Inhibition of RNA synthesis in yeast protoplasts by a peptide factor from Tetrahymena cells

Protoplasts of Schizosaccharomyces pombe, grown on a rich nutrient medium, were treated with a peptide factor isolated from cultures of the protozoan Tetrahymena pyriformis. The peptide factor is known to inhibit RNA synthesis in Tetrahymena. It has now been shown that the peptide factor also inhibits RNA synthesis in yeast protoplasts without affecting protein synthesis.     

A rapid,sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots

A rapid, sensitive method has been developed to detect antibody-antigen complexes on “Western blots.” The methods of H. Towbin, T. Staehlin, and J. Gordon were used to separate and blot the antigens onto nitrocellulose. The remaining sites of attachment were blocked and the nitrocellulose was washed with polyoxyethylenesorbitan monolaurate (Tween 20). The blot was then reacted with the antiserum or hybridoma supernate to be tested. After the antigen-antibody reaction was completed, the blot was washed and treated with anti-antibody which has been conjugated to alkaline phosphatase. The alkaline phosphatase was detected by the reduction of the tetrazolium salt to diformazan by the hydrogen ions released in the formation of indigo by the reaction of the phosphatase on the indoxyl phosphate. The advantages of this method over previously described techniques are (1) use of Tween 20 allows the blot to be stained with Coomassie blue, (2) the substrates of the alkaline phosphatase reaction are stable for long periods of time, (3) the reaction products form an intense blue color which does not fade, (4) the resolution is extremely good with little to no band broadening, (5) the reaction is sensitive to picogram quantities of antigen, and (6) the reaction is quantitative.     

The isolation of spirochaetes from the rat caecum

Spirochaetes indigenous to the conventional rat caecum were cultivated on agar medium using selective isolation techniques. The 27 axenic isolates represented four morphological types of spirochaetes differing in cell size, cell coiling pattern and number of protoplasmic flagella.     

The role of surface charge and hydrophilic groups on liposome clearance in vivo.

The effect of negative surface charge and hydrophilic groups on liposome clearance from blood was investigated in mice using liposome-entrapped 67gallium-deferoxamine as a label. The presence of negatively-charged lipids may retard or accelerate liposome clearance. Physicochemical features contributing to optimal retardation of liposome clearance include a hydrophilic carbohydrate moiety and a sterically hindered negatively-charged group. The relevance of the negative charge steric effect is suggested by the finding that phosphatidylinositol phosphate (PIP) and trisialoganglioside (GT1) are less effective than phosphatidylinositol (PI) and monosialoganglioside (GM1), respectively, in retarding liposome clearance. The need for negative charge in addition to the carbohydrate group for optimal effect on retardation of clearance is indicated by the observation that asialoganglioside (AGM1) is less effective than GM1 in this respect. The negative charge effect is observed with liposome bilayers having both low and high temperature phase-transitions. Increasing the molar fraction of negatively-charged lipid (hydrogenated PI derived from soya) from 23 to 41% resulted in a dramatic acceleration of liposome clearance. The clearance-accelerating effect of the high negative charge was specifically directed to the liver with selective reduction of spleen uptake. Increasing liposome size also had an accelerating effect on clearance but in this case it was accompanied by a non-specific concomitant increase of both liver and spleen uptake.     

Assembly of a functional F1 of the proton-translocating ATPase of Escherichia coli

Assembly of the F1 portion of the proton-translocating ATPase of Escherichia coli was examined in vivo. Analysis of strains lacking genes which specify the Fo polypeptides a, b, and c showed that the F1 subunits were able to assemble into a complex in the absence of the Fo subunits. In addition we have investigated the effects of mutations in the individual genes which specify the F1 polypeptides on the assembly process. Mutations of the uncA(alpha), uncG(gamma), or uncD(beta) genes result in a defective assembly of the F1 complex. In contrast, mutations in the uncH(delta) or uncC(epsilon) genes did not prevent assembly of the core alpha beta gamma complex. In these cases, however, the partial F1 complexes were incapable of restoring energy-linked functions to F1-depleted membranes.     

Radiation inactivation target size of rat adipocyte glucose transporters in the plasma membrane and intracellular pools

The in situ assembly states of the glucose transport carrier protein in the plasma membrane and in the intracellular (microsomal) storage pool of rat adipocytes were assessed by studying radiation-induced inactivation of the D-glucose-sensitive cytochalasin B binding activities. High energy radiation inactivated the glucose-sensitive cytochalasin B binding of each of these membrane preparations by reducing the total number of the binding sites without affecting the dissociation constant. The reduction in total number of binding sites was analyzed as a function of radiation dose based on target theory, from which a radiation-sensitive mass (target size) was calculated. When the plasma membranes of insulin-treated adipocytes were used, a target size of approximately 58,000 daltons was obtained. For adipocyte microsomal membranes, we obtained target sizes of approximately 112,000 and 109,000 daltons prior to and after insulin treatment, respectively. In the case of microsomal membranes, however, inactivation data showed anomalously low radiation sensitivities at low radiation doses, which may be interpreted as indicating the presence of a radiation-sensitive inhibitor. These results suggest that the adipocyte glucose transporter occurs as a monomer in the plasma membrane while existing in the intracellular reserve pool either as a homodimer or as a stoichiometric complex with a protein of an approximately equal size.     

Susceptibility of neuroactive peptides to aminopeptidase digestion is related to molecular size.

Peptide fragments derived from the NH₂-terminus of corticotropin were found to exhibit widely differing degrees of stability to degradation by aminopeptidase M. Corticotropin itself was 135 times more stable than its NH₂-terminal pentapeptide, and similar differences in stability were observed with peptides derived from the B-chain of bovine insulin. Enkephalin linked covalently to the A-chain of bovine insulin was at least 100 times more stable than the pentapeptide. The results demonstrate that the molecular size of a peptide is one factor that determines its NH₂-terminal stability.