BBA - Molecular Basis of Disease (v.1588, #2)
Editorial Board (ii).
Lack of association between the uncoupling protein-2 Ala55Val gene polymorphism and phenotypic features of the Metabolic Syndrome by Roland Rosmond; Claude Bouchard; Per Björntorp (103-105).
The uncoupling protein (UCP) 2 gene is expressed in adipose tissues and skeletal muscles, which are important sites for variations in energy expenditure. The objective of the current study was to examine the potential impact of a C→T substitution in exon 4, resulting in an alanine to valine substitution at codon 55, on the Metabolic Syndrome in 284 unrelated Swedish men born in 1944. The subjects were genotyped using PCR amplification of the exon 4 region of the UCP2 gene followed by digestion with the restriction enzyme EclHK1. The allelic frequencies were 0.56 for allele Ala and 0.44 for allele Val. No association was found between the Ala55Val SNP and obesity and blood levels of insulin, glucose, and lipids as well as blood pressure and circulating hormones. From these data, we conclude that the C→T substitution in exon 4 of the UCP2 gene does not contribute to the predisposition to be affected by the Metabolic Syndrome.
Keywords: Ala55Val; Obesity; UCP2; SNP; Hormone; Lipid; Blood pressure;
Expression and functional analysis of rat P23, a gut hormone-inducible isoform of trypsin, reveals its resistance to proteinaceous trypsin inhibitors by Shin-Ichi Fukuoka; Cornelio M Nyaruhucha (106-112).
Rat P23 is an isoform of trypsin (ogens) synthesized by rat acinar cells. Expression of P23 is stimulated strongly by caerulein, an analogue of cholecystokinin (CCK). However, the physiological relevance of rat P23 in healthy and pathological conditions such as caerulein-induced pancreatitis is largely unknown. Using recombinant P23 trypsinogen and reconstitution analysis of zymogen autoactivation, unique inhibitor-resistance characteristics of P23 were elucidated. P23 cDNA was expressed in Escherichia coli periplasm, yielding recombinant P23 trypsinogen. Autoactivation of zymogen granule contents from caerulein-induced rat pancreas was also studied. Activation kinetics of P23 by enterokinase was similar to those of rat anionic trypsinogen, which is a major isoform of trypsinogen. Interestingly, rat pancreatic secretory trypsin inhibitor (PSTI), which protects against deleterious activation of trypsinogens in zymogen granules, failed to inhibit P23 trypsin even with four-fold molar excess, at which concentration it effectively inhibited rat anionic trypsin to almost 100%. P23 trypsin also showed marked resistance to proteinaceous trypsin inhibitors such as soybean trypsin inhibitor and aprotinin. P23 trypsin activated by enterokinase dramatically accelerated the cascade of autoactivation of anionic trypsinogen even in the presence of PSTI. Taken together with a previous observation that P23 is specifically upregulated 14-fold by 24-h caerulein infusion, these results suggest that elevated levels of P23 should be taken into consideration in the mechanism of trypsinogens within the pancreas in pathological conditions.
Keywords: Trypsin; Gut; Rat P23;
Protective effect of d-glucose, l-leucine and fetal calf serum against oxidative stress in neonatal pancreatic islets by Luiz F Stoppiglia; Tatiane A Nogueira; Adriana R Leite; Everardo M Carneiro; Antonio C Boschero (113-118).
B-cell destruction during the onset of diabetes mellitus is associated with oxidative stress. In this work, we investigated the mechanisms of defense against oxidative stress present in neonatal islets and their modulation by d-glucose, l-leucine and fetal calf serum (FCS). Culturing neonatal rat islets in the presence of low d-glucose concentrations (2.8–5.6 mmol/l) and 1 mmol/l H2O2 increased the d-glucose uptake by islets sixfold compared to control levels. This effect was dose-dependently inhibited by d-glucose or FCS and by high concentrations of l-leucine. These supplements allowed islets to increase cytoplasmic catalase (CAT) activity only in response to H2O2, with no decrease in NO formation. Although l-leucine increased CAT activity and restored d-glucose uptake, it did not prevent damage to the islets. These data indicate that the most important H2O2 scavenger system in the islets is CAT and that this system can be modulated by metabolic substrates.
Keywords: Neonatal pancreatic islet; d-Glucose; l-Leucine; Fetal calf serum; Oxidative stress; H2O2; Catalase;
Functional characterization of PCCA mutations causing propionic acidemia by Sonia Clavero; Mª Angeles Martı́nez; Belén Pérez; Celia Pérez-Cerdá; Magdalena Ugarte; Lourdes R Desviat (119-125).
Propionic acidemia (PA, MIM 232000 and 232050) is caused by a deficiency of mitochondrial biotin-dependent propionyl-CoA carboxylase (PCC, EC 126.96.36.199), a heteropolymeric enzyme composed of α and β subunits, which are encoded by the PCCA and PCCB genes, respectively. The PCCA protein (α subunit) is responsible for the formation of carboxybiotin upon hydrolysis of ATP and contains a C-terminal biotin-binding domain and a biotin carboxylase domain, defined by homology with other biotin-dependent carboxylases, some of them characterized structurally. More than 24 mutations have been found in the PCCA gene in patients with PA, among them 14 missense mutations and one in-frame deletion, for which the precise molecular effect is unknown. In this study, we have established the pathogenicity of 11 PCCA mutations (10 missense and an in-frame deletion) by expression studies in deficient fibroblasts and in a cell-free in vitro system, and analyzed the effect of each mutation on PCC activity, protein stability and domain structure. The results show that most mutant proteins show an increased turnover and are functionally deficient, suggesting that the structural alterations they cause are incompatible with normal assembly to produce a stable, functional PCC oligomer. These results are discussed in the context of the genotype–phenotype correlations in PCCA-deficient PA patients.
Keywords: Propionic acidemia; PCCA; Mutation; Expression analysis; Genotype–phenotype correlation;
Age-related changes in cartilage endogenous osteogenic protein-1 (OP-1) by Susan Chubinskaya; Bhavna Kumar; Charis Merrihew; Katherine Heretis; David C Rueger; Klaus E Kuettner (126-134).
Articular cartilage has a poor reparative capacity. This feature is exacerbated with aging and during degenerative joint conditions, contributing to loss of motion and impairment of quality of life. This study focused on osteogenic protein-1 (OP-1) and its ability to serve as a repair-stimulating factor in articular cartilage. The purpose of this work was to develop a quantitative method for the assessment of the content of OP-1 protein in extracts from human articular cartilage and to investigate the changes in OP-1 mRNA expression and protein levels with aging of normal adult cartilage. Full thickness cartilage was dissected from femoral condyles of donors with no history of joint disease within 24 h of death. Levels of OP-1 mRNA expression were measured by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method; concentration of OP-1 protein was detected by new sandwich enzyme-linked immunosorbent assay (ELISA); qualitative changes in OP-1 forms were evaluated by Western blots with various anti-OP-1 antibodies. The sensitivity of the ELISA method allowed the detection of picogram quantities of OP-1 in cartilage extracts. We found that (1) concentration of OP-1 in normal cartilage is within the range of biological activity of OP-1 in vitro; and (2) during aging of human adult, articular cartilage, levels of OP-1 protein and message are dramatically reduced (more than 4-fold; p<0.02). The major qualitative changes affected primarily mature OP-1. The results of the current study suggest the possibility that OP-1 may be critical for chondrocytes to maintain their normal homeostasis and could also serve as a repair factor during joint disease or aging.
Keywords: Human articular cartilage; Aging; Bone morphogenetic protein; Osteogenic protein-1; ELISA; PCR;
The peak height ratio of S-sulfonated transthyretin and other oxidized isoforms as a marker for molybdenum cofactor deficiency, measured by electrospray ionization mass spectrometry by Masahiko Kishikawa; Jörn Oliver Sass; Nobuo Sakura; Toyofumi Nakanishi; Akira Shimizu; Masanori Yoshioka (135-138).
Molybdenum cofactor deficiency is a fatal neurological disorder, which follows an autosomal-recessive trait and is characterized by combined deficiency of the enzyme, sulfite oxidase, xanthine dehydrogenase and aldehyde oxidase. Early detection of molybdenum cofactor-deficient patients is essential for their proper care and genetic counseling of families at risk. We demonstrate the use of S-sulfonated transthyretin (TTR) as a marker for molybdenum cofactor deficiency. Plasma or sera obtained from 4 patients with molybdenum cofactor deficiency and 57 controls were studied by electrospray ionization mass spectrometry (ESIMS) following selective enrichment of TTR by immunoprecipitation using protein G/A agarose. The data obtained from molybdenum cofactor deficiency samples indicated a strong increase in the peak height of S-sulfonated TTR. A more significant difference was revealed if the peak height ratio of S-sulfonated TTR and the sum of the other oxidized TTR were determined. By accurate determination of the ratio, the samples of molybdenum cofactor deficiency patients could clearly be distinguished from controls without molybdenum cofactor deficiency.
Keywords: S-sulfonated; Immunoprecipitation; Protein G/A agarose; Mass spectrometry; Posttranslational modification; Molybdenum cofactor deficiency;
Selective inhibition of CYP27A1 and of chenodeoxycholic acid synthesis in cholestatic hamster liver by Yasushi Matsuzaki; Bernard Bouscarel; Tadashi Ikegami; Akira Honda; Mikio Doy; Susan Ceryak; Sugano Fukushima; Shigemasa Yoshida; Junichi Shoda; Naomi Tanaka (139-148).
The aim of this study was to explore the regulation of serum cholic acid (CA)/chenodeoxycholic acid (CDCA) ratio in cholestatic hamster induced by ligation of the common bile duct for 48 h. The serum concentration of total bile acids and CA/CDCA ratio were significantly elevated, and the serum proportion of unconjugated bile acids to total bile acids was reduced in the cholestatic hamster similar to that in patients with obstructive jaundice. The hepatic CA/CDCA ratio increased from 3.6 to 11.0 (P<0.05) along with a 2.9-fold elevation in CA concentration (P<0.05) while the CDCA level remained unchanged. The hepatic mRNA and protein level as well as microsomal activity of the cholesterol 7α-hydroxylase, 7α-hydroxy-4-cholesten-3-one 12α-hydroxylase and 5β-cholestane-3α,7α,12α-triol 25-hydroxylase were not significantly affected in cholestatic hamsters. In contrast, the mitochondrial activity and enzyme mass of the sterol 27-hydroxylase were significantly reduced, while its mRNA levels remained normal in bile duct-ligated hamster. In conclusion, bile acid biosynthetic pathway via mitochondrial sterol 27-hydroxylase was preferentially inhibited in bile duct-ligated hamsters. The suppression of CYP27A1 is, at least in part, responsible for the relative decreased production of CDCA and increased CA/CDCA ratio in the liver, bile and serum of cholestatic hamsters.
Keywords: Bile acid synthesis; Cholestasis; Cholesterol 7α-hydroxylase (CYP7A1); 7α-Hydroxy-4-cholesten-3-one 12α-hydroxylase (CYP8B1); 5β-Cholestane-3α,7α,12α-triol 25-hydroxylase (CYP3A);
The role of oxidative stress in the ochratoxin A-mediated toxicity in proximal tubular cells by G.J Schaaf; S.M Nijmeijer; R.F.M Maas; P Roestenberg; E.M de Groene; J Fink-Gremmels (149-158).
Balkan endemic nephropathy (BEN), a disease characterized by progressive renal fibrosis in human patients, has been associated with exposure to ochratoxin A (OTA). This mycotoxin is a frequent contaminant of human and animal food products, and is toxic to all animal species tested. OTA predominantly affects the kidney and is known to accumulate in the proximal tubule (PT). The induction of oxidative stress is implicated in the toxicity of this mycotoxin.In the present study, primary rat PT cells and LLC-PK1 cells, which express characteristics of the PT, were used to investigate the OTA-mediated oxidative stress response. OTA exposure of these cells resulted in a concentration-dependent elevation of reactive oxygen species (ROS) levels, depletion of cellular glutathione (GSH) levels and an increase in the formation of 8-oxoguanine.The OTA-induced ROS response was significantly reduced following treatment with α-tocopherol (TOCO). However, this chain-braking anti-oxidant did not reduce the cytotoxicity of OTA and was unable to prevent the depletion of total GSH levels in OTA-exposed cells. In contrast, pre-incubation of the cell with N-acetyl-l-cysteine (NAC) completely prevented the OTA-induced increase in ROS levels as well as the formation of 8-oxoguanine and completely protected against the cytotoxicity of OTA. In addition, NAC treatment also limited the GSH depletion in OTA-exposed PT- and LLC-PK1 cells.From these data, we conclude that oxidative stress contributes to the tubular toxicity of OTA. Subsequently, cellular GSH levels play a pivotal role in limiting the short-term toxicity of this mycotoxin in renal tubular cells.
Keywords: 8-Oxoguanine; Nephrotoxicity; Ochratoxin A; Oxidative damage; Proximal tubule cell; Reactive oxygen species;
Time-course of oxygen free radical production in acinar cells during acute pancreatitis induced by pancreatic duct obstruction by A Uruñuela; S Sevillano; A.M de la Mano; M.A Manso; A Orfao; I de Dios (159-164).
The time-course of oxygen free radicals (OFR) generation within acinar cells was studied at different stages of acute pancreatitis (AP) induced in rats by duct obstruction (PDO) for 48 h by flow cytometry, using dihydrorhodamine-123 (DHR) as fluorescent dye. Parallel measurements of the most common markers of oxidative stress such as glutathione (GSH) depletion and malondialdehyde (MDA) levels in pancreas were also performed. OFR production significantly increased within acinar cells at early stages of AP, concomitant with a marked depletion in pancreatic GSH. Lipid peroxidation was significantly enhanced 6 h after PDO, suggesting that the antioxidant defence system of the cell is overwhelmed by OFR production. Both MDA and OFR production in acinar cells decreased to normal values at late AP stages, thus allowing the recovery of pancreatic GSH levels 48 h after PDO. Among the two types of acinar cells differentiated by flow cytometry, R1 and R2, it was the R2 population that showed higher values of DHR dye. However, no differences between the two cell types were found regarding the amount of OFR generation. Our results demonstrate that individual acinar cells significantly contribute to produce large amounts of OFR at early stages of AP. The two existing populations of acinar cells displayed similar behaviour regarding oxidative stress over the course of the disease.
Keywords: Acinar cell; Acute pancreatitis; Oxidative stress; Oxygen free radical; Rat;
Human colon adenocarcinoma is associated with specific post-translational modifications of versican and decorin by Achilleas D Theocharis (165-172).
In this study, the amounts and the fine structural characteristics of versican and decorin present in human colon adenocarcinomas (HCC) were investigated and compared with those in human normal colon (HNC). HCC is characterized by significant increase in the amounts of versican and decorin (13- and 8-fold in terms of protein, respectively). These two proteoglycans (PGs) were the predominant in HCC (86% of total uronic acid). In HNC, versican and decorin contained both chondroitin sulfate/dermatan sulfate chains (CS/DS), with DS to be the predominant one (90–93%). The molecular sizes (M rs) estimated for DS and CS chains were 25–28 and 21–28 kDa, respectively. In CS/DS chains isolated from both versican and decorin, 4-sulfated disaccharides accounted for 79–86% of total disaccharide units, respectively, whereas lower amounts of 6- and non-sulfated units were also recorded. In contrast, the tumor-associated versican and decorin were of smaller hydrodynamic size with lower glycosaminoglycan (GAG) content per PG molecule as compared with those found in HNC. In HCC, both PGs contained mainly CS chains (up to 86%) and the M rs of CS and DS chains were also found to be of smaller size (12 and 16 kDa, respectively). The sulfation patterns of CS/DS chains from both PGs were also significantly different. They were composed mainly of 6-sulfated disaccharides (63–70%), whereas 4-sulfated units accounted for 23–31%. A significant increase in the proportion of non-sulfated disaccharides was also recorded. These findings indicate that the colon adenocarcinoma is characterized by a remarkable increase in the concentration of versican and decorin. Furthermore, these PGs are significantly modified at the post-translational level, i.e. the type, length and the sulfation pattern of their GAG chains. These specific structural alterations of versican and decorin may influence the biology of cancer cells in HCC.
Keywords: Colon carcinoma; Proteoglycan; Versican; Decorin; Glycosaminoglycan; Sulfation pattern;
Effect of imipramine on 1-methyl-4-phenylpyridinium ion-induced hydroxyl radical generation in rat striatum by Toshio Obata; Toru Egashira (173-178).
We examined the effect of imipramine (a tricyclic antidepressant drug) on hydroxyl radical (·OH) generation induced by 1-methyl-4-phenylpyridinium ion (MPP+) in extracellular fluid of rat striatum, using a microdialysis technique. Imipramine enhanced the formation of ·OH trapped as 2,3-dihydroxybenzoic acid (DHBA) induced by MPP+ (5 mM). Introduction of imipramine (0.1, 0.5 and 1.0 mM) dose-dependently increased the level of dopamine (DA) release. Concomitantly, imipramine enhanced DA efflux and the level of DHBA induced by MPP+, as compared with MPP+-treated control. When corresponding experiments were performed with reserpinized rats, there were small increases in the levels of DA and nonsignificant increase in the formation of DHBA. When iron (II) was administered to imipramine (1 mM)-treated animals, a marked elevation of DHBA was observed, compared with MPP+-only treated animals. A positive linear correlation was observed between iron (II) and DHBA (R 2=0.985) in the dialysate. These results indicate that imipramine enhances generation of ·OH induced by MPP+ during enhanced DA overflow.
Keywords: Imipramine; 1-Methyl-4-phenylpyridinium ion (MPP+); Dopamine; Hydroxyl radical; Parkinson's disease;
Differential expression of protein kinase C isoenzymes related to high nitric oxide synthase activity in a T lymphoma cell line by Gabriela Gorelik; Marı́a Laura Barreiro Arcos; Alicia J Klecha; Graciela A Cremaschi (179-188).
Protein kinase C (PKC) is critical for T lymphocyte activation and proliferation, while nitric oxide synthase (NOS) may function both as an activator or inhibitor of T cell apoptosis. Both enzymatic activities were studied in T lymphoma cells in comparison to normal and activated T lymphocytes. Here we show a higher translocation of PKC in BW5147 lymphoma cells than in mitogen-stimulated T lymphocytes. Tumor cells overexpressed PKC ζ isoform, while high levels of the PKC β isotype were found in mitogen-stimulated T lymphocytes. Moreover, tumoral T cells showed high NOS activity, almost undetectable in normal or stimulated T lymphocytes. PKC and NOS inhibitors or the intracellular delivery of an anti-PKC ζ antibody diminished both NO production and proliferation in tumor cells.These results suggest that atypical PKC ζ isoform expression and its association with NOS activity regulation would participate in the multistep process leading to BW5147 cell malignant transformation.
Keywords: NOS activity; PKC activity; PKC isoenzyme; T lymphocyte; T lymphoma cell line; Proliferation;
Correction of the copper transport defect of Menkes patient fibroblasts by expression of two forms of the sheep Wilson ATPase by Paul J Lockhart; Sharon La Fontaine; Stephen D Firth; Mark Greenough; James Camakaris; Julian F.B Mercer (189-194).
The Wilson disease (WD) protein (ATP7B) is a copper-transporting P-type ATPase that is responsible for the efflux of hepatic copper into the bile, a process that is essential for copper homeostasis in mammals. Compared with other mammals, sheep have a variant copper phenotype and do not efficiently excrete copper via the bile, often resulting in excessive copper accumulation in the liver. To investigate the function of sheep ATP7B and its potential role in the copper-accumulation phenotype, cDNAs encoding the two forms of ovine ATP7B were transfected into immortalised fibroblast cell lines derived from a Menkes disease patient and a normal control. Both forms of ATP7B were able to correct the copper-retention phenotype of the Menkes cell line, demonstrating each to be functional copper-transporting molecules and suggesting that the accumulation of copper in the sheep liver is not due to a defect in the copper transport function of either form of sATP7B.
Keywords: Copper metabolism; P-type ATPase; ATP7B;