BBA - Molecular Basis of Disease (v.1782, #10)
Editorial Board (ii).
Peptides and proteins in plasma and cerebrospinal fluid as biomarkers for the prediction, diagnosis, and monitoring of therapeutic efficacy of Alzheimer's disease by Christopher D. Aluise; Renã A. Sowell; D. Allan Butterfield (549-558).
Alzheimer's disease (AD) affects millions of persons worldwide. Earlier detection and/or diagnosis of AD would permit earlier intervention, which conceivably could delay progression of this dementing disorder. In order to accomplish this goal, reliable and specific biomarkers are needed. Biomarkers are multidimensional and have the potential to aid in various facets of AD such as diagnostic prediction, assessment of disease stage, discrimination from normally cognitive controls as well as other forms of dementia, and therapeutic efficacy of AD drugs. To date, biomarker research has focused on plasma and cerebrospinal fluid (CSF), two bodily fluids believed to contain the richest source of biomarkers for AD. CSF is the fluid surrounding the central nervous system (CNS), and is the most indicative obtainable fluid of brain pathology. Blood plasma contains proteins that affect brain processes from the periphery, as well as proteins/peptides exported from the brain; this fluid would be ideal for biomarker discovery due to the ease and non-invasive process of sample collection. However, it seems reasonable that biomarker discovery will result in combinations of CSF, plasma, and other fluids such as urine, to serve the aforementioned purposes. This review focuses on proteins and peptides identified from CSF, plasma, and urine that may serve as biomarkers in AD.
Keywords: Biomarker; Plasma; CSF; Alzheimer's disease;
Changes in protein structure and distribution observed at pre-clinical stages of scrapie pathogenesis by Ariane Kretlow; Qi Wang; Michael Beekes; Dieter Naumann; Lisa M. Miller (559-565).
Scrapie is a neurodegenerative disorder that involves the misfolding, aggregation and accumulation of the prion protein (PrP). The normal cellular PrP (PrPC) is rich in α-helical secondary structure, whereas the disease-associated pathogenic form of the protein (PrPSc) has an anomalously high β-sheet content. In this study, protein structural changes were examined in situ in the dorsal root ganglia from perorally 263K scrapie-infected and mock-infected hamsters using synchrotron Fourier Transform InfraRed Microspectroscopy (FTIRM) at four time points over the course of the disease (pre-clinical, 100 and 130 days post-infection (dpi); first clinical signs (∼ 145 dpi); and terminal (∼ 170 dpi)). Results showed clear changes in the total protein content, structure, and distribution as the disease progressed. At pre-clinical time points, the scrapie-infected animals exhibited a significant increase in protein expression, but the β-sheet protein content was significantly lower than controls. Based on these findings, we suggest that the pre-clinical stages of scrapie are characterized by an overexpression of proteins low in β-sheet content. As the disease progressed, the β-sheet content increased significantly. Immunostaining with a PrP-specific antibody, 3F4, confirmed that this increase was partly – but not solely – due to the formation of PrPSc in the tissue and indicated that other proteins high in β-sheet were produced, either by overexpression or misfolding. Elevated β-sheet was observed near the cell membrane at pre-clinical time points and also in the cytoplasm of infected neurons at later stages of infection. At the terminal stage of the disease, the protein expression declined significantly, likely due to degeneration and death of neurons. These dramatic changes in protein content and structure, especially at pre-clinical time points, emphasize the possibility for identifying other proteins involved in early pathogenesis, which are important for a further understanding of the disease.
Keywords: Prion; Scrapie; Dorsal root ganglia; Protein structure; Protein-folding; Infrared microspectroscopy;
ATP depletion alters the mode of cell death induced by benzyl isothiocyanate by Noriyuki Miyoshi; Etsuko Watanabe; Toshihiko Osawa; Masashi Okuhira; Yoshiyuki Murata; Hiroshi Ohshima; Yoshimasa Nakamura (566-573).
Pro-inflammatory death is presumably an undesirable event in cancer prevention process, thus biochemical comprehension and molecular definition of this process could have important clinical implications. In the present study, we examined the cytophysiological conversion of cell death mode by benzyl isothiocyanate (BITC) in human cervical cancer HeLa cells. The detailed studies using flow cytometric and morphological analyses demonstrated that the cells treated with appropriate concentration (25 μM) of BITC showed apoptotic feature, such as chromatin condensation, DNA fragmentation, and preserved plasma membrane integrity, whereas these features were disappeared by treatment with higher concentration (100 μM). The treatment with 2-deoxyglucose, an inhibitor of ATP synthesis, drastically increased in the ratio of necrotic dead cells, while it influences little that of apoptotic cells. Moreover, an analysis using the mitochondrial DNA-deficient HeLa cells demonstrated that the ρ° cells were more susceptible to the BITC-induced necrosis-like cell death compared to the wild-type (ρ+) cells, whereas the ROS production was significantly inhibited in the ρ° cells. It is likely that the BITC-induced ROS is derived from mitochondrial respiratory chain and ruled out the contribution to the mechanism of cell death mode switching. In addition, the BITC treatment resulted in a more rapid depletion of ATP in the ρ° cells than in the ρ+ cells. Furthermore, a caspase inhibitor, Z-VAD-fmk counteracted not only apoptosis, but also necrosis-like cell death induced by BITC, suggesting that increment in this cell death pattern might be due to the interruption of events downstream of a caspase-dependent pathway. The obtained data suggested that the decline in the intracellular ATP level plays an important role in tuning the mode of cell death by BITC.
Keywords: ITC; BITC; GSH; ROS; PEITC; 2-DG; PI; H2DCF-DA; LDH; ETP; CHX; GPx; TR; GST; GCLC; GAPDH; AKR1C; TR; ATM; Benzyl isothiocyanate; Apoptosis; Necrosis; HeLa cell; ATP; ROS;
Gene therapy of Hunter syndrome: Evaluation of the efficiency of muscle electro gene transfer for the production and release of recombinant iduronate-2-sulfatase (IDS) by A. Friso; R. Tomanin; A. Zanetti; C. Mennuni; F. Calvaruso; N. La Monica; O. Marin; F. Zacchello; M. Scarpa (574-580).
Mucopolysaccharidosis type II (MPSII) is an inherited disorder due to a deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). The disease is characterized by a considerable deposition of heparan- and dermatan-sulfate, causing a general impairment of physiological functions. Most of the therapeutic protocols proposed so far are mainly based upon enzyme replacement therapy which is very expensive. There is a requirement for an alternative approach, and to this aim, we evaluated the feasibility of muscle electro gene transfer (EGT) performed in the IDS-knockout (IDS-ko) mouse model. EGT is a highly efficient method of delivering exogenous molecules into different tissues. More recently, pre-treatment with bovine hyaluronidase has shown to further improve transfection efficiency of muscle EGT. We here show that, by applying such procedure, IDS was very efficiently produced inside the muscle. However, no induced IDS activity was measured in the IDS-ko mice plasma, in contrast to matched healthy controls. In the same samples, an anticipated and rapidly increasing immune response against the recombinant protein was observed in the IDS-ko vs control mice, although reaching the same levels at 5 weeks post-injection. Additional experiments performed on healthy mice showed a significant contribution of hyaluronidase pre-treatment in increasing the immune response.
Keywords: Hunter syndrome; Iduronate-2-sulfatase; Electro gene transfer; Hyaluronidase; Muscle; Gene therapy;
Impact of Tyr to Ala mutations on α-synuclein fibrillation and structural properties by Nataša Poklar Ulrih; Christopher H. Barry; Anthony L. Fink (581-585).
Substantial evidence suggests that the fibrillation of α-synuclein is a critical step in the development of Parkinson's disease. In vitro, α-synuclein forms fibrils with morphologies and a staining characteristic similar to those extracted from disease-affected brain. Monomeric α-synuclein is an intrinsically disordered protein, with three Tyr residues in the C-terminal region, one in the N-terminus, and lacking Trp. It is thought that interactions between the C-terminus and the central portion of the molecule may prevent or minimize aggregation/fibrillation. To test this hypothesis we examined the importance of the Tyr residues on the propensity for α-synuclein to fibrillate in vitro. Fibril formation of α-synuclein was completely inhibited, in the timescale over which measurements were made, by replacing the three C-terminal Tyr residues with Ala. In addition, substitution of Tyr133 by Ala also resulted in the absence of fibrillation, whereas the individual Y125A and Y136A mutants showed limited inhibition. Replacement of Tyr39 by Ala also resulted in substantial inhibition of fibrillation. Structural analysis showed that the Y133A mutant had a substantially different conformation, rich in α-helical secondary structure, as compared with the wild-type and other mutants, although the formation of any tertiary structure has not been observed as can be judged from near-UV-CD spectra. These observations suggest that the long-range intramolecular interactions between the N- and C-termini of α-synuclein are likely to be crucial to the fibrillation process.
Keywords: α-synuclein; Tyr's mutant; Fibrillation; Secondary structure; ThT fluorescence assay; Long-range intramolecular interaction;
Metabolic implications of reduced heart-type fatty acid binding protein in insulin resistant cardiac muscle by Jane Shearer; Patrick T. Fueger; ZhiZhang Wang; Deanna P. Bracy; David H. Wasserman; Jeffrey N. Rottman (586-592).
Insulin resistance is characterized by elevated rates of cardiac fatty acid utilization resulting in reduced efficiency and cardiomyopathy. One potential therapeutic approach is to limit the uptake and oxidation of fatty acids. The aims of this study were to determine whether a quantitative reduction in heart-type fatty acid binding protein (FABP3) normalizes cardiac substrate utilization without altering cardiac function. Transgenic (FABP3+/−) and wild-type (WT) littermates were studied following low fat (LF) or high fat (HF) diets, with HF resulting in obese, insulin-resistant mice. Cardiovascular function (systolic blood pressure, % fractional shortening) and heart dimension were measured at weaning and every month afterward for 3 mo. During this period cardiovascular function was the same independent of genotype and diet. Catheters were surgically implanted in the carotid artery and jugular vein for sampling and infusions in mice at 4 mo of age. Following 5 d recovery, mice underwent either a saline infusion or a hyperinsulinemic-euglycemic clamp (4 mU kg- 1 min- 1). Indices of long chain fatty acid and glucose utilization (R f, R g; μmol g wet weight- 1 min- 1) were obtained using 2-deoxy[3H]glucose and [125I]-15-ρ-iodophenyl)-3-R,S-methylpentadecanoic acid. FABP3+/− had enhanced cardiac R g compared with WT during saline infusion in both LF and HF. FABP3+/− abrogated the HF-induced decrement in insulin-stimulated cardiac R g. On a HF diet, FABP+/− but not WT had an increased reliance on fatty acids (R f) during insulin stimulation. In conclusion, cardiac insulin resistance and glucose uptake is largely corrected by a reduction in FABP3 in vivo without contemporaneous deleterious effects on cardiac function.
Keywords: Obesity; Glucose; Fatty acid; Hyperinsulinemic-euglycemic clamp;
c-Jun NH2-terminal kinase mediates leptin-stimulated androgen-independent prostate cancer cell proliferation via signal transducer and activator of transcription 3 and Akt by Toshiaki Miyazaki; Jeffrey D. Bub; Yoshiki Iwamoto (593-604).
Obesity is associated with advanced prostate cancer. Here we demonstrate that in mouse prostate cancer TRAMP-C1 cells epididymal fat extracts from high-fat diet-fed obese mice stimulate androgen-independent cell growth more significantly than those from low-fat diet-fed lean mice or genetically obese leptin-deficient ob/ob mice in correlation with leptin concentrations. This result suggests that obesity promotes androgen-independent prostate cancer cell growth via adipose leptin. We have reported that added leptin stimulates androgen-independent prostate cancer cell proliferation through c-Jun NH2-terminal kinase (JNK). As with JNK, signal transducer and activator of transcription 3 (STAT3) and Akt are implicated in androgen-independent prostate cancer. In this study, we identify novel interaction of these three molecules in leptin-stimulated androgen-independent cell proliferation. Leptin activates JNK, STAT3 and Akt in a biphasic manner with a similar time-course. Pharmacological JNK inhibition suppresses leptin-stimulated DNA binding activity, as well as Ser-727 phosphorylation, of STAT3. Since JNK upregulates STAT3 activity via Ser-727 phosphorylation, JNK mediates leptin-stimulated STAT3 activation through Ser-727 phosphorylation. Moreover, JNK inhibition impairs leptin-stimulated Ser-473 phosphorylation of Akt that is required for its activation. Thus, JNK is involved in leptin-stimulated Akt activation. These findings together indicate that JNK mediates leptin-stimulated androgen-independent prostate cancer cell proliferation via STAT3 and Akt.
Keywords: Prostate cancer; Obesity; Leptin; JNK; STAT3; Akt;
Annexin A5 increases the cell surface expression and the chloride channel function of the ΔF508-cystic fibrosis transmembrane regulator by Marie-Anne Le Drévo; Nathalie Benz; Mathieu Kerbiriou; Marie-Agnès Giroux-Metges; Jean-Pierre Pennec; Pascal Trouvé; Claude Férec (605-614).
Cystic fibrosis (CF) is caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In CF, the most common mutant ΔF508-CFTR is misfolded, is retained in the ER and is rapidly degraded. If conditions could allow ΔF508-CFTR to reach and to stabilize in the plasma membrane, it could partially correct the CF defect. We have previously shown that annexin V (anxA5) binds to both the normal CFTR and the ΔF508-CFTR in a Ca2+-dependent manner and that it regulates the chloride channel function of Wt-CFTR through its membrane integration. Our aim was to extend this finding to the ΔF508-CFTR. Because some studies show that thapsigargin (Tg) increases the ΔF508-CFTR apical expression and induces an increased [Ca2+]i and because anxA5 relocates and binds to the plasma membrane in the presence of Ca2+, we hypothesized that the Tg effect upon ΔF508-CFTR function could involve anxA5. Our results show that raised anxA5 expression induces an augmented function of ΔF508-CFTR due to its increased membrane localization. Furthermore, we show that the Tg effect involves anxA5. Therefore, we suggest that anxA5 is a potential therapeutic target in CF.
Keywords: Cystic fibrosis; Annexin A5; CFTR; Protein interaction; CFTR function;