Development of a highly sensitive immuno-PCR assay for the measurement of α-galactosidase A protein levels in serum and plasma.

Fabry disease is an X-linked genetic disorder caused by defects in the α-galactosidase A (GLA) gene, and heterogeneous mutations lead to quantitative and/or qualitative defects in GLA protein in male patients with Fabry disease. Random X-chromosomal inactivation modifies the clinical and biochemical features of female patients with Fabry disease. Functional polymorphisms have been frequently reported in recent times, and these increase the difficulty of understanding the pathogenetic basis of the disease. To date, GLA protein level has been measured using an enzyme-linked immunosorbent assay (ELISA). However, ELISA is not highly sensitive due to the high background noise. In this paper, we introduce a novel application of the immuno-polymerase chain reaction (PCR) method (termed Multiple Simultaneous Tag [MUSTag]) for measurement of the GLA protein level in blood samples. We compared the sensitivities of the MUSTag method with plates or magnetic beads with those of ELISA for recombinant human GLA and found that the apparent maximal sensitivity was higher for the former than for the latter. We then measured the GLA concentrations in serum and plasma from male patients with classic Fabry disease (Male Fabry), females with Fabry disease (Female Fabry), male subjects harboring the functional polymorphism p.E66Q (E66Q), and control (Control) subjects. Our results revealed that compared to the MUSTag plate and ELISA, the MUSTag beads assay afforded a clearer estimation of the GLA protein levels in the serum and plasma with minimal or no background noise, although all the methods could differentiate between the Male Fabry, E66Q, and Control groups. The Female Fabry group showed characteristic heterogeneity, which was consistent with the X-linked inheritance. This novel method is expected to be useful for the sensitive determination of GLA level in blood and elucidation of the pathogenetic basis of Fabry disease.

Protein-based open sandwich immuno-PCR for sensitive detection of small biomarkers.

Open sandwich (OS) immunoassay utilizes antigen-dependent stabilization of an antibody variable region to quantify various antigens, enabling noncompetitive detection of small molecules with a broad working range. For further improvement of its sensitivity, OS Immuno-PCR was attempted with recombinant fusion proteins. The maltose binding protein-fused heavy chain variable region (MBP-V(H)) of an antibody that recognizes the C-terminal fragment of human osteocalcin (bone Gla protein, BGP), a biomarker for bone-related diseases, was immobilized onto microplate wells, and the antigen together with streptavidin (SA)-fused light chain variable region of the same antibody (SA-V(L)) was added and incubated. The amount of immobilized SA-V(L) was quantified by tethered biotinylated DNA, which was used to estimate the amount of antigen by realtime PCR. When BGP C-terminal peptide was detected, the limit of detection was 100 fg/mL, which was superior than that of our previously reported phage-based OS Immuno-PCR. The developed OS Immuno-PCR system will be useful for the detection of small molecule biomarkers for disease prevention.

Cytokine marker measurement in human neuroblastoma cells with supersensitive and multiplex assay: MUSTag technology.

BACKGROUND: Recently, various sets of protein -biomarkers have been discovered in important diseases such as cancers, brain stroke and heart attack. However, clinical validation is difficult and time-consuming by individual assays or because of very low concentrations at early stages of the diseases. We have developed assay technology through an innovative modification of the immuno-PCR method for the super-sensitive and multiplex detection of target biomarkers. METHODS: In the assay technology, each different oligo-tag simultaneously detects multiplex protein targets with extremely high-level sensitivity in a dose-dependent manner by qRT-PCR (maximum: three plexes). In this study, we measured specific secreted protein concentrations in the culture supernatant of a 24-h culture of transfected SH-SY5Y cells with MUSTag. RESULTS: There was a significant increase in the protein level of tumor necrosis factor (TNF)-α measured with extremely high-level sensitivity (≥10 pg/mL). Compared with negative controls, the levels of TNF-α increased from 16.9 to 28.1 pg/mL (p = 0.011). CONCLUSION: We suggest that our assay technology might be of clinical value in treating patients with cancer, cerebral ischemia, or patients who need a prompt and predictive diagnosis for adequate treatment.

Rapid typing of influenza viruses using super high-speed quantitative real-time PCR.

The development of a rapid and sensitive system for detecting influenza viruses is a high priority for controlling future epidemics and pandemics. Quantitative real-time PCR is often used for detecting various kinds of viruses; however, it requires more than 2h per run. Detection assays were performed with super high-speed RT-PCR (SHRT-PCR) developed according to a newly designed heating system. The new method uses a high-speed reaction (18s/cycle; 40 cycles in less than 20min) for typing influenza viruses. The detection limit of SHRT-PCR was 1 copy/reaction and 10(-1) plaque-forming unit/reaction for viruses in culture supernatants during 20min. Using SHRT-PCR, 86 strains of influenza viruses isolated by the Tokyo Metropolitan Institute of Public Health were tested; the results showed 100% sensitivity and specificity for each influenza A and B virus, and swine-origin influenza virus. Twenty-seven swabs collected from the pharyngeal mucosa of outpatients were also tested, showing positive signs for influenza virus on an immunochromatographic assay; the results between SHRT-PCR and immunochromatography exhibited 100% agreement for both positive and negative results. The rapid reaction time and high sensitivity of SHRT-PCR makes this technique well suited for monitoring epidemics and pre-pandemic influenza outbreaks.

Int6/eIF3e silencing promotes functional blood vessel outgrowth and enhances wound healing by upregulating hypoxia-induced factor 2alpha expression.

BACKGROUND: We previously identified INT6/eIF3e as a novel regulator of hypoxia-inducible factor 2alpha (HIF2alpha) activity. Small interfering RNA (siRNA)-Int6 adequately stabilized HIF2alpha, even under normoxic conditions, and thereby enhanced the expression of several angiogenic factors in vitro, suggesting that siRNA-Int6 may induce angiogenesis in vivo. METHODS AND RESULTS: We demonstrated a 6- to 8-fold enhanced formation of normal arteries and veins in the subcutaneous regions of adult mice 5 days after a single siRNA-Int6 application. Subcutaneous fibroblasts were identified as the major source of secreted angiogenic factors that led to the formation of functional vessels during Int6 silencing. Fibroblasts transfected ex vivo with siRNA-Int6 induced potent neoangiogenesis when transplanted into a subcutaneous region of nude mice. Application of siRNA-Int6 promoted neoangiogenesis in the area surrounding the injury in wound healing models, including genetically diabetic mice, thereby accelerating the closure of the injury. HIF2alpha accumulation caused by siRNA-Int6 was confirmed as the unequivocal cause of the angiogenesis by an in vivo angiogenesis assay. Further analysis of the Int6 silencing-induced neoangiogenesis revealed that a negative feedback regulation of HIF2alpha stability was caused by HIF2alpha-induced transcription of Int6 via hypoxia-response elements in its promoter. Thus, siRNA-Int6 temporarily facilitates an accumulation of HIF2alpha protein, leading to hypoxia-independent transcription of angiogenic factors and concomitant neoangiogenesis. CONCLUSIONS: We suggest that the pathway involving INT6/HIF2alpha acts as a hypoxia-independent master switch of functional angiogenesis; therefore, siRNA-Int6 application might be of clinical value in treating ischemic diseases such as heart and brain ischemia, skin injury, and diseases involving obstructed vessels.

Gene expression profiling and functional assays of activated hepatic stellate cells suggest that myocardin has a role in activation.

BACKGROUND: Myofibroblast-like cells derived from transdifferentiated hepatic stellate cells (HSC) play a central role in scar formation that leads to liver fibrosis. The molecular mechanisms underlying this process are not fully understood. AIM: Our aim was to identify genes that are differentially regulated by HSC activation and to explore their function. METHODS: Using oligonucleotide microarrays, we performed transcriptional analysis of the human HSC cell line, LI90, cultured on Matrigel. Microarray data were validated by quantitative real-time polymerase chain reaction and Western blotting. The function of myocardin was assessed by myocardin RNAi and overexpression. RESULTS: Examination of Matrigel-induced deactivation of LI90 cells revealed marked downregulation of myocardin, an important transcriptional regulator in smooth and cardiac muscle development. Small interfering RNA-mediated suppression of myocardin expression in both activated LI90 and rat activated HSC resulted in loss of the phenotypic characteristics of myofibroblasts and significantly impaired the production of activated HSC markers, such as alpha-smooth muscle actin and extracellular matrix proteins like type I collagen. Overexpression of myocardin led to the upregulation of these marker genes. Myocardin was upregulated in rat primary HSC during in vitro activation and in the fibrotic liver of a dimethylnitrosamine-induced fibrosis rat model. CONCLUSIONS: This study demonstrates that myocardin is involved in the activation of HSC; myocardin may serve as a novel therapeutic target in the treatment of liver fibrosis.

[Protein biomarker measurement and simple/rapid diagnostics with supersensitive and multiplex assay, MUSTag technology].

Recently, we face the rapid progression of an aging population, and so the importance of preventive medicine is growing. We would all like to pursue a healthy life during old age through effective treatment on the basis of the early detection of diseases. In this situation, we have developed MUSTag (Multiple Simultaneous Tag) assay technology through an innovative modification of the immuno-PCR method for the super-sensitive and multiplex detection of target biomarkers. In MUSTag technology, each different oligo-tag simultaneously detects multiplex protein targets with extremely high-level sensitivity (more than 10 fg(10(-15) g)/ml) in a dose-dependent manner by qRT-PCR (maximum: 3 plexes). Herein we report our recent results of multiple cytokine assays or disease-specific biomarker assays using MUSTag technology, and, further, clinical results from patients with cancer, ischemic brain, or heart attack, who need a prompt and predictive diagnosis for adequate treatment.

[Clinical application of supersensitive and multiplex assay, MUSTag technology].

Recently, various sets of protein biomarkers have been discovered in important diseases such as cancers, brain stroke, heart attack, diabetes, and so on. Many of these biomarkers are expected to be extremely valuable as targets for clinical diagnosis and drug development; however, the clinical validation is difficult and time-consuming by individual assays or due to very low concentration in an early stage of disease. For the super-sensitive and multiplex detection of target biomarkers, we have developed MUSTag (Multiple Simultaneous Tag) assay technology with innovative modification of the immuno-PCR method. In MUSTag technology, specific antibodies against several important biomarkers were linked to 100-300bp long oligonucleotides as detection tags. Each different oligo-tag simultaneously detects multiplex protein targets with extremely high sensitivity(more than 10 fg (10(-15) g)/ml) in a dose-dependent manner by qRT-PCR-based (maximum 3 plexes) or capillary electrophoretic amplification (over 30 plexes). Here we report our recent results of multiple cytokine assay or disease-specific biomarker assay using MUSTag technology, and further, clinical results from patients with cancers, ischemic brain or heart attack, who need prompt and predictive diagnosis for adequate treatment.

X-linked inhibitor of apoptosis functions as ubiquitin ligase toward mature caspase-9 and cytosolic Smac/DIABLO.

Members of the IAP (inhibitor of apoptosis) family function as anti-apoptotic proteins by binding directly to caspase-3, -7, and -9 to inhibit their activities. During apoptosis, the activities of IAPs are relieved by a second mitochondria-derived caspase activator, named Smac/DIABLO. Some IAPs have a C-terminal RING finger domain that has been identified as the essential motif for the activity of ubiquitin ligase (E3). Here we show that X-linked IAP (XIAP) mediates the polyubiquitination of caspase-9 and Smac. The large subunit of mature caspase-9 was polyubiquitinated by XIAP in vitro, while procaspase-9 was not. Furthermore, the polyubiquitinated form of caspase-9 accumulated in an XIAP-dependent manner in intact cells. The ubiquitination of caspase-9 was significantly inhibited in the presence of mature Smac, whereas XIAP was also found to promote the polyubiquitination of cytosolic Smac both in vitro and in intact cells. These ubiquitination reactions require the RING finger domain of XIAP. These findings suggest that XIAP functions as ubiquitin ligase toward mature caspase-9 and Smac to inhibit apoptosis.