The influence of amino acid side chains on water binding to the copper(II) in bis(N,N-dimethyl-L-alpha-isoleucinato)-copper(II): an EPR and molecular mechanics study

Simulations were done of the electron paramagnetic resonance (EPR) spectra for bis(N,N-dimethyl-L-alpha-isoleucinato)copper(II) dissolved in deuterated methanol as a function of temperature. They indicated different behaviour of the complex below and above 300 degrees K. The effect was examined by the conformational analysis of the copper(II) complex with a new molecular mechanics force field.     

Oxidative stress tolerance and longevity in Arabidopsis: the late‐flowering mutant gigantea is tolerant to paraquat

Recent genetic analyses of longevity in animals have revealed that long-lived strains are more tolerant to environmental stresses. To investigate whether extended longevity in Arabidopsis also correlates with an increase in stress tolerance, the response was tested of 11 late-flowering mutants to the superoxide radical-generating herbicide paraquat. A tight correlation between flowering time and paraquat tolerance was found when plants were exposed to low doses of herbicide. Furthermore, the mutant gigantea (gi-3) with the longest delay in flowering time had a high tolerance level to paraquat-induced oxidative stress. All the tested gi alleles had an increased tolerance to paraquat toxicity compared to wild-type, although the actual levels of tolerance differed. In addition, the gi-3 mutant was more tolerant to hydrogen peroxide. These results suggest that the link between longevity and oxidative stress resistance in plants is similar to that found in animals, implying that this phenomenon may be general for all aerobic organisms.     

Maize mitochondrial seryl-tRNA synthetase recognizes Escherichia coli tRNASer in vivo and in vitro

In our studies to analyze the structure/function relationships among cytoplasmic and organellar seryl-tRNA synthetases (SerRS), we have characterized a Zea mays cDNA (SerZMm) encoding a protein with significant similarity to prokaryotic SerRS enzymes. To demonstrate the functional identity of SerZMm, the gene sequence encoding the putative mature protein was cloned. This construct complemented in vivo a temperature-sensitive Escherichia coli serS mutant strain. The mature SerZMm protein overexpressed in Escherichia coli efficiently aminoacylated bacterial tRNA^Ser in vitro, while yeast tRNA was a poor substrate. These data identify SerZMm as an organellar maize seryl-tRNA synthetase, the first plant organellar SerRS to be cloned. The analysis of its N-terminal targeting signal suggests a mitochondrial function for the SerZMm protein in maize.     

Effects of sucrose supply on growth and paraquat tolerance of the late-flowering gi-3 mutant

Mutations at the GI locus in Arabidopsis are pleiotropic: gi mutants are late-flowering, tolerant to the toxicity of the herbicide paraquat, and have an increased starch content. We tested the effects of exogenous sucrose supply on the level of paraquat tolerance and on growth and development of the gi-3 mutant. Paraquat tolerance was the highest in gi-3 seedlings grown on medium containing 1% sucrose. As expected, all measured growth parameters (root length, fresh weight, anthocyanin, and sucrose content) were influenced by the sucrose dose, but in a number of assays (effect on fresh weight and developmental characteristics) the sucrose-dependent response in gi-3 was heterochronic. Additionally, the late-flowering phenotype of the gi-3 mutant was reverted to wild type after prolonged growth in darkness on sucrose-containing media.     

Intensified antineoplastic effect by combining an HDAC‐inhibitor,an mTOR‐inhibitor and low dosed interferon alpha in prostate cancer cells

A significant proportion of men diagnosed with prostate cancer (PCa) eventually develop metastatic disease, which progresses to castration resistance, despite initial response to androgen deprivation. As anticancer therapy has become increasingly effective, acquired drug resistance has emerged, limiting efficacy. Combination treatment, utilizing different drug classes, exemplifies a possible strategy to foil resistance development. The effects of the triple application of the histone deacetylase (HDAC) inhibitor valproic acid (VPA), the mammalian target of rapamycin inhibitor everolimus and low dosed interferon alpha (IFNα) on PCa cell growth and dissemination capacity were investigated. For that purpose, the human PCa cell lines, PC‐3, DU‐145 and LNCaP were treated with the combined regimen or separate single agents. Cell growth was investigated by the MTT dye reduction assay. Flow cytometry served to analyse cell cycle progression. Adhesion to vascular endothelium or immobilized collagen, fibronectin and laminin was quantified. Migration and invasion characteristics were determined by the modified Boyden chamber assay. Integrin α and β subtypes were investigated by flow cytometry, western blotting and RT‐PCR. Integrin related signalling, Epidermal Growth Factor Receptor (EGFr), Akt, p70S6kinase and extracellular signal‐regulated kinases (ERK)1/2 activation were also assessed. The triple application of VPA, everolimus and low dosed IFNα blocked tumour cell growth and dissemination significantly better than any agent alone. Antitumour effects were associated with pronounced alteration in the cell cycle machinery, intracellular signalling and integrin expression profile. Combining VPA, everolimus and low dosed IFNα might be a promising option to counteract resistance development and improve outcome in PCa patients.     

Use of a semisynthetic epitope to probe histidine kinase activity and regulation

Histidine-aspartic acid phosphotransfer pathways are central components of prokaryotic signal transduction pathways and are also found in many eukaryotes. Tools to study histidine kinases, however, are currently quite limited. In this article, we present a new tool to study histidine-aspartic acid phosphotransfer pathways. We show that many histidine kinases will accept ATPγS as a substrate to form a stable thiophosphohistidine even when they do not form stable phosphohistidines using the natural substrate ATP. An antibody that has previously been used to detect thiophosphorylated serine, threonine, and tyrosine residues is shown to recognize thiophosphohistidine and thiophosphoaspartic acid residues. Histidine kinase autothiophosphorylation is regulated by other protein sensor domains in the same way as autophosphorylation, and thiophosphate is transferred to downstream aspartic acid containing response regulators.     

Development of Sustained Release Capsules Containing “Coated Matrix Granules of Metoprolol Tartrate”

The objective of this investigation was to prepare sustained release capsule containing coated matrix granules of metoprolol tartrate and to study its in vitro release and in vivo absorption. The design of dosage form was performed by choosing hydrophilic hydroxypropyl methyl cellulose (HPMC K100M) and hydrophobic ethyl cellulose (EC) polymers as matrix builders and Eudragit® RL/RS as coating polymers. Granules were prepared by composing drug with HPMC K100M, EC, dicalcium phosphate by wet granulation method with subsequent coating. Optimized formulation of metoprolol tartrate was formed by using 30% HPMC K100M, 20% EC, and ratio of Eudragit® RS/RL as 97.5:2.5 at 25% coating level. Capsules were filled with free flowing optimized granules of uniform drug content. This extended the release period upto 12 h in vitro study. Similarity factor and mean dissolution time were also reported to compare various dissolution profiles. The network formed by HPMC and EC had been coupled satisfactorily with the controlled resistance offered by Eudragit® RS. The release mechanism of capsules followed Korsemeyer–Peppas model that indicated significant contribution of erosion effect of hydrophilic polymer. Biopharmaceutical study of this optimized dosage form in rabbit model showed 10 h prolonged drug release in vivo. A close correlation (R² = 0.9434) was established between the in vitro release and the in vivo absorption of drug. The results suggested that wet granulation with subsequent coating by fluidized bed technique, is a suitable method to formulate sustained release capsules of metoprolol tartrate and it can perform therapeutically better than conventional immediate release dosage form.Key words: biopharmaceutical evaluation, coated granules, metoprolol tartrate, sustained release     

A new concept of olfactory biosensor based on interdigitated microelectrodes and immobilized yeasts expressing the human receptor OR17-40

This work shows the feasibility of an olfactory biosensor based on the immobilization of Saccharomyces cerevisiae yeast cells genetically modified to express the human olfactory receptor OR17-40 onto interdigitated microconductometric electrodes. This olfactory biosensor has been applied to the detection of its specific odorant (helional) with a high sensitivity (threshold 10⁻¹⁴ M). In contrast, no significant response was observed using a non-specific odorant (heptanal), which suggests a good selectivity. Thus, this work may represent a first step towards a new kind of bioelectronic noses based on whole yeast cells and allowing a real time monitoring of olfactory receptor activation. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet, France, 14–19 October, 2006.     

Butyrylcholinesterase and the control of synaptic responses in acetylcholinesterase knockout mice

At the neuromuscular junction (NMJ) acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can hydrolyze acetylcholine (ACh). Released ACh quanta are known to diffuse rapidly across the narrow synaptic cleft and pairs of ACh molecules cooperate to open endplate channels. During their diffusion through the cleft, or after being released from muscle nicotinic ACh receptors (nAChRs), most ACh molecules are hydrolyzed by AChE highly concentrated at the NMJ. Advances in mouse genomics offered new approaches to assess the role of specific cholinesterases involved in synaptic transmission. AChE knockout mice (AChE-KO) provide a valuable tool for examining the complete abolition of AChE activity and the role of BChE. AChE-KO mice live to adulthood, and exhibit an increased sensitivity to BChE inhibitors, suggesting that BChE activity facilitated their survival and compensated for AChE function. Our results show that BChE is present at the endplate region of wild-type and AChE-KO mature muscles. The decay time constant of focally recorded miniature endplate currents was 1.04 +/- 0.06 ms in wild-type junctions and 5.4 ms +/- 0.3 ms in AChE-KO junctions, and remained unaffected by BChE-specific inhibitors, indicating that BChE is not limiting ACh duration on endplate nAChRs. Inhibition of BChE decreased evoked quantal ACh release in AChE-KO NMJs. This reduction in ACh release can explain the greatest sensitivity of AChE-KO mice to BChE inhibitors. BChE is known to be localized in perisynaptic Schwann cells, and our results strongly suggest that BChE's role at the NMJ is to protect nerve terminals from an excess of ACh.     

Emergence of uropathogenic extended-spectrum beta lactamases-producing Escherichia coli strains in the community

The aim of this study was to determine the virulence characteristics and resistance pattern of the extended-spectrum/lactamases (ESBLs)-producing Escherichia coli strains isolated from urine of outpatients in the Zagreb region during a five-month period, and to compare them with the non ESBLs-producing E. coli strains isolated in the same period. Out of 2451 E. coli strains isolated from urine of nonhospitalized patients with significant bacteriuria, a total of 39 ESBLs-producing strains (1.59%) were detected by a double-disk diffusion technique and by the broth-dilution minimal inhibitory concentration reduction method. The 45 non ESBLs-producing strains were randomly chosen, and phenotype of the two groups of strains was characterized and compared. Serogroup O4, hemolysin production, expression of P- and type 1 fimbriae as well as resistance to gentamicin and amikacin were significantly more prevalent characteristics among the ESBLs-producing strains than among non ESBLs-producing strains (p < 0.01), while higher prevalence of trimethoprim-sulfamethoxazole resistance among ESBLs-producing strains was not statistically significant (p > 0.05). Chromosomal DNA analysis by pulsed-field gel electrophoresis exhibited a great genomic similarity among ESBLs-producing strains and revealed that those highly virulent and resistant E. coli strains isolated from urine of outpatients in the Zagreb region had a clonal propagation.