Unraveling the iron deficiency responsive proteome in <i>Arabidopsis?</i>shoot by iTRAQ-OFFGEL approach

Iron (Fe) is required by plants for basic redox reactions in photosynthesis and respiration, and for many other key enzymatic reactions in biological processes. Fe homeostatic mechanisms have evolved in plants to enable the uptake and sequestration of Fe in cells. To elucidate the network of proteins that regulate Fe homeostasis and transport, we optimized the iTRAQ-OFFGEL method to identify and quantify the number of proteins that respond to Fe deficiency in the model plant Arabidopsis. In this study, Fe deficiency was created using Fe-deficient growth conditions, excess zinc (Zn), and use of the irt1-1 mutant in which the IRT1 Fe transporter is disrupted. Using the iTRAQ-OFFGEL approach, we identified 1139 proteins, including novel Fe deficiency-responsive proteins, in microsomal fractions isolated from 3 different types of Fe-deficient shoots compared with just 233 proteins identified using conventional iTRAQ-CEX. Further analysis showed that greater numbers of low-abundance proteins could be identified using the iTRAQ-OFFGEL method and that proteins could be identified from numerous cellular compartments. The improved iTRAQ-OFFGEL method used in this study provided an efficient means for identifying greater numbers of proteins from microsomal fractions of Arabidopsis shoots. The proteome identified in this study provides new insight into the regulatory cross talk between Fe-deficient and excess Zn conditions.     

Salicylic acid induces vanillin synthesis through the phospholipid signaling pathway in <i>Capsicum chinense?</i>cell cultures

Signal transduction via phospholipids is mediated by phospholipases such as phospholipase C (PLC) and D (PLD), which catalyze hydrolysis of plasma membrane structural phospholipids. Phospholipid signaling is also involved in plant responses to phytohormones such as salicylic acid (SA). The relationships between phospholipid signaling, SA, and secondary metabolism are not fully understood. Using a Capsicum chinense cell suspension as a model, we evaluated whether phospholipid signaling modulates SA-induced vanillin production through the activation of phenylalanine ammonia lyase (PAL), a key enzyme in the biosynthetic pathway. Salicylic acid was found to elicit PAL activity and consequently vanillin production, which was diminished or reversed upon exposure to the phosphoinositide-phospholipase C (PI-PLC) signaling inhibitors neomycin and {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122. Exposure to the phosphatidic acid inhibitor 1-butanol altered PLD activity and prevented SA-induced vanillin production. Our results suggest that PLC and PLD-generated secondary messengers may be modulating SA-induced vanillin production through the activation of key biosynthetic pathway enzymes.     

Identification of CD133<Superscript>−</Superscript>/Telomerase<Superscript>low</Superscript> Progenitor Cells in Glioblastoma-Derived Cancer Stem Cell Lines

Glioblastoma multiforme (GBM) is paradigmatic for the investigation of cancer stem cells (CSC) in solid tumors. The CSC hypothesis implies that tumors are maintained by a rare subpopulation of CSC that gives rise to rapidly proliferating progenitor cells. Although the presence of progenitor cells is crucial for the CSC hypothesis, progenitor cells derived from GBM CSC are yet uncharacterized. We analyzed human CD133⁺ CSC lines that were directly derived from CD133⁺ primary astrocytic GBM. In these CSC lines, CD133⁺/telomerase^high CSC give rise to non-tumorigenic, CD133⁻/telomerase^low progenitor cells. The proliferation of the progenitor cell population results in significant telomere shortening as compared to the CD133⁺ compartment comprising CSC. The average difference in telomere length as determined by a modified multi-color flow fluorescent in situ hybridization was 320 bp corresponding to 4–8 cell divisions. Taken together, we demonstrate that CD133⁺ primary astrocytic GBM comprise proliferating, CD133⁻/telomerase^low progenitor cell population characterized by low telomerase activity and shortened telomeres as compared to CSC.     

Roles of Volume-sensitive Cl<Superscript>−</Superscript> Channel in Cisplatin-induced Apoptosis in Human Epidermoid Cancer Cells

The anti-cancer drug cisplatin induces apoptosis by damaging DNA. Since a stilbene-derivative blocker of Cl⁻/HCO₃⁻ exchangers and Cl⁻ channels, SITS, is known to induce cisplatin resistance in a manner independent of intracellular pH and extracellular HCO₃⁻, we investigated the relation between cisplatin-induced apoptosis and Cl⁻ channel activity in human adenocarcinoma KB cells. A stilbene derivative, DIDS, reduced cisplatin-induced caspase-3 activation and cell death, which were detected over 18 h after treatment with cisplatin. DIDS was also found to reduce sensitivity of KB cells to 5-day exposure to cisplatin. Whole-cell patch-clamp recordings showed that KB cells functionally express volume-sensitive outwardly rectifying (VSOR) Cl⁻ channels which are activated by osmotic cell swelling and sensitive to DIDS. Pretreatment of the cells with cisplatin for 12 h augmented the magnitude of VSOR Cl⁻ current. Thus, it is concluded that cisplatin-induced cytotoxicity in KB cells is associated with augmented activity of a DIDS-sensitive VSOR Cl⁻ channel and that blockade of this channel is, at least in part, responsible for cisplatin resistance induced by a stilbene derivative.     

On p21 tracking property in cancer cell unravelled bio-digitally in silico. Are apoptosis principles universal?

Upon severe DNA damage, p21 acts in a dual mode; on the one hand, it inhibits the cyclin-CDK complex for arresting the G2/M transition and on the other hand, it indirectly becomes an apoptotic factor by activating--in sequence--the retinoblastoma protein, E2F1 and APAF1 expressions. But, in a cancer cells proliferation, the mechanisms of, and participants in, the apoptosis failure remain unclear. Since the p21/p53/Mdm2 proteins network normally involves a digital response in a cancer cell, through an original design of a cell signalling-protein simulator, we demonstrate, in silico, that apoptosis phase instability is fully reciprocated by p21 mRNA irregular dynamics which operates according to a "tracking memory" principle. We show p21mRNA paradoxically ceases to act in concert with specific target genes and becomes an underlying accomplice of cancer proliferation. Here, we also identify the mechanisms for allowing the cancer cell to re-enter inside a steady stable apoptosis phase.     

Apoptosis in <Emphasis Type="Italic">Drosophila</Emphasis>: which role for mitochondria?

It is now well established that the mitochondrion is a central regulator of mammalian cell apoptosis. However, the importance of this organelle in non-mammalian apoptosis has long been regarded as minor, mainly because of the absence of a crucial role for cytochrome c in caspase activation. Recent results indicate that the control of caspase activation and cell death in Drosophila occurs at the mitochondrial level. Numerous proteins, including RHG proteins and proteins of the Bcl-2 family that are key regulators of Drosophila apoptosis, constitutively or transiently localize in mitochondria. These proteins participate in the cell death process at different levels such as degradation of Diap1, a Drosophila IAP, production of mitochondrial reactive oxygen species or stimulation of the mitochondrial fission machinery. Here, we review these mitochondrial events that might have their counterpart in human.     

Does Atmospheric NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack> Deposition Alter the Abundance and Activity of Ligninolytic Fungi in Forest Soils?

Although field studies have demonstrated an ecosystem-specific effect of experimental atmospheric nitrogen (N) deposition on litter decomposition, a mechanistic understanding of how ligninolytic microbial communities respond to atmospheric deposition is lacking. Because high levels of inorganic N suppress lignin decomposition by some basidiomycetes, it is plausible that the abundance and activity of these key microorganisms underlies differential ecosystem responses of decomposition to atmospheric N deposition. We hypothesize that: (a) atmospheric N deposition will cause an ecosystem-specific reduction in basidiomycete activity and abundance with greatest decreases in ecosystems with lignin-rich forest litter and (b) the abundance of lignin degrading basidiomycetes will be positively correlated with ligninolytic enzyme activity. To test these hypotheses, we measured the effects of experimental N deposition on the potential activity of phenol oxidase enzymes, and the abundance of basidiomycete genes encoding laccase, a primary phenol oxidase enzyme, in three hardwood forests spanning a range of leaf litter lignin content. The black oak-white oak (BOWO) contains high lignin litter, the sugar maple-basswood (SMBW) has low lignin litter, and the sugar maple-red oak (SMRO) is intermediate. An ecosystem by N deposition interaction significantly influenced phenol oxidase activity in the surface soil (P = 0.05), where phenol oxidase activity decreased with increasing experimental N deposition in the BOWO ecosystem. No consistent response to N deposition was evident for surface soil phenol oxidase activity within either the SMRO or SMBW ecosystem. This interaction did not influence laccase gene abundance. Instead, basidiomycete laccase gene abundance was reduced by experimental N deposition (main effect) in surface soil. There was only a weak correlation between basidiomycete laccase gene abundance and potential phenol oxidase enzyme activity, suggesting that the abundance of organisms possessing laccase genes may not control phenol oxidase activity in soil. Our results suggest that the regulation of laccase gene expression may mediate the decomposition response to atmospheric N deposition.     

Interference of Cranberry Constituents in Cell–Cell Signaling System of <Emphasis Type=Italic>Vibrio harveyi</Emphasis>

Cranberry juice has long been recognized in folk medicine as a therapeutic agent, mainly in urinary track infections. It acts as an antibiofilm agent against various pathogens. Quorum sensing is process where bacteria communicate with each other via signal molecules known as autoinducers. This process is strongly involved in various bacterial pathological and physiological pathways. Various strains of Vibrio harveyi bacteria were incubated with different concentrations of nondialyzable material of cranberry (NDM) with or without addition of exogenous autoinducer. Bioluminescence regulated by the autoinducers was measured in GENios reader. Effect of NDM alone or NDM supplemented with autoinducer on quorum sensing was determined as change in bioluminescence in each treated sample compared to appropriate control in every strain. Using model of V. harveyi, we found an inhibitory effect of cranberry constituents on bacterial signaling system. This effect was reversible, since exogenous autoinducer was able to recover bioluminescence which was decreased by NDM. We hypothesized that cranberry NDM interacts with V. harveyi quorum sensing by competition with autoinducer for binding to autoinducer sensor.     

Are the polygenic architectures of resistance to <Emphasis Type="Italic">Phytophthora capsici</Emphasis> and <Emphasis Type="Italic">P. parasitica</Emphasis> independent in pepper?

The pepper accession Criollo de Morelos 334 is the most efficient source of resistance currently known to Phytophthora capsici and P. parasitica. To investigate whether genetic controls of resistance to two Phytophthora species are independent, we compared the genetic architecture of resistance of CM334 to both Phytophthora species. The RIL population F5YC used to construct the high-resolution genetic linkage map of pepper was assessed for resistance to one isolate of each Phytophthora species. Inheritance of the P. capsici and P. parasitica resistance was polygenic. Twelve additive QTLs involved in the P. capsici resistance and 14 additive QTLs involved in the P. parasitica resistance were detected. The QTLs identified in this progeny were specific to these Phytophthora species. Comparative mapping analysis with literature data identified three colocations between resistance QTLs to P. parasitica and P. capsici in pepper. Whereas this result suggests presence of common resistance factors to the two Phytophthora species in pepper, which possibly derive from common ancestral genes, calculation of the colocation probability indicates that these colocations could occur by chance.     

Cellularity loss and dilman’s problem: An <Emphasis Type="Italic">in silico</Emphasis> study

We assume that prolonged trends of increasing concentration of hormones could be a consequence of deterioration of functioning of glands producing inhibitors of their synthesis. Such deterioration would result from loss of cellularity of the glands. Experiments in silico carried out using the model at show that, in principle, the diversity of hormonal effects that accompany phenoptosis of multicellular organisms can be provided with a simple “software mechanism”. This mechanism is based on the gradual loss of cellularity as a result of continuous run of apoptosis in some cells of the glands due to natural fluctuations in levels of intracellular inducers of apoptosis. The main practical sense of our work lies in the illustration of the fact that substances inhibiting cellularity loss can theoretically be effective suppressors of hormonal changes characteristic for aging.     

Changes in K<Superscript>+</Superscript>, Na<Superscript>+</Superscript>, Cl<Superscript>−</Superscript> balance and K<Superscript>+</Superscript>, Cl<Superscript>−</Superscript> fluxes in U937 cells induced to apoptosis by staurosporine: On cell dehydration in apoptosis

The K⁺, Na⁺, and Cl⁻ balance and K⁺ (Rb⁺) and ³⁶Cl⁻ fluxes in U937 cells induced to apoptosis by 0.2 or 1 μM staurosporine were studied using flame emission and radioisotope techniques. It is found that two-thirds of the total decrease in the amount of intracellular osmolytes in apoptotic cells is accounted for by monovalent ions and one-third consists of other intracellular osmolytes. A decrease in the amount of monovalent ions results from a decrease in the amount of K⁺ and Cl⁻ and an increase in the Na⁺ content. The rate of ³⁶Cl⁻, Rb⁺ (K⁺), and ²²Na⁺ equilibration between cells and the medium was found to significantly exceed the rate of apoptotic change in the cellular ion content, which indicates that unidirectional influxes and effluxes during apoptosis may be considered as being in near balance. The drift of the ion flux balance in apoptosis caused by 0.2 μM staurosporine was found to be associated with the increased ouabain-resistant Rb⁺ (K⁺) channel influx and insignificantly altered the ouabain-sensitive pump influx. Severe apoptosis induced by 1 μM staurosporine is associated with reduced pump fluxes and slightly changed channel Rb⁺ (K⁺) fluxes. In apoptotic cells, the 1.4–1.8-fold decreased Cl⁻ level is accompanied by a 1.2–1.6-fold decreased flux.     

Increased Cell Proliferation and Neurogenesis in the Hippocampal Dentate Gyrus of Old GFAP<Superscript>−/−</Superscript>Vim<Superscript>−/−</Superscript> Mice

In response to central nervous system (CNS) injury, and more discretely so also during aging, astrocytes become reactive and increase their expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. Studies of mice deficient in astrocytic intermediate filaments have provided insights into the function of reactive gliosis. Recently we demonstrated robust integrationof retinal transplants (1) and increased posttraumatic synaptic regeneration (2) in GFAP^–/–Vim^–/– mice, suggesting that modulation of astrocyte activity affects the permissiveness of the CNS environment for regeneration. Neurogenesis in the adult mammalian CNS is restricted to essentially two regions, the hippocampus and the subventricular zone. Here, we assessed neurogenesis in the hippocampus of 18-month-old GFAP^–/–Vim^–/– mice. In the granular layer of the dentate gyrus, cell proliferation/survival was 34% higher and neurogenesis 36% higher in GFAP^–/–Vim^–/– mice than in wildtype controls. These findings suggest that the adult hippocampal neurogenesis in healthy old mice can be increased by modulating astrocyte reactivity.Special issue dedicated to Lawrence. F. Eng.     

What <Emphasis Type="BoldItalic">in silico</Emphasis> molecular docking can do for the ‘bench-working biologists’

Required by an increasing amount of scientists, the in silico docking field is in full expansion, new algorithms and methods appearing at an exponential rate. The sheer range of available programs is overwhelming for the bench-working biologist, which is often discouraging by the lack of a graphical user interface, good user manual or literature to validate a given program. This mini-review attempts to present the docking problem and available solutions from a non-bioinformatician point of view and makes a selection of the available servers and programs. These tools are evaluated from several points of view, as numbers of citations, ease of usage and computer requirements. Finally, the capabilities and limitations as well as specific applications of in silico docking techniques are presented.     

A Prehistoric Mural in Spain Depicting Neurotropic <Emphasis Type="Italic">Psilocybe</Emphasis> Mushrooms?<Superscript>1</Superscript>

A Prehistoric Mural in Spain Depicting NeurotropicPsilocybeMushrooms? The Selva Pascuala mural, a work of post-Paleolithic rock art in Spain, contains fungoid figures herein hypothesized to depict neurotropic fungi, especially Psilocybe hispanica, a species that occurs in a neighboring region. This hypothesis is based on features of these figures related to fungal morphology, along with ethnographic analogy, and shamanistic explanations of rock art. If correct, this interpretation would support inference of prehistoric utilization of this fungus in the region. The mural represents the first direct evidence for possible ritual use of Psilocybe in prehistoric Europe.     

Pistil-function breakdown in a new <Emphasis Type="Italic">S</Emphasis>-allele of European pear, <Emphasis Type="Italic">S</Emphasis><Subscript><Emphasis Type="Italic">21</Emphasis></Subscript>°, confers self-compatibility

European pear exhibits RNase-based gametophytic self-incompatibility controlled by the polymorphic S-locus. S-allele diversity of cultivars has been extensively investigated; however, no mutant alleles conferring self-compatibility have been reported. In this study, two European pear cultivars, ‘Abugo’ and ‘Ceremeño’, were classified as self-compatible after fruit/seed setting and pollen tube growth examination. S-genotyping through S-PCR and sequencing identified a new S-RNase allele in the two cultivars, with identical deduced amino acid sequence as S ₂₁ , but differing at the nucleotide level. Test-pollinations and analysis of descendants suggested that the new allele is a self-compatible pistil-mutated variant of S ₂₁ , so it was named S ₂₁ °. S-genotypes assigned to ‘Abugo’ and ‘Ceremeño’ were S ₁₀ S ₂₁ ° and S ₂₁ °S ₂₅ respectively, of which S ₂₅ is a new functional S-allele of European pear. Reciprocal crosses between cultivars bearing S ₂₁ and S ₂₁ ° indicated that both alleles exhibit the same pollen function; however, cultivars bearing S ₂₁ ° had impaired pistil-S function as they failed to reject either S ₂₁ or S ₂₁ ° pollen. RT-PCR analysis showed absence of S ₂₁ °-RNase gene expression in styles of ‘Abugo’ and ‘Ceremeño’, suggesting a possible origin for S ₂₁ ° pistil dysfunction. Two polymorphisms found within the S-RNase genomic region (a retrotransposon insertion within the intron of S ₂₁ ° and indels at the 3′UTR) might explain the different pattern of expression between S ₂₁ and S ₂₁ °. Evaluation of cultivars with unknown S-genotype identified another cultivar ‘Azucar Verde’ bearing S ₂₁ °, and pollen tube growth examination confirmed self-compatibility for this cultivar as well. This is the first report of a mutated S-allele conferring self-compatibility in European pear.     

Does an electroneutral K<Superscript>+</Superscript>/Cl<Superscript>−</Superscript> antiport occur in cardiomyocyte during acute ischemia?

Electron probe microanalysis was applied to determine cytoplasmic elemental (K, Na, Cl) concentrations in cardiac cells of the rat (Wistar). Potassium, sodium and chlorine contents were measured in papillary muscle myocytes of the rat heart perfused by the Langendorff procedure. Ischemic depletion was created by perfusion with deeply deoxygenated Tyrode’s solution in the absence of glucose. It was found that the initial phase of acute ischemia is characterized by the potassium deficiency and the accumulation of sodium and chlorine in cardiac myocytes. It should be noted that changes in the total charge of the main intracellular cations (K⁺, Na⁺) do not compensate for the increased chlorine concentration. This result can be accounted for by the appearance of ionic (K⁺ and Cl⁻) transport coupled with the removal of lactate anions produced in cardiomyocytes during anaerobic glycolysis.     

Are Basidiomycete Laccase Gene Abundance and Composition Related to Reduced Lignolytic Activity Under Elevated Atmospheric NO<Subscript>3</Subscript><Superscript>−</Superscript> Deposition in a Northern Hardwood Forest?

Anthropogenic release of biologically available N has increased atmospheric N deposition in forest ecosystems, which may slow decomposition by reducing the lignolytic activity of white-rot fungi. We investigated the potential for atmospheric N deposition to reduce the abundance and alter the composition of lignolytic basidiomycetes in a regional network of four northern hardwood forest stands receiving experimental NO₃ ⁻ deposition (30 kg NO₃ ⁻−N ha⁻¹ year⁻¹) for a decade. To estimate the abundance of basidiomycetes with lignolytic potential, we used PCR primers targeting laccase (polyphenol oxidase) and quantitative fluorescence PCR to estimate gene copy number. Natural variation in laccase gene size permitted use of length heterogeneity PCR to profile basidiomycete community composition across two sampling dates in forest floor and mineral soil. Although past work has identified significant and consistent negative effects of NO₃ ⁻ deposition on lignolytic enzyme activity, microbial biomass, soil respiration, and decomposition rate, we found no consistent effect of NO₃ ⁻ deposition on basidiomycete laccase gene abundance or community profile. Rather, laccase abundance under NO₃ ⁻ deposition was lower (−52%), higher (+223%), or unchanged, depending on stand. Only a single stand exhibited a significant change in basidiomycete laccase gene profile. Basidiomycete laccase genes occurring in mineral soil were a subset of the genes observed in the forest floor. Moreover, significant effects on laccase abundance were confined to the forest floor, suggesting that species composition plays some role in determining how lignolytic basidiomycetes are affected by N deposition. Community profiles differed between July and October sampling dates, and basidiomycete communities sampled in October had lower laccase gene abundance in the forest floor, but higher laccase abundance in mineral soil. Although experimental N deposition significantly suppresses lignolytic activity in these forests, this change is not related to the abundance or community composition of basidiomycete fungi with laccase genes. Understanding the expression of laccases and other lignolytic enzymes by basidiomycete fungi and other lignin-decaying organisms appears to hold promise for explaining the consistent decline in lignolytic activity elicited by experimental N deposition. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.