A gene cloning system for the aranciamycin producer strain Streptomyces echinatus Lv 22

Streptomyces echinatus Lv 22 (=DSM 40730) produces an anthracycline antibiotic aranciamycin. Development of DNA transfer using conjugation from Escherichia coli into this strain is described. Various replicative plasmids (pKC1139, pKC1218E, pSOK1O1, pCHZ101) as well as actinophage (φC31- and VWB-based vectors pSET152 and pVWB, respectively, were transferred from E. coli ET12567 (pUB307) into the S. echinatus at a frequency ranging from 2.4 × 10^?3 to 1.6 × 10^?4. The transconjugants did not differ from wild type in their ability to produce aranciamycin and morphological features. There is one attB site for pSET152 and pVWB integrative plasmids in the S. echinatus chromosome. Developed DNA transfer system was used for expression of heterologous regulatory genes in S. echinatus cells. Expression of relA gene of ppGpp synthetase increased antibiotic production in S. echinatus. The absA2 gene of S. ghanaensis appears to play a negative role in the control of aranciamycin biosynthesis. Additional copies of absA2 leads to inhibition of aranciamycin’s production. absB and afsS had no effect on aranciamycin biosynthesis as well as on the morphological features of S. echinatus. Obtained results indicate efficiency of the developed system for gene cloning in S. echinatus.     

Conformer equilibria of triostin A and its conformer-specific interaction with nucleic acid bases

A quinoxaline antibiotic triostin A has a bicyclic octadepsipeptide structure. Proton and carbon-13 nmr spectra showed the presence of two symmetrical conformations favoring polar and nonpolar solvents, respectively. They interconvert slowly on the nmr time scale, and this slow interconversion is due to the cooperative effects of the presence of the quinoxaline ring and the N-methyl peptide bonds. Reversal of the chirality of the disulfide bond as the origin of the slow exchange was excluded by the presence of two conformers for S-benzyltriostin A. Conformer 2, which favors the polar solvent, can form hydrogen-bonded complexes with purine nucleoside derivatives in organic solvents, but conformer 1 does not. The binding sites were elucidated and a mode of interaction with DNA proposed.     

Interactions of Quinoxaline Antibiotic and DNA.: The Molecular Structure of a Triostin A—d(GCGTACGC) Complex

Abstract

The crystal structure of a DNA. octamer d(GCGTA.CGC) complexed to an antitumor antibiotic, triostin A, has been solved and refined to 2.2 Å resolution by x-ray diffraction analysis. The antibiotic molecule acts as a true bis intercalator surrouding the d(CpG) sequence at either end of the unwound right-handed DNA. double helix. A.s previously observed in the structure of triostin A.—d(CGTA.CG) complex (A.H.-J. Wang, et. al., Science, 225,1115–1121 (1984)), the alanine amino acid residues of the drug molecule form sequence-specific hydrogen bonds to guanines in the minor groove. The two central A · T base pairs are in Hoogsteen configuration with adenine in the syn conformation. In addition, the two terminal G · C base pairs flanking the quinoxaline rings are also held together by Hoogsteen base pairing. This is the first observation in an oligonucleotide of. Hoogsteen G · C base pairs where the cytosine is protonated. The principal functional components of a bis-intercalative compound are discussed.     

Fractals and the analysis of growth paths

A simple practical method exists for classifying and comparing planar curves composed of connected line segments. This method assigns, a single numberD, the fractal dimension, to each curve.D=log(n)/[log(n)+log(d/L)], where:n is the number of line segments,L is the total length of the line segments, andd is the planar diameter of the curve (the greatest distance between any two endpoints). At one end of the spectrum, for straight line curves,D=1; at the other end of the spectrum, for random walk curves,D→2. Standard statistics are done on the logarithms of the fractal dimension [log(D)]. With this measure, trails of biological movement, such as the growth paths of the cells and the paths of wandering organisms, can be analyzed to determine the likelihood that these trails are random walks and also to compare the straightness of the trails before and after experimental interventions.     

The inhibition of food intake in the dog by LDS, mescaline, psilocin, d-amphetamine and phenylisopropylamine derivatives.

Both LSD and $\text{d}$-amphetamine decreased food intake in the dog; however, the dose-response curve for amphetamine had a significantly steeper slope than that for LSD. Psilocin and mescaline also suppressed food intake with dose-response curves parallel to that of LSD. Seven ring-substituted phenylisopropylamine derivatives, that have been abused for their supposed hallucinogenic properties, also decreased food intake and produced dose-response curves parallel to that of $\text{d}$-amphetamine. It is concluded that the appetite suppressant effects of amphetamine and LSD can be distinguished by differences in slopes of the dose response curves. However, this difference does not pertain to all LSD-like drugs since some substituted amphetamines with LSD-like neuropharmacologic and behavioral profiles can show slopes for appetite suppression as steep as that of amphetamine.     

Contrasting root associated fungi of three common oak-woodland plant species based on molecular identification: host specificity or non-specific amplification?

An increasingly popular approach used to identify arbuscular mycorrhizal (AM) fungi in planta is to amplify a portion of AM fungal small subunit ribosomal DNA (SSU-rDNA) from whole root DNA extractions using the primer pair AM1-NS31, followed by cloning and sequencing. We used this approach to study the AM fungal community composition of three common oak-woodland plant species: a grass (Cynosurus echinatus), blue oak (Quercus douglasii), and a forb (Torilis arvensis). Significant diversity of AM fungi were found in the roots of C. echinatus, which is consistent with previous studies demonstrating a high degree of AM fungal diversity from the roots of various hosts. In contrast, clones from Q. douglasii and T. arvensis were primarily from non-AM fungi of diverse origins within the Ascomycota and Basidiomycota. This work demonstrates that caution must be taken when using this molecular approach to determine in planta AM fungal diversity if non-sequence based methods such as terminal restriction fragment length polymorphisms, denaturing gradient gel electrophoresis, or temperature gradient gel electrophoresis are used.     

Electrophysiological properties of pyridine receptors in the crayfish walking leg

Summary Responses of pyridine sensitive units on walking legs of the crayfishOrconectes limosus have been studied using extracellular recording techniques.Post-stimulus-time histograms were established and the mean values of the maximal frequencies were plotted in dose-response curves. The curves can be separated into two groups having a slope of 0.3 and 1 in double logarithmic plots and a sensitivity range of 4 and 2 decades, respectively. This implies two different types of pyridine receptors.Abbreviation vH van Harreveld solution     

Antibiotic susceptibility testing ofCandida albicans by flow cytometry

A flow cytofluorometric susceptibility test (FCST) for in vitro antifungal drug testing ofCandida albicans was developed. Membrane damage was indicated by increased cellular fluorescence owing to propidium iodide or rose bengal uptake. Ketoconazole caused an exponential dose-response effect, best defined by E_max, at therapeutically achievable concentrations (0.02–0.2 µg/ml). This effect was not comparable to the conventional minimum inhibitory concentration (MIC) effect elicited by higher antibiotic concentrations. Amphotericin B, on the other hand, did not elicit E_max, but caused a >1 log dose-response effect which did correspond to the MIC. 5-Fluorocytosine susceptibility was also measurable. Other cytometric data indicating abnormal growth and growth inhibition, as well as conventional growth inhibition testing, confirmed that the 10-h FCST measured useful parameters of in vitro susceptibility.     

Analysis of Colony-Forming Ability of Human Fibroblast Strains by Linear Regression

A linear regression approach is presented for the statistical analysis of dose-response curves obtained by measuring the colony-forming ability of human fibroblast strains. The crucial determination of the dose range in which the linear model can be assumed is achieved by a combination of statistical criteria and biological claims. As a basic quantitative parameter we investigate the slope of the regression line and, by taking reciprocals, we retransform it into the biologically established parameter D₀. Several methods for the combination of estimates are presented.     

Sequences of recently identified adipokinetic peptides: what do they tell us with respect to their hyperlipaemic activity in migratory locusts?

Complete dose-response curves for recently identified members of the AKH/RPCH family (four decapeptides and six octapeptides) have been measured inLocusta migratoria monitoring the lipid-mobilishing activityin vivo. In addition, dose-response curves have been produced for two octapeptide analogues which have a combination of amino acids at position 2 and 3 not occurring in naturally found AKH members. In the decapeptide members changes at position 10 from Thr to Ser are well tolerated, but the combination of Ser at position 5 and 7 around the Pro⁶ residue results in lowered activity, and efficacy of only 70%. In the octapeptides a single Leu/Val exchange at position 2 does not change the potency, however Tyr or Ile at position 2 lead to at least 3-fold loss of activity. The Ser⁵-Pro⁶-Ser⁷ combination in an octapeptide, as in the decapeptide, reduces potency. Octapeptides with 3 aromatic amino acids (Phe², Tyr⁴, Trp⁸) show no typical dose-response curve and have low efficacies. The combination of Val²-Thr³ which has never been found in an octapeptide is tolerated well, but Leu²-Val³ is not. The latter peptide is rather inactive and has a low efficacy; very likely because the hydrophilicity/hydrophobicity pattern at the N-terminus of the peptide is absent.     

EFFECTS OF ELECTRICALLY GENERATED SILVER IONS ON HUMAN CELLS AND WOUND HEALING

A method of producing local antibiotic effects by means of an iontophoretic technique using free silver ions has been evaluated in vitro and in vivo for more than two decades. The antibiotic properties of the technique have proved useful in both animal and human studies. In the course of determining the optimal clinical methodology for infected open wounds, a significant growth stimulation property resembling local tissue regeneration was noted. This has been traced to either the apparent production of dedifferentiation of normal mature cells or the stimulation of preexisting stem cells in the wound, resulting in the production of large numbers of progenitor cells. This process has now been studied in detail, and the results are presented herewith.     

Mathematical interpretation of dose-response curves

The dose-response of an individual organism can be described by a step functions if the organism survives when the dose is below a certain lethal level and dies when this level is exceeded. If, in a population of organism, the lethal dose for an individual has a unimodal distribution, the latter's properties will determine the shape of the population's response in the following manner. If the distribution is symmetric the dose-response curve has a symmetric sigmoid shape when plotted on linear coordinates. The location of the inflection point and the curve's slope around it are determined by the distribution's mode and variance. When the distribution is skewed, the dose-response curve has an asymmetric sigmoid shape which becomes reminiscent of an exponential decay when the distribution is strongly skewed to the right. The population's dose-response curve can be constructed by integration of the step changes over the distribution range. The step function representing the dose-response of an individual organism can be approximate by a Fermi function, and the distribution of an lethal doses can be represented by the Weibull distribution function. When the two functions are combined, the resulting dose-response of the populationS(X)), which is the fraction of survivors after exposure to a doseX, is given by:S(X)=∫ ₀ ¹ [1/{+exp{(X-X _c (φ))/a _i ]}]dω whereX _c (ω)={(1/b)[-ln(1-ω)]}^(1/n),n andb being the constants of the Weibull distribution anda _i an arbitrarily small number, i.e.a _i ≪[X−X _c (ϕ)], whose actual magnitude is of little significance. This model can be used to determine the underlying distributions of experimental dose-response relationship. It was applied to published survival data of microorganisms exposed to pulsed electric field, X-ray radiation and ozone to show that the different observed shapes of the dose-response curve, and shifts between them, can be expressed in terms of the correponding distribution parameters, namely the mode, variance and skewness.     

Studies on the growth metabolism of Bacillus thuringiensis and its vegetative insecticidal protein engineered strains by microcalorimetry

The metabolic power-times curves of Bacillus thuringiensis and its vegetative insecticidal protein-engineered strains were determined at 30°C using a thermal activity monitor, air Isothermal Microcalorimeter, and ampoule method. From the power-times curves, the maximum power (P _max) in the log phase, growth rate constant (k), generation times (t _G), time of the maximum power (t _max), heat effects (Q _log) for log phase, and the total heat effect in 45 h (Q _total) of. B. thuringiensis strains can be obtained. The results indicate that their power-times curves are different. The relationship between their metabolic power-times curves and character of bacteria metabolism, and thermokinetics and gene expression were analyzed and discussed. The character of the bacteria power-times curves reflected the physiologic character of gene expression. The microcalorimetric method proved to be a reliable and sensitive tool for the assessment of growth metabolism, heat output in bacteria and its engineered strains. The determination of the thermokinetic character is beneficial to the control of fermentation. The text was submitted by the authors in English.     

Receptor-Agonist Interactions in Service-Theoretic Perspective,Effects of Molecular Timing on the Shape of Dose-Response Curves

Service-theoretic concepts and methods, widely used in other fields (e.g., telecommunication and operations research), are useful also in a biochemical setting because the treatment of biocatalysts (enzymes, receptors) as servers and their ligands as customers, based on the established formal methods of service or queuing theory, may lead to insights and results unobtainable by conventional, mass-action-law-based theories. In this article, we apply the service-theoretic approach to receptor-agonist systems and show how by changing the stochastic time pattern of “operationally relevant” point events (e.g., instants of agonist arrival, instants of postclimax agonist departure) a great variety of dose-response curves may be generated, even in very simple reaction schemes, which, according to mass action kinetics, invariably lead to hyperbolic r(A) curves (r and A stand for response and agonist concentration, respectively). The molecular timing inherent to a hyperbolic response system is not optimal: for instance, at the agonist concentration A₅₀, half of the agonist molecules are rejected (“lost”) because of unfortunate timing of the arrival events. The fraction of lost arrivers can be diminished considerably if the arrivals are better timed: “sub-Poisson” arrivals improve the timing and, thus, convert hyperbolic r(A) curves into “lifted” nonhyperbolic ones. Conversely, “super-Poisson” arrivals make the nonoptimal timing in hyperbolic response systems even worse and, thus, convert hyperbolic r(A) curves into “depressed” nonhyperbolic ones. Furthermore, under special timing conditions, nonhyperbolic r(A) curves can be generated, which are partly lifted, partly depressed relative to the reference hyperbola, and which resemble in shape well-known nonhyperbolic forms of enzyme and receptor kinetics (negatively cooperative, positively cooperative, and sigmoidal kinetics). In addition unusual (undulatory and sawtooth-like) r(A) curves can be generated solely by changing the temporal pattern of arrival and service completion instants. Virtually any shape of dose-response curves may be obtained by allowing for probability distributions whose characteristic shape varies with their mean; we call such distributions “variomorphic” and apply them to the arrival process of agonist molecules.     

On the interaction between heparin and some aminoglycoside antibiotics

An interaction between the aminoglycoside antibiotics and heparin wherein charge transfer complexes are formed has been investigated to determine the degree of inhibition of antibacterial function of the antibiotic in the complexed form.Minimum inhibitory concentration (MIC) values have been obtained for the action of the aminoclycoside antibiotics tobramycin, gentamicin, amikacin, kanamycin, and streptomycin, on a sensitive strain ofE. coli. Growth curves ofE. coli determined at concentrations of these antibiotics just below the MIC demonstrated significant lengthening of the lag phase relative to control growth curves generated in the absence of antibiotic. Heparin (1 U ml^–1 and 10 U ml^–1) had no effect on control growth curves; however, particularly at the higher concentration, it reduced the effect on the lag phase produced by the aminoglycoside antibiotics. Thus kanamycin, gentamicin, and tobramycin were most affected, while amikacin and streptomycin were least affected. The rank order of inhibition of antibiotic activity by interaction with heparin was in qualitative agreement with previously published figures for the degree of complexation between antibiotics and heparin.     

Production of quinomycin A inStreptomyces lasaliensis

In addition to lasalocid, an oligoether coccidiostatic compound, other compounds are synthesized byStreptomyces lasaliensis. Mutants producing either of two antibiotics, lasalocid A or quinomycin A (an antibiotic of quinoxaline character), were obtained by natural selection and by mutagenesis. Methods of isolation, purification and estimation of both compounds were established.     

Anion modulation of the slowly activating vacuolar channel

A series of n-alkanols and phenyl-substituted n-alkanols (Φ-alkanols) of increasing chain length and phenol were characterized for their ability to block action potentials (APs) in frog sciatic nerves. APs were recorded using the single sucrose-gap method. The degree of AP attenuation when the nerve was exposed to different concentrations of an alcohol was used to construct dose-response curves. The reciprocals of the half-blocking doses (ED₅₀s) were used to obtain a measure of the potency of the alcohols. For n-alkanols and Φ-alkanols, increasing the chain length by the addition of a methylene group increased the potency on average by 3.1 for both groups of alkanols. The addition of a phenyl group caused a potency increase that ranged between the values of 77 and 122. The ED₅₀ for both groups of alkanols could not be solely predicted by the log octanol-water partition coefficient (K _OW ). Using linear solvation energy relations (LSER), the log ED₅₀ could be described as a linear combination of the intrinsic (van der Waals) molar volume (V _I ), polarity (P), and hydrogen bond acceptor basicity (β) and donor acidity (α). Size alone could not predict the ED₅₀ for both n-alkanols and Φ-alkanols. The results are consistent with the hypothesis that alkanols bind to and interact with Na channels to cause AP block. Phenyl group addition to an alkanol markedly increases the molecule's potency. Received: 11 August 2000/Revised: 21 December 2000     

Synthesis of Quinoxaline Azido and Amino Reverse Ribofuranoside and Their O-Regioisomers

A series of quinoxaline azido reverse nucleosides 3a-c and their O-regioisomers 4a-c was prepared by reaction of quinoxaline 1a-c with 3-azido-3-deoxy-1,2-O-isopropylidene-5-p-toluenesulfonyl-D-ribofuranose (2) in the presence of sodium hydride. Structure modification of these interesting structures includes reduction and the subsequent acetylation reactions to give quinoxaline amino and acetyl amino reverse nucleosides and their O-regioisomers.     

Synthesis of new 4-[2-(alkylamino)ethylthio]pyrrolo[1,2-a]quinoxaline and 5-[2-(alkylamino)ethylthio]pyrrolo[1,2-a]thieno[3,2-e]pyrazine derivatives,as potential bacterial multidrug resistance pump inhibitors

The synthesis of new 4-[2-(alkylamino)ethylthio]pyrrolo[1,2-a]quinoxaline derivatives 1a-l is described in five or six steps starting from various substituted nitroanilines 2a-e. The bioisostere 5-[2-(alkylamino)ethylthio]pyrrolo[1,2-a]thieno[3,2-e]pyrazine 1m was also prepared. The new derivatives were evaluated as efflux pump inhibitors (EPIs) in a model targeting the NorA system of Staphylococcus aureus. The antibiotic susceptibility of two strains overproducing NorA, SA-1199B and SA-1, was determined alone and in combination with the neo-synthesised compounds by the agar diffusion method and MIC determination, in comparison with reserpine and omeprazole taken as reference EPIs. A preliminary structure-activity relationship study firstly allowed to clarify the influence of the substituents at positions 7 and/or 8 of the pyrrolo[1,2-a]quinoxaline nucleus. Methoxy substituted compounds, 1b and 1g, were more potent EPIs than the unsubstituted compounds (1a and 1f), followed by chlorinated derivatives (1c-d and 1h). Moreover, the replacement of the N,N-diethylamino group (compounds 1a-e) by a bioisostere such as pyrrolidine (compounds 1f-h) enhanced the EPI activity, in contrast with the replacement by a piperidine moiety (compounds 1i-k). Finally, the pyrrolo[1,2-a]thieno[3,2-e]pyrazine compound 1m exhibited a higher EPI activity than its pyrrolo[1,2-a]quinoxaline analogue 1a, opening the way to further pharmacomodulation.