SPR-based fragment screening: advantages and applications.

Fragment-based screening has recently evolved into a promising strategy in drug discovery, and a range of biophysical methods can be employed for fragment library screening. Relevant approaches, such as X-ray, NMR and tethering are briefly introduced focussing on their suitability for fragment-based drug discovery. In particular the application of surface plasmon resonance (SPR) techniques to the primary screening of large libraries comprising small molecules is discussed in detail. SPR is known to be a powerful tool for studying biomolecular interactions in a sensitive and label-free detection format. Advantages of SPR methods over more traditional assay formats are discussed and the application of available channel and array based SPR systems to biosensing are reviewed. Today, SPR protocols have been applied to secondary screening of compound libraries and hit conformation, but primary screening of large fragment libraries for drug discovery is often hampered by the throughput of available systems. Chemical microarrays, in combination with SPR imaging, can simultaneously generate affinity data for protein targets with up to 9,216 immobilized fragments per array. This approach has proven to be suitable for screening fragment libraries of up to 110,000 compounds in a high throughput fashion. The design of fragment libraries and appropriate immobilization chemistries are discussed, as well as suitable follow-up strategies for fragment hit optimization. Finally, described case studies demonstrate the successful identification of selective low molecular weight inhibitors for pharmacologically relevant drug targets through the SPR screening of fragment libraries.

[Establishing a protein signature from prostate tissue biopsies]. / Erstellung eines Proteinprofils aus Gewebeproben der Prostata.

The prostate-specific antigen test (PSA) has been a major factor contributing to a better management of prostate cancer. The low specificity limits its use in diagnosis especially in early detection of prostate cancer. Multiply expressed proteins need to be identified to establish a disease-specific protein signature that distinguishes between cancerous and noncancerous tissue. The first aim of our study is to identify differentially expressed proteins in both tissues using two-dimensional gel electrophoresis and subsequent mass spectrometry. We elucidated whether prostate biopsies are useful. First results have shown a different protein expression pattern in cancerous and noncancerous tissue. PCR revealed an increasing amount of mRNA for some upregulated proteins. We conclude that biopsies are useful material to establish protein expression patterns.

Expression of annexin AI in conventional renal cell carcinoma (CRCC) correlates with tumour stage, Fuhrman grade, amount of eosinophilic cells and clinical outcome.

There is increasing evidence that Annexin AI (ANX AI) expression is dysregulated in several carcinomas and tumour cell lines. In order to gain insight into the putative role of ANX AI in tumorigenesis, clinical outcome and metastatic potential of conventional renal cell carcinomas (CRCCs) we investigated the expression of ANX AI in CRCCs and metastases. Furthermore, it was elucidated whether ANX AI overexpression affects migratory potential in Caki-1 cells. ANX AI immunohistochemistry was performed on 33 samples of CRCCs and 10 metastases. ANX AI expression was assessed in 12 samples by 2-dimensional gelelectrophoresis (2-DE), subsequent mass spectrometry and RT-PCR. Immunohistochemical data were statistically correlated with pathological parameters, amount of eosinophilic cells and clinical outcome. Furthermore, a haptotactic migration assay was done on Caki-1 cells transfected with ANX AI. Immunostaining for ANX AI was found in 18 tumours and all metastases investigated. Intensity of immunohistochemical staining correlated to Fuhrman grade, amount of eosinophilic cells and clinical outcome. 2-DE and RT-PCR confirmed the presence of ANX AI in neoplastic tissue. Overexpression of ANX AI did not significantly influence cell migration. From these findings ANX AI expression seems to be related to Fuhrman grade, clinical outcome and metastatic potential of CRCCs. Thus ANX AI could serve as a prognostic marker for tumour progression.

Expression of annexin II in conventional renal cell carcinoma is correlated with Fuhrman grade and clinical outcome.

Conventional renal cell carcinomas (CRCCs) were investigated for the expression of annexin II (ANX II) to determine out whether this calcium-binding protein could serve as a useful prognostic marker. CRCCs and adjacent nonneoplastic tissue from 33 patients were investigated for ANX II by immunohistochemistry, RT-PCR, and western blot analysis. ANX II expression was correlated with tumor differentiation (Fuhrman grade) and to clinical outcome. Tumors were composed of ANX II positive and negative cells. In grade I tumors only a weak membranous staining was seen in immunopositive cells. In grade II and III tumors, however, ANX II was seen in the cytoplasm and at the cell membranes of tumor cells. On serial sections membranous and cytoplasmic immunoreactivity for ANX II occurred predominantly in eosinophilic cells whereas clear cells were mostly immunonegative. The ANX II expression in CRCCs was correlated with clinical outcome and Fuhrman grade. Since ANX II expression is correlated with Fuhrman grade and clinical outcome it may be a useful marker for prognosis in CRCC.

Expression of thyroid hormone receptor isoform alpha1 in pancreatic islets.

Thyroid hormone receptors (TR) mediate the action of thyroid hormones. Genetic studies revealed that the individual TR isoforms possess different functions. In the present paper we studied the expression of the isoforms TRalpha1 and TRbeta1 in the murine pancreatic islet. TRalpha1 and TRbeta1 mRNA transcripts and proteins were detected in islets using reverse transcription-polymerase chain reaction and Western blotting analyses, respectively. In immunohistochemical studies individual cells in the periphery of islets were labelled using an anti-TRalpha1 antibody. No labelled cells were detected in the exocrine pancreas. A similar staining pattern was obtained with an anti-glucagon antibody, but not with an anti-insulin antibody, which suggests that TRalpha1 is mainly expressed in alpha-cells. In order to address a potential function of TRalpha1 in this cell type, the regulation of glucagon gene expression by triiodothyronine was studied in a glucagon-producing cell line by Northern blot analysis and transient transfection assays using glucagon promoter luciferase fusion gene constructs. In these assays, triiodothyronine did not regulate the glucagon mRNA level or the glucagon promoter activity. The predominant localization of TRalpha1 in pancreatic alpha-cells suggests that this receptor isoform mediates a specific, yet unknown, function of thyroid hormones in this cell type.

An unusual case of driving under the influence of enflurane.

An unusual case of driving under the influence of the volatile anaesthetic enflurane is reported. A markedly affected anaesthetist was sniffing at an enflurane-moistened handkerchief before he crashed into a lorry at a red traffic light. In the blood sample enflurane (2.92 mg/l) as well as diclofenac (0.28 mg/l) and 4-aminophenazone (24.4 mg/l) were found. The way of driving and the accident and the deficiency symptoms could be explained by the central suppressing effects of enflurane. The physician was considered as impaired and not suitable to drive at the time of the incidence.

Solution structure of Co(III)-bleomycin-OOH bound to a phosphoglycolate lesion containing oligonucleotide: implications for bleomycin-induced double-strand DNA cleavage.

Bleomycin (BLM) is an antitumor antibiotic that is used clinically. Its major cause of cytotoxicity is thought to be related to BLM's ability to cause double-strand (ds) DNA cleavage. A single molecule of BLM appears to cleave both strands of DNA in the presence of its required cofactors Fe(2+) and oxygen without dissociating from the helix. A mechanism for this process has been proposed based on a model structure of the hydroperoxide of Co(III)-BLM (CoBLM) bound sequence-specifically to an intact duplex containing a GTAC site, a hot spot for ds cleavage [Vanderwall, D. E., Lui, S. M., Wu, W., Turner, C. J., Kozarich, J. W., and Stubbe, J. (1997) Chem. Biol. 4, 373-387]. In this paper, we present a structural model for the second cleavage event. Two-dimensional NMR spectroscopy and molecular modeling were carried out to study CoBLM bound to d(CCAAAGXACTGGG).d(CCCAGTACTTTGG), where X represents a 3'-phosphoglycolate lesion next to a 5'-phosphate. Assignments of 729 NOEs, including 51 between the drug and the DNA and 126 within the BLM molecule, have been made. These NOEs in addition to 96 dihedral angle constraints have been used to obtain a well-defined structural model for this complex. The model reveals that the bithiazole tail is partially intercalated between the T19 and the A20 of the duplex and that the metal binding domain is poised for abstraction of the T19 H4' in the minor groove. The modeling further reveals that the predominant conformation of the bithiazole protons is trans. Two cis conformations of these protons are also observed, and ROESY experiments provide evidence for interconversion of all of these forms. The relationship of these observations to the model for ds cleavage is presented.

Rapid analysis of halothane in biological samples using headspace solid-phase microextraction and gas chromatography-mass spectrometry--a case of a double homicide.

A simple, rapid, and sensitive method for the analysis of halothane in biological samples was developed. The procedure describes the extraction of halothane from blood, liver, kidney, brain, urine, bile, and stomach contents by headspace solid-phase microextraction (HS-SPME) followed by capillary gas chromatography coupled with mass spectrometry (GC-MS). The recovery in blood samples after addition of ammonium sulfate and sulfuric acid was 72% compared to a sample prepared in water (100%). Linearity was established over a concentration range of 0.1-100 mg/kg of spiked blood samples with an excellent coefficient of correlation (0.996) and a limit of detection of 0.004 mg/kg. The time for analysis was approximately 40 min per sample including the extraction step. The procedure was used for quantitation of halothane in various samples in a case of a double homicide. HS-SPME in combination with GC-MS was an effective method for the determination and quantitation of halothane in biological material.

Rapid analysis of parathion in biological samples using headspace solid-phase micro-extraction (HS-SPME) and gas chromatography/mass spectrometry (GC/MS).

A simple and rapid method for the analysis of parathion in biological samples is presented. The method consists of the extraction of parathion from blood samples by headspace solid-phase micro-extraction (SPME), followed by capillary gas chromatography and mass spectrometry detection. The recoveries in the blood samples after addition of ammonium sulphate and sulphuric acid were between 85% and 89% compared to samples prepared in water. Linearity was established over a concentration range of 0.1-5 microg/g blood with acceptable coefficients of correlation and limits of detection reached 0.02-0.05 microg/g. The time for an analysis is 57 minutes for one sample, including the extraction step. In conclusion, HS-SPME in combination with GC/MS is an effective method for the determination and quantification of parathion-ethyl and parathion-methyl in biological material.

A portable quantitative capnometer in test.

A new hand-held quantitative capnometer (BCI Capnocheck) was tested in the animal lab setting. The end-tidal CO2 values, as measured with this device, showed good agreement with arterial (Paco2) values. This device seems suited for quantitative capnometry in the prehospital setting. It must be noted, however, that this device has no alarms.

[HELLP syndrome: recurrence in 4 consecutive pregnancies]. / HELLP-Syndrom: Wiederholtes Auftreten in vier aufeinanderfolgenden Schwangerschaften.

Recurrence in Four Consecutive Pregnancies in a Patient: We report on the recurrence of HELLP-syndrome in four consecutive pregnancies in a patient. The observation of recurrence of this rare disorder (0.2-0.8% of all pregnancies) in an otherwise healthy patient points to an immunological or genetic predisposition for the development of HELLP-syndrome. At the moment there is no possibility to identify these patients with a high recurrence risk for HELLP-syndrome before the onset of pregnancy.

Kininogen and kinin in experimental spinal cord injury.

Activation of the kallikrein-kinin system has been implicated in the pathogenesis of vasogenic brain edema and posttraumatic vascular injury. We determined the levels of kininogen and kinin in an experimental spinal cord injury model in the rat. Kininogen content in traumatized cord segments increased in a time-dependent manner. Western blot analysis showed that the kininogen in traumatized cord comigrates with 68K low-molecular-weight kininogen or T-kininogen. Trypsin treatment of the kininogen in traumatized cord released both bradykinin and T-kinin, which were separated by HPLC and quantified with a kinin radioimmunoassay. Endogenous kinin levels in the frozen spinal cord also increased up to 40-fold 2 h after injury as compared with controls. The results demonstrate an increased accumulation of kininogen and its conversion to vasoactive kinins in experimental spinal cord injury.

[Experience with timolol in consulting patients (author's transl)]. / Erfahrungen mit Timolol in der Sprechstunde.

Experience obtained in the treatment of 78 eyes of patients suffering from open-angle glaucoma with the beta-blocker Timolol 0.25% and 0.5%. Timolol was only given to patients whose intraocular pressure could no longer be adequately regulated by the medications previously employed. A synopsis is given of the medications used over many years. The intraocular pressures of the eyes that responded to Timolol remained below 23 mmHg even after prolonged use, so that a fistulizing operation was initially unnecessary.

[Cervix cerclage surgery. A way for reducing prematurity]. / Zervix-Cerclage-Operation. Ein Weg zur Senkung der Frühgeburtlichkeit.

Indications and results of 202 cerclage-operations are reported. There are prophylactic as well as therapeutic indications for a cerclage-operation. Regarding the results it is obvious that prophylactic cerclage has better prepossessions for a normally terminated delivery. Therapeutic cerclages show best results when performed within the 15th to 27th week of pregnancy. A cerclage performed after the 32nd week of pregnancy shows no statistically significant reduction of early delivery but only a postponing of the delivery-date into a phase of better survival chance for the child. A reduction of the early delivery rate is regarded to be effective by prophylactic as well as therapeutic cerclages up to the 27th week of pregnancy.

[Comparison of endoscopic biopsy and scintiscans in liver diseases (author's transl)]. / Vergleich endoskopisch-bioptischer und szintigraphischer Untersuchungen bei Lebererkrankungen.

The diagnostic possibilities of liver scintiscans with indium-113m acetylacetonate were investigated in 314 patients. The results obtained were compared with our own endoscopic biopsy investigations. The scintiscans give information on the stage of the disease, but in contradistinction to the laparoscopic biopsies, not on etiology or activity of the disease. In contrast to the endoscopic biopsies, scintigraphy carries no risk. The advantage of the indium-113m acetylacetonate we used lies in the low radiation exposure.