Modulation of activity of human alkaline phosphatases by Mg2+ and thiol compounds

Interaction of purified human liver and placental alkaline phosphatases (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) with sulfhydryl groups, sulfhydryl reagents, and Mg2+ were studied. L-Cysteine (0.1 mmol/l) or Mg2+ activated the liver enzyme 4-5-fold and the placental enzyme 2-3-fold, with optimal pH 7.5-8.0; these activations were not additive. L-Cysteine (2 mmol/l) inhibited both enzymes maximally at pH greater than 9.0; phosphate protected the enzymes. S-Methylcysteine had little effect, with or without Mg2+. Inhibition by sulfur-containing compounds paralleled their ability to bind Zn2+. Fluoresceine mercury acetate (specific for sulfhydryl groups) inhibited the isoenzymes, whereas iodoacetic acid, iodoacetamide, dithionitrobenzoic acid, and p-chloromercuribenzoate had little effect. The inhibition was reversed by L-cysteine and only slightly protected by inorganic phosphate. Thus, there are two sites on human liver and placental alkaline phosphatase that interact with L-cysteine; a Mg2+-binding site, which results in activation, and a site that involves one or both of the bound Zn2+ ions and results in inactivation. Both enzymes have a protected essential thiol group.     

Extracellular chloride regulation of Kv2.1, contributor to the major outward Kv current in mammalian outer hair cells

Outer hair cells (OHC) function as both receptors and effectors in providing a boost to auditory reception. Amplification is driven by the motor protein prestin, which is under anionic control. Interestingly, we now find that the major, 4-AP-sensitive, outward K(+) current of the OHC (I(K)) is also sensitive to Cl(-), although, in contrast to prestin, extracellularly. I(K) is inhibited by reducing extracellular Cl(-) levels, with a linear dependence of 0.4%/mM. Other voltage-dependent K(+) (Kv) channel conductances in supporting cells, such as Hensen and Deiters' cells, are not affected by reduced extracellular Cl(-). To elucidate the molecular basis of this Cl(-)-sensitive I(K), we looked at potential molecular candidates based on Cl(-) sensitivity and/or similarities in kinetics. For I(K), we identified three different Ca(2+)-independent components of I(K) based on the time constant of inactivation: a fast, transient outward current, a rapidly activating, slowly inactivating current (Ik(1)), and a slowly inactivating current (Ik(2)). Extracellular Cl(-) differentially affects these components. Because the inactivation time constants of Ik(1) and Ik(2) are similar to those of Kv1.5 and Kv2.1, we transiently transfected these constructs into CHO cells and found that low extracellular Cl(-) inhibited both channels with linear current reductions of 0.38%/mM and 0.49%/mM, respectively. We also tested heterologously expressed Slick and Slack conductances, two intracellularly Cl(-)-sensitive K(+) channels, but found no extracellular Cl(-) sensitivity. The Cl(-) sensitivity of Kv2.1 and its robust expression within OHCs verified by single-cell RT-PCR indicate that these channels underlie the OHC's extracellular Cl(-) sensitivity.     

Rapid detection of ESBL-producing Klebsiella pneumoniae in blood cultures by fluorescent in-situ hybridization

Multi-resistant Enterobacteriaceae pose a serious threat of hospital acquired infections and their rapid identification is important for better clinical outcome. This study describes the rapid identification of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae of the sulphydryl variable-type by fluorescent in-situ hybridization. The method which rapidly identifies the target genes within 1 h could be a potentially rapid bacterial diagnostic tool.     

A model of blood flow in the mesenteric arterial system

Background  

There are some early clinical indicators of cardiac ischemia, most notably a change in a person's electrocardiogram. Less well understood, but potentially just as dangerous, is ischemia that develops in the gastrointestinal system. Such ischemia is difficult to diagnose without angiography (an invasive and time-consuming procedure) mainly due to the highly unspecific nature of the disease.     

Chain scission of hyaluronan by peroxynitrite

The reaction of peroxynitrite with the biopolymer hyaluronan has been studied using stopped-flow techniques combined with detection of molecular weight changes using the combination of gel permeation chromatography and multiangle laser light scattering. From the effect of peroxynitrite on the yield of hyaluronan chain breaks, it was concluded that the chain breaks were caused by hydroxyl radicals which escape a cage containing the *OH NO*(2) radical pair. The yield of free hydroxyl radicals was determined as 5+/-1% (as a proportion of the total peroxynitrite concentration). At high peroxynitrite concentrations, it was observed that the yield of chain breaks leveled out, an effect largely attributable to the scavenging of hydroxyl radicals by nitrite ions present in the peroxynitrite preparation. These experiments also provided some support for a previous proposal that the adduct formed between ONOOH and ONOO(-) might itself produce hydroxyl radicals. The rate of this reaction would have to be of the order of 0.05 s(-1) to produce hydroxyl radical yields that would account quantitatively for chain break yields at high peroxynitrite concentrations. By carrying out experiments at higher hyaluronan concentrations, it was also concluded that an additional yield of chain breaks was produced by the bimolecular reaction of the polymer with ONOOH at a rate constant of about 10 dm(3)mol(-1)s(-1). At 5.3 x 10(-3)mol dm(-3) hyaluronan, this amounted to 3.5% chain breaks (per peroxynitrite concentration). These conclusions support the proposal that the yield of hydroxyl radicals arising from the isomerization of ONOOH to nitrate ions is relatively low.     

Studies of carotenoid one-electron reduction radicals

The relative reduction potentials of a variety of carotenoids have been established by monitoring the reaction of carotenoid radical anion (CAR1(*-)) with another carotenoid (CAR2) in hexane and benzene. This order is consistent with the reactivities of the carotenoid radical anions with porphyrins and oxygen in hexane. In addition, investigation of the reactions of carotenoids with reducing radicals in aqueous 2% Triton-X 100, such as carbon dioxide radical anion (CO2(*-)), acetone ketyl radical (AC(*-)) and the corresponding neutral radical (ACH(*)), reveals that the reduction potentials for beta-carotene and zeaxanthin lie in the range -1950 to -2100 mV and those for astaxanthin, canthaxanthin and beta-apo-8'-carotenal are more positive than -1450 mV. This illustrates that the presence of a carbonyl group causes the reducing ability to decrease. The radical cations have been previously shown to be strong oxidising agents and we now show that the radical anions are very strong reducing agents.     

Mechanistic studies of melanogenesis: the influence of N‐substitution on dopamine quinone cyclization

The influence of side‐chain structure on the mode of reaction of ortho‐quinone amines has been investigated with a view, ultimately, to developing potential methods of therapeutic intervention by manipulating the early stages of melanogenesis. Four N‐substituted dopamine derivatives have been prepared and quinone formation studied using pulse radiolysis and tyrosinase‐oximetry. Ortho‐quinones with an amide or urea side chain were relatively stable, although evidence for slow formation of isomeric para‐quinomethanes was observed. A thiourea derivative cyclized fairly rapidly (k = 1.7/s) to a product containing a seven‐membered ring, whereas a related amidine gave more rapidly (k ～ 2.5 × 10²/s) a stable spirocyclic product. The results suggest that cyclization of amides, ureas and carbamates (NHCO‐X; X = R, NHR or OR) does not occur and is not, therefore, a viable approach to the formation of tyrosinase‐activated antimelanoma prodrugs. It is also concluded that for N‐acetyldopamine spontaneous ortho‐quinone to para‐quinomethane isomerization is slow.     

Studies on the transposition rates of mobile genetic elements in a natural population of Drosophila melanogaster

In an isolated population of Drosophila melanogaster on Ishigaki Island the chromosomal distribution of several retrotransposons, including copia, 412, 297, 17.6, I, and jockey elements, was examined by in situ hybridization. In this population the cosmopolitan inversion, In(2L)t, is known to exist in high frequency. One major haplotype concerning the occupied sites of the transposable elements was identified in the In(2L)t-carrying chromosomes. This haplotype is suggested to be the ancestral one. The age of the inversion in this local population was estimated to be 1,400 generations. The transposition rates of these elements were estimated based on the age of the inversion and the number of the elements lost and gained. The excision rates were in the range from 9.13 x 10(-5) to 2.25 x 10(-4) per site per generation. They were similar each other in the copia-like elements as well as in the LINE-like elements. The rate was higher in the copia-like elements than in the LINE-like elements. Insertions occurred in the range from 6.79 x 10(-4) to 9.05 x 10(-4) per element per generation. It is herein shown that both insertions and excisions occurred at a significantly higher rate in this population than in the laboratory.