Evaluation of Cell Responses of Saccharomyces cerevisiae under Cultivation Using Wheat Bran as a Nutrient Resource by Analyses of Growth Activities and Comprehensive Gene Transcription Levels.

Wheat bran has high nutritional values and is also cheaper than yeast nitrogen base as an important component of a medium. Although its use in microbial cultivations is expected, research and development has hardly progressed so far. In this study, with experimental Saccharomyces cerevisiae BY4741, the cell responses to wheat bran as a nutrient were evaluated by analyses of cell growth, ethanol production, and comprehensive gene transcription levels. Comparing wheat bran and yeast nitrogen base, BY4741 showed specific growth rates of 0.277 ± 0.002 and 0.407 ± 0.035 as a significant difference. Additionally, wheat bran could be used as a restricted media component like yeast nitrogen base. However, in 24 h of cultivation with wheat bran and yeast nitrogen base, although conversion ratios of ethanol productions showed no significant difference at 63.0 ± 7.2% and 62.5 ± 8.2%, the ratio of cell production displayed a significant difference at 7.31 ± 0.04% and 4.90 ± 0.16%, indicating a different cell response. In fact, the comprehensive evaluation of transcription levels strongly suggested major changes in glucose metabolism. This study indicated that BY4741 could switch transcription levels efficiently to use wheat bran.

Identification of Cell-Attachment Factors Derived from Green Algal Cells Disrupted by Sonication in Fabrication of Cell Plastics.

Cell plastics which are composed of unicellular green algal cells have been proposed in previous studies. While unicellular green algae can be freely arranged using fabrication processes, a matrix is required to attach the cells together. To date, although the cell contents collected from Chlamydomonas reinhardtii show the possibility of attaching cells, but it is unclear which components can be considered attachment factors. Therefore, in this study, C. reinhardtii cells were disrupted with sonication, and the components were separated and purified with hexane. The cell plastics with only 0.5 wt% of intermediate showed similar mechanical properties to those with 17 wt% and 25 wt% of cell components that were untreated with hexane, meaning that the purified intermediates could function as matrices. The purified intermediate was composed of approximately 60 wt% of protein as the main component, and proteomic analysis was performed to survey the main proteins that remained after hexane treatment. The protein compositions of the cell content and purified intermediate were compared via proteomic analysis, revealing that the existing ratios of 532 proteins were increased in the purified intermediate rather than in the cell content. In particular, the outer structure of each of the 49 proteins-the intensity of which was increased by over 10 times-had characteristically random coil conformations, containing ratios of proline and alanine. The information could suggest a matrix of cell plastics, inspiring the possibility to endow the cell plastics with more properties and functions.

Fabrication of cell plastics composed only of unicellular green alga Chlamydomonas reinhardtii as a raw material.

Cell plastics in this study were fabricated with only unicellular green alga Chlamydomonas reinhardtii as raw materials. The sizes of cell-major axis as structures were 8.4 ± 1.2 µm, and the aspect ratios of those were 1.2 ± 0.1, showing homogeneous particle size. After optimizing extraction condition of intracellular contents, cell plastics were fabricated with the cells as ingredient components and the intracellular contents as matrix components. Those cell plastics were observed with scanning electron microscopy, displaying the smooth surfaces of the cell plastics at a low magnification level. However, the surface, especially exposed surface, were rough at high magnification level. Tensile strength test revealed that increasing the ratio of intracellular contents in the cell plastics until 21% led enhancing mechanical properties of Young's modulus and tensile strength; however, 25% of intracellular contents displayed decreases of those properties. As the optimal point, the cell plastic (21%), which contained 21% (w/w) of intracellular contents in cell plastics, showed 764 ± 100 MPa and 8.6 ± 5.2 MPa of Young's modulus and tensile strength. The cell plastics showed few plastic region and soon fractured, indicating the possibility that cells and intracellular contents could be electrostatically connected. Additionally, cells were shown as a negative charge and displayed the possibility to contribute electrically cell-gathering with intracellular ionic components. Therefore, cells and intracellular contents containing ionic metabolites could be electrostatically connected for giving the mechanical strength to cell plastics. In this study, we successfully demonstrated fabricating cell plastics with only cells for the first time and also showed the high possibility of conjugating each cell with the intracellular contents. KEY POINTS: • Cell plastics are fabricated with unicellular green algal cell directly. • Unicellular cells required to be conjugated for the fabrication with matrix. • Cells were conjugated with intracellular contents for cell-plastic fabrication.

Estimation of Carbon Metabolism in Saccharomyces cerevisiae Acclimatized to Glycerol Assimilation with Quantitative PCR.

Saccharomyces cerevisiae has the potential to produce value-added chemicals; however, this strain is restricted by using glycerol as a carbon source. Although acclimatization of S. cerevisiae as a glycerol-assimilating strain was confirmed so far, the reason why S. cerevisiae can be acclimatized was not clear in detail with limited information on the metabolic changes. In this report, glycerol-assimilating strains from S. cerevisiae BY4741 were isolated, and the biomass production, ethanol fermentation, and transcription levels related to glycolysis and the tricarboxylic acid cycle under aerobic and slightly anaerobic conditions were analyzed. As the results show, although µmax was equal to 0.15 h-1 between wildtype and glycerol-assimilating strains in an aerobic culture including glucose, the differences in max biomass production and percentage yields of ethanol and transcription levels between the two strains were shown. In slightly anaerobic culture, the differences in transcription levels downstream of glycolysis were also displayed. In the case of the glycerol-assimilating strain with glycerol under aerobic conditions, although the transcription levels related to ethanol production were sufficient, the ethanol production was not detected. Additionally, the biomass production reached a plateau even in the culture containing sufficient glycerol, indicating that the redox imbalance even in the cells of the glycerol-acclimatized strain could disturb the utilization of glycerol. The obtained knowledge will promote the use of glycerol resources with the glycerol-acclimatized S. cerevisiae in view of carbon recycling.

Development of Targeted Protein-Displaying Technology with a Novel Carbon Material.

This study reports a new carbon material and its specific display of targeted protein. The properties of the carbon materials fabricated with carbon black MOGUL® were analyzed. The carbon materials were spherical structures with 55.421 µm as a median value. The specific surface area, pore volume, average pore diameter, and total of the acidic functional group were 130 m2·g-1, 0.55 cm3·g-1, 17.2 nm, and 0.29 mEq·g-1, respectively. The adsorption-desorption isoform of the carbon materials showed type IV of the hysteresis loop as defined by IUPAC, indicating non-uniform mesoporous structures (2-50 nm). The distribution of the log differential pore volume also indicated non-uniform porous structures because (i) the difference between the average pore size and the most frequent pore size was significant and (ii) the σ value was larger than the average value regarding the pore sizes. However, 10-90% of the integrated values of the log differential pore volume were 57.4% of the total integrated values, and the distribution was similar to the Gauss distribution model. Although the value of the total of the acidic functional group was 2.5-5.4 times lower than the values of the HPLC columns, the carbon materials require good scaffold quality rather than good HPLC quality. Therefore, the amounts could be enough for the scaffold of biotin hydrazide. To demonstrate the property of displaying the targeted proteins, carbon materials displaying biotin hydrazide by covalent bonding were prepared and avidin-labeled horse radish peroxidase (HRP) was bound to the biotin region. The carbon materials were porous structures, so the unspecific adsorption of HRP was estimated. Then, the maintenance ratios of HRP activities were analyzed in the repeated-use-with-wash processes after each evaluation, resulting in the activities of HRP on the carbon materials being treated with biotin hydrazide being significantly maintained compared to that of the ones without biotin hydrazide. The study revealed the properties of the carbon materials and indicated the display of HRP, suggesting that the carbon materials could be a new material for displaying targeted proteins.

Two-way vapochromism of a luminescent platinum(ii) complex with phosphonic-acid-functionalized bipyridine ligand.

A luminescent Pt(ii) complex [Pt(CN)2(H4dpbpy)] (1P; H4dpbpy = 2,2'-bipyridine-4,4'-diphosphonic acid) bearing a phosphonic-acid-functionalized bipyridine ligand was successfully synthesized and its unique two-way vapochromic behaviour investigated. X-ray structure analyses of both the anhydrous 1P and penta-hydrated 1P·5H2O phases clearly reveal the activation of intermolecular PtPt interactions through the adsorption of water vapour. Emission spectroscopy reveals that the penta-hydrated 1P·5H2O complex exhibits an orange emission at 585 nm that shifts in two directions, to a blue-green emission at 469 nm by drying at 100 °C or to a red emission at 701 nm by drying under vacuum at room temperature. Thermogravimetric analyses and powder X-ray diffraction studies clearly reveal that anhydrous 1P, with negligible intermolecular PtPt interactions, is formed by drying at 100 °C whereas the monohydrate 1P·H2O phase, with effective PtPt interactions, is formed by drying under vacuum.

Tumor-draining lymph nodes of primary lung cancer patients: a potent source of tumor-specific killer cells and dendritic cells.

BACKGROUND: The tumor-draining lymph node tissue (TDLT) of lung cancer patients generated killer cells specific to autologous tumor cells when cultured with low dose IL-2. This production of killer cells lasted as long as 2 months after the initiation of the culture (productive phase). Even after this productive phase, TDLT supported the generation of the killer cells when these were co-cultured with peripheral blood lymphocytes (PBL) from the same patients. We tried to analyze the mechanisms of this production of killer cells from TDLT. MATERIALS AND METHODS: TDLT, tumor tissues as well as PBL were obtained from primary lung cancer patients and cultured in vitro. Cell growth, cell surface markers and specific cytotoxic activity of the lymphocytes were examined. RESULTS: The majority of the cells from TDLT or TDLT+ PBL co-culture (TDL-Pb) were CD3-positive T cells (89-99%) and a 51Cr-releasing assay showed that these cells had a stronger cytotoxic activity against autologous tumor cells than cells from PBL cultured with IL-2. Their activity against allogeneic MHC incompatible target cells was not, however, elevated. Cytotoxic activity against autologous tumor cells was blocked by anti-HLA class 1 (52.0%), class 11 (47.9%) and CD8 (46.8%) antibodies, but not by anti-CD56 antibody. The treatment of TDLT with anti-CD8, CD4, CD80 and CD83 all together completely abrogated the ability of TDLT to generate killer cells, with one of these antibodies it did so partially, while treatment with anti-CD56 antibody failed to do so at all. CONCLUSION: These results collectively suggest that TDLT contains tumor antigen-pulsed DCs as well as precursors of specific killer T cells and gives rise to the generation of killer cells when cultured in a low dose of IL-2.

[An autopsy case of pseudomesotheliomatous adenocarcinoma of the lung complicated with brain stem infarction due to nonbacterial thrombotic endocarditis].

A 53-year-old man was admitted to our hospital for back, left shoulder and upper limb pain. Chest radiography and CT on admission revealed right pleural effusion and a focal plate-like thickening of the major fissure. Pleural effusion cytology revealed adenocarcinoma, which was diagnosed as non-small-cell lung cancer with bone metastasis. The patient suffered from DIC, melena and multiple cerebral infarctions during chemotherapy and died on the eighth day of the second course of chemotherapy. Autopsy revealed a pseudomesotheliomatous adenocarcinoma covering the pleura of the right middle lobe, systemic thromboembolism and vegetations on the aortic valves due to nonbacterial endocarditis. Pseudomesotheliomatous adenocarcinoma of the lung, a rare form of lung cancer, complicated with DIC and nonbacterial endocarditis, is reported.

Positive reactions for both Cyfra21-1 and CA125 indicate worst prognosis in non-small cell lung cancer.

UNLABELLED: We examined four tumour markers [carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC), cancer antigen 125 (CA125) and cytokeratin 19 fragment (Cyfra21-1)] in 584 non-small cell lung cancer (NSCLC) patients. RESULTS: After confirmation of a significant correlation between their serum levels and clinical stages, multivariate analysis showed that Cyfra21-1 and CA125 pointed to a negative prognosis; the respective hazard ratios were 2.585 (p = 0.0008) and 2.139 (p = 0.0020) in 121 inoperable adenocarcinoma patients and 2.329 (p = 0.0004) and 1.61 (p = 0.00370) in 205 inoperable NSCLC patients. Also, patients giving positive reactions for both Cyfra21-1 and CA125 had the worst prognoses, with hazard ratios of 6.546 (p < 0.0001) in inoperable adenocarcinoma patients and 4.275 (p < 0.0001) in inoperable NSCLC patients. CONCLUSION: Cyfra21-1 or CA125 tend to imply a negative prognosis. Cyfra21-1 and CA125 together imply the worst prognosis.

[Heavy-ion therapy for non-small cell lung cancer].

Since carbon beam therapy for non-small cell lung cancer (NSCLC) was initiated in October 1996, seven trials have been conducted; three have already closed and the remaining four are ongoing. The local control rate, cause-specific survival rate, and overall survival rate of 141 patients with clinical stage I NSCLC were 82%, 58%, and 42%, respectively. Radiation pneumonia was rare (2.1%) and not serious. In the phase II clinical study, the local control rate achieved in 50 patients was 100%, with no radiation pneumonia, resulting in a 60% overall survival rate. Carbon beam therapy could be an alternative to surgery, especially for lung cancer patients of advanced age and/or with complications. For locally advanced lung cancer treated with carbon beam therapy, excellent local control comparable to that in stage INSCLC has been demonstrated and offers hopeful prospects for the treatment of lung cancer.