Variability of gene expression profiles in human blood and lymphoblastoid cell lines

Background

Infection of plants by pathogens and the subsequent disease development involves substantial changes in the biochemistry and physiology of both partners. Analysis of genes that are expressed during these interactions represents a powerful strategy to obtain insights into the molecular events underlying these changes. We have employed expressed sequence tag (EST) analysis to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe oryzae and fungal genes involved in infectious growth within the host during a compatible interaction.

Results

A cDNA library was constructed with RNA from rice leaves (Oryza sativa cv. Hwacheong) infected with M. oryzae strain KJ201. To enrich for fungal genes, subtraction library using PCR-based suppression subtractive hybridization was constructed with RNA from infected rice leaves as a tester and that from uninfected rice leaves as the driver. A total of 4,148 clones from two libraries were sequenced to generate 2,302 non-redundant ESTs. Of these, 712 and 1,562 ESTs could be identified to encode fungal and rice genes, respectively. To predict gene function, Gene Ontology (GO) analysis was applied, with 31% and 32% of rice and fungal ESTs being assigned to GO terms, respectively. One hundred uniESTs were found to be specific to fungal infection EST. More than 80 full-length fungal cDNA sequences were used to validate ab initio annotated gene model of M. oryzae genome sequence.

Conclusion

This study shows the power of ESTs to refine genome annotation and functional characterization. Results of this work have advanced our understanding of the molecular mechanisms underpinning fungal-plant interactions and formed the basis for new hypothesis.     

Characterization of a stretch‐activated potassium channel in chondrocytes

Chondrocytes possess the capacity to transduce load‐induced mechanical stimuli into electrochemical signals. The aim of this study was to functionally characterize an ion channel activated in response to membrane stretch in isolated primary equine chondrocytes. We used patch‐clamp electrophysiology to functionally characterize this channel and immunohistochemistry to examine its distribution in articular cartilage. In cell‐attached patch experiments, the application of negative pressures to the patch pipette (in the range of 20–200 mmHg) activated ion channel currents in six of seven patches. The mean activated current was 45.9 ± 1.1 pA (n = 4) at a membrane potential of 33 mV (cell surface area approximately 240 µm²). The mean slope conductance of the principal single channels resolved within the total stretch‐activated current was 118 ± 19 pS (n = 6), and reversed near the theoretical potassium equilibrium potential, E_K+, suggesting it was a high‐conductance potassium channel. Activation of these high‐conductance potassium channels was inhibited by extracellular TEA (K_d approx. 900 µM) and iberiotoxin (K_d approx. 40 nM). This suggests that the current was largely carried by BK‐like potassium (MaxiK) channels. To further characterize these BK‐like channels, we used inside‐out patches of chondrocyte membrane: we found these channels to be activated by elevation in bath calcium concentration. Immunohistochemical staining of equine cartilage samples with polyclonal antibodies to the α1‐ and β1‐subunits of the BK channel revealed positive immunoreactivity for both subunits in superficial zone chondrocytes. These experiments support the hypothesis that functional BK channels are present in chondrocytes and may be involved in mechanotransduction and chemotransduction. J. Cell. Physiol. 223: 511–518, 2010. © 2010 Wiley‐Liss, Inc.     

Inhibition of hCG-induced spawning by alpha-methylparatyrosine, a tyrosine hydroxylase inhibitor, in the catfish Heteropneustes fossilis (Bloch)

In this study, the effect of pharmacological inhibition of catecholaminergic activity on hCG-induced spawning was evaluated and correlated with tyrosine hydroxylase (TH) activity, the rate-limiting enzyme in catecholamine biosynthesis. Gravid female H. fossilis collected in both prespawning and spawning phases were given alpha-methylparatyrosine (alpha-MPT: 250 microg/g body weight, ip, an irreversible inhibitor of TH) and human chorionic gonadotropin (hCG: 100 IU/fish, ip) alone or in combination. The fish were sampled at different intervals for measuring hypothalamic and ovarian TH activity and checking spawning response. The administration of hCG resulted in ovulation and spawning in both phases with a higher response in the spawning phase. The administration of alpha-MPT did not induce any response, like the control fish. In the hCG + alpha-MPT groups, the spawning response of hCG was significantly inhibited and delayed by the inhibitor. The spawning response of hCG was accompanied by a significant increase in both hypothalamic and ovarian TH activity at 6 and 12 h of the injection. However, at 24hr the activity decreased except in the spawning phase. The alpha-MPT treatment inhibited TH activity significantly in a duration-dependent manner. In the hCG + alpha-MPT groups, enzyme activity was inhibited at all duration. The results indicate the involvement of catecholamines during the hCG-induced spawning and the specific functional nature of the involvement needs further investigation.     

Adaptive evolution of lateral plates in three‐spined stickleback Gasterosteus aculeatus: a case study in functional analysis of natural variation

The three‐spined stickleback Gasterosteus aculeatus is a model species for studying questions in ecology and evolution. The rapid diversification of G. aculeatus in post‐glacial freshwater environments, combined with recently developed molecular tools, provides a unique opportunity to study the functional basis of fitness variation in natural populations. In derived freshwater populations, a number of morphological traits have diverged in parallel from the marine ancestral state, including the number of lateral armour plates. Evolution of reduced armour in freshwater populations is due to positive selection from both abiotic and biotic mechanisms. The major effect gene (ectodysplasin‐A or Eda), along with several minor effect genetic regions, has recently been shown to control lateral plate variation. Field experiments have further determined the fitness consequences of allelic variation at the major effect locus. This work helps elucidate the mechanisms connecting genetic variation with phenotypic variation and fitness in the wild, a synthesis that should be applicable to many other phenotypic traits and species of fishes.     

Constraints on Sedge Meadow Self‐Restoration in Urban Wetlands

Invasive plants and urban run‐off constrain efforts to restore sedge meadow wetlands. We asked if native graminoids can self‐restore following the removal of Typha × glauca (hybrid cattail), and if not, what limits their recovery? After we harvested Typha and depleted its rhizome starch reserves, Carex spp. expanded vegetatively (approximately 1 m over 2 years) but not by recruiting seedlings. A seedling emergence experiment showed that seed banks were depleted where Typha had eliminated the sedge meadow over a decade ago (based on aerial photo analysis). Carex seedling emergence was 75–90% lower where Carex was absent than where it remained in the plant community, and at least 17 species that were abundant 30 years ago were absent from the seed bank and extant vegetation. By varying hydroperiod, we showed that prolonged flooding prevented emergence of Carex seedlings and that a fluctuating hydroperiod reduced emergence and ultimately killed all Carex seedlings. In contrast, Typha seedlings emerged and survived regardless of hydroperiod. Thus, slow vegetative expansion by Carex, depauperate seed banks, and altered hydroperiods all constrain self‐restoration. To compensate for multiple constraints on self‐restoration, we recommend a long‐term management approach that capitalizes on flooding and the capacity of Carex spp. to regrow vegetatively. We suggest annually harvesting swaths of Typha at the edges of clones, before or during flood events, to allow gradual, vegetative self‐restoration of Carex spp.     

Detail in network models of epidemiology: are we there yet?

Network models of infectious disease epidemiology can potentially provide insight into how to tailor control strategies for specific regions, but only if the network adequately reflects the structure of the region's contact network. Typically, the network is produced by models that incorporate details about human interactions. Each detail added renders the models more complicated and more difficult to calibrate, but also more faithful to the actual contact network structure. We propose a statistical test to determine when sufficient detail has been added to the models and demonstrate its application to the models used to create a synthetic population and contact network for the USA.     

Cost (Non)-Recovery by Platform Technology Facilities in the Bio21 Cluster

Platform technologies (PT) are techniques or tools that enable a range of scientific investigations and are critical to today''s advanced technology research environment. Once installed, they require specialized staff for their operations, who in turn, provide expertise to researchers in designing appropriate experiments. Through this pipeline, research outputs are raised to the benefit of the researcher and the host institution.¹ Platform facilities provide access to instrumentation and expertise for a wide range of users beyond the host institution, including other academic and industry users. To maximize the return on these substantial public investments, this wider access needs to be supported. The question of support and the mechanisms through which this occurs need to be established based on a greater understanding of how PT facilities operate. This investigation was aimed at understanding if and how platform facilities across the Bio21 Cluster meet operating costs. Our investigation found: 74% of platforms surveyed do not recover 100% of direct operating costs and are heavily subsidized by their home institution, which has a vested interest in maintaining the technology platform; platform managers play a major role in establishing the costs and pricing of the facility, normally in a collaborative process with a management committee or institutional accountant; and most facilities have a three-tier pricing structure recognizing internal academic, external academic, and commercial clients.     

A molecular mechanism for eflornithine resistance in African trypanosomes

Human African trypanosomiasis, endemic to sub-Saharan Africa, is invariably fatal if untreated. Its causative agent is the protozoan parasite Trypanosoma brucei. Eflornithine is used as a first line treatment for human African trypanosomiasis, but there is a risk that resistance could thwart its use, even when used in combination therapy with nifurtimox. Eflornithine resistant trypanosomes were selected in vitro and subjected to biochemical and genetic analysis. The resistance phenotype was verified in vivo. Here we report the molecular basis of resistance. While the drug's target, ornithine decarboxylase, was unaltered in resistant cells and changes to levels of metabolites in the targeted polyamine pathway were not apparent, the accumulation of eflornithine was shown to be diminished in resistant lines. An amino acid transporter gene, TbAAT6 (Tb927.8.5450), was found to be deleted in two lines independently selected for resistance. Ablating expression of this gene in wildtype cells using RNA interference led to acquisition of resistance while expression of an ectopic copy of the gene introduced into the resistant deletion lines restored sensitivity, confirming the role of TbAAT6 in eflornithine action. Eflornithine resistance is easy to select through loss of a putative amino acid transporter, TbAAT6. The loss of this transporter will be easily identified in the field using a simple PCR test, enabling more appropriate chemotherapy to be administered.     

Systematic dissection and trajectory-scanning mutagenesis of the molecular interface that ensures specificity of two-component signaling pathways

Two-component signal transduction systems enable bacteria to sense and respond to a wide range of environmental stimuli. Sensor histidine kinases transmit signals to their cognate response regulators via phosphorylation. The faithful transmission of information through two-component pathways and the avoidance of unwanted cross-talk require exquisite specificity of histidine kinase-response regulator interactions to ensure that cells mount the appropriate response to external signals. To identify putative specificity-determining residues, we have analyzed amino acid coevolution in two-component proteins and identified a set of residues that can be used to rationally rewire a model signaling pathway, EnvZ-OmpR. To explore how a relatively small set of residues can dictate partner selectivity, we combined alanine-scanning mutagenesis with an approach we call trajectory-scanning mutagenesis, in which all mutational intermediates between the specificity residues of EnvZ and another kinase, RstB, were systematically examined for phosphotransfer specificity. The same approach was used for the response regulators OmpR and RstA. Collectively, the results begin to reveal the molecular mechanism by which a small set of amino acids enables an individual kinase to discriminate amongst a large set of highly-related response regulators and vice versa. Our results also suggest that the mutational trajectories taken by two-component signaling proteins following gene or pathway duplication may be constrained and subject to differential selective pressures. Only some trajectories allow both the maintenance of phosphotransfer and the avoidance of unwanted cross-talk.     

Surgical Induction of Endolymphatic Hydrops by Obliteration of the Endolymphatic Duct

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal model of the condition and an important correlate for human Meniere''s disease. In 1965, Robert Kimura and Harold Schuknecht first described an intradural approach for obstruction of the endolymphatic duct (Kimura 1965). Although effective, this technique, which requires penetration of the brain''s protective covering, incurred an undesirable level of morbidity and mortality in the animal subjects. Consequently, Andrews and Bohmer developed an extradural approach, which predictably produces fewer of the complications associated with central nervous system (CNS) penetration.(Andrews and Bohmer 1989) The extradural approach described here first requires a midline incision in the region of the occiput to expose the underlying muscular layer. We operate only on the right side. After appropriate retraction of the overlying tissue, a horizontal incision is made into the musculature of the right occiput to expose the right temporo-occipital suture line. The bone immediately inferio-lateral the suture line (Fig 1) is then drilled with an otologic drill until the sigmoid sinus becomes visible. Medial retraction of the sigmoid sinus reveals the operculum of the endolymphatic duct, which houses the endolymphatic sac. Drilling medial to the operculum into the area of the endolymphatic sac reveals the endolymphatic duct, which is then packed with bone wax to produce obstruction and ultimately ELH. In the following weeks, the animal will demonstrate the progressive, fluctuating hearing loss and histologic evidence of ELH.Open in a separate windowClick here to view.^{(41M, flv)}