A reliable measure of similarity based on dependency for short time series: an application to gene expression networks

Background  

Microarray techniques have become an important tool to the investigation of genetic relationships and the assignment of different phenotypes. Since microarrays are still very expensive, most of the experiments are performed with small samples. This paper introduces a method to quantify dependency between data series composed of few sample points. The method is used to construct gene co-expression subnetworks of highly significant edges.     

Sphingolipid C-9 Methyltransferases Are Important for Growth and Virulence but Not for Sensitivity to Antifungal Plant Defensins in Fusarium graminearum

The C-9-methylated glucosylceramides (GlcCers) are sphingolipids unique to fungi. They play important roles in fungal growth and pathogenesis, and they act as receptors for some antifungal plant defensins. We have identified two genes, FgMT1 and FgMT2, that each encode a putative sphingolipid C-9 methyltransferase (C-9-MT) in the fungal pathogen Fusarium graminearum and complement a Pichia pastoris C-9-MT-null mutant. The ΔFgmt1 mutant produced C-9-methylated GlcCer like the wild-type strain, PH-1, whereas the ΔFgmt2 mutant produced 65 to 75% nonmethylated and 25 to 35% methylated GlcCer. No ΔFgmt1ΔFgmt2 double-knockout mutant producing only nonmethylated GlcCer could be recovered, suggesting that perhaps C-9-MTs are essential in this pathogen. This is in contrast to the nonessential nature of this enzyme in the unicellular fungus P. pastoris. The ΔFgmt2 mutant exhibited severe growth defects and produced abnormal conidia, while the ΔFgmt1 mutant grew like the wild-type strain, PH-1, under the conditions tested. The ΔFgmt2 mutant also exhibited drastically reduced disease symptoms in wheat and much-delayed disease symptoms in Arabidopsis thaliana. Surprisingly, the ΔFgmt2 mutant was less virulent on different host plants tested than the previously characterized ΔFggcs1 mutant, which lacks GlcCer synthase activity and produces no GlcCer at all. Moreover, the ΔFgmt1 and ΔFgmt2 mutants, as well as the P. pastoris strain in which the C-9-MT gene was deleted, retained sensitivity to the antifungal plant defensins MsDef1 and RsAFP2, indicating that the C-9 methyl group is not a critical structural feature of the GlcCer receptor required for the antifungal action of plant defensins.     

Mining the bitter melon (<Emphasis Type="Italic">momordica charantia</Emphasis>l.) seed transcriptome by 454 analysis of non-normalized and normalized cDNA populations for conjugated fatty acid metabolism-related genes

Background  

Seeds of Momordica charantia (bitter melon) produce high levels of eleostearic acid, an unusual conjugated fatty acid with industrial value. Deep sequencing of non-normalized and normalized cDNAs from developing bitter melon seeds was conducted to uncover key genes required for biotechnological transfer of conjugated fatty acid production to existing oilseed crops. It is expected that these studies will also provide basic information regarding the metabolism of other high-value novel fatty acids.     

Defining the role of phosphomethylethanolamine N-methyltransferase from Caenorhabditis elegans in phosphocholine biosynthesis by biochemical and kinetic analysis

In plants and Plasmodium falciparum, the synthesis of phosphatidylcholine requires the conversion of phosphoethanolamine to phosphocholine by phosphoethanolamine methyltransferase (PEAMT). This pathway differs from the metabolic route of phosphatidylcholine synthesis used in mammals and, on the basis of bioinformatics, was postulated to function in the nematode Caenorhabditis elegans. Here we describe the cloning and biochemical characterization of a PEAMT from C. elegans (gene, pmt-2; protein, PMT-2). Although similar in size to the PEAMT from plants, which contain two tandem methyltransferase domains, PMT-2 retains only the C-terminal methyltransferase domain. RNA-mediated interference experiments in C. elegans show that PMT-2 is essential for worm viability and that choline supplementation rescues the RNAi-generated phenotype. Unlike the plant and Plasmodium PEAMT, which catalyze all three methylations in the pathway, PMT-2 catalyzes only the last two steps in the pathway, i.e., the methylation of phosphomonomethylethanolamine (P-MME) to phosphodimethylethanolamine (P-DME) and of P-DME to phosphocholine. Analysis of initial velocity patterns suggests a random sequential kinetic mechanism for PMT-2. Product inhibition by S-adenosylhomocysteine was competitive versus S-adenosylmethionine and noncompetitive versus P-DME, consistent with formation of a dead-end complex. Inhibition by phosphocholine was competitive versus each substrate. Fluorescence titrations show that all substrates and products bind to the free enzyme. The biochemical data are consistent with a random sequential kinetic mechanism for PMT-2. This work provides a kinetic basis for additional studies on the reaction mechanism of PEAMT. Our results indicate that nematodes also use the PEAMT pathway for phosphatidylcholine biosynthesis. If the essential role of PMT-2 in C. elegans is conserved in parasitic nematodes of mammals and plants, then inhibition of the PEAMT pathway may be a viable approach for targeting these parasites with compounds of medicinal or agronomic value.     

Effects of 3-beta-diol, an androgen metabolite with intrinsic estrogen-like effects, in modulating the aquaporin-9 expression in the rat efferent ductules

Background  

Fluid homeostasis is critical for normal function of the male reproductive tract and aquaporins (AQP) play an important role in maintenance of this water and ion balance. Several AQPs have been identified in the male, but their regulation is not fully comprehended. Hormonal regulation of AQPs appears to be dependent on the steroid in the reproductive tract region. AQP9 displays unique hormonal regulation in the efferent ductules and epididymis, as it is regulated by both estrogen and dihydrotestosterone (DHT) in the efferent ductules, but only by DHT in the initial segment epididymis. Recent data have shown that a metabolite of DHT, 5-alpha-androstane-3-beta-17-beta-diol (3-beta-diol), once considered inactive, is also present in high concentrations in the male and indeed has biological activity. 3-beta-diol does not bind to the androgen receptor, but rather to estrogen receptors ER-alpha and ER-beta, with higher affinity for ER-beta. The existence of this estrogenic DHT metabolite has raised the possibility that estradiol may not be the only estrogen to play a major role in the male reproductive system. Considering that both ER-alpha and ER-beta are highly expressed in efferent ductules, we hypothesized that the DHT regulation of AQP9 could be due to the 3-beta-diol metabolite.     

The essential nature of sphingolipids in plants as revealed by the functional identification and characterization of the Arabidopsis LCB1 subunit of serine palmitoyltransferase

Serine palmitoyltransferase (SPT) catalyzes the first step of sphingolipid biosynthesis. In yeast and mammalian cells, SPT is a heterodimer that consists of LCB1 and LCB2 subunits, which together form the active site of this enzyme. We show that the predicted gene for Arabidopsis thaliana LCB1 encodes a genuine subunit of SPT that rescues the sphingolipid long-chain base auxotrophy of Saccharomyces cerevisiae SPT mutants when coexpressed with Arabidopsis LCB2. In addition, homozygous T-DNA insertion mutants for At LCB1 were not recoverable, but viability was restored by complementation with the wild-type At LCB1 gene. Furthermore, partial RNA interference (RNAi) suppression of At LCB1 expression was accompanied by a marked reduction in plant size that resulted primarily from reduced cell expansion. Sphingolipid content on a weight basis was not changed significantly in the RNAi suppression plants, suggesting that plants compensate for the downregulation of sphingolipid synthesis by reduced growth. At LCB1 RNAi suppression plants also displayed altered leaf morphology and increases in relative amounts of saturated sphingolipid long-chain bases. These results demonstrate that plant SPT is a heteromeric enzyme and that sphingolipids are essential components of plant cells and contribute to growth and development.     

Comprehensive analysis of the Brassica juncea root proteome in response to cadmium exposure by complementary proteomic approaches

Indian mustard (Brassica juncea L.) is known to both accumulate and tolerate high levels of heavy metals from polluted soils. To gain a comprehensive understanding of the effect of cadmium (Cd) treatment on B. juncea roots, two quantitative proteomics approaches – fluorescence two‐dimensional difference gel electrophoresis (2‐D DIGE) and multiplexed isobaric tagging technology (iTRAQ) – were implemented. Several proteins involved in sulfur assimilation, redox homeostasis, and xenobiotic detoxification were found to be up‐regulated. Multiple proteins involved in protein synthesis and processing were down‐regulated. While the two proteomics approaches identified different sets of proteins, the proteins identified in both datasets are involved in similar biological processes. We show that 2‐D DIGE and iTRAQ results are complementary, that the data obtained independently using the two techniques validate one another, and that the quality of iTRAQ results depends on both the number of biological replicates and the number of sample injections. This study determined the involvement of enzymes such as peptide methionine sulfoxide reductase and 2‐nitropropane dioxygenase in alternatives redox‐regulation mechanisms, as well as O‐acetylserine sulfhydrylase, glutathione‐S‐transferase and glutathione‐conjugate membrane transporter, as essential players in the Cd hyperaccumation and tolerance of B. juncea.     

Aggravation of myocardial dysfunction by injurious mechanical ventilation in LPS-induced pneumonia in rats

Background

Mechanical ventilation (MV) may cause ventilator-induced lung injury (VILI) and may thereby contribute to fatal multiple organ failure. We tested the hypothesis that injurious MV of lipopolysaccharide (LPS) pre-injured lungs induces myocardial inflammation and further dysfunction ex vivo, through calcium (Ca²⁺)-dependent mechanism.

Materials and methods

N = 35 male anesthetized and paralyzed male Wistar rats were randomized to intratracheal instillation of 2 mg/kg LPS or nothing and subsequent MV with lung-protective settings (low tidal volume (V_t) of 6 mL/kg and 5 cmH₂O positive end-expiratory pressure (PEEP)) or injurious ventilation (high V_t of 19 mL/kg and 1 cmH₂O PEEP) for 4 hours. Myocardial function ex vivo was evaluated in a Langendorff setup and Ca²⁺ exposure. Key mediators were determined in lung and heart at the mRNA level.

Results

Instillation of LPS and high V_t MV impaired gas exchange and, particularly when combined, increased pulmonary wet/dry ratio; heat shock protein (HSP)70 mRNA expression also increased by the interaction between LPS and high V_t MV. For the heart, C-X-C motif ligand (CXCL)1 and Toll-like receptor (TLR)2 mRNA expression increased, and ventricular (LV) systolic pressure, LV developed pressure, LV +dP/dt_max and contractile responses to increasing Ca²⁺ exposure ex vivo decreased by LPS. High V_t ventilation aggravated the effects of LPS on myocardial inflammation and dysfunction but not on Ca²⁺ responses.

Conclusions

Injurious MV by high V_t aggravates the effects of intratracheal instillation of LPS on myocardial dysfunction, possibly through enhancing myocardial inflammation via pulmonary release of HSP70 stimulating cardiac TLR2, not involving Ca²⁺ handling and sensitivity.     

The Neisseria gonorrhoeae photolyase orthologue phrB is required for proper DNA supercoiling but does not function in photo-reactivation

Neisseria gonorrhoeae (Gc) is an obligate human pathogen and the causative agent of the sexually transmitted infection, gonorrhoea. Despite the fact that the gonococcus is not normally exposed to UV irradiation or visible light, the bacterium expresses a phrB orthologue, which in other organisms encodes a DNA photolyase that repairs UV-induced pyrimidine dimers with energy provided by visible light. We show that a Gc phrB mutant is not more sensitive to UV irradiation, independent of visible light exposure, and that the Gc phrB cannot complement an Escherichia coli phrB mutant strain. The Gc phrB mutant had a reduced colony size that was not a result of a growth defect and the mutant cells exhibited an altered morphology. Although the phrB mutant exhibited increased sensitivity to oxidative killing; it showed increased survival on media containing nalidixic acid or rifampicin, but did not have an increased mutation rate to these antibiotics or spectinomycin and kasugamycin. The Gc phrB mutant showed increased negative DNA supercoiling, but while the protein bound double-stranded DNA, it did not express topoisomerase activity. We conclude that the Gc PhrB has a previously unrecognized role in maintaining DNA supercoiling that is important for normal cell physiology.