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.     

Partial purification and characterization of a factor which stimulates an increase in ornithine decarboxylase activity in the liver from tumor cell-free ascites fluid

A factor responsible for stimulating an increase in ornithine decarboxylase activity in the liver of mice was found in tumor cell-free ascites fluid of mice 3 days after inoculation of tumor cells. The factor was purified about 70-fold in 25% yield from tumor cell-free ascites fluid. As little as 1 μg of protein of purified fraction, injected intraperitoneally into normal mice, significantly increased the activity of ornithine decarboxylase in the liver. The most active preparation of the factor formed two major protein bands on analytical polyacrylamide gel electrophoresis and both these bands stained with periodic acid-Schiff's reagent. The factor was a heat-labile, alkaline-stable, acidic protein with a molecular weight of more than 300 000. It was inactivated by treatment with 10 mM dithiothreitol, 5M urea, pronase or mixed glycosidase, but was stable on treatment with DNAase, RNAase or neuraminidase.     

Effect of insulin on the glucose utilization in isolated cardiac myocytes from adult rat

The acute effects of insulin on glucose utilization in isolated rat quiescent cardiac myocytes were studied. Insulin (80 nM) increased the rate of glucose clearance by 2-3 times in the presence of glucose ranging from 0.3 microM to 5.5 mM. Glucose transport, which was measured in terms of both D-glucose uptake in the presence of 0.3 microM D-glucose and initial rate of uptake of 3-O-methylglucose, was stimulated 3-fold in the presence of insulin. At higher glucose concentrations (greater than 100 microM), a decrease in glucose clearance rate due to a shift of the rate-limiting step from glucose transport to a post-transport step in the pathway of glucose metabolism was observed. At the physiological concentration of glucose (5.5 mM), about 73% of glucose was metabolized into lactate, about 10% was oxidized into CO2 and the rest (17%) remained inside the cells. The pentose phosphate pathway did not contribute to the glucose metabolism in these cells. Insulin (80 nM) significantly increased the uptake of glucose (112%), and the conversions of glucose into lactate (16%), glycogen (64%), and triglyceride (18%), but not into CO2 (3%). Insulin transiently increased the percentage of I-form of glycogen synthase by 16% above basal, but did not affect the percentage of a-form of glycogen phosphorylase. The content of glucose 6-phosphate in the cells was increased by 46% above the basal value in the presence of insulin. These results indicate that insulin has different acute stimulatory effects on various steps in the metabolic pathway of glucose in isolated quiescent cardiac myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

The structure ofHLA-B35 suggests that it is derived fromHLA-Bw58 by two genetic mechanisms

The primary structure ofHLA-B51 andHLA-Bw52 suggested thatHLA-B51 was derived fromHLA-Bw52 by the combination of a genetic exchange withHLA-B8 and a point mutation. To investigate the evolution of theHLA-B5 cross reactive group, theHLA-B35 gene was cloned and the primary structure was determined.HLA-B35 is identical toHLA-Bw58 except in the α₁ domain. The α₁ domain ofHLA-B35 except Bw4/Bw6-associated amino acids is identical to that ofHLA-B51 ^*, which was suspected to be an intermediate gene betweenHLA-B51 andHLA-Bw52. These data suggest thatHLA-B35 has evolved fromHLA-Bw58 in two steps; an in vivo replacement of the α₁ domain withHLA-B51 and genetic exchange with one of theHLA-Bw6 genes. These three genes andHLA-Bw58 are postulated to share a common ancestor.     

Stress fibers in the mesenteric mesothelial cells of the large intestine of the bullfrog,Rana catesbeiana

Summary Actin-containing cytoplasmic fibers were visualized in the mesenteric mesothelial cells of the large intestine of bullfrog tadpoles by rhodamine-phalloidin staining of en face preparations of mesothelial cells. These fibers were concurrently stained by immunofluorescence using antibodies to myosin or -actinin. Electron microscopy showed the presence of bundles of microfilaments in the basal cytoplasm of the cells. Such fibers in the mesothelial cells may be comparable to the stress fibers present in cultured cells. The mesothelial cells initially formed axially oriented stress fibers when they changed from a rhombic to a slender spindle-like shape. On the other hand, stress fibers disappeared as cells transformed from elongated to polygonal shapes during the period of metamorphic climax. Expression of stress fibers in these cells appears to be related to the degree of tension loaded on the mesentery, which may be generated by mesenteric winding. These stress fibers in the mesothelial cells may serve to regulate cellular transformation. They may also help to maintain cellular integrity by strengthening the cellular attachment to subepithelial tissue against tensile stress exerted on the mesentery.     

Immunological identification of rat urinary inactive kallikrein as a proform of tissue kallikrein

Antibody was raised against a synthetic undecapeptide (PS 11) which corresponds to the prosegment of the rat tissue kallikrein precursor. The potential to recognize rat urinary active or inactive kallikrein was assessed by an enzyme immunoassay method for PS 11, using beta-D-galactosidase as the labeling enzyme. The active kallikrein failed to compete with the enzyme-labeled PS 11 in binding to the antibody. The inactive kallikrein displaced the enzyme-labeled PS 11 in this enzyme immunoassay, and the displacement curve was in parallel with that of PS 11. These results indicate that rat urinary inactive kallikrein contains a prosequence recognized by the antibody to PS 11. This inactive kallikrein is probably a proform of tissue kallikrein.     

Comparative Study of Glial Marker Proteins in the Hypoplastic Cerebellum of Jaundiced Gunn Rats

The behavior of marker proteins of glial cells [alpha-enolase, beta-S100 protein, and glial fibrillary acidic protein (GFAP)] was investigated quantitatively by using enzyme immunoassay systems during the development of cerebellar hypoplasia in jaundiced Gunn rats. A neuronal marker protein, gamma-enolase, was also measured as a reference. At postnatal day 8 corresponding to the early stage of cerebellar damage, the amount of beta-S100 on a protein basis was significantly higher in jaundiced homozygotes (jj) than in control nonjaundiced heterozygotes (j+), whereas no differences in alpha- and gamma-enolases and GFAP were observed between the two groups of rats. At days 15 and 30, which correspond, respectively, to the advanced and late stages of cerebellar damage, the three glial proteins, especially GFAP, were higher and the neuronal protein was lower in the jj rat cerebellum than in the control. These results are consistent with the reported histological observations that neuronal cells are vulnerable and damaged by bilirubin, whereas glial cells seem to be less sensitive. On the other hand, the amounts of beta-S100 and alpha-enolase per cerebellum were significantly lower in jj rats at days 15 and 30, as in the case of gamma-enolase, whereas that of GFAP remained at the same level as the control at day 15 and showed a slight but significant decrease at day 30. The possibility is suggested that beta-S100 and GFAP may be available as biochemical indicators of glial cells, especially in the early and advanced stages of cerebellar damage, respectively, but that alpha-enolase is less available.     

ent-Kaurene Synthesis and Endogenous Levels of Gibberellins in a Shoot Forming Tobacco Crown Gall Tissue

The in vitro ent-Mcaurene synthesizing capacity, as well asthe endogenous GA content of shoot-forming tobacco crown gallsinduced by a nopaline-type Ti plasmid, was studied. For determinationof the ent-kaurene synthesizing capacity, an HPLC procedurepreceded by sample clean-up was used and the GA content wasexamined by GC-SIM. Kaurene synthesis reached a maximum at thebeginning of the logarithmic phase of growth. There was a clearcorrelation between the ent-kaurene synthesizing capacity andthe content of C₂₀-GAs. It seems that gibberellin synthesisis related to growth and development of the tissue. The natureof the GAs identified suggests, that the GA metabolism mightbe an unusual one. (Received October 12, 1987; Accepted April 11, 1988)     

Levels of IAA, Cytokinins, ABA and Ethylene in Rice Plants as Affected by a Gibberellin Biosynthesis Inhibitor, Uniconazole-P.

Effects of uniconazole-P, a triazole-type growth retardant,on endogenous levels of IAA, cytokinins, ABA and ethylene inrice seedlings were investigated. Endogenous levels of IAA andABA were similar between control and uniconazole-P-treated riceshoots. Evolution of ethylene was promoted slightly, being 1.8times greater under 0.3 ppm uniconazole-P treatment than thatof control. The most obvious effect was the increase of trans-Zand trans-RZ in shoots. Shoots treated with uniconazole-P (10mg/m² nursery box) contained 3.4 times and 3 times more trans-Zand trans-RZ than control, respectively. No significant differencesof cytokinin levels were recognized in roots except for cis-RZ.The increase of ethylene and active forms of cytokinins, andthe decrease of gibberellin in the shoots may be the basis forphysiological phenomena caused by uniconazole-P, namely thepromotion of flowering in woody plants and the enhancement offemaleness in cucumber. (Received September 9, 1987; Accepted October 20, 1987)