Gram-scale purification of aconitine and identification of lappaconitine in Aconitum karacolicum.

Aconitum karacolicum from northern Kyrgyzstan (Alatau area) contains about 0.8-1% aconitine as well as other aconite derivatives that have already been identified. In this paper, we compare several methods for the further purification of an Aconitum karacolicum extract initially containing 80% of aconitine. Reverse-phase flash chromatography, reverse-phase semi-preparative HPLC, centrifugal partition chromatography (CPC) and recrystallization techniques were evaluated regarding first their efficiency to get the highest purity of aconitine (over 96%) and secondly their applicability in a semi-industrial scale purification process (in our case, 150g of plant extract). Even if the CPC technique shows the highest purification yield (63%), the recrystallization remains the method of choice to purify a large amount of aconitine as i) it can be easily carried out in safe conditions; ii) an aprotic solvent is used, avoiding aconitine degradation. Moreover, this study led us to the identification of lappaconitine in Aconitum karacolicum, a well-known alkaloid never found in this Aconitum species.

The role of MgBr2 to enhance the ionic conductivity of PVA/PEDOT:PSS polymer composite.

A solid polymer electrolyte system based on poly(vinyl alcohol) (PVA) and poly(3,4-Etylenedioxythiophene):poly(styrenesulfonate) ( PEDOT: PSS) complexed with magnesium bromide (MgBr2) salt was prepared using solution cast technique. The ionic conductivity is observed to increase with increasing MgBr2 concentration. The maximum conductivity was found to be 9.89 × 10(-6) S/cm for optimum polymer composite film (30 wt.% MgBr2) at room temperature. The increase in the conductivity is attributed to the increase in the number of ions as the salt concentration is increased. This has been proven by dielectric studies. The increase in conductivity is also attributable to the increase in the fraction of amorphous region in the electrolyte films as confirmed by their structural, thermal, electrical and optical properties.

Iodide-capped PbS quantum dots: full optical characterization of a versatile absorber.

Lead sulfide quantum dots represent an emerging photovoltaic absorber material. While their associated optical qualities are true for the colloidal solution phase, they change upon processing into thin-films. A detailed view to the optical key-parameters during solid-film development is presented and the limits and outlooks for this versatile and promising absorber are discussed.

Highly adherent bioactive glass thin films synthetized by magnetron sputtering at low temperature.

Thin (380-510 nm) films of a low silica content bioglass with MgO, B(2)O(3), and CaF(2) as additives were deposited at low-temperature (150°C) by radio-frequency magnetron sputtering onto titanium substrates. The influence of sputtering conditions on morphology, structure, composition, bonding strength and in vitro bioactivity of sputtered bioglass films was investigated. Excellent pull-out adherence (~73 MPa) was obtained when using a 0.3 Pa argon sputtering pressure (BG-a). The adherence declined (~46 MPa) upon increasing the working pressure to 0.4 Pa (BG-b) or when using a reactive gas mixture (~50 MPa). The SBF tests clearly demonstrated strong biomineralization features for all bioglass sputtered films. The biomineralization rate increased from BG-a to BG-b, and yet more for BG-c. A well-crystallized calcium hydrogen phosphate-like phase was observed after 3 and 15 days of immersion in SBF in all bioglass layers, which transformed monotonously into hydroxyapatite under prolonged SBF immersion. Alkali and alkali-earth salts (NaCl, KCl and CaCO(3)) were also found at the surface of samples soaked in SBF for 30 days. The study indicated that features such as composition, structure, adherence and bioactivity of bioglass films can be tailored simply by altering the magnetron sputtering working conditions, proving that this less explored technique is a promising alternative for preparing implant-type coatings.

Adrenomedullin, an autocrine/paracrine factor induced by androgen withdrawal, stimulates 'neuroendocrine phenotype' in LNCaP prostate tumor cells.

Neuroendocrine (NE) differentiation in prostate cancer (CaP) has been reported to be an early marker associated with the development of androgen independence. The mechanisms by which CaP acquires NE properties are poorly understood. In this study, a putative role of adrenomedullin (AM) in the NE differentiation was investigated. The expression of AM and AM receptors (calcitonin receptor-like receptor (CRLR)/receptor activity modifying protein-2 and -3 (RAMP2 and RAMP3) was evaluated after experimental manipulation of androgen status. Levels of AM mRNA and immunoreactive AM (ir-AM) increased four- to sevenfold in androgen-sensitive LNCaP cells after androgen withdrawal in vitro and in LNCaP xenografts in animals after castration. Treatment of LNCaP cells with androgen analogue (dihydrotestosterone; 10(-9) M) prevented the increase in AM mRNA and ir-AM levels. Interestingly, the expression of CRLR, RAMP2 and RAMP3 is not regulated by androgen status. We demonstrate that in the presence of serum, AM is able to induce an NE phenotype in LNCaP cells via CRLR/RAMP2 and RAMP3, which includes extension of neuritic processes and expression of the neuron-specific enolase (NSE), producing cGMP in a dose-dependent manner, which is mediated by a pertussis toxin-sensitive GTP-binding protein. 8-Bromo-cGMP mimicked the effects of AM on cell differentiation. We demonstrate that AM induces a G-kinase Ialpha translocation to the nucleus. The protein kinase G inhibitor KT-5823 inhibited the neurite outgrowth induced by both AM and 8-bromo-cGMP. In noncastrated animals, administration of AM enhanced expression of NSE and chromogranin A in LNCaP xenografts with a significant increase of NSE levels in serum and no changes in tumor growth. In castrated animals, intraperitoneal injection of AM resulted in a 240+/-18% (P<0.001) increase in tumor volume 36 days after treatment, indicating that the nature of effect of AM in CaP depends on the presence or absence of endogenous androgen. Together, these results demonstrate that AM may function as a mediator of NE-like differentiation in culture as well as in vivo and indicate that its production may be important for tumor resurgence following androgen ablation.

Prevalence and Clinical Features of Blastocystis hominis Infection among Patients in Sebha, Libya.

OBJECTIVE: To determine the prevalence and seasonal variation, and to assess the clinical manifestations and treatment of blastocystosis in Libyan patients. METHODS: Three thousand six hundred and forty five stool samples were screened for Blastocystis hominis using normal saline and iodine solution preparations. The clinical features of 108 patients were described, in whom B. hominis was the only parasite isolated. Fifty symptomatic patients were treated with 1500 mg metronidazole daily for 7 days and their stools were re-investigated for B. hominis. RESULTS: B. hominis was found in 969 (26.58 %) of 3645 stool specimens examined. The infection of B. hominis was significantly more (p < 0.05) in summer than in winter over a three year period. In a prospective study of 108 patients, the most common symptoms with stools positive only for B. hominis were diarrhoea (84.94 %), abdominal pain (66.66 %), flatulence (17.20 %) and vomiting (16.12 %). High concentration of B. hominis cells were found more in symptomatic patients than asymptomatic ones (9.20 cells per 40 X field versus 4.06 respectively) with statistically significant differences (p < 0.001). Patients with B. hominis responded to metronidazole and were fully cured after 7 days. CONCLUSION: The occurrence of B. hominis infections in outpatients are probably related to weather conditions, with the suggestion that the hot, dry weather of the Sebha region favors the development and transmission of this organism. B. hominis infections might have a role in some pathological conditions, resulting in gastrointestinal symptoms.

Lactococcin MMFII, a novel class IIa bacteriocin produced by Lactococcus lactis MMFII, isolated from a Tunisian dairy product.

A novel bacteriocin, lactococcin MMFII, produced by Lactococcus lactis MMFII isolated from a Tunisian dairy product had been identified. The bacteriocin was purified to homogeneity from fresh overnight M17 broth culture by sulfate ammonium precipitation, cation-exchange chromatography, sep-pack chromatography and two steps of reverse-phase chromatography. The purified bacteriocin was heat stable, pH resistant and protease sensitive. Its amino acid sequence, obtained by Edman degradation, revealed a 37-amino acid peptide with two cysteine residues in positions 9 and 14 and a calculated mass of 4144.6 Da. Laser desorption mass spectrometry analysis gave a molecular mass of 4142.6, suggesting the presence of a disulfide bond within the purified bacteriocin. Lactococcin MMFII contains the N-terminal YGNGV consensus motif and is active against Listeria. Thus, it belongs to the class IIa bacteriocins figuring the first example of such a bacteriocin produced by a lactococcal strain.

Chemical synthesis, molecular modeling, and antimicrobial activity of a novel bacteriocin, MMFII.

A new antimicrobial peptide, referred to as MMFII, was purified to homogeneity from lactic acid bacteria Lactococcus lactis, which were isolated from Tunisian dairy product. The complete amino acid sequence of the peptide has been established by amino acid analysis, Edman sequencing, and mass spectrometry and verified by solid-phase chemical synthesis. MMFII is a single-chain 37-residue polypeptide containing a single intramolecular disulfide bond, i.e., TSYGNGVHCNKSKCWIDVSELETYKAGTVSNPKDILW. It shares ca. 35% sequence identity with Leucocin A, a class IIa bacteriocin. Modeling based on the 3-D of Leucocin A shows three beta strands located in the N-terminal region (Thr1-Tyr3, Val7-Asn10, Lys13-Ile16) and an alpha helical domain from Asp17 to Asn31. When plotted as an alpha-helical wheel, the central alpha-helix of MMFII does not exhibit an amphipathic helical structure. The synthetic MMFII (sMMFII), obtained by the solid-phase method, was shown to be indistinguishable from the natural peptide. sMMFII is active against Lactococcus cremoris and Listeria ivanovii bacteria, whereas no activity was detected for any of the synthetic N-terminal truncated MMFII analogs Cys9-Trp37, Trp15-Trp37, and Val18-Trp37.

Lebetin peptides: potent platelet aggregation inhibitors.

Lebetins from Macrovipera lebetina snake venom constitute a new class of inhibitors of platelet aggregation. There are two groups of peptides: lebetin 1 (L1; 11- to 13-mer) and lebetin 2 (L2; 37- to 38-mer). The short lebetins are identical to the N-terminal segments of the longer ones. They inhibit platelet aggregation induced by various agonists (e.g. thrombin, PAF-acether or collagen). The shortest lebetin (11-mer) shows potent inhibition of rabbit (IC(50) = 7 nM) and human (IC(50) = 5 nM) platelets. They prevent collagen-induced thrombocytopenia in rats. N- and C-terminal-truncated synthetic L1gamma (sL1gamma; 11-mer) is less active in inhibiting platelet aggregation than the native peptide. Results from Ala scan studies of the sL1gamma peptide indicated that replacement of the residues (P3, G7, P8, P9 or N10) resulted in a remarkable drop in the activity, whereas replacement of residues K2, P4 or K6 by Ala resulted in enhancement of the antiplatelet activity by at least 10-fold. To examine the activity of multimeric L1gamma, several multimeric peptides were synthesized using the multiple-antigen peptide system assembled on a branched lysine core and their antiplatelet activity was evaluated in vitro. The largest multimeric peptides showed a 1,000-fold increase in antiplatelet activity.

Overexpression of the reg gene in non-obese diabetic mouse pancreas during active diabetogenesis is restricted to exocrine tissue.

We demonstrated pancreatic reg gene overexpression in non-obese diabetic (NOD) mice during active diabetogenesis. The aim of this study was to determine in which part of the pancreas (endocrine and/or exocrine) the gene(s) and the protein(s) were expressed and if their localization changed with progression of the disease. In situ hybridization analysis and immunocytochemical studies were carried out on pancreas of female and male NOD mice. Both develop insulitis but diabetes develops only in females and in males only when treated by cyclophosphamide. Our results show that whatever the age, sex, and presence of insulitis and/or diabetes, the expression of reg mRNAs and of the corresponding protein(s) was restricted to exocrine tissue. Moreover, reg remains localized in acinar cells in the two opposite situations of (a) cyclophosphamide-treated males in a prediabetic stage presenting a high level of both insulin and reg mRNAs, and (b) the overtly diabetic females with no insulin but a high level of reg mRNA. These findings suggest that overexpression of the reg gene(s) might represent a defense of the acinar cell against pancreatic aggression.

Chemical synthesis and characterization of Pi1, a scorpion toxin from Pandinus imperator active on K+ channels.

Pi1 is a 35-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the chactidae scorpion Pandinus imperator. Due to its very low abundance in the venom, we have chemically synthesized this toxin in order to study its biological activity. Enzyme-based proteolytic cleavage of the synthetic Pi1 (sPi1) demonstrates half-cystine pairings between Cys4-Cys25, Cys10-Cys30, Cys14-Cys32 and Cys20-Cys35, which is in agreement with the disulfide bridge organization initially reported on the natural toxin. In vivo, intracerebroventricular injection of sPi1 in mice produces lethal effects with an LD50 of 0.2 microgram per mouse. In vitro, the application of sPi1 induces drastic inhibition of Shaker B (IC50 of 23 nM) and rat Kv1.2 channels (IC50 of 0.44 nM) heterologously expressed in Xenopus laevis oocytes. No effect was observed on rat Kv1.1 and Kv1.3 currents upon synthetic peptide application. Also, sPi1 is able to compete with 125I-labeled apamin for binding onto rat brain synaptosomes with an IC50 of 55 pM. Overall, these results demonstrate that sPi1 displays a large spectrum of activities by blocking both SK- and Kv1-types of K+ channels; a selectivity reminiscent of that of maurotoxin, another structurally related four disulfide-bridged scorpion toxin that exhibits a different half-cystine pairing pattern.

Synthesis, 1H NMR structure, and activity of a three-disulfide-bridged maurotoxin analog designed to restore the consensus motif of scorpion toxins.

Maurotoxin (MTX) is a 34-residue toxin that has been isolated from the venom of the chactidae scorpion Scorpio maurus palmatus. The toxin displays an exceptionally wide range of pharmacological activity since it binds onto small conductance Ca(2+)-activated K(+) channels and also blocks Kv channels (Shaker, Kv1.2 and Kv1.3). MTX possesses 53-68% sequence identity with HsTx1 and Pi1, two other K(+) channel short chain scorpion toxins cross-linked by four disulfide bridges. These three toxins differ from other K(+)/Cl(-)/Na(+) channel scorpion toxins cross-linked by either three or four disulfide bridges by the presence of an extra half-cystine residue in the middle of a consensus sequence generally associated with the formation of an alpha/beta scaffold (an alpha-helix connected to an antiparallel beta-sheet by two disulfide bridges). Because MTX exhibits an uncommon disulfide bridge organization among known scorpion toxins (C1-C5, C2-C6, C3-C4, and C7-C8 instead of C1-C4, C2-C5, and C3-C6 for three-disulfide-bridged toxins or C1-C5, C2-C6, C3-C7, and C4-C8 for four-disulfide-bridged toxins), we designed and chemically synthesized an MTX analog with three instead of four disulfide bridges ([Abu(19),Abu(34)]MTX) and in which the entire consensus motif of scorpion toxins was restored by the substitution of the two half-cystines in positions 19 and 34 (corresponding to C4 and C8) by two isosteric alpha-aminobutyrate (Abu) derivatives. The three-dimensional structure of [Abu(19), Abu(34)]MTX in solution was solved by (1)H NMR. This analog adopts the alpha/beta scaffold with now conventional half-cystine pairings connecting C1-C5, C2-C6, and C3-C7 (with C4 and C8 replaced by Abu derivatives). This novel arrangement in half-cystine pairings that concerns the last disulfide bridge results mainly in a reorientation of the alpha-helix regarding the beta-sheet structure. In vivo, [Abu(19),Abu(34)]MTX remains lethal in mice as assessed by intracerebroventricular injection of the peptide (LD(50) value of 0. 25 microg/mouse). The structural variations are also accompanied by changes in the pharmacological selectivity of the peptide, suggesting that the organization pattern of disulfide bridges should affect the three-dimensional presentation of certain key residues critical to the blockage of K(+) channel subtypes.

Ion channel activation by SPC3, a peptide derived from the HIV-1 gp120 V3 loop.

SPC3 is a multibranched peptide containing eight identical GPGRAF motifs which are derived from the human immunodeficiency virus (HIV)-1 gp120 V3 loop consensus sequence. This molecule was reported to prevent the infection of CD4+ cells by various HIV-1 and HIV-2 strains. However, the molecular mode of action of SPC3 remains unclear. Here, we investigated the possibility that SPC3 could interact with alpha/beta-chemokine receptors following observations that, first, the V3 loop is likely to be involved in alpha/beta-chemokine receptor-dependent HIV entry and, second, natural ligands of these receptors are potent inhibitors of cell infection. To address this point, we examined the effects of SPC3 on Xenopus oocytes either uninjected or expressing exogenous human CXCR4 alpha-chemokine receptors. Extracellular applications of micromolar concentrations of SPC3 onto Xenopus oocytes trigger potent inward chloride currents which can be inhibited by increasing extracellular Ca2+ concentration. This effect can be blocked by chloride channel antagonists and is highly specific to SPC3 as it is not triggered by structural analogs of SPC3. The SPC3-induced chloride conductance in oocytes is alpha/beta-chemokine receptor dependent because: (i) SPC3 alters the sensitivity of this channel to external applications of human recombinant MIP-1alpha, a natural ligand of human CCR5 receptor, and (ii) the amplitude of the inward current could be increased by the expression of exogenous human CXCR4 chemokine receptor. The effect of SPC3 appears to rely on the activation of a phospholipase A2 signaling pathway, but is not affected by changes in cytosolic Ca2+ concentration, or by alterations in Gi/Go protein, adenylate cyclase, phospholipase C or protein kinase C activity. Altogether, the data indicate that SPC3 is capable of activating a surface alpha/beta-chemokine-like receptor-mediated signaling pathway in competent cells, thereby triggering, either directly or indirectly, a Ca2+-inactivated chloride conductance.

V3 loop-derived peptide SPC3 inhibits infection of CD4- and galactosylceramide- cells by LAV-2/B.

SPC3, a synthetic multibranched peptide including the GPGRAF consensus motif of the human immunodeficiency virus type 1 (HIV-1) gp120 V3-loop is a potent inhibitor of HIV infection of human CD4+ lymphocytes, macrophages and CD4-/galactosylceramide+ human colon epithelial cells and is currently tested in phase II clinical trials (FDA protocol 257 A). The antiviral property of SPC3 was further investigated for its ability to inhibit LAV-2/B, an HIV-2 clone with a CD4-independent tropism. SPC3 inhibited the LAV-2/B-mediated infection of B-cell line which does not express the CD4 and the galactosylceramide molecules on their cell surface, suggesting an SPC3-sensitive CD4/galactosylceramide-independent pathway of viral infection in HIV susceptible cells. The molecular mechanism of the peptide inhibition was also investigated. The data suggested that the SPC3-mediated inhibition does not result from a direct competition between SPC3 and gp120 binding to the cell surface of the target cell.

Anti-platelet activity of the peptides composing the lebetin 1 family, a new class of inhibitors of platelet aggregation.

We have purified from Vipera lebetina venom a family of inhibitors of platelet aggregation, named Lebetins. They are composed of two peptide groups of short (Lebetin 1: L1alpha: GDNKPPKKGPPNG; L1beta: DNKPPKKGPPNG) and long (Lebetin 2: L2alpha: GDNKPPKKGPPNGCFGHKIDRIGSHSGLGCNKVDDNKG; L2beta: DNKPPKKGPPNGCFGHKIDRIGSHSGLGCNKVDDNKG) size. The sequence presenting anti-platelet activity is mainly present within the Lebetin 1 sequence [Barbouche, R. Marrakchi, N., Mansuelle, P., Krifi, M., Fenouillet, E., Rochat, H. and El Ayeb, M. (1996) Novel anti-platelet aggregation polypeptides from Vipera lebetina venom: isolation and characterization. FEBS Lett. 392, 6-10]. Here, the peptides that compose the Lebetin 1 family were synthesized. Their respective activity was determined. Synthetic L1alpha and L1beta inhibited collagen-induced platelet aggregation in the nanomolar range. A peptide corresponding to L1beta deleted by D at its N terminus (L1gamma) also inhibited platelet aggregation potently; further truncation of L1gamma impaired its activity. Because L1 peptides efficiently inhibited fibrinogen-induced alpha-chymotrypsin treated-platelet aggregation, we tested whether they act mainly through the inhibition of platelet binding to fibrinogen and showed that they failed to inhibit platelet binding to fibrinogen-coated wells. The activity of L1 peptides was also tested in vivo: their intravenous administration strongly inhibited collagen-induced thrombocytopenia in rats.

Maurotoxin, a four disulfide bridges scorpion toxin acting on K+ channels.

Maurotoxin, a toxin from the venom of the Tunisian chactoid scorpion Scorpio maurus, has been purified to homogeneity by gel filtration/reversed-phase HPLC, and characterized. It is a basic and C-terminal amidated 34-residue polypeptide cross-linked by four disulfide bridges. From Edman sequencing results, only six different pairings between the first six half-cystines were retained whereas a disulfide bridge was predicted between the two half-cystines in positions 31 and 34. Modelling based on the structure of charybdotoxin favored two different pairings, one of which possessed two disulfides in common with the general motif of scorpion toxins. The solid-phase technique was used to obtain synthetic maurotoxin, sMTX. The half-cystine pairings of sMTX were determined by enzymatic cleavage and were found to be Cys3 Cys24, Cys9-Cys29, Cys13-Cys19, and Cys31-34, in agreement with experimental data obtained with natural maurotoxin. Both natural and synthetic maurotoxins were lethal to mice following intracerebroventricular injection (LD50, 80 ng/mouse). They blocked the Kv1.1, Kv1.2, and Kv1.3 channels expressed in Xenopus oocytes with almost identical half-effects (IC50) in the range of 40, 0.8 and 150 nM, respectively. They also competed with 125I-apamin (SKca channel blocker) and 125I-kaliotoxin (Kv channel blocker) for binding to rat brain synaptosomes with IC50 of about 5 and 0.03 nM. As the natural and synthetic maurotoxins exhibit indistinguishable physicochemical and pharmacological properties, they are likely to adopt the same half-cystine pairing pattern which is unique among known scorpion toxins. However, this disulfide organization is different from those reported for Pandinus imperator and Heterometrus spinnifer toxins 1 (Pi1 and HsTx1), two novel four-disulfide bridged K+ channel-acting scorpion toxin sharing about 50-70% sequence identity with maurotoxin.

Retroviral envelope glycoprotein processing: structural investigation of the cleavage site.

Proteolytic activation of retroviral envelope glycoprotein precursors occurs at the carboxyl side of a consensus motif consisting of the amino acid sequence (Arg/Lys)-Xaa-(Arg/Lys)-Arg. Synthetic peptides spanning the processing sites of HIV-1/2 and SIV glycoprotein precursors were examined for their ability to be cleaved by the subtilisin-like endoproteases kexin and furin. To determine the potential role of secondary structure on proteolytic activation, we examined the secondary structure of synthetic peptides by circular dichroism and NMR spectroscopy. The results indicate that (i) the peptides were correctly cleaved by kexin and furin and therefore could be used as specific substrates for the purification and characterization of the lymphocyte endoprotease(s) responsible for proteolytic processing of precursors; (ii) the regions surrounding the cleavage sites could be characterized by their flexibility in aqueous solutions. However, a loop has been shown to be a determinant for the specificity of the interaction between the enzyme and its substrate as determined by molecular modeling. Furthermore, we determine and propose a possible structure of the cleavage site which fits to the active site of the modeled furin.

Monoclonal antibodies neutralizing the toxin II from Androctonus australis hector scorpion venom: usefulness of a synthetic, non-toxic analog.

Scorpion venom contains toxins that act on ion channels. Some are responsible for the noxious effects observed when people are stung by scorpions. The study of the neutralization of these molecules and the production of monoclonal antibodies (mAbs) should prove valuable. Toxin II from Androctonus australis hector scorpion (AahII) is one of the most potent toxins and has been well-characterized and studied. Producing mAbs against such molecules is often difficult due to their toxicity. We used a synthetic, non-toxic analog, (Abu)8-AahII, to obtain mAbs which recognize and neutralize the native toxin AahII. Sets of peptides spanning the entire sequence of AahII were assayed to identify the binding sites of the mAbs. The various mAbs recognized only the largest peptides (12-17 residues). They recognized peptides corresponding to different parts of the AahII sequence, suggesting that several regions of the (Abu)8-AahII sequence mimic AahII epitopes and then elicit mAbs directed against toxin.