BBA - Molecular Basis of Disease (v.1741, #3)

A new cancer diagnostic system based on a CDK profiling technology by Hideki Ishihara; Tomokazu Yoshida; Yuko Kawasaki; Hironori Kobayashi; Masatoshi Yamasaki; Satoshi Nakayama; Erika Miki; Kei-ichiro Shohmi; Tomoko Matsushima; Sachiyo Tada; Yasuhiro Torikoshi; Masakatsu Morita; Shigeyuki Tamura; Yoko Hino; Jun Kamiyama; Yoshihiro Sowa; Yasunari Tsuchihashi; Hisakazu Yamagishi; Toshiyuki Sakai (226-233).
A series of molecular pathological investigations of the molecules that stimulate the cyclin dependent kinases (CDK1, 2, 4, and 6) have led to enormous accumulation of knowledge of the clinical significance of these molecules for cancer diagnosis. However, the molecules have yet to be applied to clinical cancer diagnosis, as there is no available technology for application of the knowledge in a clinical setting. We hypothesized that the direct measurement of CDK activities and expressions (CDK profiling) might produce clinically relevant values for the diagnosis. This study investigated the clinical relevance of CDK profiling in gastrointestinal carcinoma tissues by using originally developed expression and activity analysis methods. We have established novel methods and an apparatus for analyzing the expression and activities of the CDK molecules in lysate of tumor tissue in a clinical setting, and examined 30 surgically dissected gastrointestinal carcinomas and corresponding normal mucosal specimens. We demonstrate here that remarkably elevated CDK2 activity is evident in more than 70% of carcinoma tissues. Moreover, a G1-CDK activity profiling accurately mirrored the differences in proliferation between tumor and normal colonic tissues. Our results suggest that CDK profiling is a potent molecular–clinical approach to complement the conventional pathological diagnosis, and to further assist in the individualized medications.
Keywords: Cell cycle; CDK; Kinase; Profiling; Gastrointestinal cancer;

LRP1B functions as a receptor for Pseudomonas exotoxin by Diana V. Pastrana; Alison J. Hanson; Jane Knisely; Guojun Bu; David J. FitzGerald (234-239).
Pseudomonas aeruginosa is an opportunistic pathogen that produces several virulence factors, among them Pseudomonas Exotoxin A (PE). Previously, low-density lipoprotein receptor-related protein 1 (LRP1) was shown to be the primary receptor for PE. In this report, we show that a close family member, LRP1B, can also function as a receptor.
Keywords: Toxin; Receptor; LRP1; LRP1B; Pseudomonas; ADP-ribosylation;

Ectopic expression of alkaline phosphatase in proximal tubular brush border membrane of human renal cell carcinoma by R. Prasad; S. Lambe; P. Kaler; S. Pathania; S. Kumar; S. Attri; S.K. Singh (240-245).
The present study was conducted to find out any alteration in the expression and activity of alkaline phosphatase in the brush border membrane (BBM) from renal cell carcinoma (RCC) in comparison to normal renal BBM. The specific activity of alkaline phosphatase was drastically reduced in homogenate as well as BBM from RCC kidney when compared to ALP activity in BBM of normal kidney. Kinetic studies revealed that diminished activity of alkaline phosphatase in BBM isolated from RCC was fraternized with decrease in maximal velocity (V max) and increase in affinity constant (K m) of the enzyme. SDS-PAGE studies showed that the BBM proteins having molecular weights ranging from 95 to 170 kDa were poorly expressed in RCC BBM in relative to normal kidney BBM. Incubation of SDS-PAGE gel with BCIP/NBT dye clearly showed that the expression of ALP in tumor renal BBM was markedly reduced as compared to normal kidney. Further, Western blot analysis using anti-alkaline phosphatase antibody also confirmed the reduced expression of ALP in tumor renal BBM. Lipid composition in reference to phospholipids, glycolipids and cholesterol in tumor renal BBM was altered to that of normal renal BBM, indicating alteration in membrane fluidity of tumor renal BBM.
Keywords: Alkaline phosphatase; Renal cell carcinoma; Brush border membrane; Lipid composition;

Nanoparticle and other metal chelation therapeutics in Alzheimer disease by Gang Liu; Matthew R. Garrett; Ping Men; Xiongwei Zhu; George Perry; Mark A. Smith (246-252).
Current therapies for Alzheimer disease (AD) such as the anticholinesterase inhibitors and the latest NMDA receptor inhibitor, Namenda, provide moderate symptomatic delay at various stages of disease, but do not arrest disease progression or supply meaningful remission. As such, new approaches to disease management are urgently needed. Although the etiology of AD is largely unknown, oxidative damage mediated by metals is likely a significant contributor since metals such as iron, aluminum, zinc, and copper are dysregulated and/or increased in AD brain tissue and create a pro-oxidative environment. This role of metal ion-induced free radical formation in AD makes chelation therapy an attractive means of dampening the oxidative stress burden in neurons. The chelator desferioxamine, FDA approved for iron overload, has shown some benefit in AD, but like many chelators, it has a host of adverse effects and substantial obstacles for tissue-specific targeting. Other chelators are under development and have shown various strengths and weaknesses. In this review, we propose a novel system of chelation therapy through the use of nanoparticles. Nanoparticles conjugated to chelators show a unique ability to cross the blood–brain barrier (BBB), chelate metals, and exit through the BBB with their corresponding complexed metal ions. This method may prove to be a safe and effective means of reducing the metal load in neural tissue thus staving off the harmful effects of oxidative damage and its sequelae.
Keywords: Alzheimer disease; Chelation therapy; Metal dysregulation; Nanoparticle;

The human Burkitt lymphoma Daudi cell line expresses constitutively active nuclear factor kappaB (NF-κB) in the nucleus in spite of high levels of inhibitor kappaB-alpha (IκB-α) in the cytoplasm. The antiproliferative response of these cells to interferon-α (IFN-α) correlated with the inhibition of the constitutive NF-κB activity by the cytokine. The present study shows that IFN-α caused an increase in p53 level, inhibited cell proliferation by [3H]thymidine incorporation, and stimulated cytotoxicity and apoptosis by PARP-cleavage in the Daudi cells. In order to study the relationship between the constitutively active NF-κB and IκB-α, a dominant negative mutant IκB-α (IκB-αDN), lacking the N-terminal 36 amino acids required for the activation of NF-κB by tumor necrosis factor-α (TNF-α), was expressed in the Daudi cells. The expression of IκB-αDN protein did not inhibit the constitutive NF-κB activity, but it inhibited cell proliferation, antiproliferative response to IFN-α, and phosphorylated mitogen activated protein kinase (p-MAPK) level. Thus, our results suggest that constitutive NF-κB activity in the human Burkitt lymphoma Daudi cells is maintained by a mechanism independent of IκB-α degradation, and that the IκB-α is involved in the proliferation of these cells, possibly through the MAP kinase pathway. Therefore, in addition to IFN-α treatment, both NF-κB and IκB-α may be used as drug targets for inhibiting cell proliferation in the lymphomas.
Keywords: Constitutive NF-κB; IκB-α; Interferon-α; Cell proliferation; MAP kinase; Burkitt lymphoma;

The C2 variant of human serum transferrin retains the iron binding properties of the native protein by Paolo Zatta; Luigi Messori; Pierluigi Mauri; Susan J. van Rensburg; Johann van Zyl; Silvia Gabrielli; Chiara Gabbiani (264-270).
The tryptic digests of blood samples obtained from transferrin C1 and C2 (TfC1 and TfC2 hereafter) genotypes were analysed by Liquid Chromatography coupled to Electrospray Mass Spectrometry (LC/ESI–MS/MS). The analytical results confirmed the single base change in exon 15 of the Tf gene. The solution behaviour and the iron binding properties of the two Tf variants were studied by UV-visible spectrophotometry and by circular dichroism. It appears that TfC2 globally manifests the same spectral features as the native protein. The local conformation of the two iron binding sites is conserved in the two Tf variants as evidenced by the visible absorption and CD spectra. Also, the iron binding capacities and their pH-dependent profiles are essentially the same. Overall, our investigation points out that the single amino acid substitution in TfC2 (Pro570Ser) does not affect the general conformation of the protein nor the local structure of the iron binding sites. The implications of these results for the etiopathogenesis of Alzheimer's disease are discussed.
Keywords: Transferrin; Alzheimer's disease; Iron; Aluminum; Circular dichroism; Electrospray mass spectrometry;

Gene expression profiling of pulpal tissue reveals the molecular complexity of dental caries by Julia L. McLachlan; Anthony J. Smith; Iwona J. Bujalska; Paul R. Cooper (271-281).
High-throughput characterisation of the molecular response of pulpal tissue under carious lesions may contribute to improved future diagnosis and treatment. To identify genes associated with this process, oligonucleotide microarrays containing ∼15,000 human sequences were screened using pooled total RNA isolated from pulpal tissue from both healthy and carious teeth. Data analysis identified 445 genes with 2-fold or greater difference in expression level, with 85 more abundant in health and 360 more abundant in disease. Subsequent gene ontological grouping identified a variety of processes and functions potentially activated or down-modulated during caries. Validation of microarray results was obtained by a combination of real-time and semi-quantitative PCR for selected genes, confirming down-regulation of Dentin Matrix Protein-1 (DMP-1), SLIT2, Period-2 (PER2), Period-3 (PER3), osteoadherin, Glypican-3, Midkine, activin receptor interacting protein-1 (AIP1), osteoadherin and growth hormone receptor (GHR), and up-regulation of Adrenomedullin (ADM), Interleukin-11 (IL-11), Bone sialoprotein (BSP), matrix Gla protein (MGP), endothelial cell growth factor-1 (ECGF1), inhibin βA and orosomucoid-1 (ORM1), in diseased pulp. Real-time PCR analyses of ADM and DMP-1 in a panel of healthy and carious pulpal tissue and also in immune system cells highlighted the heterogeneity of caries and indicated increased expression of ADM in neutrophils activated by bacterial products. In contrast, DMP-1 was predominantly expressed by cells native to healthy pulpal tissue. This study has greatly extended our molecular knowledge of dental tissue disease and identified involvement of genes previously unassociated with this process.
Keywords: Pulpal tissue; Diagnosis; Treatment;

Enzymes that hydrolyze adenine nucleotides in chronic renal failure: Relationship between hemostatic defects and renal failure severity by Adriane C. Silva; André L.B. Morsch; Rafael F. Zanin; Maísa C. Corrêa; Luís C. Arantes; Maria C. Araujo; Vera M. Morsch; Maria R.C. Schetinger (282-288).
The activities of the enzymes NTPDase (E.C.3.6.1.5, apyrase, ATP diphosphohydrolase, ecto-CD39) and 5′-nucleotidase (E.C.3.1.3.5, CD73) were analyzed in platelets from patients with chronic renal failure (CRF), both undergoing hemodialysis treatment (HD) and not undergoing hemodialysis (ND), as well as from a control group. The results showed an increase in platelet NTPDase activity in CRF patients on HD treatment (52.88%) with ATP as substrate (P  < 0.0001). ADP hydrolysis was decreased (33.68% and 39.75%) in HD and ND patients, respectively. In addition, 5′-nucleotidase activity was elevated in the HD (160%) and ND (81.49%) groups when compared to the control (P  < 0.0001). Significant correlation was found among ATP, ADP and AMP hydrolysis and plasma creatinine and urea levels (P  < 0.0001). Patients were compared statistically according the time of hemodialysis treatment. We found enhanced NTPDase and 5′-nucleotidase activities between 49 and 72 months on HD patients. Our result suggests the existence of alterations in nucleotide hydrolysis in platelets of CRF patients. Possibly, this altered nucleotide hydrolysis could contribute to hemostasis abnormalities found in CRF.
Keywords: Chronic renal failure; NTPDase; 5′-nucleotidase; Platelet; Hemodialysis;

The impact and molecular mechanism of action of glucocorticoids in connective tissues is largely unclear, even though widely used, and whether factors such as injury and inflammation modulate this response has not been elucidated. This study describes the role of glucocorticoids in the regulation of mRNA levels for collagens I and III, MMP-13, biglycan, decorin, COX-2 and the glucocorticoid receptor in connective tissues of normal and injured joints in an established rabbit in vivo MCL scar model, and examines the potential mechanism(s) involved. In vitro promoter studies were performed using an MMP-13 promoter–luciferase expression construct in transient transfection assays with a rabbit synovial cell line (HIG-82) to identify sites of glucocorticoid-mediated transcriptional regulation and the promoter elements involved. The in vivo results indicate that scar tissue from different phases of healing (early inflammatory, granulation tissue and neovascular, and later remodelling phases, respectively) displays a different pattern of responsiveness to glucocorticoid treatment than uninjured tissue and that this responsiveness is gene dependent. The most significant impact was seen for genes such as collagen I, collagen III and MMP-13, all of which are involved in connective tissue structure and remodelling. The in vitro studies confirmed the apparent in vivo glucocorticoid-mediated response of MMP-13 mRNA and implicated the AP-1 site of the MMP-13 promoter in this regulation. Immunohistochemistry studies showed increased MMP-13 protein expression, consistent with the mRNA findings, following glucocorticoid treatment in injured tissue but not normal tissues. In conclusion, connective tissue responsiveness to glucocorticoid treatment varies depending on injury and the stage of healing of the tissue, and consequently, glucocorticoid-responsiveness may be modulated differently in states of injury and inflammation.
Keywords: Glucocorticoid; Medial collateral ligament; Scar; Rabbit; Injury; MMP-13;

Lysosomal leukocyte β-d-glucuronidase during enzyme replacement therapy in Fabry disease by Giancarlo Goi; Luca Massaccesi; Alessandro P. Burlina; Claudia J. Baquero Herrera; Adriana Lombardo; Guido Tettamanti; Alberto B. Burlina (300-306).
Fabry disease results from a deficiency in the activity of α-d-galactosidase A and subsequent accumulation of neutral glycosphingolipids in lysosomes. This study investigated whether lysosomal enzymes can indicate biochemical changes in the lysosomal apparatus induced by enzyme replacement therapy (ERT).Eight patients were monitored by clinical and biochemical tests and several lysosomal glycohydrolases were measured in plasma and leucocytes.Before starting ERT, β-d-glucuronidase in leukocytes was markedly increased. After 1 month of therapy, enzyme levels dropped in all patients. In the patients who regularly followed the therapy, the enzyme levels remained stable for the next 20 months. In one patient who interrupted therapy for 2 months, the enzyme levels rose again.Lysosomal enzymes can be useful for monitoring biochemical changes in patients with Fabry disease receiving ERT. Though these findings refer to only a small number of patients, the correlation between β-d-glucuronidase levels and ERT is interesting and might serve as a basis for further studies to define the potential of this enzyme in monitoring the effects of ERT in lysosomal storage disorders.
Keywords: Fabry disease; Enzyme replacement therapy; Lysosomal enzyme; β-d-Glucuronidase; Biochemical monitoring;

Inhibition of amyloid fibril formation of β-amyloid peptides via the amphiphilic surfactants by Steven S.-S. Wang; Ya-Ting Chen; Shang-Wei Chou (307-313).
β-amyloid peptide (Aβ) is the major proteinacious constituent of senile plaques in Alzheimer's disease and is believed to be responsible for the neurodegeneration process associated with the disease. While the actual size of the aggregated species responsible for Aβ neurotoxicity and fibrillogenesis mechanism(s) remain unknown, retardation of Aβ aggregation still holds assurance as an effective strategy in reducing Aβ-elicited toxicity. The research presented here is aimed at examining the inhibitory effect of two amphiphilic surfactants, di-C6-PC and di-C7-PC, on the in vitro fibrillogenesis process of Aβ(1–40) peptides at physiological pH (pH 7.2). Using ThT-induced fluorescence, turbidity, Congo red binding, and circular dichroism spectroscopy studies, our research demonstrated that the inhibition of Aβ(1–40) fibril formation was di-C6-PC and di-C7-PC concentration-dependent. The best inhibitory action on fibril formation was observed when Aβ was incubated with di-C7-PC at 100 μM over time. We believe that the outcome from this work will aid in the development and/or design of potential inhibitory agents against amyloid formation associated with Alzheimer's and other amyloid diseases.
Keywords: Alzheimer's disease; β-amyloid; Fibril; Aggregation; Inhibitor;

Renal ischemia and reperfusion activates the eIF2 alpha kinase PERK by Heather L. Montie; Foaz Kayali; Adam J. Haezebrouck; Noreen F. Rossi; Donald J. DeGracia (314-324).
Inhibition of protein synthesis occurs in the post-ischemic reperfused kidney but the molecular mechanism of renal translation arrest is unknown. Several pathways have been identified whereby cell stress inhibits translation initiation via phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α, phospho-form eIF2α(P)]. Here, we report a 20-fold increase in eIF2α(P) in kidney homogenates following 10 min of cardiac arrest-induced ischemia and 10 min reperfusion. Using immunohistochemistry, we observed eIF2α(P) in tubular epithelial cells in both cortex and medulla, where the greatest eIF2α(P) staining was found in epithelial cells of the so-called watershed area at the corticomedullary junction. We further show that increased eIF2α(P) is accompanied by activation of the PKR-like endoplasmic reticulum eIF2α kinase (PERK). These observations indicate that renal ischemia and reperfusion induce stress to the endoplasmic reticulum and activate the unfolded protein response in renal epithelial cells. As the unfolded protein response can result alternatively in a pro-survival or pro-apoptotic outcome, the present study demonstrates an new additional mechanism involved in cell damage and/or repair in ischemic and reperfused kidney.
Keywords: Renal ischemia and reperfusion; eIF2α(P); Endoplasmic reticulum stress; PERK; Protein synthesis inhibition; Unfolded protein response;

Oxidative stress and pro-apoptotic conditions in a rodent model of Wilson's disease by Alberta Samuele; Anna Mangiagalli; Marie-Thérèse Armentero; Roberto Fancellu; Eleonora Bazzini; Mariapia Vairetti; Andrea Ferrigno; Plinio Richelmi; Giuseppe Nappi; Fabio Blandini (325-330).
Wilson's disease (WD) is an inherited disorder, characterized by selective copper deposition in liver and brain, chronic hepatitis and extra-pyramidal signs. In this study, we investigated changes of biochemical markers of oxidative stress and apoptosis in liver, striatum and cerebral cortex homogenates from Long–Evans Cinnamon (LEC) rats, a mutant strain isolated from Long Evans (LE) rats, in whom spontaneous hepatitis develops shortly after birth. LEC and control (LE) rats at 11 and 14 weeks of age were used. We determined tissue levels of glutathione (GSH/GSSG ratio), lipid peroxides, protein-thiols (P-SH), nitric oxide metabolites, activities of caspase-3 and total superoxide-dismutase (SOD), striatal levels of monoamines and serum levels of hepatic amino-transferases. We observed a decrease of protein-thiols, GSH/GSSG ratio and nitrogen species associated to increased lipid peroxidation in the liver and striatum – but not in the cerebral cortex – of LEC rats, accompanied by dramatic increase in serum amino-transferases and decrease of striatal catecholamines. Conversely, SOD and caspase-3 activity increased consistently only in the cortex of LEC rats. Hence, we assume that enhanced oxidative stress may play a central role in the cell degeneration in WD, at the main sites of copper deposition, with discrete pro-apoptotic conditions developing in distal areas.
Keywords: Apoptosis; Copper-toxicosis; Long Evans Cinnamon; Peroxidation; Cell degeneration;

Analysis of pathological defects in methionine metabolism using a simple mathematical model by Anna Prudova; Mikhail V. Martinov; Victor M. Vitvitsky; Fazoil I. Ataullakhanov; Ruma Banerjee (331-338).
Derangements in methionine metabolism are a hallmark of cancers and homocystinuria, an inborn error of metabolism. In this study, the metabolic consequences of the pathological changes associated with the key pathway enzymes, methionine adenosyl transferase (MAT), glycine N-methyl transferase (GNMT) and cystathionine β-synthase (CBS) as well as an activation of polyamine metabolism, were analyzed using a simple mathematical model describing methionine metabolism in liver. The model predicts that the mere loss of allosteric regulation of CBS by adenosylmethionine (AdoMet) leads to an increase in homocysteine concentration. This is consistent with the experimental data on the corresponding genetic defects, which specifically impair allosteric activation but not basal enzyme activity. Application of the characteristics of transformed hepatocytes to our model, i.e., substitution of the MATI/III isozyme by MATII, loss of GNMT activity and activation of polyamine biosynthesis, leads to the prediction of a significantly different dependence of methionine metabolism on methionine concentrations. The theoretical predictions were found to be in good agreement with experimental data obtained with the human hepatoma cell line, HepG2.
Keywords: Methionine; Homocysteine; S-adenosylmethionine; Methylation; Cystathionine beta-synthase;

Identification of Li+ binding sites and the effect of Li+ treatment on phospholipid composition in human neuroblastoma cells: a 7Li and 31P NMR study by Brian T. Layden; Abde M. Abukhdeir; Christopher Malarkey; Lisa A. Oriti; Wajeeh Salah; Claire Stigler; Carlos F.G.C. Geraldes; Duarte Mota de Freitas (339-349).
Li+ binding in subcellular fractions of human neuroblastoma SH-SY5Y cells was investigated using 7Li NMR spin–lattice (T 1) and spin–spin (T 2) relaxation measurements, as the T 1/T 2 ratio is a sensitive parameter of Li+ binding. The majority of Li+ binding occurred in the plasma membrane, microsomes, and nuclear membrane fractions as demonstrated by the Li+ binding constants and the values of the T 1/T 2 ratios, which were drastically larger than those observed in the cytosol, nuclei, and mitochondria. We also investigated by 31P NMR spectroscopy the effects of chronic Li+ treatment for 4–6 weeks on the phospholipid composition of the plasma membrane and the cell homogenate and found that the levels of phosphatidylinositol and phosphatidylserine were significantly increased and decreased, respectively, in both fractions. From these observations, we propose that Li+ binding occurs predominantly to membrane domains, and that chronic Li+ treatment alters the phospholipid composition at these membrane sites. These findings support those from clinical studies that have indicated that Li+ treatment of bipolar patients results in irregularities in Li+ binding and phospholipid metabolism. Implications of our observations on putative mechanisms of Li+ action, including the cell membrane abnormality, the inositol depletion and the G-protein hypotheses, are discussed.
Keywords: Cell membrane; Cytosol; Lithium; Mitochondria; NMR; Phospholipid;

We have previously shown that polymorphisms in the promoter of the human platelet-derived growth factor α-receptor (PDGFRA) gene can be grouped into five distinct haplotypes, designated H1, H2α, H2β, H2γ and H2δ, and that specific combinations of these promoter haplotypes predispose to neural tube defects (NTDs). These promoter haplotypes differ strongly in their ability to drive reporter gene expression in various human cell lines, with highest activity for H2α and H2β. Here, we show that the haplotype-linked PDGFRA promoter region extends to 3.6 kb upstream from the transcription start site, and contains a total of ten polymorphic sites. For two of these polymorphic sites, i.e. −909C/A and +68GAins/del, we observed differential binding of nuclear proteins from human osteosarcoma (HOS) cells. The protein complex binding specifically to −909C, which is present in all haplotypes except the low activity haplotype H2γ, contained members of the upstream stimulatory factor (USF) family of transcription factors. Furthermore, we identified a protein complex of 125 kDa which bound specifically to the low activity haplotype H1 at position +68GAdel and may represent an H1-specific PDGFRA transcriptional repressor. The current identification of cis-acting elements in the PDGFRA promoter and the transcription factors that bind them, provides a new strategy for the identification of genes that are potentially involved in neural tube defects.
Keywords: PDGFRA; Promoter haplotype; Neural tube defect; EMSA; USF; Osteosarcoma;