Identification of a methanol-inducible promoter from Rhodococcus erythropolis PR4 and its use as an expression vector

The genus Rhodococcus exhibits a broad range of catalytic activity and is tolerant to various kinds of organic solvents. This property makes rhodococci suitable for use as a whole-cell catalyst. Various tools for genetic engineering have been developed to use Rhodococcus erythropolis as a host for bioconversion. In this study, we investigated the protein expression responses of R. erythropolis strains and found that isocitrate lyase production in R. erythropolis PR4 (ICL(Re)) was induced by methanol. By analyzing the regulation mechanisms of icl(Re) expression, the ~200-bp upstream region from the first nucleotide of the translation initiation codon of icl(Re) was shown to be sufficient for the methanol-inducible expression. Also, the ~100-bp upstream region exhibited strong constitutive promoter activity by an unknown mechanism(s). By investigating proteins that bound to the upstream region of icl(Re)in vitro, a RamB homologue of R. erythropolis PR4 (RamB(Re)) was identified. Moreover, 2 putative RamB(Re) binding sites were identified in the upstream region of icl(Re) through pull-down assays. A ramB(Re) knockout experiment suggested that RamB(Re) negatively controlled the expression of icl(Re) and that RamB(Re) regulation was dependent on the availability of a carbon source. On the basis of these findings, we were able to create novel methanol-inducible and strong constitutive expression vectors.     

Application of femtosecond laser ablation for detaching grown protein crystals from glass capillary tube

We developed a novel technique for detaching protein crystals from glass capillary tube using the counter diffusion crystallization technique by femtosecond laser irradiation. X-ray diffraction analysis demonstrated that femtosecond laser irradiation has little effect on crystallinity. This technique will contribute to progress in structural genomics as a powerful tool.     

Ammonia assimilation in Klebsiella pneumoniae F-5-2 that can utilize ammonium and nitrate ions simultaneously: purification and characterization of glutamate dehydrogenase and glutamine synthetase

The two ammonia-assimilating enzymes glutamate dehydrogenase (GDH; EC 1.4.1.4) and glutamine synthetase (GS; EC 6.3.1.2) were synthesized steadily during the cell growth of Klebsiella pneumoniae F-5-2 that can utilize NH4+ and NO3- simultaneously under aerobic conditions. The enzymes were purified to homogeneity from cell extracts and characterized. The molecular mass of the purified GDH was 300 kDa with six identical 52-kDa subunits. GDH showed its maximal activity (aminating) at pH 8.0 and was stable between pHs 5.5 and 11.5. The enzyme was NADP-specific and strongly inhibited by Ag+. It catalyzed the amination of 2-ketovalerate, 2-ketoadipate, and 2-ketobutyrate, in addition to 2-ketoglutarate. The purified GS has a molecular mass of 470 kDa with eight identical 60-kDa subunits. GS showed its maximal activity at pH 8.0 and was stable between pHs 6.0 and 7.0. The enzyme was strongly inhibited by Fe3+, Hg2+, and Cu2+.     

Novel episomal vectors and a highly efficient transformation procedure for the fission yeast Schizosaccharomyces japonicus

Schizosaccharomyces japonicus is a fission yeast for which new genetic tools have recently been developed. Here, we report novel plasmid vectors with high transformation efficiency and an electroporation method for Sz. japonicus. We isolated 44 replicating segments from 12 166 transformants of Sz. japonicus genomic fragments and found a chromosomal fragment, RS1, as a new replicating sequence that conferred high transformation activity to Sz. japonicus cells. This sequence was cloned into a pUC19 vector with ura4⁺ of Sz. pombe (pSJU11) or the kan gene on the kanMX6 module (pSJK11) as selection markers. These plasmids transformed Sz. japonicus cells in the early‐log phase by electroporation at a frequency of 123 cfu/µg for pSJK11 and 301 cfu/µg for pSJU11, which were higher than previously reported autonomously replicating sequences. Although a portion of plasmids remained in host cells by integration into the chromosome via RS1 segment, the plasmids could be recovered from transformants. The plasmid copy number was estimated to be 1.88 copies per cell by Southern blot analysis using a Sz. pombe ura4⁺ probe. The plasmid containing ade6⁺ suppressed the auxotrophic growth of the ade6‐domE mutant, indicating that the plasmid would be useful for suppressor screening and complementation assays in Sz. japonicus. Furthermore, pSJU11 transformed Sz. pombe cells with the same frequency as the pREP2 plasmid. This study is a report to demonstrate practical use of episomal plasmid vectors for genetic research in Sz. japonicus. RS1 has been submitted to the DDBJ/EMBL/GenBank database (Accession No. AB547343). Copyright © 2010 John Wiley & Sons, Ltd.     

Supplementation of the diet of dairy cows with trehalose results in milk with low lipid peroxide and high antioxidant content

The objective of this study was to investigate the effect of dietary supplementation with the disaccharides trehalose and cellobiose on antioxidant activity in rumen fluid, blood, and milk of dairy cows. Nine Holstein dairy cows housed in a free-stall barn were divided into 3 groups, with each group receiving a different dietary treatment (a control diet, a 1% trehalose-supplemented diet, or a 1% cellobiose-supplemented diet) following a 3 × 3 Latin square design. Feed intake and milk production increased in cows receiving the trehalose-supplemented diet compared with those receiving the control and cellobiose-supplemented diets. The total protozoa numbers in the rumen fluid of cows fed trehalose- or cellobiose-supplemented diets were greater than those of the control group. The C18:0 and C18:1 fatty acid content was increased in the milk of cows fed the trehalose-supplemented diet compared with that of the control group, and the C18:3n-3 fatty acid content in the milk of cows fed the cellobiose-supplemented diet was less than that of the control group. Plasma biochemical parameters were unchanged among the different treatments. In rumen fluid, 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity and superoxide dismutase activity were increased 2 h after feeding in cows receiving the cellobiose-supplemented diet compared with the control group, and the concentration of thiobarbituric acid reactive substances in the rumen fluid of cows fed the cellobiose-supplemented diet was decreased. In contrast, the values of these parameters measured in the milk of cows fed the cellobiose-supplemented diet were no different from those of control cows. Dietary supplementation with trehalose did, however, bring about an improvement of the oxidative status of milk and blood in these animals compared with controls. These results provide the first evidence supporting the use of dietary disaccharides to decrease lipid peroxide levels and increase the antioxidant content of dairy cow milk. The findings suggest that disaccharides, particularly trehalose, might be useful as supplements for reducing oxidative stress and improving the quality of milk for human consumption, as well as possibly impairing the processes that give rise to lipid oxidation odor in dairy cow milk.     

Recent advances in phosphorylation of food proteins: A review

Phosphorylation is a useful method for improving the functional properties of food proteins. In this article, various methods of phosphorylation are reviewed. Dry-heating phosphorylation, a method developed recently, is also introduced. Some characteristics of phosphate groups are involved, and the effects of phosphorylation on the structural changes, the functional properties, and the physiological functions in vitro of food proteins, are discussed. The types of phosphate linkages and the phosphopeptides from phosphorylated proteins are identified. The molten (partially unfolded) conformations of food proteins formed by phosphorylation are discussed. The phosphorylation of food proteins improved a number of functional properties, including heat stability, emulsifying properties, foaming properties, gelling properties, water absorption capacity, oil absorption capacity, and calcium phosphate-solubilizing ability. In vitro physiological function studies of protein (α-lactoalbumin) indicated that the digestibility (ovalbumin) was improved and the inflammatory response (α-lactoalbumin) was suppressed by phosphorylation. Experiments with animals are necessary to evaluate the toxicity and physiological functions of phosphorylated proteins.     

Micropatterned organoid culture of rat hepatocytes and HepG2 cells

The culture of liver cell organoids (multicellular aggregates) such as spheroids or cylindroids, which can strongly express liver functions, has been advocated as a useful technique that has advantages over monolayer culture. This paper describes a micropatterning technique for obtaining spheroids and cylindroids by using rat hepatocytes or HepG2 cells. We developed culture chips that comprised multiple, circular or rectangular microwells; the bottom surface of each microwell was modified with collagen to create a cell adhesion area, and the entire microwell, excluding the collagen-coated spots, was modified with polyethylene glycol (PEG) to create a nonadhesive area. Rat hepatocytes and HepG2 cells formed uniform spheroids and cylindroids on the circular and rectangular chips, respectively. Consequently, two-dimensional micropatterned chips containing homogeneous spheroids or cylindroids were generated. The expression of liver functions (protein secretion and ammonia removal) was greater in the spheroids and cylindroids than in the monolayer culture, and this expression was maintained for at least 2 weeks of culture. Thus, this chip technology has potential for use in various applications that involve organoid culture.     

Development of a novel aptamer-based sensing system using atomic force microscopy

Atomic force microscopy (AFM) can dynamically detect the adhesion or affinity force between a sample surface and a cantilever. This feature is useful as a detection method using aptamers--single-strand DNA that recognizes its target with very high affinity. The present study proposes a novel DNA aptamer-based sensing system using AFM. In this study, thrombin was chosen as the target molecule, and a DNA aptamer-based AFM sensing system based on competition was developed. The affinity force between the gold chip and the cantilever decreased as the concentration of thrombin increased. Moreover, a low detection limit of 0.2 nM was achieved. Therefore, the AFM sensing system used would be appropriate for the measurement of various chemical compounds.     

Liquefaction and dechlorination of hydrothermally treated waste mixture containing plastics with glass powder

Additive effects of glass powder upon the product yields and chlorine distribution after liquefaction of hydrothermally pretreated mixed waste (HMW) are compared with liquefaction of HMW with any one of water, quartz sand, or glass powder plus water. As a result, addition of either water or quartz sand did not affect liquefaction and dechlorination of HMW. Further, water (5 g) addition did not enhance liquefaction and dechlorination of HMW with glass powder. On the other hand, after liquefaction of HMW with glass powder, the yields of chlorine in the gas and water insoluble constituents decreased and the chlorine yield in the water-soluble constituent increased significantly. Because sodium in glass powder dissolved in a small amount (0.5 g) of water resulted from dehydration of HMW during liquefaction. Further, hydrogen chloride derived from polyvinylchloride in HMW was neutralized by ion exchange between H(+) and Na(+) dissolved in a small amount of water forming NaCl in the Residue (water-soluble) constituent. Therefore, most of chlorine in HMW was removed easily by water extraction of the Residue constituent after liquefaction of HMW with glass powder. Further, upgrading of HMW into the oil constituent was enhanced due to inhibition of production of chlorine containing organic compounds. Accordingly, it was clarified that glass powder was the most effective additive for liquefaction and dechlorination of HMW.