High cell density cultivation of recombinant Escherichia coli for hirudin variant 1 production

A synthetic medium, TK-25, for high cell density cultivation (HCDC) of Escherichia coli K-12 was modified to support HCDC of strain JM109. By optimizing the culture conditions, the cell concentration of 65 g/l in 14 h was obtained in the optimized medium, namely TK-10, with glucose-fed batch cultivation. When these conditions were further applied for HCDC of E. coli JM109 harboring pUC-based recombinant plasmid which expresses a hirudin variant, HV-1-fused protein under the control of trp promoter, it grew to 24 g/l of dried cells expressed as an inclusion body as 15.9% of the total protein, corresponding to 1908 mg/l hirudin-fused protein.     

Biosynthesis of astaxanthin in tobacco leaves by transplastomic engineering

The natural pigment astaxanthin has attracted much attention because of its beneficial effects on human health, despite its expensive market price. In order to produce astaxanthin, transgenic plants have so far been generated through conventional genetic engineering of Agrobacterium -mediated gene transfer. The results of trials have revealed that the method is far from practicable because of low yields, i.e. instead of astaxanthin, large quantities of the astaxanthin intermediates, including ketocarotenoids, accumulated in the transgenic plants. In the present study, we have overcome this problem, and have succeeded in producing more than 0.5% (dry weight) astaxanthin (more than 70% of total caroteniods) in tobacco leaves, which turns their green color to reddish brown, by expressing both genes encoding CrtW (β-carotene ketolase) and CrtZ (β-carotene hydroxylase) from a marine bacterium Brevundimonas sp., strain SD212, in the chloroplasts. Moreover, the total carotenoid content in the transplastomic tobacco plants was 2.1-fold higher than that of wild-type tobacco. The tobacco transformants also synthesized a novel carotenoid 4-ketoantheraxanthin. There was no significant difference in the size of the aerial part of the plant between the transformants and wild-type plants at the final stage of their growth. The photosynthesis rate of the transformants was also found to be similar to that of wild-type plants under ambient CO₂ concentrations of 1500 μmol photons m⁻² s⁻¹ light intensity.     

Suppression of cell proliferation and deoxyribonucleic acid synthesis in the cloned rat hepatoma H4-II-E cells overexpressing regucalcin.

The role of endogenous regucalcin (RC) in the regulation of cell proliferation was investigated in the cloned rat hepatoma H4-II-E cells overexpressing RC stably. H4-II-E cells were transfected with RC/pCXN2 vector and the multiple neomycin-resistant clones which overexpress stably RC were selected. The RC content of RC/pCXN2-transfected cells used in this study was 19.7-fold as compared with that of the parental wild type H4-II-E cells. Wild type H4-II-E cells, pCXN2 vector-transfected cells (mock type), and RC/pCXN2-transfected cells (transfectants) were cultured for 24, 48, and 72 h in the presence of fetal bovine serum (10% FBS). Cell numbers of wild and mock type were significantly increased with the time course of culture. Cell numbers of transfectants was significantly suppressed as compared with that of wild and mock type. Deoxyribonucleic acid (DNA) synthesis activity in the nuclear fraction of H4-II-E cells was significantly suppressed in transfectants with culture for 12-48 h. The presence of anti-RC monoclonal antibody (10-50 ng/ml) in the reaction mixture caused a significant increase in DNA synthesis activity in the nuclei of wild type and transfectants; this increase was remarkable in transfectants. The effect of anti-RC monoclonal antibody (50 ng/ml) in increasing DNA synthesis activity in transfectants was completely prevented by the addition of regucalcin (1 microM). This study demonstrates that cell proliferation is suppressed in the cloned rat hepatoma H4-II-E overexpressing RC stably.     

Determination of plasma bromvalerylurea and its main metabolite by a simple high-performance liquid chromatographic method and quantitation of bromide by energy dispersive X-ray spectrometry in carbon tetrachloride-intoxicated rats

In the present study, small volumes of plasma were used for the measurement of bromvalerylurea (BVU), its metabolite, 3-methylbutyrylurea (MVU), and bromide in carbon tetrachloride (CCl₄)-treated rats by HPLC–UV and energy dispersive X-ray spectrometry. A liquid–liquid extraction system was also investigated. BVU and MVU were extracted from 100 μl plasma samples in a single-step involving deproteination with 1 M hydrochloric acid using ethenzamide as internal standard. Samples were separated by HPLC in an acetonitrile–8 mM potassium dihydrogenphosphate buffer (35:65, v/v) mobile phase at a flow-rate of 0.4 ml/min on a 15 cm octadecylsilyl column at room temperature. Analytes were detected at a wavelength of 210 nm. The limits of quantitation for BVU, MVU and bromide are 0.1, 0.1 and 50 μg/ml, respectively. The intra-day accuracies over the range of concentrations were 95.8 to 121.1%, 97.2 to 119.7% and 96.2 to 105.8% for BVU, MVU and bromide, respectively. The inter-day accuracies were 97.7 to 115.1%, 98.3 to 111.6% and 98.3 to 102.9% for BVU, MVU and bromide, respectively. The absolute recoveries using tert.-butyl methyl ether are 96–98% for BVU and 95–98% for MVU. The decline in the plasma concentrations of BVU in olive oil-treated rats fitted a one-compartment model and the plasma MVU level reached a peak at around 1.5–2 h and then decreased gradually. The elimination of BVU in CCl₄ (1 ml/kg)-treated rats was delayed and MVU production was less than that in the olive oil-treated group. However, there was no difference in the plasma levels of bromide between CCl₄-treated rats and control rats.     

Chemoprotective effect of diallyl trisulfide from garlic against carbon tetrachloride-induced acute liver injury of rats

Diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS) are principal constituents of garlic oil. We studied the effect of these sulfides on the phase II drug-metabolizing enzymes, and on the rat model of acute liver injury induced by carbon tetrachloride (CCl4). A highly purified form of each sulfide (more than 99% purity) was administered i.p. to rats at a concentration of 10 or 100 micromol/kg body weight for 14 consecutive days. DATS (10 micromol/kg) and DADS at a 10-fold higher dose (100 micromol/kg) significantly increased the activities of glutathione S-transferase (GST) and quinone reductase (QR); whereas DAS did not. In the CCl4-induced acute liver injury model of rats, DATS (10 micromol/kg) significantly suppressed the increase in plasma lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) activities. In conclusion, hepatic phase II enzymes were induced strongly by the trisulfide and weakly by the disulfide, but not by DAS. DATS significantly reduced the liver injury caused by CCl4. DATS may be one of the important factors in garlic oil that protects our body against the injury caused by radical molecules.     

On-column refolding and characterization of soluble human interleukin-15 receptor alpha-chain produced in Escherichia coli

Interleukin-15 receptor alpha-chain (IL-15Ralpha) is a member of the new cytokine receptor family, which possesses the sushi domain. To investigate the biochemical and biophysical characteristics of soluble human IL-15Ralpha (shIL-15Ralpha), shIL-15Ralpha was recombinantly expressed in Escherichia coli. The shIL-15Ralpha containing a six histidine-tag was expressed as inclusion bodies, which were solubilized with urea, immobilized on a Ni-nitrilotriacetic acid column, and refolded by a decreasing gradient of urea concentration. The refolded shIL-15Ralpha exhibited a highly flexible structure, neutralized human interleukin-15-induced cell proliferation effectively, and bound to its ligand with the same affinity as human IL-15Ralpha on the cell surface, as demonstrated by circular dichroism, a cell proliferation assay, and surface plasmon resonance, respectively. Thus, we succeeded in refolding shIL-15Ralpha to an active form on an affinity column.     

PipeOnline 2.0: automated EST processing and functional data sorting

Expressed sequence tags (ESTs) are generated and deposited in the public domain, as redundant, unannotated, single-pass reactions, with virtually no biological content. PipeOnline automatically analyses and transforms large collections of raw DNA-sequence data from chromatograms or FASTA files by calling the quality of bases, screening and removing vector sequences, assembling and rewriting consensus sequences of redundant input files into a unigene EST data set and finally through translation, amino acid sequence similarity searches, annotation of public databases and functional data. PipeOnline generates an annotated database, retaining the processed unigene sequence, clone/file history, alignments with similar sequences, and proposed functional classification, if available. Functional annotation is automatic and based on a novel method that relies on homology of amino acid sequence multiplicity within GenBank records. Records are examined through a function ordered browser or keyword queries with automated export of results. PipeOnline offers customization for individual projects (MyPipeOnline), automated updating and alert service. PipeOnline is available at http://stress-genomics.org.     

Inhibition of hepatitis C virus NS3 protease by peptides derived from complementarity-determining regions (CDRs) of the monoclonal antibody 8D4: tolerance of a CDR peptide to conformational changes of a target

Somatic angiotensin-converting enzyme (ACE) consists of two homologous domains, each domain bearing a catalytic site. Differential scanning calorimetry of the enzyme revealed two distinct thermal transitions with melting points at 55.3 and 70.5 degrees C. which corresponded to denaturation of C- and N-domains, respectively. Different heat stability of the domains underlies the methods of acquiring either single active N-domain or active N-domain with inactive C-domain within parent somatic ACE. Selective denaturation of C-domain supports the hypothesis of independent folding of the two domains within the ACE molecule. Modeling of ACE secondary structure revealed the difference in predicted structures of the two domains, which, in turn, allowed suggestion of the region 29-133 in amino acid sequence of the N-part of the molecule as responsible for thermostability of the N-domain.     

Establishment and characterization of JHUCS-1 cell line derived from carcinosarcoma of the human uterus

The cell line designed JHUCS-1 was established from a carcinosarcoma (malignant mixed mesodermal tumor) of the uterus that was surgically removed from a 57-year-old Japanese woman. We carefully examined the histopathology of the original tumor after the cell line was established and noted differentiation into a neuroendocrine carcinoma within the tumor's epithelial components. Immunohistochemical staining of the tumorous tissue that had been heterotransplanted was positive for Leu7. Additionally, secretary granules were observed in the grafted cells as determined by electron microscopy. These results support the existence of neuroendocrine cells within the JHUCS-1 cell line.