Mycoplasma fermentans glycolipid-antigen as a pathogen of rheumatoid arthritis

Mycoplasma fermentans has been suspected as one of the causative pathogenic microorganisms of rheumatoid arthritis (RA) however, the pathogenic mechanism is still unclear. We, previously, reported that glycolipid-antigens (GGPL-I and III) are the major antigens of M. fermentans. Monoclonal antibody against the GGPL-III could detect the existence of the GGPL-III antigens in synovial tissues from RA patients. GGPL-III antigens were detected in 38.1% (32/84) of RA patient’s tissues, but not in osteoarthritis (OA) and normal synovial tissues. Immunoelectron microscopy revealed that a part of GGPL-III antigens are located at endoplasmic reticulum. GGPL-III significantly induced TNF-α and IL-6 production from peripheral blood mononulear cells, and also proliferation of synovial fibroblasts. Further study is necessary to prove that M. fermentans is a causative microorganism of RA; however, the new mechanisms of disease pathogenesis provides hope for the development of effective and safe immunotherapeutic strategies based on the lipid-antigen, GGPL-III, in the near future.     

Immunohistochemical Localization of Arginase II and Other Enzymes of Arginine Metabolism in Rat Kidney and Liver

Arginine is a precursor for the synthesis of urea, polyamines, creatine phosphate, nitric oxide and proteins. It is synthesized from ornithine by argininosuccinate synthetase and argininosuccinate lyase and is degraded by arginase, which consists of a liver-type (arginase I) and a non-hepatic type (arginase II). Recently, cDNAs for human and rat arginase II have been isolated. In this study, immunocytochemical analysis showed that human arginase II expressed in COS-7 cells was localized in the mitochondria. Arginase II mRNA was abundant in the rat small intestine and kidney. In the kidney, argininosuccinate synthetase and lyase were immunostained in the cortex, intensely in proximal tubules and much less intensely in distal tubules. In contrast, arginase II was stained intensely in the outer stripes of the outer medulla, presumably in the proximal straight tubules, and in a subpopulation of the proximal tubules in the cortex. Immunostaining of serial sections of the kidney showed that argininosuccinate synthetase and arginase II were collocalized in a subpopulation of proximal tubules in the cortex, whereas only the synthetase, but not arginase II, was present in another subpopulation of proximal tubules. In the liver, all the enzymes of the urea cycle, i.e. carbamylphosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase and lyase and arginase I, showed similar zonation patterns with staining more intense in periportal hepatocytes than in pericentral hepatocytes, although zonation of ornithine transcarbamylase was much less prominent. The implications of these results are discussed.     

Heterogeneous profile in thermal denaturation of DNA preparation from the non-sulfur purple photosynthetic bacterium Rhodopseudomonas spheroides

Thermal denaturation profiles of DNA preparations from fourstrains of Rhodopseudomonas spheroides were comparatively studied.All the melting curves in 0.1 ? SSC displayed Tm at 82 ?0.5?and a faint bimodal transition near 78?. The differential meltingrates as a function of temperature exhibited several peaks,suggesting that inter- or intra-molecular structural heterogeneitymay be present in R. spheroides DNA. ¹ Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai, Japan. (Received February 15, 1975; )     

Evaluation of the bitterness of green tea catechins by a cell-based assay with the human bitter taste receptor hTAS2R39

Catechins have a broad range of physiological functions and act as the main taste ingredient of green tea. Although catechins show a strong bitterness, the bitter taste receptor for catechins has not been fully understood. The objective of this study was to identify the receptor for the major green tea catechins such as (−)-epicatechin (EC), (−)-epicatechin gallate (ECg), (−)-epigallocatechin (EGC), and (−)-epigallocatechin gallate (EGCg). By the cell-based assay using cultured cells expressing human bitter taste receptor, a clear response of hTAS2R39-expressing cells was observed to 300 μM of either ECg or EGCg, which elicit a strong bitterness in humans. The response of hTAS2R39-expressing cells to ECg was the strongest among the tested catechins, followed by EGCg. Because the cellular response to EC and EGC is much weaker than those of ECg and EGCg, galloyl groups was strongly supposed to be involved in the bitter intensity. This finding is similar to the observations of taste intensity obtained from a human sensory study. Our results suggest the participation of hTAS2R39 in the detection of catechins in humans, indicating the possibility that bitterness of tea catechins can be evaluated by using cells expressing hTAS2R39.     

The relationship between the metabolism of sphingomyelin species and the hemolysis of sheep erythrocytes induced by Clostridium perfringens alpha-toxin

Clostridium perfringens alpha-toxin induces the hemolysis of sheep erythrocytes by activating the metabolism of sphingomyelin (SM) via a GTP binding protein in membranes. alpha-Toxin stimulated the formation of 15-N-nervonoyl sphingosine (C24:1-ceramide), which was identified by positive ion fast atom bombardment-MS and 1H-NMR spectroscopy. C24:1-ceramide stimulated the toxin-induced hemolysis of saponin-pretreated sheep erythrocytes and increased the production of sphingosine 1-phosphate (S1P) in the cells, but N-lignoceroyl sphingosine did not. These events elicited by the toxin in the presence of C24:1-ceramide were significantly attenuated by treatment with dihydrosphingosine, a sphingosine kinase inhibitor. TLC showed that the level of C24:1-ceramide was highest among the ceramides with an unsaturated bond in the fatty acyl chain in the detergent-resistant membranes (DRMs). The toxin specifically bound to DRMs rich in cholesterol, resulting in the hydrolysis of N-nervonoic sphingomyelin (C24:1-SM) in DRMs. Treatment of the cells with pertussis toxin (PT) inhibited the alpha-toxin-induced formation of C24:1-ceramide from C24:1-SM in DRMs and hemolysis, indicating that endogenous sphingomyelinase, which hydrolyzes C24:1-SM to C24:1-ceramide, is controlled by PT-sensitive GTP binding protein in membranes. These results show that the toxin-induced metabolism of C24:1-SM to S1P in DRMs plays an important role in the toxin-induced hemolysis of sheep erythrocytes.     

Stimulation of the secretion of pro-inflammatory cytokines by Bifidobacterium strains

To characterize the ability of bifidobacteria to affect the production of macrophage-derived cytokines, a murine macrophage-like cell line, J774.1, was cultured in the presence of 27 strains of heat-inactivated bifidobacteria. Bifidobacterium adolescentis and B. longum, known as adult-type bifidobacteria, induced significantly more pro-inflammatory cytokine secretion, IL-12 and TNF-alpha, by J774.1 cells, than did the infant-type bifidobacteria, B. bifidum, B. breve, and B. infantis (P<0.01). In contrast, B. adolescentis did not stimulate the production of anti-inflammatory IL-10 from J774.1 cells as the other tested bacteria did. The results suggest that the adult-type bifidobacteria, especially B. adolescentis, may be more potent to amplify but less able to down-regulate the inflammatory response.     

Insertion and assembly of human tom7 into the preprotein translocase complex of the outer mitochondrial membrane

Tom7 is a component of the translocase of the outer mitochondrial membrane (TOM) and assembles into a general import pore complex that translocates preproteins into mitochondria. We have identified the human Tom7 homolog and characterized its import and assembly into the mammalian TOM complex. Tom7 is imported into mitochondria in a nucleotide-independent manner and is anchored to the outer membrane with its C terminus facing the intermembrane space. Unlike studies in fungi, we found that human Tom7 assembles into an approximately 120-kDa import intermediate in HeLa cell mitochondria. To detect subunits within this complex, we employed a novel supershift analysis whereby mitochondria containing newly imported Tom7 were incubated with antibodies specific for individual TOM components prior to separation by blue native electrophoresis. We found that the 120-kDa complex contains Tom40 and lacks receptor components. This intermediate can be chased to the stable approximately 380-kDa mammalian TOM complex that additionally contains Tom22. Overexpression of Tom22 in HeLa cells results in the rapid assembly of Tom7 into the 380-kDa complex indicating that Tom22 is rate-limiting for TOM complex formation. These results indicate that the levels of Tom22 within mitochondria dictate the assembly of TOM complexes and hence may regulate its biogenesis.     

p53-induced adipose tissue inflammation is critically involved in the development of insulin resistance in heart failure

Several clinical studies have shown that insulin resistance is prevalent among patients with heart failure, but the underlying mechanisms have not been fully elucidated. Here, we report a mechanism of insulin resistance associated with heart failure that involves upregulation of p53 in adipose tissue. We found that pressure overload markedly upregulated p53 expression in adipose tissue along with an increase of adipose tissue inflammation. Chronic pressure overload accelerated lipolysis in adipose tissue. In the presence of pressure overload, inhibition of lipolysis by sympathetic denervation significantly downregulated adipose p53 expression and inflammation, thereby improving insulin resistance. Likewise, disruption of p53 activation in adipose tissue attenuated inflammation and improved insulin resistance but also ameliorated cardiac dysfunction induced by chronic pressure overload. These results indicate that chronic pressure overload upregulates adipose tissue p53 by promoting lipolysis via the sympathetic nervous system, leading to an inflammatory response of adipose tissue and insulin resistance.     

Possible physiological role of guanosine triphosphate and inositol 1,4,5-trisphosphate in Ca2+ release in macrophages stimulated with chemotactic peptide.

The release of Ca2+ induced by inositol 1,4,5-trisphosphate (InsP3) in the presence of GTP was examined by using saponin-permeabilized macrophages. The origin and the amount of mobilized Ca2+ in intact macrophages stimulated with chemotactic peptide were also examined to assess the physiological significance of GTP and InsP3 on Ca2+-releasing activities. The total amount of Ca2+ released by 20 microM-A23187 from the unstimulated intact macrophages was 1.4 nmol/4 x 10(6) cells, and the mitochondrial uncoupler did not cause an efflux of Ca2+ from the cells. The Ca2+ accumulation by the non-mitochondrial pool(s) was inhibited by the presence of GTP, and the total amount of releasable Ca2+ (1.4 nmol/4 x 10(6) cells) was comparable with that accumulated by the non-mitochondrial pool(s) in the presence of GTP at a free Ca2+ concentration of 0.14 microM. The mobilized and subsequently effluxed Ca2+ in cells stimulated with chemotactic peptide was estimated to be 0.3 nmol/4 x 10(6) cells. Much the same amounts were released by about the half-maximal dose of InsP3 from the non-mitochondrial pool(s) of saponin-treated macrophages that had accumulated Ca2+ at a free concentration of 0.14 microM in the presence of GTP. These results suggest that the Ca2+-releasing activity induced by GTP may play a role in the long-term regulation of Ca2+ content in the non-mitochondrial pool(s) of macrophages, and that released by InsP3 can explain, quantitatively, the chemotactic-peptide-induced mobilization of Ca2+.