LecT-Hepa facilitates estimating treatment outcome during interferon therapy in chronic hepatitis C patients.

BACKGROUND: A combination treatment of interferon and ribavirin is the standard and the commonly used treatment for chronic hepatitis C (CHC). Developing noninvasive tests like serum indicators that can predict treatment outcome at an early stage of therapy is beneficial for individualized treatment and management of CHC. A glyco-indicator based on the glyco-alteration of serum α1-acid glycoprotein, LecT-Hepa, was discovered by glycomics technologies as a robust indicator of liver fibrosis. Here, we investigated the clinical utility of LecT-Hepa for evaluation of treatment outcome. RESULTS: Firstly, ninety-seven patients with CHC were used for comparison of LecT-Hepa in serum and plasma. We found no significant difference in the concentrations of LecT-Hepa in serum and plasma. And then, 213 serum specimens from 45 patients who received 48 weeks of treatment with interferon and ribavirin were followed up for 96 weeks, and were used for evaluation of the role of LecT-Hepa. We found that LecT-Hepa might reflect the change in fibrosis regression during the treatment process. Moreover, the change of LecT-Hepa at the first 12 weeks of treatment could already predict the antiviral treatment response, which was more superior to FIB-4 index and aspartate aminotransferase-to-platelet ratio index (APRI) in this study. CONCLUSIONS: These results provide a new perspective that serum glycoprotein could be used as a joint diagnosis indicator for estimation treatment outcome of viral hepatitis at earlier stage of therapy.

Comparison of LecT-Hepa and FibroScan for assessment of liver fibrosis in hepatitis B virus infected patients with different ALT levels.

BACKGROUND: FibroScan is one of the noninvasive techniques based on the transient elastography that can assess the progression of liver fibrosis in chronic hepatitis patients in daily clinical practice. Recently, LecT-Hepa was validated as a serological glycomarker correlating well with the fibrosis stage determined by liver biopsy, and was superior to many other noninvasive biochemical markers and tests. We compared the reliability of LecT-Hepa with that of FibroScan for evaluation of liver fibrosis. METHODS: The effects of increased alanine aminotransferase (ALT) activities on LecT-Hepa and FibroScan were investigated. RESULTS: The areas under the receiver-operating characteristic curves, sensitivity and specificity for detecting cirrhosis, which is one of the outcomes of fibrosis estimation, were 0.82, 72.5% and 78.2% of LecT-Hepa, 0.85, 87.0% and 74.1% of FibroScan; these did not differ significantly. The count distribution of LecT-Hepa in non-cirrhosis group or cirrhosis group did not differ between the patients grouped according to their ALT levels, whereas that of FibroScan was substantially affected. CONCLUSION: LecT-Hepa was confirmed as a reliable noninvasive test for the evaluation of liver fibrosis in hepatitis B virus-infected patients with comparable performance to that of FibroScan and proved to be unaffected by inflammation.

Binding affinity of full-length and extracellular domains of recombinant human (pro)renin receptor to human renin when expressed in the fat body and hemolymph of silkworm larvae.

Transmembrane domains of some receptors have been found to be very important in the process of constitutive oligomerization, and in the stability and functioning of the receptor. In this study, full-length of human (pro)renin receptor (hPRR) and hPRR lacking cytoplasmic domain (hPRR-DeltaCD) were expressed in fat body of silkworm larvae, and the extracellular domain of hPRR (hPRR-DeltaTMDeltaCD) in hemolymph. Three forms of hPRR were used for investigation of the interaction between receptor and ligand using surface plasmon resonance (SPR). As a result, the cytoplasmic domain was not an essential requirement for binding affinity, but the transmembrane domain of hPRR was indispensable in the formation of functional hPRR. The dissociation equilibrium constants (K(D)) of purified hPRR and hPRR-DeltaCD were estimated to be 46 nM and 330 nM, respectively. No evidence of binding by the extracellular domain of hPRR located in hemolymph was found. However, the solubilized microsomal fraction of the extracellular domain of hPRR expressed in the fat body showed specific affinity, but lost its binding affinity after purification. Its binding affinity was recovered by mixing microsomal fraction of mock-injected fat body to the purified extracellular domain. It is probable that an artificial transmembrane domain stabilizes the extracellular domain of hPRR and native conformation may be structurally recovered. To our knowledge, these are the first findings describing the interaction of transmembrane and extracellular domains of hPRR with ligand and this may help towards the understanding of binding affinity of hPRR to ligand.

Expression of protein complex comprising the human prorenin and (pro)renin receptor in silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmids for improving biological function.

Three forms of recombinant protein complexes comprising the human prorenin (hPro) and (pro)renin receptor (hPRR) (hPRR/prorenin) were successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmids. They were localized in the fat body cells and formed a prorenin-bound hPRR complex. The expressed levels of hPro and hPRR were similar judging from Western blotting. The hPRR/prorenin complex containing 40 mug of hPRR (yield, 43%) and 30 mug of hPro (yield, 34%) was purified from 15 silkworm larvae by a series of purification using anti-FLAG and Strep-Tactin affinity chromatography. The renin activity of the purified hPRR/prorenin complex was 3.8-fold that of the mixture of hPRR and hPro expressed individually in vitro judging from the renin assay. These results show that the unstable transmembrane protein, hPRR, was coexpressed stably with ligand, hPro, and formed a stable protein, hPRR/prorenin complex that showed a high catalytic active form.

Localization of human (pro)renin receptor lacking the transmembrane domain on budded baculovirus of Autographa californica multiple nucleopolyhedrovirus.

Human (pro)renin receptor (hPRR), a construct with native transmembrane and cytoplasmic domains (hPRR-wTM), and hPRR lacking both (hPRR-w/oTM) were expressed using insect cells. The hPRR-wTM was expressed in the peripheral domains of the nucleus in infected Sf-9 cells, and its localization was observed in endoplasmic reticulum (ER). However, it could not be extracted from recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) by Triton X-100 treatment at 4 degrees C. In contrast, hPRR-w/oTM was observed in punctate domains in the cytoplasm of infected Sf-9 cells, but intracellular hPRR-w/oTM did not co-localize in the Golgi apparatus and lysosomes. This indicates that hPRR-wTM and hPRR-w/oTM is localized in the ER and cytoplasmic organelles of Sf-9 cell, respectively. Moreover, the localization of hPRR-w/oTM in budded baculovirus of recombinant AcMNPV was confirmed by Western blotting. This is the first finding of the association of a foreign protein lacking a transmembrane domain with a baculovirus. If this finding is available for double displaying system, being capable of expression on the envelope and the capsid of baculovirus, it will lead to new methodology of baculovirus display system for tissue- and cell-specific targeting and intracellular targeting.

Expression of functional human (pro)renin receptor in silkworm (Bombyx mori) larvae using BmMNPV bacmid.

The circulating RA (renin-angiotensin) system is essential for the regulation of blood pressure and electrolyte balance. Recently, plasma prorenin has been reported to significantly increase its level in diabetes and to be possibly non-proteolytically activated by binding to the PRR [(pro)renin receptor] on the cell membrane reported in several tissues during circulation. Although many pathological aspects have been researched, there is a lack of sufficient information on the biochemical structure and biological function of this hPRR (human PRR) because of the difficulty in increasing hPRR expression. In the present study, GFP(uv)-hPRR (hPRR fused with green fluorescence protein when excited with long-wave UV light) was successfully expressed by using BmMNPV (Bombyx mori multiple nucleopolyhedrovirus) bacmid DNA in silkworm (Bombyx mori) larvae. Some of the hPRR was expressed in the haemolymph of silkworm larvae and some of the hPRR was located in the fat body of silkworm larvae. The binding ability of hPRR expressed in the haemolymph and fat body with renin or prorenin was analysed by ELISA and surface plasmon resonance using a biosensor respectively. These binding assays suggest that the expressed hPRR has a functional bioactivity. hPRR preparation in silkworm larvae would, therefore, be useful for biochemical and biomedical researches related to PRR.

Efficient production of fatty acid methyl ester from waste activated bleaching earth using diesel oil as organic solvent.

Fatty acid methyl ester (FAME) production from waste activated bleaching earth (ABE) discarded by the crude oil refining industry was investigated using fossil fuel as a solvent in the esterification of triglycerides. Lipase from Candida cylindracea showed the highest stability in diesel oil. Using diesel oil as a solvent, 3 h was sufficient to obtain a yield of approximately 100% of FAME in the presence of 10% lipase from waste ABE. Kerosene was also a good solvent in the esterification of triglycerides embedded in the waste ABE. Fuel analysis showed that the FAME produced using diesel oil as a solvent complied with the Japanese diesel standard and the 10% residual carbon amount was lower than that of FAME produced using other solvents. Use of diesel oil as solvent in the FAME production from the waste ABE simplified the process, because there was no need to separate the organic solvent from the FAME-solvent mixture. These results demonstrate a promising reutilization method for the production of FAME, for use as a biodiesel, from industrial waste resources containing waste vegetable oils.