Cloning and characterization of the phosphatidylserine synthase gene of Agrobacterium sp. strain ATCC 31749 and effect of its inactivation on production of high-molecular-mass (1-->3)-beta-D-glucan (curdlan)

Genes involved in the production of the extracellular (1-->3)-beta-glucan, curdlan, by Agrobacterium sp. strain ATCC 31749 were described previously (Stasinopoulos et al., Glycobiology 9:31-41, 1999). To identify additional curdlan-related genes whose protein products occur in the cell envelope, the transposon TnphoA was used as a specific genetic probe. One mutant was unable to produce high-molecular-mass curdlan when a previously uncharacterized gene, pss(AG), encoding a 30-kDa, membrane-associated phosphatidylserine synthase was disrupted. The membranes of the mutant lacked phosphatidylethanolamine (PE), whereas the phosphatidylcholine (PC) content was unchanged and that of both phosphatidylglycerol and cardiolipin was increased. In the mutant, the continued appearance of PC revealed that its production by this Agrobacterium strain is not solely dependent on PE in a pathway controlled by the Pss(AG) protein at its first step. Moreover, PC can be produced in a medium lacking choline. When the pss(AG)::TnphoA mutation was complemented by the intact pss(AG) gene, both the curdlan deficiency and the phospholipid profile were restored to wild-type, demonstrating a functional relationship between these two characteristics. The effect of the changed phospholipid profile could occur through an alteration in the overall charge distribution on the membrane or a specific requirement for PE for the folding into or maintenance of an active conformation of any or all of the structural proteins involved in curdlan production or transport.     

The Arabidopsis sku6/spiral1 gene encodes a plus end-localized microtubule-interacting protein involved in directional cell expansion

The sku6-1 mutant of Arabidopsis thaliana exhibits altered patterns of root and organ growth. sku6 roots, etiolated hypocotyls, and leaf petioles exhibit right-handed axial twisting, and root growth on inclined agar media is strongly right skewed. The touch-dependent sku6 root skewing phenotype is suppressed by the antimicrotubule drugs propyzamide and oryzalin, and right skewing is exacerbated by cold treatment. Cloning revealed that sku6-1 is allelic to spiral1-1 (spr1-1). However, modifiers in the Columbia (Col) and Landsberg erecta (Ler) ecotype backgrounds mask noncomplementation in sku6-1 (Col)/spr1-1 (Ler) F1 plants. The SPR1 gene encodes a plant-specific 12-kD protein that is ubiquitously expressed and belongs to a six-member gene family in Arabidopsis. An SPR1:green fluorescent protein (GFP) fusion expressed in transgenic seedlings localized to microtubules within the cortical array, preprophase band, phragmoplast, and mitotic spindle. SPR1:GFP was concentrated at the growing ends of cortical microtubules and was dependent on polymer growth state; the microtubule-related fluorescence dissipated upon polymer shortening. The protein has a repeated motif at both ends, separated by a predicted rod-like domain, suggesting that it may act as an intermolecular linker. These observations suggest that SPR1 is involved in microtubule polymerization dynamics and/or guidance, which in turn influences touch-induced directional cell expansion and axial twisting.     

Chaotic mixer improves microarray hybridization

Hybridization is an important aspect of microarray experimental design which influences array signal levels and the repeatability of data within an array and across different arrays. Current methods typically require 24h and use target inefficiently. In these studies, we compare hybridization signals obtained in conventional static hybridization, which depends on diffusional target delivery, with signals obtained in a dynamic hybridization chamber, which employs a fluid mixer based on chaotic advection theory to deliver targets across a conventional glass slide array. Microarrays were printed with a pattern of 102 identical probe spots containing a 65-mer oligonucleotide capture probe. Hybridization of a 725-bp fluorescently labeled target was used to measure average target hybridization levels, local signal-to-noise ratios, and array hybridization uniformity. Dynamic hybridization for 1h with 1 or 10ng of target DNA increased hybridization signal intensities approximately threefold over a 24-h static hybridization. Similarly, a 10- or 60-min dynamic hybridization of 10ng of target DNA increased hybridization signal intensities fourfold over a 24h static hybridization. In time course studies, static hybridization reached a maximum within 8 to 12h using either 1 or 10ng of target. In time course studies using the dynamic hybridization chamber, hybridization using 1ng of target increased to a maximum at 4h and that using 10ng of target did not vary over the time points tested. In comparison to static hybridization, dynamic hybridization reduced the signal-to-noise ratios threefold and reduced spot-to-spot variation twofold. Therefore, we conclude that dynamic hybridization based on a chaotic mixer design improves both the speed of hybridization and the maximum level of hybridization while increasing signal-to-noise ratios and reducing spot-to-spot variation.     

Kinetic and stability properties of Penicillium chrysogenum ATP sulfurylase missing the C-terminal regulatory domain

ATP sulfurylase from Penicillium chrysogenum is a homohexameric enzyme that is subject to allosteric inhibition by 3'-phosphoadenosine 5'-phosphosulfate. In contrast to the wild type enzyme, recombinant ATP sulfurylase lacking the C-terminal allosteric domain was monomeric and noncooperative. All kcat values were decreased (the adenosine 5'-phosphosulfate (adenylylsulfate) (APS) synthesis reaction to 17% of the wild type value). Additionally, the Michaelis constants for MgATP and sulfate (or molybdate), the dissociation constant of E.APS, and the monovalent oxyanion dissociation constants of dead end E.MgATP.oxyanion complexes were all increased. APS release (the k6 step) was rate-limiting in the wild type enzyme. Without the C-terminal domain, the composite k5 step (isomerization of the central complex and MgPPi release) became rate-limiting. The cumulative results indicate that besides (a) serving as a receptor for the allosteric inhibitor, the C-terminal domain (b) stabilizes the hexameric structure and indirectly, individual subunits. Additionally, (c) the domain interacts with and perfects the catalytic site such that one or more steps following the formation of the binary E.MgATP and E.SO4(2-) complexes and preceding the release of MgPPi are optimized. The more negative entropy of activation of the truncated enzyme for APS synthesis is consistent with a role of the C-terminal domain in promoting the effective orientation of MgATP and sulfate at the active site.     

Sex, drugs and recombination: the wild life of Aspergillus

Throughout the eukaryotes, sexual reproduction is an almost universal phenomenon. However, within the Kingdom Fungi, this relationship is not so clear‐cut. Fungi exhibit a spectrum of reproductive modes and life‐cycles; amongst the better known species, sexual reproduction is often facultative, can be rare, and in over half of the known Ascomycota (the moulds) is unknown ( Taylor et al. 1999 ). However, over the last decade, it has become apparent that many of these asexual mitosporic taxa undergo cryptic recombination via unobserved mechanisms and that wholly asexual fungi are, in fact, a rarity ( Taylor et al. 1999, 2001 ; Heitman 2010 ). This revolution in our understanding of fungal sexuality has come about in two ways: Firstly, sexual reproduction leaves an imprint on fungal genomes by maintaining genes required for mating and by generating patterns of mutation and recombination restricted to meiotic processes. Secondly, scientists have become better at catching fungi in flagrante delicto. The genus Aspergillus is one such fungus where a combination of population genetics, genomics and taxonomy has been able to intuit the existence of sex, then to catch the fungus in the act and formally describe their sexual stages. So, why are sexy moulds exciting? One species in particular, Aspergillus flavus, is notorious for its ability to produce a diverse array of secondary metabolites, of which the polyketide aflatoxins (AF) are carcinogenic and others (such as cyclopiazonic acid) are toxigenic. Because of the predilection of A. flavus to grow on crops, such as peanuts, corn and cotton, biocontrol is widely used to mitigate infection by pre‐applying nonaflatoxigenic (AF?) strains to competitively exclude the wild‐type AF+ strains. However, the eventual fate in nature of these biocontrol strains is not known. In this issue of Molecular Ecology, Olarte et al. (2012) make an important contribution by using laboratory crosses of A. flavus to show that not only is AF highly heritable, but AF? strains can become AF+ via crossing over during meiosis. This observation has raised the spectre of cross‐breeding and non‐mendelian inheritance of AF between native and biocontrol strains of the fungus, leading to an increase in the natural diversity of the fungus with perhaps unanticipated consequences.     

Synthesis and SAR of benzyl and phenoxymethylene oxadiazole benzenesulfonamides as selective beta3 adrenergic receptor agonist antiobesity agents

Benzyl and phenoxymethylene substituted oxadiazoles are potent and orally bioavailable beta3 adrenergic receptor (AR) agonists. The 4-trifluormethoxy substituted 5-benzyl oxadiazole 5f has an EC50 of 8 nM in the beta3 AR agonist assay with 100-fold selectivity over beta1 and beta2 AR binding inhibition activity. Its oral bioavailability in dogs is 30 +/- 4%, with a half-life of 3.8 +/- 0.4 h. In the anesthetized rhesus, 5f evoked a dose-dependent glycerolemia (ED50Gly = 0.15 mg/kg). Under these conditions a heart rate increase of 15% was observed at a dose level of 10 mg/kg.     

UV and melanoma: the TP53 link

Ultraviolet radiation (UVR) is a major risk factor for melanoma development, but it has been unclear exactly how UVR leads to melanomagenesis. In a recent publication in Nature, Viros et al. identify TP53/Trp53 as a UVR-target gene in melanoma and show that UVR-induced TP53/Trp53 mutations accelerate BRAF(V600E)-driven melanomagenesis.Melanoma is the deadliest skin cancer, and its incidence has relentlessly increased over recent decades. According to the American Cancer Society''s estimates for melanoma in the United States for 2014, about 76 100 new melanomas will be diagnosed and about 9 710 people are expected to die from melanoma. It is well known that UVR is the major environmental factor contributing to melanomagenesis¹. This suggests that there is at least a component of melanoma risk which may be preventable through UVR protective strategies — an issue of immense public health importance due to the availability of sunblocks and sun-safe behaviors. Importantly, while many studies have been conducted to elucidate the link between UVR and melanoma, the precise molecular mechanism(s) by which UVR triggers melanoma formation have remained incompletely understood.Recently, a powerful UVR-induced skin inflammatory response has been shown to provoke metastasis of melanoma² and the presence of UV signature mutations has also been reported throughout the melanoma exome, including recurrent melanoma genes such as RAC1, PPP6C, and STK19^3,4. Previous mouse models for UVR-induced melanoma revealed that UVR-induced inflammation promoted melanomagenesis in neonatal mice^5,6,7. These studies underlined UVR''s significant contribution to melanoma formation. In a recent study published in Nature, Viros et al.⁸ address the role of UVR in previously established BRAF(V600E)-expressing melanocytes in vivo, and demonstrate that significantly accelerated melanoma formation often associated with mutations in TP53/Trp53. To mimic both somatic mutation acquisition and mild sunburn in humans, BRAF(V600E) was expressed at physiological levels in adult mice which were subsequently exposed to repeated low doses of UVR. In addition, certain mice were partially covered with UVR-proof cloth or topically treated with SunSense Milk Sunscreen SPF50 (2.2 mg/cm²) 30 min before UVR exposure, to assess the impact of these protective strategies.UVR was seen to significantly accelerate melanoma formation in mice whose melanocytes express BRAF(V600E), but not in BRAF wild-type mice (which unlike BRAF(V600E)-expressing mice do not develop long latency melanomas independently of UVR). Application of UVR-proof cloth or sunscreen delayed the onset of UVR-driven melanoma and partially prevented acceleration of BRAF(V600E)-driven melanomagenesis by UVR, and sunscreen-protected UVR-exposed BRAF(V600E) mice developed a reduced number of melanomas compared with unprotected UVR-exposed BRAF(V600E) mice (Figure 1). More somatic single nucleotide variants and a significantly higher proportion of C-to-T transitions at the 3′ end of pyrimidine dimers were observed in UVR-exposed melanomas, providing direct evidence of UVR-induced DNA damage. In addition, Trp53 mutations (H39Y, S124F, R245C, R270C, C272G) were detected in UVR-exposed BRAF(V600E) mouse melanomas, indicating a direct role of UVR in the induction of Trp53 mutations in melanoma. The mutated corresponding residues (S127F/S124F, R248C/R245C, R273C/R270C, C275G/C272G) were also identified in TP53 mutations in human melanoma, suggesting that TP53 mutations are linked to evidence of UVR-induced DNA damage in human melanoma. These results are consistent with previous reports that p53 deletion accelerates BRAF(V600E)-driven melanomagenesis both in mice⁹ and in zebrafish¹⁰, but demonstrate the ability of UVR to inflict UV signature mutations within the gene as has been widely observed in non-melanoma skin cancers and also in human melanomas.Open in a separate windowFigure 1A diagram depicting feasible routes of BRAF(V600E)-driven melanomagenesis.This elegant study by Viros et al. clearly helps to establish key roles of UVR in melanomagenesis, and further validates the functional importance of TP53 as a UVR-targeted tumor suppressor gene in a fraction of melanomas. The study also raises several intriguing questions worthy of follow-up analysis. For example, through which mechanism(s) did sunscreen or sunshielding delay but not prevent UVR-induced melanoma? Induction of cutaneous inflammatory changes that are less anatomically restricted to UV irradiated fields, would seem to be an attractive mechanism. This may help to explain the known risk of melanoma in both sun-exposed and less-exposed skin of lightly pigmented people. It is also valuable to better understand the role of UVB vs UVA wavelengths in melanomagenesis. Mechanistically, these distinct regions of the UV spectrum inflict largely distinctive chemical alterations on the genome. Efforts to block UVA as well as UVB in commercial sunscreen products are currently being promoted by the US Food and Drug Administration, a welcome improvement to sun protection strategies. Still, the precise role(s) of UVA in melanomagenesis remain incompletely understood and may involve both cell-autonomous and non-cell-autonomous targets. In addition to the acceleration of BRAF(V600E)-driven melanoma formation by UVR, red pigment (pheomelanin) has also been observed to accelerate BRAF(V600E)-driven melanomagenesis even in the absence of UVR¹¹. Pheomelanin has been identified as an intrinsic risk factor for melanoma with the red pigment itself producing reactive oxygen species that cause DNA damage in the skin, and consequently promote melanomagenesis independently of UVR. UVR likely exacerbates red pigment-induced BRAF(V600E)-driven melanoma, and still remains as a major contributor to melanomagenesis. Therefore, along with UV shielding by sunscreens, further preventative strategies should be investigated to diminish UVR-independent melanoma risk mechanisms.Viros et al. provide intriguing answers to several controversial questions regarding melanomagenesis: Does UVR really trigger melanoma? And can sunscreen actually prevent melanoma? The studies by Viros et al. provide experimental evidence for acceleration of BRAF(V600E)-driven melanoma by UVR-induced TP53/Trp53 mutation and demonstrate that sunscreen delayed but did not completely block UVR-driven melanoma. The current study clearly shows that UVR boosts melanoma and sunscreens may provide partial UVR protection against melanoma — evidence which matches human epidemiologic data. Nevertheless, to protect the public from melanoma, Viros et al. advise that sunscreen should be utilized in combination with additional sun avoidance strategies. In addition, measures that may prevent UV-independent melanoma formation will require additional research and may also be needed in order to optimally battle the incidence of this life-threatening malignancy.     

Drainage Size, Stream Intermittency, and Ecosystem Function in a Sonoran Desert Landscape

Understanding the interactions between terrestrial and aquatic ecosystems remains an important research focus in ecology. In arid landscapes, catchments are drained by a channel continuum that represents a potentially important driver of ecological pattern and process in the surrounding terrestrial environment. To better understand the role of drainage networks in arid landscapes, we determined how stream size influences the structure and productivity of riparian vegetation, and the accumulation of organic matter (OM) in soils beneath plants in an upper Sonoran Desert basin. Canopy volume of velvet mesquite (Prosopis velutina), as well as overall plant cover, increased along lateral upland–riparian gradients, and among riparian zones adjacent to increasingly larger streams. Foliar δ¹³C signatures for P. velutina suggested that landscape patterns in vegetation structure reflect increases in water availability along this arid stream continuum. Leaf litter and annual grass biomass production both increased with canopy volume, and total aboveground litter production ranged from 137 g m⁻² y⁻¹ in upland habitat to 446 g m⁻² y⁻¹ in the riparian zone of the perennial stream. OM accumulation in soils beneath P. velutina increased with canopy volume across a broad range of drainage sizes; however, in the riparian zone of larger streams, flooding further modified patterns of OM storage. Drainage networks represent important determinants of vegetation structure and function in upper Sonoran Desert basins, and the extent to which streams act as sources of plant-available water and/or agents of fluvial disturbance has implications for material storage in arid soils.