Continuous culture of a recombinantEscherichia coli and plasmid maintenance for tryptophan production

Summary Production of tryptophan by a temperature sensitive recombinant microorganism (Escherichia coli W3110 trpLDtrpR ^ts tna ^– (pCRT185)) was investigated. In a single-stage continous culture, at an elevated temperature, 42°C (derepressed condition), tryptophan concentration increased in an early phase of the fermentation, and then gradually decreased with time. The reduction in the production rate was mostly due to the segregation of the plasmid and subsequent increase of plasmid-free cells. However, the plasmid could be maintained stable at 37°C, with repressed condition oftrp-operon, over 200 generations. A two-stage continuous culture system, i.e. cell growth was maintained in the first stage at 37°C and gene expression was induced in the second stage at 42°C, was therefore tested to improve the performance of the fermentation system. Operation of the two-stage system showed that the plasmid stability was significantly improved, and the specific rate of tryptophan production was maintained almost constant for more than 500 hours in the second stage.     

Novel stabilization of phenylalanine ammonia-lyase catalyst during bioconversion of trans-cinnamic acid to l-phenylalanine

Summary Production of l-phenylalanine from trans-cinnamic acid using isolate SPA10 cells was reduced to 26% of that observed initially when cells were reacted a second time with fresh substrate mixture. The stability (reuseability) of Phenylalanine Ammonia-Lyase (PAL) containing cells was significantly influenced by both the trans-cinnamate concentration and initial reaction pH. Using 2% t-cinnamate, l-phenylalanine production was 7-fold greater after 3 successive runs at pH 9.0 than at the optimum of pH 10.2. Cells reacted in the presence of 5% t-cinnamate were relatively unstable. Permeabilising agents, such as toluene and xylene, stimulated l-phenylalanine production but also enhanced instability of the catalyst. Several effectors were shown to stimulate the initial rate of the PAL bioconversion, but only sorbitol, alginate, glutaraldehyde, polyethylene glycol and glycerol conferred any significant degree of stability. Sparging of cultures and bioreactors with various gases revealed that oxygen enhanced PAL inactivation, CO₂ had little effect and nitrogen conferred remarkable stability on PAL activity for several weeks in culture medium. The presence of chloride ions (from HCl) and aeration of substrate mixtures resulted in poor reuseability of catalyst. A combination of H₂SO₄ substitution for HCl and N₂-sparging resulted in excellent initial conversions and good catalyst stability at 26°C but less at 30°C. The inclusion of 1.5 M sorbitol in reaction mixtures maintained PAL stability over several successive incubations.     

Recent advances in chemiluminescence-based immunoassays for steroid hormones

Several formats of solid-phase separation techniques for the measurement of steroids and urinary steroid conjugates using chemiluminescence as an end point are described. These formats include: (1) immunoadsorption of second antibodies directed against the first antibody on solid support; (2) specific immunoadsorbents consisting of primary antibodies covalently coupled to polymer beads; (3) second antibody coupled to a polymer containing magnetic particles. In these assays a steroid-chemiluminescent marker conjugate serves as the labelled ligand, and highly specific homologous monoclonal antibodies are used to provide optimal specificity and rigorous standardization. These techniques enabled the direct measurement of steroid glucuronides in diluted urine and of steroids in plasma.     

Effect of bacterial growth-inhibiting ingredients on the Ames mutagenicity of medicinal herbs

A solvent fractionation method was introduced to screen for mutagenicity in 10 medicinal herbs being consumed in Korea. The Ames mutagenicity test result of Scutellariae and Rhei was significantly increased by eliminating growth-inhibiting substances through solvent fractionation of the crude extract. It is suggested that a physicochemical pretreatment should reduce the false-negative results which are caused by the presence of growth-inhibiting substances in complex mixtures.     

Characterization of the Stimulation of Ethylene Production by Galactose in Tomato (Lycopersicon esculentum Mill.) Fruit

We have characterized the stimulation of ethylene production by galactose in tomatoes (Lycopersicon esculentum Mill.). The effect of concentration was studied by infiltrating 0, 4, 40, 100, 200, 400, or 800 micrograms galactose for each gram of fresh fruit weight into mature green `Rutgers' fruit. Both 400 and 800 micrograms per gram fresh weight consistently stimulated a transient increase in ethylene approximately 25 hours after infiltration; the lower concentrations did not. Carbon dioxide evolution of fruit infiltrated with 400 to 800 micrograms per gram fresh weight was greater than that of lower concentrations. The ripening mutants, rin and nor, also showed the transient increase in ethylene and elevated CO₂ evolution by 400 micrograms per gram fresh weight galactose. 1-Aminocyclopropane-1-carboxylic acid (ACC) content and ACC-synthase activity increased concurrently with ethylene production. However, galactose did not stimulate ACC-synthase activity in vitro. The infiltrated galactose in pericarp tissue was rapidly metabolized, decreasing to endogenous levels within 50 hours. Infiltrated galacturonic acid, dulcitol, and mannose stimulated transient increases in ethylene production similar to that of galactose. The following sugars produced no response: sucrose, fructose, glucose, rhamnose, arabinose, xylose, raffinose, lactose, and sorbitol.     

Effect of GA3 on the Cyclic AMP Biosynthesis in Maize Seedling

The effect of GA₃ on the biosynthesis of cAMP was studied toinvestigate the mechanism of gibberellic acid action. The presenceof cAMP in germinating maize seedlings was confirmed. The concentrationgradient of cAMP in maize seedlings inclined from shoot apexto root. The amount of cAMP in maize shoot was increased about3.3 times by exogenous GA₃. These results indicate that thebiosynthesis of cAMP is stimulated by GA₃. (Received June 17, 1986; Accepted January 22, 1987)     

Radiation sensitivity of hemopoietic stroma: long-term partial recovery of hemopoietic stromal damage in mice treated during growth

We studied the ability of the hemopoietic organ stroma to recover from damage inflicted by 5 or 7 Gy gamma radiation administered during a period of stromal growth in 4-week-old mice. Irradiation resulted in an immediate depletion of femoral colony-forming fibroblastic progenitors (CFU-F) down to 10-20% of age-matched control values. A full recovery to normal numbers occurred between 120 and 240 days after irradiation and was followed by a secondary decrease 1 year after irradiation. This secondary decrease was accompanied by a decrease in the femoral CFU-S and CFU-C content. Femoral CFU-F attained normal numbers and it was demonstrated to occur from surviving CFU-F and could not be enhanced or prolonged following infusion of unirradiated bone marrow cells after irradiation. During the transient CFU-F recovery the hemopoietic stroma remained severely damaged as judged by the regenerative capacity of spleen and femur stroma after subcutaneous implantation, and the ability of the spleen to accumulate CFU-S in response to lipopolysaccharide injection. We have reported earlier that in similarly irradiated adult mice, no restoration of femoral CFU-F was observed. This difference between 4-week-old and adult mice could not be explained by a difference in in vitro radiosensitivity of CFU-F or in their in vivo regeneration kinetics following irradiation and subsequent lipopolysaccharide injection. We conclude from these observations that the recovery kinetics of the CFU-F population is different in young and adult irradiated mice, infused CFU-F do not contribute to CFU-F regeneration in an irradiated femur, CFU-F are not the sole determinants of stromal regeneration in femur and spleen following irradiation.     

Dynamics of populations with changing rates: generalization of the stable population theory

A general and complete exposition of the dynamics of populations with changing vital rates is given in the discrete time formulation. Results obtained are stronger than Lopez's or Hajnal's. In addition to the proof of existence of limits, an explicit expression for the age distribution is obtained by considering forward products of population projection matrices, while an explicit expression for the generalized reproductive values is obtained by considering backward products. The forward and backward characteristic equations respectively determine the forward and backward growth rates. The relative age distribution is compared to the alternative expression of Y.J. Kim (1986, Demography 23 (3), 451-461), which is the discrete version of S.H. Preston and A.J. Coale (1982, Pop. Index 48 (2), 217-259).     

Molecular cloning and differential expression of somatic and testis-specific H2B histone genes during rat spermatogenesis

We have cloned cDNA of a testis-specific histone, TH2B (a variant of H2B), and rat somatic H2B gene to investigate regulation of testis-specific histone genes during rat spermatogenesis. The amino acid sequences deduced from DNA sequences show extensive sequence divergence in the N-terminal third of the two histones. The rest is highly conserved. One cysteine residue was found in TH2B. No cysteine is present in somatic histones except in H3 histone. We investigated the expression of TH2B and H2B genes using the regions of sequence divergence as hybridization probes. The TH2B gene is expressed only in the testis, and the expression of this gene is detected 14 days after birth, reaching a maximum at Day 20. The level of H2B mRNA shows a reciprocal pattern. This contrasting pattern can be explained by the gradually changing proportion of spermatogonia and spermatocytes with testicular maturation. In situ cytohybridization studies show that H2B gene is expressed primarily in proliferating spermatogonia and preleptotene spermatocytes, whereas TH2B gene is expressed exclusively in pachytene spermatocytes which first appear in testis about 14 days after birth. H2B and TH2B genes appear to be ideal markers for the study of proliferation and differentiation events in spermatogenesis and their regulatory mechanisms.     

Beta B1 crystallin is an amine-donor substrate for tissue transglutaminase

The effects of tissue transglutaminase on the water-soluble proteins in bovine lens homogenates are described. Addition of liver transglutaminase and Ca2+ to calf lens homogenates resulted not only in the appearance of 50- and 57-kDa dimers, but also in a decrease in the amount of beta B1 crystallin and the almost complete disappearance of beta B3 and beta A3. This is not the result of Ca2+-induced proteolysis, since histamine completely inhibits this phenomenon. It may be concluded that these polypeptides are involved in beta-crystallin crosslinking by transglutaminase. This notion was confirmed by using beta B1- and beta Bp-specific antisera. Both sera reacted with the 57-kDa dimer; the beta Bp-specific antiserum also reacted with the 50-kDa dimer. No reaction in the region 50-57 kDa was detectable when EDTA was used instead of Ca2+. Using reconstituted mixtures of beta B1- and beta Bp-crystallin chains, and N-terminally truncated derivatives thereof, it was shown that in the beta B1/beta Bp dimer, glutamine residue -9 of beta Bp crosslinks to one of the lysine residues in the N-terminal extension of beta B1.