Quantification of 2,4-Diacetylphloroglucinol-Producing Pseudomonas fluorescens Strains in the Plant Rhizosphere by Real-Time PCR

A real-time PCR SYBR green assay was developed to quantify populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing (phlD⁺) strains of Pseudomonas fluorescens in soil and the rhizosphere. Primers were designed and PCR conditions were optimized to specifically amplify the phlD gene from four different genotypes of phlD⁺ P. fluorescens. Using purified genomic DNA and genomic DNA extracted from washes of wheat roots spiked with bacteria, standard curves relating the threshold cycles (C_Ts) and copies of the phlD gene were generated for P. fluorescens strains belonging to genotypes A (Pf-5), B (Q2-87), D (Q8r1-96 and FTAD1R34), and I (FTAD1R36). The detection limits of the optimized real-time PCR assay were 60 to 600 fg (8 to 80 CFU) for genomic DNA isolated from pure cultures of P. fluorescens and 600 fg to 6.0 pg (80 to 800 CFU, corresponding to log 4 to 5 phlD⁺ strain CFU/rhizosphere) for bacterial DNA extracted from plant root washes. The real-time PCR assay was utilized to quantify phlD⁺ pseudomonads in the wheat rhizosphere. Regression analysis of population densities detected by real-time PCR and by a previously described phlD-specific PCR-based dilution endpoint assay indicated a significant linear relationship (P = 0.0016, r² = 0.2). Validation of real-time PCR assays with environmental samples was performed with two different soils and demonstrated the detection of more than one genotype in Quincy take-all decline soil. The greatest advantage of the developed real-time PCR is culture independence, which allows determination of population densities and the genotype composition of 2,4-DAPG producers directly from the plant rhizospheres and soil.     

Oxytocin knockout mice demonstrate enhanced intake of sweet and nonsweet carbohydrate solutions

Oxytocin knockout (OT KO) mice display enhanced intake of nutritive and nonnutritive sweet solutions (i.e., sucrose and saccharin) compared with wild-type (WT) mice of the same C57BL/6 background strain. The present study further investigated the differential behavioral response of OT KO and WT mice to sucrose solutions and also examined intake preferences of OT KO and WT mice for palatable but nonsweet isocaloric solutions of carbohydrate and fat. A progressive ratio operant licking procedure demonstrated that OT KO and WT mice display a similar motivational drive to consume 10% sucrose. A series of two-bottle intake tests revealed that OT KO mice consume significantly larger amounts of both sweet and nonsweet carbohydrate solutions (i.e., sucrose, Polycose, and cornstarch) compared with WT cohorts. Intake pattern analyses revealed that OT KO mice overconsume carbohydrate solutions by initiating more drinking bouts compared with WT mice; bout sizes did not differ between the genotypes. In contrast, OT KO and WT mice did not differ in their intake of Intralipid, a palatable soybean oil emulsion. These findings indicate that the absence of OT in mice does not affect their appetitive drive to consume palatable sucrose solutions. Instead, the absence of OT may increase daily intake of palatable sweet and nonsweet solutions of carbohydrate (but not fat) by selectively blunting or masking processes that contribute to postingestive satiety.     

Survival of extrapair and within-pair young in tree swallows

In monogamous species, it is generally accepted that males seekextrapair matings to increase their reproductive success withoutadditional parental investment; however, the benefits of extrapairmatings to females are much less clear. One possibility isthat females obtain genes for enhanced offspring viabilityfrom the extrapair sires. If this is the case, then the increasedviability of extrapair young may be evident throughout the periodof embryonic development as well as later in life. Tree swallows(Tachycineta bicolor) have one of the highest known levelsof extrapair mating in birds, and females have substantialcontrol over the paternity of their offspring. We used moleculartechniques to determine the parentage of nestlings and unhatchedembryos to examine the possibility that female tree swallowsgain viability benefits for their extrapair offspring. Althoughboth extrapair paternity and mortality of embryos and nestlingswere high (89% and 54% of broods respectively), we found nodifference in the viability of within-pair and extrapair youngprior to fledging. In addition, extrapair young were not morelikely to be male. There was no bias in the sex of young atfledging, but unhatched embryos were more likely to be male.Our results do not support the idea that female tree swallowsengage in extrapair mating to increase offspring viability,at least early in life.     

Extrapair paternity is influenced by breeding synchrony and density in the common yellowthroat

The effects of breeding synchrony and density on levels of extrapair paternity in birds are controversial. We used multilocus DNAfingerprinting and microsatellite analysis to examine the effectsof breeding synchrony and density on levels of extrapair paternityin the common yellowthroat (Geothlypis trichas). As in manyNeotropical migrants, breeding synchrony was greatest at thebeginning of the breeding season. Levels of extrapair paternitywere higher after the peak in synchrony, leading to an overallnegative relationship between extrapair paternity and breeding synchrony. However, there was a significant interaction betweenbreeding synchrony and density, as levels of extrapair paternitywere higher only for males breeding when both synchrony anddensity were low. We discuss several possible explanationsfor this interaction, including lower quality males or territoriesin low density areas and greater demands on mate guarding among males with larger territories. Most studies have not consideredsimultaneously the effects of breeding synchrony and densityon extrapair paternity. Our results suggest that ecologicalcorrelates of paternity may be revealed only after testingfor interactions in multivariate analyses.     

The adipocyte as an important target cell for Trypanosoma cruzi infection

Adipose tissue plays an active role in normal metabolic homeostasis as well as in the development of human disease. Beyond its obvious role as a depot for triglycerides, adipose tissue controls energy expenditure through secretion of several factors. Little attention has been given to the role of adipocytes in the pathogenesis of Chagas disease and the associated metabolic alterations. Our previous studies have indicated that hyperglycemia significantly increases parasitemia and mortality in mice infected with Trypanosoma cruzi. We determined the consequences of adipocyte infection in vitro and in vivo. Cultured 3T3-L1 adipocytes can be infected with high efficiency. Electron micrographs of infected cells revealed a large number of intracellular parasites that cluster around lipid droplets. Furthermore, infected adipocytes exhibited changes in expression levels of a number of different adipocyte-specific or adipocyte-enriched proteins. The adipocyte is therefore an important target cell during acute Chagas disease. Infection of adipocytes by T. cruzi profoundly influences the pattern of adipokines. During chronic infection, adipocytes may represent an important long-term reservoir for parasites from which relapse of infection can occur. We have demonstrated that acute infection has a unique metabolic profile with a high degree of local inflammation in adipose tissue, hypoadiponectinemia, hypoglycemia, and hypoinsulinemia but with relatively normal glucose disposal during an oral glucose tolerance test.     

The Vh8 locus of a new gene-for-gene interaction between Venturia inaequalis and the wild apple Malus sieversii is closely linked to the Vh2 locus in Malus pumila R12740-7A

The wild apple (Malus sieversii) is a large-fruited species from Central Asia, which is used as a source of scab resistance in cultivar breeding. Phytopathological tests with races of Venturia inaequalis were performed to differentiate scab-resistance genes in Malus as well as an avirulence gene in the pathogen. A novel gene-for-gene interaction between V. inaequalis and Malus was identified. The locus of the scab-resistance gene Vh8 is linked with, or possibly allelic to, that of the Vh2 gene in Malus pumila Russian apple R12740-7A, at the lower end of linkage group 2 of Malus. Race 8 isolate NZ188B.2 is compatible with Vh8, suggesting the loss or modification of the complementary AvrVh8 gene, while isolate 1639 overcomes both Vh2 and Vh8, but is incompatible with at least one other gene not detected by any of the other race isolates tested. Our research is the first to differentiate scab-resistance genes in a putative gene cluster in apple with the aid of races of V. inaequalis.     

Photophysics of tryptophan fluorescence: link with the catalytic strategy of the citrate synthase from Thermoplasma acidophilum

The formation of all major intermediates in the reaction catalyzed by the citrate synthase from Thermoplasma acidophilum is accompanied by changes in tryptophan fluorescence. The largest change is the strong quenching observed on formation of the binary complex with substrate, oxaloacetate (OAA). The four tryptophan residues present in the enzyme have been changed to nonfluorescent ones in various combinations without major perturbations in protein stability, enzyme mechanism, or other physical properties. W348, residing in the hydrophobic core of the protein behind the active site wall ca. 9 A from OAA, is responsible for the majority of the protein's intrinsic fluorescence and all of the quenching that accompanies OAA binding. Lifetime studies show that all of the quenching results from excited-state processes. The lack of solvent isotope effects on the quantum yields excludes a quenching mechanism involving proton transfer to an acceptor. There are no significant changes in fluorescence properties in single site mutants of residues near W348 that change conformation and/or interactions when OAA binds. This result excludes these changes from a direct role. Electron transfer from the indole excited state to some acceptor is the major quenching mechanism; the reduced quenching observed in the 5F-W-substituted protein strengthens this conclusion. Using the X-ray structures of the unliganded enzyme and its OAA binary complex, hybrid quantum mechanics-molecular dynamics (QM-MM) calculations show that OAA itself is the most likely quencher with the OAA carbonyl as the electron acceptor. This conclusion is strengthened by the ability of an alpha-keto acid model compound, trimethylpyruvate, to act as a diffusional quencher of indole fluorescence in solution. The theoretical calculations further indicate that the positive electrostatic potential surrounding the OAA carbonyl within the enzymes' active site is essential to its ability to accept an electron from the excited state of W348. These same environmental factors play a major role in activating OAA to react with the carbanion of acetyl-CoA. Since carbonyl polarization plays a role in the catalytic strategies of numerous enzymes whose reactions involve this functional group, tryptophan fluorescence changes might be useful as a mechanistic probe for other systems.     

Recognition of fresh human tumor by human peripheral blood lymphocytes transduced with a bicistronic retroviral vector encoding a murine anti-p53 TCR

The p53 protein is markedly up-regulated in a high proportion of human malignancies. Using an HLA-A2 transgenic mouse model, it was possible to isolate high-avidity murine CTLs that recognize class I-restricted human p53 epitopes. We isolated the alpha- and beta-chain of a TCR from a highly avid murine CTL clone that recognized the human p53(264-272) epitope. These genes were cloned into a retroviral vector that mediated high efficiency gene transfer into primary human lymphocytes. Efficiencies of >90% for gene transfer into lymphocytes were obtained without selection for transduced cells. The p53 TCR-transduced lymphocytes were able to specifically recognize with high-avidity, peptide-pulsed APCs as well as HLA-A2.1+ cells transfected with either wild-type or mutant p53 protein. p53 TCR-transduced cells demonstrated recognition and killing of a broad spectrum of human tumor cell lines as well as recognition of fresh human tumor cells. Interestingly, both CD8+ and CD4+ subsets were capable of recognizing and killing target cells, stressing the potential application of such a CD8-independent TCR molecule that can mediate both helper and cytotoxic responses. These results suggest that lymphocytes genetically engineered to express anti-p53 TCR may be of value for the adoptive immunotherapy of patients with a variety of common malignancies.     

Ribosomal DNA sequence variation among sympatric strains of the Cyclotella meneghiniana complex (Bacillariophyceae) reveals cryptic diversity

Cyclotella meneghiniana Kützing is one of the most commonly found and intensively studied freshwater diatom species. However, it is considered taxonomically problematic because of its unusually wide ecological range and large frustule ultrastructural variation. As part of a study of morphological and genetic variation in this morphospecies, we surveyed nucleotide variation in the hypervariable D1/D2 regions of the 28S rDNA, in the ribosomal internal transcribed spacer region (containing ITS1, the 5.8S rDNA and ITS2) and in the 18S rDNA in a collection of 20 sympatric strains. High genetic variability and strong indications of genetic structure among the Cyclotella meneghiniana strains were found. Representatives of four genetically distinct--apparently reproductively isolated--groups were revealed among them. The random distribution of ITS variation within these four groups indicated that the genetic structure in Cyclotella meneghiniana can probably be explained by the presence of cryptic sexual species rather than by the lack of allogamous sexual reproduction. The morphological features traditionally used for species identification in this group cannot distinguish these putative cryptic species.     

A retinoic acid-Hox hierarchy controls both anterior/posterior patterning and neuronal specification in the developing central nervous system of the cephalochordate amphioxus

Retinoic acid (RA) mediates both anterior/posterior patterning and neuronal specification in the vertebrate central nervous system (CNS). However, the molecular mechanisms downstream of RA are not well understood. To investigate these mechanisms, we used the invertebrate chordate amphioxus, in which the CNS, although containing only about 20,000 neurons in adults, like the vertebrate CNS, has a forebrain, midbrain, hindbrain, and spinal cord and is regionalized by RA-signaling. Here we show, first, that domains of genes with expression normally limited to diencephalon and midbrain are generally not affected by altered RA-signaling, second, that contrary to previous reports, not only Hox1, 3, and 4, but also Hox2 and Hox6 are collinearly expressed in the amphioxus CNS, and third, that collinear expression of all these Hox genes is controlled by RA-signaling. Finally, we show that Hox1 is involved in mediating both the role of RA-signaling in regionalization of the hindbrain and in specification of hindbrain motor neurons. Thus, morpholino knock-down of the single amphioxus Hox1 mimics the effects of treatments with an RA-antagonist. This analysis establishes RA-dependent regulation of collinear Hox expression as a feature common to the chordate CNS and indicates that the RA-Hox hierarchy functions both in proper anterior/posterior patterning of the developing CNS and in specification of neuronal identity.