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Fatty acid binding proteins (FABPs) are intracellular lipid chaperones which mediate the uptake, transportation and signaling roles of fatty acids. Former studies suggest that FABP7 regulates proliferation and differentiation of radial glia and astrocytes and is associated with various central nervous system diseases including glioma and neuropsychiatric diseases. However, the underlying mechanism is poorly understood. To further explore the mechanistic roles of FABP7 on astrocyte function, the proteome of astrocytes cultured from Fabp7 KO mice was compared with wild-type counterparts by two-dimensional gel electrophoresis (2-DE). We found that 16 protein spots showed differential expression intensity on 2-DE gels. Among them, seven spots appeared elevated, and nine spots appeared decreased in Fabp7 KO astrocytes. Selected spots were analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis and their protein identity was subsequently revealed using protein databases. Four spots including phosphoprotein enriched in astrocytes 15 (PEA-15), GFAP, vimentin and FABP7 were selected for further confirmation by Western blotting. Consistently, there was a significant decrease in expression of PEA-15 and vimentin, both of which play significant roles in proliferation, differentiation, maturation, and survival of astrocytes. These results suggest the molecular mechanism how FABP7 controls astrocyte function and provide new insight to explain the role of FABP7 in glioma and reactive gliosis.

Active hexose-correlated compound (AHCC) is an extract of basidiomycete mushroom. It has been used as health food due to its efficacy of enhancing antitumor effects and reducing adverse effects of chemotherapy. Our previous research showed that AHCC down-regulated heat-shock protein (HSP)-27 and exhibited cytotoxic effects against gemcitabine-resistant pancreatic cancer cells. Sex-determining region Y-box 2 (SOX2) is reported to be up-regulated in other kinds of cancer cells and involved in carcinogenesis and malignancy. The aim of this study was to investigate the effects of AHCC on protein expression of SOX2 in the gemcitabine-resistant pancreatic cancer cell line KLM1-R.AHCC was applied to KLM1-R cells and expression of SOX2 was analyzed by western blotting.AHCC down-regulated SOX2 in KLM1-R cells. Nanog and Oct4, co-workers of SOX2 in maintaining pluripotency, did not exhibit any significant change in protein expression.We showed the potential of AHCC to be a candidate for combinatorial therapy in anticancer drug regimens. This result suggests that the target of AHCC in expressing therapeutic efficacy was not the pluripotent cells such as cancer stem cells (CSCs) but SOX2-specific.

Serum antibodies against pathogenic bacteria play immunologically protective roles, and can be utilized as diagnostic markers of infection. This study focused on Japanese child serum antibodies against Helicobacter pylori, a chronically-infected gastric bacterium which causes gastric cancer in adults. Serological diagnosis for H. pylori infection is well established for adults, but it needs to be improved for children. Serum samples from 24 children, 22 H. pylori (Hp)-positive and 2 Hp-negative children, were used to catalogue antigenic proteins of a Japanese strain CPY2052 by two-dimensional electrophoresis followed by immunoblot and LC-MS/MS analysis. In total, 24 proteins were identified as candidate antigen proteins. Among these, the major virulence factor, cytotoxin-associated gene A protein (CagA) was the most reactive antigen recognized by all the Hp-positive sera even from children under the age of 3 years. The major antigenic part of CagA was identified in the middle region, and two peptides containing CagA epitopes were identified using a newly developed peptide/protein-combined array chip method, modified from our previous protein chip method. Each of the epitopes was found to contain amino acid residue(s) unique to East Asian CagA. Epitope analysis of CagA indicated importance of the regional CagA antigens for serodiagnosis of H. pylori infection in children.

Lactoylglutathione lyase (GLO1), a ubiquitously expressed methylglyoxal (MG) detoxification enzyme, is implicated in the progression of various human malignant diseases. However, the role of GLO1 in the development or progression of murine fibrosarcoma is still unclear. We performed proteomic analysis to identify differences in the intracellular proteins of the regressive tumor cell line QR-32 and the inflammatory cell-promoting progressive tumor cell line QRsP-11 of murine fibrosarcoma by 2DE combined with MS. Seven upregulated proteins were identified in QRsP-11 compared to QR-32 cells, namely, GLO1, annexin A1, adenylate kinase isoenzyme 1, transcription factor BTF3, myosin light polypeptide 6, low molecular weight phosphotyrosine protein phosphatase and nucleoside diphosphate kinase B. Heat shock protein beta-1 (HspB1), a methylglyoxal-adducted protein, is concomitantly over-expressed in QRsP-11 as compared to QR-32 cells. We also found out that GLO1 is translocated into the nucleus to a higher extent in QRsP-11 compared to QR-32 cells, which can be reversed by using a MEK inhibitor (U0126). Moreover, U0126 and GLO1 siRNA can inhibit cell proliferation and migration in QRsP-11 cells. Our data suggest that overexpression and nuclear translocation of GLO1 might be associated with tumor progression in murine fibrosarcoma.

Powders of Al and C were mixed in a molar ratio of Al :C = 3:1 and were then ground in a planetary ball mill. When the thus mechanically activated mixtures were transferred into a graphite crucible and exposed to air, they spontaneously ignited and self-propagating high-temperature synthesis took place in two successive steps. The products were sliced along the depth direction and examined by X-ray diffraction. Depending on the depth of the crucible, aluminium nitride, aluminium carbide, aluminium oxynitride, aluminium oxycarbide and alpha alumina were detected. From measurements of the lattice constant, it was found that aluminium nitride with a 97% purity could be obtained at the bottom of the graphite crucible.

Bubble-free, transparent Li2B4O7 single crystals were grown by a newly developed pulling-down method. The use of a two-zone vertical furnace with a Pt-crucible realized a relatively steep temperature gradient of about 50°C/cm around the growth interface and a moderate gradient of about 13°C/cm below the interface, contributing to prevent grown crystals from cracking. Even at a pulling-down rate of 0.75mm/h, which is higher than that in the Czochralski growth, the formation of bubbles was effectively suppressed at a rotation rate of 25rpm. Any crystals grown along [110] direction involved no low-angle grain boundaries, but twinning was occasionally recognized. The dislocation density in the initial part of the crystal growth was found to be about 5×103/cm2 from an etch pit pattern, while X-ray topography of a vertical cross section revealed that the number of dislocation lines decreased as the growth proceeded.

Gemcitabine (2'-deoxy-2'-difluorodeoxycytidine) is the only clinically effective drug for pancreatic cancer. However, high levels of inherent and acquired tumor resistance to gemcitabine lead to difficulty of chemotherapy for pancreatic cancer. We have reported on a proteomic study of gemcitabine-sensitive KLM1 and -resistant KLM1-R pancreatic cancer cells, and identified some proteins which were shown to be up-regulated in KLM1-R compared to KLM1 cells. In those proteomic studies, peroxiredoxin-2 was listed as an up-regulated protein in KLM1-R cells. Peroxiredoxin-2 is a member of a family of peroxiredoxins providing a protective role for redox damage. In this study, the expression of peroxiredoxin-2 in KLM1 and KLM1-R cells was compared. It was found that peroxiredoxin-2 was significantly up-regulated in KLM1-R cells compared to KLM1 cells (p0.001). However, peroxiredoxin-1 expression was significantly down-regulated in KLM1-R cells (p0.001). These results suggest that peroxiredoxin-2 is a possible candidate biomarker for predicting the response of patients with pancreatic cancer to treatment with gemcitabine.

Lactoylglutathione lyase (GLO1), a ubiquitously expressed methylglyoxal (MG) detoxification enzyme, is implicated in the progression of various human malignant diseases. However, the role of GLO1 in the development or progression of murine fibrosarcoma is still unclear. We performed proteomic analysis to identify differences in the intracellular proteins of the regressive tumor cell line QR-32 and the inflammatory cell-promoting progressive tumor cell line QRsP-11 of murine fibrosarcoma by 2DE combined with MS. Seven upregulated proteins were identified in QRsP-11 compared to QR-32 cells, namely, GLO1, annexin A1, adenylate kinase isoenzyme 1, transcription factor BTF3, myosin light polypeptide 6, low molecular weight phosphotyrosine protein phosphatase and nucleoside diphosphate kinase B. Heat shock protein beta-1 (HspB1), a methylglyoxal-adducted protein, is concomitantly over-expressed in QRsP-11 as compared to QR-32 cells. We also found out that GLO1 is translocated into the nucleus to a higher extent in QRsP-11 compared to QR-32 cells, which can be reversed by using a MEK inhibitor (U0126). Moreover, U0126 and GLO1 siRNA can inhibit cell proliferation and migration in QRsP-11 cells. Our data suggest that overexpression and nuclear translocation of GLO1 might be associated with tumor progression in murine fibrosarcoma.

By a proteomics-based approach, we identified an overexpression of fascin in colon adenocarcinoma cells (FPCKpP-3) that developed from nontumorigenic human colonic adenoma cells (FPCK-1-1) and were converted to tumorigenic by foreign-body-induced chronic inflammation in nude mice. Fascin overexpression was also observed in the tumors arising from rat intestinal epithelial cells (IEC 6) converted to tumorigenic in chronic inflammation which was induced in the same manner. Upregulation of fascin expression in FPCK-1-1 cells by transfection with sense fascin cDNA converted the cells tumorigenic, whereas antisense fascin-cDNA-transfected FPCKpP-3 cells reduced fascin expression and lost their tumor-forming ability in vivo. The tumorigenic potential by fascin expression was consistent with their ability to survive and grow in the three-dimensional multicellular spheroids. We found that resistance to anoikis (apoptotic cell death as a consequence of insufficient cell-to-substrate interactions), which is represented by the three-dimensional growth of solid tumors in vivo, was regulated by fascin expression through caspase-dependent apoptotic signals. From these, we demonstrate that fascin is a potent suppressor to caspase-associated anoikis and accelerator of the conversion of colonic adenoma cells into adenocarcinoma cells by chronic inflammation.

QR-32 is a regressive murine fibrosarcoma cell clone which cannot grow when they are transplanted in mice; QRsP-11 is a progressive malignant tumor cell clone derived from QR-32 which shows strong tumorigenicity. A recent study showed there to be differentially expressed up-regulated and down-regulated proteins in these cells, which were identified by proteomic differential display analyses by using two-dimensional gel electrophoresis and mass spectrometry. Cofilins are small proteins of less than 20 kDa. Their function is the regulation of actin assembly. Cofilin-1 is a small ubiquitous protein, and regulates actin dynamics by means of binding to actin filaments. Cofilin-1 plays roles in cell migration, proliferation and phagocytosis. Cofilin-2 is also a small protein, but it is mainly expressed in skeletal and cardiac muscles. There are many reports showing the positive correlation between the level of cofilin-1 and cancer progression. We have also reported an increased expression of cofilin-1 in pancreatic cancer tissues compared to adjacent paired normal tissues. On the other hand, cofilin-2 was significantly less expressed in pancreatic cancer tissues. Therefore, the present study investigated the comparison of the levels of cofilin-1 and cofilin-2 in regressive QR-32 and progressive QRsP-11cells by western blotting. Cofilin-2 was significantly up-regulated in QRsP-11 compared to QR-32 cells (p0.001). On the other hand, the difference of the intensities of the bands of cofilin-1 (18 kDa) in QR-32 and QRsP-11 was not significant. However, bands of 27 kDa showed a quite different intensity between QR-32 and QRsP-11, with much higher intensities in QRsP-11 compared to QR-32 (p0.001). These results suggested that the 27-kDa protein recognized by the antibody against cofilin-1 is a possible biomarker for progressive tumor cells.

The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus.

Drugs affecting cellular morphological changes leading to tumor cell migration and invasion are desirable for cancer therapy. In the present study, we screened for small-molecule compounds that affect the cellular morphology of both unicellular yeast and mammalian HEK293 cells to identify drug candidates. The yeast formin protein Bni1 and Src homology 3 (SH3)-pleckstrin homology (PH) domain protein Boi1, which are required for proper morphogenesis, cause growth defects when overexpressed in yeast. Using this system, we screened a chemical library consisting of ～8000 compounds to identify drug candidates that suppress these growth defects. None of the screened compounds induced morphological changes in vegetatively growing yeast cells, but several compounds had inhibitory effects on pheromone-induced projection formation and actin localization, suggesting that these compounds affected a specific stage of morphogenesis. Five of the compounds also induced morphological changes in mammalian HEK293 cells. Among the identified compounds, BTB03156, 2-[(4-chlorophenyl)sulfonyl]-1-methyl-3,5-dinitrobenzene, and BTB02467, 1-[(4-chlorophenyl)sulfonyl]-2-nitro-4-(trifluoromethyl)benzene, although they have similar structures, displayed differing effects on the yeast growth defects caused by latrunculin A, an actin polymerization inhibitor. The chemical library compounds identified using this in vivo screening approach are simple, cell-permeable molecules, and therefore may be useful in the development of therapeutic drugs for cancer metastasis and other actin-related diseases.

Intrinsic or acquired resistance of pancreatic cancer to gemcitabine (2'-deoxy-2'-difluorodeoxycytidine) is an important factor in the failure of gemcitabine treatment. Proteomic analysis of gemcitabine-sensitive KLM1 pancreatic cancer cells and -resistant KLM1-R cells identified heat-shock protein-27(HSP27) as a biomarker protein which is involved in gemcitabine resistance. However, a knock-down experiment showed that HSP27 was not the only protein implicated with gemcitabine-resistance. Finding further candidate proteins is necessary for achieving effective gemcitabine therapy for patients with pancreatic cancer. DDX39 is an Asp-Glu-Ala-Asp (DEAD)-box RNA helicase reported to be overexpressed in tumor cells, such as lung squamous cell cancer, gastrointestinal stromal tumor, urinary bladder cancer and malignant pleural mesothelioma. In urinary bladder cancer cells, overexpression of this protein is intimately bound with tumorigenesis and poor prognosis. In the present study, the expression of DDX39 in gemcitabine-sensitive KLM1 and -resistant KLM1-R cells was compared. It was found that DDX39 was significantly up-regulated in gemcitabine-resistant KLM1-R cells compared to sensitive KLM1 cells. The ratio of expression of DDX39 to that of actin was significantly up-regulated in KLM1-R cells compared to KLM1 cells (p=0.0072 by Student's t-test). These results suggest that DDX39 is a possible candidate biomarker for predicting the response of patients with pancreatic cancer to treatment with gemcitabine.

Background We report here a new type of protein chip to detect antibodies in sera. This chip method was used to a prototype created to detect hepatocellular carcinoma (HCC) -related autoantibodies in the sera of hepatitis C virus (HCV) infected individuals. Results Five cysteine-tagged (Cys-tag) and green fluorescent protein (GFP)-fused recombinant heat shock protein 70 (HSP70), superoxide dismutase 2 (SOD2), and peroxiredoxin 6 (PRDX6), were spotted and immobilized on maleimide-incorporated diamond-like carbon (DLC) substrates. The antibodies in diluted sera were trapped by these proteins at each spot on the chip, and visualized by a fluorescence-conjugated anti-human IgG. The total immobilized protein level of each spot was detected with anti-GFP mouse IgG and a fluorescence-conjugated secondary anti-mouse IgG. The ratio between the two fluorescence intensities was used to quantify autoantibody levels in each serum sample. Heat treatment of the chip in a solution of denaturing and reducing agents, before serum-incubation, improved autoantibody detection. We tested serum samples from healthy individuals and HCC patients using the chips. The HSP70 autoantibodies were found at high levels in sera from HCV-positive HCC patients, but not in HCV-negative sera. Conclusion This protein chip system may have useful properties to capture a specific set of antibodies for predicting the onset of particular cancers such as HCC in HCV-infected individuals.

Three clones with differential metastatic potential were established from the parental SN12C human renal cell carcinoma (HRCC). We previously reported that in the two high metastatic SN12C clones, two isoforms of ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH‑L1) showed decreased expression by using two-dimensional electrophoresis (2‑DE) covering a pH range (pH 3.0‑10.0) followed by liquid chromatography‑tandem mass spectrometry. However, in the case of the low metastatic clone, the spot volume for UCH‑L1 was almost the same as for the parental SN12C. In the present study, we found one protein spot which was correlated with the metastatic potential of SN12C clones by using 2‑DE over a narrow pH range (pH 4.0‑7.0). The protein glyoxalase 1 (GLO1) appeared to be directly proportional to the metastatic potential of the SN12C clones. GLO1 was the only protein which consistently varied according to the metastatic potentials of SN12C clones. GLO1 was increased in high metastatic cell lines by western blot analysis. These findings suggest that GLO1 is associated with the metastatic potential of SN12C HRCC clones. We expanded our experimental range to include clones of scirrhous gastric cancer cell lines (OCUM‑2M, OCUM‑2D and OCUM‑2MLN) and similar results were obtained, thereby further strengthening our original findings.

Strong terminator regions could be used to improve metabolically engineered yeasts by increasing the target enzyme protein yields above those achieved with traditional terminator regions. We recently identified five strong terminator regions (RPL41Bt, RPL15At, DIT1t, RPL3t, and IDP1t) in a comprehensive analysis of Saccharomyces cerevisiae. The effect of the terminator regions was analyzed by measuring the protein production of a linked transgene, and was shown to be twice that of a traditional terminator region (PGK1t). Here, we investigated whether the activity of the terminator regions is affected by exchange of a strong promoter or reporter in the linked transgene, carbon source for cell growth, stress factors, host yeast strain, or stage of the growth phase. Our results indicate that the activities of all five terminator regions were twice that of PGK1t in all conditions tested. In addition, we demonstrated that the strong activity of these terminator regions could be used to improve secretory production of endoglucanase II derived from Tricoderma ressei, and that the DIT1t strain was the best of the five strains for this purpose. We therefore propose that DIT1t, and the four other terminator regions, could be applied to the development of improved metabolically engineered yeasts.

Malignant pleural mesothelioma (MPM) is a malignant tumor originating from mesothelial cells existing in pleura. Since its incidence, it is closely related to the amount and time of exposure to asbestos, and the latency period after exposure to asbestos is very long, the incidence may increase over the next two decades. Since early detection is very difficult and there is no standard curative therapy, it is important to understand the biology of MPM, and to find biomarkers and molecular targets for its therapy. DDX39 is one of the Asp-Glu-Ala-Asp (DEAD)-box RNA helicases, which are required for the export of mRNA out of the nucleus, and transcription, splicing and transport of mRNA. Some reports have shown differential expression of DDX39 in tumor cells or tissues such as lung squamous cell cancer, gastrointestinal stromal tumor and urinary bladder cancer. In the present study, the protein levels of DDX39 in the human MPM cell lines NCI-H28, NCI-H2052 and NCI-H2452, and the human pleural mesothelial cell line MeT-5A were investigated by western blotting. The protein levels of DDX39 were found to be higher in NCI-H28, NCI-H2052 and NCI-H2452 compared to MeT-5A. The intensity of the bands of DDX39 in NCI-H28, NCI-H2052 and NCI-H2452 cells were increased by 1.351-, 1.887- and 2.024-fold, respectively, compared to MPM cells. These results suggest that DDX39 is a possible candidate biomarker for molecular-targeting of MPM.

Recombinant DNAs are traditionally constructed using Escherichia coli plasmids. In the yeast Saccharomyces cerevisiae, chromosomal gene targeting is a common technique, implying that the yeast homologous recombination system could be applied for recombinant DNA construction. In an attempt to use a S. cerevisiae chromosome for recombinant DNA construction, we selected the single ura3Δ0 locus as a gene targeting site. By selecting this single locus, repeated recombination using the surrounding URA3 sequences can be performed. The recombination system described here has several advantages over the conventional plasmid system, as it provides a method to confirm the selection of correct recombinants because transformation of the same locus replaces the pre-existing selection marker, resulting in the loss of the marker in successful recombinations. In addition, the constructed strains can serve as both PCR templates and hosts for preparing subsequent recombinant strains. Using this method, several yeast strains that contained selection markers, promoters, terminators and target genes at the ura3Δ0 locus were successfully generated. The system described here can potentially be applied for the construction of any recombinant DNA without the requirement for manipulations in E. coli. Interestingly, we unexpectedly found that several G/C-rich sequences used for fusion PCR lowered gene expression when located adjacent to the start codon.

Post-transcriptional upregulation is an effective way to increase the expression of transgenes and thus maximize the yields of target chemicals from metabolically engineered organisms. Refractory elements in the 3′ untranslated region (UTR) that increase mRNA half-life might be available. In Saccharomyces cerevisiae, several terminator regions have shown activity in increasing the production of proteins by upstream coding genes; among these terminators the DIT1 terminator has the highest activity. Here, we found in Saccharomyces cerevisiae that two resident trans-acting RNA-binding proteins (Nab6p and Pap1p) enhance the activity of the DIT1 terminator through the cis element GUUCG/U within the 3′-UTR. These two RNA-binding proteins could upregulate a battery of cell-wall–related genes. Mutagenesis of the DIT1 terminator improved its activity by a maximum of 500% of that of the standard PGK1 terminator. Further understanding and improvement of this system will facilitate inexpensive and stable production of complicated organism-derived drugs worldwide.

１．Introduction Well known as safety, non-toxicity and remarkable thermal stability, LiFePO 4 (LFP) is one of a promising candidate for the cathode material of large size lithium ion batteries. However, LFP is also known as the material with poor electrochemical property at low temperature. In order to improve the low temperature properties of LFP, several attempts were made to control the particle size and the morphology of LFP particle to increase the diffusion in solid and the surface reaction area. 1 ） On the other hand, along with the increase of LFP’s specific surface area, the problems arised for the manufacturing of the electrode. The problems are the increase of the amount of binder needed and the LFP agglomeration which leads to the increase of the surface roughness of the electrode. In this research, we have studied the influence of the LFP’s primary and secondary particle size and the morphology of the secondary particle to improve the electrochemical properties. ２．Experimental LFP was synthesized by a hydrothermal process. Spherical secondary particle with the size ranging from 10 to 30 μm in diameter was observed from the spray-drying of the LFP slurry. The electrode slurry mixing ratio was LFP:AB:PVdF=90:5:5 wt.%. After the slurry was coated on Al foil current conductor, it was vacuum dried under @120℃ for 12h、and pressed by a load of 200kgf. The cross-section image of the obtained electrode was observed by SEM. The particle size of the secondary particle was observed with particle size distribution measurements. The electrochemical properties were measured with pouch cell (anode：Li4Ti5O12 electrode, separator：porous polypropylene, electrolyte：1M LiPF6/EC:DEC=1:1 +VC2wt%) ３．Result The relationship between LFP’s secondary particle size and Output – Direct Current Resistance (DCR) was shown in Fig. 1. The smaller the secondary particle size, the DCR decreased. The porous structure of the LFP’s electrode was thought to have a correlation to the electrochemical characteristics. Fig.2 shows the cross-sectional SEM images of the electrode which showed the different structure for the electrode manufactured with different particle sized LFP. The bigger the secondary particle size, the bigger the vacant spaces present inside the electrode and the small secondary particle was enclosed in the large secondary particle. The non-uniform structure was observed for the electrode made with large secondary particle compared with the uniform structure made with the small secondary particle. The main factor of DCR increase for the large secondary particle size was thought to relate to the non-uniformity of secondary particle structure and electrode structure. The relationship between the electrode structure, the secondary particle shape and the electrochemical properties will be studied further. Ref. 1) A. Yamada, S. C. Chung and K.Hinokuma , J. Electrochem. Soc., 144, p. A224 (2001) Figure 1

We report a case of non-insulin-dependent diabetes mellitus (NIDDM) complicated with idiopathic hypoparathyroidism. A 74-year-old male was hospitalized because of diplopia. He was revealed to have NIDDM. The levels of serum Ca and intact-PTH were 6.3 mg/dl and < 5 pg/ml, respectively. Brain computed tomography revealed abnormal calcification in the cerebral basal ganglia and the cerebellum. After recovery from hypocalcemia, the endogenous insulin secretion was normalized. It is suggested that the pathogenesis of NIDDM in this patient may have been related to an insulin secretory defect as a result of hypocalcemia in addition to the hereditary risk.

Important strategies against cancer are based on the understanding of the mechanisms of tumor progression. To elucidate alterations regarding tumor progression, we have performed proteomic differential display analysis for the expression of intracellular proteins in the regressive murine fibrosarcoma cell clone QR-32 and the progressive malignant tumor cell clone QRsP-11, derived from QR-32, by means of combination of two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), and we have previously reported on relevant results. However, besides the protein spots which we already reported, we identified three more particular spots of interest. In the present study, two-dimensional western blot analysis demonstrated a significantly lower expression of three isoforms of nucleophosmin in progressive, compared to regressive cell clones. These results suggest that the down-regulation of the identified nucleophosmin proteins in QRsP-11 cells compared to QR-32 cells is possibly related to tumor malignant progression.

We have estimated the effects of hyper-mannosylation of dockerin-type cellulase on cellulosome assembly by using Saccharomyces cerevisiae and 44 protein glycosylation mutants, because the heterologous protein displayed on yeast is assumed to be modified by yeast-specific hyper-mannosylation. First, we constructed the yeast strain CtminiCipA, which displays a heterologous scaffolding protein (miniCipA from Clostridium thermocellum) on its cell surface, and glycosylation mutants secreting a dockerin-type cellulase (Cel8Aenz-Cel48Sdoc: a fusion protein of the catalytic domain of C. thermocellum Cel8A and the dockerin domain of C. thermocellum Cel48S). Next, minicellulosomes were assembled by mixing the CtminiCipA strain and the dockerin-type cellulase secreted by each glycosylation mutant. By using an endoglucanase assay and flow cytometric analysis, we showed that some glycosylation mutants enhanced cellulosome assembly; in particular, disruption of glycosylation genes located in the endoplasmic reticulum showed intense enhancement. These findings suggest that inhibition of the core complex or precursor formation in protein glycosylation enhances cellulosome assembly, meaning that absence of glycosylation is more important for cellulosome assembly than reducing the size of the glycochain.

Epitaxially grown crystals of C60 are prepared on (001) vacuum-deposited MgO films by successive deposition of MgO and C60 on a heated NaCl(001) cleaved surface. C60 grows as single-crystalline triangular islands with fcc structure on the MgO films from 180 to 200°C. The epitaxial relations are (111)[110]C60//(001)[010]MgO and (111)[110]C60//(001)[100]MgO as a result of Volmer-Weber growth. The islands do not have twin-like defects like those of C60 crystals previously grown on NaCl(001) substrates. High-resolution electron microscopy shows the images of aggregates of C60 molecules as ring-like black contrast at the edge of islands prepared at room temperature and 180°C. Image simulations of one C60 molecule located on MgO crystals of 10 and 20 nm thickness are presented, and the possibility of detection of the molecule is discussed in relation to the use of imaging under off-Bragg condition.

Tungsten and carbon-related clusters supported on noise-free magnesium oxide (MgO) films were observed by high-resolution electron microscopy. The dynamic behavior of the structure change was studied in real time using a TV-video system. The surface migration of tungsten atoms and carbon-related “atom rings” was clearly observed, and the surface diffusion constant of the tungsten atoms was estimated for the first time using transmission electron microscopy. Carbon 60 (C 60 ) molecules on the MgO film, prepared by vacuum deposition, were also imaged as a ring-like contrast with a bright center region.

In order to enhance heterologous cellulase protein production in yeast, a plasmid harboring the endoglucanase gene from Clostridium thermocellum (Ctcel8A) was used to systematically transform a homozygous diploid yeast deletion strain collection. We identified 55 deletion strains that exhibited enhanced endoglucanase activity compared with that of the wild-type strain. Genes disrupted in these strains were classified into the categories of transcription, translation, phospholipid synthesis, endosome/vacuole function, ER/Golgi function, nitrogen starvation response, and cytoskeleton. The vps3Δ and vps16Δ strains, which have deletion in genes encoding components of the class C core vacuole/endosome tethering (CORVET) complex, also exhibited enhanced β-glucosidase activity when Ctcel8A was heterologously expressed. Moreover, multiple gene deletion strains were constructed by using the vps3Δ strain. Endoglucanase activity of the resulting rav1Δvps3Δ double deletion strain was exhibited higher than that of the rav1Δ or vps3Δ strains. Our genome-wide analyses using the yeast deletion strain collection identified useful genes that allow efficient expression of cellulase.

We demonstrate the value of the thermotolerant yeast Issatchenkia orientalis as a candidate microorganism for bioethanol production from lignocellulosic biomass with the goal of consolidated bioprocessing. The I. orientalis MF-121 strain is acid tolerant, ethanol tolerant, and thermotolerant, and is thus a multistress-tolerant yeast. To express heterologous proteins in I. orientalis, we constructed a transformation system for the MF-121 strain and then isolated the promoters of TDH1 and PGK1, two genes that were found to be strongly expressed during ethanol fermentation. As a result, expression of beta-glucosidase from Aspergillus aculeatus could be achieved with I. orientalis, demonstrating successful heterologous gene expression in I. orientalis for the first time. The transformant could convert cellobiose to ethanol under acidic conditions and at high temperature. Simultaneous saccharification and fermentation (SSF) was performed with the transformant, which produced 29 g l(-1) of ethanol in 72 h at 40 degrees C even without addition of beta-glucosidase when SSF was carried out in medium containing 100 g l(-1) of microcrystalline cellulose and a commercial cellulase preparation. These results suggest that using a genetically engineered thermotolerant yeast such as I. orientalis in SSF could lead to cost reduction because less saccharification enzymes are required.

The effects of several conditions for the immunization of mice was studied using an aliquot of a viomycin (VM) protein conjugate as the common primary or booster antigen. Responses of the mice were assessed by measuring mouse serum levels of total immunoglobulin G (IgG) and anti-VM antibody responses using the newly improved two assay methods. The choice of route was found to be a very important factor in immunization and intraperitoneal injection was the most optimal among the four routes studied. The effect of the concentration of Mycobacteria in Freund's complete adjuvant (FCA) was also studied, and it was found that a diluted FCA was more effective than a commercial FCA. The effect of the controlled release of the antigen was studied and three important phenomena were observed: The mice immunized by the mini-osmotic pump-aided controlled release of the antigen responded with similar small amounts of both total IgG and anti-VM antibody regardless of the presence or absence of FCA in the antigen; emulsifying the antigen with FCA was a very important condition for the effective elicitation of the specific antibody; a mixture of antigen and FCA without emulsifying produced little specific antibody and a large amount of total IgG. The more effectively immunized mice responded with a larger decrease in body weight soon after the primary injection.

Synthesis of the methyl ester of AK-toxin II, a typical host-specific toxin, toxic to Japanese white pear, and fifteen diastereoisomers as its congeners was accomplished in optically active forms starting from vitamin C as a chiral starting material. Toxicity testing of these synthetic compounds revealed that the configurations of two chiral centers in the carboxylic acid part of the toxin have an important role in the toxicity.

The yeast MAPKKK Ste11 activates three MAP kinase pathways, including pheromone signaling, osmosensing, and pseudohyphal/invasive growth pathways. We identified two chemical compounds, BTB03006 and GK03225, that suppress growth defects induced by Ste11 activation in diploid yeast cells. BTB03006, but not GK03225, was found to suppress growth defects induced by both alpha-factor and Ste4 G(beta) overexpression in the pheromone signaling pathway, suggesting that GK03225 is an osmosensing pathway-specific inhibitor. We also performed genome-wide suppressor analysis for Ste11 activation, using a yeast deletion strains collection, and identified PBS2 and HOG1, and several genes associated with chaperone functions, which represent potential target proteins of the drugs screened from Ste11 activation. GK03225 possesses an Iressa-like quinazoline ring structure, and its chemical analog, 11N-078, suppresses c-Abl human tyrosine kinase activity. These results suggest that drug screening in yeast can identify human tyrosine kinase inhibitors and other drugs for human diseases.

Repeated gene manipulations can be performed in yeast by excision of an introduced marker. Cassette modules containing a marker flanked by two direct repeat sequences of hisG or loxP have often been used for marker recycling, but these leave one copy of the repeats in the chromosome after excision. Genomic copies of a repeat can cause increased mistargeting of constructs containing the same repeats or unexpected chromosomal rearrangements via intra- or interchromosomal recombinations. Here, we describe a novel marker recycling procedure that leaves no scar in the genome, which we have designated seamless gene deletion. A 40 base sequence derived from an adjacent region to the targeted locus was placed in an integrating construct to generate direct repeats after integration. Seamless HIS3 deletion was achieved via a PCR fragment that consisted of a URA3 marker attached to a 40 base repeat-generating sequence flanked by HIS3 targeting sequences at both ends. Transformation of the designed construct resulted in his3 disruption and the generation of 40 base direct repeats on both sides of URA3 in the targeted locus. The resulting his3::URA3 disruptants were plated on 5-fluoroorotic acid medium to select for URA3 loss. All the selected colonies had lost URA3 precisely by recombination between the repeats, resulting in his3 deletion without any extraneous sequences left behind in the chromosome.

From the powder mixtures of aluminium metal and natural graphite ground in a planetary ball mill, aluminium nitride was formed in air as the consequence of self-ignition and following self-propagation reactions. The mechanism for AlN formation in air is discussed by taking into consideration the possible reactions.

Malignant pleural mesothelioma (MPM) is a malignant neoplasm arising from mesothelial cells existing in pleura. It is difficult to identify a crucial symptom in patients with MPM, and there is no standard curative therapy for this malignancy. The incidence of MPM is increasing worldwide, and many patients can not survive more than one year after diagnosis. MPM is an asbestos-related disease, and it is believed that the incidence is closely related to the amount and time of exposure to asbestos. Since the latency period after exposure to asbestos is very long, the incidence may increase over the next two decades. Therefore, it is important to understand the biology of MPM, and to find a molecular target for its therapy. Three of human malignant pleural mesothelioma (MPM) cell lines (NCIH28, NCI-H2052, NCI-H2452) and a human pleural mesothelial cell line (MeT-5A) were analyzed by proteomics with the use of twodimensional gel electrophoresis (2-DE) and liquid chromatographytandem mass spectrometry (LC-MS/MS). From the results of 2-DE between MeT-5A and three MPM cell lines, 9 protein spots showed increased expression levels in MPM cells compared to MeT-5A. These spots were analyzed by LC-MS/MS analysis, and identified by a peptide sequence tag. Identified increased spots included 78 kDa glucose regulated protein (GRP78), heat shock 70 kDa protein (HSP70), protein disulfide isomerase A3, annexin A3, peroxiredoxin-2 and 14 kDa phosphohistidine phosphatase. Western blotting using specific antibodies against GRP78 and HSP70 confirmed the elevated expression level of GRP78 and HSP70 in MPM cell lines compared to MeT-5A cells. These results suggest that GRP78 and HSP70 expression is likely to be associated with the tumorigenesis of MPM, and they may be expectable as biomarkers or therapeutic targets for MPM. Disclosure: All authors have declared no conflicts of interest.

not Available.

A new translocation involving chromosome #1, #8, and #21 in a patient with type M2 acute myeloblastic leukemia is reported. The breakpoint of #1 in this case was at band p13 and differed from that in two previously reported cases of t(1;8;21) involving the long arm of #1. A key event leading to the development of the M2 phenotype appears to be a break at band q22 of #8 with associated translocation of the terminal end of the long arm of #21.

This study was undertaken to establish the optimal immunogen dose for immunization of mice, using a viomycin-protein conjugate as a hapten immunogen. It was found that specific immunoglobulin G (IgG) formation depends on both the dose of antigen and the type of adjuvant: the optimal antigen dose for an immune response is quite different depending on whether the mice are being treated with Freund's complete adjuvant (FCA) or Freund's incomplete adjuvant (FICA). The total IgG amount depends mainly upon the type of adjuvant used. FCA gave the double the level of IgG compared to that obtained with FICA. The antigen dose was found to have little influence on the total production of IgG. Mice given a primary immunization with 10 micrograms of antigen emulsified in FCA and then given a booster with the same amount of antigen emulsified in FICA produced a strikingly high level of specific anti-viomycin antibody of over 2.5 mg/ml of the antiserum. It was also found that decreases in the weight of the mice were related to the kind of adjuvant used as well as to the level of the specific antibody formed.

Two cases of human T-cell leukemia virus (HTLV)-positive adult T-cell leukemia (ATL) in brother and sister are presented. Six of 15 members of this family were seropositive for antibodies for ATL-associated antigens (ATLA). The sister of the ATL patient developed overt ATL after 5 years and 8 months of smoldering ATL. Immunologic examinations during the smoldering phase were normal except for negative skin tests for purified protein derivative. Factors leading to the induction of ATL among HTLV carriers remain to be studied.

A 28-year-old woman manifested a hemorrhagic tendency caused by a deficiency of vitamin K—dependent coagulation factors. Her condition was diagnosed as bulimia nervosa in view of a previous history of anorexia nervosa and episodes of self-induced vomiting and purging. There were no remarkable lesions in her alimentary system. In treatment of bulimia nervosa, attention should be given not only to the loss of body fluids and electrolytes, but also to the possibility of a deficiency of vitamin K—dependent coagulation factors. ( JAMA 1983;250:792-793)

To the Editor.— Human T-cell lymphotropic virus type I (HTLV-I), the causative agent of adult T-cell leukemia, is endemic in southern Japan, 1 the Caribbean islands, 2 and parts of Africa. 3 The prevalence of antibodies to HTLV-I is highest in Okinawa, Japan, where up to 31% of adults are seropositive in some regions. 4 Moreover, a high seroprevalence rate has been found in Japanese immigrants from Okinawa and their offspring in Hawaii. 5 This prompted us to look at the antibody prevalence in a similar immigrant population in Brazil. Methods.— Serum samples were collected from 46 Japanese immigrants (issei) from Okinawa and 50 offspring (nisei) of immigrants from Okinawa in Campo Grande, Brazil. They ranged in age from 29 to 84 years (mean, 66 years) for the issei (28 men, 18 women) and from 21 to 59 years (mean, 36 years) for the nisei (38 men, 12 women). The