Reversal of HO-1 related cytoprotection with increased expression is due to reactive iron.

It is often postulated that the cytoprotective nature of heme oxygenase (HO-1) explains the inducible nature of this enzyme. However, the mechanisms by which protection occurs are not verified by systematic evaluation of the physiological effects of HO. To explain how induction of HO-1 results in protection against oxygen toxicity, hamster fibroblasts (HA-1) were stably transfected with a tetracycline response plasmid containing the full-length rat HO-1 cDNA construct to allow for regulation of gene expression by varying concentrations of doxycycline (Dox). Transfected cells were exposed to hyperoxia (95% O(2)/5% CO2) for 24 h and several markers of oxidative injury were measured. With varying concentrations of Dox, HO activity was regulated between 3- and 17-fold. Despite cytoprotection with low (less than fivefold) HO activity, high levels of HO-1 expression (greater than 15-fold) were associated with significant oxygen cytotoxicity. Levels of non-heme reactive iron correlated with cellular injury in hyperoxia whereas lower levels of heme were associated with cytoprotection. Cellular levels of cyclic GMP and bilirubin were not significantly altered by modification of HO activity, precluding a substantial role for activation of guanylate cyclase by carbon monoxide or for accumulation of bile pigments in the physiological consequences of HO-1 overexpression. Inhibition of HO activity or chelation of cellular iron prior to hyperoxic exposure decreased reactive iron levels in the samples and significantly reduced oxygen toxicity. We conclude that there is a beneficial threshold of HO-1 overexpression related to the accumulation of reactive iron released in the degradation of heme. Therefore, despite the ready induction of HO-1 in oxidant stress, accumulation of reactive iron formed makes it unlikely that exaggerated expression of HO-1 is a cytoprotective response.     

Lipid storage in cultured articular chondrocytes due to prostanoid precursors and a prostanoid synthesis inhibitor

Lapine articular chondrocytes were subcultured in the presence or absence of the prostanoid precursors, arachidonic acid or dihomo-gamma-linolenic acid, and the cyclooxygenase inhibitor indomethacin. Lipid storage was studied microscopically using the Sudan black staining method. Control chondrocyte cultures showed a weakly positive staining reaction until confluence was reached, at which point the intra-cytoplasmic lipid content decreased. Both arachidonic acid and dihomo-gamma-linolenic acid at 100 mumol/l caused a marked increase in lipid storage which continued even after confluence was achieved. 1 mumol/l concentrations were indistinguishable from controls, whereas 10 mumol/l concentrations elicited a slight increase in lipid storage compared with controls. The prostaglandin cyclooxygenase inhibitor indomethacin did not affect chondrocyte lipid storage. However, administration of a prostanoid precursor in the presence of indomethacin caused a massive increase in intra-cytoplasmic storage of lipid, eventually leading to cell death. A possible explanation is that indomethacin may alter chondrocyte lipid metabolism in the presence of substrate molecules by rechanneling lipid synthesis away from the prostaglandin pathway to other lipid synthetic pathways.     

Intracellular depletion of insulin by oleate is due to an inhibited synthesis and not to an increased secretion

In the polyclonal rat pancreatic beta-cell line INS-1, immunoreactive insulin (IRI, insulin and its precursors) and C-peptide (surrogate marker for mature insulin) were quantified after a 1-h incubation at 16.7 mM glucose with or without oleate. Oleate caused a 20% decrease (P 相似文献   

Nitric oxide inhibits prostanoid synthesis in the rat oviduct

We have studied the effect of nitric oxide (NO) on the production of arachidonic acid ([14C]-AA) metabolites in the rat oviduct. The basal synthesis of eicosanoids was measured by the conversion of ([14C]-AA) to the different radiolabeled products of cyclooxygenase (COX). The oviducts incubated for 1 h with the labeled substrate of COX were able to convert 3.3 +/- 0.3% of ([14C]-AA) to 6-ceto-PGF1alpha, 10.7 +/- 1.0% to PGF2alpha, 13.5 +/- 1.2% to PGE2 and 6.3 +/- 0.5% to TXB2. The tissues were incubated with different doses of two NO donors: SIN-1 and Spermine NONOate. The results indicated that SIN-1 produces a significant decrease (50%; P < 0.05) in all prostanoids evaluated in a dose-response fashion. The inhibitory effect was completely reversed by addition of 20 microg/ml of hemoglobin (Hb), a NO scavenger. The addition of Spermine NONOate to the incubation medium diminished significantly (65%) the synthesis of COX metabolites suggesting that NO acts by inhibiting COX activity in the rat oviduct. However, NOS inhibitors, N(G)-L-arginine-methyl-ester (L-NAME) nd N(G)-L-monomethyl-arginine (L-NMMA) had no effect on basal production of the prostanoids. These results indicate that in the rat oviduct the synthesis of COX metabolites is negatively regulated by nitric oxide.     

Adaptive cytoprotection by 0.25 M HCl is truly "cytoprotective" and may not depend upon elevated levels of prostaglandin synthesis

The ability of a mild irritant to reduce ethanol-induced damage to the rat gastric mucosa was investigated using an ex vivo gastric chamber preparation. Exposure to 0.25 M hydrochloric acid (HCl) did not cause significant damage to the surface epithelium, but did reduce both the lesion area and the extent of superficial epithelial damage caused by subsequent exposure to 40% ethanol (EtOH). "Adaptive cytoprotection" was also demonstrated by the reduction of ethanol-induced changes in transmural potential difference and net K+ efflux, and by rapid recovery of these physiological parameters following the removal of ethanol from the chamber. Pretreatment of rats with indomethacin at a dose that has been shown to significantly inhibit gastric cyclooxygenase activity did not significantly affect the ability of 0.25 M HCl to reduce the effects of ethanol on lesion area, epithelial damage, potential difference, and net K+ efflux.     

Body fat-lowering effect of conjugated linoleic acid is not due to increased lipolysis

The ability of conjugated linoleic acid (CLA) to reduce adiposity may be due to changes in energy expenditure and/or direct effects on adipocyte lipid metabolism. The aim of the present work was to analyse if CLA supplementation modifies lipolytic activity in adipose tissue from hamsters fed on high-fat diet. Hamsters were divided into two groups and fed on diets supplemented with either 0.5% linoleic acid (control) or 0.5% trans-10,cis-12 CLA. After 6 weeks, animals were fasted overnight and adipose tissues were dissected and weighed. Adipocytes were isolated by collagenase digestion and incubated in Krebs-Ringer bicarbonate buffer with or without several agents acting at different levels of the lipolytic cascade. Adipocyte diameters were measured by microscopy. Adipose tissue DNA content was assessed by spectrophotometry. Animals fed on CLA diet showed significantly reduced adipose tissue mass. No differences between both groups was found for basal lipolysis, lipolytic effects of isoproterenol, forskolin, dibutyryl-cAMP and isobutylmethylxanthine, and pD2 for isoproterenol. A similar total DNA amount was found in adipose tissue of both groups, showing that CLA diet had no effect on total cell number per fat pad. Although DNA content per gram tissue, an indirect reverse index of cell size, was significantly increased in CLA fed hamsters, microscopy did not reveal differences in medium mature adipocyte diameter, nor in cell size distribution between both groups. These results suggest that adipose tissue size reduction induced by trans-10,cis-12 CLA intake is not due to changes in lipolysis. Reduced preadipocyte differentiation into mature adipocytes may account for this fat-lowering effect.     

Aspirin-induced gastric injury in the rat: histologic changes and sucralfate cytoprotection

The effect of pretreatment with intragastric sucralfate on aspirin acid-induced gastric mucosal lesions in the rat was studied. The finding by others that sucralfate is cytoprotective and that this cytoprotective effect probably is mediated, at least in part, by stimulation of endogenous prostaglandin synthesis was confirmed. In addition, a time course study revealed that the maximum cytoprotective effect was present 1 min after sucralfate administration and persisted for at least 6 hr. Microscopic evaluation of histologic sections revealed that sucralfate significantly decreased aspirin-induced deep mucosal erosions (those extending into the parietal cell area) but not superficial mucosal damage. Superficial mucosal damage (surface cell injury and erosions involving the mucous neck cell area) could not be detected grossly. The lesions seen grossly were deeper erosions involving the parietal cell area of the mucosa.     

Elevation of hepatic epoxide hydratase activity by ethoxyquin is due to increased synthesis of the enzyme

Feeding of the antioxidant ethoxyquin to rats leads to an increase of epoxide hydratase activity in liver microsomes. The apparent half life of the increase is 3–4 days. Elevation of epoxide hydratase activity is also obtained by intraperitoneal treatment of mice with ethoxyquin. This elevation is prevented by concomitant treatment with cycloheximide. When radiolabelled leucine is incorporated into microsomal protein by liver cell fractions from either ethoxyquin-fed or untreated rats, gel electrophoresis reveals that ethoxyquin feeding increases incorporation into epoxide hydratase. These results suggest that the elevation of epoxide hydratase activity by ethoxyquin is due to increased biosynthesis of the enzyme, i.e. enzyme induction.     

beta-Carotene accumulation induced by the cauliflower Or gene is not due to an increased capacity of biosynthesis

The cauliflower (Brassica oleracea L. var. botrytis) Or gene is a rare carotenoid gene mutation that confers a high level of beta-carotene accumulation in various tissues of the plant, turning them orange. To investigate the biochemical basis of Or-induced carotenogenesis, we examined the carotenoid biosynthesis by evaluating phytoene accumulation in the presence of norflurazon, an effective inhibitor of phytoene desaturase. Calli were generated from young seedlings of wild type and Or mutant plants. While the calli derived from wild type seedlings showed a pale green color, the calli derived from Or seedlings exhibited intense orange color, showing the Or mutant phenotype. Concomitantly, the Or calli accumulated significantly more carotenoids than the wild type controls. Upon treatment with norflurazon, both the wild type and Or calli synthesized significant amounts of phytoene. The phytoene accumulated at comparable levels and no major differences in carotenogenic gene expression were observed between the wild type and Or calli. These results suggest that Or-induced beta-carotene accumulation does not result from an increased capacity of carotenoid biosynthesis.     

Alkaline ribonuclease activity is increased in rat sympathetic ganglia after nerve injury

Ribonuclease activity at pH 7.1 (alkaline ribonuclease) was determined in homogenates of rat superior cervical ganglion up to 5 days after postganglionic nerve injury under optimal conditions of assay. Measurements were performed in the presence and absence of the sulfhydryl blocking agent, N-ethylmaleimide, to assess the proportion of alkaline ribonuclease apparently bound to endogenous inhibitor. Total ribonuclease activity per ganglion was stimulated 1.3 fold by 1 day after injury and remained elevated over the 5 day period. Free ribonuclease activity accounted for about 60% of the observed increase in total activity at day 1, but had returned to control level by day 3. At day 3 the entire 90% increase in total activity was attributable to ribonuclease bound to endogenous inhibitor (i.e. latent activity). These changes are occurring at times after nerve injury when marked alterations in RNA turnover have been observed, implicating alkaline ribonucleases in the control of RNA metabolism during nerve regeneration.     

细胞更新对慢性饮酒大鼠胃黏膜适应性细胞保护的影响

目的: 以低浓度酒精作为弱刺激,通过慢性饮酒的大鼠动物模型,探讨慢性饮酒和大鼠胃粘膜适应性细胞保护作用之间的关系,以及胃粘膜细胞更新的作用.方法: 分别在饮酒不同时程的大鼠胃内灌注2 ml 100%酒精,分析胃粘膜的损伤情况.以流式细胞术、免疫组化和计算机图像处理技术观察大鼠胃粘膜的细胞增殖和凋亡,探讨胃粘膜的细胞更新情况.结果: ①纯酒精可使大鼠的胃体和胃窦出现溃疡和出血,饮用6%(v/v)酒精3～14 d的大鼠这种现象明显减轻,饮用6%(v/v)酒精1 d和28 d的大鼠则无改变.②饮用6%(v/v)酒精3～14 d的大鼠胃粘膜细胞更新加快,而饮酒28 d大鼠胃粘膜细胞凋亡增加,细胞增殖减少.结论: 细胞更新加快是适度低浓度酒精刺激引起的胃粘膜适应性细胞保护作用的重要原因,低浓度酒精刺激超过一定时限可引起胃粘膜萎缩性病变的趋势,使胃粘膜抵抗能力降低.     

The protection against gentamicin nephrotoxicity in the streptozotocin-induced diabetic rat is not related to gender.

Since gender can influence the renal toxicity of a drug in a given species, the present study was undertaken to evaluate the role of sex in the protection against gentamicin (G)-induced nephrotoxicity afforded by diabetes mellitus (DM) in the rat. We have compared the effects of administration of G (40 mg/kg/day, for 14 days) on male and female DM Sprague-Dawley rats. Non-diabetic animals of both sexes receiving identical doses of G served as controls. At the end of the experiment on day 14, both female (F) and male (M) control groups had similar and marked evidence of nephrotoxicity: elevation of plasma creatinine (F 1.7 +/- 0.7; M 2.8 +/- 0.6 mg/dl), decrease in endogenous 24-h creatinine clearance (Ccr) (F0.3 +/- 0.1; M 0.2 +/- 0.1 ml/min/100 g BW), and histological evidence of severe acute tubular necrosis. In marked contrast, the DM rats showed no functional or morphological evidence of renal damage throughout the study regardless of their gender (day 14: plasma creatinine: F 0.2 +/- 0.03; M 0.2 +/- 0.02; Ccr: F 1.2 +/- 0.1; M 1.6 +/- 0.1 ml/min/100 g BW), and they also accumulated less G in their kidney cortex than the C rats. The male controls exhibited higher renal cortex accumulation of G than the female controls (p < 0.05), whereas the opposite occurred in the DM groups (p < 0.01). Because the validity of using Ccr for the evaluation of GFR changes in experimental nephrotoxicity has been questioned, we have compared, in a separate experiment, three different methods of estimation of GFR (simultaneous short clearances of inulin and Ccr, and 24-h Ccr) in conscious female Sprague-Dawley rats undergoing the same treatment with G described above. At no time during the study did the method used for estimation of the GFR influence the results. We conclude that male and female Sprague-Dawley rats with diabetes are functionally and morphologically equally protected against G. Furthermore, no gender-related differences in the magnitude of G-induced nephrotoxicity was demonstrated in the non-diabetic control animals.     

Increased glomerular Vwf after kidney irradiation is not due to increased biosynthesis or endothelial cell proliferation.

Kuin, A., Citarella, F., Oussoren, Y. G., Van der Wal, A. F., Dewit, L. G. H. and Stewart, F. A. Increased Glomerular Vwf after Kidney Irradiation is not due to Increased Biosynthesis or Endothelial Cell Proliferation. Radiat. Res. 156, 20-27 (2001).Irradiation of the kidney induces dose-dependent, progressive renal functional impairment, which is partly mediated by vascular damage. It has previously been demonstrated that reduced renal function is preceded by an increased amount of von Willebrand factor (Vwf) in the glomerulus. The underlying mechanism and significance of this observation are unknown but, since it is an important mediator of platelet adhesion, Vwf in increased amounts could be implicated in glomerular thrombosis, resulting in impairment of renal function. Increased Vwf could be the result of increased biosynthesis by endothelial cells, or from increased numbers of endothelial cells after compensatory proliferation induced by irradiation, or it could be secondary to other events. In the present study, expression levels of mRNA for glomerular Vwf and glomerular cell proliferation rates were measured in control mouse kidneys and after irradiation with a single dose of 16 Gy. There were no significant changes in mRNA ratios for Vwf/beta-actin at 10 to 30 weeks after irradiation compared with unirradiated samples, whereas increased amounts of Vwf protein were seen in the glomeruli at these times. Labeling studies with IdU or staining for Ki67 demonstrated that glomerular proliferation was increased from 10 to 30 weeks after irradiation. Despite the increased proliferation rates, there was an absence of glomerular hyperplasia and no increase in the endothelial cell surface coverage in the glomeruli. Staining with antibodies against smooth muscle actin (SMAalpha) revealed that the observed proliferation mainly involved mesangial cells. These results indicate that the increased presence of glomerular Vwf after irradiation is not due to an increased number of endothelial cells per glomerulus, or to an increased production of Vwf. It is presumably secondary to other events, such as increased release of Vwf by damaged endothelial cells or entrapment of Vwf in the irradiated mesangial matrix.     

Stress-induced reduction of gastric acid in the rat is not associated with increased tissue somatostatin

In zero, mildly and severely stressed rats, gastric acid secretion, aortal and portal venous gastrin, venous glucagon and somatostatin in gastric, duodenal mucosa and in pancreas were examined. Serum gastrin and gastric acid secretion are reduced markedly by both kinds of stress, whereas plasma glucagon rises steadily with stress. As somatostatin in the tissues of stressed rats is not different from unstressed controls, gastrin and gastric acid reduction may not be attributed to an endocrine or paracrine action of somatostatin.     

The tolerance of the Arabidopsis defense hormone receptor mutant coi1 against the vascular pathogen Verticillium longisporum is not due to increased levels of the active hormone jasmonoyl-isoleucine

Verticillium longisporum is a soil-borne vascular pathogen found primarily on oilseed rape in Northern Europe. Infection of the model plant Arabidopsis thaliana can be achieved under laboratory conditions. In the article related to this addendum, we have shown that Arabidopsis dde2–2 mutants that are compromised in their ability to synthesize the defense hormone jasmonoyl-isoleucine (JA-Ile) are slightly more susceptible than wild-type. Contrary to the expectation that hormone biosynthesis mutants and their respective receptor mutants should have the same phenotype, we found that plants that lack the JA-Ile receptor CORONATINE INSENSITIVE1 (COI1) are more tolerant to the disease. This addendum addressed the question whether the increased JA-Ile levels found in coi1 are responsible for its tolerance phenotype. Based on the evidence that the JA-Ile-deficient dde2–2 coi1-t double mutant is as tolerant as coi1-t, we conclude that increased JA-Ile levels do not protect Arabidopsis against the fungus in the absence of COI1.     

Melanomas display increased cytoprotection to hypericin-mediated cytotoxicity through the induction of autophagy

Photodynamic therapy (PDT) as a regime for melanoma is of limited success due to factors such as the efficacy of the photosensitizer used, penetration depth and the presence of pigment. We characterised a pigmented and an unpigmented melanoma cell line with respect to their phenotypes. Cell viability was assessed after exposure to hypericin, a UVA-activated photosensitizer. Exposure to 3 μM activated hypericin induced a cytoprotective (autophagic) response from both cell lines. However, the pigmented cells accumulated a large amount of glycogen in their cytoplasm. We hypothesise that the treatment induces an initial cytoprotective response through autophagy, but with increased stress results in a different mode of cell death in pigmented melanoma cells from unpigmented cells. These results indicate that hypericin-PDT could be an adjuvant therapy for melanoma.     

Reactivity against complementary proteinase-3 is not increased in patients with PR3-ANCA-associated vasculitis

The etiology of anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitides (AAV) is unknown, but the association between infections and autoimmunity has been studied extensively. In 2004, a novel theory was proposed that could link infection and autoimmunity. This 'theory of autoantigen complementarity' was based on the serendipitous finding of antibodies against complementary-PR3 (cPR3) in patients with PR3-ANCA-associated vasculitis. cPR3 demonstrated homology to several bacterial proteins, and it was hypothesized that PR3-ANCA develop in response to anti-cPR3 antibodies, as a consequence of the anti-idiotypic network. These data have not been confirmed in other patient cohorts. We investigated the presence of anti-cPR3 antibodies in a Dutch cohort of PR3-ANCA-associated vasculitis patients. Anti-cPR3 reactivity was determined in serum using ELISA. Two separate batches of cPR3 were used to determine reactivity in two separate cohorts of PR3-ANCA-associated vasculitis patients. We found that anti-cPR3-reactivity was not increased in our PR3-ANCA-associated vasculitis patients, in comparison to control groups. Further research will be necessary to prove the concept of autoantigen complementarity in autoimmune diseases.