Naohisa Ishikawa

Stability of microcystins from cyanobacteria: effect of light on decomposition and isomerization.

Microcystins are potent hepatotoxins produced by cyanobacteria. Their geometrical isomers [6(Z)-Adda microcystin] do not essentially show hepatotoxicity and show weaker tumor-promoting activity than their parent toxins. The present study was undertaken to examine stability of microcystins during the analysis and purification and under photolysis conditions in connection with the detoxification. Microcystin LR was very stable because of limited decomposition and isomerization to its geometrical isomer during analysis and purification. While microcystins decomposed very limitedly by exposure with sunlight alone, the addition of pigments extracted from cyanobacteria accelerated their decompositions

Protein phosphatase 2A-linked and -unlinked caspase-dependent pathways for downregulation of Akt kinase triggered by 4-hydroxynonenal.

AbstractWe studied the signal pathways for regulation of serine/threonine protein kinase Akt in Jurkat cells that had been treated with 4-hydroxynonenal (HNE) for caspase-dependent apoptosis induction. Treatment of cells with HNE led to a decrease in the level of Akt activity due to the dephosphorylation at Ser473, a major regulatory phosphorylation site. HNE-mediated dephosphorylation of Akt was prevented by a protein phosphatase 2A (PP2A) inhibitor, okadaic acid, and by a caspase-3 inhibitor, DEVD-CHO. HNE treatment resulted in an increase in the total level of PP2A activity, release of active tyrosine-dephosphorylated PP2A from the cytoskeleton and PP2A-Akt association, which were all dependent on caspase-3 activation. These results suggest that the level of PP2A activity is at least in part determined by its tyrosine phosphorylation, which is dually controlled by okadaic acid-sensitive phosphatases and protein-tyrosine kinases. Possibly underlying the mechanism of caspase-mediated activation of PP2A, HNE treatment resulted in downregulation of the activity of Src kinase, as a representative caspase-sensitive kinase to phosphorylate PP2A at tyrosine. In addition, activated caspase-3 partially cleaved Akt at a late stage of the apoptosis. These results indicate the existence of two distinct caspase-dependent signal pathways for downregulation of Akt that works as a mechanism of positive feedback regulation for HNE-triggered apoptotic signals.

Protective role of nitric oxide synthase against ischemia-reperfusion injury in guinea pig myocardial mitochondria.

In guinea-pig myocardial mitochondria preparation, lowering the Ca2+ concentration or pH level in the perfusate rapidly elevated the fura-2 Ca2+ signal ([Ca2+]m). Pretreatment with 10(-4) M L-Arg inhibited the rapid [Ca2+]m influx, whereas administration of 10(-4) M L-NAME did not, suggesting some association between nitric oxide (NO*) synthase (NOS) activation and Ca2+ kinetics in mitochondria. Immunoblotting analysis showed that endothelial (e)-NOS was present in mitochondria, but not inducible (i)-NOS or brain (b)-NOS. Electron microscopy observations revealed that the e-NOS antibody-reactive site in the mitochondria was the inner cristae. The production of reactive oxygen species and NO* in isolated mitochondria was detected by the spin trapping technique with electron paramagnetic resonance (EPR) spectrometry. Pretreatment with 10(-5) M S-nitroso-N-acetyl-DL-penicillamine (SNAP) and 10(-5) M 3-[2-Hydroxy-1-(1-methylethyl)-2-nitrosohydrazino]-1-propananin e (NOC 5), which spontaneously generate NO*, completely inhibited the [Ca2+]m uptake. In addition, N-morpholino sydnonimine hydrochloride (SIN-1) (10(-5) M), which simultaneously generates NO* as well as *O2- and peroxynitrite anion (ONOO-), inhibited the increase in [Ca2+]m. ONOO- (3 x 10(-4) M) itself also inhibited this increase. Pretreatment with the *O2(-)-scavenger manganese superoxide dismutase or catalase (200 units/ml) completely inhibited the increase in [Ca2+]m caused by lowering of either the Ca2+ concentration or the pH in the perfusate. These results suggested that the formation of reactive oxygen species promoted the [Ca2+]m influx. The agents that inhibited the [Ca2+]m influx improved contractility even in Langendorff preparations after ischemia. Based on these findings, we concluded that e-NOS exists in mitochondria and that NO* may play an important protective role in reperfusion cardiac injury after ischemia, by inhibiting the Ca2+ influx into mitochondria which are otherwise damaged by *O2-.

Separation and identification of microcystins in cyanobacteria by frit fast atom bombardment liquid chromatography mass spectrometry

In order to separate and identify microcystins, a new analytical method was developed using a frit probe as an interface for fast atom bombardment mass spectral analysis of high performance liquid chromatographic (HPLC) effluents. Two types of HPLC conditions were designed for separation of standard microcystins RR, YR and LR. The HPLC conditions, for example, methanol:0.01% trifluoroacetic acid = 61:39 (containing 0.8% glycerol) as a mobile phase and 0.5 ml/min as a flow rate, provided a base line separation of standard microcystins RR, YR and LR. The HPLC conditions were also effective for separation of the non-toxic geometrical isomers of microcystins RR and LR. The total ion chromatogram of a mixture of standard microcystins showed excellent correlation with the HPLC separation using a u.v. detector. The method was subsequently applied to analysis of microcystins contained in both a culture strain and a field sample, and the procedure from toxin extraction to identification of microcystins was performed within 1 day. The mass chromatogram monitored at m/z 135 that is always observed with abundance in the FAB mass spectra of the purified microcystins, differentiated between microcystins and other types of compounds. This technique allowed the rapid identification of unknown microcystins without standard samples. Additionally, compounds other than microcystins were also found, which would not be seen by u.v. detection at 238 nm.

Protective effects of antioxidative serotonin derivatives isolated from safflower against postischemic myocardial dysfunction

N-(p-Coumaroyl)serotonin (C) and N-feruroylserotonin (F) with antioxidative activity are present in safflower oil. The protective effects of C and F were investigated in perfused guinea-pig Langendorff hearts subjected to ischemia and reperfusion. Changes in cellular levels of high phosphorous energy, NO and Ca2+ in the heart together with simultaneous recordings of left ventricular developed pressure (LVDP) were monitored by an nitric oxide (NO) electrode, fluorometry and 31P-NMR. The rate of recovery of LVDP from ischemia by reperfusion was 30.8% in the control, while in the presence of C or F a gradual increase to 63.2 or 61.0% was observed. Changes of transient NO signals (TNO) released from heart tissue in one contraction (LVDP) were observed to be upside-down with respect to transient fura-2-Ca2+ signals (TCa) and transient O2 signals detected with a pO2 electrode. At the final stage of ischemia, the intracellular concentration of Ca2+ ([Ca2+]i) and the release of NO increased with no twitching and remained at a high steady level. The addition of C increased the NO level at the end of ischemia compared with the control, but [Ca2+]i during ischemia decreased. On reperfusion, the increased diastolic level of TCa and TNO returned rapidly to the control level with the recovery of LVDP. By in vitro EPR, C and F were found to directly quench the activity of active radicals. Therefore, it is concluded that the antioxidant effects of two derivatives isolated from safflower play an important role in ischemia-reperfusion hearts in close relation with NO.

Distinct involvement of NF-κB and p38 mitogen-activated protein kinase pathways in serum deprivation-mediated stimulation of inducible nitric oxide synthase and its inhibition by 4-hydroxynonenal

Cytokine‐induced expression of inducible nitric oxide synthase (iNOS) and concomitant production of nitric oxide (NO) involve activation of mitogen‐activated protein (MAP) kinases and are in most cases mediated by the transcription factor NF‐κB. We investigated the role of p38 MAP kinase activation and IκB phosphorylation in iNOS expression in a novel iNOS‐inducing model in mouse macrophages. Deprivation of serum from the culture medium of RAW 264.7 cells up‐regulated iNOS and NO production, which were inhibited by 4‐hydroxy‐2‐nonenal (HNE), a component of oxidatively modified low‐density lipoprotein (oxLDL). Serum withdrawal induced phosphorylation of Akt, IκB, and p38 MAP kinase. Pretreatment with the potent PI3 kinase inhibitor wortmannin, the NF‐κB inhibitor PDTC or the specific p38 MAP kinase inhibitor SB203580 each partially attenuated the induction of iNOS and NO production, demonstrating that both p38 activation and IκB phosphorylation are required for iNOS expression. SB203580, however, did not prevent the phosphorylation of Akt and IκB, suggesting that the p38 MAP kinase signal contributes to iNOS gene expression through an IκB‐phosphorylation‐independent pathway. HNE, which markedly inhibited iNOS expression and NO production, prevented the serum withdrawal‐triggered IκB phosphorylation but not that of Akt or p38 MAP kinase. A high concentration of HNE stimulated dephosphorylation of IκB but promoted activation of p38 MAP kinase. Taken together, these results suggest that NF‐κB and p38 MAP kinase lie in separate signal pathways for serum deprivation‐stimulated iNOS expression and NO production. HNE selectively suppresses the former pathway, targeting a site downstream of Akt. J. Cell. Biochem. 83: 271–280, 2001.

Effects of 1‐O‐hexyl‐2,3,5‐trimethylhydroquinone on carbon tetrachloride‐induced hepatic cirrhosis in rats

Aim:  The present study was undertaken to evaluate the effects of 1‐O‐hexyl‐2,3,5‐trimethylhydroquinone (HTHQ), a synthesized vitamin E derivative, on carbon tetrachloride (CCl4)‐induced cirrhosis.

Opposing effects of isoflurane and sevoflurane on neurogenic pulmonary edema development in an animal model.

Background:The current study was undertaken to investigate the effects of pretreatment with isoflurane and sevoflurane on the development of neurogenic pulmonary edema in an animal model. Methods:Rats were exposed to room air (control), 1.5% isoflurane, or 2.5% sevoflurane for 4 h. They were then anesthetized with intraperitoneal injections of pentobarbital sodium, and fibrinogen and thrombin were injected into the cisterna magna to induce neurogenic pulmonary edema. Results:Consecutive injections of fibrinogen and thrombin caused increases in blood pressure, with the peak values obtained in the isoflurane and sevoflurane groups being lower than the control values. The incidence of significant neurogenic pulmonary edema was 58%, 100%, and 8% in the control, isoflurane, and sevoflurane groups, respectively. The lung water ratio, an index of severity of edema, was 4.86 ± 0.78, 6.15 ± 0.64, and 4.40 ± 0.32 in the control, isoflurane, and sevoflurane groups, respectively. Furthermore, immunohistochemical staining for vascular endothelial growth factor demonstrated an increase of expression in the rat lungs exposed to isoflurane. Treatment with an anti–vascular endothelial growth factor antibody during exposure to isoflurane completely inhibited the effect of isoflurane to promote neurogenic pulmonary edema in this model. Conclusion:Exposure to 1.5% isoflurane enhances the development of neurogenic pulmonary edema development in this animal model, most likely via release of vascular endothelial growth factor from bronchial epithelial cells, an effect not observed with sevoflurane.

Primary Cultures of Human Endothelial Cells Are Susceptible to Low Doses of Shiga Toxins and Undergo Apoptosis

Various endothelial cells, with the exception of those from human microvasculatures, have been known to resist Shiga toxins (Stxs) in vitro. However, freshly prepared primary cultures of human endothelial cells from the umbilical vein and artery and the saphenous vein were shown to be killed by a very low dose of Stxs. This cytotoxicity of Stxs involves apoptosis, which seems to be caused by a mechanism distinct from the well-known action of Stxs to inhibit protein synthesis, since the blockade of protein synthesis by cycloheximide could not induce apoptosis or enhance the effect of Stxs. Passaged human endothelial cells have been found to be highly resistant to Stxs, which is consistent with previous reports, and not to show any evidence of apoptosis even when they are killed by a high dose of Stxs.

Role of neuropeptide Y and its receptor subtypes in neurogenic pulmonary edema

The effect of neuropeptide Y on the number of perivascular carbon deposits, assessed as a measure of lung vascular permeability, was examined in isolated perfused lung preparations of rats. The number of carbon particle deposits after bronchial application of neuropeptide Y was increased in a dose-dependent manner. In the presence of a beta-adrenoceptor antagonist, norepinephrine augmented the effects of neuropeptide Y. Peptide YY, an analog of neuropeptide Y, demonstrated a much lower potency for increasing the number of carbon deposits, and neuropeptide Y-(18-36), which elicits a weak antagonist action on the neuropeptide Y Y3 receptor, significantly decreased the neuropeptide Y-induced increase. Furthermore, examination of the influence of neuropeptide Y-(18-36) pretreatment on fibrin-induced neurogenic pulmonary edema, in rats, revealed a reduction of the protein concentration ratio of tracheal fluid to serum. Therefore, we conclude that neuropeptide Y may elevate vascular permeability in the pulmonary circulation, conceivably through the neuropeptide Y Y3 receptor, and that neuropeptide Y may in fact play a physiological role even in the in-situ pulmonary circulation.