On disturbance of homeostasis in organ-cultured tissue

Summary The intact membranous rat mesentery was cultured in Eagle's minimum essential medium containing no serum or only low concentrations of serum. The procedure is in some important respects superior to previous organ culture techniques. To estimate the extent of disturbance of homeostasis of the tissue in culture, the spontaneous mast-cell histamine release was quantitated after preculture preparation of the specimens and after different intervals in culture. Also, the proliferation of fibroblasts and mesothelial cells that predominate in the mesentery was assessed at 48 h by cytofluorometric quantitation of DNA in single-tissue cells. Spontaneous histamine release was time dependent during cultivation, amounting to ca. 50% at 48 h, and was affected by the medium used for moistening the tissue before cultivation. Culturing also brought about great spontaneous increase in the proliferation of fibroblasts and mesothelial cells, the rate being related to the concentration of serum. Addition of the mast-cell secretagogues 48/80 or polymyxin B at 1 h caused rapid release of 50 to 60% of the histamine and was followed by augmented proliferation in the serum-containing media. The spontaneous increase of cell proliferation in tissue culture may be causally related to mast-cell secretion. Further studies are needed to define factors influencing the spontaneous mast-cell secretion and the mast-cell-dependent mitogenesis in normal tissue cells Supported by grants from the Swedish Medical Research Council (Project 5942) and State Board for Animal Experiments.     

An analysis of proton fluxes coupled to electron transport and ATP synthesis in chloroplast thylakoids

Electron transport, phosphorylation and internal proton concentration were measured in illuminated spinach chloroplast thylakoid membranes under a number of conditions. Regardless of the procedure used to vary these parameters, the data fit a simple chemiosmotic model. Protons from Photosystem II did not appear to be utilized differently from those derived from Photosystem I. The maximal phosphorylation efficiency ($\text{P}\text{e}{}_2$) for photophosphorylation in washed thylakoids under oxidizing conditions is likely to be $\text{4}\text{3}$. This value is consistent with a proton-to-electron-pair ratio of 4 for electron flow through both photosystems and a proton-to-ATP ratio of 3 for the chloroplast proton-ATPase.     

Three-step preparation and purification of phosphorus-33-labeled creatine phosphate of high specific activity

Rabbit heart mitochondria were used as a source of enzymes for the synthesis of phosphoruslabeled creatine phosphate. This method is based on the coupled reaction between mitochondrial oxidative phosphorylation and mitochondrial-bound creatine kinase. It is possible to convert more than 90% of the inorganic phosphate (P_i) to creatine phosphate. The method used only small amounts of adenine nucleotides which led to a product with only slight nucleotide contamination. This could be removed by activated charcoal extraction. For further purification, a method for the removal of residual P_i is described.     

Coordinated synthesis of the nuclear protein cyclin and DNA in serum-stimulated quiescent 3T3 cells

Quantitative two-dimensional gel electrophoretic analysis (IEF) of the nuclear polypeptide cyclin together with autoradiographic studies have revealed a coordinate synthesis of cyclin and DNA after serum stimulation of quiescent 3T3 cells. These results strengthen the notion that cyclin may be a central component of the pathway(s) that regulate cell proliferation.     

The enzymatic malonylation of 1-aminocyclopropane-1-carboxylic acid in homogenates of mung-bean hypocotyls

Homogenates of hypocotyls of light-grown mung-bean (Vigna radiata (L.) Wilczek) seedlings catalyzed the formation of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) from the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl-coenzyme A. Apparent K_m values for ACC and malonyl-CoA were found to be 0.17 mM and 0.25 mM, respectively. Free coenzyme A was an uncompetitive inhibitor with respect to malonyl-CoA (apparent K_i=0.3 mM). Only malonyl-CoA served as an effective acyl donor in the reaction. The d-enantiomers of unpolar amino acids inhibited the malonylation of ACC. Inhibition by d-phenylalanine was competitive with respect to ACC (apparent K_i=1.2 mM). d-Phenylalanine and d-alanine were malonylated by the preparation, and their malonylation was inhibited by ACC. When hypocotyl segments were administered ACC in the presence of certain unpolar d-amino acids, the malonylation of ACC was inhibited while the production of ethylene was enhanced. Thus, a close-relationship appears to exist between the malonylation of ACC and d-amino acids. The cis- as well as the trans-diastereoisomers of 2-methyl- or 2-ethyl-substituted ACC were potent inhibitors of the malonyltransferase. Treatment of hypocotyl segments with indole-3-acetic acid or CdCl₂ greatly increased their content of ACC and MACC, as well as their release of ethylene, but had little, or no, effect on their extractable ACC-malonylating activity.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - MACC 1-(malonylamino)-cyclopropane-1-carboxylic acid Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

The role of triiodothyronine in the regulation of synthesis rate and translatable mRNA level of fatty acid synthetase in a preadipocyte cell line

Triiodothyronine (T₃) effects on the activity, rate of synthesis and mRNA content of the key lipogenic enzyme, fatty acid synthetase, were studied in differentiating ob₁₇ preadipocytes cloned from ob/ob mouse epididymal adipose tissue. During differentiation in the presence of insulin, a 6–10-fold increase in both fatty acid synthetase specific activity and synthesis rate were reproducibly observed and occurred concomitantly. The relative synthesis rate exhibited a progressive elevation from 0.5% at confluence to a maximum level of 2% in the presence of insulin. The rate of the enzyme degradation determined by pulse-chase experiments was similar in differentiating cells and insulin-untreated cells of the same age ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, 40–42 h). Furthermore, the increase in the enzyme synthesis rate was preceded by a progressively elevating amount of mRNA encoding for this protein as detected by translation in a reticulocyte lysate cell-free system. It is thus suggested that the increment in total and neosynthesized fatty acid synthetase in essentially due to an increased enzyme synthesis, reflecting an increased relative content of its specific mRNA. T₃ included at a physiological concentration (1.5 nM) in the culture medium enhanced significantly both enzyme synthesis and its specific mRNA. The most important T₃ effect was an acceleration of both processes, a stimulation of the mRNA level being detected as early as day 3 post-confluence and maximum at day 5 when the effect on the synthetase synthesis rate and activity began to be enhanced. This suggests that T₃ would mainly affect fatty acid synthetase as a pretranslational level.     

Synthesis of subunit III of CF0 by thylakoid-bound polysomes from pea chloroplasts

Summary Wahsed thylakoid membranes from pea chloroplasts incorporate label from (³⁵S)-methionine into protein when supplemented with S-30 soluble factors from E. coli. One of the products associated with the thylakoids is soluble in butanol, precipitated by ether and has an apparent molecular mss of 8200D on urea-lithium dodecyl sulphate (LDS) polyacrylamide gels. In addition, the protein covalently binds dicyclohexylcarbo-diimide (DCCD) which causes it to migrate as two slower forms on gels. Based on these criteria we establish that the proteolipid or subunit III of CF₀ (the intrinsic sector of the ATPase complex) is synthesized by the thylakoid bound polysomes.     

I. Photoaffinity probes provide a general method to prepare synthetic peptide-conjugates

A general synthetic strategy is described for the preparation of peptide-conjugates where the peptides contain the NH₂ terminal, COOH terminal, or internal regions of the protein sequence. Glycoprotein D of herpes simplex virus type 1 is used as a representative protein. Ten-residue peptide fragments of the native sequence were synthesized using standard solid-phase methodology. Photoprobes stable to conditions of synthesis and HF cleavage were coupled directly to the protected-peptide resin during synthesis. This one-step procedure eliminates the potential modification of functional groups in the sequence of interest that can occur when using chemically labile bifunctional reagents. Since the photoprobe is inert until photolysis, the synthetic peptide-probe can be readily purified by high-performance liquid chromatography before cross-linking to the carrier molecule. The following photoprobe derivatives were investigated: thep-azidobenzoyl,p-nitrophenylalanyl, andp-benzoylbenzoyl groups. The benzophenone photoprobes were shown to give the highest incorporation of peptide-probe with the protein carrier over a wide range ofpH and solvent conditions. For solid-phase synthesis three benzophenone photoprobes can be used: benzoylbenzoic acid, benzoylbenzoylglycine, andN ^e-(4-benzoylbenzoyl)-N -t-butyloxycarbonyl-lysine.     

Transferrin as a donor of iron to mitochondria Effect of pyrophosphate and relationship to mitochondrial metabolism and heme synthesis

Rat liver mitochondria accumulate iron mobilized from transferrin by pyrophosphate. The uptake has a very low energy dependence, but it is highly dependent on a functioning respiratory chain. Reduction of the ferric-iron-pyrophosphate complex is not linked to any specific respiratory complex. Half of the amount of iron accumulated is passed into heme. Iron once accumulated is very little accessible to chelation by added ferric or ferrous iron chelators. Iron uptake and heme synthesis are maximal if a suitable porphyrin substrate is added simultaneously with iron. The results represent further evidence that pyrophosphate is a possible candidate for intracellular iron transport. Also, the results suggest that iron uptake is coupled to simultaneous porphyrin uptake and heme synthesis.     

Primary tissue culture of human adrenocortical Conn's adenomata

Summary Angiotensin II-induced the hypertrophy of the cytoplasmic compartment and significantly increased (5-³H)uridine incorporation into RNA species by Conn's human adult adenomatous cells in primary tissue culture. On its own, bromocriptine, while enlarging only the nucleolar compartment, also intensely stimulated the incorporation of (5-³H)uridine into RNA species by the cultured adrenocortical adenomatous cells. However, an equimolar mixture of angiotensin II and bromocriptine was totally ineffective, eliciting no change in cellular morphometry or isotope incorporation with respect to the control specimens run in parallel.The present findings support the view that bromocriptine can influence the metabolism of Conn's cells directly at the cellular level by acting as an agonist-antagonist of angiotensin.