Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: conditional gene expression at near physiological levels

Using Multisite Gateway five-DNA-fragment constructs vectors that enable expression of two tandemly situated cDNAs on a single plasmid were developed. Heterologous protein production in cells was achieved by modulating respective cDNA expression to pre-determined and different levels. Optimization of cDNA expression at near physiological protein levels was achieved using promoters from four cell cycle-dependent genes. In comparison with conventionally available promoters, EF-1alpha or CMV, the promoters used in this study were able to modulate cDNA expression levels over a magnitude of approximately 10 or 100-fold, respectively. In transiently transfected cells, two different proteins (CPalpha1 and CPbeta2), which form a heterodimer, each labeled with a different-colored fluorescent protein, were successfully synthesized at pre-determined levels from their respective cDNAs. The above vectors were designed to contain an FRT/Flp recombination site for integration onto chromosomes and for establishment of stable clones in HeLa cells by site-specific recombination. In the stable transformant cells produced only about 4% of the protein production levels measured in the transiently transformed cells. The biological significance of these observations is discussed.     

Wheat cultivar-specific proteins in grain revealed by 2-DE and their application to cultivar identification of flour

Wheat flour proteins were studied to identify the cultivar-specific proteins and use them to identify cultivars in flours. Proteins extracted from flours of Japanese wheat (cultivars Hokushin, Horoshirikomugi, Kitanokaori and Kachikei 33) and Canadian wheat (Canada Western Red Spring Wheat No. 1; 1CW) were analyzed by 2-DE with IEF gels over three pH ranges: pH 4-7, pH 5-8, and pH 6-11. This system enabled detection of more than 1600 protein spots. We recognized that among 50 protein spots showing cultivar-dependent qualitative changes, 25 proteins were wheat cultivar specific. These 50 protein spots were analyzed by N-terminal Edman degradation microsequencing and MALDI-TOF-MS; 21 protein spots were storage proteins, such as gliadin and low-molecular mass glutenin subunit. Five protein spots were identified as dehydroascorbate reductase (Triticum aestivum), triticin precursor (T. aestivum), alpha-amylase inhibitor (Oryza sativa), DNA-binding with one finger (Dof) zinc family protein (O. sativa), and nonphototropic hypocotyl 1 (NPH1) protein (Avena sativa). The other protein spots appeared to be hypothetical proteins (O. sativa or Arabidopsis thaliana) or functional unknown proteins. These specific proteins can be used as markers to identify wheat cultivars in blended flour composed of two or three flours.     

Evidence of cross-fertilization in a gonochoric population of the tadpole shrimp Triops numidicus (Crustacea: Branchiopoda: Notostraca)

Cross-fertilization was evident in a gonochoric population of the notostracan Triops numidicus, in which a thick, hard eggshell had been suspected of preventing the sperm from reaching the egg. Most of the eggs from copulated females hatched into larvae, but the eggs from virgin females did not develop. In the larvae, paternal DNA fragments were detected by AFLP. In histological sections, several spermatozoa were found in the shell of newly oviposited eggs in the brood pouches of copulated females, suggesting that the shell was still soft enough to be penetrated by spermatozoa. These results showed that copulation and subsequent fertilization achieved by penetration of sperm through the newly deposited eggshell were indispensable for the normal development of the eggs.     

Establishment of a Humanized APL Model via the Transplantation of PML-RARA-Transduced Human Common Myeloid Progenitors into Immunodeficient Mice

Recent advances in cancer biology have revealed that many malignancies possess a hierarchal system, and leukemic stem cells (LSC) or leukemia-initiating cells (LIC) appear to be obligatory for disease progression. Acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia characterized by the formation of a PML-RARα fusion protein, leads to the accumulation of abnormal promyelocytes. In order to understand the precise mechanisms involved in human APL leukemogenesis, we established a humanized in vivo APL model involving retroviral transduction of PML-RARA into CD34⁺ hematopoietic cells from human cord blood and transplantation of these cells into immunodeficient mice. The leukemia well recapitulated human APL, consisting of leukemic cells with abundant azurophilic abnormal granules in the cytoplasm, which expressed CD13, CD33 and CD117, but not HLA-DR and CD34, were clustered in the same category as human APL samples in the gene expression analysis, and demonstrated sensitivity to ATRA. As seen in human APL, the induced APL cells showed a low transplantation efficiency in the secondary recipients, which was also exhibited in the transplantations that were carried out using the sorted CD34⁻ fraction. In order to analyze the mechanisms underlying APL initiation and development, fractionated human cord blood was transduced with PML-RARA. Common myeloid progenitors (CMP) from CD34⁺/CD38⁺ cells developed APL. These findings demonstrate that CMP are a target fraction for PML-RARA in APL, whereas the resultant CD34⁻ APL cells may share the ability to maintain the tumor.     

Physical training and fasting erythrocyte activities of free radical scavenging enzyme systems in sedentary men

Effects of 10 weeks of physical training on free radical scavenging enzyme systems in erythrocytes were investigated in 7 sedentary healthy male students. The training consisted of running over 5 km, 6 times/week. Their maximum oxygen uptake and 12 min walk-run performance increased significantly after training. Of the antioxidant enzyme systems examined in the erythrocytes, both catalase activity and concentration and total glutathione reductase (GR) activity also showed significant increases following the training. The erythrocyte GR activity coefficient also increased significantly. These results suggest that chronic aerobic exercise increases riboflavin requirements and has some positive effects on antioxidative processes.     

Cross-education effect of vibration foam rolling on eccentrically damaged muscles

Objectives:Previous studies showed that vibration foam rolling (VFR) on damaged muscles improves muscle soreness and range of motion (ROM). VFR intervention can also increase the ROM and pain pressure threshold (PPT) in the non-rolling side, known as a cross-education effect. However, this is not clear for the non-rolling side. Therefore, this study aimed to investigate the cross-education effects of VFR intervention on ROM, muscle soreness, and PPT in eccentrically damaged muscles.Methods:Participants were sedentary healthy male volunteers (n=14, 21.4±0.7 y) who performed eccentric exercise of the knee extensors with the dominant leg and received 90-s VFR intervention of the quadriceps at the nondamaged side 48 h after the eccentric exercise. The dependent variables were measured before the exercise (baseline), before (preintervention), and after VFR intervention (postintervention) 48 h after the eccentric exercise. The Bonferroni post hoc test was used to determine the differences between baseline, preintervention, and postintervention.Results:Results showed that the VFR intervention on the nondamaged side 48 h after the eccentric exercise improved significantly (p<0.05) the knee flexion ROM, muscle soreness at palpation, and PPT compared to baseline.Conclusion:VFR intervention on the nondamaged side can recover ROM and muscle soreness in eccentrically damaged muscles.     

Identification and growth of Leuconostoc lactis SHO-54, producing high amounts of NAD-specific 6-phosphogluconate dehydrogenase

The G+C content of the DNA of strain SHO-54 which produces a large amount of NAD-specific 6-phosphogluconate dehydrogenase was 41.0 mol%. The extent of the DNA–DNA homology between this strain and Leuconostoc lactis NRIC 1540 ranged from 72.8% to 95.5%. A minimal medium in which strain SHO-54 grew well was determined, and a high activity of the enzyme (410 nkat/ml) was obtained.     

BCG immunization age in urban and rural areas of Akita Prefecture, Japan

BCG immunization, utilizing whole-body coordination, is a highly cost-effective means of health intervention for preventing miliary tuberculosis (TB) and TB meningitis. In this study, we investigated the appropriate age by which a child should have completed his or her BCG immunization and discuss the current BCG immunization rate in Akita Prefecture, Japan. BCG immunization rates in urban and rural areas were 90.1% and 80.7%, respectively. Our immunization data were lower than the World Health Organization's (WHO) recommended rate. Immunization coverage rates in urban settings were higher than those in rural areas among infants four months to fifteen months of age, except for those six months old. We recommend: (1) completing BCG immunization by the age of twelve months, (2) preparing and educating parents for BCG immunization by means of a health policy, and (3) changing BCG immunization methods from group to individual inoculation. Immunization coverage rates may be increased or maintained to prevent miliary TB and TB meningitis.     

Specific binding of CO to tetraheme cytochrome c3

Carbon monoxide (CO) has been identified as another bioactive molecule like NO. Binding of CO to a tetraheme cytochrome c(3) (cyt c(3)) was investigated using visible absorption spectroscopy, circular dichroism (CD), and NMR. CO was found to bind to the four hemes in different manners. CD spectra, however, indicated that only single-site CO binding can keep the protein intact. The K(d) for the single-site binding was 8.0 microM, which is a typical value for a CO sensor protein. Furthermore, NMR spectra of uniformly (15)N-labeled and specifically [(15)N]His-labeled proteins have provided evidence that CO specifically binds to the sixth coordination site of heme 2 via single-site binding. The CO-bound cyt c(3) could conduct redox reactions. In light of triheme cytochrome c(7), the CO-bound cyt c(3) may work as an electron transporter. It was reported for sulfate-reducing bacteria that CO can be used as an energy source and CO cycling is operating like H(2) cycling. Therefore, the CO-bound cyt c(3) may play a role in maintaining electron transport pathways on accumulation of toxic CO for its utilization.