Correction to: Intracerebral haemorrhage on the acute stroke unit.

The article Intracerebral haemorrhage on the acute stroke unit.

Impact of kelp cultivation on the Ecological Status of benthic habitats and Zostera marina seagrass biomass.

The Ecological Status of subtidal benthic communities within a commercial kelp farm on the southwest coast of Ireland was not impacted by macroalgal cultivation. Additionally, there was no effect on the biomass of Zostera marina, a key habitat under the EU Habitats Directive and OSPAR Commission. However, sediment grain size and total organic matter (TOM) were influenced by abiotic and biotic aspects of the farm. A temporal effect on univariate and multivariate species data, Infaunal Quality Index (IQI) and Z. marina biomass was observed. This effect was likely a community response to high storm disturbance in winter 2013/14. The use of IQI to assess the impact of macroalgal cultivation on benthic communities is a novel approach. This study supports a view that environmental impacts of macroalgal cultivation are relatively benign compared to other forms of aquaculture. Further research must be conducted to understand all interactions between aquaculture activities and the environment.

Characterisation of rat superficial superior colliculus neurones: firing properties and sensitivity to GABA.

Physiological, pharmacological and morphological properties of superficial superior colliculus neurones (n=93) were characterised using whole-cell patch-clamp recordings in rat brain slices. Six cell types (narrow- and wide-field vertical, horizontal, piriform, marginal and stellate) were identified based on Lucifer Yellow labelling but no cell type-specific spike pattern could be identified. Resting membrane potentials were homogeneous (mean: -67.1 +/- 0.7 mV, n=48), and spike frequencies ranged from 10 to 70 Hz (80 pA current injection). About 66% of the cells displayed regular and sustained spike production, throughout all neuronal categories. Rebound spikes and spontaneous activity were observed frequently in all cell types. Synaptically evoked action potentials appeared as single spikes (mean amplitude: 76.0 +/- 3.2 mV, n=34) followed by a fast after-hyperpolarising potential (mean amplitude: 25.4 +/- 1.4 mV, n=34) and variable late potentials (late after-depolarising and/or -hyperpolarising). Pharmacologically, a characterisation using GABA and its subtype-specific agonists indicated a strong inhibitory influence of this transmitter system on >90% of cells. The GABA(A) receptor agonist, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (100 microM), caused a reversible hyperpolarisation (approximately 9 mV) and spike inhibition of all neurones studied. This was more pronounced for intrinsic than for synaptically evoked spikes. Assessment of the GABA(C) receptor agonist, cis-4-aminocrotonic acid (1 mM), also revealed a hyperpolarisation (approximately 3 mV) and an inhibitory action on firing, but this was not as potent and homogeneous, compared to the GABA(A) receptor agonist. Further, the GABA(B) receptor agonist, baclofen (50-100 microM), had more variable (hyperpolarising, depolarising or no change) effects on the membrane potential. It showed little modulation of current-induced action potentials but fully blocked synaptic spikes. Assessment of GABA receptor antagonist actions revealed the presence of weak tonic and strong phasic GABA(A) receptor-mediated inhibition in the superficial superior colliculus: application of the GABA(A) receptor antagonist, bicuculline (100 microM), led to a generally enhanced excitability and depolarisation (approximately 5 mV). Intrinsic firing was somewhat enhanced, but synaptic spiking was drastically potentiated and prolonged. In contrast, 1,2,5,6-tetrahydro-(pyridin-4-yl) methylphosphinic acid (TPMPA; 100 microM), the GABA(C) receptor antagonist, produced little effect on these physiological parameters. The GABA(B) receptor antagonist, CGP35348 (200 microM), caused a partial inhibition of late after-hyperpolarising potentials (approximately 30%). Uptake of GABA contributes little to endogenous inhibition in the superior colliculus slice preparation, as suggested by the action of GABA uptake inhibitors SKF89976 (50-100 microM) and nipecotic acid (200-500 microM), both had no obvious effect on physiological parameters. However, in the presence of these compounds, sub-maximal inhibitory actions of GABA were potentiated. In conclusion, different cell types in the superficial superior colliculus do not display distinct physiological properties and are subject to strong inhibitory modulation. We therefore suggest that signal processing in this brain region does not require cell type-specific encoding of information. In line with evidence provided by previous in vivo investigations, identification of visual stimuli and orientation responses appears to be realised via the network properties of the receptive fields that form topographic maps.

Modulation of GABA(A) receptors and inhibitory synaptic currents by the endogenous CNS sleep regulator cis-9,10-octadecenoamide (cOA).

1. Cis-9,10-octadecenoamide (cOA) accumulates in the CSF of sleep-deprived cats and may represent a novel signalling molecule. Synthetic cOA has been shown to induce physiological sleep when injected into laboratory rats. Here we assess the cellular mode of action of cOA in vitro. 2. In all rat cultured cortical neurones (pyramidal cells) examined, the synthetic brain lipid (3.2-64 microM) enhanced the responses to subsaturating GABA concentrations (up to circa 2x) in a concentration-dependent manner (EC50, circa 15 microM). 3. (20 microM) cOA significantly enhanced the affinity of exogenous GABA for its receptor without changing the Hill slope or the maximal response. These effects were not voltage-dependent or secondary to shifts in E(Cl). 4. In the absence of GABA, cOA directly evoked small inhibitory currents in a subpopulation (<7%) of sensitive cells. 5. 20 microM cOA reversibly enhanced the duration of spontaneous inhibitory post synaptic currents (circa 2 fold) without significantly altering their amplitude. 6. At 32-64 microM, cOA reversibly reduced the incidence and amplitude of both inhibitory post synaptic currents (i.p.s.cs) and excitatory post synaptic currents (e.p.s.cs) in the cultured neuronal circuits in common with other depressant drugs acting at the GABA(A) receptor. 7. 32 microM Oleic acid did not modulate exogenous GABA currents or synaptic activity suggesting that cOAs actions are mediated through a specific receptor. 8. A specific, protein-dependent interaction with GABA(A) receptors was confirmed in Xenopus oocytes. Recombinant human receptors were modulated by 10 microM cOA (and diazepam) only when a gamma2 subunit was co-expressed with alpha1beta2: the cOA response was not sensitive to the specific benzodiazepine antagonist flumazenil (1 microM). 9. cOA may represent an endogenous ligand for allosteric modulatory sites on isoforms of GABA(A) receptors which are crucial for the regulation of arousal and have recently been implicated in the circadian control of physiological sleep.

Modulation of recombination human gamma-aminobutyric acidA receptors by isoflurane: influence of the delta subunit.

BACKGROUND: The gamma-aminobutyric acid (GABA)A receptor/chloride channel has a broad-spectrum anesthetic sensitivity and is a key regulator of arousal. Each receptor/channel complex is an assembly of five protein subunits. Six subunit classes have been identified, each containing one to six members; many combinations are expressed throughout the brain. Benzodiazepines and intravenous anesthetic agents are clearly subunit dependent, but the literature to date suggests that volatile anesthetics are not. The physiological role of the delta subunit remains enigmatic, and it has not been examined as a determinant of anesthetic sensitivity. METHODS: Combinations of GABA(A) receptor subunit cDNAs were injected into Xenopus laevis oocytes: alpha1beta1, alpha1beta1gamma2L, alpha1beta1delta, and alpha1beta1gamma2Ldelta. Expression of functional ion channels with distinct signalling and pharmacologic properties was demonstrated within 1-4 days by established electrophysiological methods. RESULTS: Co-expression of the delta subunit produced changes in receptor affinity; current density; and the modulatory efficacy of diazepam, zinc, and lanthanum; it also produced subtle changes in the rate of desensitization in response to GABA. Isoflurane enhanced GABA-induced responses from all combinations: alphabeta delta (>10-fold) > alphabeta > alphabeta gamma > or = alphabeta gammadelta (approximately 5-fold). Dose-response plots were bell shaped. Compared with alphabeta gamma receptors (EC50 = 225 microM), both alphabeta delta (EC50 = 372 microM) and alphabeta gammadelta (EC50 = 399 microM) had a reduced affinity for isoflurane. Isoflurane (at a concentration close to the EC50 for each subunit) increased the affinity of GABA for its receptor but depressed the maximal response (alphabeta gamma and alphabeta gammadelta). In contrast, the small currents through alphabeta delta receptors were enhanced, even at saturating agonist concentrations. CONCLUSIONS: Delta subunit expression alters GABA(A) receptor function but is not an absolute determinant of anesthetic sensitivity.

Modulation of a recombinant invertebrate gamma-aminobutyric acid receptor-chloride channel complex by isoflurane: effects of a point mutation in the M2 domain.

1. Inhalational anaesthetics modulate ligand-gated ion channels at clinical concentrations. In this paper we address submolecular mechanisms for gamma-aminobutyric acid (GABA) receptor modulation by isoflurane. 2. Wild-type Drosophila melanogaster homo-oligomeric GABA receptors were characterized and compared with an ion-channel mutant (alanine substituted to a serine in M2) by means of two-electrode voltage-clamp in membrane-invariant Xenopus oocytes. 3. Both channel receptor isoforms generated outwardly rectifying, bicuculline-insensitive currents with reversal potentials characteristic of a chloride current. 4. As previously shown, the point mutation in the M2 domain conferred a profound resistance to the blocking action of 10 microM picrotoxinin (PTX): circa 7 fold reduction at the GABA EC20. 5. Isoflurane, 195-389 microM, enhanced GABA conductance in both receptor variants by significantly increasing the affinity of the agonist for its receptor without changing Hill slope or maximal response. Relative potencies were statistically indistinguishable. 6. Isoflurane concentration-response curves (on circa GABA EC25) demonstrated that enhancement was effected at around 100-195 microM for both receptor subtypes, but a dramatic divergence was evident at concentrations above 400 microM: wild-type receptors exhibited concentration-dependent block, whilst mutant conductances continued to increase over the same concentration range, showing no tendency to saturate (up to 3330 microM). 7. The above divergence was not attributable to differential desensitization: neither wild-type nor mutant conductance desensitized significantly (P > 0.05) in the absence or presence of anaesthetic. 8. This work demonstrates that modulatory sites for anaesthetic are present on a relatively primitive insect ion channel. 9. The depression of GABA response at high isoflurane concentrations, in WT receptors, (typical of a variety of anaesthetic agents) may reflect low affinity channel block via the PTX site. 10. The non-saturable enhancement of chloride conductances, when the PTX site is mutated, is not consistent with topical proposals that inhalational anaesthetics (stereoselectively) occupy a finite number of sites on these membrane spanning proteins.

The hemocyanin of the Californian black sea hare, Aplysia vaccaria Winkler.

The hemocyanin of the Californian black sea hare. Aplysia vaccaria exists in solution largely as a di-decameric protein with a molecular weight of close to 8.0 x 10(6) and a sedimentation coefficient of about 92 S. Light-scattering measurements at pH 8.0, 0.1 M Tris, 0.05 M Mg2+, 0.01 M Ca2+ gave a molecular weight of 8.0 +/- 0.6 x 10(6), and scanning transmission electron microscopic determinations (STEM) gave a slightly higher particle mass of 8.49 +/- 0.41 x 10(6) daltons. Measurements using the STEM method gave a particle mass of 4.27 +/- 0.26 x 10(6) daltons for the dissociated half-molecules or decamers. Light-scattering measurements on the dissociated monomers at pH 11.1 and in 8.0 M urea gave molecular weights of 4.74 x 10(5). Sedimentation measurements in the presence of 0.01 M Mg2+ indicate that the hemocyanin of A. vaccaria is largely in the di-decameric form in the pH region from about 5.0 to 8.0. Above pH 8.0 the hemocyanin di-decamers are found to dissociate to half-molecules or decamers, followed by dissociation to dimers and monomers as the pH is increased above pH 9.0.

In vitro potency and mode of action of ANQ9040: a novel fast acting muscle relaxant.

1. The in vitro potency and mode of action of the novel, rapid-onset steroidal relaxant ANQ9040 were characterized in the rat isolated phrenic nerve hemidiaphragm. 2. At 32 degrees C, ANQ9040 antagonized neurally evoked contractures with EC50s of 21.5 microM for unitary twitches; 14.4 microM for 2 Hz 'trains of four'; and 7.5 microM for 50 Hz (2 s) tetanic stimulus trains. 3. (+)-Tubocurarine was 22-24 times more potent than ANQ9040 in comparative organ bath experiments. 4. Intracellular recording from endplates revealed that ANQ9040 (0.53-10.0 microM) dose-dependently and reversibly decreased the amplitude of miniature-endplate potentials (IC50 of circa 0.95 microM) without changing transmembrane potential. 5. Surmountable antagonism of subthreshold responses to exogenous (ionophoretic) acetylcholine provided evidence for a non-depolarizing and competitive blockade of post-junctional nicotinic receptors. 6. Sucrose gap recordings of phrenic nerve action potentials revealed that, at concentrations up to 32 microM, ANQ9040 produced no tonic or frequency-dependent antagonism of axonic Na+ channels. 7. We conclude that ANQ9040 is a relatively low-affinity, non-depolarizing, nicotinic antagonist. The in vitro results are discussed in relation to factors impinging on relaxant kinetics and current models for frequency-dependent fade.

Evaluation of marker-assisted selection through computer simulation.

Computer simulation was used to evaluate responses to marker-assisted selection (MAS) and to compare MAS responses with those typical of phenotypic recurrent selection (PRS) in an allogamous annual crop species such as maize (Zea mays L.). Relative to PRS, MAS produced rapid responses early in the selection process; however, the rate of these responses diminished greatly within three to five cycles. The gains from MAS ranged from 44.7 to 99.5% of the maximum potential, depending on the genetic model considered. Linkage distance between markers and quantitative trait loci (QTLs) was the factor which most limited the responses from MAS. When averaged across all models considered, flanking QTLs within two marker loci produced 38% more gain than did selection based on single markers if markers were loosely-linked to a QTL (20% recombination). Flanking markers were much less advantageous when markers were closely-linked to a QTL (5% recombination), producing an advantage over single markers of only 11%. Markers were most effective in fully exploiting the genetic potential when fewer QTLs controlled the trait. Large QTL numbers exacerbated the problem of marker-QTL recombination by requiring more generations for fixation. In annual crop species, MAS may offer a primary advantage of enabling two selection cycles per year versus the 2 years per cycle required by most PRS schemes for the evaluation of testcross progeny. MAS thus appears to allow very rapid gains for the first 2-3 years of recurrent selection, after which time conventional methods might replace MAS to achieve further responses.

c-N-ras is activated infrequently in canine malignant lymphoma.

Activated c-N-ras alleles have been detected in human lymphoma specimens. The aim of the present study was to determine the frequency of c-N-ras mutational activation in canine malignant lymphoma. DNA was isolated from 28 canine malignant lymphoma specimens collected from 28 separate dogs and examined for c-N-ras mutations by polymerase chain reaction amplification and direct sequencing. The tumors were naturally occurring and derived from 20 dogs with known exposures to the phenoxy herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) and from 8 dogs with no known exposure to the herbicide. An oncogenically activating mutation was found in 1 dog without known 2,4-D exposure. The mutation was a 13th codon, second position transition that would result in a glycine-to-aspartate amino acid substitution. The results of this study demonstrate that, similar to the human, c-N-ras mutations are uncommon in dogs with malignant lymphoma and that there is no association between 2,4-D exposure and activation of c-N-ras in the dog.

Molecular-marker-facilitated investigations of quantitative trait loci in maize : 4. Analysis based on genome saturation with isozyme and restriction fragment length polymorphism markers.

Restriction fragment length polymorphisms have become powerful tools for genetic investigations in plant species. They allow a much greater degree of genome saturation with neutral markers than has been possible with isozymes or morphological loci. A previous investigation employed isozymes as genetic markers to infer the location of genetic factors influencing the expression of quantitative traits in the maize population: (CO159×Tx303)F2. This investigation was conducted to examine the inferences that might be derived using a highly saturated map of RFLP markers and isozymes to detect quantitative trait loci (QTLs) in the same maize F2 population. Marker loci that were associated with QTL effects in this investigation generally corresponded well with previous information where such comparisons were possible. Additionally, a number of previously unmarked genomic regions were found to contain factors with large effects on some plant traits. Availability of numerous marker loci in some genomic regions allowed: more accurate localization of QTLs, resolution of linkage between QTLs affecting the same traits, and determination that some chromsome regions previously found to affect a number of traits are likely to be due to linkage of QTLs affecting different traits. Many of the factors that affected plant height quantitatively in this investigation were found to map to regions also including known sites of major genes influencing plant height. Although the data are not conclusive, they suggest that some of the identified QTLs may be allelic to known major genes affecting plant height.

Further characterization of the process of in vitro uptake of radiolabeled copper by the rat brain.

We have previously demonstrated that hypothalmic slices obtained from adult male rats accumulate 67Cu by two ligand-dependent, saturable processes: a high and low affinity process. To further establish the generality of these uptake processes, we defined the ligand requirements and the saturation kinetics of 67Cu uptake by tissue slices obtained from the newborn hypothalamus (HT); adult male hypothalamus, hippocampus, cortex, median eminence, and caudate nucleus; hypothalamus and hippocampus of castrated (14 days) males and of pregnant (19 days) and ovariectomized (14 days) females. It was found that ionic 67Cu2+ was poorly taken up by newborn HT and adult caudate, complexation with His enhanced 67Cu uptake 3-4-fold, and complexation with albumin inhibited 67Cu uptake. These ligand requirements are identical to those we have previously shown for the adult HT. When 67Cu uptake was evaluated under conditions optimal for the high or the low affinity process, for each process the dose response curves generated from these various tissues were very similar. In addition, we assessed the uptake of both components of the CuHis2 complex by incubating tissues with 67Cu3 H-His2 and found that the tissue ratio of 67Cu:3H was a sigmoidal function of the concentration of the Cu complex such that at greater than 5 microM, the ratio was about 3-fold greater than the medium ratio; indicating preferential uptake of 67Cu relative to 3H-His. The changes in isotope ratios were observed in newborn HT and adult HT, as well as caudate. These similarities in the ligand requirements and saturation kinetics of 67Cu uptake establish the generality of these two processes of in vitro uptake of copper in the rat brain.

Molecular-marker-facilitated investigations of quantitative-trait loci in maize. I. Numbers, genomic distribution and types of gene action.

Individual genetic factors which underlie variation in quantitative traits of maize were investigated in each of two F2 populations by examining the mean trait expressions of genotypic classes at each of 17-20 segregating marker loci. It was demonstrated that the trait expression of marker locus classes could be interpreted in terms of genetic behavior at linked quantitative trait loci (QTLs). For each of 82 traits evaluated, QTLs were detected and located to genomic sites. The numbers of detected factors varied according to trait, with the average trait significantly influenced by almost two-thirds of the marked genomic sites. Most of the detected associations between marker loci and quantitative traits were highly significant, and could have been detected with fewer than the 1800-1900 plants evaluated in each population. The cumulative, simple effects of marker-linked regions of the genome explained between 8 and 40% of the phenotypic variation for a subset of 25 traits evaluated. Single marker loci accounted for between 0.3% and 16% of the phenotypic variation of traits. Individual plant heterozygosity, as measured by marker loci, was significantly associated with variation in many traits. The apparent types of gene action at the QTLs varied both among traits and between loci for given traits, although overdominance appeared frequently, especially for yield-related traits. The prevalence of apparent overdominance may reflect the effects of multiple QTLs within individual marker-linked regions, a situation which would tend to result in overestimation of dominance. Digenic epistasis did not appear to be important in determining the expression of the quantitative traits evaluated. Examination of the effects of marked regions on the expression of pairs of traits suggests that genomic regions vary in the direction and magnitudes of their effects on trait correlations, perhaps providing a means of selecting to dissociate some correlated traits. Marker-facilitated investigations appear to provide a powerful means of examining aspects of the genetic control of quantitative traits. Modifications of the methods employed herein will allow examination of the stability of individual gene effects in varying genetic backgrounds and environments.

Evidence for multilocus genetic control of preferential fertilisation in maize.

Genetic segregation was studied in more than 1900 seedlings of an F2 between the maize (Zea mays L.) inbred lines T232 and CM37. Significant segregation distortion was observed at 11 of 17 segregating allozyme loci and at a single morphological marker locus distributed on 7 of the 10 chromosomes in the genome. Deviations from genotypic class expectations were small for most loci, and averaged 7.7 per cent. Percent transmission of the allele contributed by T232 varied from 47.7 per cent to 53.3 per cent. The allele donated by T232 was significantly under-represented for loci on chromosomes 1 and 8, whereas the allele contributed by CM37 was deficient for nine of the ten segregating loci on chromosomes 2, 3, and 6. In all cases, the parental origin of the deficient allele was consistent for markers on a chromosome. Evidence is presented that suggests the aberrant ratios arose from linkage of the markers with genetic factors affecting prezygotic transmission, and that a minimum of 5 such factors were operative, one on each of chromosomes 1, 2, 3, 6, and 8. In contrast to the multi-locus and multi-chromosomal distorted segregation observed in the F2, all loci in backcross progenies fit Mendelian expectations. It is suggested that this discrepancy reflects variable environmental selection pressures on genes that influence aspects of gamete competition.

Duplicated chromosome segments in maize (Zea mays L.): further evidence from hexokinase isozymes.

The genetic control of hexokinase isozymes (ATP: d-hexose-6-phosphotransferase, E.C. 2.7.7.1, HEX) in maize (Zea mays L.) was studied by starch gel electrophoresis. Genetic analysis of a large number of inbred lines and crosses indicates that the major isozymes observed are encoded by two nuclear loci, designated Hex1 and Hex2. Five active allozymes and one null variant are associated with Hex1, while Hex2 has nine active alleles in addition to a null variant. Alleles at both loci govern the presence of single bands, with no intragenic or intergenic heteromers visible, suggesting that maize HEX's are active as monomers. Organelle preparations demonstrate that the products of both loci are cytosolic. All alleles, including the nulls, segregate normally in crosses. Vigorous and fertile plants were synthesized that were homozygous for null alleles at both loci, suggesting that other hexosephosphorylating enzymes exist in maize that are undetected with our assay conditions. Linkage analyses and crosses with B-A translocation stocks place Hex1 on the short arm of chromosome 3, 27 centimorgans from Pgd2 (phosphogluconate dehydrogenase) and Hex2 on the long arm of chromosome 6, approximately 45 centimorgans from Pgd1. It is suggested that the parallel linkages among these two pairs of duplicated genes reflects an evolutionary history involving chromosome segment duplication or polyploidy.

Effect of age at first calving and milking system on the lactation performance of F1 Bos indicus/Bos taurus crossbreds in Sabah.

This paper examines the effect of age at first calving and milking system on the overall lactation performance and incidence of lactation failure of 442 F1 Bos indicus/Bos taurus dairy crossbreds in Sabah. The two types of crossbred used in the study were imported F1 Sahiwal x Friesian and locally bred Friesian x Local Indian Dairy. Heifers which did not lactate for at least 120 days or produced less than 20 kg of milk during the fourteenth week of lactation were considered to have failed to establish a lactation. The imported and locally bred heifers recorded failure rates of 23.4 and 21.4% respectively. Three milking systems were examined namely machine milking without calf, machine milking with calf and hand milking. They produced failure rates of 30, 12 and 5.6% respectively. The cows that passed recorded 120 day milk production figures of 878, 1,134 and 1,054 kg respectively. The three age groups of less than 30 months, 30 to 34 months and greater than 34 months recorded failure rates of 37, 22 and 10% respectively. The heifers machine milked without their calf which calved at less than 30 months of age recorded a failure rate of 44% while those which calved at greater than 34 months recorded a failure rate of only 17%.