Studies on the anticancer potential of high molecular weight bovine lactoferrin in targeting ovarian carcinoma

High molecular weight bovine lactoferrin (HMW-bLf) has been successfully purified from bovine colostrum.The novel bioassay based findings from this study clearly proved the potential of HMW-bLf as a strong and promising therapeutic agent for targeting different kinds of cancers such as ovarian, breast and colon cancers.

Identification of unprecedented anticancer properties of high molecular weight biomacromolecular complex containing bovine lactoferrin (HMW-bLf)

With the successful clinical trials, multifunctional glycoprotein bovine lactoferrin is gaining attention as a safe nutraceutical and biologic drug targeting cancer, chronic-inflammatory, viral and microbial diseases. Interestingly, recent findings that human lactoferrin oligomerizes under simulated physiological conditions signify the possible role of oligomerization in the multifunctional activities of lactoferrin molecule during infections and in disease targeting signaling pathways. Here we report the purification and physicochemical characterization of high molecular weight biomacromolecular complex containing bovine lactoferrin (≥250 kDa), from bovine colostrum, a naturally enriched source of lactoferrin. It showed structural similarities to native monomeric iron free (Apo) lactoferrin (∼78-80 kDa), retained anti-bovine lactoferrin antibody specific binding and displayed potential receptor binding properties when tested for cellular internalization. It further displayed higher thermal stability and better resistance to gut enzyme digestion than native bLf monomer. High molecular weight bovine lactoferrin was functionally bioactive and inhibited significantly the cell proliferation (p<0.01) of human breast and colon carcinoma derived cells. It induced significantly higher cancer cell death (apoptosis) and cytotoxicity in a dose-dependent manner in cancer cells than the normal intestinal cells. Upon cellular internalization, it led to the up-regulation of caspase-3 expression and degradation of actin. In order to identify the cutting edge future potential of this bio-macromolecule in medicine over the monomer, its in-depth structural and functional properties need to be investigated further.

Nanostructures, semicrystalline morphology, and nanoscale confinement effect on the crystallization kinetics in self-organized block copolymer/thermoset blends

This work reports the first instance of self-organized thermoset blends containing diblock copolymers with a crystallizable thermoset-immiscible block. Nanostructured thermoset blends of bisphenol A-type epoxy resin (ER) and a low-molecular-weight (Mn = 1400) amphiphilic polyethylene-block-poly(ethylene oxide) (EEO) symmetric diblock copolymer were prepared using 4,4'-methylenedianiline (MDA) as curing agent and were characterized by transmission electron microscopy (TEM), atomic force microscopy (AFM), small-angle X-ray scattering (SAXS), and differential scanning calorimetry (DSC). All the MDA-cured ER/EEO blends do not show macroscopic phase separation but exhibit microstructures. The ER selectively mixes with the epoxy-miscible PEO block in the EEO diblock copolymer whereas the crystallizable PE blocks that are immiscible with ER form separate microdomains at nanoscales in the blends. The PE crystals with size on nanoscales are formed and restricted within the individual spherical micelles in the nanostructured ER/EEO blends with EEO content up to 30 wt %. The spherical micelles are highly aggregated in the blends containing 40 and 50 wt % EEO. The PE dentritic crystallites exist in the blend containing 50 wt % EEO whereas the blends with even higher EEO content are completely volume-filled with PE spherulites. The semicrystalline microphase-separated lamellae in the symmetric EEO diblock copolymer are swollen in the blend with decreasing EEO content, followed by a structural transition to aggregated spherical micellar phase morphology and, eventually, spherical micellar phase morphology at the lowest EEO contents. Three morphological regimes are identified, corresponding precisely to the three regimes of crystallization kinetics of the PE blocks. The nanoscale confinement effect on the crystallization kinetics in nanostructured thermoset blends is revealed for the first time. This new phenomenon is explained on the basis of homogeneous nucleation controlled crystallization within nanoscale confined environments in the block copolymer/thermoset blends.

Synthesis and fractionated crystallization of organic-inorganic hybrid composite materials from an amphiphilic polyethylene-block-poly (ethylene oxide) diblock copolymer

Mesostructurally ordered inorganic–organic hybrid composite materials were successfully synthesized by utilizing a low-molecular-weight amphiphilic polyethylene-block-poly(ethylene oxide) (PE–PEO) diblock copolymer as the directing agent. The hybrid composites were formed via the sol–gel reaction of inorganic precursor tetraethoxysilane (TEOS) in an acidic ethanol/water solution with various amounts of PE–PEO. In these composite materials, the hydrophobic PE block of the PE–PEO copolymer forms separate microphase on the nanoscales within the rigid matrix of silica network. The crystallization of the PE block is strictly restricted within the microphase by the rigid silica matrix and takes place through homogeneous nucleation under the nanoscale confinement environment.

Thermal degradation of natural rubber/ZNO nanocomposites

Nanoparticies have been widely used to enhance the properties of natural rubber (NR). In the present paper a novel nanocomposite was developed by blending nano-ZnO slurry with prevulcanized NR latex, and the thermal degradation process of pure NR and NR/ZnO nanocomposites with different nano-ZnO loading was studied with a Perkin Elemer TGA-7 thermogravimetric analyzer. The thermal degradation parameters of NR/ZnO (2 parts ZnO per hundred dlY rubber) at different heating rates (Bs) were studied. The results show that the thermal degradation of pure NR and NR/ZnO nanocomposites in nitrogen is a one-step reaction. The degradation temperatures of NR/ZnO nanocomposite increase with an increasing B. The peak height (R_p) on the differential thermogravimetric curve increases with the increase of B. The degradation rates are not affected significantly by B, and the average values of thermal degradation rate C_p and C_f are 44.42 % and 81.04 %, respectively. The thermal degradation kinetic parameters are calculated with Ozawa-Flynn-Wall method. The activation energy (E) and the frequency factor (A) vary with ecomposition degree, and can be divided into three phases corresponding to the volatilization of low-molecular-weight materials, the thermal degradation ofNR main chains and the decomposition of residual carbon.

Nonribosomal peptide synthetases as technological platforms for the synthesis of highly modified peptied bioeffectors - cyclosporin synthetase as a complex example

Many microbial peptide secondary metabolites possess important medicinal properties, of which the immunosuppressant cyclosporin A is an example. The enormous structural and functional diversity of these low-molecular weight peptides is attributable to their mode of biosynthesis. Peptide secondary metabolites are assembled non-ribosomally by multi-functional enzymes, termed non-ribosomal peptide synthetases. These systems consist of a multi-modular arrangement of the functional domains responsible for the catalysis of the partial reactions of peptide assembly. The extensive homology shared among NRPS systems allows for the generalisation of the knowledge garnered from studies of systems of diverse origins. In this review we shall focus the contemporary knowledge of non-ribosomal peptide biosynthesis on the structure and function of the cyclosporin biosynthetic system, with some emphasis on the re-direction of the biosynthetic potential of this system by combinatorial approaches.

First isolation and structural determination of cyclic β-(1→2)-glucans from an alga, Chlorella pyrenoidosa

The aqueous extract of the edible green microalgae Chlorella pyrenoidosa is of interest because of its immunostimulatory activity. Some components in the extract have been identified previously, namely a unique type of arabinogalactan and a galactofuran. Further fractionation of this extract was accomplished by treating the aqueous solution of the fraction precipitated by addition of 1.5vol of 95% ethanol with cetyltrimethylammonium bromide. The residue obtained by concentration of the supernatant was fractionated further by anion-exchange chromatography and size-exclusion chromatography on Sephadex G-100. Two fractions from the latter column were retained, of which one was a starch-like alpha-(1-->4)-linked d-glucan with some alpha-(1-->6) branches, and the other contained a starch plus a mixture of beta-(1-->2)-d-glucans. ESI mass spectrometry was used to show that the mixture contained both cyclic and linear beta-(1-->2)-d-glucans in a cyclic:linear ratio of 64:36, based on intensities of mass spectral peaks. For the cyclic beta-(1-->2)-d-glucans, ring sizes ranged from 18 to 35 monosaccharides with the ring containing 21 glucose units (54% of the cyclic glucans) being greater than three times more abundant than the next most abundant component, the ring containing 22 glucose units (15%). No rings containing 20 glucose units were present. This is the first observation of cyclic beta-(1-->2)-d-glucans in algae, as far as we are aware. For the linear beta-(1-->2)-d-glucans, the component containing 20 glucoses was most abundant (35% of the linear glucans), while the component containing 21 glucose units was the next most abundant (17%). These relatively low-molecular-weight glucans had low immunostimulatory activity.

NMR determination of ionic structure in plasticized polyether-urethane polymer electrolytes

Solid polymer electrolytes based on amorphous polyether-urethane networks combined with lithium or sodium salts and a low molecular weight cosolvent (plasticizer) have been investigated in our laboratories for several years. Conductivity enhancements of up to two orders of magnitude can be obtained whilst still retaining solid elastomeric properties. In order to understand the effects of the plasticizers and their mechanism of conductivity enhancement, multinuclear NMR has been employed to investigate ionic structure in polymer electrolyte systems containing NaCF₃SO₃, LiCF₃SO₃ and LiClO₃ salts.

With increasing dimethyl formamide (DMF) and propylene carbonate (PC) concentration the increasing cation chemical shift with fixed salt concentration indicates a decreasing anion-cation association consistent with an increased number of charge carriers. ¹³C chemical shift data for the same systems suggests that whilst DMF also decreases cation-polymer interactions, PC does the opposite, presumably by shielding cation-anion interactions. Temperature dependent ⁷Li spin-lattice relaxation times indicate the expected increase in ionic mobility upon plasticization with a shift of the T₁ minimum to lower temperatures. The magnitude of T₁ at the minimum increases upon addition of DMF whereas there is a slight decrease when PC is added. This also supports the suggestion that the DMF preferentially solvates the cation whereas the action of PC is limited to coulomb screening, hence freeing the anion.

Conductivity and NMR properties of plasticized polyethers complexed with lithium salts

NMR provides a tool whereby the dynamic properties of specific nuclei can be investigated. In the present study, a poly(ethylene oxide-co-propylene oxide) network has been used as the polymer host to prepare solid polymer electrolytes (SPE) containing either LiClO₄ or LiCF₃SO₃. In addition, a low molecular weight plasticizer [propylene carbonate (PC), dimethyl formamide (DMF) or tetraglyme] has been added to several of the samples to enhance the mobility of the polymer and, thus, of the ionic species. The effects of plasticizer and salt concentration on the ionic structure and mobility in these SPEs, as measured by NMR relaxation times, and correlation to the conductivity behaviour in these systems are discussed. Temperature dependent triflate diffusion coefficients, as measured by Pulsed Field Gradient ¹⁹F-NMR, in plasticized SPEs are also reported.

13 C NMR spin–lattice relaxation times as a probeof local polymer dynamics in plasticized polyethers

¹³C NMR spin–lattice relaxation times T₁ are used to investigate the effect of low molecular weight diluents, including N,N-dimethylformamide, N-methylformamide, propylene carbonate, γ-butyrolactone, triglyme and tetraglyme, on the local polymer segmental motion in polyether–urethane networks. In all cases, an increase in the local mobility is deduced from the increasing T₁ measurements consistent with a decreasing glass transition temperature. The extent of plasticization, however, is dependent on the nature of the small molecules. Those molecules which can either form strong polymer-diluent interactions (for example through dipolar interactions) or are themselves rigid, give the least enhancement of polymer mobility and the greatest deviation from the Fox equation for T_g. In the presence of alkali metal salts, N,N-dimethylformamide and propylene carbonate are shown to have opposite effects on the local polymer motion, as seen from the T₁ measurements. In both cases, addition of the plasticizers increases the ¹³C T₁ relaxation times for the plasticizer. However, propylene carbonate decreases the polymer ¹³C T₁ whilst N,N-dimethylformamide results in the expected increase in polymer ¹³C T₁.

Positron annihilation lifetime spectroscopy as a probe of free volume in plasticized solid polymer electrolytes

A recent report on the correlation between enhanced polymer mobility and ionic conductivity at room temperature in plasticized polyether-urethane solid polymer electrolytes (Forsyth et al.[1]), has prompted the present investigation. Positron annihilation lifetime spectroscopy (PALS) has been used to study the effect of plasticizer addition on the room temperature free volume characteristics of the crosslinked polyether-urethane. The addition of low molecular weight plasticizers to the polyether-urethane results in a constant or decreasing mean free volume cavity radius, as measured by the orthoPositronium lifetime τ₃, and a decreasing relative concentration of free volume cavities as measured by the ortho-Positronium intensity, I₃. It is postulated that the plasticizers interrupt polymer-polymer interactions by occupying the inter- and intra-chain free volume. The plasticizer structure influences the polymerplasticizer interactions which affect inter- and intra-chain separation and hence the free volume of the system. The decrease in polymer-polymer interaction and the increase in polymer-plasticizer interaction in turn influence the glass transition temperature behaviour.

A 13C NMR study of the role of plasticizers in the conduction mechanism of solid polymer electrolytes

The addition of low molecular weight solvents such as dimethyl formamide (DMF) and propylene carbonate (PC) to urethane crosslinked polyethers results in enhancement of polymer segmental motion, as determined in this work from polymer ¹³C spin lattice relaxation measurements (T₁) and glass transition temperatures. The formation of salt-polyether complexes results in a decrease in T₁, even in the presence of the plasticizer, indicating that the polymer ether molecules are still involved in the alkali metal coordination. In a polymer electrolyte containing 1 mol kg⁻¹ LiClO₄ the addition of DMF and PC have significantly different affects on the polymer mobility, although they both enhance the conductivity. The conductivity enhancement therefore is not solely the result of an increased solvent mobility.

Structure-property relationships in plasticized solid polymer electrolytes

The addition of various kinds of plasticizers can enhance the conductivity of polymer electrolyte systems, in some cases by many orders of magnitude. The plasticizer may be a low molecular weight solvent, or be a low molecular weight polymer. As the plasticizer concentration increases there is an inevitable deterioration in material properties. In this work we have investigated the effect of plasticizer on the conductivity, thermal properties and matrial properties of a number of systems including urethane cross-linked polyethers and polyacrylates. In some of the systems, in particular the polyether electrolytes, the plasticizer acts to enhance conduction by acting as a cosolvent for the salt as well as increasing chain flexibility. Its efficacy is dependent on its structure and characteristics as a solvent. Although T_g is lowered in a close to linear fashion with increasing plasticizer content and thereby conductivity increased rapidly, the elastic modulus changes more slowly. This reflects the coupling of conduction to the local mobility of the molecular units of the combined solvent system and the relative decoupling of the mobility and glass transition from the material properties. In these systems the latter are a function mainly of the longer range structure of the polymer network. The changes in conductivity and materials properties are interpreted in terms of a configurational entropy model of the solution.

RAP1 controls rhoptry targeting of RAP2 in the malaria parasite Plasmodium falciparum

Rhoptry associated protein 1 (RAP1) and 2 (RAP2), together with a poorly described third protein RAP3, form the low molecular weight complex within the rhoptries of Plasmodium falciparum. These proteins are thought to play a role in erythrocyte invasion by the extracellular merozoite and are important vaccine candidates. We used gene-targeting technology in P.falciparum blood-stage parasites to disrupt the RAP1 gene, producing parasites that express severely truncated forms of RAP1. Immunoprecipitation experiments suggest that truncated RAP1 species did not complex with RAP2 and RAP3. Consistent with this were the distinct subcellular localizations of RAP1 and 2 in disrupted RAP1 parasites, where RAP2 does not traffic to the rhoptries but is instead located in a compartment that appears related to the lumen of the endoplasmic reticulum. These results suggest that RAP1 is required to localize RAP2 to the rhoptries, supporting the hypothesis that rhoptry biogenesis is dependent in part on the secretory pathway in the parasite. The observation that apparently host-protective merozoite antigens are not essential for efficient erythrocyte invasion has important implications for vaccine design.

Role of Pr160gag-pol in mediating the selective incorporation of tRNA(Lys) into human immunodeficiency virus type 1 particles

COS-7 cells transfected with human immunodeficiency virus type 1 (HIV-1) proviral DNA produce virus in which three tRNA species are most abundant in the viral tRNA population. These tRNAs have been identified through RNA sequencing techniques as tRNA(3Lys) the primer tRNA in HIV-1, and members of the tRNA(1,2Lys) isoacceptor family. These RNAs represent 60% of the low-molecular-weight RNA isolated from virus particles, while they represent only 6% of the low-molecular-weight RNA isolated from the COS cell cytoplasm. Thus, tRNA(Lys) is selectively incorporated into HIV-1 particles. We have measured the ratio of tRNA(3Lys) molecules to copies of genomic RNA in viral RNA samples and have calculated that HIV-1 contains approximately eight molecules of tRNA(3Lys) per two copies of genomic RNA. We have also obtained evidence that the Pr160gag-pol precursor is involved in primer tRNA(3Lys) incorporation into virus. First, selective tRNA(Lys) incorporation and wild-type amounts of tRNA(3Lys) were maintained in a protease-negative virus unable to process Pr55gag and Pr160gag-pol precursors, indicating that precursor processing was not required for primer tRNA incorporation. Second, viral particles containing only unprocessed Pr55gag protein did not selectively incorporate tRNA(Lys), while virions containing both unprocessed Pr55gag and Pr160gag-pol proteins demonstrated select tRNA(3Lys) packaging. Third, studies with a proviral mutant containing a deletion of most of the reverse transcriptase sequences and approximately one-third of the integrase sequence in the Pr160gag-pol precursor resulted in the loss of selective tRNA incorporation and an eightfold decrease in the amount of tRNA(3Lys) per two copies of genomic RNA. We have also confirmed herein finding of a previous study which indicated that the primer binding site is not required for the selective incorporation of tRNA(Lys).