Intersubunit Interactions at Putative Sites of Ethanol Action in the M3 and M4 Domains of the NMDA Receptor GluN1 and GluN2B Subunits

Background and Purpose: The N-methyl-D-aspartate (NMDA) receptor is an important target of alcohol action in the brain. Recent studies in this laboratory have demonstrated that alcohol-sensitive positions in the intersubunit interfaces of the M3 and M4 domains of GluN1 and GluN2A subunits interact with respect to ethanol sensitivity and receptor kinetics, and that alcohol-sensitive positions in the M domains of GluN2A and GluN2B subunits differ. In this study we tested for interactions among alcohol-sensitive positions at the M domain intersubunit interfaces in GluN1/GluN2B NMDA receptors. Experimental Approach: We used whole-cell patch-clamp recording in tsA201 cells expressing tryptophan substitution mutants at ethanol-sensitive positions in the GluN1 and GluN2B NMDA receptor subunits to test for interactions among positions. Key Results: Six pairs of positions in GluN1/GluN2B significantly interacted to regulate ethanol inhibition: Gly638/Met824, Gly638/Leu825, Phe639/Leu825, Phe639/Gly826, Met818/Phe637 and Val820/Phe637. Tryptophan substitution at Met824 or Leu825 in GluN2B did not alter ethanol sensitivity but interacted with positions in the GluN1 M3 domain to regulate ethanol action, whereas tryptophan substitution at Gly638, which is the cognate of an ethanol-sensitive position in GluN2A, did not alter ethanol sensitivity or interact with positions in GluN1. Two and three pairs of positions interacted to regulate glutamate steady-state and peak current EC50, respectively, and one pair interacted with respect to macroscopic desensitization. Conclusions: Despite highly-conserved M domain sequences and similar ethanol sensitivity in the GluN2A and GluN2B subunits, the manner in which these subunits interact with the GluN1 subunit to regulate ethanol sensitivity and receptor kinetics differs

Different Sites of Alcohol Action in the NMDA Receptor GluN2A and GluN2B Subunits

The NMDA receptor is a major target of alcohol action in the CNS, and recent behavioral and cellular studies have pointed to the importance of the GluN2B subunit in alcohol action. We and others have previously characterized four amino acid positions in the third and fourth membrane-associated (M) domains of the NMDA receptor GluN2A subunit that influence both ion channel gating and alcohol sensitivity. In this study, we found that substitution mutations at two of the four corresponding positions in the GluN2B subunit, F637 and G826, influence ethanol sensitivity and ion channel gating. Because position 826 contains a glycine residue in the native protein, we focused our attention on GluN2B(F637). Substitution mutations at GluN2B(F637) significantly altered ethanol IC50 values, glutamate EC50 values for peak (Ip) and steady-state (Iss) current, and steady-state to peak current ratios (Iss:Ip). Changes in apparent glutamate affinity were not due to agonist trapping in desensitized states, as glutamate Iss EC50 values were not correlated with Iss:Ip values. Ethanol sensitivity was correlated with values of both Ip and Iss glutamate EC50, but not with Iss:Ip. Values of ethanol IC50, glutamate EC50, and Iss:Ip for mutants at GluN2B(F637) were highly correlated with the corresponding values for mutants at GluN2A(F636), consistent with similar functional roles of this position in both subunits. These results demonstrate that GluN2B(Phe637) regulates ethanol action and ion channel function of NMDA receptors. However, despite highly conserved M domain sequences, ethanol\u27s actions on GluN2A and GluN2B subunits differ

Water Cooling Vest

Final report for Team 14 of ME450, fall 2008 semesterImagine that you are wearing a cooled vest during the hot summer time! Neurosurgeons have the same need in operating room. They are usually required to perform long surgical procedures wearing heavy lead vests underneath their surgical gowns in order to protect them from the x-ray sources used for image-guided surgery. The project is to develop a cooling system for these lead vests allowing for temperature control, giving comfort to the neurosurgeons. The system needs to be light-weight and able to maintain uniform body temperature. The project has many potential applications beyond the operating room. The military has investigated this concept extensively for combat soldiers’ clothing. It also can be used in civilian applications, such as for construction workers, or any hot-weather clothing – just thinking about carrying your own air conditioner on a hot summer day.Paul Park (Department of Neurosurgery, U of M)http://deepblue.lib.umich.edu/bitstream/2027.42/61923/1/ME450 Fall2008 Final Report - Team 14 - Cooling Vest.pd

Identification of an E3 ligase that targets the catalytic subunit of RNA Polymerase I upon transcription stress

Publisher Copyright: © 2022 The AuthorsRNA Polymerase I (Pol I) synthesizes rRNA, which is the first and rate-limiting step in ribosome biogenesis. Factors governing the stability of the polymerase complex are not known. Previous studies characterizing Pol I inhibitor BMH-21 revealed a transcriptional stress-dependent pathway for degradation of the largest subunit of Pol I, RPA194. To identify the E3 ligase(s) involved, we conducted a cell-based RNAi screen for ubiquitin pathway genes. We establish Skp–Cullin–F-box protein complex F-box protein FBXL14 as an E3 ligase for RPA194. We show that FBXL14 binds to RPA194 and mediates RPA194 ubiquitination and degradation in cancer cells treated with BMH-21. Mutation analysis in yeast identified lysines 1150, 1153, and 1156 on Rpa190 relevant for the protein degradation. These results reveal the regulated turnover of Pol I, showing that the stability of the catalytic subunit is controlled by the F-box protein FBXL14 in response to transcription stress.Peer reviewe

A sweetpotato gene index established by de novo assembly of pyrosequencing and Sanger sequences and mining for gene-based microsatellite markers

<p>Abstract</p> <p>Background</p> <p>Sweetpotato (<it>Ipomoea batatas </it>(L.) Lam.), a hexaploid outcrossing crop, is an important staple and food security crop in developing countries in Africa and Asia. The availability of genomic resources for sweetpotato is in striking contrast to its importance for human nutrition. Previously existing sequence data were restricted to around 22,000 expressed sequence tag (EST) sequences and ~ 1,500 GenBank sequences. We have used 454 pyrosequencing to augment the available gene sequence information to enhance functional genomics and marker design for this plant species.</p> <p>Results</p> <p>Two quarter 454 pyrosequencing runs used two normalized cDNA collections from stems and leaves from drought-stressed sweetpotato clone <it>Tanzania </it>and yielded 524,209 reads, which were assembled together with 22,094 publically available expressed sequence tags into 31,685 sets of overlapping DNA segments and 34,733 unassembled sequences. Blastx comparisons with the UniRef100 database allowed annotation of 23,957 contigs and 15,342 singletons resulting in 24,657 putatively unique genes. Further, 27,119 sequences had no match to protein sequences of UniRef100database. On the basis of this gene index, we have identified 1,661 gene-based microsatellite sequences, of which 223 were selected for testing and 195 were successfully amplified in a test panel of 6 hexaploid (<it>I. batatas</it>) and 2 diploid (<it>I. trifida</it>) accessions.</p> <p>Conclusions</p> <p>The sweetpotato gene index is a useful source for functionally annotated sweetpotato gene sequences that contains three times more gene sequence information for sweetpotato than previous EST assemblies. A searchable version of the gene index, including a blastn function, is available at <url>http://www.cipotato.org/sweetpotato_gene_index</url>.</p

Impact of cancer and chemotherapy on autonomic nervous system function and cardiovascular reactivity in young adults with cancer: a case-controlled feasibility study

Background Preliminary evidence suggests cancer- and chemotherapy-related autonomic nervous system (ANS) dysfunction may contribute to the increased cardiovascular (CV) morbidity- and mortality-risks in cancer survivors. However, the reliability of these findings may have been jeopardized by inconsistent participant screening and assessment methods. Therefore, good laboratory practices must be established before the presence and nature of cancer-related autonomic dysfunction can be characterized. The purpose of this study was to assess the feasibility of conducting concurrent ANS and cardiovascular evaluations in young adult cancer patients, according to the following criteria: i) identifying methodological pitfalls and proposing good laboratory practice criteria for ANS testing in cancer, and ii) providing initial physiologic evidence of autonomic perturbations in cancer patients using the composite autonomic scoring scale (CASS). Methods Thirteen patients (mixed diagnoses) were assessed immediately before and after 4 cycles of chemotherapy. Their results were compared to 12 sex- and age-matched controls. ANS function was assessed using standardized tests of resting CV (tilt-table, respiratory sinus arrhythmia and Valsalva maneuver) and sudomotor (quantitative sudomotor axon reflex test) reactivity. Cardiovascular reactivity during exercise was assessed using a modified Astrand-Ryhming cycle ergometer protocol. Our feasibility criteria addressed: i) recruitment potential, ii) retention rates, iii) pre-chemotherapy assessment potential, iv) test performance/tolerability, and v) identification and minimizing the influence of potentially confounding medication. T-tests and repeated measures ANOVAs were used to assess between- and within-group differences at baseline and follow-up. Results The overall success rate in achieving our feasibility criteria was 98.4 %. According to the CASS, there was evidence of ANS impairment at baseline in 30.8 % of patients, which persisted in 18.2 % of patients at follow-up, compared to 0 % of controls at baseline or follow-up. Conclusions Results from our feasibility assessment suggest that the investigation of ANS function in young adult cancer patients undergoing chemotherapy is possible. To the best of our knowledge, this is the first study to report CASS-based evidence of ANS impairment and sudomotor dysfunction in any cancer population. Moreover, we provide evidence of cancer- and chemotherapy-related parasympathetic dysfunction – as a possible contributor to the pathogenesis of CV disease in cancer survivors

Correction: Lack of Mucosal Immune Reconstitution during Prolonged Treatment of Acute and Early HIV-1 Infection

BACKGROUND: During acute and early HIV-1 infection (AEI), up to 60% of CD4(+) T cells in the lamina propria of the lower gastrointestinal (GI) tract are lost as early as 2–4 wk after infection. Reconstitution in the peripheral blood during therapy with highly active antiretroviral therapy (HAART) is well established. However, the extent of immune reconstitution in the GI tract is unknown. METHODS AND FINDINGS: Fifty-four AEI patients and 18 uninfected control participants underwent colonic biopsy. Forty of the 54 AEI patients were followed after initiation of antiretroviral therapy (18 were studied longitudinally with sequential biopsies over a 3-y period after beginning HAART, and 22 were studied cross sectionally after 1–7 y of uninterrupted therapy). Lymphocyte subsets, markers of immune activation and memory in the peripheral blood and GI tract were determined by flow cytometry and immunohistochemistry. In situ hybridization was performed in order to identify persistent HIV-1 RNA expression. Of the patients studied, 70% maintained, on average, a 50%–60% depletion of lamina propria lymphocytes despite 1–7 y of HAART. Lymphocytes expressing CCR5 and both CCR5 and CXCR4 were persistently and preferentially depleted. Levels of immune activation in the memory cell population, CD45RO(+) HLA-DR(+), returned to levels seen in the uninfected control participants in the peripheral blood, but were elevated in the GI tract of patients with persistent CD4(+) T cell depletion despite therapy. Rare HIV-1 RNA–expressing cells were detected by in situ hybridization. CONCLUSIONS: Apparently suppressive treatment with HAART during acute and early infection does not lead to complete immune reconstitution in the GI mucosa in the majority of patients studied, despite immune reconstitution in the peripheral blood. Though the mechanism remains obscure, the data suggest that there is either viral or immune-mediated accelerated T cell destruction or, possibly, alterations in T cell homing to the GI tract. Although clinically silent over the short term, the long-term consequences of the persistence of this lesion may emerge as the HIV-1–infected population survives longer owing to the benefits of HAART

A Polychaete’s Powerful Punch: Venom Gland Transcriptomics of Glycera Reveals a Complex Cocktail of Toxin Homologs

© The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. The article attached is the publisher's pdf

A Randomized Trial of Prophylactic Antibiotics for Miscarriage Surgery.

BACKGROUND: Surgical intervention is needed in some cases of spontaneous abortion to remove retained products of conception. Antibiotic prophylaxis may reduce the risk of pelvic infection, which is an important complication of this surgery, particularly in low-resource countries. METHODS: We conducted a double-blind, placebo-controlled, randomized trial investigating whether antibiotic prophylaxis before surgery to complete a spontaneous abortion would reduce pelvic infection among women and adolescents in low-resource countries. We randomly assigned patients to a single preoperative dose of 400 mg of oral doxycycline and 400 mg of oral metronidazole or identical placebos. The primary outcome was pelvic infection within 14 days after surgery. Pelvic infection was defined by the presence of two or more of four clinical features (purulent vaginal discharge, pyrexia, uterine tenderness, and leukocytosis) or by the presence of one of these features and the clinically identified need to administer antibiotics. The definition of pelvic infection was changed before the unblinding of the data; the original strict definition was two or more of the clinical features, without reference to the administration of antibiotics. RESULTS: We enrolled 3412 patients in Malawi, Pakistan, Tanzania, and Uganda. A total of 1705 patients were assigned to receive antibiotics and 1707 to receive placebo. The risk of pelvic infection was 4.1% (68 of 1676 pregnancies) in the antibiotics group and 5.3% (90 of 1684 pregnancies) in the placebo group (risk ratio, 0.77; 95% confidence interval [CI], 0.56 to 1.04; P = 0.09). Pelvic infection according to original strict criteria was diagnosed in 1.5% (26 of 1700 pregnancies) and 2.6% (44 of 1704 pregnancies), respectively (risk ratio, 0.60; 95% CI, 0.37 to 0.96). There were no significant between-group differences in adverse events. CONCLUSIONS: Antibiotic prophylaxis before miscarriage surgery did not result in a significantly lower risk of pelvic infection, as defined by pragmatic broad criteria, than placebo. (Funded by the Medical Research Council and others; AIMS Current Controlled Trials number, ISRCTN97143849.)