bluuebunny/crossref_metadata_2025 huggingface dataset:
Defining the transcriptional redundancy of early bodywall muscle development in C. elegans: evidence for a unified theory of animal muscle development
Authors: Fukushige Tetsunari , Brodigan Thomas M. , Schriefer Lawrence A. , Waterston Robert H. , Krause Michael
Published: December 2006

Myogenic regulatory factors (MRFs) are required for mammalian skeletal myogenesis. In contrast, bodywall muscle is readily detectable in Caenorhabditis elegans embryos lacking activity of the lone MRF ortholog HLH-1, indicating that additional myogenic factors must function in the nematode. We find that two additional C. elegans proteins, UNC-120/SRF and HND-1/HAND, can convert naïve blastomeres to muscle when overproduced ectopically in the embryo. In addition, we have used genetic null mutants to demonstrate that both of these factors act in concert with HLH-1 to regulate myogenesis. Loss of all three factors results in embryos that lack detectable bodywall muscle differentiation, identifying this trio as a set that is both necessary and sufficient for bodywall myogenesis in C. elegans. In mammals, SRF and HAND play prominent roles in regulating smooth and cardiac muscle development. That C. elegans bodywall muscle development is dependent on transcription factors that are associated with all three types of mammalian muscle supports a theory that all animal muscle types are derived from a common ancestral contractile cell type.
Feeding behavior of Aplysia: A model system for comparing cellular mechanisms of classical and operant conditioning
Authors: Baxter Douglas A. , Byrne John H.
Published: December 2006

Feeding behavior of Aplysia provides an excellent model system for analyzing and comparing mechanisms underlying appetitive classical conditioning and reward operant conditioning. Behavioral protocols have been developed for both forms of associative learning, both of which increase the occurrence of biting following training. Because the neural circuitry that mediates the behavior is well characterized and amenable to detailed cellular analyses, substantial progress has been made toward a comparative analysis of the cellular mechanisms underlying these two forms of associative learning. Both forms of associative learning use the same reinforcement pathway (the esophageal nerve, En) and the same reinforcement transmitter (dopamine, DA). In addition, at least one cellular locus of plasticity (cell B51) is modified by both forms of associative learning. However, the two forms of associative learning have opposite effects on B51. Classical conditioning decreases the excitability of B51, whereas operant conditioning increases the excitability of B51. Thus, the approach of using two forms of associative learning to modify a single behavior, which is mediated by an analytically tractable neural circuit, is revealing similarities and differences in the mechanisms that underlie classical and operant conditioning.
Knockdown of Nurr1 in the rat hippocampus: Implications to spatial discrimination learning and memory
Authors: Colón-Cesario Wanda I. , Martínez-Montemayor Michelle M. , Morales Sohaira , Félix Jahaira , Cruz Juan , Adorno Monique , Pereira Lixmar , Colón Nydia , Maldonado-Vlaar Carmen S. , Peña de Ortiz Sandra
Published: December 2006

Nurr1 expression is up-regulated in the brain following associative learning experiences, but its relevance to cognitive processes remains unclear. In these studies, rats initially received bilateral hippocampal infusions of control or antisense oligodeoxynucleotides (ODNs) 1 h prior to training in a holeboard spatial discrimination task. Such pre-training infusions of nurr1 antisense ODNs caused a moderate effect in learning the task and also impaired LTM tested 7 d later. In a second experiment, ODN infusions were given immediately after the animals had received two sessions of training, during which all animals showed normal learning. Although antisense treated rats were significantly impaired during the post-infusion stages of acquisition of the task, no group differences were observed during the LTM test given 7 d later. These animals were subjected 3 d later to reversal training in the same maze in the absence of any additional treatments. Remarkably, rats previously treated with antisense ODNs displayed perseveration: The animals were fixated with the previously learned pattern of baited holes, causing them to be significantly impaired in the extinction of acquired spatial preferences and future learning. We postulate that Nurr1 function in the hippocampus is important for normal cognitive processes.
Vectors and Agrobacterium Hosts for Arabidopsis Transformation
Authors: Weigel Detlef , Glazebrook Jane
Published: December 2006

INTRODUCTION

Arabidopsis can be stably transformed using Agrobacterium tumefaciens-mediated transfer of T-DNA. A. tumefaciens is a soil-dwelling bacterium that transforms normal plant cells into tumor-forming cells by inserting a piece of bacterial DNA (the transfer, or “T,” DNA) into the plant cell genome. The T-DNA, which is flanked by left- and right-border (LB and RB) sequences, resides on a tumor-inducing (Ti) plasmid. The Ti plasmid also carries many of the transfer functions for mobilizing the T-DNA. This article provides a brief discussion of the principles of T-DNA transformation, including consideration of T-DNA vectors and their hosts.
DNA Preparation and Needle Loading for Gene Delivery by Microinjection
Authors: Dean David A.
Published: December 2006

INTRODUCTION

For microinjection into cells, high-quality plasmid DNA is purified using standard procedures. The resulting preparation is then delivered into the microinjection needle by either a back-filling or a forward-filling approach.
Gene Delivery by Direct Injection (Microinjection) Using a Pulsed-Flow System
Authors: Dean David A.
Published: December 2006

INTRODUCTION

This protocol describes a method for pulsed-flow microinjection using the Eppendorf FemtoJet injector and Eppendorf InjectMan; this is the most common type of pulsed-flow microinjection system currently being used. The advantage of this type of system over a controlled-flow system is that much more control is available over the injection parameters, reducing variability in injections. In addition, the system allows a diagonal insertion of the needle into the cell, causing a direct piercing of the cell. This is fast and may do less damage to the cell than a traditional system, in which the cell is briefly distorted before it is pierced.
PCR Analysis of Agrobacterium
Authors: Wigge Phil
Published: December 2006

INTRODUCTION

Because transformation of Arabidopsis is a relatively long-winded process, it is a good idea to verify the presence of the T-DNA plasmid in the Agrobacterium strain before proceeding with transformation experiments. This protocol describes a method to determine the presence of plasmid DNA in an Agrobacterium culture. Compared to selection of transformed Agrobacterium, which can be ambiguous and normally takes several days for resistant colonies to appear, the approach described here is both rapid and accurate.
Root Transformation of Arabidopsis
Authors: Weigel Detlef , Glazebrook Jane
Published: December 2006

INTRODUCTION

Arabidopsis can be stably transformed using Agrobacterium tumefaciens-mediated transfer of T-DNA. We describe the generation of transgenic plants via root transformation in tissue culture, which can be useful for transforming sterile mutants.
Helianthus Nighttime Conductance and Transpiration Respond to Soil Water But Not Nutrient Availability
Authors: Howard Ava R. , Donovan Lisa A.
Published: December 2006

We investigated the response of Helianthus species nighttime conductance (g  night) and transpiration (E  night) to soil nutrient and water limitations in nine greenhouse studies. The studies primarily used wild Helianthus annuus, but also included a commercial and early domesticate of H. annuus and three additional wild species (Helianthus petiolaris Nutt., Helianthus deserticola Heiser, and Helianthus anomalus Blake). Well-watered plants of all species showed substantial g  night (0.023–0.225 mol m−2 s−1) and E  night (0.29–2.46 mmol m−2 s−1) measured as instantaneous gas exchange. Based on the potential for transpiration to increase mass flow of mobile nutrients to roots, we hypothesized that g  night and E  night would increase under limiting soil nutrients but found no evidence of responses in all six studies testing this. Based on known daytime responses to water limitation, we hypothesized that g  night and E  night would decrease when soil water availability was limited, and results from all four studies testing this supported our hypothesis. We also established that stomatal conductance at night was on average 5 times greater than cuticular conductance. Additionally, g  night and E  night varied nocturnally and across plant reproductive stages while remaining relatively constant as leaves aged. Our results further the ability to predict conditions under which nighttime water loss will be biologically significant and demonstrate that for Helianthus, g  night can be regulated.
Transposition-Based Plant Transformation
Authors: Yan Hua , Rommens Caius M.
Published: December 2006

Agrobacterium T-DNAs were used to deliver transposable Dissociation (Ds) elements into the nuclei of potato (Solanum tuberosum) cells. A double-selection system was applied to enrich for plants that only contained a transposed Ds element. This system consisted of a positive selection for the neomycin phosphotransferase (nptII) gene positioned within Ds followed by a negative selection against stable integration of the cytosine deaminase (codA) gene-containing T-DNA. Sixteen of 29 transgenic plants were found to contain a transposed element while lacking any superfluous T-DNA sequences. The occurrence of this genotype indicates that Ds elements can transpose from relatively short extrachromosomal DNA molecules into the plant genome. The frequency of single-copy Ds transformation was determined at 0.3%, which is only about 2.5-fold lower than the potato transformation frequency for backbone-free and single-copy T-DNAs. Because of the generally high expression levels of genes positioned within transposed elements, the new transformation method may find broad applicability to crops that are accessible to Agrobacterium T-DNA transfer.
Enzymatic Evidence for the Key Role of Arginine in Nitrogen Translocation by Arbuscular Mycorrhizal Fungi
Authors: Cruz Cristina , Egsgaard Helge , Trujillo Carmen , Ambus Per , Requena Natalia , Martins-Loução Maria Amélia , Jakobsen Iver
Published: December 2006

Key enzymes of the urea cycle and 15N-labeling patterns of arginine (Arg) were measured to elucidate the involvement of Arg in nitrogen translocation by arbuscular mycorrhizal (AM) fungi. Mycorrhiza was established between transformed carrot (Daucus carota) roots and Glomus intraradices in two-compartment petri dishes and three ammonium levels were supplied to the compartment containing the extraradical mycelium (ERM), but no roots. Time courses of specific enzyme activity were obtained for glutamine synthetase, argininosuccinate synthetase, arginase, and urease in the ERM and AM roots. 15NH4  + was used to follow the dynamics of nitrogen incorporation into and turnover of Arg. Both the absence of external nitrogen and the presence of l-norvaline, an inhibitor of Arg synthesis, prevented the synthesis of Arg in the ERM and resulted in decreased activity of arginase and urease in the AM root. The catabolic activity of the urea cycle in the roots therefore depends on Arg translocation from the ERM. 15N labeling of Arg in the ERM was very fast and analysis of its time course and isotopomer pattern allowed estimation of the translocation rate of Arg along the mycelium as 0.13 μg Arg mg−1 fresh weight h−1. The results highlight the synchronization of the spatially separated reactions involved in the anabolic and catabolic arms of the urea cycle. This synchronization is a prerequisite for Arg to be a key component in nitrogen translocation in the AM mycelium.
Oviposition by Pierid Butterflies Triggers Defense Responses in Arabidopsis
Authors: Little Dawn , Gouhier-Darimont Caroline , Bruessow Friederike , Reymond Philippe
Published: December 2006

Insect eggs represent a threat for the plant as hatching larvae rapidly start with their feeding activity. Using a whole-genome microarray, we studied the expression profile of Arabidopsis (Arabidopsis thaliana) leaves after oviposition by two pierid butterflies. For Pieris brassicae, the deposition of egg batches changed the expression of hundreds of genes over a period of 3 d after oviposition. The transcript signature was similar to that observed during a hypersensitive response or in lesion-mimic mutants, including the induction of defense and stress-related genes and the repression of genes involved in growth and photosynthesis. Deposition of single eggs by Pieris rapae caused a similar although much weaker transcriptional response. Analysis of the jasmonic acid and salicylic acid mutants coi1-1 and sid2-1 indicated that the response to egg deposition is mostly independent of these signaling pathways. Histochemical analyses showed that egg deposition is causing a localized cell death, accompanied by the accumulation of callose, and the production of reactive oxygen species. In addition, activation of the pathogenesis-related1∷β-glucuronidase reporter gene correlated precisely with the site of egg deposition and was also triggered by crude egg extract. This study provides molecular evidence for the detection of egg deposition by Arabidopsis plants and suggests that oviposition causes a localized response with strong similarity to a hypersensitive response.
Cell Death Suppressor Arabidopsis Bax Inhibitor-1 Is Associated with Calmodulin Binding and Ion Homeostasis
Authors: Ihara-Ohori Yuri , Nagano Minoru , Muto Shoshi , Uchimiya Hirofumi , Kawai-Yamada Maki
Published: December 2006

Cell death suppressor Bax inhibitor-1 (BI-1), an endoplasmic reticulum membrane protein, exists in a wide range of organisms. The split-ubiquitin system, overlay assay, and bimolecular fluorescence complementation analysis demonstrated that Arabidopsis (Arabidopsis thaliana) BI-1 (AtBI-1) interacted with calmodulin in yeast (Saccharomyces cerevisiae) and in plant cells. Furthermore, AtBI-1 failed to rescue yeast mutants lacking Ca2+ ATPase (Pmr1 or Spf1) from Bax-induced cell death. Pmr1 and Spf1, p-type ATPases localized at the inner membrane, are believed to be involved in transmembrane movement of calcium ions in yeast. Thus, the presence of intact Ca2+ ATPases was essential for AtBI-1-mediated cell death suppression in yeast. To investigate the effect of AtBI-1 on calcium homeostasis, we evaluated sensitivity against cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase in AtBI-1-overexpressing or knock-down transgenic Arabidopsis plants. These plants demonstrated altered CPA or ion stress sensitivity. Furthermore, AtBI-1-overexpressing cells demonstrated an attenuated rise in cytosolic calcium following CPA or H2O2 treatment, suggesting that AtBI-1 affects ion homeostasis in plant cell death regulation.
MEKK1 Is Required for flg22-Induced MPK4 Activation in Arabidopsis Plants
Authors: Suarez-Rodriguez Maria Cristina , Adams-Phillips Lori , Liu Yidong , Wang Huachun , Su Shih-Heng , Jester Peter J. , Zhang Shuqun , Bent Andrew F. , Krysan Patrick J.
Published: December 2006

The Arabidopsis (Arabidopsis thaliana) gene MEKK1 encodes a mitogen-activated protein kinase kinase kinase that has been implicated in the activation of the map kinases MPK3 and MPK6 in response to the flagellin elicitor peptide flg22. In this study, analysis of plants carrying T-DNA knockout alleles indicated that MEKK1 is required for flg22-induced activation of MPK4 but not MPK3 or MPK6. Experiments performed using a kinase-impaired version of MEKK1 (K361M) showed that the kinase activity of MEKK1 may not be required for flg22-induced MPK4 activation or for other macroscopic FLS2-mediated responses. MEKK1 may play a structural role in signaling, independent of its protein kinase activity. mekk1 knockout mutants display a severe dwarf phenotype, constitutive callose deposition, and constitutive expression of pathogen response genes. This dwarf phenotype was largely rescued by introduction into mekk1 knockout plants of either the MEKK1 (K361M) construct or a nahG transgene that degrades salicylic acid. When treated with pathogenic bacteria, the K361M plants were slightly more susceptible to an avirulent strain of Pseudomonas syringae and showed a delayed hypersensitive response, suggesting a role for MEKK1 kinase activity in this aspect of plant disease resistance. Our results indicate that MEKK1 acts upstream of MPK4 as a negative regulator of pathogen response pathways, a function that may not require MEKK1's full kinase activity.
Ca2+-Dependent and -Independent Abscisic Acid Activation of Plasma Membrane Anion Channels in Guard Cells of Nicotiana tabacum
Authors: Marten Holger , Konrad Kai R. , Dietrich Petra , Roelfsema M. Rob G. , Hedrich Rainer
Published: December 2006

Drought induces stomatal closure, a response that is associated with the activation of plasma membrane anion channels in guard cells, by the phytohormone abscisic acid (ABA). In several species, this response is associated with changes in the cytoplasmic free Ca2+ concentration. In Vicia faba, however, guard cell anion channels activate in a Ca2+-independent manner. Because of potential differences between species, Nicotiana tabacum guard cells were studied in intact plants, with simultaneous recordings of the plasma membrane conductance and the cytoplasmic free Ca2+ concentration. ABA triggered transient rises in cytoplasmic Ca2+ in the majority of the guard cells (14 out of 19). In seven out of 14 guard cells, the change in cytoplasmic free Ca2+ closely matched the activation of anion channels, while the Ca2+ rise was delayed in seven other cells. In the remaining five cells, ABA stimulated anion channels without a change in the cytoplasmic Ca2+ level. Even though ABA could activate anion channels in N. tabacum guard cells independent of a rise in the cytoplasmic Ca2+ concentration, patch clamp experiments showed that anion channels in these cells are stimulated by elevated Ca2+ in an ATP-dependent manner. Guard cells thus seem to have evolved both Ca2+-independent and -dependent ABA signaling pathways. Guard cells of N. tabacum apparently utilize both pathways, while ABA signaling in V. faba seems to be restricted to the Ca2+-independent pathway.
The Plant Structure Ontology, a Unified Vocabulary of Anatomy and Morphology of a Flowering Plant
Authors: Ilic Katica , Kellogg Elizabeth A. , Jaiswal Pankaj , Zapata Felipe , Stevens Peter F. , Vincent Leszek P. , Avraham Shulamit , Reiser Leonore , Pujar Anuradha , Sachs Martin M. , Whitman Noah T. , McCouch Susan R. , Schaeffer Mary L. , Ware Doreen H. , Stein Lincoln D. , Rhee Seung Y.
Published: December 2006

Formal description of plant phenotypes and standardized annotation of gene expression and protein localization data require uniform terminology that accurately describes plant anatomy and morphology. This facilitates cross species comparative studies and quantitative comparison of phenotypes and expression patterns. A major drawback is variable terminology that is used to describe plant anatomy and morphology in publications and genomic databases for different species. The same terms are sometimes applied to different plant structures in different taxonomic groups. Conversely, similar structures are named by their species-specific terms. To address this problem, we created the Plant Structure Ontology (PSO), the first generic ontological representation of anatomy and morphology of a flowering plant. The PSO is intended for a broad plant research community, including bench scientists, curators in genomic databases, and bioinformaticians. The initial releases of the PSO integrated existing ontologies for Arabidopsis (Arabidopsis thaliana), maize (Zea mays), and rice (Oryza sativa); more recent versions of the ontology encompass terms relevant to Fabaceae, Solanaceae, additional cereal crops, and poplar (Populus spp.). Databases such as The Arabidopsis Information Resource, Nottingham Arabidopsis Stock Centre, Gramene, MaizeGDB, and SOL Genomics Network are using the PSO to describe expression patterns of genes and phenotypes of mutants and natural variants and are regularly contributing new annotations to the Plant Ontology database. The PSO is also used in specialized public databases, such as BRENDA, GENEVESTIGATOR, NASCArrays, and others. Over 10,000 gene annotations and phenotype descriptions from participating databases can be queried and retrieved using the Plant Ontology browser. The PSO, as well as contributed gene associations, can be obtained at www.plantontology.org.
The RiceTapetum Degeneration RetardationGene Is Required for Tapetum Degradation and Anther Development
Authors: Li Na , Zhang Da-Sheng , Liu Hai-Sheng , Yin Chang-Song , Li Xiao-xing , Liang Wan-qi , Yuan Zheng , Xu Ben , Chu Huang-Wei , Wang Jia , Wen Tie-Qiao , Huang Hai , Luo Da , Ma Hong , Zhang Da-Bing
Published: December 2006

In flowering plants, tapetum degeneration is proposed to be triggered by a programmed cell death (PCD) process during late stages of pollen development; the PCD is thought to provide cellular contents supporting pollen wall formation and to allow the subsequent pollen release. However, the molecular basis regulating tapetum PCD in plants remains poorly understood. We report the isolation and characterization of a rice (Oryza sativa) male sterile mutant tapetum degeneration retardation (tdr), which exhibits degeneration retardation of the tapetum and middle layer as well as collapse of microspores. The TDR gene is preferentially expressed in the tapetum and encodes a putative basic helix-loop-helix protein, which is likely localized to the nucleus. More importantly, two genes, Os CP1 and Os c6, encoding a Cys protease and a protease inhibitor, respectively, were shown to be the likely direct targets of TDR through chromatin immunoprecipitation analyses and the electrophoretic mobility shift assay. These results indicate that TDR is a key component of the molecular network regulating rice tapetum development and degeneration.
Timing and rate of sexual maturation of European eel in brackish and freshwater environments
Authors: Bevacqua D. , Melià P. , Crivelli A. J. , De Leo G. A. , Gatto M.
Published: December 2006

Maturation rates in three European eel Anguilla anguilla populations increased from September to October and were slightly lower in fresh water than in brackish waters. Average and variance of total length at maturation were larger in females than males.
Taxes and the Efficiency‐Rent Extraction Trade‐off
Authors: ARYA ANIL , GLOVER JONATHAN , MITTENDORF BRIAN
Published: December 2006

This paper presents an adverse selection model in which progressive taxation enhances productive efficiency by encouraging a principal (buyer) to be less aggressive in contracting with an agent (seller). Wary of padded cost budgets, the buyer employs a hurdle‐rate procurement policy. With a low cost hurdle, the buyer keeps greater profits when transactions are undertaken but trade occurs less often. While the hurdle is unaffected by a flat tax, a progressive tax tilts the buyer's preference: the buyer's benefit from a lower hurdle becomes less pronounced, since the marginal increase in his profits is muted in after‐tax terms. The result is increased trade and the possibility of Pareto improvements.
The Private Provision of Public Goods under Uncertainty: A Symmetric‐Equilibrium Approach
Authors: KEENAN DONALD C. , KIM ILTAE , WARREN RONALD S.
Published: December 2006

Various studies have examined whether increased uncertainty about the non‐Nash response of others to an individual's voluntary contribution to a public good affects that individual's contribution so as to mitigate the free‐rider problem. We extend this single‐agent approach to the analysis of a symmetric equilibrium. We provide conditions on group size and endogenous relative risk aversion that imply increased equilibrium contributions in response to greater uncertainty about the productivity of each individual's contribution to the actual level of the public good. These results enable us to broaden the circumstances in which the theory predicts that increased uncertainty reduces free riding.
CRITICAL CARE ISSUES FOR THE NEPHROLOGIST: Renal Replacement Therapy in the Treatment of Acute Renal Failure—Intermittent and Continuous
Authors: John Stefan , Eckardt Kai‐Uwe
Published: December 2006

Renal replacement therapy (RRT) is increasingly used in intensive care as acute renal failure (ARF) is a common and constantly increasing complication in this setting. Different forms of RRT such as intermittent hemodialysis, continuous hemofiltration, or hybrid forms, which combine advantages of both, are available and will be discussed in this article. As a general survival benefit for neither method has been demonstrated, it is the task of the nephrologist or intensivist to choose the RRT strategy that is most advantageous for each individual patient. The choice of RRT might depend not only on the underlying disease, the time course of the disease, the etiology of ARF, the actual clinical status of the patient but also on the resources available and the cost of therapy. An adequate dose of RRT seems to result in improved survival in patients with ARF. However, clear guidelines on the dose of RRT and the timing of initiation are still lacking. Moreover, it will be discussed whether patients with sepsis and septic shock benefit from early RRT initiation, the use of increased RRT doses, and increased removal of inflammatory mediators by RRT.
CRITICAL CARE ISSUES FOR THE NEPHROLOGIST: Acid–base Disturbances in the Intensive Care Unit: Metabolic Acidosis
Authors: Schoolwerth Anton C. , Kaneko Thomas M. , Sedlacek Martin , Block Clay A. , Remillard Brian D.
Published: December 2006

This article will discuss metabolic acidosis and, to a lesser extent, metabolic alkalosis in the ICU setting. A classification and clinical approach will be the focus.
Differential sensitivity of excitatory and inhibitory synaptic transmission to modulation by nitric oxide in rat nucleus tractus solitarii
Authors: Wang Sheng , Paton Julian. F. R. , Kasparov Sergey
Published: December 2006

The nucleus tractus solitarii (NTS) is a key central link in control of multiple homeostatic reflexes. A number of studies have demonstrated that exogenous and endogenous nitric oxide (NO) within NTS regulates visceral function, but further understanding of the role of NO in the NTS is hampered by the lack of information about its intracellular actions. We studied effects of NO in acute rat brainstem slices. Aqueous NO solution (NOaq) potentiated electrically evoked excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs, respectively) in different neuronal subpopulations and, in some neurones, caused a depolarization. Similar effects were observed using the NO donor diethylamine NONOate (DEA/NO). The threshold NO concentration as determined using an NO electrochemical sensor was estimated as ∼0.4 nm (EC50∼0.9 nm) for potentiating glutamatergic EPSPs but ∼3 nm for monosynaptic GABAergic IPSPs. Bath application of the soluble guanylate cyclase (sGC) inhibitor 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ) abolished NOaq‐ and DEA/NO‐induced potentiation of evoked EPSPs, IPSPs and depolarization. All NO actions were mimicked by the non‐NO‐dependent guanylate cyclase activator Bay 41‐2272. The effects of NO on EPSPs and IPSPs persisted in cells where postsynaptic sGC was blocked by ODQ and therefore were presynaptic, owing to a direct modulation of transmitter release combined with depolarization of presynaptic neurones. Therefore, while lower concentrations of NO may be important for fine tuning of glutamatergic transmission, higher concentrations are required to directly engage GABAergic inhibition. This differential sensitivity of excitatory and inhibitory connections to NO may be important for determining the specificity of the effects of this freely diffusible gaseous messenger.
P[acman]: A BAC Transgenic Platform for Targeted Insertion of Large DNA Fragments in
D. melanogaster
Authors: Venken Koen J. T. , He Yuchun , Hoskins Roger A. , Bellen Hugo J.
Published: December 2006

We describe a transgenesis platform for
Drosophila melanogaster
that integrates three recently developed technologies: a conditionally amplifiable bacterial artificial chromosome (BAC), recombineering, and bacteriophage ΦC31–mediated transgenesis. The BAC is maintained at low copy number, facilitating plasmid maintenance and recombineering, but is induced to high copy number for plasmid isolation. Recombineering allows gap repair and mutagenesis in bacteria. Gap repair efficiently retrieves DNA fragments up to 133 kilobases long from P1 or BAC clones. ΦC31-mediated transgenesis integrates these large DNA fragments at specific sites in the genome, allowing the rescue of lethal mutations in the corresponding genes. This transgenesis platform should greatly facilitate structure/function analyses of most
Drosophila
genes.
Multiplex Quantitative Real-Time Reverse Transcriptase PCR for F
+
-Specific RNA Coliphages: a Method for Use in Microbial Source Tracking
Authors: Kirs Marek , Smith David C.
Published: December 2006

It is well documented that microbial contamination of coastal waters poses a significant risk to human health through recreational exposure and consumption of shellfish. Identifying the source of microbial contamination (microbial source tracking) plays a dominant role in enabling effective management and remediation strategies. One method used to determine the source of the contamination is quantification of the ratio of the four subgroups of F
+
-specific RNA coliphages (family
Leviviridae
) in impacted water samples. Because of typically low concentrations in the environment, enrichment assays are performed prior to detection, even though differential replication rates have been reported. These assays are also compromised by differential loss of phage infectivity among subgroups after release into the environment, thus obscuring the initial ratio. Here, a culture-independent multiplex real-time reverse transcriptase-PCR (RT-PCR) protocol for the simultaneous quantification of all four subgroups of F
+
-specific RNA coliphages using novel primer sets and molecular beacons is presented. This assay is extremely sensitive, achieving detection with as few as 10 copies of isolated coliphage RNA, and is linear for a minimum of six orders of magnitude. During survival experiments, the real-time RT-PCR technique was able to quantify coliphages in seawater when culture-based double agar layer assay failed. While infectivity was lost at different rates at the subgroup level, decay constants in seawater, calculated using the real-time RT-PCR estimates, did not vary among subgroups. The accurate determination of the in situ concentration of F
+
-specific RNA coliphages using this method will facilitate more effective remediation strategies for impacted environments.
Environmental Stress and Antibiotic Resistance in Food-Related Pathogens
Authors: McMahon M. Ann S. , Xu Jiru , Moore John E. , Blair Ian S. , McDowell David A.
Published: December 2006

This study investigated the possibility that sublethal food preservation stresses (high or low temperature and osmotic and pH stress) can lead to changes in the nature and scale of antibiotic resistance (ABR) expressed by three food-related pathogens (
Escherichia coli
,
Salmonella enterica
serovar Typhimurium, and
Staphylococcus aureus
). The study found that some sublethal stresses significantly altered antibiotic resistance. Incubation at sublethal high temperature (45°C) decreased ABR. Incubation under increased salt (>4.5%) or reduced pH (<5.0) conditions increased ABR. Some of the pathogens continued to express higher levels of ABR after removal of stress, suggesting that in some cases the applied sublethal stress had induced stable increases in ABR. These results indicate that increased use of bacteriostatic (sublethal), rather than bactericidal (lethal), food preservation systems may be contributing to the development and dissemination of ABR among important food-borne pathogens.
Molecular Characterization ofCryptosporidiumIsolates from Humans and Animals in Iran
Authors: Meamar Ahmad Reza , Guyot Karine , Certad Gabriela , Dei-Cas Eduardo , Mohraz Mino , Mohebali Mehdi , Mohammad Kazem , Mehbod Amir Ali , Rezaie Sasan , Rezaian Mostafa
Published: December 2006

Isolates ofCryptosporidiumspp. from human and animal hosts in Iran were characterized on the basis of both the 18S rRNA gene and the Laxer locus. ThreeCryptosporidiumspecies,C. hominis,C. parvum, andC. meleagridis, were recognized, and zoonotically transmittedC. parvumwas the predominant species found in humans.
Greater Diversity of Shiga Toxin-Encoding Bacteriophage Insertion Sites among
Escherichia coli
O157:H7 Isolates from Cattle than in Those from Humans
Authors: Besser Thomas E. , Shaikh Nurmohammad , Holt Nicholas J. , Tarr Phillip I. , Konkel Michael E. , Malik-Kale Preeti , Walsh Coilin W. , Whittam Thomas S. , Bono James L.
Published: December 2006

Escherichia coli
O157:H7, a zoonotic human pathogen for which domestic cattle are a reservoir host, produces a Shiga toxin(s) (Stx) encoded by bacteriophages. Chromosomal insertion sites of these bacteriophages define three principal genotypes (clusters 1 to 3) among clinical isolates of
E. coli
O157:H7. Stx-encoding bacteriophage insertion site genotypes of 282 clinical and 80 bovine isolates were evaluated. A total of 268 (95.0%) of the clinical isolates, but only 41 (51.3%) of the bovine isolates, belonged to cluster 1, 2, or 3 (
P
< 0.001). Thirteen additional genotypes were identified in isolates from both cattle and humans (four genotypes), from only cattle (seven genotypes), or from only humans (two genotypes). Two other markers previously associated with isolates from cattle or with clinical isolates showed similar associations with genotype groups within bovine isolates; the
tir
allele sp-1 and the
Q
933W
allele were under- and overrepresented, respectively, among cluster 1 to 3 genotypes. Stx-encoding bacteriophage insertion site typing demonstrated that there is broad genetic diversity of
E. coli
O157:H7 in the bovine reservoir and that numerous genotypes are significantly underrepresented among clinical isolates, consistent with the possibility that there is reduced virulence or transmissibility to humans of some bovine
E. coli
O157:H7 genotypes.
Diversity of Fungi, Bacteria, and Actinomycetes on Leaves Decomposing in a Stream
Authors: Das Mitali , Royer Todd V. , Leff Laura G.
Published: December 2006

Although fungi, bacteria, and specific bacterial taxa, such as the actinomycetes, have been studied extensively in various habitats, few studies have examined them simultaneously, especially on decomposing leaves in streams. In this study, sugar maple and white oak leaves were incubated in a stream in northeastern Ohio for 181 days during which samples were collected at regular intervals. Following DNA extraction, PCR-denaturing gradient gel electrophoresis (DGGE) was performed using fungus-, bacterium-, and actinomycete-specific primers. In addition, fungal and bacterial biomass was estimated. Fungal biomass differed on different days but not between leaves of the two species and was always greater than bacterial biomass. There were significant differences in bacterial biomass through time and between leaf types on some days. Generally, on the basis of DGGE, few differences in community structure were found for different leaf types. However, the ribotype richness of fungi was significantly greater than those of the bacteria and actinomycetes, which were similar to each other. Ribotype richness decreased toward the end of the study for each group except bacteria. Lack of differences between the two leaf types suggests that the microorganisms colonizing the leaf biofilm were primarily generalists that could exploit the resources of the leaves of either species equally well. Thus, we conclude that factors, such as the ecological role of the taxa (generalists versus specialists), stage of decay, and time of exposure, appeared to be more important determinants of microbial community structure than leaf quality.
Mixed-Valence Cytoplasmic Iron Granules Are Linked to Anaerobic Respiration
Authors: Glasauer S. , Langley S. , Boyanov M. , Lai B. , Kemner K. , Beveridge T. J.
Published: December 2006

Intracellular granules containing ferric and ferrous iron formed in
Shewanella putrefaciens
CN32 during dissimilatory reduction of solid-phase ferric iron. It is the first in situ detection at high resolution (150 nm) of a mixed-valence metal particle residing within a prokaryotic cell. The relationship of the internal particles to Fe(III) reduction may indicate a respiratory role.
Correlation of Functional Instability and Community Dynamics in Denitrifying Dispersed-Growth Reactors
Authors: Gentile M. E. , Jessup C. M. , Nyman J. L. , Criddle C. S.
Published: December 2006

Understanding the relationship between microbial community dynamics and functional instability is an important step towards designing reliable biological water treatment systems. In this study, the community dynamics of two dispersed-growth denitrifying reactors were examined during periods of functional stability and instability. In both reactors during the period of functional instability, the effluent chemistry changed over time, with periods of high nitrate concentrations followed by periods of fluctuating nitrite concentrations. Community structure was examined by clone library analysis of the 16S rRNA gene. Community dynamics were investigated with terminal restriction fragment (T-RF) length polymorphism, and the functional diversity represented by T-RFs was assessed through nitrate reduction assays of representative isolates. During the period of functional instability, the community structure changed considerably, and the dynamics correlated significantly with effluent chemistry. The nitrite concentration was significantly correlated with the relative abundances of the nitrate-reducing
Delftia
- and
Achromobacter
-like T-RFs. The isolate representing the
Acidovorax
-like T-RF reduced nitrate directly to nitrogen in batch assays without the accumulation of any intermediates. The
Acidovorax
-like T-RF relative abundance was significantly negatively correlated with nitrite concentration, indicating that it was associated with good functional performance. The results of this study reveal a clear relationship between community dynamics and functional instability and the importance of diversity among nitrate-reducing populations within a denitrifying community.
Role of Microbial Immigration in the Colonization of Apple Leaves by
Aureobasidium pullulans
Authors: McGrath Molly J. , Andrews John H.
Published: December 2006

The role of microbial immigration in the veinal colonization pattern of
Aureobasidium pullulans
on the adaxial surface of apple leaves was investigated in two experiments at two periods (early and late seasons) in 2004 by applying green fluorescent protein (GFP)-tagged blastospores to the foliage of orchard trees. Individual leaves were resampled by a semidestructive method immediately after inoculation (
t
0
) and about 1 (
t
1
), 2 (
t
2
), and 3 (
t
3
) weeks later. At
t
0
, there were no significant (
P
≤ 0.05) differences in densities (cells/mm
2
) on veinal (excluding midvein) sites and those on interveinal sites, but at all points thereafter, densities were significantly higher on veins. GFP-tagged
A. pullulans
cells remained primarily as singletons on interveinal regions (≥90% at all points), while ≥20% of cells over veins at
t
3
were in colonies of ≥4 cells. The colonies that developed from single cells placed on midveins and other veins were significantly larger than those that developed on interveinal regions of detached field and seedling leaves incubated under controlled conditions. Colonies primarily developed linearly along veins, reaching average colony sizes (72 h) of 24.4 ± 12.7 (mean ± standard deviation) cells. In contrast, colonies on interveinal regions tended to average only 2.9 ± 1.3 cells, with less linearity. To examine the potential role of
A. pullulans
growth-inhibiting factors associated with interveinal features, single GFP-tagged
A. pullulans
cells in droplets previously incubated on interveinal sites were placed on midveins and compared to midvein colonies derived from cells in a water-only suspension. No differences in colony size resulted. Our results indicate that immigration limitation and growth-inhibiting factors are not the primary factors responsible for
A. pullulans
veinal colonization patterns in the field. Rather, indirect evidence suggests that growth-promoting substances occur locally in the veinal areas.
Role of Broiler Carcasses and Processing Plant Air in Contamination of Modified-Atmosphere-Packaged Broiler Products with Psychrotrophic Lactic Acid Bacteria
Authors: Vihavainen Elina , Lundström Hanna-Saara , Susiluoto Tuija , Koort Joanna , Paulin Lars , Auvinen Petri , Björkroth K. Johanna
Published: December 2006

Some psychrotrophic lactic acid bacteria (LAB) are specific meat spoilage
organisms in modified-atmosphere-packaged (MAP), cold-stored meat
products. To determine if incoming broilers or the production plant
environment is a source of spoilage LAB, a total of 86, 122, and 447
LAB isolates from broiler carcasses, production plant air, and MAP
broiler products, respectively, were characterized using a library
of HindIII restriction fragment length polymorphism (RFLP)
patterns of the 16 and 23S rRNA genes as operational taxonomic units in
numerical analyses. Six hundred thirteen LAB isolates from the total of
655 clustered in 29 groups considered to be species specific.
Sixty-four percent of product isolates clustered either with
Carnobacterium divergens
or with
Carnobacterium
maltaromaticum
type strains. The third major product-associated
cluster (17% of isolates) was formed by unknown LAB. Representative
strains from these three clusters were analyzed for the phylogeny of
their 16S rRNA genes. This analysis verified that the two largest RFLP
clusters consisted of carnobacteria and showed that the unknown LAB
group consisted of
Lactococcus
spp. No product-associated LAB
were detected in broiler carcasses sampled at the beginning of
slaughter, whereas carnobacteria and lactococci, along with some other
specific meat spoilage LAB, were recovered from processing plant air at
many sites. This study reveals that incoming broiler chickens are not
major sources of psychrotrophic spoilage LAB, whereas the detection of
these organisms from the air of the processing environment highlights
the role of processing facilities as sources of LAB
contamination.
Chronic
Helicobacter pylori
Infection Does Not Significantly Alter the Microbiota of the Murine Stomach
Authors: Tan Mai Ping , Kaparakis Maria , Galic Maja , Pedersen John , Pearse Martin , Wijburg Odilia L. C. , Janssen Peter H. , Strugnell Richard A.
Published: December 2006

We examined the impact of
Helicobacter pylori
infection on the murine gastric microbiota by culture and terminal-restriction fragment length polymorphism and found that neither acute nor chronic
H. pylori
infection substantially affected the gastric microbial composition. Interestingly, the total
H. pylori
burden detected by real-time PCR was significantly higher than that revealed by viable counts, suggesting that the antigenic load sustaining
H. pylori
-induced gastritis could be considerably higher than previously believed.
Identification and Expression of Benzylsuccinate Synthase Genes in a Toluene-Degrading Methanogenic Consortium
Authors: Washer Cheryl E. , Edwards Elizabeth A.
Published: December 2006

Benzylsuccinate synthase (BSS) initiates anaerobic toluene biodegradation, and BSS genes have been found in several nitrate- and iron-reducing organisms. Here, two new putative
bssA
genes were identified in a methanogenic toluene-degrading culture. Transcription was upregulated with toluene but not with benzoate, consistent with the proposed function. These are the first
bss
sequences from a methanogenic culture.
Identification of Bacterial Groups Preferentially Associated with Mycorrhizal Roots of
Medicago truncatula
Authors: Offre P. , Pivato B. , Siblot S. , Gamalero E. , Corberand T. , Lemanceau P. , Mougel C.
Published: December 2006

The genetic structures of bacterial communities associated with
Medicago truncatula
Gaertn. cv. Jemalong line J5 (Myc
+
Nod
+
) and its symbiosis-defective mutants TRV48 (Myc
+
Nod
−
) and TRV25 (Myc
−
Nod
−
) were compared. Plants were cultivated in a fertile soil (Châteaurenard, France) and in soil from the Mediterranean basin showing a low fertility (Mas d'Imbert, France). Plant growth, root architecture, and the efficiency of root symbiosis of the three plant genotypes were characterized in the two soils. Structures of the bacterial communities were assessed by automated-ribosomal intergenic spacer analysis (A-RISA) fingerprinting from DNA extracted from the rhizosphere soil and root tissues. As expected, the TRV25 mutant did not develop endomycorrhizal symbiosis in any of the soils, whereas mycorrhization of line J5 and the TRV48 mutant occurred in both soils but at a higher intensity in the Mas d'Imbert (low fertility) than in the Châteaurenard soil. However, modifications of plant growth and root architecture, between mycorrhizal (J5 and TRV48) and nonmycorrhizal (TRV25) plants, were recorded only when cultivated in the Mas d'Imbert soil. Similarly, the genetic structures of bacterial communities associated with mycorrhizal and nonmycorrhizal plants differed significantly in the Mas d'Imbert soil but not in the Châteaurenard soil. Multivariate analysis of the patterns allowed the identification of molecular markers, explaining these differences, and markers were further sequenced. Molecular marker analysis allowed the delineation of 211 operational taxonomic units. Some of those belonging to the
Comamonadaceae
and
Oxalobacteraceae
(β-
Proteobacteria
) families were found to be significantly more represented within bacterial communities associated with the J5 line and the TRV48 mutant than within those associated with the TRV25 mutant, indicating that these bacterial genera were preferentially associated with mycorrhizal roots in the Mas d'Imbert soil.