Biosynthesis of a disialylated sequence in N-linked oligosaccharides: identification of an N-acetylglucosaminide (alpha 2----6)-sialyltransferase in Golgi apparatus from rat liver

Rat liver Golgi apparatus are shown to have a CMP-N-acetylneuraminate: N-acetylglucosaminide (alpha 2----6)-sialyltransferase which catalyzes the conversion of the human milk oligosaccharide LS-tetrasaccharide-a (NeuAc alpha 2----3Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1----4Glc) to disialyllacto -N- tetraose containing the terminal sequence: (formula: see text) found in N-linked oligosaccharides of glycoproteins. The N-acetylglucosaminide (alpha 2----6)-sialyltransferase has a marked preference for the sequence NeuAc alpha 2----3-Gal beta 1---- 3GlcNAc as an acceptor substrate. Thus, the order of addition of the two sialic acids in the disialylated structure shown above is proposed to be first the terminal sialic acid in the NeuAc alpha 2----3Gal linkage followed by the internal sialic acid in the NeuAc alpha 2---- 6GlcNAc linkage. Sialylation in vitro of the type 1 branches (Gal beta 1---- 3GlcNAc -) of the N-linked oligosaccharides of asialo prothrombin to produce the same disialylated sequence is also demonstrated.     

Hybrid recombinant human leukocyte interferon inhibits differentiation in murine B-16 melanoma cells

We have investigated the effects of recombinant human leukocyte interferons (IFN-alpha A and IFN-alpha D) and various hybrid recombinant human leukocyte interferons on differentiation in B-16 mouse melanoma cells. Inhibition of both spontaneous and melanocyte hormone stimulated differentiation was observed with one hybrid construct, IFN-alpha A/D (Bgl) consisting of amino acids 1 to 62 from IFN-alpha A and amino acids 64 to 166 from IFN-alpha D. In contrast, the parental human interferons, IFN-alpha A and IFN-alpha D, when used alone or in combination, as well as other hybrid human leukocyte interferons, did not cause significant inhibition of melanogenesis in B-16 mouse cells. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also inhibited B-16 differentiation and the combination of TPA with IFN-alpha A/D (Bgl) or mouse L-cell interferon was synergistic in delaying melanogenesis. These studies indicate that the IFN-alpha A/D (Bgl) hybrid that exhibits antiviral activity on mouse cells can also inhibit differentiation of murine cells.     

Biosynthesis of Protoheme and Heme a Precursors Solely from Glutamate in the Unicellular Red Alga Cyanidium caldarium

Two biosynthetic routes to the heme, chlorophyll, and phycobilin precursor, δ-aminolevulinic acid (ALA) are known: conversion of the intact five-carbon skeleton of glutamate, and ALA synthase-catalyzed condensation of glycine plus succinyl-coenzyme A. The existence and physiological roles of the two pathways in Cyanidium caldarium were assessed in vivo by determining the relative abilities of [2-¹⁴C]glycine and [1-¹⁴C]glutamate to label protoheme and heme a. Glutamate was incorporated to a much greater extent than glycine into both protoheme and heme a, even in cells that were unable to form chlorophyll and phycobilins. The small incorporation of glycine could be accounted for by transfer of label to intracellular glutamate pools, as determined from amino acid analysis. It thus appears that C. caldarium makes all tetrapyrroles, including mitochondrial hemes, solely from glutamate, and there is no contribution by ALA synthase in this organism.     

Fractionation of lymphocytes involved in the generation of cell-mediated cytotoxicity over insolubilized conjugated histamine columns.

Spleen cells from normal BALB/c mice were cultured in vitro with irradiated C57BL/6 stimulating cells. Five days later the T cell-mediated cytotoxic activity of the effector cells was assessed with a 51Cr-release assay that used H-2bEL-4 tumor cells as targets. Before the BALB/c responding lymphocytes were sensitized they were fractionated by passing the spleen cells over insolubilized histamine rabbit serum albumin Sepharose columns (H-RSA-S) or over rabbit serum albumin Sepharose (RSA-S) control columns. Fractionation of cells over the H-RSA-S columns depleted or significantly reduced the cytotoxic potential of the unretained cells. All cytotoxic potential was recovered when the cells that adhered to the H-RSA-S were eluted from the columns. In contrast, no effect on responsiveness was detected after the cells had been fractionated over the control column. The loss of response potential by the cells that did not adhere to H-RSA-S could not be accounted for by removal of macrophages nor by the concentration of cells with suppressor activity in the effluent. These cell fractionation studies raise the possiblity but do not prove that cytotoxic precursor cells may express amine receptors that could be responsible for their retention by insolubilized histamine columns.     

Charge clusters and the orientation of membrane proteins

Summary Although hydrophobic forces probably dominate in determining whether or not a protein will insert into a membrane, recent studies in our laboratory suggest that electrostatic forces may influence the final orientation of the inserted protein. A negatively charged hepatic receptor protein was found to respond totrans-positive membrane potentials as though electrophoresing into the bilayer. In the presence of ligand, the protein appeared to cross the membrane and expose binding sites on the opposite side. Similarly, a positively charged portion of the peptide melittin crosses a lipid membrane reversibly in response to atrans-negative potential. These findings, and others by Date and co-workers, have led us to postulate that transmembrane proteins would have hydrophobic transmembrane segments bracketed by positively charged residues on the cytoplasmic side and negatively charged residues on the extra-cytoplasmic side. In the thermodynamic sense, these asymmetrically placed charge clusters would create a compelling preference for correct orientation of the protein, given the inside-negative potential of most or all cells. This prediction is borne out by examination of the few transmembrane proteins (glycophorin, M13 coat protein, H-2K^b, HLA-A2, HLA-B7, and mouse Ig heavy chain) for which we have sufficient information on both sequence and orientation.In addition to the usual diffusion and pump potentials measurable with electrodes, the microscopic membrane potential reflects surface charge effects. Asymmetries in surface charge arising from either ionic or lipid asymmetries would be expected to enhance the bias for correct protein orientation, at least with respect to plasma membranes. We introduce a generalized form of Stern equation to assess surface charge and binding effects quantitatively. In the kinetic sense, dipole potentials within the membrane would tend to prevent positively charged residues from crossing the membrane to leave the cytoplasm. These considerations are consistent with the observed protein orientations. Finally, the electrostatic and hydrophobic factors noted here are combined in two hypothetical models of translocation, the first involving initial interaction of the presumptive transmembrane segment with the membrane; the second assuming initial interaction of a leader sequence.     

Metabolism of Radiolabeled β-Guaiacyl Ether-Linked Lignin Dimeric Compounds by Phanerochaete chrysosporium

Phanerochaete chrysosporium metabolized the radiolabeled lignin model compounds [γ-¹⁴C]guaiacylglycerol-β-guaiacyl ether and [4-methoxy-¹⁴C]veratrylglycerol-β-guaiacyl ether (VI) to ¹⁴CO₂ in stationary and in shaking cultures. ¹⁴CO₂ evolution was greater in stationary culture. ¹⁴CO₂ evolution from [γ-¹⁴C]guaiacyl-glycerol-β-guaiacyl ether and [4-methoxy-¹⁴C]veratrylglycerol-β-guaiacyl ether in stationary cultures was two- to threefold greater when 100% O₂ rather than air (21% O₂) was the gas phase above the cultures. ¹⁴CO₂ evolution from the metabolism of the substrates occurred only as the culture entered the stationary phase of growth. The presence of substrate levels of nitrogen in the medium suppressed ¹⁴CO₂ evolution from both substrates in stationary cultures. [¹⁴C]veratryl alcohol and 4-ethoxy-3-methoxybenzyl alcohol were formed as products of the metabolism of VI and 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether, respectively.     

Thin-layer chromatography with agarose gels: A quick,simple method for evaluating liposome size

Thin-layer gels can be made with agarose and used to assess within a few minutes the efficiency with which multilamellar vesicles are converted to small unilamellar ones by sonication. A fluorescent lipid marker or vesicle-encapsulated solute permits continuous monitoring of the chromatography. Advantages over agarose gel column chromatography include speed of analysis, small sample size, the possibility of running multiple samples simultaneously, and direct accessibility to fluorescence microscopy. This approach should also be useful in the study of liposome-lipoprotein interactions and in affinity chromatography of liposomes.     

Primary induction of metallothionein by dexamethasone in cultured rat hepatocytes

Dexamethasone or Zn⁺⁺ increase the rate of synthesis of the metal-binding protein metallothionein in hepatocyte cultures. Dexamethasone induction of the capacity to synthesize metallothionein is not blocked by cycloheximide. In contrast, the dexamethasone stimulated increase in Zn⁺⁺ uptake is inhibited by cycloheximide. Like Zn⁺⁺, dexamethasone is a “primary inducer” of metallothionein. The glucocorticoid induction of metallothionein in primary cultures of rat hepatocytes is not mediated through elevation of Zn⁺⁺ uptake.     

Fluorescent reagents for the labeling of glycoconjugates in solution and on cell surfaces

Rhodamine and fluoresceine containing hydrazides were synthesized and used for fluorescent labeling of glycoconjugates on cell surface or in solution. The procedure involves the oxidation of the glycoconjugates with sodium metaperiodate or galactose oxidase to form an aldehyde group which reacts with the respective hydrazides. The method was applied for the modification of cell surface sialic acid and galactose residues on thymocytes and nematodes as well as for the labeling of glycoproteins and gangliosides in solution. The many possible application of these highly fluorescent compounds in the study of cell surface events is considered.     

The phagocytosis stimulating peptide tuftsin: Further look into structure-function relationships

Summary Sixteen new analogs of the phagocytosis-stimulating peptide tuftsin have been synthesized. The biological activities of these synthetic peptides, in which either the C-terminal or both C- and N-terminals are chemically altered, were evaluated by studying their effects on the phagocytosis of heat-killed yeasts and on the reduction of the dye nitroblue tetrazolium by normal human polymorphonuclear leukocytes. The results demonstrate that the integrity of the guanidine side chain of arginine at position four of tuftsin is crucial for maximal activity. Modification, even in side chain length, of the guanidine leads to decreasing activity. Preservation of the positive charge of position four of tuftsin yields analogs possessing considerable activity. Simultaneous alterations of both C- and N-terminal results in diminishing activites. The results of this study are discussed in relation to the structural features of tuftsin. It appears that interaction between the carboxyl of Arg⁴ and the amino group of Thr¹ which would indicate a specific conformation such as a 4 1 -turn are not favored.