Modeling of a three-dimensional structure of cytochrome P-450 1A2 and search for its new ligands

The substances inhibiting cytochrome P450 1A2 (CYP1A2) represent a perspective class of new drugs, which application in clinical practice can become the important part in preventive maintenance in oncology. The present work is devoted to computer modelling of 3-D structure of CYP1A2 and searching of new inhibitors by database mining. The modelling of CYP1A2 was done based on homology with 4 bacterial cytochromes P450 with known 3-D structure. For optimization of CYP1A2 active site structure the models of its complexes with characteristic substrates (caffeine and 7-ethoxyresorufin) were designed. These complexes were optimized by molecular dynamics simulation in water. The models of 24 complexes of CYP1A2 with known ligands with known Kd were designed by means of DockSearch and LeapFrog programs. 3D-QSAR model with good predictive force was created based on these complexes. On a final stage the search of knew CYP1A2 ligands in testing database (more than 23.000 substances from database Maybridge and 112 known CYP1A2 ligands from database Metabolite, MDL) was executed. 680 potential ligands of CYP1A2 with Kd values, comparable with known ones were obtained. This number has included 73 compounds from 112 known ligands, introduced in tested database as the internal control.     

Adenovirus-mediated interleukin-10 gene transfer inhibits post-transplant fibrous airway obliteration in an animal model of bronchiolitis obliterans

Bronchiolitis obliterans, a form of chronic allograft rejection characterized by progressive fibrous obliteration of the airways, is the major obstacle limiting prolonged survival of lung transplant recipients. To date, no effective therapy against this fatal complication exists. Interleukin-10 (IL-10), an anti-inflammatory and immunosuppressive cytokine, inhibits various T cell and antigen-presenting cell functions. We examined the effect of IL-10 in an animal model for bronchiolitis obliterans. A heterotopic tracheal transplant model was used. IL-10 was administered to the recipient either in its recombinant form by osmotic minipump or by adenoviral-mediated IL-10 gene transfer (Ad5E1mIL-10). Successful gene transfection and expression was confirmed by measuring circulating IL-10 protein. Tracheal allografts were assessed histologically based on a scoring system. IL-10 administration (in recombinant form or by gene transfer) inhibited the development of fibrous airway obliteration 3 weeks after transplantation in comparison to untreated controls (p < 0.05). This was demonstrated only if the delivery was initiated 5 days after transplantation and not if it was started at the time of transplantation. A single administration of the gene construct was sufficient to achieve the desired effect. We have shown that IL-10 can prevent the development of airway fibro-obliteration in this model. Gene transfection at a site distant from a graft can be used to produce a desired effect on the graft. IL-10 may be of major importance in the control of post-transplant bronchiolitis obliterans. The timing of its administration is critical and further studies are required to determine the mechanisms underlying the observed effects of IL-10.     

ATP-dependent proteolysis in mitochondria. m-AAA protease and PIM1 protease exert overlapping substrate specificities and cooperate with the mtHsp70 system

To analyze protein degradation in mitochondria and the role of molecular chaperone proteins in this process, bovine apocytochrome P450scc was employed as a model protein. When imported into isolated yeast mitochondria, P450scc was mislocalized to the matrix and rapidly degraded. This proteolytic breakdown was mediated by the ATP-dependent PIM1 protease, a Lon-like protease in the mitochondrial matrix, in cooperation with the mtHsp70 system. In addition, a derivative of P450scc was studied to which a heterologous transmembrane region was fused at the amino terminus. This protein became anchored to the inner membrane upon import and was degraded by the membrane-embedded, ATP-dependent m-AAA protease. Again, degradation depended on the mtHsp70 system; it was inhibited at non-permissive temperature in mitochondria carrying temperature-sensitive mutant forms of Ssc1p, Mdj1p, or Mge1p. These results demonstrate overlapping substrate specificities of PIM1 and the m-AAA protease, and they assign a central role to the mtHsp70 system during the degradation of misfolded polypeptides by both proteases.     

Missing quality of life data in cancer clinical trials: serious problems and challenges

Measurement of quality of life (QOL) in cancer clinical trials has increased in recent years as more groups realize the importance of such endpoints. A key problem has been missing data. Some QOL data may unavoidably be missing, as for example when patients are too ill to complete forms. Other important sources are potentially avoidable and can broadly be divided into three categories: (i) methodological factors; (ii) logistic and administrative factors; (iii) patient-related factors. Logistic and administrative factors, for example, staff oversights, have proven to be most important. Since most QOL measurements require patient self-report, it is usually not possible to rectify the failure to collect baseline data or any follow-up assessments. There is strong evidence that such data are not 'missing at random', and cannot be ignored without introducing bias. Although several approaches to the analysis of partly missing data have been described, none is entirely satisfactory. Prevention of avoidable missing data is better than attempted cure. In July 1996, an international conference on missing QOL data in cancer clinical trials reported the experience of most major groups involved. This paper will serve as an introduction to the problem and provide an estimation of its magnitude, and approaches to its prevention and solution.     

Clinical and experimental studies on preventing and treating anaphylactic asthma with Zusanli point immunotherapy

We have studied on preventing and treating anaphylactic asthma with Zusanli (S36) point immunotherapy (ZPIT). Sixty-nine patients were observed. The results showed that the clinical curative effect of ZPIT was not only much higher than that of conventional desensitization therapy, but also the patients' total IgE level was reduced, anti-acarid IgE was lowered, SIgA level was raised, the absolute eosinophilic granulocyte level dropped and pulmonary function recovered. Animal experiment results showed that the ZPIT could more effectively suppress the guinea pigs' anaphylactic asthma allergized by albumin and more obviously resist the guinea pigs' bronchial spasm induced by histamine and acetylcholine than the conventional desensitization therapy and injected normal saline. The immunomodulating action of the ZPIT are elucidated from clinical study and animal experiment in the paper.     

Phospholipase A2 activity in salivary glands and saliva of the lone star tick (Acari: Ixodidae) during tick feeding

The etiology of subacute granulomatous thyroiditis (SAT) is obscure, although it is postulated to be associated with viral infections and genetic factors. In the present study, the possibility of an infectious etiology was prospectively studied in 27 consecutive patients with SAT. Special emphasis was put on the role of enteroviruses. Coupled sera (interval one month) were taken from all patients and single sera from 29 control subjects for virus antibody determinations. Stool samples were collected for virus isolation and fine-needle aspiration samples from thyroid gland for the detection of enterovirus RNA using RT-PCR were taken from SAT patients. Enteroviral antibodies were tested using three different methods: indirect EIA, heavy chain capture RIA, and standard complement fixation (CF) test. Antibodies against other common viral pathogens, including enteroviruses, were screened using the CF test and those against Mycoplasma pneumoniae and Chlamydia pneumoniae using EIA and microimmunofluorescence techniques, respectively. Common respiratory viruses were also screened from nasopharyngeal suction samples by antigen detection EIA. Based on serological findings, one patient had acute Cytomegalovirus infection. All other patients were negative in antibody tests, virus isolation, RT-PCR, and antigen detection. Enterovirus RNA was not detected by PCR in the thyroid tissue in any of the fine-needle aspiration samples. There was no evidence of recent enteroviral infections in SAT patients. The results suggest that SAT is not usually associated with acute infections. No evidence was obtained to support the proposed role of enteroviruses as an important etiologic agent of SAT.