Soluble Preparations Research Articles

Comparison of IgG4 assays using whole parasite extract and BmR1 recombinant antigen in determining antibody prevalence in brugian filariasis

BackgroundBrugia malayi is endemic in several Asian countries with the highest prevalence in Indonesia. Determination of prevalence of lymphatic filariasis by serology has been performed by various investigators using different kinds of antigen (either soluble worm antigen preparations or recombinant antigens). This investigation compared the data obtained from IgG4 assays using two different kinds of antigen in a study on prevalence of antibodies to B. malayi.MethodsSerum samples from a transmigrant population and life long residents previously tested with IgG4 assay using soluble worm antigen (SWA-ELISA), were retested with an IgG4 assay that employs BmR1 recombinant antigen (BmR1 dipstick [Brugia Rapid™]). The results obtained with the two antigens were compared, using Pearson chi-square and McNemar test.ResultsThere were similarities and differences in the results obtained using the two kinds of antigen (SWA and BmR1). Similarities included the observation that assays using both antigens demonstrated an increasing prevalence of IgG4 antibodies in the transmigrant population with increasing exposure to the infection, and by six years living in the area, antibody prevalence was similar to that of life-long residents. With regards to differences, of significance is the demonstration of similar antibody prevalence in adults and children by BmR1 dipstick whereas by SWA-ELISA the antibody prevalence in adults was higher than in children.ConclusionsResults and conclusions made from investigations of prevalence of anti-filarial IgG4 antibody in a population would be affected by the assay employed in the study.

Natural Bioactive Material: A Preparation of Soluble Eggshell Membrane Protein

AbstractThe proteins of eggshell membranes, an industrial waste product, are dissolved by reductive cleavage with aqueous 3‐mercaptopropionic acid and acetic acid. The soluble protein preparation is cast into a thin film, and its bioactivity is investigated by cell culture.Photomicrograph of NIH3T3 fibroblasts cultured on SEP film for 3 days.magnified imagePhotomicrograph of NIH3T3 fibroblasts cultured on SEP film for 3 days.

ANTIBODIES TO SCHISTOSOMA MANSONI IN HUMAN CEREBROSPINAL FLUID

Cerebrospinal fluid (CSF) and serum samples from patients suspected of having neuroschistosomiasis (NS) were evaluated by an enzyme-linked immunosorbent assay. Monoclonal antibodies of various immunoglobulin isotypes (IgM, IgA, IgE, total IgG, IgG1, IgG2, IgG3, and IgG4) were used to detect antibodies against Schistosoma mansoni soluble egg antigen (SEA) and soluble worm adult preparation (SWAP). Of the 83 CSF samples tested, 55% were reactive to SEA (26% were reactive only to SEA and 29% to both SEA and SWAP), 34% were reactive to SWAP (5% only to SWAP and 29% to both SEA and SWAP), and 40% were not reactive with any antigen. Cases that tested positive for SWAP in CSF and negative in serum were not found. Samples with high specific IgG antibody titers were selected for immunoglobulin isotype profiling. In the CSF samples, the antibodies against SEA and SWAP were mainly IgM, IgG1, and IgG4, although other immunoglobulins were also detected. Interestingly, nine patients had high levels of IgG1 only in the CSF. These results suggest that there is local synthesis of IgG1, and that this isotype could be an important immunologic marker in the diagnosis of NS.

Comparative study of hemicelluloses released during two-stage treatments with acidic organosolv and alkaline peroxide from Caligonum monogoliacum and Tamarix spp

Two-stage treatments of Caligonum monogoliacum and Tamarix spp. with acidic organosolv and alkaline peroxide were performed. Pre-treatment with ethanol–H 2O (60/40, v/v) under acid catalyst (0.2 N HCl) at 70 °C for 4 h released 18.7 and 17.8% hemicelluloses from dewaxed C. monogoliacum and Tamarix spp., respectively, Sequential treatment with 2% H 2O 2 at pH 11.5 for 16 h at 45 °C solubilized 26.2 and 26.5% hemicelluloses from pre-treated C. monogoliacum and Tamarix spp., respectively. Cellulose predominated in the insoluble residues, accounting for 36.7–37.8% of dry materials. It was found that the two alkaline peroxide soluble hemicellulosic preparations contained a much higher amount of xylose (74.1–77.3%) but lower in glucose (7.4–18.6%), galactose (3.0–8.3%), rhamnose (2.3–2.9%), mannose (0.8–2.7%), and arabinose (1.3–1.4%) than those of the two acidic organosolv soluble hemicellulosic fractions in which xylose (31.8–50.8%), glucose (21.4–30.3%), galactose (7.6–24.1%), and rhamnose (5.7–10.1%) were the major sugar components. The content of uronic acids was slightly higher in the two alkaline peroxide soluble hemicellulosic preparations (7.5–8.3%) than the two acidic organosolv soluble hemicellulosic fractions (5.0–6.5%). Furthermore, the studies showed that the two alkaline peroxide soluble hemicellulosic preparations were more linear and acidic, and had a large molecular mass ( M ̄ w, 27,220–31,410 g mol −1) than the two acidic organosolv soluble hemicellulosic fractions ( M ̄ w, 13,820–18,380 g mol −1). Lignin content and its composition associated in the four isolated hemicelluloses were determined by alkaline nitrobenzene oxidation and monitored with HPLC. No significant differences in lignin content (5.94–9.82%) and its composition were found. Further comparative study of the four hemicellulosic preparations and two cellulosic fractions was carried by both degraded methods such as acid hydrolysis and thermal analysis and non-degradation techniques such as FT-IR and 13C-NMR spectroscopy.

Immune responses of elk to Mycobacterium bovis bacillus Calmette Guerin vaccination

Although rare, detection of Mycobacterium bovis infection of captive or free-ranging elk ( Cervus elaphus) elicits serious concern due to regulatory and zoonotic implications. Few studies, however, have evaluated the immune response of elk to M. bovis or other pathogens. To model natural infection, elk were vaccinated with live M. bovis bacillus Calmette Guerin (BCG, Pasteur strain) for evaluation of immune responsiveness to this attenuated live vaccine. Peripheral blood mononuclear cells (PBMC) of vaccinated elk proliferated in response to stimulation with a soluble mycobacterial antigen preparation (i.e. M. bovis purified protein derivative, PPDb). Greater numbers of sIgM + cells (i.e. B cells) proliferated in this response than did either CD4 +, γδTCR + or CD8 + cells. The in vivo response (i.e. delayed type hypersensitivity, DTH) to PPDb by vaccinated elk exceeded both the response by non-vaccinated elk and BCG-vaccinated cattle at 24, 48, and 72 h post-administration of PPD. In vivo responses to PPDb by vaccinated elk diminished after 72 h as compared to responses at 24 and 48 h. Serum was also collected periodically and evaluated by ELISA for immunoglobulin (i.e. IgG heavy and light chains) reactivity to crude mycobacterial antigens. Two weeks post-vaccination and throughout the duration of the study, serum immunoglobulin reactivity to PPDb and to a proteinase K-digested whole cell sonicate of BCG exceeded that of serum from non-vaccinated elk. Intradermal administration of PPD for measurement of hypersensitive responses boosted the serum antibody response. These findings demonstrate that BCG vaccination of elk induces a serum antibody response to crude M. bovis antigens, a B cell in vitro proliferative response, and in vivo trafficking of mononuclear cells to sites of mycobacterial antigen administration (i.e. delayed type hypersensitivity). A predominant B cell in vitro proliferative response by elk PBMC to crude mycobacterial test antigens will likely impact the development of improved diagnostic tests of tuberculosis infection for this species.

Immune correlate study on human Schistosoma japonicum in a well-defined population in Leyte, Philippines: II. Cellular immune responses to S. japonicum recombinant and native antigens

Cellular immune responses to specific Schistosoma japonicum recombinant and native antigens were investigated in a defined study population of 155 individuals in the Philippines, where data collected from a 3-year observation of exposure, infection and reinfection pattern were used to categorically classify putative ‘resistant’ and ‘susceptible’ individuals. Using peripheral blood mononuclear cells (PBMC) of individuals enrolled in the study, in vitro lymphocyte proliferation and cytokine (IFN-γ, IL-5 and IL-10) production in response to defined recombinant antigens (97 kDa paramyosin, 22 kDa tegumental antigen, 37 kDa glyceraldehyde-3-phosphate dehydrogenase, 14 kDa fatty acid binding protein and 28 kDa gluthathione- S-tranferase) and native antigen soluble worm antigen preparation (SWAP) were measured. Cellular responses to the recombinant and SWAP antigens suggest that Th1 type of response appear to be important in predicting resistance in this population. Of the five recombinant antigens tested, rPMY induced significant levels of IFN-γ. The production of IL-10, a Th2-type cytokine was strongly implicated in immune regulation. Of importance was the evidence found for SWAP and rPMY induced IFN-γ responses in predicting ‘resistance’. It was noted that these associations were significant even after the effect of age and sex were accounted for in a multivariate analysis.

Losses of vitamin A and E in parenteral nutrition suitable for premature infants.

To assess the bioavailability of vitamins A and E administered parenterally with either water-soluble or lipid-soluble preparations. A water soluble preparation (MVI Pediatric) administered with a glucose-amino acid solution and a lipid soluble preparation (Vitalipid N Infant) infused with a lipid emulsion were subjected to phototherapy light, different flow rates, light protection, different tubing materials and tubing sizes, and concentrations in the effluents were determined. Recovery of retinol in glucose-amino acid solution was poor under all conditions (16-30% without; 21-42% with light protection tubing) and increased to 61% with polyethylene and to 44% with polyurethane tubings. Polyurethane tubings with reduced volume improved retinol delivery to 56%. Retinylpalmitate (Vitalipid) losses were low, with recovery of 86 and 77% with and without light protection, respectively. Recoveries of alpha-tocopherylacetate in GLUC-AA were 103-107% without and 94-102% with light protection and of alpha-tocopherol in LIPID 89% without and 85% with light protection. Parenteral vitamin A delivery is improved by the infusion of retinylpalmitate with lipids. Light protecting tubings provide only a marginal benefit with artificial light and none with phototherapy light. Polyethylene and polyvinylchloride tubings adsorb less retinol than polyurethane tubings. Small tubing diameters resulting in higher flow rates enhance retinol delivery.

Down-regulation of specific antigen-driven cytokine production in a population with endemic Schistosoma japonicum infection.

Schistosome antigen-driven cytokine responses and antischistosome antibody levels of residents of a Schistosoma japonicum endemic island in Poyang Lake, Jiangxi Province were studied before and 45 days after treatment with praziquantel. IL-4, IL-5, IL-10 and INF-gamma were all detected in the supernatants of whole-blood cultures after stimulation with schistosome soluble egg antigen (SEA) and soluble worm antigen preparation (SWAP). The percentages of subjects producing detectable amounts of each cytokine assayed were higher in the group who were negative by stool examination at the start of the study than in those who were initially stool positive. After praziquantel treatment the percentages of subjects producing both type I and type II cytokines increased. This suggests that the production of both types of cytokine was down-regulated in the presence of live, egg-laying S. japonicum adult worms but that this was reversible by treatment. In contrast, the antibody studies showed higher levels of SWAP and SEA-specific antibodies (IgE, total IgG, IgG4, IgM) in subjects who were originally stool-positive than in those who were stool-negative. After treatment specific IgE responses were elevated, but total IgG and IgG4 anti-SEA and IgM anti-SWAP antibody levels all fell significantly.

Infection with Schistosoma mansoni correlates with altered immune responses to Ascaris lumbricoides and hookworm

Studies were performed on humoral and cellular immune responses of patients from areas in Brazil endemic for hookworm and Ascaris lumbricoides, and either endemic or non-endemic for Schistosoma mansoni. Humoral and cellular responses were evaluated by enzyme-linked immunosorbant assay (ELISA) and peripheral blood mononuclear cell (PBMC) proliferation assays against larval hookworm antigens, A. lumbricoides egg antigens, and soluble egg antigens (SEA) or soluble whole adult antigenic preparation (SWAP) from S. mansoni. Patients from S. mansoni-endemic areas, who currently had only hookworm or Ascaris infections, expressed lower humoral and cellular responses to hookworm or Ascaris antigens, respectively, than did their counterparts from areas not endemic for S. mansoni. Individuals from S. mansoni endemic area, although without detectable S. mansoni infection, do mount humoral and cellular responses to SEA and SWAP. This group of individuals has been probably in contact with S. mansoni antigens, since the groups harboring A. lumbricoides or hookworm infections from non- S. mansoni endemic areas do not have detectable anti- S. mansoni responses. PBMC proliferative responses discriminated well between patients with active hookworm infections versus ascariasis, if they were from areas not endemic for S. mansoni.

Inhibitory Potency and Specificity of Subtilase-like Pro-protein Convertase (SPC) Prodomains

The SPCs (subtilisin-like pro-protein convertases) are a family of enzymes responsible for the proteolytic processing of numerous precursor proteins of the constitutive and regulated secretory pathways. SPCs are themselves synthesized as inactive zymogens. Activation of SPCs occurs via the intramolecular autocatalytic removal of the prodomain. SPC prodomains have been proposed as templates in the development of potent and specific SPC inhibitors. In this study, we investigated the specificity and potency of complete prodomains and short C-terminal prodomain peptides of each SPC on highly purified, soluble enzyme preparations of human SPC1, SPC6, and SPC7. Progress curve kinetic analysis of prodomain peptides and complete prodomains showed competitive inhibitory profiles in the low nanomolar range. Complete prodomains were 5-100 times more potent than C-terminal prodomain peptides, suggesting that N-terminal determinants are involved in the recognition process. However, complete prodomains and prodomain peptides exhibit only a partial specificity toward their cognate enzyme. Ala-scan structure activity studies indicated the importance of basic residues in the P(4), P(5), and P(6) positions for inhibition of SPC1. In contrast, hydrophobic residues in P(6) and P(7), as well as basic residues in P(4) and P(5), were critical for inhibition of SPC7. Our data demonstrated that the use of prodomains as specific inhibitors acting in trans would be of limited usefulness, unless modified into more specific compounds.

Phentermine inhibition of recombinant human liver monoamine oxidases A and B

Recent studies with rat tissue preparations have suggested that the anorectic drug phentermine inhibits serotonin degradation by inhibition of monoamine oxidase (MAO) A with a K I value of 85–88 μM, a potency suggested to be similar to that of other reversible MAO inhibitors (Ulus et al., Biochem Pharmacol 2000;59:1611–21). Since there are known differences between rats and humans in substrate and inhibitor specificities of MAOs, the interactions of phentermine with recombinant human purified preparations of MAO A and MAO B were determined. Human MAO A was competitively inhibited by phentermine with a K I value of 498±60 μM, a value approximately 6-fold weaker than that observed for the rat enzyme. Phentermine was also observed to be a competitive inhibitor of recombinant human liver MAO B with a K I value of 375±42 μM, a value similar to that observed with the rat enzyme (310–416 μM). In contrast to the behavior with rat tissue preparations, no slow time-dependent behavior was observed for phentermine inhibition of purified soluble human MAO preparations. Difference absorption spectral studies showed similar perturbations of the covalent FAD moieties of both human MAO A and MAO B, which suggests a similar mode of binding in both enzymes. These data suggest that phentermine inhibition of human MAO A (or of MAO B) is too weak to be of pharmacological relevance.

PH-dependent Ca2+ binding to the F0 c-subunit affects proton translocation of the ATP synthase from Synechocystis 6803.

It was shown before (Wooten, D. C., and Dilley, R. A. (1993) J. Bioenerg. Biomembr. 25, 557-567; Zakharov, S. D., Li, X., Red'ko, T. P., and Dilley, R. A. (1996) J. Bioenerg. Biomembr. 28, 483-493) that pH dependent reversible Ca2+ binding near the N- and C-terminal end of the 8 kDa subunit c modulates ATP synthesis driven by an applied pH jump in chloroplast and E. coli ATP synthase due to closing a "proton gate" proposed to exist in the F0 H+ channel of the F0F1 ATP synthase. This mechanism has further been investigated with the use of membrane vesicles from mutants of the cyanobacterium Synechocystis 6803. Vesicles from a mutant with serine at position 37 in the hydrophilic loop of the c-subunit replaced by the charged glutamic acid (strain plc 37) has a higher H+/ATP ratio than the wild type and therefore shows ATP synthesis at low values of deltamuH+. The presence of 1 mM CaCl2 during the preparation and storage of these vesicles blocked acid-base jump ATP formation when the pH of the acid side (inside) was between pH 5.6 and 7.1, even though the deltapH of the acid-base jump was thermodynamically in excess of the necessary energy to drive ATP formation at an external pH above 8.28. That is, in the absence of added CaCl2, ATP formation did occur under those conditions. However, when the base stage pH was 7.16 and the acid stage below pH 5.2, ATP was formed when Ca2+ was present. This is consistent with Ca2+ being displaced by H+ ions from the F0 on the inside of the thylakoid membrane at pH values below about 5.5. Vesicles from a mutant with the serine of position 3 replaced by a cysteine apparently already contain some bound Ca2+ to F0. Addition of 1 mM EGTA during preparation and storage of those vesicles shifted the otherwise already low internal pH needed for onset of ATP synthesis to higher values when the external pH was above 8. With both strains it was shown that the Ca2+ binding effect on acid-base induced ATP synthesis occurs above an internal pH of about 5.5. These results were corroborated by 45Ca2+-ligand blot assays on organic solvent soluble preparations containing the 8 kDa F0 subunit c from the S-3-C mutant ATP synthase, which showed 5Ca2+ binding as occurs with the pea chloroplast subunit III. The phosphorylation efficiency (P/2e), at strong light intensity, of Ca2+ and EGTA treated vesicles from both strains were almost equal showing that Ca2+ or EGTA have no other effect on the ATP synthase such as a change in the proton to ATP ratio. The results indicate that the Ca2+ binding to the F0 H+ channel can block H+ flux through the channel at pH values above about 5.5, but below that pH protons apparently displace the bound Ca2+, opening the CF0 H+ channel between the thylakoid lumen and H+ conductive channel.

Detection and characterization of the S. typhimurium HilA protein

BackgroundVirulence genes on Salmonella pathogenicity island 1 (SPI1) are coordinately regulated by HilA, a member of the OmpR/ToxR family of transcription factors. Although a great deal is known about the complex regulation of hilA gene expression, very little is known about the HilA protein.ResultsIn order to detect and localize the HilA protein in S. typhimurium, we raised polyclonal antiserum against purified His-tagged HilA. This allowed us to study the effect of environmental conditions on the production of HilA. We also used the antiserum to examine the fractionation properties and SDS-PAGE mobility of native HilA. Our results indicate that S. typhimurium initiates translation of HilA from the first AUG codon in the hilA open-reading frame (ORF), producing a soluble 553 amino acid (63 kDa) protein product.ConclusionMaterials and methods are now available to study the environmental regulation of the HilA protein in S. typhimurium. Our results also indicate that future in vitro studies of the interaction between HilA and DNA should utilize soluble preparations of HilA. Previous analyses used preparations of HilA in which the protein fractionated with the membrane, greatly limiting the types of experiments that could be conducted.

Signal transduction events in human peripheral blood mononuclear cells stimulated by schistosoma mansoni antigens

Activation of protein tyrosine kinases (PTKs) is a common step of T cell stimulation. However, the relationship between PTKs and activation of peripheral blood mononuclear cells (PBMC) from intestinal chronic schistosomiasis patients has not been explored yet. In this study, we investigated the participation of Lck and ZAP-70 protein tyrosine kinases (PTKs), as well as PLC-γ1 and Shc proteins in PBMC activation by Shistosoma mansoni antigens. PBMC were stimulated with SEA (soluble egg antigen) or SWAP (soluble worm preparation), lysed, precipitated with specific antibodies and the level of tyrosine phosphorylation evaluated. Our results show that Lck and Shc were phosphorylated upon stimulation of the cells with SWAP, as well as with SEA. However, the phosphorylation level was more pronounced in SWAP than in SEA-stimulated cells. Phosphorylation of ZAP-70 was observed only in SWAP stimulated cells. Additionally, PLC-γ1 phosphorylation was not observed in PBMC stimulated with SEA. Together, these results indicate that SEA and SWAP induce PBMC proliferation through distinct intracellular signaling pathways. Moreover, the weaker response of PBMC to SEA compared to SWAP stimulation suggests down-regulation of cells from intestinal chronic schistosomiasis patients to SEA, which may occur during immunomodulation to S. mansoni response.

Stabilization of pancreatic ribonuclease A by immobilization on Sepharose-linked antibodies that recognize the labile region of the enzyme

The stabilizing potential of the antibodies recognizing the labile region of pancreatic ribonuclease A (RNase) has been investigated. The dodecapeptide SRNLTKDRAKPV corresponding to the labile region 32–43 on RNase was synthesized by the solid-phase method. Antiserum raised against the dodecapeptide-bovine serum albumin conjugate showed good cross-reactivity with the peptide and native RNase. RNase immobilized on Sepharose support precoupled either with the antipeptide immunoglobulin (IgG) or anti-RNase IgG proved to be more resistant to thermal inactivation than the soluble enzyme. Besides, stability against inactivation by trypsin at 55°C was markedly high when enzyme was immobilized on the antipeptide IgG support, as compared to the soluble and other immobilized preparations. These results suggest that matrices bearing antibodies recognizing specific labile regions of enzyme may be useful in selectively improving their stability against specific forms of inactivation.

Characterization of murine immunoglobulin G antibodies against human amyloid-β 1–42

It has been demonstrated that immunization of transgenic mouse models of Alzheimer's disease (AD) with amyloid-β 1–42 peptide (Aβ 1–42) results in amelioration of AD-like pathology, including reduced soluble and deposited β-amyloid and decreased cognitive impairment. Based on the proposed importance of immunoglobulin G (IgG) anti-Aβ antibodies (Abs) in these effects, we sought to characterize these Abs in splenocytes from mice immunized with Aβ 1–42. Data show that a more aggregated preparation of Aβ 1–42 gives a robust IgG anti-Aβ Ab response, while these Abs are almost undetectable when a less aggregated preparation of Aβ 1–42 is used as the immunogen. Importantly, IgG anti-Aβ Ab production is detected after just 12 weeks of Aβ 1–42 treatment. Analysis of anti-Aβ Ab IgG isotypes reveals that the majority of these Abs are IgG1, with significantly fewer Abs of the IgG2a or IgG2b isotypes (IgG1>IgG2a>IgG2b), suggesting a T lymphocyte helper type II response after Aβ 1–42 immunization. To determine the epitope of Aβ recognized by IgG anti-Aβ Abs, intact Aβ and Aβ peptide fragments were analyzed for their ability to bind these Abs. Data show that these Abs specifically recognize an amino-terminal epitope of Aβ between amino acids one and twelve, with higher affinity for a more soluble preparation of Aβ 1–42. These data further indicate the immunogenic potential of Aβ 1–42 and offer insight into the nature of the IgG anti-Aβ Ab response.

Linker DNA destabilizes condensed chromatin

The contribution of the linker region to maintenance of condensed chromatin was examined in two model systems, namely sea urchin sperm nuclei and chicken red blood cell nuclei. Linkerless nuclei, prepared by extensive digestion with micrococcal nuclease, were compared with Native nuclei using several assays, including microscopic appearance, nuclear turbidity, salt stability, and trypsin resistance. Chromatin in the Linkerless nuclei was highly condensed, resembling pyknotic chromatin in apoptotic cells. Linkerless nuclei were more stable in low ionic strength buffers and more resistant to trypsin than Native nuclei. Analysis of histones from the trypsinized nuclei by polyacrylamide gel electrophoresis showed that specific histone H1, H2B, and H3 tail regions stabilized linker DNA in condensed nuclei. Thermal denaturation of soluble chromatin preparations from differentially trypsinized sperm nuclei demonstrated that the N-terminal regions of histones Sp H1, Sp H2B, and H3 bind tightly to linker DNA, causing it to denature at a high temperature. We conclude that linker DNA exerts a disruptive force on condensed chromatin structure which is counteracted by binding of specific histone tail regions to the linker DNA. The inherent instability of the linker region may be significant in all eukaryotic chromatins and may promote gene activation in living cells.Key words: chromatin condensation, sea urchin sperm, chicken red blood cell, nuclei, linker DNA, histone variants, micrococcal nuclease, nucleosome, trypsin, gel electrophoresis.

A strategy for optimizing the monodispersity of fusion proteins: application to purification of recombinant HPV E6 oncoprotein.

Recombinant production of HPV oncoprotein E6 is notoriously difficult. The unfused sequence is produced in inclusion bodies. By contrast, fusions of E6 to the C-terminus of carrier proteins such as maltose-binding protein or glutathione-S-transferase are produced soluble. However, it has not yet been possible to purify E6 protein from such fusion constructs. Here, we show that this was due to the biophysical heterogeneity of the fusion preparations. We find that soluble MBP-E6 preparations contain two subpopulations. A major fraction is aggregated and contains exclusively misfolded E6 moieties ('soluble inclusion bodies'). A minor fraction is monodisperse and contains the properly folded E6 moieties. Using monodispersity as a screening criterion, we optimized the expression conditions, the purification process and the sequence of E6, finally obtaining stable monodisperse MBP-E6 preparations. In contrast to aggregated MBP-E6, these preparations yielded fully soluble E6 after proteolytic removal of MBP. Once purified, these E6 proteins are stable, folded and biologically active. The first biophysical measurements on pure E6 were performed. This work shows that solubility is not a sufficient criterion to check that the passenger protein in a fusion construct is properly folded and active. By contrast, monodispersity appears as a better quality criterion. The monodispersity-based strategy presented here constitutes a general method to prepare fusion proteins with optimized folding and biological activity.

Aspirin: past, present and future

Many folk remedies used since prehistoric times have depended upon salicylates for their effect. One hundred years ago aspirin was formulated from salicylic and acetic acids. It was the first drug to be synthesised and its formulation is regarded as the foundation of the modern pharmaceutical industry.The benefit of low-dose aspirin as a prophylactic after a thrombotic event was first reported 25 years ago. Its use after coronary or cerebral thrombosis is virtually mandatory, unless there are signs of intolerance. A ‘loading dose' of soluble aspirin should be given on first contact with a patient who may be suffering from myocardial infarction. Patients considered to be at increased risk of a vascular event should also be advised to carry their own aspirin and, if they experience sudden severe chest pain, to chew and swallow a 300mg tablet or a soluble preparation immediately.The current phase of the aspirin story is, however, not over, and its possible value in a variety of conditions, including dementia and certain cancers, seems likely to ensure that it will long continue to play a remarkable part in clinical practice.

The schistosome egg: development and secretions.

We have investigated the development of the schistosome egg and its secretions in order to understand how it migrates through gut tissues and also initiates pathology in the liver. We show by electron microscopy that the subshell envelope is absent in the newly deposited egg, but appears very early and differentiates as development progresses. In the mature egg, this nucleated envelope contains extensive endoplasmic reticulum, suggestive of a protein synthetic capacity. Furthermore, Reynolds' layer only appears between the envelope and the egg-shell in the mature egg and may represent its accumulated secretions. We have biosynthetically labelled and collected the secretions (ESP) released by mature but not immature eggs during culture. Their fractionation by SDS-PAGE reveals a simple pattern of 6 bands, differing markedly in composition from soluble egg antigen preparations. Electrophoresis in casein substrate gels demonstrates the presence of 2 distinct proteases in the egg secretions. By immunocytochemistry, ESP localized predominantly to the envelope of the mature egg, suggesting that this layer rather than the miracidium is the source of egg secretions.