TARGETING OF PHOTOTOXIC DRUGS TO ANTIGEN-SPECIFIC T LYMPHOCYTES in vitro USING ANTIGEN-PRESENTING CELL MEMBRANES

We have used the complex of antigen with class II major histocompatibility proteins (la) in membrane-bound form to target a phototoxic compound to antigen-specific T cell hybridomas in vitro. The iodoacetamidyl ester of phototoxic pyrene was bound covalently to antigen-presenting cells (APC), and protein antigens were added to the cells for processing, presentation and targeting of the drug to three different T hybridomas specific for myelin basic protein (MBP), ovalbumin (OVA) and keyhole limpet hemocyanin (KLH). The B hybridoma LS102.9 was used as APC to present MBP, KLH and either a tryptic digest of OVA or the synthetic peptide OVA323–339to these T cells. A transformed B lymphoma, which expresses trinitrophenol (TNP)-specific surface IgM, A20-HL, was used to present TNP conjugates of KLH and OVA to T cells. Either the antigen-bearing intact APC or Ia⁺ membranes shed spontaneously from them were used as drug carriers to target pyrene to the T cells. In the dark, or in the absence of pyrene, both the intact APC or the shed membranes stimulated interleukin-2 (IL-2) production by the T cells in an antigen-specific way. After UVA (320–400nm) irradiation, both forms of these drug carriers had an antigen-specific toxic effect on the T hybridoma cells with receptors for the antigen that they carried. Both spontaneous T cell proliferation and antigen-induced IL-2 production were inhibited. The shed membranes had a more antigen-specific toxic effect than the intact APC, which tend to settle out with the T cells in the microtiter plates, possibly causing nonspecific contact. These results indicate that the antigen-la complex in membrane-bound form can be used to antigen-target cytotoxic drugs to antigen-specific T cells. The Ia⁺ membranes shed from APC may be useful to target drugs to antigen-specific clones of T cells in vivo.     

Enzymatic digestion and chromatographic analysis of arsenic species released from proteins

A method combining gel filtration chromatography (GFC), protease digestion, and ion pair chromatography with inductively coupled plasma mass spectrometry detection was developed for the determination of arsenic species bound to proteins. The method was first established by examining the interactions of two model proteins, metallothionein (MT) and hemoglobin, with three reactive trivalent arsenic species. It was then successfully applied to the speciation of arsenic in red blood cells of rats. Inorganic arsenite (iAs^III), monomethylarsonous acid (MMA^III), and dimethylarsinous acid (DMA^III) were efficiently released from the proteins by protease digestion at pH 8.0, with the recovery ranging from 93% to 106%. There was no oxidation of iAs^III or MMA^III during the protease digestion process. Up to 61% DMA^III (the least stable arsenic species) was unchanged, and the rest was oxidized to the pentavalent dimethylarsinic acid (DMA^V). The arsenic species in the red blood cells of control rats was present as DMA^III complex with hemoglobin. The method enabling the determination of the specific arsenic species that bind to cellular proteins is potentially useful for studying arsenic distribution, metabolism, and toxicity.     

High-yield bacillus subtilis protease production by solid-state fermentation

A Bacillus subtilis isolate was shown to be able to produce extracellular protease in solid-state fermentations (SSF) using soy cake as culture medium. A significant effect of inoculum concentration and physiological age on protease production was observed. Maximum activities were obtained for inocula consisting of exponentially growing cells at inoculum concentrations in the range of 0.7–2.0 mg g⁻¹. A comparative study on the influence of cultivation temperature and initial medium pH on protease production in SSF and in submerged fermentation (SF) revealed that in SSF a broader pH range (5–10), but the same optimum temperature (37°C), is obtained when compared to SF. A kinetic study showed that enzyme production is associated with bacterial growth and that enzyme inactivation begins before biomass reaches a maximum level for both SF and SSF. Maximum protease activity and productivity were 960 U g⁻¹ and 15.4 U g⁻¹ h⁻¹ for SSF, and 12 U mL⁻¹ and 1.3 U mL⁻¹ h⁻¹ for SF. When SSF protease activity was expressed by volume of enzyme extract, the enzyme level was 10-fold higher and the enzyme productivity 45% higher than in SF. These results indicate that this bacterial strain shows a high biotechnological potential for protease production in solid-state fermentation.     

An Estimation Technique of Molecular Weights of Bio-Oligomers by Reversed-Phase Liquid Chromatography

Abstract

A new simple and accurate method for molecular weight estimation of oligomers by reversed-phase liquid chromatography was developed and has been proposed to use in the investigation of bio-oligomers. Capacity factors (k′) of 24 peptides and proteins ranging in molecular weight from 200 to 70000 were measured independently under isocratic eluting conditions with slightly different acetonitrile contents in the mobile phase. Profiles of plots of the log k' values of the peptides and proteins against the acetonitrile contents were on straight lines with different slopes. These slopes were characteristic for each oligomer and found to be proportional to the two/thirds power of the molecular weight. This elution behavior of oligomers in reversed-phase liquid chromatography can be explained in terms of the solvophobic theory. The estimated molecular weights of several bio-oligomers were more accurate than those obtained by size-exclusion chromatography. when the range of molecular weights was limited from 10³ to 2×10⁴.     

Comparison of 125I-Labeled and 14C-Labeled Peptides of the Major outer Membrane Protein of Chlamydia Trachomatis Strain L2/434 separated by High-Performance Liquid Chromatography

Abstract

The objective of this study was to determine if in-gel chloramine-T radioiodination adequately labels OM proteins to allow for accurate and precise structural comparison of these molecules. Therefore, intrinsically ¹⁴C-amino acid labeled proteins and ¹²⁵I-labeled proteins were cleaved with two endopeptidic reagents and the peptide fragments separated by HPLC. A comparison of retention times of the fragments, as determined by differential radiation counting, thus indicated whether ¹²⁵I-labeling identified of all the peptide peaks seen in the ¹⁴C-labeled proteins. Results demonstrated that radioiodination yields complete and accurate information about the primary structure of outer membrane proteins. In addition, it permits the use of extremely small amounts of protein allowing for method optimization and multiple separations to insure reproducibility.     

Analysis of Cyanide in Blood by Headspace-Isotope-Dilution-GC-MS

Abstract

An uncomplicated, rapid, automated procedure for the analysis of low cyanide concentrations in whole blood is reported. The analysis was performed by headspace gas chromatography and mass spectrometry in the (¹H¹²C¹⁴N) and m/z 29 (¹H¹³C¹⁵N). Carryover from cyanide adsorption onto the surface of the needle was prevented by developing a new method that enabled automated flushing of the needle in between each cyanide analysis. Results were compared of ordinary calibrations and those of isotope dilutions. The total time of analysis was 18 min for a single cyanide analysis.     

New silica hybrids elaborated by sol-gel process from bifunctional thiadiazole and 1,2,4-triazole precursors

New hybrid materials were synthesized from an inorganic precursor, (tetraethoxysilane: TEOS) and bifunctional organic precursors through the sol-gel process. Indeed, monolithic and transparent hybrid gels were obtained by the reaction of tetraethoxysilicate (TEOS) with 1,2,4-thiadiazole-3,5-diamine (1), 5-amino-1,3,4-thiadiazole-2-thiol (2), and 1H-1,2,4-triazole-3-thiol (3) in ethanol as solvent, using chloric acid (HCl 10^?1M) as catalyst. It is shown that TEOS has been cross-linked with bi-functional heterocyclic compounds: (1), (2) and (3) leading to the formation of transparent and colorous gels. The characterization was realized by infrared, ²⁹Si and ¹³C CP MAS NMR. The xerogels morphology and texture were studied by scanning electron microscopy (SEM) and Brunauer – Emmet – Teller method (BET). The obtained results show that organic adducts (1), (2) and (3) react with TEOS leading to gels in which Si–N and Si–S bridges were formed. According to N₂-physisorption results, xerogels are mesoporous with specific surface area varied from 105 to 312 m²g^?1 and their isotherms are classified as type IV. The optic and magnetic properties of hybrids were studied respectively by UV-Vis spectroscopy and RPE. This study showed that all materials are paramagnetic semiconductors.     

Carbazole-driven ratiometric fluorescence turn on for dual ion recognition of Zn2+ and Hg2+ by thiophene-pyridyl conjugate in HEPES buffer medium: spectroscopy,computational, microscopy and cellular studies

Carbazole-based thiophene-pyridyl conjugate (L) was synthesised and characterised. The complexation between L with Zn²⁺ and Hg²⁺ was studied in HEPES buffer medium by fluorescence, absorption and visual colour change with the detection limit of ~3.7 and ~4.8 μM, respectively. The L detects Zn²⁺ by bringing ratiometric change in the fluorescence signals at 418 and 515 nm, but in the case of Hg²⁺, the signals are observed at 418 and 365 nm, while no new band is observed at 515 nm. The structure of L has been established by single-crystal XRD and that of complexes [ZnL] and [HgL] by density functional theory calculations. TDDFT calculations were performed in order to demonstrate the electronic properties of receptors and their zinc and mercury complexes. The isolated fluorescent complexes [ZnL] and [HgL] were found to be sensitive and selective towards phosphate-bearing ions and sulfide ions, respectively, among the other anions studied. The nanostructural features such as shape and size obtained using atomic force microscopy distinguish L from its complexes formed between L and Zn²⁺ from that formed with Hg²⁺. Moreover, the utility of the conjugate L in showing the zinc recognition in live cells has also been demonstrated using RAW cells as monitored by fluorescence imaging.     

Purification and Biochemical Characterization of a Novel Alkaline Protease Produced by Penicillium nalgiovense

Penicillium nalgiovense PNA9 produces an extracellular protease during fermentation with characteristics of growth-associated product. Enzyme purification involved ammonium sulfate precipitation, dialysis, and ultrafiltration, resulting in 12.1-fold increase of specific activity (19.5 U/mg). The protein was isolated through a series of BN-PAGE and native PAGE runs. ESI-MS analysis confirmed the molecular mass of 45.2 kDa. N-Terminal sequencing (MGFLKLLKGSLATLAVVNAGKLLTANDGDE) revealed 93 % similarity to a Penicillium chrysogenum protease, identified as major allergen. The protease exhibits simple Michaelis-Menten kinetics and K _m (1.152 mg/ml), V _max (0.827 mg/ml/min), and k _cat (3.2?×?10²) (1/s) values against azocasein show that it possesses high substrate affinity and catalytic efficiency. The protease is active within 10–45 °C, pH 4.0–10.0, and 0–3 M NaCl, while maximum activity was observed at 35 °C, pH 8.0, and 0.25 M NaCl. It is active against the muscle proteins actin and myosin and inactive against myoglobin. It is highly stable in the presence of non-ionic surfactants, hydrogen peroxide, BTNB, and EDTA. Activity was inhibited by SDS, Mn²⁺ and Zn²⁺, and by the serine protease inhibitor PMSF, indicating the serine protease nature of the enzyme. These properties make the novel protease a suitable candidate enzyme in meat ripening and other biotechnological applications.     

Binding Modes of Thioflavin T on the Surface of Amyloid Fibrils Studied by NMR

The mechanism for the interaction of thioflavin T (ThT) with amyloid fibrils at the molecular level is not known. Here, we used ¹H NMR spectroscopy to determine the binding mode of ThT on the surface of fibrils from lysozyme and insulin. Relayed rotating‐frame Overhauser enhancements in ThT were observed, indicating that the orientation of ThT is orthogonal to the fibril surface. Importantly, the assembly state of ThT on both surfaces is different. On the surface of insulin fibrils, ThT is oligomeric, as indicated by rapid ¹H spin‐lattice relaxation rate in the rotating frame (R_1ρ), presumably due to intermolecular dipole–dipole interactions between ThT molecules. In contrast, ThT on the surface of lysozyme fibrils is a monomer, as indicated by slower ¹H R_1ρ. These results shed new light into the mechanism for the enhancement of ThT fluorescence and may lead to more efficient detectors of amyloid assemblies, which have escaped detection by ThT in monomer form.     

Adding Value to the Oil Cake as a Waste from Oil Processing Industry: Production of Lipase and Protease by <Emphasis Type="Italic">Candida utilis</Emphasis> in Solid State Fermentation

Olive oil cake is a by-product from the olive oil processing industry and can be used for the lipase and protease production by Candida utilis in solid state fermentation. Different carbon and nitrogen sources were evaluated, and the results showed that the supplementation of the substrate with maltose and starch as carbon sources and yeast extract as a nitrogen source significantly increased the lipase production. The best results were obtained with maltose, whereas rather low lipase and protease activities were found with glucose and oleic acid. Response surface methodology and a five-level–three-factor central composite rotatable design were used to evaluate the effects of the initial moisture content, inoculum size and fermentation time on both lipase and protease activity levels. A lipase activity value of ≈25 U g^-1 and a protease activity value of 110 U g^-1 were obtained under the optimized fermentation conditions. An alkaline treatment of the substrate appeared to be efficient, leading to increases of 39% and 133% in the lipase and protease production, respectively. The results showed that the olive cake could be a good source for enzyme production by solid state fermentation.     

Photochemical properties and sensor applications of modified yellow fluorescent protein (YFP) covalently attached to the surfaces of etched optical fibers (EOFs)

Fluorescent proteins have the inherent ability to act as sensing components which function both in vitro and inside living cells. We describe here a novel study on a covalent site-specific bonding of fluorescent proteins to form self-assembled monolayers (SAMs) on the surface of etched optical fibers (EOFs). Deposition of fluorescent proteins on EOFs gives the opportunity to increase the interaction of guided light with deposited molecules relative to plane glass surfaces. The EOF modification is carried out by surface activation using 3-aminopropylthrimethoxysilane (APTMS) and bifunctional crosslinker sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC) which exposes sulfhydryl-reactive maleimide groups followed by covalent site-specific coupling of modified yellow fluorescent protein (YFP). Steady-state and fluorescence lifetime measurements confirm the formation of SAM. The sensor applications of YPF SAMs on EOF are demonstrated by the gradual increase of emission intensity upon addition of Ca²⁺ ions in the concentration range from a few tens of micromolars up to a few tens of millimolars. The studies on the effect of pH, divalent cations, denaturing agents, and proteases reveal the stability of YFP on EOFs at normal physiological conditions. However, treatments with 0.5% SDS at pH 8.5 and protease trypsin are found to denaturate or cleave the YFP from fiber surfaces.     

Characterization of modified silica powders by fourier transform infrared spectroscopy and cross-polarization magic angle spinning NMR

Silica gel and Cab-O-Sil were chemically modified (silylated) with 3-aminopropyltriethoxysilane and 3-methacryloxypropyltrimethoxysilane under carefully controlled conditions. Subsequently the products were investigated by elemental analysis, Fourier transform IR spectroscopy, and ¹³C and ²⁹Si cross-polarization magic angle spinning NMR (CP-MAS NMR). The influence of the reaction conditions of the silylation and the effect of subsequent heat treatment and water addition were studied. The resulting differences shed new light on the combined effects of reaction conditions and silica surface structures on the course of the reactions. Some assignments of ²⁹Si NMR signals to specific structures were confirmed, while in one case a reassignment was proposed.     

Surface markers on mouse cells transformed after exposure to HeLa chromosomes are mycoplasma membrane proteins

Antisera were raised against HeLa cells and mouse cells transformed after exposure to HeLa chromosomes (ME-ch.HeLa). The antisera were positive in indirect immunofluorescence assays on both HeLa and ME-ch.HeLa cells, but were negative on normal mouse cells. Immunoprecipitation of ¹²⁵I-labelled cell extracts showed that ME-ch.HeLa cells contain at their surface 3 proteins of apparent molecular weights of 185,000, 105,000 and 45,000 daltons, which were also present on the surface of our HeLa cells but not on other mouse cell lines tested. However, further study has shown that these proteins are not normal constituents of HeLa plasma membranes but are in fact surface proteins of Mycoplasma orale.     

New self-assemblies with zinc meso-tetra[3-(1H-imidazol-1-yl)phenyl] porphyrin for use in supramolecular solar cells

In this work, a new zinc meso-tetra[3-(1H-imidazol-1-yl)phenyl]porphyrin (ZnP) was synthesized. Further, the porphyrin ZnP was immobilized by metal-ligand axial coordination (ZnP-A) and a metal-ligand edged binding approach (ZnP-Zn-A) on the nanostructured TiO₂ electrode surface modified with coordinating ligand functionality, isonicotinic acid (A). The performances of the assemblies-sensitized solar cells were performed under irradiance of 100 mW?cm^?2 AM 1.5G sunlight. Photo-electrochemical studies reveal significantly improved performance of the assembly ZnP-A. These assemblies can afford a fertile base for further design and fabrication of new supramolecular solar cells in future.     

Purification, Characterization, and Specificity Determination of a New Serine Protease Secreted by Penicillium waksmanii

The purpose of this work was to purify a protease from Penicillium waksmanii and to determine its biochemical characteristics and specificity. The extracellular protease isolated that was produced by P. waksmanii is a serine protease that is essential for the reproduction and growth of the fungus. The protease isolated showed 32 kDa, and has optimal activity at pH 8.0 and 35 °C towards the substrate Abz-KLRSSKQ-EDDnp. The protease is active in the presence of CaCl₂, KCl, and BaCl, and partially inhibited by CuCl₂, CoCl₂ and totally inhibited by AlCl₃ and LiCl. In the presence of 1 M urea, the protease remains 50 % active. The activity of the protease increases 60 % when it is exposed to 0.4 % nonionic surfactant-Triton X-100 and loses 10 % activity in the presence of 0.4 % Tween-80. Using fluorescence resonance energy transfer analysis, the protease showed the most specificity for the peptide Abz-KIRSSKQ-EDDnp with k _cat/K _m of 10,666 mM^?1?s^?1, followed by the peptide Abz-GLRSSKQ-EDDnp with a k _cat/K _m of 7,500 mM^?1?s^?1. Basic and acidic side chain-containing amino acids performed best at subsite S₁. Subsites S₂, S₃, S^′ ₂, and S^′ ₁, S^′ ₃ showed a preference for binding for amino acids with hydrophobic and basic amino acid side chain, respectively. High values of k _cat/K _m were observed for the subsites S₂, S₃, and S^′ _2. The sequence of the N-terminus (ANVVQSNVPSWGLARLSSKKTGTTDYTYD) showed high similarity to the fungi Penicillium citrinum and Penicillium chrysogenum, with 89 % of identity at the amino acid level.     

Purification and characterization of organic solvent stable serine alkaline protease from newly isolated Bacillus circulans M34

A protease from newly isolated Bacillus circulans M34 was purified by Q‐Sepharose anion exchange chromatography and Sepharose–bacitracin affinity chromatography followed by (NH₄)₂SO₄ precipitation. The molecular mass of the purified enzyme was determined using SDS–PAGE. The optimum pH and temperature for protease activity were 11 and 50°C, respectively. The effect of various metal ions on protease activity was investigated. Alkaline protease from Bacillus circulans M34 wase activated by Zn²⁺, Cu²⁺ and Co²⁺ up to 31%. The purified protease was found to be stable in the organic solvents, surfactants and oxidizing agent. The substrate specificity of purified protease was investigated towards different substrates. The protease was almost completely inhibited by the serine protease inhibitor phenylmethanesulfonyl fluoride. The kinetic parameters of the purified protease, maximum rate (V_max) and Michaelis constant (K_m), were determined using a Lineweaver–Burk plot. Copyright © 2015 John Wiley & Sons, Ltd.     

NMR parameters of the tetrahedrane [Fe2(CO)6(μ-SNH)], studied by experiment and DFT

The intramolecular dynamic behavior of the tetrahedrane-type cluster [Fe₂(CO)₆(μ-SNH)] 1 was studied by ¹³C NMR spectroscopy. The ⁵⁷Fe chemical shift of 1 and the coupling constants ¹ J(⁵⁷Fe,¹³C) were measured. These NMR parameters, and also ¹ J(⁵⁷Fe,¹⁵N), were found to be in good agreement with data calculated by using density functional theory (DFT) methods (B3LYP), based on the geometry calculated at the 6-311+G(d,p) level of theory. The isolobal replacement of the Fe(CO)₃ with BH fragments leads to the tetrahedranes [Fe(CO)₃(BH)(μ-SNH)] 2 and [(HB)₂(μ-SNH)] 3. Both were identified by calculations as minima on the respective potential energy surface (PES). However, the tetrahedrane-type structure of 3 is much higher in energy when compared with the planar cyclic isomers 3a and 3b.     

A new member of the cationic dinitrosyl iron complexes family incorporating N-ethylthiourea is effective against human HeLa and MCF-7 tumor cell lines

A new analog of the active site of mononuclear dinitrosyl [1Fe–2S] proteins, [C₃N₂H₈SFe(NO)₂Cl][Fe(NO)₂(C₃N₂H₈S)₂]⁺Cl^? (I), has been synthesized by reacting NO with an aqueous mixture of iron(II) sulfate and N-ethylthiourea in acidic medium. The structure and properties of the complex were studied by X-ray diffraction, IR, Mössbauer, and EPR spectroscopy, in addition to quantum chemical calculations. Complex I spontaneously generates NO in protic media. The cytotoxicity of I was investigated against human cervical carcinoma (HeLa), breast cancer (MCF7), and non-immortalized (FetMCS) cell lines. The cytotoxicity of I against HeLa is similar to that of anticancer agents currently used clinically (platinum complexes), but I is 10 times less toxic in normal cells. The cytotoxicity of MCF7 cells to I is low.     

Screening and Characteristics of Marine Bacillus velezensis Z-1 Protease and Its Application of Enzymatic Hydrolysis of Mussels to Prepare Antioxidant Active Substances

Bacillus velezensis is a type of microorganism that is beneficial to humans and animals. In this work, a protease-producing B. velezensis strain Z-1 was screened from sludge in the sea area near Qingdao (deposit number CGMCC No. 25059). The response surface methodology was used to analyze protease production, and the optimal temperature was 37.09 °C and pH 7.73 with the addition of 0.42% NaCl, resulting in maximum protease production of 17.64 U/mL. The optimum reaction temperature and pH of the protease of strain Z-1 were 60 °C and 9.0, respectively. The protease had good temperature and pH stability, and good stability in solvents such as methanol, ethanol and Tween 80. Ammonium, NH₄⁺,and Mn²⁺ significantly promoted enzyme activity, while Zn²⁺ significantly inhibited the enzyme activity. The protease produced by strain Z-1 was used for the enzymolysis of mussel meat. The mussel hydrolysate exhibited good antioxidant function, with a DPPH free radical removal rate of 75.3%, a hydroxyl free radical removal rate of 75.9%, and a superoxide anion removal rate of 84.4%. This study provides a reference for the application of B. velez protease and the diverse processing applications of mussel meat.