Morganella morganii bacteria produces phenol as the sex pheromone of the New Zealand grass grub from tyrosine in the colleterial gland.

Costelytra zealandica (Coleoptera: Scarabeidae) is a univoltine endemic species that has colonised and become a major pest of introduced clover and ryegrass pastures that form about half of the land area of New Zealand. Female beetles were previously shown to use phenol as their sex pheromone produced by symbiotic bacteria in the accessory or colleterial gland. In this study, production of phenol was confirmed from the female beetles, while bacteria were isolated from the gland and tested for attractiveness towards grass grub males in traps in the field. The phenol-producing bacterial taxon was identified by partial sequencing of the 16SrRNA gene, as Morganella morganii. We then tested the hypothesis that the phenol sex pheromone is biosynthesized from the amino acid tyrosine by the bacteria. This was shown to be correct, by addition of isotopically labelled tyrosine ((13)C) to the bacterial broth, followed by detection of the labelled phenol by SPME-GCMS. Elucidation of this pathway provides specific evidence how the phenol is produced as an insect sex pheromone by a mutualistic bacteria.

Formulations for delivering Trichoderma atroviridae spores as seed coatings, effects of temperature and relative humidity on storage stability.

AIMS: We aimed to evaluate different formulations for their ability to adhere Trichoderma atroviridae spores to wheat seeds, and promote survival during storage at a range of temperatures and relative humidities (RH). METHODS AND RESULTS: We tested a range of formulations for their ability to adhere T. atroviridae spores to wheat seeds. Treated seeds were stored for 6 months at a range of temperatures and RH, and spore viability among formulation was compared over time. Spore survival within formulations interacted significantly with environmental conditions. Notably, under optimum conditions (low temperatures and RH) best spore survival was recorded with a xanthan-gum-based formulation. Conversely under suboptimum conditions (high temperatures and RH), survival of spores was best in a waxy-starch formulation, but very poor in the xanthan-gum formulation. CONCLUSIONS: These results indicate that T. atroviridae spores can be effectively delivered on to seeds and that a xanthan-gum formulation is promising when optimal storage conditions can be maintained. SIGNIFICANCE AND IMPACT OF THE STUDY: Most published formulation papers/patents only report survival of organisms over time at a single or limited number of temperatures and RH. For the first time, this study shows how different formulations are better suited to certain temperature and RH combinations.

Can medical students teach? A near-peer-led teaching program for year 1 students.

The General Medical Council states that United Kingdom graduates must function effectively as educators. There is a growing body of evidence showing that medical students can be included as teachers within a medical curriculum. Our aim was to design and implement a near-peer-led teaching program in an undergraduate medical curriculum and assess its acceptability among year 1 students. Students received six tutorials focusing on aspects of cardiac, respiratory, and blood physiology. Tutorials ran alongside standard module teaching. Students were taught in groups of ~30 students/group, and an active teaching approach was used in sessions where possible. Using anonymous evaluations, student feedback was collected for the program overall and for each tutorial. The program was voluntary and open to all first-year students, and 94 (of 138) medical students from year 1 at Brighton and Sussex Medical School were recruited to the study. The tutorial program was popular among students and was well attended throughout. Individual tutorial and overall program quantitative and qualitative feedback showed that students found the tutorials very useful in consolidating material taught within the module. Students found the small group and active teaching style of the near-peer tutors very useful to facilitating their learning experience. The end-of-module written examination scores suggest that the tutorials may have had a positive effect on student outcome compared with previous student attainment. In conclusion, the present study shows that a near-peer tutorial program can be successfully integrated into a teaching curriculum. The feedback demonstrates that year 1 students are both receptive and find the additional teaching of benefit.

Multilocus sequence analysis (MLSA) of 'Rickettsiella costelytrae' and 'Rickettsiella pyronotae', intracellular bacterial entomopathogens from New Zealand.

AIMS: Larvae of scarab beetles live in the soil and are frequently hosts for microbial pathogens. In New Zealand, larvae of the grass grub, Costelytrae zealandica (Coleoptera: Scarabaeidae), and manuka beetles, Pyronota spp. (Coleoptera: Scarabaeidae), have been collected from field populations showing loss of vigour and a whitened appearance. Diagnosis indicated an intracellular infection of fat body tissues by Rickettsiella-like micro-organisms. Rickettsiella bacteria are under evaluation as a possible new source of insect bio-control agents for important agricultural pests as, e.g. scarabaeid and elaterid larvae. The present study aimed at the unequivocal molecular taxonomic identification and comparison of the bacteria associated with Costelytra and Pyronota. METHODS AND RESULTS: Electron microscopy and phylogenetic reconstruction using a multilocus sequence analysis approach based on the 16S ribosomal RNA gene together with four protein-encoding markers (ftsY, gidA, rpsA, and sucB) demonstrated that both bacteria from New Zealand are phylogenetically closely related, but not identical, and belong to the taxonomic genus Rickettsiella. CONCLUSIONS: The bacteria under study should be referred to as pathotypes 'Rickettsiella costelytrae' and 'Rickettsiella pyronotae', respectively. Moreover, on the basis of the currently accepted systematic organization of the genus Rickettsiella, both pathotypes should be considered synonyms of the nomenclatural type species, Rickettsiella popilliae. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates that Rickettsiella bacteria are geographically widespread pathogens of scarabaeid larvae. Implications of the phylogenetic findings presented for the stability of host adaptation by Rickettsiella bacteria are critically discussed.

First Report of Goss's Bacterial Wilt and Leaf Blight (Clavibacter michiganensis subsp. nebraskensis) of Corn in Texas.

In September 2009, the University of Nebraska-Lincoln Plant and Pest Diagnostic Clinic received leaf samples of hybrid corn (Zea mays L.) displaying long, necrotic lesions with wavy margins. The lesions had discontinuous water-soaked spots that are indicative of Goss's bacterial wilt and leaf blight. The symptomatic leaves were submitted from Dallam County, located in the Texas Panhandle (northwest Texas). According to the USDA Farm Service Agency and the National Agricultural Statistics Service, in 2009 Dallam County had 54,025 ha planted to corn. This is approximately 19% of the total corn planted in the 26 counties in the Texas Panhandle and 6% of the total corn planted in the state of Texas. Extracts from the infected leaf tissue tested positive for Clavibacter michiganensis subsp. nebraskensis with a commercially available ELISA test (Neogen Inc., Scotland, UK). Isolation from the infected tissue onto CNS selective media (1) resulted in round, dark orange, mucoid colonies that tested gram positive with the Gram-stain test. BLAST nucleotide sequence alignments of the amplified 500-bp 16S rRNA region of the suspect culture's genome (2) revealed a 96% similarity for C. michiganensis subsp. nebraskensis (NCBI BLAST Accession No. U09381.1). To fulfill Koch's postulates, three sweet corn plants (Golden Cross Bantam) at growth stage V3 to V4 were inoculated in the greenhouse with a suspension of approximately 1 × 109 CFU/ml from suspect cultures grown on CNS for 5 days. Wounds approximately 6.5 cm long were created with sterile scissors on the fifth leaf from the bottom running parallel to the veins on either side of the midrib at the leaf apex. The leaf apex was dipped into 150 ml of the inoculum suspension for 5 s. Approximately 6 days after inoculation, discontinuous, water-soaked spots consistent with the symptoms on the original symptomatic leaves appeared on all the inoculated leaves near the site of infection. Colonies consistent with C. michiganensis subsp. nebraskensis (dark orange, mucoid) were reisolated onto CNS, completing Koch's postulates. To our knowledge, this is the first report of Goss's bacterial wilt and leaf blight on corn in Texas and because it is a residue-borne pathogen, the probability of it becoming a resident disease is relatively high. References: (1) D. C. Gross and A. K. Vidaver. Phytopathology 69:82, 1979. (2) X. Li and S. H. De Boer. 1995. Phytopathology 85:837, 1995.

Spatial trends and historical deposition of mercury in eastern and northern Canada inferred from lake sediment cores.

Recent and historical deposition of mercury (Hg) was examined over a broad geographic area from southwestern Northwest Territories to Labrador and from the U.S. Northeast to northern Ellesmere Island using dated sediment cores from 50 lakes (18 in midlatitudes (41-50 degrees N), 14 subarctic (51-64 degrees N) and 18 in the Arctic (65-83 degrees N)). Distinct increases of Hg overtime were observed in 76% of Arctic, 86% of subarctic and 100% of midlatitude cores. Subsurface maxima in Hg depositional fluxes (microg m(-2) y(-1)) were observed in only 28% of midlatitude lakes and 18% of arctic lakes, indicating little recent reduction of inputs. Anthropogenic Hg fluxes adjusted for sediment focusing and changes in sedimentation rates (deltaF(adj,F)) ranged from -22.9 to 61 microg m(-2) y(-1) and were negatively correlated (r = -0.57, P < 0.001) with latitude. Hg flux ratios (FRs; post-1990)/pre-1850) ranged from 0.5 to 7.7. The latitudinal trend for Hg deltaF(adj,F) values showed excellent agreement with predictions of the global mercury model, GRAHM for the geographic location of each lake (r = 0.933, P < 0.001). The results are consistent with a scenario of slow atmospheric oxidation of mercury, and slow deposition of reactive mercury emissions, declining with increasing latitude away from emission sources in the midlatitudes, and support the view that there are significant anthropogenic Hg inputs in the Arctic.

Phenotypic changes and the fate of digestive enzymes during induction of amber disease in larvae of the New Zealand grass grub (Costelytra zealandica).

Amber disease of the New Zealand grass grub Costelytra zealandica (Coleoptera: Scarabaeidae) is caused by ingestion of pADAP plasmid carrying isolates of Serratia entomophila or Serratia proteamaculans (Enterobacteriaceae) and causes infected larvae to cease feeding and clear their midgut to a pale amber colour where midgut serine protease activities are virtually eliminated. Using bacterial strains and mutants expressing combinations of the anti-feeding (afp) and gut clearance (sep) gene clusters from pADAP, we manipulated the disease phenotype and demonstrated directly the relationship between gene clusters, phenotype and loss of enzyme activity. Treatment with afp-expressing strains caused cessation of feeding without gut clearance where midgut protease activity was maintained at levels similar to that of healthy larvae. Treatment with strains expressing sep-genes caused gut clearance followed by a virtual elimination of trypsin and chymotrypsin titre in the midgut indicating both the loss of pre-existing enzyme from the lumen and a failure to replenish enzyme levels in this region by secretion from the epithelium. Monitoring of enzymatic activity through the alimentary tract during expression of disease showed that loss of serine protease activity in the midgut was matched by a surge of protease activity in the hindgut and frass pellets, indicating a flushing and elimination of the midgut contents. The blocking of enzyme secretion through amber disease appears to be selective as leucine aminopeptidase and alpha-amylase were still detected in the midgut of diseased larvae.

Autochthonous bacterial flora indicated by PCR-DGGE of 16S rRNA gene fragments from the alimentary tract of Costelytra zealandica (Coleoptera: Scarabaeidae).

AIMS: To locate and identify putative autochthonous bacteria within the grass grub gut that may have a role in symbiosis. METHODS AND RESULTS: Polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) fingerprinting was used to investigate bacterial diversity in the grass grub larval gut. The microbial community profiles from five geographically distinct populations were compared and the influence of feeding was analysed. Bacterial community in the midgut was highly variable between locations and was affected by feeding. The hindgut contained a more diverse but stable bacterial community that was less affected by external conditions. Forty-seven distinct DGGE bands, representing different bacterial genotypes, could be distinguished from all samples, with 34 different bands occurring in the hindgut. The 22 most common bands were isolated and DNA was sequenced. Sequence analysis revealed that most bacteria (16/22) were affiliated to the Clostridiales with the predominant bacteria affiliated to the genus Clostridium. The remaining bacteria were aligned to the Betaproteobacteria, Deltaproteobacteria and Bacteroidetes. CONCLUSIONS: The grass grub larva has an autochthonous microflora with predominance of Clostridium spp. in the hindgut. SIGNIFICANCE AND IMPACT OF THE STUDY: Occurrence of an autocthonous microflora in the grass grub hindgut suggests a symbiotic relationship which could help explain the ability of larval scarabs to feed on recalcitrant organic matter.

Serratia entomophila inoculation causes a defect in exocytosis in Costelytra zealandica larvae.

Rapid elimination of midgut luminal proteinase activity and gut clearance are the two major symptoms of amber disease in Costelytra zealandica larvae because of the three-subunit protein toxin complex produced in Serratia entomophila and Serratia proteamaculans. Quantitative PCR analysis of mRNA from the major serine proteinase gene families showed that loss of proteinase activity did not result from transcriptional downregulation. Unexpectedly, protein levels and rates of protein synthesis increased, rather than decreased, in the midgut of diseased insects. Proteomic analysis of midgut tissues showed marked differences between healthy and diseased midguts. Large increases in soluble forms of both actin and tubulin were identified from 2D-gels, together with concurrent decreases in the levels of polymeric actin-associated proteins: actin depolymerizing factor and cyclophilin. These results suggest that the Serratia toxin acts to cause degradation of the cytoskeletal network and prevent secretion of midgut gut digestive proteinases as both the actin cytoskeleton and microtubules are involved in exocytosis. Proteinases synthesized in the diseased midgut must be rapidly degraded because they do not accumulate in an inactive form.

Serine proteases identified from a Costelytra zealandica (White) (Coleoptera: Scarabaeidae) midgut EST library and their expression through insect development.

Costelytra zealandica larvae are pests of New Zealand pastures causing damage by feeding on the roots of grasses and clovers. The major larval protein digestive enzymes are serine proteases (SPs), which are targets for disruption in pest control. An expressed sequence tag (EST) library from healthy, third instar larval midgut tissue was constructed and analysed to determine the composition and regulation of proteases in the C. zealandica larval midgut. Gene mining identified three trypsin-like and 11 chymotrypsin-like SPs spread among four major subgroups. Representative SPs were examined by quantitative PCR and enzyme activity assayed across developmental stages. The serine protease genes examined were expressed throughout feeding stages and downregulated in nonfeeding stages. The study will improve targeting of protease inhibitors and bacterial disruptors of SP synthesis.

Interactions Between the Soybean Cyst Nematode and Fusarium solani f. sp. glycines Based on Greenhouse Factorial Experiments.

ABSTRACT The soybean cyst nematode, Heterodera glycines, and the fungus that causes sudden death syndrome (SDS) of soybean, Fusarium solani f. sp. glycines, frequently co-infest soybean (Glycine max) fields. The interactions between H. glycines and F. solani f. sp. glycines were investigated in factorial greenhouse experiments with different inoculum levels of both organisms on a soybean cultivar susceptible to both pathogens. Measured responses included root and shoot dry weights, H. glycines reproduction, area under the SDS disease progress curve, and fungal colonization of roots. Both H. glycines and F. solani f. sp. glycines reduced the growth of soybeans. Reproduction of H. glycines was suppressed by high inoculum levels but not by low levels of F. solani f. sp. glycines. The infection of soybean roots by H. glycines did not affect root colonization by the fungus, as determined by real-time polymerase chain reaction. Although both pathogens reduced the growth of soybeans, H. glycines did not increase SDS foliar symptoms, and statistical interactions between the two pathogens were seldom significant.

The incidence and use of Oryctes virus for control of rhinoceros beetle in oil palm plantations in Malaysia.

The rhinoceros beetle, Oryctes rhinoceros, has emerged as a serious pest of oil palm since the prohibition of burning as a method for maintaining estate hygiene in the 1990s. The abundance of beetles is surprising given that the Malay peninsula was the site of first discovery of the Oryctes virus, which has been used to effect good as a biological control agent in other regions. A survey of adult beetles was carried out throughout Malaysia using pheromone traps. Captured beetles were examined for presence of virus using both visual/microscopic examination and PCR detection methods. The survey indicated that Oryctes virus was common in Malaysia among the adult beetles. Viral DNA analysis was carried out after restriction with HindIII enzyme and indicated at least three distinct viral genotypes. Bioassays were used to compare the viral strains and demonstrate that one strain (type B) is the most virulent against both larvae and adults of the beetle. Virus type B has been cultured and released into healthy populations where another strain (type A) forms the natural background. Capture and examination of beetles from the release site and surrounding area has shown that the spread and persistence of the applied virus strain is accompanied by a reduction in palm frond damage.

Heterodera glycines Infectivity and Egg Viability Following Nonhost Crops and During Overwintering.

The most effective management program for soybean cyst nematode, Heterodera glycines, is a crop rotation that uses nonhost crops and resistant soybean cultivars. However, little is known about the effects of rotation crops and overwintering on H. glycines biology. These experiments were initiated to determine the effects of seven alternative crops on H. glycines' ability to infect and mature on subsequent soybean crops, and to assess the viability of eggs during the overwintering months. Rotation studies were conducted for 2 years in each of two naturally infested fields, and overwintering tests were conducted in three consecutive growing seasons in one naturally infested field. Rotation crop and fallow treatments did not have a consistent effect on the ability of H. glycines to infect soybean or mature. Soybean yields were often higher following fallow or a nonhost crop than following soybean, although not usually significantly so. Heterodera glycines egg viability did not differ (P < 0.05) between overwintering months at 0-to-10 or 10-to-20-cm soil depths. These results suggest that H. glycines' ability to infect a subsequent soybean crop and develop to maturity is not diminished by nonhost crops or during the winter months.

Detection and Quantification of Fusarium solani f. sp. glycines in Soybean Roots with Real-Time Quantitative Polymerase Chain Reaction.

Fusarium solani f. sp. glycines is the causal organism of soybean sudden death syndrome (SDS). This organism is difficult to detect and quantify because it is a slow-growing fungus with variable phenotypic characteristics. Reliable and fast procedures are important for detection of this soybean pathogen. Protocols were optimized for extraction of DNA from pure fungal cultures and fresh or dry roots. A new procedure to test polymerase chain reaction (PCR) inhibitors in DNA extracts was developed. Novel real-time quantitative PCR (QPCR) assays were developed for both absolute and relative quantification of F. solani f. sp. glycines. The fungus was quantified based on detection of the mitochondrial small-subunit rRNA gene, and the host plant based on detection of the cyclophilin gene of the host plant. DNA of F. solani f. sp. glycines was detected in soybean plants both with and without SDS foliar symptoms to contents as low as 9.0 × 10-5 ng in the absolute QPCR assays. This is the first report of relative QPCR using the comparative threshold cycle (Ct) method to quantify the DNA of a plant pathogen relative to its host DNA. The relative QPCR assay is reliable if care is taken to avoid reaction inhibition and it may be used to further elucidate the fungus-host interaction in the development of SDS or screen for resistance to the fungus.

Races of Phytophthora sojae in Arkansas Soybean Fields and Their Effects on Commonly Grown Soybean Cultivars.

Isolates of Phytophthora sojae were collected during 1995 to 1998 from soil samples collected in 23 Arkansas soybean fields in 14 counties, and characterized by race. A total of seven races (races 2, 10, 14, 15, 24, 26, and 38) were found. Races 10, 24, and 15 were the most common and comprised 47, 22, and 9% of the 32 isolates, respectively. A single isolate each of races 2, 14, 26, and 38 also was found. Three of the isolates collected could not be characterized to race due to inconsistent results. In 1997 and 1998, a portion of a single soybean field at the University of Arkansas Southeast Research and Experiment Center near Rohwer, AR was surveyed intensively for P. sojae. The area was planted each year to the P. sojae-susceptible cv. Williams and both plants and soil were collected to assay for P. sojae from 16 and 28 plots (4.9 by 7.6 m) in 1997 and 1998, respectively. A total of 83 isolates were collected (11 from plants and 72 from soil), and found to represent 13 pathotypes, including 6 with virulence formulae that have not been described previously. Nine commercial soybean cultivars representing a range of reported resistance and tolerance to Phytophthora root and stem rot were screened for resistance to races 10, 15, and 26 of P. sojae using both hypocotyl injection and inoculum layer techniques. Cvs. Manokin, Hartz Variety 5545, and Riverside 499 were consistently resistant to all of the races using both inoculation methods. These results indicate that, although considerable pathogenic variability in P. sojae exists in soybean fields in Arkansas, cultivars with effective resistance are available to help growers manage Phytophthora root and stem rot.

Fibroblast growth factor activation of the rat PRL promoter is mediated by PKCdelta.

Fibroblast growth factors play a critical role in cell growth, development, and differentiation and are also implicated in the formation and progression of tumors in a variety of tissues including pituitary. We have previously shown that fibroblast growth factor activation of the rat PRL promoter in GH4T2 pituitary tumor cells is mediated via MAP kinase in a Ras/Raf-1-independent manner. Herein we show using biochemical, molecular, and pharmacological approaches that PKCdelta is a critical component of the fibroblast growth factor signaling pathway. PKC inhibitors, or down-regulation of PKC, rendered the rat PRL promoter refractory to subsequent stimulation by fibroblast growth factors, implying a role for PKC in fibroblast growth factor signal transduction. FGFs caused specific translocation of PKCdelta from cytosolic to membrane fractions, consistent with enzyme activation. In contrast, other PKCs expressed in GH4T2 cells (alpha, betaI, betaII, and epsilon) did not translocate in response to fibroblast growth factors. The PKCdelta subtype-selective inhibitor, rottlerin, or expression of a dominant negative PKCdelta adenoviral construct also blocked fibroblast growth factor induction of rat PRL promoter activity, confirming a role for the novel PKCdelta isoform. PKC inhibitors selective for the conventional alpha and beta isoforms or dominant negative PKCalpha adenoviral expression constructs had no effect. Induction of the endogenous PRL gene was also blocked by adenoviral dominant negative PKCdelta expression but not by an analogous dominant negative PKCalpha construct. Finally, rottlerin significantly attenuated FGF-induced MAP kinase phosphorylation. Together, these results indicate that MAP kinase-dependent fibroblast growth factor stimulation of the rat PRL promoter in pituitary cells is mediated by PKCdelta.

Pathobiology of amber disease, caused by Serratia Spp., in the New Zealand grass grub, Costelytra zealandica.

Amber disease in the New Zealand grass grub (Costelytra zealandica) is caused by some strains of Serratia entomophila or Serratia proteamaculans (Enterobacteriaceae). When treated with pathogenic isolates, larvae ceased feeding within 48 h, developed an amber coloration after 72 h, and entered a long chronic phase without feeding. An acute dose of 2-4 x 10(4) pathogenic bacteria was sufficient to produce disease in 50% of treated larvae. Time to death was directly related to temperature. At 15 degrees C, infected larvae remained in a chronic, nonfeeding state for more than 4 months prior to death. Nonpathogenic isolates, lacking the disease-causing plasmid (pADAP), had no effect on either feeding or disease. Twenty-four hours after ingestion, bacteria were found predominantly in the hindgut and growth occurred primarily within the fermentation chamber and in the head section of the larvae. Nonpathogenic strains did not multiply in treated larvae. Treatment of diseased larvae with antibiotic eliminated Serratia cells from the insects but did not result in restoration of feeding or the dark gut characteristic of the healthy larva.

Nuclear receptor conformation, coregulators, and tamoxifen-resistant breast cancer.

The development of tamoxifen resistance and consequent disease progression are common occurrences in breast cancers, often despite the continuing expression of estrogen receptors (ER). Tamoxifen is a mixed antagonist, having both agonist and antagonist properties. We have suggested that the development of tamoxifen resistance is associated with an increase in its agonist-like properties, resulting in loss of antagonist effects or even inappropriate tumor stimulation. Nuclear receptor function is influenced by a family of transcriptional coregulators, that either enhance or suppress transcriptional activity. Using a mixed antagonist-biased two-hybrid screening strategy, we identified two such proteins: the human homolog of the nuclear receptor corepressor, N-CoR, and a novel coactivator, L7/SPA (Switch Protein for Antagonists). In transcriptional studies, N-CoR suppressed the agonist properties of tamoxifen and RU486, and L7/SPA increased agonist effects. We speculated that the relative levels of these coactivators and corepressors may determine the balance of agonist and antagonist properties of mixed antagonists, such as tamoxifen. Using quantitative RT-PCR, we, therefore, measured the levels of transcripts encoding these coregulators, as well as the corepressor SMRT, and the coactivator SRC-1, in a small cohort of tamoxifen-resistant and sensitive breast tumors. The results suggest that tumor sensitivity to mixed antagonists may be governed by a complex set of transcription factors, which we are only now beginning to understand.

Plasmid-located pathogenicity determinants of Serratia entomophila, the causal agent of amber disease of grass grub, show similarity to the insecticidal toxins of Photorhabdus luminescens.

Serratia entomophila and Serratia proteamaculans cause amber disease in the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. Larval disease symptoms include cessation of feeding, clearance of the gut, amber coloration, and eventual death. A 115-kb plasmid, pADAP, identified in S. entomophila is required for disease causation and, when introduced into Escherichia coli, enables that organism to cause amber disease. A 23-kb fragment of pADAP that conferred disease-causing ability on E. coli and a pADAP-cured strain of S. entomophila was isolated. Using insertion mutagenesis, the pathogenicity determinants were mapped to a 17-kb region of the clone. Sequence analysis of the 17-kb region showed that the predicted products of three of the open reading frames (sepA, sepB, and sepC) showed significant sequence similarity to components of the insecticidal toxin produced by the bacterium Photorhabdus luminescens. Transposon insertions in sepA, sepB, or sepC completely abolished both gut clearance and cessation of feeding on the 23-kb clone; when recombined back into pADAP, they abolished gut clearance but not cessation of feeding. These results suggest that SepA, SepB, and SepC together are sufficient for amber disease causation by S. entomophila and that another locus also able to exert a cessation-of-feeding effect is encoded elsewhere on pADAP.

Thoughts on tamoxifen resistant breast cancer. Are coregulators the answer or just a red herring?

The antiestrogen tamoxifen is an effective treatment for estrogen receptor positive breast cancers, slowing tumor growth and preventing disease recurrence, with relatively few side effects. However, many patients who initially respond to treatment, later become resistant to treatment. Tamoxifen has both agonist and antagonist activities, which are manifested in a tissue-specific pattern. Development of tamoxifen resistance can be characterized by an increase in the partial agonist properties of the antiestrogen in the breast, resulting in loss of growth inhibition and even inappropriate tumor stimulation. Nuclear receptor function is modulated by transcriptional coregulators, which either enhance or repress receptor activity. Using a mixed antagonist-biased two-hybrid screening strategy, we identified two such proteins: the human homolog of the nuclear receptor corepressor, N-CoR, and a novel coactivator, L7/SPA (Switch Protein for Antagonists). In transcriptional studies N-CoR suppressed the agonist properties of tamoxifen and RU486, while L7/SPA increased agonist effects. We speculated that the relative level of these coactivators and corepressors might determine the balance of agonist and antagonist properties of mixed antagonists such as tamoxifen. Using quantitative RT-PCR we therefore measured the levels of transcripts encoding these coregulators, as well as the corepressor SMRT, and the coactivator SRC-1, in a small cohort of tamoxifen resistant and sensitive breast tumors. The results suggest that tumor sensitivity to mixed antagonists may be governed by a complex set of transcription factors, which we are only now beginning to understand.