The Batten disease gene product (CLN3p) is a Golgi integral membrane protein

Batten disease (juvenile neuronal ceroid lipofuscinosis) is a recessive neurodegenerative disorder of childhood. The gene, CLN3, was recently identified and found to encode a novel 438 amino acid protein of unknown function. In order to gain insight into the function of the Batten disease protein (CLN3p), we investigated its subcellular localization. Protein constructs incorporating CLN3p fused to the green fluorescence protein or an eight amino acid peptide tag were transiently expressed in fibroblasts, HeLa and COS-7 cells. A juxtanuclear, asymmetric localization pattern was observed that correlated with the Golgi apparatus in all three cell types. However, a proportion of transiently transfected cells exhibited a punctate vesicular distribution throughout the cytoplasm in addition to or without the Golgi localization. In order to account for localization patterns arising from intracellular protein transport disruption due to exaggerated overexpression in transiently transfected cells, we isolated a stably transfected cell line expressing only one copy of the CLN3 -GFP DNA construct. Fluorescence and biochemical analyses using this cell line demonstrated that CLN3p is an integral membrane protein that localizes primarily in the Golgi apparatus. The functional implications of this finding are discussed.      

Partial purification of penicillin acylase from Escherichia coli in poly(ethylene glycol)-sodium citrate aqueous two-phase systems

Studies on the partition and purification of penicillin acylase from Escherichia coli osmotic shock extract were performed in poly(ethylene glycol)-sodium citrate systems. Partition coefficient behavior of the enzyme and total protein are similar to those described in other reports, increasing with pH and tie line length and decreasing with PEG molecular weight. However, some selectivity could be attained with PEG 1000 systems and long tie line at pH 6.9. Under these conditions 2.6-fold purification with 83% yield were achieved. Influence of pH on partition shows that is the composition of the system and not the net charge of the enzyme that determines the behaviour in these conditions. Addition of NaCl to PEG 3350 systems significantly increases the partition of the enzyme. Although protein partition also increased, purification conditions were possible with 1.5 M NaCl where 5.7-fold purification and 85% yield was obtained. This was possible due to the higher hydrophobicity of the enzyme compared to that of most contaminants proteins.     

Preparation,characterization and properties of gradient polymer blends: discussion of poly(vinyl chloride)/poly(methyl methacrylate) blend films containing a wide compositional gradient phase

We prepared several types of thick ≈200 μm films of a poly(vinyl chloride)/poly-(methyl methacrylate) (PVC/PMMA) gradient polymer blend, which contained a widely waried compositional gradient phase, using a dissolution and diffusion method. We discussed the preparation conditions by classifying those gradient blend films into 5 types based on the results of differential scanning calorimetry (DSC) measurements and found the optimum conditions for preparing the ideal gradient blend consisting of only one extremely wide compositional gradient phase. Further, the gradient structures, which were evaluated by the results of DSC measurements, were confirmed by means of Fourier transform infrared/attenuated total reflection (FTIR-ATR). Finally, the ideal blend was found to have significantly new or improved properties (e.g. a large half-width of temperature of the dissipation factor tan δ) which cannot be observed in any PVC, PMMA homogeneously (that is ordinarily) miscible blend or a laminate system.     

New chitin-based polymer hybrids, 3. Miscibility of chitin-graft-poly(2-ethyl-2-oxazoline) with poly(vinyl alcohol)

The miscibility of blends of poly(vinyl alcohol) (PVA) with chitin-graft-poly(2-ethyl-2-oxazoline) ( 1 ) and poly(2-ethyl-2-oxazoline) homopolymer (PEtOZO) was investigated. Calorimetric results showed a single glass transition temperature (T_g) in the entire range of compositions for both blend systems, which indicated that PVA is miscible with both the graft copolymer 1 and PEtOZO. The T_g of PVA is also shifted to lower temperature upon blending with the graft copolymer 1 . IR analysis revealed the existence of specific interactions via hydrogen bonding between the hydroxyl groups in PVA and the carbonyl groups in the poly(2-ethyl-2-oxazoline) side chain of graft copolymer 1 . The results show that the interaction of graft copolymer 1 with PVA is increased by introduction of longer poly(2-ethyl-2-oxazoline) side chains. Thermal decomposition (TG) measurements supported the compatibility of PVA with graft copolymer 1 and with PEtOZO, and showed that the thermal stability of PVA is improved upon blending with 1 or PEtOZO.     

Polyarylenes and poly(arylenevinylene)s, 9 The oxidized states of a (1,4-phenylene) ladder polymer

The oxidized states of double-stranded, planar (1,4-phenylene)-type ladder polymers are characterized by means of UV/VIS/NIR

1 NIR: Near infrared.

spectroscopy and cyclic voltammetry. The oxidation with antimony pentachloride or iron trichloride proceeds as a two-step process; first, radical cationic (polaronic) species are generated, followed by the formation of dicationic (bipolaronic) ones. Especially the polymeric radical cations formed possess an outstanding chemical stability. The findings of cyclovoltammetric investigations are in full agreement with the chemical oxidation experiments.     

On the hydrophilicity of polyzwitterion poly (N,N-dimethyl-N-(3-(methacrylamido)propyl)ammoniopropane sulfonate) in water,deuterated water,and aqueous salt solutions

A series of zwitterionic model polymers with defined molar masses up to 150,000?Da and defined end groups are prepared from sulfobetaine monomer N,N-dimethyl-N-(3-(methacrylamido)propyl)ammoniopropanesulfonate (SPP). Polymers are synthesized by reversible addition-fragmentation chain transfer polymerization (RAFT) using a functional chain transfer agent labeled with a fluorescent probe. Their upper critical solution temperature-type coil-to-globule phase transition in water, deuterated water, and various salt solutions is studied by turbidimetry. Cloud points increase with polyzwitterion concentration and molar mass, being considerably higher in D₂O than in H₂O. Moreover, cloud points are strongly affected by the amount and nature of added salts. Typically, they increase with increasing salt concentration up to a maximum value, whereas further addition of salt lowers the cloud points again, mostly down to below freezing point. The different salting-in and salting-out effects of the studied anions can be correlated with the Hofmeister series. In physiological sodium chloride solution and in phosphate buffered saline (PBS), the cloud point is suppressed even for high molar mass samples. Accordingly, SPP-polymers behave strongly hydrophilic under most conditions encountered in biomedical applications. However, the direct transfer of results from model studies in D₂O, using, e.g. ¹H NMR or neutron scattering techniques, to ‘normal’ systems in H₂O is not obvious.     

Co-effect of aqueous solubility of drugs and glycolide monomer on in vitro release rates from poly(D,L-lactide-co-glycolide) discs and polymer degradation

The objective of this study was to investigate the effect of aqueous solubility of model drugs and glycolide monomer (GM) from poly(D,L-lactide-co-glycolide) (PLGA) discs on in vitro release rates and polymer degradation. 5-Fluorouracil (5-FU), a water-soluble compound, and dexamethasone in a water-insoluble base form were selected as model drugs. Glycolide monomer, that has moderate solubility in water, was a non-toxic and biodegradable additive as a derivative material of hydrolysis of PLGA in order to obtain desirable drugs release rates. PLGA discs with or without GM were formulated by means of compression molding method. The prepared polymeric discs were incubated at 37 degrees C in phosphate-buffered saline (PBS, pH 7.4) and characterized at scheduled time points for water uptake, mass loss, diameter and morphology change, molecular weight and composition change using scanning electron microscopy (SEM), gel-permeation chromatography (GPC), and H-NMR, respectively. The supernatants were taken out of the sample vials and were analyzed for drug release. The 5-FU release was found to be increasing in proportion to the drug loading amount with an initial burst for 5 days, while dexamethasone release showed inverse relationship with the increasing drug loading amount. However, the release behaviors of 5-FU and dexamethasone polymeric discs containing GM showed faster release rates than control discs (without GM) and did not show lag periods during the in vitro release test due to adding GM, which acted as a channeling agent that has moderate solubility in water. Polymer degradation was found to be affected by aqueous solubility of drugs and GM. In conclusion, we observed that drugs release rates were influenced by their aqueous solubility and loading amount and also GM plays a major role in controlling drug release rates regardless of solubility of drugs. This system appears to be promising for controlled drug delivery aimed at local therapy.     

Interaction between polynucleotides in aqueous NaCl solution, 4. Poly(riboadenylic acid) — poly(2-riboinosinic acid) system

Solution properties of poly(A) and poly(I) solution at a mole ratio of poly(A) to poly(I) of 1/2 were studied by means of calorimetry and a modified differential scanning calorimetry (DSC) with the help of circular dichroism (CD) and ultraviolet (UV) spectral methods. It was found that the heat of mixing, ΔH^M, depends sharply on the ionic strength. At [NaCl] < 10^?2 mol · dm^?3 ΔH^M is nearly zero. At higher ionic strength ΔH^M decreases and reaches a minimum value at [NaCl] ≈? 2 · 10^?2 mol ? dm^?3. Above this ionic strength ΔH^M increases again. This behavior is explained on the basis of the formation of a triplex poly(A) · poly(2I) and the transition of poly(I) from a disordered to an ordered structure with increasing ionic strength. The heat of formation, ΔH, of the poly(A) · poly(21) triplex was estimated to be ca ?41 kJ per mole of base triplet. Above an NaCl concentration of 2 · 10^?1 mol · dm^?3 the triplex formation may be represented by the following scheme:      

LCST phase separation in biodegradable polymer blends: poly(D,L‐lactide) and poly(ϵ‐caprolactone)

A lower critical solution temperature (LCST) phase transition is reported for blends of the biodegradable polymers poly(D,L ‐lactide) (PDLA) and poly(ε‐caprolactone) (PCL). From light scattering measurements the cloud point curve is determined to have a critical temperature of 86°C and a critical concentration of mass fraction 36 wt.‐% PCL. Optical microscopy of phase‐separated films indicates a spinodal morphology at the critical concentration, and droplet phases at off‐critical concentrations. After quenching phase separated blends below the melting temperature of PCL (60°C), the crystallization of PCL is used to positively identify PCL‐rich and PDLA‐rich phases. When cystallization of PCL follows LCST phase separation, the size, shape, and distribution of crystalline regions can be adjusted by the degree of PCL/PDLA phase separation. Thus, the LCST phase separation offers a novel method to control microphase structure in biodegradable materials. Applications to control of mechanical and physical properties in tissue engineering scaffolds are discussed in light of the results.     

Urea-induced “denaturation” of a polymer membrane with poly(L-glutamic acid) domains for permeation

The effect of urea on the permeability was investigated for potassium ion, phenyl-1,2-ethanediol, and sugars for a membrane from the graft copolymer 3a containing poly(butyl methacrylate) and poly(L-glutamic acid) moieties and having polypeptide domains for permeation. The membrane treated with urea shows an increase in permeability and diminishes its ability to recognize the molecular size of permeating sugars, owing to a conformational change of the poly(L-glutamic acid) chains induced by urea.     

Insulin release from islets of Langerhans entrapped in a poly(N-isopropylacrylamide-co-acrylic acid) polymer gel

Abstraet-A copolymer of N-isopropylacrylamide (98 mol% in feed) and acrylic acid, poly(N-isopropylacrylamide-co-acrylic acid) (P(NIPAAm-co-AAc)), was prepared by free radical polymerization for development of a thermally reversible polymer to entrap islets of Langerhans for a refillable biohybrid artificial pancreas. A 5 wt% solution of the polymer in Hanks' balanced salt solution forms a gel at 37°C that exhibits no syneresis. Diffusion of fluorescein isothiocyanate (FITC) dextrans having molecular weights of 4400 and 70000 were used to evaluate mass transport in the gel at 37°C. Insulin secretion from islets in the polymer gel was also investigated in both static and dynamic systems. The polymer gel exhibited excellent diffusion of FITC dextran 4400 and FITC dextran 70 000 with diffusion ratios, D/D₀ (ratio of diffusion in the gel to diffusion in water), of 0.20 ± 0.04 and 0.35±0.17, respectively. Human islets entrapped in the polymer gel showed prolonged insulin secretion in response to basal (5.5 mM) glucose concentration compared to free human islets. Rat islets showed prolonged insulin secretion in response to high (16.5 mM) glucose concentrations compared to free rat islets. Rat islets in the polymer gel maintained insulin secretion in response to the higher glucose concentration for over 26 days. Rat islets entrapped by the polymer also released higher quantities of insulin more rapidly in response to changes in concentrations of glucose and other stimulants than rat islets entrapped in an alginate control. These results suggest that this material would provide adequate diffusion for rapid insulin release in an application as a synthetic extracellular matrix for a biohybrid artificial pancreas.     

Experimental approaches to generate compositional gradients in the fully biodegradable polymer blend system based on poly(3‐hydroxybutyric acid)

The fully biodegradable polymer blend films with compositional gradients from the surface to the inside of the films were prepared by the solution‐diffuse technique. A solution of bacterial poly(3‐hydroxybutyric acid) (PHB) in hexafluoro‐2‐propanol (HFIP), which is also a good solvent for poly(vinyl alcohol) (PVA), was cast on a PVA film and the solvent HFIP was evaporated. By controlling the solvent evaporation rate, the PHB/PVA blend film with a compositional gradient was obtained. The phase structure and biodegradation profile of the PHB/PVA blend film were found to be different from those of the corresponding blend film prepared by the conventional solution‐cast method.     

Synthesis and viscosity behavior of poly(γ-p-biphenylmethyl-L-glutamate) in benzene/dichloroacetic acid mixtures,a comparison with poly(γ-benzyl-L-glutamate)

The synthesis of poly(γ-p-biphenylmethyl-L-glutamate), PBPLG, (poly{L-imino-1-[2-(4-biphenylylmethoxycarbonyl)ethyl]-2-oxoethylene}), ( 1d ) is described. The viscosity behavior of this polymer in benzene/dichloroacetic acid mixtures (c=0,2.10^—3—1,4.10^—3 g/cm³) at 25°CC is investigated. The results are compared with measurements on poly(γ-benzyl-L-glutamate), PBLG, (poly[L-imino-1-(2-benzyloxycarbonylethyl)-2-oxoethylene]), ( 1c ) under the same conditions. A transition from a rigid hydrogen bonded helix to a random solvated coil occurs in two stages for both: PBPLG (first stage 0—55%, second stage 55—100% dichloroacetic acid) and PBLG (first stage 0—70%, second stage 70—100% dichloroacetic acid). Therefore, the introduction of a p-phenyl substituent in PBLG leads to a less stable helix in benzene/dichloroacetic acid mixtures. The stability and viscosity behavior of PBLG in benzene/dichloroacetic acid mixtures (c=0,2.10^—3—1,4.10^—3g/cm³) is quite similar to the behavior of PBLG in m-cresol/dichloroacetic acid mixtures (c=1,0.10^—3—4,0.10^—3 g/cm³).     

Fabrication of capillary-like network in a matrix of water-soluble polymer using poly(methyl methacrylate) microfibers

Poly(methyl methacrylate) (PMMA) microfibers were used as a template for development of a capillary-like network in agarose hydrogel. Microfibers with diameter 10-20 μm, which is comparable to the diameter of native capillary vessels, were fabricated using a wet spinning technique. The microfibers were embedded in agarose gel and dissolved by immersing the gel in dichloromethane. The resultant microchannels in the gel had the same diameter as the microfibers, and allowed an aqueous solution to be perfused through the gel. The methodology is promising for fabricating a capillary-like network in tissue engineering scaffolds of various water-soluble polymers.     

Steric effects in 3-substituted polythiophenes: comparing band gap,nonlinear optical susceptibility and conductivity of poly(3-cyclohexylthiophene) and poly(3-hexylthiophene)

We present the synthesis, characterization (NMR, mass spectroscopy, IR) and polymerization of 3-cyclohexylthiophene. Although anodic polymerization is ineffective, it was possible to polymerize 3-cyclohexylthiophene with FeCI₃ as the oxidizing reagent, the resulting poly(3-cyclohexylthiophene) (PC6T) having a peak molar mass of 73 000 g · mol^?1. NMR-Investigations reveal that the coupling ocurred mainly via the 2,5-positions, and the band gap, as determined from UV-vis spectroscopy and cyclic voltammetry, was 2,3–2,4 eV. The nonlinear susceptibility χ⁽³⁾(?3ω; ω ω ω) of the neutral polymer and the electrical conductivity of the CIO₄^? and PF^?₆ salts were determined. The effect of the steric requirements of the cyclohexyl substituent was estimated by comparing the polymer to a poly(3-hexylthiophene) (PN6T) of the same molar mass and with a comparable number of defects (mainly head-to-head and tail-to-tail coupling of the subunits). In the case of PC6T the band gap of the neutral polymer is larger by 0,3 to 0,4 eV, the nonlinear susceptibility has approximately half the value, and the electrical conductivity of the oxidized polymer is two orders of magnitude lower than the corresponding values of PN6T. These effects are explained by the increased sterical requirements of the cyclohexyl substituent, which forces the thiophene rings out of a coplanar conformation and thus reduces the conjugation length.     

Photoconductivity of 3,5-dinitrobenzoate of poly[1-phenyl-1-penten-3-yn-5-ol] (DN-PPPYO) blended with poly[4-(p-N,N-diphenylaminophenyl)-1-phenyl-1-buten-3-yne] (PPAPBEY) as a hole transporting polymer

The 3,5-dinitrobenzoate of poly[1-phenyl-1-penten-3-yn-5-ol] (DN-PPPYO) blended with a charge transporting polymer, poly[4-(p-N,N-diphenylaminophenyl)-1-phenyl-1-buten-3-yne] (PPAPBEY), and polystyrene shows high photoconductivity due to the high concentration of the triphenylamine (TPA) moiety and the fully conjugated backbone as verified by xerographic measurements.     

Transporter (TAP)-independent processing of a multiple membrane-spanning protein,the Epstein-Barr virus latent membrane protein 2

Antigen presentation to CD8⁺ cytotoxic T lymphocytes (CTL) usually involves proteolytic cleavage of antigen in the cytosol and the delivery of epitope peptides onto major histocompatibility complex class I molecules in the endoplasmic reticulum (ER) via the heterodimeric peptide transporter TAP1/TAP2. In the few exceptional cases where TAP-independent presentation of an endogenously expressed protein has been observed, the epitope-containing domain of the protein either has naturally accessed or has been directed into the ER lumen where it is thought to become susceptible to ER proteases. Here, we describe a novel example of TAP-independent processing involving the Epstein-Barr virus (EBV) latent membrane protein LMP2, a multiple membrane-spanning protein with minimal projection into the ER. Expression of LMP2 in the TAP⁻ T2 cell line, whether from the resident EBV genome or from a recombinant vaccinia virus vector vacc-LMP2, rendered the cells sensitive to recognition by CTL clones specific for two HLA-A2.1-restricted peptide epitopes, LMP2 329–337 or 426–434. Vacc-LMP2-mediated sensitization to lysis required expression of the antigen de novo in T2 cells and was blocked by brefeldin A. In the same experiments, two other EBV-specific CTL epitopes, one derived from LMP2 but restricted through a different HLA allele (A11), the other restricted through A2.1 but derived from a different viral protein (BMLF1), did not display TAP-independent processing. The results are discussed in relation to the unusual topology of LMP2 in the membrane and the position of the epitope peptides within that structure.     

Improved injectability and in vitro degradation of a calcium phosphate cement containing poly(lactide-co-glycolide) microspheres

An injectable calcium phosphate cement (CPC) containing 30 wt.% poly(lactide-co-glycolide) (PLGA) microspheres was developed in the present study. Sodium citrate solution was used as the cement liquid phase. The effects of sodium citrate concentration on the injectability, rheological properties, mechanical strength and self-setting properties of CPC containing PLGA microspheres were systematically investigated. The in vitro degradation behavior of the composite during immersion in phosphate buffer solution was also studied. With an increase in sodium citrate concentration, the viscosity and yield stress of the paste were reduced, thereby improving the injectability. At a sodium citrate concentration of 15%, the injectability of the paste reached 95%. The compressive strength of the specimen was also enhanced by the addition of sodium citrate. The specimens had a compressive strength of 32.24+/-2.72 MPa at 15% sodium citrate concentration, compared to 22.15+/-3.60 MPa for the specimen without sodium citrate. The in vitro degradation results demonstrate that incorporated PLGA microspheres can provide the required high strength to CPC in the early stage, which would gradually degrade to create macropores for bone ingrowth. In conclusion, an in situ macropore-generable CPC exhibited excellent injectability and high early strength, and should be a promising material for bone repair and bone reconstruction.