Acetylcholinesterase mediated susceptibility of soldiers and workers of formosan subterranean termite (Isoptera: Rhinotermitidae) to chlorpyrifos

Target site studies were undertaken to examine the difference in susceptibility of Formosan subterranean termite, Coptotermes formosanus Shiraki, workers and soldiers to chlorpyrifos. Workers exhibited significantly greater acetylcholinesterase activity per insect than soldiers (118.63 +/- 48.51 versus 47.98 +/- 22.59 mOD/min/insect equivalent). Likewise, enzyme activity (mean +/- SD) per milligram of protein was greater in workers than soldiers (440.30 +/- 267.43 versus 311.53 +/- 149.83 mOD/min/mg protein). The enzyme of soldiers was more sensitive to the acetylcholinesterase (AChE) inhibitors eserine and chlorpyrifos-oxon than that of workers. The I50s of chlorpyrifos-oxon were 2.66 and 4.59 nM for soldiers and workers, respectively, whereas the I50s of eserine were 16.56 and 25.41 nM for soldiers and workers, respectively. The amount of protein was significantly higher in workers than in soldiers with mean values of 0.270 +/- 0.102 and 0.154 +/- 0.054 mg/insect equivalent, respectively. We suggest that the differential response of workers and soldiers to chlorpyrifos may be due to the difference in AChE sensitivity to inhibition and the amount of protein between them.     

Identification and characterization of coenzyme B12-dependent glycerol dehydratase- and diol dehydratase-encoding genes from metagenomic DNA libraries derived from enrichment cultures

To isolate genes encoding coenzyme B(12)-dependent glycerol and diol dehydratases, metagenomic libraries from three different environmental samples were constructed after allowing growth of the dehydratase-containing microorganisms present for 48 h with glycerol under anaerobic conditions. The libraries were searched for the targeted genes by an activity screen, which was based on complementation of a constructed dehydratase-negative Escherichia coli strain. In this way, two positive E. coli clones out of 560,000 tested clones were obtained. In addition, screening was performed by colony hybridization with dehydratase-specific DNA fragments as probes. The screening of 158,000 E. coli clones by this method yielded five positive clones. Two of the plasmids (pAK6 and pAK8) recovered from the seven positive clones contained genes identical to those encoding the glycerol dehydratase of Citrobacter freundii and were not studied further. The remaining five plasmids (pAK2 to -5 and pAK7) contained two complete and three incomplete dehydratase-encoding gene regions, which were similar to the corresponding regions of enteric bacteria. Three (pAK2, -3, and -7) coded for glycerol dehydratases and two (pAK4 and -5) coded for diol dehydratases. We were able to perform high-level production and purification of three of these dehydratases. The glycerol dehydratases purified from E. coli Bl21/pAK2.1 and E. coli Bl21/pAK7.1 and the complemented hybrid diol dehydratase purified from E. coli Bl21/pAK5.1 were subject to suicide inactivation by glycerol and were cross-reactivated by the reactivation factor (DhaFG) for the glycerol dehydratase of C. freundii. The activities of the three environmentally derived dehydratases and that of glycerol dehydratase of C. freundii with glycerol or 1,2-propanediol as the substrate were inhibited in the presence of the glycerol fermentation product 1,3-propanediol. Taking the catalytic efficiency, stability against inactivation by glycerol, and inhibition by 1,3-propanediol into account, the hybrid diol dehydratase produced by E. coli Bl21/pAK5.1 exhibited the best properties of all tested enzymes for application in the biotechnological production of 1,3-propanediol.     

Laboratory animal allergy: an update

Allergic reactions are among the most common conditions affecting the health of workers involved in the care and use of research animals. Between 11 and 44% of the individuals working with laboratory animals report work-related allergic symptoms. Of those who become symptomatic, 4 to 22% may eventually develop occupational asthma that can persist even after exposure ceases. Allergic symptoms consist of rashes where animals are in contact with the skin, nasal congestion and sneezing, itchy eyes, and asthma (cough, wheezing, and chest tightness). The generation of immunoglobulin E (IgE) antibodies is a prerequisite for the production of allergic symptoms. The mechanism by which IgE antibodies develop is becoming clearer. The propensity to produce IgE is genetically determined, and pre-existing allergy may be a risk factor for the development of laboratory animal allergy (LAA). However, exposure to animal allergens is the major risk factor for the development of LAA. Techniques to measure the airborne concentration of laboratory animal allergens have been developed. Research on animal allergens themselves indicates that many of the mouse and rat urinary proteins belong to a family of proteins called lipocalins, which share sequence homology with antigens of the parasitic agent that causes schistosomiasis. The fact that parasite infections also trigger IgE antibody responses may account for the development of LAA in persons who have never had any previous allergy. The prevention of LAA should be a major goal of an effective health and safety program in the animal research facility, and it can be accomplished by education and training of employees, reduction of exposure (including the use of personal protective gear), and changes in facility design. Medical surveillance programs can also play a role in improving health of individuals working with laboratory research animals. Early recognition of symptoms and evidence of sensitization can lead to interventions to reduce exposure and thereby avoid the long-term health consequences of LAA.     

Characterization of peptide-amphiphiles possessing cellular activation sequences

Numerous approaches have been described for modifying biomaterials to incorporate extracellular matrix components. "Peptide-amphiphiles", whereby monoalkyl hydrocarbon chains are covalently linked to peptide sequences, have been shown previously to (a) form specific molecular architecture with enhanced stability and (b) promote cell adhesion, spreading, and signaling. The present study has examined the use of chimeric peptide-amphiphiles for inducing protein-like structures and peptide-amphiphile mixtures for enhancing surface bioactivity. The alpha-helical propensity of a 21 residue peptide, incorporating the SPARC(119-122) angiogenesis-inducing sequence and either unmodified or acylated with a C(6), C(10), C(14), C(16), C(18), C(18:1), or C(18:1-OH) monoalkyl hydrocarbon chain, has been examined. Peptide and peptide-amphiphile structures were characterized by circular dichroism and one- and two-dimensional NMR spectroscopic techniques. The 21 residue peptide alone does not form a distinct structure in solution, whereas N-terminal acylation by monoalkyl hydrocarbon chains results in the 21 residue peptide-amphiphile adopting a predominantly alpha-helical structure in solution. The thermal stability of the alpha-helix increases with increasing hydrocarbon chain length. The SPARC(119-122) peptide-amphiphiles were then screened for promotion of endothelial cell adhesion and spreading. The greatest activity was achieved by using a mixture of the alpha-helical SPARC(119-122) peptide-amphiphile, a triple-helical peptide-amphiphile incorporating the alpha2beta1 integrin binding site from type I collagen, and a pseudolipid. The pseudolipid is most likely required for a spatial distribution of the peptide-amphiphiles that allows for optimal cellular interactions. Overall, we have found that incorporation of bioactive sequences within peptide-amphiphiles results in the induction of an ordered structure of the bioactive sequence and that mixtures of peptide-amphiphiles can be used to promote endothelial cell behaviors comparable to extracellular matrix components.     

Targeted disruption of Aldh1a1 (Raldh1) provides evidence for a complex mechanism of retinoic acid synthesis in the developing retina

Genetic studies have shown that retinoic acid (RA) signaling is required for mouse retina development, controlled in part by an RA-generating aldehyde dehydrogenase encoded by Aldh1a2 (Raldh2) expressed transiently in the optic vesicles. We examined the function of a related gene, Aldh1a1 (Raldh1), expressed throughout development in the dorsal retina. Raldh1(-/-) mice are viable and exhibit apparently normal retinal morphology despite a complete absence of Raldh1 protein in the dorsal neural retina. RA signaling in the optic cup, detected by using a RARE-lacZ transgene, is not significantly altered in Raldh1(-/-) embryos at embryonic day 10.5, possibly due to normal expression of Aldh1a3 (Raldh3) in dorsal retinal pigment epithelium and ventral neural retina. However, at E16.5 when Raldh3 is expressed ventrally but not dorsally, Raldh1(-/-) embryos lack RARE-lacZ expression in the dorsal retina and its retinocollicular axonal projections, whereas normal RARE-lacZ expression is detected in the ventral retina and its axonal projections. Retrograde labeling of adult Raldh1(-/-) retinal ganglion cells indicated that dorsal retinal axons project to the superior colliculus, and electroretinography revealed no defect of adult visual function, suggesting that dorsal RA signaling is unnecessary for retinal ganglion cell axonal outgrowth. We observed that RA synthesis in liver of Raldh1(-/-) mice was greatly reduced, thus showing that Raldh1 indeed participates in RA synthesis in vivo. Our findings suggest that RA signaling may be necessary only during early stages of retina development and that if RA synthesis is needed in dorsal retina, it is catalyzed by multiple enzymes, including Raldh1.     

A genetic analysis of the London strain of rainbow trout

The London strain of rainbow trout (Oncorhynchus mykiss) was created by interbreeding three other strains of rainbow trout and therefore was expected to have higher levels of genetic variation than other strains of rainbow trout. We examined 129 London strain rainbow trout from Indiana by allozyme electrophoresis to assess levels of genetic variation and to examine the relationship between the London strain and other hatchery strains. When using the same loci to compare with other hatchery strains the London strain showed levels of genetic variation within the range of other hatchery strains: mean heterozygosity of 0.053 (0.031-0.099), 1.27 (1.20-1.60) alleles per locus and 20.0% (20.0-40.0%) of the loci were polymorphic. The London strain is somewhat distinct from other hatchery strains (D=0.009-0.072), in part because of the high frequency of the sIDHP*40 allele.     

Body mass and individual fitness in female ungulates: bigger is not always better

In female vertebrates, differences in fitness often correspond to differences in phenotypic quality, suggesting that larger females have greater fitness. Variation in individual fitness can result from variation in life span and/or variation in yearly reproductive success, but no study has yet assessed the relationships between the components of fitness and phenotypic quality while controlling for life span. We tried to fill this gap using data from long-term monitoring (23 years) of marked roe deer and bighorn sheep, two ungulates with very different life histories. In both species, we found a strong positive relationship between an adult female's mass and her probability of reaching old age: over the long term, bigger is indeed better for ungulate females. On the other hand, we found no evidence in either species that heavier females had higher fitness when differences in life span were accounted for: over the short term, bigger is not necessarily better. Our results indicate that, while broad differences in phenotypic quality affect individual fitness, when differences in life span are accounted for phenotypic quality has no residual effect on fitness. Therefore, within a given range of phenotypic quality, bigger is not always better, for reasons which may differ between species.     

Mass- and density-dependent reproductive success and reproductive costs in a capital breeder

For capital breeders, mass may affect reproductive potential. Reproductive expenditure may reduce future reproductive potential, particularly when resources are scarce. To test the hypothesis that reproductive success and the costs of reproduction vary according to mass and population density, we analyzed 25 yr of data on bighorn ewes (Ovis canadensis). The number of adult females was first limited by yearly removals, then allowed to triple. We found no survival costs of reproduction for ewes aged 4-7 yr. For ewes aged 8-14 yr, survival was density dependent for barren ewes but not for ewes that weaned lambs. Failure to lamb was rare and negatively correlated with fertility the following year. At low population density, lactation had a negative effect on mass gain but had a limited reproductive cost. At high density, heavy ewes had higher reproductive success than light ewes, and the reproductive cost and somatic costs of reproduction increased. The cost of reproduction was greater for light than for heavy ewes. Survival of weaned lambs to 1 yr was affected by population density but not by maternal mass or previous reproductive success. In large mammals, manipulations of reproductive effort are problematic, but long-term monitoring of individual mass and reproductive success under varying conditions of resource availability can provide insights into the evolution of life histories.     

A Mixed Culture Recovery Method Indicates that Enteric Bacteria Do Not Enter the Viable but Nonculturable State

A new method, called the mixed culture recovery (MCR) method, has been developed to determine whether recovery of culturable bacterial cells from a population of largely nonculturable cells is due to resuscitation of the nonculturable cells from a viable but nonculturable state or simply to growth of residual culturable cells. The MCR method addresses this issue in that it involves the mixing of two easily distinguishable strains (e.g., lactose positive and negative) in such a way that large numbers of nonculturable cells of both strains are present together with a small number of culturable cells of only one strain, performing a nutrient addition resuscitation procedure, and then plating the cells to determine whether both cell types are recoverable. In repeated experiments with strains of Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Enterobacter aerogenes, and Salmonella choleraesuis, only cells of the culturable strain were recovered after application of various resuscitation techniques. These results suggest that the nonculturable cells were dead and that the apparent resuscitation was merely due to the growth of the remaining culturable cells.