The diagnostic value of Western blot method in patients with cystic echinococcosis

Cystic echinococcosis (CE) is the larval cystic stage (called echinococcal cysts) of a small taeniid-type tapeworm (Echinococcus granulosus). Carnivores such as dogs are usually definitive hosts. Intermediate hosts are typically herbivores such as sheep and cattle. CE can be detected using various imaging techniques such as ultrasonography or radiology. Moreover the primary diagnosis has to be confirmed by serological tests since the clinical signs of the disease are non-specific. This study examined the antigenic band patterns useful for serologic diagnosis of hydatidosis. We also report on the post-operative evolution of patients treated for this disease and also determined the diagnostic performance of Western blot IgG kit. Twenty-five (16 females and 9 males) non-operated patients with hydatid cysts (NOP) and 33 (21 females and 12 males) operated patients with hydatid cysts (OP) were included as study group and 22 healthy individuals (14 females and 8 males) with no known chronic diseases were included as a control group. The ages of the patients and control group individuals were between 16-83 years. Patient and control groups were matched for age and sex. Cyst hydatid IgG antibodies were detected in the sera from all patient groups but no antibodies were found in the sera from the control group using ELISA IgG method. Twenty-three (92%) non-operated patients and 18 (54.5%) operated patients exhibited positive results when Western blot IgG kit was used. The P7 band pattern was detected in the sera from all operated and non-operated patients. Twenty-seven of these positive cases had p7 and (p7+p16/18), (p7+p24/26) or (p7+p16/18+p24/26). No antibodies against p7, p16/18 ve p24/26 band patterns were seen in sera from the control group A statistically significant difference was detected between operated and nonoperated patients for Western blot positivity.(p<0.01). p: 0.018- X2=5,604- OR: 0.176- 95% CI: 0.037- 0.841. The sensitivity, specificity, positive prediction and negative prediction values of Echinococcus granulosus Western blot kit for 25 cases with CE and 22 healthy controls were calculated as 92%, 100%, 100% and 91.7%, respectively. In conclusion, we suggest that monitoring p7 in all non-operated patients may be useful to determine the efficiacy of medical treatment and that monitoring p7 antibodies using serological and Western blot methods in operated patients may be useful for the screening of post-operative evolution in patients with hydatid cyst.     

Microchamber array based DNA quantification and specific sequence detection from a single copy via PCR in nanoliter volumes

A novel method for DNA quantification and specific sequence detection in a highly integrated silicon microchamber array is described. Polymerase chain reaction (PCR) mixture of only 40 nL volume could be introduced precisely into each chamber of the mineral oil layer coated microarray by using a nanoliter dispensing system. The elimination of carry-over and cross-contamination between microchambers, and multiple DNA amplification and detection by TaqMan chemistry were demonstrated, for the first time, by using our system. Five different gene targets, related to Escherichia coli were amplified and detected simultaneously on the same chip by using DNA from three different serotypes as the templates. The conventional method of DNA quantification, which depends on the real-time monitoring of variations in fluorescence intensity, was not applied to our system, instead a simple method was established. Counting the number of the microchambers with a high fluorescence signal as a consequence of TaqMan PCR provided the precise quantification of trace amounts of DNA. The initial DNA concentration for Rhesus D (RhD) gene in each microchamber was ranged from 0.4 to 12 copies, and quantification was achieved by observing the changes in the released fluorescence signals of the microchambers on the chip. DNA target could be detected as small as 0.4 copies. The amplified DNA was detected with a CCD camera built-in to a fluorescence microscope, and also evaluated by a DNA microarray scanner with associated software. This simple method of counting the high fluorescence signal released in microchambers as a consequence of TaqMan PCR was further integrated with a portable miniaturized thermal cycler unit. Such a small device is surely a strong candidate for low-cost DNA amplification, and detected as little as 0.4 copies of target DNA.     

Cell-surface protein disulfide isomerase is required for transnitrosation of metallothionein by S-nitroso-albumin in intact rat pulmonary vascular endothelial cells

S-nitrosation of the metal binding protein, metallothionein (MT) appears to be a critical link in affecting endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO)-induced changes in cytoplasmic and nuclear labile zinc, respectively. Although low molecular weight S-nitrosothiols also appear to affect this signaling system, less is known about the ability of extracellular protein nitrosothiols to transnitrosate MT. Accordingly, we synthesized fluorescently labeled S-nitroso-albumin (SNO-albumin, a major protein S-nitrosothiol in plasma) and determined, via confocal microscopy in fixed tissue, that it is transported into cultured rat pulmonary vascular endothelial cells in a temperature sensitive fashion. The cells were transfected with an expression vector that encodes human MT-IIa cDNA sandwiched between enhanced cyan (donor) and yellow (acceptor) fluorescent proteins (FRET-MT) that can detect conformational changes in MT through fluorescence resonance energy transfer (FRET). SNO-albumin and the membrane-permeant low molecular weight S-nitroso-l-cysteine ethyl ester (l-SNCEE) caused a conformational change in FRET-MT as ascertained by full spectral laser scanning confocal microscopy in live rat pulmonary vascular endothelial cells, a result which is consistent with transnitrosation of the reporter molecule. Transnitrosation of FRET-MT by SNO-albumin, but not l-SNCEE, was sensitive to antisense oligonucleotide-mediated inhibition of the expression of cell surface protein disulfide isomerase (csPDI). These results extend the original observations of Ramachandran et al. (Ramachandran N, Root P, Jiang XM, Hogg PJ, Mutus B. Proc Natl Acad Sci U S A 98: 9539-9544, 2001) and suggest that csPDI-mediated denitrosation helps to regulate the ability of the major plasma NO carrier (SNO-albumin) to transnitrosate endothelial cell molecular targets (e.g. MT).     

Role of Lysyl Oxidase Propeptide in Secretion and Enzyme Activity

Lysyl oxidase (LOX) is secreted as a proenzyme (proLOX) that is proteolytically processed in the extracellular milieu to release the propeptide and mature, active LOX. LOX oxidizes lysyl residues of a number of protein substrates in the extracellular matrix and on the cell surface, which impacts several physiological and disease states. Although the LOX propeptide (LOX‐PP) is glycosylated, little is known about the role of this modification in LOX secretion and activity. To gain insight into this issue, cells were transfected with native, full‐length LOX cDNA (pre‐pro‐LOX), the N‐glycosylation null pre‐[N/Q]pro‐LOX cDNA and the deletion mutant pre‐LOX cDNA, referred to as secretory LOX, in which mature LOX is targeted to the secretory pathway without its N‐terminal propeptide sequence. The results show that glycosylation of the LOX‐PP is not required for secretion and extracellular processing of pro‐LOX but it is required for optimal enzyme activity of the resulting mature LOX. Complete deletion of the propeptide sequence prevents mature LOX from exiting the endoplasmic reticulum (ER). Taken together, our study points out the requirement of the LOX‐PP for pro‐LOX exit from the ER and is the first to highlight the influence of LOX‐PP glycosylation on LOX enzyme activity. J. Cell. Biochem. 111: 1231–1243, 2010. © 2010 Wiley‐Liss, Inc.     

Mass-spectrometry based oxidative lipidomics and lipid imaging: applications in traumatic brain injury

Lipids, particularly phospholipids, are fundamental to CNS tissue architecture and function. Endogenous polyunsaturated fatty acid chains of phospholipids possess cis-double bonds each separated by one methylene group. These phospholipids are very susceptible to free-radical attack and oxidative modifications. A combination of analytical methods including different versions of chromatography and mass spectrometry allows detailed information to be obtained on the content and distribution of lipids and their oxidation products thus constituting the newly emerging field of oxidative lipidomics. It is becoming evident that specific oxidative modifications of lipids are critical to a number of cellular functions, disease states and responses to oxidative stresses. Oxidative lipidomics is beginning to provide new mechanistic insights into traumatic brain injury which may have significant translational potential for development of therapies in acute CNS insults. In particular, selective oxidation of a mitochondria-specific phospholipid, cardiolipin, has been associated with the initiation and progression of apoptosis in injured neurons thus indicating new drug discovery targets. Furthermore, imaging mass-spectrometry represents an exciting new opportunity for correlating maps of lipid profiles and their oxidation products with structure and neuropathology. This review is focused on these most recent advancements in the field of lipidomics and oxidative lipidomics based on the applications of mass spectrometry and imaging mass spectrometry as they relate to studies of phospholipids in traumatic brain injury.     

HIF-1alpha has an anti-apoptotic effect in human airway epithelium that is mediated via Mcl-1 gene expression

Hypoxia-inducible factor-1alpha (HIF-1alpha) and myeloid cell leukemia-1 (Mcl-1) proteins have been shown to regulate apoptosis in some cell systems but have not been studied in this context in airway epithelium. Using a model of anoxia/reoxygenation (A/R), the present study employed RNA interference (RNAi) targeting HIF-1alpha and Mcl-1 to evaluate their possible anti-apoptotic effects on HBE1 cells, an immortalized human bronchial epithelial cell line. The cells were either cultured under normoxic conditions or were transfected with small interfering RNA (siRNA) duplexes targeting HIF-1alpha or Mcl-1 mRNA and then immediately exposed to A/R. As controls, non-transfected HBE1 cells and cells transfected with scrambled RNA duplexes were subjected to A/R. Apoptosis was evaluated by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and RNAi was assessed by knockdown of HIF-1alpha and Mcl-1 mRNA and protein expression using real-time quantitative RT-PCR (Q-PCR), immunohistochemistry, and Western blots. HBE1 cells transfected with siRNA duplexes targeting either HIF-1alpha or Mcl-1 and subjected to A/R manifested considerable apoptosis, a finding not observed in either non-transfected cells or cells transfected with scrambled RNA duplexes. Specific knockdown of mRNA and protein expression by RNAi in HBE1 cells after A/R was shown for siRNA duplexes targeting either HIF-1alpha or Mcl-1. Unexpectedly, knockdown of HIF-1alpha induced parallel knockdown of Mcl-1 mRNA and protein expression, whereas Mcl-1 knockdown had no noticeable effect on HIF-1alpha expression. Thus, although both of these proteins were shown to be anti-apoptotic, the action of HIF-1alpha appeared to be mediated in part via Mcl-1.     

Peroxidase activity and structural transitions of cytochrome c bound to cardiolipin-containing membranes

During apoptosis, cytochrome c (cyt c) is released from intermembrane space of mitochondria into the cytosol where it triggers the caspase-dependent machinery. We discovered that cyt c plays another critical role in early apoptosis as a cardiolipin (CL)-specific oxygenase to produce CL hydroperoxides required for release of pro-apoptotic factors [Kagan, V. E., et al. (2005) Nat. Chem. Biol. 1, 223-232]. We quantitatively characterized the activation of peroxidase activity of cyt c by CL and hydrogen peroxide. At low ionic strength and high CL/cyt c ratios, peroxidase activity of the CL/cyt c complex was increased >50 times. This catalytic activity correlated with partial unfolding of cyt c monitored by Trp(59) fluorescence and absorbance at 695 nm (Fe-S(Met(80)) band). The peroxidase activity increase preceded the loss of protein tertiary structure. Monounsaturated tetraoleoyl-CL (TOCL) induced peroxidase activity and unfolding of cyt c more effectively than saturated tetramyristoyl-CL (TMCL). TOCL/cyt c complex was found more resistant to dissociation by high salt concentration. These findings suggest that electrostatic CL/cyt c interactions are central to the initiation of the peroxidase activity, while hydrophobic interactions are involved when cyt c's tertiary structure is lost. In the presence of CL, cyt c peroxidase activity is activated at lower H(2)O(2) concentrations than for isolated cyt c molecules. This suggests that redistribution of CL in the mitochondrial membranes combined with increased production of H(2)O(2) can switch on the peroxidase activity of cyt c and CL oxidation in mitochondria-a required step in execution of apoptosis.     

Mitochondria targeting of non-peroxidizable triphenylphosphonium conjugated oleic acid protects mouse embryonic cells against apoptosis: role of cardiolipin remodeling

Peroxidation of cardiolipin in mitochondria is essential for the execution of apoptosis. We suggested that integration of oleic acid into cardiolipin generates non-oxidizable cardiolipin species hence protects cells against apoptosis. We synthesized mitochondria-targeted triphenylphosphonium oleic acid ester. Using lipidomics analysis we found that pretreatment of mouse embryonic cells with triphenylphosphonium oleic acid ester resulted in decreased contents of polyunsaturated cardiolipins and elevation of its species containing oleic acid residues. This caused suppression of apoptosis induced by actinomycin D. Triacsin C, an inhibitor of acyl-CoA synthase, blocked integration of oleic acid into cardiolipin and restored cell sensitivity to apoptosis.     

Polymorphisms in Toll-Like Receptor 4 Underlie Susceptibility to Tumor Induction by the Mouse Polyomavirus

PERA/Ei (PE) mice are susceptible to tumor induction by polyomavirus (Py), while C57BR/cdJ (BR) mice are resistant. Antigen-presenting cells from BR mice respond to the virus with interleukin-12 (IL-12) and those from PE mice with IL-10. These polarized cytokine responses underlie the development of effective antitumor immunity in BR mice and the lack thereof in PE mice. An ex vivo cytokine production assay using spleen cells from infected [PE × BR] F2 mice together with a genome-wide SNP (single-nucleotide polymorphism)-based QTL (quantitative trait locus) analysis was used to map the determinant of cytokine production to a region of chromosome 4 carrying the Toll-like receptor 4 (TLR4) gene. Genotyping of infected F2 mice showed concordance of TLR4 allele-specific DNA sequences with the cytokine profile. Cytokine responses elicited by Py are MyD88 dependent. Bacterial lipopolysaccharide (LPS), a known TLR4 ligand, induced the same polarized responses as the virus in these host strains. Spleen cells from C3H/HeJ and C57BL/10ScNJ LPS-nonresponsive mice challenged in vitro with Py showed an impaired IL-12 response but were unaffected in IL-10 production. TLR4s of strains PE and BR differ by 3 amino acid substitutions, 2 in the extracellular domain and 1 in the intracellular domain. cDNAs encoding the TLR4s signaled equally to an NF-κB reporter in 293 cells in a ligand-independent manner. When introduced into TLR2/TLR4 double-knockout macrophages, the TLR4 cDNA from BR mice conferred a robust IL-12 response to Py and no IL-10 response. The TLR4 cDNA from PE mice failed to confer a response with either cytokine. These results establish TLR4 as a key mediator of the cytokine response governing susceptibility to tumor induction by Py.     

Effects of point substitutions on the structure of toxic Alzheimer's β-amyloid channels: atomic force microscopy and molecular dynamics simulations

Alzheimer's disease (AD) is a misfolded protein disease characterized by the accumulation of β-amyloid (Aβ) peptide as senile plaques, progressive neurodegeneration, and memory loss. Recent evidence suggests that AD pathology is linked to the destabilization of cellular ionic homeostasis mediated by toxic pores made of Aβ peptides. Understanding the exact nature by which these pores conduct electrical and molecular signals could aid in identifying potential therapeutic targets for the prevention and treatment of AD. Here using atomic force microscopy (AFM) and molecular dynamics (MD) simulations, we compared the imaged pore structures with models to predict channel conformations as a function of amino acid sequence. Site-specific amino acid (AA) substitutions in the wild-type Aβ(1-42) peptide yield information regarding the location and significance of individual AA residues to its characteristic structure-activity relationship. We selected two AAs that our MD simulation predicted to inhibit or permit pore conductance. The substitution of Phe19 with Pro has previously been shown to eliminate conductance in the planar lipid bilayer system. Our MD simulations predict a channel-like shape with a collapsed pore, which is supported by the AFM channel images. We suggest that proline, a known β-sheet breaker, creates a kink in the center of the pore and prevents conductance via blockage. This residue may be a viable target for drug development studies aiming to inhibit Aβ from inducing ionic destabilization toxicity. The substitution of Phe20 with Cys exhibits pore structures indistinguishable from the wild type in AFM images. MD simulations predict site 20 to face the solvated pore. Overall, the mutations support the previously predicted β-sheet-based channel structure.