Microdissection and Measurement of Polytene Chromosomes Using the Atomic Force Microscope

A method to isolate specific regions of the Drosophila polytene chromosome using an atomic force microscope (AFM) was explored. The AFM was used for the microdissection of the locus of interest with much greater precision than standard microdissection techniques. The amplification of DNA isolated in this fashion by the polymerase chain reaction (PCR) is discussed. A study of the effect of hydration level on gross chromosome structure was carried out. It was shown that chromosome swelling is dependent upon humidity or the buffered medium. The significance of this swelling with respect to studies of chromosome structure under physiological conditions is considered

Analyzing Chromosomes, Ion Channels and Novel Nucleic Acid Structures by AFM

The atomic force microscope (AFM) is proving to be a powerful tool for analysis of biological samples. We provide three examples of the application of AFM to the study of biological questions. First, polytene chromosomes from Drosophila are imaged and manipulated by the AFM. Second, the localization of calcium channels on the release face of a nerve terminal is described. Finally, analyses of a new form of DNA, the G-wire, is presented. These examples illustrate the wide variety of biological questions to which AFM can contribute

Predictors of contemporary coronary artery bypass grafting outcomes

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Post-intervention Status in Patients With Refractory Myasthenia Gravis Treated With Eculizumab During REGAIN and Its Open-Label Extension

OBJECTIVE: To evaluate whether eculizumab helps patients with anti-acetylcholine receptor-positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension. METHODS: Patients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study. RESULTS: A total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1-4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected. CONCLUSION: Eculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population. CLINICALTRIALSGOV IDENTIFIER: REGAIN, NCT01997229; REGAIN open-label extension, NCT02301624. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo

Minimal Symptom Expression' in Patients With Acetylcholine Receptor Antibody-Positive Refractory Generalized Myasthenia Gravis Treated With Eculizumab

The efficacy and tolerability of eculizumab were assessed in REGAIN, a 26-week, phase 3, randomized, double-blind, placebo-controlled study in anti-acetylcholine receptor antibody-positive (AChR+) refractory generalized myasthenia gravis (gMG), and its open-label extension

Drosophila Raf's N Terminus Contains a Novel Conserved Region and Can Contribute to Torso RTK Signaling

Drosophila Raf (DRaf) contains an extended N terminus, in addition to three conserved regions (CR1–CR3); however, the function(s) of this N-terminal segment remains elusive. In this article, a novel region within Draf's N terminus that is conserved in BRaf proteins of vertebrates was identified and termed conserved region N-terminal (CRN). We show that the N-terminal segment can play a positive role(s) in the Torso receptor tyrosine kinase pathway in vivo, and its contribution to signaling appears to be dependent on the activity of Torso receptor, suggesting this N-terminal segment can function in signal transmission. Circular dichroism analysis indicates that DRaf's N terminus (amino acids 1–117) including CRN (amino acids 19–77) is folded in vitro and has a high content of helical secondary structure as predicted by proteomics tools. In yeast two-hybrid assays, stronger interactions between DRaf's Ras binding domain (RBD) and the small GTPase Ras1, as well as Rap1, were observed when CRN and RBD sequences were linked. Together, our studies suggest that DRaf's extended N terminus may assist in its association with the upstream activators (Ras1 and Rap1) through a CRN-mediated mechanism(s) in vivo

Phylogenetic Analyses and Characterization of RNase X25 from Drosophila melanogaster Suggest a Conserved Housekeeping Role and Additional Functions for RNase T2 Enzymes in Protostomes

Ribonucleases belonging to the RNase T2 family are enzymes associated with the secretory pathway that are almost absolutely conserved in all eukaryotes. Studies in plants and vertebrates suggest they have an important housekeeping function in rRNA recycling. However, little is known about this family of enzymes in protostomes. We characterized RNase X25, the only RNase T2 enzyme in Drosophila melanogaster. We found that RNase X25 is the major contributor of ribonuclease activity in flies as detected by in gel assays, and has an acidic pH preference. Gene expression analyses showed that the RNase X25 transcript is present in all adult tissues and developmental stages. RNase X25 expression is elevated in response to nutritional stresses; consistent with the hypothesis that this enzyme has a housekeeping role in recycling RNA. A correlation between induction of RNase X25 expression and autophagy was observed. Moreover, induction of gene expression was triggered by oxidative stress suggesting that RNase X25 may have additional roles in stress responses. Phylogenetic analyses of this family in protostomes showed that RNase T2 genes have undergone duplication events followed by divergence in several phyla, including the loss of catalytic residues, and suggest that RNase T2 proteins have acquired novel functions. Among those, it is likely that a role in host immunosuppression evolved independently in several groups, including parasitic Platyhelminthes and parasitoid wasps. The presence of only one RNase T2 gene in the D. melanogaster genome, without any other evident secretory RNase activity detected, makes this organism an ideal system to study the cellular functions of RNase T2 proteins associated with RNA recycling and maintenance of cellular homeostasis. On the other hand, the discovery of gene duplications in several protostome genomes also presents interesting new avenues to study additional biological functions of this ancient family of proteins.This is an article from PLoS ONE 9 (2014): 1, doi:10.1371/journal.pone.0105444. Posted with permission.</p

Juan manuel martínez fonseca.paternalismo y resistencia: los trabajadores de bavaria; 1889-1930.bogotá: rodríguez quito editores, 2007. 205 páginas.

<div><p>Ribonucleases belonging to the RNase T2 family are enzymes associated with the secretory pathway that are almost absolutely conserved in all eukaryotes. Studies in plants and vertebrates suggest they have an important housekeeping function in rRNA recycling. However, little is known about this family of enzymes in protostomes. We characterized RNase X25, the only RNase T2 enzyme in <i>Drosophila melanogaster</i>. We found that RNase X25 is the major contributor of ribonuclease activity in flies as detected by <i>in gel</i> assays, and has an acidic pH preference. Gene expression analyses showed that the <i>RNase X25</i> transcript is present in all adult tissues and developmental stages. <i>RNase X25</i> expression is elevated in response to nutritional stresses; consistent with the hypothesis that this enzyme has a housekeeping role in recycling RNA. A correlation between induction of <i>RNase X25</i> expression and autophagy was observed. Moreover, induction of gene expression was triggered by oxidative stress suggesting that RNase X25 may have additional roles in stress responses. Phylogenetic analyses of this family in protostomes showed that RNase T2 genes have undergone duplication events followed by divergence in several phyla, including the loss of catalytic residues, and suggest that RNase T2 proteins have acquired novel functions. Among those, it is likely that a role in host immunosuppression evolved independently in several groups, including parasitic Platyhelminthes and parasitoid wasps. The presence of only one RNase T2 gene in the <i>D. melanogaster</i> genome, without any other evident secretory RNase activity detected, makes this organism an ideal system to study the cellular functions of RNase T2 proteins associated with RNA recycling and maintenance of cellular homeostasis. On the other hand, the discovery of gene duplications in several protostome genomes also presents interesting new avenues to study additional biological functions of this ancient family of proteins.</p></div

Analyzing Chromosomes, Ion Channels and Novel Nucleic Acid Structures by AFM

The atomic force microscope (AFM) is proving to be a powerful tool for analysis of biological samples. We provide three examples of the application of AFM to the study of biological questions. First, polytene chromosomes from Drosophila are imaged and manipulated by the AFM. Second, the localization of calcium channels on the release face of a nerve terminal is described. Finally, analyses of a new form of DNA, the G-wire, is presented. These examples illustrate the wide variety of biological questions to which AFM can contribute.This is a proceeding from NATO Advanced Research Workshop: "Scanning Probe Microscopies and Molecular Materials" (1994): 1. </p