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.

Threonine-77 Is a Determinant of the Low-Temperature Conditioning of the Most Abundant Isoform of Tropomyosin in Atlantic Salmon.

The Atlantic salmon Salmo salar survives below 10 °C. The main skeletal muscle is composed of a single isoform of tropomyosin (classified as Tpm1 α-fast) that is >92% identical to the mammalian homologue. How salmon Tpm1 maintains flexibility is investigated by reversing the only full charge substitution; threonine-77(g) in salmon and lysine in other vertebrates. The mutation (Thr-77 to Lys), which falls within a known destabilizing alanine cluster, (i) yields a useful electrophoretic shift in the absence and presence of an anionic detergent, (ii) increases the Tms of both cooperative transitions (calorimetry, 0.1 M salt, pH 7) [35 °C (minor) and 44 °C (major); ΔTm1 = 5 °C, ΔTm2 = 3.5 °C], (iii) increases the Tm of CN1A (residues 11-127) to 53 °C (ΔTm = 13 °C), a value similar to that of mammalian CN1A, (iv) markedly reduces the rate of proteolysis at Leu-169, and (v) weakens the affinity of salmon Tpm1 for troponin-Sepharose. Glu-82(e), the interstrand ionic partner of Lys-77(g), is conserved. The change in ionic interactions at this locus is postulated to be "sensed" in actin period 5 (residues 166-207) and likely beyond. Wild type (acetylated) salmon Tmp1 binds more tightly to F-actin at 4 °C than at 22 °C, which is the opposite of the long-known relationship displayed by the mammalian homologue. All of the evidence indicates that the presence of a neutral 77th amino acid destabilizes a sizable portion of salmon Tpm1 that includes the midregion. Threonine-77 is a key factor in rescuing the thin filament from the peril of cold-induced rigidity.

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.

Normal targeting of a tagged Kv1.5 channel acutely transfected into fresh adult cardiac myocytes by a biolistic method.

The transfection of cardiac myocytes is difficult, and so most of the data regarding the regulation of trafficking and targeting of cardiac ion channels have been obtained using heterologous expression systems. Here we apply the fast biolistic transfection procedure to adult cardiomyocytes to show that biolistically introduced exogenous voltage-gated potassium channel, Kv1.5, is functional and, like endogenous Kv1.5, localizes to the intercalated disc, where it is expressed at the surface of that structure. Transfection efficiency averages 28.2 +/- 5.7% of surviving myocytes at 24 h postbombardment. Ventricular myocytes transfected with a tagged Kv1.5 exhibit an increased sustained current component that is approximately 40% sensitive to 100 microM 4-aminopyridine and which is absent in myocytes transfected with a fluorescent protein-encoding construct alone. Kv1.5 deletion mutations known to reduce the surface expression of the channel in heterologous cells similarly reduce the surface expression in transfected ventricular myocytes, although targeting to the intercalated disc per se is generally unaffected by both NH(2)- and COOH-terminal deletion mutants. Expressed current levels in wild-type Kv1.5, Kv1.5DeltaSH3(1), Kv1.5DeltaN209, and Kv1.5DeltaN135 mutants were well correlated with apparent surface expression of the channel at the intercalated disc. Our results conclusively demonstrate functionality of channels present at the intercalated disc in native myocytes and identify determinants of trafficking and surface targeting in intact cells. Clearly, biolistic transfection of adult cardiac myocytes will be a valuable method to study the regulation of surface expression of channels in their native environment.

Ethnopharmacological Use and Biological Activities of Tragia involucrata L.

Plants have been utilized as medicines to treat various ailments since ancient times. Formulations made by plant materials have been used in traditional, complementary, and alternative medicine and remain widespread in both developing and developed countries. In developing countries, traditional medicines are widely practiced due to its accessibility and affordability, while in developed countries, complementary and alternative medicine are widely popular due to the adverse effects of chemical drugs. Tragia involucrata Linn. (family: Euphorbiaceae) is a highly used medicinal plant used in both Sri Lankan and Indian traditional medical systems. Since this plant is a weed, it is being extensively destroyed due to the lack of knowledge regarding the medicinal value of this plant. Hence, the objective of this study was to collect data on the medicinal value of this plant by correlating its scientifically validated biological activities with its ethnopharmacological uses. An attempt was made to gather as much information available regarding the ethnopharmacological uses and scientifically validated biological activities of Tragia involucrata through authentic traditional texts, scientific journals, and other authentic texts regarding medicinal plants. Thus, the review provides an insight to the capability of Tragia involucrata to be used as a monoherbal formulation for diseases pertaining to multiple systems of the body. With all the scientifically validated biological activities and the ethnopharmacological uses, Tragia involucrata may qualify as a potent candidate to be developed into a phytomedicine to be utilized as both a preventive and as a therapeutic agent.

Effect of removing the amino-terminal hexapeptide of tropomyosin on the properties of the thin filament.

The role of the amino-terminal region of alpha-striated muscle tropomyosin has been analyzed by reconstituting thin filaments with a version of the protein lacking the first six amino acids. While Omp T-digested tropomyosin (residues 7-284) does not bind significantly to F-actin at micromolar concentrations, an interaction is induced by skeletal troponin. At a moderate ionic strength (50 mM KCl), the apparent Kd values are 0.26 microM (with EGTA) and 1.6 microM (with Ca2+). At higher neutral salt (140 mM KCl), reconstitution is observed in the micromolar range only at high pCa (Kd = 1.3 microM). However, when chloride is replaced by acetate, the binding isotherms reach saturation under both extremes of Ca2+ [apparent Kd values of 0.32 microM (with EGTA) and 2.6 microM (with Ca2+)]. The induction of binding of truncated tropomyosin to F-actin by troponin is attributable, in part, to troponin-I, but whereas the amino-terminal fragment of troponin-T (N-Tn-T, residues 1-158) enhances the effect of troponin-I in the case of other tropomyosin products specifically, unacetylated tropomyosin (residues 1-284), and carboxypeptidase-digested tropomyosin (residues 1-273) [Heeley, D. H., et al. (1987) J. Biol. Chem. 262, 9971-9978], it is ineffective with regard to Omp T-digested tropomyosin, suggesting that cleavage has disrupted a binding site for this section of troponin-T. Thin filaments (with Ca2+) containing Omp T-digested tropomyosin activate the steady-state myosin-MgATPase to a greater extent than the integral system, consistent with the interaction between N-Tn-T and the amino-terminal region of tropomyosin having a regulatory function. At high pCa, the truncated system exhibits a less cooperative interaction with myosin-S1-ADP but the affinity for the ligand is stronger. In context with the current methodologies, the consequences of shortening tropomyosin at one end as opposed to the other are the reverse of each other.

Characterization of Daboia russelii and Naja naja venom neutralizing ability of an undocumented indigenous medication in Sri Lanka.

BACKGROUND: Indigenous medicinal practice in Sri Lanka talks about powerful compounds extracted from native plants for treating venomous snake bites which are hardly documented in literature but are used by the indigenous doctors for thousand years. OBJECTIVE: We screened the neutralizing ability of a herbal preparation practiced in indigenous medicine of Sri Lanka, consisting of Sansevieria cylindrica, Jatropha podagrica and Citrus aurantiifolia, for its ability to neutralize venom toxins of Naja naja (Common Cobra) and Daboia russelii (Russell's viper). MATERIALS AND METHODS: The venom toxicity was evaluated using a 5-day old chicken embryo model observing the pathophysiology and the mortality for six hours, in the presence or absence of the herbal preparation. The known toxin families to exist in snake venom, such as Phospholipase A2, Snake venom Metalloprotease, were evaluated to understand the mechanism of venom neutralizing ability of the herbal preparation. RESULTS: The LD50 of D. russelii venom, as measured using the 5-day old chicken embryo model, was 4.8 ± 0.865 ug (R2 = 84.8%, P = 0.079). The pre-incubation of venom with the herbal preparation increased the LD50 of D. russelii venom to 17.64 ± 1.35 µg (R2 = 81.0%, P = 0.100), showing a clear neutralizing action of D. russelii venom toxicity by the herbal medicine. Whereas the pre-incubation of venom with the 1× venom neutralizing dose of commercially available polyvalent anti-venom serum shifted the LD50 venom only up to 5.5 ± 1.35 µg (R2 = 98.8%, P = 0.069). In the presence of the herbal preparation, Phospholipase A2 activity of D. russelii venom was significantly reduced from 9.2 × 10-3 mM min-1 to 8.0 × 10-3 mM min-1 and that of N. naja from 2.92 × 10-2 mM min-1 to 0.188 × 10-2 mM min-1. Further, the pre-incubation of N. naja venom with the herbal preparation significantly reduced its Metalloprotease activity from 0.069 units min-1 to 0.019 units min-1. CONCLUSION: The herbal preparation shows a clear neutralizing action against the toxicities of D. russelii and N. naja venoms demonstrating the potential to be used as a plant based antidote for snake envenomation.

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.

Systematic Bioinformatic Approach for Prediction of Linear B-Cell Epitopes on Dengue E and prM Protein.

B-cell epitopes on the envelope (E) and premembrane (prM) proteins of dengue virus (DENV) were predicted using bioinformatics tools, BepiPred, Ellipro, and SVMTriP. Predicted epitopes, 32 and 17 for E and prM proteins, respectively, were then characterized for their level of conservations. The epitopes, EP4/E (48-55), epitope number 4 of E protein at amino acids 48-55, EP9/E (165-182), EP11/E (218-233), EP20/E (322-349), EP21/E (326-353), EP23/E (356-365), and EP25/E (380-386), showed a high intraserotype conservancy with very low pan-serotype conservancy, demonstrating a potential target as serotype specific diagnostic markers. EP3 (30-41) located in domain-I and EP26/E (393-409), EP27/E (416-435), EP28/E (417-430) located in the stem region of E protein, and EP8/prM (93-112) from the prM protein have a pan-serotype conservancy higher than 70%. These epitopes indicate a potential use as universal vaccine candidates, subjected to verification of their potential in viral neutralization. EP2/E (16-21), EP5/E (62-123), EP6/E (63-89), EP19/E (310-329), and EP24/E (371-402), which have more than 50% pan-serotype conservancies, were found on E protein regions that are important in host cell attachment. Previous studies further show evidence for some of these epitopes to generate cross-reactive neutralizing antibodies, indicating their importance in antiviral strategies for DENV. This study suggests that bioinformatic approaches are attractive first line of screening for identification of linear B-cell epitopes.

Cholesterol and cardiac arrhythmias.

Cardiac arrhythmias are a leading cause of morbidity and mortality in the Western world. Ventricular arrhythmias are reportedly responsible for the majority of sudden cardiac deaths and atrial fibrillation is responsible for 15% of all strokes in the USA. Recent evidence suggests a role for cholesterol in the development of these arrhythmias. In addition to its association with atherosclerotic plaques, high cholesterol has been shown to cause changes in membrane properties, including the function of hormone receptors, ion channels and pumps. These effects are mediated through direct interactions between cholesterol and the membrane proteins, through changes in membrane fluidity and/or an association with lipid rafts. Cholesterol-lowering therapy, therefore, may prove an effective method for the treatment of cardiac arrhythmias. Statins, a class of cholesterol-lowering drugs, have been frequently shown to protect against ventricular arrhythmias and atrial fibrillation. Some of this protection may stem from their cholesterol-lowering activities.

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.