Postischemic Cerebral Lipid Peroxidation In Vitro: Modification by Dietary Vitamin E

Using an in vitro system, we studied the effect of postischemic reoxygenation on cerebral lipid peroxidation in relation to the dietary intake of vitamin E (VE) in rats. Homogenates prepared from VE-deficient, -normal, and -supplemented brains, which were previously rendered ischemic for 30 min by decapitation, were incubated under air or nitrogen gas for 60 min. The extent of peroxidation in brain tissue was estimated by a thiobarbituric acid (TBA) test and by diene conjugation in total lipid extracts. The brain levels of alpha-tocopherol and of total and free fatty acids (FAs) were also determined. Aerobic incubation increased TBA reactants in all dietary groups; the effect was largest in the VE-deficient group, intermediate in the VE-normal group, and smallest in the VE-supplemented group. In contrast, nitrogen incubation did not alter the basal levels of TBA reactants except for a small rise associated with VE deficiency. Conjugated dienes changed in parallel with TBA reactants. alpha-Tocopherol decreased after aerobic incubation and also, to a lesser degree, after nitrogen incubation in each dietary group. Only in the reoxygenated samples of the VE-deficient group was there a significant fall in total polyunsaturated FAs. The levels of free FAs continuously increased throughout ischemia and subsequent incubation. However, the level of free polyunsaturated FAs was similar after aerobic and nitrogen incubation in each dietary group, and was not affected by VE. Thus, cerebral reoxygenation after ischemia propagates peroxidative reactions within esterified polyunsaturated FAs. The modification by VE of reoxygenation-induced lipid peroxidation suggests free radical mediation.     

Kinetics and Thermostability of NADP-Isocitrate Dehydrogenase from Cephalosporium acremonium

NADP-isocitrate dehydrogenase [isocitrate:NADP(sup+) oxidoreductase (decarboxylating); EC 1.1.1.42] was purified from Cephalosporium acremonium as a single species. The enzyme is a dimer of 140 kDa with identical subunits of 75 kDa. The existence of a monomer-dimer equilibrium is apparent as revealed by an enzyme dilution approach. The chelate complex of the tribasic form of isocitrate and Mg(sup2+) is the true substrate. The V(infmax) depends on a basic form of an ionizable group of the enzyme-substrate complex with a pK(infes) (pK of the enzyme-substrate complex) of 6.9 and a (Delta)H(infion) (activation enthalpy) of -2 (plusmn) 0.4 kcal mol(sup-1) (ca. 8 (plusmn) 2 kJ mol(sup-1)). The enzyme showed maximum activity at 60(deg)C, an unusually high temperature for a nonthermophilic fungus. The thermodynamic parameters for isocitrate oxidative decarboxylation and for the binding of isocitrate and NADP(sup+) were calculated. We analyzed the kinetic thermal stability of the enzyme at pH 6.5 and 7.6. It was inactivated above 40(deg)C following a first-order kinetics. The presence of 12 mM Mg(sup2+) plus 10 mM dl-isocitrate led to 100% protection of enzyme activity against inactivation at 60(deg)C for 120 min. Removal of either or both compounds led to activity loss. A greater stabilizing role for Mg(sup2+) was seen at pH 6.5 than at pH 7.6, whereas the stabilizing effect of isocitrate was not dependent on pH.     

Differing Neurochemical and Morphological Sequelae of Global Ischemia: Comparison of Single- and Multiple-Insult Paradigms

The purpose of this investigation was to investigate pathomechanisms responsible for the deleterious effects of repeated episodes of brief forebrain ischemia. Halothane-anesthetized male Wistar rats were subjected to either (a) a single 15-min period or (b) three 5-min periods (separated by 1 h) of global forebrain ischemia by bilateral carotid artery occlusions plus hypotension (50 mm Hg), followed by various periods of recirculation. Brain temperature was normothermic throughout. In one series of rats, extracellular levels of glutamate, glycine, and gamma-aminobutyric acid (GABA) were measured in the dorsolateral striatum (n = 6-8 per group) and lateral thalamus (n = 4-6 per group) by microdialysis and HPLC before and during ischemia and during 3-5 h of recirculation. In a parallel series of rats (n = 6 per group), ischemic cell change was quantified at 2 (dark neurons), 24, or 72 h following either single or multiple ischemic insults. A single 15-min ischemic period led to massive glutamate release (13-fold increase; p = 0.001), which returned to normal by 20-30 min of recirculation and remained normal thereafter. By contrast, in rats with three 5-min periods of ischemia, the glutamate level rise with each repeated insult (four- to 4.5-fold; p < or = 0.02) was smaller than that observed during the single 15-min insult, but a late sustained rise (five- to six-fold; p < 0.05) occurred at 2-3 h of recirculation. Brief ischemia-induced elevations of glycine and GABA levels were detected in both the single- and multiple-insult groups, with normalization during recirculation. In contrast, the excitotoxic index, a composite measure of neurotransmitter release ([glutamate] x [glycine]/[GABA]), differed markedly following single versus multiple insults (p = 0.002 by repeated-measures analysis of variance) and increased by seven- to 12-fold (p < 0.05) at 1-3 h following the third insult. The total amount of glutamate released was 3.3-fold higher in the multiple-insult than in the single-insult group (p < 0.02). At 2 h of recirculation, histopathological analysis of dorsolateral striatum showed a significantly greater frequency of dark neurons in the multiple- than in the single-insult group (p < 0.05 by analysis of variance). In the thalamus, a higher frequency of ischemic neurons was seen in the multiple-than in the single-insult group at all intervals studied. Thus, in rats with multiple ischemic insults, accelerated ischemic damage was found in the striatum, and severe ischemic injury was documented in the thalamus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Potentiation of lipid peroxides by ischemia in rat brain

Post-ischemic changes in energy metabolites and natural antioxidant compounds have been measured in rat brain in vitro concurrent with two different assays for peroxidized lipids. No exogenous free radical initiators were employed. In vitro oxygenation of minced brain preparations for periods of 10 minutes to 4 hours, following 5 minutes of preparatory ischemia, yielded increased levels of lipid conjugated dienes and TBA-reactive material, in contrast to anaerobically incubated preparations. However, either aerobic or anaerobic incubation of brain minces facilitated increased ratios of lactate: pyruvate and glutathione (oxidized): glutathione (reduced), as well as increased total ubiquinone content and loss of -tocopherol. Observation of lipid radical formation in vivo was then attempted using rats given embolic stroke in one hemisphere and left in the post-ischemic condition for times up to 24 hours. Conjugated dienes were found in lipids extracted from the ipsilateral hemisphere but not from the contralateral hemisphere. These observations of conjugated dienes in vivo (formed presumably during post-ischemic reperfusion) and in vitro (facilitated by oxygenation of brain minces), indicate that lipid radical intermediates and associated chain peroxidation processes are potentiated by ischemia and occur during tissue reoxygenation.     

VIP and PACAP receptors coupled to adenylyl cyclase in human lung cancer: a study in biopsy specimens

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are important neuropeptides in the control of lung physiology. Both of these commonly bind to specific G protein coupled receptors named VPAC(1)-R and VPAC(2)-R, and PAC(1)-R (with higher affinity for PACAP). VIP and PACAP have been implicated in the control of cell proliferation and tumor growth. This study examined the presence of VIP and PACAP receptors in human lung cancer samples, as well as the functionality of adenylyl cyclase (AC) stimulated by both peptides. Results from RT-PCR and immunoblot experiments showed the expression of VPAC(1)-, VPAC(2)- and PAC(1)-R in lung cancer samples. Immunohistochemical studies showed the expression of VPAC(1) and VPAC(2) receptors. These receptors were positively coupled to AC, but the enzyme activity was impaired as compared to normal lung. There were no changes in Galpha(s) or Galpha(i) levels. Present results contribute to a better knowledge of VIP/PACAP actions in lung cancer and support the interest for the development of VIP/PACAP analogues with therapeutic roles.     

Synthesis of conformationally constrained hydroxy-α-amino acids by intramolecular conjugate addition

Summary. An efficient and easily applicable method for the synthesis of a variety of hydroxy-α-amino acids analogues of serine and phenylalanine has been established. The method involves the stereoselective intramolecular conjugate addition of the benzamide group to cyclohexenone promoted by Lewis acid. Subsequent transformations of functional groups pro-vide the conformationally constrained 2-hydroxy- and 2,4-dihydroxy-6-phenylcyclohexane-α-amino acids. Received February 5, 1999, Accepted May 16, 1999     

Accumulated bending energy elicits neutral sphingomyelinase activity in human red blood cells

We propose that accumulated membrane bending energy elicits a neutral sphingomyelinase (SMase) activity in human erythrocytes. Membrane bending was achieved by osmotic or chemical processes, and SMase activity was assessed by quantitative thin-layer chromatography, high-performance liquid chromatography, and electrospray ionization-mass spectrometry. The activity induced by hypotonic stress in erythrocyte membranes had the pH dependence, ion dependence, and inhibitor sensitivity of mammalian neutral SMases. The activity caused a decrease in SM contents, with a minimum at 6 min after onset of the hypotonic conditions, and then the SM contents were recovered. We also elicited SMase activity by adding lysophosphatidylcholine externally or by generating it with phospholipase A(2). The same effect was observed upon addition of chlorpromazine or sodium deoxycholate at concentrations below the critical micellar concentration, and even under hypertonic conditions. A unifying factor of the various agents that elicit this SMase activity is the accumulated membrane bending energy. Both hypo-and hypertonic conditions impose an increased curvature, whereas the addition of surfactants or phospholipase A(2) activation increases the outer monolayer area, thus leading to an increased bending energy. The fact that this latent SMase activity is tightly coupled to the membrane bending properties suggests that it may be related to the general phenomenon of stress-induced ceramide synthesis and apoptosis.     

Autoreactive B cells discriminate CpG-rich and CpG-poor DNA and this response is modulated by IFN-alpha

Autoreactive B cells are activated by DNA, chromatin, or chromatin-containing immune complexes (ICs) through a mechanism dependent on dual engagement of the BCR and TLR9. We examined the contribution of endogenous DNA sequence elements to this process. DNA sequence can determine both recognition by the BCR and by TLR9. DNA fragments containing CpG islands, a natural source of unmethylated CpG dinucleotides, promote the activation of DNA-reactive B cells derived from BCR transgenic mice as well as DNA-reactive B cells present in the normal repertoire. ICs containing these CpG island fragments are potent ligands for AM14 IgG2a-reactive B cells. In contrast, ICs containing total mammalian DNA, or DNA fragments lacking immunostimulatory motifs, fail to induce B cell proliferation, indicating that BCR crosslinking alone is insufficient to activate low-affinity autoreactive B cells. Importantly, priming B cells with IFN-alpha lowers the BCR activation threshold and relaxes the selectivity for CpG-containing DNA. Taken together, our findings underscore the importance of endogenous CpG-containing DNAs in the TLR9-dependent activation of autoreactive B cells and further identify an important mechanism through which IFN-alpha can contribute to the pathogenesis of systemic lupus erythematosus.     

Seasonal stable isotope variations of the modern Amazonian freshwater bivalve Anodontites trapesialis

In a floodplain lake of the Amazon River near the city of Iquitos, northeastern Peru, a one-year monitoring experiment was conducted during which water samples and living bivalves (Anodontites trapesialis) were collected with the aim to investigate seasonal δ¹⁸O variation in and fractionation between bivalve aragonite and host water. Both host water and molluscan growth increments show more than 8‰ seasonal variation in δ¹⁸O. In the floodplain lake under study the δ¹⁸O variation of the water is controlled by contrasting dry and wet season evaporation-precipitation regimes. Molluscan δ¹⁸O appears to be in equilibrium with the host water. Although an approximately 4.0‰ offset occurs, δ¹³C records of water and bivalves are in good agreement, suggesting that both δ¹⁸O and δ¹³C of the shells of freshwater bivalve A. trapesialis are good recorders of (palaeo-)environmental conditions. The δ¹³C of Dissolved Inorganic Carbon (DIC) is governed by plant growth and/or by changes in aquatic chemistry, affecting the DIC pool.