[On freedom of scientific research]. / Freiheit in der Forschung.

Debates about science and, more specifically, about scientific research quickly bring up the question about its freedom. Science is readily blamed for technological disasters or criticized for nursing fantasies of omnipotence and commercial gain. This prompts the call for a restriction of its freedom. At the same time, society's demands on science are enormous, to the effect that science and technology have acquired the status of a deus-ex-machina: they are expected to furnish short-term, affordable, and convenient solutions to a wide range of problems, including issues of health, transportation, food and, more generally, a comfortable life. What kind of freedom is required to meet these expectations? Who is in a position to grant it? What does freedom for science mean and how is it linked to responsibility? The paper examines the current situation of freedom in scientific research and of its restrictions, many of which are mentally or economically conditioned. It calls for the involvement of an informed, self-confident bourgeoisie in research decisions and for the educational measures this necessitates. Finally, it demands a greater appreciation of education (rather than training) as the basis of social trust, and the recognition of continuous education as a productive investment of time and a crucial element in the employment of social goods.

Adenovirus E1A functions as a cofactor for retinoic acid receptor beta (RAR beta) through direct interaction with RAR beta.

Transcription regulation by DNA-bound activators is thought to be mediated by a direct interaction between these proteins and TATA-binding protein (TBP), TFIIB, or TBP-associated factors, although occasionally cofactors or adapters are required. For ligand-induced activation by the retinoic acid receptor-retinoid X receptor (RAR-RXR) heterodimer, the RAR beta 2 promoter is dependent on the presence of E1A or E1A-like activity, since this promoter is activated by retinoic acid only in cells expressing such proteins. The mechanism underlying this E1A requirement is largely unknown. We now show that direct interaction between RAR and E1A is a requirement for retinoic acid-induced RAR beta 2 activation. The activity of the hormone-dependent activation function 2 (AF-2) of RAR beta is upregulated by E1A, and an interaction between this region and E1A was observed, but not with AF-1 or AF-2 of RXR alpha. This interaction is dependent on conserved region III (CRIII), the 13S mRNA-specific region of E1A. Deletion analysis within this region indicated that the complete CRIII is needed for activation. The putative zinc finger region is crucial, probably as a consequence of interaction with TBP. Furthermore, the region surrounding amino acid 178, partially overlapping with the TBP binding region, is involved in both binding to and activation by AF-2. We propose that E1A functions as a cofactor by interacting with both TBP and RAR, thereby stabilizing the preinitiation complex.

Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling.

The thymidine kinase (TK) genes from herpes simplex virus (HSV) types 1 and 2 were recombined in vitro with a technique called DNA family shuffling. A high-throughput robotic screen identified chimeras with an enhanced ability to phosphorylate zidovudine (AZT). Improved clones were combined, reshuffled, and screened on increasingly lower concentrations of AZT. After four rounds of shuffling and screening, two clones were isolated that sensitize Escherichia coli to 32-fold less AZT compared with HSV-1 TK and 16,000-fold less than HSV-2 TK. Both clones are hybrids derived from several crossover events between the two parental genes and carry several additional amino acid substitutions not found in either parent, including active site mutations. Kinetic measurements show that the chimeric enzymes had acquired reduced K(M) for AZT as well as decreased specificity for thymidine. In agreement with the kinetic data, molecular modeling suggests that the active sites of both evolved enzymes better accommodate the azido group of AZT at the expense of thymidine. Despite the overall similarity of the two chimeric enzymes, each contains key contributions from different parents in positions influencing substrate affinity. Such mutants could be useful for anti-HIV gene therapy, and similar directed-evolution approaches could improve other enzyme-prodrug combinations.

Swiss Canine Cancer Registry 1955-2008: Occurrence of the Most Common Tumour Diagnoses and Influence of Age, Breed, Body Size, Sex and Neutering Status on Tumour Development.

This study is based on the Swiss Canine Cancer Registry, comprising 121,963 diagnostic records of dogs compiled between 1955 and 2008, in which 63,214 (51.83%) animals were diagnosed with tumour lesions through microscopical investigation. Adenoma/adenocarcinoma (n = 12,293, 18.09%) was the most frequent tumour diagnosis. Other common tumour diagnoses were: mast cell tumour (n = 4,415, 6.50%), lymphoma (n = 2,955, 4.35%), melanocytic tumours (n = 2,466, 3.63%), fibroma/fibrosarcoma (n = 2,309, 3.40%), haemangioma/haemangiosarcoma (n = 1,904, 2.80%), squamous cell carcinoma (n = 1,324, 1.95%) and osteoma/osteosarcoma (n = 842, 1.24%). The relative occurrence over time and the most common body locations of those tumour diagnoses are presented. Analyses of the influence of age, breed, body size, sex and neutering status on tumour development were carried out using multiple logistic regression. In certain breeds/breed categories the odds ratios (ORs) for particular tumours were outstandingly high: the boxer had higher ORs for mast cell tumour and haemangioma/haemangiosarcoma, as did the shepherd group for haemangioma/haemangiosarcoma, the schnauzer for squamous cell carcinoma and the rottweiler for osteoma/osteosarcoma. In small dogs, the risk of developing mammary tumours was three times higher than in large dogs. However, small dogs were less likely to be affected by many other tumour types (e.g. tumours of the skeletal system). Examination of the influence of sex and neutering status on tumour prevalence showed that the results depend on the examination method. In all sampling groups the risk for female dogs of developing adenoma/adenocarcinoma was higher than for male dogs. Females had a lower risk of developing haemangioma/haemangiosarcoma and squamous cell carcinoma than males. Neutered animals were at higher risk of developing specific tumours outside the genital organs than intact animals. The sample size allows detailed insight into the influences of age, breed, body size, sex and neutering status on canine tumour development. In many cases, the analysis confirms the findings of other authors. In some cases, the results are unique or contradict other studies, implying that further investigations are necessary.

Swiss Feline Cancer Registry 1965-2008: the Influence of Sex, Breed and Age on Tumour Types and Tumour Locations.

Cancer registries are valuable sources for epidemiological research investigating risk factors underlying different types of cancer incidence. The present study is based on the Swiss Feline Cancer Registry that comprises 51,322 feline patient records, compiled between 1965 and 2008. In these records, 18,375 tumours were reported. The study analyses the influence of sex, neutering status, breed, time and age on the development of the most common tumour types and on their locations, using a multiple logistic regression model. The largest differences between breeds were found in the development of fibrosarcomas and squamous cell carcinomas, as well as in the development of tumours in the skin/subcutis and mammary gland. Differences, although often small, in sex and neutering status were observed in most analyses. Tumours were more frequent in middle-aged and older cats. The sample size allowed detailed analyses of the influence of sex, neutering status, breed and age. Results of the study are mainly consistent with previous analyses; however, some results cannot be compared with the existing literature. Further investigations are necessary, since feline tumours have not been investigated in depth to date. More accurate comparisons would require the definition of international standards for animal cancer registries.

Folding and self-assembly of herpes simplex virus type 1 thymidine kinase.

Thymidine kinase from herpes simplex virus type 1 (HSV1 TK) has been postulated to be a homodimer throughout the X-ray crystallography literature. Our study shows that HSV1 TK exists as a monomer-dimer equilibrium mixture in dilute aqueous solutions. In the presence of 150 mM NaCl, the equilibrium is characterized by a dissociation constant of 2.4 microm; this constant was determined by analytical ultracentrifugation and gel filtration experiments. Dimerization seems to be unfavorable for enzymatic activity: dimers show inferior catalytic efficiency compared to the monomers. Moreover, soluble oligomers formed by self-assembly of TK in the absence of physiological salt concentrations are even enzymatically inactive. This study investigates enzymatic and structural relevance of the TK dimer in vitro. Dissociation of the dimers into monomers is not accompanied by large overall changes in secondary or tertiary structure as shown by thermal and urea-induced unfolding studies monitored by circular dichroism and fluorescence spectroscopy. A disulfide-bridge mutant TK (V119C) was designed bearing two cysteine residues at the dimer interface in order to crosslink the two subunits covalently. Under reducing conditions, the properties of V119C and wild-type HSV1 TK (wt HSV1 TK) were identical in terms of expression yield, denaturing SDS PAGE gel electrophoresis, enzyme kinetics, CD spectra and thermal stability. Crosslinked V119C (V119Cox) was found to have an increased thermal stability with a t(m) value of 59.1(+/-0.5) degrees C which is 16 deg. C higher than for the wild type protein. This is thought to be a consequence of the conformational restriction of the dimer interface. Furthermore, enzyme kinetic studies on V119Cox revealed a K(m) for thymidine of 0.2 microm corresponding to wt HSV1 TK, but a significantly higher K(m) for ATP. The present findings raise the question whether the monomer, not the dimer, might be the active species in vivo.

The retinoic acid receptor-beta 2 contains two separate cell-specific transactivation domains, at the N-terminus and in the ligand-binding domain.

In contrast to other members of the steroid/thyroid hormone superfamily, not much is known about the regions involved in transactivation of the receptors for retinoic acid. To determine the transactivation function of RARs, fusion proteins between the DNA-binding domain of the yeast transcription factor GAL4 and retinoic acid receptor-alpha (RAR alpha) or RAR beta were made. Transfection of these constructs resulted in RA-induced activation of a GAL4-responsive element-containing promoter. Deletion analysis revealed that RAR beta-2 has two transcription activation functions (TAFs). TAF-1 activates transcription constitutively and was mapped to the first 32 amino acids of the A-region. TAF-2 is located in the ligand-binding domain between amino acids 137 and 410 and activated transcription only in the presence of RA. The presence of two TAFs was confirmed by cotransfection of RAR beta deletion constructs with the human RAR beta-2 promoter as reporter, showing that the absence of RAR beta TAF-1 causes a decrease in transactivation, whereas truncation of TAF-2 completely blocks this function. Internal deletions in the ligand-binding domain in both GAL-RAR beta and RAR beta expression constructs resulted in a nonfunctional receptor, indicating that the complete ligand-binding domain is required for its transactivation function. Furthermore, we have shown that the contribution of the two TAFs in transcription activation varies among different cell lines, suggesting that they act in a cell-specific manner.

Identification of a novel human organic anion transporting polypeptide as a high affinity thyroxine transporter.

Transport of various amphipathic organic compounds is mediated by organic anion transporting polypeptides (OATPs in humans, Oatps in rodents), which belong to the solute carrier family 21A (SLC21A/Slc21a). Several of these transporters exhibit a broad and overlapping substrate specificity and are expressed in a variety of different tissues. We have isolated and functionally characterized OATP-F (SLC21A14), a novel member of the OATP family. The cDNA (3059 bp) contains an open reading frame of 2136 bp encoding a protein of 712 amino acids. Its gene containing 15 exons is located on chromosome 12p12. OATP-F exhibits 47-48% amino acid identity with OATP-A, OATP-C, and OATP8, the genes of which are clustered on chromosome 12p12. OATP-F is predominantly expressed in multiple brain regions and Leydig cells of the testis. OATP-F mediates high affinity transport of T(4) and reverse T(3) with apparent K(m) values of approximately 90 nM and 128 nM, respectively. Substrates less well transported by OATP-F include T(3), bromosulfophthalein, estrone-3-sulfate, and estradiol-17beta-glucuronide. Furthermore, OATP-F-mediated T(4) uptake could be cis-inhibited by L-T(4) and D-T(4), but not by 3,5-diiodothyronine, indicating that T(4) transport is not stereospecific, but that 3',5'-iodination is important for efficient transport by OATP-F. Thus, in contrast to most other family members, OATP-F has a more selective substrate preference and may play an important role in the disposition of thyroid hormones in brain and testis.

E1A functions as a coactivator of retinoic acid-dependent retinoic acid receptor-beta 2 promoter activation.

The retinoic acid (RA) receptor (RAR) beta 2 promoter is strongly activated by RA in embryonal carcinoma (EC) cells. We examined this activation in the P19 EC-derived END-2 cell line and in E1A-expressing counterparts and found strong RA-dependent RAR beta 2 promoter activation in the E1A-expressing cells, which was not observed in the parental cell line, indicating a possible role for E1A in RAR beta 2 activation. In transient transfection assays, E1A functioned as a coactivator of RA-dependent RAR beta 2 promoter activation and, moreover, was able to restore this activation in cells lacking RAR beta 2 activation. By deletion analysis, two regions in the RAR beta 2 promoter were identified that mediate the stimulatory effect of E1A: the RA response element and TATA box-containing region and a more up-stream region between -180 and -63, in which a cAMP response element-related motif was identified as a target element for E1A. In addition, determination of endogenous E1A-like activity by measuring E2A promoter activity in transient transfection assays in EC and differentiated cells revealed a correlation between RA-dependent RAR beta 2 promoter activation and the presence of this activity, suggesting an important role for the cellular equivalent of E1A in regulation of the RAR beta 2 promoter.

Swiss Feline Cancer Registry: A Retrospective Study of the Occurrence of Tumours in Cats in Switzerland from 1965 to 2008.

Cancer is one of the leading causes of death in companion animals. Information on the epidemiology of cancer is instrumental for veterinary practitioners in patient management; however, spontaneously arising tumours in companion animals also resemble those in man and can provide useful data in combating cancer. Veterinary cancer registries for cats are few in number and have often remained short-lived. This paper presents a retrospective study of tumours in cats in Switzerland from 1965 to 2008. Tumour diagnoses were coded according to topographical and morphological keys of the International Classification of Oncology for Humans (ICD-O-3). Correlations between breed, sex and age were then examined using a multiple logistic regression model. A total of 18,375 tumours were diagnosed in 51,322 cats. Of these, 14,759 (80.3%) tumours were malignant. Several breeds had significantly lower odds ratios for developing a tumour compared with European shorthair cats. The odds of a cat developing a tumour increased with age, up to the age of 16 years, and female cats had higher risk of developing a tumour compared with male cats. Skin (4,970; 27.05%) was the most frequent location for tumours, followed by connective tissue (3,498; 19.04%), unknown location (2,532; 13.78%) and female sexual organs (1,564; 8.51%). The most common tumour types were epithelial tumours (7,913; 43.06%), mesenchymal tumours (5,142; 27.98%) and lymphoid tumours (3,911; 21.28%).

The Swiss Canine Cancer Registry: a retrospective study on the occurrence of tumours in dogs in Switzerland from 1955 to 2008.

Diagnostic records are a key feature of any cancer epidemiology, prevention or control strategy for man and animals. Therefore, the information stored in human and animal cancer registries is essential for undertaking comparative epidemiological, pathogenic and therapeutic research. This study presents the Swiss Canine Cancer Registry, containing case data compiled between 1955 and 2008. The data consist of pathology diagnostic records issued by three veterinary diagnostic laboratories in Switzerland. The tumours were classified according to the guidelines of the International Classification of Oncology for Humans on the basis of tumour type, malignancy and body location. The dogs were classified according to breed, age, sex, neuter status and place of residence. The diagnostic data were correlated with data on the Swiss general dog population and the incidence of cancer in dogs was thus investigated. A total of 67,943 tumours were diagnosed in 121,963 dogs and 47.07% of these were malignant. The most common tumour location was the skin (37.05%), followed by mammary glands (23.55%) and soft tissue (13.66%). The most common tumour diagnoses were epithelial (38.45%), mesenchymal (35.10%) and lymphoid tumours (13.23%). The results are compared with data in other canine registries and similarities in tumour distribution and incidence are noted. It is hoped that this study will mark the beginning of continuous registration of dog tumours in Switzerland, which, in turn, will serve as a reference for research in the fields of animal and human oncology.

The structures of thymidine kinase from herpes simplex virus type 1 in complex with substrates and a substrate analogue.

Thymidine kinase from Herpes simplex virus type 1 (TK) was crystallized in an N-terminally truncated but fully active form. The structures of TK complexed with ADP at the ATP-site and deoxythymidine-5'-monophosphate (dTMP), deoxythymidine (dT), or idoxuridine-5'-phosphate (5-iodo-dUMP) at the substrate-site were refined to 2.75 A, 2.8 A, and 3.0 A resolution, respectively. TK catalyzes the phosphorylation of dT resulting in an ester, and the phosphorylation of dTMP giving rise to an anhydride. The presented TK structures indicate that there are only small differences between these two modes of action. Glu83 serves as a general base in the ester reaction. Arg163 parks at an internal aspartate during ester formation and binds the alpha-phosphate of dTMP during anhydride formation. The bound deoxythymidine leaves a 35 A3 cavity at position 5 of the base and two sequestered water molecules at position 2. Cavity and water molecules reduce the substrate specificity to such an extent that TK can phosphorylate various substrate analogues useful in pharmaceutical applications. TK is structurally homologous to the well-known nucleoside monophosphate kinases but contains large additional peptide segments.

The effect of substrate binding on the conformation and structural stability of Herpes simplex virus type 1 thymidine kinase.

The structure of Herpes simplex virus type 1 thymidine kinase (TK(HSV1)) is known at high resolution in complex with a series of ligands and exhibits important structural similarities to the nucleoside monophosphate (NMP) kinase family, which are known to show large conformational changes upon binding of substrates. The effect of substrate binding on the conformation and structural stability of TK(HSV1), measured by thermal denaturation experiments, far-UV circular dichroism (CD) and fluorescence is described, and the results indicate that the conformation of the ligand-free TK(HSV1) is less ordered and less stable compared to the ligated enzyme. Furthermore, two crystal structures of TK(HSV1) in complex with two new ligands, HPT and HMTT, refined to 2.2 A are presented. Although TK(HSV1):HPT does not exhibit any significant deviations from the model of TK(HSV1):dT, the TK(HSV1):HMTT complex displays a unique conformationally altered active site resulting in a lowered thermal stability of this complex. Moreover, we show that binding affinity and binding mode of the ligand correlate with thermal stability of the complex. We use this correlation to propose a method to estimate binding constants for new TK(HSV1)substrates using thermal denaturation measurements monitored by CD spectroscopy. The kinetic and structural results of both test substrates HPT and HMTT show that the CD thermal denaturation system is very sensitive to conformational changes caused by unusual binding of a substrate analog.

Identification of steroid sulfate transport processes in the human mammary gland.

Circulating hormones and local biotransformation of steroid precursors are both sources of estrogen in human mammary tissue. Estrone-3-sulfate (E(1)S) is an important estrogenic form in premenopausal women, and dehydroepiandrosterone sulfate (DHEAS) constitutes a major adrenal precursor. Membrane transport systems that govern delivery of these anionic steroid conjugates to the mammary gland were investigated. RNA was screened by RT-PCR and Northern blotting for expression of organic anion transporting polypeptide (OATP) (solute carrier family 21A) and organic anion transporter (OAT) (solute carrier family 22A) gene families. OATP-B (SLC21A9) was the major carrier expressed; OATP-D (SLC21A11) and OATP-E (SLC21A12) were less abundant. In normal sections, OATP-B immunolocalized to the myoepithelium that surrounds the ductal epithelial cells. In invasive carcinoma, ductal epithelial cells were positive. OATP-B was characterized in stable transfected Chinese hamster ovary cells. E(1)S affinity constant (K(m)) [K(m) = 5 micro mol/liter, maximum velocity (V(max)) V(max) = 777 pmol/mg.min] and DHEAS (K(m) = 9 micro mol/liter, V(max) = 85 pmol/mg.min) were substrates. The prostaglandins (PG) A(1) and PGA(2) stimulated uptake of E(1)S and DHEAS by increasing V(max) 2-fold but not changing K(m). The effect of PGA was selectively blocked by the lipophilic thiol reagent N-ethylmaleimide but not by the hydrophilic acetamido-4'(iodoacetyl)aminostilbene-2,2'-disulfonic acid, suggesting an interaction between the electrophilic cyclopentenone ring and specific cysteine residues of OATP-B.

The three-dimensional structure of thymidine kinase from herpes simplex virus type 1.

Recombinant thymidine kinase from Herpes simplex virus type 1 (ATP:thymidine 5'-phosphotransferase; EC 2.7.1.21), an enzyme of therapeutic importance, was purified and crystallized in an N-terminally truncated but still fully active form. The three-dimensional structure was solved by X-ray diffraction analysis at 3.0 A resolution using isomorphous replacement. The chain fold is presented together with the bound substrates thymidine and ATP. Three chain segments at the surface could not be located. The chain fold, the location of the substrates and presumbly also the catalytic mechanism resemble the well-known adenylate kinases.

Use of fluorescence polarization to monitor MHC-peptide interactions in solution.

We describe here fluorescence polarization-based methods to investigate class I MHC-peptide interactions in solution. Fluorescein-labelled peptides were used to determine MHC/peptide complex association and dissociation constants as well as the equilibrium binding constant (KD). Furthermore, we developed a competition assay for the determination of IC50 values of nonlabelled compounds. Both kinetic and equilibrium parameters are of prime importance for the development of immunomodulating compounds. The assays described here show a good reproducibility and require only picomolar amounts of labelled tracers. A high ratio between the experimental values obtained for bound and free labelled ligand as well as a low standard deviation, permits the detection of class I MHC ligands with low affinity. Fluorescence polarization allows the direct measurement of the ratio between free and bound labelled ligand in solution without any separation step. Thus, in combination with microtiter-plates, the time for analysis is significantly decreased to 10 s per sample. Our assays represent versatile tools for characterizing the binding of single ligands as well as for rapid screening of large numbers of compounds.

Protein-based virtual screening of chemical databases. 1. Evaluation of different docking/scoring combinations.

Three different database docking programs (Dock, FlexX, Gold) have been used in combination with seven scoring functions (Chemscore, Dock, FlexX, Fresno, Gold, Pmf, Score) to assess the accuracy of virtual screening methods against two protein targets (thymidine kinase, estrogen receptor) of known three-dimensional structure. For both targets, it was generally possible to discriminate about 7 out of 10 true hits from a random database of 990 ligands. The use of consensus lists common to two or three scoring functions clearly enhances hit rates among the top 5% scorers from 10% (single scoring) to 25-40% (double scoring) and up to 65-70% (triple scoring). However, in all tested cases, no clear relationships could be found between docking and ranking accuracies. Moreover, predicting the absolute binding free energy of true hits was not possible whatever docking accuracy was achieved and scoring function used. As the best docking/consensus scoring combination varies with the selected target and the physicochemistry of target-ligand interactions, we propose a two-step protocol for screening large databases: (i) screening of a reduced dataset containing a few known ligands for deriving the optimal docking/consensus scoring scheme, (ii) applying the latter parameters to the screening of the entire database.

Nonapeptide analogues containing (R)-3-hydroxybutanoate and beta-homoalanine oligomers: synthesis and binding affinity to a class I major histocompatibility complex protein.

Crystal structures of antigenic peptides bound to class I MHC proteins suggest that chemical modifications of the central part of the bound peptide should not alter binding affinity to the MHC restriction protein but could perturb the T-cell response to the parent epitope. In our effort in designing nonpeptidic high-affinity ligands for class I MHC proteins, oligomers of (R)-3-hydroxybutanoate and(or) beta-homoalanine have been substituted for the central part of a HLA-B27-restricted T-cell epitope of viral origin. The affinity of six modified peptides to the B2705 allele was determined by an in vitro stabilization assay. Four out of the six designed analogues presented an affinity similar to that of the parent peptide. Two compounds, sharing the same stereochemistry (R,R,S,S) at the four stereogenic centers of the nonpeptidic spacer, bound to B2705 with a 5-6-fold decreased affinity. Although the chiral spacers do not strongly interact with the protein active site, there are configurations which are not accepted by the MHC binding groove, probably because of improper orientation of some lateral substituents in the bound state and different conformational behavior in the free state. However we demonstrate that beta-amino acids can be incorporated in the sequence of viral T-cell epitopes without impairing MHC binding. The presented structure-activity relationships open the door to the rational design of peptide-based vaccines and of nonnatural T-cell receptor antagonists aimed at blocking peptide-specific T-cell responses in MHC-associated autoimmune diseases.

Gene technology-based antimetabolite design: the use of an in vitro protein expression system to facilitate antimetabolite design for virally-induced human diseases and malignant conditions.

A precondition for the chemotherapeutic treatment of a variety of virally-induced human diseases and malignant conditions is a highly selective interaction of the drug molecule to be used with it's biological target. To ensure the development of novel, effective drugs, it is essential that the biological target is well characterised with regard to it's structure and activity. Such characterisation relies upon adequate amounts of pure target being available. One of the most important enzymatic importers for antimetabolites is the enzyme thymidine kinase. In this article an in vitro protein expression system is described which facilitates the production of milligram amounts of pure and biologically active thymidine kinase, from a number of important biological sources. Results have shown that the in vitro produced enzyme has the exact biochemical propeties of the in vivo enzyme. Thus the in vitro protein expression system is an ideal vechicle to facilitate an in depth investigation of the enzyme's biological properties.