Comparative evaluation of five protocols for protein extraction from stony corals (Scleractinia) for proteomics

Corals especially the reef‐building species are very important to marine ecosystems. Proteomics has been used for researches on coral diseases, bleaching and responses to the environment change. A robust and versatile protein extraction protocol is required for coral proteomics. However, a comparative evaluation of different protein extraction protocols is still not available for proteomic analysis of stony corals. In the present study, five protocols were compared for protein extraction from stony corals. The five protocols were TRIzol, phenol‐based extraction (PBE), trichloroacetic acid (TCA)‐acetone, glass bead‐assisted extraction (GBAE) and a commercially available kit. PBE, TRIzol and the commercial kit were more robust for extracting proteins from stony corals. The protein extraction efficiency and repeatability, two dimensional electrophoresis (2‐DE) and matrix‐assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF MS) were employed to evaluate the protocols. The results indicated that PBE protocol had the better protein extraction efficiency than the others. Protein extraction coverage varied among the procedures. Each protocol favored for certain proteins. Therefore, it is very important for coral proteomic analysis to select a suitable protein protocol upon the experimental design. In general, PBE protocol can be the first choice for extracting proteins from stony corals.     

Synthesis and characterization of poly(butylene terephthalates) modified by hydroquinone bis(2-hydroxyethyl)ether

A series of thermoplastic poly(butylene-co-hydroquinone bis(2-hydroxyethyl)ether terephthalates) (PBHT), with different molar ratios of hydroquinone bis(2-hydroxyethyl)ether (HQEE)/1,4-butanediol 9/91, 18/82 and 27/73, were synthesized via melt polycondensation. The compositions, thermodynamics and crystallization properties of the obtained copolyesters were characterized in detail by ¹H NMR, differential scanning calorimeters (DSC), thermogravimetric analysis (TGA) and X-ray diffraction (XRD). These results showed that the PBHTs were successfully synthesized, and the incorporation of the HQEE group significantly improved thermal properties of the polymers. However, HQEE did not change the crystal structure of PBT. The T_m values of the copolymers decreased (from 208?°C to 174?°C) with increasing content of HQEE segments, on the contrary, T_g values increased (from 37?°C to 43?°C). The temperatures for 5% weight loss did not decrease and appeared at a range of 373–377?°C.     

Sugarcane proteomics: Establishment of a protein extraction method for 2‐DE in stalk tissues and initiation of sugarcane proteome reference map

Sugarcane is an important commercial crop cultivated for its stalks and sugar is a prized commodity essential in human nutrition. Proteomics of sugarcane is in its infancy, especially when dealing with the stalk tissues, where there is no study to date. A systematic proteome analysis of stalk tissue yet remains to be investigated in sugarcane, wherein the stalk tissue is well known for its rigidity, fibrous nature, and the presence of oxidative enzymes, phenolic compounds and extreme levels of carbohydrates, thus making the protein extraction complicated. Here, we evaluated five different protein extraction methods in sugarcane stalk tissues. These methods are as follows: direct extraction using lysis buffer (LB), TCA/acetone precipitation followed by solubilization in LB, LB containing thiourea (LBT), and LBT containing tris, and phenol extraction. Both quantitative and qualitative protein analyses were performed for each method. 2‐DE analysis of extracted total proteins revealed distinct differences in protein patterns among the methods, which might be due to their physicochemical limitations. Based on the 2‐D gel protein profiles, TCA/acetone precipitation‐LBT and phenol extraction methods showed good results. The phenol method showed a shift in pI values of proteins on 2‐D gel, which was mostly overcome by the use of 2‐D cleanup kit after protein extraction. Among all the methods tested, 2‐D cleanup‐phenol method was found to be the most suitable for producing high number of good‐quality spots and reproducibility. In total, 30 and 12 protein spots commonly present in LB, LBT and phenol methods, and LBT method were selected and subjected to eLD‐IT‐TOF‐MS/MS and nESI‐LC‐MS/MS analyses, respectively, and a reference map has been established for sugarcane stalk tissue proteome. A total of 36 nonredundant proteins were identified. This is a very first basic study on sugarcane stalk proteome analysis and will promote the unexplored areas of sugarcane proteome research.     

Systematic re-evaluation of the bis(2-hydroxyethyl)disulfide (HEDS) assay reveals an alternative mechanism and activity of glutaredoxins

The reduction of bis(2-hydroxyethyl)disulfide (HEDS) by reduced glutathione (GSH) is the most commonly used assay to analyze the presence and properties of enzymatically active glutaredoxins (Grx), a family of central redox proteins in eukaryotes and glutathione-utilizing prokaryotes. Enzymatically active Grx usually prefer glutathionylated disulfide substrates. These are converted via a ping-pong mechanism. Sequential kinetic patterns for the HEDS assay have therefore been puzzling since 1991. Here we established a novel assay and used the model enzyme ScGrx7 from yeast and PfGrx from Plasmodium falciparum to test several possible causes for the sequential kinetics such as pre-enzymatic GSH depletion, simultaneous binding of a glutathionylated substrate and GSH, as well as substrate or product inhibition. Furthermore, we analyzed the non-enzymatic reaction between HEDS and GSH by HPLC and mass spectrometry suggesting that such a reaction is too slow to explain high Grx activities in the assay. The most plausible interpretation of our results is a direct Grx-catalyzed reduction of HEDS. Physiological implications of this alternative mechanism and of the Grx-catalyzed reduction of non-glutathione disulfide substrates are discussed.     

Uncatalyzed,One-pot Synthesis of 3,3'-(Benzylene)bis(4-hydroxy-2H-chromen-2-one)Derivatives under Thermal Solvent-free Conditions

A series of 3,3′‐(benzylene)bis(4‐hydroxy‐2H‐chromen‐2‐one) derivatives have been synthesized from condensation reactions of 4‐hydroxycoumarin with benzaldehydes under thermal solvent‐free conditions. This environmentally friendly synthesis is uncatalyzed and affords the desired products in excellent yields.     

Improved resolution in the acidic and basic region of 2‐DE of insect antennae proteins using hydroxyethyl disulfide

Olfaction is essential for processing chemical signals in insects, but characterizing the proteins implicated in this process has proved challenging. We optimized 2‐DE gel resolution of insect proteins by using a buffer containing two reducing agents, DTT and hydroxyethyl disulfide. This buffer clearly improved resolution and decreased spot streaking and spot trains of 2‐DE in comparison to DTT alone. We described for the first time that the buffer with DTT and hydroxyethyl disulfide further to reducing streaking in the basic part of the gel eliminates false spots in the acidic gel regions that appeared when only DTT was used as reducing agent.     

Trans‐bis(saccharinato)cadmium(II) Complexes with 2‐Aminomethylpyridine and 2‐Aminoethylpyridine — Synthesis,Crystal Structures,Spectral and Thermal Characterization of trans‐[Cd(sac)2(ampy)2] and trans‐[Cd(sac)2(aepy)2]

Two trans‐bis(saccharinato) (sac) complexes of cadmium(II ) with 2‐aminomethylpyridine (ampy) and 2‐aminoethylpyridine (aepy) were synthesized and characterized by means of elemental analysis, FT‐IR spectroscopy and thermal analysis. In addition, their solid‐state structures were determined by single crystal X‐ray diffraction studies. The [Cd(sac)₂(ampy)₂] ( 1 ) and [Cd(sac)₂(aepy)] ( 2 ) complexes consist of neutral monomeric units and crystallize in the orthorhombic (Pbca) and monoclinic (P2₁/c) crystal systems, respectively. The cadmium(II ) ions in 1 and 2 sit on inversion centres andexhibit distorted octahedral coordination by two sac anions and two aminopyridine ligands. The sac ligands in both complexes are N‐coordinated and located in trans positions, while the ampy and aepy ligands act as a bidentate ligand forming two symmetrically chelate rings around cadmium(II ). IR spectra and thermal decompositions of the complexes are also discussed.     

A polarographic study of the complexes formed in alkaline solution by the cadmium(II) ion with ethylenediamine,N-(2-hydroxyethyl)ethylenediamine,and N,N′-bis(2-hydroxyethyl)ethylenediamine

A multiple regression analysis of polarographic data has been used to determine the formulas and formation constants of complexes formed in alkaline solution by reaction of cadmium(II) ion and hydroxide ion with ethylenediamine (en), N-(2-hydroxyethyl)-ethylenediamine (hn) and N,N′-bis(2-hydroxyethyl)ethylenediamine (2hn). The complexes formed are designated by the general formula Cd(A)_p(OH)_p^2?q and the formation constants are given as log β_pq. The complexes found and their formation constants are: for en, 1 : 2 (10.3), 1 : 3 (12.3), and 1 : 2 : 1 (12.2); for hn, 1 : 2 (9.5), 1 : 2 : 1 (12.2), and 1 : 2 : 2 (12.6); and for 2hn, 1 : 2 (8.9), 1 : 1 : 2 (11.1), 1 : 2 : 1 (11.2), and 1 : 2 : 2 (12.6). It is concluded that in each case for which the hydroxide ion is reacted, a proton is removed from an alcoholic hydroxyl group which is coordinated to form a five-membered chelate ring linking a nitrogen atom and oxygen atom to the cadmium(II) ion.     

Synthesis,crystal structures,and redox behavior of bis(2-phenylindenyl) nickel and bis(2-phenylindenyl)zirconium dichloride

Redox reactions of the sandwich jn⁵-(Ph)Ind]₂Ni complex (1) (where (Ph)Ind is the 2-plrenylindenyl anion) in CH₂CI₂ and of the bent-sandwich (⁵-(Plr)Ind]₂ZrCl₂ complex (2) in THF have been studied by low-temperature cyclic voltammetry. Complex ( undergoes quasi-reversible two-step oxidation to cation i+ and dication 12+, which are stable at room temperature within the cyclic voltammetry time scale. Two-electron reduction of complex 1 is irreversible up to -50 °C, and this process is accompanied by destruction of the sandwich structure of the complex. Redaction of complex 2 is described by the conventional scheme for bent-sandwich complexes. According to this scheme, further one-electron reduction of radical anion 2- generates dianion 2^2–, which eliminates two CI^– ions to afford bisindcnyl sandwich complex (⁵-(Ph)lnd]₂Zr (3). This complex is stable at T < -45 °C within the cyclic voltammetry time scale and is capable of undergoing one-electron reduction to the corresponding radical anion 3-. Synthesis and crystal structures of complexes 1 and 2 are reported.Translated fromIzvestiya Akademii Nauk. Seriya Kidmicheskaya, No. 6, pp. 1529–1536, June, 1996.     

Metal Derivatives of Heterocyclic‐2‐thiones: Crystal Structures of [Bis(diphenylphosphanyl)propane][bis(pyrimidine‐2‐thiolato)]ruthenium(II) and [Bis(diphenylphosphanyl)methane][bis(pyridine‐2‐thiolato)]ruthenium(II)

Ruthenium(II) Complexes containing pyrimidine‐2‐thiolate (pymS^–) and bis(diphenylphosphanyl)alkanes [Ph₂P–(CH₂)_m–PPh₂, m = 1, dppm; m = 2, dppe; m = 3, dppp; m = 4, dppb] are described. Reactions of [RuCl₂L₂] (L = dppm, dppp) and [Ru₂Cl₄L₃] (L = dppb) with pyrimidine‐2‐thione (pymSH) in 1:2 molar ratio in dry benzene in the presence of Et₃N base yielded the [Ru(pymS)₂L] complexes (pymS^– = pyrimidine‐2‐thiolate; L = dppm ( 1 ); dppp ( 3 ); dppb ( 4 )). The complex [Ru(pymS)₂(dppe)] ( 2 ) was indirectly prepared by the reaction of [Ru(pymS)₂(PPh₃)₂] with dppe. These complexes were characterized using analytical data, IR, ¹H, ¹³C, ³¹P NMR spectroscopy, and X‐ray crystallography (complex 3 ). The crystal structure of the analogous complex [Ru(pyS)₂(dppm)] ( 5 ) with the ligand pyridine‐2‐thiolate (pyS^–) was also described. X‐ray crystallographic investigation of complex 3 has shown two four‐membered chelate rings (N, S donors) and one six‐membered ring (P, P donors) around the metal atom. Compound 5 provides the first example in which Ru^II has three four‐membered chelate rings: two made up by N, S donor ligands and one made up by P, P donor ligand. The arrangement around the metal atoms in each complex is distorted octahedral with cis:cis:trans:P, P:N, N:S, S dispositions of the donor atoms. The ³¹P NMR spectroscopic data revealed that the complexes are static in solution, except 2 , which showed the presence of more than one species.     

1H and 13C NMR assignments for new heterocyclic TAM leuco dyes, (2Z,2'E)-2,2'-(2-phenyl propane-1,3-diylidene) bis(1,3,3-trimethylindoline) derivatives. Part II

The (1)H and (13)C NMR spectra of the novel heterocyclic Leuco-TAM dyes, (2Z, 2'E)-2,2'-(2-phenyl propane-1,3-diylidene) bis(1,3,3-trimethylindoline) derivatives 1-4 as precursors of triarylmethane (TAM)(+) (Malachite Green FB-analog) dyes were completely assigned by 1D and 2D NMR experiments, including DEPT, COSY, HSQC, HMBC, and NOESY. Especially, the diastereotopic gem-dimethyl protons at the C3 and C3' positions of the FB rings were definitively assigned. The (Z,E) isomers adopt the energetically favored three-bladed propeller conformation in solution.     

Synthesis, structural characterization, and properties of heavier alkaline earth complexes containing bis(pyrazolyl)borate or bis(3,5-diisopropylpyrazolyl)borate ligands

Treatment of a solid mixture of KBH₄ with six equivalents of 3,5-diisopropylpyrazole (iPr₂pzH) at 180 °C afforded KTp^iPr2(iPr₂PzH)₃ in 53% yield. KBp^iPr2 was synthesized in 56% yield by treatment of a 1:2 M ratio of KBH₄ and iPr₂PzH in refluxing dimethylacetamide. Treatment of MI₂ (M = Ca, Sr, Ba) with two equivalents of KBp or KBp^iPr2 in tetrahydrofuran afforded MBp₂(THF)₂ (M = Ca, 64%, M = Sr, 81%), BaBp₂(THF)₄ (32%), and M(Bp^iPr2)₂(THF)₂ (M = Ca, 63%; M = Sr, 61%, M = Ba, 48%) as colorless crystalline solids upon workup. These complexes were characterized by spectral and analytical techniques and by X-ray crystal structure determinations of all complexes except KBp^iPr2. KTp^iPr2(iPr₂PzH)₃ contains one κ³-N,N,N-Tp^iPr2 ligand and three κ¹-iPr₂pzH ligands, with overall distorted octahedral geometry about the K ion. The iPr₂PzH nitrogen-hydrogen bonds are engaged in intramolecular hydrogen bonding to the 2-nitrogen atoms of the Tp^iPr2 ligand. The solid state structures of MBp₂(THF)₂, BaBp₂(THF)₄, and M(Bp^iPr2)₂(THF)₂ contain κ³-N,N,H Bp and Bp^iPr2 ligands, which form through metal-nitrogen bond formation to the 2-nitrogen atoms of the pyrazolyl fragments and metal-hydrogen bond formation to one boron-bound hydrogen atom per Bp ligand. SrBp₂(THF)₂has the shortest metal-hydrogen interactions among the series. A combination of preparative sublimations, solid state decomposition temperatures, and thermogravimetric analysis demonstrated that MBp₂(THF)₂, BaBp₂(THF)₄, and M(Bp^iPr2)₂(THF)₂ undergo solid state decomposition at moderate temperatures.     

Sub- and supercritical glycolysis of polyethylene terephthalate (PET) into the monomer bis(2-hydroxyethyl) terephthalate (BHET)

Sub- and supercritical glycolysis of polyethylene terephthalate (PET) with ethylene glycol (EG) to bis(2-hydroxyethyl) terephthalate (BHET) was investigated for the purpose of developing a PET recycling process. Supercritical glycolysis was carried out at 450 °C and 15.3 MPa while subcritical glycolysis was carried out at 350 °C and 2.49 MPa or at 300 °C and 1.1 MPa. High yields (gt; 90%) of the monomer BHET were obtained in both super- and subcritical cases. For the same PET/EG weight ratio of about 0.06, the optimum reaction time was 30 min for supercritical glycolysis and 75 and 120 min for two cases of subcritical glycolysis. GPC, RP-HPLC, ¹H NMR and ¹³C NMR, and DSC were used to characterize the polymer and reaction products. Supercritical glycolysis will be suitable to a process requiring a high throughput due to its short reaction time.     

Poly(2‐oxazoline) hydrogels crosslinked with aliphatic bis(2‐oxazoline)s: Properties,cytotoxicity, and cell cultivation

A series of hydrogels from 2‐ethyl‐2‐oxazoline and three bis(2‐oxazoline) crosslinkers—1,4‐butylene‐2,2′‐bis(2‐oxazoline), 1,6‐hexamethylene‐2,2′‐bis(2‐oxazoline), and 1,8‐octamethylene‐2,2′‐bis(2‐oxazoline)—are prepared. The hydrogels differ by the length of aliphatic chain of crosslinker and by the percentage of crosslinker (2–10%). The influence of the type and the percentage of the crosslinker on swelling properties, mechanical properties, and state of water is studied. The equilibrium swelling degree in water ranges from 2 to 20. With a proper selection of the crosslinker, Young's modulus can be varied from 10 kPa to almost 100 kPa. To evaluate the potential for medical applications, the cytotoxicity of extracts and the contact toxicity toward murine fibroblasts are measured. The hydrogels with the crosslinker containing a shorter aliphatic exhibit low toxicity toward fibroblast cells. Moreover, the viability and the proliferation of pancreatic β‐cells incubated inside hydrogels for 12 days are analyzed. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 1548–1559     

Electrospinning of the hydrophilic poly (2-hydroxyethyl methacrylate) and its copolymers with 2-ethoxyethyl methacrylate

The goal was to electrospin 2-hydroxyethyl methacrylate — based biocompatible polymers and prepare submicron fibres (nanofibers) for biomedicinal applications. Syntheses of poly(2-hydroxyethyl methacrylate) (HEMA) and its copolymer with 2-ethoxyethyl methacrylate (EOEMA), and their characterization by viscometry and molecular weight are described. Their relation to electrospinning is discussed. Electrospinning of HEMA homopolymer from water-ethanol is successful for molecular weights 6.31 × 10⁵ and 1.80 × 10⁶ g/mol. Electrospinning of HEMA/EOEMA copolymers is feasible from ethanol.      

Protein extraction for 2-DE from the lamina of Ecklonia kurome (laminariales): recalcitrant tissue containing high levels of viscous polysaccharides

Extraction of proteins from the tissues of laminarialean algae, i.e. kelp, is difficult due to high levels of nonprotein interfering compounds, mainly viscous polysaccharides. To establish proteomic analysis of kelp species, an ethanol/phenol extraction method was developed and compared to other popular methods. Proteins were extracted with phenol from crude protein powder, obtained by homogenizing the kelp tissues in ice-cold ethanol. The ethanol/phenol method produced high-quality proteins of the highest purity from the lamina of Ecklonia kurome, one of the Japanese dominant laminarialean algae. This method gave well-resolved 1-D SDS-PAGE or 2-DE images with low background and the highest number of bands or spots. In particular, proteins with neutral to basic pI's were efficiently extracted. Furthermore, 27 spots on the 2-DE gel were extensively identified by MALDI-TOF/TOF analysis. To the best of our knowledge, this is the first report of a protocol for protein extraction from kelp tissues that gives satisfactory 2-D protein profiles. It is expected that the protocol can be applied to other algae tissues or other recalcitrant plant tissues containing high levels of nonprotein interfering compounds.     

Assignment of human plasma polypeptides on a nondenaturing 2-D gel using MALDI-MS and PMF and comparisons with the results of intact protein mapping

Human plasma proteins were separated by 2-DE under nondenaturing conditions followed by the assignment of the CBB-stained spots using MALDI-MS and PMF, aiming to correlate the information of intact proteins with that of constituent polypeptides. A microgel system was employed to facilitate the analysis. Totally 157 spots on a nondenaturing micro-2-DE gel were numbered, the spots were excised, the proteins in the gel pieces were subjected to in-gel digestion with trypsin followed by polypeptide analysis using MALDI-MS and PMF. Two PMF algorithms, MASCOT (with Swiss-Prot database) and ProFound (with NCBInr database) were employed. A total of 153 spots out of the 157 provided significant match (p <0.05) with polypeptides in databases. Eighty spots were assigned to contain multiple (2-4) polypeptides, suggesting (i) noncovalent interaction between proteins/polypeptides, (ii) disulfide bonding of polypeptides, or (iii) overlapping of the protein locations on the gel. The results of polypeptide assignment coincided very well with the results of protein mapping previously reported, in which 33 plasma proteins were identified using blotting-immunochemical staining (Manabe, T., Takahashi, Y., Higuchi, N., Okuyama, T., Electrophoresis 1985, 6, 462-467). Further, 19 polypeptides in 25 spots were newly assigned. These results demonstrate that the techniques of MALDI-MS and PMF can be applied for analysis of proteins separated on nondenaturing 2-DE gels, providing information on their polypeptide structure. The integrated information on proteins and polypeptides would help the comprehensive understanding on the functions of complex protein systems.     

Synthesis and Structural Characterization of Bis(2,2'-bpy)bis(azide)cobalt(III) nitrate dihydrate

The title complex [CoIII(2, 2?-bpy)2(N3)2]@NO3@2H2O was obtained by an auto-oxidization reaction of cobalt nitrate with 2, 2?-bpy and sodium azide in aqueous solution at room temperature, and violet single crystals were prepared in ethanol solution. The structure was determined by X-ray crystallography. The crystal is of triclinic, space group P ī with a = 8.285(4), b = 11.990(8), c = 12.596(7) ?, ( = 86.630(3), ( = 86.280(5), ( = 71.130(10)°, C20H20CoN11O5, Mr = 553.40, Z = 2, V = 1180.6(12) ?3, F(000) = 568, Dc = 1.557 g/cm3, ( = 0.784 mm-1, R = 0.0403 and wR = 0.1008. The title complex consists of a [CoⅢ(2, 2?-bpy)2(N3)2]+ cation, a NO3- anion and two lattice water molecules. The center CoⅢ ion coordinated by two chelating 2, 2?-bpy ligands and two terminal azide groups with a CoN6 coordination environment exhibits a distorted octahedral geometry.     

Syntheses and crystal structures of diorganotin(IV) bis(2‐pyridinethiolato‐N‐oxide) complexes

The complexes di‐n‐butyldi(2‐pyridinethiolato‐N‐oxide)tin(IV) (1), diphenyldi(2‐pyridinethiolato‐N‐oxide)tin(IV) ( 2 ) and dibenzyldi(2‐pyridinethiolato‐N‐oxide)tin(IV) ( 3 ) are synthesized and characterized by elemental analyses, IR, ¹H, ¹³C, ¹¹⁹Sn NMR spectroscopy, and their structures are determined by X‐ray crystallography. In complex 1 the coordination geometry at tin is a skew‐trapezoidal bipyramid, with cis‐S,S and cis‐O,O atoms occupying the trapezoidal plane and two n‐butyl groups occupying the apical positions, which also exhibits strong π–π stacking interactions. In complexes 2 and 3 the geometry at tin is distorted cis‐octahedral, with cis‐O,O and cis‐C,C atoms occupying the equatorial plane and trans‐S,S atoms occupying the apical positions. Their in vitro cytotoxicity against two human tumour cell lines, MCF‐7 and WiDr is reported. The ID₅₀ values found are comparable to those found for cis‐platin, but lower than for many other diorganotin compounds. Copyright © 2003 John Wiley & Sons, Ltd.