Tumor necrosis factor receptor p55 mediates induction of HIV type 1 expression in chronically infected U1 cells.

Tumor necrosis factor alpha (TNF-alpha) is a potent inducer of human immunodeficiency virus type 1 (HIV-1) expression in chronically infected cells. The aim of this study was to investigate the role played by the two known TNF-alpha receptors, TNFR-p55 and TNFR-p75, in the activation of HIV-1 expression. As a model system the latently infected human promonocytic cell line U1 was stimulated with wild-type TNF-alpha, with TNF-alpha muteins that specifically bind to one or the other receptor or with receptor-specific monoclonal antibodies. Induction of HIV-1 expression, measured by p24 core antigen capture enzyme-linked immunosorbent assay (ELISA), was found to be exclusively triggered by TNFR-p55 stimulation. However, our results also showed that the addition of TNFR-p75-specific ligands negatively modulated the HIV-1 expression induced via TNFR-p55.

Mice lacking the tumour necrosis factor receptor 1 are resistant to TNF-mediated toxicity but highly susceptible to infection by Listeria monocytogenes.

Tumour necrosis factor (TNF), jointly referring to TNF alpha and TNF beta, is a central mediator of immune and inflammatory responses; its activities are mediated by two distinct receptors, TNFR1 (p55) and TNFR2 (p75) (reviewed in refs 1-3). The cytoplasmic domains of the TNFRs are unrelated, suggesting that they link to different intracellular signalling pathways. Although most TNF responses have been assigned to one or the other of the TNF receptors (mostly TNFR1), there is no generally accepted model for the physiological role of the two receptor types. To investigate the role of TNFR1 in beneficial and detrimental activities of TNF, we generated TNFR1-deficient mice by gene targeting. We report here that mice homozygous for a disrupted Tnfr1 allele (Tnfr1(0)) are resistant to the lethal effect of low doses of lipopolysaccharide after sensitization with D-galactosamine, but remain sensitive to high doses of lipopolysaccharide. The increased susceptibility of Tnfr1(0)/Tnfr1(0) mutant mice to infection with the facultative intracellular bacterium Listeria monocytogenes indicates an essential role of TNF in nonspecific immunity.

The 55-kD tumor necrosis factor receptor on human keratinocytes is regulated by tumor necrosis factor-alpha and by ultraviolet B radiation.

In previous studies we showed that cultured human keratinocytes expressed the 55-kD TNF receptor (TNFR) and that its expression the important for TNF alpha-mediated upregulation of intercellular adhesion molecule-1 (ICAM-1) expression on keratinocytes. Because factors that either reduce or enhance TNFR expression are likely to have a major impact on the biological effects of TNF alpha on keratinocytes, these studies were conducted to determine the factors that regulate its expression on keratinocytes. Using reverse transcriptase polymerase chain reaction, human keratinocytes were shown to lack 75-kD TNFR expression, indicating that TNF responsiveness of human keratinocytes critically depended on regulation of 55-kD TNFR expression. Human keratinocyte 55-kD TNFR surface and mRNA expression was found to be regulated in vitro by recombinant human (rh) TNF alpha. Stimulation of keratinocytes with rhTNF alpha initially decreased, but later increased, 55-kD TNFR surface expression. This biphasic modulation of 55-kD TNFR surface expression was associated with concomitant changes in 55-kD TNFR mRNA expression. Ultraviolet B (UVB) radiation, a well-known inducer of synthesis and secretion of TNF alpha by human keratinocytes, was found to mimic TNF alpha-induced modulation of 55-kD TNFR surface and mRNA expression via a TNF alpha-mediated autocrine regulatory mechanism. Production of soluble 55-kD TNFR by human keratinocytes remained unaffected by TNF alpha stimulation or UVB irradiation. These studies provide clear evidence that membrane expression of the human 55-kD TNFR may be regulated in human keratinocytes by the ligand itself: TNF alpha. Since in previous studies UVB irradiation transiently inhibited TNF alpha-induced human keratinocyte ICAM-1 expression, it is proposed that UVB radiation-induced biphasic modulation of human keratinocyte 55-kD TNFR expression may affect the capacity of these cells to respond to TNF alpha.

Monoclonal antibody-coated magnetite particles as contrast agents in magnetic resonance imaging of tumors.

A highly specific and powerful magnetic resonance imaging contrast agent has been prepared by coating magnetite (Fe3O4) particles with monoclonal antibodies directed against a tumor antigen. The preparation maintains both the immunoreactivity of the monoclonal antibody and the full relaxing capability of the magnetite particle. MRI image contrast by spin-echo methods can be easily induced in a concentration range of 1-10 nM of the antibody-coated magnetite particles.

Purification of the N-acetylglucosaminide alpha(1-3/4)fucosyltransferase of human milk.

The N-acetylglucosaminide alpha(1-3/4)fucosyltransferase has been purified 1.8 x 10(6)-fold from human milk by ion-exchange chromatography, affinity chromatography on GDP-agarose and HPLC. The alpha(1-3/4)fucosyltransferase behaves in gel filtration-HPLC as a molecule of M(r) 98,000, and differs from the alpha(1-3)fucosyltransferase which behaves like a molecule of about M(r) 47,000. The enzyme is a glycoprotein, and the purified preparation appears in SDS polyacrylamide gel electrophoresis as a band of M(r) 44,000. The results present the first purification of human milk alpha(1-3/4)fucosyltransferase to apparent homogeneity, and suggest that the alpha(1-3/4)- and alpha(1-3)fucosyltransferase of human milk differ in their native molecular sizes, the former being a dimer of two subunits.

Modification of the F0 portion of the H+-translocating adenosinetriphosphatase complex of Escherichia coli by the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and effect on the proton channeling function.

1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), a water-soluble carbodiimide, inhibited ECF1-F0 ATPase activity and proton translocation through F0 when reacted with Escherichia coli membrane vesicles. The site of modification was found to be in subunit c of the F0 portion of the enzyme but did not involve Asp-61, the site labeled by the hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD). EDC was not covalently incorporated into subunit c in contrast to DCCD. Instead, EDC promoted a cross-link between the C-terminal carboxyl group (Ala-79) and a near-neighbor phosphatidylethanolamine as evidenced by fragmentation of subunit c with cyanogen bromide followed by high-pressure liquid chromatography and thin-layer chromatography.

Inhibition of the adenosinetriphosphatase activity of Escherichia coli F1 by the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide is due to modification of several carboxyls in the beta subunit.

Reaction of the ATPase of Escherichia coli (ECF1) with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) resulted in a time- and concentration-dependent inhibition of ATPase activity. The inactivation was greatly reduced by Mg2+ ions. Close to 13 mol of EDC per mol of ECF1 was incorporated into the enzyme at 95% inhibition of ATPase activity. Two-thirds of the label was found to be associated with subunit beta with a stoichiometry of about 3 mol of EDC per mol of beta. Cleavage of EDC-modified subunit beta with cyanogen bromide and fractionation of the peptides by high-pressure liquid chromatography revealed a short segment of 33 amino acids (CB8, residues 162-194) containing 3 mol of EDC per mol of peptide. In tryptic peptide maps, two EDC-labeled fragments could be identified (T18, residues 166-183, and T20, residues 186-202). The analyses were complicated by significant internal cross-linking within the beta subunit induced by EDC. The results show that EDC modifies multiple sites in a short segment of subunit beta which includes the glutamic acids modified by dicyclohexylcarbodiimide in F1 from both E. coli and PS3. In addition to covalent modification, EDC also promoted the formation of intersubunit cross-links. The predominant cross-linked product was identified as a beta-epsilon complex by antibody binding experiments.

Interconversion of high and low adenosinetriphosphatase activity forms of Escherichia coli F1 by the detergent lauryldimethylamine oxide.

The amphipathic detergent lauryldimethylamine oxide (LDAO) stimulated ATP hydrolytic activity of Escherichia coli membranes and isolated ECF1 and ECF1-F0 ATPase complexes in a concentration-dependent manner. The enzyme was maximally activated 3-fold in membranes and 5-6-fold for isolated ECF1 or the ECF1-F0 complex. The maximal specific activity of activated ECF1 was 140-160 mumol of ATP hydrolyzed min-1 mg-1. The activation by LDAO was reversible. LDAO specifically released subunit delta from ECF1, generating a four subunit enzyme (alpha, beta, gamma, and epsilon subunits). The removal of subunit delta was not responsible for the stimulation of ATPase activity as evidenced by the full activation of the four subunit enzyme by LDAO. Treatment of ECF1 with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide generated a beta-epsilon cross-link in high yield [Lötscher, H.R., DeJong, C., & Capaldi, R. A. (1984) Biochemistry (accompanying paper in this issue)]. The formation of this cross-link was greatly reduced in the presence of LDAO, indicating that the detergent perturbated the interaction between epsilon and beta subunits although epsilon was not removed from the ECF1 complex. The results suggest that the interconversion of ECF1 from a low to a high ATPase activity form by LDAO is in major part due to a release of the inhibitory action of subunit epsilon on subunit beta.

Structural asymmetry of the F1 of Escherichia coli as indicated by reaction with dicyclohexylcarbodiimide.

Dicyclohexylcarbodiimide (DCCD) inhibits the ATPase activity of F1 from Escherichia coli by covalent modification of a single glutamic acid in the beta subunit. 95% inhibition was obtained after incorporation of around 1 mole of DCCD per mole F1, i.e. 1 mole of reagent per 3 beta subunits; and up to 2 moles of DCCD per mole F1 were readily incorporated into the protein. One of the 3 beta subunits per F1 can be crosslinked to the epsilon subunit by 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide (EDC). This beta subunit (beta 1) is here shown to be shielded from reaction with DCCD, presumably by its association with epsilon and also possibly the gamma subunit. Thus the three beta subunits are not equivalent in the enzyme complex.

ADP-ribosylation in inner membrane of rat liver mitochondria.

NAD+ glycohydrolase activity is found at high levels in submitochondrial particles. It leads to the reaction products ADP-ribose, nicotinamide, and small amounts of 5'-AMP. Furthermore, submitochondrial particles catalyze the exchange reaction: [adenosine-14C]ADP-ribose + NAD+ in equilibrium [adenosine-14C]-NAD+ + ADP-ribose. When submitochondrial particles are incubated with NAD+, mono(ADP-ribosyl)ation of protein molecules migrating with an apparent molecular weight of 30,000 in sodium dodecyl sulfate/polyacrylamide gel electrophoresis is demonstrable. Inhibitor studies suggest attachment of ADP-ribose to arginine residues. ADP-ribose bound to submitochondrial particles is rapidly turning over. The release of ADP-ribose from the protein is probably enzyme catalyzed. The rapid turnover, the specificity of the modification, and the inhibition of ADP-ribosylation by ATP and nicotinamide suggest a regulatory role of mono(ADP-ribosyl)ation of a protein in the inner mitochondrial membrane.

ATP prevents both hydroperoxide-induced hydrolysis of pyridine nucleotides and release of calcium in rat liver mitochondria.

The mechanism of the hydroperoxide-induced release of calcium from rat liver mitochondria was investigated. In calcium-loaded mitochondria both the hydroperoxide-induced hydrolysis of pyridine nucleotides and the release of calcium is inhibited by intramitochondrial ATP. In submitochondrial particles, in the absence of ATP a hydrolytic product of NAD+, probably ADP-ribose, binds covalently to a protein of the inner mitochondrial membrane. It is proposed that a compound deriving from NAD(P)+ is an important factor in the calcium release mechanism in rat liver mitochondria.

Hydroperoxide-induced loss of pyridine nucleotides and release of calcium from rat liver mitochondria.

It has been previously reported (Lötscher, H. R., Winterhalter, K. H., Carafoli, E., and Richter, C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4340-4344) that in Ca2+-loaded mitochondria hydroperoxides induce a release of Ca2+ from mitochondria and an irreversible oxidation of mitochondrial pyridine nucleotides. Here we show that in the presence of Ca2+ oxidized mitochondrial pyridine nucleotides are hydrolyzed inside mitochondria and that nicotinamide is released from mitochondria. The extent of the hydrolysis of NAD(P)+ is dependent on the amount of both hydroperoxide and Ca2+. The hydrolysis is reversible in the presence of added nicotinamide. The release of Ca2+ from mitochondria is electroneutral, and is directly or indirectly dependent on oxidized mitochondrial pyridine nucleotides. By contrast, the uptake of Ca2+ most probably does not require the present of reduced pyridine nucleotides. Control experiments show that even under the most drastic conditions employed in this study (100 nmol of Ca2+ and 85 nmol of t-butylhydroperoxide/mg of protein) mitochondria retain a considerable degree of functional integrity.

The energy-state of mitochondria during the transport of Ca2+.

The fluctuations of the membrane potential during mitochondrial Ca2+ transport have been monitored with an electrode sensitive to tetraphenylphosphonium. The following conclusions have been reached. 1. The membrane becomes depolarized during the influx of Ca2+. When the bulk of the Ca2+ pulse has been taken up, it repolarizes, but not completely. 2. If all of the accumulated Ca2+ is released from mitochondria and cycling is inhibited, the membrane repolarizes completely. 3. The accumulation of Ca2+ alone does not induce mitochondrial damage. In the presence of inorganic phosphate, the uptake of Ca2+ may lead to complete and irreversible depolarization, depending on the amount of Ca2+ and phosphate accumulated. The irreversible damage observed in the presence of phosphate is prevented by Mg2+.

Hydroperoxides can modulate the redox state of pyridine nucleotides and the calcium balance in rat liver mitochondria.

When rats are fed a selenium-deficient diet, the glutathione peroxidase activity in liver mitochondria decreases within 5 weeks to 0-6% of that of control animals fed on a diet supplemented with 0.5 ppm of selenium as sodium selenite. Analysis of the temperature dependence of energy-linked Ca(2+) uptake by means of Arrhenius plots reveals two breaks (at around 11 degrees C and 24 degrees C) in mitochondria isolated from selenium-supplemented animals, whereas in selenium-deficient rats the break at 11 degrees C is absent. Ca(2+)-loaded mitochondria of selenium-supplemented rats-i.e., with active glutathione peroxidase in the matrix-lose Ca(2+) rapidly, with a concomitant oxidation of endogenous NAD(P)H, when exposed to t-butyl hydroperoxide or H(2)O(2). In contrast, in selenium deficiency, t-butyl hydroperoxide and H(2)O(2) induce neither a release of Ca(2+) nor an oxidation of NAD(P)H. The peroxide-induced oxidation of NAD(P)H is reversible in the presence of succinate when no Ca(2+) has been taken up. When Ca(2+) has previously been accumulated, however, the oxidation of NAD(P)H is irreversible. Enzymatic analysis of mitochondrial pyridine nucleotides reveals that the peroxide-induced oxidation of NAD(P)H in Ca(2+)-loaded mitochondria leads to a loss of NAD(+) and NADP(+). It is proposed that the redox state of mitochondrial pyridine nucleotides can be or is in part controlled by glutathione peroxidase and glutathione reductase and is a factor in the balance of Ca(2+) between mitochondria and medium.