Sugar specific cellular lectins of Phallusia mamillata hemocytes: Purification, characterization and evidence for cell surface localization

Cellular lectins (CLs) of Phallusia mamillata were demonstrated in protein preparations obtained by salt fractionation from hemocytes sonicated in a suitable medium. Since the lectins from the precipitated fraction bind sugars containing D-galactosyl groups, they were purified by affinity chromatography on Sepharose. SDS-PAGE under reducing conditions showed that CLs are formed of two components of apparent MWs approximately 36,900 and 35,090 and thus differ from serum lectins (SLs) (MW about 62,200). The "shrinkage" observed when SLs were examined under nonreducing conditions suggest the presence of intrachain disulphide bonds which can affect the molecular structure of the SLs. CL-SL differences were also revealed by the nonidentity reaction of the immuno-precipitate in immunodiffusion using an anti-SL immune serum. The capacity of hemocytes to form rosettes or clumps with erythrocytes demonstrated that they possess α-lactose specific CLs on their surfaces. © 1989

Bright spots in the darkness of cancer: A review of starfishes-derived compounds and their anti-tumor action

The fight against cancer represents a great challenge for researchers and, for this reason, the search for new promising drugs to improve cancer treatments has become inevitable. Oceans, due to their wide diversity of marine species and environmental conditions have proven to be precious sources of potential natural drugs with active properties. As an example, in this context several studies performed on sponges, tunicates, mollusks, and soft corals have brought evidence of the interesting biological activities of the molecules derived from these species. Also, echinoderms constitute an important phylum, whose members produce a huge number of compounds with diverse biological activities. In particular, this review is the first attempt to summarize the knowledge about starfishes and their secondary metabolites that exhibited a significant anticancer effect against different human tumor cell lines. For each species of starfish, the extracted molecules, their effects, and mechanisms of action are described

Natural and Synthetic Ligand-Binding Induce Different Pilin Assemblies in vitro and Control P. aeruginosa Bioactivities in vivo, and Development of Bacterial-Motility Enabled Binding Assays

Proteins found on bacterial cells surfaces are capable of sensing and transducing signals from the environment to elicit a biological response. Fibrous appendages formed by assembly of pilin proteins on P. aeruginosa are surface proteins that are necessary for host colonization, adhesion on abiotic surfaces, controlling motilities, formation of biofilm, horizontal gene transfer and virulence production of the bacterium. Upon contact with stimuli, pili appendages extend and retract on the cell surface, driven by the assembly and disassembly of pili at inner membrane of the bacteria cells. The dynamic response of this protein assembly is likely caused by a conformational change in the pilin monomers at the tip of the pili appendage upon making contact with a ligand or surface, as well as caused by chemical signals within the bacterium. Despite all the functions of pili identified, the natural and synthetic ligands specific for pilin proteins remain elusive. In this research, we report natural and synthetic ligands of pilin that control P. aeruginosa bioactivities. For biochemical and structural studies on pilin protein, we used a common technique of recombinantly expressing truncated pilin in E. coli. The truncation of pilin from the N’-terminal α-helix retains the perceived binding region within the disulfide loop and yields a soluble pilin. Our approach of expressing truncated variants of the P. aeruginosa PA1244N3(pPAC46) and single amino acid mutants have demonstrated that pilin binds to the natural and synthetic ligands and that the disulfide loops plays an important role for this function. Using a novel bacterial motility-enabled binding assay, we demonstrated that spreading expressed pilin monomers on the hydrated gel surface can inhibit the swarming motilities of the wild-type P. aeruginosa by binding and sequestering rhamnolipids secreted by the bacterium, or reactivate the swarming motility of the bacteria by sequestering the synthetic inhibitor added in the hydrated gel. Separating the components of rhamnolipids reveal that monorhamnolipid is more active than dirhamnolipid at controlling the swarming motility of P. aeruginosa. Ligand-induced changes to pilin structures were detected by circular dichroism, nuclear magnetic resonance (NMR) and fluorescence spectroscopy. Pilin monomers bind to the rhamnolipids at picomolar ranges and induce pilin proteins to form linear nano-assemblies. About one pico-molar of the ligands causes the transition of fluorescence signal to plateau for 100 nM of pilin monomers. This 10-5 equivalence effect is likely due to tight ligand-receptor binding rather that a catalytic effect based on titration studies. The mechanism of the assembly appears to be isodesmic and does not require a critical aggregation concentration to form linear assemblies. A class of synthetic ligands consists of saturated farnesol tethered with disaccharide also binds directly to pilin proteins at picomolar range by intrinsic fluorescence, and to dominate rhamnolipids resulting in complete inhibition of swarming motilities, and to induce the pilin proteins to form an amorphous assembly. The nonamphiphilic chromonic salt, disodium cromoglycate (5’DSCG) was used to conduct preliminary crystallization studies on pilin from P. aeruginosa. We demonstrated that the 5’DSCG molecules demix in the presence of peptides and form isodesmic assemblies. This demixing phenomenon was then further explored to precipitate and crystallize pilin proteins. The resulting precipitates include radial precipitates for the native 1244 pilin and needle-like crystals for the truncated pilin. These results, along with past findings, suggest that 5’DSCG isodesmic assemblies has the potential to assist in protein purification and crystallization. This work presents the use of a label-free bacterial motility enabled assay, together with biophysical techniques to provide a mechanistic understanding of the ligand binding between natural and synthetic ligands to pilin. The findings and methods in this study have potential use for the development and screening of therapeutics targeting the protein receptor that control the bioactivites of P. aeruginosa

D-Galactose binding lectins from the tunicate Ascidia malaca: Subunit characterization and hemocyte surface distribution

D-galactose specific lectins purified from Ascidia malaca serum contain a major protein component with an apparent molecular weight of about 58,000 daltons, which moves more rapidly under non-reducing conditions. Intramolecular disulfide linkages can explain this behaviour, suggesting a compact protein structure. Membrane lectins have been demonstrated on the surface of about 34% hemocytes by immunofluorescent methods using a rabbit antiserum against the isolated serum lectins. Small, medium and large hemocytes can be positive, as also shown by binding on Sepharose spherules or by rosette formation with sheep and rabbit erythrocytes. Binding is inhibited by the same sugars specific for the serum lectins. Finally, antibodies to the serum lectins specifically agglutinate the hemocytes. This evidence supports the hypothesis that a lectin with the same specificity and certain structural similarities can be found free in the serum and present on hemocyte surfaces. © 1988

Echinoderm Antimicrobial Peptides to Contrast Human Pathogens

Increased attention has been focused in marine invertebrates as a source of bioactive molecules for biomedical applications. Many bioactive molecules are part of the innate immune system. Some more recently isolated compounds, mainly from sea urchin and sea cucumber are antimicrobial peptides (AMP) active against Gram positive, Gram negative and fungi. In this review we described the most recent studies on AMP isolated from echinoderms. The AMP are little peptides <10 kDa with cationic charge and amphipathic structure. Recently, it was demonstrated that in the coelomocyte lysates of Paracentrotus. lividus and Holothuria tubulosa AMP are present with activity against staphylococcal and Pseudomonas aeruginosa antibiofilm. The data show the great potential of application of AMPs in biotechnology for developing novel therapeutic agents and as complements to conventional antibiotic therapy to combat the multi - resistant bacterial strains

In Vitro Release of Lectins From Phallusia mamillata Hemocytes After Their Fractionation on a Density Gradient

Hemocytes were fractionated by centrifugation on a discontinuous Percoll density gradient from the hemolymph of Phallusia mamillata. Results obtained from microcultures of the fractionated hemocytes, sugar-inhibition experiments, SDS-PAGE, and immunoblotting indicate that "compartment cells" release cellular-type (CL) lectins that are specific for a-lactose and lactulose. The released lectins have the same properties as the CL lectins that were previously isolated from sonicated unfractionated hemocytes, but they differ in terms of some molecular and immunological properties from the lectins (SL) purified from the serum. SLs were never found in the supernatants from microcultures ofthe fractionated hemocytes

In vitro release of lectins by Phallusia mamillata hemocytes

α-Lactose specific lectins are released from Phallusia mamillata hemocytes during short-term cultures. The molecular weight of the subunits, the immunological cross-reaction and the sugar specificity suggest that the released lectins are similar to those isolated from the sonicated hemocytes. Because lectin release appears to take place independently of active protein synthesis, the possibility exists that lectins are pre-formed, stored in hemocytes and released when in vitro conditions stimulate the cells. © 1991

The performance of New Zealand initial public offerings (IPOs): 1995-2000 / Arizza Sapuan

This study will focus on the long-term performance of initial public offerings in New Zealand for five years or 60 periods and three years of 36 periods from 1995 to 2004. This paper will analyze on 40 of IPOs companies that listed in 60 periods and 19 of IPOs companies that listed in 36 periods of New Zealand Stock Exchange (NZSE). NZSE 40 was used as a benchmark in evaluating share price performance. The objective of this study is to determine the long term performance of IPOs. This is in order to find out whether the New Zealand IPOs are under perform or over perform the market for the long run. Abnormal Return and Buy-and-hold Abnormal Return has been used to determine the long run performance of IPOs. Also used t-statistics skewness adjusted to see the significant of the IPOs. The result show that the performance of returns on all IPOs companies has no significant to the returns on the market for each period either 60 periods or 36 periods. So, I conclude that all IPOs companies do not under perform the market and it's comparable to the market as a benchmark

Fragments of -thymosin from the sea urchin Paracentrotus lividus as potential antimicrobial peptides against staphylococcal biofilms

The immune mediators in echinoderms can be a potential source of novel antimicrobial peptides (AMPs) applied toward controlling pathogenic staphylococcal biofilms that are intrinsically resistant to conventional antibiotics. The peptide fraction <5 kDa from the cytosol of coelomocytes of the sea urchin Paracentrotus lividus (5-CC) was tested against a group of Gram-positive and Gram-negative pathogen reference strains. The 5-CC of P. lividus was active against all planktonic-tested strains but also showed antibiofilm properties against staphylococcal strains. Additionally,wedemonstrated the presenceof three smallpeptides in the5-CCbelonging tosegment 9-41of aP. lividus -thymosin. The smallest of these peptides in particular, showed the common chemical\u2013physical characteristics of AMPs. This novel AMP from -thymosin has high potential activity as an antibiofilm agent, acting on slow-growing bacterial cells that exhibit a reduced susceptibility to conventional antibiotics and represent a reservoir for recurrent biofilm-associated infections