BBA - Molecular Basis of Disease (v.1639, #3)

Phagocytosis of apoptotic cells and the resolution of inflammation by Paola Maderna; Catherine Godson (141-151).
Clearance of apoptotic cells by phagocytic cells plays a significant role in the resolution of inflammation, protecting tissue from harmful exposure to the inflammatory and immunogenic contents of dying cells. Apoptosis induces cell surface changes that are important for recognition and engulfment of cells by phagocytes. These changes include alterations in surface sugars, externalization of phosphatidylserine and qualitative changes in the adhesion molecule ICAM-3. Several studies have contributed to clarify the role of the receptors on the surface of phagocytes that are involved in apoptotic cell clearance. The phagocytic removal of apoptotic cells does not elicit pro-inflammatory responses; in contrast, apoptotic cell engulfment appears to activate signals that suppress release of pro-inflammatory cytokines. Therefore, clearance of apoptotic leucocytes is implicated in the resolution of inflammation and mounting evidence suggests that defective clearance of apoptotic cells contributes to inflammatory and autoimmune diseases. Defining the ligands on apoptotic cells and the corresponding receptors on phagocytes with which they engage, is likely to lead to the development of novel anti-inflammatory pro-resolution drugs. In this article, we will review the recognition and signaling mechanisms involved in the phagocytosis of apoptotic cells as well as the role of endogenous compounds that play a relevant role in the modulation of inflammation. We will also discuss what is currently known about diseases that may reflect impaired phagocytosis and the consequences on inflammation and immune responses.
Keywords: Apoptotic cell; Phagocytosis; Resolution of inflammation;

In situ identification of protein structural changes in prion-infected tissue by Janina Kneipp; Lisa M. Miller; Marion Joncic; Martin Kittel; Peter Lasch; Michael Beekes; Dieter Naumann (152-158).
Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative disorders characterized by the conversion of the normal prion protein (PrPC) into aggregates of its pathological conformer (PrPSc). The mechanism behind this structural conversion is unclear. We report the identification of disease-related protein structural differences directly within the tissue environment. Utilizing a synchrotron infrared (IR) light source, IR images of protein structure were obtained at a subcellular resolution, revealing regions of decreased α-helical content and elevated β-sheet structure in and around infected neurons in the 263 K scrapie hamster model. PrPSc immunostaining of the same tissue demonstrated that the elevated β-sheet regions correspond to regions where the misfolded structure of PrPSc is located. No evidence of these structural changes was observed in normal neurons.
Keywords: Prion; Transmissible spongiform encephalopathy (TSE); 263 K scrapie; Synchrotron Fourier-transform infrared (FTIR) microspectroscopy; Dorsal root ganglion; Syrian hamster;

Akt/PKB kinase phosphorylates separately Thr212 and Ser214 of tau protein in vitro by Hanna Ksiezak-Reding; Han Kyoung Pyo; Boris Feinstein; Giulio M. Pasinetti (159-168).
Microtubule-associated protein tau contains a consensus motif for protein kinase B/Akt (Akt), which plays an essential role in anti-apoptotic signaling. The motif encompasses the AT100 double phospho-epitope (Thr212/Ser214), a specific marker for Alzheimer's disease (AD) and other neurodegenerations, raising the possibility that it could be generated by Akt. We studied Akt-dependent phosphorylation of tau protein in vitro. We found that Akt phosphorylated both Thr212 and Ser214 in the longest and shortest tau isoforms as determined using phospho site-specific antibodies against tau. Akt did not phosphorylate other tau epitopes, including Tau-1, AT8, AT180, 12E8 and PHF-1. The Akt-phosphorylated tau retained its initial electrophoretic mobility. Immunoprecipitation studies with phospho-specific Thr212 and Ser214 antibodies revealed that only one of the two sites is phosphorylated per single tau molecule, resulting in tau immunonegative for AT100. Mixed kinase studies showed that prior Ser214 phosphorylation by Akt blocked protein kinase A but not GSK3β activity. On the other hand, GSK3β selectively blocked Ser214 phosphorylation, which was prevented by lithium. The results suggest that Akt may be involved in AD-specific phosphorylation of tau at the AT100 epitope in conjunction with other kinases. Our data suggest that phosphorylation of tau by Akt may play specific role(s) in Akt-mediated anti-apoptotic signaling, particularly relevant to AD and other neurodegenerations.
Keywords: Tau; Akt/protein kinase B; Protein kinase A; Glycogen synthase kinase 3β; AT100 epitope; Alzheimer's disease;

Administration of the soluble complement inhibitor, Crry-Ig, reduces inflammation and aquaporin 4 expression in lupus cerebritis by Jessy J. Alexander; Lihua Bao; Alexander Jacob; Damien M. Kraus; V.Michael Holers; Richard J. Quigg (169-176).
Changes in brain water and cerebral volume can lead to brain edema that may be one of the underlying causes of death in many neurological diseases. Cerebral water content is regulated by aquaporin 4 (AQ4) present in astrocytic end feet and around blood vessels. In systemic lupus erythematosus (SLE), magnetic resonance imaging (MRI) studies of the brain have demonstrated lesions with the prominent appearance of edema. Activation of complement may play a significant role in the pathogenesis of lupus cerebritis by causing inflammation that can lead to edema. In this study, the well-established MRL/lpr lupus mouse model was used to evaluate the role of complement in lupus cerebritis. IgG and C1q colocalized in perivascular deposits indicating that the blood–brain barrier was compromised. Both RNA and protein expressions of AQ4 were significantly increased in brains of MRL/lpr mice. Chronic administration of the soluble complement inhibitor, Crry-Ig, reduced inflammation as measured by decreased accumulation of IgG. In contrast to control MRL/lpr mice, AQ4 expression in complement inhibited MRL/lpr mice was not changed relative to untreated congenic controls. These results illustrate that complement activation in brains of lupus mice leads to enhanced AQ4 expression and inflammation. It is conceivable that increased AQ4 expression results in cerebral edema and hence complement inhibition may provide a new therapeutic option in inflammatory cerebral disorders such as lupus cerebritis.
Keywords: Crry-Ig; Inflammation; Aquaporin 4 expression; Lupus cerebitis;

Although oxygen free radicals (OFR) are considered to be one of the pathophysiological mechanisms involved in acute pancreatitis (AP), the contribution of acinar cells to their production is not well established. The aim of the present study was to determine the effect of N-acetylcysteine (NAC) in the course of AP induced by pancreatic duct obstruction (PDO) in rats, directly analysing by flow cytometry the quantity of OFR generated in acinar cells. NAC (50 mg/kg) was administered 1 h before and 1 h after PDO. Measurements by flow cytometry of OFR generated in acinar cells were taken at different PDO times over 24 h, using dihydrorhodamine-123 as fluorescent dye. Histological studies of pancreas and measurements of neutrophil infiltration in the pancreas, pancreatic glutathione (GSH), malondialdehyde (MDA) levels, plasma amylase activity and hemoconcentration were carried out in order to assess the severity of AP at different stages. NAC effectively blunted GSH depletion at early AP stages and prevented OFR generation found in acinar cells as a consequence of AP induced by PDO. This attenuation of the redox state impairment reduced cellular oxidative damage, as reflected by less severe pancreatic lesions, normal pancreatic MDA levels, as well as diminished neutrophil infiltration in pancreas. Hyperamylasemia and hemoconcentration following AP induction were ameliorated by NAC administration at early stages, when oxidative stress seems to be critical in the development of pancreatitis. In conclusion, NAC reinforces the antioxidant defences in acinar cells, preventing OFR generation therefore attenuating oxidative damage and subsequently reducing the severity of PDO-induced AP at early stages of the disease.
Keywords: Acinar cell; Acute pancreatitis; N-Acetylcysteine; Oxidative stress; Oxygen free radical;

Thyroid hormone status influences calcium metabolism. To elucidate the mechanism of action of thyroid hormones on transcellular transport of calcium in rat intestine, Ca2+ influx and efflux studies were carried out in brush border membrane vesicles (BBMV) and across the basolateral membrane (BLM) of enterocytes, respectively. Steady-state uptake of Ca2+ into BBMV as well as Ca2+ efflux from the BLM enterocytes was significantly increased in hyperthyroid (Hyper-T) rats and decreased in hypothyroid (Hypo-T) rats as compared to euthyroid (Eu-T) rats. Kinetic studies revealed that increase in steady state Ca2+ uptake into BBMV from hyper-T rats was fraternized with decrease in Michaelis Menten Constant (K m), indicating a conformational change in Ca2+ transporter. Further, this finding was supported by significant changes in transition temperature and membrane fluidity. Increased Ca2+ efflux across enterocytes was attributed to sodium-dependent Ca2+ exchange activity which was significantly higher in Hyper-T rats and lower in Hypo-T rats as compared to Eu-T rats. However, there was no change in Ca2+-ATPase activity of BLMs of all groups. Kinetic studies of Na+/Ca2+ exchanger revealed that alteration in Na+-dependent Ca2+ efflux was directly associated with maximal velocity (V max) of exchanger among all the groups. cAMP, a potent activator of Na+/Ca2+ exchanger, was found to be significantly higher in intestinal mucosa of Hyper-T rats as compared to Eu-T rats. Therefore, the results of this study suggest that Ca2+ influx across BBM is possibly modulated by thyroid hormones by mediating changes in membrane fluidity. Thyroid hormones activated the Na+/Ca2+ exchange in enterocytes possibly via cAMP-mediated pathway.
Keywords: Thyroid hormones; Calcium metabolism; Enterocyte; Brush border membrane; Basolateral membrane; cAMP; Ca2+-ATPase; Na+/Ca2+ exchange;

Endomorphins, endogenous opioid peptides, provide antioxidant defense in the brain against free radical-induced damage by Xin Lin; Ding-Jian Yang; Wen-Qing Cai; Qian-Yu Zhao; Yan-Feng Gao; Qiang Chen; Rui Wang (195-202).
Oxidative stress has been considered to be a major cause of cellular injuries in a variety of chronic health problems, such as carcinogenesis and neurodegenerative disorders. The brain appears to be more susceptible to oxidative damage than other organs. Therefore, the existence of antioxidants may be essential in brain protective systems. The antioxidative and free radical scavenging effects of endomorphin 1 (EM1) and endomorphin 2 (EM2), endogenous opioid peptides in the brain, have been investigated in vitro. The oxidative damage was initiated by a water-soluble initiator 2,2′-azobis(2-amidinopropane hydrocholoride) (AAPH) and hydrogen peroxide (H2O2). The linoleic acid peroxidation, DNA and protein damage were monitored by formation of hydroperoxides, by plasmid pBR 322 DNA nicking assay and single-cell alkaline electrophoresis, and by SDS-polyacrylamide gel electrophoresis. Endomorphins can inhibit lipid peroxidation, DNA strand breakage, and protein fragmentation induced by free radical. Endomorphins also reacted with galvinoxyl radicals in homogeneous solution, and the pseudo-first-order rate constants were determined spectrophotometrically by following the disappearance of galvinoxyl radicals. In all assay systems, EM1 was more potent than EM2 and GSH, a major intracellular water-soluble antioxidant. We propose that endomorphins are one of the protective systems against free radical-induced damage in the brain.
Keywords: Endomorphin; Antioxidant; Comet assay; Lipid peroxidation; Free radical;

Human paraoxonase gene cluster polymorphisms as predictors of coronary heart disease risk in the prospective Northwick Park Heart Study II by Kirsty S. Robertson; Emma Hawe; George J. Miller; Philippa J. Talmud; Steve E. Humphries (203-212).
The anti-atherogenic effect of HDL has been suggested to be partly due to the action of HDL-associated paraoxonase (PON). Three distinct enzymes have been identified, encoded by PON1, PON2 and PON3, clustered on chromosome 7q21–q22. Two cSNPs in PON1 (L55M and Q192R) and one in PON2 (S311C) have been implicated as independent risk factors for coronary heart disease (CHD) in some, but not all, studies. A PON3 SNP (A99A) was identified and the effect of these four PON SNPs on HDL levels and CHD risk was examined in the prospective Northwick Park Heart Study II (NPHSII). Genotype frequencies did not differ between cases and controls but the CHD risk associated with smoking was significantly modified by PON1 L55M genotype. Compared to LL non-smokers, LL smokers had a hazard ratio (HR) of 1.30 (95% CI 0.81–2.06) while M-allele carriers had a HR of 1.76 (1.17–2.67). When genotypes were analysed in combination, men with the genotype PON1 55 LM/MM+PON2 311 CC, had HR of 3.54 (1.81–6.93) compared to PON1 LL+PON2 SS/SC men (interaction P=0.004). These effects were independent of classical risk factors. These data demonstrate the importance of stratifying by environmental factors and the use of multiple SNPs for genetic analysis.
Keywords: Paraoxonase; Polymorphism; PON1; PON2; PON3;

P-glycoprotein-mediated multidrug resistance phenotype of L1210/VCR cells is associated with decreases of oligo- and/or polysaccharide contents by R. Fiala; Z. Sulová; A.H. El-Saggan; B. Uhrík; T. Liptaj; I. Dovinová; E. Hanušovská; Z. Drobná; M. Barančík; A. Breier (213-224).
Multidrug resistance of murine leukaemic cell line L1210/VCR (obtained by adaptation of parental drug-sensitive L1210 cells to vincristine) is associated with overexpression of mdr1 gene product P-glycoprotein (Pgp)—the ATP-dependent drug efflux pump. 31P-NMR spectra of L1210 and L1210/VCR cells (the latter in the presence of vincristine) revealed, besides the decrease of ATP level, a considerable lower level of UDP-saccharides in L1210/VCR cells. Histochemical staining of negatively charged cell surface binding sites (mostly sialic acid) by ruthenium red (RR) revealed a compact layer of RR bound to the external coat of sensitive cells. In resistant cells cultivated in the absence or presence of vincristine, the RR layer is either reduced or absent. Consistently, resistant cells were found to be less sensitive to Concanavalin A (ConA). Moreover, differences in the amount and spectrum of glycoproteins interacting with ConA-Sepharose were demonstrated between sensitive and resistant cells. Finally, the content of glycogen in resistant cells is lower than in sensitive cells. All the above facts indicate that multidrug resistance of L1210/VCR cells mediated predominantly by drug efflux activity of Pgp is accompanied by a considerable depression of oligo- and/or polysaccharides biosynthesis.
Keywords: Multidrug resistance; P-glycoprotein; L1210 cell line; Concanavalin A; Ruthenium red; Oligo- (poly-)saccharides;

Sulfation patterns in heparin and heparan sulfate: effects on the proliferation of bovine pulmonary artery smooth muscle cells by Hari G. Garg; Lunyin Yu; Charles A. Hales; Toshihiko Toida; Tasneem Islam; Robert J. Linhardt (225-231).
Heparin's (HP's) antiproliferative effect on smooth muscle cells is potentially important in defining new approaches to treat pulmonary hypertension. The commercially available HP and heparan sulfate (HS) are structurally heterogenous polymers. In order to examine which sulfonate groups are required for endogenous antiproliferative activity, we prepared the following six chemically modified porcine mucosal HP and HS, which fell into three groups. One group consisted of fully O-sulfonated-N-acetylated, the second group consisted of de-N-sulfonated and re-N-acetylated, and the third group consisted of 6-O-desulfonated HP and HS derivatives. These six preparations were assayed for their antiproliferative potency on bovine pulmonary artery smooth muscle cells. The results of this assay show that (a) over-O-sulfonation of both HP and HS increases antiproliferative activity, (b) substitution of hexosamine with N-acetyl diminishes antiproliferative activity in both HP and HS, and (c) 6-O-desulfonation of HP and HS diminishes antiproliferative potency. Surprisingly, the type of uronic acid residue present at a given level of sulfation is unimportant for antiproliferative potency. In conclusion, only the level of O- and N-sulfo group substitution correlates well with HP and HS antiproliferative activity.
Keywords: Heparin; Heparan sulfate; Smooth muscle cell; Proliferation; Post-column derivatization HPLC; 1H NMR;

Inhibition of brain energy metabolism by the α-keto acids accumulating in maple syrup urine disease by Angela M. Sgaravatti; Rafael B. Rosa; Patrícia F. Schuck; César A.J. Ribeiro; Clóvis M.D. Wannmacher; Angela T.S. Wyse; Carlos S. Dutra-Filho; Moacir Wajner (232-238).
Neurological dysfunction is a common finding in patients with maple syrup urine disease (MSUD). However, the mechanisms underlying the neuropathology of brain damage in this disorder are poorly known. In the present study, we investigated the effect of the in vitro effect of the branched chain α-keto acids (BCKA) accumulating in MSUD on some parameters of energy metabolism in cerebral cortex of rats. [14CO2] production from [14C] acetate, glucose uptake and lactate release from glucose were evaluated by incubating cortical prisms from 30-day-old rats in Krebs–Ringer bicarbonate buffer, pH 7.4, in the absence (controls) or presence of 1–5 mM of α-ketoisocaproic acid (KIC), α-keto-β-methylvaleric acid (KMV) or α-ketoisovaleric acid (KIV). All keto acids significantly reduced 14CO2 production by around 40%, in contrast to lactate release and glucose utilization, which were significantly increased by the metabolites by around 42% in cortical prisms. Furthermore, the activity of the respiratory chain complex I–III was significantly inhibited by 60%, whereas the other activities of the electron transport chain, namely complexes II, II–III, III and IV, as well as succinate dehydrogenase were not affected by the keto acids. The results indicate that the major metabolites accumulating in MSUD compromise brain energy metabolism by blocking the respiratory chain. We presume that these findings may be of relevance to the understanding of the pathophysiology of the neurological dysfunction of MSUD patients.
Keywords: Maple syrup urine disease; α-Ketoisocaproic acid; α-Keto-β-methylvaleric acid; α-Ketoisovaleric acid; Energy metabolism;