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91.
Genetic evidence in Arabidopsis (Arabidopsis thaliana) suggests that the auxin precursor indole-3-butyric acid (IBA) is converted into active indole-3-acetic acid (IAA) by peroxisomal β-oxidation; however, direct evidence that Arabidopsis converts IBA to IAA is lacking, and the role of IBA-derived IAA is not well understood. In this work, we directly demonstrated that Arabidopsis seedlings convert IBA to IAA. Moreover, we found that several IBA-resistant, IAA-sensitive mutants were deficient in IBA-to-IAA conversion, including the indole-3-butyric acid response1 (ibr1) ibr3 ibr10 triple mutant, which is defective in three enzymes likely to be directly involved in peroxisomal IBA β-oxidation. In addition to IBA-to-IAA conversion defects, the ibr1 ibr3 ibr10 triple mutant displayed shorter root hairs and smaller cotyledons than wild type; these cell expansion defects are suggestive of low IAA levels in certain tissues. Consistent with this possibility, we could rescue the ibr1 ibr3 ibr10 short-root-hair phenotype with exogenous auxin. A triple mutant defective in hydrolysis of IAA-amino acid conjugates, a second class of IAA precursor, displayed reduced hypocotyl elongation but normal cotyledon size and only slightly reduced root hair lengths. Our data suggest that IBA β-oxidation and IAA-amino acid conjugate hydrolysis provide auxin for partially distinct developmental processes and that IBA-derived IAA plays a major role in driving root hair and cotyledon cell expansion during seedling development.The auxin indole-3-acetic acid (IAA) controls both cell division and cell expansion and thereby orchestrates many developmental events and environmental responses. For example, auxin regulates lateral root initiation, root and stem elongation, and leaf expansion (for review, see Davies, 2004). Normal plant morphogenesis and environmental responses require modulation of auxin levels by controlling biosynthesis, regulating transport, and managing storage forms (for review, see Woodward and Bartel, 2005a). In some storage forms, the carboxyl group of IAA is conjugated to amino acids or peptides or to sugars, and free IAA can be released by hydrolases when needed (Bartel et al., 2001; Woodward and Bartel, 2005a). A second potential auxin storage form is the side chain-lengthened compound indole-3-butyric acid (IBA), which can be synthesized from IAA (Epstein and Ludwig-Müller, 1993) and is suggested to be shortened into IAA by peroxisomal β-oxidation (Bartel et al., 2001; Woodward and Bartel, 2005a).Genetic evidence suggests that the auxin activity of both IAA-amino acid conjugates and IBA requires free IAA to be released from these precursors (Bartel and Fink, 1995; Zolman et al., 2000). Mutation of Arabidopsis (Arabidopsis thaliana) genes encoding IAA-amino acid hydrolases, including ILR1, IAR3, and ILL2, reduces plant sensitivity to the applied IAA-amino acid conjugates that are substrates of these enzymes, including IAA-Leu, IAA-Phe, and IAA-Ala (Bartel and Fink, 1995; Davies et al., 1999; LeClere et al., 2002; Rampey et al., 2004), which are present in Arabidopsis (Tam et al., 2000; Kowalczyk and Sandberg, 2001; Kai et al., 2007).Unlike the simple one-step release of free IAA from amino acid conjugates, release of IAA from IBA is suggested to require a multistep process (Zolman et al., 2007, 2008). Conversion of IBA to IAA has been demonstrated in a variety of plants (Fawcett et al., 1960; for review, see Epstein and Ludwig-Müller, 1993) and may involve β-oxidation of the four-carbon carboxyl side chain of IBA to the two-carbon side chain of IAA (Fawcett et al., 1960; Zolman et al., 2000, 2007). Mutation of genes encoding the apparent β-oxidation enzymes INDOLE-3-BUTYRIC ACID RESPONSE1 (IBR1), IBR3, or IBR10 results in IBA resistance, but does not alter IAA response or confer a dependence on exogenous carbon sources for growth following germination (Zolman et al., 2000, 2007, 2008), consistent with the possibility that these enzymes function in IBA β-oxidation but not fatty acid β-oxidation.Both conjugate hydrolysis and IBA β-oxidation appear to be compartmentalized. The IAA-amino acid hydrolases are predicted to be endoplasmic reticulum localized (Bartel and Fink, 1995; Davies et al., 1999) and enzymes required for IBA responses, including IBR1, IBR3, and IBR10, are peroxisomal (Zolman et al., 2007, 2008). Moreover, many peroxisome biogenesis mutants, such as peroxin5 (pex5) and pex7, are resistant to exogenous IBA, but remain IAA sensitive (Zolman et al., 2000; Woodward and Bartel, 2005b).Although the contributions of auxin transport to environmental and developmental auxin responses are well documented (for review, see Petrášek and Friml, 2009), the roles of various IAA precursors in these processes are less well understood. Expansion of root epidermal cells to control root architecture is an auxin-regulated process in which these roles can be dissected. Root epidermal cells provide soil contact and differentiate into files of either nonhair cells (atrichoblasts) or hair cells (trichoblasts). Root hairs emerge from trichoblasts as tube-shaped outgrowths that increase the root surface area, thus aiding in water and nutrient uptake (for review, see Grierson and Schiefelbein, 2002). Root hair length is determined by the duration of root hair tip growth, which is highly sensitive to auxin levels (for review, see Grierson and Schiefelbein, 2002). Mutants defective in the ABCG36/PDR8/PEN3 ABC transporter display lengthened root hairs and hyperaccumulate [3H]IBA, but not [3H]IAA, in root tip auxin transport assays (Strader and Bartel, 2009), suggesting that ABCG36 functions as an IBA effluxer and that IBA promotes root hair elongation. The related ABCG37/PDR9 transporter also can efflux IBA (Strader et al., 2008b; Růžička et al., 2010) and may have some functional overlap with ABCG36 (Růžička et al., 2010). In addition to lengthened root hairs, abcg36/pdr8/pen3 mutants display enlarged cotyledons, a second high-auxin phenotype. Both of these developmental phenotypes are suppressed by the mildly peroxisome-defective mutant pex5-1 (Strader and Bartel, 2009), suggesting that IBA contributes to cell expansion by serving as a precursor to IAA, which directly drives the increased cell expansion that underlies these phenotypes. However, whether IBA-derived IAA contributes to cell expansion events during development of wild-type plants is not known.Here, we directly demonstrate that peroxisome-defective mutants are defective in the conversion of IBA to IAA, consistent with previous reports that these genes are necessary for full response to applied IBA. We found that a mutant defective in three suggested IBA-to-IAA conversion enzymes displays low-auxin phenotypes, including decreased root hair expansion and decreased cotyledon size. We further found that these mutants suppress the long-root-hair and enlarged cotyledon phenotypes of an abcg36/pdr8 mutant, suggesting that endogenous IBA-derived IAA drives root hair and cotyledon expansion in wild-type seedlings.  相似文献   
92.

Background  

The genus Campylobacter includes many species, some of which are known human and animal pathogens. Even though studies have repeatedly identified domestic dogs as a risk factor for human campylobacteriosis, our understanding of Campylobacter ecology in this reservoir is limited. Work to date has focused primarily on a limited number of species using culture-based methods. To expand our understanding of Campylobacter ecology in dogs, a collection of fecal samples from 70 healthy and 65 diarrheic pet dogs were examined for the presence and levels of 14 Campylobacter species using quantitative PCR.  相似文献   
93.
Cancer is the consequence of sequential acquisition of mutations within somatic cells. Mutations alter the relative reproductive fitness of cells, enabling the population to evolve in time as a consequence of selection. Cancer therapy itself can select for or against specific subclones. Given the large population of tumor cells, subclones inevitably emerge and their fate will depend on the evolutionary dynamics that define the interactions between such clones. Using a combination of in vitro studies and mathematical modeling, we describe the dynamic behavior of two cell lines isolated from the same patient at different time points of disease progression and show how the two clones relate to one another. We provide evidence that the two clones coexisted at the time of initial presentation. The dominant clone presented with biopsy-proven cardiac AL amyloidosis. Initial therapy selected for the second clone that expanded leading to a change in the diagnosis to multiple myeloma. The evolutionary dynamics relating the two cell lines are discussed and a hypothesis is generated in regard to the mechanism of one of the phenotypic characteristics that is shared by these two cell lines.Key words: multiple myeloma, amyloidosis, chemotherapy, clonal evolution, selection  相似文献   
94.
ABSTRACT.   Little is known about the genetic mating systems of tropical passerines and how they vary among species. We studied the Lesser Elaenia ( Elaenia chiriquensis ) and the Yellow-bellied Elaenia ( E. flavogaster ) near Gamboa, Panama. These species breed in the same habitat, but Lesser Elaenias are intratropical migrants with seasonal territories and Yellow-bellied Elaenias are permanent residents that remain paired and defend territories throughout the year. Lesser Elaenias exhibited greater breeding synchrony (15–18 %) than Yellow-bellied Elaenias (9–10%). For Lesser Elaenias, 10 of 15 (67%) nests contained extra-pair young and 14 of 38 (37%) young resulted from extra-pair fertilizations (EPFs). In contrast, only one extra-pair nestling (4%, N = 24 nestlings) was found in 13 Yellow-bellied Elaenia nests. Neither species exhibited strong mate guarding. The higher rate of EPFs in Lesser Elaenias is consistent with the hypothesis that year-round territorial tropical passerines with low breeding synchrony have little or no extra-pair behavior compared with species that breed seasonally. Although the low singing rates of Lesser Elaenias (7 songs/h) suggest that this not an important cue for female extra-pair mate choice, the role of conspicuous male dawn song remains to be investigated. Further studies of tropical passerines are needed to help disentangle the effects of synchrony, density, and other ecological and behavioral factors that have influenced the evolution of extra-pair mating systems in passerines.  相似文献   
95.
A process was developed for production of a candidate vaccine antigen, recombinant C-terminal heavy chain fragment of the botulinum neurotoxin serotype E, rBoNTE(H(c)) in Pichia pastoris. P. pastoris strain GS115 was transformed with the rBoNTE(H(c)) gene inserted into pHILD4 Escherichia coli-P. pastoris shuttle plasmid. The clone was characterized for genetic stability, copy number, and BoNTE(H(c)) sequence. Expression of rBoNTE(H(c)) from the Mut(+) HIS4 clone was confirmed in the shake-flask, prior to developing a fed-batch fermentation process at 5 and 19 L scale. The fermentation process consists of a glycerol growth phase in batch and fed-batch mode using a defined medium followed by a glycerol/methanol transition phase for adaptation to growth on methanol and a methanol induction phase resulting in the production of rBoNTE(H(c)). Specific growth rate, ratio of growth to induction phase, and time of induction were critical for optimal rBoNTE(H(c)) production and minimal proteolytic degradation. A computer-controlled exponential growth model was used for process automation and off-gas analysis was used for process monitoring. The optimized process had an induction time of 9 h on methanol and produced up to 3 mg of rBoNTE(H(c)) per gram wet cell mass as determined by HPLC and Western blot analysis.  相似文献   
96.
The ubiquitin-like protein RELATED TO UBIQUITIN (RUB) is conjugated to CULLIN (CUL) proteins to modulate the activity of Skp1-Cullin-F-box (SCF) ubiquitylation complexes. RUB conjugation to specific target proteins is necessary for the development of many organisms, including Arabidopsis (Arabidopsis thaliana). Here, we report the isolation and characterization of e1-conjugating enzyme-related1-1 (ecr1-1), an Arabidopsis mutant compromised in RUB conjugation. The ecr1-1 mutation causes a missense change located two amino acid residues from the catalytic site cysteine, which normally functions to form a thioester bond with activated RUB. A higher ratio of unmodified CUL1 relative to CUL1-RUB is present in ecr1-1 compared to wild type, suggesting that the mutation reduces ECR1 function. The ecr1-1 mutant is resistant to the auxin-like compound indole-3-propionic acid, produces fewer lateral roots than wild type, displays reduced adult height, and stabilizes a reporter fusion protein that is degraded in response to auxin, suggesting reduced auxin signaling in the mutant. In addition, ecr1-1 hypocotyls fail to elongate normally when seedlings are grown in darkness, a phenotype shared with certain other RUB conjugation mutants that is not general to auxin-response mutants. The suite of ecr1-1 molecular and morphological phenotypes reflects roles for RUB conjugation in many aspects of plant growth and development. Certain ecr1-1 elongation defects are restored by treatment with the ethylene-response inhibitor silver nitrate, suggesting that the short ecr1-1 root and hypocotyl result from aberrant ethylene accumulation. Further, silver nitrate supplementation in combination with various auxins and auxin-like compounds reveals that members of this growth regulator family may differentially rely on ethylene signaling to inhibit root growth.  相似文献   
97.
Tailed bacteriophages and large eukaryotic viruses employ powerful molecular motors to translocate dsDNA into a preassembled capsid shell. The phage T4 motor is composed of a dodecameric portal and small and large terminase subunits assembled at the special head-tail connector vertex of the prohead. The motor pumps DNA through the portal channel, utilizing ATP hydrolysis energy provided by an ATPase present in the large terminase subunit. We report that the ATPase motors of terminases, helicases, translocating restriction enzymes, and protein translocases possess a common coupling motif (C-motif). Mutations in the phage T4 terminase C-motif lead to loss of stimulated ATPase and DNA translocation activities. Surprisingly, the mutants can catalyze at least one ATP hydrolysis event but are unable to turn over and reset the motor. This is the first report of a catalytic block in translocating ATPase motor after ATP hydrolysis occurred. We suggest that the C-motif is an ATP hydrolysis sensor, linking product release to mechanical motion. A novel terminase-driven mechanism is proposed for translocation of dsDNA in viruses.  相似文献   
98.
Arboviruses (arthropod-borne viruses) represent quintessential generalists, with the ability to infect and perform well in multiple hosts. However, antagonistic pleiotropy imposed a cost during the adaptation to persistent replication of vesicular stomatitis virus in sand fly cells and resulted in strains that initially replicated poorly in hamster cells, even when the virus was allowed to replicate periodically in the latter. Once a debilitated strain started replicating continuously in mammalian cells, fitness increased significantly. Fitness recovery did not entail back mutations or compensatory mutations, but instead, we observed the replacement of persistence-adapted genomes by mammalian cell-adapted strains with a full set of new, unrelated sequence changes. These mammalian cell-adapted genomes were present at low frequencies in the populations with a history of persistence for up to a year and quickly became dominant during mammalian infection, but coexistence was not stable in the long term. Periodic acute replication in mammalian cells likely contributed to extending the survival of minority genomes, but these genomes were also found in strictly persistent populations.  相似文献   
99.
Recombinant poxvirus vectors are undergoing intensive evaluation as vaccine candidates for a variety of infectious pathogens. Avipoxviruses, such as canarypox virus, are replication deficient in mammalian cells by virtue of a poorly understood species-specific restriction. Highly attenuated vaccinia virus strains such as modified vaccinia virus Ankara (MVA) are similarly unable to complete replication in most mammalian cells but have an abortive-late phenotype, in that the block to replication occurs post-virus-specific DNA replication. In this study, an identical expression cassette for human immunodeficiency virus gag, pro, and env coding sequences was placed in canarypox virus and MVA vector backbones in order to directly compare vector-borne expression and to analyze differences in vector-host cell interactions. Antigen production by recombinant MVA was shown to be greater than that from recombinant canarypox virus in the mammalian cell lines and in the primary human cells tested. This observation was primarily due to a longer duration of antigen production in recombinant MVA-infected cells. Apoptosis induction was found to be more profound with the empty canarypox virus vector than with MVA. Remarkably, however, the inclusion of a gag/pro/env expression cassette altered the kinetics of apoptosis induction in recombinant MVA-infected cells to levels equal to those found in canarypox virus-infected cells. Antigen production by MVA was noted to be greater in human dendritic cells and resulted in enhanced T-cell stimulation in an in vitro antigen presentation assay. These results reveal differences in poxvirus vector-host cell interactions that should be relevant to their use as immunization vehicles.  相似文献   
100.
We examined two expression systems for studying the Na+/H+ exchanger in the mammalian myocardium. Mammalian NHE1 with a hemagglutinin (HA) tag and was cloned behind the alpha myosin heavy chain promoter. Transgenic mice were made with wild type NHE1 protein or with a hyperactive NHE1 protein mutated at the calmodulin-binding domain. Three lines of transgenic mice were made of each cDNA with expression levels of each type varying from high to low. Higher levels and activity of the Na+/H+ exchanger were associated with decreased long-term survival of mice, and with dilated or hypertrophic cardiomyopathy. The exogenous NHE1 protein was present in freshly made cardiomyocytes from transgenic mice, however, expression from the alpha myosin heavy chain promoter declined rapidly and little exogenous NHE1 was apparent on the fourth day after cardiomyocyte isolation. To express NHE1 protein in isolated cardiomyocytes, we transferred a mutated form of the protein into an adenoviral expression system. Infection of neonatal rat cardiomyocytes resulted in robust expression of the exogenous NHE1 protein. The mutant form of the NHE1 protein could be distinguished from the endogenous Na+/H+ exchanger by its resistance to inhibition by amiloride analogs. Our results suggest that for in vivo studies on intact hearts and animals, expression in transgenic mice is an appropriate system, however for long-term studies on cardiomyocytes, this model is inappropriate due to waning expression from the alpha myosin heavy chain promoter. Therefore, infection by adenovirus is a superior system for long-term studies on cardiomyocytes in culture.  相似文献   
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