Application of Deuterated A-Tocopherols to the Biokinetics and Bioavailability of Vitamin E

-Tocopherol, a superior chain-breaking, peroxyl radical-trapping antioxidant and the most active component of vitamin E, is elevated in liver tumor cells, contributing to their greater resistance towards lipid peroxidation compared to cells from normal tissues. Also, in regenerating rat liver the level of vitamin E has been found to fluctuate in phase with the rate of cell division. In order to study the biokinetcis and mechanisms of the distribution of vitamin E in organs and within tissues of animals, deuterated forms of -tocopherol have been synthesized and their uptake into blood and tissues has been measured by gas chromatography-mass spectrometry. Measurement of the competitive uptake from a mixture of the RRR-and SRR--tocopherol stereoisomers labelled with different amounts of deuterium shows that the liver exerts a strong preference for secretion of the natural (RRR) stereoisomer into the plasma. It is suggested that a tocopherol-binding protein plays a key role in this process.     

Impact of sar and agr on methicillin resistance in Staphylococcus aureus

Abstract The global regulators agr and sar control expression of cell wall and extracellular proteins. Inactivation of either sar and/or agr in a typical heterogeneously methicillin-resistant Staphylococcus aureus resulted in a small but reproducible decrease in the number of cells in the subpopulation expressing high methicillin resistance. The amount of low affinity penicillin-binding protein PBP2', the prerequisite for methicillin resistance, was apparently not affected, however, a reduction in PBP1 and PBP3 production was observed, suggesting that these resident PBPs of the cells might be involved somehow together with PBP2' in high level methicillin resistance.     

Refolding of trypsin-subtilisin inhibitor from marine turtle eggwhite

Trypsin-subtilisin inhibitor from marine turtle eggwhite refolded quantitatively from its fully reduced state atpH 8.5 in the presence of reduced and oxidized glutathione. The refolding process was studied by following the accompanying changes in inhibitory activity, fluorescence, sulfhydryl group titer, and hydrodynamic volume. The refolding process followed second-order kinetics with rate constants of 4.80×10² M^–1 sec^–1 for trypsin-inhibiting domain and 0.77× 10² M^–1 sec^–1 for subtilisin-inhibiting domain of the inhibitor at 30°C and their respective activation energies of the refolding process were 15.9 and 21.6 kcal/mol. Fluorescence intensity of the reduced inhibitor decreased with time of refolding until it corresponded to the intensity of the native inhibitor. The inhibitor contained 1–2%-helix, 40–42%-sheet, and 57–58% random coil structure. Refolded inhibitor gave a circular dichroic spectrum identical to that of the native inhibitor. A number of principal intermediates were detected as a function of the refolding time. Size-exclusion chromatography separated the intermediates differing in hydrodynamic volume (Stokes radius). The Stokes radius ranged from 23 Å (fully reduced inhibitor) to 18.8 Å (native inhibitor). Results indicated the independent refolding of two domains of the inhibitor and multiple pathways of folding were followed rather than an ordered sequential pathway.     

Signalling by protein kinase C isoforms in the heart

Understanding transmembrane signalling process is one of the major challenge of the decade. In most tissues, since Fisher and Krebs's discovery in the 1950's, protein phosphorylation has been widely recognized as a key event of this cellular function. Indeed, binding of hormones or neurotransmitters to specific membrane receptors leads to the generation of cytosoluble second messengers which in turn activate a specific protein kinase. Numerous protein kinases have been so far identified and roughly classified into two groups, namely serine/threonine and tyrosine kinases on the basis of the target amino acid although some more recently discovered kinases like MEK (or MAP kinase kinase) phosphorylate both serine and tyrosine residues.Protein kinase C is a serine/threonine kinase that was first described by Takai et al. [1] as a Ca- and phospholipid-dependent protein kinase. Later on, Kuo et al. [2] found that PKC was expressed in most tissues including the heart. The field of investigation became more complicated when it was found that the kinase is not a single molecular entity and that several isoforms exist. At present, 12 PKC isoforms and other PKC-related kinases [3] were identified in mammalian tissues. These are classified into three groups. (1) the Ca-activated -, -,and -PKCs which display a Ca-binding site (C2); (2) the Ca-insensitive -, -, -, -, and -PKCs. The kinases that belong to both of these groups display two cystein-rich domains (C1) which bind phorbol esters (for recent review on PKC structure, see [4]). (3) The third group was named atypical PKCs and include , , and -PKCs that lack both the C2 and one cystein-rich domain. Consequently, these isoforms are Ca-insensitive and cannot be activated by phorbol esters [5]. In the heart. evidence that multiple PKC isoforms exist was first provided by Kosaka et al. [6] who identified by chromatography at least two PKC-related isoenzymes. Numerous studies were thus devoted to the biochemical characterization of these isoenzymes (see [7] for review on cardiac PKCs) as well as to the identification of their substrates.This overview aims at updating the present knowledge on the expression, activation and functions of PKC isoforms in cardiac cells. (Mol Cell Biochem 157: 65–72, 1996)     

Engineering the lac permease for purification and crystallization

The lactose permease is being used as a model system for the rational redesign of a membrane protein with the goal of increasing the likelihood of crystallization. Various modifications to the protein have been added for the purposes of purification, stability, and potential for crystallization. The addition of six consecutive histidines at the C-terminus of the protein allows for the rapid purification by nickel-chelate chromatography, and the insertion of an entire protein domain into one of the inner cytoplasmic loops of the permease gives the resulting protein a larger hydrophilic surface area. The increase in polar surface area makes the fusion protein easier to handle and more likely to crystallize. In particular, the introduction of cytochromeb562 ofE. coli into the central hydrophilic domain of the lac permease results in a fusion protein with the transport activity of the permease and the visible absorbance spectrum of the cytochrome. The red permease is very easy to monitor through the steps of expression, purification, concentration, and crystallization.     

An endochitinase gene expressed at high levels in the stylar transmitting tissue of tomatoes

A gene (pMON9617; Chi2;1) identified by screening a tomato pistil cDNA library has been found to encode a protein similar in sequence to class II chitinases. Using a baculovirus expression system we show that the Chi2;1 protein is an active endochitinase. The Chi2;1 protein is most similar in sequence to a previously described stylar chitinase (SK2) isolated from potato. Both proteins lack the diagnostic N-terminal cysteine-rich regions and the C-terminal vacuolar targeting signals of class I chitinases and appear to define a novel type of class II endochitinases associated with flowers. Chi2;1 is expressed predominantly in floral organs and its expression within these organs is temporally regulated. The highest level of expression is found in the transmitting tissue of the style where Chi2;1 mRNA begins to accumulate just prior to anthesis. In vegetative tissue, low levels of Chi2;1 mRNA were detected, and these levels did not increase in response to wounding or treatment with ethephon. mRNA from Chi2;1 orthologs was not detected in most other angiosperms tested, even including some members of the Solanaceae, and it is therefore unlikely that Chi2;1 is essential for stylar function. A fragment containing 1.4 kilobase pairs of 5-flanking DNA from the Chi2;1 gene was shown to drive high-level expression of an attached reporter gene in the styles of transgenic tomatoes. This fragment has utility for engineering expression of other coding regions in styles.     

Immunohistochemical characteristics of the constituents of senile plaques and amyloid angiopathy in aged cynomolgus monkeys

Abstract: In this study, we immunohistochemically examined the several constituents of senile plaques (SPs) and cerebral amyloid angiopathy (CAA) in aged cynomolgus monkeys. Apolipoprotein E (apoE) deposited in all mature plaques and CAA, and in half of the diffuse plaques. Alpha-1-antichymotripsin (αACT) deposited in half of the mature plaques and in one third of the CAA. Amyloid precursor protein (APP), ubiquitin (Ub), and microtubule-associated protein-2 (MAP-2) accumulated in the swollen neurites of mature plaques. Glial fibrillary acidic protein (GFAP) was detected in the astrocytes and their processes surrounding the mature plaques. Tau was detected in neither the SPs nor CAA. Therefore, mature plaques involved extracellular Aβ, apoE, and αACT, and also astrocytes and swollen neurites. However, diffuse plaques involved only extracellular Aβ and apoE. Since these features, except for tau, were consistent with those in humans, this animal model will be useful for studying the pathogenesis of cerebral amyloid deposition.     

Modulation of ion currents and regulation of transmitter release in short-term synaptic plasticity: The rise and fall of the action potential

Up and down-regulation of calcium and potassium conductances are associated with several forms of short-term synaptic modulation. Detailed investigation of synaptic plasticity in the marine gastropodAplysia, and in other mollusks, indicates that synaptic transmission can be influenced in a number of ways by modulatory neurotransmitters acting through several second-messenger cascades. Modulation at the synapse itself occurs by means of the regulation of calcium current as well as through effects on processes directly involved in transmitter mobilization and exocytosis. Modulation of potassium current plays a major role in controlling neuronal excitability and may contribute to a lesser extent to the regulation of transmitter release through actions on the resting potential and on action potential configuration.     

A cell-based reporter assay for the identification of protein kinase C activators and inhibitors

Transmission of extra cellular signals across biological membranes results in the generation of lipid metabolites which in turn influence specific cellular events such as cell growth or differentiation. Many of these lipid messengers can activate protein kinase C (PKC) isozymes of which one function is to perpetuate the extracellular signals to the nucleus by phosphorylating other targets proteins. We have engineered mammalian cell lines to identify and evaluate activators and inhibitors of PKC-dependent and independent signal transduction pathways. The A31 mouse fibroblast cell line, has been stably transfected with a construct containing a triplet repeat of the TPA response element (TRE) upstream of a thymidine kinase promoter fused to the human growth hormone (hGH) gene. A31 cells containing this reporter construct exhibit significant increases in hGH secretion following stimulation by phorbol esters or other mitogens. The levels of hGH secretion are modulated in this system using different pharmacological agents. We demonstrate that this assay can be used to identify specific and general inhibitors as well as activators of the signal transduction pathway mediated by PKC isozymes. (Mol Cell Biochem141: 129–134, 1994)