Molecular cloning and immunological characterization of porcine kidney ferredoxin.

Porcine renodoxon is a kidney mitochondrial iron-sulfur protein (ISP) that functions to transfer electron to cytochromes P450 of the vitamin D pathway. A full-length cDNA clone to porcine renodoxin was isolated in the current investigation and used to study the protein's primary structure and immunological properties. The cysteine ligands for the iron-sulfur center, and the surface protein-binding and phosphorylation sites occupied identical positions in both porcine renodoxin and bovine adrenodoxin. Furthermore, porcine renodoxin was functionally indistinguishable from bovine adrenodoxin and the mature forms of both proteins had the same encoded length and shared approximately 91% sequence similarity. A synthetic peptide to the surface protein-binding region was used to demonstrate the antigenicity of the domain in both the porcine and the bovine ISPs. However, porcine renodoxin displayed only limited immunological identity to other regions of bovine adrenodoxin as measured by competitive enzyme-linked immunosorbent assay. Part of this immunological distinction was attributed to the COOH-terminal processing of porcine renodoxin, an action which negated expression of a COOH-terminal antigenic site that is present in bovine adrenodoxin. Other antigenic differences were linked to charged-residue substitutions that were located in predicted surface domains. The highest frequency of surface-residue substitutions in ferredoxin proteins was predicted for porcine renodoxin, which could provide a basis for understanding why the pig protein appears more antigenically divergent than other ferredoxins.     

Contents Volume 11 1990

Contents Volume 11 1990     

Cationic and PEGylated Amphiphilic Cyclodextrins: Co-Formulation Opportunities for Neuronal Sirna Delivery

Optimising non-viral vectors for neuronal siRNA delivery presents a significant challenge. Here, we investigate a co-formulation, consisting of two amphiphilic cyclodextrins (CDs), one cationic and the other PEGylated, which were blended together for siRNA delivery to a neuronal cell culture model. Co-formulated CD-siRNA complexes were characterised in terms of size, charge and morphology. Stability in salt and serum was also examined. Uptake was determined by flow cytometry and toxicity was measured by MTT assay. Knockdown of a luciferase reporter gene was used as a measure of gene silencing efficiency. Incorporation of a PEGylated CD in the formulation had significant effects on the physical and biological properties of CD.siRNA complexes. Co-formulated complexes exhibited a lower surface charge and greater stability in a high salt environment. However, the inclusion of the PEGylated CD also dramatically reduced gene silencing efficiency due to its effects on neuronal uptake. The co-formulation strategy for cationic and PEGylated CDs improved the stability of the CD.siRNA delivery systems, although knockdown efficiency was impaired. Future work will focus on the addition of targeting ligands to the co-formulated complexes to restore transfection capabilities.     

Species co‐occurrence networks show reptile community reorganization under agricultural transformation

Agricultural transformation represents one of the greatest threats to biodiversity, causing degradation and loss of habitat, leading to changes in the richness and composition of communities. These changes in richness and composition may, in turn, lead to altered species co‐occurrence, but our knowledge of this remains limited. We used a novel co‐occurrence network approach to examine the impact of agricultural transformation on reptile community structure within two large (> 172 000 km²; 224 sites) agricultural regions in southeastern Australia. We contrasted assemblages from sites surrounded by intact and modified landscapes and tested four key hypotheses that agricultural transformation leads to (H1) declines in species richness, (H2) altered assemblages, (H3) declines in overall co‐occurrence, and (H4) complex restructuring of pairwise associations. We found that modified landscapes differed in composition but not richness compared with intact sites. Modified landscapes were also characterized by differences in co‐occurrence network structure; with species sharing fewer sites with each other (reduced co‐occurrence connectance), fewer highly‐connected species (truncation of the frequency distribution of co‐occurrence degree) and increased modularity of co‐occurrence networks. Critically, overall loss of co‐occurrence was underpinned by complex changes to the number and distribution of pair‐wise co‐occurrence links, with 41–44% of species also gaining associations with other species. Change in co‐occurrence was not correlated with changes in occupancy, nor by functional trait membership, allowing a novel classification of species susceptibility to agricultural transformation. Our study reveals the value of using co‐occurrence analysis to uncover impacts of agricultural transformation that may be masked in conventional studies of species richness and community composition.     

An RNA-binding protein recognizes a mammalian selenocysteine insertion sequence element required for cotranslational incorporation of selenocysteine.

In mammalian selenoprotein mRNAs, the recognition of UGA as selenocysteine requires selenocysteine insertion sequence (SECIS) elements that are contained in a stable stem-loop structure in the 3' untranslated region (UTR). In this study, we investigated the SECIS elements and cellular proteins required for selenocysteine insertion in rat phospholipid hydroperoxide glutathione peroxidase (PhGPx). We developed a translational readthrough assay for selenoprotein biosynthesis by using the gene for luciferase as a reporter. Insertion of a UGA or UAA codon into the coding region of luciferase abolished luciferase activity. However, activity was restored to the UGA mutant, but not to the UAA mutant, upon insertion of the PhGPx 3' UTR. The 3' UTR of rat glutathione peroxidase (GPx) also allowed translational readthrough, whereas the PhGPx and GPx antisense 3' UTRs did not. Deletion of two conserved SECIS elements in the PhGPx 3' UTR (AUGA in the 5' stem or AAAAC in the terminal loop) abolished readthrough activity. UV cross-linking studies identified a 120-kDa protein in rat testis that binds specifically to the sense strands of the PhGPx and GPx 3' UTRs. Direct cross-linking and competition experiments with deletion mutant RNAs demonstrated that binding of the 120-kDa protein requires the AUGA SECIS element but not AAAAC. Point mutations in the AUGA motif that abolished protein binding also prevented readthrough of the UGA codon. Our results suggest that the 120-kDa protein is a significant component of the mechanism of selenocysteine incorporation in mammalian cells.     

The migration route and behaviour of Eastern Curlews Numenius madagascariensis

Eastern Curlews Numenius madagascariensis were satellite-tracked onto breeding grounds in north-eastern Russia from south-eastern Queensland over a distance of 12 000 km. They made initial non-stop, long distance flights across the Western Pacific Ocean towards the coastlines of China and Korea followed by shorter flights, over a period of more than a month. The return journey involved a major flight-leg as well, south from the Yellow Sea region. Many birds attempted to migrate but returned to the non-breeding grounds over periods of up to several months. Islands of the Western Pacific region, the southern coastline of New Guinea and north-eastern coastline of Australia are important, particularly for birds that stop migrating. Eastern Curlews that stopped migrating generally survived, which suggests that the species has adapted to deal with adverse conditions en route and/or a physical inability to complete the migration. Such a capacity is perhaps characteristic of a large wader with low annual mortality.     

Evidence against somatic association in hexaploid wheat

In mitotic interphase nuclei, nucleolus-organizing regions of homologous chromosomes lay no closer to each other than nucleolus-organizing regions of non-homologous chromosomes. — Mitotic metaphase studies of distances between satellites and also between telocentrics showed no greater nearness of homologues than of non-homologues. — These studies fail to support the concept of somatic association in hexaploid wheat.