Effects of cultural conditions on the production of phenylalanine from a plasmid-harboring E. coli strain

Summary Phenylalanine production from E. coli KA 197/pJN6 (plasmid harboring genes for aro F, phe A^FBR, Amp^R and Tc^R) was studied under varying nutritional conditions in batch and continuous cultures. In batch culture experiments where growth was deliberately interrupted by limiting concentrations of sulphate and phosphate the phenylalanine production continued from the non-growing cells. However, the depletion of phosphate resulted in an immediate cessation of phenylalanine production but thereafter a low specific rate of phenylalanine formation resumed, while the decrease in specific rate of product formation was less after sulphate depletion. In the chemostat experiments, however, phosphate limitation was the only case where the specific rate of phenylalanine formation remained constant, while at the corresponding time in sulphate and glucose limited chemostats it was declining respectively had ceased.     

Preparation of cell envelopes of large numbers of individual bacterial strains with the use of an automatic cell disruptor

Analysis of the cell envelopes of large numbers of bacterial strains is used for the epidemiological and taxonomic investigation of clinical, veterinarian, and ecological isolates. Isolation of cell envelopes requires lysis of the bacteria. We developed an apparatus to disrupt bacterial cells of 200 different isolates in suspension by ultrasonication automatically. It is composed of modified standard laboratory equipment (fraction collector, cooling unit, pump), a standard ultrasonifier, and a newly designed control unit, which includes a sampler. This apparatus was applied to the analysis of cell envelope proteins of 96 Haemophilus influenzae strains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis within 3 days after the first culture.     

Hydrogen exchange kinetics of bovine pancreatic trypsin inhibitor beta-sheet protons in trypsin-bovine pancreatic trypsin inhibitor, trypsinogen-bovine pancreatic trypsin inhibitor, and trypsinogen-isoleucylvaline-bovine pancreatic trypsin inhibitor

Hydrogen exchange rates of six beta-sheet peptide amide protons in bovine pancreatic trypsin inhibitor (BPTI) have been measured in free BPTI and in the complexes trypsinogen-BPTI, trypsinogen-Ile-Val-BPTI, bovine trypsin-BPTI, and porcine trypsin-BPTI. Exchange rates in the complexes are slower for Ile-18, Arg-20, Gln-31, Phe-33, Tyr-35, and Phe-45 NH, but the magnitude of the effect is highly variable. The ratio of the exchange rate constant in free BPTI to the exchange rate constant in the complex, k/kcpIx, ranges from 3 to much greater than 10(3). Gln-31, Phe-45, and Phe-33 NH exchange rate constants are the same in each of the complexes. For Ile-18 and Tyr-35, k/kcpIx is much greater than 10(3) for the trypsin complexes but is in the range 14-43 for the trypsinogen complexes. Only the Arg-20 NH exchange rate shows significant differences between trypsinogen-BPTI and trypsinogen-Ile-Val-BPTI and between porcine and bovine trypsin-BPTI.     

Effect of zearalenone and estradiol benzoate on serum concentrations of LH, FSH and prolactin in ovariectomized gilts

Six ovariectomized gilts were given zearalenone (Z), estradiol benzoate (EB) or vehicle in a replicated 3 x 3 Latin square design. Zearalenone was added to 2.3 kg of a corn-soybean ration at a dose of 1 mg Z/kg body weight; EB was given intramuscularly at 0.1 mg EB/kg body weight. Control gilts received vehicle solvent for both Z and EB. Blood samples were collected from indwelling jugular cannulas at 6-h intervals for 48 h before Z, EB or vehicle was given. After treatment, blood samples were drawn at 6-h intervals for an additional 84 h. Serum concentrations of luteinizing hormone (LH) decreased (P<0.001) from 4.67 ng/ml to 0.29 ng/ml within 6 h of EB. From 54 to 84 h after EB, serum concentrations of LH rose to 15.60 ng/ml (P<0.001). Serum concentrations of LH were reduced (P<0.001) in a similar pattern after Z (3.70 ng/ml to 0.49 ng/ml), but a rise in serum LH was not observed 54 to 84 h after Z (1.30 ng/ml). Serum concentrations of LH remained unchanged (P=0.55) in gilts given vehicle. Serum concentrations of follicle stimulating hormone (FSH) were suppressed (P<0.03) at 6 h in EB (19.10 vs 11.35 ng/ml) and Z gilts (16.16 vs 11.41 ng/ml) but remained unchanged in vehicle gilts. Serum concentrations of FSH did not change in EB or Z gilts during the next 36 h. These data indicate that the suppressive action of Z on serum concentrations of LH and FSH was similar to that of EB, while the biphasic stimulatory effect of EB for LH was not manifested by Z.     

Seed and tuber banks of aquatic macrophytes in the Askö area, northern Baltic proper

Sediment samples from 5 stations in the northern Baltic proper, 6.5 o/oo S, were collected in April 1987 and the emergence of seedlings was recorded over 120 days in a greenhouse at 20°C. Total seedling densities varied from 0 to 3328 m^-2: and of seven species, Zannichellia palustris and Chara spp. were the most abundant among seedlings and sporelings, respectively. Several common macrophytes in the area were rare as seedlings and no seedlings were recorded for the most abundant angiosperms, Potamogeton pectinatus, Potamogeton perfoliatus and Ranunculus baudotii. Except for the few annual species, reproduction by seeds contributed little to the dynamics of the vegetation in the area and no correlation was found between vegetation composition and the seed bank. For perennial species the winter survival of vegetative propagules is the most important factor for vegetation dynamics.     

The primary structure of histone H3 from wheat

Wheat embryo histone H3 has been isolated and purified and the elucidation of the complete amino-acid sequence is described. Peptides were generated by cleavages with CNBr, S. aureus V8 proteinase, endoproteinase Lys-C and trypsin. The peptides were purified by HPLC and the sequence determined by solid-state and gas-phase sequencing methodology. The amino-acid sequence of the protein is identical to pea embryo histone H3 and the sequence deduced from the nucleotide sequence of a wheat embryo histone gene (Tabata T. et al. (1984) Mol. Gen. Genet. 196, 397-400).     

Characterization of pregnant mare's serum gonadotropin-stimulated rat ovarian aromatase and its inhibition by 10-propargylestr-4-ene-3,17-dione

Aromatase, the important regulatory enzyme that converts androgens to estrogens, is found in relatively high levels in the human placenta. However, since the ovary is the major source of the estrogens in females, we undertook studies to compare the rodent ovarian enzyme with that from human placenta. Pregnant mare's serum gonadotropin (PMSG) markedly increases aromatase activity in the ovaries of immature rats, and this model was used in order to reproducibly obtain high enzyme levels. An injection of PMSG resulted in a specific stimulation of aromatase activity 12 times the increase in ovarian weight in 48 h. Kinetic studies demonstrated that, although the PMSG-stimulated ovarian microsomes had one-tenth the specific activity of the human placenta, the Km values were similar (about 33 and 44 nM, respectively). The potent inhibitor of placenta aromatase, 10-propargylestr-4-ene-3,17-dione, was used to further characterize the enzyme. It inhibited the rat aromatase with an I50 of 36 nM and exhibited time-dependent inhibition with a half-life of inactivation of 16 min and a Ki of 15 nM. These values are similar to those we obtained with the human enzyme (10 nM, 12 min, and 5 nM, respectively). The enzyme parameters in the presence and absence of the inhibitor suggest that the enzymes from the two sources are kinetically quite similar.     

On the activity of γ-aminobutyric acid and glutamate transporters in chick embryonic neurons and rat synaptosomes

The uptake of radioactive -aminobutyric acid (GABA) andd-aspartate and the effect of SKF 89976-A, a non-substrate inhibitor of the GABA transporter, on this uptake have been investigated. Neuronal cultures from eight-day-old chick embryos grown for three or six days in vitro, were used as a model. For comparison, we also used the P₂-fraction from rat. Neuronal cultures grown for three and six days expressed high-affinity uptake systems for [³H]GABA and ford-[³H]aspartate with an increasing V_max during this period. The lipophilic non-substrate GABA uptake inhibitor, SKF 89976-A, inhibited transporter mediated uptake of GABA both in cell cultures from chicken, and in P₂-fractions from rat. The results also showed that SKF 89976-A was a poor inhibitor of the uptake ofd-aspartate. We found no non-saturable uptake ofd-aspartate.     

A method for estimating oxygen availability of stationary plant cell cultures in liquid media

A method for estimating the oxygen availability in plant cell cultures grown in stationary liquid media (e.g. many protoplast cultures) was developed. The method is based on short-term measurements of respiration rate versus oxygen concentration on a sample of cells, suspended in liquid media. From such data it is possible to estimate the oxygen concentration at the bottom of a stagnant liquid culture, by calculating the amount of oxygen reaching the cells by diffusion. As an example, rape (Brassica napus L. cv. Omega) hypocotyl protoplasts were grown with different oxygen concentrations at the site of the cells, obtained by varying the cell density, the height of the liquid layer and the oxygen content of the gas phase. The number of surviving calli was positively correlated with the estimated oxygen availability in the range between 60 and 350 M O₂, below 60 M all cells died. This indicates that oxygen availability can be a limiting factor in the range usually encountered in protoplast cultures, and that the method can be useful when designing optimal growth conditions for stationary cultures of plant cells.Abbreviations C₁ bulk oxygen concentration in agitated medium - C_o oxygen concentration in medium at the gas-liquid interface, in equilibrium with the gas - C_x oxygen concentration at cell level - D diffusion constant of oxygen in water - K_La oxygen transfer rate - l height of liquid above cells - n number of cells per ml - R_x respiration rate per cell