BBA - Molecular Basis of Disease (v.1689, #3)

Interaction of ACE2 and integrin β1 in failing human heart by Qishan Lin; Rebecca S. Keller; Barbara Weaver; Lawrence S. Zisman (175-178).
ACE2 purified from failing human heart was found to form a complex with integrin β1 by immunoprecipitation, Western blotting, activity assay, and ESI tandem mass spectroscopy. The ACE2/integrin complex showed a K m of 6.8 μM and a V max of 2.13 pmol/min/μl purified enzyme. Activity was optimal at pH 7.5 with Ang II substrate.
Keywords: Angiotensin-converting enzyme 2; Angiotensin-(1–7); Heart Failure; Cardiomyopathy; Integrin β1;

Cytosolic carbonic anhydrase activity in chronic myeloid disorders with different clinical phenotype by Giuseppe Bonapace; Francesco Iuliano; Stefano Molica; Antonio Peta; Pietro Strisciuglio (179-181).
Carbonic anhydrase family (CAs) plays an important role in the extracellular acidification and several studies suggest a possible involvement of such enzymes in the increased tumor progression due to the acidic extracellular pH. We measured the activities of carbonic anhydrase I and II isoforms in a group of patients affected by four specific chronic haematological diseases, sharing a common origin but characterized by a different neoplastic evolution: agnogenic myeloid metaplasia (AMM), essential thrombocythemia (ET), chronic myeloid leukemia (CML) and polycythemia vera (PV) in order to understand the correlation between CAs activities and neoplastic outcome. In comparison to controls, our data demonstrate an increase of CAI and CAII activities in all our patients with a specific increase of the CAI activity in the group of the diseases with major malignancy (CML and AMM). These results suggest a possible role of such isozymes in the progression of the myeloid disorders and CAs specific inhibitors should be useful in slowing the progression of the disease.
Keywords: Carbonic anhydrase; Myeloid; Phenotype;

Oxidant stress in type I autoimmune hepatitis: the link between necroinflammation and fibrogenesis? by Philip W. Pemberton; Ali Aboutwerat; Alexander Smith; Peter C. Burrows; Raymond F.T. McMahon; Thomas W. Warnes (182-189).
Autoimmune hepatitis (AIH) is a chronic liver disease of unknown aetiology characterized by circulating autoantibodies, hyperglobulinaemia and interface hepatitis. The mechanisms of progression from initial autoimmune attack to fibrosis and cirrhosis are unclear but oxidant stress may be involved. Markers of lipid peroxidation, antioxidant status, hepatic fibrogenesis and liver function were measured in blood and urine in 35 controls and in 33 patients with type-1 AIH; histology was assessed in 18 patients. In AIH, markers of lipid peroxidation were significantly elevated (8-isoprostane in both plasma and urine P<0.001; plasma malondialdehyde P=0.017). Total antioxidant capacity in protein-free serum and total glutathione in both whole blood and plasma were significantly reduced (P=0.007, P=0.037, P<0.001, respectively). The antioxidants selenium, vitamin A and vitamin E were significantly decreased (P=0.007, P<0.001, P=0.025, respectively); vitamin C was unchanged. Urinary 8-isoprostane correlated positively with interface hepatitis and necroinflammatory score and with hepatic fibrogenesis (type III procollagen peptide). Interface hepatitis correlated negatively with vitamin A and whole blood total glutathione. Oxidant stress, as reflected in blood and urine by a wide range of pro- and antioxidant markers, is a significant feature of AIH and provides a probable mechanism linking hepatic necroinflammation to fibrogenesis and disease progression.
Keywords: Oxidant stress; Autoimmune hepatitis; Lipid peroxidation; Antioxidant; 8-Isoprostane; Cirrhosis;

In the present study, the role of finger millet feeding on skin antioxidant status, nerve growth factor (NGF) production and wound healing parameters in healing impaired early diabetic rats is reported. Hyperglycemic rats received food containing 50 g/100 g finger millet (FM). Non-diabetic controls and diabetic controls received balanced nutritive diet. Full-thickness excision skin wounds were made after 2 weeks prior feeding of finger millet diet. The rate of wound contraction, and the levels of collagen, hexosamine and uronic acid in the granulation tissue were determined. The skin antioxidant status and lipid peroxide concentration were also monitored during the study. In hyperglycemic rats fed with finger millet diet, the healing process was hastened with an increased rate of wound contraction. Skin levels of glutathione (GSH), ascorbic acid and  α-tocopherol in alloxan-induced diabetic rat were lower as compared to non-diabetics. Altered activities of superoxide dismutase (SOD) and catalase (CAT) were also recorded in diabetics. Interestingly, thiobarbituric acid reactive substances (TBARS) were elevated in the wound tissues of all the groups, when compared to normal (unwounded) skin tissues. However, in diabetic rats the TBARS levels of both normal and wounded skin tissues were significantly elevated (P<0.001) when compared with control (non-diabetic) and diabetics fed with FM. Impaired production of NGF, determined by ELISA, in diabetic rats was improved upon FM feeding and further confirmed by immunocytochemical observations reflects the increased expression of NGF in hyperglycemic rats supplemented with FM-enriched diet. Histological and electron microscopical evaluations revealed the epithelialization, increased synthesis of collagen, activation of fibroblasts and mast cells in FM-fed animals. Thus, increased levels of oxidative stress markers accompanied by decreased levels of antioxidants play a vital role in delaying wound healing in diabetic rats. However, FM feeding to the diabetic animals, for 4 weeks, controlled the glucose levels and improved the antioxidant status, which hastened the dermal wound healing process.
Keywords: Finger millet; Wound healing; Diabetic;

Involvement of caspase-10 in advanced glycation end-product-induced apoptosis of bovine retinal pericytes in culture by Marc Lecomte; Ulriche Denis; Daniel Ruggiero; Michel Lagarde; Nicolas Wiernsperger (202-211).
Apoptosis appears to be the death mechanism of pericyte loss observed in diabetic retinopathy. We have previously shown that advanced glycation end-products (AGE-MGX) induce apoptosis of retinal pericytes in culture associated with diacylglycerol (DAG)/ceramide production. In the present study, we investigated possible caspase involvement in this process. Bovine retinal pericytes (BRP) were cultured with AGE-MGX and apoptosis examined after annexin V staining. Effects of peptidic inhibitors of caspases were determined on DAG/ceramide production and apoptosis. Pan-caspase inhibitor z-VAD-fmk (50 μM) was able to inhibit both DAG/ceramide production and apoptosis, whereas caspase-3-like inhibitor z-DEVD-fmk (50 μM) or caspase-9 inhibitor z-LEHD-fmk (50 μM) was only active on apoptosis. This differential effect strongly suggests involvement of initiator caspase(s) upstream and effector caspase(s) downstream DAG/ceramide production in AGE-mediated apoptosis. Pericyte treatment with caspase-8 inhibitor z-IETD-fmk (50 μM) did not protect cells against AGE-induced apoptosis and we failed to detect caspase-8 in pericytes by immunoblotting assay. Interestingly, one inhibitor of caspase-10 and related caspases z-AEVD-fmk (50 μM) inhibited both AGE-MGX-induced apoptosis and DAG/ceramide formation in pericytes. Cleavage of caspase-10 precursor into its active subunits was demonstrated by immunoblotting assay in pericytes incubated with AGE-MGX. These results strongly suggest that caspase-10, but not caspase-8, might be involved in the early phase of AGE-induced pericyte apoptosis, in contrast to caspase-9 and -3-like enzymes involved after DAG/ceramide production. This finding may provide new therapeutic perspectives for early treatment in diabetic retinopathy.
Keywords: Advanced glycation end-product; Apoptosis; Caspase-10; Pericyte; Diabetic retinopathy;

Studies have shown that increased gut-derived norepinephrine (NE) release plays an important role in producing hepatocellular dysfunction at the early stage of sepsis. Although the gut has been demonstrated to be the major source of NE in sepsis, it remains unknown whether the increased NE is associated with up-regulation of intestinal NE biosynthesis enzymes such as tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH). To determine this, adult male rats were subjected to sepsis by cecal ligation and puncture (CLP) followed by fluid resuscitation. Small intestinal samples were harvested at 2 h (i.e., early sepsis) or 20 h (late sepsis) after CLP or sham-operation. Protein levels of TH and DBH were determined by Western blot analysis and immunohistochemistry. Their gene expression was assessed by RT-PCR technique. The results indicate that intestinal TH protein levels increased significantly at 2 and 20 h after CLP, while DBH was not altered under such conditions. Immunohistochemical examination shows that both TH and DBH were located in intestinal sympathetic nerve fibers and TH staining was markedly increased in septic animals. TH gene expression increased significantly at 2 h but not at 20 h after CLP, while DBH gene expression was not altered in sepsis. Thus, the increased TH gene and protein expression appears to be responsible for the increased gut-derived NE in sepsis.
Keywords: Catecholamines; Small intestine; Tyrosine hydroxylase; Dopamine β-hydroxylase; Sympathetic nerve; Cecal ligation and puncture;

N-Octyl-β-valienamine up-regulates activity of F213I mutant β-glucosidase in cultured cells: a potential chemical chaperone therapy for Gaucher disease by Hou Lin; Yuko Sugimoto; Yuki Ohsaki; Haruaki Ninomiya; Akira Oka; Miyako Taniguchi; Hiroyuki Ida; Yoshikatsu Eto; Seiichiro Ogawa; Yuji Matsuzaki; Miwa Sawa; Takehiko Inoue; Katsumi Higaki; Eiji Nanba; Kousaku Ohno; Yoshiyuki Suzuki (219-228).
Gaucher disease (GD) is the most common form of sphingolipidosis and is caused by a defect of β-glucosidase (β-Glu). A carbohydrate mimic N-octyl-β-valienamine (NOV) is an inhibitor of β-Glu. When applied to cultured GD fibroblasts with F213I β-Glu mutation, NOV increased the protein level of the mutant enzyme and up-regulated cellular enzyme activity. The maximum effect of NOV was observed in F213I homozygous cells in which NOV treatment at 30 μM for 4 days caused a ∼6-fold increase in the enzyme activity, up to ∼80% of the activity in control cells. NOV was not effective in cells with other β-Glu mutations, N370S, L444P, 84CG and RecNciI. Immunofluorescence and cell fractionation showed localization of the F213I mutant enzyme in the lysosomes of NOV-treated cells. Consistent with this, NOV restored clearance of 14C-labeled glucosylceramide in F213I homozygous cells. F213I mutant β-Glu rapidly lost its activity at neutral pH in vitro and this pH-dependent loss of activity was attenuated by NOV. These results suggest that NOV works as a chemical chaperone to accelerate transport and maturation of F213I mutant β-Glu and may suggest a therapeutic value of this compound for GD.
Keywords: Gaucher disease; β-glucosidase; Valienamine; Glucosylceramide; Chaperone;

Inactivation and intracellular retention of the human I183N mutated melanocortin 3 receptor associated with obesity by Mohamed Rached; Anna Buronfosse; Martine Begeot; Armelle Penhoat (229-234).
Melanocortins are known to be involved in the regulation of feeding behavior. These hormones mediate their effects through G protein-coupled receptors (GPCRs) by stimulating adenylate cyclase. The melanocortin 3 receptor (MC3R) in the melanocortin receptor (MCR) family has been identified as a neural receptor subtype mainly expressed in the brain in mammals. Until now, only one heterozygous mutation (I183N) has been identified in the coding region of this receptor in two obese patients of the same family. In this study, we reported the functional characterization of the I183N mutated MC3R compared with that of the wild-type MC3R after transfection in HEK293 cells.Our results showed that the I183N mutation totally abolished the activity of the mutated receptor to generate intracellular cAMP. Furthermore, confocal microscopy observation revealed that the mutation induced an intracellular retention of the mutated receptor. Moreover, we demonstrated for the first time by co-transfection studies that the mutated receptor could reduce the wild-type receptor activity through a dominant negative effect.
Keywords: Melanocortin-3 receptor; Obesity; Mutation; Dominant negative effect;

Mouse liver PMP70 and ALDP: homomeric interactions prevail in vivo by Carla P. Guimarães; Pedro Domingues; Patrick Aubourg; Françoise Fouquet; Aurora Pujol; Gerardo Jimenez-Sanchez; Clara Sá-Miranda; Jorge E. Azevedo (235-243).
ALDP, ALDPR, PMP70 and PMP70R are half ATP-binding cassette (ABC) transporters of the mammalian peroxisomal membrane. By analogy with other members of this family, it is assumed that peroxisomal ABC transporters must dimerize to become functional units. However, not much is known regarding the type of dimers (i.e., homodimers and/or heterodimers) that are formed in vivo under normal expression conditions. In this work, we have characterized the quaternary structure of mouse liver PMP70 and ALDP. The PMP70 protein complex was purified to apparent homogeneity using a two-step purification protocol. The ALDP-containing protein complex was characterized by preparative immunoprecipitation experiments. In both cases, no evidence for the existence of heteromeric interactions or for the presence of accessory proteins in these ABC transporter protein complexes could be obtained. Our data indicate that the majority (if not all) of mouse liver PMP70 and ALDP are homomeric proteins.
Keywords: Half ABC transporter; Peroxisome; Adrenoleukodystrophy; X-ALD; Membrane protein purification;

Apolipoprotein E isoprotein-specific interactions with tissue plasminogen activator by Susan J. Biehle; Janice Carrozzella; Rakesh Shukla; Jonathan Popplewell; Marcus Swann; Neville Freeman; Joseph F. Clark (244-251).
Apolipoprotein E (Apo E) is an important genetic risk factor for multiple neurological, vascular and cardiovascular diseases. Previously, we reported Apo E isoprotein-specific modulation of tissue plasminogen activator (tPA) using an in vitro blood-clotting assay. Here, we studied the conformational changes of Apo E2, E3 and E4 in the presence of tPA and vice versa using circular dichroism (CD) and dual polarization interferometry (DPI). We report isoprotein and state-specific intermolecular interactions between the Apo E isoforms and tPA. Apo E2 interaction with immobilized tPA leads to significant conformational changes which are not observed with Apo E3 or E4. Additionally, tPA induces changes in helicity of lipidated Apo E2 whereas no detectible changes were observed in Apo E3 or E4. The Tukey's test for interaction indicated a significant (P<0.001) interaction between tPA and Apo E2 in the lipidated environment. These results may be important regarding the mechanism by which Apo E has isoprotein-specific effects on many biological processes and diseases involving blood clotting, proteolysis and perfusion.
Keywords: Apolipoprotein E; Tissue plasminogen activator; Thrombolysis; Protein–protein interaction; Mechanism; Dual polarization interferometry;

Luminal Ca2+-binding proteins play a central role in mediating between Ca2+-uptake and Ca2+-release during the excitation–contraction–relaxation cycle in muscle fibres. In the most commonly inherited neuromuscular disorder, Duchenne muscular dystrophy (DMD), the reduced expression of key Ca2+-binding proteins causes abnormal Ca2+-buffering in the sarcoplasmic reticulum (SR) of skeletal muscle. The heart is also affected in dystrophinopathies, as manifested by the pathological replacement of cardiac fibres by connective and fatty tissue. We therefore investigated whether similar changes occur in the abundance of luminal Ca2+-regulatory elements in dystrophin-deficient cardiac fibres. Two-dimensional immunoblotting of total cardiac extracts was employed to unequivocally determine potential changes in the expression levels of SR components. Interestingly, the expression of the histidine-rich Ca2+-binding protein was increased in the dystrophic heart. In contrast, the major Ca2+-reservoir protein of the terminal cisternae, calsequestrin (CSQ), and the Ca2+-shuttle and ion-binding protein of the longitudinal tubules, sarcalumenin, were drastically reduced in cardiac mdx fibres. This result agrees with the recently reported decrease in the Ca2+-release channel and Ca2+-ATPase in the mdx heart. Abnormal Ca2+-handling appears to play a major role in the molecular pathogenesis of the cardiac involvement in X-linked muscular dystrophy.
Keywords: Muscular dystrophy; Cardiomyopathy; mdx; Dystrophin; Calsequestrin; Sarcalumenin;

Cisplatin up-regulates the in vivo biosynthesis and degradation of renal polyamines and c-Myc expression by Agnieszka Stachurska; Magdalena Dudkowska; Alicja Czopek; Małgorzata Manteuffel-Cymborowska; Barbara Grzelakowska-Sztabert (259-266).
Time-dependent changes in polyamine metabolism and c-Myc expression are reported in kidney of mice treated with cisplatin, a widely used anticancer drug. We show that cisplatin significantly induces the expression of two enzymes critical to proper homeostasis of cellular polyamines, ornithine decarboxylase (ODC) and spermidine/spermine N 1-acetyltransferase (SSAT). We also document the cross-talk between signalling pathway(s) induced by cisplatin injury to renal tubules and the testosterone/androgen receptor pathway. Their interaction results in a decrease in testosterone-induced ODC activity and ODC mRNA level, and in differential modulation of SSAT expression. Moreover, cisplatin and antifolate CB 3717, another nephrotoxic drug examined, severalfold up-regulate expression of c-Myc mRNA, albeit with different kinetics. However, cisplatin, contrary to CB 3717, does not induce renal hepatocyte growth factor (HGF)/c-Met expression being without effect on HGF mRNA level and significantly down-regulating c-Met transmembrane receptor message. In conclusion, these in vivo studies document significant cisplatin-induced modulation of polyamine biosynthesis/degradation and up-regulation of c-Myc expression, and suggest that c-Myc transcription factor is involved in the induction of ODC in kidney injured with antifolate, but not with cisplatin.
Keywords: Cisplatin; Antifolate CB 3717; Testosteron; Ornithine decarboxylase; Spermidine/spermine N 1-acetyltransferase; c-Myc; HGF/c-Met;