A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Mist trickling bioreactor for <Emphasis Type="Italic">Centaurium erythraea</Emphasis> Rafn growth of shoots and production of secoiridoids

Shoots of Centaurium erythraea Rafn were cultivated in 5 l mist trickling bioreactor for 21 and 28 days increasing their dry weight from 0.54 g to 13.7 g and 18.3 g, respectively. About 6880 shoots from 223 initial shoot-tips in 21-day bioreactor producing cycle were produced. The shoots could be successfully rooted and transferred to soil. Secoiridoid accumulation (expressed as a sum of gentiopicroside, sweroside and swertiamarin) in shoots after 21 days of culture reached about 303 mg l⁻¹.     

A homology model of restriction endonuclease SfiI in complex with DNA

Background

Restriction enzymes (REases) are commercial reagents commonly used in recombinant DNA technologies. They are attractive models for studying protein-DNA interactions and valuable targets for protein engineering. They are, however, extremely divergent: the amino acid sequence of a typical REase usually shows no detectable similarities to any other proteins, with rare exceptions of other REases that recognize identical or very similar sequences. From structural analyses and bioinformatics studies it has been learned that some REases belong to at least four unrelated and structurally distinct superfamilies of nucleases, PD-DxK, PLD, HNH, and GIY-YIG. Hence, they are extremely hard targets for structure prediction and homology-based inference of sequence-function relationships and the great majority of REases remain structurally and evolutionarily unclassified.

Results

SfiI is a REase which recognizes the interrupted palindromic sequence 5'GGCCNNNN^NGGCC3' and generates 3 nt long 3' overhangs upon cleavage. SfiI is an archetypal Type IIF enzyme, which functions as a tetramer and cleaves two copies of the recognition site in a concerted manner. Its sequence shows no similarity to other proteins and nothing is known about the localization of its active site or residues important for oligomerization. Using the threading approach for protein fold-recognition, we identified a remote relationship between SfiI and BglI, a dimeric Type IIP restriction enzyme from the PD-DxK superfamily of nucleases, which recognizes the 5'GCCNNNN^NGGC3' sequence and whose structure in complex with the substrate DNA is available. We constructed a homology model of SfiI in complex with its target sequence and used it to predict residues important for dimerization, tetramerization, DNA binding and catalysis.

Conclusions

The bioinformatics analysis suggest that SfiI, a Type IIF enzyme, is more closely related to BglI, an "orthodox" Type IIP restriction enzyme, than to any other REase, including other Type IIF REases with known structures, such as NgoMIV. NgoMIV and BglI belong to two different, very remotely related branches of the PD-DxK superfamily: the α-class (EcoRI-like), and the β-class (EcoRV-like), respectively. Thus, our analysis provides evidence that the ability to tetramerize and cut the two DNA sequences in a concerted manner was developed independently at least two times in the evolution of the PD-DxK superfamily of REases. The model of SfiI will also serve as a convenient platform for further experimental analyses.     

The Effect of Growth Regulators on Quality Parameters and Ginsenosides Accumulation in Panax quinquefolium L. Roots

American ginseng (Panax quinquefolium L.) is a perennial medicinal herb originally grown in Canada and USA, and recently also in China, Australia, Holland and Poland. Several commercial preparations are produced from ginseng roots, that are known for their antifatigue, antitumor, antistress and immune system stimulating functions. The medicinal properties are due mainly to the active components – ginsenosides. In this work, the results of field cultivation experiments are presented that examine the effects of foliar application of several growth regulators on quality parameters and ginsenoside content of P. quinuefolium roots. The growth regulators tested, i.e., kinetin, daminozide, mixture of gibberellic acid (GA₃) with potassium salt of α-naphthalene acetic acid (kNAA) and new preparation – IPO-1 – benzimidazole derivative (obtained from the Institute of Organic Industry in Warsaw – at present during the process of patent), were applied at a concentration of 100 or 200 mg l⁻¹ in the middle of June in the 2nd year of vegetation. After 4 years of cultivation, the roots were dug up and dried, and subsequently the quantitative analysis of individual saponins (R_b1, R_b2, R_c, R_d, R_e, R_g1) by HPLC was performed. Growth regulators significantly affected quality parameters, morphological features and accumulation of individual and total ginsenosides in ginseng roots. Regardless of doses, the plant roots treated with growth regulators had a higher content of total ginsenosides in comparison to the control. The growth regulators also affected individual ginsenosides level and narrowed the ratio of R_b:R_g group. The application of kinetin, daminozide and benzimidazole derivative for foliar spray during 2nd year of American ginseng vegetation caused a significant increase in air dry weight of roots and aboveground parts whereas the mixture of GA₃ and kNAA showed a decreasing effect. An increase of roots size was observed using higher doses (200 mg l⁻¹) of kinetin and daminozide while a decreasing tendency appeared with the application of the other preparations.     

Effect of emulsified feeding of oily substrate via submerged ceramic membranes on surfactant production in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> fermentation

Aqueous two-phase systems often face mass transfer limitations due to very poor miscibility of the fluids, and to enhance the homogeneity (or emulsification) in the reaction volume, high energy inputs are required which result in high shear forces in the culture medium. For the purposes of emulsification, microporous systems have advantages over other conventional methods due to mild operating conditions and narrow droplet-size distribution. In this study, emulsification within the culture volume was achieved by feeding the oily substrate (dispersed phase) into the aqueous medium (dispersion phase) via ceramic membranes integrated in the bioreactor. The method was investigated for bioprocesses aimed at producing rhamnolipids and polyhydroxyalkanoates (PHA). Better homogenization of the mixed volume and hence improved consumption of oily substrate was successful. Surfactants are produced by various bacterial cultures, especially Pseudomonas aeruginosa species, when oil is present as the only carbon source. Are surfactants produced only as a result of bacteria feeding on the oily substrate, or as a requirement to feed on the oily substrate, owing to their surface-active characteristics? This paper also intends to draw some conclusions in this respect.     

Appropriateness of Diagnostic Coronary Angiography as a Measure of Cardiac Ischemia Testing in Non-Emergency Patients – A Retrospective Cross-Sectional Analysis

BackgroundAdequate application of guidelines concerning non-invasive ischemia testing (NIIT) could avoid inappropriate invasive testing in non-emergency situations. Hardly any data exists regarding frequency and appropriateness of diagnostic coronary angiography (CA). The aim of this study was to evaluate the proportion and predictors of patients without NIIT prior to elective purely diagnostic CA without therapeutic intervention.MethodsRetrospective cross-sectional analysis of insurance claims data from 2012 and 2013. Patients <18 years, acute cardiac ischemia and emergency procedures and patients insured in a managed care model were excluded from analysis. The proportion of patients with NIIT procedures (stress-ECG, transthoracic echocardiography, stress echocardiography, scintigraphy, computer tomography, heart MRI) undertaken within two months before diagnostic CA was assessed. Multiple logistic regression analysis was applied to investigate independent determinants for receiving NIIT.Findings2714 patients were included for analysis. 37.5% (1018) did not receive any NIIT before CA. When high risk patients (patients having received therapeutic cardiac intervention within one month after or 18 months prior to diagnostic CA, n = 766) were excluded 34.3% (669) did not receive NIIT before CA. High risk status as well as >6 chronic comorbidities were independently associated with a lower proportion of NIIT (p<0.0001, OR 0.607 and p = 0.0041, OR 0.648), when additionally controlled for age, sex, language area, insurance coverage, inpatient treatment, cardiovascular medication and lower number of chronic comorbidities. Age (p<0.05, OR 1.009) and intake of oral antiplatelet therapy (p<0.0001, OR 1.914) were independently associated with a higher proportion of NIIT when controlled for the mentioned cofactors.ConclusionsOur data show that despite the existence of guidelines a substantial overuse of a potentially harmful and inappropriate diagnostic intervention is performed suggesting the need for improvement of diagnostic pathways prior to invasive testing.