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The bis-azo dye Direct Blue 1 (Chicago Sky Blue 6B) has been studied in solution and on cellophane and cotton surfaces using NMR, resonance Raman, infrared, and UV–visible spectroscopy. The data indicate that Direct Blue 1 is present as the hydrazone tautomer in all of these media, with distinct changes in the spectra with medium showing that the dye is affected by interactions with its environment. In DMF and DMSO solutions, the dye is present as a monomer that is internally hydrogen-bonded. It is also monomeric at low concentrations in aqueous solution, with subtle changes in the Raman spectra from those in DMF and DMSO being attributed to external hydrogen bonding with water. Direct Blue 1 is also present as a hydrazone monomer at low concentrations on cellophane and cotton: the Raman spectra indicate that there is hydrogen bonding with cellulose, and the UV–visible spectra indicate that it experiences an apolar environment which is attributed to its adsorption onto cellulose surfaces.

A series of free base and zinc porphyrins, mono meso-substituted with a pyridyl group via an amide link, have been prepared by stepwise functionalisation of free base tetraphenylporphyrin, H₂tpp. Four [W(CO)₅L] (L = pyridyl-porphyrin) complexes have been synthesised; [M{tpp-NHCO-py-W(CO)₅}], where M = 2H or Zn, one phenyl group is substituted at the 4-position and the pyridine is substituted at the 3- or 4-position. The spectroscopic data of the [W(CO)₅L] complexes have been compared with those of the porphyrin ligands alone and with those of the model compounds, [W(CO)₅(3-H₂NCO-py)] and [W(CO)₅(4-H₂NCO-py)]. The UV-Vis spectra of the model compounds show that the MLCT and LF absorption bands overlap and no emission is observed at room temperature in solution. The UV-Vis absorption and emission spectra of the porphyrins with peripheral W(CO)₅ are dominated by porphyrin bands. Time-resolved emission was observed from the porphyrin moiety in the [W(CO)₅L] complexes and fitted to mono-exponential kinetics to give lifetimes of ≤10 ns for the zinc porphyrins and of ca. 22 ns for the free base porphyrins. Transients characteristic of the porphyrin 3(π–π*) excited state have been observed by time-resolved absorption and their decay kinetics have been fitted to an appropriate rate law. Long wavelength photolysis of [W(CO)₅(3-H₂NCO-py)] and [W(CO)₅(4-H₂NCO-py)] in THF resulted in conversion to [W(CO)₅(THF)]. The [W(CO)₅L] complexes behaved similarly with photolysis at λ > 395 nm but were photostable with λ > 495 nm.

We have previously identified a novel family of proteins called the GGAs (Golgi-localized, gamma-ear-containing, ADP-ribosylation factor-binding proteins). These proteins consist of an NH(2)-terminal VHS domain, followed by a GAT domain, a variable domain, and a gamma-adaptin ear homology domain. Studies from our own laboratory and others, making use of both yeast and mammals cells, indicate that the GGAs facilitate trafficking from the trans-Golgi network to endosomes. Here we have further investigated the function of the GGAs. We find that GGA-deficient yeast are not only defective in vacuolar protein sorting but they are also impaired in their ability to process alpha-factor. Using deletion mutants and chimeras, we show that the VHS domain is required for GGA function and that the VHS domain from Vps27p will not substitute for the GGA VHS domain. In contrast, the gamma-adaptin ear homology domain contributes to GGA function but is not absolutely required, and full function can be restored by replacing the GGA ear domain with the gamma-adaptin ear domain. Deleting the gamma-adaptin gene together with the two GGA genes exacerbates the phenotype in yeast, suggesting that they function on parallel pathways. In mammalian cells, the association of GGAs with the membrane is extremely unstable, which may account for their absence from purified clathrin-coated vesicles. Double- and triple-labeling immunofluorescence experiments indicate that the GGAs and AP-1 are associated with distinct populations of clathrin-coated vesicles budding from the trans-Golgi network. Together with results from other studies, our findings suggest that the GGAs act as monomeric adaptors, with the four domains involved in cargo selection, membrane localization, clathrin binding, and accessory protein recruitment.

The autoxidation of six esters, neopentyl butanoate, 2,2-dimethylpropanoate, 3,3-dimethylbutanoate, 2,2-dimethylbutanoate, 2-methylbutanoate and 1,1-[²H₂]-neopentyl butanoate, has been studied at 438 K. The reaction products were determined for each system and key reactions leading to the formation and further reactions of the primary products have been identified. Primary products include a range of hydroperoxides which lead to the formation of keto- and hydroxy-esters. Large amounts of neopentanol and the parent carboxylic acid are formed from each ester. It is shown that these are principally oxidation, and not hydrolysis, products. The relative rates of autoxidation of the first five esters mirror the relative rates of attack that occur on reaction with alkoxyl radicals; the sites of attack are on both the alkyl and acyl groups, with the α-alkyl hydrogen atoms on the ester showing particular vulnerability compared to the acyl hydrogen atoms. The analysis of products from the deuterated ester supports this conclusion.

EPR experiments involving the attack of oxygen-centred free radicals on polysaccharides in aqueous solution reveal two sources of regioselectivity in C–H abstraction. For SO₄⁻&z.rad; (derived from one-electron reduction of S₂O₈²⁻), hydrogen-atom abstraction is encouraged by SOMO–σ* interactions involving β-C–O bonds which eclipse the orbital of the unpaired electron at the developing radical centre. In addition, for a series of cyclodextrins, the reactivity observed with SO₄⁻&z.rad; points to the selective reaction of the latter within the cyclodextrin cavity; these findings are in contrast to those obtained with &z.rad;OH (from H₂O₂).

The oxidation of ethylbenzene with dioxygen catalysed by iron(III) porphyrins in a solvent free system has been studied over the temperature range 30–110 °C. The time dependence of the formation of the three main products, 1-phenylethanol, acetophenone and 1-phenylethyl hydroperoxide, and the fate of the iron porphyrin are interpreted in terms of a free radical autoxidation mechanism. The yields of the oxidation products are determined by the rate of reaction and by the lifetime of the catalyst. Catalyst degradation is shown to involve reaction of the porphyrin ligand with 1-phenylethoxyl and 1-phenylethylperoxyl radicals. The disadvantages of increased induction periods and longer reaction times of the oxidations observed at lower reaction temperatures are counter balanced by increased catalyst turnovers. Less extensive studies on the oxidations of toluene, cumene, (2-methylpropyl)benzene and tert-butylbenzene support the overall mechanism proposed for ethylbenzene. A comparative study using the catalysts iron(III) 2,3,7,8,12,13,17,18-octachloro-5,10,15,20-tetrakis(2,6-dichlorophenyl)porphyrin and iron(III) tetrakis(pentafluorophenyl)porphyrin and five of its derivatives reveals that halogenation of the β-pyrrole positions markedly increases the activity of the catalysts but not the stability of the porphyrin towards degradation. The highest yields were obtained with the μ-oxodimer of iron(III) tetrakis(pentafluorophenyl)porphyrin and iron(III) tetrakis(4-dimethylamino-2,3,5,6-tetrafluorophenyl)porphyrin.

Six neopentyl esters have been selected, as models for pentaerythrityl esters used as lubricants, to study the selectivity of hydrogen abstraction by alkoxyl radicals. Kinetic and product data for the reactions of two oxygenated radicals, tert-butoxyl and cumyloxyl, with the six neopentyl esters between 408 and 438 K have been determined. They show that attack by the radicals occurs at the α- and subsequent positions on the acyl moiety as well as at the alkyl group. The selectivity of the reactions is discussed in terms of bond dissociation, steric and polar effects.

Product studies on the oxidation of two 1-arylazo-2-hydroxynaphthalene-6-sulfonate dyes by peroxy acids and by tert-butyl hydroperoxide catalysed by iron(III) 5,10,15,20-tetra(2,6-dichloro-2-sulfonatophenyl)porphyrin in solution have been carried out. In single turnover experiments at pH 11.88, 9.30 and 6.93, dye bleaching by peroxy acids is a two-step process; an initial fast reaction, during which the oxoiron(IV) porphyrin is also formed, is followed by a slow phase involving the regeneration of the iron(III) porphyrin from the oxoiron(IV) porphyrin. The peroxy acid∶dye stoichiometry is 1∶1 for the initial fast dye bleaching and 2∶1 for the slow phase; the latter is equivalent to a 4∶1 stoichiometry of oxoiron(IV) porphyrin to dye. In multiple turnover reactions the dye bleaching occurs in two sequential oxidations by oxoiron(IV) porphyrin π-cation radical, each with a peroxy acid∶substrate stoichiometry of 1∶1. Evidence is presented to show that, in multi-turnover reactions at pH 11.88 where the substrate is the dye anion, the initial product arises from the hydroxylation of the 1-position of the naphthol ring which in the second step is oxidised further to give an azoxy compound. At pH 9.30, where the dyes exist predominantly as the hydrazone tautomers, the initial oxidation generates an azoxy compound which following a Wallach rearrangement is further oxidised on the naphthol ring. More extensive oxidation results in the cleavage of the N–N bond. The iron(III) porphyrin-catalysed dye oxidation with excess of tert-butyl hydroperoxide at pH 6.93, which occurs by hydrogen atom-abstraction, has a hydroperoxide∶dye stoichiometry approaching 2∶1 and gives the same azoxy product as obtained at pH 11.88 with the peroxyacids. Mechanisms to account for the observed products and stoichiometries are discussed.

Attack by SO₄⁻&z.rad; (generated by photolysis and one-electron transfer) on a number of carbohydrates and some model compounds has been followed directly by EPR spectroscopy. SO₄⁻&z.rad; is a reactive, electrophilic species but is more selective (less reactive) than HO&z.rad;. We present evidence for the importance of polar effects and radical stabilisation, especially in determining trends in selectivity. Examples include the preference for C–H abstraction α to a ring oxygen atom in a five-membered ring (e.g. THF, sucrose, β-D-fructofuranose) and abstraction of the axial C¹ hydrogen atom from β-D-glucose. The reaction of SO₄⁻&z.rad; with α-D-glucose is selective towards the C², C⁵ and C⁶ positions. This reflects the activating effect of a β-oxygen substituent where the radical orbital can eclipse the β-C–O bond providing a SOMO–σ* interaction which stabilises the developing radical centre. For myo-inositol, abstraction is also observed from axial C–H bonds for which the resulting radicals are stabilised by an axial β-OH substituent but, due to the absence of a ring oxygen atom, more rapid abstraction occurs from the equatorial C–H bond.

A systematic synthesis designed to connect a metalloporphyrin to a metal carbonyl moiety is demonstrated through a zinc tetraphenyl porphyrin substituted with Re(CO)3(4′-methyl-2,2′-bipyridyl)Br via an amide bond; the emission and excited state absorption spectra are dominated by porphyrin transitions.

The axial ligand of a series of iron(III) 5,10,15,20-tetramesitylporphyrin phenolate complexes was found to exchange with added carboxylic acids and alcohols. The rates of axial ligand exchange with carboxylic acids were found to be dependent on acidity of the carboxylic acid, basicity of the phenolate, and the steric bulk of both the carboxylic acid and the phenolate axial ligand. The rates of axial ligand exchange with alcohols were found to be dependent on the nucleophilicity of the alcohol, leaving group ability of the phenolate, and the steric bulk of both the alcohol and the phenolate axial ligand. Different mechanisms for ligand exchange are proposed for carboxylic acids and alcohols.

Leukemia inhibitory factor [LIF] is a glycoprotein that is able to induce the differentiation of M1 myeloid leukemic cells and prevent the differentiation of murine embryonic stem [ES] cells. This report presents a simple method for the production and purification of large amounts of recombinant murine and human LIF in Escherichia coli. LIF is expressed initially as a fusion product with glutathione Stransferase, separated by a thrombin cleavage site. The fusion product can be rapidly purified on a glutathione-agarose affinity matrix and fully biologically active LIF released from the matrix by cleavage with thrombin. In this form LIF is suitable for many in vitro cell culture uses and in particular for ES cell culture. Further purification to homogeneity of thrombin-released LIF can be achieved by a single fractionation by reversed-phase high performance liquid chromatography; the purified product contains no detectable bacterial endotoxin. Evidence is presented that the intact fusion product has little or no biological activity but biologically active LIF is released by endogenous proteolytic cleavage.

Irradiation (λ > 390 nm) of acidic anaerobic aqueous solutions of iron(III) tetrakis(2-N-methylpyridyl)porphyrin, in the presence of mono- or di-basic amino acids or their N-acylated derivatives, generates the iron(II) porphyrin and the corresponding acyloxyl radical; subsequent decarboxylation of the latter gives the corresponding ammonioalkyl or amidoalkyl radical. The rate and course of each reaction have been monitored by UV–VIS spectroscopy and EPR spin-trapping techniques. The large differences in the observed rates of iron(II) porphyrin formation are controlled by two factors: the binding affinity of the carboxy group for the iron(III) porphyrin to form the photoactive complex and the competitive reactions of the acyloxyl radical [decarboxylation and regeneration of the iron(III) porphyrin complex], following photolysis. With cationic carboxylate ligands, such as glycine or L-alanine, charge repulsion with the cationic porphyrin results in the former effect predominating, whereas with neutral ligands, for example N-acetylglycine, it is the latter that determines the overall rate of reaction. In aqueous base, the amino acids ligate to the iron(III) porphyrin via the amino rather than the carboxy group. Subsequent irradiation brings about an electron transfer from the ligand to give the iron(II) porphyrin and the amino acid cation–radical which reacts further to give an α-amino radical either by decarboxylation or proton loss.

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The photo-decarboxylations of a selection of alkyl-, arylmethyl- and cycloalkyl-carboxylate complexes of iron(III) tetra(2-N-methylpyridyl)porphyrin pentachloride in aqueous solution have been studied. An analysis of the influence of substituent electronic effects, σ* for alkyl- and σ for arylmethyl-carboxylic acids, and α,α-deuteration on the rates of reaction indicates that the reactions involve the discrete formation of a carboxyl radical. Thus after the initial photo-excitation, there is a rate-determining homolytic cleavage of the iron–carboxylate bond followed by rapid decarboxylation of the resultant carboxyl radical. Support for this two-step mechanism, over an alternative synchronous process, comes from a study of the influence of ring size on the reactions of five cycloalkylcarboxylic acids.

Electrospray mass spectrometry has been used to identify the formation of oxo-manganese(V) species in the oxidation of 4-methoxyphenol by H2O2 catalysed by manganese 1,4,7-triazacyclononane complexes in aqueous solution.

Iron(III) 5-(pentafluorophenyl)-10,15,20-tris(2,6-dichlorophenyl)porphyrin has been synthesised and used to catalyse hydrocarbon oxidation by iodosylbenzene. In homogeneous solution it is shown to be a stable and effective catalyst for alkene epoxidation and alkane hydroxylation with a selectivity and reactivity closer to iron(III) tetrakis(pentafluorophenyl)porphyrin than to iron(III) tetrakis(2,6-dichlorophenyl)porphyrin. The new sterically hindered iron porphyrin has also been covalently bound, by nucleophilic aromatic substitution to aminopropylated silica. The resulting heterogenised catalyst is also stable towards oxidation but is less reactive than its homogeneous analogue.

EPR spectroscopy has been employed directly to monitor the initial stages in the free-radical copolymerisation of carbohydrates and methacrylic acid, initiated by a metal–peroxide couple. For the addition of a variety of oxygen-conjugated substrate-derived radicals (including those from myo-inositol, α- and β-D-glucose, D-fructose and sucrose) the rate constants are in the range (0.3–3.8) 4; 4;106 dm3 mol–1 s–1; in contrast &z.rad;CH2OH has a value of 6 4; 4;106 and α,α-dioxygen-substituted radicals have values of ca. 107 dm3 mol–1 s–1.The reaction is clearly assisted by the +M electronic effect of the α-oxygen and by bending at the radical centre (for α-dioxygen-substituted species). The reaction rate is, however, reduced by β-oxygen substituents, especially through a SOMO–σ* (β-C–O) interaction, giving a substantial stereoelectronic retardation for axial β-oxygen substituents and for acyclic β-OH groups where eclipsing geometry (with respect to the radical centre) is favoured.

Embryonic stem (ES) cells, the totipotent outgrowths of bias-tocysts1,2, can be cultured and manipulated in vitro and then returned to the embryonic environment where they develop normally and can contribute to all cell lineages3–9. Maintenance of the stem-cell phenotype in vitro requires the presence of a feeder layer of fibroblasts1,2,10or of a soluble factor, differentiation inhibitory activity (DIA) produced by a number of sources5,11,12; in the absence of DIA the ES cells differentiate into a wide variety of cell types. We recently noted several similarities between partially purified DIA and a haemopoietic regulator, myeloid leukaemia inhibitory factor (LIF), a molecule which induces differentiation in Ml myeloid leukaemic cells and which we have recently purified, cloned and characterized13–18. We demonstrate here that purified, recombinant LIF can substitute for DIA in the maintenance of totipotent ES cell lines that retain the potential to form chimaeric mice.

The technique of E.S.R. spectroscopy, when employed in conjunction with a continuous flow system, provides direct evidence for the nature of free radicals formed from organic substrates in the presence of FeII and H2O2 in aqueous solution. It is shown, both via the identification of hydroxyl-radical adducts to alkenes and via the observed site-selectivity of radical attack, that the hydroxyl radical is formed as the reactive intermediate in the presence of various chelators (e.g. EDTA, DTPA). This approach also allows the rate constants for the FeII-H2O2 reaction in the presence of the different chelates to be determined; values obtained are in reasonable agreement with most of those measured by other methods. Examples of radical oxidation (by FeIII) and reduction (by FeII) are revealed.

The oxidation of 1-(substituted phenylazo)-2-hydroxynaphthalene-6-sulfonate dyes by oxoiron(IV) tetra(2,6-dichloro-3-sulfonatophenyl)porphyrin (OFeIVTDCSPP) in aqueous solution is first-order in the concentration of the dye and of the oxoiron(IV) species over the pH range 6.93–12.68. The pH dependence of the second-order rate constant, kobs, which shows a minimum between pH 8.5 and 9.5, can be simulated using two pH-dependent equilibria [OFeIVTDCSPP(OH2)/OFeIVTDCSPP(OH) and dye/dye anion] and the rate constants for three oxidations, dye by OFeIVTDCSPP(OH2) and OFeIVTDCSPP(OH) and dye anion by OFeIVTDCSPP(OH). The fourth combination, the oxidation of the dye anion by OFeIVTDCSPP(OH2), makes an insignificant contribution to the overall rate of reaction. The mechanisms of the three oxidation processes have been studied using Hammett correlations of substituent effects and by comparisons with the oxidations of isomeric 1-aryl-4-hydroxynaphthalenesulfonate dyes, of an O-methylated dye and of a deuterated 1-phenylazo-2-hydroxynaphthalene-6-sulfonate dye, bearing in mind that the dyes in aqueous solution exist as an equilibrium between azo and hydrazone tautomers.In strongly basic solution the dominant reaction is an electron-transfer oxidation between the dye anion and OFeIVTDCSPP(OH) whereas at neutral pH the major reaction is hydrogen atom-abstraction by OFeIVTDCSPP(OH2) from the azo tautomer of the dye. The mechanism at the pH–rate minimum, between OFeIVTDCSPP(OH) and dye, is less clear and alternatives are discussed.

1. The urinary metabolites of [14C]chlorobenzene administered orally to rabbits have been examined.2. The identified metabolites which account for 98-8% of the radioactivity in the urine include four previously unreported metabolites, namely, quinol, 3-chlorocatechol and o- and m-chlorophenylmercapturic acids.3. The distribution of isomeric mercapturic acids, free phenols and phenolic conjugates is reported.4. The major metabolites are r-chlorophenylmercapturic acid and the conjugates of 4-chlorocatechol.5. The metabolism of chlorobenzene is discussed in terms of an initial epoxidation to 3,4-chlorobenzene oxide.

Previous to the introduction of heparin it had been discovered that certain mammalian tissues contain one or more anticoagulants. McLean,1 in Howell's laboratory in 1916, first isolated an active fraction which in 1918 Howell and Holt2 named heparin and which was at first isolated exclusively from the liver tissue of the dog. Later, Charles and Scott3 showed that this substance is present in varying amounts in the lung, liver and skeletal muscles of the ox, the lung containing the largest amount. During the past five years the anticoagulant properties of heparin have been available for use outside the laboratory, owing to the work of Best4 and his co-workers in Toronto, Canada, and also to that of Jorpes and his school5 in Stockholm, Sweden. Jorpes described heparin as a polysaccharide of high molecular weight which also contains nitrogen and sulfur. The purest preparation contains 40 per

A selection of 5-substituted salicylaldehydes have been reacted with cis,cis-triaminocyclohexane to prepare a series of tri-imine and tri-amine ligands; cyclic voltammetry has been used to show that the electronic effect of the substituent has a marked and systematic effect on the Mn^III/Mn^IV and Mn^III/Mn^II redox potentials of their manganese complexes.

A study has been undertaken of patients having a nylon darn for inguinal hernia at St. Thomas's Hospital with a follow-up of 5 to 12 years and a comparison made with the results of other methods of repair previously studied at the hospital. One hundred and eighty-six repairs were performed with an overall recurrence rate of 7.5%. The recurrence rate for oblique hernia was 4.2% and for direct hernia 13.2%.The use of the nylon repair has produced an improvement on methods previously in use at this hospital.An analysis of the type of recurrence was made and some suggestions have been made to improve technique and subsequent results.

A novel manganese(iii) complex with a water soluble cis,cis-1,3,5-triaminocyclohexane-based ligand was synthesised and shown to exhibit superoxide dismutase activity.

The photochemical and electrochemical properties of a Zn-porphyrin appended rhenium(i) tricarbonyl bipyridine 3-Me-pyridine complex have been investigated; visible-light sensitisation of electron transfer results in ligand substitution at a site remote from the chromophore.

• An 81-year-old woman with a 13-year history of hypoparathyroidism developed dysarthria and dysphagia. Cranial computed tomography demonstrated extensive calcification involving the basal ganglia, corona radiata, and deep cerebellar structures. The cerebral small-vessel calcification that occurs in chronic hypoparathyroidism may produce the syndrome of progressive dysarthria and dysphagia.

We report a case of autoimmune polyglandular syndrome type 1 (APS1) complicated by severe vascular insufficiency due to diffuse vascular calcification. APS1 is characterised clinically by multiple autoimmune conditions and development of at least two components of the triad of mucocutaneous candidiasis, hypoparathyroidism, and autoimmune adrenal insufficiency. We highlight the problems in current serum calcium monitoring methods and suggest that fluctuations in serum calcium concentrations due to difficulties treating hypoparathyroidism may have contributed to the vascular calcification seen in this case.

Gas-chromatographic conditions are reported for the quantitative analysis of mixtures of each of seven groups of isomeric monosubstituted phenols and for α- and β-naphthol. Tris (2,4-xylenyl) phosphate is a suitable liquid phase for the more volatile phenols (cresols, fluorophenols and chlorophenols), but its volatility makes it unsuitable for the higher-boiling phenols. Both diethylenegylcol adipate polyester and polyethylenegylcol monostearate, which can be used at higher temperatures, are suitable for the methoxyphenols. For the nitrophenols, cyanophenols, dihydroxybenzenes and naphthols, most of which are not only very high-boiling but also strongly polar substances, the problems of peak-tailing and long retention times are surmounted by using very low concentrations of diethylenegylcol adipate polyester on glass beads.