Vitamin E,catalase, manganous or cobaltous ions and dithiothreitol protect against Tween 20-induced hemolysis of vitamin E-deficient chick and kid erythrocytes

Summary The effective Tween 20 concentration at which 70% hemolysis was achieved in vitro correlated with the plasma vitamin E content of chicks (r=0.85). Addition of catalase, MnCl₂, CoCl₂ or dithiothreitol in vitro showed significant protection against the hemolysis induced by Tween 20 in vitamin E-deficient chick and kid erythrocytes.     

Induction of nuclear styrene monooxygenase and epoxide hydrolase in rat liver

Summary The apparent K_m and V_max of styrene monooxygenase and styrene epoxide hydrolase were determined in intact nuclear preparations from male rat liver after in vivo treatment with phenobarbital and -naphthoflavone, which are known to induce microsomal cytochrome P-450 and cytochrome P-448 respectively. Treatment with phenobarbital does not alter the apparent K_m, but greatly increases the V_max of both nuclear styrene monooxygenase and styrene epoxide hydrolase. Almost the same pattern is observed for styrene monooxygenase after treatment with -naphthoflavone, whereas the same treatment slightly increases both the V_max and K_m value of styrene epoxide hydrolase.This work was supported by the CNR (National Research Council) Contract No. 78.02864.96 within the special program Control of Cancer Growth.     

Development of in vitro toxicity tests with cultures of freshly isolated rat hepatocytes

Summary Freshly isolated and cultured hepatocytes were analyzed by two-parameter flow cytometry. The combined analysis of DNA and cellular protein content allowed the contribution of ploidy classes and of subpopulations within a ploidy class to be defined. Analysis of hepatocytes during exposure to dimethylsulfoxide (DMSO), phenobarbital (PB), low oxygen tension (5% O₂) or fetal calf serum (FCS), provided insight into the dynamic response of individual ploidy classes as a function of culture time. By analogy with the age-dependent ploidy shifts in vivo, hepatocyte-cultures shift towards adult animals during exposure to DMSO and towards young animals when cultured at low pO₂ (4% O₂). FCS and phenobarbital disturb this constitutive ploidy balance. FCS increased the 2 N cell population, where stem cells probably respond to the proliferative stimuli provided by growth factors in the serum. Phenobarbital affects the liver-specific 4 N hepatocytes, which agrees with effects seen in liver after exposure in vivo. It is suggested that drug-induced pathological alterations in ploidy in hepatocyte cultures could serve as indicators of compounds, such as liver tumor promoters, which interfere with cell differentiation in liver. The heterotypic cell-cell interaction of freshly isolated hepatocytes with isolated, in vitro cultured, rat liver epithelial cells in co-cultures proved to be a valuable concept in toxicity testing: aldrin epoxidase, an enzyme system involved in xenobiotic metabolism, was stabilized for more than two weeks. After exposure to the three chemicals, 2-acetylaminofluoren, procarbazine and cyproterone-acetate, a preferential toxicity for each compound and cell population was established. Thus heterotypic cell cultures can considerably increase the amount of information available from in vitro studies.The final concept, combining monitoring of cellular DNA (ploidy) and protein content in hepatocyte cultures during and after exposure to a given test compound at tissue oxygen tension with the heterotypic cell-cell interaction, would create a more in vivo-like culture system. This would enhance the predictability of hepatocyte cultures and contribute to a more widespread use of the test system and as a result help to reduce the number of whole-animal tests.     

Autophagic activity in cortical neurons under acute oxidative stress directly contributes to cell death

Primary neurons undergo insult-dependent programmed cell death. We examined autophagy as a process contributing to cell death in cortical neurons after treatment with either hydrogen peroxide (H₂O₂) or staurosporine. Although caspase-9 activation and cleavage of procaspase-3 were significant following staurosporine treatment, neither was observed following H₂O₂ treatment, indicating a non-apoptotic death. Autophagic activity increased rapidly with H₂O₂, but slowly with staurosporine, as quantified by processing of endogenous LC3. Autophagic induction by both stressors increased the abundance of fluorescent puncta formed by GFP-LC3, which could be blocked by 3-methyladenine. Significantly, such inhibition of autophagy blocked cell death induced by H₂O₂ but not staurosporine. Suppression of Atg7 inhibited cell death by H₂O₂, but not staurosporine, whereas suppression of Beclin 1 prevented cell death by both treatments, suggesting it has a complex role regulating both apoptosis and autophagy. We conclude that autophagic mechanisms are activated in an insult-dependent manner and that H₂O₂ induces autophagic cell death.     

The effect of hydrogen peroxide on the cyclin D expression in fibroblasts

Activation of mitogen-activated protein (MAP) kinase is essential for cyclin D1 expression and provides a link between mitogenic signalling and cell cycle progression. Hydrogen peroxide (H₂ O₂ ) activates MAP kinase; however, it is not known whether this leads to cyclin D expression. Sustained expression of cyclin D1 and D2 was observed when Her14 fibroblasts were incu-bated with 3 mM or higher H₂ O₂ concentrations. Similar results were obtained when cells were incubated in the presence of serum (FCS). However, the sustained expres-complex sion of cyclin D1 and D2 upon H₂ O₂ treatment was not due to the MAP kinase pathway, because MAP kinase kinase inhibitors did not inhibit cyclin D expression. Furthermore, cyclin D1 and D2 levels remained constant even after addition of a protein synthesis inhibitor, indicating that the effect of H₂ O₂ was not due to induction of protein synthesis. These results indicate that H₂ O₂ reversibly inhibits the ubiquitin-proteasome dependent degra-dation of cyclin D1 and D2, probably by transiently in-hibiting ubiquitination and/or the proteasome. Received 12 March 2001; received after revision 5 April 2001; accepted 9 April 2001     

Hydrogen peroxide protects tobacco from oxidative stress by inducing a set of antioxidant enzymes

Tolerance against oxidative stress generated by high light intensities or the catalase inhibitor aminotriazole (AT) was induced in intact tobacco plants by spraying them with hydrogen peroxide (H₂O₂). Stress tolerance was concomitant with an enhanced antioxidant status as reflected by higher activity and/or protein levels of catalase, ascorbate peroxidase, guaiacol peroxidases, and glutathione peroxidase, as well as an increased glutathione pool. The induced stress tolerance was dependent on the dose of H₂O₂ applied. Moderate doses of H₂O₂ enhanced the antioxidant status and induced stress tolerance, while higher concentrations caused oxidative stress and symptoms resembling a hypersensitive response. In stress-tolerant plants, induction of catalase was 1.5-fold, that of ascorbate peroxidase and glutathione peroxidase was 2-fold, and that of guaiacol peroxidases was approximately 3-fold. Stress resistance was monitored by measuring levels of malondialdehyde, an indicator of lipid peroxidation. The levels of malondialdehyde in all H₂O₂-treated plants exposed to subsequent high light or AT stress were similar to those of unstressed plants, whereas lipid peroxidation in H₂O₂-untreated plants stressed with either high light or AT was 1.5- or 2-fold higher, respectively. Although all stress factors caused increases in the levels of reduced glutathione, its levels were much higher in all H₂O₂-pretreated plants. Moreover, significant accumulation of oxidized glutathione was observed only in plants that were not pretreated with H₂O₂. Extending the AT stress period from 1 to 7 days resulted in death of tobacco plants that were not pretreated with H₂O₂, while all H₂O₂-pretreated plants remained little affected by the prolonged treatment. Thus, activation of the plant antioxidant system by H₂O₂ plays an important role in the induced tolerance against oxidative stress. Received 11 December 2001; received after revision 25 January 2002; accepted 4 February 2002     

Cytosolic free Ca2+ in insulin secreting cells and its regulation by isolated organelles

Summary The role of Ca²⁺ in secretagogue-induced insulin release is documented not only by the measurements of⁴⁵Ca fluxes in pancreatic islets, but also, by direct monitoring of cytosolic free Ca²⁺, [Ca²⁺]_i. As demonstrated, using the fluorescent indicator quin 2, glyceraldehyde, carbamylcholine and alanine raise [Ca²⁺]_i in the insulin secreting cell line RINm5F, whereas glucose has a similar effect in pancreatic islet cells. The regulation of cellular Ca²⁺ homeostasis by organelles from a rat insulinoma, was investigated with a Ca²⁺ selective electrode. The results suggest that both the endoplasmic reticulum and the mitochondria participate in this regulation, albeit at different Ca²⁺ concentrations. By contrast, the secretory granules do not appear to be involved in the short-term regulation of [Ca²⁺]_i. Evidence is presented that inositol 1,4,5-trisphosphate, which is shown to mobilize Ca²⁺ from the endoplasmic reticulum, is acting as an intracellular mediator in the stimulation of insulin release.     

Arachidonic acid signaling is involved in the mechanism of imidazoline-induced K<Subscript>ATP</Subscript> channel-independent stimulation of insulin secretion

The mechanism by which the novel, pure glucose-dependent insulinotropic, imidazoline derivative BL11282 promotes insulin secretion in pancreatic islets has been investigated. The roles of K_ATP channels, α₂-adrenoreceptors, the I₁-receptor-phosphatidylcholine-specific phospholipase (PC-PLC) pathway and arachidonic acid signaling in BL11282 potentiation of insulin secretion in pancreatic islets were studied. Using SUR1^(-/-) deficient mice, the previous notion that the insulinotropic activity of BL11282 is not related to its interaction with K_ATP channels was confirmed. Insulinotropic activity of BL11282 was not related to its effect on α₂-adrenoreceptors, I₁-imidazoline receptors or PC-PLC. BL11282 significantly increased [³H]arachidonic acid production. This effect was abolished in the presence of the iPLA₂ inhibitor, bromoenol lactone. The data suggest that potentiation of glucose-induced insulin release by BL11282, which is independent of concomitant changes in cytoplasmic free Ca²⁺ concentration, involves release of arachidonic acid by iPLA₂ and its metabolism to epoxyeicosatrienoic acids through the cytochrome P-450 pathway. Received 5 July 2007; received after revision 18 September 2007; accepted 20 September 2007     

Time reversal operations,representations of the Lorentz group,and the direction of time

A theory is usually said to be time reversible if whenever a sequence of states S₁(t₁), S₂(t₂), S₃(t₃) is possible according to that theory, then the reverse sequence of time reversed states S₃^T(t₁), S₂^T(t₂), S₁^T(t₃) is also possible according to that theory; i.e., one normally not only inverts the sequence of states, but also operates on the states with a time reversal operator T. David Albert and Paul Horwich have suggested that one should not allow such time reversal operations T on states. I will argue that time reversal operations on fundamental states should be allowed. I will furthermore argue that the form that time reversal operations take is determined by the type of fundamental geometric quantities that occur in nature and that we have good reason to believe that the fundamental geometric quantities that occur in nature correspond to irreducible representations of the Lorentz transformations. Finally, I will argue that we have good reason to believe that space-time has a temporal orientation.     

Developmental changes of aldehyde dehydrogenase isozymes in human livers: Mitochondrial ALDH2 isozyme is expressed in fetal livers

Summary Previous reports suggested that the major cytosolic aldehyde dehydrogenase (ALDH₁) was present in fetal and infant livers, but the major mitochondrial isozyme (ALDH₂) was absent or severely diminished. Re-examination by means of starch gel electrophoresis followed by enzyme activity staining, and by means of dot blot immuno-hybridization of liver samples with known genotypes of theALDH ₂ locus, indicated that bothALDH ₁ andALDH ₂ genes are expressed in fetal and infant livers. In addition, ALDH₄ isozyme was also observed. The results imply that a fetus with the usual homozygousALDH ₂ ¹ /ALDH ₂ ¹ genotype, but not one with the atypicalALDH ₂ ¹ /ALDH ₂ ² orALDH ₂ ² /ALDH ₂ ² , is capable of detoxifying acetaldehyde transferred from the mother.     

EP2 receptor stimulation promotes calcium responses in astrocytes via activation of the adenylyl cyclase pathway

Astrocytes are a heterogeneous population of cells that are endowed with a great variety of receptors for neurotransmitters and neuromodulators. Recently prostaglandin E₂ has attracted great interest since it is not only released by astrocytes but also activates receptors coupled to either phospholipase C or adenylyl cyclase. We report that EP₂ receptor stimulation triggers cAMP production but also causes release of Ca²⁺ from intracellular stores. This effect is shared by other receptors similarly coupled to adenylyl cyclase and elicited by direct stimulation of the enzyme or application of cAMP analogues. However, the stimulation of the Ca²⁺ response by cAMP is not mediated by protein kinase A, since a specific antagonist of this kinase had no effect. Such a cross-talk between cAMP and Ca²⁺ was not observed in all astrocytes. It might therefore reflect a specific resource of either a subpopulation or astrocytes in a specific functional state. Received 6 June 2006; received after revision 25 July 2006; accepted 31 August 2006     

Relaxation of isolated rabbit veins mediated by latent histamine H2-receptors

Summary Isolated rabbit veins preconstricted by either norepinephrine, methoxamine or potassium were relaxed by histamine in the presence of mepyramine, a histamine H₁-antagonist. The relaxation was not antagonized by atropine, propranolol and indomethacin but by an H₂-antagonist cimetidine. It is likely that histamine relaxes the rabbit veins through H₂-receptors.     

Oxidative stress triggers neuronal caspase-independent death: Endonuclease G involvement in programmed cell death-type III

To characterize neuronal death, primary cortical neurons (C57/Black 6 J mice) were exposed to hydrogen peroxide (H₂O₂) and staurosporine. Both caused cell shrinkage, nuclear condensation, DNA fragmentation and loss of plasma membrane integrity. Neither treatment induced caspase-7 activity, but caspase-3 was activated by staurosporine but not H₂O₂. Each treatment caused redistribution from mitochondria of both endonuclease G (Endo G) and cytochrome c. Neurons knocked down for Endo G expression using siRNA showed reduction in both nuclear condensation and DNA fragmentation after treatment with H₂O₂, but not staurosporine. Endo G suppression protected cells against H₂O₂-induced cell death, while staurosporine-induced death was merely delayed. We conclude that staurosporine induces apoptosis in these neurons, but severe oxidative stress leads to Endo G-dependent death, in the absence of caspase activation (programmed cell death-type III). Therefore, oxidative stress triggers in neurons a form of necrosis that is a systematic cellular response subject to molecular regulation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Kondensationsprodukte von Oxyketonen und Aminoketonen mit 2,4,5-Triamino-6-oxy-pyrimidin

Summary By condensing 2:4:5-triamino-6-hydroxy-pyrimidine with dihydroxyacetone (diacetate), diaminoacetone or acetone-1,3-di (p-formylaminobenzoic acid) not the expected 8- or 9-oxymethyl resp. -aminomethyl-pteridines but 8-or 9-methyl-pteridines were obtained. With p-tolyl-d-isoglucosamine not a tetrahydroxybutyl-pteridine but a trihydroxybutyl-pteridine was formed. For an explanation of these results it is supposed that from the dihydro-pteridines formed at first by intramolecular splitting off of H₂O or R·NH₂ aromatization takes place.     

P2X4 and P2X6 receptors associate with VE-cadherin in human endothelial cells

We investigated the expression of P2X₄ and P2X₆ receptors on human umbilical vein endothelial cells (HUVECs) and found that both P2X receptor subtypes on plasma membranes are largely restricted to areas of cell-cell contact. Co-labelling experiments at the confocal and electron microscopy levels revealed that P2X₄ and P2X₆ receptors are strongly co-localised with the cell adhesion molecule VE-cadherin. The P2X₄ and P2X₆ receptors on plasma membranes at cellular junctions are rapidly (within 5 min) internalised specifically after decreasing extracellular [Ca²⁺]. Disruption of microfilaments, microtubules and integrin-mediated adhesion or stimulation of P2 receptors with ATP did not alter P2X₄ and P2X₆ receptor expression on HUVEC plasma membranes. Membraneous P2X₄ and P2X₆ receptors resisted extraction with Triton-X 100, whereas cytoplasmic P2X receptors were Triton-X 100 soluble. P2X₄ receptors, but not P2X₆ receptors, could be co-immunoprecipitated with VE-cadherin and vice versa. We conclude that P2X₄ and P2X₆ receptors are associated with VE-cadherin at HUVEC adherens junctions. Received 15 March 2002; revised 15 March 2002; accepted 19 March 2002     

Protease-activated-receptor-2 affects protease-activated-receptor-1-driven breast cancer

Mammalian protease-activated-receptor-1 and -2 (PAR₁ and PAR₂) are activated by proteases found in the flexible microenvironment of a tumor and play a central role in breast cancer. We propose in the present study that PAR₁ and PAR₂ act together as a functional unit during malignant and physiological invasion processes. This notion is supported by assessing pro-tumor functions in the presence of short hairpin; shRNA knocked-down hPar2 or by the use of a truncated PAR₂ devoid of the entire cytoplasmic tail. Silencing of hPar2 by shRNA-attenuated thrombin induced PAR₁ signaling as recapitulated by inhibiting the assembly of Etk/Bmx or Akt onto PAR₁-C-tail, by thrombin-instigated colony formation and invasion. Strikingly, shRNA-hPar2 also inhibited the TFLLRN selective PAR₁ pro-tumor functions. In addition, while evaluating the physiological invasion process of placenta extravillous trophoblast (EVT) organ culture, we observed inhibition of both thrombin or the selective PAR₁ ligand; TFLLRNPNDK induced EVT invasion by shRNA-hPar2 but not by scrambled shRNA-hPar2. In parallel, when a truncated PAR₂ was utilized in a xenograft mouse model, it inhibited PAR₁–PAR₂-driven tumor growth in vivo. Similarly, it also attenuated the interaction of Etk/Bmx with the PAR₁-C-tail in vitro and decreased markedly selective PAR₁-induced Matrigel invasion. Confocal images demonstrated co-localization of PAR₁ and PAR₂ in HEK293T cells over-expressing YFP-hPar2 and HA-hPar1. Co-immuno-precipitation analyses revealed PAR₁-PAR₂ complex formation but no PAR₁-CXCR4 complex was formed. Taken together, our observations show that PAR₁ and PAR₂ act as a functional unit in tumor development and placenta-uterus interactions. This conclusion may have significant consequences on future breast cancer therapeutic modalities and improved late pregnancy outcome.     

Differential vascular effects of neuropeptide Y(NPY) selective receptor agonists

Neuropeptide Y (NPY) increases blood pressure either directly or indirectly by potentiating the effect of various vasoconstrictors. Only one (the Y₁-receptor) of two subtypes of receptors (Y₁ and Y₂) is thought to mediate the vascular smooth muscle contraction. To test this hypothesis we challenged isolated rat mesenteric arteries that had a functional endothelium with (1–36) NPY and with specific Y₁-receptor ([Leu³¹, Pro³⁴] NPY) and Y₂-receptor ([Ahx^5–24, -Glu²--Lys³⁰] NPY) agonists. The Y₁-receptor agonist elicited a contractile response similar to that of NPY, whereas the Y₂-receptor agonist had no effect on wall tension. We also found that the presence of a functional endothelium has no influence on the contractile response to NPY. From these data we conclude that the direct contractile effect of NPY in the mesenteric artery is mediated by stimulation of Y₁-receptors and is not endothelium-dependent.     

A VEGF-A splice variant defective for heparan sulfate and neuropilin-1 binding shows attenuated signaling through VEGFR-2

The development of functional blood and lymphatic vessels requires spatio-temporal coordination of the production and release of growth factors such as vascular endothelial growth factors (VEGFs). VEGF family proteins are produced in multiple isoforms with distinct biological properties and bind to three types of VEGF receptors. A VEGF-A splice variant, VEGF-A₁₆₅b, has recently been isolated from kidney epithelial cells. This variant is identical to VEGF-A₁₆₅ except for the last six amino acids encoded by an alternative exon. VEGF-A₁₆₅b and VEGF-A₁₆₅ bind VEGF receptors 1 and 2 with similar affinity. VEGF-A₁₆₅b elicits drastically reduced activity in angiogenesis assays and even counteracts signaling by VEGF-A₁₆₅. VEGF-A₁₆₅b weakly binds to heparan sulfate and does not interact with neuropilin-1, a coreceptor for VEGF receptor 2. To determine the molecular basis for altered signaling by VEGF-A₁₆₅b we measured VEGF receptor 2 and ERK kinase activity in endothelial cells in culture. VEGF-A₁₆₅ induced strong and sustained activation of VEGF receptor 2 and ERK-1 and −2, while activation by VEGF-A₁₆₅b was only weak and transient. Taken together these data show that VEGF-A₁₆₅b has attenuated signaling potential through VEGF receptor 2 defining this new member of the VEGF family as a partial receptor agonist. Received 31 May 2006; received after revision 26 June 2006; accepted 14 July 2006     

Effect of hydrogen peroxide on the binding of Merocyanine 540 to human erythrocytes

The fluorescent dye Merocyanine 540 (MC540) is often used as a probe to monitor the molecular packing of phospholipids in the outer leaflet of biomembranes. In a previous study we showed that the increased staining of erythrocytes with a perturbed membrane structure was mainly due to an increase in the fluorescence yield of cell-bound MC540, rather than to an increase of the number of bound molecules. Erythrocytes and ghosts exposed to continuous fluxes of H₂O₂ exhibited pronounced lipid peroxidation. Further, red blood cells subjected to this form of oxidative stress also showed increased staining with MC540. It appeared that this was caused by a strong increase in binding of MC540, together with a slight red shift of the fluorescence emission maximum and a small increase in the fluorescence yield of bound MC540. The changed MC540 binding characteristics were not observed when lipid peroxidation was suppressed by the presence of the antioxidant BHT in the incubation medium. However, open ghosts exposed to H₂O₂ showed no increase of MC540 binding, excluding a direct involvement of lipid peroxidation. Measurement of fluorescence emission spectra and gel filtration studies showed that MC540 can bind to H₂O₂-exposed hemoglobin. Experiments with erythrocytes lysed in hypotonic medium after exposure to H₂O₂ revealed that peroxidation of lipids with H₂O₂ induced a non-specific permeabilization of the plasma membrane to MC540, thereby allowing MC540 to bind to the oxidatively denatured, more hydrophobic hemoglobin. These results indicate that conclusions about packing of phospholipids in the outer leaflet of the membrane based on increased MC540-staining should be drawn with care. Received 27 September 1996; received after revision 5 November 1996; accepted 27 November 1996     

15-Deoxy-Δ12,14-prostaglandin-J2 reveals a new pVHL-independent, lysosomal-dependent mechanism of HIF-1α degradation

Hypoxia-inducible factor-1α (HIF-1α) protein is degraded under normoxia by its association to von Hippel-Lindau protein (pVHL) and further proteasomal digestion. However, human renal cells HK-2 treated with 15-deoxy-Δ^12,14-prostaglandin-J₂ (15d-PGJ₂) accumulate HIF-1α in normoxic conditions. Thus, we aimed to investigate the mechanism involved in this accumulation. We found that 15d-PGJ₂ induced an over-accumulation of HIF-1α in RCC4 cells, which lack pVHL and in HK-2 cells treated with inhibitors of the pVHL-proteasome pathway. These results indicated that pVHL-proteasome-independent mechanisms are involved, and therefore we aimed to ascertain them. We have identified a new lysosomal-dependent mechanism of HIF-1α degradation as a target for 15d-PGJ₂ based on: (1) HIF-1α colocalized with the specific lysosomal marker Lamp-2a, (2) 15d-PGJ₂ inhibited the activity of cathepsin B, a lysosomal protease, and (3) inhibition of lysosomal activity did not result in over-accumulation of HIF-1α in 15d-PGJ₂-treated cells. Therefore, expression of HIF-1α is also modulated by lysosomal degradation.