Inhibition of carrageenin-induced paw edema by a superoxide dismutase derivative that circulates bound to albumin.

Although the possible involvement of superoxide radical and its metabolite(s) in the pathogenesis of various types of edema have been suggested, direct evidence supporting this concept is lacking. Since intravenously administered Cu2+Zn2(+)-type superoxide dismutase (SOD) rapidly disappeared from the circulation with a half-life of 4 min, the enzyme could not be used to test whether superoxide radicals played a critical role in the modulation of vascular permeability. We previously synthesized a SOD derivative (SM-SOD) by linking poly(styrene co-maleic acid butyl ester) (SM) to the enzyme (Ogino, T., Inoue, M., Ando, Y., Awai, M., Maeda, H. and Morino Y. (1988) Int. J. Pept. Protein Res. 32, 1583-1588); SM-SOD circulates bound to albumin with a half-life of 6 h. To test whether superoxide radicals play an important role in the regulation of vascular permeability, the effect of SM-SOD on experimental paw edema was studied in the rat. Subcutaneous injections of carrageenin to the paw rapidly induced local edema by increasing vascular permeability. Intravenous administration of SM-SOD markedly inhibited the carrageenin-induced increase in vascular permeability and suppressed the development of paw edema. In contrast, the same dose of SOD showed no such inhibitory effect. These results suggest that superoxide radical and/or its metabolite(s) might play a critical role in the pathogenesis of carrageenin-induced vasogenic edema.     

Inhibition of stress-induced gastric mucosal injury by a long acting superoxide dismutase that circulates bound to albumin

To determine whether oxygen-derived free radicals play an important role in the pathogenesis of stress-induced tissue injury, the effect of a superoxide dismutase derivative, which binds to albumin and circulates with a half-life of 6 h in intact rats, on acute gastric mucosal lesion was observed in rats which were given water-immersion-restraint. This enzyme derivative also circulated bound to albumin with a half-life of 8 h in rats which were challenged with water-immersion-restraint. This treatment significantly perturbed systemic circulation of animals by decreasing the effective volume of circulating blood, increased vascular permeability of the gastric mucosa, and induced acute gastric mucosal lesion. Intravenous administration of this enzyme derivative normalized both systemic circulation and vascular permeability of the gastric mucosa and prevented the occurrence of stress-induced gastric injury. These findings suggest that the superoxide radical and/or its metabolite(s) plays an important role in the pathogenesis of stress-induced acute gastric mucosal lesion.     

Synthesis of a superoxide dismutase derivative that circulates bound to albumin and accumulates in tissues whose pH is decreased

Protection of tissues from oxidative stress is one of the major prerequisites for aerobic life. Since intravenously injected Cu2+/Zn2+-type superoxide dismutase (SOD) disappears from the circulation with a short half-life of 5 min, its clinical use as a scavenger for superoxide radical is limited. We synthesized a human erythrocyte type SOD derivative (SM-SOD) by linking 2 mol of hydrophobic organic anion, alpha-4-[( 6-(N-maleimido)hexanoyloxymethyl]cumyl]half-butyl-esterified poly(styrene-co-maleic acid) (SM), to the cysteinyl residues of the dimeric enzyme without decreasing enzymic activity. SM-SOD, but not SOD, bound to an albumin-Sepharose column; the bound SM-SOD was eluted by a buffer solution containing 0.5% sodium dodecyl sulfate or 10 mM warfarin, suggesting that SM-SOD reversibly binds to the warfarin site on albumin. Due to the amphipathic nature of the SMI moiety, SM-SOD bound also to cell membranes particularly when the pH was decreased. In vivo analysis in the rat revealed that intravenously injected SM-SOD circulated bound to albumin with a half-life of 6 h. Postischemic reperfusion arrhythmias were almost completely prevented by a single dose of SM-SOD, but not SOD. Thus, the prolonged half-life of SM-SOD in the circulation and its preferential accumulation in an injured site with decreased pH appeared to be responsible for preventing myocardial injury. These results suggest that superoxide radical and/or its metabolite(s) would play an important role in the pathogenesis of postischemic reperfusion arrhythmias and that SM-SOD may be useful for decreasing tissue injury in ischemic heart disease.     

Polynitroxyl albumin plus tempol attenuates liver injury and inflammation after hepatic ischemia and reperfusion

PNA+Tempol, albumin containing conjugated (polynitroxyl albumin; PNA) and free (4-hydroxyl-2,2,6,6-tetramethyl-piperidinyl-1-oxyl; Tempol) nitroxide may protect against injury caused by reactive oxygen species. Therefore, the actions of PNA+Tempol on liver injury and inflammation induced by hepatic ischemia and reperfusion (I/R) were examined. Rats were subjected to 1 h ischemia followed by 24 h reperfusion in the absence (I/R) or presence of PNA+Tempol (25%; 15 mL/kg, i.v.) (I/R+PNA+Tempol) or human serum albumin (23%; 13.5 mL/kg, i.v.) (I/R+HSA). Test solutions were administered prior to and for 2 h during reperfusion. Sham-operated rats underwent surgery with neither ischemia nor infusion. I/R+PNA+Tempol rats had significantly less liver injury and inflammation than I/R rats. I/R+PNA+Tempol livers exhibited focal lesions whereas I/R livers exhibited global necrosis. Likewise, plasma ALT activity was significantly lower in I/R+PNA+Tempol rats. PNA+Tempol reduced I/R-induced neutrophil accumulation and intercellular adhesion molecule-1 (ICAM-1) expression. HSA did not alter I/R-induced liver injury or inflammation. Sham-operated rats exhibited normal liver morphology and no inflammation. Attenuation of I/R liver injury by PNA+Tempol may be mediated by its effect on inflammation, the major contributor to I/R injury. Reduction of inflammation by PNA+Tempol is most likely due to the antioxidative nature of the nitroxides.     

Inhibition of inflammation and oxidative stress by an imidazopyridine derivative X22 prevents heart injury from obesity

Inflammation and oxidative stress plays an important role in the development of obesity‐related complications and cardiovascular disease. Benzimidazole and imidazopyridine compounds are a class of compounds with a variety of activities, including anti‐inflammatory, antioxidant and anti‐cancer. X22 is an imidazopyridine derivative we synthesized and evaluated previously for anti‐inflammatory activity in lipopolysaccharide‐stimulated macrophages. However, its ability to alleviate obesity‐induced heart injury via its anti‐inflammatory actions was unclear. This study was designed to evaluate the cardioprotective effects of X22 using cell culture studies and a high‐fat diet rat model. We observed that palmitic acid treatment in cardiac‐derived H9c2 cells induced a significant increase in reactive oxygen species, inflammation, apoptosis, fibrosis and hypertrophy. All of these changes were inhibited by treatment with X22. Furthermore, oral administration of X22 suppressed high‐fat diet‐induced oxidative stress, inflammation, apoptosis, hypertrophy and fibrosis in rat heart tissues and decreased serum lipid concentration. We also found that the anti‐inflammatory and anti‐oxidative actions of X22 were associated with Nrf2 activation and nuclear factor‐kappaB (NF‐κB) inhibition, respectively, both in vitro and in vivo. The results of this study indicate that X22 may be a promising cardioprotective agent and that Nrf2 and NF‐κB may be important therapeutic targets for obesity‐related complications.     

Antioxidant activity of liver growth factor,a bilirubin covalently bound to albumin

We previously reported that treatment of spontaneously hypertensive rats (SHR) with liver growth factor (LGF), an albumin–bilirubin complex with a covalent bond, reduces blood pressure, improves nitric oxide (NO)-dependent vasodilatation, and exerts vascular antifibrotic actions. Because bilirubin, albumin, and albumin-bound bilirubins have antioxidant properties, we hypothesize that LGF might exert its cardiovascular actions through an antioxidant mechanism. We have tested in vitro the capacity of LGF to scavenge ABTS cation and peroxyl and hydroxyl radicals and to protect vascular NO from degradation by superoxide anion. We have also compared the antioxidant capacity of LGF with that of its molecular components albumin and bilirubin and the reference antioxidant trolox. LGF exhibited antioxidant capacity against all free radicals tested at lower concentrations than albumin, bilirubin, and trolox. LGF, bilirubin, and albumin were also able to protect endothelial NO from superoxide anion degradation in a fashion similar to that of superoxide dismutase or tiron, but at much lower concentrations. These data, together with our previous results in SHR, suggest that LGF might exert its cardiovascular regenerative actions, at least in part, through an antioxidant mechanism and that LGF could be a relevant circulating antioxidant in situations of oxidative stress.     

Inhibition of posthemorrhagic transfusion-induced gastric injury by a long-acting superoxide dismutase derivative

Although oxygen-free radicals have been postulated to play an important role in the pathogenesis of gastric mucosal injury induced by posthemorrhagic blood transfusion, direct evidence supporting this hypothesis is lacking. Superoxide dismutase (SOD) has been shown to inhibit oxygen toxicity in vitro in various types of cell injury. However, in some cases, oxidative tissue injury cannot be decreased efficiency predominantly due to its rapid elimination by renal glomerular filtration. To overcome such frustrating situations, we have synthesized a SOD derivative that circulates bound to albumin with a half-life of 6 hr. When blood was withdrawn from the rat (22 ml/kg) for 30 min followed by transfusion of the extracted blood, marked gastric mucosal lesions occurred within 30 min after transfusion. Intravenously injected SOD derivative markedly decreased gastric mucosal injury. Kinetic analysis using 125I-labeled albumin revealed that the vascular permeability of the stomach increased significantly after transfusion by a SOD derivative inhibitable mechanism. Thus, superoxide radical and/or its metabolite(s) play a critical role in the pathogenesis of posthemorrhagic transfusion-induced gastric injury.     

Comparison of kinetic study of the photochemical changes of (ZZ)-bilirubin IX alpha bound to human serum albumin with that bound to rat serum albumin.

It has been stated by McDonagh, Palma & Lightner [(1982) J. Am. Chem. Soc. 104, 6867-6871] that complexing of bilirubin with serum albumin has a marked species-dependent influence on bilirubin photoisomerization in vitro and in vivo. Therefore the kinetics for the quantitatively important reaction: (Formula: see text) of the photochemical interconversion between bilirubin and its photoisomers bound to human or rat serum albumin in aqueous solution, assayed by h.p.l.c., was used to elucidate the observed species-dependent difference. The relative rate constants for bilirubin bound to human serum albumin, except for k4, the rate of interconversion from (ZZ)-bilirubin into (EZ)-bilirubin, proved to be considerably larger than those for bilirubin bound to rat serum albumin. In accordance with these rate constants, the formation of photoisomers of bilirubin bound to human serum albumin, except for (EZ)-bilirubin, is very rapid and much greater than that for bilirubin bound to rat serum albumin.     

Inhibition of the U1A-RNA complex by an aminoacridine derivative

The RNA recognition motif (RRM) is one of the most common RNA binding domains. There have been few investigations of small molecule inhibitors of RRM-RNA complexes, although these inhibitors could be valuable tools for probing biological processes involving RRM-RNA complexes and would have the potential to be effective drugs. In this paper, the inhibition by small molecules of the complex formed between the N-terminal RRM of the U1A protein and stem loop 2 of U1 snRNA has been investigated. An aminoacridine derivative has been found to promote dissociation of the U1A-stem loop 2 RNA complex with an IC(50) value of 1 microM. Fluorescence experiments indicate that two aminoacridine ligands bind to each RNA target site. RNase A footprinting suggests that one binding site may be near the base pair that closes the loop and the other may be in a more flexible region of the loop. The addition of the aminoacridine derivative to stem loop 2 RNA increases the susceptibility of other portions of the loop to digestion by RNase A, which implies that binding of the ligand changes the conformation or dynamics of the stem loop target site. Either direct binding to the RNA or indirect alteration of the structure or dynamics of the loop would be likely to inhibit binding of the U1A protein to this RNA.     

Inhibition of Rho kinase by fasudil hydrochloride attenuates lung injury induced by intestinal ischemia and reperfusion

AimThe aim of this study is to evaluate the role of Rho-kinase in the pathogenesis of lung injury induced by intestinal ischemia/reperfusion (I/R) and the preconditioning effects of fasudil hydrochloride. The novel therapeutic approach of using Rho-kinase inhibitors in the treatment of intestinal I/R is introduced.MethodsSprague–Dawley (SD) rats were divided into 4 groups: intestinal I/R group, two fasudil pretreatment groups (7.5 mg/kg and 15 mg/kg), and controls. Intestinal and lung histopathology was evaluated; myeloperoxidase (MPO) and superoxide dismutase (SOD) levels in lung parenchyma were determined. Serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured. eNOS and P-ERM expression were measured by Western Blot.ResultsLung and intestinal injury were induced by intestinal I/R, characterized by histological damage and a significant increase in BALF protein. Compared to controls, serum TNF-α, IL-6, and lung MPO activity increased significantly in the I/R group, while SOD activity decreased. A strongly positive P-ERM expression was observed, while eNOS expression was weak. After fasudil administration, injury was ameliorated. Serum TNF-α, IL-6, lung MPO and P-ERM expression decreased significantly as compared to the I/R group, while SOD activity and eNOS expression increased significantly.SignificanceRho-kinase plays a key role in the pathogenesis of lung injury induced by intestinal I/R. The inhibition of the Rho-kinase pathway by fasudil hydrochloride may prevent lung injury.     

Inhibition of lipid oxidation by serum and albumin

The influence of serum and albumin on enzymatic and non-enzymatic lipid oxidation was investigated. Intensity of oxidation was measured as the amount of oxygen consumed by the sample and by quantitation of malonaldehyde formed during breakdown of lipid peroxides. Non-enzymatic lipid oxidation was stimulated by ascorbic acid or ferrous ions and enzymatic by NADPH-dependent oxidase, 15-lipoxygenase and 12-lipoxygenase. Albumin inhibits lipid oxidation only when pure fatty acid (arachidonic or linoleic) is the substrate for this oxidation. Serum was a stronger inhibitor than an equivalent amount of albumin and it also inhibited oxidation of a mixture of lipids from liver microsomes. It is concluded that serum contains two antioxidant factors: albumin which binds fatty acids and probably another factor which is a true antioxidant.     

Inhibition of cathepsin S attenuates myocardial ischemia/reperfusion injury by suppressing inflammation and apoptosis

Myocardial ischemia/reperfusion (I/R) injury leads to high mortality and morbidity due to the incomplete understanding of the underlying mechanism and the consequent lack of effective therapy. The present study revealed and validated key candidate genes in relation to inflammation and apoptosis pathways underlying myocardial I/R injury. Cathepsin S was identified as the top hub protein based on the protein–protein interaction analysis, and, thus, its role during myocardial I/R injury was further investigated. Myocardial I/R in mice resulted in significantly increased levels of myocardial injury biomarkers (cardiac troponin I, lactic dehydrogenase, and creatinine kinase‐MB) and inflammatory cytokines (interleukin‐1β [IL‐1β], IL‐6, and tumor necrosis factor‐α), elevated apoptosis rate, and upregulated protein expression of cleaved caspase‐8, cleaved caspase‐3, and cleaved poly ADP‐ribose polymerase. These abovementioned changes were blocked by two different selective cathepsin S inhibitors, LY3000328 or MIV‐247. Moreover, Kaplan–Meier survival plot showed that cathepsin S inhibition improved 21‐day survival rate following myocardial I/R injury. This study demonstrated that the inhibition of cathepsin S alleviated myocardial I/R‐induced injury by suppressing inflammation and apoptosis, which may be used in clinical applications of cardioprotection.     

In vitro and in vivo evidence that the C-terminus of preproenkephalin-A circulates as an 8500-dalton molecule

One of the major immunoreactive components of the enkephalin precursor BAM-22P in extracts of adrenal medulla is a molecule of approximately 8500 Da. A similar component is detected, after gel-permeation chromatography, in extracts of bovine plasma and in culture medium harvested from acetylcholine-stimulated bovine adrenomedullary cells in culture. Simultaneous detection of the C-terminal epitome of preproenkephalin-A: Met-enkephalin-Arg6-Phe7-OH in this secreted high-molecular-weight zone suggest that the approximately 8500-Da form of BAM-22P is the C-terminal of preproenkephalin A.     

Acetylation of ribosome-associated proteins in vitro by an acetyltransferanse bound to rat liver ribosomes.

Incubation of rat liver ribosomes with [1-14-C]acetyl-coenzyme A results in the incorporation of [14-C]acetyl into a material insoluble in cold trichloroacetic acid. The acetyltransferase involved in the self-acetylation of ribosomes can be released by high salt washing of the ribosomes; the activity of the solubilized enzyme can be assayed using histones as acetyl acceptors. Electrophoretic analysis of acetylated risosomes or ribosomal proteins indicated that the acetyl radicals are associated with a group of relatively basic proteins, having molecular weights ranging from 10,000 to 45,000. Chromatographic analysis of the enzymatic hydrolsates of proteins extracted from acetylated ribosomes indicates that acetylation is mainly or exclusively NH2 terminal. Almost 80% of the acetyl proteins are released from the ribosomes by high salt treatment. Most of the acetyl radicals not solubilized by the high salt treatment were found in the 60S subunit, associated with a protein(s) having an apparent molecular weight of 43,000. This acetyl protein(s) was released from the 60S subunit by EDTA treatment and was found in a ribonucleoprotein complex having a bouyant density of 1.56.     

Recombinant human erythropoietin attenuates hepatic injury induced by ischemia/reperfusion in an isolated mouse liver model

Introduction Apoptosis is a central mechanism of cell death following reperfusion of the ischemic liver. Recombinant human erythropoietin (rhEPO) have an important role in the treatment of myocardial ischemia/reperfusion (I/R) injury, by preventing apoptosis. The aim of the study was to investigate the effect of different regimens of rhEPO in preventing apoptosis following I/R-induced hepatic injury. Material and methods Isolated mouse livers were randomly divided into five groups: (1) control group, perfused for the whole study period (105 min); (2) 30-min perfusion followed by 90 min of ischemia and 15 min of reperfusion; (3), (4) and (5) like group 2, but with administration of rhEPO 5,000 units/kg i.p. at 30 min, 24 h, or both 30 min and 24 h respectively, before induction of ischemia. Perfusate liver enzyme levels and intrahepatic caspase-3 activity were measured, and apoptotic cells were identified by morphological criteria, TUNEL assay, and immunohistochemistry for caspase-3. Using immunoblot the expression of the proapoptotic JNK and inhibitor of NFκB (IκBα) were also evaluated. von Willebrand factor (vWF) immunohistochemistry was used as a marker of endothelial cells. Results Compared to the I/R livers, all 3 rhEPO pretreated groups showed: a significant reduction in liver enzyme levels (P < 0.05) and intrahepatic caspase-3 activity (P < 0.05), fewer apoptotic hepatocytes (P < 0.05) and positive vWF staining in numerous endothelial cells lining the sinusoids. EPO decreased JNK phosphorylation and the degradation of the inhibitor of NFκB (IκBα) during I/R. There was no added benefit of the multiple- over the single-dose rhEPO regimen. Conclusion Pretreatment with one dose of rhEPO can attenuate post-I/R hepatocyte apoptotic liver damage. NFκB and JNK activation is likely to play a pivotal role in the pathophysiology of I/R hepatic injury and might have a key role in EPO-mediated protective effects. This effect is associated with the increase in sinusoidal vWF immunostaining suggests an additional effect of rhEPO in liver angiogenesis recovery. These findings have important implications for the potential use of rhEPO in I/R injury during liver transplantation. Edith Hochhauser and Orit Pappo are first two coauthors.     

Fatty acids bound to alpha-fetoprotein and albumin during rat development

The time-course levels and composition of the fatty acids bound to rat alpha-fetoprotein (AFP) and albumin from several sources, were determined throughout development, and related to the intake of lipids from milk and the compositional changes in brain and liver fatty acids. The major fatty acids bound to AFP were acids bound to AFP were polyunsaturated and mainly docosahexaenoic acid (22:6(n-3], either from fetal serum (23.1%) or whole fetuses (21.6%), whereas palmitic (34.1%) and oleic (29.9%) acids were the main acids bound to albumin from the same sources. Amniotic fluid AFP contained less fatty acids (0.8 mol/mol protein) than that of fetal serum (1.4 mol/mol protein), and especially noticeable was a reduced amount of 22:6 (9.6%). Both AFP-concanavalin A microforms showed identical fatty acid composition. Levels of 22:6 bound to AFP decreased quickly after birth until a minimum at 8-10 days, increasing moderately thereafter. This minimum is coincident in time with a maximal accumulation of this fatty acid by brain and a loss of 22:6 by liver. Except for colostrum, levels of 22:6 in milk lipids were low and fairly constant, but always greater than those of its precursor, linolenic acid (18:3 (n-3]. These results support a specialized role of AFP in the plasma transport and tissue delivery of polyunsaturated fatty acids, and mainly docosahexaenoic acid.