Analysis of drug interactions with very low density lipoprotein by high-performance affinity chromatography

High-performance affinity chromatography (HPAC) was utilized to examine the binding of very low density lipoprotein (VLDL) with drugs, using R/S-propranolol as a model. These studies indicated that two mechanisms existed for the binding of R- and S-propranolol with VLDL. The first mechanism involved non-saturable partitioning of these drugs with VLDL, which probably occurred with the lipoprotein’s non-polar core. This partitioning was described by overall affinity constants of 1.2 (±0.3)?×?10⁶ M^?1 for R-propranolol and 2.4 (±0.6)?×?10⁶ M^?1 for S-propranolol at pH 7.4 and 37 °C. The second mechanism occurred through saturable binding by these drugs at fixed sites on VLDL, such as represented by apolipoproteins on the surface of the lipoprotein. The association equilibrium constants for this saturable binding at 37 °C were 7.0 (±2.3)?×?10⁴ M^?1 for R-propranolol and 9.6 (±2.2)?×?10⁴ M^?1 for S-propranolol. Comparable results were obtained at 20 and 27 °C for the propranolol enantiomers. This work provided fundamental information on the processes involved in the binding of R- and S-propranolol to VLDL, while also illustrating how HPAC can be used to evaluate relatively complex interactions between agents such as VLDL and drugs or other solutes.     

Neutral Serine Protease from Penicillium italicum. Purification,Biochemical Characterization,and Use for Antioxidative Peptide Preparation from Scorpaena notata Muscle

In the present study, purification and properties of an extracellular neutral serine protease from the fungus Penicillium italicum and its potential application as an antioxidant peptides producer are reported. The protease was purified to homogeneity using ammonium sulfate precipitation, Sephacryl S-200 gel filtration, diethylaminoethanol (DEAE)-Sepharose ion exchange chromatography, and TSK-HPLC gel filtration with a 10.2-fold increase in specific activity and 25.8 % recovery. The purified enzyme appeared as single protein band with a molecular mass of 24 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature for the proteolytic activity were pH 7.0 and 50 °C, respectively. The enzyme was stable in the pH range of 6.0–9.0. The protease was activated by divalent cations such as Ca²⁺ and Mg²⁺. Complete inhibition of the purified enzyme by phenylmethylsulfonyl fluoride confirmed that the protease was of serine-type. The purified enzyme revealed high stability and relatively broad specificity. Scorpaena notata muscle protein hydrolysates prepared using purified serine protease (protease from P. italicum (Prot-Pen)) showed good in vitro antioxidative activities. The antioxidant activities of Scorpaena muscle protein hydrolyzed by Prot-Pen (SMPH-PP) were evaluated using various antioxidant assays: 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, reducing power, ferrous chelating activity, and DNA nicking assay. SMPH-PP showed varying degrees of antioxidant activity and almost the same strongest protection against hydroxyl radical induced DNA breakage.     

Identification of Heat-Related ESTs in Moth Bean Through Suppression Subtraction Hybridization

Moth bean (Vigna aconitifolia (Jacq.) Marechal), an important grain-legume crop grown in hot desert regions of Thar, under scorching sun rays, was investigated for heat tolerance at molecular level. In the present study, we constructed a forward suppression subtractive hybridization (SSH) cDNA library of heat tolerant genotype RMO-40 to identify genes expressing under delayed response to elevated temperature. Heat induction was carried out by exposing 14-day-old seedlings to elevated temperature of 42 °C for 30 min. A total of 125 unigenes (33 contigs and 92 singletons) were derived by cluster assembly and sequence alignment of 200 ESTs; out of 125 unigenes, 21 (16 %) were found to be novel to moth bean. Gene ontology functional classification terms were retrieved for 98 (78.4 %) unigenes of which 73 (58.4 %) ESTs were functionally annotated (GO consensus) where 19 unigenes were annotated with 11 enzyme commission (EC) codes and were mapped to 25 different KEGG pathways. We have identified a majority of heat-shock proteins (constituting 35 % of the present library) aiding heat stress tolerance to moth bean. An expression level of 22 ESTs generated from the above SSH cDNA library was studied through semiquantitative RT-PCR assay simultaneously under 5 and 30 min of heat stress at 42 °C.     

Use of hydroxylapatite composite membranes for analysis of bisphenol A

This study evaluates solid-phase micro-extraction (SPME) coupled with gas chromatography–mass spectrometry (GC–MS) to determine trace levels of bis-phenol A in water and leached from plastic containers. In our study, we used very thin composite membranes prepared in the laboratory. The extraction using headspace post-derivatization with bis(trimethylsilyl) trifluoroacetamide (BSTFA), containing 1 % trimethylchlorosilane (TMCS) vapor, following SPME was compared with extraction without derivatization. The SPME experimental procedures to extract bis-phenol A in water were optimized with a relatively polar polyacrylate (PA)-coated fiber, an extraction time of 50 min, and desorption at 300 °C for 2 min. Headspace derivatization following SPME was performed using 7 μL of BSTFA with 1 % TMCS at 65 °C for 30 s. The precision was 5.2 % without derivatization and 9.0 % headspace derivatization. The detection limit was determined to be at the nanogram per liter level. When SPME was used following headspace derivatization, the detection limit was one order of magnitude better than that achieved without derivatization. The results of this study reveal the adequacy of the SPME–GC–MS method for analyzing bisphenol A leached from plastic containers. The concentrations of bisphenol A leached from plastic containers into water ranged from 0.7 to 78.5 μg L^?1.     

Over-Expression of a Proline Specific Aminopeptidase from Aspergillus oryzae JN-412 and Its Application in Collagen Degradation

A strain that exhibited intracellular proline-specific aminopeptidase (PAP) activity was isolated from soy sauce koji and identified as Aspergillus oryzae JN-412. The gene coding PAP was cloned and efficiently expressed in Escherichia coli BL21 in a biologically active form. The highest specific activity reached 52.28 U mg^?1 at optimum cultivation conditions. The recombinant enzyme was purified 3.3-fold to homogeneity with a recovery of 36.7 % from cell-free extract using Ni-affinity column chromatography. It appeared as a single protein band on SDS-PAGE with molecular mass of approximately 50 kDa. The purified enzyme exhibited the highest activity at 60 °C and pH 7.5. The enzyme activity was inhibited by PMSF and ions like Zn²⁺ and Cu²⁺. DTT, β-mercaptoethanol, EDTA, and ions like Co²⁺, Mg²⁺, Mn²⁺, and Ca²⁺ had no influence on enzyme activity, whereas Ni²⁺ enhanced the enzyme activity. By using collagen as a substrate, the purified recombinant prolyl aminopeptidase contributed to the hydrolysis of collagen when used in combination with neutral protease, and free amino acids in collagen hydrolysates was significantly increased.     

New EDTA determination method based on ion chromatography with suppressed conductimetric detection

A novel direct method for the determination of EDTA in alkaline radioactive evaporator residue solution was developed and validated based on ion chromatography with suppressed conductimetric detection and anion exchange columns (A Supp 4, 4 mm × 250 mm and A Supp 5, 4 mm × 150 mm). The yttrium-EDTA complex resulted one single chromatographic peak in the eluent and allowed the correct determination of EDTA in an alkaline, high concentration radioactive waste water. Depending on coexisting substances, suitable eluent is 10.0 mM carbonate buffer/pH 10.6 or 10.75 (t _R,Y–EDTA = 7.01 and 6.4 min, respectively). For 10.0 mM carbonate buffer/pH 10.6 and isocratic flow rate of 1.0 cm³/min, a linear calibration curve was obtained from 5 to 40 mg/dm³ (r > 0.999) EDTA. Good resolution was achieved from commonly coexisting anions (chloride, nitrite, nitrate, sulphate, phosphate, bromide and citrate). The developed simple ion chromatographic method was applied for the assay of EDTA in various radioactive alkaline solutions.     

A colorimetric assay for d-Penicillamine in urine and plasma samples based on the aggregation of gold nanoparticles

We report herein the development of a highly sensitive colorimetric method for detection of d-Penicillamine using citrate-capped gold nanoparticles (AuNPs). This assay relies upon the distance-dependent of gold nanoparticles surface plasmon resonance band of gold nanoparticles. By replacing the thiol-containing chelator drug, d-Penicillamine, with citrate on the gold nanoparticles surface, a new peak appearing at a longer wavelength intensifies and shifts further to the red from the original peak position due to aggregation of gold nanoparticles which depends on ionic strength, gold nanoparticles and d-Penicillamine concentration. During this process, the plasmon band at 521 nm decreases gradually along with the formation of a new red-shifted band at 630 nm. The calibration curve which is derived from the ratio intensities of absorbance at longer wavelength (630 nm) to original wavelength (521 nm) displays a linear relation in the range of 5.0 × 10^?6–3.0 × 10^?4 M d-Penicillamine. Lower limit of detection for d-Penicillamine, at the signal-to-noise ratio of 3 (3σ), was 3.8 × 10^?6 M. The developed methodology was successfully applied for the determination of d-Penicillamine in human urine and plasma.     

Interactions of collagen and cellulose in their blends with 1-ethyl-3-methylimidazolium acetate as solvent

Collagen/cellulose blended solutions with collagen/cellulose mass ratio (Col/Cel) of 0, 1/40, 1/20, 1/10 and 1/5 were prepared using [Emim]Ac as solvent. The interactions between the two polymers before and after regeneration were investigated. In steady shear flow, all of the experimental viscosity values were greater than those of the estimated values calculated from the log-additivity rule for each sample, suggesting interactions between the two polymers in solutions. All solutions exhibited shear thinning behavior and the flow curves could be described by Cross model. Zero shear viscosity (η ₀) versus Col/Cel was examined and a linear increase (from 8.73 to 16.39 Pa·s) can be observed for η ₀ as Col/Cel ≤ 1/10, while there was only a slight increase (from 16.39 to 18.42 Pa·s) in η ₀ as Col/Cel increased to 1/5. Dynamic rheology results suggested the existence of aggregates in solution with Col/Cel = 1/10. Furthermore, the activation energy of solution was 84.5 kJ mol^?1 as Col/Cel = 1/10, higher than that of cellulose solution (44.2 kJ mol^?1). Regenerated films were prepared and characterized to trace back the interactions between the two polymers in [Emim]Ac. Fourier transform infrared spectroscopy indicated the hydrogen-bond interaction between collagen and cellulose in films. The denaturation temperature of collagen in films with Col/Cel ≤ 1/10 could be improved, but it was decreased with the increase of collagen content, and finally was reduced to be close to that of collagen as Col/Cel = 1/5. The features of dynamic mechanical analysis for films were indicative of the lack of homogeneity between collagen and cellulose as Col/Cel = 1/5. Atomic force microscopy images further confirmed the phase-separation when Col/Cel = 1/5.     

Biosynthesis and Characterization of Pd and Pt Nanoparticles Using Piper betle L. Plant in a Photoreduction Method

An extremely rapid green approach that generates bulk quantities of nanocrystals of noble metals such as palladium (Pd) and platinum (Pt) nanoparticles (NPs) with a small sizes of 3.8 ± 0.2 and 2.1 ± 0.4 nm by using Piper betle L. (Piperaceae) leaf extract is described. The highly stable and monodispersed Pd and Pt NPs were obtained at 10 min of continuous sunlight exposure. The bio-reduced Pd and Pt NPs were further characterized by using UV–Visible spectroscopy, transmission electron microscopy, selected area electron diffraction, X-ray diffraction, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis and cyclic voltammetry measurements. The particles, although discrete, were predominately associated with the P. betle plant proteins, which makes them stable over long time periods. These synthesized biogenic Pd and Pt NPs were evaluated for their acute toxicity studies against aquatic organism, Daphnia magna.     

Graft copolymers via combination of cationic polymerization and atom transfer radical polymerization and their phase separation into spherical/worm-like nanostructures

Poly(p-chloromethyl styrene)-graft-poly(methyl methacrylate) (PCMS-g-PMMA) and poly(p-chloromethyl styrene)-graft-poly(benzyl methacrylate) (PCMS-g-PBzMA) graft copolymers with asymmetric branches are synthesized via the combination of cationic polymerization and atom transfer radical polymerization (ATRP). The process involves first, the preparation of poly(p-chloromethyl styrene) (PCMS-CH₂Cl) macroinitiator without any cross-linking or side reactions through pendant benzyl chloride (?CH₂Cl) functionality by cationic polymerization using a simple FeCl₃-based initiating system at 25 °C. The as-synthesized PCMS-CH₂Cl, without any transformation, is then used as the macroinitiator to graft PMMA and PBzMA branches by ATRP to produce PCMS-g-PMMA and PCMS-g-PBzMA graft copolymers of varying compositions with controlled molecular weight and moderately narrow polydispersities (M _w/M _n?≤?1.32). The resulting PCMS₂₁ -g-PMMA₂₃₂ graft copolymer in thin film form phase separates into spherical morphology with an average diameter of 170?±?72 nm. Whereas the PCMS₂₁ -g-PBzMA₁₅₆ graft copolymer gives worm-like nanostructures with an average length of 94 nm and width of 31 nm due to phase separation as visualized through atomic force microscopy. On the other hand, the phase-separated morphology is not very well-defined for other graft copolymers (PCMS₁₁₃ -g-PMMA₂₂₇ and PCMS₁₁₃ -g-PBzMA₁₅₄) thin films containing longer PCMS chains. This approach represents a rapid and convenient route to prepare unique spherical/worm-like polymer nanostructures. Figure

Well-defined poly(p-chloromethyl styrene)-graft-poly(methyl methacrylate) (PCMS-g-PMMA) and poly(p-chloromethyl styrene)-graft-poly(benzyl methacrylate) (PCMS-g-PBzMA) graft copolymers with asymmetric branches are synthesized by the combination of living cationic polymerization and atom transfer radical polymerization (ATRP). The resulting PCMS₂₁ -g-PMMA₂₃₂ and PCMS₂₁ -g-PBzMA₁₅₆ graft copolymers phase separate into nanostructured spherical and worm-like morphologies, respectively, in thin film form. The phase-separated morphology is not very well-defined for graft copolymers (PCMS₁₁₃ -g-PMMA₂₂₇ and PCMS₁₁₃ -g-PBzMA₁₅₄) thin films containing longer PCMS chains.     

Production and Characterization of a Novel Thermostable Extracellular Agarase from Pseudoalteromonas hodoensis Newly Isolated from the West Sea of South Korea

A Gram-negative, aerobic, motile, rod-shaped, agarolytic bacterium, designated as H7, was isolated from a coastal seawater sample. This strain grows at pH 6.0–8.0, temperature of 15–40 °C, and at an NaCl concentration of 1–7 % (w/v). Ubiquinone-8 was the predominant respiratory quinone, and the DNA G+C content was 45.82 mol%. Analysis of the 16S rRNA sequence suggests that strain H7 belongs to the genus Pseudoalteromonas. DNA-DNA hybridization analysis showed DNA relatedness of as low as 55.42 and 40.27 % with its nearest phylogenetic neighbors Pseudoalteromonas atlantica IAM12927^T and Pseudoalteromonas espejiana NCIMB2127^T, respectively, which led us to name H7 Pseudoalteromonas hodoensis sp. nov. The type strain is H7^T (=DSM25967^T = KCTC23887^T). An agarase (AgaA7) was purified to homogeneity from the cell-free culture broth of H7 through many steps of chromatography. Purified AgaA7 had an apparent molecular weight of 35 kDa, with a distinct NH₂-terminal sequence of Ala-Asp-Ala-Thr-X-Pro (X, any amino acid) from the reported proteins, implying that it is a novel enzyme. The optimum pH and temperature for agarase activity were 7.0 and 45 °C, respectively. Thin-layer chromatography analysis, mass spectrometry, and enzyme assay using p-nitrophenyl-α/β-D-galactopyranoside revealed that AgaA7 is both an exo- and endo-type β-agarase that degrades agarose into neoagarotetraose, neoagarohexaose, and neoagarooctaose (minor).     

Purification and Characterization of a Chymosin from Rhizopus microsporus var. rhizopodiformis

Purification and characterization of a chymosin from Rhizopus microsporus var. rhizopodiformis were investigated in the present study. A newly isolated R. microsporus var. rhizopodiformis F518 produced a high level of milk-clotting activity (1,001 SU/mL). A chymosin from the fungus was purified 3.66-fold with a recovery yield of 33.2 %. The enzyme appeared as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of 37.0 kDa. It was optimally active at 60 °C and was stable up to 40 °C. The purified enzyme was an acid protease with an optimum pH of 5.2 and retained 80 % of residual activity within pH 2.0–8.0. The inhibition of 96 and 100 % by pepstatin A at 0.01 and 0.02 mM, respectively, revealed that the enzyme is an aspartic protease. Thus, high milk-clotting activity of the chymosin with good stability will strengthen the potential use of the chymosin as a substitute for calf rennet in cheese manufacturing.     

Ultrasensitive detection and identification of BRAF V600 mutations in fresh frozen,FFPE, and plasma samples of melanoma patients by E-ice-COLD-PCR

A number of molecular diagnostic methods have been developed for the detection and identification of mutations in tumor samples, which are important for the choice of treatment in the context of personalized medicine. For the treatment of metastatic melanoma, Vemurafenib is recommended for patients with BRAF V600 activating mutations. However, the different assays developed to date for the detection of these mutations lack sensitivity or specificity or do not allow a sequencing-based identification or validation of the mutation. Recently, enhanced improved and complete enrichment co-amplification at lower denaturation temperature-polymerase chain reaction (E-ice-COLD-PCR) has been developed as a sensitive method for the detection and identification of mutations in KRAS codons 12/13. Here, we present the first E-ice-COLD-PCR assay for the detection and identification of BRAF codon 600 mutations, which has a large dynamic range, as 25 pg to 25 ng can be used as DNA input without any reduction in mutation enrichment efficiency, and which can detect down to 0.01 % of mutated alleles in a wild-type background. The assay has been validated on fresh frozen, formalin-fixed paraffin-embedded (FFPE), and plasma samples of melanoma patients and has allowed the detection and identification of BRAF mutations present in samples appearing as wild type using standard pyrosequencing, endpoint genotyping, or Sanger sequencing. Thus, the BRAF V600 E-ice-COLD-PCR assay is currently one of the most powerful molecular diagnostic tools for the ultrasensitive detection and identification of BRAF codon 600 mutations.     

Dehydrogenation of 4-aryl(hetaryl) spinacine derivatives with dimethyl sulfoxide

Aromatization of 4-aryl(hetaryl)tetrahydroimidazo[4,5-c]pyridine-6-carboxylic acids and their lithium salts by the action of dimethyl sulfoxide has been revealed for the first time. Heating of these compounds in DMSO for 5–7 h at 90–95°C leads to the formation of 4-aryl(hetaryl)imidazo[4,5-c]pyridine derivatives as a result of dehydrogenation and decarboxylation. Heating of the corresponding lithium salts generated in situ (DMSO, 90–95°C, 3–5 h) affords difficultly accessible 4-aryl(hetaryl)imidazo[4,5-c]pyridine-6-carboxylic acids.     

The Prognostic Value of Histidine-Rich Glycoprotein RNA in Breast Tissue Using Unmodified Gold Nanoparticles Assay

The aim of is this study is to explore the role of tissue histidine-rich glycoprotein (HRG) RNA as a promising clinically useful biomarker for breast cancer patients prognosis using nanogold assay. Expression of the HRG RNA was assessed by gold nanoparticles and conventional RT-PCR after purification by magnetic nanoparticles in breast tissue samples. The study included 120 patients, 60 of which were histologically proven breast carcinoma cases, 30 had benign breast lesions and 30 were healthy individuals who had undergone reductive plastic surgery. ER, PR and HER2 status were also investigated. The prognostic significance of tissue HRG RNA expression in breast cancer was explored. The magnetic nanoparticles coated with specific thiol modified oligonucleotide probe were used successfully in purification of HRG RNA from breast tissue total RNAs with satisfactory yield. The developed HRG AuNPs assay had a sensitivity and a specificity of 90 %, and a detection limit of 1.5 nmol/l. The concordance rate between the HRG AuNPs assay with RT-PCR after RNA purification using magnetic nanoparticles was 93.3 %. The median follow-up period was 60 months. Among traditional prognostic biomarkers, HRG was a significant independent prognostic marker in relapse-free survival (RFS). HRG RNA is an independent prognostic marker for breast cancer and can be detected using gold NPs assay, which is rapid, sensitive, specific, inexpensive to extend the value for breast cancer prognosis.     

Miscibility and crystallization behaviors of stereocomplex-type poly(l- and d-lactide)/poly(methyl methacrylate) blends

Stereocomplex-poly(l- and d-lactide) (sc-PLA) and poly(methyl methacrylate) (PMMA) blends were prepared by solution blending at PMMA loadings from 20 to 80 mass%. The miscibility and crystallization behaviors of the blends have been studied in detail by differential scanning calorimeter. The single-glass transition temperatures (T _g) of the blends demonstrated that the obtained system was miscible in the amorphous state. It was observed that the crystallization peak temperature of sc-PLA/PMMA blends was marginally lower than that of neat sc-PLA at various cooling rates, indicating the dilution effect of PMMA on the sc-PLA component to restrain the overall crystallization process. In the study of isothermal crystallization kinetics, the reciprocal value of crystallization peak time ( \( t_{\text{p}}^{ - 1} \) ) decreased with increasing PMMA content, indicating that the addition of non-crystalline PMMA inhibited the isothermal crystallization of sc-PLA at an identical crystallization temperature (T _c). Moreover, the negative value of Flory–Huggins interaction parameter (χ ₁₂ = ?0.16) of the blend further indicated that sc-PLA and PMMA formed miscible blends.     

PET imaging of sterile inflammation with a 18F-labeled bis(zinc(II)-dipicolylamine) complex

Small-molecular probe ¹⁸F-labeled bis(zinc(II)-dipicolylamine) complex (¹⁸F-FB-DPAZn2) was evaluated for PET imaging of sterile inflammation. In comparison with ¹⁸F-2-deoxy-β-d-glucose (¹⁸F-FDG), ¹⁸F-radiolabeled Annexin V (¹⁸F-FB-Annexin V) showed rapid clearance of radioactivity from the kidney and low uptake in most tissues. Both the lower radioactivity accumulation in brain and heart and the higher uptakes in the lung, liver, and intestine were observed for the biodistribution of ¹⁸F-FB-DPAZn2. In PET imaging, ¹⁸F-FDG showed significantly higher tumor and inflammation uptake than did of ¹⁸F-FB-DPAZn2 and ¹⁸F-FB-Annexin V in the S-180 fibrosarcoma mouse model and sterile inflammation mouse model. Both ¹⁸F-FB-DPAZn2 and ¹⁸F-FB-Annexin V performed the specifically localization in inflammation, and the ratios of inflammatory lesion-to-muscle and tumor-to-muscle were 1.83 ± 0.20 and 0.90 ± 0.12 (P < 0.05) for ¹⁸F-FB-DPAZn2, and 1.51 ± 0.14 and 1.21 ± 0.12 (P > 0.05) for ¹⁸F-FB-Annexin V, respectively. Terminal deoxynucleotide end-labeling (TUNEL) assays performed on the dissected tissues showed the significantly more TUNEL-positive nuclei in the inflammatory muscle than in tumor and normal muscle, and these TUNEL results correlated with the uptake of ¹⁸F-FB-DPAZn2 in dissected tissues. Biodistribution and PET imaging studies showed that the ¹⁸F-FB-DPAZn2 is suitable for imaging sterile inflammation in vivo and is capable of the differentiating tumor from inflammation.     

Synthesis,characterization and self-assembly of amphiphilic block copolymer poly(4-vinyl benzene chloride)-b-poly(N-vinylpyrrolidone) in aqueous solution

The synthesis, characterization and self-assembly of a novel amphiphilic block copolymer containing a poly(N-vinylpyrrolidone) as a segment of hydrophilic and poly(4-vinyl benzene chloride) (PVBC) arms are reported. The copolymer was characterized by FT-IR spectroscopy ¹H NMR. The composition and the molecular weights of the block copolymers were established using gel permeation chromatography and ¹H NMR. The water-soluble fraction of poly(N-vinyl-2-pyrrolidone) (PVP)/PVBC block copolymers formed micelles which were investigated at 25 °C in water at 5 mg/ml concentration using a tensiometer. The morphology of micelles in aqueous solution was determined by the AFM, SANS, and SAXS methods.