Purification and characterization of the flagellar basal body of Rhodobacter sphaeroides

Flagellar hook-basal body (HBB) complexes were purified from Rhodobacter sphaeroides. The HBB was more acid labile but more heat stable than that of Salmonella species, and protein identification revealed that HBB components were expressed only from one of the two sets of flagellar gene clusters on the R. sphaeroides genome, under the heterotrophic growth conditions tested here.

Purification and molecular cloning of antimicrobial peptides from Scots pine seedlings

A novel protocol for rapid and efficient purification of antimicrobial peptides from plant seedlings has been developed. Two peptides with antimicrobial activity, designated p1 and p2, were purified nearly to homogeneity from Scots pine seedlings by a combination of sulfuric acid extraction, ammonium sulfate precipitation, heat-inactivation and ion-exchange chromatography on phosphocellulose. Purified proteins had molecular masses of 11 kDa (p1) and 5.8 kDa (p2) and were identified by mass spectrometry as defensin and lipid-transfer protein, respectively. We demonstrated their growth inhibitory effects against a group of phytopathogenic fungi. Furthermore, we report for the first time molecular cloning and characterization of defensin I cDNA from Scots pine. A cDNA expression library from 7 days Scots pine seedlings was generated and used to isolate a cDNA clone corresponding to Scots pine defensin, termed PsDef1. The full-length coding sequence of PsDef1 is 252 bp in length and has an open reading frame capable to encode a protein of 83 amino residues. The deduced sequence has the typical features of plant defensins, including an endoplasmic reticulum signal sequence of 33 aa, followed by a characteristic defensin domain of 50 amino acids representing its active form. The calculated molecular weight of the mature form of PsDef1 is 5601.6 Da, which correlates well with the results of SDS-PAGE analysis. Finally, the antimicrobial properties of PsDef1 against a panel of fungi and bacteria define it as a member of the morphogenic group of plant defensins. (C) 2009 Elsevier Inc. All rights reserved.

Purification and functional characterisation of Rhinocerase, a novel serine protease from the venom of Bitis gabonica rhinoceros

Background: Serine proteases are a major component of viper venoms and are thought to disrupt several distinct elements of the blood coagulation system of envenomed victims. A detailed understanding of the functions of these enzymes is important both for acquiring a fuller understanding of the pathology of envenoming and because these venom proteins have shown potential in treating blood coagulation disorders. Methodology/Principal Findings: In this study a novel, highly abundant serine protease, which we have named rhinocerase, has been isolated and characterised from the venom of Bitis gabonica rhinoceros using liquid phase isoelectric focusing and gel filtration. Like many viper venom serine proteases, this enzyme is glycosylated; the estimated molecular mass of the native enzyme is approximately 36kDa, which reduces to 31kDa after deglycosylation. The partial amino acid sequence shows similarity to other viper venom serine proteases, but is clearly distinct from the sequence of the only other sequenced serine protease from Bitis gabonica. Other viper venom serine proteases have been shown to exert distinct biological effects, and our preliminary functional characterization of rhinocerase suggest it to be multifunctional. It is capable of degrading α and β chains of fibrinogen, dissolving plasma clots and of hydrolysing a kallikrein substrate. Conclusions/Significance: A novel multifunctional viper venom serine protease has been isolated and characterised. The activities of the enzyme are consistent with the known in vivo effects of Bitis gabonica envenoming, including bleeding disorders, clotting disorders and hypotension. This study will form the basis for future research to understand the mechanisms of serine protease action, and examine the potential for rhinocerase to be used clinically to reduce the risk of human haemostatic disorders such as heart attacks and strokes.

Synthesis and characterization of thermally stable second-order nonlinear optical side-chain polyimides containing thiazole and benzothiazole push-pull chromophores

Push-pull nonlinear optical (NLO) chromophores containing thiazole and benzothiazole acceptors were synthesized and characterized. Using these chromophores a series of second-order NLO polyimides were Successfully prepared from 4,4'-(hexafluoroisopropylidene) diphthalic anhydride (6FDA), pyromellitic dianhydride (PMDA) and 3,3'4,4'-benzophenone tetracarboxylic dianhydride (BTDA) by a standard condensation polymerization technique. These polyimides exhibit high glass transition temperatures ranging from 160 to 188 degrees C. UV-vis spectrum of polyimide exhibited a slight blue shift and decreases in absorption due to birefringence. From the order parameters, it was found that chromophores were aligned effectively. Using in situ poling and temperature ramping technique, the optical temperatures for corona poling were obtained. It was found that the optimal temperatures of polyimides approach their glass transition temperatures. These polyimides demonstrate relatively large d(33) values range between 35.15 and 45.20 pm/V at 532 nm. (C) 2008 Elsevier B.V. All rights reserved.

Overproduction, purification and preliminary X-ray diffraction analysis of YncE, an iron-regulated Sec-dependent periplasmic protein from Escherichia coli

The yncE gene of Escherichia coli encodes a predicted periplasmic protein of unknown function. The gene is de-repressed under iron restriction through the action of the global iron regulator Fur. This suggests a role in iron acquisition, which is supported by the presence of the adjacent yncD gene encoding a potential TonB-dependent outer-membrane transporter. Here, the preliminary crystallographic structure of YncE is reported, revealing that it consists of a seven-bladed beta-propeller which resembles the corresponding domain of the `surface-layer protein' of Methanosarcina mazei. A full structure determination is under way in order to provide insight into the function of this protein.

Purification and electron cryomicroscopy of coronavirus particles

Intact, enveloped coronavirus particles vary widely in size and contour, and are thus refractory to study by traditional structural means such as X-ray crystallography. Electron microscopy (EM) overcomes some problems associated with particle variability and has been an important tool for investigating coronavirus ultrastructure. However, EM sample preparation requires that the specimen be dried onto a carbon support film before imaging, collapsing internal particle structure in the case of coronaviruses. Moreover, conventional EM achieves image contrast by immersing the specimen briefly in heavy-metal-containing stain, which reveals some features while obscuring others. Electron cryomicroscopy (cryo-EM) instead employs a porous support film, to which the specimen is adsorbed and flash-frozen. Specimens preserved in vitreous ice over holes in the support film can then be imaged without additional staining. Cryo-EM, coupled with single-particle image analysis techniques, makes it possible to examine the size, structure and arrangement of coronavirus structural components in fully hydrated, native virions. Two virus purification procedures are described.

Synthesis and characterization of fluorescent poly(aromatic amide) dendrimers

The synthesis of a series of poly(aromatic amide) dendrimers up to the second generation is described herein. The AB, building block used throughout the synthesis of the dendrimers was the allyl ester of 3,5-diaminocinnamic acid, which has been synthesized from 3,5-dinitrobenzoic acid in good yield with use of a four-step procedure. Dendron synthesis was achieved via a convergent approach with use of a sequence of deprotection/coupling steps. Two commercially available alcohols, L-menthol and citronellol, were coupled to the AB(2) monomer by using an alkyl diacid spacer and two core units; 1,7-diaminoheptane and tris(2-aminoethyl)amine have been used to produce the final dendrimers. Characterization was carried out by NMR and IR spectroscopies, MALDI-TOF mass spectrometry, GPC, and DSC. The novel monomer and dendritic derivatives exhibited a strong fluorescence emission in the visible region (lambda approximate to 500 nm) of the spectrum and a weak emission in the near-infrared (lambda approximate to 850 nm) upon excitation in the near-UV region. The fluorescence emission characteristics were found to be solvent and dendrimer generation dependent.

Synthesis and characterization of hyperbranched polyesters incorporating the AB(2) monomer 3,5-bis(3-hydroxylprop-1-ynyl)benzoic acid

The AB, monomer, 3,5-bis(3-hydroxylprop-1-ynyl)benzoic acid 1, has been synthesized using a Sonogashira cross-coupling with a palladium catalyst system developed for use with deactivated aryl halides. Numerous condensation methods have then been assessed in the homopolymerization of the acid-diol monomer 1 to afford hyperbranched polyesters. However, as a result of the thermal instability of the monomer, direct thermal polymerizations could not be employed. Alternative approaches using carbodiimide-coupling reagents enabled the production of soluble polyesters possessing molecular weights and degrees of branching ranging from 2500 to 11,000 and 0.22 to 0.33, respectively. (C) 2003 Elsevier Ltd. All rights reserved.

Soluble redox-active polymetallic chains [{Ru-0(CO)(L)(bpy)}(m)](n) (bpy=2,2 '-bipyridine, L = PrCN, Cl-; m=0,-1): electrosynthesis and characterization

Electrochemical and spectroelectrochemical techniques were employed to study in detail the formation and so far unreported spectroscopic properties of soluble electroactive molecular chains with nonbridged metal-metal backbones, namely, [{Ru-0(CO)(PrCN)(bpy)}(m)](n) (m = 0, -1) and [{Ru-0(CO)(bpy)Cl}(m)](n) (m = -1, -2; bpy = 2,2'-bipyridine). The precursors cis-(Cl)-[Ru-II(CO)(MeCN)(bpy)Cl-2] (in PrCN) and mer-[Ru-II(CO)(bpy)Cl-3](-) (in tetrahydrofuran (THF) and PrCN) undergo one-electron reductions to reactive radicals [Ru-II(CO)(MeCN)(bpy(center dot-))Cl-2](-) and [Ru-II(CO)(bpy(center dot-))Cl-3](2-), respectively. Both [bpy(center dot-)]-containing species readily electropolymerize on concomitant dissociation of two chloride ligands and consumption of a second electron. Along this path, mer-to-fac isomerization of the bpy-reduced trichlorido complex (supported by density functional theory calculations) and a concentration-dependent oligomerization process contribute to the complex reactivity pattern. In situ spectroelectrochemistry (IR, UV/vis a has revealed that the charged polymer [{Ru-0(CO)(bpy)Cl}(-)](n) is stable in THF, but in PrCN it converts readily to [Ru-0(CO)(PrCN)(bpy)](n). An excess of chloride ions retards this substitution at low temperatures. Both polymetallic chains are completely soluble in the electrolyte solution and can be reduced reversibly to the corresponding [bpy(center dot-)]-containing species.

Synthesis and characterization of carbamoyl methyl pyrazole(CMPz) compounds of palladium(II) chloride: the crystal and molecular structure of [PdCl2{(C3H3N2CH2CONBu2)-Bu-i}(2)]

Carbamoyl methyl pyrazole compound of palladium(II) chloride of the type [PdCl2L2] (where L = C5H7N2CH2CON(C4H9)(2), C5H7N2CH2CON((C4H9)-C-i)(2), C3H3N2CH2CON(C4H9)(2), or C3H3N2CH2CON((C4H9)-C-i)(2)) has been synthesized and characterized by IR and H-1 NMR spectroscopy. The structure of the compound [PdCl2{(C3H3N2CH2CONBu2}2)-Bu-i] has been determined by single crystal X-ray diffraction and shows that the ligands are bonded through the soft pyrazolyl nitrogen atom to the palladium(II) chloride in a trans disposition. (c) 2007 Elsevier B.V. All rights reserved.

Synthesis and characterization of biocompatible, thermoresponsive ABC and ABA triblock copolymer gelators

The synthesis of doubly thermoresponsive PPO-PMPC-PNIPAM triblock copolymer gelators by atom transfer radical polymerization using a PPO-based macroinitiator is described. Provided that the PPO block is sufficiently long, dynamic light scattering and differential scanning calorimetry studies confirm the presence of two separate thermal transitions corresponding to micellization and gelation, as expected. However, these ABC-type triblock copolymers proved to be rather inefficient gelators: free-standing gels at 37 degrees C required a triblock copolymer concentration of around 20 wt%. This gelator performance should be compared with copolymer concentrations of 6-7 wt% required for the PNIPAM-PMPC-PNIPAM triblock copolymers reported previously. Clearly, the separation of micellar self-assembly from gel network formation does not lead to enhanced gelator efficiencies, at least for this particular system. Nevertheless, there are some features of interest in the present study. In particular, close inspection of the viscosity vs temperature plot obtained for a PPO43-PMPC160-PNIPAM(81) triblock copolymer revealed a local minimum in viscosity. This is consistent with intramicelle collapse of the outer PNIPAM blocks prior to the development of the intermicelle hydrophobic interactions that are a prerequisite for macroscopic gelation.

Synthesis and characterization of packed mesoporous tungsteno-silicates: application to the catalytic dehydrogenation of 2-propanol

The synthesis of highly ordered mesoporous tungsteno-silicas in which a high percentage of tungsten is introduced into a silica framework is reported hereafter. Powder XRD and TEM have been used to characterize the materials synthesized at room temperature. The materials are shown to be homogeneous as there is no evidence for any crystalline species other than the silica framework. The pore diameter and the surface area of the materials, evaluated from the nitrogen adsorption isotherms and unit cell parameter indicate a pore diameter of about 2 nm and a surface area of 1400 m(2) g(-1) for a content of 10% tungsten. Catalyzed dehydration of 2-propanol has been investigated and the activity of the materials synthesized is significant, even for low tungsten content W-MCM-41 materials. (C) 2003 Elsevier B.V. All rights reserved.

One-pot synthesis and characterization of soluble poly(aryl ether-ketone)s having pendant carboxyl groups

Aromatic poly(ether-ketone)s having pendant carboxyl groups have been obtained by direct, one-pot, Friedel-Crafts copolycondensation of 4,4'-diphenoxybenzophenone with a mixture of terephthaloyl chloride (TC) and trimellitic anhydride acid chloride (TAAC), over a wide range of TAAC/TC molar ratios, in the presence of anhydrous aluminum chloride. The syntheses were performed as precipitation-polycondensations, and the polymers were obtained in particulate form. Besides globular particles of polymer, small quantities of elongated, needlelike particles were observed when the mole ratio TAAC/TC was less than 1. Use of X-ray microdiffraction with synchrotron radiation has revealed that the needlelike material consists of a cyclic compound containing 10 phenylene units, i.e., the crystals are of a [2 + 2] macrocyclic dimer. The polymers obtained are soluble in strong acids and in mixtures of methanesulfonic acid or trifluoroacetic acid with chlorinated hydrocarbons. The molecular structures of the polymers were confirmed by H-1 and C-13 NMR spectroscopy. Reaction of TAAC with 4,4'-diphenoxybenzophenone produced mainly meta-orientation of the resulting ketone linkages. The size of the polymer particles, their molecular weights, and the melting behavior of the products obtained depend on the TAAC/TC ratio used. Ortho-keto acid residues, formed during reaction of anhydride groups of TAAC with 4,4'-diphenoxybenzophenone, exhibit ring-chain tautomerism. A carboxyl-containing aromatic polyketone derived from p-terphenyl, and thus having with no ether linkages in the main chain, was prepared by analogous chemistry, and functional derivatives of carboxy-substituted polyketones were also obtained and characterized.