Alkyl-dihydroxyacetone phosphate synthase and dihydroxyacetone phosphate acyltransferase form a protein complex in peroxisomes.

Dihydroxyacetone phosphate (GrnP) acyltransferase and alkyl-GrnP synthase are the key enzymes involved in the biosynthesis of ether phospholipids. Both enzymes are located on the inside of the peroxisomal membrane. Here we report evidence for a direct interaction between these enzymes obtained by the use of chemical cross-linking. After cross-linking and immunoblot analysis alkyl-GrnP synthase could be detected in a 210-kDa complex which was located entirely on the lumenal side of the peroxisomal membrane. Two-dimensional SDS/PAGE demonstrated that GrnP-acyltransferase is also cross-linked in a 210-kDa complex. Co-immunoprecipitation confirmed that the two enzymes interact, in a heterotrimeric complex. Furthermore, alkyl-GrnP synthase can form a homotrimeric complex in the absence of GrnP-acyltransferase as was demonstrated by immunoblot analysis after cross-linking experiments with either GrnP-acyltransferase deficient human fibroblast homogenates or recombinant (His)6-tagged alkyl-GrnP synthase. We conclude that alkyl-GrnP synthase interacts selectively with GrnP-acyltransferase in a heterotrimeric complex and in the absence of GrnP-acyltransferase can also form a homotrimeric complex.     

Local and relational judgements of surface colour: constancy indices and discrimination performance

Colour constancy is generally assumed to arise from a combination of perceptual constancy mechanisms operating to partially discount illumination changes and relational mechanisms involved in judging the colour relationships between object surfaces. Here we examined the characteristics of these mechanisms using a 'yes/no' task. Subjects judged whether a target colour patch embedded in an array of coloured patches (a) stayed the same across a simulated temporal illuminant change (local colour judgement), or (b) changed in a manner consistent with the illuminant change (relational colour judgement). The colour of the target patch remained constant in one-third of the trials, changed in accord with the illuminant shift in another third, and shifted partially with the illuminant change in the remaining third. We found that perceptual constancy was relatively weak and relational constancy strong, as assessed using a modified colour constancy index. Randomising the spatial positions of coloured patches across the illuminant change did not affect subjects' constancy indices. Application of signal detection analysis revealed some otherwise hidden effects. In the case of relational judgements, subjects adopted more conservative criteria (fewer true and false positives) with randomisation, maintaining a constant level of discrimination performance (d'). For local judgements, randomisation led to small increases in performance but no changes in criteria. We conclude that signal detection theory provides a useful tool to supplement conventional approaches to understanding colour constancy.     

Comparative pathogenesis of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus in noctuid hosts of different susceptibility

Neonate larvae of the noctuid moth Spodoptera exigua were susceptible to an infection by Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV). Biological activity (LD(50),ST(50)) of the virus was considerably reduced as compared to its activity in the homologous host, H. armigera. Pathogenesis was studied using a recombinant HaSNPV carrying a green fluorescent protein gene, which induces fluorescence in infected cells to mark infection. In larvae of H. armigera, fluorescence was pronounced in the fat body after 2.9 days post infection and could also be detected in several other tissues. In contrast, fluorescence was not observed in tissues of S. exigua until 9 days post infection and was restricted almost exclusively to cells of the ganglia. Examination of serial sections of wildtype HaSNPV-infected S. exigua-larvae revealed a similar pattern of tissue tropism. Apparently, HaSNPV does not undergo the usual steps in host invasion and infection in this insect species, but targets specifically to nervous tissue.     

Structural basis for the RNA binding selectivity of oligonucleotide analogues containing alkylsulfide internucleoside linkages and 2'-substituted 3'-deoxyribonucleosides.

In this report we describe the synthesis of oligonucleotides containing sulfide-linked dinucleoside units, namely rT(2'OH)sdT, rT(2'OMe)sdT, dTsrU(2'OMe) and dT(2'OMe)srU(2'OMe). We also describe the interactions of such oligomers with complementary DNA and RNA targets, and provide the structural basis for their remarkable RNA binding selectivity. In all cases, the Tm values of the S/P-chimera duplexes were lower than those of the corresponding unmodified duplexes. We attribute this to steric interactions between the 5'sulfur and the atoms of the nearby base/sugar residues. The 2'-substituents (i.e., 2'OH or 2'OMe) vicinal to the alkylsulfide internucleoside linkage significantly perturb the structure and stability of the duplexes formed with DNA, and more so than with RNA. The introduction of three rT(2'OH)sdTp (or rT(2'OMe)sdTp) units into an oligodeoxynucleotide sequence was sufficient to abolish binding to complementary DNA but not RNA. The same three substitutions with dTsrU(2'OMe)p and dT(2'OMe)srU(2'OMe)p did not abolish binding to DNA but the resulting complexes had poor thermal stability. The RNA-binding 'selectivity' exhibited by these oligomers is attributed to the tendency of the 2'-substituted (branched) furanoses to adopt the C3'-endo pucker, a conformation that is inconsistent with the B-form structure of helical DNA. The preference of these sugars to exist often exclusively in the C3'-endo form is attributed to stereoelectronic effects, namely gauche and anomeric effects. Our findings support the hypothesis that nucleoside analogues puckered exclusively in the C3'-endo form may result in them being especially good binders of targeted mRNA [S.H. Kawai (1991), Ph.D. Thesis, McGill University; Kawasaki et al. (1993) J. Med. Chem. 36, 831-841].     

PPARalpha /gamma ragaglitazar eliminates fatty liver and enhances insulin action in fat-fed rats in the absence of hepatomegaly

Peroxisome proliferator-activated receptor (PPAR)alpha and PPARgamma agonists lower lipid accumulation in muscle and liver by different mechanisms. We investigated whether benefits could be achieved on insulin sensitivity and lipid metabolism by the dual PPARalpha/gamma agonist ragaglitazar in high fat-fed rats. Ragaglitazar completely eliminated high-fat feeding-induced liver triglyceride accumulation and visceral adiposity, like the PPARalpha agonist Wy-14643 but without causing hepatomegaly. In contrast, the PPARgamma agonist rosiglitazone only slightly lessened liver triglyceride without affecting visceral adiposity. Compared with rosiglitazone or Wy-14643, ragaglitazar showed a much greater effect (79%, P < 0.05) to enhance insulin's suppression of hepatic glucose output. Whereas all three PPAR agonists lowered plasma triglyceride levels and lessened muscle long-chain acyl-CoAs, ragaglitazar and rosiglitazone had greater insulin-sensitizing action in muscle than Wy-14643, associated with a threefold increase in plasma adiponectin levels. There was a significant correlation of lipid content and insulin action in liver and particularly muscle with adiponectin levels (P < 0.01). We conclude that the PPARalpha/gamma agonist ragaglitazar has a therapeutic potential for insulin-resistant states as a PPARgamma ligand, with possible involvement of adiponectin. Additionally, it can counteract fatty liver, hepatic insulin resistance, and visceral adiposity generally associated with PPARalpha activation, but without hepatomegaly.     

Phylogenetic Analysis of Orgyia pseudotsugata Single-nucleocapsid Nucleopolyhedrovirus

The Douglas-fir tussock moth Orgyia pseudotsugata （Lepidoptera： Lymantriidae） is a frequent defoliator of Douglas-fir and true firs in western USA and Canada. A single nucleopolyhedrovirus （SNPV） isolated from O. pseudotsugata larvae in Canada （OpSNPV） was previously analyzed via its polyhedrin gene, but is phylogenetic status was ambiguous. Sequences of four conserved baculovirus genes, polyhedrin, lef-8, pif-2 and dpol, were amplified from OpSNPV DNA in polymerase chain reactions using degenerate primer sets and their sequences were analyzed phylogenetically. The analysis revealed that OpSNPV belongs to group Ⅱ NPVs and is most closely related to SNPVs that infect O. ericae and O. anartoides, respectively. These results show the need for multiple, concatenated gene phylogenies to classify baculoviruses.