Natural immunogenic properties of bioinformatically predicted linear B-cell epitopes of dengue envelope and pre-membrane proteins.

BACKGROUND: The natural antibody responses to B-cell epitopes from dengue structural proteins were assessed using immune sera from people having well-defined past dengue infections with one of the four serotypes. METHOD: Based on an immune-computational analysis previously conducted, nineteen epitopes from the envelope (E) and eight epitopes from pre-membrane (prM), which were more than 50% conserved across all the four DENV serotypes, were selected. Peptides to represent these B-cell epitopes were obtained from commercially available arrays, and were subjected to enzyme linked immunosorbent assay with sera obtained from dengue seropositive healthy volunteers (DENV1 n = 12: DENV2 n = 12: DENV3 n = 12 and DENV4 n = 12), and 10 dengue seronegative healthy volunteers from Sri Lanka. The cut-off value for the positive antibody response was set by taking the mean response of a peptide to the negative sera plus three standard deviations. The peptides (N = 7) showing the broad immune responses were used to generate antibodies in three mice (Balb/c) batches. The mice antisera were then subjected to microneutralization assays against all the four DENV serotypes. An EC50 viral neutralization ≥ 40 times the serum dilution was considered as neutralizing. RESULTS: Five of the E-peptide and two prM peptides were recognised by most individuls exposed to infections with each of the four serotypes, showing a serotype cross-reactive broad antibody response. The mice immune sera against the peptides representing the five E protein epitopes neutralized all the four DENV serotypes. Two of these five epitopes are from the Domain II, whereas one of them includes the whole bc-loop region. CONCLUSION: The antibody responses of highly conserved epitopes across the serotypes, were broadly responsive with sera of all four DENV serotypes collected from individuals infected with only one DENV serotype. Weakly conserved epitopes showed rather specific antibody responses dominated by one or few serotypes.

Further Investigation into the Biochemical Effects of Phosphorylation of Tropomyosin Tpm1.1(α). Serine-283 Is in Communication with the Midregion.

The phosphorylated and unphosphorylated forms of tropomyosin Tpm1.1(α) are prepared from adult rabbit heart and compared biochemically. Electrophoresis confirms the high level of enrichment of the chromatography fractions and is consistent with a single site of phosphorylation. Covalently bound phosphate groups at position 283 of Tpm1.1(α) increase the rate of digestion at Leu-169, suggestive of a conformational rearrangement that extends to the midregion. Such a rearrangement, which is supported by ellipticity measurements between 25 and 42 °C, is consistent with a phosphorylation-mediated tightening of the interaction between various myofilament components. In a nonradioactive, co-sedimentation assay [30 mM KCl, 1 mM Mg(II), and 4 °C], phosphorylated Tpm1.1(α) displays a higher affinity for F-actin compared to that of the unphosphorylated control (Kd, 0.16 µM vs 0.26 µM). Phosphorylation decreases the concentration of thin filaments (pCa 4 plus ATP) required to attain a half-maximal rate of release of product from a pre-power stroke complex [myosin-S1-2-deoxy-3-O-(N-methylanthraniloyl)ADP-Pi], as investigated by double-mixing stopped-flow fluorescence, suggestive of a change in the proportion of active (turned on) and inactive (turned off) conformers, but similar maximum rates of product release are observed with either type of reconstituted thin filament. Phosphorylated thin filaments (pCa 4 and 8) display a higher affinity for myosin-S1(ADP) versus the control scenario without affecting isotherm steepness. Specific activities of ATP and Tpm1.1(α) are determined during an in vitro incubation of rat cardiac tissue [12 day-old, 50% phosphorylated Tpm1.1(α)] with [32P]orthophosphate. The incorporation of an isotope into tropomyosin lags behind that of ATP by a factor of approximately 10, indicating that transfer is a comparatively slow process.

Medicinal plants commonly used against cancer in traditional medicine formulae in Sri Lanka.

Cancer is a global burden. In low- and middle-income countries around 70% of deaths are due to cancer. For a number of years natural products have been a good source of agents for combatting cancer and plants have played a huge role in anti-cancer product development. For many centuries, indigenous cultures around the world have used traditional herbal medicine to treat a myriad of diseases including cancer. In Sri Lanka, a number of plants have been reported to have anti-cancer properties and some of the commonly used plants are described in this review with an account of their compounds and modes of action. Only a small number of the plants in Sri Lanka have been tested for their bioactivity and more research is required to determine their medicinal activity with the aim of developing novel drugs to fight this disease.

Natural antibody responses to the capsid protein in sera of Dengue infected patients from Sri Lanka.

This study aims to characterize the antigenicity of the Capsid (C) protein and the human antibody responses to C protein from the four dengue virus (DENV) serotypes. Parker hydrophilicity prediction, Emini surface accessibility prediction and Karplus & Schulz flexibility predictions were used to bioinformatically characterize antigenicity. The human antibody response to C protein was assessed by ELISA using immune sera and an array of overlapping DENV2 C peptides. DENV2 C protein peptides P1 (located on C protein at 2-18 a.a), P11 (79-95 a.a) and P12 (86-101 a.a) were recognized by most individuals exposed to infections with only one of the 4 DENV serotypes as well as people exposed to infections with two serotypes. These conserved peptide epitopes are located on the amino (1-40 a.a) and carboxy (70-100 a.a) terminal regions of C protein, which were predicted to be antigenic using different bioinformatic tools. DENV2 C peptide P6 (39-56 a.a) was recognized by all individuals exposed to DENV2 infections, some individuals exposed to DENV4 infections and none of the individuals exposed to DENV1 or 3 infections. Thus, unlike C peptides P1, P11 and P12, which contain epitopes, recognized by DENV serotype cross-reactive antibodies, DENV2 peptide P6 contains an epitope that is preferentially recognized by antibodies in people exposed to this serotype compared to other serotypes. We discuss our results in the context of the known structure of C protein and recent work on the human B-cell response to DENV infection.

Conformational properties of striated muscle tropomyosins from some salmonid fishes.

The conformational stability of tropomyosins from salmonids fishes has been investigated under a variety of conditions (salt, pH and osmolyte) using electronic circular dichroism. Every salmonid tropomyosin (from: fast skeletal muscle; slow skeletal muscle and heart) is less resistant to heat-induced denaturation than rabbit alpha-striated tropomyosin. Induction of unfolding, by application of a linear temperature gradient, yields a distinct profile for each protein (0.1 M salt, pH 7, plus dithiothreitol): fast tropomyosin (Tms 24 and 40 degrees C major); cardiac tropomyosin (Tm, 36 degrees C) and slow tropomyosin (Tms, 39 major and 47 degrees C). Correlation of these results, and others obtained under different solvent conditions, with the known sequences (Jackman DM, Waddleton DM, Younghusband B, Heeley DH (1996) Further characterisation of fast, slow and cardiac muscle tropomyosins from salmonid fish. Eur. J. Biochem. 242:363-371) provides insight into how the coiled-coil may have adapted to cold. The most variable sections of sequence encompass residues 9-49, 73-87 and 172-216. In two of these regions there is a pair of closely-spaced glycines, namely at residues 24 and 27 in fast skeletal tropomyosin and residues 83 and 87 in cardiac tropomyosin. A region of low stability is located in the carboxy-terminal half of the isoform from fast skeletal muscle. This segment cooperatively unfolds in the 20 degrees range and accounts for 20% of the total far-UV ellipticity change under reducing conditions. It is unresponsive to increasing ionic strength and the presence of osmolyte but is sensitive to oxidation at cysteine 190. A likely contributor to the relative instability is a substitution at position 179 whereby fast skeletal tropomyosin, but not the other tropomyosins under examination, has lost a "d" position alanine in the fifth cluster and gained a polar side-chain.

Some binding properties of Omp T digested muscle tropomyosin.

Cleavage of vertebrate muscle tropomyosin by bacterial Omp T produces an amino-terminally truncated product (residues 7-284). The proteolysed protein, which is resolved from the parent by electrophoresis in the presence of sodium dodecylsulphate, can be generated from a variety of striated and smooth muscle tropomyosins, including ones from mammal, bird and fish. Edman-based sequencing and mass analysis confirm that the main site of chain hydrolysis is the peptide bond between Lys 6 and Lys 7. Loss of the hexapeptide, together with the blocking group, from tropomyosin weakens its affinity for troponin. Compared to wild type, the shortened forms of rabbit skeletal tropomyosin and Atlantic salmon fast skeletal tropomyosin, as well as the unacetylated (full-length) version of the latter, all display reduced affinity for both troponin and the amino-terminal fragment of troponin-T (residues 1-158), as judged by affinity chromatography. This is consistent with the view that the amino terminal region is required for full interaction with troponin-T. Truncated tropomyosin fails to bind to F-actin at micromolar concentration, as expected. Interestingly, binding is restored by troponin in the presence of either added Ca(2+) or EGTA. Digestion of muscle tropomyosin by Omp T, which can be carried out on quantitative amounts of protein, is concluded to yield a product that has useful biochemical applications.