Conservation of a phenomenon: rapid,reversible colour change in both sexes of one of the world’s most threatened damselflies

Physiological colour change is rare in insects. Unusually, both the males and females of Spesbona angusta (Odonata: Platycnemididae), Red Listed as Endangered, are capable of rapid and reversible colour change. There is only one known population of this species, which occurs in a unique habitat in the Cape Floristic Region, South Africa. Appreciation of this unusual phenomenon of distinct physiological colour change helps us appreciate that we need to conserve phenomena in the insect world as well as the species themselves. Using controlled experiments, we evaluated the importance of ambient temperature as the possible primary cue for physiological colour change. We found that S. angusta responds rapidly to short-term changes in ambient temperature, even in the absence of additional environmental stimuli and without the body temperature matching the ambient temperature. Colour change is reversible when temperature returns to its earlier level. The reason why S. angusta shows this rapid and reversible colour change may be a combination of reproductive enhancement, competitive advantage and thermoregulation. This colour change appears to have strong selective advantage in a very particular habitat type, meaning that careful conservation of its habitat in all respects is important, and must be considered in any possible future translocations.     

Scyliorhinin I and II: two novel tachykinins from dogfish gut

Two peptides with tachykinin-like ability to contract longitudinal muscle from the guinea pig ileum were isolated from the intestine of the common dogfish, Scyliorhinus caniculus. The amino acid sequence of scyliorhinin I was established as Ala-Lys-Phe-Asp-Lys-Phe-Tyr-Gly-Leu-Met-NH2 and this peptide cross-reacted with antisera directed against the C-terminal region fo substance P. The amino acid sequence of scyliorhinin II was established as Ser-Pro-Ser-Asn-Ser-Lys-Cys-Pro-Asp-Gly-Pro-Asp-Cys-Phe-Val-Gly-Leu-Met- NH2 and this peptide cross-reacted with antisera directed against the C-terminal region of neurokinin A. The mammalian peptides substance P and neurokinin A were absent from the dogfish intestinal tissue.     

Sensitivity enhancement of optical immunosensors by the use of a surface plasmon resonance fluoroimmunoassay

Optical immunosensors employing evanescent wave techniques have the potential to address the requirements of the 'alternative site' market; however, this potential has yet to be realised. The development of 'direct' sensors, such as those using surface plasmon resonance (SPR), has been hampered by problems of non-specific binding and poor sensitivity to small molecules. 'Indirect' sensors (for example, those employing a fluorescently labelled reagent) overcome many of the problems of direct sensors but require more sophisticated instrumentation because of the low light levels detected. In an attempt to combine the best features of the two techniques, an indirect SPR fluoroimmunoassay (SPRF) technique has been investigated. The surface field intensity enhancement produced by SPR is used to boost the emission from a fluorescently labelled immunoassay complex at a metal surface. The potential of the method is demonstrated by assaying for human Chorionic Gonadotrophin (hCG) in serum. Enhanced sensitivity over conventional total internal reflection fluorescence (TIRF) and SPR techniques was achieved.     

An assay for human chorionic gonadotrophin using the capillary fill immunosensor

Recently there has been much research effort directed towards the development of immunosensors. Optical technologies are currently proving very attractive for the construction of such sensors. The fluorescence capillary fill device (FCFD) has been designed to fulfil these needs. The development of an assay for human chorionic gonadotrophin (hCG) in the FCFD for a variety of body fluids (whole blood, serum, urine and saliva) demonstrates the versatility and assay performance of the device.     

Neuropeptide K-(1-24)-peptide: storage and release by carcinoid tumors

An antiserum directed against the COOH-terminal region of neuropeptide K-(1-24)-peptide that shows only 0.5% reactivity with neuropeptide K has been used in radioimmunoassay to study the posttranslation processing of human beta-preprotachykinin. A primary midgut carcinoid tumor contained high concentration of substance P (2970 pmol/g), neurokinin A (3660 pmol/g) and neuropeptide K-(1-24)-peptide (3430 pmol/g) but only a very low concentration (less than 5 pmol/g) of intact neuropeptide K. Neuropeptide K-(1-24)-peptide was also detected in extracts of metastatic tumor tissue from four patients with midgut carcinoid tumors. The amino acid sequence of tumor neuropeptide K-(1-24)-peptide was identical to that predicted from the nucleotide sequence of a human beta-preprotachykinin cDNA. The fasting plasma concentration of neuropeptide K-(1-24)-peptide was elevated in a patient with the carcinoid syndrome (821 fmol/ml compared with less than 18 fmol/ml in healthy subjects) and rose approximately 2-fold after intravenous pentagastrin. The study has demonstrated that the Lys25-Arg26 bond in neuropeptide K (corresponding to Lys96-Arg97 in the precursor) is an important processing site in human beta-preprotachykinin.     

Localisation of the Vacuolar Proton Pump (V-H+-ATPase) and Carbonic Anhydrase II in the Human Eccrine Sweat Gland

The localisation of the vacuolar proton pump (V-H+ -ATPase) and the enzyme carbonic anhydrase II (CAII) was investigated in the human eccrine sweat gland employing standard immunohistochemical techniques after antigen retrieval using microwave heat treatment and high pressure. The high-pressure antigen retrieval unmasked the presence of V-H+ -ATPase in the clear cells of the secretory coil, with a distribution similar to that previously observed for CAII. However, the dark cells were unreactive to both antibodies. In addition, heat and high-pressure antigen retrieval demonstrated the presence of CAII in the apical zone of luminal cells of the reabsorptive duct, a location not previously reported. The localisation of V-H+ -ATPase and CAII in the secretory coil clear cells suggests that the formation of HCO3- and H+ by carbonic anhydrase II and the transport of H+ by V-H+ -ATPase may play an role in sweat fluid secretion. Their presence at the apex of the duct cells indicates involvement in ductal ion reabsorption.     

Control of the take-all fungus byMicrodochium bolleyi,and interactions involvingM. bolleyi,Phialophora graminicola andPericonia macrospinosa on cereal roots

Summary In glasshouse experiments,Microdochium bolleyi (Mb) significantly reduced infection of wheat roots by the take-all fungus,Gaeumannomyces graminis vartritici (Ggt), when inocula were dispersed in soil at ratios of 10∶1 (Mb:Ggt) or more. Spread of take-all lesions up roots from a layer of inoculum also was reduced when Mb was inoculated immediately below the crown. In contrast,Periconia macrospinosa did not control take-all even at an inoculum ratio of 100∶1. M. bolleyi interfered with growth on roots byPhialophora graminicola, a known biocontrol agent of take-all. It is suggested that this phenomenon and control of take-all by these fungi occur by competition for cortical cells that senesce in the normal course of root development.     

Shotgun Crystallization Strategy for Structural Genomics II: Crystallization Conditions that Produce High Resolution Structures for T. maritima Proteins

Currently, 119 high resolution structures of Thermotoga maritima proteins have been determined by the Joint Center for Structural Genomics (JCSG, www.jcsg.org). Sixty-seven of these were solved using the first implementation of the multi-tiered crystallization strategy at the JCSG for the efficient crystallization of large numbers of protein targets. Previously, we reported the analysis of all proteins crystallized using this multi-tiered strategy [Lesley, S.A. et al. (2002) Proc. Natl. Acad. Sci. USA 99, 11664–11669; Page, R. et al. (2003) Acta Crystallogr. D Biol. Crystallogr. 59, 1028–1037]. Here, we extend the analysis and describe the crystallization characteristics of those proteins that produced diffraction quality crystals, ultimately resulting in high resolution structures. First, we found that over 77% (52) of the crystals used for structure determination were produced directly from high-throughput coarse screens, indicating that less than one quarter of the crystals (15) required fine screening. In addition, as observed for the proteome screen [Page, R. et al. (2003) Acta Crystallogr. D Biol. Crystallogr. 59, 1028–1037], the majority of conditions that produced crystals for natively expressed proteins, whose structures have been determined, were distinct from those of their more extensively purified and selenomethionine-labeled counterparts. Finally, 99% of the proteins whose structures were solved crystallized in conditions contained in the JCSG Minimal Core Screen [Page, R. et al. (2003) Acta Crystallogr. D Biol. Crystallogr. 59, 1028–1037; Page, R. and Stevens, R.C. (2004) Methods 34, 373–389], a set of 67 conditions previously identified as those most likely to produce crystals of a diverse set of proteins, confirming its success for rapid identification of proteins with a natural propensity to crystallize.     

A protocol for high-throughput phenotyping, suitable for quantitative trait analysis in mice

Whole-genome genetic association studies in outbred mouse populations represent a novel approach to identifying the molecular basis of naturally occurring genetic variants, the major source of quantitative variation between inbred strains of mice. Measuring multiple phenotypes in parallel on each mouse would make the approach cost effective, but protocols for phenotyping on a large enough scale have not been developed. In this article we describe the development and deployment of a protocol to collect measures on three models of human disease (anxiety, type II diabetes, and asthma) as well as measures of mouse blood biochemistry, immunology, and hematology. We report that the protocol delivers highly significant differences among the eight inbred strains (A/J, AKR/J, BALBc/J, CBA/J, C3H/HeJ, C57BL/6 J, DBA/2 J, and LP/J), the progenitors of a genetically heterogeneous stock (HS) of mice. We report the successful collection of multiple phenotypes from 2000 outbred HS animals. The phenotypes measured in the protocol form the basis of a large-scale investigation into the genetic basis of complex traits in mice designed to examine interactions between genes and between genes and environment, as well as the main effects of genetic variants on phenotypes.