The impact of nitric oxide toxicity on the evolution of the glutathione transferase superfamily: A proposal for an evolutionary driving force

Background: Why do ancestral GSTs utilize cysteine/serine as catalytic residues, whereas more recently evolved GSTs utilize tyrosine? Results: Only the more recently evolved GSTs display enough affinity to bind and make harmless the toxic DNDGIC (a natur

Pyrazines from bacteria and ants: convergent chemistry within an ecological niche

Ants use pheromones to coordinate their communal activity. Volatile pyrazines, for instance, mediate food resource gathering and alarm behaviors in different ant species. Here we report that leaf-cutter ant-associated bacteria produce a family of pyrazines that includes members previously identified as ant trail and alarm pheromones. We found that L-threonine induces the bacterial production of the trail pheromone pyrazines, which are common for the host leaf-cutter ants. Isotope feeding experiments revealed that L-threonine along with sodium acetate were the biosynthetic precursors of these natural products and a biosynthetic pathway was proposed

Selvamicin, an atypical antifungal polyene from two alternative genomic contexts

The bacteria harbored by fungus-growing ants produce a variety of small molecules that help maintain a complex multilateral symbiosis. In a survey of antifungal compounds from these bacteria, we discovered selvamicin, an unusual antifungal polyene macrolide, in bacterial isolates from two neighboring ant nests. Selvamicin resembles the clinically important antifungals nystatin A1 and amphotericin B, but it has several distinctive structural features: a noncationic 6-deoxymannose sugar at the canonical glycosylation site and a second sugar, an unusual 4-O-methyldigitoxose, at the opposite end of selvamicin’s shortened polyene macrolide. It also lacks some of the pharmacokinetic liabilities of the clinical agents and appears to have a different target. Whole genome sequencing revealed the putative type I polyketide gene cluster responsible for selvamicin’s biosynthesis including a subcluster of genes consistent with selvamicin’s 4-O-methyldigitoxose sugar. Although the selvamicin biosynthetic cluster is virtually identical in both bacterial producers, in one it is on the chromosome, in the other it is on a plasmid. These alternative genomic contexts illustrate the biosynthetic gene cluster mobility that underlies the diversity and distribution of chemical defenses by the specialized bacteria in this multilateral symbiosis.National Institutes of Health/[R01 GM086258]/NIH/Estados UnidosNational Institutes of Health/[U19 AI09673]/NIH/Estados UnidosUniversidad de Costa Rica//UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM

Genome Sequence of Streptomyces sp. Strain RTd22, an Endophyte of the Mexican Sunflower

We report here the complete genome sequence of Streptomyces sp. strain RTd22, an endophytic actinobacterium that was isolated from the roots of the Mexican sunflower Tithonia diversifolia. The bacterium’s 11.1-Mb linear chromosome is predicted to encode a large number of unknown natural products

The Roman Bridge: a "double pulley – suture bridges" technique for rotator cuff repair

<p>Abstract</p> <p>Background</p> <p>With advances in arthroscopic surgery, many techniques have been developed to increase the tendon-bone contact area, reconstituting a more anatomic configuration of the rotator cuff footprint and providing a better environment for tendon healing.</p> <p>Methods</p> <p>We present an arthroscopic rotator cuff repair technique which uses suture bridges to optimize rotator cuff tendon-footprint contact area and mean pressure.</p> <p>Results</p> <p>Two medial row 5.5-mm Bio-Corkscrew suture anchors (Arthrex, Naples, FL), which are double-loaded with No. 2 FiberWire sutures (Arthrex, Naples, FL), are placed in the medial aspect of the footprint. Two suture limbs from a single suture are both passed through a single point in the rotator cuff. This is performed for both anchors. The medial row sutures are tied using the double pulley technique. A suture limb is retrieved from each of the medial anchors through the lateral portal, and manually tied as a six-throw surgeon's knot over a metal rod. The two free suture limbs are pulled to transport the knot over the top of the tendon bridge. Then the two free suture limbs that were used to pull the knot down are tied. The end of the sutures are cut. The same double pulley technique is repeated for the other two suture limbs from the two medial anchors, but the two free suture limbs are used to produce suture bridges over the tendon, by means of a Pushlock (Arthrex, Naples, FL), placed 1 cm distal to the lateral edge of the footprint.</p> <p>Conclusion</p> <p>This technique maximizes the advantages of two techniques. On the one hand, the double pulley technique provides an extremely secure fixation in the medial aspect of the footprint. On the other hand, the suture bridges allow to improve pressurized contact area and mean footprint pressure. In this way, the bony footprint in not compromised by the distal-lateral fixation, and it is thus possible to share the load between fixation points. This maximizes the strength of the repair and provides a barrier preventing penetration of synovial fluid into the healing area of tendon and bone.</p

M1 and M2 macrophage recruitment during tendon regeneration induced by amniotic epithelial cell allotransplantation in ovine

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Investigating the catalytic mechanism of the meta-cleavage product hydrolases

The meta-cleavage product (MCP) hydrolases are members of the α/β-hydrolase superfamily that utilize a Ser-His-Asp triad to catalyze the hydrolysis of a C-C bond. The catalytic mechanism of the MCP hydrolases is poorly defined and particularly interesting due to a requisite substrate ketonization that precedes hydrolysis. To resolve the catalytic mechanism of the MCP hydrolases, two enzymes were studied: tetrameric BphDLB400 from Burkholderia xenovorans LB400 and dimeric DxnB2 from Sphingomonas witichii RW1. Both efficiently hydrolyze 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to produce 2-hydroxypenta-2,4-dienoic acid (HPD) and benzoic acid. A series of experiments established that BphDLB400 uses an histidine-independent nucleophilic mechanism of catalysis and is half-site reactive. Benzoylation of Ser112 was demonstrated by LC ESI/MS/MS analyses and a pre-steady-state kinetic burst of HPD formation indicated the reactivity. While acylation during HOPDA hydrolysis by BphDLB400 occurred on a similar timescale for the WT and H265Q variant, esterase activity was abrogated in the histidine variant. Thus, alternative mechanisms of nucleophile activation are employed for C-C and C-O bond cleavage. A covalent mechanism of catalysis was inferred for DxnB2, however, the turnover of HOPDA was 1:1 with respect to enzyme concentration. A solvent kinetic isotope effect suggested that a proton transfer, and therefore, substrate ketonization determines the rate of acylation in the MCP hydrolases. Substrate ketonization, and therefore acylation, can be indirectly observed as consumption of ESred, an intermediate named for its bathochromically-shifted absorption spectrum. A proton transfer to ESred allowed the assignment of this species to an enzyme-bound HOPDA dianion. An extended Brønsted analysis revealed a linear correlation between substrate basicity and the rate constant determined for the ketonization reaction. Finally, the MCP hydrolase P-subsite, which contacts the MCP dienoate moiety, was definitively linked to substrate ketonization. In DxnB2 Asn43 and Arg180 variants, ESred formation was found to limit this proton transfer reaction. A substrate-assisted nucleophilic mechanism of catalysis has been proposed for the MCP hydrolases. Therein, the electron-rich dienoate moiety substitutes for the His-Asp pair as the general base for nucleophile activation. Overall, definition of the chemical mechanism of the MCP hydrolases has implications for environmental bioremediation strategies and the rational design of therapeutics.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat