The turnip mutant of Arabidopsis reveals that LEAFY COTYLEDON1 expression mediates the effects of auxin and sugars to promote embryonic cell identity

The transition from embryonic to vegetative growth marks an important developmental stage in the plant life cycle. The turnip (tnp) mutant was identified in a screen for modifiers of POLARIS expression, a gene required for normal root growth. Mapping and molecular characterization of tnp shows that it represents a gain-of-function mutant of LEAFY COTYLEDON1 (LEC1), due to a promoter mutation. This results in the ectopic expression of LEC1, but not of other LEC genes, in vegetative tissues. The LEC class of genes are known regulators of embryogenesis, involved in the control of embryonic cell identity by currently unknown mechanisms. Activation of the LEC-dependent pathway in tnp leads to the loss of hypocotyl epidermal cell marker expression and loss of SCARECROW expression in the endodermis, the ectopic accumulation of starch and lipids, and the up-regulation of early and late embryonic genes. tnp also shows partial deetiolation during dark growth. Penetrance of the mutant phenotype is strongly enhanced in the presence of exogenous auxin and sugars, but not by gibberellin or abscisic acid, and is antagonized by cytokinin. We propose that the role of LEC1 in embryonic cell fate control requires auxin and sucrose to promote cell division and embryonic differentiation.     

Novel phosphorus-containing 17beta-side chain mifepristone analogues as progesterone receptor antagonists

A novel series of steroidal compounds were designed and synthesized with various phosphorus-containing groups on the 17beta-side chain as progesterone receptor antagonists. The structure-activity relationships of these compounds are discussed. Selected compounds were tested in an rat progesterone-sensitive assay. Some of these compounds are more potent than mifepristone, with a better selectivity profile in differentiating progesterone receptor from glucocorticoid receptor.     

Standardising bull breeding soundness evaluations and reporting in Australia

There is substantial variation in bull breeding soundness evaluation procedures and reports in Australia; the situation is compounded by difficulties in interpretation and the validity of many reports. In an effort to overcome this, the scientific literature was reviewed [Fordyce G. In: Fordyce G, editor. Bull fertility: selection and management in Australia. Eight Mile Plains, Australia: Australian Cattle Vets; 2002] and the needs of stakeholders were considered in preparing a manual, Evaluating and Reporting Bull Fertility [Entwistle KW, Fordyce G. Evaluating and reporting bull fertility. Eight Mile Plains, Australia: Australian Cattle Vets; 2003.] that outlined standards for assessing and reporting bull breeding soundness. A new recording and reporting system, called Bull Reporter, is based on standards from this manual and groups bull fertility traits into five summary categories: Scrotum, Physical, Crush-side Semen, Sperm Morphology, and Serving. The client will generally select which categories they wish to have included in the evaluation to suit their specific purposes. While there is adequate room for comments, the veterinarian is not required to make an overall judgment of whether the bull has normal capacity to sire calves under natural mating management, but ensures the standards for each selected category are met. Professional, standardised, easy-to-read reports are produced either electronically [Entwistle KW, Fordyce G. Evaluating and reporting bull fertility. Eight Mile Plains, Australia: Australian Cattle Vets; 2003.] or manually. A bull owner or their agent signs the certificate to affirm that bulls have not undergone procedures to rectify faults which may have otherwise caused them to fail the standards. An accreditation system for assessing sperm morphology was established because of its demonstrated relationship with pregnancy rates and because of the difficulties in achieving consistent and accurate assessments among laboratories. It is considered that Bull Reporter is applicable to beef and dairy bulls across all levels of management, genotypes and environments throughout Australia, with substantial potential for application elsewhere in the world.     

Molecular Diversity of nifH Genes from Bacteria Associated with High Arctic Dwarf Shrubs

Biological nitrogen fixation is the primary source of new N in terrestrial arctic ecosystems and is fundamental to the long-term productivity of arctic plant communities. Still, relatively little is known about the nitrogen-fixing microbes that inhabit the soils of many dominant vegetation types. Our objective was to determine which diazotrophs are associated with three common, woody, perennial plants in an arctic glacial lowland. Dryas integrifolia, Salix arctica, and Cassiope tetragona plants in soil were collected at Alexandra Fiord, Ellesmere Island, Canada. DNA was extracted from soil and root samples and a 383-bp fragment of the nifH gene amplified by the polymerase chain reaction. Cloned genotypes were screened for similarity by restriction fragment length polymorphism (RFLP) analysis. Nine primary RFLP phylotypes were identified and 42 representative genotypes selected for sequencing. Majority of sequences (33) were type I nitrogenases, whereas the remaining sequences belonged to the divergent, homologous, type IV group. Within the type I nitrogenases, nifH genes from posited members of the Firmicutes were most abundant, and occurred in root and soil samples from all three plant species. nifH genes from posited Pseudomonads were found to be more closely associated with C. tetragona, whereas nifH genes from putative alpha-Proteobacteria were more commonly associated with D. integrifolia and S. arctica. In addition, 12 clones likely representing a unique clade within the type I nitrogenases were identified. To our knowledge, this study is the first to report on the nifH diversity of arctic plant-associated soil microbes.     

Chelator-induced dispersal and killing of Pseudomonas aeruginosa cells in a biofilm

Biofilms consist of groups of bacteria attached to surfaces and encased in a hydrated polymeric matrix. Bacteria in biofilms are more resistant to the immune system and to antibiotics than their free-living planktonic counterparts. Thus, biofilm-related infections are persistent and often show recurrent symptoms. The metal chelator EDTA is known to have activity against biofilms of gram-positive bacteria such as Staphylococcus aureus. EDTA can also kill planktonic cells of Proteobacteria like Pseudomonas aeruginosa. In this study we demonstrate that EDTA is a potent P. aeruginosa biofilm disrupter. In Tris buffer, EDTA treatment of P. aeruginosa biofilms results in 1,000-fold greater killing than treatment with the P. aeruginosa antibiotic gentamicin. Furthermore, a combination of EDTA and gentamicin results in complete killing of biofilm cells. P. aeruginosa biofilms can form structured mushroom-like entities when grown under flow on a glass surface. Time lapse confocal scanning laser microscopy shows that EDTA causes a dispersal of P. aeruginosa cells from biofilms and killing of biofilm cells within the mushroom-like structures. An examination of the influence of several divalent cations on the antibiofilm activity of EDTA indicates that magnesium, calcium, and iron protect P. aeruginosa biofilms against EDTA treatment. Our results are consistent with a mechanism whereby EDTA causes detachment and killing of biofilm cells.     

The kinetics of Theileria parva infection and lymphocyte transformation in vitro

Theileriaparva is an intracellular protozoan parasite that causes a fatal lymphoproliferative disease of cattle known as East Coast Fever. The parasite infects host lymphocytes causing their transformation and uncontrolled proliferation. Infiltration of major organs with parasitized lymphoblasts results in most cases in death within 3 weeks. Although both T and B lymphocytes are susceptible to infection, the majority of cell lines arising from infection of peripheral blood mononuclear cells in vitro are of T cell lineage. To explore the basis of this phenotypic bias we have followed the very early stages of parasite development in vitro at the single cell level. Peripheral blood mononuclear cells were infected and stained for both surface phenotype and intracellular parasite antigen and analysed by flow cytometry. Although the parasite antigen was detected intracellularly as early as 6h p.i., our data indicate that parasite infection does not lead to cell transformation in all instances. Rather, specific cell types appear to undergo selection very early after infection and expansion of particular cell subsets results in survival and growth of only a small proportion of the cells originally parasitized.     

Tyr-95 and Ile-172 in transmembrane segments 1 and 3 of human serotonin transporters interact to establish high affinity recognition of antidepressants

In previous studies examining the structural determinants of antidepressant and substrate recognition by serotonin transporters (SERTs), we identified Tyr-95 in transmembrane segment 1 (TM1) of human SERT as a major determinant of binding for several antagonists, including racemic citalopram ((RS)-CIT). Here we described a separate site in hSERT TM3 (Ile-172) that impacts (RS)-CIT recognition when switched to the corresponding Drosophila SERT residue (I172M). The hSERT I172M mutant displays a marked loss of inhibitor potency for multiple inhibitors such as (RS)-CIT, clomipramine, RTI-55, fluoxetine, cocaine, nisoxetine, mazindol, and nomifensine, whereas recognition of substrates, including serotonin and 3,4-methylenedioxymethamphetamine, is unaffected. Selectivity for antagonist interactions is evident with this substitution because the potencies of the antidepressants tianeptine and paroxetine are unchanged. Reduced cocaine analog recognition was verified in photoaffinity labeling studies using [(125)I]MFZ 2-24. In contrast to the I172M substitution, other substitutions at this position significantly affected substrate recognition and/or transport activity. Additionally, the mouse mutation (mSERT I172M) exhibits similar selective changes in inhibitor potency. Unlike hSERT or mSERT, analogous substitutions in mouse dopamine transporter (V152M) or human norepinephrine transporter (V148M) result in transporters that bind substrate but are deficient in the subsequent translocation of the substrate. A double mutant hSERT Y95F/I172M had a synergistic impact on (RS)-CIT recognition ( approximately 10,000-fold decrease in (RS)-CIT potency) in the context of normal serotonin recognition. The less active enantiomer (R)-CIT responded to the I172M substitution like (S)-CIT but was relatively insensitive to the Y95F substitution and did not display a synergistic loss at Y95F/I172M. An hSERT mutant with single cysteine substitutions in TM1 and TM3 resulted in formation of a high affinity cadmium metal coordination site, suggesting proximity of these domains in the tertiary structure of SERT. These studies provided evidence for distinct binding sites coordinating SERT antagonists and revealed a close interaction between TM1 and TM3 differentially targeted by stereoisomers of CIT.     

Antibiotic inducibility of the MexXY multidrug efflux system of Pseudomonas aeruginosa: involvement of the antibiotic-inducible PA5471 gene product

The MexXY components of the MexXY-OprM multidrug efflux system of Pseudomonas aeruginosa are encoded by a MexZ repressor-regulated operon that is inducible by antibiotics that target the ribosome. Mutant strains disrupted in a gene, PA5471, were shown to be compromised for drug-inducible mexXY expression and, therefore, MexXY-OprM-mediated antimicrobial resistance. The PA5471 gene was inducible by the same ribosome-targeting agents that induce mexXY expression. Moreover, vector-driven expression of cloned PA5471 was sufficient to promote mexXY expression and MexXY-mediated resistance in the absence of antibiotic exposure, consistent with PA5471 directly or indirectly activating mexXY expression following its own upregulation in response to antibiotics. The requirement for PA5471 for mexXY expression and antimicrobial resistance was, however, obviated in mutants lacking the MexZ repressor of mexXY expression, suggesting that PA5471 directly or indirectly modulates MexZ activity in effecting mexXY expression. While the recruitment of PA5471 and MexXY in response to ribosome disruption by antimicrobials is consistent with their genes playing a role in protecting cells from the adverse consequences of disrupting the translation process, reminiscent of trans-translation, these genes appear to operate independently in their contribution to resistance: mutants defective in trans-translation showed a much more modest (twofold) decrease in resistance to ribosome-targeting agents than those lacking PA5471 or MexXY, and this decrease was observed whether functional PA5471/MexXY was present or not.     

Distinct properties of the five UDP-D-glucose/UDP-D-galactose 4-epimerase isoforms of Arabidopsis thaliana

Plant genomes contain genetically encoded isoforms of most nucleotide sugar interconversion enzymes. Here we show that Arabidopsis thaliana has five genes encoding functional UDP-D-glucose/UDP-D-galactose 4-epimerase (named UGE1 to UGE5). All A. thaliana UDP-d-glucose 4-epimerase isoforms are dimeric in solution, maximally active in vitro at 30-40 degrees C, and show good activity between pH 7 and pH 9. In vitro, UGE1, -3, and -5 act independently of externally added NAD+, whereas cofactor addition stimulates the activity of UGE2 and is particularly important for UGE4 activity. UGE1 and UGE3 are most efficiently inhibited by UDP. The five isoforms display kcatUDP-Gal values between 23 and 128 s(-1) and KmUDP-Gal values between 0.1 and 0.3 mm. This results in enzymatic efficiencies ranging between 97 and 890 mm(-1) s(-1) for UGE4 = UGE1 < UGE3 < UGE5 < UGE2. The KmUDP-Glc values, derived from the Haldane relationship, were 0.76 mm for UGE1, 0.56 mm for UGE4, and between 0.13 and 0.23 mm for UGE2, -3, and -5. The expression of UGE isoforms is ubiquitous and displays developmental and cell type-dependent variations. UGE1 and -3 expression patterns globally resemble enzymes involved in carbohydrate catabolism, and UGE2, -4, and -5 expression is more related to carbohydrate biosynthesis. UGE1, -2, and -4 are present in the cytoplasm, whereasUGE4 is additionally enriched close to Golgi stacks. All UGE genes tested complement the UGE4rhd1 phenotype, confer increased galactose tolerance in planta, and complement the galactose metabolization deficiency in the Saccharomyces cerevisiae gal10 mutant. We suggest that plant UGE isoforms function in different metabolic situations and that enzymatic properties, gene expression pattern, and subcellular localization contribute to the differentiation of isoform function.