Synthesis and evaluation of Triticum durum — T. monococcum amphiploids

Summary Nine Triticum durum — T. monococcum amphiploids (AABBA^mA^m) were synthesized by chromosome doubling of sterile triploid F₁ hybrids involving nine T. durum (AABB) cultivars and a T. monococcum (A^mA^m) line. The triploid F₁ hybrids had a range of 4–7 bivalents and 7–13 univalents per PMC. The synthetic amphiploids, however, showed a high degree of preferential pairing of chromosomes of the A genomes of diploid and tetraploid wheats. The amphiploids were meiotically stable and fully fertile. Superiority of four amphiploids for tiller number per plant, 100-grain weight, protein content and resistance to Karnal bunt demonstrated that these could either be commercially exploited as such after overcoming certain inherent defects or used to introgress desirable genes into durum and bread wheat cultivars. Methods for improvement of these amphiploids are discussed.     

Secretion of a heat-stable fungalβ-glucanase from transgenic,suspension-cultured barley cells

Transgenic plant cell cultures have a potential for production and secretion of important proteins and peptides. To assess the possibilities of using a stable barley suspension culture for secretion of heterologous proteins in active form, we expressed the cDNA of the thermostable-glucanase (EGI) ofTrichoderma reesei in barley suspension cells. The cDNA coding for EGI and its signal sequence was placed under the control of the CaMV 35S promoter and the construction was transferred to the cells by particle bombardment. Stably transformed lines were obtained by selecting for a cotransformed antibiotic resistance marker. The expression of EGI cDNA led to accumulation of EGI in the culture medium, as shown by analysis with EGI-specific antibodies. Enzymatic assays confirmed that the EGI secreted by the suspension cells retained its activity and thermostable character. Furthermore, it was shown that the enzyme produced by the transgenic suspension culture could be used for degradation of soluble-glucans during mashing.     

Mature ferredoxin-NADP reductase with a glutaminyl residue at N-terminus from spinach chloroplasts

When 35%-acetone extract of spinach chloroplasts was separated by SDS-PAGE, ferredoxin-NADP reductase (FNR) appeared as a single band at a molecular mass of 35 kDa. After the polypeptides on the SDS-PAGE plate were electroblotted onto PVDF membrane, the FNR band was cut out and analyzed for N-terminal structure in a gas-phase protein sequencer. Two different FNR peptides were identified: one with glutamine at its N-terminus (Gln-FNR) and the other with -pyroglutamic acid (tFNR) fraction was extracted from chloroplasts with their loosely bound FNR (lFNR) fraction removed in advance. The tFNR fraction contained Gln-FNR only. The Gln-FNR could be highly purified by affinity chromatography using a ferredoxin column. The purified Gln-FNR was digested with arginyl endopeptidase for peptide mapping and partial sequence analysis. Primary structure of Gln-FNR differed from that of lFNR loosely bound FNR - tFNR tightly bound FNR - -pyroglutamic acid at N-terminus     

Potentiation of antitumor effects of tumor necrosis factor α and interferon γ by macrophage-colony-stimulating factor in a MmB16 melanoma model in mice

The efficacy of systemic infusion of recombinant human macrophage-colony-stimulating factor (M-CSF) in combination with local treatment with human recombinant tumor necrosis factor (TNF) and mouse recombinant interferon (IFN) was studied in vivo on a subclone of B16 melanoma (MmB16) in mice. Short-term intravenous administration of M-CSF at a dose of 10⁶ units daily had no antitumor effect in vivo. Similarly, local treatment of tumor with TNF (5 g daily) did not produce any therapeutic effect. However, simultaneous administration of the same dose of TNF with IFN (1000 units daily) resulted in a synergistic effects manifested by the retardation of tumor growth. Addition of systemic infusion of M-CSF to the local therapy with TNF and IFN induced further augmentation of antitumor efficacy and delayed progression of MmB16 melanoma. The strengthened antitumor effect of combination therapy including M-CSF, TNF and IFN was most probably due to the increased release of monocytes from the bone marrow, their recruitment into the site of tumor growth and subsequent local stimulation of their antitumor activity.     

Adenine nucleotide contents and energy charge of Azotobacter vinelandii grown at low phosphate concentration

The effect of a low phosphate concentration on intracellular adenine nucleotide content, oxygen consumption and poly--hydroxybutyrate deposition was investigated with N-free and NH ₄ ⁺ batch cultures of Azotobacter vinelandii. When the microorganisms were cultured under low-phosphate concentrations the cells contained much larger amounts of poly--hydroxybutyrate, but displayed lower oxygen consumption activities and energy charge values than did control cells. Also, the ratio ATP to ADP was much higher in control cells and the intracellular levels of ATP were lower in low-phosphate cells.     

Sequestration of arginine by polyphosphate in vacuoles of yeast (Saccharomyces cerevisiae)

The conditions for synthesis, purification, and properties of tryptophanase by a marine organism (Vibrio K-7) were studied. Tryptophanase was induced by tryptophan and its analogs, and partially repressed by 0.5% glucose or glycerol. NaCl (0.4M) was required for optimal growth and tryptophanase activity in whole cells. The enzyme was purified to 92% homogeneity by heat treatment, hydroxyapatite chromatography and fractionation with ammonium sulfate. This tryptophanase has been found to have kinetic properties similar to the tryptophanase from other microorganisms. It carries out both , -elimination reactions (using tryptophan, serine, cysteine and S-methyl-cysteine as substrates) and -replacement reactions (forming tryptophan from indole and serine, cysteine or S-methyl-cysteine). The enzyme has a sedimentation coefficient of 9.2S and requires pyridoxal 5-phosphate as a cofactor. The optimal pH for the tryptophanase reaction is pH 8.0.Nonstandard Abbreviations PLP pyridoxal 5-phosphate - TPase tryptophanase - TSase tryptophan synthase - DHase dehydratase - TCA tricarboxylic acid - BSA bovine serum albumin Preliminary reports of this work have been presented (M. J. Klug and R. D. DeMoss, Bacteriol. Proc. 1971, p. 132; D. D. Whitt and R. D. DeMoss, Abstr. Annu. Meet. Am. Soc. Microbiol. 1973, p. 148)     

Large-scale production of GDP-fucose and Lewis X by bacterial coupling

A large-scale production system of GDP-fucose (GDP-Fuc) and fucosylated oligosaccharides was established by the combination of recombinant Escherichia coli cells overexpressing GDP-Fuc biosynthetic genes and Corynebacterium ammoniagenes cells. E. coli cells overexpressed the genes for glucokinase, phosphomannomutase, mannose-1-phosphate guanylyltransferase, GDP-mannose (GDP-Man) dehydratase, and GDP-4-keto-6-deoxy-mannose (GKDM) epimerase/reductase as well as phosphoglucomutase and phosphofructokinase. C. ammoniagenes contributed to the formation of GTP from GMP. GDP-Fuc accumulated to 29 mM (18.4 g l⁻¹) after a 22-h reaction starting with GMP and mannose through introducing the two-step reaction to overcome the inhibition of GDP-Fuc on GDP-Man dehydratase activity. When E. coli cells overexpressing the α1,3-fucosyltransferase gene of Helicobacter pylori were put into the GDP-Fuc production system, Lewis X [Galβ1–4(Fucα1–3)GlcNAc] was produced at an amount of 40 mM (21 g l⁻¹) for 30 h from GMP, mannose, and N-acetyl lactosamine. The production system through bacterial coupling can be applied to the industrial manufacture of fucosylated oligosaccharides. Journal of Industrial Microbiology & Biotechnology (2000) 25, 213–217. Received 01 May 2000/ Accepted in revised form 20 July 2000