Effects of Furosemide on Cochlear Neural Activity,Central Hyperactivity and Behavioural Tinnitus after Cochlear Trauma in Guinea Pig

Cochlear trauma causes increased spontaneous activity (hyperactivity) to develop in central auditory structures, and this has been suggested as a neural substrate for tinnitus. Using a guinea pig model we have previously demonstrated that for some time after cochlear trauma, central hyperactivity is dependent on peripheral afferent drive and only later becomes generated intrinsically within central structures. Furosemide, a loop diuretic, reduces spontaneous firing of auditory afferents. We investigated in our guinea pig model the efficacy of furosemide in reducing 1) spontaneous firing of auditory afferents, using the spectrum of neural noise (SNN) from round window recording, 2) hyperactivity in inferior colliculus, using extracellular single neuron recordings and 3) tinnitus at early time-points after cochlear trauma. Tinnitus was assessed using gap prepulse inhibition of acoustic startle (GPIAS). Intraperitoneal furosemide, but not saline, caused a marked decrease in both SNN and central hyperactivity. Intracochlear perfusion with furosemide similarly reversed central hyperactivity. In animals in which GPIAS measurements suggested the presence of tinnitus (reduced GPIAS), this could be reversed with an intraperitoneal injection with furosemide but not saline. The results are consistent with furosemide reducing central hyperactivity and behavioural signs of tinnitus by acting peripherally to decrease spontaneous firing of auditory afferents. The data support the notion that hyperactivity may be involved in the generation of tinnitus and further suggest that there may be a therapeutic window after cochlear trauma using drug treatments that target peripheral spontaneous activity.     

Spectrophotofluorometric evidence of negatively cooperative binding of warfarin and phenylbutazone to human serum albumin

Competition between drugs for common binding sites on plasma proteins is an often discussed mechanism of drug interaction. In this study, the validity of the concept that phenylbutazone displaces warfarin from human serum albumin (HSA) by direct competition for the same sites was investigated. The fluorescence enhancement titration procedure of Kolb & Weber, Biochemistry $\text{14}$, 4471–4476 (1975) was used to characterise the warfarin-HSA interaction under the following conditions: excitation and emission wavelenghts - 310 & 390 nm respectively; buffer 0.1 M phosphate, pH 7.0 (25°C) and [HSA] - 10^?6M. The titrations were then repeated in the presence of varying concentrations of phenylbutazone (10^?6M ? 5 × 10^?4M). Bjerrum plots of the data showed a shift of the warfarin-HSA binding curves to higher free levels of warfarin with increasing concentrations of phenylbutazone. However this shift was saturable, contrary to expectation for directly competitive antagonism. Together with indirect evidence from the literature, the results suggest that displacement of warfarin by phenylbutazone is via a negatively cooperative mechanism rather than by direct competition.     

Molecular systematics of threadfin breams and relatives (Teleostei,Nemipteridae)

Threadfin breams and relatives of the family Nemipteridae comprise 69 currently recognized species in five genera. They are found in the tropical and subtropical Indo‐West Pacific and most are commercially important. Using recently developed molecule‐based approaches exploiting DNA sequence variation among species/specimens, this study reconstructed a comprehensive phylogeny of the Nemipteridae, examined the validity of species and explored the cryptic diversity of the family, and tested previous phylogenetic hypotheses. A combined data set (105 taxa from 41 morphospecies) with newly determined sequences from two nuclear genes (RAG1 and RH) and one mitochondrial gene (COI), and a data set with only COI gene sequences (329 newly obtained plus 328 from public databases from a total of 53 morphospecies) were used in the phylogenetic analysis. The latter was further used for species delimitation analyses with two different tools to explore species diversity. Our phylogenetic results showed that all the currently recognized genera were monophyletic. The monotypic genus Scaevius is the sister group of Pentapodus and they together are sister to Nemipterus. These three genera combined to form the sister group of the clade comprising Parascolopsis and Scolopsis. The validity of most of the examined species was confirmed except in some cases. The combined evidence from the results of different analyses revealed a gap in our existing knowledge of species diversity in the Nemipteridae. We found several currently recognized species contain multiple separately evolving metapopulation lineages within species; some lineages should be considered as new species for further assignment. Finally, some problematic sequences deposited in public databases (probably due to misidentification) were also revised in this study to improve the accuracy for prospective DNA barcoding work on nemipterid fishes.     

Labour party doctrine and devolution: The Welsh experience

David Coleman and John Salt (eds) ETHNICITY IN THE 1991 CENSUS, VOLUME ONE: DEMOGRAPHIC CHARACTERISTICS OF THE ETHNIC MINORITY POPULATIONS, London: HMSO, 1996, 290 pp., £25.00     

Simplified Paper Format for Detecting HIV Drug Resistance in Clinical Specimens by Oligonucleotide Ligation

Human immunodeficiency virus (HIV) is a chronic infection that can be managed by antiretroviral treatment (ART). However, periods of suboptimal viral suppression during lifelong ART can select for HIV drug resistant (DR) variants. Transmission of drug resistant virus can lessen or abrogate ART efficacy. Therefore, testing of individuals for drug resistance prior to initiation of treatment is recommended to ensure effective ART. Sensitive and inexpensive HIV genotyping methods are needed in low-resource settings where most HIV infections occur. The oligonucleotide ligation assay (OLA) is a sensitive point mutation assay for detection of drug resistance mutations in HIV pol. The current OLA involves four main steps from sample to analysis: (1) lysis and/or nucleic acid extraction, (2) amplification of HIV RNA or DNA, (3) ligation of oligonucleotide probes designed to detect single nucleotide mutations that confer HIV drug resistance, and (4) analysis via oligonucleotide surface capture, denaturation, and detection (CDD). The relative complexity of these steps has limited its adoption in resource-limited laboratories. Here we describe a simplification of the 2.5-hour plate-format CDD to a 45-minute paper-format CDD that eliminates the need for a plate reader. Analysis of mutations at four HIV-1 DR codons (K103N, Y181C, M184V, and G190A) in 26 blood specimens showed a strong correlation of the ratios of mutant signal to total signal between the paper CDD and the plate CDD. The assay described makes the OLA easier to perform in low resource laboratories.