Engineering sugarcane cultivars with bovine pancreatic trypsin inhibitor (aprotinin) gene for protection against top borer (Scirpophaga excerptalis Walker)

The inhibitory activity of bovine pancreatic trypsin inhibitor (aprotinin), a natural polypeptide and a proteinase inhibitor, was demonstrated on gut proteinases of three lepidopteran borers of sugarcane using commercially available aprotinin. A synthetic gene coding for aprotinin, designed and codon optimized for better expression in plant system (Shantaram 1999), was transferred to two sugarcane cultivars namely CoC 92061 and Co 86032 through particle bombardment. Aprotinin gene expression was driven by maize ubiquitin promoter and the plant selection marker used was hygromycin resistance. The integration, expression and functionality of the transgene was confirmed by Southern, Western and insect bioassay, respectively. Southern analysis showed two to four integration sites of the transgene in the transformed plants. Independent transgenic events showed varied levels of transgene expression resulting in different levels (0.16–0.50%) of aprotinin. In in vivo bioassay studies, larvae of top borer Scirpophaga excerptalis Walker (Lepidoptera: Pyralidae) fed on transgenics showed significant reduction in larval weight which indicated impairment of their development. Results of this study show the possibility of deploying aprotinin gene for the development of transgenic sugarcane cultivars resistant to top borer.     

Assessing conditions influencing the longitudinal distribution of exotic brown trout (<Emphasis Type="Italic">Salmo trutta</Emphasis>) in a mountain stream: a spatially-explicit modeling approach

Trout species often segregate along elevational gradients, yet the mechanisms driving this pattern are not fully understood. On the Logan River, Utah, USA, exotic brown trout (Salmo trutta) dominate at low elevations but are near-absent from high elevations with native Bonneville cutthroat trout (Onchorhynchus clarkii utah). We used a spatially-explicit Bayesian modeling approach to evaluate how abiotic conditions (describing mechanisms related to temperature and physical habitat) as well as propagule pressure explained the distribution of brown trout in this system. Many covariates strongly explained redd abundance based on model performance and coefficient strength, including average annual temperature, average summer temperature, gravel availability, distance from a concentrated stocking area, and anchor ice-impeded distance from a concentrated stocking area. In contrast, covariates that exhibited low performance in models and/or a weak relationship to redd abundance included reach-average water depth, stocking intensity to the reach, average winter temperature, and number of days with anchor ice. Even if climate change creates more suitable summer temperature conditions for brown trout at high elevations, our findings suggest their success may be limited by other conditions. The potential role of anchor ice in limiting movement upstream is compelling considering evidence suggesting anchor ice prevalence on the Logan River has decreased significantly over the last several decades, likely in response to climatic changes. Further experimental and field research is needed to explore the role of anchor ice, spawning gravel availability, and locations of historical stocking in structuring brown trout distributions on the Logan River and elsewhere.     

Modification of the Stewart biphasic colorimetric assay for stable and accurate quantitative determination of Pluronic and Tetronic block copolymers for application in biological systems

Block copolymers are increasingly being applied in areas such as transfection, membrane sealing, site-specific targeting, and bionanoengineering yet there is a relative paucity of assays available for simple, stable and reproducible colorimetric determination of copolymer concentration in solution. We have focused on improving the accuracy and reproducibility of a modified version of the Stewart biphasic colorimetric assay for quantitative determination of Pluronic (poloxamer) and Tetronic (poloxamine) macromolecules. The optimized assay achieved linear response ranges in chloroform for commonly used copolymers such as poloxamine 904 (20-300 microg/ml), poloxamine 908 (10-400 microg/ml), poloxamer 402 (20-400 microg/ml), and poloxamer 407 (10-400 microg/ml). Variation in the type of chlorinated solvent used significantly increased assay sensitivity, presumably through macromolecular reorientation, affording increased access for copolymer-ferrothiocyanate complexation. This was found to be optimally favored by the planar geometry of the solvent cis 1,2-dichloroethylene. For application to biological systems copolymer-protein interactions were for the first time determined and were found to be dependent on the fraction of hydrophobic constituents of the block copolymers and protein type. For instance serum albumin was found to interact with copolymers of low hydrophilic-lipophilic balance values and poly(propylene oxide) contaminants, whereas this interaction was not significant with the relatively hydrophilic IgG. In such systems the colorimetric assay directly determines the fraction of unbound (free) copolymer in the presence of proteins.     

Initiation of poliovirus negative-strand RNA synthesis requires precursor forms of p2 proteins

The replication proteins encoded in the P2 region of the poliovirus genome induce extensive rearrangement of cellular membranes into vesicles and are a required component of viral RNA replication complexes. To identify distinct viral protein(s) from the P2 region of the genome that were required to form functional RNA replication complexes, the P2 proteins were expressed in addition to P3 in HeLa S10 translation-RNA replication reactions. Membrane-associated preinitiation replication complexes were isolated from these reactions and used to measure negative-strand synthesis. The formation of replication complexes capable of initiating negative-strand synthesis was observed when either P23 or when P2 and P3 were expressed in the HeLa S10 translation-replication reactions. The amount of negative-strand RNA synthesized with P2 and P3 was approximately 50% of that observed with P23. Negative-strand synthesis was not observed when the processed forms of the P2 proteins (e.g., 2A, 2B, 2C, 2AB, and 2BC) were used in various combinations in place of P2. In contrast, the expression of 2A and 2BCP3 supported negative-strand synthesis at the same level observed with P23. Therefore, functional replication complexes were formed in reaction mixtures that contained either 2A and 2BCP3 or P2 and P3. Genetic complementation analysis of P23 RNA that contained a lethal mutation in 2C confirmed these results. The expression of 2BCP3 in trans restored the replication of P23-2C(P131N) RNA to wild-type levels. The expression of P2 and P3 also complemented the replication of this mutant RNA, although very inefficiently. Complementation was not observed in reactions that contained P2 alone, 2BC, or 2C. Based on these results, we propose that RNA replication complexes are initially formed with the primary cleavage products of P23 (i.e., P2 and P3 or 2A and 2BCP3), and that 2A and 2BCP3 are preferentially used in this process.     

Potential problems in the use of oligonucleotide probes for staphylococcal enterotoxin genes

Oligonucleotide probes unique to the five major enterotoxin genes of Staphylococcus aureus were synthesized and used to detect DNA sequences homologous to these genes in 27 non-clinical isolates of Staph. aureus isolated from nasal swabs of 74 healthy human volunteers. Genomic DNA from all 27 isolates reacted with at least one of the probes. In a phenotypic assay for toxin production by a reverse passive latex agglutination test however, only 15 of the 27 isolates produced enterotoxin in culture. The results raise the possibility that a number of Staph. aureus isolates harbour DNA sequences that are apparently silent or mutant copies of the enterotoxin genes. This complicates the identification of enterotoxin producers by tests which depend on oligonucleotide or DNA hybridization.     

Antimalarial drugs impact chemical messenger secretion by blood platelets

BackgroundAdvances in antimalarial drug development are important for combating malaria. Among the currently identified antimalarial drugs, it is suggested that some interact directly with the malarial parasites while others interact indirectly with the parasites. While this approach leads to parasite elimination, little is known about how these antimalarial drugs impact immune cells that are also critical in malarial response.MethodsHerein, the effects of two common antimalarial drugs, chloroquine and quinine, on platelets were explored at both the bulk level, using high performance liquid chromatography, and the single cell level, using carbon-fiber microelectrode amperometry, to characterize any changes in chemical messenger secretion.ResultsThe data reveal that both drugs cause platelet activation and reduce the number of platelet exocytosis events as well as delay fusion pore opening and closing.ConclusionsThis work demonstrates how chloroquine and quinine quantitatively and qualitatively impact in vitro platelet function.General significanceOverall, the goal of this work is to promote understanding about how antimalarial drugs impact platelets as this may affect antimalarial drug development as well as therapeutic approaches to treat malarial infection.     

Dishonest signalling in a fiddler crab

Animal communication theory predicts that low-frequency cheating should be common in generally honest signalling systems. However, perhaps because cheats are designed to go undetected, there are few examples of dishonest signals in natural populations. Here we present what we believe is the first example of a dishonest signal which is used commonly by males to attract mates and fight sexual rivals. After losing their large claw male fiddler crabs (Uca annulipes) grow a new one which has less mass, is a less effective weapon and costs less to use in signalling than an equivalent-length claw of the original form. Males with original claws do not differentially fight males with regenerated claws even though they are likely to win. Regenerated claws effectively bluff fighting ability and deter potential opponents before they fight. During mate searching, females do not discriminate against males with low-mass, regenerated claws, indicating that they are deceived as to the true costs males pay to produce sexual signals. Up to 44% of males in natural populations have regenerated claws, a level unanticipated by current signalling theory. The apparent rarity of cheating may be an artefact of the usual difficulty of detecting cheats and dishonesty may be quite common.