BBA - Molecular Basis of Disease (v.1588, #3)

Quantitative evaluation of expression of iron-metabolism genes in ceruloplasmin-deficient mice by Kanji Yamamoto; Kunihiro Yoshida; Yuko Miyagoe; Aki Ishikawa; Kazunori Hanaoka; Shozo Nomoto; Kazuma Kaneko; Shu-ichi Ikeda; Shin'ichi Takeda (195-202).
Aceruloplasminemia is an autosomal recessive disorder caused by mutations in the ceruloplasmin (CP) gene, and is characterized by a unique combination of neurovisceral iron overload and iron deficiency anemia. We generated CP-deficient (CP −/−) mice to investigate the functional involvement of CP in iron metabolism. The mice showed a marked iron overload in the liver and mild iron deficiency anemia. We examined the expression of iron-metabolism genes in the duodenum and liver using TaqMan RT-PCR. The divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1), and hephaestin (HEPH) genes were not up-regulated in the duodenum from CP −/− mice. These data suggest that the mechanism of hepatic iron overload in aceruloplasminemia is quite different from that in hemochromatoses and atransferrinemia. In the liver, CP −/− mice showed no increase of gene expression for DMT1 and transferrin receptors (TFR and TFR2), indicating that none of the known pathways of iron uptake is activated in hepatocytes of CP −/− mice. This result supports the hypothesis that CP mainly acts to release iron from cells in the liver.
Keywords: Ceruloplasmin; Iron overload; Divalent metal transporter 1; Ferroportin 1; Hephaestin; Transferrin receptor;

Uptake of recombinant iduronate-2-sulfatase into neuronal and glial cells in vitro by A Daniele; R Tomanin; G.R.D Villani; F Zacchello; M Scarpa; P Di Natale (203-209).
Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is a congenital storage disorder resulting from mutations on the iduronate-2-sulfatase (IDS) gene. The disease shows variable clinical phenotypes from severe to mild with progressive neurological dysfunction. The therapeutic options for treatment of MPS II are limited and currently no specific therapies are available; the problem is further compounded by difficulties in delivering therapeutic agents to the central nervous system (CNS). In this work, as a potential treatment for this disease, the transfer of the recombinant IDS enzyme into brain cells has been studied in vitro. Two different approaches to obtain recombinant IDS have been utilized: production of the recombinant enzyme by a transfected human clone (Bosc 23 cells); production of the recombinant enzyme by adenoviral transduction of neuronal (SK-N-BE) or glial (C6) cells. Our data indicate that the transfected as well as the infected cells produce a large amount of the IDS enzyme, which is efficiently endocytosed into neuronal and glial cells through the mannose 6-phosphate (M6P) receptor system. Somatic gene therapy appears therefore to be suitable to correct IDS deficiency in brain cells.
Keywords: Mucopolysaccharidosis type II; Hunter syndrome; Iduronate-2-sulfatase; Somatic gene therapy; Neuronal and glial cells;

Molecular and functional effects of the T14709C point mutation in the mitochondrial DNA of a patient with maternally inherited diabetes and deafness by D. Perucca-Lostanlen; R.W. Taylor; H. Narbonne; B. Mousson de Camaret; C.M. Hayes; A. Saunieres; V. Paquis-Flucklinger; D.M. Turnbull; B. Vialettes; C. Desnuelle (210-216).
A heteroplasmic T to C transition at nucleotide position 14709 in the mitochondrial tRNA glutamic acid (tRNAGlu) gene has previously been associated with maternally inherited diabetes and deafness (MIDD). To investigate the pathogenic mechanism of the T14709C mutation, we have constructed transmitochondrial cell lines by transferring fibroblasts mitochondria from a patient with the mutation into human cells lacking mitochondrial DNA (mtDNA) (rho° cells). Clonal cybrid cell lines were obtained containing various levels of the heteroplasmic mutation, or exclusively mutated or wild-type mtDNA. Measurement of respiratory chain enzymatic activities failed to detect a difference between the homoplasmic mutant and homoplasmic wild-type cybrid cell lines. However, a subtle decrease in the steady-state levels of tRNAGlu transcripts in some mutant clones. Our studies suggest that the T14709C mutation is insufficient to lead impairment of mitochondrial function in homoplasmic osteosarcoma cybrid clones, and that we cannot exclude that the T14709C mutation affects mitochondrial function by a yet unidentified mechanism.
Keywords: Mitochondrial diabetes; Mitochondrial DNA; Point mutation; Transmitochondrial cybrid;

Ethanol-induced augmentation of annexin IV in cultured cells and the enhancement of cytotoxicity by overexpression of annexin IV by ethanol by Hiroko Ohkawa; Hitoshi Sohma; Rie Sakai; Yoshio Kuroki; Eri Hashimoto; Shinji Murakami; Toshikazu Saito (217-225).
The annexins are a family of highly homologous Ca2+ and phospholipid binding proteins. The expressive amounts of several annexins have been shown to alter in certain pathological states such as brain ischemia and Alzheimer's disease. It has been demonstrated that ethanol induces cytotoxicity, which results in brain damage. In this study, we examined the relationship between ethanol-induced cytotoxicity and the intrinsic amount of annexins using cell lines (rat glioma C6 cells and human adenocarcinoma A549 cells). A decrease in the mitochondrial enzyme (dehydrogenase) activity, which is widely used to measure cytotoxicity, was observed with a high concentration of ethanol (200 mM or more) after a 24-h exposure in both C6 and A549 cells. Western blot analysis revealed that the amount of annexin IV was augmented in both cells by ethanol, whereas levels of annexins I and V were unchanged. The amount of annexin IV was augmented with increasing concentration of ethanol. The overexpression of annexin IV in C6 cells by transfection with annexin IV-DNA enhanced ethanol-induced cell lesion and was accompanied by NFκB activation. Thus, it might be indicated that the amount of annexin IV is selectively augmented and this augmentation facilitates the development of cell lesion by ethanol.
Keywords: Annexin IV; Ethanol; Cytotoxicity; NFκB; Cell line;

Expression and regulation by insulin of low-density lipoprotein receptor-related protein mRNA in human skeletal muscle by Philippe Boucher; Pierre-Henri Ducluzeau; Paul Davelu; Fabrizio Andreelli; Paulette Vallier; Jean-Paul Riou; Martine Laville; Hubert Vidal (226-231).
Evidence suggests that increased hydrolysis and/or uptake of triglyceride-rich lipoprotein particles in skeletal muscle can be involved in insulin resistance. We determined the steady state mRNA levels of the low-density lipoprotein-related receptor (LRP) and lipoprotein lipase (LPL) in skeletal muscle of eight healthy lean control subjects, eight type 2 diabetic patients and eight nondiabetic obese individuals. The regulation by insulin of LRP and LPL mRNA expression was also investigated in biopsies taken before and at the end of a 3 h euglycemic hyperinsulinemic clamp (insulinemia of about 1 nM). LRP mRNA was expressed in human skeletal muscle (1.3±0.1 amol/μg total RNA in control subjects). Type 2 diabetic patients, but not nondiabetic obese subjects, were characterized by a reduced expression of LRP (0.8±0.2 and 1.3±0.3 amol/μg total RNA in diabetic and obese patients, respectively; P<0.05 in diabetic vs. control subjects). Insulin infusion decreased LRP mRNA levels in muscle of the control subjects but not in muscle of type 2 diabetic and nondiabetic obese patients. Similar results were found when investigating the regulation of the expression of LPL. Taken together, these results did not support the hypothesis that a higher capacity for clearance or hydrolysis of circulating triglycerides in skeletal muscle is present during obesity- or type 2 diabetes-associated insulin resistance.
Keywords: Gene expression; Insulin resistance; Lipoprotein lipase; Triglyceride; Hyperinsulinemic clamp; RT-PCR;

Antibacterial peptide PR-39 affects local nitric oxide and preserves tissue oxygenation in the liver during septic shock by Melanie Madhani; Aaron Barchowsky; Linda Klei; Chris R Ross; Simon K Jackson; Harold M Swartz; Philip E James (232-240).
The effects of the antibacterial peptide PR-39 on nitric oxide (NO) and liver oxygenation (pO2) in a mouse model of endotoxaemia have been explored. In vivo electron paramagnetic resonance (EPR) spectroscopy was used to make direct measurements of liver NO and pO2. Measurements of pO2 were made at two different anatomical locations within hepatic tissue to assess effects on blood supply (hence oxygen supply) and lobule oxygenation; selectively from the liver sinusoids or an average pO2 across the liver lobule. PR-39 induced elevated levels of liver NO at 6 h following injection of lipopolysaccharide (LPS) as a result of increased iNOS expression in liver, but had no effect on eNOS or circulatory NO metabolites. Sinusoidal oxygenation was preserved, and pO2 across the hepatic tissue bed improved with PR-39 treatment. We propose that the beneficial effects of PR-39 on liver in this septic model were mediated by increased levels of local NO and preservation of oxygen supply to the liver sinusoids.
Keywords: PR-39; Endotoxaemia; Liver; NO; pO2;

Isolation of differentially expressed genes in human heart tissues by Guifeng Sun; Siu Yuen Chan; Yihua Yuan; Kin Wang Chan; Guangrong Qiu; Kailai Sun; Maurice Ping Leung (241-246).
We applied RNA arbitrarily primed-PCR (RAP-PCR) to screen the genes differentially expressed between common congenital heart defects (CHD) [atrial septal defect, ventricular septal defect, Tetrology of Fallot (TOF)] and normal human heart samples. Three of these differentially amplified fragments matched cDNA sequences coding for proteins of unknown function in humans: hCALO (human homologue of calossin), NP79 (coding for a nuclear protein of 79KD) and SUN2 (Sad-1 unc-84 domain protein 2). The other four fragments were from known human genes: apolipoprotein J, titin, dystrophin and protein kinase C-delta. Northern blot analysis confirmed that all of these genes are expressed in the human heart. The results of RAP-PCR were reconfirmed by quantitative RT-PCR in TOF and control heart samples. Both techniques showed the levels of expression of hCALO, NP79 and SUN2 to be comparable in TOF and control samples and the level of expression of dystrophin and titin, both coding for cytoskeletal proteins, to be significantly upregulated in TOF samples. In summary, we have shown that the RAP-PCR technique is useful in the identification of differentially expressed gene from biopsy samples of human CHD tissues. In this manner, we have identified three novel genes implicated in the normal function of the human heart and two known genes upregulated in TOF samples.
Keywords: RNA arbitrarily primed-PCR; Congenital heart defect; Gene expression; Titin; Dystrophin;

Novel mutations (Asn 484 Lys, Thr 500 Ala, Gly 438 Glu) in Morquio B disease by Richard D Bagshaw; Sunqu Zhang; Alina Hinek; Marie-Anne Skomorowski; Donald Whelan; Joe T.R Clarke; John W Callahan (247-253).
Primary deficiency of β-galactosidase results in GM1 gangliosidosis and Morquio B disease. Of the more than 40 disease-causing mutations described in the Gal gene to date, about 75% are of the missense type and are scattered along the length of the gene. No single, major common mutation has been associated with GM1 gangliosidosis. However, a Trp 273 Leu mutation has been commonly found in the majority of patients with Morquio B disease defined genotypically to date.We now report three new mutations in three Morquio B patients where the Trp 273 Leu mutation is absent. Two of the mutations, C1502G (Asn 484 Lys) and A1548G (Thr 500 Ala), were found in twins (one male, one female) who display a mild form of Morquio B disease and keratan sulfate in the urine. In their fibroblasts, residual activity was 1.9% and 2.1% of controls. On Western blots, the 84-kDa precursor and the 64-kDa mature protein were barely detectable. The occurrence of a 45-kDa degradation product indicates that the mutated protein reached the lysosome but was abnormally processed. In the third case, we identified only a G1363A (Gly 438 Glu) mutation (a major deletion on the second allele has not been ruled out). This female patient too displays a very mild form of the disease with a residual activity of 5.7% of control values. In fibroblasts from this case, the 84-kDa precursor and the 45-kDa degradation product were present, while the mature 64-kDa form was barely detectable. The occurrence of these three mutations in the same area of the protein may define a domain involved in keratan sulfate degradation.
Keywords: β-Galactosidase; Missense mutation; Western blot; Leupeptin;

A regulating element essential for PDGFRA transcription is recognized by neural tube defect-associated PRX homeobox transcription factors by Paul H.L.J Joosten; Mascha Toepoel; Dirk van Oosterhout; Gijs B Afink; Everardus J.J van Zoelen (254-260).
We have previously shown that deregulated expression of the platelet-derived growth factor α-receptor (PDGFRA) can be associated with neural tube defects (NTDs) in both men and mice. In the present study, we have investigated the transcription factors that control the up-regulation of PDGFRA expression during differentiation of early embryonic human cells in culture. In Tera-2 embryonal carcinoma cells, PDGFRA expression is strongly enhanced upon differentiation induced by retinoic acid and cAMP treatment. Here we show that the corresponding increase in promoter activity is controlled by an ATTA-sequence-containing element located near the transcription initiation site, which is bound by a transcriptional complex that includes PBX and PRX homeobox transcription factors. Mutation of the putative binding sites for these transcription factors results in strong impairment of PDGFRA promoter activity in differentiated cells. Since functional inactivation of Prx genes has been associated with NTDs in mice, these data support a model in which improper PDGFRA expression as a result of mutations in or altered binding of its upstream regulators may be causally related to NTDs.
Keywords: Embryonal carcinoma; Differentiation; Spina bifida; Platelet-derived growth factor alpha-receptor; Promoter analysis;

Author Index (261-263).

Cumulative Contents (264-266).