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BBA - Molecular Basis of Disease (v.1772, #3)
Alzheimer disease: Amyloidogenesis, the presenilins and animal models by M. Newman; F.I. Musgrave; M. Lardelli (pp. 285-297).
Alzheimer’s disease is the most prevalent form of dementia. Neuropathogenesis is proposed to be a result of the accumulation of amyloid beta peptides in the brain together with oxidative stress mechanisms and neuroinflammation. The presenilin proteins are central to the gamma-secretase cleavage of the amyloid prescursor protein (APP), releasing the amyloid beta peptide. Point mutations in the presenilin genes lead to cases of familial Alzheimer’s disease by increasing APP cleavage resulting in excess amyloid beta formation. This review discusses the molecular mechanism of Alzheimer’s disease with a focus on the presenilin genes. Alternative splicing of transcripts from these genes and how these may function in several disease states is discussed. There is an emphasis on the importance of animal models in elucidating the molecular mechanisms behind the development of Alzheimer’s disease and how the zebrafish, Danio rerio, can be used as a model organism for analysis of presenilin function and Alzheimer’s disease pathogenesis.
Keywords: Alzheimer’s disease; Amyloid-beta; Animal models; Oxidative stress; Presenilin; Zebrafish
Effects of genetic backgrounds on hyperbilirubinemia in radixin-deficient mice due to different expression levels of Mrp3 by Kanehisa Fukumoto; Shojiro Kikuchi; Norio Itoh; Atsushi Tamura; Masaki Hata; Hisakazu Yamagishi; Shoichiro Tsukita; Sachiko Tsukita (pp. 298-306).
ERM (ezrin/radixin/moesin) proteins are organizers of apical actin cortical layer in general. We previously reported that the knockout of radixin resulted in Rdx− /− mice with displacement/loss of the canalicular transporter Mrp2, giving rise to Dubin–Johnson syndrome-like conjugated hyperbilirubinemia in the mixed genetic background (C57BL/6-129/Sv) (Kikuchi, et al. (2002) Nature Genetics 31, 320–325). However, when these mice were kept under mixed genetic background for years (late mixed backgrounds; LMB), the conjugated hyperbilirubinemia gradually became inconspicuous, while evidence of liver injury increased. We examined the effect of genetic background by backcrossing LMB Rdx− /− mice to C57BL/6 and 129/Sv wild type mice with the result that the Rdx− /− congenic mice regained hyperbilirubinemia with reduced hepatocellular damage. As revealed by immunofluorescence and western blots, the localization/expression of apical transporters, Mrp2, CD26, P-gps, and Bsep were not influenced by backcrossing, though those of a basolateral transporter, Mrp3, were strikingly increased by backcrossing.
Keywords: Abbreviations; ERM; ezrin/radixin/moesin; mAb; monoclonal antibody; pAb; polyclonal antibody; Mrp; multidrug resistance protein; Rdx; radixin; Bsep; bile salt export pump; P-gps; P-glycoprotein; EMB; early mixed background; LMB; late mixed backgroundERM; Mrp2; Mrp3; Hyperbilirubinemia; Genetic background
Functioning of oxidative phosphorylation in liver mitochondria of high-fat diet fed rats by Jolita Ciapaite; Stephan J.L. Bakker; Gerco Van Eikenhorst; Marijke J. Wagner; Tom Teerlink; Casper G. Schalkwijk; Mariann Fodor; D. Margriet Ouwens; Michaela Diamant; Robert J. Heine; Hans V. Westerhoff; Klaas Krab (pp. 307-316).
We proposed that inhibition of mitochondrial adenine nucleotide translocator (ANT) by long chain acyl-CoA (LCAC) underlies the mechanism associating obesity and type 2 diabetes. Here we test that after long-term exposure to a high-fat diet (HFD): (i) there is no adaptation of the mitochondrial compartment that would hinder such ANT inhibition, and (ii) ANT has significant control of the relevant aspects of oxidative phosphorylation. After 7 weeks, HFD induced a 24±6% increase in hepatic LCAC concentration and accumulation of the oxidative stress marker Nε-(carboxymethyl)lysine. HFD did not significantly affect mitochondrial copy number, oxygen uptake, membrane potential (Δ ψ), ADP/O ratio, and the content of coenzyme Q9, cytochromes b and a+ a3. Modular kinetic analysis showed that the kinetics of substrate oxidation, phosphorylation, proton leak, ATP-production and ATP-consumption were not influenced significantly. After HFD-feeding ANT exerted considerable control over oxygen uptake (control coefficient C=0.14) and phosphorylation fluxes ( C=0.15), extra- ( C=0.23) and intramitochondrial ( C=−0.56) ATP/ADP ratios, and Δ ψ ( C=−0.11). We conclude that although HFD induces accumulation of LCAC and Nε-(carboxymethyl)lysine, oxidative phosphorylation does not adapt to these metabolic challenges. Furthermore, ANT retains control of fluxes and intermediates, making inhibition of this enzyme a more probable link between obesity and type 2 diabetes.
Keywords: Abbreviations; ANT; adenine nucleotide translocator; Ap5A; P1(P5-adenosine-5′ pentaphosphate); BSA; bovine serum albumin; C; ANT; Δ; Ψ; concentration control coefficient quantifying control of Δ; ψ; by ANT; C; ANT; ATP; in; /ADP; in; concentration control coefficient quantifying control of intramitochondrial ATP/ADP ratio by ANT; C; ANT; ATP; out; /ADP; out; concentration control coefficient quantifying control of extramitochondrial ATP/ADP ratio by ANT; C; ANT; J; o; flux control coefficient quantifying control of oxygen uptake flux; J; o; by ANT; C; ANT; J; p; flux control coefficient, quantifying control of phosphorylation flux; J; p; by ANT; CML; N; ε; -(carboxymethyl)lysine; Co Q; 9; coenzyme Q; 9; FA; fatty acid; HFD; high-fat diet; HNE; 4-hydroxy-2-nonenal; LCAC; long-chain fatty acyl-CoA esters; LFD; low-fat diet; MCA; metabolic control analysis; ROS; reactive oxygen species; TCA; tricarboxylic acid cycle; TPP; +; tetraphenylphosphonium ion; Δ; ψ; membrane potential, i.e. electric potential across the inner mitochondrial membrane (out minus in)High fat diet; Mitochondria; Long chain acyl-CoA esters; Oxidative stress; Obesity and type 2 diabetes
Altered expression of FHL1, CARP, TSC-22 and P311 provide insights into complex transcriptional regulation in pacing-induced atrial fibrillation by Chien-Lung Chen; Jiunn-Lee Lin; Ling-Ping Lai; Chun-Hsu Pan; Shoei K. Stephen Huang; Chih-Sheng Lin (pp. 317-329).
Atrial fibrillation (AF) is the most common progressive disease in patients with cardiac arrhythmia. AF is accompanied by complex atrial remodeling and changes in gene expression, but only a limited number of transcriptional regulators have been identified. Using a low-density cDNA array, we identified 31 genes involved in transcriptional regulation, signal transduction or structural components, which were either significantly upregulated or downregulated in porcine atria with fibrillation (induced by rapid atrial pacing at a rate of 400–600 bpm for 4 weeks that was then maintained without pacing for 2 weeks). The genes for four and a half LIM domains protein-1 ( FHL1), transforming growth factor-β (TGF-β)-stimulated clone 22 ( TSC-22), and cardiac ankyrin repeat protein ( CARP) were significantly upregulated, and chromosome 5 open reading frame gene 13 ( P311) was downregulated in the fibrillating atria. FHL1 and CARP play important regulatory roles in cardiac remodeling by transcriptional regulation and myofilament assembly. Induced mRNA expression of both FHL1 and CARP was also observed when cardiac H9c2 cells were treated with an adrenergic agonist. Increasing TSC-22 and marked P311 deficiency could enhance the activity of TGF-β signaling and the upregulated TGF-β1 and - β2 expressions were identified in the fibrillating atria. These results implicate that observed alterations of underlying molecular events were involved in the rapid-pacing induced AF, possibly via activation of the β-adrenergic and TGF-β signaling.
Keywords: Atrial fibrillation; Low-density cDNA array; Transcriptional regulator; Transforming growth factor-β signaling
The magnetic susceptibilities of iron deposits in thalassaemic spleen tissue by S. Hackett; W. Chua-anusorn; P. Pootrakul; T.G. St Pierre (pp. 330-337).
The iron-specific magnetic susceptibility of tissue iron deposits is used in the field of non-invasive measurement of tissue iron concentrations. It has generally been assumed to be a constant for all tissue and disease types. The iron-specific magnetic susceptibilities χFe for spleen tissue samples from 7 transfusion dependent β-thalassaemia (β-thal) patients and 11 non-transfusion dependent β-thalassaemia/Haemoglobin E (β/E) patients were measured at 37 °C. Both groups of patients were iron loaded with no significant difference in the distribution of spleen iron concentrations between the two groups. There was a significant difference between the mean χFe of the spleen tissue from each group. The non-transfusion dependent β/E patients had a higher mean (±standard deviation) spleen χFe (1.55±0.23×10−6 m3/kg Fe) than the transfusion dependent β-thal patients (1.16±0.25×10−6 m3/kg Fe). Correlations were observed between χFe of the spleen tissue and the fraction of magnetic hyperfine split sextet in the57Fe Mössbauer spectra of the tissues at 78 K (Spearman rank order correlation r=−0.54, p=0.03) and between χFe of the spleen tissue and the fraction of doublet in the spectra at 5 K ( r=0.58, p=0.02) indicating that χFe of the spleen tissue is related to the chemical speciation of the iron deposits in the tissue.
Keywords: Abbreviations; W–M–W; Wilcoxon–Mann–WhitneyHaemosiderin; Ferritin; Magnetic susceptibility; Thalassaemia; Iron
Endothelin-1 and angiotensin II contribute to BNP but not c- fos gene expression response to elevated load in isolated mice hearts by Jarkko Piuhola; István Szokodi; Heikki Ruskoaho (pp. 338-344).
The early events in the cardiac hypertrophic process induced by hemodynamic load include activation of B-type natriuretic peptide (BNP) and c- fos gene expression. However, it is unknown whether stretch acts directly or through local paracrine factors to trigger changes in cardiac gene expression. Herein we studied the involvement of endothelin-1 (ET-1) and angiotensin II (Ang II) in load-induced activation of left ventricular BNP and c- fos gene expression using an in vitro stretch model in isolated perfused adult mice hearts. Two-hour stretch induced by increasing coronary flow rate from 2 to 5 ml/min increased the expression of BNP and c- fos genes by 1.9- and 1.5-fold, respectively ( P<0.001 and P<0.05). A mixed ETA/B receptor antagonist bosentan attenuated the BNP gene expression response to load by 58% ( P<0.005). A similar 53% inhibition was observed with the selective ETA receptor blocker BQ-123 ( P<0.05). Type 1 Ang II receptor antagonist CV-11974 decreased the activation of BNP gene expression by 50% ( P<0.05). In contrast, the activation of c- fos gene expression was not inhibited by antagonists of ETA/B and AT1 receptors. Our results show that ET-1 and Ang II play a key role in the induction of BNP, but not c- fos gene expression in response to load in intact adult murine hearts.
Keywords: Vasoactive peptides; Ventricular stretch; Hypertrophy; Cardiac overload
Regulated ion transport in mouse liver cyst epithelial cells by R. Brian Doctor; Sylene Johnson; Kelley S. Brodsky; Claudia R. Amura; Vincent Gattone; J. Gregory Fitz (pp. 345-354).
Derived from bile duct epithelia (BDE), secretion by liver cyst-lining epithelia is positioned to drive cyst expansion but the responsible ion flux pathways have not been characterized. Cyst-lining epithelia were isolated and cultured into high resistance monolayers to assess the ion secretory pathways. Electrophysiologic studies showed a marked rate of constitutive transepithelial ion transport, including Cl− secretion and Na+ absorption. Na+ absorption was amiloride-sensitive, suggesting the activation of epithelial sodium channels (ENaC). Further, both cAMPi and extracellular ATP induced robust secretory responses. Western blotting and immunohistologic analysis of liver cyst epithelia demonstrated expression of P2X4, a potent purinergic receptor in normal BDE. Luminometry and bioassaying measured physiologically relevant levels of ATP in a subset of liver cyst fluid samples. Liver cyst epithelia also displayed a significant capacity to degrade extracellular ATP. In conclusion, regulated ion transport pathways are present in liver cyst epithelia and are positioned to direct fluid secretion into the lumen of liver cysts and promote increases in liver cyst expansion and growth.
Keywords: Autosomal dominant polycystic kidney disease (ADPKD); Ussing analysis; Purinergic signaling; cAMP; Epithelial sodium channel
The effect of APOA5 and APOC3 variants on lipid parameters in European Whites, Indian Asians and Afro-Caribbeans with type 2 diabetes by Birgit Dorfmeister; Jackie A. Cooper; Jeffrey W. Stephens; Helen Ireland; Steven J. Hurel; Steve E. Humphries; Philippa J. Talmud (pp. 355-363).
Common variants in APOA5 and APOC3 have been associated with differences in plasma triglyceride (TG) levels in healthy individuals. The aim of this study was to examine the association of APOA5 (−1131T>C, S19W) and APOC3 (−482C>T, 1100C>T) polymorphisms in patients with type 2 diabetes (T2D) of European White (EW) ( n=931), Indian Asian (IA) ( n=610) and Afro-Caribbean (AC) ( n=167) origin, with lipid and T2D parameters. Rare allele frequencies and linkage disequilibrium differed significantly amongst ethnic groups. Compared to APOA5 −1131T and 19S homozygotes, −1131C and 19W carriers had higher TGs in all groups, but this effect was only statistically significant for the −1131C in the EWs ( P=0.04) and 19W in the IAs ( P<0.001). APOC3 SNPs showed no significant association with lipid levels in any ethnic group. While haplotypes carrying −1131C allele showed significant TG-raising in the EWs only, the 19W defined haplotype showed significant TG-raising in both IAs and EWs. Comparing all four SNPs in EW T2D subjects with healthy EWs ( n=2579), the APOC3 1100C>T frequency was significantly higher in T2D [0.26 (0.24, 0.28)] vs. healthy EWs [0.22 (0.20, 0.23)], P=0.001. While the variable size effects of the two APOA5 SNPs on TG levels may result from ethnically different gene–gene or gene–environment interactions, APOA5 and APOC3 variants did not affect parameters of T2D. However, comparison between EWs with T2D and healthy EWs suggest APOC3 1100C>T is associated with increased risk of diabetes probably through mechanisms other than direct effects on TG.
Keywords: Apolipoprotein A5; Apolipoprotein C3; Triglycerides; Single nucleotide polymorphism; Type 2 diabetes
High glucose downregulates the number of caveolae in monocytes through oxidative stress from NADPH oxidase: Implications for atherosclerosis by Toshio Hayashi; Packiasamy A.R. Juliet; Asaka Miyazaki; Louis J. Ignarro; Akihisa Iguchi (pp. 364-372).
Atherosclerosis, an inflammatory disease, is closely associated with hyperglycemia, major sign of diabetes mellitus. Caveolae are vesicular invaginations of the plasma membrane that mediate the intracellular transport of lipids such as cholesterol. We evaluated the relationship between the expression of caveolin-1 and the number of caveolae in macrophages under conditions of high glucose concentration. Increased superoxide production, induction of inducible nitric oxide synthase (iNOS), and decreased caveolin-1 were observed in a concentration-dependent manner in THP-1 derived macrophages with high glucose concentrations. Mannitol, used as an osmotic control, showed no effects. Furthermore, co-localization of the NADPH oxidase component, p47phox, and caveolin was confirmed by confocal microscopy. An atomic force microscopy (AFM) study showed that high glucose concentrations reduced the number and size of the caveolae. The percentage of cells with fragmented DNA was increased in cells grown in hyperglycemic media. Taken together, high glucose concentrations suppress the levels of caveolin-1 expression and reduce the number of caveolae. This might be due to the actions of superoxide via the activation of NADPH oxidase by translocation of its component and uncoupling of induced iNOS in macrophages. Furthermore, the apoptosis of macrophages might occur with high glucose concentrations, leading to the spreading of lipids from macrophages into intracellular spaces in the vessel wall.
Keywords: Abbreviations; PMA; phorbol myristate acetate; NO; nitric oxide; LDL; low-density lipoprotein; INF-γ; human interferon gamma; TNF-α; tumor necrosis factor alpha; SOD; superoxide dismutaseCaveolae; Glucose; Nitric oxide; Macrophage; NADPH oxidase
Superoxide production is inversely related to complex I activity in inherited complex I deficiency by Sjoerd Verkaart; Werner J.H. Koopman; Sjenet E. van Emst-de Vries; Leo G.J. Nijtmans; Lambertus W.P.J. van den Heuvel; Jan A.M. Smeitink; Peter H.G.M. Willems (pp. 373-381).
Deficiency of NADH:ubiquinone oxidoreductase or complex I (CI) is the most common cause of disorders of the oxidative phosphorylation system in humans. Using life cell imaging and blue-native electrophoresis we quantitatively compared superoxide production and CI amount and activity in cultured skin fibroblasts of 7 healthy control subjects and 21 children with inherited isolated CI deficiency. Thirteen children had a disease causing mutation in one of the nuclear-encoded CI subunits, whereas in the remainder the genetic cause of the disease is not yet established. Superoxide production was significantly increased in all but two of the patient cell lines. An inverse relationship with the amount and residual activity of CI was observed. In agreement with this finding, rotenone, a potent inhibitor of CI activity, dose-dependently increased superoxide production in healthy control cells. Also in this case an inverse relationship with the residual activity of CI was observed. In sharp contrast, however, rotenone did not decrease the amount of CI. The data presented show that superoxide production is increased in inherited CI deficiency and that this increase is primarily a consequence of the reduction in cellular CI activity and not of a further leakage of electrons from mutationally malformed complexes.
Keywords: Abbreviations; CI; complex I or NADH:ubiquinone oxidoreductase; CII; complex II or succinate dehydrogenase-ubiquinone oxidoreductase; CIII; complex III or ubiquinol:cytochrome; c; oxidoreductase; CIV; complex IV or cytochrome; c; oxidase; OXPHOS; oxidative phosphorylation; HEt; hydroethidine; Δ; ψ; mitochondrial membrane potentialFibroblast; Mitochondria; Hydroethidine; Rotenone; Fluorescence microscopy
Elevation of rat plasma P-selectin in acute lung injury by Mayumi Hirose; Toshiyuki Murai; Hiroto Kawashima (pp. 382-389).
Acute lung injury in the rat caused by intravenous (i.v.) infusion of cobra venom factor (CVF) or lipopolysaccharide (LPS) is mediated by P-selectin-dependent neutrophil infiltration into the lung. In these lung injury models, P-selectin expression is induced on lung vascular endothelial cells after the CVF or LPS infusion, suggesting soluble P-selectin derived from inflamed sites might also be elevated. Here we established a sensitive enzyme-linked immunosorbent assay (ELISA) to measure soluble P-selectin in plasma, a potential marker of lung injury. Nine anti-rat P-selectin monoclonal antibodies that we established previously were first classified into 5 groups based on real-time biospecific interaction analyses, and used to develop a sandwich ELISA for accurately measuring the amount of soluble P-selectin in plasma. We then used this ELISA to measure the plasma P-selectin levels in Long Evans, Wistar, and Sprague–Dawley rats after the i.v. infusion of CVF or LPS. The elevation in P-selectin levels was significantly different among the strains, but it consistently correlated with the extent of lung inflammation, measured by myeloperoxidase levels in the lung tissues. Thus, our results indicate that the soluble P-selectin in plasma could serve as a sensitive biomarker reflecting lung inflammation, which is of clinical importance for detecting and preventing severe lung injury.
Keywords: P-selectin; Lung injury; ELISA; Monoclonal antibody; Plasma