Effects of injecting exogenous lipid transfer protein into rats

Rats were injected intravenously with preparations of partially purified lipid transfer protein isolated from human plasma. Cholesteryl ester transfer activity disappeared from the plasma of recipient rats with a t1/2 of about 10 h and after 24 h had fallen to a level comparable to that in human plasma. By contrast there was no measurable cholesteryl ester transfer activity in the plasma of control rats. Plasma collected from rats 24 h after the injection was subjected to ultracentrifugation at 1.225 g/ml; lipoproteins in the 1.225 g/ml supernatant were subsequently separated by both gel filtration chromatography and gradient gel electrophoresis. The major change in the treated animals was a total loss of the large, cholesteryl ester-rich, apolipoprotein E-rich high-density lipoproteins, HDL1, which are prominent in the plasma of control rats. This loss of HDL1 unmasked an obvious peak of low-density lipoproteins that had been obscured in the control rats. Other changes in the treated rats included an increase in the relative cholesteryl ester content of very-low-density lipoproteins and the emergence of a peak of triacylglycerol in the high-density lipoproteins.     

In vitro synthesis of polyribonucleotides

Polyribonucleotide: orthophosphate nucleotidyl transferase, commonly known as polynucleotide phosphorylase catalyzes the reversible polymerization of ribonucleoside diphosphates with the liberation of orthophosphate. The equilibrium constant is approximately 1.0. Although isolated from a variety of sources, the enzyme occurs essentially as two types: one which does not require a primer for reaction initiation and a second which does. A parallel study of an E. coli preparation representing the first type and an M. lysodeikticus preparation representing the second showed differences other than the primer requirement. Rates of polymerization were different as were the K_ms. The E. coli preparation catalyzed the synthesis of polyguanylic acid while the M. lysodeikticus preparation did not although synthesis of hetoropolymers containing guanylic acid was catalyzed by the M. lysodeikticus enzyme. Use of repurified commercial substrates made the validity of some primer-requirement experiments suspect. End group analysis of product polymers served only to raise questions concerning the reaction-initiating compounds and the reaction mechanism. A study of hetero-polymer synthesis showed not only that the rate of polymerization was different, from that of homopolymers but that uncompetitive inhibition rather than competitive inhibition occurred when two ribonucleoside diphosphates were present in the reaction mixture. Furthermore, the experiments showed a preferential uptake of one substrate over another and an “enrichment” which was constant. It has also been shown that RNA polymerase, a DNA-RNA directed polymerase, can be used to synthesize polyribonucleotides if the appropriate template is provided.     

Effect of synthetic polyribonucleotides on the immunological and colony-forming activity of irradiated bone marrow cells

The experimental data are presented concerning the effect of polyribonucleotides on the immunologic and colony forming ability of bone marrow or irradiated mice. All the compounds under study exhibited a pronounced, but to a different degree, colony-forming and immunostimulating action. The comparative study of the influence of polyribonucleotides on the number of endogenous colonies and antibody-forming cells showed an inverse relationship between these parameters: The preparations exerting the most pronounced immunostimulating effect had an insignificant colony-forming action and vice versa. This is evidently indicative of the capacity of these preparations to turn the differentiation of haemopoietic stem cells towards the immunopoiesis.     

Trichinella spiralis: effect of synthetic double-stranded polyribonucleotides

Administration of double-stranded synthetic polynucleotide, polyribosinic-polyribocytidylic acid [poly(I · C)] to animals sensitized once to Trichinella spiralis antigen + complete adjuvant (Difco's H37 Ra) did not affect induction or transfer of delayed hypersensitivity. Poly (I · C) did not enhance antibody production in animals sensitized only once when tested by passive cutaneous anaphylaxis (PCA). However, it appeared to enhance antibody production in hypersensitized animals. Several of the undiluted serum samples from hypersensitized animals treated with 3 mg of poly(I · C) were anaphylactogenic and inhibitory when tested by PCA, but not when serum samples were diluted 1:80. Possible reasons for these phenomena are discussed.     

Arginine-rich intracellular delivery peptides noncovalently transport protein into living cells

Plasma membranes of plant or animal cells are generally impermeable to peptides or proteins. Many basic peptides have previously been investigated and covalently cross-linked with cargoes for cellular internalization. In the current study, we demonstrate that arginine-rich intracellular delivery (AID) peptides are able to deliver fluorescent proteins or beta-galactosidase enzyme into animal and plant cells, as well as animal tissue. Cellular internalization and transdermal delivery of protein could be mediated by effective and nontoxic AID peptides in a neither fusion protein nor conjugation fashion. Therefore, noncovalent AID peptides may provide a useful strategy to have active proteins function in living cells and tissues in vivo.     

A procedure to introduce protein molecules into living mammalian cells

Although several methods are now available by which to introduce macromolecules into cultured living mammalian cells, each has limitations on its adoption as a general means, for a variety of purposes. We describe here a simple procedure to introduce protein molecules into various living mammalian cells. This procedure is based upon the finding that mammalian cells, after exposure to a low concentration of a phospholipid (L-alpha-lysophosphatidylcholine) in the presence of high (hypertonic) concentrations of glycerol became permeable to protein molecules and that a significant portion of the exposed cells regain their viability following incubation in the appropriate growth medium. We have demonstrated that diphtheria toxin (A fragment), horseradish peroxidase and antibodies against SV40 T-antigens are incorporated into living mouse erythroleukemia (Friend) cells, baby hamster kidney (BHK) cells and mouse fibroblasts (C3H), respectively. The volume introduced into a single cell (mouse Friend cells) is approx. 3 X 10(-15) liter, which is comparable to those with other systems. Parameters affecting permeability to protein molecules and viability of the treated cells were also investigated with these and other cell lines.     

Interaction of ethidium bromide with synthetic double-stranded polyribonucleotides

The interaction of ethidium bromide (EtBr) with double helical synthetic polyribonucleotides poly(G).poly(C), poly(A).poly(U) and poly(I).poly(C) has been investigated by the method of isothermal microcalorimetry and according to the character of changes on the spectra of circular dichroism, absorption and fluorescence at binding. The calculations showed that at binding of EtBr with poly(A).poly(U) the saturation stechiometry was one EtBr molecule per 2 base pairs with binding constant (2.5 +/- 0.5).10(6) M-1 at 30 degrees C and 0.1 M. NaCl. In the case of binding of EtBr with poly(G).poly(C) and poly(I).poly(C) the saturation stechiometry was not less than 0.2 EtBr molecule per 1 base pair with binding constant (4 +/- 1).10(3) M-1 and (1.5 +/- 0.3).10(4) M-1 respectively, at 25 degrees C and 0.1 M NaCl. The binding enthalpies of EtBr with poly(A).poly(U) and poly(G).poly(C) have been determined to be (-7.5 +/- 0.5) Kcal per 1 mol of bound EtBr in average for both polymers. It has been shown that the observed strong selectivity of EtBr binding with polyribonucleotides is of entropic origin.     

Dephosphorylation of tau protein by calcineurin triturated into neural living cells

Alzheimer disease and related dementia are characterized by the presence of hyperphosphorylated tau aggregated into filaments. The role of tau phosphorylation in the fibrillogenesis has not yet been unraveled. Therefore, it is important to know which phosphatases can dephosphorylate tau protein in vivo. The effect of recombinant purified calcineurin (CN(PP2B)) and several calcineurin mutants on tau phosphorylation was studied in two neuronal like cell lines PC12 and SH-SY5Y. The modulation of tau phosphorylation at Ser199/Ser202, Ser396/Ser404, Ser262/Ser356, and Thr181 sites was examined in these cell lines using the phosphorylation state-dependent antitau antibodies Tau 1, PHF1, 12E8, and AT270. The results have shown that CN directly dephosphorylates all of those sites of tau protein. Recombinant calcineurin introduced into cells that have previously been treated with okadaic acid and cyclosporin A, which are inhibitors of phosphatases (PP1/PP2A and PP2B), has a direct effect on the phosphorylation status on all phosphorylation sites studied. We conclude that calcineurin is (besides PP2A) a important modulator of tau phosphorylation in vivo.     

Delivery of antibody-captured proteins into living cells using PTD-fused protein A

Protein transduction domain (PTD)-mediated protein delivery into animal cells is a useful technique for regulating cellular functions. Proteins captured by antibodies were delivered into living cells using an antibody/PTD-fused protein A complex. As a model protein, fluorescent-modified antibodies, captured by their respective primary antibody, were analyzed by fluorescence-activated cell sorting (FACS) which showed that the fluorescent-modified antibodies were directly delivered into cells. Peroxidase, captured by its specific antibody, was also delivered into cells and retained its activity.     

The synthesis of polyribonucleotides by cytoplasmic enzymes

1. The possibility that the cell cytoplasm contains enzymes catalysing the biosynthesis of RNA was investigated in fractions obtained by differential centrifugation of homogenates of Landschutz ascites-tumour cells. 2. The microsomal fraction was shown to be most active in incorporating UMP residues from [alpha-(32)P]UTP into polyribonucleotide material. 3. The same fraction also incorporated [(3)H]CTP, [(3)H]ATP and [(3)H]GTP separately and independently of the presence of complementary ribonucleoside 5'-triphosphates. 4. The reaction was promoted by the addition of RNA and showed an absolute requirement for Mg(2+) ions. 5. Analysis of alkaline hydrolysates of the reaction products after the incorporation of [alpha-(32)P]UTP showed that most of the radioactivity was recovered in (2',3')-UMP residues irrespective of whether CTP, ATP and GTP were present in the reaction mixture. 6. Extraction of RNA from the reaction mixtures after the incorporation of [(3)H]ATP, [(3)H]GTP or [(3)H]CTP and analysis by sucrosedensity-gradient centrifugation showed no labelling of the ribosomal RNA. Radioactive material appeared between the 4s region and the meniscus of the sucrose gradient. In agreement with this observation, determinations of the chain length of the product showed that only short sequences of polynucleotides were synthesized. It is concluded that only homopolyribonucleotide synthesis is catalysed by the microsomal fractions and that there is little or no synthesis of RNA-like heteropolymers.     

Microinjection of gelsolin into living cells

Gelsolins are actin-binding proteins that cap, nucleate, and sever actin filaments. Microinjection of cytoplasmic or plasma gelsolin into living fibroblasts and macrophages did not affect the shape, actin distribution, deformability, or ruffling activity of the cells. Gelsolin requires calcium for activity, but the NH2-terminal half is active without calcium. Microinjection of this proteolytic fragment had marked effects: the cells rounded up, stopped ruffling, became soft, and stress fibers disappeared. These changes are similar to those seen with cytochalasin, which also caps barbed ends of actin filaments. Attempts to raise the cytoplasmic calcium concentration and thereby activate the injected gelsolin were unsuccessful, but the increases in calcium concentration were minimal or transient and may not have been sufficient. Our interpretation of these results is that at the low calcium concentrations normally found in cells, gelsolin does not express the activities observed in vitro at higher calcium concentrations. We presume that gelsolin may be active at certain times or places if the calcium concentration is elevated to a sufficient level, but we cannot exclude the existence of another molecule that inhibits gelsolin. Microinjection of a 1:1 gelsolin/actin complex had no effect on the cells. This complex is stable in the absence of calcium and has capping activity but no severing and less nucleation activity as compared with either gelsolin in calcium or the NH2-terminal fragment. The NH2-terminal fragment-actin complex also has capping and nucleating activity but no severing activity. On microinjection it had the same effects as the fragment alone. The basis for the difference between the two complexes is unknown. The native molecular weight of rabbit plasma gelsolin is 82,500, and the extinction coefficient at 280 nm is 1.68 cm2/mg. A new simple procedure for purification of plasma gelsolin is described.     

Effects of retinoic acid on protein synthesis in cultured melanoma cells

Retinoic acid reduces the growth rate of mouse S91 melanoma cells in culture and increases the proportion of cells in the G₁ phase of the cell cycle. Because of the integral role protein synthesis has been shown to play in growth control we studied the effect of retinoic acid on the protein synthesis machinery with a cell-free system developed from the melanoma cells. This system was capable of translating endogenous mRNA, exogenous globin mRNA, and the synthetic template poly(U). Of the above activities of the protein synthesis system only the translation of endogenous mRNA was reduced significantly in the cell-free system prepared from retinoic acid-treated cells. Analyses of the amount and function of RNA revealed that treatment with retinoic acid leads to reductions in total RNA content, in the proportion of ribosomes in polysomes, in the amount of poly(A)RNA, and in the amount of polysome-associated mRNA. All these effects of retinoic acid contribute to the decrease in protein synthesis activity of treated cells. Two-dimensional electrophoresis anlaysis of L-[³⁵S]methionine-labeled proteins produced by untreated and treated cells revealed only a few quantitative differences. We suggest that retinoic acid-induced suppression of protein synthesis activity may be the cause for growth inhibition.