Tensile properties of Arabidopsis cell walls depend on both a xyloglucan cross-linked microfibrillar network and rhamnogalacturonan II-borate complexes

The mechanical properties of plant organs depend upon anatomical structure, cell-cell adhesion, cell turgidity, and the mechanical properties of their cell walls. By testing the mechanical responses of Arabidopsis mutants, it is possible to deduce the contribution that polymers of the cell wall make to organ strength. We developed a method to measure the tensile parameters of the expanded regions of turgid or plasmolyzed dark-grown Arabidopsis hypocotyls and applied it to the fucose biosynthesis mutant mur1, the xyloglucan glycosyltransferase mutants mur2 and mur3, and the katanin mutant bot1. Hypocotyls from plants grown in the presence of increasing concentrations of dichlorobenzonitrile, an inhibitor of cellulose synthesis, were considerably weakened, indicating the validity of our approach. In order of decreasing strength, the hypocotyls of mur2 > bot1 and mur1 > mur3 were each found to have reduced strength and a proportionate reduction in modulus compared with wild type. The tensile properties of the hypocotyls and of the inflorescence stems of mur1 were rescued by growth in the presence of high concentrations of borate, which is known to cross-link the pectic component rhamnogalacturonan II. From comparison of the mechanical responses of mur2 and mur3, we deduce that galactose-containing side chains of xyloglucan make a major contribution to overall wall strength, whereas xyloglucan fucosylation plays a comparatively minor role. We conclude that borate-complexed rhamnogalacturonan II and galactosylated xyloglucan contribute to the tensile strength of cell walls.     

In vivo recruitment of XPC to UV-induced cyclobutane pyrimidine dimers by the DDB2 gene product

The initial step in mammalian nucleotide excision repair (NER) of the major UV-induced photoproducts, cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs), requires lesion recognition. It is believed that the heterodimeric proteins XPC/hHR23B and UV-DDB (UV-damaged DNA binding factor, composed of the p48 and p127 subunits) perform this function in genomic DNA, but their requirement and lesion specificity in vivo remains unknown. Using repair-deficient xeroderma pigmentosum (XP)-A cells that stably express photoproduct-specific photolyases, we determined the binding characteristics of p48 and XPC to either CPDs or 6-4PPs in vivo. p48 localized to UV-irradiated sites that contained either CPDs or 6-4PPs. However, XPC localized only to UV-irradiated sites that contained 6-4PPs, suggesting that XPC does not efficiently recognize CPDs in vivo. XPC did localize to CPDs when p48 was overexpressed in the same cell, signifying that p48 activates the recruitment of XPC to CPDs and may be the initial recognition factor in the NER pathway.     

Direct interference with rhamnogalacturonan I biosynthesis in Golgi vesicles

Pectin is a class of complex cell wall polysaccharides with multiple roles during cell development. Assigning specific functions to particular polysaccharides is in its infancy, in part, because of the limited number of mutants and transformants available with modified pectic polymers in their walls. Pectins are also important polymers with diverse applications in the food and pharmaceutical industries, which would benefit from technology for producing pectins with specific functional properties. In this report, we describe the generation of potato (Solanum tuberosum L. cv Posmo) tuber transformants producing pectic rhamnogalacturonan I (RGI) with a low level of arabinosylation. This was achieved by the expression of a Golgi membrane-anchored endo-alpha-1,5-arabinanase. Sugar composition analysis of RGI isolated from transformed and wild-type tubers showed that the arabinose content was decreased by approximately 70% in transformed cell walls compared with wild type. The modification of the RGI was confirmed by immunolabeling with an antibody recognizing alpha-1,5-arabinan. This is the first time, to our knowledge, that the biosynthesis of a plant cell wall polysaccharide has been manipulated through the action of a glycosyl hydrolase targeted to the Golgi compartment.     

Alphavirus replicon particles as candidate HIV vaccines

Replicon particles based on Venezuelan equine encephalitis virus (VEE) contain a self-replicating RNA encoding the VEE replicase proteins and expressing a gene of interest in place of the viral structural protein genes. Structural proteins for packaging of replicon RNA into VEE replicon particles (VRPs) are expressed from separate helper RNAs. Aspects of the biology of VEE that are exploited in VRP vaccines include 1) expression of very high levels of immunogen, 2) expression of immunizing proteins in cells in the draining lymph node, and 3) the ability to induce mucosal immunity from a parental inoculation. Results of experiments with VRPs expressing green fluorescent protein or influenza virus hemagglutinin (HA) demonstrated that specific mutations in the VRP envelope glycoproteins affect both targeting in the draining lymph node and efficiency of the immune response in mice. VRPs expressing either the matrix-capsid portion of Gag, the full-length envelope gp160, or the secreted gp140 of cloned SIVsm H-4i were mixed in a cocktail and used to immunize macaques at 0, 1, and 4 months. Neutralizing antibodies against SIVsm H-4 were induced in 6 of 6 vaccinates and CTL in 4 of 6. An intrarectal challenge with the highly pathogenic SIVsm E660 was given at 5 months. A vaccine effect was seen in reduced peak virus loads, reduced virus loads both at set point and at 41 weeks postchallenge, and preserved or increased CD4 counts compared to controls. A candidate VRP HIV vaccine expressing Clade C Gag contains a sequence that is very close to the South African Clade C consensus and was selected from a recent seroconverter in the Durban cohort to represent currently circulating genotypes in South Africa. A GMP lot of this vaccine has been manufactured and tested for a phase I trial in the first months of 2002.     

Genome sequence of the dissimilatory metal ion-reducing bacterium Shewanella oneidensis

Shewanella oneidensis is an important model organism for bioremediation studies because of its diverse respiratory capabilities, conferred in part by multicomponent, branched electron transport systems. Here we report the sequencing of the S. oneidensis genome, which consists of a 4,969,803-base pair circular chromosome with 4,758 predicted protein-encoding open reading frames (CDS) and a 161,613-base pair plasmid with 173 CDSs. We identified the first Shewanella lambda-like phage, providing a potential tool for further genome engineering. Genome analysis revealed 39 c-type cytochromes, including 32 previously unidentified in S. oneidensis, and a novel periplasmic [Fe] hydrogenase, which are integral members of the electron transport system. This genome sequence represents a critical step in the elucidation of the pathways for reduction (and bioremediation) of pollutants such as uranium (U) and chromium (Cr), and offers a starting point for defining this organism's complex electron transport systems and metal ion-reducing capabilities.     

Galpha3 and protein kinase A represent cross-talking pathways for gene expression in Dictyostelium discoideum

Heterotrimeric G proteins and protein kinase A (PKA) are regulators of development in Dictyostelium discoideum. It has been reported that disruption of the Dictyostelium Galpha3 gene (galpha3-) blocks development and expression of several early development genes, characteristics that are reminiscent of mutants lacking the catalytic subunit of PKA (pkac-). The hypothesis that Galpha3 and PKA signaling pathways may interact to control developmental gene expression was tested by comparing the regulation of seven genes expressed early in development in the wild-type and in galpha3- and pkac- mutants, and comparing PKA activity in the wild-type and in a galpha3- mutant. The expression patterns of six genes were affected similarly by the Galpha3 and PKA mutations, while the expression of only one gene, the cAMP receptor 1 (cAR1), differed between the mutants. PKA activity, measured by phosphorylation of the PKA-specific substrate Kemptide, was higher in galpha3- cells than in wild-type cells, suggesting that Galpha3 normally exerts an inhibitory effect on PKA activity. Although some early development genes appear to require both Galpha3 and PKA for expression, the differing response of cAR1 expression and the inhibitory effect of Galpha3 on PKA activity suggest that Galpha3 and PKA are members of interacting pathways controlling gene expression early in development.     

Differential sensitivity of inward rectifier K+ channels to metabolic inhibitors

Inhibition of inward rectifier K(+) channels under ischemic conditions may contribute to electrophysiological consequences of ischemia such as cardiac arrhythmia. Ischemia causes metabolic inhibition, and the use of metabolic inhibitors is one experimental method of simulating ischemia. The effects of metabolic inhibitors on the activity of inward rectifier K(+) channels K(ir)2.1, K(ir)2.2, and K(ir)2.3 were studied by heterologous expression in Xenopus oocytes and two-electrode voltage clamp. 10 microm carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) inhibited K(ir)2.2 and K(ir)2.3 currents but was without effect on K(ir)2.1 currents. The rate of decline of current in FCCP was faster for K(ir)2.3 than for K(ir)2.2. K(ir)2.3 was inhibited by 3 mm sodium azide (NaN(3)), whereas K(ir)2.1 and K(ir)2.2 were not. K(ir)2.2 was inhibited by 10 mm NaN(3). All three of these inward rectifiers were inhibited by lowering the pH of the solution perfusing inside-out membrane patches. K(ir)2.3 was most sensitive to pH (pK = 6.9), whereas K(ir)2.1 was least sensitive (pK = 5.9). For K(ir)2.2 the pK was 6.2. These results demonstrate the differential sensitivity of these inward rectifiers to metabolic inhibition and internal pH. The electrophysiological response of a particular cell type to ischemia may depend on the relative expression levels of different inward rectifier genes.     

Marine biofouling: a sticky problem

Organisms that colonise underwater surfaces, such as barnacle larvae and spores of algae, use a diverse array of biological 'glues' to provide both temporary and more permanent adhesion. The practical consequence of colonisation by these organisms is biofouling - something that has plagued mariners for years - causing increased drag and, in extreme cases, corrosion. Might there be a biological solution to this biological problem?