Low-dose expression of a human apolipoprotein E transgene in macrophages restores cholesterol efflux capacity of apolipoprotein E-deficient mouse plasma

Apolipoprotein E- (apoE) deficient (E−/−) mice develop severe hyperlipidemia and diffuse atherosclerosis. Low-dose expression of a human apoE3 transgene in macrophages of apoE-deficient mice (E−/−hTgE+/0), which results in about 5% of wild-type apoE plasma levels, did not correct hyperlipidemia but significantly reduced the extent of atherosclerotic lesions. To investigate the contribution of apoE to reverse cholesterol transport, we compared plasmas of wild-type (E+/+), E−/−, and E−/−hTgE+/0 mice for the appearance of apoE-containing lipoproteins by electrophoresis and their capacity to take up and esterify 3H-labeled cholesterol from radiolabeled fibroblasts or J774 macrophages. Wild-type plasma displayed lipoproteins containing apoE that were the size of high density lipoprotein and that had either electrophoretic α or γ mobilities. Similar particles were also present in E−/−hTgE+/0 plasma. Depending on incubation time, E−/− plasma released 48–74% less 3H-labeled cholesterol from fibroblasts than E+/+ plasma, whereas cholesterol efflux into E−/−hTgE+/0 plasma was only 11–25% lower than into E+/+ plasma. E−/−hTgE+/0 plasma also released 10% more 3H-labeled cholesterol from radiolabeled J774 macrophages than E−/− plasma. E+/+ and E−/−hTgE+/0 plasma each esterified significantly more cell-derived 3H-labeled cholesterol than E−/− plasma. Moreover, E−/− plasma accumulated much smaller proportions of fibroblast-derived 3H-labeled cholesterol in fractions with electrophoretic γ and α mobility than E+/+ and E−/−hTgE+/0 plasma. Thus, low-dose expression of apoE in macrophages nearly restored the cholesterol efflux capacity of apoE-deficient plasma through the formation of apoE-containing particles, which efficiently take up cell-derived cholesterol, and through the increase of cholesterol esterification activity. Thus, macrophage-derived apoE may protect against atherosclerosis by increasing cholesterol efflux from arterial wall cells.

Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins

Fast excitation-driven fluctuations in the fluorescence emission of yellow-shifted green fluorescent protein mutants T203Y and T203F, with S65G/S72A, are discovered in the 10−6–10−3-s time range, by using fluorescence correlation spectroscopy at 10−8 M. This intensity-dependent flickering is conspicuous at high pH, with rate constants independent of pH and viscosity with a minor temperature effect. The mean flicker rate increases linearly with excitation intensity for at least three decades, but the mean dark fraction of the molecules undergoing these dynamics is independent of illumination intensity over ≈6 × 102 to 5 × 106 W/cm2. These results suggest that optical excitation establishes an equilibration between two molecular states of different spectroscopic properties that are coupled only via the excited state as a gateway. This reversible excitation-driven transition has a quantum efficiency of ≈10−3. Dynamics of external protonation, reversibly quenching the fluorescence, are also observed at low pH in the 10- to 100-μs time range. The independence of these two bright–dark flicker processes implies the existence of at least two separate dark states of these green fluorescent protein mutants. Time-resolved fluorescence measurements reveal a single exponential decay of the excited state population with 3.8-ns lifetime, after 500-nm excitation, that is pH independent. Our fluorescence correlation spectroscopy results are discussed in terms of recent theoretical studies that invoke isomerization of the chromophore as a nonradiative channel of the excited state relaxation.

The Biosynthesis of Erucic Acid in Developing Embryos of Brassica rapa1

The prevailing hypothesis on the biosynthesis of erucic acid in developing seeds is that oleic acid, produced in the plastid, is activated to oleoyl-coenzyme A (CoA) for malonyl-CoA-dependent elongation to erucic acid in the cytosol. Several in vivo-labeling experiments designed to probe and extend this hypothesis are reported here. To examine whether newly synthesized oleic acid is directly elongated to erucic acid in developing seeds of Brassica rapa L., embryos were labeled with [14C]acetate, and the ratio of radioactivity of carbon atoms C-5 to C-22 (de novo fatty acid synthesis portion) to carbon atoms C-1 to C-4 (elongated portion) of erucic acid was monitored with time. If newly synthesized 18:1 (oleate) immediately becomes a substrate for elongation to erucic acid, this ratio would be expected to remain constant with incubation time. However, if erucic acid is produced from a pool of preexisting oleic acid, the ratio of 14C in the 4 elongation carbons to 14C in the methyl-terminal 18 carbons would be expected to decrease with time. This labeling ratio decreased with time and, therefore, suggests the existence of an intermediate pool of 18:1, which contributes at least part of the oleoyl precursor for the production of erucic acid. The addition of 2-[{3-chloro-5-(trifluromethyl)-2-pyridinyl}oxyphenoxy] propanoic acid, which inhibits the homodimeric acetyl-CoA carboxylase, severely inhibited the synthesis of [14C]erucic acid, indicating that essentially all malonyl-CoA for elongation of 18:1 to erucate was produced by homodimeric acetyl-CoA carboxylase. Both light and 2-[{3-chloro-5-(trifluromethyl)-2-pyridinyl}oxyphenoxy]-propanoic acid increased the accumulation of [14C]18:1 and the parallel accumulation of [14C]phosphatidylcholine. Taken together, these results show an additional level of complexity in the biosynthesis of erucic acid.

Action spectra for phytochrome A- and B-specific photoinduction of seed germination in Arabidopsis thaliana.

We have examined the seed germination in Arabidopsis thaliana of wild type (wt), and phytochrome A (PhyA)- and B (PhyB)-mutants in terms of incubation time and environmental light effects. Seed germination of the wt and PhyA-null mutant (phyA) was photoreversibly regulated by red and far-red lights of 10-1,000 micromol m-2 when incubated in darkness for 1-14 hr, but no germination occurred in PhyB-null mutant (phyB). When wt seeds and the phyB mutant seeds were incubated in darkness for 48 hr, they synthesized PhyA during dark incubation and germinated upon exposure to red light of 1-100 nmol m-2 and far-red light of 0.5-10 micromol m-2, whereas the phyA mutant showed no such response. The results indicate that the seed germination is regulated by PhyA and PhyB but not by other phytochromes, and the effects of PhyA and PhyB are separable in this assay. We determined action spectra separately for PhyA- and PhyB-specific induction of seed germination at Okazaki large spectrograph. Action spectra for the PhyA response show that monochromatic 300-780 nm lights of very low fluence induced the germination, and this induction was not photoreversible in the range examined. Action spectra for the PhyB response show that germination was photoreversibly regulated by alternate irradiations with light of 0.01-1 mmol m-2 at wavelengths of 540-690 nm and 695-780 nm. The present work clearly demonstrated that PhyA photoirreversibly triggers the germination upon irradiations with ultraviolet, visible and far-red light of very low fluence, while PhyB controls the photoreversible effects of low fluence.

Interactions of maize bushy stunt phytoplasma with the leafhopper vector, Dalbulus maidis (Delong and Wolcott) (Hemiptera: Cicadellidae) and associated microbiota

Phytoplasmas are bacteria with a persistent propagative transmission by insect vectors that generates direct and indirect interactions among them. In order to understand these interactions for maize bushy stunt phytoplasma (MBSP) and the leafhopper vector Dalbulus maidis (Hemiptera: Cicadellidae), two research lines were addressed. The first one aimed to determine the indirect effects of maize infection by MBSP on some biological and behavioral parameters of the vector, whereas a second line investigated direct interactions of the phytoplasma with D. maidis during its movement through the vector body following acquisition from plants, and associated microbiota. Indirect effects were investigated in choice experiments in which alighting and oviposition preferences by D. maidis were compared on healthy vs. MBSP-infected plants with variable incubation time (diseased plants with early and advanced symptoms, or still asymptomatic). Likewise, indirect effect of MBSP on the D. maidis biology was determined in two life table experiments in which the vector was reared on healthy vs. MBSP-infected plants expressing advanced disease symptoms or still asymptomatic. Choice experiments showed that alighting and oviposition preferences of D. maidis on MBSP-infected plants compared to healthy plants depend on the pathogen incubation period in the plant. The leafhopper preferred MBSP-infected plants over healthy ones during the asymptomatic phase of the disease, but rejected infected plants with advanced symptoms. The vector was able to acquire MBSP from asymptomatic infected plants shortly (3 days) after inoculation, but transmission efficiency increased when acquisition occurred at later stages of the pathogen incubation period (≥14 days) in the source plants and the test plants showed disease symptoms faster. These results suggest that MBSP modulates D. maidis preference for asymptomatic infected plants in the early stages of the crop, allowing rapid spread of this pathogen. Maize infection by the phytoplasma had a neutral effect on most life table parameters of D. maidis; a lower net reproductivity rate (Ro) was observed in the cohort reared on MBSP-infected plants with advanced symptoms, which was compensated to some extent by a higher sexual ratio. MBSP acquisition by all vector nymphal stadia was confirmed by PCR, and the pathogen as detected in both male and female reproductive organs. Concerning direct MBSP-vector interactions, transmission electron microscopy analyses showed phytoplasma-like cells in the midgut lumen, microvilli and epithelial cells, suggesting that MBSP enters the epithelium midgut through the microvilli wall. Within the epithelial cells, mitochondria and bacteria-like cells (possibly endosymbionts) were observed together with masses of phythoplasma-like cells. In the hemocoel, phytoplasma-like cells grouped into a matrix were also observed in association with bacteria-like cells similar to those observed in the midgut epithelium. Similar associations were found in the salivary gland. Interestingly, in-situ hybridization (FISH) technique revealed a variation in diversity and abundance of the microbiota in intestine and salivary glands of D. maidis adults over time after MBSP acquisition from plants. Sulcia sp., Cardinium sp. and eubacteria increased their abundance over time, whereas Rickettsia sp. decreased. The frequent association of the vector microbiota with the phytoplasma in some tissues of D. maidis suggests that endosymbiotic bacteria may play some role in MBSP-vector interactions.

S-Nitrosocaptopril: In vitro characterization of pulmonary vascular effects in rats

1 On rat isolated pulmonary arteries, vasorelaxation by S-nitrosocaptopril (SNOcap) was compared with S-nitrosoglutathione (GSNO) and nitroprusside, and inhibition by SNOcap of contractions to angiotensin I was compared with the angiotensin converting enzyme (ACE) inhibitor, captopril. 2 SNOcap was equipotent as a vasorelaxant on main (i.d. 2-3 mm) and intralobar (i.d. 600 mum)pulmonary arteries (pIC(50) values: 5.00 and 4.85, respectively). Vasorelaxant responses reached equilibrium rapidly (2-3 min). 3 Pulmonary vasorelaxant responses to SNOcap, like GSNO, were (i) partially inhibited by the soluble guanylate cyclase inhibitor, ODQ (1H-(1,2,4) oxadiazolo(4,3-a)-quinoxalin-1-one; 3 muM) whereas responses to nitroprusside were abolished and (ii) potentiated by hydroxocobalamin (HCOB; NO. free radical scavenger; 100 muM) whereas responses to nitroprusside were inhibited. 4 The relative potencies for pulmonary vasorelaxation compared with inhibition of platelet aggregation were: SNOcap 7: 1; GSNO 25: 1; nitroprusside > 2000:1. 5 SNOcap, like captopril, concentration-dependently and time-dependently increased the EC50 for angiotensin I but not angiotensin II. The dependence on incubation time was independent of the presence of tissue but differed for SNOcap and captopril. This difference reflected the slow dissociation of SNOcap and instability of captopril, and precluded a valid comparison of the potency of the two drugs. After prolonged incubation (greater than or equal to 5.6 h) SNOcap was more effective than captopril. 6 Thus, in pulmonary arteries SNOcap (i) possesses NO donor properties characteristic of S-nitrosothiols but different from nitroprusside and (ii) inhibits ACE at least as effectively as captopril. These properties suggest that SNOcap could be valuable in the treatment of pulmonary hypertension.

A PCR method for the identification of methicillin-resistant Staphylococcus aureus (MRSA) from screening swabs

Aim: The aim of this study was to assess the discriminatory power and potential turn around time ( TAT) of a PCR-based method for the detection of methicillin-resistant Staphylococcus aureus (MRSA) from screening swabs. Methods: Screening swabs were examined using the current laboratory protocol of direct culture on mannitol salt agar supplemented with oxacillin (MSAO-direct). The PCR method involved pre-incubation in broth for 4 hours followed by a multiplex PCR with primers directed to mecA and nuc genes of MRSA. The reference standard was determined by pre-incubation in broth for 4 hours followed by culture on MSAO (MSAO-broth). Results: A total of 256 swabs was analysed. The rates of detection of MRSA using MSAO-direct, MSAO-broth and PCR were 10.2, 13.3 and 10.2%, respectively. For PCR, the sensitivity, specificity, positive predictive value and negative predictive values were 66.7% (95% CI 51.9 - 83.3%), 98.6% ( 95% CI 97.1 - 100%), 84.6% ( 95% CI 76.2 - 100%) and 95.2% ( 95% CI 92.4 - 98.0%), respectively, and these results were almost identical to those obtained from MSAO-direct. The agreement between MSAO-direct and PCR was 61.5% ( 95% CI 42.8 - 80.2%) for positive results, 95.6% ( 95% CI 93.0 - 98.2%) for negative results and overall was 92.2% ( 95% CI 88.9 - 95.5%). Conclusions: ( 1) The discriminatory power of PCR and MSAO-direct is similar but the level of agreement, especially for true positive results, is low. ( 2) The potential TAT for the PCR method provides a marked advantage over conventional methods. ( 3) Further modifications to the PCR method such as increased broth incubation time, use of selective broth and adaptation to real-time PCR may lead to improvement in sensitivity and TAT.

Assessing the cellular transmembrane electrical potential difference on the hepatic uptake of palmitate

Understanding the driving forces for the hepatic uptake of endogenous and exogenous substrates in isolated cells and organs is fundamental to describing the underlying hepatic physiology/pharmacology. In this study we investigated whether uptake of plasma protein-bound [H-3]-palmitate across the hepatocyte wall is governed by the transmembrane electrical potential difference (PD). Uptake was studied in isolated hepatocytes and isolated perfused rat livers (IPL). Protein-binding and vasoactive properties of the different perfusates were determined using in vitro heptane/buffer partitioning studies and the multiple indicator dilution (MID) technique in the IPL, respectively. Altering hepatocyte PD by perfusate ion substitution resulted in either a substantial depolarization (-14 +/- 1 mV, n = 12, mean +/- S.E., substituting choline for Na+) or hyperpolarization (-46 +/- 3 mV, n = 12, mean +/- S.E., substituting nitrate for Cl-). Perfusate ion substitution also affected the equilibrium binding constant for the palmitate-albumin complex. IPL studies suggested that, other than with gluconate buffer, hepatic [H-3]-palmitate extraction was not affected by the buffer used, implying PD was not a determinant of extraction. [H-3]-Palmitate extraction was much lower (p < 0.05) when gluconate was substituted for Cl- ion. This work contrasts with that for the extraction of [H-3]-alanine where hepatic extraction fraction was significantly reduced during depolarization. Changing the albumin concentration did not affect hepatocyte PD, and [H-3]-palmitate clearance into isolated hepatocytes was not affected by the buffers used. MID studies with vascular and extravascular references revealed that, with the gluconate substituted buffer, the extravascular volume possibly increased the diffusional path length thus explaining reduced [H-3]-palmitate extraction fraction in the IPL.

Development of a rapid biological assay for determination of potency of Newcastle disease vaccine (strain I-2)

A rapid biological assay based on incubation time has been developed for determination of the potency of Newcastle disease virus strain I-2 vaccine. It is based on the observation that the interval between inoculation and the first detection of haemagglutinin (HA) depends on the titre of the vaccine inoculated. Chicken embryonated eggs were inoculated with different titres (10(9), 10(6) and 10(3) EID50/0.1 ml) of vaccine and incubated for 24 h. At hourly intervals, 5 eggs from each vaccine titre were tested for the presence of HA. The results showed that the HA activity was detected from 5, 11 and 15 h after inoculation with vaccine doses of 10(9), 10(6) and 10(3) EID50, respectively. On the basis of these results it is suggested that if there is no HA detected from 5 to 11 h after inoculation of eggs with the vaccine virus, the vaccine should not be used to vaccinate chickens as it might have an infectivity titre of less than 10(6) EID50/0.1 ml, which is equivalent to the recommended single chicken dose. It is concluded that measuring the time between inoculation of the vaccine virus and the onset of HA activity might provide an estimate of the titre of the vaccine within 24 h.

EPIPOX:immunoinformatic characterization of the shared T-cell epitome between variola virus and related pathogenic orthopoxviruses

Concerns that variola viruses might be used as bioweapons have renewed the interest in developing new and safer smallpox vaccines. Variola virus genomes are now widely available, allowing computational characterization of the entire T-cell epitome and the use of such information to develop safe and yet effective vaccines. To this end, we identified 124 proteins shared between various species of pathogenic orthopoxviruses including variola minor and major, monkeypox, cowpox, and vaccinia viruses, and we targeted them for T-cell epitope prediction. We recognized 8,106, and 8,483 unique class I and class II MHC-restricted T-cell epitopes that are shared by all mentioned orthopoxviruses. Subsequently, we developed an immunological resource, EPIPOX, upon the predicted T-cell epitome. EPIPOX is freely available online and it has been designed to facilitate reverse vaccinology. Thus, EPIPOX includes key epitope-focused protein annotations: time point expression, presence of leader and transmembrane signals, and known location on outer membrane structures of the infective viruses. These features can be used to select specific T-cell epitopes suitable for experimental validation restricted by single MHC alleles, as combinations thereof, or by MHC supertypes.

Interactions of different arsenic species with thols: Chemical and biological implications

Arsenic has been classified as a group I carcinogen. It has been ranked number one in the CERCLA priority list of hazardous substances due to its frequency, toxicity and potential for human exposure. Paradoxically, arsenic has been employed as a successful chemotherapeutic agent for acute promyelocytic leukemia and has found some success in multiple myeloma. Since arsenic toxicity and efficacy is species dependent, a speciation method, based on the complementary use of reverse phase and cation exchange chromatography, was developed. Inductively coupled plasma mass spectrometer (ICP-MS), as an element specific detector, and electrospray ionization mass spectrometer (ESI-MS), as a molecule specific detector, were employed. Low detection limits in the µg. L−1 range on the ICP-MS and mg. L−1 range on the ESI-MS were obtained. The developed methods were validated against each other through the use of a Deming plot. With the developed speciation method, the effects of both pH on the stability of As species and reduced glutathione (GSH) concentration on the formation and stability of arsenic glutathione complexes were studied. To identify arsenicals in multiple myeloma (MM) cell lines post arsenic trioxide (ATO) and darinaparsin (DAR) incubation, an extraction method based on the use of ultrasonic probe was developed. Extraction tools and solvents were evaluated and the effect of GSH concentration on the quantitation of arsenic glutathione (As-GSH) complexes in MM cell extracts was studied. The developed method was employed for the identification of metabolites in DAR incubated cell lines where the effect of extraction pH, DAR incubation concentration and incubation time on the relative distribution of the As metabolites was assessed. A new arsenic species, dimethyarsinothioyl glutathione (DMMTA V-GS), a pentavalent thiolated arsenical, was identified in the cell extracts through the use of liquid chromatography tandem mass spectrometry. The formation of the new metabolite in the extracts was dependent on the decomposition of s-dimethylarsino glutathione (DMA(GS)). These results have major implications in both the medical and toxicological fields of As because they involve the metabolism of a chemotherapeutic agent and the role sulfur compounds play in this mechanism.

Ammonium excretion and respiration rate of tropical copepods and euphausiids measured during METEOR cruise M93, M97 and Maria S. Merian cruise MSM22

Respiration and ammonium excretion rates at different oxygen partial pressure were measured for calanoid copepods and euphausiids from the Eastern Tropical South Pacific and the Eastern Tropical North Atlantic. All specimens used for experiments were caught in the upper 400 m of the water column and only animals appearing unharmed and fit were used for experiments. Specimens were sorted, identified and transferred into aquaria with filtered, well-oxygenated seawater immediately after the catch and maintained for 1 to 13 hours prior to physiological experiments at the respective experimental temperature. Maintenance and physiological experiments were conducted in darkness in temperature-controlled incubators at 11, 13 or 23 degree C (±1). Before and during experiments, animals were not fed. Respiration and ammonium excretion rate measurements (both in µmol h-1 gDW-1) at varying oxygen concentrations were conducted in 12 to 60 mL gas-tight glass bottles. These were equipped with oxygen microsensors (ø 3 mm, PreSens Precision Sensing GmbH, Regensburg, Germany) attached to the inner wall of the bottles to monitor oxygen concentrations non-invasively. Read-out of oxygen concentrations was conducted using multi-channel fiber optic oxygen transmitters (Oxy-4 and Oxy-10 mini, PreSens Precision Sensing GmbH, Regensburg, Germany) that were connected via optical fibers to the outside of the bottles directly above the oxygen microsensor spots. Measurements were started at pre-adjusted oxygen and carbon dioxide levels. For this, seawater stocks with adjusted pO2 and pCO2 were prepared by equilibrating 3 to 4 L of filtered (0.2 µm filter Whatman GFF filter) and UV - sterilized (Aqua Cristal UV C 5 Watt, JBL GmbH & Co. KG, Neuhofen, Germany) water with premixed gases (certified gas mixtures from Air Liquide) for 4 hours at the respective experimental temperature. pCO2 levels were chosen to mimic the environmental pCO2 in the ETSP OMZ or the ETNA OMZ. Experimental runs were conducted with 11 to 15 trial incubations (1 or 2 animals per incubation bottle and three different treatment levels) and three animal-free control incubations (one per experimental treatment). During each run, experimental treatments comprised 100% air saturation as well as one reduced air saturation level with and without CO2. Oxygen concentrations in the incubation bottles were recorded every 5 min using the fiber-optic microsensor system and data recording for respiration rate determination was started immediately after all animals were transferred. Respiration rates were calculated from the slope of oxygen decrease over selected time intervals. Chosen time intervals were 20 to 105 min long. No respiration rate was calculated for the first 20 to 60 min after animal transfer to avoid the impact of enhanced activity of the animal or changes in the bottle water temperature during initial handling on the respiration rates and oxygen readings. Respiration rates were obtained over a maximum of 16 hours incubation time and slopes were linear at normoxia to mild hypoxia. Respiration rates in animal-free control bottles were used to correct for microbial activity. These rates were < 2% of animal respiration rates at normoxia. Samples for the measurement of ammonium concentrations were taken after 2 to 10 hours incubation time. Ammonium concentration was determined fluorimetrically (Holmes et al., 1999). Ammonium excretion was calculated as the concentration difference between incubation and animal-free control bottles. Some specimens died during the respiration and excretion rate measurements, as indicated by a cessation of respiration. No excretion rate measurements were conducted in this case, but the oxygen level at which the animal died was noted.

Impact of salinity and growth phase on alkenone distributions in coastal haptophytes

Batch cultures of Isochrysis galbana (strain CCMP 1323) and Chrysotila lamellosa (strain CCMP 1307) were grown at salinity ca. 10 to ca. 35 and the alkenone distributions determined for different growth phases. UK'37 values decreased slightly with salinity for C. lamellosa but were largely unaffected for I. galbana except during the decline phase. The values decreased with incubation time in both species. The proportion of C37:4, used as proxy for salinity, increased in both species at 0.16-0.20% per salinity unit, except during the stationary phase for I. galbana. C37:4 was much more abundant in C. lamellosa (30-44%) than in I. galbana (4-12%). Although our results suggest that salinity has a direct effect on alkenone distributions, growth phase and species composition will also have a marked impact, complicating the use of alkenone distributions as a proxy for salinity in the marine environment.

Investigation of escape response of Norwegian Pecten maximus

The ongoing process of ocean acidification already affects marine life and, according to the concept of oxygen- and capacity limitation of thermal tolerance (OCLTT), these effects may be exacerbated at the boarders of the thermal tolerance window. We studied the effects of elevated CO2 concentrations on clapping performance and energy metabolism of the commercially important scallop Pecten maximus. Individuals were exposed for at least 30 days to 4°C (winter) or to 10°C (spring/summer) at either ambient (0.04 kPa, normocapnia) or predicted future PCO2 levels (0.11 kPa, hypercapnia). Cold (4°C) exposed groups revealed thermal stress exacerbated by PCO2 indicated by a high mortality overall and its increase from 55% under normocapnia to 90% under hypercapnia. We therefore excluded the 4°C groups from further experimentation. Scallops at 10°C showed impaired clapping performance following hypercapnic exposure. Force production was significantly reduced although the number of claps was unchanged between normo- and hypercapnia exposed scallops. The difference between maximal and resting metabolic rate (aerobic scope) of the hypercapnic scallops was significantly reduced compared to normocapnic animals, indicating a reduction in net aerobic scope. Our data confirm that ocean acidification narrows the thermal tolerance range of scallops resulting in elevated vulnerability to temperature extremes and impairs the animal's performance capacity with potentially detrimental consequences for its fitness and survival in the ocean of tomorrow.

Characterisation of a secretory serine protease inhibitor (SjB6) from Schistosoma japonicum

BACKGROUND: Proteins belonging to the serine protease inhibitor (serpin) superfamily play essential physiological roles in many organisms. In pathogens, serpins are thought to have evolved specifically to limit host immune responses by interfering with the host immune-stimulatory signals. Serpins are less well characterised in parasitic helminths, although some are thought to be involved in mechanisms associated with host immune modulation. In this study, we cloned and partially characterised a secretory serpin from Schistosoma japonicum termed SjB6, these findings provide the basis for possible functional roles.

METHODS: SjB6 gene was identified through database mining of our previously published microarray data, cloned and detailed sequence and structural analysis and comparative modelling carried out using various bioinformatics and proteomics tools. Gene transcriptional profiling was determined by real-time PCR and the expression of native protein determined by immunoblotting. An immunological profile of the recombinant protein produced in insect cells was determined by ELISA.

RESULTS: SjB6 contains an open reading frame of 1160 base pairs that encodes a protein of 387 amino acid residues. Detailed sequence analysis, comparative modelling and structural-based alignment revealed that SjB6 contains the essential structural motifs and consensus secondary structures typical of inhibitory serpins. The presence of an N-terminal signal sequence indicated that SjB6 is a secretory protein. Real-time data indicated that SjB6 is expressed exclusively in the intra-mammalian stage of the parasite life cycle with its highest expression levels in the egg stage (p < 0.0001). The native protein is approximately 60 kDa in size and recombinant SjB6 (rSjB6) was recognised strongly by sera from rats experimentally infected with S. japonicum.

CONCLUSIONS: The significantly high expression of SjB6 in schistosome eggs, when compared to other life cycle stages, suggests a possible association with disease pathology, while the strong reactivity of sera from experimentally infected rats against rSjB6 suggests that native SjB6 is released into host tissue and induces an immune response. This study presents a comprehensive demonstration of sequence and structural-based analysis of a secretory serpin from a trematode and suggests SjB6 may be associated with important functional roles in S. japonicum, particularly in parasite modulation of the host microenvironment.