In vitro reconstruction of the biosynthetic pathway of peptidoglycan cytoplasmic precursor in Pseudomonas aeruginosa

Bacterial peptidoglycan is the cell wall component responsible for maintaining cell integrity against osmotic pressure. Biosynthesis of the cytoplasmic precursor UDP-N-acetylmuramyl pentapeptide is catalyzed by the Mur enzymes. Genomic analysis of the three regions encoding Mur proteins was achieved. We have cloned and over-expressed the murA, -B, -D, -E and -F genes of Pseudomonas aeruginosa in pET expression system by adding a His-Tag to the C-termini of the proteins. Mur proteins were purified to homogeneity by a single chromatographic step on affinity nickel columns. Protein identities were verified through N-terminal sequencing. Enzyme activity was proved by the identification of the pathway's final product.     

FEV1 inversely correlates with metalloproteinases 1, 7, 9 and CRP in COPD by biomass smoke exposure

Background

Matrix metalloproteinases (MMPs) and C-reactive protein (CRP) are involved in chronic obstructive pulmonary disease (COPD) pathogenesis. The aim of the present work was to determine plasma concentrations of MMPs and CRP in COPD associated to biomass combustion exposure (BE) and tobacco smoking (TS).

Methods

Pulmonary function tests, plasma levels of MMP-1, MMP-7, MMP-9, MMP-9/TIMP-1 and CRP were measured in COPD associated to BE (n = 40) and TS (n =40) patients, and healthy non-smoking (NS) healthy women (controls, n = 40).

Results

Plasma levels of MMP-1, MMP-7, MMP-9, and MMP-9/TIMP-1 and CRP were higher in BE and TS than in the NS healthy women (p <0.01). An inverse correlation between MMP-1, MMP-7, MMP-9, MMP-9/TIMP-1 and CRP plasma concentrations and FEV₁ was observed.

Conclusions

Increase of MMPs and CRP plasma concentrations in BE suggests a systemic inflammatory phenomenon similar to that observed in COPD associated to tobacco smoking, which may also play a role in COPD pathogenesis.     

Evaluation of inhibition of the carbenicillin-hydrolyzing β-lactamase PSE-4 by the clinically used mechanism-based inhibitors

Characterization of the biochemical steps in the inactivation chemistry of clavulanic acid, sulbactam and tazobactam with the carbenicillin-hydrolyzing β-lactamase PSE-4 from Pseudomonas aeruginosa is described. Although tazobactam showed the highest affinity to the enzyme, all three inactivators were excellent inhibitors for this enzyme. Transient inhibition was observed for the three inactivators before the onset of irreversible inactivation of the enzyme. Partition ratios (k_cat/k_inact) of 11, 41 and 131 were obtained with clavulanic acid, tazobactam and sulbactam, respectively. Furthermore, these values were found to be 14-fold, 3-fold and 80-fold lower, respectively, than the values obtained for the clinically important TEM-1 β-lactamase. The kinetic findings were put in perspective by determining the computational models for the pre-acylation complexes and the immediate acyl-enzyme intermediates for all three inactivators. A discussion of the pertinent structural factors is presented, with PSE-4 showing subtle differences in interactions with the three inhibitors compared to the TEM-1 enzyme.     

A Study on the Safety and Effects of Amorpha fruticosa Fruit Extract on Spontaneously Hypertensive Rats with Induced Type 2 Diabetes

Metabolic syndrome is characterized by a variety of diagnostic criteria: obesity, dyslipidemia, type 2 diabetes, and arterial hypertension. They contribute to the elevated risk of cardiovascular morbidity and mortality. The potential for Amorpha fruticosa L. (Fabaceae) to improve diabetes and metabolic disease is promising, based on in vitro tests. This is why a further investigation of the species is needed. Additionally, a toxicity review in relation to safety revealed that to date, there are no published data regarding the toxicity of A. fruticosa towards humans. This species could provide abundant and cheap resources because it is an aggressive invasive plant that grows almost unrestrictedly. The objective of this study was to evaluate the acute toxicity of a purified extract of A. fruticosa (EAF), and to assess its antioxidant, antihypertensive, and antihyperglycemic activity in streptozotocin-induced diabetic spontaneously hypertensive rats (SHRs). The EAF was slightly toxic (LD₅₀ = 2121 mg/kg, b.w.) when administered orally, and moderately toxic (LD₅₀ = 316 mg/kg, b.w.) at intraperitoneal administration, both in mice. The oral administration of EAF (100 mg/kg) for 35 days to SHRs caused significant decreases in the systolic pressure, blood glucose levels, and MDA quantity. It also increased the hepatic level of the endogenous antioxidant GSH, not only in diabetic SHRs, but also in the control group. An additional potential benefit to human health might be conferred through the environmental management of A. fruticosa based on its large-scale use for medicinal purposes, as this aggressive invasive species brings problems to natural habitats in many European countries.     

Structural and functional characterization of tnpI, a recombinase locus in Tn21 and related beta-lactamase transposons

A novel discrete mobile DNA element from Tn21 from the plasmid R100.1 is described, and its mobilization function was confirmed experimentally. In addition, the element behaves as a recombinase-active locus (tnpI) which facilitates insertions of antibiotic resistance genes as modules or cassettes at defined hot spots or integration sites. A similar tnpI sequence was detected by DNA hybridization in a series of beta-lactamase transposons and plasmids and localized on their physical maps. The genetic function of the locus cloned from Tn21 into pACYC184 was tested for conduction and integration into the plasmids R388 and pOX38Km, and the results suggested recombinase-integrase activity and recA independence. DNA sequence analysis of the tnpI locus revealed no inverted or direct terminal repeats or transposition features of class I and class II transposons. The coding capacity revealed three putative open reading frames encoding 131, 134, and 337 amino acids. Orf3 encoded a putative polypeptide product of 337 amino acids that shared highly significant identity with the carboxyl region of integrase proteins. A comparison and an alignment of the tnpI locus from Tn21 and its flanking sequences identified similar sequences in plasmids and in transposons. The alignment revealed discrete nucleotide changes in these tnpI-like loci and a conserved 3' and 5' GTTA/G hot spot as a duplicated target site. Our data confirm the remarkable ubiquity of tnpI associated with antibiotic resistance genes. We present a model of transposon modular evolution into more complex multiresistant units via tnpI and site-specific insertions, deletions, and DNA rearrangements at this locus.