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BBA - Molecular Basis of Disease (v.1762, #4)
Ex vivo biochemical analysis of CFTR in human rectal biopsies by Andrea van Barneveld; Frauke Stanke; Manfred Ballmann; Hassan Y. Naim; Burkhard Tümmler (pp. 393-397).
This report describes the first biosynthetic analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) in freshly excised human rectal biopsies. Expression of functional CFTR was assessed by intestinal current measurement (ICM) prior to biosynthetic studies. Several structural features of CFTR are found to be comparable to those established in CFTR-expressing cell lines. Interestingly, maturation of CFTR increases substantially in tissue incubated at 26 °C. Our data provide a solid basis for future studies on the characterisation of CFTR in pathological cases.
Keywords: CFTR; Metabolic labelling; Immunoblot; Human tissue
Autoantibody biomarker opens a new gateway for cancer diagnosis by M. Nesterova; N. Johnson; C. Cheadle; Y.S. Cho-Chung (pp. 398-403).
The list of cancer markers of current interest has grown considerably, but none of the markers used in clinical work is a true tumor marker. These cancer biomarkers are based on the determination of tumor antigens. Here, we report a single method of autoantibody enzyme immunoassay (EIA) screens for a spectrum of serum tumor markers. A comparison of the autoantibody-based EIA to conventional antigen EIA kits, using receiver operating characteristic (ROC) plots, showed that the autoantibody EIA can significantly enhance the sensitivity and specificity of tumor markers. The detection of serum autoantibodies for a spectrum of serum tumor markers, as demonstrated here, suggests that most, if not all, serum cancer biomarkers produce autoantibodies. A unique autoantibody biomarker screening method, as presented here, might therefore facilitate achieving the accurate and early diagnosis of cancer.
Keywords: Extracellular protein kinase A; Autoantibody; Cancer biomarker; Cancer diagnosis; Enzyme immunoassay
Emerging roles of chloride channels in human diseases by Livia Puljak; Gordan Kilic (pp. 404-413).
In the past decade, there has been remarkable progress in understanding of the roles of Cl− channels in the development of human diseases. Genetic studies in humans have identified mutations in the genes encoding Cl− channels which lead to a loss of Cl− channel activity. These mutations are responsible for the development of a variety of deleterious diseases in muscle, kidney, bone and brain including myotonia congenita, dystrophia myotonica, cystic fibrosis, osteopetrosis and epilepsy. Recent studies indicate that some diseases may develop as a result of Cl− channel activation. There is growing evidence that the progression of glioma in the brain and the growth of the malaria parasite in red blood cells may be mediated through Cl− channel activation. These findings suggest that Cl− channels may be novel targets for the pharmacological treatment of a broad spectrum of diseases. This review discusses the proposed roles of abnormal Cl− channel activity in the pathogenesis of human diseases.
Keywords: Cl; −; channels; Cystic fibrosis; Osteopetrosis; Epilepsy; Glioma; Malaria
Animal models with enhanced erythropoiesis and iron absorption by Gladys O. Latunde-Dada; Andrew T. McKie; Robert J. Simpson (pp. 414-423).
The regulation of iron absorption is of considerable interest in mammals since excretion is minimal. Recent advances in iron metabolism have expounded the molecular mechanisms by which iron absorption is attuned to the physiological demands of the body. The pinnacle was the discovery and identification of hepcidin, a hepatic antimicrobial peptide that regulates absorption to maintain iron homeostasis. While the intricacies of its expression and regulation by HFE, transferrin receptor 2 and hemojuvelin are still speculative, hepcidin responsiveness has correlated negatively with iron absorption in different models and disorders of iron metabolism. Consequently, hepcidin expression is repressed to enhance iron absorption during stimulated erythropoiesis even in situations of elevated iron stores. Animal models have been crucial to the advances in understanding iron metabolism and the present review focuses on phenylhydrazine treated and hypotransferrinaemic rodents. These, respectively, experimental and genetic models of enhanced erythropoiesis highlight the shifting focus of iron absorption regulation from the marrow to the liver.
Keywords: Abbreviations; PHZ; phenylhydrazine; TIBC; total iron binding capacity; EPO; erythropoietin; TFR; transferrin receptor; DMT1; divalent metal transporter (nRamp2, DCT1, SCL11a3); NTBI; non-transferrin bound iron; RE; reticuloendothelial; RBC; red blood cell; Dcytb; duodenal cytochrome b; KO; selective gene knockout; Hjv; Hemojuvelin; trfhpx/hpx; homozygous hypotransferrinaemic mouse; hbd; haemoglobin deficite mice; Hfe; haemochromatosis type 1 gene; USF; upstream stimulatory factorErythropoiesis; Phenylhydrazine-induced anaemia; Hypotransferrinaemic mice; Hepcidin
Serum amyloid A (SAA) activates human mast cells which leads into degradation of SAA and generation of an amyloidogenic SAA fragment by Katri Niemi; Marc H. Baumann; Petri T. Kovanen; Kari K. Eklund (pp. 424-430).
Serum amyloid A (SAA) is a precursor for the amyloid A in AA type of amyloidosis. Distribution of mast cells in tissues is similar to the distribution of amyloid deposits in secondary AA-amyloidosis. Therefore, we studied whether mast cells could be involved in SAA metabolism. Human mast cell line (HMC-1) cells were cultured with recombinant human apoSAA (rhSAA), and the production of tumour necrosis factor (TNF)-α and interleukin (IL)-1β was determined by ELISA. RhSAA and human SAA (huSAA) were incubated with human chymase, tryptase or with intact human mast cell (huMC) in cultures, and degradation of SAA was followed by gel electrophoresis, liquid chromatography and mass spectrometry. SAA induced dose-dependent production of TNF-α and IL-1β in HMC-1 cells. Tryptase, chymase, and huMC granules degraded efficiently the SAA protein. Degradation of SAA by tryptase, but not by chymase, released a highly amyloidogenic N-terminal fragment of SAA. Finally, incubation of huMC with rhSAA alone resulted in degradation of SAA and formation of protofibrillar intermediates. These results suggest a pathogenic role for mast cells in AA-amyloidosis.
Keywords: Serum amyloid A; AA-amyloidosis; Amyloid fibril formation; HMC-1; Mast cell proteases
Keratoconus: Matrix metalloproteinase-2 activation and TIMP modulation by V.A. Smith; F.J. Matthews; M.A. Majid; S.D. Cook (pp. 431-439).
Keratoconus is an ocular condition that causes corneal thinning, cone formation and scarring. In view of a hypothesis that activated MMP-2 may initiate or facilitate disease progression, the MMP-2/TIMP systems of stromal cells derived from normal and keratoconic corneas have been compared. To achieve this, stromal cell cultures were established from normal, clear keratoconic (KCS-1) and scarred keratoconic (KCS-2) corneas. The secreted MMP-2 was assayed using [3H]Type IV collagen and analysed by zymography. Optimally maintained and nutrient deprived cells were subsequently incubated with [3H]lysine. The secreted radiolabelled macromolecules were separated and quantified. The results obtained indicated that optimally maintained KCS-1 stromal cells produced more MMP-2 than normal stromal cells but not TIMP. Nutrient deprivation induced MMP-2 activation and cell death. Surviving cells upregulated TIMP-1 synthesis and in this respect became similar to the KCS-2 stromal cells that did not excessively generate activated MMP-2 or die as a consequence of nutrient deprivation. From these results, it was concluded that KCS-1 stromal cells over-expressed MMP-2 without increasing TIMP production. This may facilitate MMP-2 activation in vivo and hence advance the keratoconic condition. KCS-2 cultures over-expressed both MMP-2 and TIMP-1. Because TIMP-1 inhibits MMP-2 activity and protects against cell death it may be of significance in initiating repair processes and curtailing keratoconus.
Keywords: Matrix metalloproteinases (MMPs); Tissue inhibitors of matrix metalloproteinases (TIMPs); Cornea; Keratoconus; Keratocytes
Genetic analysis of the diabetes-prone C57BLKS/J mouse strain reveals genetic contribution from multiple strains by Hui Z. Mao; Evanthia T. Roussos; Miklós Péterfy (pp. 440-446).
The C57BLKS/J (BKS) inbred mouse strain is a widely used animal model of type 2 diabetes. In the presence of the diabetes ( db) mutation, obese BKS- db mice develop severe diabetes. Genetic studies of diabetes-susceptibility in this strain are facilitated by the fact that BKS is a genetic composite between the diabetes-resistant C57BL/6J (B6) and susceptible DBA/2J (DBA) strains. On this basis, it has been hypothesized that diabetes-susceptibility in BKS is conferred by DBA-derived alleles. However, recent studies revealed non-B6/non-DBA genetic material in BKS. To identify the origin of this genetic component, we generated a genomic map of BKS using 537 microsatellite markers. Our results demonstrate that, in addition to B6 and DBA, BKS contains alleles from at least three other strains, including 129, C57BL/10 and an unidentified mouse strain. We also analyzed two congenic strains, B6- db and BKS- db, which are widely used for the genetic mapping of diabetes-susceptibility loci. We identified several donor-derived genomic regions introduced during the generation of these congenic strains. In summary, our study reveals novel aspects of the genetic fine-structure of BKS and related strains and facilitates the identification of diabetes-susceptibility loci in this mouse model.
Keywords: Abbreviations; B6; C57BL/6J; B10; C57BL/10J; BKS; C57BLKS/J; Chr; chromosome; db; diabetes; mutation; QTL; quantitative trait locus; SNP; single nucleotide polymorphismType 2 diabetes mellitus; Animal model; C57BLKS mouse strain; Leptin receptor mutation; Microsatellite marker
Luteinizing hormone modulates cognition and amyloid-β deposition in Alzheimer APP transgenic mice by Gemma Casadesus; Kate M. Webber; Craig S. Atwood; Miguel A. Pappolla; George Perry; Richard L. Bowen; Mark A. Smith (pp. 447-452).
Until recently, the study of hormonal influences in Alzheimer disease was limited to the role of sex steroids. Despite numerous epidemiological studies supporting a protective role for estrogen in Alzheimer disease, recent studies show that estrogen administration in elderly women increases the risk of disease. Reconciling these contradictory reports, we previously hypothesized that other hormones of the hypothalamic–pituitary–gonadal axis, such as luteinizing hormone, may be involved in the onset and development of the disease. In this regard, luteinizing hormone is elevated in Alzheimer disease and is known to modulate amyloidogenic processing of amyloid-β protein precursor. Therefore, in this study, to evaluate the therapeutic potential of luteinizing hormone ablation, we administered a gonadotropin-releasing hormone analogue, leuprolide acetate, to an aged transgenic mouse model of Alzheimer disease (Tg 2576) and measured cognitive Y-maze performance and amyloid-β deposition after 3 months of treatment. Our data indicate that luteinizing hormone ablation significantly attenuated cognitive decline and decreased amyloid-β deposition as compared to placebo-treated animals. Importantly, leuprolide acetate-mediated reduction of amyloid-β correlated with improved cognition. Since both cognitive loss and amyloid-β deposition are features of Alzheimer disease, leuprolide acetate treatment may prove to be a useful therapeutic strategy for this disease.
Keywords: Alzheimer disease; Amyloid-β; Cognition; Hippocampal function; Luteinizing hormone; Therapeutics
The lack of effect of glucosamine sulphate on aggrecan mRNA expression and35S-sulphate incorporation in bovine primary chondrocytes by Cheng-Juan Qu; Hannu M. Karjalainen; Heikki J. Helminen; Mikko J. Lammi (pp. 453-459).
Glucosamine and glucosamine sulphate have been promoted as a disease-modifying agent to improve the clinical symptoms of osteoarthritis. The precise mechanism of the action of the suggested positive effect of glucosamine or glucosamine sulphate on cartilage proteoglycans is not known, since the level of glucosamine in plasma remains very low after oral administration of glucosamine sulphate. We examined whether exogenous hexosamines or their sulphated forms would increase steady-state levels of aggrecan and hyaluronan synthase (HAS) or glycosaminoglycan synthesis using Northern blot and35S-sulphate incorporation analyses. Total RNA was extracted from bovine primary chondrocytes which were cultured either in 1 mM concentration of glucosamine, galactosamine, mannosamine, glucosamine 3-sulphate, glucosamine 6-sulphate or galactosamine 6-sulphate for 0, 4, 8 and 24 h, or in three different concentrations (control, 100 μM and 1 mM) of glucosamine sulphate salt or glucose for 24 or 72 h. Northern blot assay showed that neither hexosamines nor glucosamine sulphate salt stimulated aggrecan and HAS-2 mRNA expression. Glycosaminoglycan synthesis remained at a control level in the treated cultures, with the exception of mannosamine which inhibited35S-sulphate incorporation in low-glucose DMEM treatment. In our culture conditions, hexosamines or their sulphated forms did not increase aggrecan expression or35S-sulphate incorporation.
Keywords: Osteoarthritis; Glucosamine sulphate; Bovine; Proteoglycan
Brain-specific change in alternative splicing of Tau exon 6 in myotonic dystrophy type 1 by Olivier Leroy; Junning Wang; Claude-Alain Maurage; Michel Parent; Thomas Cooper; Luc Buée; Nicolas Sergeant; Athena Andreadis; Marie-Laure Caillet-Boudin (pp. 460-467).
Alternative splicing is altered in myotonic dystrophy of type 1 (DM1), a syndrome caused by an increase of CTG triplet repeats in the 3′ untranslated region of the myotonic dystrophy protein kinase gene. Previously, we reported the preferential skipping of Tau exon 2 in DM1 brains. In this study, we analyze the alternative splicing of Tau exon 6 which can be inserted in three different forms (c, p and d) depending on the 3′ splice site used. In fact, inclusion of exon 6c decreases in DM1 brains compared to control brains whereas inclusion of 6d increases. Alteration of exon 6 splicing was not observed in DM1 muscle although this exon was inserted in RNAs from normal muscle and DM1 splicing alterations were first described in this organ. In contrast, alteration of exon 2 of Tau mRNA was observed in both muscle and brain. However, co-transfections of a minigene containing exon 6 with CELF or MBNL1 cDNAs, two splicing factor families suspected to be involved in DM1, showed that they influence exon 6 splicing. Altogether, these results show the importance of determining all the exons and organs targeted by mis-splicing to determine the dysregulation mechanisms of mis-splicing in DM1.
Keywords: Tauopathy; Myotonic dystrophy of type 1; Microtubule-associated Tau; Alternative splicing; Muscle; Brain
Lipopolysaccharide-induced liver apoptosis is increased in interleukin-10 knockout mice by Jian Zhong; Ion V. Deaciuc; Ravshan Burikhanov; Willem J.S. de Villiers (pp. 468-477).
Although IL-10 down-regulates pro-inflammatory cytokine secretion by hepatic Kupffer cells, the mechanisms underlying its hepatoprotective effects are not fully clear. This study tested the hypothesis that IL-10 protects the liver against pro-inflammatory cytokines by counteracting their pro-apoptotic effects. Wild type and IL-10 knockout mice were treated with bacterial lipopolysaccharide and sacrificed 1, 4, 8, and 12 h later. Plasma ALT activity was measured as a marker of liver injury. Liver pathology and TUNEL response were assessed by histology. Plasma levels and whole liver mRNA levels were measured for TNF-α, IL-1β, TGF-β1, IL-10, and their respective receptors. Hepatic mRNA levels were measured for several pro-apoptotic adaptors/regulators, including FasL, Fas receptor, FADD, TRADD, Bad, Bak, Bax, and Bcl-XS, and anti-apoptotic regulators, including Bcl-w, Bcl-XL, Bcl-2, and Bfl-1. Caspase-3 activity in the liver was determined as well as immunohistochemistry for IL-1RII, TGF-βRII and Fas receptor. At all time points the livers from IL-10 knockout mice displayed a significantly increased number of apoptotic nuclei compared to wild type mice. Changes in plasma cytokine levels and their liver mRNA levels were consistent with suppression by IL-10 of pro-inflammatory cytokine secretion. In addition, pro-inflammatory cytokine receptor mRNA levels (TNF-α, TGF-β, and IL-1β) were markedly up-regulated by LPS at all time points in IL-10 knockout mice as compared to wild type mice. Expression of the pro-inflammatory cytokine receptor IL-1RII was similarly increased as shown by immunostaining. The mRNA levels of a typical pro-apoptotic cytokine, TRAIL, were increased and LPS also up-regulated the mRNA expression of other apoptotic factors to a larger extent in IL-10 knockout mice than in their wild type counterparts, suggestive of an IL-10 anti-apoptotic effect. In the livers of knockout mice, markedly increased caspase-3 activity was already evident at the 1-h time point following LPS administration, while in the wild type animals this increase was delayed. Immunostaining also indicated that LPS increased hepatic expression of the pro-apoptotic receptors Fas and TGF-βRII in IL-10 knockout mice. The data presented in this study show that: (i) IL-10 modulates not only the secretion of pro-inflammatory cytokines, but also the receptors of these cytokines, and ii) IL-10 protects the liver against LPS-induced injury at least in part by counteracting pro-inflammatory cytokine-induced liver apoptosis.
Keywords: Interleukin-10; Liver; Apoptosis; Lipopolysaccharide; Kupffer cell
Identification of nine new IDS alleles in mucopolysaccharidosis II. Quantitative evaluation by real-time RT-PCR of mRNAs sensitive to nonsense-mediated and nonstop decay mechanisms by Susanna Lualdi; Maja Di Rocco; Fabio Corsolini; Marco Spada; Bruno Bembi; Giovanna Cotugno; Roberta Battini; Marina Stroppiano; Maria Gabriela Pittis; Mirella Filocamo (pp. 478-484).
The present study aimed to characterize mutant alleles in Mucopolysaccharidosis II and evaluate possible reduction of mRNA amount consequent to nonsense-mediated or nonstop mRNA decay pathways. A combination of different approaches, including real-time RT-PCR, were used to molecularly characterize seventeen patients. Fifteen alleles were identified and nine of them were new. The novel alleles consisted of three missense mutations (p.S71R, p.P197R, p.C432R), two nonsense (p.Q66X, p.L359X), two frameshifts (p.V136fs75X, p.C432fs8X), one allele carrying two in-cis mutations [p.D252N;p.S369X], and a large deletion (p.G394_X551). Analysing these results it emerged that most of the alterations resulted in mutants leading to mRNAs with premature termination codons, and therefore, potentially sensitive to mRNA surveillance pathway. By using real-time RT-PCR, the mRNAs resulting (i) from substitutions that changed one amino acid to a stop codon (L359X, and S369X), or caused the shifted reading frame with premature introduction of a stop codon (C432fs8X), (ii) from large deletion (p.G394_X551) that included the termination codon, seemed to be subject to degradation by nonsense-mediated (i) or nonstop decay (ii) mechanisms, as mRNA was strongly underexpressed. On the contrary, two mutations (Q66X and V136fs75X) produced transcripts evading mRNA surveillance pathway despite both of them fulfilled the known criteria. These results confirm the wide variability of the mRNA expression levels previously reported and represent a further exception to the rules governing susceptibility to nonsense-mediated decay. A close examination of the molecular basis of the disease is becoming increasingly important for optimising the choices of available or forthcoming therapies such as, enzyme replacement therapy or enzyme enhancement therapy.
Keywords: Hunter; Mucopolysaccharidosis; IDS; real-time RT-PCR; Genotype–phenotype correlation; mRNA decay
Correction of a mouse model of Menkes disease by the human Menkes gene by Roxana M. Llanos; Bi-Xia Ke; Magali Wright; Yolanda Deal; Francois Monty; David R. Kramer; Julian F.B. Mercer (pp. 485-493).
The brindled mouse is an accurate model of the fatal human X-linked copper deficiency disorder, Menkes disease. Males carrying the mutant allele of the Menkes gene orthologue Atp7a die in the second week of life. To determine whether the genetic defect in the brindled mice could be corrected by expression of the human Menkes gene, male transgenic mice expressing ATP7A from the chicken β-actin composite promoter (CAG) were mated with female carriers of the brindled mutation ( Atp7a Mo-br). Mutant males carrying the transgene survived and were fertile but the copper defect was not completely corrected. Unexpectedly males corrected with one transgenic line (T25#5) were mottled and resembled carrier females, this effect appeared to be caused by mosaic expression of the transgene. In contrast, males corrected with another line (T22#2) had agouti coats. Copper concentrations in tissues of the rescued mutants also resembled those of the heterozygous females, with high levels in kidney (84.6±4.9 μg/g in corrected males vs. 137.0±44.3 μg/g in heterozygotes) and small intestine (15.6±2.5 μg/g in corrected males vs. 15.7±2.8 μg/g in heterozygotes). The results show that the Menkes defect in mice is corrected by the human Menkes gene and that adequate correction is obtained even when the transgene expression does not match that of the endogenous gene.
Keywords: Gene correction; Menkes disease; Copper-transporting ATPase; Menkes protein; Brindled mutant mouse; CAG-promoter
Increased guanidino species in murine and human succinate semialdehyde dehydrogenase (SSADH) deficiency by Erwin E.W. Jansen; Nanda M. Verhoeven; Cornelis Jakobs; Andreas Schulze; Henry Senephansiri; Maneesh Gupta; O. Carter Snead; K. Michael Gibson (pp. 494-498).
Mice with targeted deletion of the GABA-degradative enzyme succinate semialdehyde dehydrogenase (SSADH; Aldh5a1; OMIM 271980) manifest globally elevated GABA and regionally decreased arginine in brain extracts. We examined the hypothesis that arginine–glycine amidinotransferase catalyzed the formation of guanidinobutyrate (GB) from increased GABA by quantifying guanidinoacetate (GA), guanidinopropionate (GP) and GB in brain extracts employing stable isotope dilution gas chromatographic-mass spectrometry. GA and GB were up to 4- and 22-fold elevated, respectively, in total and regional (cerebellum, hippocampus, cortex) brain extracts derived from SSADH−/− mice. Corresponding analyses of urine and cerebrospinal fluid derived from SSADH-deficient patients revealed significant ( P<0.05) elevations of GA and GB in urine, as well as GB levels in CSF. These data suggest that GB may be an additional marker of SSADH deficiency, implicate additional pathways of pathophysiology, and identify the second instance of elevated GB in a human inborn error of metabolism.
Keywords: Succinate semialdehyde dehydrogenase (SSADH) deficiency; Gamma-aminobutyrate (GABA); Arginine; Guanidinoacetate; Guanidinobutyrate; Creatine