Direct refolding of recombinant human growth differentiation factor 5 for large-scale production process

An efficient downstream process for the production of recombinant human growth differentiation factor 5 (rhGDF5) has been developed for industrial application utilizing a novel "direct refolding" method. In this method, the starting material is an inclusion body produced in Escherichia coli, and the critical step is the direct refolding step that follows directly after solubilization of the inclusion body. rhGDF5 can be refolded at a markedly high concentration of 2.4 mg.ml(-1), which is 24 times that hitherto achieved by the proteins of the TGF-beta superfamily. The refolding yield is 63%, and after purification by diafiltration, isoelectric precipitation and reverse-phase chromatography, the final purification yield is 20% with purity higher than 99%. The yield is more than twice that of a conventionally established process having three chromatography steps and the purity is equivalent. The first pilot-scale trial shows a refolding yield of 51% and a final yield of 11%. The final yield is 1.4 times that of the conventional process, and further optimization at pilot-scale is expected to bring this figure up to or above that of laboratory-scale. As a result, the calculated production cost of rhGDF5 has been reduced dramatically. This type of efficient and simple process is beneficial particularly in the large-scale production of recombinant proteins in which high yield and quality are required.     

Experimental Study of the Relation Between Heat Transfer and Flow Behavior in a Single Microtube

The flow boiling heat transfer in microchannels have become important issue because it is extremely high-performance heat exchanger for electronic devices. For a detailed study on flow boiling heat transfer in a microtube, we have used a transparent heated microtube, which is coated with a thin gold film on its inner wall. The gold film is used as a resistance thermometer to directly evaluate the inner wall temperature averaged over the entire temperature measurement length. At the same time, the transparency of the film enables the observation of fluid behavior. Flow boiling experiments have been carried out using the microtube under the following conditions; mass velocity of 105 kg/m² s, tube diameter of 1 mm, heat flux in the range of 10 ~ 380 kW/m² s, and the test fluid used is ionized water. Under low heat flux conditions, the fluctuations in the inner wall temperature and mass velocity are closely related; the frequency of these fluctuations is the same. However, the fluctuations in the inner wall temperature and heat transfer coefficient are found to be independent of the fluctuation in the mass velocity under high heat flux conditions.     

Inference of common genetic network using fuzzy adaptive resonance theory associated matrix method

Inferring genetic networks from gene expression data is the most challenging work in the post-genomic era. However, most studies tend to show their genetic network inference ability by using artificial data. Here, we developed the fuzzy adaptive resonance theory associated matrix (F-ART matrix) method to infer genetic networks and applied it to experimental time series data, which are gene expression profiles of Saccharomyces cerevisiae responding under oxidative stresses such as diamide, heat shock and H2O2. We preprocessed them using the fuzzy adaptive resonance theory and successfully identified genetic interactions by drawing a 2-dimensional matrix. The identified interactions between diamide and heat shock stress were confirmed to be the common interactions for two stresses, compared with the KEGG metabolic map, BRITE protein interaction map, and gene interaction data of other papers. In the predicted common genetic network, the hit ratio was 60% for the KEGG map. Several gene interactions were also drawn, which have been reported to be important in oxidative stress. This result suggests that F-ART matrix has the potential to function as a new method to extract the common genetic networks of two different stresses using experimental time series microarray data.     

Personalized assessment of oxidative cellular damages associated with diabetes using erythrocytes adhesion assay

Increased generation of reactive oxygen species (ROS) is recognized as a prominent feature of vascular dysfunction with diabetes. For self-monitoring of the risk of diabetic complications, we proposed a simple adhesion test of erythrocytes using positively charged surfaces and its adhesion property was found to correlate HbA_1c level in diabetic patients.     

Gingival and dermal fibroblasts: their similarities and differences revealed from gene expression

Gene expression profiles in normal human gingival and dermal fibroblasts were investigated using DNA microarrays. Their fundamental characteristics were almost identical, but 5% of their genes were uniquely expressed. These results help us to choose an optimal cell source for effective fibroblast-based cell therapy that is dependent on differential gene expression profiles.     

The Use of Transition-Metal Silicides to Reduce the Contact Resistance Between the Electrode and Sintered n-Type Mg2Si

The electrical and thermoelectric characteristics of n-type Mg₂Si equipped with electrodes of Ni and the transition-metal silicides CoSi₂, CrSi₂, TiSi₂, and NiSi were examined. To form the electrodes on the Mg₂Si matrix, a monobloc sintering method, i.e., simultaneous sintering of the electrode material during Mg₂Si sintering, was used. To obtain dense electrodes and to keep an appropriately low sintering temperature for the Mg₂Si matrix, a Ni binder was used for the CoSi₂, CrSi₂, and TiSi₂ monobloc sintering. The mixture ratio between the transition-metal silicide and the Ni was 50:50 in wt.%. The room-temperature I?CV characteristics of the fabricated CoSi₂, CrSi₂, and TiSi₂ electrodes with the Ni binder and NiSi electrodes were considered to be adequate for practical applications in as much as ohmic contacts were obtained. The contact resistance at the Mg₂Si/electrode interface decreased by 35% and 28%, respectively, for the CoSi₂ and CrSi₂ electrodes compared with our standard Ni electrode. The thermoelectric power output was measured at the practical operating temperature of 600?K, with ??T?=?500?K. The observed output powers for 3.0?mm?×?3.0?mm?×?7.5?mm samples equipped with CoSi₂, CrSi₂, and NiSi electrodes were 153?mW, 149?mW, and 125?mW, respectively, representing increases of 27%, 24%, and 4%, respectively, compared with the 120?mW measured for the sample with Ni electrodes.     

Construction of membrane-anchoring fusion protein of Thermococcus kodakaraensis glycerol kinase and its application to repetitive batchwise reactions

We previously demonstrated the stoichiometric conversion of glycerol to glycerol-3-phosphate (G3P) using Escherichia coli recombinants producing the ATP-dependent glycerol kinase of the hyperthermophile Thermococcus kodakaraensis (TkGK) and the polyphosphate kinase of Thermus thermophilus HB27 (TtPPK). TtPPK was associated with the membrane fraction of E. coli recombinants, whereas TkGK was released from the cells during the reaction at 70°C. In this study, TkGK was fused with either TtPPK or an E. coli membrane-intrinsic protein, YedZ, to minimize the heat-induced leakage of TkGK. When the E. coli recombinants having these fusion proteins were incubated at 70°C for 2h, more than 80% of TkGK activity was retained in the heated E. coli cells. However, the yields of G3P production by E. coli having the fusion proteins of TtPPK and TkGK were only less than 35%. Polyphosphate is a strong chelator for metal ions and has an inhibitory effect on TkGK which requires magnesium. Insufficient space between TtPPK and TkGK might enhance the inhibitory effect of polyphosphate on TkGK activity of the fusion protein. The mixture of E. coli cells having TtPPK and those having TkGK fused with YedZ converted 80% of glycerol into G3P. These recombinant cells could be easily recovered from the reaction mixture by centrifugation and repeatedly used without a significant loss of enzyme activities.     

From Terpenoids to Aliphatic Acids: Further Evidence for Late-Instar Switch in Osmeterial Defense as a Characteristic Trait of Swallowtail Butterflies in the Tribe Papilionini

We compared the chemical compositions of the osmeterial secretions of fourth and fifth (last) instars of eight swallowtail species of the tribe Papilionini. Four species (Papilio demoleus, P. polytes, P. paris, and P. macilentus) are Asian Rutaceae-feeding swallowtails. The other four (Chilasa epicydes, C. agestor, P. troilus, and P. glaucus) represent more distant clades within the Papilionini and species with larval hosts in other plant families. We conducted a quantitative analysis for six species, but only qualitative analysis for P. glaucus and C. agestor. In all eight species, regardless of larval host plant, secretions of the fourth instar principally consisted of mono- and sesquiterpene hydrocarbons, whereas those of the fifth instar comprised aliphatic acids and their esters. Consistent with earlier findings, our results suggest that this “heterogeneous” pattern of osmeterial chemistry, not seen in other tribes, may characterize the Papilionini as a whole. Unlike those of most Papilio species, the fourth and fifth instars of Chilasa species resemble each other in body coloration. Thus, the heterogeneous osmeterial pattern is not necessarily associated with color change in papilionid larvae. The major terpenoids identified in fourth instar larval secretions from the six species were α-pinene, sabinene, β-myrcene, limonene, β-phellandrene, (Z)-β-ocimene, (E)-β-ocimene, p-mentha-1,4(8)-diene, β-elemene, β-caryophyllene, (E)-β-farnesene, (3Z,6E)-α-farnesene, (Z)-α-bisabolene, germacrene-A, (E)-α-bisabolene, and germacrene-B. The profiles for individual species differed both qualitatively and quantitatively from one another, and certain species also secreted methyl 3-hydroxy-n-butyrate and oxygenated sesquiterpenes in relatively large proportions. Secretions from fifth instars were composed of varying proportions of isobutyric, 2-methylbutyric, and acetic acids, and methyl and ethyl (minor) esters of both isobutyric and 2-methylbutyric acids. The heterogeneity of osmeterial chemistry in the tribe Papilionini may represent fine-tuning of chemical defense in response to shifting predation pressures as the larvae age and grow.     

In Planta production of immunogenic poliovirus peptide using tobacco mosaic virus-based vector system

The tobacco mosaic virus (TMV) provides an attractive means of producing foreign peptides in plants. In this study, a TMV-based vector was designed such that a fragment encoding 15 amino acids of the poliovirus peptide (PVP) derived from the viral capsid proteins VP3 and VP1 of poliovirus type 1 Sabin was inserted downstream of the six-base 3' context nucleotide sequence of the TMV coat protein (CP) gene. This design allowed readthrough at the amber stop codon, thereby producing the chimeric TMV particle with both intact CP and CP-fusion protein (CP-PVP) in Nicotiana tabacum cv. Samsun infected with the TMV vector. The TMVCP-PVP virus particle induced antibodies against PVP as well as TMVCP in mice after intraperitoneal immunization. These data illustrate the potential of the readthrough translation system with TMVCP for antigen presentation and vaccine production.     

Peptide array-based interaction assay of solid-bound peptides and anchorage-dependant cells and its effectiveness in cell-adhesive peptide design

Peptide array, the designable peptide library covalently synthesized on cellulose support, was applied to assay peptide-cell interaction, between solid-bound peptides and anchorage-dependant cells, to study objective peptide design. As a model case, cell-adhesive peptides that could enhance cell growth as tissue engineering scaffold material, was studied. On the peptide array, the relative cell-adhesion ratio of NIH/3T3 cells was 2.5-fold higher on the RGDS (Arg-Gly-Asp-Ser) peptide spot as compared to the spot with no peptide, thus indicating integrin-mediated peptide-cell interaction. Such strong cell adhesion mediated by the RGDS peptide was easily disrupted by single residue substitution on the peptide array, thus indicating that the sequence recognition accuracy of cells was strictly conserved in our optimized scheme. The observed cellular morphological extension with active actin stress-fiber on the RGD motif-containing peptide supported our strategy that peptide array-based interaction assay of solid-bound peptide and anchorage-dependant cells (PIASPAC) could provide quantitative data on biological peptide-cell interaction. The analysis of 180 peptides obtained from fibronectin type III domain (no. 1447-1629) yielded 18 novel cell-adhesive peptides without the RGD motif. Taken together with the novel candidates, representative rules of ineffective amino acid usage were obtained from non-effective candidate sequences for the effective designing of cell-adhesive peptides. On comparing the amino acid usage of the top 20 and last 20 peptides from the 180 peptides, the following four brief design rules were indicated: (i) Arg or Lys of positively charged amino acids (except His) could enhance cell adhesion, (ii) small hydrophilic amino acids are favored in cell-adhesion peptides, (iii) negatively charged amino acids and small amino acids (except Gly) could reduce cell adhesion, and (iv) Cys and Met could be excluded from the sequence combination since they have less influence on the peptide design. Such rules that are indicative of the nature of the functional peptide sequence can be obtained only by the mass comparison analysis of PIASPAC using peptide array. By following such indicative rules, numerous amino acid combinations can be effectively screened for further examination of novel peptide design.