BBA - Molecular Basis of Disease (v.1740, #1)
Editorial Board (ii).
Stability of RNA isolated from human trabecular bone at post-mortem and surgery by Julia S. Kuliwaba; Nicola L. Fazzalari; David M. Findlay (1-11).
To determine the reliability of gene expression studies in human post-mortem bone, it is important to evaluate the stability of RNA isolated from such tissues as a function of the post-mortem interval. The stability of total RNA and bone-specific mRNA species was examined in bone samples obtained from routine autopsies and at surgery. The optimal temperature for any storage and transport of the bone before RNA isolation was shown to be 4 °C, and RT-PCR analysis is the preferred technique for the analysis of gene expression in post-mortem bone as it tolerates partial RNA degradation. For gene expression studies in bone, post-mortem cases, with a post-mortem interval of less than 48 h, should be selected, and the time that bone is stored after retrieval at autopsy or surgery should be kept to a minimum. Overall, our findings indicate that with appropriate storage and handling, RNA can be reliably isolated from human bone obtained at post-mortem and surgery to study ex vivo the pattern of gene expression in healthy individuals and in patients with musculoskeletal diseases such as osteoporosis and osteoarthritis.
Keywords: Bone; mRNA; Post-mortem; RNA stability; Surgery;
Homocysteine attenuates the expression of osteocalcin but enhances osteopontin in MC3T3-E1 preosteoblastic cells by Wataru Sakamoto; Haruo Isomura; Katsutoshi Fujie; Yoshiaki Deyama; Akihisa Kato; Jun Nishihira; Hiroshi Izumi (12-16).
It has been pointed out that very high plasma levels of homocysteine are characteristic of homocystinuria, a rare autosomal recessive disease accompanied by the early onset of generalized osteoporosis. However, it is unclear by which mechanism hyperhomocysteine induces osteoporosis, although it is known to interfere eith the formation of cross-links in collagen, an essential process in bone formation. Therefore, we investigated the effect of homcysteine on expression of osteocalcin and osteopontin in MC3T3-E1 preosteoblastic cells. Confluent cells were grown in RPMI 1640 containing 10% fetal calf serum with or without homocysteine in an atmosphere of 95% humidified air, 5% CO2 at 37 °C. The secretion of osteocalcin from the cells increased time-dependently until the end of culture (day 34), but 500 μM homocysteine led to an approximately 61% decrease for osteocalcin after 19 days of culture as compared with the control. On the other hand, osteopontin was not inhibited by 500 μM homocysteine but rather activated, and ranged from 134%–209% of the control level in the period from 10 days until the end of culture. From analysis of RT-PCR for mRNA of osteocalcin and osteopontin at the end of the culture, homocysteine levels of 100 and 500 μM significantly increased the expression of osteopontin mRNA with the control (p <0.05). In contrast, the expression of osteopontin mRNA was suppressed in a dose-dependent manner, showing a mirror image of the effect on osteopontin mRNA. These findings suggest that hyperhomocystenemia appears to be an independent risk factor for osteoporosis by disturbing osteoblast function.
Keywords: Homocysteine; Osteocalcin; Osteopontin; MC3T3-E1 cells;
Erythropoietin hypersensitivity in primary familial and congenital polycythemia: Role of tyrosines Y285 and Y344 in erythropoietin receptor cytoplasmic domain by Murat O. Arcasoy; Aysen F. Karayal (17-28).
Erythropoietin receptor (EPOR) gene mutations leading to truncations of the cytoplasmic, carboxy-terminal region of EPOR have been described in some patients with primary familial and congenital polycythemia (PFCP), a disorder characterized by isolated erythrocytosis and increased sensitivity of erythroid progenitors to Epo. We studied the role of EPOR in the pathogenesis of PFCP and the requirement for intracytoplasmic tyrosine residues Y285 and Y344 in generation of Epo hypersensitivity phenotype. Interleukin-3-dependent hematopoietic cells were engineered to express variant human EPORs using retrovirus-mediated gene transfer. We introduced tyrosine to phenylalanine substitutions in EPOR-ME, a naturally occurring, mutant human EPOR (G5881T), truncated by 110 carboxy-terminal amino acids and associated with autosomal dominantly inherited PFCP. Cells expressing EPOR-ME exhibited increased Epo sensitivity compared to cells expressing wild type EPOR. Mutation of Y285 alone had a relatively minor effect on Epo hypersensitivity whereas mutation of Y344 resulted in loss of increased Epo sensitivity. Expression of a tyrosine-null truncated EPOR conferred further decrease of Epo-mediated proliferation suggesting that both Y285 and Y344 may contribute to proliferation signals. In the context of EPOR-ME, Y344 was required for Epo-induced Stat5 tyrosine phosphorylation. The positive effect of either Y285 or Y344 on cellular proliferation was associated with Epo-induced tyrosine phosphorylation of Stat1. These findings suggest that both tyrosine residues Y285 and Y344 in the cytoplasmic domain of EPOR-ME may contribute to increased Epo sensitivity that is characteristic of PFCP phenotype.
Keywords: Erythropoietin; Erythropoietin receptor; Familial polycythemia; Primary erythrocytosis; Janus kinase; Signal transducers; Activators of transcription;
Altered renal elimination of organic anions in rats with chronic renal failure by Adriana Mónica Torres; Myriam Mac Laughlin; Angélica Muller; Anabel Brandoni; Naohiko Anzai; Hitoshi Endou (29-37).
The progress of chronic renal failure (CRF) is characterized by the development of glomerular and tubular lesions. However, little is known about the expression of organic anions renal transporters. The objective of this work was to study, in rats with experimental CRF (5/6 nephrectomy), the expression of the organic anion transporter 1 (OAT1) and organic anion transporter 3 (OAT3) and their contribution to the pharmacokinetics and renal excretion of p-aminohippurate (PAH). Two groups of animals were used: Sham and CRF. Six months after surgery, systolic blood pressure and plasma creatinine concentrations were significantly higher in CRF groups. CRF rats showed a diminution in: the filtered, secreted and excreted load of PAH; the systemic clearance of PAH; the renal OAT1 expression; and the renal Na–K-ATPase activity. No remarkable modifications were observed in the OAT3 expression from CRF kidneys. The diminution in the systemic depuration and renal excretion of PAH may be explained by the decrease in its filtered and secreted load. The lower OAT1 expression in remnant renal mass of CRF rats or/and the lower activity of Na–K-ATPase might justify, at least in part, the diminished secreted load of this organic anion.
Keywords: Organic anion; Chronic renal failure; Renal depuration; Organic anion transporter 1; Organic anion transporter 3;
Budding, vesiculation and permeabilization of phospholipid membranes—evidence for a feasible physiologic role of β2-glycoprotein I and pathogenic actions of anti-β2-glycoprotein I antibodies by Aleš Ambrožič; Borut Božič; Tanja Kveder; Janja Majhenc; Vesna Arrigler; Saša Svetina; Blaž Rozman (38-44).
The in vivo physiologic role of β2-glycoprotein I (β2GPI) is presumed to be related to its interactions with negatively charged phospholipid membranes. Increased quantities of procoagulant microparticles derived by the vesiculation of blood cells have been detected in patients with antiphospholipid syndrome (APS) frequently associated with antibodies against β2GPI (anti-β2GPI). We investigated the influence of β2GPI and anti-β2GPI on giant phospholipid vesicles (GPVs). GPVs composed of phosphatidylserine and phosphatidylcholine were formed in an aqueous medium and individually transferred to a compartment containing either β2GPI, anti-β2GPI, or β2GPI along with anti-β2GPI. Shape changes of a single GPV were observed by a phase contrast microscope. Most GPVs transferred to the solution containing only β2GPI budded moderately. Upon the transfer of GPVs to the solution containing β2GPI and anti-β2GPI either from patient with APS or mouse monoclonal anti-β2GPI Cof-22, the budding was much more pronounced, generating also daughter vesicles. No such effects were seen when GPV was transferred to the solution containing anti-β2GPI without β2GPI. Our results suggest a significant physiologic role of β2GPI in the budding of phospholipid membranes, which may be explained by the insertion of the C-terminal loop of β2GPI into membranes, thus increasing the surface of the outer layer of a phospholipid bilayer. Anti-β2GPI, recognizing domains I to IV of β2GPI, enhanced the budding and vesiculation of GPVs in the presence of β2GPI. This might be a novel pathogenic mechanism of anti-β2GPI, promoting in vivo the expression of proadhesive and procoagulant phospholipid surfaces in APS.
Keywords: β2-glycoprotein I; Anti-β2-glycoprotein I; Antiphospholipid syndrome; Phospholipid membrane; Giant phospholipid vesicle;
Dermatan sulfate is the predominant antithrombotic glycosaminoglycan in vessel walls: Implications for a possible physiological function of heparin cofactor II by Ana M.F. Tovar; Diogo A. de Mattos; Mariana P. Stelling; Branca S.L. Sarcinelli-Luz; Rômulo A. Nazareth; Paulo A.S. Mourão (45-53).
The role of different glycosaminoglycan species from the vessel walls as physiological antithrombotic agents remains controversial. To further investigate this aspect we extracted glycosaminoglycans from human thoracic aorta and saphenous vein. The different species were highly purified and their anticoagulant and antithrombotic activities tested by in vitro and in vivo assays. We observed that dermatan sulfate is the major anticoagulant and antithrombotic among the vessel wall glycosaminoglycans while the bulk of heparan sulfate is a poorly sulfated glycosaminoglycan, devoid of anticoagulant and antithrombotic activities. Minor amounts of particular a heparan sulfate (< 5% of the total arterial glycosaminoglycans) with high anticoagulant activity were also observed, as assessed by its retention on an antithrombin-affinity column. Possibly, this anticoagulant heparan sulfate originates from the endothelial cells and may exert a significant physiological role due to its location in the interface between the vessel wall and the blood. In view of these results we discuss a possible balance between the two glycosaminoglycan-dependent anticoagulant pathways present in the vascular wall. One is based on antithrombin activation by the heparan sulfate expressed by the endothelial cells. The other, which may assume special relevance after vascular endothelial injury, is based on heparin cofactor II activation by the dermatan sulfate proteoglycans synthesized by cells from the subendothelial layer.
Keywords: Vascular glycosaminoglycan; Anticoagulant glycosaminoglycan; Thrombosis; Dermatan sulfate; Heparan sulfate; Heparin cofactor II;
Identification of novel mutations of the human N-acetylglutamate synthase gene and their functional investigation by expression studies by Eva Schmidt; Jean-Marc Nuoffer; Johannes Häberle; Silke Pauli; Nathalie Guffon; Christine Vianey-Saban; Bendicht Wermuth; Hans Georg Koch (54-59).
The mitochondrial enzyme N-acetylglutamate synthase (NAGS) produces N-acetylglutamate serving as an allosteric activator of carbamylphosphate synthetase 1, the first enzyme of the urea cycle. Autosomal recessively inherited NAGS deficiency (NAGSD) leads to severe neonatal or late-onset hyperammonemia. To date few patients have been described and the gene involved was described only recently. In this study, another three families affected by NAGSD were analyzed for NAGS gene mutations resulting in the identification of three novel missense mutations (C200R [c.598T>C], S410P [c.1228T>C], A518T [c.1552G>A]). In order to investigate the effects of these three and two additional previously published missense mutations on enzyme activity, the mutated proteins were overexpressed in a bacterial expression system using the NAGS deficient E. coli strain NK5992. All mutated proteins showed a severe decrease in enzyme activity providing evidence for the disease-causing nature of the mutations. In addition, we expressed the full-length NAGS wild type protein including the mitochondrial leading sequence, the mature protein as well as a highly conserved core protein. NAGS activity was detected in all three recombinant proteins but varied regarding activity levels and response to stimulation by l-arginine. In conclusion, overexpression of wild type and mutated NAGS proteins in E. coli provides a suitable tool for functional analysis of NAGS deficiency.
Keywords: NAGS; Hyperammonemia; NAGS deficiency;
The type of collagen cross-link determines the reversibility of experimental skin fibrosis by Annemarie J. van der Slot-Verhoeven; Ernst A. van Dura; Joline Attema; Bep Blauw; Jeroen DeGroot; Tom W.J. Huizinga; Anne-Marie Zuurmond; Ruud A. Bank (60-67).
Fibrotic processes in humans are characterised by an excessive accumulation of collagen containing increased levels of hydroxyallysine-derived cross-links. The occurrence of these cross-links appears to be an important criterion in assessing the irreversibility of fibrosis. We hypothesise that increased hydroxyallysine cross-linking results in a collagenous matrix that is less susceptible to proteolytic degradation and therefore the collagen deposition is no longer reversible. In this report, we show that collagen matrices with increased hydroxyallysine cross-link levels were less susceptible to matrix metalloproteinase 1 degradation than are collagen matrices containing low hydroxyallysine levels. These data indicate that the type of collagen cross-link influences collagen catabolism. In vivo evidence for the importance of the cross-linking type in determining the reversibility of the fibrotic process was found using the bleomycin-induced skin fibrosis mouse model. The analysis of the accumulated collagen in the fibrotic skin of bleomycin-treated mice did not reveal an increase in hydroxyallysine cross-link levels. In concurrence with our hypothesis, the collagen accumulation resolved in time when the mice were no longer receiving bleomycin treatment, showing the reversibility of the fibrosis. In conclusion, our data indicate that the type of collagen cross-linking is an important factor in determining whether the outcome of the fibrotic process is reversible or not.
Keywords: Fibrosis; Hydroxyallysine-derived cross-link; Collagen accumulation; Collagen degradation; MMP-1;
Oxidative stress in patients with phenylketonuria by L.R. Sirtori; C.S. Dutra-Filho; D. Fitarelli; A. Sitta; A. Haeser; A.G. Barschak; M. Wajner; D.M. Coelho; S. Llesuy; A. Belló-Klein; R. Giugliani; M. Deon; C.R. Vargas (68-73).
Phenylketonuria (PKU) is an autossomal recessive disease caused by phenylalanine-4-hydroxylase deficiency, which is a liver-specific enzyme that catalyzes the hydroxylation of l-phenylalanine (Phe) to l-tyrosine (Tyr). The deficiency of this enzyme leads to the accumulation of Phe in the tissues and plasma of patients. The clinical characterization of this disease is mental retardation and other neurological features. The mechanisms of brain damage are poorly understood. Oxidative stress is observed in some inborn errors of intermediary metabolism owing to the accumulation of toxic metabolites leading to excessive free radical production and may be a result of restricted diets on the antioxidant status. In the present study we evaluated various oxidative stress parameters, namely thiobarbituric acid-reactive species (TBA-RS) and total antioxidant reactivity (TAR) in the plasma of PKU patients. The activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were also measured in erythrocytes from these patients. It was observed that phenylketonuric patients present a significant increase of plasma TBA-RS measurement, indicating a stimulation of lipoperoxidation, as well as a decrease of plasma TAR, reflecting a deficient capacity to rapidly handle an increase of reactive species. The results also showed a decrease of erythrocyte GSH-Px activity. Therefore, it is presumed that oxidative stress is involved in the pathophysiology of the tissue damage found in PKU.
Keywords: Phenylketonuria; Oxidative stress; Free radical;
Islet amyloid polypeptide gene promoter polymorphisms are not associated with Type 2 diabetes or with the severity of islet amyloidosis by Christopher Esapa; Jennifer H. Moffitt; Anna Novials; Catherine M. McNamara; Jonathan C. Levy; Marku Laakso; Ramon Gomis; Anne Clark (74-78).
The over-expression of the islet amyloid polypeptide (IAPP) gene could be a causal factor for islet amyloidosis and β-cell destruction in Type 2 diabetes (T2DM). An IAPP gene promoter polymorphism, IAPP-132G to A, has been associated with T2DM in Spain. To investigate this polymorphism in other cohorts and in relation to therapy, DNA from 425 T2DM and 279 unrelated, non-diabetic UK subjects (ND) and 102 T2DM and 80 ND Finnish subjects was examined. The relationship of amyloid severity (percent amyloid/islet) to prevalence (number of islets affected) and the association of IAPP-132G/A with amyloid was determined in post-mortem pancreas from 38 T2DM subjects. The -132G/A was not associated with T2DM in the UK cohorts (4.5% T2DM; 3.2% ND) or associated with requirement for insulin therapy by 6 years. The mutation was and undetected in the Finnish samples but a new variant, -166T/C, was identified in 2 Finnish T2DM subjects. -132G/A was found in 2/38 diabetic, amyloid-containing and 3/19 ND, amyloid-free subjects. The islet amyloid severity was linearly correlated with the prevalence in T2DM. The IAPP-132G/A promoter polymorphism is not associated with T2DM, a requirement for insulin therapy or with the degree of islet amyloidosis in cohorts from the UK or Finland.
Keywords: Islet amyloid; Type 2 diabetes; Islet; Genetic; Gene promoter; Polymorphism;
Altered expression of iron transport proteins in streptozotocin-induced diabetic rat kidney by D.T. Ward; K Hamilton; R. Burnand; C.P. Smith; D.R. Tomlinson; D. Riccardi (79-84).
Diabetes mellitus is associated with altered iron homeostasis in both human and animal diabetic models. Iron is a metal oxidant capable of generating reactive oxygen species (ROS) and has been postulated to contribute to diabetic nephropathy. Two proteins involved in iron metabolism that are expressed in the kidney are the divalent metal transporter, DMT1 (Slc11a2), and the Transferrin Receptor (TfR). Thus, we investigated whether renal DMT1 or TfR expression is altered in diabetes, as this could potentially affect ROS generation and contribute to diabetic nephropathy. Rats were rendered diabetic with streptozotocin (STZ-diabetes) and renal DMT1 and TfR expression studied using semi-quantitative immunoblotting and immunofluorescence. In STZ-diabetic Sprague–Dawley rats, renal DMT1 expression was significantly reduced and TfR expression increased after 2 weeks. DMT1 downregulation was observed in both proximal tubules and collecting ducts. Renal DMT1 expression was also decreased in Wistar rats following 12 weeks of STZ-diabetes, an effect that was fully corrected by insulin-replacement but not by cotreatment with the aldose reductase inhibitor, sorbinil. Increased renal TfR expression was also observed in STZ-diabetic Wistar rats together with elevated cellular iron accumulation. Together these data demonstrate renal DMT1 downregulation and TfR upregulation in STZ-diabetes. Whilst the consequence of altered DMT1 expression on renal iron handling and oxidant damage remains to be determined, the attenuation of the putative lysosomal iron exit pathway in proximal tubules could potentially explain lysosomal iron accumulation reported in human diabetes and STZ-diabetic animals.
Keywords: Kidney; Iron; Streptozotocin; Divalent metal transporter 1; Transferrin receptor;
Partial characterisation of the human GFAT promoter: Effect of single nucleotide polymorphisms on promoter function by Davina Burt; Katrin Brodbeck; Hans U. Häring; Erwin D. Schleicher; Cora Weigert (85-90).
The 5′-flanking region of the human glutamine:fructose-6-phosphate amidotransferase (GFAT) gene was characterised as a functional active promoter and the GFAT gene contained multiple transcription start sites. A novel single nucleotide polymorphism identified at position −1412 (G to C) had a functional effect on promoter activity and EMSA revealed specific binding of nuclear proteins to this region.
Keywords: GFAT; Promoter; Hexosamine; Single nucleotide polymorphism; Mesangial cell; Transcriptional;