Purification of Ribulose 5-phosphate Kinase and Minor Polypeptides of Pyrenoid from the Green Alga Bryopsis maxima

Ribulose 5-phosphate (Ru5P) kinase (ATP:D-ribulose 5-phosphate1-phosphotrans- ferase; EC 2.7.1.19 [EC] ), an enzyme in the reductivepentose phosphate cycle, was purified from the green alga Bryopsismaxima and its activity and peptide composition were studied.The specific activity of purified Ru5P kinase was 20 µmoleRuBP formed (mg protein)^–1 min^–1 corresponding toa 490-fold purification from the supernatant of chloroplasts.The K_m values of Ru5P kinase for ATP and Ru5P were 69 µMand 330 µM, respectively. The molecular size of Ru5P kinase was estimated as 90 kDa bygel filtration and that of its polypeptide as 41 kDa by SDS-polyacrylamidegel electrophoresis. A small portion of the Ru5P kinase wasfound in a large molecular state (500 kDa) which was consideredto be an inactive form of the enzyme. Ru5P kinase activity has been reported in the pyrenoid of Eremosphaeraviridis as well as ribulose 1,5-bisphosphate carboxylase-oxygenase(RuBisCO) and ribose 5-phosphate isomerase activity (Holdsworth1971). In Bryopsis maxima, among the pyrenoid polypeptides otherthan that of RuBisCO, we found a polypeptide of 42 kDa, similarto that of Ru5P kinase in molecular size and ratio to RuBisCO.A peptide map of the 42 kDa pyrenoid polypeptide, however, showedthat it differed from that of Ru5P kinase. In conclusion, Ru5Pkinase may be not involved in the pyrenoid of this alga. (Received January 19, 1985; Accepted May 15, 1985)     

Expression of a xylanase gene of Bacillus pumilus in Escherichia coli and Bacillus subtilis

Summary A hybrid plasmid, pOXN29 (10.4 Mdal), coding the xylanase (xynA) and -xylosidase (xynB) genes of Bacillus pumilus IPO was constructed by the ligation of pBR322 and a 7.7 Mdal PstI-fragment of chromosomal DNA as reported in our previous paper (Panbangred et al. 1983). A deletion plasmid of pOXN29, pOXN293 (9.2 Mdal), which contains xynA and xynB, was ligated with pUB110 at an EcoRI site, and used to transform B. subtilis MI111. Two selected clones of B. subtilis as xylanase hyper-producers contained plasmids pOXW11 (4.2 Mdal) and pOXW12 (4.0 Mdal), both consisting of only pUB110, xynA, and its flanking regions, as the result of spontaneous deletion. These B. subtilis clones produced 2.7–3.0 times as much xylanase as B. pumilus. Escherichia coli and B. subtilis clones harbouring the hybrid plasmids synthesized xylanase and -xylosidase constitutively, whereas both enzymes were induced by xylose in B. pumilus.Xylanase synthesized by B. subtilis harbouring pOXW11 or pOXW12 was excreted into the medium like that of B. pumilus IPO, but xylanase synthesized in E. coli harbouring pOXN29, 293 or pOXW1 coding xynA was intracellular. In a previous investigation (Panbangred et al. 1983), xylanase was found to be located in the cytoplasm, not the periplasm nor the membrane fraction in E. coli cells harbouring pOXN29 derivatives. In spite of the abnormal location of xylanase synthesized in E. coli, the signal peptide was processed in the same way as in B. pumilus, with the same molecular weight and the same amino terminal sequences of xylanase prepared from E. coli cells and B. pumilus culture fluid.     

SB subregion of the human major histocompatibility complex: gene organization, allelic polymorphism and expression in transformed cells

The SB region of the human major histocompatibility complex (MHC) has been cloned from cosmid and lambda phage libraries made from the human B-lymphoblastoid cell line Priess (DR4/4, DC4/4, SB3/4). Two alpha genes and two beta genes are encoded in the 100 kb long SB region in the order SB alpha-SB beta-SX alpha-SX beta. The SB alpha and SB beta genes encode the alpha and beta subunits of the SB subset of class II MHC molecules. Both the SX alpha and the SX beta genes are pseudogenes in the haplotype examined. From the isolated clones, the two haplotypes of the Priess cell line, SB3 and SB4, are distinguished by nucleotide sequencing and blot hybridization analyses. Restriction site polymorphisms between the SB3 and SB4 clones were observed only in relatively small regions of the SB beta and SX beta genes. A mouse macrophage cell line was transfected with one of the cosmid clones containing both SB alpha and SB beta genes. Expression of the alpha and beta genes was detected by fluorescene-activated cell sorting (FACS) and two-dimensional gel electrophoresis using SB-specific monoclonal antibodies.     

Arterial perfusion of frog tongue for intracellular recording of taste cell receptor potential

The frog tongue was perfused through its artery with a Ringer solution using a peristaltic pump, and a method was developed to record stable intracellular receptor potentials of taste cells. Perfusing at 0.05 ml/min with a Ringer solution containing 5% dextran did not cause tongue edema, but perfusing at the same rate with Ringer without dextran caused edema. After perfusion at 0.05 ml/min with 100 mM K Ringer, the membrane potential of taste cells gradually decreased and reached a constant level in about 30 min, indicating that the intercellular fluid of the tongue could be replaced within this time period. While the artery of the frog tongue was perfused at 0.05 ml/min with Ringer containing 5% dextran, intracellular receptor potentials of taste cells elicited by four basic taste stimuli (1 M NaCl, 10 mM quinine-HCl (Q-HCl), 1 mM acetic acid and 1 M galactose) were similar to those obtained from the control taste cells under normal blood flow.     

Antitumor activities and tumor necrosis factor producibility of traditional Chinese medicines and crude drugs

Summary The antitumor activities and capacity for tumor necrosis factor (TNF) production of traditional Chinese herbal preparations (Zhu-ling-tang, Xiao-chai-hu-tang), crude drugs (Polyporus, Hoelen, Bupleuri radix, Angelica radix, Cnidii rhizoma, Cinnamomum cortex), and Krestin (PSK) were investigated. These drugs were given to DDY mice in the drinking water before and after transplantation of Ehrlich tumors, and the development of the intradermally transplanted Ehrlich tumors and survival rate were observed. A good survival rate and sometimes a complete cure were found in the groups administered Bupleuri radix, Xiao-chai-hu-tang, Angelica radix, or Cinnamomum cortex, while the group given Hoelen showed poor results. To examine the capacity for TNF production these drugs were given to DDY mice PO as initial stimulating agents, to stimulate the reticuloendothelial system (RES) prior to lipopolysaccharide injection. The TNF activity was tested from the cytotoxicity against L cells. Significant differences in capacity for TNF production were observed among the drugs. Relatively high levels of TNF activity were noted in the groups given Angelica radix, Bupleuri radix, Cnidii rhizoma, or Cinnamomum cortex, very low activities in the groups given Xiao-chai-hu-tang, Zhu-ling-tang, or Krestin, and no TNF activities in the groups given Polyporus or Hoelen. The TNF capacity for production broadly paralleled the survival rate of the mice transplanted to Ehrlich tumors. Our findings suggest that one mechanism underlying the antitumor activities of these drugs is based on stimulation of the RES and is closely related of TNF production.This work was supported in part by a grant-in-aid from the Ministry of Education, Japan     

DNA-DNA hybridization analysis of nylon oligomer-degradative plasmid pOAD2: identification of the DNA region analogous to the nylon oligomer degradation gene.

Fine structure of the gene of 6-aminohexanoic acid cyclic dimer hydrolase, one of the enzymes responsible for the degradation of the nylon oligomer (6-aminohexanoic acid cyclic dimer), on the plasmid pOAD2 harbored in Flavobacterium sp. KI72 was determined by constructing miniplasmids from plasmid pNDH5 (a hybrid plasmid consisting of pBR322 and a 9.1-kilobase-pair HindIII fragment of pOAD2 ). The 6-aminohexanoic acid cyclic dimer hydrolase produced by cells of Escherichia coli C600 harboring pNDH5 or its miniplasmid was examined immunologically and electrophoretically and was found to be identical to that of Flavobacterium sp. KI72 . A fragment of pOAD2 (17.2- to 19.1-kilobase-pair region on pOAD2 ) was detected as hybridized fragment by Southern blotting experiments, indicating the presence of the DNA region analogous to the 6-aminohexanoic acid cyclic dimer hydrolase gene on the plasmid.     

Production of transgenic fish: introduction and expression of chicken delta-crystallin gene in medaka embryos

To produce a model of transgenic fish, recombinant plasmids containing chicken delta-crystallin gene were microinjected into the oocyte nucleus of a small teleost, medaka (Oryzias latipes). About 50% of the microinjected oocytes developed to 7-day-old embryos. By Southern blotting delta-crystallin gene was detected in 4 of 8 embryos, and, by Western blotting, delta-crystallin polypeptides in 5 of 16. In 1 of 6 examined histologically, delta-crystallin DNA was detected in all the tissues, and delta-crystallin polypeptides, in many of the tissues including the lens. Thus, the exogenous gene and/or its products were detected in 10 of 30 embryos examined. This is the first report of successful production of transgenic fish.