Expression,purification, and evidence for the interaction of the two nucleotide-binding folds of the sulphonylurea receptor

The ATP-sensitive potassium channel is made up of four pore forming Kir6.2 subunits, surrounded by four regulatory sulphonylurea receptor (SUR) subunits. The latter subunit contains two nucleotide-binding folds (NBFs) that confer the ability on the channel to sense changes in the metabolic status ([ATP]/[ADP]) of the cell and couple the changes to the membrane potential of the cell. In an attempt to better understand the mechanisms by which NBFs influence the activity of the channel, we have expressed the NBF domains with C-terminally added epitopes (FLAG to NBF1 and His(6) to NBF2) in Escherichia coli and the rabbit reticulocyte lysate system and examined the ability of these domains to interact with each other and with Kir6.2. Both NBFs could be expressed to high levels in E. coli and purified to homogeneity from inclusion bodies. Re-folding of the proteins proved to be unsuccessful. However, we were able to obtain small amounts of radio-labelled NBFs in a soluble state. Using co-immunoprecipitation, we demonstrate that the radio-labelled NBF1 and NBF2 interact with each other. Neither of the NBFs bound to Kir6.2 expressed in the presence of canine microsomes.     

Collection of wind-borne haematophagous insects in the Torres Strait,Australia

Circumstantial evidence has implicated wind-borne mosquitoes (Diptera: Culicidae) in the introduction of Japanese encephalitis (JE) virus into Australia from the New Guinea mainland. A study was initiated on Saibai Island in the northern Torres Strait, during January and February 2000, to identify the potential source of insects collected in aerial (kytoon) and surface-level traps. Wind speed and direction were recorded to determine wind profiles during insect sampling. Northerly winds capable of carrying insects from New Guinea to Saibai Island were only present on three out of 18 nights sampled. Only three male mosquitoes, comprising two Verrallina funerea (Theobald) and one Ochlerotatus vigilax (Skuse), were collected in aerial samples, and were most likely of local origin. Culicoides midges were also collected in aerial nets and included gravid/parous C. bundyensis Lee and Reye, and one parous C. histrio Johannsen. Highest densities of arthropods (up to 1562/million m3) were on 30 January 2000 when NW winds, sustained for six hours, probably introduced midges from the New Guinea mainland. Adult mosquitoes (including three female Ve. funerea and a single female Ficalbia) and Culicoides (including two gravid C. bundyensis and one parous C. cordiger Macfie) were also collected in 2 m high mast nets during northerly surface winds. Although the results do not provide evidence that wind-blown mosquitoes introduced JE from New Guinea into Australia, they do not preclude that strong N winds associated with low pressure systems SW of the Torres Strait could have done so. However, results suggest that Culicoides were more likely than mosquitoes to reach high altitude and travel long distances during the light N winds experienced during the study.     

Quantitative assessment of IgG antibodies to Helicobacter pylori and outcome of ischaemic heart disease

Criticisms of serological studies on Helicobacter pylori and ischaemic heart disease (IHD) include: undiagnosed heart disease in live controls; no assessment of severity or outcome of IHD; and qualitative not quantitative measurements of IgG to the bacteria. The aim was to assess quantitatively IgG levels specific for H. pylori (ng ml(-1)) among patients who survived a myocardial infarction (MI) with those who died of IHD. Sera were from four groups: (1) men who survived one MI; (2) men matched for age and socioeconomic background to group 1; (3) individuals who died suddenly of IHD; (4) accidental deaths matched for age and sex to group 3. Levels of IgG to H. pylori increased with age (P<0.005) but were not associated with smoking or socioeconomic groups. There was a correlation between IgG to the bacteria and decreasing socioeconomic levels only among group 1 (P<0.01). IgG levels were higher for subjects who died of heart disease (median=151 ng ml(-1)) compared with survivors (median=88 ng ml(-1)) (P=0.034) and higher for survivors compared with their controls (median=58 ng ml(-1)) (P=0.039). Future serological studies of H. pylori in relation to IHD should be quantitative and severity of disease considered in analyses.     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

Fish can show emotional fever: stress-induced hyperthermia in zebrafish

Whether fishes are sentient beings remains an unresolved and controversial question. Among characteristics thought to reflect a low level of sentience in fishes is an inability to show stress-induced hyperthermia (SIH), a transient rise in body temperature shown in response to a variety of stressors. This is a real fever response, so is often referred to as ‘emotional fever’. It has been suggested that the capacity for emotional fever evolved only in amniotes (mammals, birds and reptiles), in association with the evolution of consciousness in these groups. According to this view, lack of emotional fever in fishes reflects a lack of consciousness. We report here on a study in which six zebrafish groups with access to a temperature gradient were either left as undisturbed controls or subjected to a short period of confinement. The results were striking: compared to controls, stressed zebrafish spent significantly more time at higher temperatures, achieving an estimated rise in body temperature of about 2–4°C. Thus, zebrafish clearly have the capacity to show emotional fever. While the link between emotion and consciousness is still debated, this finding removes a key argument for lack of consciousness in fishes.     

The genus Teliocrinus (Crinoidea,Echinodermata): a key taxon among pentacrinid stalked crinoids

The main characters of the stalked crinoids of the family Pentacrinitidae attributed to the genus Teliocrinus are re‐evaluated from a quantitative study of phenotype variation, new observations on arm and stalk articulations, and observation of ontogenetic trends. All of the specimens collected in the northern Indian Ocean belong to the same species, i.e. Teliocrinus springeri (Clark, 1909). However, two phenotypes living at different depths remain valid as subspecies: Teliocrinus springeri springeri (Clark, 1909) and Teliocrinus springeri liliaceus (Clark, 1909). Teliocrinus shares several ontogenetic trends with Endoxocrinus, especially in nonfunctional brachial articulations and stalk symplexies. Its assignment to the Diplocrininae is confirmed. A discussion of its affinities with pentacrinid fossil genera in which the crown is well preserved suggests that Diplocrininae could have first appeared during the Lower Cretaceous. A shortening of brachitaxes and a paedomorphic trend of stalk symplexies are the main other evolutionary traits. Nonfunctional articulations are frequently found at the paedomorphic pole of the heterochronic gradient, without clear derived characters. Classification of pentacrinids mainly based on such symplesiomorphy or paedomorphic characters must be definitively abandoned. However, in post‐Palaeozoic stalked crinoids the scarcity of well‐preserved fossils, the high frequency of paedomorphy, and convergent adaptive characters makes phylogenetic reconstruction only based on morphological characters very difficult and speculative. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 155 , 22–39.     

Ultrastructure of Kunjin virus-infected cells: colocalization of NS1 and NS3 with double-stranded RNA, and of NS2B with NS3, in virus-induced membrane structures.

The subcellular location of the nonstructural proteins NS1, NS2B, and NS3 in Vero cells infected with the flavivirus Kunjin was investigated using indirect immunofluorescence and cryoimmunoelectron microscopy with monospecific antibodies. Comparisons were also made by dual immunolabelling using antibodies to double-stranded RNA (dsRNA), the putative template in the flavivirus replication complex. At 8 h postinfection, the immunofluorescent patterns showed NS1, NS2B, NS3, and dsRNA located in a perinuclear rim with extensions into the peripheral cytoplasm. By 16 h, at the end of the latent period, all patterns had changed to some discrete perinuclear foci associated with a thick cytoplasmic reticulum. By 24 h, this localization in perinuclear foci was more apparent and some foci were dual labelled with antibodies to dsRNA. In immuno-gold-labelled cryosections of infected cells at 24 h, all antibodies were associated with clusters of induced membrane structures in the perinuclear region. Two important and novel observations were made. First, one set of induced membranes comprised vesicle packets of smooth membranes dual labelled with anti-dsRNA and anti-NS1 or anti-NS3 antibodies. Second, adjacent masses of paracrystalline arrays or of convoluted smooth membranes, which appeared to be structurally related, were strongly labelled only with anti-NS2B and anti-NS3 antibodies. Paired membranes similar in appearance to the rough endoplasmic reticulum were also labelled, but less strongly, with antibodies to the three nonstructural proteins. Other paired membranes adjacent to the structures discussed above enclosed accumulated virus particles but were not labelled with any of the four antibodies. The collection of induced membranes may represent virus factories in which translation, RNA synthesis, and virus assembly occur.     

Cloning and characterization of DNA complementary to rat liver UDP-glucuronosyltransferase mRNA

UDP-glucuronosyltransferase (transferase) clones were isolated from a cDNA bank constructed in pBR322 using transferase-enriched mRNA from the livers of phenobarbital-treated rats. The enrichment of mRNA was accomplished by polysome immunoadsorption with antibody to purified mouse liver transferase. This antibody was shown to bind specifically to rat transferase by Ouchterlony double diffusion analysis, immunoadsorption of glucuronidating activities, and selective inhibition of the immunoadsorption of in vitro synthesized enzyme by purified rat liver transferase. The isolated clones were verified to contain DNA complementary to transferase mRNA by hybrid translation-selection. Three classes of transferase cDNAs were characterized by restriction endonuclease mapping, and the largest insert-containing clone of each class was designated pUDPGTr-1, pUDPGTr-2, and pUDPGTr-3. Their insert sizes were approximately 2,400, 2,000, and 2,000 bp, respectively. All three cDNAs hybridized with a 2,300 +/- 150 bp mRNA, and each selected the translation of a 52,000-dalton polypeptide. Immunoadsorption of the 35S-labeled translation product could be competitively inhibited in each case by the addition of purified rat liver transferase. pUDPGTr-1 and pUDPGTr-3 inserts shared extensive sequence homology. This was demonstrated by Southern blot analysis using purified inserts and electron microscopic heteroduplex analysis. Southern blot analysis revealed that these cDNAs hybridized to overlapping genomic fragments. pUDPGTr-2 shared less sequence homology with the other two classes of cDNAs, based on the above criteria. In addition, mRNA corresponding to pUDPGTr-2 was elevated 5-fold by phenobarbital treatment, whereas the other mRNAs levels were unaffected. These studies demonstrate that in rat liver there are a minimum of three distinct transferase mRNAs, two of which may be associated with a common gene or gene family.