Toad atrial natriuretic peptide : cDNA cloning and functional analysis in isolated perfused kidneys

Complementary DNA (cDNA) encoding Bufo marinus (toad) preproatrial natriuretic peptide (preproANP) was isolated by reverse-transcription polymerase chain reaction. Sequence analysis of toad preproANP cDNA revealed an open reading frame of 150 amino acid residues, which shared 72% and 66% identity with Rana catesbeiana and Xenopus laevis preproANP, respectively. The deduced amino acid sequence of toad ANP that corresponded to ANP 1–24 of R. catesbeiana and Rana ridibunda was identical, but it differed by four residues from that of X. laevis. ANP mRNA transcripts were also shown to be expressed in the toad kidney. Subsequently, the effect of frog ANP (1–24) on renal function in toad was examined using a perfused kidney preparation. The arterial infusion of frog ANP caused a dose-dependent decrease in the arterial perfusion pressure that was associated with an increase in the glomerular filtration rate (GFR) and a renal natriuresis and diuresis. The renal natriuresis and diuresis resulted predominantly from an increased GFR rather than from direct tubular effects. This study demonstrates that ANP can regulate renal function, which suggests it may be involved in overall fluid volume regulation.

Photoaffinity labeling of the N-methyltransferase domains of cyclosporin synthetase

The multifunctional polypeptide cyclosporin synthetase (CySyn) remains one of the most complex nonribosomal peptide synthetase described. In this study we used a highly specific photoaffinity labeling procedure with the natural cofactor S-adenosyl-l-methionine (AdoMet), 14C-isotopically labeled at the Sδ methyl group to probe the concerted AdoMet-binding interaction of the N-methyltransferase (N-MTase) centers of CySyn. The binding stoichiometry for the enzyme–AdoMet complex was determined to be 1:7, which is in agreement with inferences made from analysis of the complementary DNA sequence of the simA gene encoding the CySyn polypeptide. The photolabeling of the AdoMet-binding sites displayed homotropic negative cooperativity, characterized by a curvilinear Scatchard plot with upward concavity. Although, the process of N-methyl transfer is not a critical event for peptide elongation, the destabilizing homotropic interactions between N-MTase centers that were observed may represent a mechanism whereby the enzyme preserves the proficiency of the substrate-channeling process of cyclosporin peptide assembly over a broad range of cofactor concentrations. Furthermore, we demonstrated the utility of the photolabeling procedure for tracking the enzyme during purification.

Primary T-lymphocytes rescue the replication of HIV-1 DIS RNA mutants in part by facilitating reverse transcription

The dimerization initiation site (DIS) stem-loop within the HIV-1 RNA genome is vital for the production of infectious virions in T-cell lines but not in primary cells. In comparison to peripheral blood mononuclear cells (PBMCs), which can support the replication of both wild type and HIV-1 DIS RNA mutants, we have found that DIS RNA mutants are up to 100 000-fold less infectious than wild-type HIV-1 in T-cell lines. We have also found that the cell-type-dependent replication of HIV-1 DIS RNA mutants is largely producer cell-dependent, with mutants displaying a greater defect in viral cDNA synthesis when viruses were not derived from PBMCs. While many examples exist of host–pathogen interplays that are mediated via proteins, analogous examples which rely on nucleic acid triggers are limited. Our data provide evidence to illustrate that primary T-lymphocytes rescue, in part, the replication of HIV-1 DIS RNA mutants through mediating the reverse transcription process in a cell-type-dependent manner. Our data also suggest the presence of a host cell factor that acts within the virus producer cells. In addition to providing an example of an RNA-mediated cell-type-dependent block to viral replication, our data also provides evidence which help to resolve the dilemma of how HIV-1 genomes with mismatched DIS sequences can recombine to generate chimeric viral RNA genomes.

The A-rich RNA sequences of HIV-1 pol are important for the synthesis of viral cDNA

The bias of A-rich codons in HIV-1 pol is thought to be a record of hypermutations in viral genomes that lack biological functions. Bioinformatic analysis predicted that A-rich sequences are generally associated with minimal local RNA structures. Using codon modifications to reduce the amount of A-rich sequences within HIV-1 genomes, we have reduced the flexibility of RNA sequences in pol to analyze the functional significance of these A-rich ‘structurally poor’ RNA elements in HIV-1 pol. Our data showed that codon modification of HIV-1 sequences led to a suppression of virus infectivity by 5–100-fold, and this defect does not correlate with, viral entry, viral protein expression levels, viral protein profiles or virion packaging of genomic RNA. Codon modification of HIV-1 pol correlated with an enhanced dimer stability of the viral RNA genome, which was associated with a reduction of viral cDNA synthesis both during HIV-1 infection and in a cell free reverse transcription assay. Our data provided direct evidence that the HIV-1 A-rich pol sequence is not merely an evolutionary artifact of enzyme-induced hypermutations, and that HIV-1 has adapted to rely on A-rich RNA sequences to support the synthesis of viral cDNA during reverse transcription, highlighting the utility of using ‘structurally poor’ RNA domains in regulating biological process.

Single-walled carbon nanotubes with DNA recognition

Two methods for attaching DNA to oxidized single-walled carbon nanotubes either in organic solvent or aqueous solution are described. The sites of DNA attachment to the nanotubes have been verified by binding gold nanoparticles modified with DNA of complementary sequence to the DNA-modified nanotubes, and imaging with TEM. The gold nanoparticles appear on the tips of the nanotubes, and at isolated positions (defects) on the sidewalls. The methods provide versatility for the modification of nanotubes with DNA for their directed assembly, or for their composites with gold nanoparticles, into nanoscale devices.

Macrocyclic Zn complexes for DNA detection

Researchers from Monash University have developed an electrocatalytic method based on a charge transport through DNA films, which allows detection of complementary over non-complementary and mismatched DNA sequences in fully hybridized duplexes.

Azide photochemistry for facile modification of graphitic surfaces : preparation of DNA-coated carbon nanotubes for biosensing

A facile, two-step method for chemically attaching single-stranded DNA to graphitic surfaces, represented here by carbon nanotubes, is reported. In the first step, an azide-containing compound, N-5-azido-nitrobenzoyloxy succinimide (ANB-NOS), is used to form photo-adducts on the graphitic surfaces in a solid-state photochemical reaction, resulting in active ester groups being oriented for the subsequent reactions. In the second step, pre-synthesized DNA strands bearing a terminal amine group are coupled in an aqueous solution with the active esters on the photo-adducts. The versatility of the method is demonstrated by attaching pre-synthesized DNA to surfaces of carbon nanotubes in two platforms—as vertically-aligned multi-walled carbon nanotubes on a solid support and as tangled single-walled carbon nanotubes in mats. The reaction products at various stages were characterized by x-ray photoelectron spectroscopy. Two different assays were used to check that the DNA strands attached to the carbon nanotubes were able to bind their partner strands with complementary base sequences. The first assay, using partner DNA strands tethered to gold nanoparticles, enabled the sites of DNA attachment to the carbon nanotubes to be identified in TEM images. The second assay, using radioactively labelled partner DNA strands, quantified the density of functional DNA strands attached to the carbon nanotubes. The diversity of potential applications for these DNA-modified carbon-nanotube platforms is exemplified here by the successful use of a DNA-modified single-walled carbon-nanotube mat as an electrode for the specific detection of metal ions.