Increased water drinking induced by sodium depletion in sheep.

Forty-eight hours of sodium depletion by acute cannulation of a parotid duct, via the buccal papilla, in the sheep, resulted in a progressive decrease in salivary secretion rate, salivary, urinary and plasma [Na] and no change in plasma [K]. In the first 24 h of Na depletion water intake was significantly increased. As normal sheep parotid saliva [Na] is higher than plasma [Na] and salivary loss over the first 24 h represented Na loss in excess of water relative to extracellular proportions, increased water intake was not osmotically induced. However, the animals did not replace their water deficit on either of the 2 days of Na depletion. This would appear to be valuable experimental model of increased water intake probably induced by hypovaolaemia, but uncomplicated concurrent osmotic stimuli, or any other factors which might result with the other commonly used experimental stimuli of thirst such as haemorrhage.     

On the mechanism of protein-synthesis inhibition by abrin and ricin. Inhibition of the GTP-hydrolysis site on the 60-S ribosomal subunit.

The mechanism of protein synthesis inhibition by the toxic lectins, abrin and ricin, has been studied in crude and in purified cell-free systems from rabbit reticulocytes and Krebs II ascites cells. In crude systems abrin and ricin strongly inhibited protein synthesis from added aminoacyl-tRNA, demonstrating that the toxins act at some point after the charging of tRNA. Supernatant factors and polysomes washed free of elongation factors were treated separately with the toxins and then neutralizing amounts of anti-toxins were added. Recombination experiments between toxin-treated ribosomes and untreated supernatant factors and vice versa showed that the toxin-treated ribosomes had lost most of their ability to support polyphenylalanine synthesis, whereas treatment of the supernatant factors with the toxins did not inhibit polypeptide synthesis. Recombination experiments between toxin-treated isolated 40-S subunits and untreated 60-S subunits and vice versa showed that only when the 60-S subunits had been treated with the toxins was protein synthesis inhibited in the reconstituted system. The incorporation of [3H]puromycin into nascent peptide chains was unaffected by the toxins, indicating that the peptidyl transferase is not inhibited. Both the EF-1-catalyzed and the EF-2-catalyzed ability of the ribosomes to hydrolyze [gamma-32P]GTP was inhibited by abrin and ricin. An 8-S complex released from the 60-S subunit by EDTA treatment possessed both GTPase and ATPase activity, while the particle remaining after the EDTA treatment had lost most of its GTPase activity. Both enzyme activities of the 8-S complex were inhibited by abrin and ricin. The present data indicate that there is a common site on the 60-S subunits for EF-1- and EF-2- stimulated GTPase activity and they suggest that abrin and ricin inhibit protein synthesis by modifying this site.     

Sites of action of D2O in intact and skinned crayfish muscle fibers

Summary The effect on tension development of replacing 90% of the H₂O of the bathing saline with D₂O was studied on intact single fibers, and on skinned fibers before and after the latter were treated so as to eliminate Ca-accumulation by the sarcoplasmic reticulum (SR). Excitation-contraction coupling (ECC) of intact fibers is not abolished, but is depressed by D₂O so that higher depolarizations are required to elicit a given tension. The reduction in tension at a given level of depolarization is not due to inhibition of the contractile system. The latter showed an enhanced Ca sensitivity; that is, skinned fibers respond to Ca concentrations that are 1–2 orders of magnitude smaller in D₂O than in H₂O saline. When bathed in D₂O saline, intact fibers or skinned fibers with functional SR can still accumulate and release Ca in sufficient quantities to allow repeated induction of maximum tensions. Relaxation is slowed in all three types of preparation, perhaps because of an increased affinity of troponin to Ca in D₂O salines.     

The comparison of rat liver rough endoplasmic reticulum membrane proteins before and after in vitro removal of its bound ribosomes

The comparison of the proteins of rat liver rough membrane after stripping with EDTA or KCl-puromycin by two dimensional gel electrophoresis is described. By stripping the membrane with EDTA, most of the basic ribosomal proteins are still attached to the membrane; in contrast to the EDTA stripping method, treatment with KCl-puromycin removes most of the ribosomal proteins and does not remove any of the membranal proteins.     

Role and Regulation of Bacillus subtilis Glutamate Dehydrogenase Genes

The complete Bacillus subtilis genome contains two genes with the potential to encode glutamate dehydrogenase (GlutDH) enzymes. Mutations in these genes were constructed and characterized. The rocG gene proved to encode a major GlutDH whose synthesis was induced in media containing arginine or ornithine or, to a lesser degree, proline and was repressed by glucose. A rocG null mutant was impaired in utilization of arginine, ornithine, and proline as nitrogen or carbon sources. The gudB gene was expressed under all growth conditions tested but codes for a GlutDH that seemed to be intrinsically inactive. Spontaneous mutations in gudB that removed a 9-bp direct repeat within the wild-type gudB sequence activated the GudB protein and allowed more-efficient utilization of amino acids of the glutamate family.     

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale expansion, may become cumbersome, laborious and time-consuming when large numbers of cells are required. To address this need, the performance of a new multi-layered cell culture vessel to facilitate easy scale up of cells from single layered T-flasks will be discussed. The flasks tested are available in 3- and 5-layer format and enable culture and complete recovery of three and five times the number of cells respectively, compared to T-175 flasks. A key feature of the BD Multi-Flask is a mix/equilibration port that allows rapid in-vessel mixing as well as uniform distribution of cells and reagents within and between layers of each vessel and consistently produce cells that can be cultured in an environment that is congruent to T-175 flasks.The design of these Multi-Flasks also allows for convenient pipette access for adding reagents and cells directly into the flasks as well as efficient recovery of valuable cells and reagents and reduces risk of contamination due to pouring. For applications where pouring is preferred over pipetting, the design allows for minimal residual liquid retention so as to reduce wastage of valuable cells and reagents.