Terpenoid metabolism in plastids : sites of phytoene synthetase activity and synthesis in plant cells

The biosynthesis of phytoene from prephytoene pyrophosphate has been localized exclusively in the plastid compartment of ruptured protoplasts derived from Triticum leaves and Capsicum fruits.

The phytoene synthetase activity in Triticum leaves deficient in plastid ribosomes was comparable to those obtained in normal leaves. In addition, the stimulation of phytoene synthetase activity observed in green Capsicum fruit after 2-(4-chlorophenylthio)triethylamine hydrochloride treatment was not abolished by chlororamphenicol and lincomycin, in contrast to the inhibition observed after cycloheximide treatment.

These data conclusively show that phytoene synthetase is localized exclusively in the plastid compartment in higher plants and that its synthesis is not performed on 70S ribosomes.

     

Identification of the sugars involved in mycobacterial cell aggregation

Abstract Incubation of Mycobacterium tuberculosis and M. smegmatis cells with the sugar components of their surface-exposed glycans demonstrated that d-arabinose, but not α-d-glucose or d-mannose, led to the dispersion of the large clumps formed by the bacilli in stationary liquid cultures. These results confirm the presence of arabinose-containing glycans on the mycobacterial cell surface and demonstrate the implication of selective sugars in cell aggregation, suggesting that the clumping of mycobacterial cells is probably mediated by lectin-carbohydrate interactions.     

Identification of the first eubacterial endonuclease coded by an intein allele in the pps1 gene of mycobacteria

A survey of a vast range of mycobacterial strains led us to discover a new Pps1 intein allele in Mycobacterium gastri which differs from those of Mycobacterium tuberculosis and Mycobacterium leprae in both its sequence and insertion site. While little is known about Pps1, except that it belongs to the YC24 family of ABC transporters, we show that, unlike the other inteins described so far from Eubacteria, the MgaPps1 intein possesses a specific endonuclease activity. The intein is the first eubacterial intein to be characterised as an endonuclease. Like other intein endonucleases, its minimal sequence for recognition and cleavage is quite large, with 22 bp spanning the Pps1-c site. The fact that an active endonuclease is found among the mycobacterial inteins supports the concept of a cyclical model of invasion by horizontal transfer of these genes, followed by degeneration and loss until a new invasion event, thus explaining their long-term persistence in closely related eubacterial species.     

Plastid enzymes of terpenoid biosynthesis. Purification and characterization of gamma-tocopherol methyltransferase from Capsicum chromoplasts

gamma-Tocopherol methyltransferase was solubilized and purified from Capsicum chromoplast membranes by a combination of standard fractionation techniques. The purified enzyme was electrophoretically homogeneous, and its molecular weight, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 33,000. In the absence of detergent, the enzyme formed high molecular weight aggregates. Several properties of the enzyme have been determined. The Km values were 2.5 and 13.7 microM for S-adenosylmethionine and gamma-tocopherol, respectively. The enzyme was able to transfer the methyl group S-adenosylmethionine to N-4-azido-2-nitrophenyl-beta-alanyl-gamma-tocopherol. The rate of transfer was less efficient compared to gamma-tocopherol. In the presence of ultraviolet light, this analog inhibited the gamma-tocopherol methyltransferase activity.     

Segmental duplication implicated in the genesis of inversion 2Rj of Anopheles gambiae

The malaria vector Anopheles gambiae maintains high levels of inversion polymorphism that facilitate its exploitation of diverse ecological settings across tropical Africa. Molecular characterization of inversion breakpoints is a first step toward understanding the processes that generate and maintain inversions. Here we focused on inversion 2Rj because of its association with the assortatively mating Bamako chromosomal form of An. gambiae, whose distinctive breeding sites are rock pools beside the Niger River in Mali and Guinea. Sequence and computational analysis of 2Rj revealed the same 14.6 kb insertion between both breakpoints, which occurred near but not within predicted genes. Each insertion consists of 5.3 kb terminal inverted repeat arms separated by a 4 kb spacer. The insertions lack coding capacity, and are comprised of degraded remnants of repetitive sequences including class I and II transposable elements. Because of their large size and patchwork composition, and as no other instances of these insertions were identified in the An. gambiae genome, they do not appear to be transposable elements. The 14.6 kb modules inserted at both 2Rj breakpoint junctions represent low copy repeats (LCRs, also called segmental duplications) that are strongly implicated in the recent (approximately 0.4N(e) generations) origin of 2Rj. The LCRs contribute to further genome instability, as demonstrated by an imprecise excision event at the proximal breakpoint of 2Rj in field isolates.     

A Common Mechanism of Inhibition of the Mycobacterium tuberculosis Mycolic Acid Biosynthetic Pathway by Isoxyl and Thiacetazone

Isoxyl (ISO) and thiacetazone (TAC), two prodrugs once used in the clinical treatment of tuberculosis, have long been thought to abolish Mycobacterium tuberculosis (M. tuberculosis) growth through the inhibition of mycolic acid biosynthesis, but their respective targets in this pathway have remained elusive. Here we show that treating M. tuberculosis with ISO or TAC results in both cases in the accumulation of 3-hydroxy C₁₈, C₂₀, and C₂₂ fatty acids, suggestive of an inhibition of the dehydratase step of the fatty-acid synthase type II elongation cycle. Consistently, overexpression of the essential hadABC genes encoding the (3R)-hydroxyacyl-acyl carrier protein dehydratases resulted in more than a 16- and 80-fold increase in the resistance of M. tuberculosis to ISO and TAC, respectively. A missense mutation in the hadA gene of spontaneous ISO- and TAC-resistant mutants was sufficient to confer upon M. tuberculosis high level resistance to both drugs. Other mutations found in hypersusceptible or resistant M. tuberculosis and Mycobacterium kansasii isolates mapped to hadC. Mutations affecting the non-essential mycolic acid methyltransferases MmaA4 and MmaA2 were also found in M. tuberculosis spontaneous ISO- and TAC-resistant mutants. That MmaA4, at least, participates in the activation of the two prodrugs as proposed earlier is not supported by our biochemical evidence. Instead and in light of the known interactions of both MmaA4 and MmaA2 with HadAB and HadBC, we propose that mutations affecting these enzymes may impact the binding of ISO and TAC to the dehydratases.     

Modulation of lung allergic response by renal ischemia and reperfusion injury

The Th1/Th2 balance represents an important factor in the pathogenesis of renal ischemia-reperfusion injury (IRI). In addition, IRI causes a systemic inflammation that can affect other tissues, such as the lungs. To investigate the ability of renal IRI to modulate pulmonary function in a specific model of allergic inflammation, C57Bl/6 mice were immunized with ovalbumin/albumen on days 0 and 7 and challenged with an ovalbumin (OA) aerosol on days 14 and 21. After 24 h of the second antigen challenge, the animals were subjected to 45 minutes of ischemia. After 24 h of reperfusion, the bronchoalveolar lavage (BAL) fluid, blood and lung tissue were collected for analysis. Serum creatinine levels increased in both allergic and non-immunized animals subjected to IRI. However, BAL analysis showed a reduction in the total cells (46%) and neutrophils (58%) compared with control allergic animals not submitted to IRI. In addition, OA challenge induced the phosphorylation of ERK and Akt and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lung homogenates. After renal IRI, the phosphorylation of ERK and expression of COX-2 and iNOS were markedly reduced; however, there was no difference in the phosphorylation of Akt between sham and ischemic OA-challenged animals. Mucus production was also reduced in allergic mice after renal IRI. IL-4, IL-5 and IL-13 were markedly down-regulated in immunized/challenged mice subjected to IRI. These results suggest that renal IRI can modulate lung allergic inflammation, probably by altering the Th1/Th2 balance and, at least in part, by changing cellular signal transduction factors.