Cell-type selective phototoxicity achieved with chlorophyll-a derived photosensitizers in a co-culture system of primary human tumor and normal lung cells

The ATP-dependent transporter ABCG2 exports certain photosensitizers (PS) from cells, implying that the enhanced expression of ABCG2 by cancer cells may confer resistance to photodynamic therapy (PDT) mediated by those PS. In 35 patient-derived primary cultures of lung epithelial and stromal cells, PS with different subcellular localization and affinity for ABCG2 displayed cell-type specific retention both independent and dependent on ABCG2. In the majority of cases, the ABCG2 substrate 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH) was lost from fibroblastic cells more rapidly than from their epithelial counterparts, even in the absence of detectable ABCG2 expression, facilitating selective eradication by PDT of epithelial over fibroblastic cells in tumor/stroma co-cultures. Pairwise comparison of normal and transformed epithelial cells also identified tumor cells with elevated or reduced retention of HPPH, depending on ABCG2. Enhanced ABCG2 expression led to the selective PDT survival of tumor cells in tumor/stroma co-cultures. This survival pattern was reversible through HPPH derivatives that are not ABCG2 substrates or the ABCG2 inhibitor imatinib mesylate. PS retention, not differences in subcellular distribution or cell signaling responses, was determining cell type selective death by PDT. These data suggest that up-front knowledge of tumor characteristics, specifically ABCG2 status, could be helpful in individualized PDT treatment design.     

Synthesis,rheology, and morphology of nylon‐11/layered silicate nanocomposite

Exfoliated nylon‐11/layered silicate nanocomposites were prepared via in situ polymerization by dispersing organoclay in 11‐aminoundecanoic acid monomer. The original clay was modified by a novel method with 11‐aminoundecanoic acid. In situ Fourier transform infrared spectroscopy results show that stronger hydrogen bonds exist between nylon‐11 and organoclay than that of between nylon‐11 and original clay. The linear dynamic viscoelasticity of organoclay nanocomposites was investigated. Before taking rheological measurements, the exfoliated and intercalating structures and the thermal properties were characterized using X‐ray diffraction, transmission electron microscopy, differential scanning calorimetry, and thermogravimetric analysis. The results show that the clay was uniformly distributed in nylon‐11 matrix during in situ polymerization of clay with 4 wt % or less. The presence of clay in nylon‐11 matrix increased the crystallization temperature and the thermal stability of nanocomposites prepared. Rheological properties such as storage modulus, loss modulus, and relative viscosity have close relationship with the dispersion favorably compatible with the organically modified clay. Comparing with neat nylon‐11, the nanocomposites show much higher dynamic modulus and stronger shear thinning behavior. © 2006 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 2161–2172, 2006     

Molecular composites prepared by in situ direct synthesis of wholly aromatic rigid‐rod polyamides via the phosphorylation reaction in a dissolved nylon‐6 matrix

Wholly aromatic rigid‐rod polyamides such as poly(p‐phenyleneterephthalamide) (PPD‐T) were synthesized in situ in a solution of nylon‐6 via the phosphorylation polycondensation method to form nanocomposites or so‐called “molecular composites.” The incorporation of PPD‐T into a nylon‐6 matrix was achieved by this approach in a more compatibilized form than that obtained by the conventional coagulation method that entails precipitation of a blend of PPD‐T and nylon‐6 in a solvent, for example, concentrated sulfuric acid. Gelation occurred during the synthesis, presumably because of the formation of interpenetrating networks accompanied by some block‐copolymer formation. The transparency and tensile properties of the resultant composite films from the rigid‐rod aromatic polyamide/nylon‐6 combination were improved over those of nylon‐6 film alone. Rainbow‐colored intense birefringence was observed for the composite films under crossed polarizers. These properties are discussed in context with the in situ synthesized rigid‐rod polyamides uniformly incorporated in nylon‐6. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 1014–1026, 2003     

Reactive compatibilization of nylon copolymer/EPDM blends: experimental aspects and their comparison with theory

In situ reactive compatibilization was first time applied to a low melting nylon (nylon 6 and 66 copolymer) and EPDM blend system. The effects of in situ compatibilization and concentration of compatibilizer on the morphology and mechanical properties of nylon/EPDM blends have been investigated. The influence of EPM‐g‐MA on the phase morphology was examined by the scanning electron microscopy (SEM) after preferential extraction of the minor phase. The SEM micrographs were quantitatively analyzed for domain size measurements. The compatibilizer concentrations used were 0, 1, 2.5, 5, and 10 wt%. The graft copolymer (nylon‐g‐EPM) formed at the interface showed relatively high emulsifying activity. A maximum phase size reduction was observed when 2.5 wt% of compatibilizer was added to the blend system. This was followed by a leveling‐off at higher loadings indicating interfacial saturation. The conformation of the compatibilizer at the interface was deduced based on the area occupied by the compatibilizer at the blend interface. The experimental compatibilization results were compared with theoretical predictions of Noolandi and Hong. It was concluded that the molecular state of compatibilizer at interface changes with concentration. The in situ compatibilized blends showed considerable improvement in mechanical properties. Measurement of tensile properties shows increased elongation as well as enhanced modulus and strength up on compatibilization. At higher concentrations of compatibilizer, a leveling‐off of the tensile properties was observed. A good correlation has been observed between the mechanical properties and morphological parameters. Copyright © 2007 John Wiley & Sons, Ltd.     

Acid hydrolysis followed by matrix‐assisted laser desorption/ionization mass spectrometry for the rapid diagnosis of serum protein biomarkers in patients with major depression

We have developed a technique combining acid hydrolysis with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) for the rapid study of the changes in the levels of positive and negative acute phase protein biomarkers in the sera of patients suffering from major depression. The serum proteins were first precipitated using an organic solvent; after separation, they were subjected to hydrochloric acid hydrolysis (6 M HCl) for 10 min. The resulting peptides were characterized using MALDI‐TOF MS. Short‐term treatment of the serum proteins with HCl efficiently removed interference from the abundant protein – albumin – and produced abundant peptide ion signals in the range of m/z 4000–10 000. This approach allowed us to rapidly detect the peptide ions originating from transferrin (a common negative acute phase protein) and fibrinogen (a common positive acute phase protein). The average ratios and (standard deviations) of the ion signals derived from transferrin/fibrinogen were 3.58 (±1.93) for the healthy control subjects and 1.02 (±0.52) for the patients suffering from major depression. The differences in transferrin/fibrinogen ratios between healthy controls and patients suggest that major depression will induce internal inflammation and cause either an increase in the level of fibrinogen or a decrease in the level of transferrin. Copyright © 2009 John Wiley & Sons, Ltd.     

Preliminary study to prepare a reference material of styrene metabolites – mandelic acid and phenylglyoxylic acid – in human urine

 A preliminary batch of the reference material was prepared by freeze-drying pooled urine samples obtained from healthy persons occupationally exposed to styrene. Tests for homogeneity and stability were performed by determining urine concentrations of mandelic (MA) and phenylglyoxylic acids (PGA). The urinary MA and PGA concentrations were followed over an 8-month period using high performance liquid chromatography (HPLC). No changes of the concentration values were found. Pure PA and PGA from Merck and Fluka, respectively, were used for traceability purposes, because certified or standard reference materials for MA and PGA do not exist. Control material ClinChek-Urine Control (Recipe) was analysed simultaneously. The mean values of MA and PGA compared well with the means of control samples and fell within the control range. The certified values and their uncertainties were evaluated from the results of interlaboratory comparisons, homogeneity (277.0 ± 7.4 mg L⁻¹ for MA and 148.0 ± 4.7 mg L⁻¹ for FGA) and stability tests. The values are unweighted arithmetical averages of accepted results and their uncertainties are combined uncertainties enlarged by coefficient k=1, evaluated from the standard uncertainties of the interlaboratory comparison, homogeneity and stability tests. Received: 17 September 2002 Accepted: 1 November 2002 Acknowledgement This work was supported by the Internal Grant Agency of Ministry of Health of the Czech Republic (Grant NJ/6784–3). Presented at CERMM-3, Central European Reference Materials and Measurements Conference: The function of reference materials in the measurement process, May 30–June 1, 2002, Rogaška Slatina, Slovenia Correspondence to I. Šperlingová     

Swelling and biocompatibility of sodium alginate/poly(γ‐glutamic acid) hydrogels

Sodium alginate (Alg) hydrogel films were crosslinked with either calcium poly(γ‐glutamate) (Ca‐PGA) or CaCl₂. The hydrophilicity of the resulting hydrogel films was evaluated through swelling tests, water retention capacity tests, and water vapor permeation tests. The swelling ratio, water retention capacity, and the water vapor transmission rate (WVTR) of Alg/Ca‐PGA were higher than those of Ca‐Alg. The swelling ratio of Alg/Ca‐PGA was 651 and 190% at pH 7.4 and pH 1.2, respectively. The tensile strength of Alg/Ca‐PGA hydrogel was lower than that of Ca‐Alg. The results of hemocompatibility test showed that Alg/Ca‐PGA caused shorter activated partial thromboplastin time (APTT) than Ca‐Alg. Both Ca‐Alg and Alg/Ca‐PGA exhibited almost no adsorption of human serum albumin (HSA), whereas the adsorption of human plasma fibrinogen (HPF) of Ca‐Alg was 10 times of that of Alg/Ca‐PGA. In addition, Alg/Ca‐PGA exhibited platelet adhesion higher than Ca‐Alg. Furthermore, both Alg/Ca‐PGA and Ca‐Alg exhibited no cytotoxicity. Copyright © 2009 John Wiley & Sons, Ltd.     

Inoculum studies in production of penicillin g acylase by Bacillus megaterium ATCC 14945

This article reports studies concerning the production of penicillin G acylase (PGA) by Bacillus megaterium. This enzyme has industrial use in the hydrolysis of penicillin G to obtain 6-aminopenicillanic acid, an essential intermediate for the production of semisynthetic β-lactam antibiotics. Although most microorganisms produce the enzyme intracellularly, B. megaterium provides extracellular PGA. The enzyme production by microorganisms involves several steps, resulting in a many operational variables to be studied. The study of the inoculum is an important step to be accomplished, before addressing other issues such as culture optimization and downstream processing. In this study, using a standard inoculum as reference, several runs were performed aiming at the definition of operational conditions in the PGA production. Cell concentration and PGA activity in the production medium were measured after 24, 48, and 72 h of the beginning of the production phase. This study encompasses the duration of the inoculum germination phase and the concentration of cells used to startup the germination. Based on these results, PGA productivity during the production phase was maximized. The selected values for these variables were 1.5 × 10⁷ spores/mL of germination medium, germination during 24 h, and 72 h for the production phase.     

Nonisothermal crystallization kinetics of in situ nylon 6/graphene composites by differential scanning calorimetry

The nonisothermal crystallization kinetics was investigated by differential scanning calorimetry for the nylon 6/graphene composites prepared by in situ polymerization. The Avrami theory modified by Jeziorny, Ozawa equation, and Mo equation was used to describe the nonisothermal crystallization kinetics. The analysis based on the Avrami theory modified by Jeziorny shows that, at lower cooling rates (at 5, 10, and 20 K/min), the nylon 6/graphene composites have lower crystallization rate than pure nylon 6. However, at higher cooling rates (at 40 K/min), the nylon 6/graphene composites have higher crystallization rate than pure nylon 6. The values of Avrami exponent m and the cooling crystallization function F(T) from Ozawa plots indicate that the mode of the nucleation and growth at initial stage of the nonisothermal crystallization may be as follows: two‐dimensional (2D), then one‐dimensional (1D) for all samples at 5–10 °C/min; three‐dimensional (3D) or complicated than 3D, then 2D and 1D at 10–20 and 20–40 °C/min. The good linearity of the Mo plots indicated that the combined approach could successfully describe the crystallization processes of the nylon 6 and nylon 6/graphene composites. The activation energies (ΔE) of the nylon 6/graphene composites, determined by Kissinger method, were lower than those of pure nylon 6. © 2011 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 49: 1381–1388, 2011     

Reactive melt blends of thermoplastic polyolefins,MAH‐g‐PP and nylon 6

Reactive melt blends of an ethylene‐propylene‐diene terpolymer (EPDM) based thermoplastic elastomer (TPE), maleic anhydride grafted polypropylene (MAH‐g‐PP), and nylon 6 were prepared in a single screw extruder and evaluated in terms of morphological, rheological, thermal, dynamic mechanical, and mechanical properties of the blends. It was found that MAH‐g‐PP‐co‐nylon 6 copolymers were in situ formed and acted as effective compatibilizers for polypropylene (PP) and nylon 6. Phase separation of PP and EPDM in TPE increased with the addition and increasing amount of MAH‐g‐PP and nylon 6, leading to decreased glass transition temperature (T_g) of TPE and increased crystalline melting temperature (T_m) of PP. Copyright © 2004 John Wiley & Sons, Ltd.     

INVOLVEMENT OF THYLAKOID MEMBRANE-DEPENDENT PHOTOSENSITIZATION IN PHOTOINHIBITION OF THE CALVIN CYCLE ACTIVITY IN SPINACH CHLOROPLASTS

Photoinhibition of the light-regulated key enzymes of the photosynthetic carbon reduction (PCR) cycle was investigated using chloroplasts isolated from spinach leaves. Light quality dependence of the light-induced activity change (activation or inactivation) of key PCR enzymes in situ demonstrated that, while light activation is promoted mainly by red light (Λ.> 600 nm), inactivation takes place largely in the region of blue light (Λ < 500 nm). Inactivation was suppressed by a lipid soluble singlet oxygen (¹O_2,¹Δ_g) quencher. When “stromal protein” was subjected to a severe photoinhibitory treatment, no significant loss of activity was observed for any PCR enzyme assayed. However, the inclusion of thylakoids in the photolysis system resulted in a substantial inactivation of the enzymes; this inactivation was significantly diminished in the presence of imidazole and enhanced to some extent by a partial deuteration of medium. In contrast, superoxide dismutase did not exert any effect. The blue light-induced inactivation of the enzymes was remarkably decreased in the presence of thylakoids whose Fe-S centers were destroyed. The results obtained in this study suggest that photoinactivation of the PCR enzymes in situ is mediated mainly by ¹O₂, which is photoproduced primarily by the Fe-S centers of thylakoids and diffuses into the stroma.     

New polymers syntheses. 104. Synthesis of poly(ester‐imide)s from nylon 6, nylon 11, or nylon 12

Nylon 6 was reacted with trimellitic anhydride (TMA) at 230 °C so that a complete degradation to N‐(5‐carboxy‐pentamethylene) trimellitimide was obtained. The crude imide dicarboxylic acid was reacted in situ with 4,4′‐bisacetoxy biphenyl whereby an enantiotropic smectic polyesterimide was obtained. Analogous degradation and polycondensation reactions were also performed with nylon 11 and nylon 12. Parallel syntheses were conducted with isolated imide dicarboxylic acids. Furthermore, the crude imide dicarboxylic acid obtained from nylons 6, 11, and 12 were polycondensed in situ with diacetates of hydroquinone or substituted hydroquinone in combination with various amounts of acetoxy benzoic acid or 6‐acetoxy‐2‐naphthoic acid. In this way enantiotropic nematic copoly(ester‐imide)s were prepared. The phase transition of all LC‐poly(ester‐imide)s were characterized by DSC measurement and optical microscopy. In addition, a series of isotropic poly(ester‐imides)s was prepared using nonmesogenic bisphenols, such as bisphenol A, as comonomers. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 1630–1638, 2000     

Intramarrow injection of β-catenin-activated,but not na?ve mesenchymal stromal cells stimulates self-renewal of hematopoietic stem cells in bone marrow

Bone marrow mesenchymal stromal cells (MSCs) have been implicated in the microenvironmental support of hematopoietic stem cells (HSCs) and often co-transplanted with HSCs to facilitate recovery of ablated bone marrows. However, the precise effect of transplanted MSCs on HSC regeneration remains unclear because the kinetics of HSC self-renewal in vivo after co-transplantation has not been monitored. In this study, we examined the effects of intrafemoral injection of MSCs on HSC self-renewal in rigorous competitive repopulating unit (CRU) assays using congenic transplantation models in which stromal progenitors (CFU-F) were ablated by irradiation. Interestingly, naïve MSCs injected into femur contributed to the reconstitution of a stromal niche in the ablated bone marrows, but did not exert a stimulatory effect on the in-vivo self-renewal of co-transplanted HSCs regardless of the transplantation methods. In contrast, HSC self-renewal was four-fold higher in bone marrows intrafemorally injected with β-catenin-activated MSCs. These results reveal that naïve MSCs lack a stimulatory effect on HSC self-renewal in-vivo and that stroma must be activated during recoveries of bone marrows. Stromal targeting of wnt/β-catenin signals may be a strategy to activate such a stem cell niche for efficient regeneration of bone marrow HSCs.     

Common Pathway for K562 Cells Endocytosis and Release of Gac-Tf and Ga2-Tf via a Transferrin Receptor

There has been an increasing interest in the use of gallium in anticancer activity. However, whether the uptakes of two species of transferrin, including digallium transferrin (Ga₂‐Tf) and the C‐terminal monogallium transferrin (Ga_C‐Tf) by cells, are different is not well understood. In this work the mechanism of both species passing in and out K562 cells has been established by using ¹²⁵I‐labeled transferrin. There were about (1.5±0.08)×10⁵ binding sites per cell surface. Both Ga₂‐Tf and Ga_C‐Tf were recycled to the cell exterior with a protracted endocytic cycle compared to apotransferrin (apoTf). The cycling time from the internalization to release was calculated about t_1/2= (3.15±0.055) min for apoTf, t_1/2= (4.69±0.09) min for Ga₂‐Tf and t_1/2= (4.78±0.15) min for Ga_C‐Tf. The result implies that metal dissociating from transferrin in acidic endosomes was likely to be the key step. Both Ga₂‐Tf and Ga_C‐Tf into K562 cells are transferrin receptor‐mediated process with a similar rate of endocytosis and release. Our present observations provide useful information for better targeted drugs in specific therapy.     

A conjoint multi metal-ion iminodiacetic acid monolith microfluidic chip for structural-based protein pre-fractionation

PDMS-based multichannel microfluidic chip was designed and fabricated in a simple approach using readily available tools. UV-initiated in situ polymerization of poly(2-hydroxy ethyl methacrylate-co-di(ethylene glycol) diacrylate-co-N,Nʹ-diallyl l -tartardiamide) in an Eppendorf tube was achieved within 40 min. This polymerization process was successfully translated to a microfluidic chip format without any further modifications. Iminodiacetic acid was successfully immobilized on aldehyde functional monoliths via Schiff base reaction and confirmed by FT-IR spectroscopy. Four transition metal ions (Co (II), Zn (II), Ni (II), and Cu (II)) were chelated individually on four IDA-monolith microfluidic chips. The conjoint metal-ion monolith microfluidic chip has displayed high permeability (9.40 × 10^–13 m²) and a porosity of 32.8%. This affinity microfluidic chip has pre-fractioned four human plasma proteins (fibrinogen, immunoglobulin, transferrin, and human serum albumin) based on their surface-exposed histidine surface topography. A protein recovery of approximately 95% (Bradford assay data) was achieved. The multimonolith microchip can be reusable even after three protein adsorption-desorption cycles.     

The application of an in situ karyotyping technique for mesenchymal stromal cells: a validation and comparison study with classical G-banding

The cytogenetic analysis of mesenchymal stromal cells (MSCs) is essential for verifying the safety and stability of MSCs. An in situ technique, which uses cells grown on coverslips for karyotyping and minimizes cell manipulation, is the standard protocol for the chromosome analysis of amniotic fluids. Therefore, we applied the in situ karyotyping technique in MSCs and compared the quality of metaphases and karyotyping results with classical G-banding and chromosomal abnormalities with fluorescence in situ hybridization (FISH). Human adipose- and umbilical cord-derived MSC cell lines (American Type Culture Collection PCS-500-011, PCS-500-010) were used for evaluation. The quality of metaphases was assessed by analyzing the chromosome numbers in each metaphase, the overlaps of chromosomes and the mean length of chromosome 1. FISH was performed in the interphase nuclei of MSCs for 6q, 7q and 17q abnormalities and for the enumeration of chromosomes via oligo-FISH in adipose-derived MSCs. The number of chromosomes in each metaphase was more variable in classical G-banding. The overlap of chromosomes and the mean length of chromosome 1 as observed via in situ karyotyping were comparable to those of classical G-banding (P=0.218 and 0.674, respectively). Classical G-banding and in situ karyotyping by two personnel showed normal karyotypes for both cell lines in five passages. No numerical or structural chromosomal abnormalities were found by the interphase-FISH. In situ karyotyping showed equivalent karyotype results, and the quality of the metaphases was not inferior to classical G-banding. Thus, in situ karyotyping with minimized cell manipulation and the use of less cells would be useful for karyotyping MSCs.     

Nylon 610/functionalized multiwalled carbon nanotube composite prepared from in‐situ interfacial polymerization

Pristine multiwalled carbon nanotubes (P‐MWNTs) were functionalized with 4‐chlorobenzoic acid via “direct” Friedel‐Crafts acylation in polyphosphoric acid (PPA)/phosphorous pentoxide (P₂O₅) medium. The resultant 4‐chlorobenzoyl‐functionalized MWNTs (F‐MWNTs) were soluble in chlorinated solvents such as dichloromethane, chloroform, and carbon tetrachloride. A large scale of nylon 610/F‐MWNT composite could be conveniently prepared by in situ interfacial polymerization of 1, 6‐hexamethylenediamine (HMDA) in an aqueous phase, and sebacoyl chloride with F‐MWNTs in an organic phase. Similarly, nylon 610/P‐MWNT composite was also prepared for comparison. The state of F‐MWNTs dispersion in nylon 610 matrix was distinctively better than that of P‐MWNTs, which could be clearly discerned by both naked eye and scanning electron microcopy (SEM). As a result, the tensile strength of nylon 610/F‐MWNT composite was 4.9‐fold higher than that of nylon 610/P‐MWNT composite. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 6041–6050, 2008     

Ethanol production from dilute-acid softwood hydrolysate by co-culture

Dilute-acid softwood hydrolysate, with glucose and xylose as the dominant sugars was fermented to ethanol by co-cultures. The strains used include Saccharomyces cerevisiae 2.535 (1#), Pachysolen tannophilis ATCC 2.1662 (2#), and recombinant Escherichia coli (3#) constructed in our laboratory carrying both pdc and adhB genes derived from Zymomonas mobilis. Before fermentation, the co-cultures were adapted for five batches. Observation under light microscope showed aggregation of adapted strains, which could possibly improve their ability to degrade inhibitors. In addition, we tried to detoxify the dilute-acid softwood hydrolysate with a combined method before fermentation. Our study showed that fermentation of detoxified hydrolysate by adapted co-culture (1# + 2@) generated an exceptionally high ethanol yield on total sugar of 0.49 g/g, corresponding to 96.1% of the maximal theoretical value after 48h; fermentation of detoxified hydrolysate by adapted co-culture (1# + 3#) is faster (24h) and could reach a high ethanol yield (0.45 g/g total sugar). These experiments suggest that both adaptation and detoxification significantly improve hydrolysate fermentation and ethanol production.     

Synthesis of Polyelectrolytes Bearing Phosphorylcholine Moieties

We report on the modification of the polyelectrolytes poly(L ‐glutamic acid) (PGA), poly(acrylic acid) (PAA), and poly(L ‐lysine) (PLL) with side groups bearing phosphorylcholine (PC) groups. Two different side chains were synthesized from monoamino tri‐ or tetraethylene glycol, both with PC group linked to the OH end: the tert‐butyloxycarbonyl protected compounds BOCNH(EO)_nPC (n = 2, 3) and the succinylated compound SucNH(EO)₃PC. BOCNH(EO)_nPC was deprotected and the resulting amine was condensed with PGA or PAA and SucNH(EO)₃PC was directly coupled to PLL. Degrees of substitution as high as 80% were obtained with the method described. These polyelectrolytes are potential candidates to fabricate multilayers with protein repellent properties.