Measurement of sulfidopeptide leukotrienes and their metabolism in human synovial fluid of patients with rheumatoid arthritis

Leukotriene (LT)C4 in the synovial fluid of patients with osteoarthritis deformans (OA) and rheumatoid arthritis (RA) was measured by radioimmunoassay (RIA) after extraction with Sep-Pak C18 cartridge. The amounts of immunoreactive LTC4 (i-LTC4) in samples from patients with OA and RA were not significantly different, being 0.198 +/- 0.018 pmol/ml (n = 11) and 0.179 +/- 0.016 pmol/ml (n = 12), respectively. After separation by high performance liquid chromatography (HPLC) and measurement by RIA, the levels of other sulfidopeptide LTs, such as LTD4 and LTE4, in synovial fluid from patients with RA were found to be significantly higher than those in fluid from patients with OA. The leukocyte number in synovial fluids did not correlate with the i-LTC4 level. The metabolic activities of these synovial fluids were determined by incubating them with 3H-LTC4 and then separating sulfidopeptide LTs by HPLC. The conversion of LTC4 to LTD4 in synovial fluids of patients with OA and RA were similar, but the dipeptidase activity converting LTD4 to LTE4 was higher in fluid from patients with RA. It is suggested that a high level of LTE4 may contribute to exudation of synovial fluid, since LTE4 increases vascular permeability.     

Heterogeneous distribution of the precursor of type I and type III collagen and fibronectin in the rough endoplasmic reticulum of palatal mesenchymal cells of the mouse embryo cultured in ascorbate-depleted medium

Summary In order to examine the intracellular distribution of precursors of type I and type III collagen and fibronectin in the palatal mesenchymal (MEPM) cells of the mouse embryo cultured under ascorbate-deficient conditions, immuno-electron-microscopic studies were carried out by use of affinity purified antibodies for these proteins. MEPM cells were obtained from the palatal shelves of 14-day-old mouse fetuses and cultured for 3–7 days in medium, either with or without 50 ng/dish/day ascorbic acid. Results obtained were as follows: (1) Although the rough endoplasmic reticulum (rER) of MEPM cells cultured for 5 days in ascorbate-supplemented medium was flattened, that in cells cultured in ascorbate-deficient medium had a distended or vesicular appearance. (2) Vesicular or distended rER showed heterogeneous staining for both type I and type III collagen, namely, some parts of rER showed positive staining for both types of collagen, while others showed negative staining. (3) Both type I and type III collagen showed codistribution in the same vesicular rER. (4) Vesicular rER showed negative or very faint labelling for fibronectin. These results may suggest regional differences in the function of rER.     

Induction of interdigitating reticulum cell-like differentiation in human monocytic leukemia cells by conditioned medium from IL-2-stimulated helper T-cells

Monocytic leukemia (MoL) cells were obtained from the peripheral blood of a patient in whom the leukemic cells infiltrating various lymphoreticular organs exhibited features intermediate between interdigitating reticulum cells (IDC) and ordinary phagocytic macrophages, whereas the leukemic cells in the peripheral blood were essentially monocytic and lacked such features. Peripheral blood CD4+ T-cells were established as an interleukin-2-dependent T-cell line. When the MoL cells were exposed for a few days to conditioned medium from the T-cell line, they extended several dendritic cytoplasmic projections and became intensely positive for HLA-DR antigen, cytoplasmic S-100β protein, and CD1 antigen. Functionally, the conditioned medium significantly down-regulated Fc-mediated and Fc-independent phagocytic activities, and the levels of lysosomal enzymes such as lysozyme and nonspecific esterase in the MoL cells. Moreover, the conditioned medium significantly up-regulated the accessory cell function of the MoL cells as measured by the primary allogenic mixed leukocyte reaction (MLR). Furthermore, the conditioned medium significantly down-regulated the expression of CD14 antigen. Biochemical analysis indicated that the factor responsible for these changes is a protein which is distinct from known human cytokines and whose molecular weight is approximately 31 kDa. These findings suggest that IDC are closely related the monocytic lineage and that helper T-cells play an important role in constructing the microenvironment of T-lymphoid tissues which is necessary for the differentiation and maturation of IDC.     

Characterization of smooth muscle caldesmon as a microtubule-associated protein.

We have previously shown that nonmuscle caldesmon copurified with brain microtubules binds to microtubules in vitro [Ishikawa et al.: FEBS Lett. 299:54-56, 1992]. To explore the role of caldesmon in the functions of microtubules, further characterization was performed using smooth muscle caldesmon, whose molecular structure and function have been best-characterized in all caldesmon species. Smooth muscle caldesmon bound to microtubules with a stoichiometry of five tubulin dimers to one molecule of caldesmon with the binding constant of 1.1 x 10(6) M-1. The binding of caldesmon to microtubules was inhibited in the presence of Ca2+ and calmodulin. Partial digestion of the caldesmon with alpha-chymotrypsin revealed that the binding site of the caldesmon for microtubules lay in the 34-kDa C-terminal domain. When the caldesmon was in the dimeric form in the absence of a reducing agent, the caldesmon cross-linked microtubules to form bundles. Further, the caldesmon potentiated the polymerization of tubulin, and inhibited the in vitro movement of microtubules on dynein. These results suggest that caldesmon may be involved in the regulation by Ca2+ of the functions of microtubules.     

Formation of glycine conjugate and (-)-(R)-enantiomer from (+)-(S)-2-phenylpropionic acid suggesting the formation of the CoA thioester intermediate of (+)-(S)-enantiomer in dogs.

It has been proposed that the chiral inversion of the 2-arylpropionic acids is due to the stereospecific formation of the (-)-R-profenyl-CoA thioesters which are putative intermediates in the inversion. Accordingly, amino acid conjugation, for which the CoA thioesters are obligate intermediates, should be restricted to those optical forms which give rise to the (-)-R-profenyl-CoA, i.e., the racemates and the (-)-(R)-isomers. We have examined this problem in dogs with respect to 2-phenylpropionic acid(2-PPA). Regardless of the optical configuration of 2-phenylpropionic acid administered, the glycine conjugate was the major urinary metabolite and this was shown to be exclusively the (+)-(S)-enantiomer by chiral HPLC. Both (-)-(R)- and (+)-(S)-2-phenylpropionic acid were present in plasma after the administration of either antipode, and further evidence of the chiral inversion of both enantiomers was provided by the presence of some 25% of the opposite enantiomer in the free 2-phenylpropionic acid and its glucuronide excreted in urine after administration of (-)-(R)- and (+)-(S)-2-phenylpropionic acid. The (+)-(S)-enantiomer underwent chiral inversion to the (-)-(R)-antipode when incubated with dog hepatocytes. These data suggests that both enantiomers of 2-phenylpropionic acid are substrates for canine hepatic acyl CoA ligase(s) and thus undergo chiral inversion, but that the CoA thioester of only (+)-(S)-2-phenylpropionic acid is a substrate for the glycine N-acyl transferase. These studies are presently being extended to the structure and species specificity of the reverse inversion and amino acid conjugation of profen NSAIDs.     

Intra- and interspecific variation in body size ofProtohermes (Megaloptera: Corydalidae)

The life history of three populations ofProtohermes grandis and two populations ofProtohermes immaculatus (Megaloptera: Corydalidae) was compared. In general, the larvae lived in stream riffles for 2 years and the adults appeared in summer. Adult body size differed between these closely related species and also between the populations ofP. grandis. Dwarfism occurred inP. immaculatus, a species that is endemic to the small, isolated island, Amami Island. The population ofP. grandis on Yaku Island, located between Amami Island and the mainland Kyushu, had an intermediate body size between that ofP. immaculatus and the mainland population ofP. grandis. Despite being an insular population,P. grandis on Tsushima Island had a similar body size to mainlandP. grandis. In these populations with large adults, some larvae lived in the streams for 3 years. The size distribution of benthic animals, which are the prey available toProtohermes larvae, differed between the streams studied. The density of large prey was lowest on Amami Island, intermediate on Yaku Island, and highest on the mainland and Tsushima Island. Different size distributions of available prey may be caused by the differences of benthic fauna; most of Ecdyonuridae and Ephemerellidae (large mayflies) and Perlidae (large stoneflies) were not found on Amami and Yaku Islands. Thus, there is a tendency to dwarfism in the populations ofProtobermes inhabiting streams where the density of large prey is low.     

Effect of dietary alpha-linolenate/linoleate balance on lipopolysaccharide-induced tumor necrosis factor production in mouse macrophages

We examined the effect of dietary alpha-linolenate (18:3n-3)/linoleate (18:2n-6) balance on lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) production in mouse macrophages. Resident and casein-induced peritoneal macrophages from mice fed a high alpha-linolenate diet produced a higher amount of TNF than in the high linoleate diet group. However, TNF production was not affected by the dietary alpha-linolenate/linoleate balance when thioglycollate- and complete Freund's adjuvant-induced macrophages were stimulated with LPS. Serum TNF levels of mice intraperitoneally injected with LPS was also higher in the high alpha-linolenate group than in the high linoleate group. These diets affected the n-3/n-6 ratios of 20 and 22 carbon highly unsaturated fatty acids in macrophage lipids. Thus, the dietary enrichment with alpha-linolenate was found to enhance TNF production of macrophages isolated under limited conditions.     

Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli β-glucuronidase gene and analysis of its expression in Aspergillus oryzae

Summary Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae R11340 was conducted using the cloned Taka-amylase A (TAA) gene as a probe. The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source. In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding -glucuronidase (GUS) downstream of the TAA promoter and introduced the resultant fusion gene into the A. oryzae genome. Production of a functional GUS protein was induced by starch, but not by glucose. When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter.