NMDA Receptor Involvement in Toxicity to Dopamine Neurons In Vitro Caused by the Succinate Dehydrogenase Inhibitor 3-Nitropropionic Acid

Abstract: Exposure of mesencephalic dopamine neurons to an irreversible inhibitor of succinate dehydrogenase (SDH), 3-nitropropionic acid (3-NPA), for 24 h on day 12 in vitro, produced a dose-dependent loss of high-affinity dopamine uptake when measured 48 h following 3-NPA removal. ATP concentrations in the cultures were reduced by 57% after 3 h of treatment with the highest concentration of 3-NPA tested (500 µ M ). To determine whether glutamate receptors mediated the dopamine toxicity by 3-NPA, cultures were examined for their sensitivity to excitatory amino acid-induced toxicity. Mesencephalic cultures exposed to either 100 µ M NMDA or kainate, on day 12 for 24 h, showed complete loss of dopamine uptake following 48 h of recovery. The NMDA and non-NMDA antagonists, MK-801 (1 µ M ) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 15 µ M ), completely prevented the effects of NMDA or kainate, respectively, when present at the time of toxin exposure. In cultures treated with 3-NPA, MK-801, but not CNQX, significantly attenuated the loss of dopamine uptake. Direct measurement of the effect of 3-NPA on SDH activity showed that 3-NPA dose-dependently inhibited SDH in vitro in a manner commensurate with the loss of dopamine uptake by 3-NPA. MK-801 had no effect on basal SDH activity or on 3-NPA inhibition of SDH. These data are consistent with the interpretation that metabolic inhibition in dopamine neurons can trigger a secondary excitotoxicity that is mediated by NMDA receptors.     

Assessment of rpoB and 16S rRNA genes as targets for PCR-based identification of Pasteurella pneumotropica

Diagnosis of Pasteurella pneumotropica in laboratory animals relies on isolation of the organism, biochemical characterization, and, more recently, DNA-based diagnostic methods. 16S rRNA and rpoB gene sequences were examined for development of a real-time PCR assay. Partial sequencing of rpoB (456 bp) and 16S rRNA (1368 bp) of Pasteurella pneumotropica isolates identified by microbiologic and biochemical assays indicated that either gene sequence can be used to distinguish P. pneumotropica from other members of the Pasteurellaceae family. However, alignment of rpoB sequences from the Pasteurella pneumotropica Heyl (15 sequences) and Jawetz (16 sequences) biotypes with other Pasteurellaceae sequences from GenBank indicated that although rpoB DNA sequencing could be used for diagnosis, development of diagnostic primers and probes would be difficult, because the sequence variability between Heyl and Jawetz biotypes is not clustered in any particular region of the rpoB sequence. In contrast, alignment of 16S rRNA sequences revealed a region with unique and stable nucleotide motifs sufficient to permit development of a specific fluorogenic real-time PCR assay to confirm P. pneumotropica isolated by culture and to differentiate Heyl and Jawetz biotypes.     

Comparative Hazard Identification by a Single Dose Lung Exposure of Zinc Oxide and Silver Nanomaterials in Mice

Comparative hazard identification of nanomaterials (NMs) can aid in the prioritisation for further toxicity testing. Here, we assessed the acute lung, systemic and liver responses in C57BL/6N mice for three NMs to provide a hazard ranking. A silver (Ag), non-functionalised zinc oxide (ZnO) and a triethoxycaprylylsilane functionalised ZnO NM suspended in water with 2% mouse serum were examined 24 hours following a single intratracheal instillation (I.T.). An acute pulmonary inflammation was noted (marked by a polymorphonuclear neutrophil influx) with cell damage (LDH and total protein) in broncho-alveolar lavage fluid (BALF) after administration of both non-functionalised and functionalised ZnO. The latter also induced systemic inflammation measured as an increase in blood neutrophils and a decrease in blood lymphocytes. Exposure to Ag NM was not accompanied by pulmonary inflammation or cytotoxicity, or by systemic inflammation. A decrease in glutathione levels was demonstrated in the liver following exposure to high doses of all three nanomaterials irrespective of any noticeable inflammatory or cytotoxic effects in the lung. By applying benchmark dose (BMD) modeling statistics to compare potencies of the NMs, we rank functionalised ZnO ranked the highest based on the largest number of affected endpoints, as well as the strongest responses observed after 24 hours. The non-functionalised ZnO NM gave an almost similar response, whereas Ag NM did not cause an acute response at similar doses.     

Molecular analyses of a Lesch-Nyhan syndrome mutation (hprt Montreal) by use of T-lymphocyte cultures

Summary The frequency of hprt mutants in peripheral blood T-lymphocytes of two putative Lesch-Nyhan individuals and their parents was determined by a cell cloning assay to quantify the frequency of thioguanine-resistant mutants. The results confirmed the Lesch-Nyhan diagnosis and demonstrated that the mother has an elevated mutant frequency consistent with being heterozygous for an hprt mutation. Mass cultures of T-lymphocytes from both the children and their mother, as well as cultures of hprt mutant clones from the mother, were employed as sources of mRNA for cDNA sequence analysis. These hprt mutants show a single base substitution (TC transition) at position 170 (exon 3). The predicted amino acid change is the substitution of threonine for methionine₅₆. We have designated this new Lesch-Nyhan mutation hprt _Montreal. The use of T-lymphocyte cultures allows rapid sequence analyses of hprt mutations, as well as family studies to define the origin of a particular mutation.     

SveDem,the Swedish Dementia Registry – A Tool for Improving the Quality of Diagnostics,Treatment and Care of Dementia Patients in Clinical Practice

BackgroundThe Swedish Dementia Registry (SveDem) was developed with the aim to improve the quality of diagnostic work-up, treatment and care of patients with dementia disorders in Sweden.MethodsSveDem is an internet based quality registry where several indicators can be followed over time. It includes information about the diagnostic work-up, medical treatment and community support (www.svedem.se). The patients are diagnosed and followed-up yearly in specialist units, primary care centres or in nursing homes.ResultsThe database was initiated in May 2007 and covers almost all of Sweden. There were 28 722 patients registered with a mean age of 79.3 years during 2007–2012. Each participating unit obtains continuous online statistics from its own registrations and they can be compared with regional and national data. A report from SveDem is published yearly to inform medical and care professionals as well as political and administrative decision-makers about the current quality of diagnostics, treatment and care of patients with dementia disorders in Sweden.ConclusionSveDem provides knowledge about current dementia care in Sweden and serves as a framework for ensuring the quality of diagnostics, treatment and care across the country. It also reflects changes in quality dementia care over time. Data from SveDem can be used to further develop the national guidelines for dementia and to generate new research hypotheses.     

Resting Energy Expenditure Changes With Weight Loss: Racial Differences

It is controversial whether weight loss reduces resting energy expenditure (REE) to a different magnitude in black and white women. This aim of this study was to determine whether changes in REE with weight loss were different between black and white postmenopausal women, and whether changes in body composition (including regional lean and fat mass) were associated with REE changes within each race. Black (n = 26) and white (n = 65) women (age = 58.2 ± 5.4 years, 25 < BMI < 40 kg/m²) completed a 20‐week weight‐loss intervention. Body weight, lean and fat mass (total body, limb, and trunk) via dual‐energy X‐ray absorptiometry, and REE via indirect calorimetry were measured before and after the intervention. We found that baseline REE positively correlated with body weight, lean and fat mass (total, limb, and trunk) in white women only (P < 0.05 for all). The intervention decreased absolute REE in both races similarly (1,279 ± 162 to 1,204 ± 169 kcal/day in blacks; 1,315 ± 200 to 1,209 ± 185 kcal/day in whites). REE remained decreased after adjusting for changes in total or limb lean mass in black (1,302–1,182 kcal/day, P = 0.043; 1,298–1,144 kcal/day, P = 0.006, respectively), but not in white, women. Changes in REE correlated with changes in body weight (partial r = 0.277) and fat mass (partial r = 0.295, 0.275, and 0.254 for total, limb, and trunk, respectively; P < 0.05) independent of baseline REE in white women. Therefore, with weight loss, REE decreased in proportion to the amount of fat and lean mass lost in white, but not black, women.     

Chromosome micromanipulation

The attachment of individual chromosomes to the spindle has been studied by micromanipulation in functionally normal grasshopper spermatocytes. Prometaphase to anaphase I chromosomes can be repeatedly stretched with a microneedle without much increase in the distance between the kinetochores and the poles. Individual chromosomes can, however, be displaced laterally (prometaphase-anaphase) or toward the pole (anaphase) without loss of spindle attachment and without greatly disturbing other chromosomes. It is concluded that chromosomes are firmly and individually attached to the spindle by chromosomal spindle fibers which are capable of bearing any normal mitotic load, including the stretching of dikinetic (dicentric) chromosomes in anaphase. Prolonged or severe manipulation can produce a small — three or four micron — increase in the kinetochore-to-pole distance. Anaphase motion continues normally in spite of lateral or poleward displacements or of small increases in the kinetochore-to-pole distance. In late anaphase, chromosomes can be displaced to the opposite pole. An unusual, rapid motion back toward the original pole follows such displacements, but repeated displacements eventually result in non-disjunction. No evidence for firm interzonal connections between anaphase chromosomes was obtained. Prometaphase and metaphase bivalents can be detached from the spindle by manipulations other than bivalent stretching, but half-bivalents in anaphase are never detached by these manipulations.This investigation was supported in part by research grants GM-8480 and GM-13745 from the Division of General Medical Sciences, United States Public Health Service.