Comparison of CE and HPLC Methods for Determining Lovastatin and Its Oxidation Products after Exposure to an Oxidative Atmosphere

Lovastatin is a cholesterol-lowering agent, which competitively inhibits the enzyme HMG-CoA reductase. Several HPLC methods for its analysis have been developed but there is no report of its determination using capillary electrophoresis (CE). In this paper, we report the development of a simple CE method for lovastatin determination, which is selective with respect to its degradation products and useful for routine analyses. Since the molecule of lovastatin in its lactone form is uncharged and is only slightly soluble in water, base hydrolysis was used to open the lactone ring and transform the compound into a water-soluble acid form, which is negatively charged. Different solvents, different amounts of NaOH added, different hydrolysis times and different temperatures for sample preparation were tested. The CE and HPLC methods are compared in terms of susceptibility, precision, linearity and accuracy. HPLC method was found to be more susceptible and more precise

The Coding Region of the Equinatoxin II Gene Lacks Introns

Sea anemones produce several toxic peptides and proteins. Equi- natoxins (Eqt) isolated from the sea anemone Actinia equina are basic cytolytic proteins with molecular masses of approximately 20 kDa. Of the three Eqt purified so far, Eqtll is the most abundant and well characterized. Its gene organization has not yet been studied. In order to obtain the first information about the Eqtll gene structure and sequence, genomic DNA was isolated from A. equina and the target DNA fragment amplified by the polymerase chain reaction (PCR) using three different pairs of oligonucleotide primers deduced from the preserved regions of Eqt cDNA clones. The sequence of the PCR product obtained after amplification of genomic DNA, using an oligonucleotide specific for Eqtll, was almost indistinguishable from that of Eqtll cDNA. As the DNA fragments derived from PCR of genomic DNA were of the same length as those from control PCR reactions performed on an A. equina cDNA library and Eqtll cDNA, the Eqtll gene was proved to be in- tronless, at least within the amplified preproprotein region. The presence of such an intronless gene coding for this cytotoxic protein might be explained by the relative low position of cnidarians in the evolutionary tree or by the advantage provided by a potentially higher rate of gene expression

Студија молекулског докинга са биомолекулима изолованим из ендофитних гљива

Recently, growing interest has been devoted to the investigation of compounds with antimicrobial activity due to rising cases of resistance of mic-robes to known therapies. A reliable and versatile source of novel drug disco-very was recently found among endophytic fungi. Hitherto, the research usu-ally enclosed the in vitro evaluation of antimicrobial activity and chemical structure elucidation of biomolecules extracted from fungal material. There-fore, this research was designed as an extension to previous investigations of endophytic fungi growing on conifer needles by means of conducting a mole-cular docking study. The in silico methods were used with the main goal to make a contribution to the understanding of the mechanisms underlying the interaction of biomolecules isolated from fungus Phomopsis species and eight different types of receptors that belong to usually multidrug resistant bacterial pathogens. The results revealed valuable interactions with receptors 3G7B (Staphylococcus aureus’s gyrase B), 1F0K (1.9 Å structure of Escherichia coli’s transferase) and 1SHV (Klebsiella pneumoniae’s SHV-1 β-lactamase) thus pointing out the receptors that trigger antibiotic response upon activation by the most potent compounds 325-3, 325-5, phomoenamide and phomol. These findings also recommended further discovery of novel potent and broad-spectrum antibiotics based on the structure of selected molecules.У последње време, као одговорна повећање резистенције микроорганизама на познату терапију, све већа пажња се поклања истраживању једињења са антимикробном активношћу. Ендофитне гљиве су недавно представљене као поуздан и богат извор за развој нових лекова. До сада, истраживања су се углавном ограничавала на in vitro процену антимикробне активности и разоткривање хемијске структуре биомолекула изолованих из материјала гљива. Из тог разлога, ово истраживање је осмишљено као проширење претходно спроведених испитивања ендофита које расту на иглицама четинара путем in silico студије молекулског докинга. Главни циљ употребе in silico метода је био да се направи прилог разумевању механизама који стоје иза интеракције биомолекула изолованих из гљиве Phomopsis species са осам различитих типова рецептора који припадају патогеним бактеријама у обичајеном ултирезистентних на лекове. Резултати су указали на важне интеракције са рецепторима 3G7B (Staphylococcus aureus гиразаБ), 1F0K (структура Escherichia Coli трансферазе величине 1,9 Å) и 1SHV (SHV-1 β-лакта-маза Klebsiella pneumoniae) указујући на тај начин на рецепторе путем којих се започиње антибиотски одговор након активације најпотентнијим једињењима, 325-3, 325-5, фомо-енамидом и фомолом. Овим открићем се такође препоручује будући развој нових моћних антибиотика са широким спектром деловања базиран на структури изабраних молекула

Improved determination of plasmid copy number using quantitative real-time PCR for monitoring fermentation processes

<p>Abstract</p> <p>Background</p> <p>Recombinant protein production in <it>Escherichia coli </it>cells is a complex process, where among other parameters, plasmid copy number, structural and segregational stability of plasmid have an important impact on the success of productivity. It was recognised that a method for accurate and rapid quantification of plasmid copy number is necessary for optimization and better understanding of this process. Lately, qPCR is becoming the method of choice for this purpose. In the presented work, an improved qPCR method adopted for PCN determination in various fermentation processes was developed.</p> <p>Results</p> <p>To avoid experimental errors arising from irreproducible DNA isolation, whole cells, treated by heating at 95°C for 10 minutes prior to storage at -20°C, were used as a template source. Relative quantification, taking into account different amplification efficiencies of amplicons for chromosome and plasmid, was used in the PCN calculation. The best reproducibility was achieved when the efficiency estimated for specific amplicon, obtained within one run, was averaged. It was demonstrated that the quantification range of 2 log units (100 to 10000 bacteria per well) enable quantification in each time point during fermentation. The method was applied to study PCN variation in fermentation at 25°C and the correlation between PCN and protein accumulation was established.</p> <p>Conclusion</p> <p>Using whole cells as a template source and relative quantification considering different PCR amplification efficiencies are significant improvements of the qPCR method for PCN determination. Due to the approaches used, the method is suitable for PCN determination in fermentation processes using various media and conditions.</p

Isolation and Determination of Fomentariol: Novel Potential Antidiabetic Drug from Fungal Material

Diabetes mellitus is one of the leading world's public health problems. Therefore, it is of a huge interest to develop new antidiabetic drugs. Apart from traditional therapy of diabetes, nowadays, importance is given to natural substances with antidiabetic potential. Fomes fomentarius is a mushroom widely used for different purposes, due to its range of already confirmed activities. Fomentariol is a constituent of Fomes fomentarius, responsible for its antidiabetic potential. In that respect, it is important to develop a method for isolation and quantification of fomentariol from fungal material, which will be simple and efficient. Multistep, complex extraction applied in the previously reported studies was avoided with ethanol, providing rapid single-step extraction. The presence of fomentariol in ethanolic extract was confirmed by high-resolution mass spectrometry. Semipreparative HPLC method was developed and applied for isolation from ethanol extract and purification of the active compound fomentariol. It was a gradient reversed-phase method with a mobile phase consisting of acetonitrile and 0.1% formic acid in water and total run time of 15 minutes. The amount of 6.5 mg of high-purity fomentariol was determined by quantitative NMR with toluene as internal standard. The isolated and determined amount of substance can be further used for the quantitative estimation of activity of fomentariol