Evidence for the Addition of the Superoxide Anion to the Anti-Oxidant n-Propyl Gallate in Aqueous Solution

n-Propyl gallate reacts with the superoxide radical anion in aqueous solution (k = 5.1 × 10⁵ mol^-1 dm³s^-1). The spectrum of the transient species so formed has been measured (absorbance maximum at 550nm, ε = 1360mol^-1dm³cm^-1). Electron or H atom transfer processes as well as proton abstraction have been excluded as possible mechanisms, and it is proposed that an addition reaction takes place.     

Isoform-specific O-glycosylation by murine UDP-GalNAc:polypeptide N- acetylgalactosaminyltransferase-T3, in vivo

Multiple isoforms of UDP-GalNAc:polypeptide N-acetylgalactosaminyl- transferase (ppGaNTase) have been cloned and expressed from a variety of organisms. In general, these isoforms display different patterns of tissue-specific expression, but exhibit overlapping substrate specificities, in vitro . A peptide substrate, derived from the sequence of the V3 loop of the HIV gp120 protein (HIV peptide), has previously been shown to be glycosylated in vitro exclusively by the ppGaNTase-T3 (Bennett et al. , 1996). To determine if this isoform- specificity is maintained in vivo , we have examined the glycosylation of this substrate when it is expressed as a reporter peptide (rHIV) in a cell background (COS7 cells) which lacks detectable levels of the ppGaNTase-T3. Glycosylation of rHIV was greatly increased by coexpression of a recombinant ppGaNTase-T3. Overexpression of ppGaNTase- T1 yielded only partial glycosylation of the reporter. We have also determined that the introduction of a proline residue at the +3 position flanking the potential glycosylation site eliminated ppGaNTase- T3 selectivity toward rHIV observed both in vivo and in vitro .      

Activated 3',5'-cyclic AMP-dependent protein kinase is sufficient to induce neuroendocrine-like differentiation of the LNCaP prostate tumor cell line

Neuroendocrine (NE) differentiation within prostate tumors is proposed to be a contributing factor in disease progression. However, the cellular origin and molecular mechanism controlling differentiation of prostatic NE cells are unresolved. The prostate tumor cell line, LNCaP, can reversibly acquire many NE characteristics in response to treatment with beta-adrenergic receptor agonists and activators of adenylate cyclase. In this study, we demonstrate that these treatments induce protein kinase A (PKA) activation in LNCaP cells and that ectopic expression of a constitutively activated form of the PKA catalytic subunit, CIalpha, results in acquisition of NE characteristics, including the extension of neuritic processes, cessation of mitotic activity, and production of neuron-specific enolase. Forskolin-, epinephrine-, and isoproterenol-dependent NE differentiation of LNCaP cells was significantly inhibited by expressing a dominant negative mutant of the PKA regulatory subunit, RIalpha. These results demonstrate that prostatic NE differentiation in response to these agents depends on PKA activation, and this signaling pathway may provide a therapeutic target for treating advanced forms of prostate cancer.     

Potassium permanganate and tetraethylammonium chloride are a safe and effective substitute for osmium tetroxide in solid-phase fluorescent chemical cleavage of mismatch.

Whilst chemical cleavage of mismatch (CCM) detects all point mutations in DNA, its widespread use has been hampered by the complex multistage methodology and the need for toxic chemicals, in particular osmium tetroxide. Here we show that osmium tetroxide can be replaced by potassium permanganate, giving the same spectrum of mutation detection, but with greater sensitivity. The use of potassium permanganate is compatible with solid phase capture and fluorescent detection, giving a safer method of mutation detection. We present here a comparison of CCM with osmium tetroxide and with potassium permanganate, tested on a complete set of single base pair mismatches and a number of small insertion/deletions.     

The SO4(.-)-induced chain reaction of 1,3-dimethyluracil with peroxodisulphate

The sulphate radical SO4(.-) reacts with 1,3-dimethyluracil (1,3-DMU) (k = 5 X 10(9) dm3 mol-1 s-1) thereby forming with greater than or equal to 90 per cent yield the 1,3-DMU C(5)-OH adduct radical 4 as evidenced by its absorption spectrum and its reactivity toward tetranitromethane. Pulse-conductometric experiments have shown that a 1,3-DMU-SO4(.-) aduct 3 as well as the 1,3-DMU radical cation 1, if formed, must be very short-lived (t1/2 less than or equal to 1 microsecond). The 1,3-DMU C(5)-OH adduct 4 reacts slowly with peroxodisulphate (k = 2.1 X 10(5) dm3 mol-1 s-1). It is suggested that the observed new species is the 1,3-DMU-5-OH-6-SO4(.-) radical 7. At low dose rates a chain reaction is observed. The product of this chain reaction is the cis-5,6-dihydro-5,6-dihydroxy-1,3-dimethyluracil 2. At a dose rate of 2.8 X 10(-3) Gys-1 a G value of approximately 200 was observed ([1,3-DMU] = 5 X 10(-3) mol dm-3; [S2O8(2-)] = 10(-2) mol dm-3; [t-butanol] = 10(-2) mol dm-3). The peculiarities of this chain reaction (strong effect of [1,3-DMU], smaller effect of [S2O(2-)8]) is explained by 7 being an important chain carrier. It is proposed that 7 reacts with 1,3-DMU by electron transfer, albeit more slowly (k approximately 1.2 X 10(4) dm3 mol-1 s-1) than does SO4(.-). The resulting sulphate 6 is considered to hydrolyse into 2 and sulphuric acid which is formed in amounts equivalent to those of 2. Computer simulations provide support for the proposed mechanism. The results of some SCF calculations on the electron distribution in the radical cations derived from uracil and 1-methyluracil are also presented.     

The reactivity of various free radicals with hyaluronic acid: steady-state and pulse radiolysis studies

The reactions of the primary water radicals with the biopolymer hyaluronic acid have been studied by pulse radiolysis. Bimolecular rate constants, expressed in terms of the disaccharide repeating sub-unit of hyaluronic acid, for OH., H. and eaq- were found to be 7 X 10(8) M-1 X s-1, 5 X 10(7) M-1 X s-1 and less than 5 X 10(6) M-1 X s-1, respectively. By comparing the viscosities of samples, gamma-irradiated in the steady state under a variety of conditions, with unirradiated controls, the efficiencies with which selected radicals cause chain breakage have been determined. Efficiencies of 30%, 15%, 0%, 0.2% and 5% were estimated for OH., H., eaq-, methanol radicals and tert-butanol radicals, respectively. The presence of oxygen during irradiation increased the extent of chain breakage by a factor of 1.75.     

Pulse radiolytic studies on uracil and uracil derivatives. Protonation of their electron adducts at oxygen and carbon

The electron adducts of uracil, 1,3-dimethyluracil and 1,3-dimethylthymine, known to protonate rapidly in aqueous solution at oxygen, are now shown to undergo a slower protonation at C(6) producing a radical centred at C(5), a reaction which can be catalysed by buffer.