Characteristic features of kynurenine aminotransferase allosterically regulated by (alpha)-ketoglutarate in cooperation with kynurenine

Kynurenine aminotransferase from Pyrococcus horikoshii OT3 (PhKAT), which is a homodimeric protein, catalyzes the conversion of kynurenine (KYN) to kynurenic acid (KYNA). We analyzed the transaminase reaction mechanisms of this protein with pyridoxal-5'-phosphate (PLP), KYN and α-ketoglutaric acid (2OG) or oxaloacetic acid (OXA). 2OG significantly inhibited KAT activities in kinetic analyses, suggesting that a KYNA biosynthesis is allosterically regulated by 2OG. Its inhibitions evidently were unlocked by KYN. 2OG and KYN functioned as an inhibitor and activator in response to changes in the concentrations of KYN and 2OG, respectively. The affinities of one subunit for PLP or 2OG were different from that of the other subunit, as confirmed by spectrophotometry and isothermal titration calorimetry, suggesting that the difference of affinities between subunits might play a role in regulations of the KAT reaction. Moreover, we identified two active and allosteric sites in the crystal structure of PhKAT-2OG complexes. The crystal structure of PhKAT in complex with four 2OGs demonstrates that two 2OGs in allosteric sites are effector molecules which inhibit the KYNA productions. Thus, the combined data lead to the conclusion that PhKAT probably is regulated by allosteric control machineries, with 2OG as the allosteric inhibitor.     

水稻(Oryza sativa L.)苹果酸酶(OsNADP-ME3)基因在逆境下的表达特性研究

水稻苹果酸酶（NADP-ME）是多基因家族,由3个胞质型NADP-ME和1个质体型NADP-ME构成。本研究针对水稻胞质型成员（命名为NADP-ME3）（NM₀01061367）进行初步的功能解析。克隆获得的NADP-ME3基因的cDNA序列全长为2240bp,其中5’非翻译区为151bp,3’非翻译区为376bp,开放读码框（ORF）长1713bp,编码570个氨基酸。为研究NADP-ME3在逆境胁迫下的表达量变化,Northern blot检测结果显示,在NaCl、NaHCO3和PEG胁迫条件下,NADP-ME3随胁迫处理时间的不同表达量呈现不同程度的变化,推断NADP-ME3可能与非生物胁迫有应答关系,将NADP-ME3转入拟南芥中并通过观察转基因拟南芥在非生物胁迫下表型变化,发现NADP-ME3能够在一定程度上提高植物对非生物胁迫的耐受性。     

Molecular cloning, expression, and characterization of a Ca2+-dependent nuclease of Arabidopsis thaliana

Yeast Pichia pastoris has been widely utilized to express heterologous recombinant proteins. P. pastoris expressed recombinant porcine interleukin 3 (IL3) has been used for porcine stem cell mobilization in allo-hematopoietic cell transplantation models and pig-to-primate xeno-hematopoietic cell transplantation models in our lab for many years. Since the yeast glycosylation mechanism is not exactly the same as those of other mammalian cells, P. pastoris expressed high-mannose glycoprotein porcine IL3 has been shown to result in a decreased serum half-life. Previously this was avoided by separation of the non-glycosylated porcine IL3 from the mixture of expressed glycosylated and non-glycosylated porcine IL3. However, this process was very inefficient and lead to a poor yield following purification. To overcome this problem, we engineered a non-N-glycosylated version of porcine IL3 by replacing the four potential N-glycosylation sites with four alanines. The codon-optimized non-N-glycosylated porcine IL3 gene was synthesized and expressed in P. pastoris. The expressed non-N-glycosylated porcine IL3 was captured using Ni-Sepharose 6 fast flow resin and further purified using strong anion exchange resin Poros 50 HQ. In vivo mobilization studies performed in our research facility demonstrated that the non-N-glycosylated porcine IL3 still keeps the original stem cell mobilization function.     

Identification of Actinomycetes Producing Phospholipase D with High Transphosphatidylation Activity

Previously we isolated six actinomycetes strains, 9-4, 10-1, 10-2, 10-3, 10-6, and 21-4, that produce phospholipase D (PLD) with high transphosphatidylation activity. In this study, we identified these strains, and the PLD activities were compared with those of reference strains. 16S rDNA sequences and DNA–DNA hybridization tests indicated taxonomic affiliations of strain 9-6 with Streptomyces senoensis, strains 10-1 and 10-6 with S. vinaceus, and strains 10-2 and 10-3 with S. racemochromogenes. Strain 21-4, though identified as a Streptomyces sp., could not be identified with any known species. Meanwhile, most of the culture supernatants of reference strains demonstrated no or very weak PLD activity, while those of our strains exhibited significantly higher activity. All of the strains in this study were identified as Streptomyces species. The PLD activity of our strains exceeded most of the reference Streptomyces strains. The findings in this study imply that the Streptomyces strains, although they are members of the same species, can produce different quantities of PLD enzyme.     

Characterization of T helper (Th)1- and Th2-type immune responses caused by baculovirus-expressed protein derived from the S2 domain of feline infectious peritonitis virus, and exploration of the Th1 and Th2 epitopes in a mouse model

Feline infectious peritonitis virus (FIPV) may cause a lethal infection in cats. Antibody-dependent enhancement (ADE) of FIPV infection has been recognized, and cellular immunity is considered to play an important role in preventing the onset of feline infectious peritonitis. In the present study, whether or not the T helper (Th)1 epitope was present in the spike (S)2 domain was investigated, the ADE epitope being thought to be absent from this domain. Three kinds of protein derived from the C-terminal S2 domain of S protein of the FIPV KU-2 strain were developed using a baculovirus expression system. These expressed proteins were the pre-coil region which is the N-terminal side of the putative fusion protein (FP), the region from FP to the heptad repeat (HR)2 (FP-HR2) region, and the inter-helical region which is sandwiched between HR1 and HR2. The ability of three baculovirus-expressed proteins to induce Th1- and Th2-type immune responses was investigated in a mouse model. It was shown that FP-HR2 protein induced marked Th1- and Th2-type immune responses. Furthermore, 30 peptides derived from the FP-HR2 region were synthesized. Five and 16 peptides which included the Th1 and Th2 epitopes, respectively, were identified. Of these, four peptides which included both Th1 and Th2 epitopes were identified. These findings suggest that the identification of Th1 epitopes in the S2 domain of FIPV has important implications in the cat.     

Expression of the AKT1-type K+ channel gene from Puccinellia tenuiflora, PutAKT1, enhances salt tolerance in Arabidopsis

Potassium channels are important for many physiological functions in plants, one of which is to regulate plant adaptation to stress conditions. In this study, a K⁺ channel PutAKT1 cDNA was isolated from the salt-tolerant plant Puccinellia tenuiflora. A phylogenetic analysis showed that PutAKT1 belongs to the AKT1-subfamily in the Shaker K⁺ channel family. PutAKT1 was localized in the plasma membrane and it was preferentially expressed in the roots. The expression of PutAKT1 was induced by K⁺-starvation stress in the roots and was not down-regulated by the presence of excess Na⁺. Arabidopsis plants over-expressing PutAKT1 showed enhanced salt tolerance compared to wild-type plants as shown by their shoot phenotype and dry weight. Expression of PutAKT1 increased the K⁺ content of Arabidopsis under normal, K⁺-starvation, and NaCl-stress conditions. Arabidopsis expressing PutAKT1 also showed a decrease in Na⁺ accumulation both in the shoot and in the root. These results suggest that PutAKT1 is involved in mediating K⁺ uptake (i) both in low- and in high-affinity K⁺ uptake range, and (ii) unlike its homologs in rice, even under salt-stress condition.     

A method for detecting effect of beneficial mutations in natural populations of Drosophila melanogaster.

An experimental method is proposed for detecting the effects of positive natural selection on DNA polymorphisms. Since beneficial mutations are expected to increase in frequency faster than neutral mutations, variants which have reached high frequencies in a relatively short period could be linked to some beneficial mutation. D. melanogaster has a cosmopolitan polymorphic inversion -In(2L)t- whose age in some local populations has been estimated. Setting the age of In(2L)t as the upper limit for the age of variants, we searched for variants whose frequencies were possibly influenced by positive natural selection. We detected a single candidate whose frequency and distribution met the requirements imposed by our method.     

Continuous column culture system for adherent cells

A new continuous column culture system for adherent cells was developed using beads. The beads were packed in a column and an appropriate medium was continuously passed through. The whole system was kept under closed conditions. L cells and C6 cells were cultured by this new system. The number of cells increased linearly up to 16 days and reached a maximum at around 18 days. As the heat production remained constant for 16 days, it can be concluded that cells grown in this system had identical characteristics. The final concentration of cells reached was 1.0 × 10⁸ml^?1. The cells could grow both in the upward and the downward direction. Advantages of this system are: (1) Cells can be recovered in their adherent form on the beads; (2) cells can easily be collected from the column by trypsinization, and (3) cells remaining in the column after trypsinization can grow again.     

A stimulatory effect of somatostatin: enhancement of activin A-mediated FSH secretion in rat pituitary cells

The interaction between somatostatin and activin A was studied in terms of FSH secretion in rat pituitary cells in primary culture. Incubation of pituitary cells with 1 nM activin A for 48 hrs resulted in an increase in FSH release into incubation medium. The effect of activin A was dependent on cell-density and the higher the density, the smaller the stimulatory action of activin A. Somatostatin, by itself, did not affect the FSH secretion. When 100 nM somatostatin was included together with activin A or the cells were pretreated with somatostatin for 2 hrs, the activin A-induced FSH secretion was enhanced. This potentiation effect of somatostatin was inversely dependent on the cell-density. These results indicate that somatostatin enhances, rather than inhibits, the activin A action in pituitary cells.     

Enhancement of Newcastle Disease Virus-Induced Fusion of Mouse L Cells by Sodium Vanadate

Sodium vanadate enhanced Newcastle disease virus (NDV)-induced cell fusion in L cells, and there was a direct correlation between the degree of cell fusion and the dose of vanadate added. When anti-F protein of NDV monospecific antiserum was added to the culture fluid of L cells infected with NDV, the enhancement of cell fusion was suppressed. In contrast, neither anti-HN nor anti-M protein monospecific antiserum inhibited the enhancement. Incubation at low temperature (4 C) and addition of sodium azide to the culture fluid suppressed the enhancement. The suppression by azide was seen only when the drug was added within 5 min after the beginning of incubation of NDV-infected L cells with vanadate. On the other hand, incubation at low temperature inhibited the enhancement at any time during incubation with vanadate. Cytochalasin D also inhibited the enhancement if it was added at any time during incubation with vanadate.