Clubroot (Plasmodiophora brassicae Woronin): an agricultural and biological challenge worldwide

Clubroot disease and the causal microbe Plasmodiophora brassicae offer abundant challenges to agriculturists and biological scientists. This microbe is well fitted for the environments which it inhabits. Plasmodiophora brassicae exists in soil as microscopic well protected resting spores and then grows actively and reproduces while shielded inside the roots of host plants. The pathogen is active outside the host for only short periods. Consequently, scientific studies are made challenging by the biological context of the host and pathogen and the technology required to investigate and understand that relationship. Controlling clubroot disease is a challenge for farmers, crop consultants and plant pathology practitioners because of the limited options which are available. Full symptom expression happens solely in members of the Brassicaceae family. Currently, only a few genes expressing strong resistance to P. brassicae are known and readily available. Agrochemical control is similarly limited by difficulties in molecule formulation which combines efficacy with environmental acceptability. Manipulation of husbandry encouraging improvements in soil structure, texture, nutrient composition and moisture content can reduce populations of P. brassicae. Integrating such strategies with rotation and crop management will reduce but not eliminate this disease. There are indications that forms of biological competition may be mobilised as additions to integrated control strategies. The aim of this review is to chart key themes in the development of scientific biological understanding of this host-pathogen relationship by offering signposts to grapple with clubroot disease which devastates crops and their profitability. Particular attention is given to the link between soil and nutrient chemistry and activity of this microbe.

Identification of novel cholesteatoma-related gene expression signatures using full-genome microarrays

This study demonstrates that the expression profile of cholesteatoma is similar to a metastatic tumour and chronically inflamed tissue. Based on the investigated profiles we present novel protein-protein interaction and signal transduction networks, which include cholesteatoma-regulated transcripts and may be of great value for drug targeting and therapy development.

Early membrane estrogenic effects required for full expression of slower genomic actions in a nerve cell line.

Interpretations of steroid hormone actions as slow, nuclear, transcriptional events have frequently been seen as competing against inferences of rapid membrane actions. We have discovered conditions where membrane-limited effects potentiate later transcriptional actions in a nerve cell line. Making use of a two-pulse hormonal schedule in a transfection system, early and brief administration of conjugated, membrane-limited estradiol was necessary but not sufficient for full transcriptional potency of the second estrogen pulse. Efficacy of the first pulse depended on intact signal transduction pathways. Surprisingly, the actions of both pulses were blocked by a classical estrogen receptor (ER) antagonist. Thus, two different modes of steroid hormone action can synergize.

Comparison of the global gene expression profiles produced by methylparaben, n-butylparaben and 17 beta-oestradiol in MCF7 human breast cancer cells

Since the alkyl esters of p-hydroxybenzoic acid (parabens) can be measured intact in the human breast and possess oestrogenic properties, it has been suggested that they could contribute to an aberrant burden of oestrogen signalling in the human breast and so play a role in the rising incidence of breast cancer. However, although parabens have been shown to regulate a few single genes (reporter genes, pS2, progesterone receptor) in a manner similar to that of 17 beta-oestradiol, the question remains as to the full extent of the similarity in the overall gene profile induced in response to parabens compared with 17 beta-oestradiol. The GE-Amersham CodeLink 20 K human expression microarray system was used to profile the expression of 19881 genes in MCF7 human breast cancer cells following a 7-day exposure to 5 x 10(-4) m methylparaben, 10(-5) m n-butylparaben and 10(-8) m 17 beta-oestradiol. At these concentrations, the parabens gave growth responses in MCF7 cells of similar magnitude to 17 beta-oestradiol. The study identified genes which are upregulated or downregulated to a similar extent by methylparaben, n-butylparaben and 17 beta-oestradiol. However, the majority of genes were not regulated in the same way by all three treatments. Some genes responded differently to parabens from 17 beta-oestradiol, and furthermore, differences in expression of some genes could be detected even between the two individual parabens. Therefore, although parabens possess oestrogenic properties, their mimicry in terms of global gene expression patterns is not perfect and differences in gene expression profiles could result in consequences to the cells that are not identical to those following exposure to 17 beta-oestradiol. Copyright (c) 2006 John Wiley & Sons, Ltd.

Differential changes in transforming growth factor β isoform expression during postnatal cardiac growth

Transforming growth factor-β (TGF-β) is synthesised as an inactive precursor protein; this is cleaved to produce the mature peptide and a latency associated protein (LAP), which remains associated with the mature peptide until activation by LAP degradation. Isoform specific antibodies raised against the LAPs for TGF-β2and -β3were used to determine the myocardial levels of LAP (activatable TGF-β) and full length precursor (inactive TGF-β) forms during post-natal development in the rat. TGF-β2was present predominantly as the precursor in 2 day old myocardium. There was an age-dependent shift from precursor protein to LAP between 2 and 28 days. A corresponding increase in the level of mature (activatable) TGF-β2was found. TGF-β3was detected in significant quantities only as LAP. However, a four-fold increase in the expression of TGF-β3LAP was observed between 2 and 28 days. The substantial increases in activatable forms of TGF-β2and -β3that occur in myocardium during the first 28 days of life in the rat support a role for these proteins in post-natal cardiac development.

Cloning, expression, and characterization of human cytosolic aminopeptidase P: a single manganese(II)-dependent enzyme

The mammalian bradykinin-degrading enzyme aminopeptidase P (AP-P; E. C. 3.4.11.9) is a metal-dependent enzyme and is a member of the peptidase clan MG. AP-P exists as membrane-bound and cytosolic forms, which represent distinct gene products. A partially truncated clone encoding the cytosolic form was obtained from a human pancreatic cDNA library and the 5' region containing the initiating Met was obtained by 5' rapid accumulation of cDNA ends (RACE). The open reading frame encodes a protein of 623 amino acids with a calculated molecular mass of 69,886 Da. The full-length cDNA with a C-terminal hexahistidine tag was expressed in Escherichia coli and COS-1 cells and migrated on SDS-PAGE with a molecular mass of 71 kDa. The expressed cytosolic AP-P hydrolyzed the X-Pro bond of bradykinin and substance P but did not hydrolyze Gly-Pro-hydroxyPro. Hydrolysis of bradykinin was inhibited by 1,10-phenanthroline and by the specific inhibitor of the membrane-bound form of mammalian AP-P, apstatin. Inductively coupled plasma atomic emission spectroscopy of AP-P expressed in E. coli revealed the presence of 1 mol of manganese/mol of protein and insignificant amounts of cobalt, iron, and zinc. The enzymatic activity of AP-P was promoted in the presence of Mn(II), and this activation was increased further by the addition of glutathione. The only other metal ion to cause slight activation of the enzyme was Co(II), with Ca(II), Cu(II), Mg(II), Ni(II), and Zn(II) all being inhibitory. Removal of the metal ion from the protein was achieved by treatment with 1,10-phenanthroline. The metal-free enzyme was reactivated by the addition of Mn(II) and, partially, by Fe(II). Neither Co(II) nor Zn(II) reactivated the metal-free enzyme. On the basis of these data we propose that human cytosolic AP-P is a single metal ion-dependent enzyme and that manganese is most likely the metal ion used in vivo.

Codon and mRNA sequence optimization of microdystrophin transgenes improves expression and physiological outcome in dystrophic mdx mice following AAV2/8 gene transfer

Duchenne muscular dystrophy is a fatal muscle-wasting disorder. Lack of dystrophin compromises the integrity of the sarcolemma and results in myofibers that are highly prone to contraction-induced injury. Recombinant adenoassociated virus (rAAV)-mediated dystrophin gene transfer strategies to muscle for the treatment of Duchenne muscular dystrophy (DMD) have been limited by the small cloning capacity of rAAV vectors and high titers necessary to achieve efficient systemic gene transfer. In this study, we assess the impact of codon optimization on microdystrophin (ΔAB/R3-R18/ΔCT) expression and function in the mdx mouse and compare the function of two different configurations of codon-optimized microdystrophin genes (ΔAB/R3-R18/ΔCT and ΔR4-R23/ΔCT) under the control of a muscle-restrictive promoter (Spc5-12). Codon optimization of microdystrophin significantly increases levels of microdystrophin mRNA and protein after intramuscular and systemic administration of plasmid DNA or rAAV2/8. Physiological assessment demonstrates that codon optimization of ΔAB/R3-R18/ΔCT results in significant improvement in specific force, but does not improve resistance to eccentric contractions compared with noncodon-optimized ΔAB/ R3-R18/ΔCT. However, codon-optimized microdystrophin ΔR4-R23/ΔCT completely restored specific force generation and provided substantial protection from contraction-induced injury. These results demonstrate that codon optimization of microdystrophin under the control of a muscle-specific promoter can significantly improve expression levels such that reduced titers of rAAV vectors will be required for efficient systemic administration.

Bmp4 regulates chick Ebf2 and Ebf3 gene expression in somite development

The chick Early B-cell Factor-2 and 3 (cEbf2 and cEbf3) genes are members of EBF family of helix loop helix transcription factors. The expression, regulation and importance of these genes have been extensively studied in lymphatic, nervous and muscular tissues. Recently, a new role for some members of EBF in bone development has been investigated. However, the expression profile and regulation in the axial skeleton precursor, the somite, have yet to be elucidated. Therefore, this study was aimed to investigate the expression and regulation of cEbf2 and cEbf3 genes in the developing chick embryo somite from HH4 to HH28. The spatiotemporal expression study revealed predominant localization of cEbf2 and cEbf3 in the lateral sclerotomal domains and later around vertebral cartilage anlagen of the arch and the proximal rib. Subsequently, microsurgeries, ectopic gene expression experiments were performed to analyze which tissues and factors regulate cEbf2 and cEbf3 expression. Lateral barriers experiments indicated the necessity for lateral signal(s) in the regulation of cEbf2 and cEbf3 genes. Results from tissue manipulations and ectopic gene expression experiments indicate that lateral plate-derived Bmp4 signals are necessary for the initiation and maintenance of cEbf2 and cEbf3 genes in somites. In conclusion, cEbf2 and cEbf3 genes are considered as lateral sclerotome markers which their expression is regulated by Bmp4 signals from the lateral plate mesoderm.

Regulation of gene and protein expression in cardiac myocyte hypertrophy and apoptosis

Considerable efforts have been expended in elucidating the inter-cellular and intra-cellular signaling pathways which elicit cardiac myocyte hypertrophy or apoptosis, and in identifying the changes which are associated with the end-stage of the response. The challenge now is to link the two. Although some of the signaling effects will be the acute modulation of existing protein function, long-term effects which bring about and maintain the hypertrophic state or which culminate in cell death are mediated at the level of gene and protein expression. With the advances in micro-array technology and genome sequencing, it is now possible to obtain a picture of the global gene expression profile in myocytes or in whole heart which dictates the proteins which could be made. This is not the final picture since additional regulation at the level of translation modulates the relative proportions of each protein that can be made from the transcriptome. Even here, further regulation of protein stability and turnover means that ultimately it is still necessary to examine the proteome to determine what may cause the functional changes in a cell. Thus, in order to gain a full picture of events which regulate the response and gain some insight into possible points of intervention for therapy, it is necessary to examine gene expression, mRNA translation and protein expression in concert.