RDR1 and SGS3, Components of RNA-Mediated Gene Silencing, Are Required for the Regulation of Cuticular Wax Biosynthesis in Developing Inflorescence Stems of Arabidopsis

The cuticle is a protective layer that coats the primary aerial surfaces of land plants and mediates plant interactions with the environment. It is synthesized by epidermal cells and is composed of a cutin polyester matrix that is embedded and covered with cuticular waxes. Recently, we have discovered a novel regulatory mechanism of cuticular wax biosynthesis that involves the ECERIFERUM7 (CER7) ribonuclease, a core subunit of the exosome. We hypothesized that at the onset of wax production, the CER7 ribonuclease degrades an mRNA specifying a repressor of CER3, a wax biosynthetic gene whose protein product is required for wax formation via the decarbonylation pathway. In the absence of this repressor, CER3 is expressed, leading to wax production. To identify the putative repressor of CER3 and to unravel the mechanism of CER7-mediated regulation of wax production, we performed a screen for suppressors of the cer7 mutant. Our screen resulted in the isolation of components of the RNA-silencing machinery, RNA-DEPENDENT RNA POLYMERASE1 and SUPPRESSOR OF GENE SILENCING3, implicating RNA silencing in the control of cuticular wax deposition during inflorescence stem development in Arabidopsis (Arabidopsis thaliana).     

A new way of measuring apoptosis by absolute quantitation of inter-nucleosomally fragmented genomic DNA

Several critical events of apoptosis occur in the cell nucleus, including inter-nucleosomal DNA fragmentation (apoptotic DNA) and eventual chromatin condensation. The generation of apoptotic DNA has become a biochemical hallmark of apoptosis because it is a late 'point of no return' step in both the extrinsic (cell-death receptor) and intrinsic (mitochondrial) apoptotic pathways. Despite investigators observing apoptotic DNA and understanding its decisive role as a marker of apoptosis for over 20 years, measuring it has proved elusive. We have integrated ligation-mediated PCR and qPCR to design a new way of measuring apoptosis, termed ApoqPCR, which generates an absolute value for the amount (picogram) of apoptotic DNA per cell population. ApoqPCR's advances over current methods include a 1000-fold linear dynamic range yet sensitivity to distinguish subtle low-level changes, measurement with a 3- to 4-log improvement in sample economy, and capacity for archival or longitudinal studies combined with high-throughput capability. We demonstrate ApoqPCR's utility in both in vitro and in vivo contexts. Considering the fundamental role apoptosis has in vertebrate and invertebrate health, growth and disease, the reliable measurement of apoptotic nucleic acid by ApoqPCR will be of value in cell biology studies in basic and applied science.     

Impact of ivermectin on onchocerciasis transmission: assessing the empirical evidence that repeated ivermectin mass treatments may lead to elimination/eradication in West-Africa

BACKGROUND: The Onchocerciasis Control Program (OCP) in West Africa has been closed down at the end of 2002. All subsequent control will be transferred to the participating countries and will almost entirely be based on periodic mass treatment with ivermectin. This makes the question whether elimination of infection or eradication of onchocerciasis can be achieved using this strategy of critical importance. This study was undertaken to explore this issue. METHODS: An empirical approach was adopted in which a comprehensive analysis was undertaken of available data on the impact of more than a decade of ivermectin treatment on onchocerciasis infection and transmission. Relevant entomological and epidemiological data from 14 river basins in the OCP and one basin in Cameroon were reviewed. Areas were distinguished by frequency of treatment (6-monthly or annually), endemicity level and additional control measures such as vector control. Assessment of results were in terms of epidemiological and entomological parameters, and as a measure of inputs, therapeutic and geographical coverage rates were used. RESULTS: In all of the river basins studied, ivermectin treatment sharply reduced prevalence and intensity of infection. Significant transmission, however, is still ongoing in some basins after 10-12 years of ivermectin treatment. In other basins, transmission may have been interrupted, but this needs to be confirmed by in-depth evaluations. In one mesoendemic basin, where 20 rounds of four-monthly treatment reduced prevalence of infection to levels as low as 2-3%, there was significant recrudescence of infection within a few years after interruption of treatment. CONCLUSIONS: Ivermectin treatment has been very successful in eliminating onchocerciasis as a public health problem. However, the results presented in this paper make it almost certain that repeated ivermectin mass treatment will not lead to the elimination of transmission of onchocerciasis from West Africa. Data on 6-monthly treatments are not sufficient to draw definitive conclusions.     

A New Species of Amphirhagatherium (Choeropotamidae, Artiodactyla, Mammalia) from the Late Eocene Headon Hill Formation of Southern England and Phylogeny of Endemic European 'anthracotherioids'

A new species of artiodactyl, Amphirhagatherium edwardsi sp. nov., is described from the Late Eocene (Priabonian) Headon Hill Formation of the Hampshire Basin, southern England. The Haplobunodontidae, in which Amphirhagatherium is usually placed, has recently been combined with the monotypic Choeropotamidae, both essentially European endemic families. New anatomical information is forthcoming from both the new species and recently published data on related species. A cladistic analysis of taxa included in the two families, the possible anthracotheriid Thaumastognathus and the enigmatic Tapirulus , was conducted to test the relationships implied by observed morphological similarities. The genus Anthracobunodon is shown to be paraphyletic and is here synonymized with Amphirhagatherium . Choeropotamus and Thaumastognathus are sister taxa nested with three species of Haplobunodon and Haplobunodon is paraphyletic and polyphyletic, but this clade is too weakly resolved internally for reliable taxonomic changes. Lophiobunodon Tapirulus are sister taxa nested with a fourth species of Haplobunodon . The synonymy of the Haplobunodontidae with the Choeropotamidae is upheld and close relationship of the family with the Anthracotheriidae is argued to be unlikely. Choeropotamids are inferred to have had mixed frugivorous and browsing herbivorous diets. They seem to have diversified in the northern parts of Europe, some terminal taxa having originated following southward dispersal.     

Expression of functional human coagulation factor XIII A-domain in plant cell suspensions and whole plants

Coagulation factor XIII, a zymogen present in blood as a tetramer (A2B2) of A- and B-domains, is one of the components of many "wound sealants" which are proposed for use or currently in use as effective hemostatic agents, sealants, and tissue adhesives in surgery. After activation by alpha-thrombin cleavage, coagulation factor XIII A-domain, a transglutaminase, is formed and catalyzes the covalent cross-linking of the alpha- and gamma-chains of linear fibrin to form homopolymers, which can quickly stop bleeding. We have successfully expressed the A-domain of factor XIII in both plant cell cultures and whole plants. Transgenic plant cell culture allows a rapid method for testing production feasibility while expression in whole plants demonstrates an economic production system for recombinant human plasma-based proteins. The expressed factor XIII A-domain had a similar size as that of human plasma-derived factor XIII. Crude plant extract containing recombinant factor XIII A-domain showed transglutaminase activity with monodansylcadaverine and casein as substrates and cross-linking activity in the presence of linear fibrin. The expression of factor XIII A-domain was not affected by plant leaf position.     

Gaseous emissions from burning diesel, crude and prime bleachable summer yellow cottonseed oil in a burner for drying seedcotton

Cottonseed oil has been used as a fuel source either as a blend with diesel in varying proportions or undiluted (100%) in numerous studies evaluating its potential use in internal combustion engines. However, limited research is available on the use of cottonseed oil as a fuel source in a multi-fueled burner similar to those used by cottonseed oil mills and cotton gins in their drying operations. The purpose of this study was to evaluate emissions from five fuel oil treatments while firing a multi-fueled burner in a setup similar to those used for drying operations of both cottonseed oil mills and cotton gins. For each treatment, gaseous emissions were measured while firing the burner at three fuel flow rates. The five fuel oil treatments evaluated were: (1) No. 2 diesel at 28.3 degrees C, (2) prime bleachable summer yellow (PBSY) cottonseed oil at 28.3 degrees C (PBSY-28), (3) crude cottonseed oil at 28.3 degrees C (Crude-28), (4) PBSY at 60 degrees C (PBSY-60), and (5) crude at 60 degrees C (Crude-60). Results indicate that PBSY treatments had the lowest overall emissions of all treatments. The other treatments varied in emission rates based on treatment and fuel flow rate. Preheating the oil to 60 degrees C resulted in higher NO(x) emissions but displayed varying results in regards to CO. The CO emissions for the crude treatments were relatively unaffected by the 60 degrees C preheat temperature whereas the preheated PBSY treatments demonstrated lower CO emissions. Overall, both cottonseed oils performed well in the multi-fueled burner and displayed a promising potential as an alternative fuel source for cottonseed oil mills and cotton gins in their drying operations.     

Expression of the wax-specific condensing enzyme CUT1 in Arabidopsis

The Arabidopsis thaliana gene CUT1 encodes a very-long-chain fatty acid-condensing enzyme required for the production of epicuticular wax in bolting stems. We have examined the expression pattern of CUT1 in Arabidopsis at different developmental stages and under different environmental conditions. RNA blot analysis showed that CUT1 was highly expressed in shoots, but not in roots. CUT1 expression was detectable throughout development. Light was required for CUT1 expression, and expression was increased by salt and drought treatments. The promoter region of the CUT1 gene was cloned, and 1.2 kb of the sequence 5' to the translation start codon was used to direct beta-glucuronidase (GUS) expression in transgenic plants. Histochemical and fluorometric (quantitative) GUS assays confirmed that the CUT1 promoter directed epidermal-specific expression and was highly active in Arabidopsis and in tobacco. A construct using the CUT1 promoter to drive CUT1 expression (CUT1p-CUT1) was used to transform Arabidopsis. Transgenic plants which had somewhat increased (overexpression) or greatly reduced (co-suppression) wax loads were recovered. Thus, the CUT1 promoter should be useful for genetic engineering applications that require epidermis-specific expression of genes.