Analysis of the flanking regions of the human alpha-lactalbumin gene responsible for position-effect independent expression

Transgenic rats with the 130 kb bacterial artificial chromosome construct bLA, including the alpha-lactalbumin gene, had position-independent and copy number-dependent expression, which confirmed previous experiments using the 210 kb yeast artificial construct, yLALBA. To identify elements that confer a position effect, we compared the yLALBA and bLA sequences. yLALBA was chimeric. A common 32 kb region was identified and the total nucleotide sequence was determined. We previously analyzed transgenic rats using polymerase chain reaction to compare the integrity and expression of the transgenes. The -6 to +9 kb region is considered to be necessary for position-independent expression. Transgenic rats lacking the -3.4 to -0.85 kb region had a severe position effect. This 2.5 kb region contains two DNaseI hypersensitive sites at -1.0 and -2.8 kb. The 2.5 kb region is proposed to be a locus control region of the human alpha-lactalbumin gene.     

The NB protein of influenza B virus is not necessary for virus replication in vitro

The NB protein of influenza B virus is thought to function as an ion channel and therefore would be expected to have an essential function in viral replication. Because direct evidence for its absolute requirement in the viral life cycle is lacking, we generated NB knockout viruses by reverse genetics and tested their growth properties both in vitro and in vivo. Mutants not expressing NB replicated as efficiently as the wild-type virus in cell culture, whereas in mice they showed restricted growth compared with findings for the wild-type virus. Thus, the NB protein is not essential for influenza B virus replication in cell culture but promotes efficient growth in mice.     

Production of recombinant human relaxin 3 in AtT20 cells

Relaxin 3 has been reported recently as a member of the insulin/IGF/relaxin family. To clarify the function of relaxin 3, we prepared recombinant human relaxin 3 using a mouse adrenocorticotrophic hormone (ACTH)-secreting cell line, AtT20. To detect a mature form of recombinant human relaxin 3, a competitive enzyme immunoassay (EIA) was developed using a monoclonal antibody (mAb; HK4-144-10), which was raised for the N-terminal peptide of human relaxin 3 A-chain. We detected immunoreactive (ir-) relaxin 3 in the culture supernatant of AtT20 cells stably transfected with human relaxin 3 cDNA. After treatment with 5 microM forskolin for 3 days, the concentration of the ir-relaxin 3 in the culture supernatant reached 12 nM. Ir-relaxin 3 was purified from the culture supernatant by a combination of various chromatographies. By analyses of N-terminal amino acid sequence and electrospray ionization mass spectrometry (ESI-MS), we confirmed that the purified material was a mature form of human relaxin 3. The recombinant human relaxin 3 thereby obtained increased intracellular cAMP production in THP-1 cells. Our results demonstrate that the expression of relaxin 3 cDNA in AtT20 cells is a useful tool to produce a bioactive and mature form of relaxin 3.     

Simple maxillary reconstruction using free tissue transfer and prostheses

Prosthetic rehabilitation is essential for maintaining postoperative oral function after maxillary reconstruction. However, the maxillary prosthesis becomes unstable in some patients because of extensive palatomaxillary resection and drooping of the transferred flap. In such patients, maintaining sufficient oral function is difficult, especially if the patient is edentulous. To achieve prosthetic retention, the authors performed microvascular maxillary reconstruction with a slit-shaped fenestration in the midline of the hard palate. Maxillary defects after subtotal or total maxillectomy were reconstructed with rectus abdominis musculocutaneous flaps in five patients. Defects of the nasal lining and palate were reconstructed with the single cutaneous portion of the flap, and a slit-shaped fenestration was left between the cutaneous portion of the flap and the edge of the remaining hard palate. Postoperatively, patients were fitted with maxillary prostheses that had a flat projection for the palatal fenestration. In all patients, the prosthesis was stable enough for mastication and prevented nasal regurgitation. Speech function was rated as excellent on Hirose's scoring system for Japanese speech ability. The authors believe that their method of palatomaxillary reconstruction is both simple and reliable.     

Effects of intake of a mixture of thiamin, arginine, caffeine, and citric acid on adiposity in healthy subjects with high percent body fat

We assessed the effects of intake of thiamin, arginine, caffeine, and citric acid (TACC) on lipid metabolism in healthy subjects. Thirty-one subjects with high percent body fat (> or = 25.0%) were randomly assigned to a 12-wk intervention with daily intake of TACC-supplemented tea (1.1, 1240, 52, and 540 mg, respectively; n=16) or control tea (n=15). The percent body fat decreased significantly during the intervention in both groups, especially in the TACC group. A percentage decrease in triceps skinfold was significantly greater in the TACC group than in the control group. The decrease in abdominal visceral fat in obese subjects was significantly greater in the TACC group than in the control group. Serum triglyceride was significantly lower during intervention than that during the non-intervention period in the TACC group. These results suggest that TACC may be effective in reducing body fat in obese subjects.     

Preparation and characterization of recombinant murine p65/L-plastin expressed in Escherichia coli and high-titer antibodies against the protein

We previously identified a 65-kDa protein (p65) that was phosphorylated in activated macrophages. It has turned out to be a murine homologue of human L-plastin, which was identified as a novel protein in human cancer cells. p65/L-plastin is characterized by a series of Ca(2+)-, calmodulin-, and actin-binding domains, and is thought to play a crucial role in leukocytes and cancer cells. We have expressed a recombinant (r) p65/L-plastin in Escherichia coli that binds to beta-actin and prepared high-titer antibodies using large amounts of the protein as immunogen. Anti-rp65/L-plastin antibodies recognize native p65/L-plastin as well as rp65/L-plastin and have enabled us to detect the fine structures of intracellular p65/L-plastin, and it was found that its localization was extensively changed by stimulation with bacterial components. We further developed an enzyme-linked immunosorbent assay system and a flow cytometry method using these reagents, which made it possible to measure antibodies, including autoantibodies, against p65/L-plastin and to evaluate the maturation-dependent expression of the protein in leukocytes.     

Putative cis-elements in the promoter region of the carrot phenylalanine ammonia-lyase gene induced during anthocyanin synthesis

Deletion mutants of the carrot phenylalanine ammonia-lyase gene promoter were used to survey cis-elements for their effect on expression of promoter activity by transient expression. Two putative cis-elements were required to give full activity, but a third might be the most important in regulation of the promoter by 2,4-dichlorophenoxyacetic acid. Electronic Publication     

Integration of Life Cycle assessment and population balance model for assessing environmental impacts of product population in a social scale case studies for the global warming potential of air conditioners in Japan

Scope  In this study, a dynamic model was built in which LCA and PBM were integrated to quantitatively assess the total environmental impacts induced by the product population in a society over time. Specifically, a determination was carried out concerning how Japan’s air conditioner population is used (lifetime distribution, number of units, etc.) and an assessment was made concerning the Global Warming Potential (GWP) associated with the air conditioner population. Methods  The proposed dynamic model was applied to air conditioners for analyzing the total GWP caused by the air conditioner population in Japan from 1990 to 2010. To create a trend forecast model for future environmental load, scenarios for air conditioner production up to 2010 were formulated and the total GWP from the air conditioner population was predicted. Conducted also were sensitivity analyses whose parameters were air conditioner performance, lifetime and the rate of refrigerant recovery when retired units are processed. Results and Discussion  Applying the PBM to the air conditioner population in 2000, it was found that 81.5 million units consumed 5.94 x 10^p10 kWh in that year, which was a 6.1 % increase in the total annual power consumption in 1990. In both a stationary scenario and a steady growth (1.5% annual increase), it was found that the total GWP would be 27.7% higher than in 1990 under the stationary scenario and 37.8% higher under the steady growth scenario. The improvements in air conditioner performance will have a small effect on reducing the total GWP from that population. Furthermore, in connection with the average lifetime, it was found that the GWP, due to refrigerant releases when units are disposed of, would be relatively large in 2000 and the following years. Conclusions  Thus, shorter product lifetimes will spur a replacement of air conditioners with new units, a situation that will only lead to the reduction of GWP if the recovery rate of refrigerant is to be achieved to more than 50% under the stationary scenario. Recommendations and Outlook  To meet COP3 targets for Japan in 2010 (i.e. to reach the same level as in 1990 for household appliances), our study shows that it will be vital to raise the refrigerant recovery rate. If the number of air conditioners in use remains unchanged, recovery would have to be 45.7%, but under the steady growth scenario it would have to be at least 60.4%. Therefore, it will be difficult to meet COP3 targets unless the refrigerant recovery rate is strongly increased. This method is applicable to assess not only the GWP of air conditioners, but also other environmental impacts caused by a variety of product populations, which will be quite effective for setting targets of products’ performance, policymaking, etc.