Spatial variability in carbon- and nitrogen-related traits in apple trees: the effects of the light environment and crop load.

Photosynthetic carbon assimilation rates are highly dependent on environmental factors such as light availability and on metabolic limitations such as the demand for carbon by sink organs. The relative effects of light and sink demand on photosynthesis in perennial plants such as trees remain poorly characterized. The aim of the present study was therefore to characterize the relationships between light and fruit load on a range of leaf traits including photosynthesis, non-structural carbohydrate content, leaf structure, and nitrogen-related variables in fruiting ('ON') and non-fruiting ('OFF') 'Golden Delicious' apple trees. We show that crop status (at the tree scale) exerts a greater influence over leaf traits than the local light environment or the local fruit load. High rates of photosynthesis were observed in the ON trees. This was correlated with a high leaf nitrogen content. In contrast, little spatial variability in photosynthesis rates was observed in the OFF trees. The lack of variation in photosynthesis rates was associated with high leaf non-structural carbohydrate content at the tree level. Taken together, these results suggest that low carbon demand leads to feedback limitation on photosynthesis resulting in a low level of within-tree variability. These findings provide new insights into carbon and nitrogen allocations within trees, which are heavily dependent on carbon demand.

Non-structural carbohydrates in woody plants compared among laboratories.

Non-structural carbohydrates (NSC) in plant tissue are frequently quantified to make inferences about plant responses to environmental conditions. Laboratories publishing estimates of NSC of woody plants use many different methods to evaluate NSC. We asked whether NSC estimates in the recent literature could be quantitatively compared among studies. We also asked whether any differences among laboratories were related to the extraction and quantification methods used to determine starch and sugar concentrations. These questions were addressed by sending sub-samples collected from five woody plant tissues, which varied in NSC content and chemical composition, to 29 laboratories. Each laboratory analyzed the samples with their laboratory-specific protocols, based on recent publications, to determine concentrations of soluble sugars, starch and their sum, total NSC. Laboratory estimates differed substantially for all samples. For example, estimates for Eucalyptus globulus leaves (EGL) varied from 23 to 116 (mean = 56) mg g(-1) for soluble sugars, 6-533 (mean = 94) mg g(-1) for starch and 53-649 (mean = 153) mg g(-1) for total NSC. Mixed model analysis of variance showed that much of the variability among laboratories was unrelated to the categories we used for extraction and quantification methods (method category R(2) = 0.05-0.12 for soluble sugars, 0.10-0.33 for starch and 0.01-0.09 for total NSC). For EGL, the difference between the highest and lowest least squares means for categories in the mixed model analysis was 33 mg g(-1) for total NSC, compared with the range of laboratory estimates of 596 mg g(-1). Laboratories were reasonably consistent in their ranks of estimates among tissues for starch (r = 0.41-0.91), but less so for total NSC (r = 0.45-0.84) and soluble sugars (r = 0.11-0.83). Our results show that NSC estimates for woody plant tissues cannot be compared among laboratories. The relative changes in NSC between treatments measured within a laboratory may be comparable within and between laboratories, especially for starch. To obtain comparable NSC estimates, we suggest that users can either adopt the reference method given in this publication, or report estimates for a portion of samples using the reference method, and report estimates for a standard reference material. Researchers interested in NSC estimates should work to identify and adopt standard methods.

Synthesis, biological activity, and evaluation of the mode of action of novel antitubercular benzofurobenzopyrans substituted on A ring.

The 8-, 9-, 10-, and 11-halo, hydroxy, and methoxy derivatives of the antimycobacterial 3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran were synthesized by condensation of the diazonium salts of 2-chloroanilines (13-17) with 1,4-benzoquinone (18), reduction of the intermediate phenylbenzoquinones 19-22 to dihydroxybiphenyls, cyclisation to halo-2-hydroxydibenzofurans 24-27, and construction of the pyran ring by thermal rearrangement of the corresponding dimethylpropargyl ethers 35-38. Palladium catalyzed nucleophilic aromatic substitution permitted conversion of the halo to the corresponding hydroxy derivatives which were methylated to methoxy-3,3-dimethyl-3H-benzofuro[3,2-f][1]benzopyran. All compounds substituted on the A ring were found more potent than the reference compound 1 against Mycobacterium bovis BCG and the virulent strain Mycobacterium tuberculosis H37Rv. The effect of the most active derivatives on mycolate synthesis was explored in order to confirm the preliminary hypothesis of an effect on mycobacterial cell wall biosynthesis. The linear 9-methoxy-2,2-dimethyl-2H-benzofuro[2,3-g][1]benzopyran (46) exhibiting a good antimycobacterial activity and devoid of cytotoxicity appeared to be the most promising compound.

Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis.

New drugs are required to counter the tuberculosis (TB) pandemic. Here, we describe the synthesis and characterization of 1,3-benzothiazin-4-ones (BTZs), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo, and in mouse models of TB. Using genetics and biochemistry, we identified the enzyme decaprenylphosphoryl-beta-d-ribose 2'-epimerase as a major BTZ target. Inhibition of this enzymatic activity abolishes the formation of decaprenylphosphoryl arabinose, a key precursor that is required for the synthesis of the cell-wall arabinans, thus provoking cell lysis and bacterial death. The most advanced compound, BTZ043, is a candidate for inclusion in combination therapies for both drug-sensitive and extensively drug-resistant TB.

Diversity-oriented synthesis of furo[3,2-f]chromanes with antimycobacterial activity.

We previously reported the synthesis and the antimycobacterial activity of 4-(7,7-dimethyl-7H-furo[3,2-f]chromen-2-yl)pyridine. From this result, we sought to design simple synthetic accesses to related structures allowing the preparation of a diverse set of analogues. Two approaches were investigated. From 3-(2-bromo-7,7-dimethyl-8,9-dihydro-7H-furo[3,2-f]chromen-1-yl)propyl acetate, we prepared 2-arylated derivatives via Suzuki-Miyaura reactions between this bromine-bearing compound and various arylboronates. Moreover, and even more simple, we prepared the ((6-hydroxy-2,2,7,8-tetramethylchroman-5-yl)methyl)triphenylphosphonium salt via a selective bromination of 2,2,5,7,8-pentamethylchroman-6-ol. From this salt, a two stage Wittig reaction with an array of activated acids allowed the quick preparation of many analogues. The biological evaluation of the effect of these compounds on the growth of Mycobacterium bovis as well as Mycobacterium tuberculosis pointed out a fourfold improvement of the antimycobacterial properties for one of the compounds made. However, the many analogues which inhibited the growth of M. tuberculosis in the 0.6-5 microg/mL range turned out to be also cytotoxic on VERO cells growth at the same concentration range.

A new synthetic access to furo[3,2-f]chromene analogues of an antimycobacterial.

From the structure of 3,3-dimethyl-3H-benzofuro[3,2-f][1]-benzopyran, a selective in vitro inhibitor of mycobacterial growth, we have undertaken a structure-activity relationship investigation. We wish to report here our results on the use of [2+3] cycloadditions between 2-formylbenzoquinone and various enol derivatives to give various 4-formyl-5-hydroxy benzofurans. In the next step, an ytterbium triflate-catalysed reaction with 2-methylpropene allowed the preparation of various original furo[3,2-f]chromenes derivatives. Their biological evaluation on the growth of Mycobacterium smegmatis as well as Mycobacterium tuberculosis pointed out that some analogues were four times more active than the initial hit.

A novel drug discovery concept for tuberculosis: inhibition of bacterial and host cell signalling.

The Mycobacterium tuberculosis genome encodes for eleven eukaryotic-like Ser/Thr protein kinases. At least three of these (PknA, PknB and PknG) are essential for bacterial growth and survival. PknG is secreted by pathogenic mycobacteria, in macrophages to intervene with host cell signalling pathways and to block the fusion of the lysosomes with the phagosome by a still unknown mechanism. Based on our previously published results, we have initiated a drug discovery program, aiming to improve the potency against PknG and the physiochemical properties of the initially identified hit compound, AX20017, from the class of the tetrahydrobenzothiophenes. We have established a radioactive biochemical PknG kinase assay to test the novel analogues around AX20017. We have developed lead molecules with IC50 values in nanomolar range, and demonstrated their antituberculotic effects on human macrophages. Selected leads might ultimately serve the purpose of inducing phagosomal-lysosomal fusion and therefore destroy the residence of the intracellular mycobacteria. It is unclear at this time if these "homeless" mycobacteria are getting killed by the host, but they will be at least vulnerable to the activity of antimycobacterial agents. Released mycobacteria rely on the essential function of PknB for survival, which is our second molecular kinase target. PknB is a transmembrane protein, responsible for the cell growth and morphology. We have screened our library and synthesized novel compounds for the inhibition of PknB. A pharmacophore model was built and 70,000 molecules from our synthesizable virtual library have been screened to identify novel inhibitor scaffolds for the generation of templated compound libraries. Currently, we are using a radioactive kinase assay employing GarA as the putative, physiological substrate of PknB kinase. We have identified hits and generated optimised hit compounds with IC50 values for the inhibition of PknB in the nanomolar range. Yet those promising hits are not potent enough to yield meaningful "minimum inhibitory concentrations" in mycobacterial growth assays. In the course of our future work, we will increase the potency of the next generation of PknB inhibitors in order to improve their antibacterial activity.

Synthesis and antimycobacterial evaluation of benzofurobenzopyran analogues.

We recently reported that 3,3-dimethyl-3H-benzofuro[3,2,f][1]-benzopyran and its hydrogenated analogue are selective in vitro inhibitors of mycobacterial growth. However, their lack of in vivo activity on a murine model of Mycobacterium tuberculosis infection due to their poor bioavailability led to a structure-activity relationship investigation. We wish to report here the preparation of some structural analogues along with their biological effect on the growth of Mycobacterium smegmatis, M. tuberculosis, as well as on VERO cells for the most active compound.

Inactivation of Rv2525c, a substrate of the twin arginine translocation (Tat) system of Mycobacterium tuberculosis, increases beta-lactam susceptibility and virulence.

The twin arginine translocation (Tat) system is used by many bacteria to export fully folded proteins containing cofactors. Here, we show genetically that this system is essential for Mycobacterium tuberculosis, as the tatAC operon and tatB genes could be inactivated only in partially diploid strains. Using comparative genomics, the rv2525c gene of M. tuberculosis was identified as encoding a histidine-rich protein, with a twin arginine signal peptide, and orthologous genes were shown to be present in several but not all actinobacterial species. Conservation of this gene by Mycobacterium leprae, which has undergone reductive evolution, suggested an important role for rv2525c. An rv2525c knockout mutant was constructed, and biochemical analysis indicated that the mature Rv2525c protein is secreted. Upon exposure to antituberculous drugs, rv2525c expression is significantly up-regulated together with those of other genes involved in cell wall biogenesis. Phenotypic comparison of the mutant with the parental strain revealed an increase in susceptibility to some beta-lactam antibiotics and, despite slower growth in vitro, enhanced virulence in both cellular and murine models of tuberculosis. The Tat system thus contributes in multiple ways to survival of the tubercle bacillus.

The Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth.

The receptor-like protein kinase PknB from Mycobacterium tuberculosis is encoded by the distal gene in a highly conserved operon, present in all actinobacteria, that may control cell shape and cell division. Genes coding for a PknB-like protein kinase are also found in many more distantly related gram-positive bacteria. Here, we report that the pknB gene can be disrupted by allelic replacement in M. tuberculosis and the saprophyte Mycobacterium smegmatis only in the presence of a second functional copy of the gene. We also demonstrate that eukaryotic Ser/Thr protein kinase inhibitors, which inactivate PknB in vitro with a 50% inhibitory concentration in the submicromolar range, are able to kill M. tuberculosis H37Rv, M. smegmatis mc(2)155, and Mycobacterium aurum A+ with MICs in the micromolar range. Furthermore, significantly higher concentrations of these compounds are required to inhibit growth of M. smegmatis strains overexpressing PknB, suggesting that this protein kinase is the molecular target. These findings demonstrate that the Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth and support the development of protein kinase inhibitors as new potential antituberculosis drugs.

Structure and mechanism of the alkyl hydroperoxidase AhpC, a key element of the Mycobacterium tuberculosis defense system against oxidative stress.

The peroxiredoxin AhpC from Mycobacterium tuberculosis (MtAhpC) is the foremost element of a NADH-dependent peroxidase and peroxynitrite reductase system, where it directly reduces peroxides and peroxynitrite and is in turn reduced by AhpD and other proteins. Overexpression of MtAhpC in isoniazid-resistant strains of M. tuberculosis harboring mutations in the catalase/peroxidase katG gene provides antioxidant protection and may substitute for the lost enzyme activities. We report here the crystal structure of oxidized MtAhpC trapped in an intermediate oligomeric state of its catalytic cycle. The overall structure folds into a ring-shaped hexamer of dimers instead of the usual pentamer of dimers observed in other reduced peroxiredoxins. Although the general structure of the functional dimer is similar to that of other 2-Cys peroxiredoxins, the alpha-helix containing the peroxidatic cysteine Cys61 undergoes a unique rigid-body movement to allow the formation of the disulfide bridge with the resolving cysteine Cys174. This conformational rearrangement creates a large internal cavity enclosing the active site, which might be exploited for the design of inhibitors that could block the catalytic cycle. Structural and mutagenesis evidence points to a model for the electron transfer pathway in MtAhpC that accounts for the unusual involvement of three cysteine residues in catalysis and suggests a mechanism by which MtAhpC can specifically interact with different redox partners.

Effect of katG mutations on the virulence of Mycobacterium tuberculosis and the implication for transmission in humans.

The usefulness of isoniazid (INH), a key component of short-course chemotherapy of tuberculosis, is threatened by the emergence of drug-resistant strains of Mycobacterium tuberculosis with mutations in the katG gene. It is shown here that the most commonly occurring KatG mutation, where Ser 315 is replaced by Thr (S315T), is associated with clinically significant levels of INH resistance. In contrast to another resistant isolate, in which Pro replaces Thr 275, the S315T mutant produces active catalase-peroxidase and is virulent in the mouse model of the disease, indicating that a significant loss of bacterial fitness does not result from this frequent mutation. The implications of this finding for the transmission and reactivation of multidrug-resistant strains of M. tuberculosis are severe.

Are the PE-PGRS proteins of Mycobacterium tuberculosis variable surface antigens?

Mycobacterium tuberculosis H37Rv contains 67 PE-PGRS genes, with multiple tandem repetitive sequences, encoding closely related proteins that are exceptionally rich in glycine and alanine. As no functional information was available, 10 of these genes were selected and shown to be expressed in vitro by reverse transcription-polymerase chain reaction (RT-PCR). Antibodies against five PE-PGRS proteins, raised in mice by DNA vaccination, detected single proteins when the same plasmid constructs used for immunization were expressed in epithelial cells or in reticulocyte extracts, confirming that the PE-PGRS proteins are antigenic. As expected from the conserved repetitive structure, the antibodies cross-reacted with more than one PE-PGRS protein, suggesting that different proteins share common epitopes. PE-PGRS proteins were detected by West-ern blotting in five different mycobacterial species (M. tuberculosis, M. bovis BCG, M. smegmatis, M. marinum and M. gordonae) and 11 clinical isolates of M. tuberculosis. Whole-genome comparisons of M. tuberculosis predicted allelic diversity in the PE-PGRS family, and this was confirmed by immunoblot studies as size variants were detected in clinical strains. Subcellular fractionation studies and immunoelectron microscopy localized many PE-PGRS proteins in the cell wall and cell membrane of M. tuberculosis. The data suggest that some PE-PGRS proteins are variable surface antigens.