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We investigated the role of FAD2, which was predicted to encode a fatty acid desaturase of the n-alkane-assimilating yeast Yarrowia lipolytica. Northern blot analysis suggested that FAD2 transcription was upregulated at low temperature or in the presence of n-alkanes or oleic acid. The FAD2 deletion mutant grew as well as the wild-type strain on glucose, n-alkanes, or oleic acid at 30 °C, but grew at a slower rate at 12 °C, when compared to the wild-type strain. The growth of the FAD2 deletion mutant at 12 °C was restored by the addition of 18:2, but not 18:1, fatty acids. The amount of 18:2 fatty acid in the wild-type strain was increased by the incubation at 12 °C and in the presence of n-octadecane. In contrast, 18:2 fatty acid was not detected in the deletion mutant of FAD2, confirming that FAD2 encodes the Δ12-fatty acid desaturase. These results suggest that Δ12-fatty acid desaturase is involved in the growth of Y. lipolytica at low temperature.

Yarrowia lipolytica possesses twelve ALK genes, which encode cytochromes P450 in the CYP52 family. In this study, using a Y. lipolytica strain from which all twelve ALK genes had been deleted, strains individually expressing each of the ALK genes were constructed and their roles and substrate specificities were determined by observing their growth on n-alkanes and analyzing fatty acid metabolism. The results suggested that the twelve Alk proteins can be categorized into four groups based on their substrate specificity: Alk1p, Alk2p, Alk9p, and Alk10p, which have significant activities to hydroxylate n-alkanes; Alk4p, Alk5p, and Alk7p, which have significant activities to hydroxylate the ω-terminal end of dodecanoic acid; Alk3p and Alk6p, which have significant activities to hydroxylate both n-alkanes and dodecanoic acid; and Alk8p, Alk11p, and Alk12p, which showed faint or no activities to oxidize these substrates. The involvement of Alk proteins in the oxidation of fatty alcohols and fatty aldehydes was also analyzed by measuring viability of the mutant deleted for twelve ALK genes in medium containing dodecanol and by observing growth on dodecanal of a mutant strain, in which twelve ALK genes were deleted along with four fatty aldehyde dehydrogenase genes. It was suggested that ALK gene(s) is/are involved in the detoxification of dodecanol and the assimilation of dodecanal. These results imply that genes encoding CYP52-family P450s have undergone multiplication and diversification in Y. lipolytica for assimilation of various hydrophobic compounds.

Question: A 68year-old man with severe alcoholic liver cirrhosis underwent a left lobe adult-to-adult living donor liver transplantation. The volume of the donor’s liver was relatively low for the recipient; therefore, portacaval shunting was performed between the right branch of the portal vein and the inferior vena

In the n-alkane assimilating yeast Yarrowia lipolytica, n-alkanes are oxidized to fatty acids via fatty alcohols and fatty aldehydes, after which they are utilized as carbon sources. Here, we show that four genes (HFD1-HFD4) encoding fatty aldehyde dehydrogenases (FALDHs) are involved in the metabolism of n-alkanes in Y. lipolytica. A mutant, in which all of four HFD genes are deleted (Δhfd1-4 strain), could not grow on n-alkanes of 12-18 carbons; however, the expression of one of those HFD genes restored its growth on n-alkanes. Production of Hfd2Ap or Hfd2Bp, translation products of transcript variants generated from HFD2 by the absence or presence of splicing, also supported the growth of the Δhfd1-4 strain on n-alkanes. The FALDH activity in the extract of the wild-type strain was increased when cells were incubated in the presence of n-decane, whereas this elevation in FALDH activity by n-decane was not observed in Δhfd1-4 strain extract. Substantial FALDH activities were detected in the extracts of Escherichia coli cells expressing the HFD genes. Fluorescent microscopic observation suggests that Hfd3p and Hfd2Bp are localized predominantly in the peroxisome, whereas Hfd1p and Hfd2Ap are localized in both the endoplasmic reticulum and the peroxisome. These results suggest that the HFD multigene family is responsible for the oxidation of fatty aldehydes to fatty acids in the metabolism of n-alkanes, and raise the possibility that Hfd proteins have diversified by gene multiplication and RNA splicing to efficiently assimilate or detoxify fatty aldehydes in Y. lipolytica.

CTP:phosphoethanolamine cytidylyltransferase (ECT) is a key enzyme in the CDP-ethanolamine branch of the Kennedy pathway, which is the primary pathway of phosphatidylethanolamine (PE) synthesis in mammalian cells. Here, the enzymatic properties of recombinant human ECT (hECT) were characterized. The catalytic reaction of hECT obeyed Michaelis–Menten kinetics with respect to both CTP and phosphoethanolamine. hECT is composed of two tandem cytidylyltransferase (CT) domains as ECTs of other organisms. The histidines, especially the first histidine, in the CTP-binding motif HxGH in the N-terminal CT domain were critical for its catalytic activity in vitro, while those in the C-terminal CT domain were not. Overexpression of the wild-type hECT and hECT mutants containing amino acid substitutions in the HxGH motif in the C-terminal CT domain suppressed the growth defect of the Saccharomyces cerevisiae mutant of ECT1 encoding ECT in the absence of a PE supply via the decarboxylation of phosphatidylserine, but overexpression of hECT mutants of the N-terminal CT domain did not. These results suggest that the N-terminal CT domain of hECT contributes to its catalytic reaction, but C-terminal CT domain does not.

In the n-alkane assimilating yeast Yarrowia lipolytica, the expression of ALK1, encoding a cytochrome P450 that catalyzes terminal mono-oxygenation of n-alkanes, is induced by n-alkanes. The transcription of ALK1 is regulated by a heterocomplex that comprises the basic helix-loop-helix transcription activators, Yas1p and Yas2p, and binds to alkane-responsive element 1 (ARE1) in the ALK1 promoter. An Opi1 family transcription repressor, Yas3p, represses transcription by binding to Yas2p. Yas3p localizes in the nucleus when Y. lipolytica is grown on glucose but localizes to the endoplasmic reticulum (ER) upon the addition of n-alkanes. In this study, we showed that recombinant Yas3p binds to the acidic phospholipids, phosphatidic acid (PA) and phosphoinositides (PIPs), in vitro. The ARE1-mediated transcription was enhanced in vivo in mutants defective in an ortholog of the Saccharomyces cerevisiae gene PAH1, encoding PA phosphatase, and in an ortholog of SAC1, encoding PIP phosphatase in the ER. Truncation mutation analyses for Yas3p revealed two regions that bound to PA and PIPs. These results suggest that the interaction with acidic phospholipids is important for the n-alkane-induced association of Yas3p with the ER membrane.

Interaction between interleukin (IL)-5 and its receptor (IL-5R) is important for the regulation of immunity against worm infections, allergic reactions and B cell response in mammals. In this study, we identified a full-length cDNA encoding chicken IL-5R α-chain (chIL-5Rα). The deduced amino acid sequence showed 41-43% identity to mammalian homologues. It has four well-conserved cysteines and a WSXWS motif in the extracellular region, and a PPXP motif in the cytoplasmic region. Quantitative RT-PCR analysis revealed that chIL-5Rα mRNA expression was markedly high in bone marrow and relatively high in spleen and lung. Recombinant proteins of soluble chIL-5Rα and cytokines (artificially produced chIL-5 (achIL-5) and another IL-5-like molecule KK34) were expressed by 293F cells to examine the cytokine-receptor interactions. Interaction assay using a Biacore biosensor showed that chIL-5Rα has the capability to bind with monomeric achIL-5, but not with KK34. In conclusion, chicken has an IL-5Rα homologue but KK34 does not complement the IL-5/IL-5R system.

The yeast Yarrowia lipolytica effectively utilizes hydrophobic substrates such as fatty acids and n-alkanes. To identify a gene(s) regulating fatty acid utilization in Y. lipolytica, we first studied homologous genes to OAF1 and PIP2 of Saccharomyces cerevisiae, but their disruption did not change growth on oleic acid at all. We next characterized a Y. lipolytica gene, POR1 (primary oleate regulator 1), an ortholog of farA encoding a transcriptional activator that regulates fatty acid utilization in Aspergillus nidulans. The deletion mutant of POR1 was defective in the growth on various fatty acids, but not on glucose, glycerol, or n-hexadecane. It exhibited slight defect on n-decane. The transcriptional induction of genes involved in β-oxidation and peroxisome proliferation by oleate was distinctly diminished in the Δpor1 strains. These data suggest that POR1 encodes a transcriptional activator widely regulating fatty acid metabolism in Y. lipolytica.

Background Tumor necrosis factor (TNF)-α is a cytokine that initiates liver regeneration after hepatectomy (HTx), although extensive HTx can cause liver failure with significant rise in serum TNF-α levels. To test our hypothesis that modulation of endogenous TNF-α attenuates liver failure even after extensive HTx, we used ONO-SM362, a novel TNF-α inhibitor, in mice subjected to 85% HTx. Methods ICR mice were divided into 5 groups: 70% HTx, 85% HTx, 85% HTx plus ONO-SM362, 85% HTx plus monoclonal TNF-α antibody (mAb), and 85% HTx plus FR167653, a TNF-α inhibitor. We analyzed the survival rate, blood ammonia (NH 3 ), serum TNF-α levels, TNF-α mRNA expression in the liver and spleen by real-time polymerase chain reaction, histologic changes, polymorphonuclear neutrophils (PMNs) infiltration, and proliferating cell nuclear antigen labeling index (PCNA LI) in the 5 groups. Results The survival rate at 7 days after surgery was 100%, 0%, 100%, 50%, and 0%, for the 70% HTx, 85% HTx, 85% HTx + ONO-SM362, 85% HTx + mAb, and 85% HTx + FR167653, respectively. Mice that underwent 85% HTx died from liver failure associated with a significant rise in serum TNF-α level. ONO-SM362 and mAb improved animal survival and enhanced PCNA LI. In addition, ONO-SM362 inhibited TNF-α mRNA expression in the remnant liver and suppressed PMNs infiltration. Conclusions Suppression of excessive TNF-α production using ONO-SM362 ameliorated liver failure after 85% HTx.

Mammalian interleukin-13 (IL-13) is an important regulatory T2 cytokine secreted by activated T lymphocytes. The IL-13 receptor (IL-13R) has two different chains, IL-13Ralpha1 and IL-13Ralpha2. Although the chicken IL-13 gene is well characterized, little is known about IL-13Rs. We cloned a cDNA encoding the 380 amino acid pro-peptide of chicken IL-13Ralpha2 (chIL-13Ralpha2) and developed a monoclonal antibody (mAb), HU13-1, against it. The chIL-13Ralpha2 amino acid sequence showed 37-39% sequence identity with mammalian homologs. High levels of chIL-13Ralpha2 mRNA were expressed in liver, testis, ovary, brain, and lipopolysaccharide (LPS)-stimulated IN24 cells. HU13-1 specifically recognized recombinant chIL-13Ralpha2 in ELISAs, and western blots identified a 45-kDa glycoprotein or a 41-kDa non-glycosylated protein in LPS-stimulated IN24 cell lysates. LPS induced a gradual increase in HU13-1-positive IN24 cells over 20 h. These results indicate that mAb HU13-1 recognizes native chIL-13Ralpha2 and will be valuable for further studies of chIL-13Rs.

Rare earth ternary borides, RRh 3 B x (R = La, Gd, Lu and Sc) have been synthesized by arc melting method. Borides RRh 3 B x (R = La, Gd, Lu and Sc) have perovskite-type cubic structure: space group Pm 3 m ; Z = 1. The lattice parameters a of the stoichiometric RRh 3 B for R = La, Gd, Lu and Sc are 0.4251(1), 0.4183(1), 0.4126(1) and 0.4080(1) nm, respectively. LaRh 3 B x does not have boron-nonstoichiometry as x = 0. In GdRh 3 B x and LuRh 3 B x , boron- nonstoichiometry ranges between 0.55 ≦ x ≦ 1 and 0.30 ≦ x ≦ 1, respectively. The boron-nonstoichiometry range is the widest, 0 ≦ x ≦ 1, for R = Sc. Boron-nonstoichiometry increases with decreasing atomic radius of R. The microhardness of the stoichiometric RRh 3 B for R = La, Gd, Lu and Sc is 4.2 ± 0.1, 6.8 ± 0.1, 7.7 ± 0.5 and 9.9 ± 0.1 GPa, respectively. As a result, microhardness increases with decreasing atomic size of R in RRh 3 B; R is positioned at the eight corners of the cube in the perovskite-type structure. Thus, hardness is strongly dependent on R element. The hardness changes almost linearly with boron concentration x for R = Gd and Lu in RRh 3 B x , while no linear dependency is found for R = Sc. Ab initio calculations have been performed to obtain the equilibrium lattice constants and the bulk moduli. The calculated lattice constants are in excellent agreement with experimental results.

Perovskite-type RRh 3 B and RRh 3 C (R = Y, Sc) form a continuous solid solution, RRh 3 B x C 1-x , in the range of 0?x?1 with cubic structure (space group: Pm3m, Z= 1). The values of the microhardness of YRh 3 B x C 1-x for x = 0, 0.25, 0.50, 0.75 and 1.00 are investigated as 4.4 ± 0.1, 4.9 ± 0.1, 5.5 ± 0.2, 6.4 ± 0.2 and 7.5 ± 0.15 GPa, respectively. On the other hand, the values of the microhardness of ScRh 3 B x C 1-x for x = 0, 0.25, 0.50, 0.75 and 1.00 are 4.5 ± 0.2,6.1 ± 0.2, 7.4 ± 0.2, 8.9 ± 0.2 and 9.6 ± 0.1 GPa, respectively. Thus, the microhardness of RRh 3 B x C 1-x continuously becomes larger with increasing boron content. The oxidation onset temperatures of YRh 3 B x C 1-x for x=0, 0.25, 0.50, 0.75 and 1.00 are 604, 631, 655, 687 and 978 K, respectively. On the other hand, the oxidation onset temperatures of ScRh 3 B x C 1-x for x=0, 0.25, 0.50, 0.75 and 1.00 are 674, 675, 695, 725 and 753 K, respectively. Thermogravimetric analysis of the phase indicates that the oxidation onset temperature also increases with boron content. Thus, it appears that both mechanical strength and chemical stability of the RRh 3 B x C 1-x phase essentially depend on its boron content. Ab initio calculations have been performed to obtain the equilibrium lattice constants and the bulk moduli. The calculated lattice constants are in excellent agreement with experimental results.

Interleukin-6 (IL-6) is a multifunctional cytokine that plays roles in regulating immune responses, acute phase reactions and hematopoiesis. IL-6 signaling is regulated by two receptors, a specific alpha chain (IL-6Ralpha) and a signal transducer, gp130. In this study, cDNA encoding the 445 amino acid propeptide of chicken IL-6Ralpha (chIL-6Ralpha) was identified. The predicted 445 amino acids showed approximately 40% sequence identity with mammalian homologues. In a domain search, chIL-6Ralpha had a signal peptide of 20 residues, an immunoglobulin-like (IG) domain of 71 residues and a fibronectin-type III (FN III) domain of 85 residues. On comparison with mammalian homologues, four conserved cysteine residues and the WSXWS motif were observed in the N- and C-terminal regions of the FN III domain, respectively. Expression analysis revealed that chIL-6Ralpha is strongly expressed in liver and the chicken hepatoma cell line LMH. These findings indicate that the identified chicken cDNA sequence encodes a chIL-6Ralpha homologue.

Tributyltin, an environmental pollutant, affected adrenal steroid hormone biosynthesis by two modes of action. Treatment of bovine adrenal cultured cells with 10-100 nM tributyltin for 48 h suppressed cortisol and androstenedione secretion, but induced the accumulation of 17alpha-hydroxyprogesterone and deoxycortisol, indicating that the P450(C21) and P450(11beta) activities were specifically suppressed. Direct inhibition of the enzymatic activities due to tributyltin was not observed in isolated organelles of untreated cells at concentrations less than 10 microM. Western blotting experiments using specific antibodies against steroidogenic enzymes showed that treatment with 1-100 nM tributyltin caused a decrease in cellular P450(C21) and P450(11beta) protein levels, and real-time PCR experiments showed that the decrease in protein content was attributable to decreases in mRNA of the enzymes. Tributyltin at concentrations higher than 100 nM suppressed all steroid biosynthesis in the adrenal cells. This suppression was closely correlated to the decrease in steroidogenic acute regulatory protein. Since nanomolar concentrations of tributyltin disturbed steroidogenesis in mammalian cells, there is the possibility that steroid hormone synthesis in polluted wild animals is affected by this compound.

When compared with mammalian IgG, chicken IgY is advantageous in terms of cross-reactivity. In this study, two plasmids were constructed for expression of recombinant chicken IgY derived from a chicken hybridoma. The first was for expression of the light (L) chain, and the other was for the heavy (H) chain with a histidine (His) tag at the carboxy-terminal. After transfection of recombinant chicken IgY gene into Chinese hamster ovary cells, a transfectant designated HF33 that secreted the specific antibody was selected. HF33 cells produced recombinant IgY with His tag at 10-15 microg/10(6) cells/24 h. On Western blotting analysis, the recombinant IgY was detected as one band for the H chain and two bands for the L chain. The recombinant IgY was successfully purified in a one-step procedure using a nickel-affinity resin. These results indicate that the present recombinant chicken IgY is useful for further applications.

The plaA gene encoding a protein that contains the cytosolic Phospholipase A(2) (cPLA(2)) motif is cloned for the first time from the filamentous fungus, Aspergillus nidulans. The translated 837 amino acid protein product of plaA comprises conserved lipase regions that are present in most mammalian cPLA(2) homologs. High expression of plaA was observed in glucose-lactose medium by Northern blot analyses. Deletion mutants of plaA grew and formed conidia similar to the wild-type strain, but showed decreased PLA(2) activity. Expression of the N-terminal truncated form of plaA in yeast cells resulted in increased Ca(2+)-dependent PLA(2) activity with (14)C-labeled phosphatidylcholine (PC) and phosphatidylethanolamine (PE) as substrates, compared with vector-transformed cells. In conclusion, we have identified and cloned a phospholipid-hydrolyzing novel cPLA(2) protein from A. nidulans for the first time.

To easily and rapidly recover exogenous gene products from chicken egg yolk, we constructed pVTG-catD (VTG, vitellogenin; catD, cathepsin D), a vector cassette carrying two catD-recognition signal peptides (catD-RSPs) in addition to the cloning site. An enhanced green fluorescence protein (EGFP)-encoding DNA fragment was ligated into the pVTG-catD. When the resultant construct pVTG-EGFP-catD containing histidine- and myc-tags was transfected into the chicken hepatoma cell line LMH, EGFP-expression at 24h post-cultivation was confirmed by fluorescence microscopy. Because a signal peptide (NTVLAEF) encoded in pVTG-EGFP-catD is recognized by catD, the VTG-EGFP fusion protein digested with catD was detectable by Western blotting. Digested exogenous gene product was recovered with nickel resin. These results indicate that catD-recognition sites bearing pVTG-catD and His-tags are functional in chicken LMH cells. Therefore, the system described here may be of use in making excision exogenous gene products in the chicken and in creating homozygous knock-in chickens.

The chicken is a useful animal for preparation of antibodies that are reactive with highly conserved mammalian molecules. For further clinical application of chicken antibodies, we constructed the novel expression vectors for chicken-human chimeric antibodies, pcSLCgamma1, pcSLCgamma4 and pcSLCkappa. These vectors had the following characteristics: (1) any chicken variable regions from hybridomas or a phage display library can be easily introduced; (2) the variable regions are able to be expressed in different immunoglobulin isotypes; and (3) the chimeric antibodies can be highly expressed in either transiently or stably transfected eukaryotic cells (COS-7 and CHO-K1 cells). Western blot analysis of the chimeric antibodies revealed that the expressed products were of the predicted size, structure and specificity. These results indicate that these vectors are useful tools for the chimerization of chicken antibodies.

We previously reported the development of chicken monoclonal antibodies (mAb) against mammalian-conserved molecules by cell fusion and phage display using the mouse mAb expression vector pPDS. However, chicken hybridomas produce relatively small amounts of antibody when compared with mouse hybridomas, and application of the pPDS may be limited in two-antibody assays with a mouse mAb because it contains mouse Ckappa as a detection tag. To circumvent the above problems, two expression vectors were established and used to produce a functional recombinant chicken mAb. These vectors, which were designed to accommodate a single chain fragment of the variable region (scFv) of the antibody, contained a chicken Clambda and FLAG with or without 6 x histidine sequences in the 3' terminus of the scFv to serve as detection and purification tags. In this study, a prion protein (PrP)-specific chicken mAb (HUC2-13) was expressed as phage-displayed and soluble scFv mAb forms by using these vectors. The scFv mAbs expressed by these vectors exhibited the same antigen-binding specificity to PrP as that of the original HUC2-13, could be purified with ease, and used in combination with a mouse mAb. These results indicate that the methods described herein offer an alternative to chicken mAb production from hybridomas and immunized chicken splenocytes, and may contribute to the use of chicken mAb reagents in numerous fields.

A rapid downshift in the growth temperature of Saccharomyces cerevisiae from 30 to 10 degrees C resulted in an increase in transcript levels of the antioxidation genes SOD1 [encoding Cu-Zn superoxide dismutase (SOD)], CTT1 (encoding catalase T), and GSH1 (encoding gamma-glutamylcysteine synthetase). The cellular activities of SOD and catalase were also increased, indicating that the temperature downshift caused an antioxidant response. In support of this, a simultaneous increase in the intracellular level of H(2)O(2) was observed. The level of YAP1 mRNA, encoding a transcription factor critical for the oxidative stress response in this yeast, was also increased by the temperature downshift. However, deletion of YAP1 did not reduce the elevated mRNA levels of the antioxidant genes. This suggests that the temperature downshift-induced increase in the mRNA level of anti-oxidant genes is YAP1-independent.

The germinal center (GC) develops after antigenic stimulation and is thought to occur at the site of various immune responses. We separated a single GC from chicken spleen after antigenic stimulation. Flow cytometric analysis of the cells derived from a single GC and RT-PCR analysis of Ig mRNA expression in GC was performed. Direct evidence indicates that: (1) there was a considerable difference in the cell population of each GC, (2) the ratio of CD3(+) cells in a GC remains constant at 10-20%, (3) the highest proportion of sIgY(+) cells in a GC occurs 1 week after the time of highest proportion of sIgM(+) cells, and (4) RT-PCR analysis was used to detect IgY mRNA expression in a GC. The continuous existence of CD3(+) cells, the alterations in sIgM(+) and sIgY(+) cell ratios, and the expression of IgY mRNA strongly suggest that Ig class switching occurs in the GC during an immune response.

The YlALK1 gene, which encodes cytochrome P450ALK1, plays a primary role in the assimilation of n-decane by yeast Yarrowia lipolytica and is inducible by n-decane at the transcriptional level. Deletion analysis of the YlALK1 promoter revealed that a 95-bp region on the YlALK1 promoter (from the position -400 to -304 upstream of the ATG codon) is essential for the induction by n-decane and we named this region ARR1 (alkane-responsive region). ARR1 was found to be made up of two different elements, ARE1 (alkane-responsive element 1; from -394 to -371) and ARE2 (from -325 to -305). By electrophoretic mobility shift assay, we found that the respective elements gave specific shift bands with the extracts from Y. lipolytica cells grown on n-alkane, but not much evidently from the cells grown on glycerol or glucose. This suggests that proteins that specifically bind to these elements are present and their binding or synthesis is dependent on n-alkane.

Polycrystalline samples of NdRh 3 B x have been synthesized by arc melting technique. The crystal structure of NdRh 3 B x is the perovskite-type cubic system (space group Pm 3 m ) for nominal boron concentration in the range of 0.706≤ x ≤1.000 (15–20 mol.% B). The lattice parameter a varies linearly from 0.41749(7) nm ( x =0.706) to a =0.42136(6) nm ( x =1.000). Thermogravimetric analysis indicates that the oxidation onset temperature for NdRh 3 B 1.000 is 663 K. The weight gain of the sample by heating in air up to 1473 K is 10.00% for NdRh 3 B 1.000 , and oxidized products are Rh and NdBO 3 . The micro-Vickers hardness is 4.9±0.05 GPa for NdRh 3 B 1.000 . NdRh 3 B 1.000 shows a metallic temperature dependence of the resistivity down to 0.5 K. Magnetic susceptibilities for NdRh 3 B 1.000 show Curie-like paramagnetic temperature dependence due to Nd 3+ moments. No trace of magnetic phase transitions and superconductivity is found.

Avian thrombocytes are nucleated blood cells homologous in function to mammalian platelets. In the present study, we obtained a cDNA from chicken thrombocyte polyadenylated RNA [Poly(A)+RNA], which coded for the chicken PDGF-B chain. The sequence was 1083-bp long and had an open reading frame (ORF) of 753-bp. At the amino acid level, the predicted mature protein showed 69% homology with the processed coding region of human PDGF-B. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that PDGF-B mRNA was expressed at high levels in thrombocytes and in the lung. The expression of PDGF-B chain mRNA in thrombocytes reached its maximum level 12 h following type 1 collagen treatment. These results suggest that chicken PDGF-B chain may play an important role in the vascular system and in healing wounded tissue.

The relationship between the structure of perovskite-type BaXO3 and the ionic radius of X was investigated. The size of the X ion was controlled by an appropriate combination of a pair of tetravalent ions such as (Ce, Tb), (Pr, Tb), (Pr, Zr) and (Tb, Zr). The phases examined were prepared by solid–solid reaction at 1500°C. Structural characterization was carried out by the powder X-ray diffraction technique. As a result, the change from orthorhombic to a rhombohedral-like structure takes place at 0.0745 nm for X, and another change from rhombohedral-like to a cubic structure occurs at around 0.0825 nm. These structural characteristics are discussed compared with perovskite-type (Ndx, Sm1−x)AlO3, which shows similar phenomena to BaXO3.

Platelet-derived growth factor (PDGF) affects cell proliferation and differentiation during mammalian embryogenesis. In a number of avian species, PDGF-alpha receptors and PDGF-A chain (PDGF-A) are present during chicken limb and lens development. However, little is understood about the chicken PDGF-A gene. The present study identified short form type 1 (S1), long form (L) and short form type 2 (S2) cDNA clones encoding chicken PDGF-A chain (PDGF-A). These clones were isolated from a chicken hepatoma cell line (LMH) mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) and cDNA library cloning. Genomic sequencing and Southern blotting revealed that these forms were generated by alternative splicing. The mRNAs of S1 and L contained two transcription start sites on one exon. At the amino acid level, the mature protein encoded by the L clone showed 90 and 85% homology with the processed coding regions of the long form of human and Xenopus PDGF-A, respectively. The putative mature peptides of all forms of chicken PDGF-A encompassed the eight cysteine residues conserved in all known forms of PDGF. We examined the expression of the three forms in chicken tissues and cells using RT-PCR. Expression of these forms varied among tissues and cells. Levels of PDGF mRNAs were very low in chicken thrombocytes, which are analogous to mammalian platelets. However, the level of PDGF-A chain mRNA expression in chicken thrombocytes peaked 4 h after exposure to type 1 collagen or thrombin, and then decreased gradually with continued incubation. These results suggest that chicken PDGF in thrombocytes plays an important role in the vascular system and in healing damaged tissue.

Localization and role of bursin during Bursa of Fabricius (BF) ontogeny were examined by immunohistochemical staining and by in ovo injection with anti-bursin antibody. Mouse monoclonal anti-bursin antibody HU2 was generated by immunization with synthetic bursin. It recognized reticular cells (REC), follicular associated epithelium (FAE), FAE-supporting cells, and the basal layer of interfollicular epithelium (IFE) in the mature BF. Bu-1+ cells were first detectable in the mesenchyme area at 13 days of embryogenesis (E13) before bud formation, then lined up along the bud, and homed into the bud at around E15. IgM+ cells were detected in the bud after E13. Bursin was first observed at the under edge of the bud. Injection of HU2 into embryonal vein at E13 suppressed the appearance of IgM+ cells in the Bursa at E17. These results indicate that bursin exists beneath the bud and may act on the appearance of IgM+ cells during BF ontogeny.

Using cDNA subtraction screening, we identified five Saccharomyces cerevisiae genes whose expressions is up-regulated when culture temperature was down-shifted from 30 to 10°C. Among these LOT ( lo w t emperature-responsive) genes, three (LOT1, LOT2, and LOT3) were identical to FBA1, RPL2B, and NOP1, encoding a fructose biphosphate aldolase, a ribosomal protein L2B, and a nucleolar protein for rRNA processing, respectively. No functions were assigned for LOT5 and LOT6, which are identical to YKL183w and YLR011w, respectively. Northern hybridization analysis revealed that these genes are not uniformly regulated in response to the change of growth temperature. In addition, all the LOT genes, except for LOT1/FBA1, were induced by a low concentration of cycloheximide. The data indicate that multiple mechanisms, including translational functionality may be involved in the regulation of LOT gene expression in yeast.

Polycrystalline samples of ScRh 3 B x (0≤ x ≤1.0) were synthesized by the arc melting method. The crystal structure is the perovskite-type cubic structure (space group Pm3m ) for boron content in the range 0≤ x ≤1.0 (0–20 at.% B). The lattice parameter a varies linearly from a =0.3903(1) nm ( x =0) to 0.40799(3) nm ( x =1.0). The micro-Vickers hardness increases with increasing boron content from 1.9 (±0.1) GPa for ScRh 3 (0 at.% B) to 9.9 (±0.1) GPa for ScRh 3 B 1.0 (20 at.% B). Thermogravimetric analysis indicates that the oxidation onset temperature for stoichiometric ScRh 3 B is 868 K. A sharp exothermic peak is observed at 1068 K by differential thermal analysis. The weight gain of the sample by heating in air up to 1473 K is 12.7%. The weight gain increases with increasing boron content. All samples in the range 0≤ x ≤1.0 show a metallic temperature dependence of the resistivity down to 0.5 K. Magnetic susceptibilities also show Pauli paramagnetic behavior and no trace of magnetic transitions on superconductivity in any of the samples.

Overproduction of delta(pro), a mutated secretory proteinase derived from a filamentous fungus Rhizopus niveus, results in formation of gross aggregates (delta(pro) aggregates) in the yeast endoplasmic reticulum (ER) lumen, activation of the unfolded protein response (UPR) and ER membrane proliferation. To investigate the roles of the UPR against the delta(pro) aggregates, we constructed an IRE1-deleted ((delta)ire1) strain. In contrast to wild-type cells, (delta)ire1 cells ceased to grow several hours after the overproduction of (delta)pro. Two lines of evidence argued against the possibility that the growth defect was due to the inability to make extra ER membrane which accommodates the (delta)pro aggregates. First, by electron microscopy, ER membrane proliferation was observed in (delta)ire1 cells overproducing (delta)pro. Second, disruption of the OPI1 gene in the (delta)ire1 mutant, which is considered to derepress the activities of phospholipid-synthesizing enzymes, did not restore the growth upon the overproduction of (delta)pro. Instead, the growth was restored when an extra copy of the KAR2 gene, which encodes yeast BiP, was introduced, indicating that an increase in the amount of BiP is essential for cell growth when the (delta)pro aggregates accumulate in the ER. Since BiP is included in the insoluble (delta)pro aggregates, it is likely that the amount of free BiP in the ER lumen is insufficient without the UPR to fully exert its functions. Consistently, overproduction of (delta)pro impaired protein translocation and folding in (delta)ire1 cells but not in wild-type cells. The tunicamycin sensitivity of (delta)ire1 cells was also suppressed by extra expression of KAR2, suggesting that BiP plays a principal role in protecting cell growth against misfolded proteins accumulated in the ER.

HoRh 3 B is synthesized by the arc melting method. Its chemical and magnetic properties are studied by XPS and magnetization measurements. Although the spectral features Ho4d, Ho4f and Ho5p between the compound and the Ho metal are very similar, each binding energy in the compound is smaller by about 0.3 eV than that in the metal. The density of states at the Fermi level of the compound are contributed mainly from Rh levels. The XP spectra for HoRh 3 B and HoRh 3 B 0.706 coincide with each other except for the intensity of B is level. Both HoRh 3 B and HoRh 3 B 0.706 exhibit normal paramagnetic behavior. The effective magnetic moments of 10.44 μ B for HoRh 3 B and 10.40 μ B for HoRh 3 B 0.706 are in agreement with the Hund value of 10.61 μ B for Ho 3+ .

Single crystals of a new compound Nb5Sn2Ga were obtained by the flux method using molten tin as a solvent. The crystal structure represents a tetragonal symmetry, space group D4h18I4/mcm, ordered W5Si3-type structure. Lattice parameters are a=1.0586(2) nm and c=0.5177(1) nm. According to XPS study, the peaks of Nb, Sn and Ga 3dXP spectra negatively shift when the three elements of Nb, Sn and Ga form the compound of Nb5Sn2Ga. The compound shows superconductivity at Tc=1.75 K and ΔTc=140 mK. The residual resistivity ratio, RRR=ρ (293 K)/ρ (4.2 K), is 12. The Micro-Vickers hardness (MVH) value for the (100) or (110) face is 8.9–8.5 GPa, and for the (001) face is 10.1–9.1 GPa. Oxidation of the Nb5Sn2Ga starts at 562°C. Weight gain of the compound heated up to 1200°C in air is 37.3%. Final oxidation product contains NbO2, Nb2O5, Nb12O29, SnO2 and Ga2O3.

Single crystals of ternary borides ErRh3B (cubic,Pm3m), ErRh3B2 (monoclinic, C2/m) and ErRh4B4 (tetragonal, P42/nmc) have been grown from copper solution by slow cooling method. The electrical resistivity, oxidation resistivity and Vickers microhardness were studied. The electrical resistivities at room temperature of the (100) face of ErRh3B, (001) face of ErRh3B2 and (100) face of ErRh4B4 are 25.6 μΩ·cm, 50.0 μΩ·cm and 106.8 μΩ·cm, respectively. According to thermogravimetric and differential thermal analyses, the oxidation of ErRh3B, ErRh3B2 and ErRh4B4 start at 1030°C, 373°C and 690°C, respectively. The weight gain of the same compounds after heating in air up to 1200°C is 0.7%, 15.44% and 5.4%, respectively. The values of the Vickers microhardness for the (100) face of ErRh3B, the (100) face of ErRh3B2 and the (110) face of ErRh4B4 are 4.8–5.0 GPa, 10.4–10.9 GPa and 10.9–11.3 GPa, respectively. The effect of boron content and crystal structure of each compound on the electrical resistivity, oxidation resistivity and Vickers microhardness are discussed.

Phase transition of solid solution phases of (Nd x ,Sm 1− x )AlO 3 was investigated by powder X-ray diffraction technique. The transition temperature T c from orthorhombic to trigonal structure is linearly related to x in (Nd x ,Sm 1− x )AlO 3 with the relation of T c (°C)=−1043.4 x +785.8 as an approximation at ambient pressure. This transition is reversible against the change of temperature. At room temperature, the structural change from orthorhombic to trigonal system takes place at around x =0.73 when x varies from 0.0 to 1.0 in (Nd x ,Sm 1− x )AlO 3 . Thus, the average ionic radius of R 3+ plays an important role to decide the structure of RAlO 3 , and an appropriate selection of ionic radius r R for R will function so as to control temperature and/or pressure for the structural change. As a result, structural diagram of RAlO 3 given by temperature condition and atomic number of R, which implies a phase diagram under pressure and temperature conditions, was proposed. Change of molar volume of a series of RAlO 3 was also discussed based on both effects of temperature and ionic radius R 3+ .

AcsmAgene that encodes chitin synthase with a myosin motor-like domain was isolated from the filamentous fungusAspergillus nidulans.Initially, we obtained thecsmAas a homolog of theAspergillus fumigatus chsE-partial fragment. A large open reading frame encoding a polypeptide of 1,852 a.a. was identified by determining the cDNA sequences. The chitin synthase conserved region was situated at the C-terminus and classified into class V as reported previously. On the other hand, the N-terminal region showed significant similarity to myosin motors and could not be classified into any types of myosins identifided so far. Thus, it is suggested that this is the first report of unconventional myosin fused to a metabolic enzyme. The finding of this new type of chitin synthase gene suggests that localization of chitin synthesis may be guided by association with cytoskeletal structures.

Structure change from orthorhombic to trigonal system of (Nd x , Sm 1− x )AlO 3 , which takes place by substituting Sm 3+ for Nd 3+ , was investigated by X-ray powder diffraction method. The samples were prepared by a solid state reaction. Their structures are based on a perovskite-type structure with slightly deformed lattices from an ideal cubic structure. The lattice deformation from an ideal cubic lattice is minimum at around x = 0.0 and it systematically increases by the amount of substitution of Sm 3+ for Nd 3+ , and the structure changes from orthorhombic to trigonal system at around x = 0.7. Thus, the substitution of Sm 3+ for Nd 3+ apparently plays a role of the changes of temperature and/or pressure for the cause of a first-order phase transition.

Rhizopus niveus aspartic proteinase-I (RNAP-I) is secreted by Saccharomyces cerevisiae extracellularly (Horiuchi, H., Ashikari, T., Amachi, T., Yoshizumi, H., Takagi, M., and Yano, K. (1990) Agric. Biol. Chem. 54, 1771-1779). The prosequence of RNAP-I has the function to promote correct folding of its mature part. Deletion (Deltapro) and amino acid substitutions (M1) in the prosequence block secretion of RNAP-I (Fukuda, R., Horiuchi, H., Ohta, A., and Takagi, M. (1994) J. Biol. Chem. 269, 9556-9561). In this study, little accumulation of Deltapro was observed in Western blot analysis of the cell extracts of the transformants producing Deltapro using anti-RNAP-I antisera. In contrast, M1 was accumulated in the yeast cells. Pulse-chase analysis revealed that they were synthesized at almost the same rates and that Deltapro was degraded in the cells more rapidly than M1. In subcellular fractionation analysis, Deltapro was found in the fraction that contained most of the activity of an endoplasmic reticulum (ER) marker enzyme, NADPH-cytochrome c reductase. In indirect immunofluorescence microscopy, Deltapro was observed in the ER. Similar result was also observed in a mutant which is deficient of the two vacuolar proteases, proteinase A and proteinase B. So, the vacuolar proteases are not involved in degradation of Deltapro. From these results, we concluded that RNAP-Is with the mutated prosequences, which probably could not be folded correctly, were retained and degraded in the ER.

Single crystals of CrSi2 were obtained in the form of hexagonal prisms by the solution growth method using molten tin as a flux. The maximum size of the crystal is about 0.3 mm in diameter and 25 mm in length. The crystal structure of CrSi2 has hexagonal symmetry with space group P6222 and the lattice parameters are a=0.425(2) nm and c=0.6375(1) nm, respectively. The crystals are semiconducting. The value of the micro-Vickers hardness for the { 1 1 0 0 } face with hexagonal symmetry is 11.2±0.4 GPa. Weight gain of the crystals heated up to 1473 K in air is negligible.

Single crystals of RA1O3 (R = La-Lu) were successfully obtained at atmospheric pressure by a flux method using KF. According to the R element, solute was prepared by three different processes. Single crystals of RA1O3 (R = Ce, Pr and Tb) were grown in He atmosphere to prevent oxidation, and others were grown in air. Single crystals are transparent cubes. In the case of the growth experiment of NdA1O3, the solute was prepared from alcoholates and it was very smoothly dissolved in the KF flux. As a result, crystals of NdA1O3 were grown up to 300–600 μm in size compared with 30–60 μm for other phases. Single crystals of LuAlO3 were successfully obtained for the first time by this flux method at ambient pressure in air. RA1O3 for R = La, Pr and Nd shows trigonal symmetry with a rhombohedral lattice, and CeAlO3 crystallizes in tetragonal symmetry. Others show orthorhombic symmetry. The lattice distortion from an ideal cubic perovskite structure is smaller in the aluminates of rare earth elements with lower atomic numbers in each structure group.

Promoter analysis was performed on the Rhizopus niveus 3-phosphoglycerate kinase 2-encoding gene (pgk2), one of the two pgk genes (pgk1 and pgk2) from this filamentous fungus sequenced so far. Deletion mutants of the promoter region were fused to the Escherichia coli uidA gene (which codes for beta-glucuronidase; GUS), and introduced into R. niveus to measure the intracellular GUS activities of the transformants. Deletion of the sequence between nt -174 to -133 (numbers indicate the position from the putative translation start codon) caused a significant decrease in the ratio of the GUS activity of the transformant cultured in glucose medium compared to that in glycerol medium. In this region, a 21-nt sequence which is well conserved between pgk1 and pgk2 is present. When it was inserted into the promoter region of the uninducible gene encoding RNase Rh of R. niveus, ligated in front of uidA and introduced into R. niveus, the GUS activity of the transformant was greatly induced by glucose, but less by glycerol. We therefore suggest that the 21-nt sequence is a glucose-inducible transcriptional activator of R. niveus. This is the first report on a transcriptional activator in zygomycetes.

C 60 and C 70 single crystals free from solvent contamination were grown from their vapor. Large C 60 crystals up to a size of about 5 x 3 x 3 mm 3 and C 70 crystals of about 1 x 1 x 1 mm 3 were obtained. Morphological measurement and X-ray diffraction analysis of C 60 crystals showed two types of morphological faces, namely {111} and {100},frequently with twinning on {111} faces. C 70 crystals obtained have an hcp structure with a = 10.1 A and c = 18.7 A.

Geochemical and Sr, Pb, O and C isotopic data are reported for carbonatite samples from five locations in southeast Brazil. Elemental abundances and δ13CPDB data (between −5.8 and −7.2‰) prove that all the samples are derived from the mantle. Dupal isotopic characteristics in the all carbonatite samples from five locations in southeast Brazil are found in this study, characteristics that have not previously been recognized in carbonatites. Brazilian carbonatites possess average Δ8 4 values of between 101 and 145, average Δ7 4 values of between 5.2 and 10.3, and initial Sr isotopic compositions of between 0.7046 and 0.7062. The Brazilian carbonatites comprise two groups: The northern group is coincident with the passage of the Trindade non-Dupal hotspot at ca. 80 Ma, while the southern group mainly corresponds to the passage of the Tristan de Cunha Dupal hotspot at ca. 130 Ma. Although we expected a geochemical correlation between the Brazilian carbonatites and the South Atlantic hotspots, the enriched isotopic signature (EM1) of all the carbonatite samples is similar to that of alkali basalts on Tristan de Cunha. The combined OSr isotopic diagram indicates that the southern group carbonatites have negligible or only slight crustal contamination. The northern group samples show significantly higher δ18OSMOW values of 9–14‰, more radiogenic Pb isotopic ratios, and a 87 Sr 86 Sr value of 0.705. Even if these signatures are derived from the contamination of a lower crustal component with mantle sources, it is clear that the parental magma also has inherent EM1 isotopic characteristics. The interpretation of the origin of EM1 in the Brazilian carbonatites (subcontinental lithospheric mantle vs. asthenosphere) is dependent on the model of Parana volcanism at ca. 130 Ma, which remains controversial. One possibility is that both the northern and southern carbonatites came from enriched SCLM under a part of Gondwanaland. In this case, a hotspot would provide the thermal energy to melt the lithospheric source region for both the Parana flood basalts and the alkali carbonatitic volcanism. Another possibility is that the source of the northern carbonatites is also the Tristan plume carbonate-rich material which had once been trapped under the crust and reactivated by the Trindade hotspot, on the assumption that Parana volcanism at ca. 130 Ma was mainly triggered by the huge Tristan plume activity. If anything, we favour the latter and believe an asthenospheric mantle plume origin for both the ultimate carbonate source and the Dupal anomaly in the Brazilian carbonatites.

Photo-illumination on the surface of C60 single crystals and films leads to instability of the surface, resulting polymerization. The polymerized surface exhibits a high resistance to thermal sublimation so that the skin remained after sublimation of the inner part of the crystals. Studies of TEM, X-ray diffraction, Raman scattering and IR spectra on the skins thus obtained indicated that it is composed of the photopolymerized C60, which still keeps the fcc structure similar to pristine C60 but is non-evaporable, and in addition an amorphous matter which comes from the decomposed C60 during illumination or during the subsequent heat treatment.

The nucleotide sequence of the upstream region of the bphC gene from Pseudomonas sp. strain KKS102 was determined. Four genes were found in this region. Deduced amino acid sequences of the first, second, third and fourth genes showed significant homology with a large subunit of iron-sulfur protein, a small subunit of iron-sulfur protein, ferredoxin and dihydrodiol dehydrogenase, respectively, from other bacteria which degrade biphenyl/polychlorinated biphenyls, toluene and benzene. E. coli, in which the four genes, bphC and the gene for ferredoxin reductase from benzene degrading bacterium were expressed, was able to produce meta-cleavage compounds from chlorinated biphenyls. These results show that these gene products are functional in both biphenyl and polychlorinated biphenyls degradation.

Extracellular Rhizopus niveus aspartic proteinase-I (RNAP-I) was secreted effectively by Saccharomyces cerevisiae when RNAP-I with its preprosequence was synthesized in this organism (Horiuchi, H., Ashikari, T., Amachi, T., Yoshizumi, H., Takagi, M., and Yano, K. (1990) Agric. Biol. Chem. 54, 1771-1779). Certain deletions (delta pro, delta 1, delta 2), and amino acid substitutions (M1) in the prosequence blocked secretion of RNAP-I, although the protease protection assay revealed that even delta pro could be translocated across the membrane of the endoplasmic reticulum. When delta pro or M1 was synthesized simultaneously with the wild-type preprosequence in S. cerevisiae, secretion of RNAP-I was recovered. Therefore, the physical linkage of the prosequence to the mature region is not a prerequisite for secretion of active RNAP-I. Purified RNAP-I with the prosequence once denatured in 6 M guanidine HCl could be renatured and activated to have its enzymatic activity by removing guanidine HCl in vitro, but RNAP-I without the prosequence could not. Furthermore, the wild-type prosequence helped the recovery of the activity of the denatured RNAP-I in trans, but the prosequences of M1 with which secretion of RNAP-I was not observed in vivo, did not. From these results we concluded that the prosequence of RNAP-I supports correct folding of RNAP-I in the endoplasmic reticulum lumen and its subsequent secretion in S. cerevisiae. The functional role of the prosequence of an aspartic proteinase was elucidated.

Decomposition of the low- T c phase of Bi-based oxide superconductor (Bi 2 Sr 2 CaCu 2 O 8 ) was investigated in situ to study the phase relation. Thin plate crystals were observed to grow from the solution produced by the partial melting of the low- T c phase. They were identified using micro-area X-ray diffractometry and chemical analysis by EDX. The results showed that they were composed from the high- T c phase (Bi 2 Sr 2 Ca 2 Cu 3 O 10 ). The sequence of the crystallization of various solid phases was clarified.

A new ternary intermetallic compound Nb6Sn1.5Ge1.5 has been synthesized by the self-flux method using molten tin as a solvent. Well- developed facets of the crystal are [110] planes. The crystal structure is tetragonal (space group D 4h 18 I4mcm : this ternary compound basically has a W5Si3-type structure. The lattice parameters are a = 10.45 (0) A and c = 5.192(0) A , V = 566.97(9) A 3 , Z = 4, and the X-ray density Dx is 8.80 g cm−3 at room remperature. This compound does not show superconductivity down to 1.4 K.

A systematic study of Va-Sn-Ga (Va = Ta, Nb, V) ternary systems has been made from the point of view of crystal growth, crystal structure and physical properties. Applying the self-component flux method where molten tin acted as a solvent, single crystals of new ternary intermetallic compounds Ta5SnGa2, Nb5Sn2Ga and V5Sn5Ga3, were successfully synthesized. The crystal structures of Ta5SnGa2 and Nb5Sn2Ga are very similar each other: both of them belong to the tetragonal system (space group: 14/mcm) and are isostructural with W5Si3. In contrast to them, V5Sn5Ga3, is orthorhombic (space group: Acam) and crystallizes in a new structural type. Superconducting transitions have been observed in Ta5SnGa2 and Nb5Sn2Ga crystals at 1.80 and 1.75 K.