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PubMed2002

Multielemental analysis of Mn-Fe nodules by ICP-MS: optimisation of analytical method.

Two acid digestion procedures (microwave-assisted and room temperature) were developed for the quantitative analysis of ferromanganese nodules by inductively coupled plasma double focusing sector field mass spectrometry (ICP-SFMS). Different compositions of the acid mixture, dilution factors and corrections for spectral interferences were tested. A combination of nitric, hydrochloric and hydrofluoric acids is necessary for complete sample digestion, with lowest acid to sample ratios (v/m) of 15 and 1.5, respectively, for the last two acids. Sample dilution factors higher than 2 x 10(4) should be used in order to decrease matrix effects and provide robust long-term instrumental operation. In spite of high dilution, method detection limits in the sub-microg g(-1) range were obtained for 54 out of 71 elements tested, due to the high detection capability of ICP-SFMS, as well as the special care taken to ensure the purity of reagents, to clean the instrument sample introduction system and to minimise sample handling. Owing to the presence of unresolved (at the resolution available) spectral interferences, accurate determination of Au, Hg, Os, Pd, Re and Rh is impossible without matrix separation. The accuracy of the entire analytical method was tested by the analysis of two nodule reference materials. The results generated agreed to within +/-2% for about 10, within +/-10% for more than 40 and within +/-20% for about 50 of 53 elements for which certified, recommended or literature values are available. A precision better than 3%, expressed as the between-digestion relative standard deviation (n = 4), was obtained for the majority of elements, except in cases limited by low analyte concentrations.

Journal Article↗
PubMed1996

Prevention by (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin of both catalepsy and the rises in rat striatal dopamine metabolism caused by haloperidol.

1. The influence of (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) on haloperidol-induced increases in the dopamine metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 4-hydroxy-3-methoxyphenylacetic acid (HVA), was measured in three microdissected brain regions of the rat following a quantitative assessment of catalepsy. 2. Haloperidol alone (2.66 mumol kg-1, i.p.) caused a robust cataleptic response. Given 30 min after haloperidol, 8-OH-DPAT (76 or 760 nmol kg-1, s.c.) prevented catalepsy in 30% and 100% of rats, respectively. 3. Haloperidol significantly increased the DOPAC (by 2 to 4 fold) and HVA (by 3 to 7 fold) contents of the caudate-putamen, nucleus accumbens and medial prefrontal cortex. Given alone, only the lower dose of 8-OH-DPAT caused a significant biochemical change, a doubling of cortical DOPAC. 4. In the cases where catalepsy was prevented by either dose of 8-OH-DPAT, the haloperidol-induced increases in DOPAC and HVA were consistently lower in the caudate-putamen. This pattern was true for the rise in cortical HVA but only in response to the lower dose of 8-OH-DPAT. In contrast, neither dose of 8-OH-DPAT was able to influence the haloperidol-induced rises in cortical DOPAC. In the nucleus accumbens, 8-OH-DPAT did not affect the haloperidol-induced increases in the dopamine metabolites, irrespective of the dose employed or the resulting behaviour. When catalepsy was not prevented, 8-OH-DPAT did not alter the neurochemical responses to haloperidol in any region. 5. These results suggest that part of the mechanism by which 8-OH-DPAT prevents haloperidol-induced catalepsy is reflected by a reversal of the compensatory increase in meso-striatal and/or meso-cortical dopamine neuronal activity that normally accompanies postsynaptic dopamine receptor blockade with haloperidol.

3,4-Dihydroxyphenylacetic Acid↗
PubMed2004

Sleep rhythmicity and homeostasis in mice with targeted disruption of mPeriod genes.

In mammals, sleep is regulated by circadian and homeostatic mechanisms. The circadian component, residing in the suprachiasmatic nucleus (SCN), regulates the timing of sleep, whereas homeostatic factors determine the amount of sleep. It is believed that these two processes regulating sleep are independent because sleep amount is unchanged after SCN lesions. However, because such lesions necessarily damage neuronal connectivity, it is preferable to investigate this question in a genetic model that overcomes the confounding influence of circadian rhythmicity. Mice with disruption of both mouse Period genes (mPer)1 and mPer2 have a robust diurnal sleep-wake rhythm in an entrained light-dark cycle but lose rhythmicity in a free-run condition. Here, we examine the role of the mPer genes on the rhythmic and homeostatic regulation of sleep. In entrained conditions, when averaged over the 24-h period, there were no significant differences in waking, slow-wave sleep (SWS), or rapid eye movement (REM) sleep between mPer1, mPer2, mPer3, mPer1-mPer2 double-mutant, and wild-type mice. The mice were then kept awake for 6 h (light period 6-12), and the mPer mutants exhibited increased sleep drive, indicating an intact sleep homeostatic response in the absence of the mPer genes. In free-run conditions (constant darkness), the mPer1-mPer2 double mutants became arrhythmic, but they continued to maintain their sleep levels even after 36 days in free-running conditions. Although mPer1 and mPer2 represent key elements of the molecular clock in the SCN, they are not required for homeostatic regulation of the daily amounts of waking, SWS, or REM sleep.

Alleles↗
PubMed2003

Cost-effectiveness of fluticasone propionate in the treatment of chronic obstructive pulmonary disease: a double-blind randomized, placebo-controlled trial.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a debilitating disease and places a large financial burden on health-care systems and society. We prospectively evaluated the cost-effectiveness offluticasone propionate (FP) treatment in patients with moderate-to-severe COPD, who were symptomatic on regular bronchodilator therapy. METHODS: An economic analysis was performed in a 6-month, randomized, double-blind clinical trial comparing FP 1,000 microg/day with placebo in 281 patients aged 45-79 years with symptomatic moderate-to-severe COPD. Data on clinical efficacy, health-care resource use and productivity loss associated with the management of COPD were prospectively collected. The main outcome measures were the incremental cost-effectiveness of achieving a > or = 10% improvement in FEV1 and of remaining exacerbation-free throughout the study. The economic evaluation was costed from the perspective of the NHS (direct costs) and of society (direct and indirect costs). RESULTS: FP was significantly more effective than placebo in terms of the proportions of patients demonstrating a > or = 10% improvement in FEV1 (32 vs. 19%; P = 0.02) and remaining free of moderate/severe exacerbations (75 vs. 63%; P = 0.02). The difference between the groups in total costs was not significantly different. Incremental cost-effectiveness analyses showed that the additional clinical benefits of FP relativeto placebo, in terms of a > or = 10% improvement in FEV1 or an increased number of patients free of moderate/severe exacerbations, were achieved at minimal additional costs from an NHS perspective (additional 0.25 pounds per day for bath) or at a net saving from a societal perspective. Sensitivity analysis showed that these results were robust to changes in the underlying assumptions. CONCLUSIONS: Treatment with FP was associated with statistically significant clinical benefits in patients with moderate-to-severe COPD currently symptomatic on regular bronchodilator therapy. As the differences in direct and total costs compared with placebo were small and non-significant, this treatment can be considered cost-effective in this patient population.

Aged↗
PubMed2005

Combined tetanus, diphtheria, and 5-component pertussis vaccine for use in adolescents and adults.

CONTEXT: Increasing reports of pertussis among US adolescents, adults, and their infant contacts have stimulated vaccine development for older age groups. OBJECTIVE: To assess the immunogenicity and reactogenicity of a tetanus-diphtheria 5-component (pertussis toxoid, filamentous hemagglutinin, pertactin, and fimbriae types 2 and 3) acellular pertussis vaccine (Tdap) in adolescents and adults. DESIGN, SETTING, AND PARTICIPANTS: A prospective, randomized, modified double-blind, comparative trial was conducted in healthy adolescents and adults aged 11 through 64 years from August 2001 to August 2002 at 39 US clinical centers. INTERVENTIONS: A single 0.5-mL intramuscular dose of either Tdap or tetanus-diphtheria vaccine (Td). MAIN OUTCOME MEASURES: Antibody titers to diphtheria and tetanus toxoids for Tdap and Td were measured in sera collected from subsets of adolescents and adults, before and 28 days after vaccination. For pertussis antigens, titers in sera from Tdap vaccinees were assessed vs those from infants who received analogous pediatric diphtheria-tetanus-acellular pertussis vaccine (DTaP) in a previous efficacy trial. Safety was assessed via solicited local and systemic reactions for 14 days and adverse events for 6 months following vaccination. RESULTS: A total of 4480 participants were enrolled. For both Tdap and Td, more than 94% and nearly 100% of vaccinees had protective antibody concentrations of at least 0.1 IU/mL for diphtheria and tetanus, respectively. Geometric mean antibody titers to pertussis toxoid, filamentous hemagglutinin, pertactin, and fimbriae types 2 and 3 exceeded (by 2.1 to 5.4 times) levels in infants following immunization at 2, 4, and 6 months with DTaP. The incidence of solicited local and systemic reactions and adverse events was generally similar between the Tdap and Td groups. CONCLUSIONS: This Tdap vaccine elicited robust immune responses in adolescents and adults to pertussis, tetanus, and diphtheria antigens, while exhibiting an overall safety profile similar to that of a licensed Td vaccine. These data support the potential routine use of this Tdap vaccine in adolescents and adults.

Adolescent↗
PubMed2000

Age-related loss of the AMPA receptor subunits GluR2/3 in the human nucleus basalis of Meynert.

Magnocellular cholinergic neurons in the basal forebrain have long been recognized as vulnerable to the pathology of Alzheimer's disease. Despite numerous anatomical, pharmacological, behavioral, and physiological investigations of these neurons the cellular mechanism that underlines their selective vulnerability remains unclear. As part of an ongoing investigation into the molecular mechanism(s) underlying neuronal vulnerability in Alzheimer's disease and normal aging, we employed immunocytochemical techniques and examined the cellular localization of the alpha-amino-3-hydroxy-5-methyl-4-isoaxolepropionate (AMPA) glutamate receptor subunits GluR1 and GluR2/3 in the basal forebrain of eight nondemented elderly human subjects (66-102 years). For each case we observed GluR1-positive magnocellular cells darkly labeled within all main divisions of the basal forebrain (Ch1-Ch4). Double-labeling immunohistochemical techniques confirmed that the overwhelming majority (94%) of these neurons were also positive for the p75NGFr antibody, thus substantiating the cholinergic nature of these neurons. In contrast, GluR2/3 immunolabeling upon magnocellular neurons was relatively faint or nonexistent. The latter observations were most apparent in cases of advanced age and in the posterior part of the nucleus basalis of Meynert (NBM) (i.e., Ch4). In contrast, in adjacent structures (e.g., globus pallidus), a number of robustly labeled GluR2/3-positive cells were observed. In addition to the eight elderly subjects, we examined GluR1 and GluR2/3 immunostaining in the NBM of five younger cases, 5, 33, 36, 47, and 48 years of age. Although practical considerations limited our observations to the Ch4 region, we observed both GluR1 and GluR2/3 labeling upon NBM neurons in this latter region. On average, the distribution of labeled cells and intensity of immunoreaction were comparable between GluR1 and GluR2/3. The presence of GluR2/3- and GluR1-labeled neurons in the Ch4 region of younger cases but primarily GluR1 in cases of advanced age suggests an age-related decrease in GluR2/3. Functionally, the loss of GluR2 from the AMPA receptor complex results in ion channels highly permeable to Ca(2+). These alterations in cation permeability of the AMPA receptor together with the occurrence of a number of other intrinsic and extrinsic events (i.e., decrease Ca(2+)-binding protein) likely contribute to the vulnerability of these neurons in aging and in AD.

Adult↗
PubMed1990

Differential effects of axotomy on substance P-containing and nicotinic acetylcholine receptor-containing retinal ganglion cells: time course of degeneration and effects of nerve growth factor.

The time course of degeneration of chick retinal ganglion cells was examined with Nissl stains and immunohistochemical methods for detection of substance P-like immunoreactive and nicotinic acetylcholine receptor immunoreactive neurons. Small lesions were made in the retinae, adjacent to the optic nerve head, and were subsequently sectioned parallel to the vitreal surface, permitting direct comparison of normal and axotomized retinal ganglion cells distal to the site of axon damage. At four and six days after surgery, a large number of degenerating cells with clear cytoplasm and pyknotic nuclei were seen. After eight, 10 and 14 days, many retinal ganglion cells displayed a chromatolytic response with dispersed Nissl granules, eccentric nuclei and the cells appeared crenulated. The number of apparently normal neurons in the ganglion cell layer in the axotomized region was reduced by about 50% six days following surgery, by about 70% on the 10th day and by about 75% on the 17th day. The remaining neurons in the ganglion cell layer were identified as displaced amacrine cells. From day 2 onwards, increased numbers of glial cells were present in the optic fibre, ganglion cell and inner plexiform layers. Many glial cells were enlarged and displayed extensive cytoplasmic processes, while others showed mitotic activity. Somata and proximal dendrites of retinal ganglion cells were intensely stained for substance P-like immunoreactivity at two and four days following surgery. At six, eight and 10 days, staining intensity was markedly reduced though still evident and at 14 and 17 days, substance P-like immunoreactivity had virtually disappeared. The persistence of limited substance P-like immunoreactive ganglion cells 10 days after surgery indicates that these cells have a relatively protracted response to axotomy. Nicotinic acetylcholine receptor-like immunoreactivity in the ganglion cells at two and four days following axotomy was substantially reduced. The majority of faintly stained nicotinic acetylcholine receptor-like immunoreactive ganglion cells, as visualized in counterstained sections, did not exhibit pyknosis in the immediate period following axotomy. Double label studies demonstrated that substance P-like immunoreactive ganglion cells were distinct from the nicotinic acetylcholine receptor-like immunoreactive ganglion cells. In a second set of experiments, nerve growth factor was then placed into the vitreous humor following intra-retinal axotomy. The somata, dendrites and proximal axons of lesioned substance P-like immunoreactive ganglion cells in these retinae were more intensely stained for a longer period of time and appeared more robust than cells from untreated retinae.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2002

Isolation and functional analysis of a gene, tcsB, encoding a transmembrane hybrid-type histidine kinase from Aspergillus nidulans.

We cloned and characterized a novel Aspergillus nidulans histidine kinase gene, tcsB, encoding a membrane-type two-component signaling protein homologous to the yeast osmosensor synthetic lethal N-end rule protein 1 (SLN1), which transmits signals through the high-osmolarity glycerol response 1 (HOG1) mitogen-activated protein kinase (MAPK) cascade in yeast cells in response to environmental osmotic stimuli. From an A. nidulans cDNA library, we isolated a positive clone containing a 3,210-bp open reading frame that encoded a putative protein consisting of 1,070 amino acids. The predicted tcsB protein (TcsB) has two probable transmembrane regions in its N-terminal half and has a high degree of structural similarity to yeast Sln1p, a transmembrane hybrid-type histidine kinase. Overexpression of the tcsB cDNA suppressed the lethality of a temperature-sensitive osmosensing-defective sln1-ts yeast mutant. However, tcsB cDNAs in which the conserved phosphorylation site His(552) residue or the phosphorelay site Asp(989) residue had been replaced failed to complement the sln1-ts mutant. In addition, introduction of the tcsB cDNA into an sln1delta sho1delta yeast double mutant, which lacked two osmosensors, suppressed lethality in high-salinity media and activated the HOG1 MAPK. These results imply that TcsB functions as an osmosensor histidine kinase. We constructed an A. nidulans strain lacking the tcsB gene (tcsBdelta) and examined its phenotype. However, unexpectedly, the tcsBdelta strain did not exhibit a detectable phenotype for either hyphal development or morphology on standard or stress media. Our results suggest that A. nidulans has more complex and robust osmoregulatory systems than the yeast SLN1-HOG1 MAPK cascade.

Amino Acid Sequence↗
PubMed1999

Dehydroepiandrosterone treatment of midlife dysthymia.

BACKGROUND: This study evaluated the efficacy of the adrenal androgen, dehydroepiandrosterone, in the treatment of midlife-onset dysthymia. METHODS: A double-blind, randomized crossover treatment study was performed as follows: 3 weeks on 90 mg dehydroepiandrosterone, 3 weeks on 450 mg dehydroepiandrosterone, and 6 weeks on placebo. Outcome measures consisted of the following. Cross-sectional self-ratings included the Beck Depression Inventory, and visual analogue symptom scales. Cross-sectional objective ratings included the Hamilton Depression Rating Scale, the Cornell Dysthymia Scale and a cognitive test battery. Seventeen men and women aged 45 to 63 years with midlife-onset dysthymia participated in this study. Response to dehydroepiandrosterone or placebo was defined as a 50% reduction from baseline in either the Hamilton Depression Rating Scale or the Beck Depression Inventory. RESULTS: In 15 patients who completed the study, a robust effect of dehydroepiandrosterone on mood was observed compared with placebo. Sixty percent of the patients responded to dehydroepiandrosterone at the end of the 6-week treatment period compared with 20% on placebo. A significant response was seen after 3 weeks of treatment on 90 mg per day. The symptoms that improved most significantly were anhedonia, loss of energy, lack of motivation, emotional "numbness," sadness, inability to cope, and worry. Dehydroepiandrosterone showed no specific effects on cognitive function or sleep disturbance, although a type II error could not be ruled out. CONCLUSIONS: This pilot study suggests that dehydroepiandrosterone is an effective treatment for midlife-onset dysthymia.

Adaptation, Psychological↗
PubMed2002

Acetaminophen for altering body temperature in acute stroke: a randomized clinical trial.

BACKGROUND AND PURPOSE: Mild alterations in temperature have prominent effects on ischemic cell injury and stroke outcome. Elevated core body temperature (CBT), even if mild, may exacerbate neuronal injury and worsen outcome, whereas hypothermia is potentially neuroprotective. The antipyretic effects of acetaminophen were hypothesized to reduce CBT. METHODS: This was a randomized, controlled clinical trial at 2 university hospitals. Patients were included if they had stroke within 24 hours of onset of symptoms, National Institutes of Health Stroke Scale (NIHSS) score > or =5, initial CBT <3 8.5 degrees C, and white blood cell count < 12 600 cells/mm(3); they were excluded if they had signs of infection, severe medical illness, or contraindication to acetaminophen. CBT was measured every 30 minutes. Patients were randomized to receive acetaminophen 650 mg or placebo every 4 hours for 24 hours. The primary outcome measure was mean CBT during the 24-hour study period; the secondary outcome measure was the change in NIHSS. RESULTS: Thirty-nine patients were randomized. Baseline CBT was the same: 36.96 degrees C for acetaminophen versus 36.95 degrees C for placebo (P=0.96). During the study period, CBT tended to be lower in the acetaminophen group (37.13 degrees C versus 37.35 degrees C), a difference of 0.22 degrees C (95% CI, -0.08 degrees C to 0.51 degrees C; P=0.14). Patients given acetaminophen tended to be more often hypothermic <36.5 degrees C (OR, 3.4; 95% CI, 0.83 to 14.2; P=0.09) and less often hyperthermic >37.5 degrees C (OR, 0.52; 95% CI, 0.19 to 1.44; P=0.22). The change in NIHSS scores from baseline to 48 hours did not differ between the groups (P=0.93). CONCLUSIONS: Early administration of acetaminophen (3900 mg/d) to afebrile patients with acute stroke may result in a small reduction in CBT. Acetaminophen may also modestly promote hypothermia <36.5 degrees C or prevent hyperthermia >37.5 degrees C. These effects are unlikely to have robust clinical impact, and alternative or additional methods are needed to achieve effective thermoregulation in stroke patients.

Acetaminophen↗
PubMed1997

Impact of interferon beta-1a on neurologic disability in relapsing multiple sclerosis. The Multiple Sclerosis Collaborative Research Group (MSCRG).

BACKGROUND AND OBJECTIVE: A phase III double-blind, placebo-controlled clinical trial demonstrated that interferon beta-1a (IFN beta-1a) (Avonex, Biogen) significantly delayed progression of disability in relapsing MS patients. The primary clinical outcome was time from study entry until disability progression, defined as > or = 1.0 point worsening from baseline Kurtzke Expanded Disability Status Scale (EDSS) score persisting for at least two consecutive scheduled visits separated by 6 months. The objective of this study was to examine the magnitude of benefit on EDSS and its clinical significance. METHODS: Post hoc analyses related to disability outcomes using data collected during the double-blind, placebo-controlled phase III clinical trial. RESULTS: (1) Clinical efficacy related to disability did not depend on the definition of disability progression. A significant benefit in favor of IFN beta-1a was observed when > or = 2.0 point worsening from baseline EDSS was required or when worsening was required to persist for > or = 1.0 year. (2) Placebo recipients who reached the primary clinical outcome worsened by a larger amount from baseline EDSS than did IFN beta-1a recipients who reached the primary study outcome. (3) Significantly fewer IFN beta-1a recipients progressed to EDSS milestones of 4.0 (relatively severe impairment) or 6.0 (unilateral assistance needed to walk). (4) Cox proportional hazards models demonstrated that the only baseline characteristic strongly correlated with longer time to disability progression was IFN beta-1a treatment. CONCLUSIONS: The primary clinical outcome for the IFN beta-1a clinical trial underestimated clinical benefits of treatment. Results in this report demonstrate that IFN beta-1a treatment is associated with robust, clinically important beneficial effects on disability progression in relapsing MS patients.

Adolescent↗
PubMed2005

Intracellular detection of macrophage migration inhibitory factor in peripheral blood leukocytes.

The oxidoreductase MIF is currently discussed as a new promising target of immunomodulatory therapy in patients with severe sepsis. An increasing body of evidence attributes an important role especially to intracellular MIF for regulation of endotoxin responsiveness as well as regulation of nuclear transcription factors. Up to now, measurement of MIF relied on ELISA techniques, lacking the ability to directly measure intracellular MIF and distinguish between different leukocyte subpopulations. Therefore, we developed a sensitive and robust flow cytometry-based method to reliably detect intracellular levels of MIF. This method can readily be applied in cultured cells as well as in subsets of human leukocytes in whole blood. Intracellular MIF content of whole-blood leukocyte subsets is detected simultaneously, and is individually determined for T-lymphocytes, B-lymphocytes, macrophages, and granulocytes, respectively. When tested in an ex vivo whole-blood stimulation system using PMA/Ionomycin the intracellular MIF content doubled in CD3+ T-lymphocytes and increased threefold in CD14+ macrophages. Baseline intracellular MIF levels of different leukocyte subpopulations were quantified in 22 healthy blood donors. Baseline intracellular MIF levels in T-lymphocytes are twice as high compared to those in B-lymphocytes and macrophages.

Enzyme-Linked Immunosorbent Assay↗
PubMed2005

Allergenic potential of novel foods.

Concerns have been expressed that the introduction of novel foods into the diet might lead to the development of new food allergies in consumers. Novel foods can be conveniently divided into GM and non-GM categories. Decision-tree approaches (e.g. International Life Sciences Institute-International Food Biotechnology Council and WHO/FAO) to assess the allergenic potential of GM foods were developed following the discovery, during product development, of the allergenic potential of GM soyabean expressing a gene encoding a storage protein from Brazil nut (Bertolletia excelsa). Within these decision trees considerations include: the source of the transgene; amino acid homology with known allergens; cross-reactivity with IgE from food-allergic individuals; resistance to proteolysis; prediction using animal models of food allergy. Such decision trees are under constant review as new knowledge and improved models emerge, but they provide a useful framework for the assessment of the allergenic potential of GM foods. For novel non-GM foods the assessment of allergenic potential is more subjective; some foods or food ingredients will need no assessment other than a robust protein assay to demonstrate the absence of protein. Where protein is present in the novel non-GM food, hazard and risk assessments need to be made in terms of the quantity of protein that might be consumed, the identity of individual protein components and their relationships to known food allergens. Where necessary, this assessment would extend to serum screening for potential cross-reactivities, skin-prick tests in previously-sensitised individuals and double-blind placebo-controlled food challenges.

Decision Trees↗
PubMed2005

The yeast chromatin remodeler RSC complex facilitates end joining repair of DNA double-strand breaks.

Repair of chromosome double-strand breaks (DSBs) is central to cell survival and genome integrity. Nonhomologous end joining (NHEJ) is the major cellular repair pathway that eliminates chromosome DSBs. Here we report our genetic screen that identified Rsc8 and Rsc30, subunits of the Saccharomyces cerevisiae chromatin remodeling complex RSC, as novel NHEJ factors. Deletion of RSC30 gene or the C-terminal truncation of RSC8 impairs NHEJ of a chromosome DSB created by HO endonuclease in vivo. rsc30Delta maintains a robust level of homologous recombination and the damage-induced cell cycle checkpoints. By chromatin immunoprecipitation, we show recruitment of RSC to a chromosome DSB with kinetics congruent with its involvement in NHEJ. Recruitment of RSC to a DSB depends on Mre11, Rsc30, and yKu70 proteins. Rsc1p and Rsc2p, two other RSC subunits, physically interact with yKu80p and Mre11p. The interaction of Rsc1p with Mre11p appears to be vital for survival from genotoxic stress. These results suggest that chromatin remodeling by RSC is important for NHEJ.

Amino Acid Sequence↗
PubMed2005

Behavioral and cardiovascular effects of 7.5% CO2 in human volunteers.

The study of carbon dioxide (CO2) inhalation in psychiatry has a long and varied history, with recent interest in using inhaled CO2 as an experimental tool to explore the neurobiology and treatment of panic disorder. As a consequence, many studies have examined the panic-like response to the gas either using the single or double breath 35% CO2 inhalation or 5-7% CO2 inhaled for 15-20 min, or rebreathing 5% CO2 for a shorter time. However, this lower dose regime produces little physiological or psychological effects in normal volunteers. For this reason we have studied the effects of a higher concentration of CO2, 7.5%, given over 20 min. Twenty healthy volunteers were recruited to a double blind, placebo-controlled study where air and 7.5% CO2 were inhaled for 20 min. Cardiovascular measures and subjective ratings were obtained. When compared to air, inhaling 7.5% CO2 for 20 min increases systolic blood pressure and heart rate, indicating increased autonomic arousal. It also increases ratings of anxiety and fear and other subjective symptoms associated with an anxiety state. The inhalation of 7.5% CO2 for 20 min is safe for use in healthy volunteers and produces robust subjective and objective effects. It seems promising as an anxiety provocation test that could be beneficial in the study of the effects of anxiety on sustained performance, the discovery of novel anxiolytic agents, and the study of brain circuits and mechanisms of anxiety.

Administration, Inhalation↗
PubMed2024

Replication of single viruses across the kingdoms, Fungi, Plantae, and Animalia.

It is extremely rare that a single virus crosses host barriers across multiple kingdoms. Based on phylogenetic and paleovirological analyses, it has previously been hypothesized that single members of the family Partitiviridae could cross multiple kingdoms. Partitiviridae accommodates members characterized by their simple bisegmented double-stranded RNA genome; asymptomatic infections of host organisms; the absence of an extracellular route for entry in nature; and collectively broad host range. Herein, we show the replicability of single fungal partitiviruses in three kingdoms of host organisms: Fungi, Plantae, and Animalia. Betapartitiviruses of the phytopathogenic fungusRosellinia necatrix could replicate in protoplasts of the carrot (Daucus carota), Nicotiana benthamiana and Nicotiana tabacum, in some cases reaching a level detectable by agarose gel electrophoresis. Moreover, betapartitiviruses showed more robust replication than the tested alphapartitiviruses. One of the fungal betapartitiviruses, RnPV18, could persistently and stably infect carrot plants regenerated from virion-transfected protoplasts. Both alpha- and betapartitiviruses, although with different host preference, could replicate in two insect cell lines derived from the fall armyworm Spodoptera frugiperda and the fruit fly Drosophila melanogaster. Our results indicate the replicability of single partitiviruses in members of three kingdoms and provide insights into virus adaptation, host jumping, and evolution.

Animals↗
PubMed2004

Functional consequences of phosphomimetic mutations at key cAMP-dependent protein kinase phosphorylation sites in the type 1 inositol 1,4,5-trisphosphate receptor.

Regulation of Ca(2+) release through inositol 1,4,5-trisphosphate receptors (InsP(3)R) has important consequences for defining the particular spatio-temporal properties of intracellular Ca(2+) signals. In this study, regulation of Ca(2+) release by phosphorylation of type 1 InsP(3)R (InsP(3)R-1) was investigated by constructing "phosphomimetic" charge mutations in the functionally important phosphorylation sites of both the S2+ and S2- InsP(3)R-1 splice variants. Ca(2+) release was investigated following expression in Dt-40 3ko cells devoid of endogenous InsP(3)R. In cells expressing either the S1755E S2+ or S1589E/S1755E S2- InsP(3)R-1, InsP(3)-induced Ca(2+) release was markedly enhanced compared with nonphosphorylatable S2+ S1755A and S2- S1589A/S1755A mutants. Ca(2+) release through the S2- S1589E/S1755E InsP(3)R-1 was enhanced approximately 8-fold over wild type and approximately 50-fold when compared with the nonphosphorylatable S2- S1589A/S1755A mutant. In cells expressing S2- InsP(3)R-1 with single mutations in either S1589E or S1755E, the sensitivity of Ca(2+) release was enhanced approximately 3-fold; sensitivity was midway between the wild type and the double glutamate mutation. Paradoxically, forskolin treatment of cells expressing either single Ser/Glu mutation failed to further enhance Ca(2+) release. The sensitivity of Ca(2+) release in cells expressing S2+ S1755E InsP(3)R-1 was comparable with the sensitivity of S2- S1589E/S1755E InsP(3)R-1. In contrast, mutation of S2+ S1589E InsP(3)R-1 resulted in a receptor with comparable sensitivity to wild type cells. Expression of S2- S1589E/S1755E InsP(3)R-1 resulted in robust Ca(2+) oscillations when cells were stimulated with concentrations of alpha-IgM antibody that were threshold for stimulation in S2- wild type InsP(3)R-1-expressing cells. However, at higher concentrations of alpha-IgM antibody, Ca(2+) oscillations of a similar period and magnitude were initiated in cells expressing either wild type or S2- phosphomimetic mutations. Thus, regulation by phosphorylation of the functional sensitivity of InsP(3)R-1 appears to define the threshold at which oscillations are initiated but not the frequency or amplitude of the signal when established.

Animals↗
PubMed2004

Unsupported standing with minimized ankle muscle fatigue.

In the past, limited unsupported standing has been restored in patients with thoracic spinal cord injury through open-loop functional electrical stimulation of paralyzed knee extensor muscles and the support of intact arm musculature. Here an optimal control system for paralyzed ankle muscles was designed that enables the subject to stand without hand support in a sagittal plane. The paraplegic subject was conceptualized as an underactuated double inverted pendulum structure with an active degree of freedom in the upper trunk and a passive degree of freedom in the paralyzed ankle joints. Control system design is based on the minimization of a cost function that estimates the effort of ankle joint muscles via observation of the ground reaction force position, relative to ankle joint axis. Furthermore, such a control system integrates voluntary upper trunk activity and artificial control of ankle joint muscles, resulting in a robust standing posture. Figures are shown for the initial simulation study, followed by disturbance tests on an intact volunteer and several laboratory trials with a paraplegic person. Benefits of the presented methodology are prolonged standing sessions and in the fact that the subject is able to maintain voluntary control over upper body orientation in space, enabling simple functional standing.

Algorithms↗