Integrated Nanosensors to Determine Levels and Functional Activity of Human Telomerase

Telomerase is a key oncogenic enzyme, and a number of novel telomerase inhibitors are currently under development. Because inhibition can be achieved either at the protein or at the enzymatic activity level, independent measurements of these parameters are important in the development of effective therapeutic agents. In the current study, we have developed a set of functional magnetic nanosensors capable of measuring the concentration of telomerase, as well as its enzymatic activity in parallel. The method is based on a magnetic relaxation switch assay, which can be performed in crude tissue samples and is fast and extremely sensitive. Using this method, we were able to detect different amounts of telomerase protein and activity in various cancer and normal cell lines. Furthermore, we were able to study the effect of phosphorylation on telomerase activity. This system not only could provide a rapid assay for the evaluation of antitelomerase therapies but could also be implemented to the study of other cancer markers.     

Efficacy of anticoagulation in resolving left atrial and left atrial appendage thrombi: A transesophageal echocardiographic study

BACKGROUND: Transesophageal echocardiography (TEE) is the gold standard for evaluation of the left atrium and the left atrial appendage (LAA) for the presence of thrombi. Anticoagulation is conventionally used for patients with atrial fibrillation to prevent embolization of atrial thrombi. The mechanism of benefit and effectiveness of thrombi resolution with anticoagulation is not well defined. METHODS AND RESULTS: We used a TEE database of 9058 consecutive studies performed between January 1996 and November 1998 to identify all patients with thrombi reported in the left atrium and/or LAA. One hundred seventy-four patients with thrombi in the left atrial cavity (LAC) and LAA were identified (1.9% of transesophageal studies performed). The incidence of LAA thrombi was 6.6 times higher than LAC thrombi (151 vs 23, respectively). Almost all LAC thrombi were visualized on transthoracic echocardiography (90.5%). Mitral valve pathology was associated with LAC location of thrombi (P <.0001), whereas atrial fibrillation or flutter was present in most patients with LAA location of thrombi. Anticoagulation of 47 +/- 18 days was associated with thrombus resolution in 80.1% of the patients on follow-up TEE. Further anticoagulation resulted in limited additional benefit. CONCLUSIONS: LAC thrombi are rare and are usually associated with mitral valve pathology. Transthoracic echocardiography is effective in identifying these thrombi. LAA thrombi occur predominantly in patients with atrial fibrillation or flutter. Short-term anticoagulation achieves a high rate of resolution of LAA and LAC thrombi but does not obviate the need for follow-up TEE.     

Diagnostic chorionic biopsies in the first trimester. Report of experiences over a 2 year period

We report about 58 ultrasonographically guided transcervical chorionic villus biopsies from January 1985 to November 1987. Maternal age greater than 35 years (n = 28), followed by trisomy 21 or 18 (n = 10) were the mean indications. Biochemically evaluation of storage diseases (n = 6) and genomically DNA-analysis because of phenylketonuria (n = 2) were combined in each case with cytogenetic diagnosis. In the other cases certain indications were the reasons for biopsy. In 52 of 58 cases we were successful in biopsies and diagnoses. In the other 6 biopsy specimen we didn't found chorionic villi. 3 abortions we observed up to day 3 after operation (n = 3) and after 6 weeks (n = 1). Pathological findings were 1 trisomy 16, 1,47,XYY-karyotype and 1 embryo with phenylketonuria. Another reason for termination of pregnancy was male karyotype in a Morbus Duchenne-risk and 1 risk for Rett-syndrome. Meanwhile 30 healthy babies were born.     

A mouse model for Sorsby fundus dystrophy

PURPOSE: Sorsby fundus dystrophy (SFD) is a rare, late-onset macular dystrophy caused by mutations in the tissue inhibitor of metalloproteinases-3 (TIMP3) gene. The known mutations introduce potentially unpaired cysteine residues in the C terminus of the protein and result in the formation of higher-molecular-weight protein complexes of as yet unknown composition and functional consequences in the pathologic course of SFD. To facilitate in vivo investigation of mutant TIMP3, the authors generated a knock-in mouse carrying a disease-related Ser156Cys mutation in the orthologous murine Timp3 gene. METHODS: Site-directed mutagenesis and homologous recombination in embryonic stem (ES) cells was used to generate mutant ES cells carrying the Timp3(S156C) allele. Chimeric animals were obtained, of which two displayed germline transmission of the mutated allele. Molecular genetic, biochemical, electron microscopic, and electrodiagnostic techniques were used for characterization. RESULTS: At 8 months of age, knock-in mice showed abnormalities in the inner aspect of Bruch's membrane and in the organization of the adjacent basal microvilli of the retinal pigment epithelium (RPE). Changes resembling those in the mutant animals were also present to some extent in normal littermates, but only at an advanced age of 30 months. Long-term electrodiagnostic recordings indicated normal retinal function throughout life. The biochemical characteristics of the mutant protein appear similar in humans and knock-in mice, suggesting common molecular pathways in the two species. The localization of the mutant protein in the eye is normal, although there is evidence of increased Timp3 levels in Bruch's membrane of mutant animals. CONCLUSIONS: The knock-in mice display early features of age-related changes in Bruch's membrane and the RPE that may represent the primary clinical manifestations of SFD. In addition, our immunolabeling studies and biochemical data support a model proposing that site-specific excess rather than absence or deficiency of functional Timp3 may be the primary consequence of the known Timp3 mutations.     

Neurobiology of relapse to heroin and cocaine seeking: a review

The objective of this article is to review data from studies that used a reinstatement model in rats to elucidate the neural mechanisms underlying relapse to heroin and cocaine seeking induced by exposure to the self-administered drug (drug priming), conditioned drug cues, and stressors. These factors were reported to contribute to relapse to drug use in humans following prolonged abstinence periods. In the reinstatement model, the ability of acute exposure to drug or nondrug stimuli to reinstate drug seeking is determined following training for drug self-administration and subsequent extinction of the drug-reinforced behavior. We will review studies in which pharmacological agents were injected systemically or intracranially to block (or mimic) reinstatement by drug priming, drug cues, and stressors. We also will review studies in which brain lesions, in vivo microdialysis and electrochemistry, and gene expression methods were used to map brain sites involved in relapse to drug seeking. Subsequently, we will discuss theoretical issues related to the processes underlying relapse to drugs and address methodological issues in studies on reinstatement of drug seeking. Finally, the implications of the findings from the studies reviewed for addiction theories and treatment will be discussed. The main conclusion of this review is that the neuronal mechanisms involved in relapse to heroin and cocaine seeking induced by drug priming, drug cues, and stressors are to a large degree dissociable. The data reviewed also suggest that the neuronal events mediating drug-induced reinstatement are to some degree dissociable from those mediating drug reinforcement.