Characterization of a meta-Fluorotyrosine-Tolerant Cell Culture of Eschscholtzia californica Cham

A cell line of Eschscholtzia californica selected for meta-fluorotyrosine (MFT) tolerance was found to have 10-fold increased levels of phenylalanine and tyrosine compared to the parent line, while most other amino acids were only increased 2-fold. Tracer experiments with shikimic acid in the presence of MFT showed that the biosynthesis of the aromatic amino acids was not impaired in the tolerant line. Feeding experiments with phenylalanine, tyrosine, or shikimic acid also revealed a reduced turnover of the pools of the aromatic amino acids in the variant. Thus undisturbed de novo biosynthesis of the aromatic amino acids and dilution of toxic effects of MFT by the enlarged pool sizes seemed to be the main reason for the acquired tolerance. Despite the enlarged availability of the precursor tyrosine, formation of the benzophenanthridine alkaloids was enhanced neither in the growth nor in the production medium.     

Comparative biochemical and immunological studies of the glycine betaine synthesis pathway in diverse families of dicotyledons

Members of the Chenopodiaceae can accumulate high levels (>100 mol·(g DW)^-1) of glycine betaine (betaine) in leaves when salinized. Chenopodiaceae synthesize betaine by a two-step oxidation of choline (cholinebetaine aldehyde betaine), with the second step catalyzed by betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8). High betaine levels have also been reported in leaves of species from several distantly-related families of dicotyledons, raising the question of whether the same betaine-synthesis pathway is used in all cases.Fast atom bombardment mass spectrometry showed that betaine levels of >100 mol·(g DW)^-1 are present in Lycium ferocissimum Miers (Solanaceae), Helianthus annuus L. (Asteraceae), Convolvulus arvensis L. (Convolvulaceae), and Amaranthus caudatus L. (Amaranthaceae), that salinization promotes betaine accumulation in these plants, and that they can convert supplied choline to betaine aldehyde and betaine. Nicotiana tabacum L. and Lycopersicon lycopersicum (L.) Karst. ex Farw. (Solanaceae), Lactuca sativa L. (Asteraceae) and Ipomoea purpurea L. (Convolvulaceae) also contained betaine, but at a low level (0.1–0.5 mol·(g DW)^-1. Betaine aldehyde dehydrogenase activity assays, immunotitration and immunoblotting demonstrated that the betaine-accumulating species have a BADH enzyme recognized by antibodies raised against BADH from Spinacia oleracea L. (Chenopodiaceae), and that the M_r of the BADH monomer is in all cases close to 63 000. These data indicate that the cholinebetaine aldehydebetaine pathway may have evolved by vertical descent from an early angiosperm ancestor, and might be widespread (albeit not always strongly expressed) among flowering plants. Consistent with these suggestions, Magnolia x soulangiana was found to have a low level of betaine, and to express a protein of M_r 63 000 which cross-reacted with antibodies to BADH from Spinacia oleracea.Abbreviations BADH Betaine aldehyde dehydrogenase - DCIMS desorption chemical ionization mass spectrometry - FABMS fast atom bombardment mass spectrometry - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TLC thin-layer chromatography     

The effect of low- and high-density lipoprotein cholesterol on steroid hormone production and ACTH-induced differentiation of rat adrenocortical cells in primary culture

Summary The choroid plexus consists of the choroidal epithelium, a derivative of the neural tube, and the choroidal stroma, which originates from the embryonic head mesenchyme. This study deals with epithelio-mesenchymal interactions of these two components leading to the formation of the organ. Grafting experiments of the prospective components have been performed using the quail-chicken marker technique. Prospective choroidal epithelium of quail embryos, forced to interact with mesenchyme of the body wall of chicken embryos, gives rise to a choroid plexus showing normal morphogenesis and differentiation. The choroidal epithelium induces the differentiation of organtypical fenestrated capillaries, which are highly permeable to intravenously injected horseradish peroxidase. The choroidal epithelium of the grafts constitutes a blood-cerebrospinal fluid barrier. On top of the choroidal epithelium, there are epiplexus cells displaying a typical ultrastructure. The experimental results show that these cells do not originate from the transplanted neural epithelium. Prospective choroidal stroma of chicken embryos does not exert a choroid plexus-inducing influence upon a quail embryo's neural epithelium isolated from parts of the brain that normally do not develop a choroid plexus. The experiments show that the choroidal epithelial cells are determined at least three days before the first organ anlage is detectable.This work was supported by the Deutsche Forschungsgemeinschaft (grant Ch 44/7-1)     

Plasmids in the bacterial assemblage of a dystrophic Lake: Evidence for plasmid-encoded nickel resistance

Sixty-two aerobic bacterial strains isolated from the unproductive dystrophic Lake Skärshultsjön (South Sweden) were screened for plasmids. The lake is considered to be an extreme environment because of its high concentration of persistent but nontoxic humic compounds. One-third of the isolates harbored multiple plasmids usually of similar high molecular weights (>25 Mdal). The plasmid-bearing strains were members of the common aquatic taxaPseudomonas spp.,Acinetobacter sp.,Alcaligenes sp.,Aeromonas/Vibrio group, andEnterobacteriaceae (taxonomy is tentative). The majority of isolates displayed multiple resistance to antibiotics and heavy metals. Some of them were capable of degrading aromatic compounds. Three isolates were chosen for curing experiments. Only strain S-68, anAlcaligenes sp., could be cured of one of its two plasmids. It harbored the two cryptic plasmids pQQ32 and pQQ70 of 32 and ca. 70 Mdal, and the latter was segregated during ethidium bromide treatment. Parental strain S-68 was capable of degrading some of nonchlorinated phenolic compounds and displayed resistance to a broad spectrum of antibiotics and the heavy metals Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, and Hg²⁺. Derivative strain S-68-41 lost its resistance to nickel, suggesting segregated plasmid PQQ70 coded for nickel resistance. Transformation experiments to restore nickel resistance in the cured derivative strain were not successful.     

Characterisation of neuropeptides of the AKH/RPCH-family from corpora cardiaca of Coleoptera

Summary Extracts of corpora cardiaca from two members of the family Tenebrionidae,Zophobas rugipes andTenebrio molitor, from one member of the Chrysomelidae,Leptinotarsa decemlineata, and from three members of the Scarabaeidae,Pachnoda marginata, P. sinuata andMelolontha hippocastani, were assayed for adipokinetic and hypertrehalosaemic activity in acceptor locusts (Locusta migratoria) and cockroaches (Periplaneta americana), respectively. All corpus cardiacum material tested, except that from the cockchafer,M. hippocastani, gave positive bioassay results. Biological activities of corpus cardiacum extracts from all species investigated can be resolved on reversed-phase high performance liquid chromatography (RP-HPLC). Gland extracts from the two tenebrionid species each show a single peak of biological activity associated with a single peak of UV absorbance having an identical retention time in both species. The two biologically active fractions from the corpora cardiaca of the potato beetle,L. decemlineata, coelute with exogenous (synthetic) hypertrehalosaemic hormones I and II of the American cockroach. The two species of the genusPachnoda contain two active compounds in their glands; compound I of each species is more abundant and elutes just ahead of the (synthetic) hypertrehalosaemic hormone of the cockroachBlaberus discoidalis. The gland material ofM. hippocastani exhibits and absorbance peak with the same retention time as the major peak from thePachnoda-species; however, this peak material does not elicit biological activity in the assays used here. After fractionation by RP-HPLC the main biologically active compounds were subjected to amino acid analyses. All factors are peptidic and contain 8 amino acid residues. The peptides from the tenebrionid species have the amino acid residues Asx₍₂₎, Glx₍₁₎, Ser₍₁₎, Pro₍₁₎, Leu₍₁₎, Phe₍₁₎ and Trp_(i), whereas the main peptide from corpora cardiaca ofP. marginata contains the residues Asx₍₂₎, Glx₍₁₎, Ser₍₁₎, Pro₍₁₎, Tyr₍₁₎, Leu₍₁₎ and Trp₍₁₎. Amino acid composition analyses of the two active fractions fromL. decemlineata reveal the residues Asx₍₂₎, Glx₍₁₎, Ser₍₁₎, Pro₍₁₎, Val₍₁₎, Phe₍₁₎ and Trp₍₁₎ for compound I and Asx₍₁₎, Glx₍₁₎, Thr₍₂₎, Pro₍₁₎, Leu₍₁₎, Phe₍₁₎ and Trp₍₁₎ for compound II.     

Glucose as a substrate in recombinant strain fermentation technology

Summary Glucose supplements to complex growth media of Escherichia coli affect the production of a recombinant model protein under the control of a temperature-sensitive expression system. The bacterial Crabtree effect, which occurs in the presence of glucose under aerobic conditions, not only represses the formation of citric acid cycle enzymes, but also represses the formation of the plasmid-encoded product even though the synthesis of this protein is under the control of the temperature-inducible lambda P _R-promoter/cl857-repressor expression system. When the recombinant E. coli is grown at a moderate temperature (35° C) with protein hydrolysate and glucose as substrates, a biphasic growth and production pattern is observed. In the first phase, the cells grow with a high specific growth rate, utilizing glucose and forming glutamate as a byproduct. The intracellular level of recombinant protein is very low in this phase. Later, glutamate is consumed, indicating an active citric acid cycle. The degradation of glutamate is accompanied by the intracellular accumulation of high amounts of recombinant protein.     

Glucocorticoid receptor binds cooperatively to adjacent recognition sites.

In order to define the mechanism of synergistic induction mediated by multiple glucocorticoid response elements (GRE), the affinity of the glucocorticoid receptor to a single or duplicated GRE was analyzed by gel retardation, nitrocellulose filter binding and by footprinting experiments. Direct measurement of the relative affinity and indirect determination by competition showed greater than 10-fold higher affinity of the glucocorticoid receptor to a duplicated GRE when compared to a single element. Maximal stability of the GRE-receptor complex was obtained using two closely spaced GREs positioned on the same side of the DNA helix. Increasing the distance or changing the helical position of the GREs considerably increased the off rate of the receptor. DNase I footprinting shows in addition to the protection of the GRE region, an altered pattern in the nonprotected intervening DNA indicating structural alteration of the DNA helix by the receptor bound to adjacent GREs.     

Reactive extraction of penicillin G from mycel-containing broth in a countercurrent extraction decanter

Penicillin was recovered from mycel-containing fermentation broth by direct reactive extraction into a counter-current extraction decanter, Type CA 226-290 of the Westfalia Separator Co., at room temperature via steady state operation. Penicillin concentrations in the feed varied from 3 to 41 g L(-1), Amberlite LA-2 carrier concentrations from 7 to 20 g L(-1)and/or DITDA carrier concentrations from 7.2 to 84 g L(-1), the LA-2-to-penicillin mole concentration ratio from 4 to 6.4, and/or the DITDA-to-penicillin mole concentration ratio was maintained at 2. The throughputs of the fermentation broth (520 to 880 L h(-1)) of the solvent phase (200 to 860 L h(-1)) and the over all throughput (800 to 1750 L h(-1)) were high. Extraction degrees of 72 to 96% were achieved between pH 4.6 and 5.1. Without carriers in the same pH range, extraction degrees of only 17 to 19% were attained. By reducing the pH to 2.3 and in the absence of carriers, the degree of extraction was increased to 61%. However, during the extraction, 6.5% of the penicillin decomposed. At these high throughputs, the steady state was attained within 1 to 4 min. Through the mechanical stress, the length of the hyphae was reduced and the protein content of the broth was increased by 50 to 100%. However, this protein content had no appreciable influence on the phase separation.