Characterization of an oxidoreductase from the arylamine N-acetyltransferase operon in Mycobacterium smegmatis

Mycobacterium?tuberculosis, the most successful bacterial pathogen, causes tuberculosis, a disease that still causes more than 2 million deaths per year. Arylamine N-acetyltransferase is an enzyme that is conserved in most Mycobacterium spp. The nat gene belongs to an operon that is important for the intracellular survival of M. tuberculosis within macrophages. The nat operon in Mycobacterium smegmatis and other fast-growing mycobacterial species has a unique organization containing genes with uncharacterized function. Here, we describe the biochemical, biophysical and structural characterization of the MSMEG_0308 gene product (MS0308) of the M. smegmatis nat operon. While characterizing the function of MS0308, we validated the oxidoreductase property; however, we found that the enzyme was not utilizing dihydrofolate as its substrate, hence we first report that MS0308 is not a dihydrofolate reductase, as annotated in the genome. The structure of this oxidoreductase was solved at 2.0 ? in complex with the cofactor NADPH and has revealed the hydrophobic pocket where the endogenous substrate binds.     

PolarCath cryoplasty enhances smooth muscle cell apoptosis in a rabbit iliac artery model: an experimental in vivo controlled study

Purpose

To in vivo investigate the histological response after single and double cryoplasty therapy in a rabbit iliac artery model.

Materials and methods

In total, 40 New Zealand White rabbits underwent percutaneous transluminal angioplasty of the iliac artery using either PolarCath balloon or a conventional angioplasty balloon of equal diameter. Arterial injury, inflammatory response and smooth muscle cells (SMC) apoptosis with the TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) immunohistochemical assay were analyzed. Rabbits were divided between single or double balloon inflation and histological results were compared between cryoplasty and control angioplasty both at 30 min and 72 h.

Results

Arterial injury and wall inflammation scores were low and similar between cryoplasty and control groups after single and double balloon inflation. Compared to conventional balloon angioplasty, Polarcath cryoplasty demonstrated superior SMC apoptosis after single inflation at 30 min [12.0 ± 1.2 cells/(0.025 mm)2 vs 7.0 ± 1.5 cells/(0.025 mm)², p = 0.002], single inflation at 72 h [9.0 ± 1.0 cells/(0.025 mm)² vs 5.4 ± 1.4 cells/(0.025 mm)², p = 0.001], double inflation at 30 min [11.6 ± 1.5 cells/(0.025 mm)² vs 6.8 ± 1.4 cells/(0.025 mm)², p = 0.001] and double inflation at 72 h [9.2 ± 0.8 cells/(0.025 mm)² vs 5.0 ± 0.7 cells/(0.025 mm)², p = 0.001]. There were no significant differences in apoptosis between single and double cryoplasty application at 30 min and 72 h.

Conclusion

Cryoplasty demonstrated superior rates of SMC apoptosis at 30 min and 72 h and was associated to relatively low arterial injury and inflammation scores. An immediate second PolarCath inflation did not achieve superior apoptosis.     

Oral nicotinamide riboside raises NAD+ and lowers biomarkers of neurodegenerative pathology in plasma extracellular vesicles enriched for neuronal origin

Declining nicotinamide adenine dinucleotide (NAD⁺) concentration in the brain during aging contributes to metabolic and cellular dysfunction and is implicated in the pathogenesis of aging-associated neurological disorders. Experimental therapies aimed at boosting brain NAD⁺ levels normalize several neurodegenerative phenotypes in animal models, motivating their clinical translation. Dietary intake of NAD⁺ precursors, such as nicotinamide riboside (NR), is a safe and effective avenue for augmenting NAD⁺ levels in peripheral tissues in humans, yet evidence supporting their ability to raise NAD⁺ levels in the brain or engage neurodegenerative disease pathways is lacking. Here, we studied biomarkers in plasma extracellular vesicles enriched for neuronal origin (NEVs) from 22 healthy older adults who participated in a randomized, placebo-controlled crossover trial (NCT02921659) of oral NR supplementation (500 mg, 2x /day, 6 weeks). We demonstrate that oral NR supplementation increases NAD⁺ levels in NEVs and decreases NEV levels of Aβ42, pJNK, and pERK1/2 (kinases involved in insulin resistance and neuroinflammatory pathways). In addition, changes in NAD(H) correlated with changes in canonical insulin–Akt signaling proteins and changes in pERK1/2 and pJNK. These findings support the ability of orally administered NR to augment neuronal NAD⁺ levels and modify biomarkers related to neurodegenerative pathology in humans. Furthermore, NEVs offer a new blood-based window into monitoring the physiologic response of NR in the brain.     

Turnover-based in vitro selection and evolution of biocatalysts from a fully synthetic antibody library

This report describes the selection of highly efficient antibody catalysts by combining chemical selection from a synthetic library with directed in vitro protein evolution. Evolution started from a naive antibody library displayed on phage made from fully synthetic, antibody-encoding genes (the Human Combinatorial Antibody Library; HuCAL-scFv). HuCAL-scFv was screened by direct selection for catalytic antibodies exhibiting phosphatase turnover. The substrate used was an aryl phosphate, which is spontaneously transformed into an electrophilic trapping reagent after cleavage. Chemical selection identified an efficient biocatalyst that then served as a template for error-prone PCR (epPCR) to generate randomized repertoires that were subjected to further selection cycles. The resulting superior catalysts displayed cumulative mutations throughout the protein sequence; the ten-fold improvement of their catalytic proficiencies (>10(10) M(-1)) resulted from increased kcat values, thus demonstrating direct selection for turnover. The strategy described here makes the search for new catalysts independent of the immune system and the antibody framework.     

A thermostable beta-ketothiolase of polyhydroxyalkanoates (PHAs) in Thermus thermophilus: purification and biochemical properties

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates (HAs) synthesised by numerous bacteria as intracellular carbon and energy storage compounds which accumulate as granules in the cytoplasm of the cells. The biosynthesis of PHAs, in the thermophilic bacterium T. thermophilus grown in a mineral medium supplemented with sodium gluconate as sole carbon source has been recently reported. Here, we report the purification at apparent homogeneity of a beta-ketoacyl-CoA thiolase from T. thermophilus, the first enzyme of the most common biosynthetic pathway for PHAs. B-Ketoacyl-CoA thiolase appeared as a single band of 45.5-kDa molecular mass on SDS/PAGE. The enzyme was purified 390-fold with 7% recovery. The native enzyme is a multimeric protein of a molecular mass of approximately of 182 kDa consisting of four identical subunits of 45.5 kDa, as identified by an in situ renaturation experiment on SDS-PAGE. The enzyme exhibited an optimal pH of approximately 8.0 and highest activity at 65 degrees C for both direction of the reaction. The thiolysis reaction showed a substrate inhibition at high concentrations; when one of the substrates (acetoacetyl CoA or CoA) is varied, while the concentrations of the second substrates (CoA or acetoacetyl CoA respectively) remain constant. The initial velocity kinetics showed a pattern of a family of parallel lines, which is in accordance with a ping-pong mechanism. beta-Ketothiolase had a relative low Km of 0.25 mM for acetyl-CoA and 11 microM and 25 microM for CoA and acetoacetyl-CoA, respectively. The enzyme was inhibited by treatment with 1 mM N-ethylmaleimide either in the presence or in the absence of 0.5 mM of acetyl-CoA suggesting that possibly a cysteine is located at/or near the active site of beta-ketothiolase.     

Deacylated tRNA is released from the E site upon A site occupation but before GTP is hydrolyzed by EF-Tu

The presence or absence of deacylated tRNA at the E site sharply influences the activation energy required for binding of a ternary complex to the ribosomal A site indicating the different conformations that the E-tRNA imparts on the ribosome. Here we address two questions: (i) whether or not peptidyltransferase—the essential catalytic activity of the large ribosomal subunit—also depends on the occupancy state of the E site and (ii) at what stage the E-tRNA is released during an elongation cycle. Kinetics of the puromycin reaction on various functional states of the ribosome indicate that the A-site substrate of the peptidyltransferase center, puromycin, requires the same activation energy for peptide-bond formation under all conditions tested. We further demonstrate that deacylated tRNA is released from the E site by binding a ternary complex aminoacyl-tRNA•EF-Tu•GDPNP to the A site. This observation indicates that the E-tRNA is released after the decoding step but before both GTP hydrolysis by EF-Tu and accommodation of the A-tRNA. Collectively these results reveal that the reciprocal linkage between the E and A sites affects the decoding center on the 30S subunit, but does not influence the rate of peptide-bond formation at the active center of the 50S subunit.     

Essential oil composition is related to the natural habitats: Coridothymus capitatus and Satureja thymbra in NATURA 2000 sites of Crete

The study of essential oils obtained from Coridothymus capitatus and Satureja thymbra collected from different natural habitat types of 11 NATURA 2000 sites scattered all over Crete has shown that they are characterized either by a high amount of carvacrol (up to 75.7%) or thymol (up to 65.6%) or by a more or less equal amount of the two phenols. The results of a discriminant analysis with predefined groups the natural habitat types wherefrom the plants were collected have shown that the oils of both species collected from the dry dwarf-shrub formations of the lowland have a high carvacrol content whereas those collected from the more mesic timber or highland formations have a high thymol content. Furthermore, the results of this study introduce the use of natural habitat unit as a tool for the assessment of essential oil variation.     

A peptide carrier with a built-in vaccine adjuvant: construction of immunogenic conjugates

A multifunctional carrier combining B/T cell epitopes (i), a built-in vaccine adjuvant (ii), and a universal T cell epitope (iii) for the construction of potent and specific immunogenic conjugates is presented. The IL-1beta(163-171) fragment known to reproduce the immunostimulatory and adjuvant effects of the whole IL-1beta without possessing any of the pro-inflammatory properties of IL-1beta was covalently anchored to the N-terminus of the Sequential Oligopeptide Carrier, SOC(n), formed by the repeating tripeptide unit Lys-Aib-Gly. A promiscuous T cell epitope derived from the tetanus toxin, TT(593-599), was also positioned in the carboxy terminus of SOC(n) as a universal immunogen to provide broad immunogenicity. Selected B/T cell epitopes from the Sm and La/SSB autoantigens, against which is directed the humoral autoimmunity in patients with systemic lupus erythematosus and Sj?gren's Syndrome, respectively, were coupled to the Lys-N(epsilon)H2 groups of the carrier, and the formulated constructs were administered in animals following the conventional immunization protocol of complete/incomplete Freund's adjuvant. The induced immune responses were compared with that produced when the Sm- and La/SSB-reconstituted immunogenic conjugates were injected alone. High titers of specific antibodies recognizing the priming construct, as well as the cognate autoantigen, were obtained when administered alone without the assistance of Freund's adjuvant. It is concluded that our approach provides the conceptual and experimental framework for the development of multifunctional immunogenic conjugates eliciting enhanced, specific, and prolonged humoral response for usage as human vaccine candidates.     

PI3Kgamma inhibition blocks glomerulonephritis and extends lifespan in a mouse model of systemic lupus

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease generated by deregulation of T cell-mediated B-cell activation, which results in glomerulonephritis and renal failure. Disease is treated with immunosuppressants and cytostatic agents that have numerous side effects. Here we examine the use of inhibitors of phosphoinositide 3-kinase (PI3K) gamma, a lipid kinase that regulates inflammation, in the MRL-lpr mouse model of SLE. Treatment reduced glomerulonephritis and prolonged lifespan, suggesting that P13Kgamma may be a useful target in the treatment of chronic inflammation.