Pantothenate Kinase from the Thermoacidophilic Archaeon Picrophilus torridus

Pantothenate kinase (CoaA) catalyzes the first step of the coenzyme A (CoA) biosynthetic pathway and controls the intracellular concentrations of CoA through feedback inhibition in bacteria. An alternative enzyme found in archaea, pantoate kinase, is missing in the order Thermoplasmatales. The PTO0232 gene from Picrophilus torridus, a thermoacidophilic euryarchaeon, is shown to be a distant homologue of the prokaryotic type I CoaA. The cloned gene clearly complements the poor growth of the temperature-sensitive Escherichia coli CoaA mutant strain ts9, and the recombinant protein expressed in E. coli cells transfers phosphate to pantothenate at pH 5 and 55°C. In contrast to E. coli CoaA, the P. torridus enzyme is refractory to feedback regulation by CoA, indicating that in P. torridus cells the CoA levels are not regulated by the CoaA step. These data suggest the existence of two subtypes within the class of prokaryotic type I CoaAs.Coenzyme A (CoA) is an essential cofactor synthesized from pantothenate (vitamin B₅), cysteine, and ATP (1, 20, 30). The thiol group derived from the cysteine moiety in a CoA molecule forms a thioester bond, which is a high-energy bond, with carboxylates including fatty acids. The resulting compounds are called acyl-CoAs (CoA thioesters) and function as the major acyl group carriers in numerous metabolic and energy-yielding pathways. Since it is thought that the pantetheine moiety in CoA existed when life first came about on Earth (25) and at present, a CoA, acyl-CoA, or 4′-phosphopantethein moiety that is common to CoA and acyl carrier proteins is utilized by about 4% of all enzymes as a substrate (6), these compounds are thought to play a crucial role in the earliest metabolic system.Bacteria, fungi, and plants can produce pantothenate, which is the starting material of CoA biosynthesis, although animals must take it from their diet (41). The canonical CoA biosynthetic pathway consists of five enzymatic steps: i.e., pantothenate kinase (CoaA in prokaryotes and PanK in eukaryotes; EC 2.7.1.33), phosphopantothenoylcysteine synthetase (CoaB; EC 6.3.2.5), phosphopantothenoylcysteine decarboxylase (CoaC: EC 4.1.1.36), phosphopantetheine adenylyltransferase (CoaD; EC 2.7.7.3), and dephospho-CoA kinase (CoaE; EC 2.7.1.24). The organisms belonging to the domains Bacteria and Eukarya have this pathway (20, 30). CoaB, CoaC, CoaD, and CoaE are detectable in the complete genome sequences as orthologs of the counterparts from E. coli and humans (15, 16, 32). However, there is diversity among the CoaAs and PanKs, depending on their primary structures, and to date, three types of CoaA in bacteria and one type of PanK in eukaryotes have been identified. CoaAs and PanK catalyze the phosphorylation of pantothenate to produce 4′-phosphopantothenate at the first step of the pathway. First, the Escherichia coli CoaA (CoaA_Ec) was cloned as a prokaryotic type I CoaA after characterization of the properties enzymatically (42-44, 48). Thereafter, the eukaryotic PanK isoforms were isolated from Aspergillus nidulans (AnPanK), mice (mPanK), and humans (hPanK) (10, 17, 28, 29, 33, 34, 54-56). These enzyme activities were clearly regulated by end products of the biosynthetic pathway such as CoA, acetyl-CoA, and malonyl-CoA, and the pantothenate kinases governed the intracellular concentrations of CoA and acyl-CoAs (10, 17, 28, 29, 33, 34, 43, 44, 48, 54, 55). However, CoaAs insensitive to CoA and acyl-CoAs were recently identified from Staphylococcus aureus (CoaA_Sa), Pseudomonas aeruginosa (CoaA_Pa), and Helicobacter pylori (CoaA_Hp) as prokaryotic type II and III CoaAs (9, 11, 18, 27). The structural and functional diversity among pantothenate kinases suggests that they are key indicators of the regulation of the CoA biosynthesis. In archaea neither CoaA nor pantothenate synthetase (PanC; EC 6.3.2.1), which catalyzes the condensation of pantoate and β-alanine to produce pantothenate, had been identified biochemically until very recently. COG1829 and COG1701 were assigned as the respective candidates based on comparative genomic analysis (15). COG1701 was reported to be PanC (36), and later the enzyme was revised to phosphopantothenate synthetase, which catalyzed the condensation of phosphopantoate and β-alanine (52). Together with the identification of COG1701, COG1829 was found to be pantoate kinase, responsible for the phosphorylation of pantoate (52). Homologues of pantoate kinase and phosphopantothenate synthetase are found in most archaeal genomes, thus establishing a noncanonical CoA biosynthetic pathway involving the two novel enzymes. However, homologues of the two novel enzymes are missing in the order Thermoplasmatales.Hence, we proceeded with a search for the kinase genes of the remaining archaea to elucidate the regulatory mechanism(s) underlying archaeal CoA biosynthesis. The PTO0232 gene in the complete genome sequence of Picrophilus torridus was identified as encoding a distant homologue of CoaA_Ec by a BLAST search. The recombinant protein phosphorylated pantothenate, but the activity was not inhibited at all by CoA or CoA thioesters despite its classification as prokaryotic type I CoaA. This functional difference between P. torridus CoaA (CoaA_Pt) and CoaA_Ec can be accounted for by an amino acid substitution at position 247 which possibly interacts with CoA. Here we describe the existence of a second subtype in the class of prokaryotic type I CoaAs.     

Analysis of the Relationships between Growth, Photosynthesis and Carbohydrate Metabolism Using Quantitative Trait Loci (QTLs) in Young Maize Plants Subjected to Water Deprivation

One hundred to 120 maize recombinant inbred lines at the mature fourth leaf stage derived from F-2 and Io parental lines were grown in a glasshouse and were deprived of water for 9 days in order to detect pertinent markers of the physiological response to water stress which may be used for breeding. Carbohydrate metabolism QTLs were compared to photosynthesis gas exchange QTLs. The locations of these QTLs were further compared with those of morphological trait QTLs when water availability varied. The traits ranged from three enzyme activities (invertase, sucrose-P synthase, ADP glucose pyrophosphorylase) and hexose, sucrose, starch content to CO₂ uptake and stomatal conductance, water status, leaf size, root/shoot ratio, and ABA (leaf, root and xylem sap). Four main results were obtained (1) only 14 % of QTLs were common to both drought and watered treatments, confirming the existence of stress specific chromosome regions, (2) the QTLs tended to form clusters, frequently consisting of QTLs from different classes (growth, photosynthesis, water status, carbohydrate metabolism and ABA), (3) carbohydrate metabolism trait QTLs were more frequently co-located with growth trait QTLs than photosynthesis related ones, especially in control conditions, (4) one co-location was observed between the three enzyme activities implied in sucrose and starch metabolism and a corresponding structural gene, which can be considered as a candidate gene for explaining part of the variability of each enzymatic trait (invertase, sucrose-P synthase, ADPglucose pyrophosphorylase). It is concluded that, carbohydrate metabolism provides valuable traits for understanding and improving maize responses to water stress.     

Toxicity and mode of action of Bacillus thuringiensis Cry proteins in the Mediterranean corn borer, Sesamia nonagrioides (Lefebvre)

Sesamia nonagrioides is one of the most damaging pests of corn in Spain and other Mediterranean countries. Bt corn expressing the Bacillus thuringiensis Cry1Ab toxin is being grown on about 58,000 ha in Spain. Here we studied the mode of action of this Cry protein on S. nonagrioides (binding to specific receptors, stability of binding, and pore formation) and the modes of action of other Cry proteins that were found to be active in this work (Cry1Ac, Cry1Ca, and Cry1Fa). Binding assays were performed with (125)I- or biotin-labeled toxins and larval brush border membrane vesicles (BBMV). Competition experiments indicated that these toxins bind specifically and that Cry1Aa, Cry1Ab, and Cry1Ac share a binding site. Cry1Ca and Cry1Fa bind to different sites. In addition, Cry1Fa binds to Cry1A's binding site with very low affinity and vice versa. Binding of Cry1Ab and Cry1Ac was found to be stable over time, which indicates that the observed binding is irreversible. The pore-forming activity of Cry proteins on BBMV was determined using the voltage-sensitive fluorescent dye DiSC(3)(5). Membrane permeability increased in the presence of the active toxins Cry1Ab and Cry1Fa but not in the presence of the nonactive toxin Cry1Da. In terms of resistance management, based on our results and the fact that Cry1Ca is not toxic to Ostrinia nubilalis, we recommend pyramiding of Cry1Ab with Cry1Fa in the same Bt corn plant for better long-term control of corn borers.     

Role of Autochthonous Filamentous Fungi in Bioremediation of a Soil Historically Contaminated with Aromatic Hydrocarbons

Nine fungal strains isolated from an aged and heavily contaminated soil were identified and screened to assess their degradative potential. Among them, Allescheriella sp. strain DABAC 1, Stachybotrys sp. strain DABAC 3, and Phlebia sp. strain DABAC 9 were selected for remediation trials on the basis of Poly R-478 decolorization associated with lignin-modifying enzyme (LME) production. These autochthonous fungi were tested for the abilities to grow under nonsterile conditions and to degrade various aromatic hydrocarbons in the same contaminated soil. After 30 days, fungal colonization was clearly visible and was confirmed by ergosterol determination. In spite of subalkaline pH conditions and the presence of heavy metals, the autochthonous fungi produced laccase and Mn and lignin peroxidases. No LME activities were detected in control microcosms. All of the isolates led to a marked removal of naphthalene, dichloroaniline isomers, o-hydroxybiphenyl, and 1,1′-binaphthalene. Stachybotrys sp. strain DABAC 3 was the most effective isolate due to its ability to partially deplete the predominant contaminants 9,10-anthracenedione and 7H-benz[DE]anthracen-7-one. A release of chloride ions was observed in soil treated with either Allescheriella sp. strain DABAC 1 or Stachybotrys sp. strain DABAC 3, suggesting the occurrence of oxidative dehalogenation. The autochthonous fungi led to a significant decrease in soil toxicity, as assessed by both the Lepidium sativum L. germination test and the Collembola mortality test.     

Time-Series Evolution of Toxic Organisms and Related Environmental Factors in a Brackish Ecosystem of the Mediterranean Sea

In the framework of the EU Project STRATEGY, a short-term study was carried out in the Marinello ecosystem, a small brackish area located on the Tyrrhenian coast of Sicily (Italy). The investigation was aimed at understanding the dynamics of phytoplankton toxic blooms in relation to other planktonic species and environmental conditions. The study started on 10 March 2003, in coincidence with the first detection of Alexandrium minutum, a dinoflagellate known as a producer of Paralyzing Shellfish Toxins (PST) and lasted until 4 June 2003, when the bloom collapsed. The specific identity of A. minutum was confirmed on field mixed samples, through the use of species-specific PCR-primers targeting the 5.8S rDNA-ITS regions. Water samples and phytoplankton net hauls were taken approximately at 10 days intervals in the Verde Pond, one of the five basins of the Marinello ecosystem, in order to evaluate the incidence of toxic and non-toxic dinoflagellate species over the whole planktonic community. The evolution of the main environmental and trophic parameters (temperature, salinity, dissolved oxygen, POC, C/N, DIN, PO₄–P) was simultaneously investigated. Alexandrium blooms were mostly characterized by A. minutum (max. 6 × 10⁵ cells l⁻¹ on April 11) and Alexandrium tamarense as an associated species (max. 2.5 × 10⁴ cells l⁻¹ on March 25). During the bloom, dinoflagellates or small flagellates dominated over the other taxa, with a minimum incidence of diatoms. The load of dissolved inorganic nitrogen was maximum in the pre-bloom phase (29 μM on March 19), after which it decreased sharply. An oxygen supersaturation event was registered in coincidence with the A. minutum bloom. The amounts of POC ranged between 266 and 658 μg l⁻¹ showing a discontinuous temporal trend. A recent introduction of A. minutum into the Verde Pond is suggested on the basis of the absence of this species in past years.     

Cytokine gene polymorphisms and atopic disease in two European cohorts. (ECRHS-Basel and SAPALDIA)

Background

Avoidance of allergens is still recommended as the first and best way to prevent allergic illnesses and their comorbid diseases. Despite a variety of attempts there has been very limited success in the area of environmental control of allergic disease. Our objective was to identify a non-invasive, non-pharmacological method to reduce indoor allergen loads in atopic persons' homes and public environments. We employed a novel in vivo approach to examine the possibility of using aluminum sulfate to control environmental allergens.

Methods

Fifty skin test reactive patients were simultaneously skin tested with conventional test materials and the actions of the protein/glycoprotein modifier, aluminum sulfate. Common allergens, dog, cat, dust mite, Alternaria, and cockroach were used in the study.

Results

Skin test reactivity was significantly reduced by the modifier aluminum sulfate. Our studies demonstrate that the effects of histamine were not affected by the presence of aluminum sulfate. In fact, skin test reactivity was reduced independent of whether aluminum sulfate was present in the allergen test material or removed prior to testing, indicating that the allergens had in some way been inactivated.

Conclusion

Aluminum sulfate was found to reduce the in vivo allergic reaction cascade induced by skin testing with common allergens. The exact mechanism is not clear but appears to involve the alteration of IgE-binding epitopes on the allergen. Our results indicate that it may be possible to diminish the allergenicity of an environment by application of the active agent aluminum sulfate, thus producing environmental control without complete removal of the allergen.     

Prostaglandin F2α promotes ovulation in prepubertal heifers

The objective was to determine the effects of exogenous prostaglandin F_2α (PGF), with or without progesterone treatment, on first ovulation in prepubertal heifers. We tested the hypothesis that PGF has a luteolysis-independent ovulatory effect in cattle. Crossbred Angus heifers (12 to 14 mo old, 250 kg body weight, and an average body condition score of 3 out of 5) were examined by transrectal ultrasonography on two occasions, 11 days apart. Heifers in which a CL was not detected at either examination were considered prepubertal. Heifers were assigned randomly to three experimental groups: (1) PG group (N = 14); heifers were treated with a PGF analog (500 μg cloprostenol im) 5 days after the emergence of a spontaneous (i.e., naturally occurring, noninduced) follicular wave; (2) PPG group (N = 12); heifers were given an intravaginal progesterone-releasing insert (CIDR; Pfizer Animal Health, Montreal, QC, Canada), and a follicular wave was induced with 50 mg of progesterone + 2 mg of estradiol benzoate im, and a PGF analog was given at the time of CIDR removal, on Day 5 of the follicular wave (on average, 8.6 ± 0.5 days after CIDR insertion); and (3) control group heifers were given no treatment (N = 14). Heifers were examined daily by transrectal ultrasonography from the start of the experiment to confirmation that ovulation had occurred, or to 5 days after PGF injection (PG and PPG groups) or until dominant follicles of the next follicular wave reached 8 mm (control group). The percentage of heifers that ovulated within 10 days after wave emergence was higher in PPG (10/12; 83.3%) and PG (11/14; 78.5%) groups than in control (1/14; 7.1%; P < 0.0001). Ovulations occurred 69.6 ± 6 h and 93.8 ± 5 h after PGF treatment in PPG and in PG groups, respectively, whereas only one heifer in the control group ovulated 96 h after Day 5 of follicular wave (P = 0.13). In summary, PGF treatment was associated with ovulation in prepubertal heifers whether or not exogenous progesterone was used as a pretreatment. The hypothesis that PGF will induce ovulation by a luteolysis-independent mechanism was supported.     

Systems analysis of primary Sjögren's syndrome pathogenesis in salivary glands identifies shared pathways in human and a mouse model

Bone tissue has an exceptional quality to regenerate to native tissue in response to injury. However, the fracture repair process requires mechanical stability or a viable biological microenvironment or both to ensure successful healing to native tissue. An improved understanding of the molecular and cellular events that occur during bone repair and remodeling has led to the development of biologic agents that can augment the biological microenvironment and enhance bone repair. Orthobiologics, including stem cells, osteoinductive growth factors, osteoconductive matrices, and anabolic agents, are available clinically for accelerating fracture repair and treatment of compromised bone repair situations like delayed unions and nonunions. Preclinical and clinical studies using biologic agents like recombinant bone morphogenetic proteins have demonstrated an efficacy similar or better than that of autologous bone graft in acute fracture healing. A lack of standardized outcome measures for comparison of biologic agents in clinical fracture repair trials, frequent off-label use, and a limited understanding of the biological activity of these agents at the bone repair site have limited their efficacy in clinical applications.     

Acyl-sulfamates target the essential glycerol-phosphate acyltransferase (PlsY) in Gram-positive bacteria

PlsY is the essential first step in membrane phospholipid synthesis of Gram-positive pathogens. PlsY catalyzes the transfer of the fatty acid from acyl-phosphate to the 1-position of glycerol-3-phosphate to form the first intermediate in membrane biogenesis. A series of non-metabolizable, acyl-sulfamate analogs of the acyl-phosphate PlsY substrate were prepared and evaluated as inhibitors of Staphylococcus aureus PlsY and for their Gram-positive antibacterial activities. From this series phenyl (8-phenyloctanoyl) sulfamate had the best overall profile, selectively inhibiting S. aureus phospholipid biosynthesis and causing the accumulation of both long-chain fatty acids and acyl-acyl carrier protein intermediates demonstrating that PlsY was the primary cellular target. Bacillus anthracis was unique in being more potently inhibited by long chain acyl-sulfamates than other bacterial species. However, it is shown that Bacillus anthracis PlsY is not more sensitive to the acyl-sulfamates than S. aureus PlsY. Metabolic profiling showed that B. anthracis growth inhibition by the acyl-sulfamates was not specific for lipid synthesis illustrating that the amphipathic acyl-sulfamates can also have off-target effects in Gram-positive bacteria. Nonetheless, this study further advances PlsY as a druggable target for the development of novel antibacterial therapeutics, through the discovery and validation of the probe compound phenyl (8-phenyloctanoyl) sulfamate as a S. aureus PlsY inhibitor.     

Spatial patterns of red fox (Vulpes vulpes) dens in a semi-arid landscape of North Africa

Different patterns of the use of space by red foxes ( Vulpes vulpes ) depend mainly on the distribution and availability of food patches. Nevertheless, other key resources such as suitable areas for dens can also influence the territory size and social organization of this predator. In fact, landscape modifications such as habitat patchiness made by human activities (agricultural practices, urbanization) should create ample unfavourable zones for den settlement. Several studies focused on the distribution and use of dens by the red fox in different habitat types but we do not have any data on the den settlement and habitat composition in the semi-arid regions of North Africa. This study was carried out at Djerba island (SE Tunisia), virtually deprived of surface water and with a semi-arid climate because of the bordering Sahara desert. We used line and random transects to find fox dens. The spatial pattern of dens varied mainly in different habitat types and geoclimatic regions. Highly fragmented areas were also occupied by foxes that built dens close together inside small suitable patches. Fox dens had fewer entrances in more arid regions and they mainly faced the south. Habitat selection was influenced by water availability and irrigated tree plantations that modify soil textures allowing digging of dens. Moreover, asphalt roads limit den settlements. We suggest that the choice of denning sites by foxes depends on persisting harsh conditions and human activities.