Increased fermentative adenosine production by gene-targeted Bacillus subtilis mutation.

Adenosine, which is produced mainly by microbial fermentation, plays an important role in the therapy of cardiovascular disease and has been widely used as an antiarrhythmic agent. In this study, guanosine 5'-monophosphate (GMP) synthetase gene (guaA) was inactivated by gene-target manipulation to increase the metabolic flux from inosine 5'-monophosphate (IMP) to adenosine in B. subtilis A509. The resulted mutant M3-3 showed an increased adenosine production from 7.40 to 10.45 g/L, which was further enhanced to a maximum of 14.39 g/L by central composite design. As the synthesis of succinyladenosine monophosphate (sAMP) from IMP catalysed by adenylosuccinate synthetase (encoded by purA gene) is the rate-limiting step in adenosine synthesis, the up-regulated transcription level of purA was the potential underlying mechanism for the increased adenosine production. This work demonstrated a practical strategy for breeding B. subtilis strains for industrial nucleoside production.

Emergence of Imipenem-Resistant Pseudomonas aeruginosa Clinical Isolates from Egypt Coharboring VIM and IMP Carbapenemases.

Pseudomonas aeruginosa is an important human pathogen and the leading cause of nosocomial infections. P. aeruginosa is characterized by massive intrinsic resistance to a multiple classes of antibiotics with carbapenems being the most potent inhibitor of P. aeruginosa and considered the first choice for its treatment. Therefore, it is crucial to investigate novel mechanisms of resistance of P. aeruginosa to carbapenems for achieving successful therapy. A total of 114 P. aeruginosa isolates from two university hospitals in Egypt were recruited in this study. Antimicrobial susceptibility testing revealed that 50 isolates (43.8%) exhibited multidrug-resistant (MDR) phenotype, of them 14 isolates (12.2%) were imipenem (IPM)-resistant. Of these 14 isolates, 13 isolates (11.4%) exhibited the metallo-ß-lactamase (MBL) phenotype. MBLs encoding genes, VIM and IMP, were identified by PCR. PCR results revealed that four isolates harbored the VIM gene alone, one isolate harbored IMP gene alone, and four isolates harbored both genes. The correct size of PCR products of VIM and IMP genes (390 and 188 bp, respectively) were sequenced to confirm results of PCR and to look for any possible polymorphism among MBL genes of tested isolates. Data analysis of these sequences showed 100% identity of nucleotide sequences of MBL genes among tested Egyptian patients. To our knowledge, this is the first report of IMP carbapenemase-encoding gene in Africa and the first detection of the emergence of P. aeruginosa coproducing VIM and IMP genes in Egypt.

Two distinct determinants of ligand specificity in T1R1/T1R3 (the umami taste receptor).

Umami taste perception in mammals is mediated by a heteromeric complex of two G-protein-coupled receptors, T1R1 and T1R3. T1R1/T1R3 exhibits species-dependent differences in ligand specificity; human T1R1/T1R3 specifically responds to L-Glu, whereas mouse T1R1/T1R3 responds more strongly to other L-amino acids than to L-Glu. The mechanism underlying this species difference remains unknown. In this study we analyzed chimeric human-mouse receptors and point mutants of T1R1/T1R3 and identified 12 key residues that modulate amino acid recognition in the human- and mouse-type responses in the extracellular Venus flytrap domain of T1R1. Molecular modeling revealed that the residues critical for human-type acidic amino acid recognition were located at the orthosteric ligand binding site. In contrast, all of the key residues for the mouse-type broad response were located at regions outside of both the orthosteric ligand binding site and the allosteric binding site for inosine-5'-monophosphate (IMP), a known natural umami taste enhancer. Site-directed mutagenesis demonstrated that the newly identified key residues for the mouse-type responses modulated receptor activity in a manner distinct from that of the allosteric modulation via IMP. Analyses of multiple point mutants suggested that the combination of two distinct determinants, amino acid selectivity at the orthosteric site and receptor activity modulation at the non-orthosteric sites, may mediate the ligand specificity of T1R1/T1R3. This hypothesis was supported by the results of studies using nonhuman primate T1R1 receptors. A complex molecular mechanism involving changes in the properties of both the orthosteric and non-orthosteric sites of T1R1 underlies the determination of ligand specificity in mammalian T1R1/T1R3.

Remote reperfusion lung injury is associated with AMP deaminase 3 activation and attenuated by inosine monophosphate.

BACKGROUND: Remote reperfusion lung injury occurs in patients with vascular occlusion and surgical procedures. Inosine monophosphate (IMP) produced by adenosine monophosphate deaminase (AMPD) 3 is involved in the remote reperfusion injury. The purpose of the present study was to identify whether IMP administration attenuated the remote reperfusion lung injury in a skeletal muscle ischemia-reperfusion model. METHODS AND RESULTS: A remote reperfusion lung injury was created using reperfusion after the bilateral ligation of the hind-limb. AMPD activity, myeloperoxidase (MPO) activity, IMP, AMPD3 mRNA and tumor necrosis factor (TNF)-alpha in the lungs before and after reperfusion were analyzed. Furthermore, the effects of IMP on these parameters were examined. AMPD3 mRNA, AMPD activity and IMP production in the lungs significantly increased after ischemia-reperfusion with increases in MPO activity, TNF-alpha level and decreased oxygen saturation (SpO(2)). Histological examination of the lungs demonstrated significant neutrophil infiltration and accumulation. IMP administration significantly reduced MPO activity, TNF-alpha and neutrophil infiltration, with ameliorated SpO(2). CONCLUSIONS: Along with the activation of AMPD3, ischemia-reperfusion-induced lung inflammation is associated with increased MPO activity and TNF-alpha level. IMP significantly decreased the lung injury, MPO activity, TNF-alpha and increased SpO(2). These findings may lead to the development of a new therapeutic strategy for remote reperfusion lung injury.

Kinetic characterization of inosine monophosphate dehydrogenase of Leishmania donovani.

Trypanosomatid protozoan pathogens are purine auxotrophs that are highly dependent on the enzyme inosine monophosphate dehydrogenase (IMPDH) for the synthesis of guanylate nucleotides. Enzymatic characterization of the Leishmania donovani IMPDH (LdIMPDH) overexpressed in E. coli revealed that this enzyme was highly specific for the substrates IMP and NAD(+) with K(m)(app) values of 33 and 390 microM, respectively. In contrast to other IMPDHs, LdIMPDH exhibits no substrate inhibition in high concentrations of NAD(+). Kinetic studies revealed that XMP and GMP were inhibitors with K(i) values of approximately 26 and 210 microM, respectively, suggesting that these nucleotides may regulate LdIMPDH activity. Mycophenolic acid was also a potent inhibitor of L. donovani IMPDH with a K(i) value of approximately 25 nM. Confocal immunofluorescence microscopy and subcellular fractionation localized LdIMPDH to the glycosome. Protein-protein interaction assays revealed that LdIMPDH associated tightly with glycosomal protein sorting receptor LdPEX5.

Interactions of the human, rat, Saccharomyces cerevisiae and Escherichia coli 3-methyladenine-DNA glycosylases with DNA containing dIMP residues.

In DNA, the deamination of dAMP generates 2'-deoxy-inosine 5'-monophosphate (dIMP). Hypoxanthine (HX) residues are mutagenic since they give rise to A.T-->G.C transition. They are excised, although with different efficiencies, by an activity of the 3-methyl-adenine (3-meAde)-DNA glycosylases from Escherichia coli (AlkA protein), human cells (ANPG protein), rat cells (APDG protein) and yeast (MAG protein). Comparison of the kinetic constants for the excision of HX residues by the four enzymes shows that the E.coli and yeast enzymes are quite inefficient, whereas for the ANPG and the APDG proteins they repair the HX residues with an efficiency comparable to that of alkylated bases, which are believed to be the primary substrates of these DNA glycosylases. Since the use of various substrates to monitor the activity of HX-DNA glycosylases has generated conflicting results, the efficacy of the four 3-meAde-DNA glycosylases of different origin was compared using three different substrates. Moreover, using oligo-nucleotides containing a single dIMP residue, we investigated a putative sequence specificity of the enzymes involving the bases next to the HX residue. We found up to 2-5-fold difference in the rates of HX excision between the various sequences of the oligonucleotides studied. When the dIMP residue was placed opposite to each of the four bases, a preferential recognition of dI:T over dI:dG, dI:dC and dI:dA mismatches was observed for both human (ANPG) and E.coli (AlkA) proteins. At variance, the yeast MAG protein removed more efficiently HX from a dI:dG over dI:dC, dI:T and dI:dA mismatches.

Isolation and characterization of a purC(orf)QLF operon from Lactococcus [correction of Lactobacillus] lactis MG1614.

We have isolated genes encoding enzymes of the de novo purine nucleotide biosynthesis pathway from Lactococcus lactis MG1614 by colony hybridization using DIG-labeled DNA probes. The organization of the genes needed for the de novo biosynthesis of purine nucleotides in L. lactis differs from that found in other organisms. In L. lactis there is a gene cluster, which contains five out of the 11 genes needed for the de novo biosynthesis of IMP, namely purC, orf, purQ, purL and purF. These genes were shown to be transcribed as a single transcription unit by Northern hybridization analysis. The 5' end of the transcript of the purC(orf)QLF operon was determined by primer extension analysis using fluorescently end-labeled probes. The purC(orf)QLF operon of L. lactis is transcribed in Escherichia coli, and the gene product of the purF gene, glutamine phosphoribosylpyrophosphate amidotransferase (glutamine PRPP ATase, EC 2.4.2.14), can functionally complement the E. coli purF mutant strain TX158. We also show that the promoter of the purC(orf)QLF operon is regulated in response to exogenously added purines.

Rho-dependent termination within the trp t' terminator. I. Effects of rho loading and template sequence.

About one-half of the terminators of the Escherichia coli genome require transcription termination factor rho to function. Here we use the very "diffuse" trp t' terminator of E. coli to show that both template sequence and transcript secondary structure are involved in controlling the template positions and efficiencies of rho-dependent termination. Termination begins in the wild-type trp t' terminator sequence approximately 97 bps downstream of the promoter under our standard reaction conditions, and termination efficiencies for individual positions on three related templates have been determined in the form of quantitative patterns of rho-dependent RNA release. Comparison of these patterns shows that the rho-dependent termination efficiency at individual template positions depends primarily on the nucleotide sequence at and near the putative 3' end of the transcript, although these efficiencies can also be influenced by RNA sequence elements located further upstream. The amplitudes of the peaks of the RNA release patterns at specific template positions are controlled primarily by the effectiveness of the binding of the rho hexamer to the "rho loading site" of the transcript. Introduction of a stable element of secondary structure into the nascent RNA within the loading site both shifts the position of initial rho-dependent termination downstream and decreases the amplitudes of the peaks of the RNA release pattern at the corresponding sequences. These results and others are consistent with the view that rho-dependent terminators contain two essential components: (i) an upstream rho loading site on the RNA that is 70-80 nucleotide residues in length, essentially devoid of secondary structure, and which contains sufficient numbers of rC residues to activate the RNA-dependent ATPase of rho; and (ii) a downstream sequence within which termination actually occurs. In this study we use the trp t' terminator to characterize the involvement of each of these sequence components in detail in order to provide the parameters required to define a quantitative mechanistic model for the function of rho in transcript termination.