BBA - General Subjects (v.1573, #1)

Oxalic acid is available as a natural antioxidant in some systems by Tomoko Kayashima; Tetsuyuki Katayama (1-3).
Oxalic acid is found in a wide variety of plants. This study showed that oxalic acid suppressed in vitro lipid peroxidation in a concentration-dependent manner. Furthermore, oxalic acid reduced the rate of ascorbic acid oxidation in the presence of hydrogen peroxide and Cu2+. These results suggest that oxalic acid is available as a natural antioxidant.
Keywords: Oxalic acid; Antioxidant; Lipid peroxidation; Ascorbic acid oxidation; Chelating action;

An increased antioxidant response (catalase, glutathione S-transferase (GST) and glutathione reductase (GRd) activities in liver and GST activity in head kidney) was observed in carp parasitized by Ptychobothrium sp. compared to healthy fish. In case of a copper contamination of these fish, the decrease in enzymatic activities observed was less pronounced in parasitized than in healthy carp.
Keywords: Antioxidant response; Copper; Carp;

The role of transhepatic bile salt flux in the control of hepatic secretion of triacylglycerol-rich lipoproteins in vivo in rodents by Baukje M Elzinga; Rick Havinga; Julius F.W Baller; Henk Wolters; Vincent Bloks; Arjen R Mensenkamp; Folkert Kuipers; Henkjan J Verkade (9-20).
Bile salts (BS) have been shown to suppress the secretion of very-low-density lipoprotein-triglyceride (VLDL-TG) in rat and human hepatocytes in vitro. In the present study, we investigated whether the transhepatic BS flux affects VLDL-TG concentration and hepatic VLDL-TG secretion in vivo. In rats, the transhepatic BS flux was quantitatively manipulated by 1-week chronic bile diversion (BD), followed by intraduodenal infusion with taurocholate (TC) or saline for 6 h. In mice, the transhepatic BS flux was manipulated by a 3-week dietary supplementation with TC (0.5 wt.%) or cholestyramine (2 wt.%). In rats, BD followed by saline or TC infusion did not affect plasma triacylglycerol (TG) concentration, hepatic TG production rate or VLDL lipid composition, compared to control rats. In mice supplemented for 3 weeks with TC or cholestyramine, the transhepatic BS flux was increased by 335% and decreased by 48%, respectively, compared to controls. Among the three experimental groups of mice, an inverse relationship between transhepatic BS flux and either plasma TG concentration (R 2=0.89) or VLDL-TG production rate (R 2=0.87) was observed, but differences were relatively small. Present data support the concept that BS can reduce VLDL-TG concentration and inhibit hepatic TG secretion in vivo; however, this occurs only at supraphysiological transhepatic BS fluxes in mice.
Keywords: Very-low-density lipoprotein; Taurocholate; Cholestyramine; Lipid; Liver;

Regulation of cadmium uptake by Saccharomyces cerevisiae by D.S Gomes; L.C Fragoso; C.J Riger; A.D Panek; E.C.A Eleutherio (21-25).
In this work, we verified that yeast cells deleted in ZRT1 were not capable of transporting cadmium, suggesting that the transport of this metal into the cell would be carried out through this zinc transporter. On the other hand, cadmium absorption shown by a Δgsh1 strain (a mutant not able of synthesizing glutathione) was twofold higher than in the control strain. Moreover, the deletion of YCF1 (which encodes a vacuolar glutathione S-conjugate pump) impaired the transport of this metal significantly. Using a mutant strain deficient in YAP1, which codifies a transcription factor that controls the expression of both GSH1 and YCF1, we also observed a twofold increase in cadmium uptake, the same behavior shown by Δgsh1 cells. Cadmium is compartmentalized in vacuoles through the Ycf1 transporter, in the form of a bis-glutathionato–cadmium complex. We propose that gsh1 cells are unable to form the Cd–GS2 complex, while ycf1 cells would accumulate high levels of this complex in the cytoplasm. In face of these results we raised the hypothesis that Cd–GS2 complex controls cadmium uptake through the Zrt1 protein.
Keywords: Cadmium; ZRT1; YCF1; YAP1; Glutathione; Saccharomyces cerevisiae;

2-Benzyloxybenzaldehyde inhibits formyl-methionyl-leucyl-phenylalanine stimulation of phospholipase D activation in rat neutrophils by Jih-Pyang Wang; Ling-Chu Chang; Mei-Feng Hsu; Li-Jiau Huang; Sheng-Chu Kuo (26-32).
2-Benzyloxybenzaldehyde (CCY1a) inhibited the formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated phospholipase D (PLD)-mediated products, phosphatidic acid (PA) and phosphatidylethanol (PEt) formation in rat neutrophils in a concentration-dependent manner with IC50 values of 15.8±2.5 and 13.9±2.0 μM, respectively. The underlying cellular signaling mechanism of CCY1a inhibition was investigated. CCY1a inhibited the plateau phase but not the initial Ca2+ spike of fMLP-stimulated Ca2+ signal. CCY1a did not inhibit the [Ca2+]i change in Ca2+-free medium in response to fMLP, but inhibited the [Ca2+]i change by the subsequent addition of Ca2+. In addition, CCY1a treatment attenuated the fMLP-induced protein tyrosine phosphorylation. The membrane translocation of ADP-ribosylation factor (ARF) and Rho A proteins in neutrophils stimulated with fMLP was attenuated by CCY1a in a concentration-dependent manner. In a cell-free system, neither the membrane association of ARF and Rho A caused by GTPγS nor the phorbol myristate acetate-stimulated membrane translocation of Rho A was suppressed significantly by CCY1a. These results indicate that the attenuation of protein tyrosine phosphorylation, blockade of Ca2+ entry, and the suppression of ARF and Rho A membrane translocation are probably obligatory for the CCY1a inhibition of PLD activity in rat neutrophils in response to fMLP.
Keywords: Phospholipase D; Protein tyrosine phosphorylation; Intracellular free Ca2+; ADP-ribosylation factor; Rho A; Neutrophil;

Protective effect of metallothionein-III on DNA damage in response to reactive oxygen species by Ho Jin You; Deuk-hee Oh; Chul Yung Choi; Dong Gun Lee; Kyung-Soo Hahm; Ae Ran Moon; Hye Gwang Jeong (33-38).
Metallothionein (MT)-III is a member of a brain-specific MT family, in contrast to MT-I and MT-II that are found in most tissues and are implicated in metal ion homeostasis and as an antioxidant. To investigate the defensive role of MT-III in terms of hydroxyl radical-induced DNA damage, we used purified human MT-III. DNA damage was detected by single-strand breaks of plasmid DNA and deoxyribose degradation. In this study, we show that MT-III is able to protect against the DNA damage induced by ferric ion-nitrilotriacetic acid and H2O2, and that this protective effect is inhibited by the alkylation of the sulfhydryl groups of MT-III by treatment with EDTA and N-ethylmaleimide. MT-III was also able to efficiently remove the superoxide anion, which was generated from the xanthine/xanthine oxidase system. These results strongly suggest that MT-III is involved in the protection of reactive oxygen species-induced DNA damage, probably via direct interaction with reactive oxygen species, and that MT-III acts as a neuroprotective agent.
Keywords: Metallothionein-III; Reactive oxygen species; DNA damage; Radical scavenging;

An original technique of use of two-phase polymer systems as an analytical research method is described. The technique is based on the absorbance change of two-phase systems in visible spectrum during formation of the phases. Dynamics of this process was demonstrated as the kinetic curves. Addition of studied objects (macromolecules or cells) to the two-phase system modified the shape of the kinetic curve, depending on their individual surface properties. The technique has the following advantages as compared with traditional procedures of the particle surface analysis with the help of two-phase polymer systems: examination of particles with partition coefficients approaching zero; multiple analyses of the same samples; use of interphase as a matrix for study of spontaneous formation of studied particle complexes. The opportunities of the technique were demonstrated in a series of previous authors' works.
Keywords: Aqueous two-phase polymer system; Kinetics; Technique;

This study describes the occurrence of 3-hydroxy-5-hydroperoxy-2-methyl-5,6-dihydropyran-4-one (HMDP) in plasma obtained from normal subjects and patients with type 2 diabetes. We have shown previously that HMDP is a novel hydrophilic hydroperoxide formed in the in vitro Maillard reaction that could be analyzed using ultrasensitive chemiluminescence detection–high-performance liquid chromatography (CL-HPLC). The HMDP concentration was 273±227 nmol/l in normal subjects and 656±535 nmol/l in patients with diabetes. The HMDP concentration was proportional to the plasma glucose concentration level (r=0.640; P<0.01) but not with the glycated hemoglobin level. To investigate the in vivo effects of HMDP, a range of concentrations of the compound was incubated for different time periods with human serum albumin and lipoproteins. HMDP was found to induce denaturation of these macromolecules by modifying lysine residues and causing cross-linking and polymerization of proteins. In the presence of metal ions such as iron and copper, HMDP induced peroxidative degradation of lipoprotein lipids as measured by an elevation in thiobarbituric acid reactive substances (TBARS) concentration. These results suggested that HMDP is produced as a consequence of both hyperglycemia and increased oxidative stress, and may have the potential to contribute to the pathogenesis of arterial complications in diabetes mellitus.
Keywords: 3-Hydroxy-5-hydroperoxy-2-methyl-5,6-dihydropyran-4-one; Maillard reaction; Glycation; Diabetes; Chemiluminescence;

In vivo detection of aflatoxin-induced lipid free radicals in rat bile by R.A Towner; R.P Mason; L.A Reinke (55-62).
Aflatoxin B1 (AFB1), a potent hepatotoxin and hepatocarcinogen, is metabolized in the liver via cytochrome P-450 to an AFB1-8,9-epoxide intermediate. The formation of the AFB1-8,9-epoxide correlates with the pathological changes observed in numerous mammalian species. Oxidative damage has been postulated to play a major role in the mechanisms associated with AFB1-induced cytotoxicity and carcinogenecity in mammalian species. The aim of this study was to detect and identify free radical intermediates from the hepatic metabolism of AFB1 in vivo. Rat bile ducts were cannulated and rats were treated simultaneously with AFB1 (3 mg/kg i.p.) and the spin trapping agent 4-POBN (α-(4-pyridyl-1-oxide)-N-tert-butyl nitrone) (1 g/kg i.p.), and bile was collected over a period of 2 h at 20-min intervals. ESR spectroscopy was used to detect a carbon-centered radical adduct of 4-POBN in rat bile. The effect of metabolic inhibitors, such as deferoxamine mesylate (DFO), an iron chelator, and SKF 525A, a cytochrome P-450 inhibitor, on in vivo aflatoxin-induced free radical formation were also studied. It was found that there was a significant decrease in free radical formation by pre-treatment with both DFO and SKF 525A. This indicates that oxidation of AFB1 generates free radical species via CYP metabolism and an iron-mediated redox mechanism.
Keywords: Aflatoxin B1; Spin trapping; Lipid radical; Desferoxamine mesylate; SKF 525A;

Increased dopamine peroxidation in postmortem Parkinsonian brain by Angela De Iuliis; Alessandro P Burlina; Roberta Boschetto; Pamela Zambenedetti; Paola Arslan; Lauro Galzigna (63-67).
We have previously reported the presence, in human midbrain, of an enzymatic activity which catalyzes the formation of dopaminochrome from dopamine (DA) and hydrogen peroxide. Here, we report, for the first time, an increased DA peroxidizing activity in the midbrain and basal ganglia of autoptic Parkinsonian brains. The crude activity was determined spectrophotometrically in extracts of paraffin-embedded slices obtained from autopsied brain. No addition of substrate was necessary since endogenous substrates such as DA and hydrogen peroxide were present in the samples. In Parkinson's patients' midbrain, this activity was substantially increased compared to normal midbrain. Moreover, the DA peroxidizing activity, which was absent in basal ganglia of normal people, was detectable in all our Parkinson's patients. These observations suggest that a peroxidizing pathway of DA may be present in human brain. The increased peroxidizing activity in Parkinson's patients generates the toxic compound dopaminochrome which may play a role in the pathogenesis of this disease.
Keywords: Parkinson's disease; Dopaminochrome; Dopamine peroxidation; Dopamine; Midbrain; Basal ganglia;

Glycogen depletion and resynthesis during 14 days of chronic low-frequency stimulation of rabbit muscle by C Prats; C Bernal; J.A Cadefau; J.A Frias; M Tibolla; R Cussó (68-74).
Electro-stimulation alters muscle metabolism and the extent of this change depends on application intensity and duration. The effect of 14 days of chronic electro-stimulation on glycogen turnover and on the regulation of glycogen synthase in fast-twitch muscle was studied. The results showed that macro- and proglycogen degrade simultaneously during the first hour of stimulation. After 3 h, the muscle showed net synthesis, with an increase in the proglycogen fraction. The glycogen content peaked after 4 days of stimulation, macroglycogen being the predominant fraction at that time.Glycogen synthase was determined during electro-stimulation. The activity of this enzyme was measured at low UDPG concentration with either high or low Glu-6-P content. Western blots were performed against glycogen synthase over a range of stimulation periods. Activation of this enzyme was maximum before the net synthesis of glycogen, partial during net synthesis, and low during late synthesis. These observations suggest that the more active, dephosphorylated and very low phosphorylated forms of glycogen synthase may participate in the first steps of glycogen resynthesis before net synthesis is observed, while partially phosphorylated forms are most active during glycogen elongation.
Keywords: Glycogen; Glycogen synthase; Rabbit muscle; Electro-stimulation;

Nonviral gene delivery to the lung with copolymer-protected and transferrin-modified polyethylenimine by C Rudolph; U Schillinger; C Plank; A Gessner; P Nicklaus; R.H Müller; J Rosenecker (75-83).
Polyethylenimine (PEI) has been shown to efficiently mediate topical gene transfer to the lungs after either direct intratracheal instillation or nebulisation. Recently, the protection of polyplexes with novel copolymers of poly(ethylene glycol) (PEG) via electrostatic interaction has been reported. In this study, such coated PEI polyplexes were investigated for their stability and interaction with human plasma and bronchoalveolar lavage fluid (BALF). Further, their potential for gene delivery to the mouse lungs in vivo was examined. Plasma protein and mucin adsorption was effectively inhibited when polyplexes were coated with the novel copolymers. Gene transfer efficiency of the coated PEI polyplexes decreased as compared with uncoated PEI polyplexes when administered intratracheally to the lung. The higher the molecular weight of the copolymerized PEG was, the stronger the observed gene transfer reduction. Gene transfer decreased presumably due to reduced interaction of the coated gene vectors with the cell surface. To circumvent this problem, transferrin was combined with PEI/DNA polyplexes for specific binding to the cell surface. In this case, gene transfer efficiency decreased. Gene transfer of the copolymer-protected and transferrin-modified gene vectors increased as compared with the copolymer-protected gene vectors alone but did not reach the level of uncoated gene vectors. These data show that copolymers could be used to effectively shield polyplexes from interaction with components of the airway surface liquid (ASL). Increased gene delivery was found upon transferrin modification of the coated PEI polyplexes suggesting a targeting effect.
Keywords: Gene delivery; Polyethylenimine; Transferrin; Lung; Bronchoalveolar lavage fluid; Gene transfer;

Mechanism of potent antiperoxidative effect of capsaicin by Kentaro Kogure; Satoru Goto; Miki Nishimura; Mina Yasumoto; Kazutoyo Abe; Chie Ohiwa; Hironori Sassa; Takenori Kusumi; Hiroshi Terada (84-92).
The effect of a pungent ingredient of red pepper, capsaicin, on lipid peroxidation of rat liver mitochondria (RLM) induced by ADP/Fe2+ was studied. Capsaicin inhibited the lipid peroxidation significantly, being more effective than the well-known antioxidant α-tocopherol. Capsaicin was also found to scavenge 1,1′-diphenyl-2-picrylhydrazyl (DPPH) radicals both in solution and in membranes, especially the latter. Capsaicin was found to scavenge radicals both at/near the membrane surface and in the interior of the membrane. The phenolic OH-group of capsaicin remained intact after reaction with DPPH radicals, indicating that the hydroxyl group is not associated with the radical scavenging reaction. From the results of quantum chemical calculations of various radical intermediates derived from the model compound N-vanillylacetamide, and the findings that vanillin and 8-methyl-6-noneamide were major reaction products of capsaicin with DPPH radicals, it was concluded that the radical scavenging site of capsaicin is the C7-benzyl carbon.
Keywords: Capsaicin; Lipid peroxidation; Antioxidant; Radical scavenging;

Zigzag motions of the myosin-coated beads actively sliding along actin filaments suspended between immobilized beads by Jun'ichi Wakayama; Makoto Shohara; Chiharu Yagi; Hanako Ono; Norihito Miyake; Yuki Kunioka; Takenori Yamada (93-99).
The motions of myosin filaments actively sliding along suspended actin filaments were studied. By manipulating a double-beam laser tweezers, single actin filaments were suspended between immobilized microbeads. When another beads coated with myosin filaments were dragged to suspended actin filaments, the beads instantly and unidirectionally slid along the actin filaments. The video image analysis showed that the beads slid at a velocity of ca. 3–5 μm/s accompanied with zigzag motions. When beads were densely coated with myosin filaments, the sliding motions became straight and smooth. The obtained results indicate that (1) during the sliding motions, the interaction between myosin heads and actin filaments is weak and susceptible to random thermal agitations, (2) the effects of thermal agitations to the sliding motions of myofilaments are readily suppressed by mechanical constraints imposed to the filaments, and (3) the active sliding force is produced almost in parallel to the filaments axis.
Keywords: Myosin; Actin; Sliding motion; Brownian motion; Motility assay; Optical tweezer;

Available information on the fate and insulinotropic action of l-alanine in isolated pancreatic islets is restricted to data collected in obese hyperglycemic mice. Recent data, however, collected mostly in tumoral islet cells of either the RINm5F line or BRIN-BD11 line, have drawn attention to the possible role of Na+ co-transport in the insulinotropic action of l-alanine. In the present study conducted in islets prepared from normal adult rats, l-alanine was found (i) to inhibit pyruvate kinase in islet homogenates, (ii) not to affect the oxidation of endogenous fatty acids in islets prelabelled with [U-14C]palmitate, (iii) to stimulate 45Ca uptake in islets deprived of any other exogenous nutrient, and (iv) to augment insulin release evoked by either 2-ketoisocaproate or l-leucine, whilst failing to significantly affect glucose-induced insulin secretion. The oxidation of l-[U-14C]alanine was unaffected by d-glucose, but inhibited by l-leucine. Inversely, l-alanine decreased the oxidation of d-[U-14C]glucose, but failed to affect l-[U-14C]leucine oxidation. It is concluded that the occurrence of a positive insulinotropic action of l-alanine is restricted to selected experimental conditions, the secretory data being compatible with the view that stimulation of insulin secretion by the tested nutrient(s) reflects, as a rule, their capacity to augment ATP generation in the islet B cells. However, the possible role of Na+ co-transport in the secretory response to l-alanine cannot be ignored.
Keywords: Pancreatic islet; l-alanine; Insulin secretion; Pyruvate kinase;