Flexible memory controls sperm competition responses in male Drosophila melanogaster.

Males of many species use social cues to predict sperm competition (SC) and tailor their reproductive strategies, such as ejaculate or behavioural investment, accordingly. While these plastic strategies are widespread, the underlying mechanisms remain largely unknown. Plastic behaviour requires individuals to learn and memorize cues associated with environmental change before using this experience to modify behaviour. Drosophila melanogaster respond to an increase in SC threat by extending mating duration after exposure to a rival male. This behaviour shows lag times between environmental change and behavioural response suggestive of acquisition and loss of memory. Considering olfaction is important for a male's ability to assess the SC environment, we hypothesized that an olfactory learning and memory pathway may play a key role in controlling this plastic behaviour. We assessed the role of genes and brain structures known to be involved in learning and memory. We show that SC responses depend on anaesthesia-sensitive memory, specifically the genes rut and amn We also show that the γ lobes of the mushroom bodies are integral to the control of plastic mating behaviour. These results reveal the genetic and neural properties required for reacting to changes in the SC environment.

Experimental and Numerical Analysis of Initial Plasticity in P91 Steel Small Punch Creep Samples.

To date, the complex behaviour of small punch creep test (SPCT) specimens has not been completely understood, making the test hard to numerically model and the data difficult to interpret. This paper presents a novel numerical model able to generate results that match the experimental findings. For the first time, pre-strained uniaxial creep test data of a P91 steel at 600 ∘C have been implemented in a conveniently modified Liu and Murakami creep damage model in order to simulate the effects of the initial localised plasticity on the subsequent creep response of a small punch creep test specimen. Finite element (FE) results, in terms of creep displacement rate and time to failure, obtained by the modified Liu and Murakami model are in good agreement with experimental small punch creep test data. The rupture times obtained by the FE calculations which make use of the non-modified creep damage model are one order of magnitude shorter than those obtained by using the modified constitutive model. Although further investigation is needed, this novel approach has confirmed that the effects of initial localised plasticity, taking place in the early stages of small punch creep test, cannot be neglected. The new results, obtained by using the modified constitutive model, show a significant improvement with respect to those obtained by a 'state of the art' creep damage constitutive model (the Liu and Murakami constitutive model) both in terms of minimum load-line displacement rate and time to rupture. The new modelling method will potentially lead to improved capability for SPCT data interpretation.

Evaluation of a digital microfluidic real-time PCR platform to detect DNA of Candida albicans in blood.

Species of Candida frequently cause life-threatening infections in neonates, transplant and intensive care unit (ICU) patients, and others with compromised host defenses. The successful management of systemic candidiasis depends upon early, rapid diagnosis. Blood cultures are the standard diagnostic method, but identification requires days and less than half of the patients are positive. These limitations may be eliminated by using real-time polymerase chain reaction (PCR) to detect Candida DNA in the blood specimens of patients at risk. Here, we optimized a PCR protocol to detect 5-10 yeasts in low volumes of simulated and clinical specimens. We also used a mouse model of systemic candidiasis and determined that candidemia is optimally detectable during the first few days after infection. However, PCR tests are often costly, labor-intensive, and inconvenient for routine use. To address these obstacles, we evaluated the innovative microfluidic real-time PCR platform (Advanced Liquid Logic, Inc.), which has the potential for full automation and rapid turnaround. Eleven and nine of 16 specimens from individual patients with culture-proven candidemia tested positive for C. albicans DNA by conventional and microfluidic real-time PCR, respectively, for a combined sensitivity of 94%. The microfluidic platform offers a significant technical advance in the detection of microbial DNA in clinical specimens.

Identification of a heparin-binding hemagglutinin present in mycobacteria.

Adherence to mammalian host tissues is an important virulence trait in microbial pathogenesis, yet little is known about the adherence mechanisms of mycobacteria. Here, we show that binding of mycobacteria to epithelial cells but not to macrophages can be specifically inhibited by sulfated carbohydrates. Using heparin-Sepharose chromatography, a 28-kD heparin-binding protein was purified from culture supernatants and cell extracts of Mycobacterium bovis and Mycobacterium tuberculosis. This protein, designated heparin-binding hemagglutinin (HBHA), promotes the agglutination of rabbit erythrocytes, which is specifically inhibited by sulfated carbohydrates. HBHA also induce mycobacterial aggregation, suggesting that it can mediate bacteria-bacteria interactions as well. Hemagglutination, mycobacterial aggregation, as well as attachment to epithelial cells are specifically inhibited in the presence of anti-HBHA antibodies. Immunoelectron microscopy using anti-HBHA monoclonal antibodies revealed that the protein is surface exposed, consistent with a role in adherence. Immunoblot analyses using antigen-specific antibodies indicated that HBHA is different from the fibronectin-binding proteins of the antigen 85 complex and p55, and comparison of the NH2-terminal amino acid sequence of purified HBHA with the protein sequence data bases did not reveal any significant similarity with other known proteins. Sera from tuberculosis patients but not from healthy individuals were found to recognize HBHA, indicating its immunogenicity in humans during mycobacterial infections. Identification of putative mycobacterial adhesins, such as the one described in this report, may provide the basis for the development of new therapeutic and prophylactic strategies against mycobacterial diseases.

Connections between the Ras-cyclic AMP pathway and G1 cyclin expression in the budding yeast Saccharomyces cerevisiae.

We have identified two processes in the G1 phase of the Saccharomyces cerevisiae cell cycle that are required before nutritionally arrested cells are able to return to proliferative growth. The first process requires protein synthesis and is associated with increased expression of the G1 cyclin gene CLN3. This process requires nutrients but is independent of Ras and cyclic AMP (cAMP). The second process requires cAMP. This second process is rapid, is independent of protein synthesis, and produces a rapid induction of START-specific transcripts, including CLN1 and CLN2. The ability of a nutritionally arrested cell to respond to cAMP is dependent on completion of the first process, and this is delayed in cells carrying a CLN3 deletion. Mating pheromone blocks the cAMP response but does not alter the process upstream of Ras-cAMP. These results suggest a model linking the Ras-cAMP pathway with regulation of G1 cyclin expression.

Interactions between adenylate cyclase and the yeast GTPase-activating protein IRA1.

The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains many proteins, including the CYR1 polypeptide, which is responsible for catalyzing the formation of cyclic AMP from ATP, RAS1 and RAS2 polypeptides, which mediate stimulation of cyclic AMP synthesis by guanine nucleotides, and the yeast GTPase-activating protein analog IRA1. We have previously reported that adenylate cyclase is only peripherally bound to the yeast membrane. We have concluded that IRA1 is a strong candidate for a protein involved in anchoring adenylate cyclase to the membrane. We base this conclusion on the following criteria: (i) a disruption of the IRA1 gene produced a mutant with very low membrane-associated levels of adenylate cyclase activity, (ii) membranes made from these mutants were incapable of binding adenylate cyclase in vitro, (iii) IRA1 antibodies inhibit binding of adenylate cyclase to the membrane, and (iv) IRA1 and adenylate cyclase comigrate on Sepharose 4B.

Insecticidal proteins from Bacillus thuringiensis protect corn from corn rootworms.

Field tests of corn co-expressing two new delta-endotoxins from Bacillus thuringiensis (Bt) have demonstrated protection from root damage by western corn rootworm (Diabrotica virgifera virgifera LeConte). The level of protection exceeds that provided by chemical insecticides. In the bacterium, these proteins form crystals during the sporulation phase of the growth cycle, are encoded by a single operon, and have molecular masses of 14 kDa and 44 kDa. Corn rootworm larvae fed on corn roots expressing the proteins showed histopathological symptoms in the midgut epithelium.

Controlled drug delivery to the lung: Influence of hyaluronic acid solution conformation on its adsorption to hydrophobic drug particles.

This work reports investigations into the interaction and adsorption of the hydrophilic polymer hyaluronic acid (HA) onto the surface of the hydrophobic corticosteroid drug fluticasone propionate (FP). The eventual aim is to formulate a bioadhesive pulmonary drug delivery system with prolonged action that avoids rapid clearance from the lungs by the mucociliary escalator. Adsorption isotherms detailing the adsorption of HA from aqueous HA solution concentrations ranging from 0.14 to 0.0008% (w/v) to a fixed FP particle concentration of 0.1% (w/v) were investigated. The method of preparing FP particles with HA molecules adsorbed on their surfaces (FP/HA particles) involved suspension of the FP either in hydrated HA solution or in water followed by addition of solid HA, centrifugation of the solids to form a pellet, washing the pellet several times with water until no HA was found in the supernatant and then freeze drying the suspension obtained by dispersing the final pellet. The freeze dried powder was then analysed for adsorbed HA using a Stains-all assay. The influence of order of addition of HA to FP, time for the adsorption process, and temperature of preparation on the adsorption isotherms was investigated. The non-equilibrium adsorption isotherms produced generally followed the same trend, in that as the HA solution concentration increased, the amount of HA adsorbed increased to a maximum at a solution concentration of approximately 0.1% (w/v) and then decreased. The maxima in the adsorption isotherms were close to the change from secondary to tertiary conformation in the HA solutions. Below the maxima, adsorption occurred via interaction of FP with the hydrophobic patches along the HA chains in the secondary structures. Above the maxima, secondary HA molecules aggregate in solution to form tertiary network structures. Adsorption from tertiary structure was reduced because strong interactions between the HA molecules limited the availability of hydrophobic patches for adsorption of HA onto FP. The influence of preparation variables on adsorption was also related to the availability of hydrophobic patches for adsorption.

Physical ageing and thermal analysis of PLGA microspheres encapsulating protein or DNA.

PLGA microspheres undergo physical ageing but their ageing kinetics have not been reported, nor the effect of encapsulated protein or plasmid DNA on any associated changes to the glass transition. Differential scanning calorimetry (DSC) was used to measure the rate of ageing of various PLGA microsphere formulations, with temperature-modulated DSC used to accurately measure the associated glass transition. The Cowie-Ferguson model was applied to determine the parameters describing the enthalpy relaxation kinetics. We show that encapsulated proteins had no significant effect on the glass transition of the microspheres, whereas DNA and PVA were mild antiplasticising agents, particularly with high Mw PLGA. Physical ageing occurred through a range of enthalpy relaxation times (or modes) and was independent of both encapsulated protein and surfactant used during microsphere preparation. Analysis of accelerated ageing at 35 degrees C gave calculated enthalpy relaxation times to thermal equilibrium of 280-400 h. No ageing was observed < or = 10 degrees C and at 25 degrees C estimated relaxation times were at least one order of magnitude greater than at 35 degrees C. Ageing of PLGA microspheres therefore occurs at temperatures >10 degrees C, but relaxation will be far from equilibrium unless storage times and/or temperatures are prolonged or nearing the glass transition, respectively.

Changes in gene expression in the Ras/adenylate cyclase system of Saccharomyces cerevisiae: correlation with cAMP levels and growth arrest.

Levels of cyclic 3',5'-cyclic monophosphate (cAMP) play an important role in the decision to enter the mitotic cycle in the yeast, Saccharomyces cerevisiae. In addition to growth arrest at stationary phase, S. cerevisiae transiently arrest growth as they shift from fermentative to oxidative metabolism (the diauxic shift). Experiments examining the role of cAMP in growth arrest at the diauxic shift show the following: 1) yeast lower cAMP levels as they exhaust their glucose supply and shift to oxidative metabolism of ethanol, 2) a reduction in cAMP is essential for traversing the diauxic shift, 3) the decrease in adenylate cyclase activity is associated with a decrease in the expression of CYR1 and CDC25, two positive regulators of cAMP levels and an increase in the expression of IRA1 and IRA2, two negative regulators of intracellular cAMP, 4) mutants carrying disruptions in IRA1 and IRA2 were unable to arrest cell division at the diauxic shift and were unable to progress into the oxidative phase of growth. These results indicate that changes cAMP levels are important in regulation of growth arrest at the diauxic shift and that changes in gene expression plays a role in the regulation of the Ras/adenylate cyclase system.

Bench-scale study using PVA gel as a biocarrier in a UASB reactor treating corn steep liquor wastewater.

PVA-gel beads were used as a biocarrier for treatment of corn steep liquor wastewater containing high levels of volatile fatty acids (VFA), where retention of biomass could be either solely in the porous microstructure of the gel or by granule formation using a gel bead as a nucleus. With stable COD removal efficiencies of 90% or greater, continuous treatment was demonstrated over a four month period, with organic loading rates being increased stepwise from 2.5 to 22.5 kg COD/m3 d. In addition, VFA in the effluent were, with few exceptions maintained close to zero. Gas production increased over the course of the study and reached a level of 0.38 m3/kg COD consisting of 65% methane with the remainder being mostly carbon dioxide. Biomass granules containing methane producing bacteria progressively formed around the PVA-gel beads during the study. In contrast, very few small natural granules developed apart from PVA-gel nuclei indicating that PVA gel may serve well as a seeding material to enhance granulation when natural occurrence is lacking.

A pilot-plant study of a moving-bed biofilm reactor system using PVA gel as a biocarrier for removals of organic carbon and nitrogen.

A pilot-plant study was conducted to evaluate the performance of a moving-bed biofilm reactor process using PVA-gel beads as a biocarrier. Real primary-settled wastewater was fed to the pre-denitrification system and removals of nitrogenous and organic contaminants were evaluated over a 1-year period. The results demonstrated that at a total nitrogen (TN) loading of 18 mg/L.h, a TN removal efficiency in keeping with and even exceeding the theoretical maximum efficiency based on the level of internal recycle, was possible and a nitrification rate of 15 mg/L.h was sustained with a HRT of only 2.5 h at 15 degrees C. Furthermore, soluble COD and BOD5 in the effluent of the pilot plant were reduced to levels well below most regulatory discharge limits. In addition, the possibility of using this biocarrier in a system, including the elimination of waste organic sludge, was discussed.

Predisposition to efficient mammary tumor metastatic progression is linked to the breast cancer metastasis suppressor gene Brms1.

Tumor metastasis is one of the most important clinical aspects of neoplastic disease because patient mortality is frequently attributable to disseminated rather than primary tumors. However, it still is not possible to definitively distinguish those individuals at high risk for disseminated disease, who would benefit from aggressive adjuvant therapy, from the low-risk patients who might be spared the side effects of additional anticancer therapy. To identify factors that predispose toward metastatic disease, we have used a genetic approach. Using a highly metastatic model of mammary cancer, we identified previously inbred mouse strains (DBA/2J, NZB/B1NJ, and I/LnJ) that harbor genetic factors that significantly suppress metastatic efficiency. In this study, we report the results of four experiments to localize the genetic map locations of the metastasis efficiency modifier genes. One statistically significant locus was identified on proximal Chr 19 designated Mtes1. Secondary candidate intervals were detected on Chrs 6, 9, 13, and 17. Interestingly, Mtes1 colocalizes with the murine orthologue of the human breast cancer metastasis suppressor gene Brms1, suggesting that allelic variants of Brms1 might be responsible for the metastasis suppression observed.

Purification and partial amino acid sequencing of rat bone tumor (UMR106) alkaline phosphatase.

Cultured rat osteosarcoma (UMR106) alkaline phosphatase was purified to apparent homogeneity by sequential application of polyclonal antibody affinity, DEAE-cellulose, and Sepharose CL-6B chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme preparation treated with sodium dodecyl sulfate and mercaptoethanol showed the presence of a dominant band (using silver staining) corresponding to a molecular weight of 80,000. The amino acid composition was similar to those of various alkaline phosphatases. The N-terminal amino acid sequence was determined as follows: Phe-Val-Pro-Glu-Lys-Glu-Lys- Asp-Pro-Ser-Tyr-Trp-Arg-Gln-Gln-Ala-Gln-Glu-Thr-Leu- Lys-Asn-Ala-Leu-Lys-?-Gln-Lys-?-Asn-Val-Asn-Ala-Lys.

Bovine parathyroid glands secrete a 26-kDa N-terminal fragment of chromogranin-A which inhibits parathyroid cell secretion.

Chromogranin-A (CgA) is a ubiquitous protein which colocalizes in secretory granules of multiple endocrine tissues and cosecretes with peptide hormones from these tissues. Although the function of CgA has remained unknown, there has been recent interest in its potential role as a prohormone for smaller, biologically active peptides. We isolated and characterized a 26-kDa N-terminal fragment of CgA which is a natural breakdown product of bovine parathyroid CgA in storage. A similar, if not identical, fragment of CgA is secreted by bovine parathyroid glands. The secreted fragment elutes on HPLC and migrates on both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and acid-urea gels in the same position as the 26-kDa N-terminal fragment. When added to the incubation medium of dispersed bovine parathyroid cells, the 26-kDa N-terminal fragment of CgA inhibits the low calcium-stimulated secretion of both PTH and CgA. This N-terminal fragment is homologous to betagranin, which is a 21-kDa N-terminal fragment of CgA that is generated from CgA in rat insulin granules. Thus, a naturally occurring betagranin-like N-terminal fragment of bovine parathyroid CgA is not only secreted itself, but can inhibit the secretion of PTH and intact CgA by bovine parathyroid cells. The processing of intact CgA to fragments such as the N-terminal fragment that we describe may be important in the autocrine or paracrine regulation of secretion.

Biochemical characterization of recombinant factor IX.

Mature human factor IX is a 55,000-d glycoprotein with a modular domain structure and numerous posttranslational modifications. A recombinant form of human factor IX (rFIX) has been produced from a Chinese hamster ovary cell line that was engineered for high-level protein processing and expression. To ensure that the recombinant molecule contains the requisite structural and functional features of the plasma-derived form, rFIX was subjected to detailed biochemical and biophysical characterization. The laboratory studies showed that the posttranslational modifications and primary, secondary, and tertiary structures of rFIX were similar to those of plasma-derived factor IX (pdFIX). In addition, rFIX displayed a high degree of purity and a product release specification for specific activity that is > or = 200 IU/mg.

SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1.

A class of pyridinyl imidazoles inhibit the MAP kinase homologue, termed here reactivating kinase (RK) [Lee et al. (1994) Nature 372, 739-746]. We now show that one of these compounds (SB 203580) inhibits RK in vitro (IC50 = 0.6 microM), suppresses the activation of MAPKAP kinase-2 and prevents the phosphorylation of heat shock protein (HSP) 27 in response to interleukin-1, cellular stresses and bacterial endotoxin in vivo. These results establish that MAPKAP kinase-2 is a physiological RK substrate, and that HSP27 is phosphorylated by MAPKAP kinase-2 in vivo. The specificity of SB 203580 was indicated by its failure to inhibit 12 other protein kinases in vitro, and by its lack of effect on the activation of RK kinase and other MAP kinase cascades in vivo. We suggest that SB 203580 will be useful for identifying other physiological roles and targets of RK and MAPKAP kinase-2.

Identification of human serum interferants in the recombinant P-selectin glycoprotein ligand-1 clinical ELISA using MALDI MS and RP-HPLC.

A colorimetric enzyme-linked immunosorbent assay (ELISA) was developed to detect circulating levels of rPSGL to permit pharmacokinetic analysis of clinical samples. The ELISA is an asymmetric sandwich utilizing a monoclonal antibody pair. Initial validation studies indicated that 57% of normal individuals scored above the limit of detection of the assay. Specificity experiments indicated that the signal was not due to circulating endogenous P-selectin glycoprotein ligand-1 (PSGL-1). Using matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and sampling within the individual microplate wells, the interferant was detected in the vicinity of 6.6 kDa in lipemic and normal human sera, but not delipidized sera. These results were consistent with the ELISA data where 97.5% of known lipemic, 57% of normal, and 0% of delipidized sera scored above detectable limits in the ELISA. Preparative isolations of the 6.6 kDa species were performed using reversed-phase high performance liquid chromatography (RP-HPLC) with UV and MS detection. Edman N-terminal sequencing identified the 6.6 kDa unknown as Apolipoprotein C-I. Additional apolipoproteins were found by MALDI and RP-HPLC. Digestion of sera with liposome lipase and extraction of sera with anti-apolipoprotein C-I, C-II, and C-III antibody beads significantly reduced the ELISA interference. These experiments combined with the MALDI detection of phosphatidylcholine-type lipids from NHS eluate suggested that lipoprotein particles or remnants were causing the interference. A method combining Triton-X 100 with sonication was developed to overcome this interference without altering rPSGL recovery in the ELISA.

Injection of reagent ions into the selvedge region in fast-atom bombardment mass spectrometry.

A divided probe that incorporates a potassium aluminosilicate glass target and an analyte/glycerol matrix target, spatially separated, was used to inject potassium ions (K(+)) into the high-pressure "selvedge" region formed above the analyte/glycerol matrix target during fast-atom bombardment (FAB); [M+K](+) adduct ions that represent the types of gas-phase neutral molecules present in the selvedge region are observed. Computer modeling assisted in designing the divided target and an additional ion optical element for the FAB ion source to optimize interactions between K(+) ions and the desorbed neutral molecules. The capability of injecting K(+) ions into the FAB experiment has utility in both mechanistic studies and analyses. Experimental results here are consistent with a model for the desorption/ionization processes in FAB in which some types of neutral analyte molecules are desorbed intact and are subsequently protonated by glycerol chemical ionization. Unstable protonated molecules undergo unimolecular decomposition to yield observed fragment ions. The use of K(+) cationization of analytes for molecular weight confirmation is demonstrated, as well as its utility in FAB experiments in which mixtures are encountered.

Nitrogen pollution: an assessment of its threat to amphibian survival.

The potential for nitrate to affect amphibian survival was evaluated by examining the areas in North America where concentrations of nitrate in water occur above amphibian toxicity thresholds. Nitrogen pollution from anthropogenic sources enters bodies of water through agricultural runoff or percolation associated with nitrogen fertilization, livestock, precipitation, and effluents from industrial and human wastes. Environmental concentrations of nitrate in watersheds throughout North America range from < 1 to > 100 mg/L. Of the 8,545 water quality samples collected from states and provinces bordering the Great Lakes, 19.8% contained nitrate concentrations exceeding those which can cause sublethal effects in amphibians. In the laboratory lethal and sublethal effects in amphibians are detected at nitrate concentrations between 2.5 and 100 mg/L. Furthermore, amphibian prey such as insects and predators of amphibians such as fish are also sensitive to these elevated levels of nitrate. From this we conclude that nitrate concentrations in some watersheds in North America are high enough to cause death and developmental anomalies in amphibians and impact other animals in aquatic ecosystems. In some situations, the use of vegetated buffer strips adjacent to water courses can reduce nitrogen contamination of surface waters. Ultimately, there is a need to reduce runoff, sewage effluent discharge, and the use of fertilizers, and to establish and enforce water quality guidelines for nitrate for the protection of aquatic organisms.