[The ecology of ticks, tick-borne diseases and biological tick control in Baden-Württemberg]. / Ökologie von Zecken als Überträger von Krankheitserregern in Baden-Württemberg und biologische Zeckenbekämpfung.

Ticks and tick-borne diseases are of great significance for the health of humans and animals. However, the factors influencing their distribution and dynamics are inadequately known. In a project financed by the Baden-Württemberg Ministry of the Environment, Climate and Energy Industry, as part of the program BWPLUS, interdisciplinary specialists work together to determine the influence of weather, (micro)climate, habitat, land use, human activities, and the population dynamics of host animals on the distribution and abundance of ticks and the diseases that they transmit in Baden-Württemberg. The project comprises four modules: the large-scale distribution of ticks in Baden-Württemberg (module 1), detailed studies of host-tick-pathogen interaction in relation to the microclimate (module 2), and the spatial occurrence of important tick-borne pathogens (module 3). The fourth module involves the comprehensive analysis and synthesis of all data in order to determine the relative importance of the factors studied and to develop a risk model. Recently, intensive investigations into tick control have been undertaken using various entomopathogenic fungi and nematodes as well as a parasitoid wasp. Our aim was to determine whether these natural enemies could be used to effectively reduce the number of free-living ticks.

A compendium of antibiotic-induced transcription profiles reveals broad regulation of Pasteurella multocida virulence genes.

The transcriptional responses of Pasteurella multocida to eight antibiotics with known mode of actions (MoAs) and one novel antibiotic compound with an unknown MoA were collected to create a compendium of transcriptional profiles for MoA studies. At minimal inhibitory concentration the three bactericidal compounds enrofloxacin, cefquinome and the novel compound had a minor impact on gene regulation with approximately 1% of the P. multocida genome affected, whilst the bacteriostatic compounds florfenicol, tilmicosin, rifampin, trimethoprim and brodimoprim regulated 20% of the genome. Novobiocin was special in that it regulated 40% of all P. multocida genes. Regulation of target genes was observed for novobiocin, rifampin, florfenicol and tilmicosin and signature genes were identified for most antibiotics. The transcriptional profile induced by the novel compound was unrelated to the compendium profiles suggesting a new MoA. The transcription of many P. multocida virulence factors, particularly genes involved in capsule synthesis and export, LPS synthesis, competence, adherence and iron transport were altered in the presence of antibiotics. Virulence gene transcription was mainly negatively affected, however the opposite effect was also observed in the case of rifampin where the up-regulation of the tad locus involved in tight adherence was seen. Novobiocin and trimethoprim caused a marked reduction in the transcription of capsule genes, which correlated with a concomitant reduction of the capsular layer on the surface of P. multocida. The broad negative impact on virulence gene transcription supports the notion that the therapeutic effect of some antibiotics could be a combination of growth and virulence inhibition.

[Antibiotic resistance patterns of enterococci isolated from estuarine waters]. / Patrones de resistencia a antibióticos de enterococos aislados de aguas estuarinas.

The introduction of antibiotic-resistant bacteria to the environment, affects its hygienic-sanitary quality. The objective of this work is to study the prevalence and the antibiotic resistance patterns of enterococci species isolated from Bahía Blanca estuarine waters. One hundred and three isolates were biochemically identified as Enterococcus spp. The diffusion technique was implemented, by using disks of: vancomycin (Van 30 microg), gentamicin (GenH 120 microg), streptomycin (StrH 300 microg), teicoplanin (T 30 microg), ampicillin (Am 10 microg) and ciprofloxacin (CIP 5 microg) according to the Clinical and Laboratory Standards Institute. Seven Enterococcus species were identified, being Enterococcus faecium and Enterococcus faecalis the most frequent. High level resistance to aminoglycosides was shown by 1.9% of the enterococci whereas 12.6% of the isolates were resistant to CIP. No isolates showed simultaneous resistance to StrH and GenH. Neither resistance to glycopeptides nor to Am was detected. Thirty four per cent of the isolates exhibited susceptibility to all antibiotics tested. Surveillance studies on antimicrobial resistance are usually based upon microorganisms isolated from clinical samples. The findings of this work constitute relevant data for the control of resistant strains, which were believed to be circumscribed to the hospital environment, but are also widespread in the natural sites.

Influence of Ca2+ depletion on cytoskeleton and nucleolus morphology in Trypanosoma brucei.

Trypanosoma brucei bloodstream forms were incubated in a calcium-free medium containing 10 microM ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Under these conditions, addition of 5 microM calcium ionophore A23187 led to striking morphological alterations, as judged by light and electron microscopy. The cytoskeleton of trypanosomes consists of a subpellicular corset of microtubules. Characteristically four of these microtubules are attached invariantly to an extension of the endoplasmic reticulum at the flagellar attachment site. Specifically in this area calcium depletion led to the polymerization of additional microtubules and to a retraction of the endoplasmic reticulum extension from its usual position. Additionally, A23187 led to nucleolus segregation, as revealed by immunocytochemistry using antibodies against DNA and fibrillarin, respectively. Nucleolus segregation, but not microtubule accumulation, was also obtained by using 20 microM camptothecin, a specific inhibitor of topoisomerase I. Our data suggest that intracellular calcium regulation might be important for specific depolymerization/polymerization reactions during the course of cell division and the formation of functional ribosomes.

Down regulation of S-adenosyl-L-methionine decarboxylase activity of Trypanosoma brucei during transition from long slender to short stumpy-like forms in axenic culture.

Long slender trypanosomes, isolated from infected mouse blood or from cryopreserved stabilates, respectively, were unable to grow in conditioned media (cMEM), prepared from the declining phase of axenic bloodstream form cultures. Additionally, mixtures of fresh medium and cMEM led to decreased growth rates and, in accordance to the amount of cMEM used, to a decreased S-adenosyl-L-methionine decarboxylase (Ado-MetDC; E.C. 4.1.1.50) activity. Since addition of polyamines could not overcome the process of transition from dividing to non-dividing cells and the intracellular S-adenosyl-L-methionine (AdoMet), ornithine and putrescine concentrations seemed unaltered during the course of cultivation, we questioned if polyamine metabolism is involved in this transition process. Activities of two key enzymes of polyamine metabolism, AdoMetDC and ornithine decarboxylase (ODC; E.C. 4.1.1.17) were therefore monitored during different growth stages. Our results revealed a specific activity of 44 pmol min-1 mg protein-1 for AdoMetDC and a KM of 10 mu M for AdoMet. Methylglyoxal bis(guanylhydrazone) showed a Ki of 6 mu M. The constant activity of the enzyme during a 7 h time-course in the presence of cycloheximide indicates a t1/2 of more than 7 h for the trypanosomal enzyme. Enzyme activity in trypanosomes isolated from infected laboratory animals and from logarithmic phase bloodstream or procyclic form cultures was high according to a high dividing rate, whereas enzyme activity in parasites isolated from the stationary phase of bloodstream from culture was negligible. In these cultures, AdoMetDC activity decreased with a t1/2 of 7 h during transition from long slender to short stumpy-like forms as soon as the stationary phase was reached. ODC activity was high (approximately 300 pmol min-1 mg protein-1) in dividing trypanosomes isolated from infected animals as well as from logarithmic phase bloodstream or procyclic form cultures and decreased also during transition with a t1/2 of 10 h.

Characterisation of Tc-cpl-1, a cathepsin L-like cysteine protease from Toxocara canis infective larvae.

Cysteine proteases play vital biological roles in both intracellular and extracellular environments. A cysteine protease migrating at 30 kDa was identified in somatic extracts of Toxocara canis larvae (TEX), by its binding to the biotinylated inhibitor Phe-Ala-CH2F. TEX proteases readily cleaved the cathepsin L- and B-specific peptide substrate Z-Phe-Arg-AMC and to a lesser extent, the cathepsin B-specific peptide Z-Arg-Arg-AMC. Excretory/secretory (TES) products of T. canis larvae did not cleave either substrate. Partial sequence encoding the 5' end of a cysteine protease cDNA from infective T. canis larvae was then obtained from an expressed sequence tag (EST) project. The entire cDNA (termed Tc-cpl-1) was subsequently sequenced and found to encode a preproenzyme similar to cathepsin L-like proteases (identities between 36 and 69%), the closest homologues being two predicted proteins from Caenorhabditis elegans cosmids, a cathepsin L-like enzyme from Brugia pahangi and a range of parasite and plant papain-like proteases. Sequence alignment with homologues of known secondary structure indicated several charged residues in the S1 and S2 subsites involved in determining substrate specificity. Some of these are shared with human cathepsin B, including Glu 205 (papain numbering), known to permit cleavage of Arg-Arg peptide bonds. The recombinant protease (rTc-CPL-1) was expressed in bacteria for immunisation of mice and the subsequent antiserum shown to specifically react with the 30 kDa native protease in TEX. Sera from mice infected with the parasite also contained antibodies to rTc-CPL-1 as did sera from nine patients with proven toxocariasis; control sera did not. Larger scale studies are underway to investigate the efficacy of rTc-CPL-1 as a diagnostic antigen for human toxocariasis, the current test for which relies on whole excretory/secretory antigens of cultured parasites.

A novel cultivation technique for long-term maintenance of bloodstream form trypanosomes in vitro.

We used an axenic cultivation system to grow African trypanosomes in vitro. Long-term cultivation for more than 60 days has been achieved by replacing the culture medium at regular intervals between 6 and 48 h. In contrast to a control culture without medium replacement, increasing amounts of maximum cell concentrations have been obtained, ranging from 5 x 10(6) to 2 x 10(7) trypanosomes ml-1, whereas the generation doubling time remained constant (about 6 h). Higher cell concentrations have only been obtained by total medium replacement; neither addition of fresh medium nor serum led to a higher cell yield, suggesting that a trypanosome-derived factor or metabolite accumulated in the medium rather than medium was depleted of an essential nutrient. Most interestingly, however, successive waves have been obtained which eventually led to a damped oscillation curve with a constant high population density after about 40 days of cultivation. Cultures were started with a homogeneous population of the long-slender form. As judged by light microscopy, cells showed a stumpy morphology during the declining phase and became slender again in the following growth phase. At later time points, when cells remained in a stationary phase at high population density, many different morphological stages have been observed, similar to those described by early authors as intermediate forms [Ormerod, W. E. (1979) In: Biology of the Kinetoplastida, Vol. 2, pp. 340-393], although many dividing forms are still present at that time. In contrast, identically treated procyclic cultures were unable to produce cyclic growth waves. Based on these results, a novel concept considering a possible differentiation mechanism is discussed.

Fast calculation of molecular polar surface area as a sum of fragment-based contributions and its application to the prediction of drug transport properties.

Molecular polar surface area (PSA), i.e., surface belonging to polar atoms, is a descriptor that was shown to correlate well with passive molecular transport through membranes and, therefore, allows prediction of transport properties of drugs. The calculation of PSA, however, is rather time-consuming because of the necessity to generate a reasonable 3D molecular geometry and the calculation of the surface itself. A new approach for the calculation of the PSA is presented here, based on the summation of tabulated surface contributions of polar fragments. The method, termed topological PSA (TPSA), provides results which are practically identical with the 3D PSA (the correlation coefficient between 3D PSA and fragment-based TPSA for 34 810 molecules from the World Drug Index is 0.99), while the computation speed is 2-3 orders of magnitude faster. The new methodology may, therefore, be used for fast bioavailability screening of virtual libraries having millions of molecules. This article describes the new methodology and shows the results of validation studies based on sets of published absorption data, including intestinal absorption, Caco-2 monolayer penetration, and blood-brain barrier penetration.

Optimizing strategies for laser angioplasty.

The intrinsic optical properties of normal and diseased vascular tissues and their interaction with continuous wave (cw) and pulsed laser light were investigated to determine the optimal source for laser angioplasty. Both intima and atheromatous plaque demonstrated increasing spectral absorbance at shorter wavelengths (in the near ultraviolet). The relative differences in absorbance between diseased and nondiseased tissues were not sufficient to allow selective ablation of plaque. Atheromatous plaque appears more resistant than normal intima to damage by cw argon laser. The interaction of tissue with a high-power, pulsed ultraviolet laser showed a nonlinear response as pulse repetition rate and pulse energy were varied. From theoretical considerations and our experimental results, we propose that a pulsed ultraviolet laser with 50 millijoules of power per pulse and a repetition rate of 10 pps would be safer and more effective for recanalization than the cw argon laser.

Rapid access to infrared reference spectra of arbitrary organic compounds: scope and limitations of an approach to the simulation of infrared spectra by neural networks

Substance identification by infrared spectroscopy is performed by comparison of the experimental spectrum with a reference spectrum from a printed compilation or a database. If the analyzed compound can not be found in a database the corresponding reference spectrum has to be simulated. In order to achieve this, several reasonable candidates of structures for the compound at hand have to be conceived and for all these, infrared spectra have to be developed. The simulated spectrum that is most similar to the experimental suggests the correct structure. A rapid spectrum prediction method based on neural networks has been developed that supplies reference spectra for any organic compound. The scope and limitations of this method will be discussed on a test set of 16 compounds representing a broad range of organic chemistry.

MR and conventional angiography: work in progress toward assessing utility in radiology.

RATIONALE AND OBJECTIVES: The authors assessed health-related quality of life changes associated with peripheral x-ray angiography and magnetic resonance (MR) angiography. MATERIALS AND METHODS: Utility (the desirability or preference that individuals exhibit for a particular health state) was assessed in 30 patients with peripheral vascular disease referred for angiography by using a rating scale, additional categoric scaling questions to separate preference from experience, a willingness-to-pay technique, functional and cognitive status questions, and a time trade-off technique. All patients underwent both MR angiography and x-ray angiography. RESULTS: Patients reported significantly (P < .05) less anxiety after the test, less pain after the test, fewer new physical limitations, and less effect on performance of daily activities with MR angiography. Findings from the overall rating scale and categoric scaling questions also significantly (P < .05) favored MR angiography. Patients were willing to pay a mean of 2.12% of annual income to avoid MR angiography and a mean of 7.41% to avoid x-ray angiography. The median quality-adjusted life gain required by patients to undergo the procedures was 52.5-60 days for x-ray angiography and 10.5 days for MR angiography, without discounting. CONCLUSION: X-ray angiography has more profound short-term adverse effects on life than does MR angiography. Preference-based measures can be adapted to elicit patient values for short-term health states as seen in radiology.

Web-based cheminformatics and molecular property prediction tools supporting drug design and development at Novartis.

Web-based tools offer many advantages for processing chemical information, most notably ease of use and high interactivity. Therefore more and more pharmaceutical companies are using web technology to deliver sophisticated molecular processing tools directly to the desks of their chemists, to assist them in the process of designing and developing new drugs. In this paper, the web-based cheminformatics system developed at Novartis and currently used by more than thousand users is described. The system allows various molecular modeling and molecular processing tasks, including the calculation of molecular and substituent properties, property-based virtual screening, visualization of molecules, bioisosteric design, diversity analysis, and support of combinatorial chemistry. The methodology to calculate various molecular properties relevant to drug design is described, including the prediction of intestinal absorption, blood-brain barrier penetration, efflux, and water solubility. Information about the web technology used is also provided.

Decision support systems for chemical structure representation, reaction modeling, and spectra simulation.

The choice of an appropriate structure coding scheme is the secret to success in QSAR studies. Depending on the problem at hand, 2D or 3D descriptors have to be chosen; the consideration of electronic effects might be crucial, conformational flexibility has to be of special concern. Artificial neural networks, both with unsupervised and with supervised learning schemes, are powerful tools for establishing relationships between structure and physical, chemical, or biological properties. The EROS system for the simulation of chemical reactions is briefly presented and its application to the degradation of s-triazine herbicides is shown. It is further shown how the simulation of chemical reactions can be combined with the simulation of infrared spectra for the efficient identification of the structure of degradation products.

A combined bioinformatics and chemoinformatics approach for the development of new antiparasitic drugs.

A modern concept for the development of novel antiparasitic drugs is the combination of bioinformatics and chemoinformatics approaches. This covers, for example, the identification of target proteins serving as molecular points of attack for parasiticides--the idea is that, owing to some essential role, inhibition of a target protein should eradicate the parasite. To prevent toxicity problems for vertebrate host organisms, it is advantageous that these proteins show significant differences from their vertebrate counterparts. In the present work, we identified potential target proteins in parasitic nematodes (Ascaris suum, Brugia malayi, and Haemonchus contortus) and arthropods (Boophilus microplus and Rhipicephalus appendiculatus) using bioinformatic sequence comparison methods on expressed sequence tags. Interesting target proteins (e.g., S-adenosyl-l-methionine synthetase) were characterized in detail by subjecting them to in-depth bioinformatic analysis. S-Adenosyl-l-methionine synthetase was also used to elucidate chemoinformatics approaches like homology modeling and docking, which represent appropriate methods for generating valuable data for the development of new drug candidates.

Radiative spectral transfer in ruby.

The first direct observation of radiative spectral transfer within an inhomogeneously broadened line (the R(1) line of ruby) is reported. As opposed to nonradiative transfer, the temperature dependence of the radiative process is consistent with exponential, in agreement with recent predictions. A temperature-independent, resonant process is also directly observed. The implication of these results for the concept of microscopic strain broadening is discussed.

A combined application of reaction prediction and infrared spectra simulation for the identification of degradation products of s-triazine herbicides.

Substance identification in analytical chemistry is usually performed by comparing an experimental spectrum with a reference spectrum. Especially in environmental chemistry, reference spectra from databases are only available for a limited number of compounds. The combination of the reaction prediction system EROS and of infrared spectra simulation is a powerful tool for computer-assisted substance identification. First, possible degradation products of a chemical are predicted and then the infrared spectra of all these compounds are simulated. Comparison of the simulated infrared spectra with experimental spectra allows one to identify the structure of compounds. The method is demonstrated with the example of s-triazine herbicides.

Pulsed dye laser applied to modulated spectroscopy magnetic circular dichroism.

A tunable jet-stream dye laser pumped by a cavity-dumped argon laser has been synchronized to a photoelastic modulator to measure magnetic circular dichroism. Gated signal amplification takes advantage of the laser's pulsed operation to detect weak dichroic signals. Comparative data show that the dye laser and gated amplification give a SNR better than that obtained with the previously used lamp, monochromator, and lockin detector. Other applications of the dye laser and gated amplification are discussed.

Target-based drug discovery for the development of novel antiinfectives.

In the 20th century and especially during the last 50 years, antiinfectives have been increasingly used to control and prevent infectious diseases. Unfortunately the resistance of microorganisms to these pharmaceuticals has increased as well. At the same time the discovery process for novel antiinfectives, the so-called "conventional" screening approach, involves testing natural products or derivatives of known compounds in in vitro cultures. By now it is obvious that this screening approach did not meet the expectations to generate a sufficient number of novel drug candidates. Consequently, studies for selective antiinfectives with new modes of action, which are able to break resistance, are highly desirable for human and animal health. The enormous advance in sequencing technologies--leading to a constantly growing number of known microbial genomes--together with the rapid development of computer power and bioinformatic software tools, now makes it possible to identify genes and gene products that are essential to the pathogenic organisms and are therefore considered to be novel targets for the development of new antiinfectives. When these potential targets have been validated by sophisticated laboratory methods, large diverse compound libraries can be tested in in vitro assays using high-throughput screening. This approach will most likely generate an increasing number of novel lead structures that will be specifically optimized by modern combinatorial chemistry and subsequently lead to new antiinfective candidates strengthening the armoury of weapons available to fight infectious diseases in humans and animals.

Expression and alteration of the S2 subsite of the Leishmania major cathepsin B-like cysteine protease.

The mature form of the cathepsin B-like protease of Leishmania major (LmajcatB) is a 243 amino acid protein belonging to the papain family of cysteine proteases and is 54% identical to human-liver cathepsin B. Despite the high identity and structural similarity with cathepsin B, LmajcatB does not readily hydrolyse benzyloxycarbonyl-Arg-Arg-7-amino-4-methyl coumarin (Z-Arg-Arg-AMC), which is cleaved by cathepsin B enzymes. It does, however, hydrolyse Z-Phe-Arg-AMC, a substrate typically cleaved by cathepsin L and B enzymes. Based upon computer generated protein models of LmajcatB and mammalian cathepsin B, it was predicted that this variation in substrate specificity was attributed to Gly234 at the S2 subsite of LmajcatB, which forms a larger, more hydrophobic pocket compared with mammalian cathepsin B. To test this hypothesis, recombinant LmajcatB was expressed in the Pichia pastoris yeast expression system. The quality of the recombinant enzyme was confirmed by kinetic characterization, N-terminal sequencing, and Western blot analysis. Alteration of Gly234 to Glu, which is found at the corresponding site in mammalian cathepsin B, increased recombinant LmajcatB (rLmajcatB) activity toward Z-Arg-Arg-AMC 8-fold over the wild-type recombinant enzyme (kcat/Km=3740+/-413 M-1.s-1 versus 472+/-72.4 M-1.s-1). The results of inhibition assays of rLmajcatB with an inhibitor of cathepsin L enzymes, K11002 (morpholine urea-Phe-homoPhe-vinylsulphonylphenyl, kinact/Ki=208200+/-36000 M-1. s-1), and a cathepsin B specific inhibitor, CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l- prolin e, kinact/Ki=199200+/-32900 M-1.s-1], support the findings that this protozoan protease has the P2 specificity of cathepsin L-like enzymes while retaining structural homology to mammalian cathepsin B.