Intermolecular relations between the glucocorticoid receptor, ZAP-70 kinase, and Hsp-90

The glucocorticoid receptor (GR) participates in both genomic and non-genomic glucocorticoid hormone (GC) actions by interacting with other cytoplasmic signalling proteins. Previously, we have shown that high dose Dexamethasone (DX) treatment of Jurkat cells causes tyrosine phosphorylation of ZAP-70 within 5 min in a GR-dependent manner. By using co-immunoprecipitation and confocal microscopy, here we demonstrate that the liganded GR physically associates with ZAP-70, in addition to its phosphorylation changes. The association of the ligand-bound GR and ZAP-70 was also observed in HeLa cells transfected with ZAP-70, suggesting that this co-clustering is independent of lymphocyte specific factors. Furthermore, the ZAP-70 was found to also co-precipitate with Hsp-90 chaperone both in Jurkat and transgenic HeLa cells, independent of the presence of DX. These findings raise the possibility that ZAP-70 may serve as an important link between GC and TcR-induced signaling, thereby transmitting non-genomic GC action in T-cells.     

The biology and ecology of lotic nematodes

• 1 Morphological structures for identifying freshwater nematodes, e.g. buccal cavity, sensory receptors, oesophagus, reproductive organs and tail are described.
• 2 Most freshwater nematodes belong to the Adenophorea and are characterised by the presence of setae, adhesive glands and conspicuous amphids.
• 3 Methods for collecting nematodes from the sediments of running water (e.g. corer, pumps), within plants and aufwuchs are listed. Methods for fixation, extracting and preparing nematodes for identification are described.
• 4 Life history parameters (e.g. generation time, eggs per female) are not available for lotic nematodes but are summarised for free‐living nematodes in soil, lakes and seas. Field studies indicate that, in contrast to laboratory experiments with nematode cultures, many species will have a generation time of several months.
• 5 Abundance and species diversity of nematodes of lotic habitats are provided; more than 100 nematode species inhabit lotic habitats and densities can reach 230 individuals per ml.
• 6 Links between meiobenthic nematodes and the micro‐ and macrobenthos are unclear at present. Evidence such as the increased bacterial activity due to nematode grazing suggests that such interactions may be significant.

     

Population biology and biological control of nematodes.

We studied the population biology of the nematophagous fungus Hirsutella rhossiliensis to understand its potential as a biological control agent. Because the fungus is an infectious and transmissible parasite, we framed our study within an epidemiological context. Field observations, theory, and experiments demonstrated that (i) parasitism of nematodes by H. rhossiliensis is dependent on nematode density, (ii) local populations of the fungus will go extinct unless supplied with some minimum number of nematodes (the host threshold density), and (iii) natural epidemics of this fungus in populations of nematodes develop slowly and only after long periods of high host density. Additional in-depth research on population biology is needed to explain other biological control systems and to guide future research. The most effective research will combine field observation, theory, and experimentation.     

Synthetic biology in multicellular organisms: Opportunities in nematodes

Synthetic biology has mainly focused on introducing new or altered functionality in single cell systems: primarily bacteria, yeast, or mammalian cells. Here, we describe the extension of synthetic biology to nematodes, in particular the well-studied model organism Caenorhabditis elegans, as a convenient platform for developing applications in a multicellular setting. We review transgenesis techniques for nematodes, as well as the application of synthetic biology principles to construct nematode gene switches and genetic devices to control motility. Finally, we discuss potential applications of engineered nematodes.     

Molecular biology of reproduction and development in parasitic nematodes: progress and opportunities

Molecular biological research on the development and reproduction of parasites is of major significance for many fundamental and applied areas of medical and veterinary parasitology. Together with knowledge of parasite biology and epidemiology, the application of molecular tools and technologies provides unique opportunities for elucidating developmental and reproductive processes in helminths. This article focuses specifically on recent progress in studying the molecular mechanisms of development, sexual differentiation and reproduction in parasitic nematodes of socio-economic importance and comparative analyses, where appropriate, with the free-living nematode Caenorhabditis elegans. It also describes the implications of such work for understanding reproduction, tissue migration, hypobiosis, signal transduction and host-parasite interactions at the molecular level, and for seeking new means of parasite intervention.     

Functional genomics of hsp-90 in parasitic and free-living nematodes

Heat shock protein 90 (Hsp-90) is a highly conserved essential protein in eukaryotes. Here we describe the molecular characterisation of hsp-90 from three nematodes, the free-living Caenorhabditis elegans (Ce) and the parasitic worms Brugia pahangi (Bp) and Haemonchus contortus (Hc). These molecules were functionally characterised by rescue of a Ce-daf-21 (hsp-90) null mutant. Our results show a gradient of rescue: the C. elegans endogenous gene provided full rescue of the daf-21 mutant, while Hc-hsp-90 provided partial rescue. In contrast, no rescue could be obtained using a variety of Bp-hsp-90 constructs, despite the fact that Bp-hsp-90 was transcribed and translated in the mutant worms. daf-21 RNA interference (RNAi) experiments were carried out to determine whether knock-down of the endogenous daf-21 mRNA in N2 worms could be complemented by expression of either parasite gene. However neither parasite gene could rescue the daf-21 (RNAi) phenotypes. These results indicate that factors other than the level of sequence identity are important for determining whether parasite genes can functionally complement in C. elegans.     

Hsp-72, a candidate prognostic indicator of heatstroke

Exposure of rats to environmental heat enhances the expression of heat shock protein-72 (Hsp-72) in most of their organs proportionally to heat stress severity. Pre-induction or over-expression of Hsp-72 prevents organ damage and lethality, suggesting that heat shock proteins (Hsps) may have a pathogenic role in this condition. We investigated the expression profile of Hsps in baboons subjected to environmental heat stress until the core temperature attained 42.5°C (moderate heatstroke) or occurrence of hypotension associated with core temperature ≥43.5°C (severe heatstroke). Western blot analysis demonstrated a differential induction of Hsp-72 among organs of heat-stressed animals with the highest induction in the liver and the lowest in lung. Hsp-60 and Hsc-70 expression was similar between control and heat-stressed animals. ELISA studies indicated a marked release of Hsp-72 into the circulation of baboons with severe heatstroke with a peak at 24 h post-heatstroke onset and remained sustained up to 72 h. Hsp-72 release was not associated with core temperature or systolic blood pressure, but correlated with markers of liver, myocardium, and skeletal muscle tissue necrosis. Non-survivors displayed significantly higher Hsp-72 levels than survivors. No Hsp-60 was detected in the circulation. These findings add further evidence that increased expression of Hsp-72 may be an important component of the host response to severe heatstroke. They also suggest that extracellular Hsp-72 is a marker of multiple organs tissue damage. Whether extracellular Hsp-72 plays a role in the host immune response to heat stress merits further studies.     

Effect of Supplemental Trace Minerals on Hsp-70 mRNA Expression in Commercial Broiler Chicken

The effects of supplementing the organic forms of selenium (Se), chromium (Cr), and zinc (Zn) on Hsp-70 mRNA expression and body weight in broiler chickens were evaluated. 200 chicks were equally distributed into stainless steel battery brooders at the rate of 5 birds per pen and reared under heat stress condition up to 42^nd day. The chicks were fed with three experimental diets supplemented with organic forms of Se (0.30?mg/kg), Cr (2?mg/kg), and Zn (40?mg/kg) during the starter and finisher phases and a control diet without any supplementation. On the 21st and 42nd day, 20 birds from each period were sacrificed and samples were collected for analysis. Organic Se, Cr, and Zn supplementation significantly (P?Hsp-70 mRNA levels. The Hsp-70 mRNA expression levels were significantly (P?Hsp-70 mRNA expression level was not affected by age of the birds. The study concluded that organic trace mineral (oTM) supplementation resulted in low Hsp-70 mRNA expression, indicating reduced heat stress in broilers.     

Weather and the ecology of bursate nematodes

The free-living stages of bursate nematodes are affected in different ways by meteorological factors. The eggs and 1st and 2nd stage larvae live in the feces, and these factors act on them through the feces. The embryo in the egg is protected by the eggshell. The 1st and 2nd stage larvae feed on micro-organisms, and are affected by the species of microorganisms and their metabolic products. The 3rd stage, infective larvae do not feed but leave the feces and migrate onto the vegetation, where they are more directly affected by meteorological factors than the earlier stages. They are partially protected by their sheath. The degree day concept can be used to predict the length of time required for development to the infective stage. Under constant conditions at 100% relative humidity and 20–35° C,Trichostrongylus colubriformis requires 90–115 degree days (temperature times number of days) to reach the infective stage, andHaemonchus contortus requires 95–144 degree days. The great majority of eggs dies within the 1st day after reaching pasture, altho most of them would develop to the infective stage in the laboratory. Only 0.03% ofH. contortus andT. colubriformis eggs placed on pasture were recovered from the vegetation as infective larvae. When larvae ofH. contortus were placed on pasture, 1.6% (about 50 times as many but still only aout 1/30 of their potential) were later recovered. The cause of this die-off is unknown. Two types of potential worm burden prediction must be made-general and specific. The former can be made on the basis of temperature and moisture, but the latter requires information on many modifying factors.Presented at the Eighth International Congress of Biometeorology, 9–14 September 1979. Shefayim, Israel.     

The host plant and the nematodes

Parasite selection by the host would be expected and is frequently found amongst the specialized plant parasitic nematodes and on perennial plants. Hijink & Oostenbrink (1968) showed that annual cropping could establish distinct nematode communities and such communities can become quickly established (Green, 1975). In carrot crops Aphelenchoides and Rotylenchus species became a larger proportion of the community very early in the growth of the crop (Green, 1976). Stabilization of communities in this way indicates a very close relationship between the nematodes and the host plants even when specialized feeding is not apparent. Cropping alters the plant community so that even these nematodes with a wide host range become dependant on the one host, provided weed control is efficient. Spacing of the crop further restricts the nematodes so they become more dependant on individual plants. Nematode attacks on crops are notable for two features. The hosts appear under-exploited, that is there are few individual hosts with many parasites but many with few and, the hosts are rarely killed.     

Application and commercialization of nematodes

While nematodes are most commonly known for their negative impact on plants, animals, and humans, there are a number of species which are commercially explored. This review highlights some of the most important success stories for the application of nematodes. They are used as bioindicators in ecological and toxicity studies, as model organisms for elucidating fundamental biological questions and for high throughput screening of drugs. Besides these indirect uses, direct applications include the use of Beddingia siricidicola against a major forest pest and the commercialization of Steinernema, Heterorhabditis, and Phasmarhabditis as biological pest control products. New directions for the commercialization of nematodes are the use as living food, specifically loaded with essential nutrients for various fish and shrimp larvae. Even human parasites or closely related species have been successfully used for curing autoimmune disorders and are currently in the process of being developed as drugs. With the striving development of life sciences, we are likely to see more applications for nematodes in the future. A prerequisite is that we continue to explore the vast number of yet undiscovered nematode species.     

Discovery of aminoquinolines as a new class of potent inhibitors of heat shock protein 90 (Hsp90): Synthesis, biology, and molecular modeling

The molecular chaperone Hsp90 plays important roles in maintaining malignant phenotypes. Recent studies suggest that Hsp90 exerts high-affinity interactions with multiple oncoproteins, which are essential for the growth of tumor cells. As a result, research has focused on finding Hsp90 probes as potential and selective anticancer agents. In a high-throughput screening exercise, we identified quinoline 7 as a moderate inhibitor of Hsp90. Further hit identification, SAR studies, and biological investigation revealed several synthetic analogs in this series with micromolar activities in both fluorescent polarization (FP) assay and a cell-based Western blot (WB) assay. These compounds represent a new class of Hsp90 inhibitors with simple chemical structures.