Identification and biochemical characterization of macrophage migration inhibitory factor-2 (MIF-2) homologue of human lymphatic filarial parasite, Wuchereria bancrofti

Homologues of human macrophage migration inhibitory factor (hMIF) have been reported from vertebrates, invertebrates and prokaryotes, as well as plants. Filarial parasites produce two homologues of hMIF viz., MIF-1 and MIF-2, which play important role in the host immune modulation. Earlier, we have characterized MIF-1 (Wba-mif-1) from Wuchereria bancrofti, the major causal organism of human lymphatic filariasis. Here, we are reporting the molecular and biochemical characterization of MIF-2 from this parasite (Wba-mif-2). The complete Wba-mif-2 gene and its cDNA were amplified, cloned and sequenced. The size of Wba-mif-2 gene and cDNA were found to be 4.275 kb and 363 bp, respectively. The gene annotation revealed the presence of a large intron of 3.912 kb interspersed with two exons of 183 bp and 180 bp. The alignment of derived amino acid sequences of Wba-MIF-2 with Wba-MIF-1 showed 44% homology. The conserved CXXC oxido-reductase catalytic site present in Wba-mif-1 was found absent in Wba-mif-2 coding sequence. The amplified Wba-mif-2 cDNA was cloned into an expression vector pRSET-B and transformed into salt inducible Escherichia coli strain GJ1158. The expressed recombinant Wba-MIF-2 protein showed tautomerase activity against l-dopachrome methyl ester and the specific activity was determined to be 18.57 ± 0.77 μmol/mg/min. Three known inhibitors of hMIF tautomerase activity significantly inhibited the tautomerase activity of recombinant Wba-MIF-2. Although the conserved CXXC oxido-reductase motif is absent in Wba-mif-2, the recombinant protein showed significant oxido-reductase activity in the insulin reduction assay, possibly because of the presence of vicinal cysteine residues.     

PTHrP potentiating estradiol-induced vitellogenesis in sea bream (Sparus auratus, L.)

In fish, vitellogenin is an important nutritional precursor protein produced solely in the liver and released into the blood where it binds calcium. In the gilthead sea bream (Sparus auratus) 17beta-Estradiol (E2) plays an important role in the synthesis of vitellogenin, but also the pituitary hormones prolactin (PRL) and growth hormone (GH) can stimulate vitellogenin induction in fish. Considering the emerging involvement of PTHrP in fish calcium metabolism and the importance of calcium regulation in reproduction, we investigated the possible role of PTHrP in vitellogenesis. E2-na?ve and E2-primed sea bream hepatocytes were used in an in vitro primary hepatocyte culture and stimulated with a recombinant sea bream PTHrP (sbPTHrP) to establish the contribution of sbPTHrP alone or in combination with E2 to the regulation of hepatic vitellogenin synthesis. Hepatocytes stimulated solely with sbPTHrP were not affected in their vitellogenesis. However, in hepatocytes stimulated with E2 in combination with sbPTHrP a higher vitellogenin production was seen than with E2 alone. It is concluded that sbPTHrP has a potentiating effect on estradiol stimulation of vitellogenin production by sea bream hepatocytes. The sea bream provides a unique model where vitellogenesis regulation can be studied on E2-na?ve liver cells, both in vivo and in vitro.     

Ontogeny of parathyroid hormone-related protein in the ovine parathyroid gland.

PTH-related protein (PTHrP) has been implicated in calcium regulation during fetal life. In this study the ontogeny of PTHrP was examined in ovine parathyroid glands. Immunohistochemical techniques, Western blot analysis, and a RIA with antisera raised against synthetic fragments of human (h) PTHrP (i.e. 1-34, 1-40, 50-69, and 107-141) were used to detect the presence of immunoreactive PTHrP in parathyroid glands from fetal and neonatal lambs and maternal ewes. Positive immunostaining for PTHrP was observed in fetal (from 116 days of gestation) and lamb (up to 180 days post birth) but not maternal parathyroid glands with the PTHrP(50-69) antiserum. Fetal and lamb parathyroid glands consisted entirely of one cell type in which PTHrP immunoreactivity to PTHrP(50-69) antiserum was found. In contrast, immunoreactivity to PTHrP could not be detected in sections of fetal, lamb, or maternal parathyroid glands with antisera raised against PTHrP(1-34) or PTHrP(107-141). However, PTHrP immunoreactivity in urea/acid extracts of newborn lamb parathyroid glands could be detected by Western blot analysis and RIA with antisera raised against the N-terminal portion of PTHrP. Western blot analysis with the PTHrP(1-34) antisera revealed that urea/acid extracts of newborn lamb parathyroid glands contained a substance with a mol wt of 14.4K, which corresponded in size to that of hPTHrP(1-84). Newborn lamb parathyroid glands contained 0.35 ng PTHrP/micrograms extract, whereas maternal parathyroid glands contained only 0.035 ng PTHrP/micrograms extract when tested in a RIA employing recombinant hPTHrP(1-84) as standard and an antibody raised against hPTHrP(1-40). The detection of immunoreactive PTHrP in the developing ovine parathyroid gland provides further evidence to support the suggestion that PTHrP produced in the parathyroid gland is involved in the normal hormonal regulation of calcium metabolism in the mammalian fetus and neonate.     

Single-column purification and bio-characterization of recombinant human parathyroid hormone-related protein (1-139)

Recombinant human parathyroid hormone-related protein (hPTHrP) (1-139) was expressed using the IMPACT T7 (intein-mediated purification with an affinity chitin-binding tag) system, allowing purification of free recombinant peptide in a single chromatographic step. This system utilizes an intein, which is a protein splicing element from the Saccharomyces cerevisiae VMA1 gene. The intein has been modified so that it undergoes a self-cleavage reaction at its N-terminus at low temperatures in the presence of 1,4-dithiothreitol (DTT). The cDNA encoding hPTHrP (1-139) was cloned into the pTYB1 vector to create an in-frame fusion at the N-terminus of the intein gene. The cDNA for the chitin-binding domain from Bacillus circulans is present at the C-terminus of intein for affinity purification of the three-part fusion protein on a chitin column. The recombinant plasmid was transfected into E. coli ER2566 cells and synthesis of the PTHrP fusion protein was induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). This system produced pure hPTHrP (1-139) and an N-terminally truncated analogue, hPTHrP (27-139), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blot analysis, N-terminal sequence analysis and mass spectroscopy. hPTHrP (1-139) stimulated cAMP accumulation in ROS 17/2.8 osteoblastic bone cells, whereas hPTHrP (27-139) failed to elicit a response. hPTHrP (1-139) also inhibited the growth of the breast cancer cell line MDA-MB-231; the magnitude of the response was comparable with that of synthetic hPTHrP (1-34) and (1-86). Neutralization of endogenous PTHrP and added hPTHrP (1-139) and N-terminal species with an anti-PTHrP antiserum completely abolished the growth inhibitory effects. These results indicate that the added peptides modulate cell growth by acting at the cell surface. Availability of recombinant hPTHrP (1-139) will allow further study of its biological function, as well as its structure.     

Differential expression of PTHrP and its receptor in pituitary gland and gills in estradiol-treated gilthead sea bream (Sparus auratus, L.)

In the gilthead sea bream (Sparus auratus) 17β-estradiol (E₂) plays an important role in the synthesis of vitellogenin. During vitellogenesis, vitellogenin as a nutritional precursor protein is loaded with calcium, which requires elevated plasma calcium levels. This is accomplished via E₂-dependent processes. Reports have shown that hypercalcemic effects of E₂ are possibly mediated by another hypercalcemic factor, viz. parathyroid hormone related protein (PTHrP). To further investigate the possibility of PTHrP as a mediator of E₂-induced hypercalcemia, we investigated the local expression levels of the pthrp mRNA and of the gene coding for the PTHrP receptor, PTH1R (pth1r) in two tissues involved in the calcium regulation (gills, pituitary gland) of the sea bream treated with E₂. Compared to control, treatment with E₂ resulted in: significantly increased total calcium and plasma PTHrP levels (P < 0.01), a down-regulation of pthrp mRNA in the pituitary gland (P < 0.01), and up-regulation of expression levels for both pthrp and pth1r in the branchial system (P < 0.05). These findings provide direct evidence for a mediating role of PTHrP in E₂ induced hypercalcemia, and in addition support the idea for the presence of two independent systems, an endocrine pituitary PTHrP system and a peripheral paracrine branchial PTHrP system.     

Characterization of a Trichinella spiralis 31 kDa protein and its potential application for the serodiagnosis of trichinellosis

The Trichinella spiralis 31 kDa protein (Ts31) was screened from the excretory-secretory (ES) proteins of muscle larvae (ML) by immunoproteomics using serum from mice infected with T. spiralis at 18 days post infection (dpi). The aim of this study was to characterize the Ts31 protein and to evaluate the potential of the recombinant Ts31 protein (rTs31) for serodiagnosis of human trichinellosis. Ts31 gene was cloned and rTs31 was produced in an E. coli expression system. An anti-rTs31serum recognized the native protein migrating in a 25–55 kDa range by Western blotting of ML crude or ES antigens. Expression of Ts31 gene was observed at all developmental stages of T. spiralis (adult worms, newborn larvae, pre-encapsulated larvae and ML). An immunolocalization analysis identified Ts31 in the cuticle and stichocytes of the parasite. The sensitivity of rTs31-ELISA and ES antigen ELISA for detecting anti-Trichinella IgG antibodies in sera of patients with trichinellosis was 97.83% (45/46) and 86.78% (39/46), respectively (P > 0.05); The specificity of rTs31-ELISA was 99.13% (114/115), which was significantly higher than 85.22% (98/115) of ES antigen ELISA (P < 0.01). The rTs31 protein of T. spiralis could be considered as a potential diagnostic antigen for trichinellosis.     

Determination of tissue and plasma concentrations of PTHrP in fish: development and validation of a radioimmunoassay using a teleost 1-34 N-terminal peptide

A specific and sensitive radioimmunoassay (RIA) for the N-terminus of sea bream (Sparus auratus) and flounder (Platichthys flesus) parathyroid hormone-related protein (PTHrP) was developed. A (1-34) amino-terminal sequence of flounder PTHrP was synthesized commercially and used as the antigen to generate specific antiserum. The same sequence with an added tyrosine (1-35(Tyr)) was used for iodination. Human (1-34) parathyroid hormone (PTH), human (1-34) PTHrP, and rat (1-34) PTHrP did not cross-react with the antiserum or displace the teleost peptide. Measurement of PTHrP in fish plasma was only possible after denaturing by heat treatment due to endogenous plasma binding activity. The minimum detectable concentration of (1-34) PTHrP in the assay was 2.5 pg/tube. The level of immunoreactive (1-34) PTHrP in plasma was 5.2+/-0.44 ng/ml (mean+/-SEM, n=20) for flounder and 2.5+/-0.29 ng/ml (n=64) for sea bream. Dilution curves of denatured fish plasma were parallel to the assay standard curve, indicating that the activity in the samples was indistinguishable immunologically from (1-34) PTHrP. Immunoreactivity was present, in order of abundance, in extracts of pituitary, oesophagus, kidney, head kidney, gills, intestine, skin, muscle, and liver. The pituitary gland and oesophagus contained the most abundant levels of PTHrP, 37.7+/-6.1 ng/g wet tissue and 2.3+/-0.7 ng/g wet tissue, respectively. The results suggest that in fish PTHrP may act in a paracrine and/or autocrine manner but may also be a classical hormone with the pituitary gland as a potential major source of the protein.     

Application of signaling protein 14-3-3 and 26 kDa glutathione-S-transferase to serological diagnosis of Schistosomiasis japonica

Schistosomiasis japonica is currently one of the most serious parasitic diseases and over 670 000 people are infected in China by the end of 2006. In order to establish an effective diagnostic method, the gene coding for Sj14-3-3 and Sj26 kDa GST were cloned and expressed separately in Escherichia coli as fusion protein with His-tag. The rSj14-3-3 and 26 kDa rSjGST were combinedly used as antigens for enzyme-linked immunosorbent assays (ELISA) to diagnose acute and chronic S. japonica. Our results showed that the sensitivity in diagnoses of both acute and chronic schistosomiasis was 94.4% (67/71) and 80.7% (96/119), respectively. The specificity was 94.7% applying 132 sera from people living in S. japonicum-free areas. The data also showed that the recombinant proteins cross-react with Clonorchis sinensis and hookworms at a rate of 11.8% and 5.3% respectively. Parallel tests were conducted among ELISA, indirect hemagglutination assay (IHA) and circular ovum precipitin test (COPT) to determine anti-S. japonicum antibodies in sera of patients with schistosomiasis, healthy control, and those infected with other parasites and the results showed no significant difference in sensitivity for acute schistosomiasis between ELISA and IHA assays (χ² = 1.33, P > 0.05), but significant between ELISA and COPT assays (χ² = 6.72, P < 0.01). Our results also revealed significant difference in positive rate between ELISA and IHA (χ² = 24.74, P < 0.005), as well as between ELISA and COPT (χ² = 58.14, P < 0.005). These results suggest that the rSj14-3-3 and r26 kDa SjGST would be effective diagnostic antigens for detection of antibodies to S. japonicum in human. Due to the easy production, high sensitivity and specificity, the recombinant proteins tested in this study can be considered as candidate reagent for immunological diagnosis of human schistosomiasis.     

Actions of parathyroid hormone-related protein on the rat kidney in vivo

Peptides containing residues 1-34 of parathyroid hormone-related protein (PTHrP) and of bovine parathyroid hormone (bPTH), and recombinant full-length PTHrP(1-141) were infused i.v. into anaesthetized thyroparathyroidectomized rats to compare their action and potency on the renal handling of calcium, phosphate and cyclic AMP (cAMP) in vivo. All three peptides decreased the excretion of calcium and increased the excretion of phosphate and cAMP in the urine, with PTHrP(1-34) and PTHrP(1-141) having virtually equipotent effects. Thus the essential requirements for the major physiological activity of PTHrP on the kidney are contained within the 34 amino-terminal amino acids. For all three peptides, the lowest infusion rate that increased phosphate and cAMP excretion was 0.01 nmol/kg per h, whereas the lowest infusion rate that decreased calcium excretion was 0.025 nmol/kg per h for the PTHrP peptides and 0.1 nmol/kg per h for bPTH(1-34). The response to the PTHrP peptides was maximal at an infusion rate of 0.1 nmol/kg per h for both calcium and phosphate. Since the kidney is either equally sensitive to PTHrP and bPTH(1-34), or more sensitive to PTHrP than to bPTH(1-34), the hypercalcaemia of humoral hypercalcaemia of malignancy may develop because uncontrolled secretion of PTHrP increases the renal reabsorption of calcium to such an extent that even a modest increase in the inflow of calcium into the blood raises plasma calcium concentration.     

Signaling by N- and C-terminal sequences of parathyroid hormone-related protein in hippocampal neurons.

Parathyroid hormone-related protein (PTHrP) is synthesized in the brain, and a single type of cloned receptor for the N-terminal portion of PTHrP and PTH is present in the central nervous system. Nothing is known about the physiological actions or signaling pathways used by PTHrP in the brain. Using cultured rat hippocampal neurons, we demonstrate that N-terminal PTHrP[1-34] and PTH[1-34] signal via cAMP and cytosolic calcium transients. The cAMP response showed strong acute (< or = 6 h) homologous and heterologous desensitization after preincubation with PTHrP or PTH. In contrast, the acute calcium response did not desensitize after preincubation with PTHrP; in fact, preincubation dramatically recruited additional responsive neurons. Unexpectedly, C-terminal PTHrP[107-139], which does not bind or activate the cloned PTH/PTHrP receptor, signaled in neurons via cytosolic calcium but not cAMP. Although some neurons responded to both PTHrP[1-34] and PTHrP[107-139], others responded only to PTHrP[1-34]. We conclude that certain hippocampal neurons exhibit dual signaling in response to PTHrP[1-34] and that some neurons have a receptor for C-terminal PTHrP that signals only via cytosolic calcium.     

Molecular cloning and characterization of heat shock protein 70 from Trichinella spiralis

A cDNA encoding heat shock protein 70 of Trichinella spiralis (Ts-Hsp70) was identified by immunoscreening the adult T. spiralis cDNA library with rabbit antisera against T. spiralis adult extracts. The open reading frame of Ts-Hsp70 cDNA encoded a 623-amino acid peptide with a predicted molecular weight of 68.7 kDa, which shares a high degree of sequence conservation with HSP70s from other parasites. Recombinant Ts-Hsp70 was expressed in Escherichia coli and purified with nickel column chromatography. Western blot analysis showed that recombinant Ts-Hsp70 could be recognized not only by trichinellosis patient sera, but also by T. spiralis-infected sera from rabbits, swine, and mice. Mice vaccinated with recombinant Ts-Hsp70 formulated with Freund's adjuvant exhibited strong humoral immune responses indicated by high titer of IgG antibody and significant muscle larval reduction (37%) after being challenged with T. spiralis larvae. The present results indicate that Ts-Hsp70 is a possible candidate vaccine against T. spiralis infection.     

Regulation of PTH/PTH-related protein receptor expression by endogenous PTH-related protein in the rat osteosarcoma cell line ROS 17/2.8

We have utilized clonal lines of the rat osteoblastic cell line ROS 17/2.8 stably transfected with full-length parathyroid hormone-related protein (PTHrP) cDNA in a sense or an antisense orientation to examine the effects of alteration in the production of endogenous PTHrP on expression of the PTH/PTHrP receptor. In the stably transfected clonal cell lines, changes in PTH/PTHrP receptor expression were evaluated by Northern blot analysis, whole-cell ligand binding of ¹²⁵I-[Tyr³⁶] PTHrP (1–36), and exogenous PTHrP (1–34)-stimulated cyclic adenosine monophosphate (cAMP) accumulation. Compared to control (vector-transfected) cells, PTHP-overproducing (sense-transfected) cells exhibited a marked decrease in the expression of PTH/PTHrP receptor mRNA and PTHrP ligand binding, as well as a corresponding decrease in the PTHrP (1–34)-stimulated cAMP response. By contrast, the antisense-transfected cells showed a marked increase in expression of PTH/PTHrP receptor mRNA and PTHrP (1–34) ligand binding, but a significant increase in the PTHrP (1–34)-stimulated cAMP response was not detected. Using antisense-transfected ROS cells, PTH/PTHrP receptor mRNA expression and ¹²⁵I-[Tyr³⁶] PTHrP (1–36) binding were downregulated by treatment for 24 h with exogenous PTHrP (1–36), forskolin, or dibutyryl cAMP. The findings extend those of earlier studies showing receptor downregulation by exogenous PTH by indicating that endogenous PTHrP, as well as circulating PTH, may help regulate receptor production; and suggesting that even very low concentrations of the peptide may influence receptor production.     

Antifilarial activity in vitro and in vivo of some flavonoids tested against Brugia malayi

We evaluated the antifilarial activity of 6 flavonoids against the human lymphatic filarial parasite Brugia malayi using an in vitro motility assay with adult worms and microfilariae, a biochemical test for viability (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT)-reduction assay), and two animal models, Meriones unguiculatus (implanted adult worms) and Mastomys coucha (natural infections). In vitro, naringenin and hesperetin killed the adult worms and inhibited (>60%) MTT-reduction at 7.8 and 31.2 μg/ml concentration, respectively. Microfilariae (mf) were killed at 250-500 μg/ml. The half maximal inhibitory concentration (IC₅₀) of naringenin for motility of adult females was 2.5 μg/ml. Flavone immobilized female adult worms at 31.2 μg/ml (MTT > 80%) and microfilariae at 62.5 μg/ml. Rutin killed microfilariae at 125 μg/ml and inhibited MTT-reduction in female worms for >65% at 500 μg/ml. Naringin had adulticidal effects at 125 μg/ml while chrysin killed microfilariae at 250 μg/ml. In vivo, 50 mg/kg of naringenin elimiated 73% of transplanted adult worms in the Meriones model, but had no effect on the microfilariae in their peritoneal cavity. In Mastomys, the same drug was less effective, killing only 31% of the naturally acquired adult worms, but 51%, when the dose was doubled. Still, effects on the microfilariae in the blood were hardly detectable, even at the highest dose. In summary, all 6 flavonoids showed antifilarial activity in vitro, which can be classed, in a decreasing order: naringenin > flavone = hesperetin > rutin > naringin > chrysin. In jirds, naringenin and flavone killed or sterilized adult worms at 50 mg/kg dose, but in Mastomys, where the parasite produces a patent infection, only naringenin was filaricidal. Thus naringenin and flavone may provide a lead for design and development of new antifilarial agent(s). This is the first report on antifilarial efficacy of flavonoids.     

Antibody specificities of children living in a malaria endemic area to inhibitory and blocking epitopes on MSP-119 of Plasmodium falciparum

Merozoite surface protein-1₁₉ (MSP-1₁₉) specific antibodies which include processing inhibitory, blocking and neutral antibodies have been identified in individuals exposed to Plasmodium falciparum. Here we intend to look at the effect of single and multiple amino acid substitutions of MSP-1₁₉ on the recognition by polyclonal antibodies from children living in Igbo-Ora, Nigeria. This would provide us with information on the possibility of eliciting mainly processing inhibitory antibodies with a recombinant MSP-1₁₉ vaccine. Blood was collected from children in the rainy season and binding of anti-MSP-1₁₉ antibodies to modified mutants of MSP-1₁₉ was analysed by ELISA. The MSP-1₁₉ mutant proteins with single substitutions at positions 22 (Leu → Arg), 43 (Glu → Leu) and 53 (Asn → Arg) and the MSP-1₁₉ mutant protein with multiple substitutions at positions 27 + 31 + 34 + 43 (Glu → Tyr, Leu → Arg, Tyr → Ser, Glu → Leu); which had inhibitory epitopes; had the highest recognition. Children recognised both sets of mutants with different age groups having different recognition levels. The percentage of malaria positive individuals (32-80%) with antibodies that bound to the mutants MSP-1₁₉ containing epitopes that recognise only processing inhibitory and not blocking antibodies, were significantly different from those with antibodies that did not bind to these mutants (21-28%). The amino acid substitutions that abolished the binding of blocking antibodies without affecting the binding of inhibitory antibodies are of particular interest in the design of MSP-1₁₉ based malaria vaccines. Although these MSP-1₁₉ mutants have not been found in natural population, their recognition by polyclonal antibodies from humans naturally infected with malaria is very promising for the future use of MSP-1₁₉ mutants in the design of a malaria vaccine.     

Stimulation of cortisol release by the N terminus of teleost parathyroid hormone-related protein in interrenal cells in vitro

The mode of action of PTHrP in the regulation of sea bream (Sparus auratus) interrenal cortisol production was studied in vitro using a dynamic superfusion system. Piscine (1-34)PTHrP (10(-6)-10(-11) M) stimulated cortisol production in a dose-dependent manner. The ED50 of (1-34)PTHrP was 2.8 times higher than that of (1-39)ACTH, and maximum increase in cortisol production in response to 10(-8) M of (1-34)PTHrP was approximately 7-fold lower than for 10(-8) M of (1-39)ACTH. In contrast to (1-34)PTHrP, piscine (10-20)PTHrP, (79-93)PTHrP, and (100-125)PTHrP (10(-9)-10(-7) M) did not stimulate cortisol production. The effect of piscine (1-34)PTHrP on cortisol production was abolished by N-terminal peptides in which the first amino acid (Ser) was absent and by simultaneous addition of inhibitors of the adenylyl cyclase-protein kinase A and phospholipase C-protein kinase C intracellular pathways but not by each separately. The PTHrP-induced signal transduction was further investigated by measurements of cAMP production and [H3]myo-inositol incorporation in an interrenal cell suspension. Piscine (1-34)PTHrP increased cAMP and total inositol phosphate accumulation, which is indicative that the mechanism of action of PTHrP in interrenal tissue involves the activation of both the adenylyl cyclase-cAMP and phospholipase C-inositol phosphate signaling pathways. These results, together with the expression of mRNA for PTHrP and for PTH receptor (PTHR) type 1 and PTHR type 3 receptors in sea bream interrenal tissue, suggest a specific paracrine or autocrine steroidogenic action of PTHrP mediated by the PTHRs.     

Diarylheptanoid compounds from Alnus nepalensis express in vitro and in vivo antifilarial activity

A large number of medicinal plants remain to be explored for antifilarial compounds. In the present study a crude methanolic extract of leaves of Alnus nepalensis, chloroform- and n-butanol-partitioned fractions from the crude extract and 6 bioactivity-guided isolated compounds including two new diarylheptanoid from the fractions were assayed for microfilaricidal, macrofilaricidal and female worm sterilizing activity using the lymphatic filariid Brugia malayi in in vitro and in vivo systems. In vitro, the crude methanolic extract exerted better microfilaricidal (LC100: 15.63 μg/ml, IC50: 6.00 μg/ml) than macrofilaricidal (LC100: >250; IC50: 88 μg/ml) activity whereas chloroform and n-butanol fractions were more macrofilaricidal (LC100: 125 and 31.25 μg/ml; IC50: 13.14 and 11.84, respectively) than microfilaricidal (LC100: 250–500 μg/ml, IC50: 44.16 μg/ml). In addition, n-butanol fraction also caused 74% inhibition in MTT reduction potential of the adult worms. In vivo (doses: crude: 100–200 mg/kg; fractions: 100 mg/kg, i.p. × 5 days) the chloroform fraction exerted >50% macrofilaricidal activity whereas methanolic extract and n-butanol fraction produced 38–40% macrofilaricidal action along with some female sterilizing efficacy. Of the 5 diarylheptanoid compounds isolated, alnus dimer, and (5S)-5-hydroxy-1-(4-hydroxyphenyl)-7-(3,4-dihydroxyphenyl)-3-heptanone were found to show the most potent with both macrofilaricidal (LC100: 15.63 μg/ml, IC50: 6.57–10.31 μg/ml) and microfilaricidal (LC100: 31.25–62.5 μg/ml, IC50: 11.05–22.10 μg/ml) activity in vitro. These findings indicate that the active diarylheptanoid compounds may provide valuable lead for design and development of new antifilarial agent(s).     

Identification and expression of mRNAs encoding bursicon in the plesiomorphic central nervous system of Homarus gammarus

Ecdysis in arthropods is a complex process, regulated by many neurohormones, which must be released in a precisely coordinated manner. In insects, the ultimate hormone involved in this process is the cuticle tanning hormone, bursicon. Recently, this hormone has been identified in crustaceans. To further define the distribution of bursicon in crustacean nervous systems, and to compare hormone structures within the sub-phylum, cDNAs encoding both bursicon subunits were cloned and sequenced from the nervous system of the European lobster, Homarus gammarus, and expression patterns including those for CCAP determined using in-situ hybridisation, quantitative RT-PCR and immunohistochemistry. Full-length cDNAs encoded bursicon subunits of 121 amino acids (Average M_r: 13365.48) for Burs α, 115 amino acids (Average M_r: 12928.54) for Burs β. Amino acid sequences were most closely related to those of crabs, and for Burs β the sequence was identical to that of the American lobster, Homarus americanus. Complete co-localisation with CCAP in the VNC was seen. Copy numbers burs α, burs β and CCAP mRNAs were between 0.5 and 1.5 × 10⁵ for both bursicon subunits, 0.5-6 × 10⁵ per cdn neurone for CCAP. The terminal abdominal ganglia (AG 6-8) contained about 52 cdn-type neurons, making it the largest bursicon producing region in the CNS. Double labelling IHC using recombinant Carcinus Burs α and CCAP antisera demonstrated complete co-localisation in the VNC. On the basis of the results obtained, it is proposed that CCAP and bursicon release occur simultaneously during ecdysis in crustaceans.     

Outcome and treatment quality of transfer primary percutaneous intervention in older patients with acute ST-elevation myocardial infarction (STEMI)

The aim of this study was to evaluate the outcome and treatment quality of transfer percutaneous coronary intervention (PCI) in older patients with acute STEMI. In this prospective study all patients with diagnosed acute (pain-to-balloon ≤ 12 h) STEMI transferred to our institution for primary PCI (n = 400) between January 2005 and October 2007 were under investigation. Overall 125 older patients with age ≥70 years were included (mean age 77.5 ± 4.9 years; 77 males). Pre-hospital delays were more common in older patients with longer pain-to-balloon: median (range) = 85 (5-629) vs. 66 (1-688) p = 0.031, and pain-to-first medical-contact-times: median: 206 (84-711) vs. 172 (45-720); p = 0.001. A trend towards a higher (non-significant) rate of major 5/125 (5%) vs. 5/275 (1.8%), p = 0.195 and minor 10/125 (8%) vs. 14/275 (5.1%). p = 0.256 bleeding complications in older patients was evident. In-hospital mortality was significantly higher in older patients compared to the younger patients group: 13/125, 10.4% vs. 8/275, 2.9%, p = 0.002). Overall mortality at 30-day follow-up was 11.2% in older and 3.3% in younger patients: 14/125 vs. 9/275, p = 0.002. Transfer PCI is an effective treatment strategy for older patients with acute ST-elevation myocardial infarction. Overall-30-day mortality in older STEMI-patients transferred for primary PCI is comparably low.