Synthesis of a novel prostaglandin H2(pgH2) analogue

We describe a five-step synthesis of a PGH₂ analogue from (R)-glyceraldehyde acetonide via formation of 1,2(S)-0-isopropylidene-hex-3(E)-en-5-one, conjugate addition of prostanoid C₁₃- C₂₀ side-chain as the cuprate with C₁-C₇ side-chain used to quench the resultant enolate, and finaily acid-catalysed ketal exchange to provide the desired analogue.     

Characterization of a Nudix hydrolase from <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> with a marked specificity for (deoxy)ribonucleoside 5'-diphosphates

Background  

Nudix hydrolases form a protein family whose function is to hydrolyse intracellular nucleotides and so regulate their levels and eliminate potentially toxic derivatives. The genome of the radioresistant bacterium Deinococcus radiodurans encodes 25 nudix hydrolases, an unexpectedly large number. These may contribute to radioresistance by removing mutagenic oxidised and otherwise damaged nucleotides. Characterisation of these hydrolases is necessary to understand the reason for their presence. Here, we report the cloning and characterisation of the DR0975 gene product, a nudix hydrolase that appears to be unique to this organism.     

Impact of prefractionation using Gradiflow on two-dimensional gel electrophoresis and protein identification by matrix assisted laser desorption/ionization-time of flight-mass spectrometry

Prefractionation of protein samples prior to two-dimensional electrophoresis (2-DE) has the potential to increase the dynamic detection range for proteomic analysis. We evaluated a membrane-based electrophoretic separation technique (Gradiflow) for its ability to fractionate an exoproteome sample from the filamentous fungus Trichoderma reesei. The sample was separated on the basis of size and charge. Buffer optimization was found to be necessary for successful size fractionation. Fractionation by charge was used to resolve the sample into four fractions that were subjected to analysis by two-dimensional electrophoresis (2-DE). Enhanced detection of low-abundance proteins with selective removal of high-abundance species was achieved. Fractionated and unfractionated samples were examined for differences in the ability to identify proteins following 2-DE using trypsin in-gel digestion followed by peptide mass fingerprinting using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Fractionated samples showed marked improvement in protein identification ability and sequence coverage. This study demonstrates the utility of the Gradiflow for fractionation, resulting in an enhancement of resolution and characterization of a moderately complex proteome.     

Determination of alcohol polyether average molar oligomer value/distribution via supercritical fluid chromatography coupled with UV and MS detection

Supercritical fluid chromatography (SFC) was used for the analysis of ethoxylated and propoxylated surfactants. Samples were derivatized to phenylated silyl ethers with a disilazane-chlorosilane mixture. Addition of a phenyl group to the surfactant allowed UV-absorbance detection of each oligomer. Acetonitrile and methanol were evaluated as mobile phase modifiers. Better peak shape was realized with methanol-modified CO2 on an octadecyl silica bonded phase than with acetonitrile-modified CO2. Peak assignments were made via SFC coupled with electrospray ionization-mass spectrometry (ESI-MS) in the positive ion mode. A sulfonamide-embedded alkyl stationary phase was also evaluated for separation of the derivatized samples. SFC-UV and SFC-ESI-MS data were jointly used for calculation of average molar oligomer values which were then compared to values calculated from 1H NMR data of non-derivatized samples. The derivatization or separation method using the sulfonamide embedded phase required no preliminary cleanup and yielded reproducible oligomer values that were consistent with those of the manufacturer's nominal values.     

ADRIAMYCIN RESISTANCE IN CHINESE HAMSTER FIBROBLASTS FOLLOWING OXIDATIVE STRESS INDUCED BY PHOTODYNAMIC THERAPY

Photodynamic therapy (PDT) generates reactive oxygen species that are responsible for the initial cytotoxic events produced by this treatment. An extended (16 h) porphyrin incubation prior to light irradiation increased expression of the 75, 78 and 94 kDa glucose-regulated stress proteins (GRP), as well as the cognate form of the 70 kDa heat shock protein. However, these stress proteins were not induced following isoeffective PDT doses using a short (1 h) porphyrin incubation protocol. In the current study, Chinese hamster fibroblasts were used to examine sensitivity to adjunctive PDT and adriamycin as previous reports indicate a correlation between stress protein synthesis and a decrease in adriamycin cytotoxicity. Treatments that either induced GRP ( i.e . PDT with an extended porphyrin incubation or exposure to the calcium ionophore A23187) or did not induce GRP ( i.e . PDT with a short porphyrin incubation or UV irradiation) were followed at increasing time intervals with a 1 h adriamycin incubation. A time-dependent decrease in adriamycin cytotoxicity was observed when cells were first exposed to either of the PDT protocols or to A23187. Alterations in intracellular drug levels did not account for the change in adriamycin sensitivity. Likewise, intracellular glutathione concentrations and antioxidant enzyme activities were not significantly altered following PDT or A23187. Parameters associated with altered adriamycin sensitivity included a decrease in the percentage of S phase cells following PDT and A23187 as well as a depletion of intracellular ATP after PDT using the extended porphyrin incubation. These results demonstrate that PDT can be added to the growing list of diverse stresses producing transient resistance to adriamycin and that stress protein induction is not universally associated with all oxidative treatments inducing this resistance.     

High-resolution spectroscopy of induced chiral dimers: a study of the dimers of ethanol by Fourier transform microwave spectroscopy

We present the first recording of the high-resolution spectrum of an induced chiral dimer. Three conformers of the induced chiral dimers of ethanol have been observed using a pulsed molecular-beam Fourier transform microwave spectrometer. The rotational constants of the normal isotopomers of the three species have been determined to be (a) A=5113.826(5), B=1329.7214(4), and C=1257.5151(3) MHz, (b) A=5086.459(5), B=1316.6508(4), and C=1243.6329(4) MHz, and (c) A=4851.608(5), B=1369.7558(6), and C=1243.4184(4) MHz. The observed species have been assigned to calculated structures via Kraitchman double substitution analyses and ab initio calculations. The Kraitchman analyses and the fitted centrifugal distortion parameters suggest that the deuterium bond is significantly stronger than the hydrogen bond in the dimers of ethanol.     

Templated ceramic microstructures by using the breath-figure method

We describe the synthesis of two cyclobutadiene(cyclopentadienyl)cobalt-containing poly(p-phenylene ethynylene)s (PPEs) and their use as precursors for stable ceramic surface coatings. Organometallic PPEs were shaped into hexagonally ordered assemblies by using the breath-figure method. Such breath figures can be washed away with an appropriate solvent. Upon pyrolysis at 500 degrees C under either nitrogen or air, the bubble arrays persist as ceramics and are insoluble in organic solvents or water. The formed pyrolyzed bubble arrays were analyzed by optical and scanning electron microscopy, as well as energy dispersive X-ray microanalysis (EDX). The composition of the ceramic materials is discussed based on EDX and IR data.     

Synthesis of mixed metal decaosmium carbido clusters: The X-ray structures of the monoanions [Os10C(CO)24Cu(NCMe)]− and [Os10C(CO)24AuPPh3]−

The high nuclearity mixed metal cluster monoanions [Os₁₀C(CO)₂₄Cu(NCMe)]^? (I) and [Os₁₀C(CO)₂₄AuPPh₃]^? (II) have been obtained by reaction of the carbido-dianion [Os₁₀C(CO)₂₄]^2? (III) with one equivalent of [Cu(NCMe)₄] [BF₄] and Ph₃PAuCl, respectively, in CH₂Cl₂. X-ray analysis of the [PPh₃Me]⁺ salts of I and II show that the Cu and Au ligands have added to capping tetrahedra of the dianion III in μ₃- and μ₂-bridging positions, respectively.     

Synthesis,characterisation and ferric ion complexation studies of chelating probes for oxidative stress

The ferric ion binding characteristics of a novel chelating probe for detecting reactive oxygen and nitrogen species are reported. The probe comprises a polyaminocarboxylic acid chelator (EDTA) conjugated with aromatic amino acids. The probe (i) complexes redox-active and non-redox active metal ions and (ii) differentiates between different reactive oxygen/nitrogen species based upon the reaction products with same. The ferric ion binding characteristics were measured using species distribution and electronic absorption spectroscopic investigations. Over the physiological pH range a favoured one to one complex exists.