Resolving genetic diversity in Australasian Culex mosquitoes: Incongruence between the mitochondrial cytochrome c oxidase I and nuclear acetylcholine esterase 2

Insects that vector pathogens are under constant surveillance in Australasia although the repertoire of genetic markers to distinguish what are often cryptic mosquito species remains limited. We present a comparative assessment of the second exon–intron region of the acetylcholine esterase 2 gene (ace-2) and the mitochondrial DNA cytochrome c oxidase I (COI) using two closely related Australasia mosquitoes Culex annulirostris and Culex palpalis. The COI revealed eight divergent lineages of which four were confirmed with the ace-2. We dissect out the nuclear chromosomal haplotypes of the ace-2 as well as the exon–intron regions by assessing the protein’s tertiary structure to reveal a hypervariable 5’-exon that forms part of an external protein loop and displays a higher polymorphic rate than the intron. We retrace the evolutionary history of these mosquitoes by phylogenetic inference and by testing different evolutionary hypotheses. We conclude that DNA barcoding using COI may overestimate the diversity of Culex mosquitoes in Australasia and should be applied cautiously with support from the nuclear DNA such as the ace-2. Together the COI and ace-2 provide robust evidence for distinct cryptic Culex lineages—one of which correlates exactly with the southern limit of Japanese encephalitis virus activity in Australasia.     

Efficiency clustering for low-density microarrays and its application to QPCR

Background  

Pathway-targeted or low-density arrays are used more and more frequently in biomedical research, particularly those arrays that are based on quantitative real-time PCR. Typical QPCR arrays contain 96-1024 primer pairs or probes, and they bring with it the promise of being able to reliably measure differences in target levels without the need to establish absolute standard curves for each and every target. To achieve reliable quantification all primer pairs or array probes must perform with the same efficiency.     

Development of macroporous poly(ethylene glycol) hydrogel arrays within microfluidic channels

The mass transport of solutes through hydrogels is an important design consideration in materials used for tissue engineering, drug delivery, and protein arrays used to quantify protein concentration and activity. We investigated the use of poly(ethylene glycol) (PEG) as a porogen to enhance diffusion of macromolecules into the interior of polyacrylamide and PEG hydrogel posts photopatterned within microfluidic channels. The diffusion of GST-GFP and dextran-FITC into hydrogels was monitored and effective diffusion coefficients were determined by fitting to the Fickian diffusion equations. PEG-diacrylate (M(r) 700) with porogen formed a macroporous structure and permitted significant penetration of 250 kDa dextran. Proteins copolymerized in these macroporous hydrogels retained activity and were more accessible to antibody binding than proteins copolymerized in nonporous gels. These results suggest that hydrogel macroporosity can be tuned to regulate macromolecular transport in applications such as tissue engineering and protein arrays.     

Structure-specific tRNA determinants for editing a mischarged amino acid

Alanyl-tRNA synthetase efficiently aminoacylates tRNAAla and an RNA minihelix that comprises just one domain of the two-domain L-shaped tRNA structure. It also clears mischarged tRNAAla using a specialized domain in its C-terminal half. In contrast to full-length tRNAAla, minihelixAla was robustly mischarged and could not be edited. Addition in trans of the missing anticodon-containing domain did not activate editing of mischarged minihelixAla. To understand these differences between minihelixAla and tRNAAla, several chimeric full tRNAs were constructed. These had the acceptor stem of a non-cognate tRNA replaced with the stem of tRNAAla. The chimeric tRNAs collectively introduced multiple sequence changes in all parts but the acceptor stem. However, although the acceptor stem in isolation (as the minihelix) lacked determinants for editing, alanyl-tRNA synthetase effectively cleared a mischarged amino acid from each chimeric tRNA. Thus, a covalently continuous two-domain structure per se, not sequence, is a major determinant for clearance of errors of aminoacylation by alanyl-tRNA synthetase. Because errors of aminoacylation are known to be deleterious to cell growth, structure-specific determinants constitute a powerful selective pressure to retain the format of the two-domain L-shaped tRNA.     

alpha-bromoacetophenone derivatives as neutral protein tyrosine phosphatase inhibitors: structure-Activity relationship

A series of alpha-haloacetophenone derivatives was tested for inhibition of protein tyrosine phosphatases SHP-1 and PTP1B. The results show that the bromides are much more potent than the corresponding chlorides, whereas the phenyl ring is remarkably tolerant to modifications. Derivatization of the phenyl ring with a tripeptide Gly-Glu-Glu resulted in a potent, selective inhibitor against PTP1B.     

Distribution and evolution of the Anopheles punctulatus group (Diptera: Culicidae) in Australia and Papua New Guinea

The members of the Anopheles punctulatus group are major vectors of malaria and Bancroftian filariasis in the southwest Pacific region. The group is comprised of 12 cryptic species that require DNA-based tools for species identification. From 1984 to 1998 surveys were carried out in northern Australia, Papua New Guinea and on islands in the southwest Pacific to determine the distribution of the A. punctulatus group. The results of these surveys have now been completed and have generated distribution data from more than 1500 localities through this region. Within this region several climatic and geographical barriers were identified that restricted species distribution and gene flow between geographic populations. This information was further assessed in light of a molecular phylogeny derived from the ssrDNA (18S). Subsequently, hypotheses have been generated on the evolution and distribution of the group so that future field and laboratory studies may be approached more systematically. This study suggested that the ability for widespread dispersal was found to have appeared independently in species that show niche-specific habitat preference (Anopheles farauti s.s. and A. punctulatus) and conversely in species that showed diversity in their larval habitat (Anopheles farauti 2). Adaptation to the monsoonal climate of northern Australia and southwest Papua New Guinea was found to have appeared independently in A. farauti s.s., A. farauti 2 and Anopheles farauti 3. Shared or synapomorphic characters were identified as saltwater tolerance (A. farauti s.s. and Anopheles farauti 7) and elevational affinities above 1500 m (Anopheles farauti 5, Anopheles farauti 6 and A. farauti 2).     

Comparison of static and dynamic medium environments for culturing of pre-implantation mouse embryos

The ability to manipulate mouse pre-implantation embryos in vitro has become a valuable tool in many scientific disciplines. However, fewer embryos maintain viability following in vitro manipulation compared with embryos in vivo. It has been suggested that use of dynamic medium environments may improve viability by simulating in utero environment. The objective of the study reported here was to compare a microdrop in vitro culture system with a microdevice in vitro culture system containing a static and two dynamic medium environments (0.1 and 0.5 microl/h) for culturing of mouse pre-implantation embryos. Results indicated that the static medium environment, in silicon-glass microdevices, was not significantly different from the microdrop control environment in proportion of embryos developing from the two-cell to the blastocyst stage. However, the static microdevice environment produced significantly (P < 0.05) more morulas than did that of the control group. Both of these treatment groups, under the presented conditions, consistently had significantly higher proportions of blastocysts (P < 0.05) and morulas (P < 0.05) and lower proportions of abnormal (P < 0.05) and eight-cell embryos (P < 0.05), compared with those of the high flow rate dynamic environment microdevice treatment groups. Studies exploring slower or pulsatile rates of medium delivery in a dynamic medium environment are indicated.     

Comparative analysis of the zeta-crystallin/quinone reductase gene in guinea pig and mouse

zeta-Crystallin is a novel nicotinamide adenine dinucleotide phosphate:quinone reductase, present at enzymatic levels in various tissues of different species, which is highly expressed in the lens of some hystricomorph rodents and camelids. We report here the complementary DNA (cDNA) cloning of zeta-crystallin from liver libraries in guinea pig (Cavia porcellus), where zeta-crystallin is highly expressed in the lens, and in the laboratory mouse (Mus musculus), where expression in the lens occurs only at enzymatic levels. A 5' untranslated sequence different from the one previously reported for the guinea pig lens cDNA was found in these clones. We also report the isolation of genomic clones including the complete guinea pig zeta-crystallin gene and the 5' region of this gene in mouse. These results show the presence of two promoters in the guinea pig zeta-crystallin gene, one responsible for expression at enzymatic levels and the other responsible for the high expression in the lens. The guinea pig lens promoter is not present in the mouse gene. This is the first example in which the recruitment of an enzyme as a lens crystallin can be explained by the acquisition of an alternative lens- specific promoter.      

Blood pressure of rats as affected by diet and concentration of NaCl in drinking water.

In the first experiment, weanling rats were fed a grain ration or one of three semipurified diets high in fat, sucrose, or cornstarch. Rats in each dietary group were divided into two subgroups, one of which drank distilled deionized water whereas the other group drank 2% NaCl solution. Blood pressure and sodium intake were individually measured for each rat at weekly intervals for a 10-week period. Rats receiving the salt solution had higher mean blood pressures (127-178 mmHg) than rats offered distilled water (108-127 mmHg). When drinking solutions were the same, more severe rises in blood pressure occurred in rats fed the semipurified diets than in those rats fed grain. In a second experiment, rats were fed one of the four diets used in the first experiment; however, they received a 1% NaCl drinking solution for 9 weeks followed by a 1.5% NaCl solution for an additional 9 weeks. At Week 18, pressures among these groups of rats ranged from 136-140 mmHg, regardless of diet.