Novel inhibitors of poly(ADP-ribose) polymerase/PARP1 and PARP2 identified using a cell-based screen in yeast.

Multicellular organisms must have means of preserving their genomic integrity or face catastrophic consequences such as uncontrolled cell proliferation or massive cell death. One response is a modification of nuclear proteins by the addition and removal of polymers of ADP-ribose that modulate the properties of DNA-binding proteins involved in DNA repair and metabolism. These ADP-ribose units are added by poly(ADP-ribose) polymerase (PARP) and removed by poly(ADP-ribose) glycohydrolase. Although budding yeast Saccharomyces cerevisiae does not possess proteins with significant sequence similarity to the human PARP family of proteins, we identified novel small molecule inhibitors against two family members, PARP1 and PARP2, using a cell-based assay in yeast. The assay was based on the reversal of growth inhibition caused by the heterologous expression of either PARP1 or PARP2. Validation of the assay was achieved by showing that the growth inhibition was relieved by a mutation in a single residue in the catalytic site of PARP1 or PARP2 or exposure of yeast to a known PARP1 inhibitor, 6(5H)-phenanthridinone. In separate experiments, when a putative protein regulator of PARP activity, human poly(ADP-ribose) glycohydrolase, was coexpressed with PARP1 or PARP2, yeast growth was restored. Finally, the inhibitors identified by screening the yeast assay are active in a mammalian PARP biochemical assay and inhibit PARP1 and PARP2 activity in yeast cell extracts. Thus, our data reflect the strength of using yeast to identify small molecule inhibitors of therapeutically relevant gene families, including those that are not found in yeast, such as PARP. The resultant inhibitors have two critical uses (a) as leads for drug development and (b) as tools to dissect cellular function.

Motoneuron differentiation of immortalized human spinal cord cell lines.

Human motoneuron cell lines will be valuable tools for spinal cord research and drug discovery. To create such cell lines, we immortalized NCAM(+)/neurofilament(+) precursors from human embryonic spinal cord with a tetracycline repressible v-myc oncogene. Clonal NCAM(+)/neurofilament(+) cell lines differentiated exclusively into neurons within 1 week. These neurons displayed extensive processes, exhibited immunoreactivity for mature neuron-specific markers such as tau and synaptophysin, and fired action potentials upon current injection. Moreover, a clonal precursor cell line gave rise to multiple types of spinal cord neurons, including ChAT(+)/Lhx3(+)/Lhx4(+) motoneurons and GABA(+) interneurons. These neuronal restricted precursor cell lines will expedite the elucidation of molecular mechanisms that regulate the differentiation, maturation and survival of specific subsets of spinal cord neurons, and the identification and validation of novel drug targets for motoneuron diseases and spinal cord injury.

Immortalized human dorsal root ganglion cells differentiate into neurons with nociceptive properties.

A renewable source of human sensory neurons would greatly facilitate basic research and drug development. We had established previously conditionally immortalized human CNS cell lines that can differentiate into functional neurons (). We report here the development of an immortalized human dorsal root ganglion (DRG) clonal cell line, HD10.6, with a tetracycline-regulatable v-myc oncogene. In the proliferative condition, HD10.6 cells have a doubling time of 1.2 d and exhibit a neuronal precursor morphology. After differentiation of clone HD10.6 for 7 d in the presence of tetracycline, v-myc expression was suppressed, and >50% of the cells exhibited typical neuronal morphology, stained positively for neuronal cytoskeletal markers, and fired action potentials in response to current injection. Furthermore, this cell line was fate-restricted to a neuronal phenotype; even in culture conditions that promote Schwann cell or smooth muscle differentiation of neural crest stem cells, HD10.6 differentiated exclusively into neurons. Moreover, differentiated HD10.6 cells expressed sensory neuron-associated transcription factors and exhibited capsaicin sensitivity. Taken together, these data indicate that we have established an immortalized human DRG cell line that can differentiate into sensory neurons with nociceptive properties. The cell line HD10.6 represents the first example of a human sensory neuronal line and will be valuable for basic research, as well as for the discovery of novel drug targets and clinical candidates.

Application of ex vivo gene therapy in the treatment of Parkinson's disease.

Ex vivo gene therapy approaches hold great promise for the treatment of neurodegenerative diseases where there is currently no cure or adequate treatment for affected individuals. In this review we have focused on the use of ex vivo gene transfer techniques in Parkinson's disease models; however, the issues and approaches outlined are applicable to other neurodegenerative disorders. In utilizing the ex vivo strategy two considerations are critical for delivery of therapeutic levels of transgene product to the target: (i) the vector system and (ii) the cell type for grafting. We describe herein different vector systems that are currently available and briefly review the various cell types that have been transduced and grafted into the striatum of animals with experimental Parkinson's disease. The strategies for application of gene therapy techniques to a treatment for Parkinson's disease have expanded beyond the classical dopamine replacement toward the use of neurotrophic factors in enhancing cell function or preventing cell death. In addition, we explore the utility of CNS-derived neural progenitors as alternative cell types for ex vivo gene therapy in an animal model of Parkinson's disease.

[Mask physiotherapy for prevention of pulmonary complications after heart surgery. A controlled study]. / Fysioterapi med maskebehandling til forebyggelse af pulmonale komplikationer efter hjertekirurgi. Et kontrolleret studie.

The objective of this prospective, consecutive, randomized, controlled study was to investigate the effects of mask physiotherapy on postoperative complications after heart surgery. Sixty-six low-risk male patients undergoing coronary artery by-pass graft surgery were evaluated. The patients were treated with routine chest physiotherapy alone or supplied with either positive expiratory pressure (PEP), or inspiratory resistance-positive expiratory pressure (IR-PEP). Postoperative pulmonary complications were assessed by forced vital capacity (FVC), arterial oxygen tension (PaO2), and chest X-ray examination. There was an almost equal decrease and subsequent rise in spirometric and blood gas values in all three groups, but patients treated with the PEP mask had a borderline significantly higher increase in PaO2 from day three to day six compared with patients treated with no mask. There was an almost equal frequency of atelectasis in the three treatment groups. It is concluded that no significant differences in outcome were found between the three groups.

Mask physiotherapy in patients after heart surgery: a controlled study.

OBJECTIVE: Investigate the effects of mask physiotherapy on post-operative complications after thoracic surgery. DESIGN: A prospective, consecutive, randomized, controlled study. SETTING: Department of Thoracic and Heart Surgery at a University Hospital. The treatments were performed by experienced and specially trained physiotherapists. PATIENTS: 97 low-risk male patients undergoing coronary artery by-pass graft surgery were evaluated. 66 patients completed the study. INTERVENTIONS: The patients were treated with routine chest physiotherapy alone or supplied with either positive expiratory pressure (PEP), or inspiratory resistance-positive expiratory pressure (IR-PEP). MEASUREMENTS AND RESULTS: Post-operative pulmonary complications were assessed by forced vital capacity (FVC), arterial oxygen tension (PaO2), and chest X-ray examination, all measured pre-operatively and on the third and sixth post-operative day. There was an almost equal decrease and subsequent rise in spirometric and blood gas values, but patients treated with the PEP mask had a borderly significantly higher increase in PaO2 from day 3 to day 6 compared with patients treated with no mask. There was an almost equal frequency of atelectasis in the 3 treatments. The patients filled in a questionaire expressing their opinion about their treatment. Most patients liked their treatment and found it helpful but a little less so in the IR-PEP group. CONCLUSION: We did not find any significant difference between the three groups; however, a tendency to decreased risk of having post-operative complications was observed in the groups having positive expiratory pressure (PEP) and inspiratory resistance-positive expiratory pressure (IR-PEP).

Resolution and conservation of mismatches in DNA end joining.

DNA end joining is a major pathway for the elimination of double-strand breaks from chromosomal DNA of higher eucaryotic cells. Extracts of Xenopus laevis eggs rejoin such breaks even when their short single-stranded termini are expected to form imperfectly matched overlaps. However, end-joined products cloned in Escherichia coli, necessarily give rise to perfectly matched products. Therefore it has not been possible to determine whether the end joining process creates mismatched products, perfectly matched (resolved) products or both. To investigate whether mismatch resolution was the result of the X. laevis end joining process or of activities of the bacterial host we used denaturing gradient gel electrophoresis to analyse joined products. We found that the end joining process does include mismatch resolution, the degree of which varies with regard to the nature of the original overlap structure. Mismatches 3' to a gap are completely resolved, mismatches 3' to a nick and 5' to a nick or gap are resolved to some extent but are generally conserved. Mismatches between base matches are always conserved. These findings suggest competing processes of ligation, DNA fill-in synthesis or exonucleolytic excision of mismatched bases next to a gap or nick. At mismatches 3' to a nick the probability of ligation is greater than that of excision while at mismatches 3' to a gap the probability of excision is greater than elongation of a given mismatch. At mismatches 5' to nicks or gaps it appears that ligation or elongation and ligation, respectively, are the most probable pathways but products resulting from mismatch excision, elongation and ligation are also detected.

Mechanisms of overlap formation in nonhomologous DNA end joining.

Rejoining of nonhomologous DNA termini plays a central role in processes of illegitimate recombination. In Xenopus egg extracts, DNA ends with noncomplementary 4-nucleotide antiparallel single-strand protrusions are assumed to be joined by formation of short mismatched overlap intermediates. The extents of these overlaps may be set by single fortuitously matching base pairs and determine the patterns of subsequent gap filling and nick ligation. Under conditions of alternative overlap settings, rules for the most probable joining pathway and the effects of mismatches on junction formation were analyzed. We show that in certain cases, fill-in and ligation converting overlap intermediates into covalently closed junctions may proceed in the presence of unrepaired mismatches, whereas in other cases, completion of junction formation is preceded by removal of mismatches. Results are discussed in relation with "alignment" proteins postulated to structurally support overlap heteroduplexes during junction formation.

A novel pathway of DNA end-to-end joining.

Repair mechanisms related to illegitimate recombination can join nonhomologous DNA ends that terminate as protruding single strands (PSS). Here we analyze in Xenopus egg extracts joining reactions between 3' PSS termini and various partner termini. In junctions, 3' PSS termini are preserved by fill-in DNA synthesis, although their 5' recessed ends cannot serve as a primer. Alternative priming from a partner terminus ligated to the 3' PSS end appears unlikely, because no single strand-specific DNA ligases are detectable. We show that fill-in of 3' PSS termini precedes ligation and can even be primed in the absence of any ligation. Therefore, priming requires precise alignment of terminus pairs by a novel mechanism. We postulate that this is achieved by unique DNA binding proteins that align ends in various types of joining reactions.

Laryngo-tracheal laser surgery and general anesthesia.

The use of the carbon dioxide laser in treatment of laryngo-tracheal lesions is extremely effective when combined with general anesthesia. However, the potential for intraoperative complication is increased due to a pathological reduction in the size of the airway and the presence of an endotracheal tube. To avoid the hazard of endotracheal tube ignition, specific techniques are employed.