Demonstration of neutralizing autoantibodies against IL-1 alpha in sera from patients with rheumatoid arthritis

We have recently reported the presence of IgG which has a potent inhibitory activity against IL-1 alpha in some sera from patients with rheumatoid arthritis. The mechanism of this inhibition by IgG against IL-1 alpha is now elucidated. IgG with IL-1 alpha-inhibitory activity inhibited the binding of 125I-IL-1 alpha to receptors on rheumatoid synovial cells. In addition, preincubation of synovial cells with the inhibitory IgG did not block the binding of 125I-IL-1 alpha to receptors, suggesting a direct interaction between IgG and IL-1 alpha. To examine which region of the IgG, namely Fab or Fc region, has the inhibitory activity, the IgG was digested with papain, and Fab and Fc fragments were purified. Fab fragments, but not Fc fragments, inhibited both IL-1 alpha-induced thymocyte-proliferation and the binding of 125I-IL-1 alpha to receptors. We further demonstrated that the inhibitory IgG which was bound to protein A Sepharose could bind a significant amount of 125I-IL-1 alpha, whereas only a negligible binding of the radiolabeled ligand was detected when IgG without the inhibitory activity was used as control. Moreover, the binding of 125I-IL-1 alpha to IgG with the inhibitory activity was clearly blocked by Fab fragments of IgG having the inhibitory activity. Finally, affinity-purified IgG over an IL-alpha affinity column showed approximately 100-fold more potent inhibitory activity on IL-1 alpha-induced thymocyte proliferation compared with untreated IgG. From these results, we conclude that IgG molecules with IL-1-alpha-inhibitory activity are neutralizing autoantibodies against IL-1 alpha.     

Optical single-channel analysis of the aerolysin pore in erythrocyte membranes.

Scanning microphotolysis (Scamp), a recently developed photobleaching technique, was used to analyze the transport of two small organic anions and one inorganic cation through single pores formed in human erythrocyte membranes by the channel-forming toxin aerolysin secreted by Aeromonas species. The transport rate constants of erythrocyte ghosts carrying a single aerolysin pore were determined to be (1.83 +/- 0.43) x 10(-3) s-1 for Lucifer yellow, (0.33 +/- 0.10) x 10(-3) s-1 for carboxyfluorescein, and (8.20 +/- 2.30) x 10(-3) s-1 for Ca2+. The radius of the aerolysin pore was derived from the rate constants to be 19-23 A, taking steric hindrance and viscous drag into account. The size of the Ca2+ rate constant implies that at physiological extracellular Ca2+ concentrations (> 1 mM) the intracellular Ca2+ concentration would be elevated to the critical level of > 1 microM in much less than a second after formation of a single aerolysin pore in the plasma membrane. Thus changes in the levels of Ca2+ or other critical intracellular components may be more likely to cause cell death than osmotic imbalance.     

Role of rat thymus chromatin lipids in response to the harmful effect of gamma-radiation

In vitro incubation of thymocytes with [2-14C]acetate results in a more intensive label incorporation into chromatin lipids as compared to nuclear lipids. The deleterious effect of gamma-irradiation leads to specific changes in [2-14C]acetate incorporation into the total fraction of phosphatidylcholine-phosphatidylserine and chromatin sphingomyelin. This is paralleled with an increase in the ratio of specific radioactivities of chromatin cardiolipin and nuclear cardiolipin. The changes in specific radioactivities of sphingomyelin, phosphatidylcholine-phosphatidylserine and cardiolipin suggest a role of phospholipids in the mechanisms of regulating the activity of intranuclear structures.     

Production, crystallization, and preliminary X-ray analysis of rabbit skeletal muscle troponin complex consisting of troponin C and fragment (1-47) of troponin I.

Troponin is a ternary protein complex consisting of subunits TnC. TnI, and TnT, and plays a key role in calcium regulation of the skeletal and cardiac muscle contraction. In the present study, a partial complex (CI47) was prepared from Escherichia coli-expressed rabbit skeletal muscle TnC and fragment 1-47 of TnI, which is obtained by chemical cleavage of an E. coli-expressed mutant of rabbit skeletal muscle TnI. Within the ternary troponin complex, CI47 is thought to form a core that is resistant to proteolytic digestion, and the interaction within CI47 likely maintains the integrity of the troponin complex. Complex CI47 was crystallized in the presence of sodium citrate. The addition of trehalose improved the diffraction pattern of the crystals substantially. The crystal lattice belongs to the space group P3(1)(2)21, with unit cell dimensions a = b = 48.2 A, c = 162 A. The asymmetric unit presumably contains one CI47 complex. Soaking with p-chloromercuribenzenesulfonate (PCMBS) resulted in loss of isomorphism, but enhanced the quality of the crystals. The crystals diffracted up to 2.3 A resolution, with completeness of 91% and R(merge) = 6.4%. The crystals of PCMBS-derivative should be suitable for X-ray studies using the multiple-wavelength anomalous diffraction technique. This is the first step for elucidating the structure of the full troponin complex.     

Molecular cloning of a human serotonin receptor (S12) with a pharmacological profile resembling that of the 5-HT1D subtype.

We report the molecular cloning of a fragment of human genomic DNA called S12, containing an open reading frame of 1170 nucleotides, which encodes a receptor for serotonin of 390 amino acids. The receptor function of the S12 protein was demonstrated by functional expression in mouse LS12 cells obtained by stable transfection of Ltk- cells, and LM5S12 cells, derived from LM5 cells (Ltk- cells previously transfected with the M5 muscarinic acetylcholine receptor). Adenylyl cyclase studies showed that the S12 receptor is able to mediate inhibition of adenylyl cyclase in response to serotonin in both types of cells. As studied in LM5S12 cells, the S12 receptor did not promote Ca2+ mobilization from internal stores, nor did it significantly modulate the sustained increase in [Ca2+]i elicited by stimulation of the phospholipase C stimulating M5 acetylcholine receptor. The pharmacologic profile of S12 as seen in adenylyl cyclase assays is as follows: (EC50 in nM): serotonin, full agonist (37 nM), 5-carboxamidotryptamine, full agonist (10 nM), sumatriptan, full agonist (50 nM), metergoline, partial agonist (10 nM), methysergide, partial agonist (40 nM), yohimbine, partial agonist (150 nM), metitepin, antagonist (KB = 0.7 to 1.2 nM). We propose that the human S12 serotonin receptor is a receptor of the 5-hydroxytryptamine1D subtype.     

Intermolecular triplex formation distorts the DNA duplex in the regulatory region of human papillomavirus type-11.

A conformational distortion in the DNA duplex at the regulatory region of human papillomavirus type-11 next to an intermolecular triplex, formed with a synthetic oligonucleotide, was investigated with several chemical probes. The sequence targeted for triplex formation borders on the binding sites for the regulatory proteins encoded by the viral E2 open reading frame. Dimethyl sulfate, diethyl pyrocarbonate, and OsO4 all react to a greater extent with nucleotides in the duplex that are immediately adjacent to the triplex as compared to other bases throughout the duplex. This hypermodification was observed on both the polypurine and polypyrimidine strands of the duplex DNA. Similar hyperreactivity of bases flanking a triplex also was seen when the contiguous target polypurine tract was effectively extended by mutating interrupting pyrimidines in the human papillomavirus type-11 sequence to purines. We propose that this hyperreactivity is due to a structural distortion caused by the junction between the triplex and the duplex tracts.     

The toxicity of methanol

Methanol toxicity in humans and monkeys is characterized by a latent period of many hours followed by a metabolic acidosis and ocular toxicity. This is not observed in most lower animals. The metabolic acidosis and blindness is apparently due to formic acid accumulation in humans and monkeys, a feature not seen in lower animals. The accumulation of formate is due to a deficiency in formate metabolism which is, in turn, related, in part, to low hepatic tetrahydrofolate (H4 folate). An excellent correlation between hepatic H4 folate and formate oxidation rates has been shown within and across species. Thus, humans and monkeys possess low hepatic H4 folate levels, low rates of formate oxidation and accumulation of formate after methanol. Formate, itself, produces blindness in monkeys in the absence of metabolic acidosis. In addition to low hepatic H4 folate concentrations, monkeys and humans also have low hepatic 10-formyl H4 folate dehydrogenase levels, the enzyme which is the ultimate catalyst for conversion of formate to carbon dioxide. This review presents the basis for the role of folic acid-dependent reactions in the regulation of methanol toxicity.