Critical Point Mutations for Hepatitis C Virus NS3 Proteinase

AbstractThe hepatitis C virus NS3 proteinase plays an essential role in processing of HCV nonstructural precursor polyprotein. To detect its processing activity, we developed a simpletrans-cleavage assay. Two recombinant plasmids expressing the NS3 proteinase region and a chimeric substrate polyprotein containing the NS5A/5B cleavage site between maltose binding protein and protein A were co-introduced intoEscherichia colicells. The proteinase processed the substrate at the single site during their polyprotein expression. Deletion analysis indicated that the functionally minimal domain of the NS3 proteinase was composed of 146 amino acids, 1059 to 1204. We isolated several cDNA clones encoding the functional domain of the NS3 proteinase from the sera of patients chronically infected with HCV and determined their proteinase activity by thistrans-cleavage assay. Both active and inactive clones existed in the same patients. Comparative sequence analyses of these clones suggested that certain point mutations seemed to be related to the loss of proteolytic activity. This was confirmed by back mutation experiments. Among the critical mutations, Pro-1168 to Thr and Arg-1135 to Gly were intriguing. These amino acids, which are situated near the oxyanion hole, seem to be essential for maintaining the conformation of the active center of the NS3 proteinase

AKARI Infrared Camera Survey of the Large Magellanic Cloud. I. Point Source Catalog

We present a near- to mid-infrared point source catalog of 5 photometric bands at 3.2, 7, 11, 15 and 24 um for a 10 deg2 area of the Large Magellanic Cloud (LMC) obtained with the Infrared Camera (IRC) onboard the AKARI satellite. To cover the survey area the observations were carried out at 3 separate seasons from 2006 May to June, 2006 October to December, and 2007 March to July. The 10-sigma limiting magnitudes of the present survey are 17.9, 13.8, 12.4, 9.9, and 8.6 mag at 3.2, 7, 11, 15 and 24 um, respectively. The photometric accuracy is estimated to be about 0.1 mag at 3.2 um and 0.06--0.07 mag in the other bands. The position accuracy is 0.3" at 3.2, 7 and 11um and 1.0" at 15 and 24 um. The sensitivities at 3.2, 7, and 24 um are roughly comparable to those of the Spitzer SAGE LMC point source catalog, while the AKARI catalog provides the data at 11 and 15 um, covering the mid-infrared spectral range contiguously. Two types of catalog are provided: a Catalog and an Archive. The Archive contains all the detected sources, while the Catalog only includes the sources that have a counterpart in the Spitzer SAGE point source catalog. The Archive contains about 650,000, 140,000, 97,000, 43,000, and 52,000 sources at 3.2, 7, 11, 15, and 24 um, respectively. Based on the catalog, we discuss the luminosity functions at each band, the color-color diagram, and the color-magnitude diagram using the 3.2, 7, and 11 um band data. Stars without circumstellar envelopes, dusty C-rich and O-rich stars, young stellar objects, and background galaxies are located at distinct regions in the diagrams, suggesting that the present catalog is useful for the classification of objects towards the LMC.Comment: 59 pages, 12 figures, accepted for the Astronomical Journa

Research Report on Miyako Ryukyuan : General Study for Research and Conservation of Endangered Dialects in Japan

National Institute for Japanese Language and LinguisticsFrench National Centre for Scientific ResearchKyoto UniversityHiroshima UniversityUniversity of the RyukyusHokusei Gakuen UniversityHitotsubashi UniversityHitotsubashi UniversityHitotsubashi UniversityFirst Published: August 1, 2012 (in Japanese

Role of Elf3 in diabetic nephropathy

Diabetic nephropathy (DN) is among the most serious complications of diabetes mellitus, and often leads to end-stage renal disease ultimately requiring dialysis or renal transplantation. The loss of podocytes has been reported to have a role in the onset and progression of DN. Here, we addressed the activation mechanism of Smad3 signaling in podocytes. Expression of RII and activation of Smad3 were induced by AGE exposure (P<0.05). Reduction of the activation of RII-Smad3 signaling ameliorated podocyte injuries in Smad3-knockout diabetic mice. The bone morphogenetic protein 4 (BMP4) significantly regulated activation of RII-Smad3 signalings (P<0.05). Moreover, the epithelium-specific transcription factor, Elf3was induced by AGE stimulation and, subsequently, upregulated RII expression in cultured podocytes. Induction of Elf3 and activation of RII-Smad3 signaling, leading to a decrease in WT1 expression, were observed in podocytes in diabetic human kidneys. Moreover, AGE treatment induced the secretion of Elf3-containing exosomes from cultured podocytes, which was dependent on the activation of the TGF-β-Smad3 signaling pathway. In addition, exosomal Elf3 protein in urine could be measured only in urinary exosomes from patients with DN. The appearance of urinary exosomal Elf3 protein in patients with DN suggested the existence of irreversible injuries in podocytes. The rate of decline in the estimated Glomerular Filtration Rate (eGFR) after measurement of urinary exosomal Elf3 protein levels in patients with DN (R2 = 0.7259) might be useful as an early non-invasive marker for podocyte injuries in DN

Calibration of the AKARI Far-Infrared Imaging Fourier Transform Spectrometer

The Far-Infrared Surveyor (FIS) onboard the AKARI satellite has a spectroscopic capability provided by a Fourier transform spectrometer (FIS-FTS). FIS-FTS is the first space-borne imaging FTS dedicated to far-infrared astronomical observations. We describe the calibration process of the FIS-FTS and discuss its accuracy and reliability. The calibration is based on the observational data of bright astronomical sources as well as two instrumental sources. We have compared the FIS-FTS spectra with the spectra obtained from the Long Wavelength Spectrometer (LWS) of the Infrared Space Observatory (ISO) having a similar spectral coverage. The present calibration method accurately reproduces the spectra of several solar system objects having a reliable spectral model. Under this condition the relative uncertainty of the calibration of the continuum is estimated to be $\pm$ 15% for SW, $\pm$ 10% for 70-85 cm^(-1) of LW, and $\pm$ 20% for 60-70 cm^(-1) of LW; and the absolute uncertainty is estimated to be +35/-55% for SW, +35/-55% for 70-85 cm^(-1) of LW, and +40/-60% for 60-70 cm^(-1) of LW. These values are confirmed by comparison with theoretical models and previous observations by the ISO/LWS.Comment: 22 pages, 10 figure

Assessment of Macular Function by Multifocal Electroretinography and Optical Coherence Tomography before and after Panretinal Photocoagulation in Diabetic Retinopathy

We evaluated macular function before and after panretinal photocoagulation (PRP) in diabetic retinopathy using a multifocal electroretinogram (mfERG) and optical coherence tomogram (OCT). In mfERGs, the 1st positive wave (P1) minus the 1st negative wave (N1) amplitude (P1 ? N1 amplitude), the P1 peak latency and the response density were measured in 7, 19, 37 and 103 hexagonal areas or elements (Areas 1, 2, 3 and 4) within a central radius of 5, 7, 10 and 20 degrees, respectively. The mean retinal thickness was estimated from 9 calculation points at the foveal region within 5 degrees; the central and each of the other 4 points at a distance of 250 ?m and 500 ?m from the central por tion on horizontal and vertical sections on OCT. The P1 peak latencies from the 4 areas were remarkably prolonged in 14 eyes of 9 patients with preproliferative or early proliferative diabetic retinopathy showing no clinically significant macular edema before PRP as compared with those in 15 normal control eyes, without a tendency of recovery throughout the course after PRP except for area 1. The P1-N1 amplitudes and the mean response density levels from the 4 areas were remarkably decreased in the diabetic eyes before PRP as compared with those in the control eyes, followed by a maximum decrease in both parameters at 3 months after PRP. However, remarkable recoveries were detected in both decreased parameters from the 4 areas at 6 months after PRP. The mean foveal retinal thickness on OCT was remarkably increased in the diabetic eyes before PRP as compared with the thickness in 16 normal control eyes. Most remarkably, a transient increase in thickness was detected in diabetic eyes 1 month after PRP, followed by a tendency of recovery 3 to 6 months after PRP. These results indicate that mfERG and OCT examinations are useful in the assessment of macular function before and after PRP in diabetic retinopathy, especially within 5 degrees of the central portion, and that the effects of PRP on macular function in this entity seem to be reversible at the foveal region, although we need to do further investigation in relation to the outcome of visual acuity

Diagnosis of renal AL amyloidosis using DAPI

Amyloidosis is often overlooked because its clinical manifestations can mimic those of more-common diseases. It is important to get a precise diagnosis as early as possible for the prevention of further organ damages. Amyloidosis is a disorder caused by deposition of insoluble abnormal amyloid. The kidney is a frequent site of amyloid deposition. The amyloid fibrils have a characteristic appearance and generate birefringence under polarized light when stained with the Congo red dye. Classification of amyloidosis is based on the precursor protein that forms the amyloid fibrils and the distribution of amyloid deposits as either systemic or localized. Involvement of amyloid fibrils in kidneys mainly occurs as amyloid light-chain (AL) or amyloid A (AA) amyloidosis. The potassium permanganate method with Congo red staining was once used widely to discriminate AL and AA amyloidoses, but thismethod has a problem of false positive results. We found that extracellular and cytoplasmic glomerular 4’,6-diamidino-2-phenylindole (DAPI)-positive areas were clearly consistent with amyloid deposition in AL amyloidosis. In contrast, the overlapping staining was not seen in AA amyloidosis. Therefore, we propose that DAPI staining readily distinguishes AL renal amyloidosis from AA renal amyloidosis as a simple and reproducible histochemical method