Testing the “methanochondrion concept”: are nucleotides transported across internal membranes in Methanobacterium thermoautotrophicum?

In order to test the Methanochondrion concept, uptake of adenine nucleotides in various membrane preparations of Methanobacterium thermoautotrophicum was studied. The uptake showed properties which are in general interpreted as indicative of a transport mechanism: (i) kinetics in the time range of minutes, (ii) temperature dependence, (iii) substrate specificity and (iv) failure to remove the substrate by extensive washing.However, nucleotide transport as an interpretation of this uptake can definitely be excluded. Not only an exchange mechanism of the mitochondrial type, but also a general exchange or an uniport mechanism was ruled out. In contrast, the nucleotide uptake was shown to be actually a tight and specific binding of ADP and ATP to binding sites at the interior side of the cell membrane. This was conclusively demonstrated in protoplasts obtained from M. thermoautotrophicum cells. In these protoplasts which do not contain internal membranes also nucleotide binding was observed, but only after disruption of the plasma membrane by osmotic lysis, which leads to the exposure of binding sites.     

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.     

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)     

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.     

Toxicity and isolation of the cyanobacterium Nodularia spumigena from the southern Baltic Sea in 1986

Three water bloom samples were collected in August 1986 from the southern Baltic Sea. Acute toxicity of the samples was determined by mouse bioassay and the toxins were further studied by HPLC. The bloom samples contained equal amounts of cyanobacteria Nodularia spumigena and Aphanizomenon flos-aquae and were hepatotoxic. Two hepatotoxic Nodularia spumigena strains were isolated from the samples. The isolates produce a toxic peak indistinguishable from the bloom material in the HPLC analysis. The toxicity of the fractions was verified by mouse bioassay. Thus the toxicity of the bloom samples was in all likelihood caused by Nodularia spumigena.     

The 76 kD cell-adhesion factor from crayfish haemocytes promotes encapsulation in vitro

Summary Semigranular cells from the crayfish, Pacifastacus leniusculus, were separated by Percoll gradient centrifugation and were used to study the encapsulation of foreign particles. The semigranular cells were found strongly to encapsulate glass beads coated with haemocyte lysate in which the prophenoloxidase-activating system had been activated with laminarin or with a low concentration of calcium ions. The granular cells only weakly encapsulated these particles. The encapsulationpromoting factor was purified from haemocyte lysates and found to be a 76 kD protein which was recognized by an antiserum to the previously described 76 kD cell-adhesion factor. After the last step in purification (Con A-Sepharose chromatography), the flowthrough consisted of several proteins, which had some, but less, encapsulation-promoting activity and contained a 30 kD band that was also recognized by the antiserum to the 76 kD cell-adhesion factor. If the haemocyte lysate prepared in low [Ca²⁺] was incubated with a -1,3-glucan prior to purification, no 76 kD protein could be isolated but only a 30 kD protein. The 30 kD protein thus seems to be a degradation product of the 76 kD cell-adhesion factor. We conclude that the 76 kD protein which is released from degranulating haemocytes, and to a lesser extent its 30 kD fragment, can promote encapsulation. Phenoloxidase did not have any encapsulation-promoting activity.     

Effects of endogenous and exogenous cholesterol on the ultrastructure and steroid secretion of undifferentiated rat adrenocortical cells in primary culture

Summary The present study was undertaken to define the effects of lipoprotein-derived cholesterol and endogenous, de novo synthesized cholesterol on the ultrastructure and function of undifferentiated rat adrenocortical cells [lipoprotein (HDL₃ and LDL) receptor-negative, zona glomerulosa-like adrenocortical cells] in primary culture. For this purpose human plasma high density lipoprotein (HDL₃) or low density lipoprotein (LDL) was added to culture medium devoid of cholesterol. Steroid secretion remained at the low basal level even after addition of lipoproteins, and the amount of intracellular lipid droplets did not increase. When mevinolin (0.96 µg/ml), an inhibitor of cholesterol synthesis, was added to the culture medium, a low secretion of corticosterone was measured both in serum-free and serum-containing media. Ultrastructurally, lipid droplets disappeared after treatment with mevinolin in both media used. At this concentration of mevinolin cell proliferation was similar to that in the controls, but at higher concentrations (4.8 or 9.6 µg/ml) proliferation was inhibited to 42% and 26% in serum-free medium, and 20% and 12% in serum-supplemented medium, respectively. This study demonstrates that cell proliferation and synthesis of corticosterone by undifferentiated rat adrenocortical cells is identical in the absence or presence of exogenous lipoprotein cholesterol. Inhibition of de novo cholesterol synthesis by mevinolin over a period of 7 days does not inhibit corticosterone secretion or proliferation of cells but decreases the amount of intracellular lipid droplets, thus suggesting utilization of intracellular cholesterol esters. However, higher concentrations of mevinolin inhibit proliferation of cells both in serum-free and serum-containing media.