BBA - General Subjects (v.1621, #2)

Inactivation of mprF affects vancomycin susceptibility in Staphylococcus aureus by Alexey Ruzin; Anatoly Severin; Soraya L Moghazeh; Jerome Etienne; Patricia A Bradford; Steven J Projan; David M Shlaes (117-121).
A chemically generated mutant of Staphylococcus aureus RN4220, GC6668, was isolated that had a fourfold increase in resistance to vancomycin. This phenotype reverted back to susceptibility by insertional mutagenesis with Tn917. In a selected set of revertants, Tn917 insertion was mapped to a unique chromosomal region upstream of mprF, a recently described gene that determines staphylococcal resistance to several host defense peptides. The genetic linkage between the vancomycin susceptibility and Tn917 insertion was then confirmed by transduction backcrosses into both GC6668 and GISA isolates, MER-S12 and HT2002 0127. Northern blot analysis, insertional inactivation and complementation experiments showed that mprF mediates vancomycin susceptibility in S. aureus. The inactivation of mprF by Tn917 insertion in HT2002 0127 caused a significant increase in the binding of vancomycin to the cell membranes. This observation serves as a likely mechanism of the increased vancomycin susceptibility associated with mprF inactivation.
Keywords: Staphylococcus aureus; Vancomycin; Inactivation;

Despite the description of the ways of acetone metabolism, its real role(s) is (are) still unknown in metabolic network. In this article, a trial is made to ascertain a comprehensive overview of acetone research extending discussion from chemistry to clinical implications. Mammals are quite similar regarding their acetone metabolism, even if species differences can also be observed. By reviewing experimental data, it seems that plasma concentration of acetone in different species is in the order of 10 μm range and the concentration-dependent acetone metabolism is common to all mammals. At low concentrations of plasma acetone, the C3 pathways are operative, while at higher concentrations, the metabolism through acetate becomes dominant. Glucose formation from acetone may also contribute to the maintenance of a constant blood glucose level, but it seems to be only a minor source for that. From energetical point of view, an interorgan cooperation is suggested because transportable C3 fragments produced in the liver can serve as alternative sources of energy for the peripheral tissues in the short of circulating glucose. The degradation of acetoacetate to acetone contributes to the maintenance of pH buffering capacity, as well. Special attention is paid to the discussion of acetone production in diseases amongst which endogenous and exogenous acetonemiae have been defined. Acetonemiae of endogenous origin are due to the increased rate of acetone production followed by an increase of degrading capacity as cytochrome P450IIE1 (CYPIIE1) isozymes become induced. Exogenous acetonemiae usually resulted from intoxications caused by either acetone itself or other exogenous compounds (ethanol, isopropyl alcohol). It is highlighted that, on the one hand, isopropanol is also a normal constituent of metabolism and, on the other hand, the flat opinion that the elevation of its plasma level is a sign of alcoholism cannot further be held. The possible future directions of research upon acetone are depicted by emphasizing the need for the clear-cut identification of mammalian acetoacetate decarboxylase, and the investigation of race differences and genetic background of acetone metabolism.
Keywords: Acetone; Acetol; Methylglyoxal; Isopropyl alcohol; Fasting; Diabetes mellitus; Gluconeogenesis;

Heterogeneous inhibition of horseradish peroxidase activity by cadmium by Jacqueline Keyhani; Ezzatollah Keyhani; Nahid Einollahi; Dariush Minai-Tehrani; Sekineh Zarchipour (140-148).
Inhibition of horseradish peroxidase (HRP) activity by cadmium was studied under steady-state kinetic conditions after preincubation of the enzyme with millimolar concentrations of Cd2+ for various periods of time. The H2O2-mediated oxidation of o-dianisidine by HRP was used to assess the enzymatic activity. Cd2+ was found to be either a noncompetitive inhibitor of HRP or a mixed inhibitor of HRP depending both on the duration of incubation with HRP and on Cd2+ concentration. Furthermore, for the same inhibition type, K i values dropped as incubation time increased. These results suggested that Cd2+ would slowly bind to the enzyme and progressively induce conformational changes. Spectrophotometric analysis showed that indeed Cd2+ altered the heme Soret absorption band on binding HRP and exhibited a K d which decreased as the incubation time of HRP with Cd2+ increased. Hill plots suggested a cooperative binding of up to three Cd2+ ions per molecule of HRP. Thus, Cd2+ binding to HRP resulted in progressive inhibition of enzymatic activity with a change in the inhibition type as the number of Cd2+ ions per HRP molecule increased. Results also illustrated the potential danger of long-term exposure to heavy metals, even for enzymes with low affinity for them.
Keywords: Cadmium; Enzyme inhibition; Cooperativity; Horseradish peroxidase; O-dianisidine;

Cathepsin B in osteoblasts by Maria Cristina Aisa; Tommaso Beccari; Egidia Costanzi; Dario Maggio (149-159).
Active cathepsin B has been found in cell extract and medium of human osteoblast-like cells and MG-63 cells. The released form is stable at neutral and alkaline pH and, in both cell types, intracellular and extracellular cathepsin B activities are increased by interleukin-1 beta (IL-1β) and parathyroid hormone (PTH). To evaluate the physiological role of cathepsin B in osteoblasts, we investigated the production and secretion of this enzyme in normal human synovial fibroblasts and modulation by IL-1β and PTH. Lactate secretion concurrent with release of cathepsin B and comparable responses in osteoblasts were also examined. Our data show that synovial fibroblasts respond differently to treatment with the two agents, suggesting a cell-specific regulation of cathepsin B and possible involvement in osteoblast physiology. Cathepsin B involvement was then evaluated in the activation of plasminogen activator (PA) in MG-63 cells using two specific inhibitors of cathepsin B, CA074 and CA074-Me, in constitutive conditions and after treatment with IL-1β. As results of PA activity obtained in the presence of IL-β were in contrast with previous reports, we examined the activities of PA, pro-PA activated with trypsin, and plasmin in cell extract and media of MG-63 cells after 24-h treatment with IL-1β. Results show that in normal conditions and in the presence of IL-1β, cathepsin B is involved in the activation of PA. Moreover, IL-1β stimulates PA, pro-PA activated by trypsin, and plasmin activity in medium, whereas in cell extract it stimulates pro-PA activated by trypsin and plasmin activity. IL-1β has no effect on cell extract-associated PA.
Keywords: Cathepsin B; Plasminogen activator; MG-63 cells; Plasmin; Interleukin-1 beta; CA074; CA074-Me;

Ozone impact on the photosynthetic apparatus and the protective role of polyamines by E. Navakoudis; C. Lütz; C. Langebartels; U. Lütz-Meindl; K. Kotzabasis (160-169).
One of the primary plant mechanisms protecting leaf cells against enhanced atmospheric ozone is the accumulation of polyamines, generally observed as an increase in putrescine level, and in particular its bound form to thylakoid membranes. Ozone-sensitive plants of tobacco (cultivar Bel W3) in contrast to ozone-tolerant Bel B, are not able to increase their endogenous thylakoid membrane-bound putrescine when they are exposed to an atmosphere with enhanced ozone concentration, resulting in reduction of their photosynthetic rates and consequently reduction in plant biomass formation. In comparison to the tolerant cultivar Bel B, a prolongation of ozone exposure thus can lead to typical visible symptoms (necrotic spots) in leaves of the sensitive plant. Exogenously manipulated increase of the cellular putrescine levels of the ozone-sensitive Bel W3 is sufficient to revert these effects, whereas a reduction in endogenous putrescine levels of the tolerant cultivar Bel B renders them sensitive to ozone treatment. The results of this work reveal a regulator role for polyamines in adaptation of the photosynthetic apparatus and consequently to its protection in an environment polluted by ozone.
Keywords: Bel B and Bel W3 tobacco cultivar; Ozone; Polyamine; Photosynthesis;

A trypsin inhibitor from Peltophorum dubium seeds active against pest proteases and its effect on the survival of Anagasta kuehniella (Lepidoptera: Pyralidae) by Maria Lı́gia Rodrigues Macedo; Maria das Graças Machado Freire; Elaine Cristina Cabrini; Marcos H Toyama; José Camillo Novello; Sérgio Marangoni (170-182).
A novel trypsin inhibitor was purified from the seeds of Peltophorum dubium (Spreng.). SDS-PAGE under reducing conditions showed that the inhibitor consisted of a single polypeptide chain (ca. 20 kDa). The dissociation constants of 4×10−10 and 1.6×10−10 M were obtained with bovine and porcine trypsin, respectively. This constant was lower (2.6×10−7 M) for chymotrypsin. The inhibitory activity was stable over a wide range of temperature and pH and in the presence of DTT. The N-terminal sequence of the P. dubium inhibitor showed a high degree of homology with other Kunitz-type inhibitors. When fed to the insect Anagasta kuehniella, in an artificial diet (inhibitor concentration 1.6%), the inhibitor produced ∼56% and delayed the development of this lepidopteran. The concentration of inhibitor in the diet necessary to cause a 50% reduction in the weight (ED50) of fourth instar larvae was ∼1%. The action of the P. dubium trypsin inhibitor (PDTI) on A. kuehniella may involve inhibition of the trypsin-like activity present in the larval midgut, resistance of the inhibitor to digestion by midgut enzymes and bovine trypsin, and association of the inhibitor with a chitin column and chitinous structures in the peritrophic membrane and/or midgut of the insect.
Keywords: Trypsin inhibitor; Anagasta kuehniella; Peltophorum dubium; Pest protease; Resistance;

Spectroscopic studies of the interaction of bichromophoric cyanine dyes with DNA. Effect of ionic strength by Fábio A. Schaberle; Vladimir A. Kuz'min; Iouri E. Borissevitch (183-191).
Spectroscopic characteristics of a cyanine dye with two chromophores (biscyanine dye, BCD) in aqueous solutions and effects of NaCl and DNA upon these characteristics have been studied by optical absorption, circular dichroism (CD) and fluorescence spectroscopies. In homogeneous solutions, BCD is characterized by intense optical absorption (ε=1.33×105 M−1 cm−1) and weak fluorescence (ϕ fl=0.018) in the wavelength region greater than 600 nm. The dye forms H-aggregates at low concentrations (10−6 M). NaCl stimulates the formation of both H- and J-aggregates of the dye at much lower dye concentrations, while DNA in low concentrations (<10−6 M) stimulated the formation of just J-aggregates on the surface of the DNA molecule. Higher DNA concentrations induce the dye to disaggregate, and there exists an equilibrium between three dye forms: free monomers, J-aggregates and bound monomers, the maximum content of J-aggregates was observed at [DNA]/[BCD]=0.6±0.2 and total disaggregation at [DNA]/[BCD]=190±20. J-aggregates are characterized by ϕ fl=0.05 and bound monomers by ϕ fl=0.44. In the presence of NaCl, total disaggregation was observed at [DNA]/[BCD]=570±10 due to competition between Na+ and the dye molecules for DNA electronegative binding sites.
Keywords: Bichromophoric cyanine dye; Photodynamic therapy; DNA; Ionic strength;

In humans, thromboxane (TX) A2 signals through two TXA2 receptor (TP) isoforms, termed TPα and TPβ, that diverge exclusively within the carboxyl terminal cytoplasmic domains. The amino terminal extracellular region of the TPs contains two highly conserved Asn (N)-linked glycosylation sites at Asn4 and Asn16. While it has been established that impairment of N-glycosylation of TPα significantly affects ligand binding/intracellular signalling, previous studies did not ascertain whether N-linked glycosylation was critical for ligand binding per se or whether it was required for the intracellular trafficking and the functional expression of TPα on the plasma membrane (PM). In the current study, we investigated the role of N-linked glycosylation in determining the functional expression of TPα, by assessment of its ligand binding, G protein coupling and intracellular signalling properties, correlating it with the level of antigenic TPα protein expressed on the PM and/or retained intracellularly. From our data, we conclude that N-glycosylation of either Asn4 or Asn16 is required and sufficient for expression of functionally active TPα on the PM while the fully non-glycosylated TPαN4,N16–Q4,Q16 is almost completely retained within the endoplasmic reticulum (ER) and remains functionally inactive, failing to associate with its coupling G protein Gαq and, in turn, failing to mediate phospholipase (PL) Cβ activation.
Keywords: Thromboxane receptor; N-linked glycosylation; Surface expression; G protein interaction; Effector signalling;

Two distinct extracellular α-l-arabinofuranosidases (AFases; EC 3.2.1.55) were purified from the culture filtrate of Penicillium chrysogenum 31B. The molecular masses of the enzymes were estimated to be 79 kDa (AFQ1) and 52 kDa (AFS1) by SDS-PAGE. Both enzymes had their highest activities at 50 °C and were stable up to 50 °C. Enzyme activities of AFQ1 and AFS1 were highest at pH 4.0 to 6.5 and pH 3.3 to 5.0, respectively. Addition of 10 mg/ml arabinose to the reaction mixture decreased the AFS1 activity but hardly affected AFQ1. Both enzymes displayed broad substrate specificities; they released arabinose from branched arabinan, debranched arabinan, arabinoxylan, arabinogalactan, and arabino-oligosaccharides. AFS1 also showed low activity towards p-nitrophenyl-β-d-xylopyranoside. An exo-arabinanase, which catalyzes the release of arabinobiose from linear arabinan at the nonreducing terminus, acted synergistically with both enzymes to produce l-arabinose from branched arabinan.
Keywords: Arabinofuranosidase; Penicillium chrysogenum; Arabinan;

DNA damage measured by liquid chromatography-mass spectrometry in mouse fibroblast cells exposed to oxidative stress by Jean B. Dawidzik; Helen B. Patrzyc; Herbert Iijima; Edwin E. Budzinski; Alan J. Higbee; Han-Chun Cheng; Harold C. Box (211-217).
Oxidative DNA damage can result from environmental factors, such as radiation, as well as from the untoward consequences of normal metabolic processes. It is of interest to assay oxidative DNA damage in cells and tissues because this damage has been implicated in human disease, particularly cancer. Eleven indicators of oxidative DNA damage have been measured by Liquid Chromatography-Mass Spectrometry (LC-MS) in DNA extracted from cells exposed to oxidative stress. Mouse fibroblast cells were exposed to hydrogen peroxide and to UVC light and to the combined action of both agents. Significant increases of the 8-oxo-7,8-dihydropurine lesions over background were detected. Significant increases of the formamido lesions resulting from breakdown of pyrimidine bases were also observed. Of especial interest was the observation of double lesions, tandem combinations of both aforementioned lesions, in cells exposed to oxidative stress.
Keywords: DNA damage; Liquid chromatography-mass spectrometry; Oxidative stress;

Induction of hsp70 in transgenic Drosophila: biomarker of exposure against phthalimide group of chemicals by Aamir Nazir; Daya Krishna Saxena; Debapratim Kar Chowdhuri (218-225).
The expression of stress genes is suggested to be a potentially sensitive indicator of any chemical or physical assault. This led us to explore the possibility of using expression of one of the major stress genes, hsp70, in Drosophila as a biomarker against phthalimide group of chemicals, which may accordingly provide an early indication of exposure to these hazardous chemicals. We exposed third instar larvae of transgenic Drosophila melanogaster (hsp70lacZ) Bg 9 to different concentrations of the test chemicals (Captan, Captafol and Folpet) for various time intervals (2–48 h) to evaluate expression of hsp70 by X-gal staining, ONPG assay and whole organ in situ immunohistochemistry. The study was further extended to examine the effect of the said chemicals on development of the organism and tissue damage occurring in them, thus raising the possibility of evaluating comparative deleterious effect inducing potential of the test chemicals. Our results showed a strong hsp70 expression in the Captafol-exposed larvae followed by weaker expression in Captan- and Folpet-treated larvae. The effect was further reflected on development as revealed by a delay in emergence of the flies by 3 days in 200 ppm Captafol-exposed group. Hsp70 was found not to be induced at 0.0002 ppm Captafol and at 0.002 ppm Captan and Folpet. The present study suggests that (a) hsp70 induction is sensitive enough to be used as a biomarker against phthalimide group of chemicals, (b) amongst the three test chemicals, Captafol is the most deleterious compound followed by Captan and Folpet, (c) 0.0002 ppm for Captafol and 0.002 ppm for Captan and Folpet, respectively, can be regarded as no observed adverse effect level (NOAEL).
Keywords: Phthalimide; hsp70; Transgenic Drosophila; Biomarker;

Glutathione S-transferases (GSTs) are multi-functional enzymes, known to conjugate xenobiotics and degrade peroxides. Herein, we report on the potential of four Zea mays GST isoforms (Zm GST I–I, Zm GST I–II, Zm GST II–II and Zm GST III–III) to act as binding and protection proteins. These isoforms bind protoporphyrin IX (PPIX), mesoporphyrin, coproporphyrin, uroporphyrin and Mg-protoporpyhrin, but do not form a glutathione conjugate. The binding is non-covalent and inhibits GSTs enzymatic activity, dependent on the type of the porphyrin and GST isoform tested. I 50 values are in the range of 1 to 10 μM for PPIX, the inhibition by mesoporphyrin and Mg-protoporphyrin (Mg-PPIX) is two to five times less. The mode of binding is non-competitive for the hydrophobic substrate and competitive for glutathione. Binding affinities (K D values) of the GST isoforms are between 0.3 and 0.8 μM for coproporphyrin and about 2 μM for mesoporphyrin. Zm GST III–III prevents the nonenzymatic autoxidation of protoporphyrinogen to the phytotoxic PPIX. Zm GST II–II can reduce the oxidative degradation of hemin. This points to a specific ligand role of distinct GST isoforms to protect tetrapyrroles in the plant cell.
Keywords: Glutathione S-transferase; Non-covalent binding; Protection; Heme; Protoporphyrinogen; Tetrapyrrole;