DNA nanoparticles: detection of long-term transgene activity in brain using bioluminescence imaging

In this study, we used bioluminescence imaging (BLI) to track long-term transgene activity following the transfection of brain cells using a nonviral gene therapy technique. Formulations of deoxyribonucleic acid (DNA) combined with 30-mer lysine polymers (substituted with 10 kDa polyethylene glycol) form nanoparticles that transfect brain cells in vivo and produce transgene activity. Here we show that a single intracerebral injection of these DNA nanoparticles (DNPs) into the rat cortex, striatum, or substantia nigra results in long-term and persistent luciferase transgene activity over an 8- to 11-week period as evaluated by in vivo BLI analysis, and single injections of DNPs into the mouse striatum showed stable luciferase transgene activity for 1 year. Compacted DNPs produced in vivo signals 7- to 34-fold higher than DNA alone. In contrast, ex vivo BLI analysis, which is subject to less signal quenching from surrounding tissues, demonstrated a DNP to DNA alone ratio of 76- to 280-fold. Moreover, the ex vivo BLI analysis confirmed that signals originated from the targeted brain structures. In summary, BLI permits serial analysis of luciferase transgene activity at multiple brain locations following gene transfer with DNPs. Ex vivo analysis may permit more accurate determination of relative activities of gene transfer vectors.     

Enhanced toxicity of Bacillus thuringiensis subspecies kurstaki and aizawai to black cutworm larvae (Lepidoptera: Noctuidae) with Bacillus sp. NFD2 and Pseudomonas sp. FNFD1

Bacillus thuringiensis subspecies kurstaki and aizawai are important control agents for lepidopteran pests. Bioassays were designed to test B. t. kurstaki and aizawai against second- and-fourth instar black cutworm larvae with and without Bacillus sp. NFD2 and Pseudomonas sp. FNFD1 bacteria. B. thuringiensis subsp. aizawai (XenTari) was more toxic to both second- and fourth-instar black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), larvae than B. t. kurstaki (DiPel) at 7 d after treatment (DAT). When DiPel was combined with NFD2 or FNFD1 versus second instars, the LC50s were 5.0X and 4.7X lower, respectively, than with DiPel alone. DiPel combined with both NFD2 and FNFD1 versus second instars resulted in an LC50 value 7.7X lower than with DiPel alone. When XenTari was combined with NFD2 or FNFD1 versus second instars, the LC50s were 5.2X and 3.8X lower, respectively, than with XenTari alone. XenTari combined with both NFD2 and FNFD1 versus second instars resulted in an LC50 9.7X lower than with XenTari alone. When DiPel was combined with NFD2 or FNFD1 versus fourth instars, the LC50s were 4.4X and 3.4X lower, respectively, than with DiPel alone. DiPel combined with both NFD2 and FNFD1 versus fourth instars resulted in an LC50 5.0X lower than with DiPel alone. When XenTari was combined with NFD2 or FNFD1 versus fourth instars, the LC50s were 5.7X and 3.3X lower, respectively, than with XenTari alone. XenTari combined with both NFD2 and FNFD1 versus fourth instars resulted in an LC50 6.7X lower than with XenTari alone.     

A one step separation of immunoglobulin g from bovine serum by pseudobioaffinity chromatography on histidine grafted to epoxy activated sepharose

The selective adsorption of proteins and macromolecules on activated matrix grafted with histidine has been shown to be dependent on certain separation parameters like, pH and buffer type. In the present study, the significant potential of the histidine ligand to separate bovine IgG from other bovine serum proteins has been demonstrated. The successful separation was carried out by pseudobioaffinty chromatography on histidine grafted-epoxy activated sepharose. The method was applied to sterile bovine serum. Bovine IgG was completely separated in the form of a single peak with 25 mM MES buffer containing 0.2 M NaCl at pH 5. The purity of the separated bovine IgG was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Ouchterlony radial immunodiffusion (ODD) assay showed that bovine IgG was the main component present in the elution fraction.     

Protocols for hyperlactatemia induction in the lactate minimum test adapted to swimming rats

The lactate minimum test (LACmin) has been considered an important indicator of endurance exercise capacity and a single session protocol can predict the maximal steady state lactate (MLSS). The objective of this study was to determine the best swimming protocol to induce hyperlactatemia in order to assure the LACmin in rats (Rattus norvegicus), standardized to four different protocols (P) of lactate elevation. The protocols were P1: 6 min of intermittent jumping exercise in water (load of 50% of the body weight — bw); P2: two 13% bw load swimming bouts until exhaustion (tlim); P3: one tlim 13% bw load swimming bout; and P4: two 13% bw load swimming bouts (1st 30 s, 2nd to tlim), separated by a 30 s interval. The incremental phase of LACmin beginning with initial loads of 4% bw, increased in 0.5% at each 5 min. Peak lactate concentration was collected after 5, 7 and 9 min (mmol L^− 1) and differed among the protocols P1 (15.2 ± 0.4, 14.9 ± 0.7, 14.8 ± 0.6) and P2 (14.0 ± 0.4, 14.9 ± 0.4, 15.5 ± 0.5) compared to P3 (5.1 ± 0.1, 5.6 ± 0.3, 5.6 ± 0.3) and P4 (4.7 ± 0.2, 6.8 ± 0.2, 7.1 ± 0.2). The LACmin determination success rates were 58%, 55%, 80% and 91% in P1, P2, P3 and P4 protocols, respectively. The MLSS did not differ from LACmin in any protocol. The LACmin obtained from P4 protocol showed better assurance for the MLSS identification in most of the tested rats.     

Experimental analysis of a paternally inherited extrachromosomal factor

Virtually all known cases of extrachromosomal inheritance involve cytoplasmic inheritance through the maternal line. Recently, a paternally transmitted factor that causes the production of all-male families has been discovered in a parasitic wasp. The wasp has haplodiploid sex determination: male offspring are haploid and usually develop from unfertilized eggs, whereas females are diploid and usually develop from fertilized eggs. It has been postulated that this paternal sex-ratio factor (psr) is either (1) an infectious agent (a venereal disease) that is transmitted to the female reproductive tract during copulation with an infected male and, subsequently, causes all-male families or (2) a male cytoplasmic factor that is transmitted by sperm to eggs upon egg fertilization and, somehow, causes loss of the paternal set of chromosomes.—Experimental evidence is presented which shows that the factor requires egg fertilization for transmission to the next generation; therefore, it is likely to be a cytoplasmic factor. Significant potential intragenomic conflict results from the presence of this factor and two other sex-ratio distorters in this wasp species.     

Activation of the constitutive androstane receptor decreases HDL in wild-type and human apoA-I transgenic mice

The nuclear hormone receptor constitutive androstane receptor (CAR, NR1I3) regulates detoxification of xenobiotics and endogenous molecules, and has been shown to be involved in the metabolism of hepatic bile acids and cholesterol. The goal of this study was to address potential effects of CAR on the metabolism of HDL particles, key components in the reverse transport of cholesterol to the liver. Wild-type (WT) mice, transgenic mice expressing human apolipoprotein A-I (HuAITg), and CAR-deficient (CAR(-/-)) mice were treated with the specific CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). CAR activation decreased HDL cholesterol and plasma apolipoprotein A-I (apoA-I) levels in both WT and HuAITg mice, but not CAR(-/-) mice. Both mouse apoA-I and human apoA-I were decreased by more than 40% after TCPOBOP treatment, and kinetic studies revealed that the production rate of HDL is reduced in TCPOBOP-treated WT mice. In transient transfections, TCPOBOP-activated CAR decreased the activity of the human apoA-I promoter. Although loss of CAR function did not alter HDL levels in normal chow-fed mice, HDL cholesterol, apoA-I concentration, and apoA-I mRNA levels were increased in CAR(-/-) mice relative to WT mice when both were fed a high-fat diet. We conclude that CAR activation in mice induces a pronounced decrease in circulating levels of plasma HDL, at least in part through downregulation of apoA-I gene expression.     

Enhanced toxicity of Bacillus thuringiensis japonensis strain Buibui toxin to oriental beetle and northern masked chafer (Coleoptera: Scarabaeidae) larvae with Bacillus sp. NFD2

Bacillus thuringiensisjaponensis strain Buibui (Btj) has the potential to be an important control agent for pest scarabs. Bioassays using autoclaved and nonautoclaved soil showed there were always lower LC, values associated with nonautoclaved soil. We identified five other bacteria found in the hemolymph of insects killed by Btj and used them in bioassays to see whether we could enhance the control achieved with Btj alone. One bacterium, designated NFD2 and later identified as a Bacillus sp., showed the greatest enhancement of Btj in preliminary experiments and was used in bioassays with Btj versus oriental beetle, Anomala orientalis (Waterhouse), and northern masked chafer, Cyclocephala borealis Arrow (Coleoptera: Scarabaeidae), larvae. This bacterium alone was nontoxic to grubs in bioassays. A combination of this bacterium with Btj in nonautoclaved soil resulted in a significantly lower LC50 value (0.23 microg toxin per g soil) from all other treatments for A. orientalis with one exception; the LC50 where NFD2 was added back into autoclaved soil (0.29 microg toxin per g soil). A combination of this bacterium with Btj in nonautoclaved soil resulted in a significantly lower LC50 value (48.29 microg toxin per g soil) from all other treatments for C. borealis with the exception of the treatment where Bacillus sp. NFD2 was added back to autoclaved soil (96.87 microg toxin per g soil) with Btj. This research shows that other soil bacteria can be used to enhance the toxicity of Btj and possibly other Bts.     

Specificity and interactions of the protein OppA: partitioning solvent binding effects using mass spectrometry

Mass spectrometry (MS) was used to characterise the binding of the 58 kDa protein OppA to 11 peptides with diverse properties. Peptides with two, three and five amino acid residues were added to OppA, and the mass spectra showed that the highest-affinity complexes are formed between OppA and tripeptide ligands. Lower-affinity complexes were observed for OppA and dipeptide ligands, and no complex formation was detected with pentapeptides or a tripeptide in which the N-terminal amino group was acetylated. Tripeptides containing a single d amino acid residue were found not to bind to native OppA. Evidence from the peak width and the, charge in the spectra of the complexes suggests that the bound peptides are encapsulated by the protein in a solvent-filled cavity in the gas phase of the mass spectrometer. Analysis of the proportions of peptide-bound and free proteins under low-energy MS conditions shows a good correlation with solution-phase K(d) measurements where available. Increasing the internal energy of the gas-phase complex led to dissociation of the complex. The ease of dissociation is interpreted in terms of the intrinsic stability of the complex in the absence of the stabilising effects of bulk solvent. The results from this study demonstrate insensitivity to the hydrophobic and ionic properties, of the side-chains of the peptides, in contrast to the investigation of other protein ligand systems by MS. Moreover, these findings are in accord with the physiological role of this protein in allowing into the cell di- and tripeptides containing naturally occurring amino acids, regardless of their sequence, while barring access to potentially harmful peptide mimics.