NADH-activated cell-free transfer between Golgi apparatus and plasma membranes of rat liver.

This report concerns development of a cell-free system from rat liver to study transport of membrane constituents from the Golgi apparatus to the plasma membrane. Highly purified Golgi apparatus as donor and a mixture of sheets and vesicles as plasma membrane acceptor fractions were combined to analyze requirements for lipid and protein transport. In the reconstituted system, the Golgi apparatus donor was in suspension. To measure transfer, membrane constituents of the donor membranes were radiolabeled with [3H]acetate (lipids) or [3H]leucine (proteins). The plasma membrane vesicles were used as the acceptor and were unlabeled and immobilized on nitrocellulose for ease of recovery and analysis. The reconstituted cell-free transfer was dependent on temperature, but even at 37 degrees C, the amount of transfer did not increase with added ATP, was not specific for any particular membrane fraction or subfraction nor was it facilitated by cytosol. ATP was without effect both in the presence or absence of a cytosolic fraction capable of the support of cell-free transfer in other systems. In contrast to results with ATP, NADH added to the reconstituted system resulted in an increased amount of transfer. A further increase in transfer was obtained with NADH plus a mixture of ascorbate and dehydroascorbate to generate ascorbate free radical. The transfer of labeled membrane constituents from the Golgi apparatus to the plasma membrane supported by NADH plus ascorbate radical was stimulated by a cytosol fraction enriched in less than 10 kDa components. This was without effect in the absence of NADH/ascorbate radical or with ATP as the energy source. Specific transfer was inhibited by both N-ethylmaleimide and GTP gamma S. The findings point to the possibility of redox activities associated with the trans region of the Golgi apparatus as potentially involved in the transport of membrane vesicles from the Golgi apparatus to the cytoplasmic surface of the plasma membrane.     

Rat metanephric organ culture in terato-embryology

The development of the permanent mammalian kidney, or metanephros, depends on mesenchymal-epithelial interactions, leading to branching morphogenesis of the ureteric bud that forms the collecting ducts and to conversion of the metanephric mesenchyme into epithelium that forms the nephrons. Rat metanephric organ culture in which these interactions are maintained is a valuable in vitro model system for investigating normal and abnormal renal organogenesis. Methods were designed to evaluate either the capacity of the ureteric bud to branch or that of the mesenchyme to form nephrons. Both are based on specific staining of the ureteric bud and the glomeruli with lectins. Using this approach, we have shown that retinoids are potent stimulating factors of nephrogenesis, acting through an increase in the branching capacity of the ureteric bud. On the other hand, several drugs such as gentamicin and cyclosporin A were found to reduce the number of nephrons formed in vitro. While gentamicin affects the early branching pattern of the ureteric bud, cyclosporin may affect the capacity of the mesencyme to convert into epithelium. This methodology therefore appears a potentially useful tool for toxicological studies new drugs.     

Recruitment of plasma membrane voltage-dependent calcium-permeable channels in carrot cells.

Numerous biological assays and pharmacological studies have led to the suggestion that depolarization-activated plasma membrane Ca2+ channels play prominent roles in signal perception and transduction processes during growth and development of higher plants. The recent application of patch-clamp techniques to isolated carrot protoplasts has led to direct voltage-clamp evidence for the existence of Ca2+ channels activated by physiological depolarizations in the plasma membrane of higher plant cells. However, these voltage-dependent Ca2+ channels were not stable and their activities decreased following the establishment of whole-cell recordings. We show here that large pre-depolarizing pulses positive to 0 mV induced not only the recovery of Ca2+ channel activities, but also the activation of initially quiescent voltage-dependent Ca2+ channels in the plasma membrane (recruitment). This recruitment was dependent on the intensity and duration of membrane depolarizations, i.e. the higher and longer the pre-depolarization, the greater the recruitment. Pre-depolarizing pulses to +118 mV during 30 s increased the initial calcium currents 5- to 10-fold. The recruited channels were permeable to Ba2+ and Sr2+ ions. The data suggested that voltage-dependent Ca(2+)-permeable channels are regulated by biological mechanisms which might be induced by large pre-depolarizations of the plasma membrane. In addition, this study provides evidence for the existence in the plasma membrane of higher plant cells of a large number of voltage-dependent Ca2+ channels of which a major part are inactive and quiescent. It is suggested that quiescent Ca2+ channels can be rapidly recruited for Ca(2+)-dependent signal transduction.     

Value of silver staining technics for the study of acrocentric ring chromosomes and supernumerary microchromosomes

The silver staining techniques was used to study two cases of ring chromosomes (ring chromosome 15 and ring chromosome 22) and two cases of small extra chromosomes. This technique allows identification of the breakpoints and provides some information about the origin of small extra chromosomes.     

In vivo incorporation of acetate into the acyl moieties of polar lipids from etiolated leek seedlings

Seven-day-old leek seedlings actively synthesize lipids in vivo from [1-¹⁴C]acetate, both in the light and in the dark. In the dark, phospholipid synthesis is more effective than galactolipid synthesis. Whatever the time of acetate incorporation by the etiolated seedlings, very long chain fatty acids having from 20 to 26 carbon atoms are found in all the polar lipids, including the acyl-CoAs. All of the labelled very long chain fatty acids incorporated into the polar lipids are saturated. On the other hand, the labelled C₁₈-fatty acids are unsaturated in phospholipids and galactolipids and almost no label is found in the saturated or unsaturated C₁₈-fatty acids of the acyl-CoAs.     

Induction of prophage lambda does not require full induction of RecA protein synthesis

In mitomycin C-treated lambda lysogens, even though the rate of synthesis of RecA protein was greatly reduced by a low concentration of rifampicin (4 microgram/ml), induction of prophage lambda occurred readily as assessed by (i) cell lysis of the lysogens, (ii) production of progeny phage, and (iii) extensive cleavage of lambda repressor. The extent and the rate of cleavage of lambda repressor were not significantly affected by the low rate of synthesis of RecA protein resulting from rifampicin action. However, the yield of phage progeny was reduced and lysis of the cells was slightly delayed. We conclude that in RecA+ bacteria, induction of prophage lambda does not require full induction of RecA protein synthesis.