Cloning of rat brain succinyl-CoA:3-oxoacid CoA-transferase cDNA. Regulation of the mRNA in different rat tissues and during brain development.

3-Oxoacid CoA-transferase, which catalyses the first committed step in the oxidation of ketone bodies, is uniquely regulated in developing rat brain. Changes in 3-oxoacid CoA-transferase activity in rat brain during the postnatal period are due to changes in the relative rate of synthesis of the enzyme. To study the regulation of this enzyme, we identified, with a specific polyclonal rabbit anti-(rat 3-oxoacid CoA-transferase), two positive cDNA clones (approx. 800 bp) in a lambda gtll expression library, constructed from poly(A)+ RNA from brains of 12-day-old rats. One of these clones (lambda CoA3) was subcloned into M13mp18 and subjected to further characterization. Labelled single-stranded probes prepared by primer extension of the M13mp18 recombinant hybridized to a 3.6 kb mRNA. Rat brain mRNA enriched by polysome immunoadsorption for a single protein of size 60 kDa which corresponds to the precursor form of 3-oxoacid CoA-transferase was also found to be similarly enriched for the hybridizable 3.6 kb mRNA complementary to lambda CoA3. Affinity-selected antibody to the lambda CoA3 fusion protein inhibited 3-oxoacid CoA-transferase activity present in rat brain mitochondrial extracts. The 3.6 kb mRNA for 3-oxoacid CoA-transferase was present in relative abundance in rat kidney and heart, to a lesser extent in suckling brain and mammary gland and negligible in the liver. The specific mRNA was also found to be 3-fold more abundant in the brain from 12-day-old rats as compared with 18-day-old foetuses and adult rats, corresponding to the enzyme activity and relative rate of synthesis profile during development. These data suggest that 3-oxoacid CoA-transferase enzyme activity is regulated at a pretranslational level.     

Differential regulation by dexamethasone of glucocorticoid receptor messenger RNA concentrations in neuronal cultures derived from fetal rat hypothalamus and cerebral cortex

1. Differential regulation, by dexamethasone, of glucocorticoid receptor gene expression was studied in three different neuronal cultures derived from hypothalamus amygdala, and cerebral cortex. 2. Cellular glucocorticoid receptor (GR) mRNA concentration was measured by hybridization using a 32P-labeled RNA probe complementary to a 2.2-kb fragment of the glucocorticoid receptor mRNA. Changes in the amount of GR mRNA were evaluated in relation to the content of beta-actin mRNA. 3. In cells derived from either hypothalamus or cerebral cortex, we observed a complex pattern of GR mRNA concentrations which were characterized by cyclic variations of GR mRNA content during continuous treatment with dexamethasone for up to 72 hr. 4. In contrast to cells derived from the hypothalamus where a persistent 30-40% reduction in GR mRNA levels was seen for up to a least 72 hr, we observed, in cells derived from the cerebral cortex, a sustained increased (1.4-fold) of the GR mRNA at this same time interval.     

Selective channelling of arachidonic and linoleic acids into glycerolipids of rat hepatocytes in primary culture.

Rat hepatocytes in primary culture were incubated with a mixture of linoleic and arachidonic acid at various total fatty acid/serum albumin molar ratios. Mixed fatty acids were taken up at the same rate and distributed with the same pattern as fatty acids added separately. The rates of total uptake, incorporation into hepatocyte and secreted triacylglycerols and beta-oxidation were linearly related to the fatty acid/albumin ratios, whereas the rate of incorporation into phospholipids was saturable. Neither the uptake rate nor the distribution of both fatty acids considered together varied with the arachidonic acid/linoleic acid molar ratio. Changes in this ratio and in the uptake rate led to significant variations in the respective fate of the fatty acids. The preferential channelling of arachidonic acid versus linoleic acid into beta-oxidation and phosphatidylinositol was greatest at a low uptake rate and then decreased as the uptake rose. Conversely, the preferential channelling of arachidonic acid versus linoleic acid into phosphatidylcholine, but not phosphatidylethanolamine, increased with the uptake rate. Moreover, both arachidonic acid and linoleic acid were preferentially incorporated into the 1-palmitoyl molecular species of phosphatidylcholine and phosphatidylethanolamine at a low uptake rate, and of phosphatidylcholine at a high uptake rate. This could be related to the synthesis of biliary phosphatidylcholine, of which 1-palmitoyl-2-linoleoyl and 1-palmitoyl-2-arachidonoyl are the main molecular species. Linoleic and arachidonic acid were selectively distributed into distinct metabolic pools of triacylglycerol, the intrahepatocyte pool which preferentially incorporated linoleic acid at a low uptake rate and the secreted pool in which the relative enrichment of arachidonic acid increased with the uptake rate. This strengthens the central role of hepatic secretion in the supply of arachidonic acid to peripheral tissues.     

The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.     

The atrioventricular node, His bundle and bundle branches-a new histologic technique.

A technique for examination of the conducting system of the heart is described. A block of tissue embracing the ostium of the coronary sinus, the pars membranacea, the septal leaflet of the tricuspid valve, and appropriate amounts of interatrial and interventricular septum is flattened and fixed in a Kaiserling I solution. Blocks are subsequently cleared in methyl salicylate and trimmed. Sections are cut from the blocks after paraffin embedding beginning from the endocardial surfaces of the right heart chambers. The sections are mounted and stained on 35 mm unperforated leader film and covered with an acrylic preservative. For examination of the conducting system many fewer sections are required than with previous techniques.     

Linking mosquito surveillance to dengue fever through Bayesian mechanistic modeling

Our ability to effectively prevent the transmission of the dengue virus through targeted control of its vector, Aedes aegypti, depends critically on our understanding of the link between mosquito abundance and human disease risk. Mosquito and clinical surveillance data are widely collected, but linking them requires a modeling framework that accounts for the complex non-linear mechanisms involved in transmission. Most critical are the bottleneck in transmission imposed by mosquito lifespan relative to the virus’ extrinsic incubation period, and the dynamics of human immunity. We developed a differential equation model of dengue transmission and embedded it in a Bayesian hierarchical framework that allowed us to estimate latent time series of mosquito demographic rates from mosquito trap counts and dengue case reports from the city of Vitória, Brazil. We used the fitted model to explore how the timing of a pulse of adult mosquito control influences its effect on the human disease burden in the following year. We found that control was generally more effective when implemented in periods of relatively low mosquito mortality (when mosquito abundance was also generally low). In particular, control implemented in early September (week 34 of the year) produced the largest reduction in predicted human case reports over the following year. This highlights the potential long-term utility of broad, off-peak-season mosquito control in addition to existing, locally targeted within-season efforts. Further, uncertainty in the effectiveness of control interventions was driven largely by posterior variation in the average mosquito mortality rate (closely tied to total mosquito abundance) with lower mosquito mortality generating systems more vulnerable to control. Broadly, these correlations suggest that mosquito control is most effective in situations in which transmission is already limited by mosquito abundance.     

Association between pygmy right whales (Caperea marginata) and areas of high marine productivity off Australia and New Zealand

Abstract

Opportunistic sightings and strandings of Caperea marginata (n=196) from the vicinity of Australia and New Zealand (1884 to early 2007) were used to relate geographic and temporal patterns to oceanographic and broad-scale climatic variability. Records were not uniformly distributed along the coast and more (69%) were from Australia than New Zealand. Seven coastal whale ‘hotspots’ were identified which accounted for 61% of records with locality data. Half of the hotspot records were from southeast (37) and northwest (20) Tasmania—others each had 9–15 events. Upwelling and/or high zooplankton abundance has been documented near all whale hotspots. Records of C. marginata occurred in all months, with 75% in spring and summer. Inter-annual variability showed broad agreement between increased whale records (usually in spring/summer) and strongly positive ‘Niño 3.4’ during 1980–1995 but not thereafter. Coastal upwelling and productivity increase during climatic phenomena such as El Niño and are likely to be quickly beneficial to plankton-feeding whales such as C. marginata.