Purification, characterization and immobilization of an NADPH-dependent enzyme involved in the chiral specific reduction of the keto ester M, an intermediate in the synthesis of an anti-asthma drug, Montelukast, from Microbacterium campoquemadoensis (MB5614)

(S)(E)-2-{3-[3-[2-(7-chloro-2-quinolinyl)ethenyl]-phenyl]-3-hydroxypropyl} benzoic acid methyl ester,␣a key intermediate in the synthesis of the anti-asthma drug, Montelukast, was prepared from the corresponding ketone (keto ester M) by microbial transformation. The biotransforming organism, Microbacterium campoquemadoensis (MB5614), was discovered as a result of an extensive screening program and was used for the isolation and purification of the responsible enzyme. The enzyme is a soluble cytoplasmic protein which was purified as a complex with a low-molecular-mass molecule that had a visible-light absorption maximum at 460 nm. The purified enzyme has an apparent molecular mass of 60 kDa, when denatured, and is isolated in the native state as an oligomer. The isolated enzyme requires NADPH for its activity and reduces the keto ester M to the desired (S)-hydroxy ester with an enantiomeric excess greater than 95% at the optimum temperature of 30 °C and pH 8. The enzyme was immobilized on oxirane-activated acrylamide beads with some loss of activity, but it was fully active in a two-phase (water/hexane 25:75) solvent system, both as a free solution and in an immobilized form. Received: 31 October 1997 / Received revision: 8 January 1998 / Accepted: 24 January 1998     

Inositol monophosphate phosphatase genes of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Background  

Mycobacteria use inositol in phosphatidylinositol, for anchoring lipoarabinomannan (LAM), lipomannan (LM) and phosphatidylinosotol mannosides (PIMs) in the cell envelope, and for the production of mycothiol, which maintains the redox balance of the cell. Inositol is synthesized by conversion of glucose-6-phosphate to inositol-1-phosphate, followed by dephosphorylation by inositol monophosphate phosphatases (IMPases) to form myo-inositol. To gain insight into how Mycobacterium tuberculosis synthesises inositol we carried out genetic analysis of the four IMPase homologues that are present in the Mycobacterium tuberculosis genome.     

SIRT1 deacetylase protects against neurodegeneration in models for Alzheimer's disease and amyotrophic lateral sclerosis

A progressive loss of neurons with age underlies a variety of debilitating neurological disorders, including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), yet few effective treatments are currently available. The SIR2 gene promotes longevity in a variety of organisms and may underlie the health benefits of caloric restriction, a diet that delays aging and neurodegeneration in mammals. Here, we report that a human homologue of SIR2, SIRT1, is upregulated in mouse models for AD, ALS and in primary neurons challenged with neurotoxic insults. In cell-based models for AD/tauopathies and ALS, SIRT1 and resveratrol, a SIRT1-activating molecule, both promote neuronal survival. In the inducible p25 transgenic mouse, a model of AD and tauopathies, resveratrol reduced neurodegeneration in the hippocampus, prevented learning impairment, and decreased the acetylation of the known SIRT1 substrates PGC-1alpha and p53. Furthermore, injection of SIRT1 lentivirus in the hippocampus of p25 transgenic mice conferred significant protection against neurodegeneration. Thus, SIRT1 constitutes a unique molecular link between aging and human neurodegenerative disorders and provides a promising avenue for therapeutic intervention.     

Quantification of global transcription patterns in prokaryotes using spotted microarrays

We describe an analysis, applicable to any spotted microarray dataset produced using genomic DNA as a reference, that quantifies prokaryotic levels of mRNA on a genome-wide scale. Applying this to Mycobacterium tuberculosis, we validate the technique, show a correlation between level of expression and biological importance, define the complement of invariant genes and analyze absolute levels of expression by functional class to develop ways of understanding an organism's biology without comparison to another growth condition.     

A GAF domain in the hypoxia/NO-inducible Mycobacterium tuberculosis DosS protein binds haem

The majority of the Mycobacterium tuberculosis response to hypoxia and nitric oxide is through the DosRS (DevRS) two-component regulatory system. The N-terminal input domain of the DosS sensor contains two GAF domains. We demonstrate here that the proximal GAF domain binds haem, and identified histidine 149 of DosS as critical to haem-binding; the location of this histidine residue is similar to the cGMP-binding site in a crystal structure of cyclic nucleotide phosphodiesterase 2A. GAF domains are frequently involved in binding cyclic nucleotides, but this is the first GAF domain to be identified that binds haem. In contrast, PAS domains (similar to GAF domains in structure but not primary sequence) frequently use haem cofactors, and these findings further illustrate how the functions of these domains overlap. We propose that the activation of the DosS sensor is controlled through the haem binding of molecular oxygen or nitric oxide.     

Trypanosoma brucei thymidine kinase is tandem protein consisting of two homologous parts, which together enable efficient substrate binding

Trypanosoma brucei causes African sleeping sickness, a disease for which existing chemotherapies are limited by their toxicity or lack of efficacy. We have found that four parasites, including T. brucei, contain genes where two or four thymidine kinase (TK) sequences are fused into a single open reading frame. The T. brucei full-length enzyme as well as its two constituent parts, domain 1 and domain 2, were separately expressed and characterized. Of potential interest for nucleoside analog development, T. brucei TK was less discriminative against purines than human TK1 with the following order of catalytic efficiencies: thymidine > deoxyuridine ≫ deoxyinosine > deoxyguanosine. Proteins from the TK1 family are generally dimers or tetramers, and the quaternary structure is linked to substrate affinity. T. brucei TK was primarily monomeric but can be considered a two-domain pseudodimer. Independent kinetic analysis of the two domains showed that only domain 2 was active. It had a similar turnover number (k_cat) as the full-length enzyme but could not self-dimerize efficiently and had a 5-fold reduced thymidine/deoxyuridine affinity. Domain 1, which lacks three conserved active site residues, can therefore be considered a covalently attached structural partner that enhances substrate binding to domain 2. A consequence of the non-catalytic role of domain 1 is that its active site residues are released from evolutionary pressure, which can be advantageous for developing new catalytic functions. In addition, nearly identical 89-bp sequences present in both domains suggest that the exchange of genetic material between them can further promote evolution.     

Serum Iron,Zinc, and Copper Concentration in Premature Graying of Hair

Premature graying of hair with unclear etiology, which is known as premature canities, is a common cause of referrals to the dermatologists. We assessed the relationship between serum iron, copper, and zinc concentrations with premature canities. This study was conducted on patients under 20 years old suffering from premature canities, having a minimum of ten gray hair fibers, and referring to university hospitals of Isfahan (Iran). The results were compared with age–sex-matched controls. Demographic data and disease characteristics were recorded for two groups. We studied serum iron, copper, and zinc concentrations of 66 patients and 66 controls using atomic absorption and Ferrozine methods. The mean age of studied cases was 17.8 ± 2.0 years, and the mean age of the onset of canities was 15.5 ± 3.2 years with no significant difference between males and females (P > 0.05). Serum copper concentration was significantly lower in patients compared with controls (90.7 ± 37.4 vs. 105.3 ± 50.2 μg/dL, P = 0.048), but serum iron concentration was significantly lower in controls compared to patients (88.8 ± 39.5 vs. 108.3 ± 48.4 μg/dL, P = 0.008). Also, there was no significant difference between patients and controls in serum zinc concentration (114.8 ± 67.8 vs. 108.2 ± 49.9 μg/dL, P = 0.285). According to these results, among copper, zinc, and iron, a low serum copper concentration may play a role in premature graying of hairs in our society. Further studies are needed to find the underlying mechanism of this relationship.     

Adaptive Neuro-Fuzzy Methodology for Noise Assessment of Wind Turbine

Wind turbine noise is one of the major obstacles for the widespread use of wind energy. Noise tone can greatly increase the annoyance factor and the negative impact on human health. Noise annoyance caused by wind turbines has become an emerging problem in recent years, due to the rapid increase in number of wind turbines, triggered by sustainable energy goals set forward at the national and international level. Up to now, not all aspects of the generation, propagation and perception of wind turbine noise are well understood. For a modern large wind turbine, aerodynamic noise from the blades is generally considered to be the dominant noise source, provided that mechanical noise is adequately eliminated. The sources of aerodynamic noise can be divided into tonal noise, inflow turbulence noise, and airfoil self-noise. Many analytical and experimental acoustical studies performed the wind turbines. Since the wind turbine noise level analyzing by numerical methods or computational fluid dynamics (CFD) could be very challenging and time consuming, soft computing techniques are preferred. To estimate noise level of wind turbine, this paper constructed a process which simulates the wind turbine noise levels in regard to wind speed and sound frequency with adaptive neuro-fuzzy inference system (ANFIS). This intelligent estimator is implemented using Matlab/Simulink and the performances are investigated. The simulation results presented in this paper show the effectiveness of the developed method.     

Histopathological Changes of Hepatorenal Toxicity Induced by Gentamicin in Killifish,Aphanius hormuzensis (Aphaniidae) and its Kidney Regeneration Through Nephron Neogenesis

Journal of Ichthyology - The histopathological changes in liver and kidney toxicity after induction by gentamicin are studied in killifish, Aphanius hormuzensis, and its kidney regeneration through...     

Nitric Oxide and Protein Disulfide Isomerase Explain the Complexities of Unfolded Protein Response Following Intra-hippocampal Aβ Injection

Several pathways involved in regulation of intracellular protein integrity are known as the protein quality control (PQC) system. Molecular chaperones as the main players are engaged in various aspects of PQC system. According to the importance of these proteins in cell survival, in the present study, we traced endoplasmic reticulum-specific markers and chaperone-mediated autophagy (CMA)-associated factors as two main arms of PQC system in intra-hippocampal amyloid beta (Aβ)-injected rats during 10 days running. Data analysis from Western blot indicated that exposure to Aβ activates immunoglobulin heavy-chain-binding protein (Bip) which is the upstream regulator of unfolded protein responses (UPR). Activation of UPR system eventually led to induction of pro-apoptotic factors like CHOP, calpain, and caspase-12. Moreover, our data revealed that protein disulfide isomerase activity dramatically decreased after Aβ injection, which could be attributed to the increased levels of nitric oxide. Besides, Aβ injection induced levels of 2 members of heat shock proteins (Hsp) 70 and 90. Elevated levels of Hsps family members are accompanied by increased levels of lysosome-associated membrane protein type-2A (Lamp-2A) that are involved in CMA. Despite the reduction in CHOP, calpain, caspase-12, and Lamp-2A protein levels, the levels of molecular chaperones Bip, Hsps70, and 90 increased 10 days after Aβ injection in comparison to the control group. Based on our results, 10 days after Aβ injection, despite the activation of protective chaperones, markers associated with neurotoxicity were still elevated.