending aortic aneurysms.

Hypertension and congenital aortic valve malformations are frequent causes of ascending aortic aneurysms. The molecular mechanisms of aneurysm formation under these circumstances are not well understood. Reference genes for gene activity studies in aortic tissue that are not influenced by aortic valve morphology and its hemodynamic consequences, aortic dilatation, hypertension, or antihypertensive medication are not available so far. This study determines genes in ascending aortic tissue that are independent of these parameters. Tissue specimens from dilated and undilated ascending aortas were obtained from 60 patients (age ≤70 years) with different morphologies of the aortic valve (tricuspid undilated n = 24, dilated n = 11; bicuspid undilated n = 6, dilated n = 15; unicuspid dilated n = 4). Of the studied individuals, 36 had hypertension, and 31 received ACE inhibitors or AT1 receptor antagonists. The specimens were obtained intraoperatively from the wall of the ascending aorta. We analyzed the expression levels of 32 candidate reference genes by quantitative RT-PCR (RT-qPCR). Differential expression levels were assessed by parametric statistics. The expression analysis of these 32 genes by RT-qPCR showed that EIF2B1, ELF1, and PPIA remained constant in their expression levels in the different specimen groups, thus being insensitive to aortic valve morphology, aortic dilatation, hypertension, and medication with ACE inhibitors or AT1 receptor antagonists. Unlike many other commonly used reference genes, the genes EIF2B1, ELF1, and PPIA are neither confounded by aortic comorbidities nor by antihypertensive medication and therefore are most suitable for gene expression analysis of ascending aortic tissue.

Glucose represses connexin36 in insulin-secreting cells.

The gap-junction protein connexin36 (Cx36) contributes to control the functions of insulin-producing cells. In this study, we investigated whether the expression of Cx36 is regulated by glucose in insulin-producing cells. Glucose caused a significant reduction of Cx36 in insulin-secreting cell lines and freshly isolated pancreatic rat islets. This decrease appeared at the mRNA and the protein levels in a dose- and time-dependent manner. 2-Deoxyglucose partially reproduced the effect of glucose, whereas glucosamine, 3-O-methyl-D-glucose and leucine were ineffective. Moreover, KCl-induced depolarization of beta-cells had no effect on Cx36 expression, indicating that glucose metabolism and ATP production are not mandatory for glucose-induced Cx36 downregulation. Forskolin mimicked the repression of Cx36 by glucose. Glucose or forskolin effects on Cx36 expression were not suppressed by the L-type Ca(2+)-channel blocker nifedipine but were fully blunted by the cAMP-dependent protein kinase (PKA) inhibitor H89. A 4 kb fragment of the human Cx36 promoter was identified and sequenced. Reporter-gene activity driven by various Cx36 promoter fragments indicated that Cx36 repression requires the presence of a highly conserved cAMP responsive element (CRE). Electrophoretic-mobility-shift assays revealed that, in the presence of a high glucose concentration, the binding activity of the repressor CRE-modulator 1 (CREM-1) is enhanced. Taken together, these data provide evidence that glucose represses the expression of Cx36 through the cAMP-PKA pathway, which activates a member of the CRE binding protein family.

A regulatory network for the efficient control of transgene expression.

BACKGROUND: Expression of heterologous genes in mammalian cells or organisms for therapeutic or experimental purposes often requires tight control of transgene expression. Specifically, the following criteria should be met: no background gene activity in the off-state, high gene expression in the on-state, regulated expression over an extended period, and multiple switching between on- and off-states. METHODS: Here, we describe a genetic switch system for controlled transgene transcription using chimeric repressor and activator proteins functioning in a novel regulatory network. In the off-state, the target transgene is actively silenced by a chimeric protein consisting of multimerized eukaryotic transcriptional repression domains fused to the DNA-binding tetracycline repressor. In the on-state, the inducer drug doxycycline affects both the derepression of the target gene promoter and activation by the GAL4-VP16 transactivator, which in turn is under the control of an autoregulatory feedback loop. RESULTS: The hallmark of this new system is the efficient transgene silencing in the off-state, as demonstrated by the tightly controlled expression of the highly cytotoxic diphtheria toxin A gene. Addition of the inducer drug allows robust activation of transgene expression. In stably transfected cells, this control is still observed after months of repeated cycling between the repressed and activated states of the target genes. CONCLUSIONS: This system permits tight long-term regulation when stably introduced into cell lines. The underlying principles of this network system should have general applications in biotechnology and gene therapy.

Measuring limits of telomere movement on nuclear envelope.

The dynamic behavior of the decondensed chromatin can be monitored by real-time fluorescence confocal microscopy. It can be observed that different chromosomal sites enjoy different degrees of freedom during a certain period, exploring larger or smaller portions of nuclear volume. Here we measure the accessible surface for two chromosomal sites (yeast telomeres Tel3R and Tel6R) that both exhibit strong preferential association with the nuclear membrane in galactose-containing media, but differ significantly in gene activity. Telomere Tel6R, which harbors an inducible gene with high levels of transcription, explores a much larger surface than the telomere Tel3R, which is adjacent to inactive chromatin. Thus, our results distinguish two perinuclear movements characteristic of different transcriptional state, allowing for a better understanding of the correlation between activity of genes and chromatin dynamics.

Patch-clamp detection of macromolecular translocation along nuclear pores

The present paper reviews the application of patch-clamp principles to the detection and measurement of macromolecular translocation along the nuclear pores. We demonstrate that the tight-seal 'gigaseal' between the pipette tip and the nuclear membrane is possible in the presence of fully operational nuclear pores. We show that the ability to form a gigaseal in nucleus-attached configurations does not mean that only the activity of channels from the outer membrane of the nuclear envelope can be detected. Instead, we show that, in the presence of fully operational nuclear pores, it is likely that the large-conductance ion channel activity recorded derives from the nuclear pores. We conclude the technical section with the suggestion that the best way to demonstrate that the nuclear pores are responsible for ion channel activity is by showing with fluorescence microscopy the nuclear translocation of ions and small molecules and the exclusion of the same from the cisterna enclosed by the two membranes of the envelope. Since transcription factors and mRNAs, two major groups of nuclear macromolecules, use nuclear pores to enter and exit the nucleus and play essential roles in the control of gene activity and expression, this review should be useful to cell and molecular biologists interested in understanding how patch-clamp can be used to quantitate the translocation of such macromolecules into and out of the nucleus

The use of genes for performance enhancement: doping or therapy?

Recent biotechnological advances have permitted the manipulation of genetic sequences to treat several diseases in a process called gene therapy. However, the advance of gene therapy has opened the door to the possibility of using genetic manipulation (GM) to enhance athletic performance. In such ‘gene doping’, exogenous genetic sequences are inserted into a specific tissue, altering cellular gene activity or leading to the expression of a protein product. The exogenous genes most likely to be utilized for gene doping include erythropoietin (EPO), vascular endothelial growth factor (VEGF), insulin-like growth factor type 1 (IGF-1), myostatin antagonists, and endorphin. However, many other genes could also be used, such as those involved in glucose metabolic pathways. Because gene doping would be very difficult to detect, it is inherently very attractive for those involved in sports who are prepared to cheat. Moreover, the field of gene therapy is constantly and rapidly progressing, and this is likely to generate many new possibilities for gene doping. Thus, as part of the general fight against all forms of doping, it will be necessary to develop and continually improve means of detecting exogenous gene sequences (or their products) in athletes. Nevertheless, some bioethicists have argued for a liberal approach to gene doping.

Formation and differentiation of multiple mesenchymal lineages during lung development is regulated by {beta}-catenin signaling.

BACKGROUND: The role of ss-catenin signaling in mesodermal lineage formation and differentiation has been elusive. METHODOLOGY: To define the role of ss-catenin signaling in these processes, we used a Dermo1(Twist2)(Cre/+) line to target a floxed beta-catenin allele, throughout the embryonic mesenchyme. Strikingly, the Dermo1(Cre/+); beta-catenin(f/-) conditional Knock Out embryos largely phenocopy Pitx1(-/-)/Pitx2(-/-) double knockout embryos, suggesting that ss-catenin signaling in the mesenchyme depends mostly on the PITX family of transcription factors. We have dissected this relationship further in the developing lungs and find that mesenchymal deletion of beta-catenin differentially affects two major mesenchymal lineages. The amplification but not differentiation of Fgf10-expressing parabronchial smooth muscle progenitor cells is drastically reduced. In the angioblast-endothelial lineage, however, only differentiation into mature endothelial cells is impaired. CONCLUSION: Taken together these findings reveal a hierarchy of gene activity involving ss-catenin and PITX, as important regulators of mesenchymal cell proliferation and differentiation.

Pollen ultrastructure in anther cultures of Datura innoxia. I. Division of the presumptive vegetative cell.

Ultrastructural features of embryogenic pollen in Datura innoxia are described, just prior to, during, and after completion of the first division of the presumptive vegetative cell. In anther cultures initiated towards the end of the microspore phase and incubated at 28 degrees C in darkness, the spores divide within 24 h and show features consistent with those of dividing spores in vivo. Cytokinesis is also normal in most of the spores and the gametophytic cell-plate curves round the presumptive generative nucleus in the usual highly ordered way. Further differentiation of the 2 gametophytic cells does not take place and the pollen either switches to embryogenesis or degenerates. After 48-72 h, the remaining viable pollen shows the vegetative cell in division. The cell, which has a large vacuole and thin layer of parietal cytoplasm carried over from the microspore, divides consistently in a plane parallel to the microspore division. The dividing wall follows a less-ordered course than the gametophytic wall and usually traverses the vacuole, small portions of which are incorporated into the daughter cell adjacent to the generative cell. The only structural changes in the vegetative cell associated with the change in programme appear to be an increase in electron density of both plastids and mitochondria and deposition of an electron-dense material (possibly lipid) on the tonoplast. The generative cell is attached to the intine when the vegetative cell divides. Ribosomal density increases in the generative cell and exceeds that in the vegetative cell. A thin electron-dense layer also appears in the generative-cell wall. It is concluded that embryogenesis commences as soon as the 2 gametophytic cells are laid down. Gene activity associated with postmitotic synthesis of RNA and protein in the vegetative cell is switched off. The data are discussed in relation to the first division of the embryogenic vegetative cells in Nicotiana tabacum.

1,8-cineol potentiates IRF3-mediated antiviral response in human stem cells and an ex vivo model of rhinosinusitis

Common cold is one of the most frequent human inflammatory diseases caused by viruses and can facilitate bacterial super-infections resulting in sinusitis or pneumonia. The active ingredient of the drug Soledum, 1,8-cineole, is commonly applied for treating inflammatory diseases of the respiratory tract. However, the potential of 1,8-cineole for treating primary viral infections of the respiratory tract remains unclear. In the present study, we demonstrate for the first time that 1,8-cineole potentiates Poly(I:C)-induced activity of the anti-viral transcription factor Interferon Regulatory Factor 3, while simultaneously reducing pro-inflammatory NF-κB-activity in human cell lines, inferior turbinate stem cells (ITSCs) and ex vivo cultivated human nasal mucosa. Co-treatment of cell lines with Poly(I:C) and 1,8-cineole resulted in significantly increased IRF3 reporter gene activity compared to Poly(I:C) alone, whereas NF-κB-activity was reduced. Accordingly, 1,8-cineole- and Poly(I:C)-treatment led to increased nuclear translocation of IRF3 in ITSCs and a human ex vivo model of rhinosinusitis compared to the Poly(I:C)-treated approach. Nuclear translocation of IRF3 was significantly increased in ITSCs and slice cultures treated with LPS and 1,8-cineole compared to the LPS-treated cells mimicking bacterial infection. Our findings strongly suggest that 1,8-cineole potentiates the antiviral activity of IRF3 in addition to its inhibitory effect on pro-inflammatory NF-κB-signalling and may thus broaden its field of application.

Chromosomal studies in four species of genus Chaunus (Bufonidae, Anura): localization of telomeric and ribosomal sequences after fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) using telomeric and ribosomal sequences was performed in four species of toad genus Chaunus: C. ictericus, C. jimi, C. rubescens and C. schneideri. Analyses based on conventional, C-banding and Ag-NOR staining were also carried out. The four species present a 2n = 22 karyotype, composed by metacentric and submetacentric chromosomes, which were indistinguishable either after conventional staining or banding techniques. Constitutive heterochromatin was predominantly located at pericentromeric regions, and telomeric sequences (TTAGGG)(n) were restricted to the end of all chromosomes. Silver staining revealed Ag-NORs located at the short arm of pair 7, and heteromorphism in size of NOR signals was also observed. By contrast, FISH with ribosomal probes clearly demonstrated absence of any heteromorphism in size of rDNA sequences, suggesting that the difference observed after Ag-staining should be attributed to differences in chromosomal condensation and/or gene activity rather than to the number of ribosomal cistrons.