Effects of several variable factors on the isotope ratio by HRGC-MS

In the isotope ratio (Ir) analysis using GC-MS, several variable factors in sampling incidental to any food analysis were investigated for yuzu fruit. The Irs of ten monoterpene hydrocarbons in yuzu essential oils from each of six fruiting positions of three trees were measured. The sign test following t-test of all the Ir values demonstrated that there was no significant difference between both sampling years of 2001 and 2002. There was also no significant variation in the Ir values among the three trees and six fruiting positions in the individual two years.     

The cytotoxicity of Bacillus thuringiensis subsp. coreanensis A1519 strain against the human leukemic T cell

A novel cytotoxic protein was isolated from the crystal produced by Bacillus thuringiensis subsp. coreanensis A1519 strain. Upon treatment of the crystal proteins by proteinase K, the significant cytotoxicity toward the leukemic T cell, MOLT-4, was exhibited. The microscopic observation indicated that the cell death was accompanied by no extensive rupture of the cell membrane. It was, therefore, suggested that the cell death of MOLT-4 was induced through a mechanism other than the colloid-osmotic swelling and cell lysis as caused by hitherto known B. thuringiensis crystal proteins. The 29-kDa polypeptide proved to be an active component of the proteinase K-digested A1519 crystal proteins. EC(50) of the purified 29-kDa polypeptide was 0.078 microg/ml. The N-terminal amino acid sequence of the 29-kDa polypeptide shared no significant homology with all the known proteins, suggesting that this polypeptide belong to a new family of B. thuringiensis crystal proteins. In the ligand blotting analysis, specific binding proteins for the 29-kDa polypeptide were detected from the cell membrane of MOLT-4.     

New human myelodysplastic cell line,TER-3: G-CSF specific downregulation of Ca2+/calmodulin-dependent protein kinase IV

We have established a new hematopoietic cell line from a patient with myelodysplastic syndrome (MDS), which was refractory anemia with excess blasts (RAEB). This cell line, designated TER-3, depends on several cytokines for long-term survival and growth, and requires interleukin-3 (IL-3) for continuous growth. Cytochemical analysis revealed that TER-3 cells are weakly dianisidine positive and nonspecific esterase positive, but peroxidase negative. The surface marker profile shows that the TER-3 cells are strongly positive for myeloid, lymphoid, and megakaryocytic antigens such as CD15, CD19, and CD61, and negative for some common multilineage antigens such as CD13, CD33, and CD34. Thus, this cell line has a multilineage phenotype, suggesting that the transformation event occurred in multipotent stem cells. Dianisidine- and nonspecific esterase-positive TER-3 cells increase with granulocyte-colony stimulating factor (G-CSF) rather than with IL-3. These results suggest that the cell line is useful for understanding the mechanism underlying G-CSF-associated hematopoietic cell differentiation and activation in the patient with MDS.     

Rice receptor for chitin oligosaccharide elicitor does not couple to heterotrimeric G-protein: Elicitor responses of suspension cultured rice cells from Daikoku dwarf (d1) mutants lacking a functional G-protein α-subunit

We present evidence that the rice receptor for N -acetylchitooligosaccharide elicitor does not couple to heterotrimeric G-protein (G-protein), one of the most important signal transducers from the cell surface to down-stream effectors in various cellular responses of many organisms. Using mutant rice cells lacking functional G-protein α-subunit, cellular responses of suspension-cultured rice cells derived from Daikoku dwarf ( d1 ) mutants that were shown to contain mutations in the coding region of the G-protein α-subunit (Fujisawa et al. 1999) to N -acetylchitooligosaccharide (oligochitin) elicitor were compared with those of the corresponding parent cell lines. All the elicitor-induced cellular responses, such as medium alkalinization, generation of reactive oxygen species, expression of early responsive genes, PAL and PR genes, and production of phytoalexin, were basically identical in the mutant and wild type cell lines. Considering the reported presence of a single copy gene for the G-protein α-subunit in many plant species including rice, these results strongly support the above conclusion.     

Development of mast cells in vitro. II. Biologic function of cultured mast cells.

Mast cells were obtained by long term culture of rat thymus cells on rat embryonic fibroblast monolayers. Pure mast cell preparations obtained culture were incubated with 125I-labeled rat E myeloma protein to study receptors for IgE on their surface. When the cells were obtained after 35 to 45 days culture, the average number of receptors per mast cell was 100,000 to 400,000. An equilibrium constant of the binding reaction between their receptor and rat IgE was in the order of 108 M-1. The histamine content of the cultured mast cells was 0.2 to 5 mug/106 cells. The measurement of histamine content in mast cells recovered after different periods of culture suggested that the histamine content increased with maturation. Even after 45 to 50 days culture, the histamine content of cultured mast cells was significantly lower than that in rat peritoneal mast cells. The cultured mast cells were passively sensitized in vitro with rat IgE antibody against Nippostrongylus brasiliensis. The sensitized cells released histamine upon incubation with the antigen. It was also found that cultured mast cells released histamine upon exposure to compound 48/80. These results indicated that cultured mast cells have physiologic functions similar to those of normal rat mast cells, but they have not reached full maturation.     

Reaginic antibody formation in the mouse. VII. Depression of the ongoing IgE antibody formation by suppressor T cells.

The ongoing IgE antibody formation against ovalbumin (OA) in high responder mice was depressed by i.v. injections of either native or urea-denatured ovalbumin (UD-OA). Adoptive transfer experiments to determine the helper function of spleen cells from the treated animals showed that helper function for both IgE and IgG antibody responses diminished after treatment. Evidence was obtained that treatment suppressed the expansion of IgE-G memory cells. When the same treatment with OA or UD-OA was given to OA-primed mice before the appearance of IgE antibody in their serum, OA-specific splenic suppressor T cells were demonstrable. Thus, the transfer of splenic T cells from treated mice into normal mice suppressed the primary IgE and IgG antibody responses of the recipeints to DNP-OA. It was also found that the transfer of the splenic T cells from UD-OA-treated mice into OA-primed mice depressed ongoing IgE antibody formation in the recipients. The results suggested strongly that the decrease of helper function and the depression of ongoing IgE antibody formation by repeated injections of UD-OA was caused by generation of antigen (OA)-specific suppressor T cells.     

Toxoplasma gondii-derived heat shock protein 70 induces lethal anaphylactic reaction through activation of cytosolic phospholipase A2 and platelet-activating factor via Toll-like receptor 4/myeloid differentiation factor 88

Toxoplasma gondii-derived heat shock protein 70 (T.g.HSP70) was proven to induce IFN-gamma-dependent lethal anaphylactic reaction in T. gondii-infected mice through an alternative PAF-mediated pathway, but not the classical immunoglobulin (Ig)E-dependent pathway. Although marked IFN-gamma production was observed by CD11b(+), CD11c(+), CD4(+) and CD8(+) splenocytes, CD11b(+) and CD11c(+) cells were shown to be the key effecter cells which generated pro-inflammatory lipid such as PAF and caused T.g.HSP70-induced anaphylactic reaction. In the present study, we found that the T.g.HSP70-induced anaphylactic reaction was not observed in TLR 4-deficient ((-/-)) mice, whereas it was observed in WT and TLR2(-/-) mice. The mRNA expression of PAF-AH, the main enzyme for PAF degradation, increased in T. gondii-infected WT and TLR2(-/-) but not in TLR4(-/-) mice after T.g.HSP70 injection. Furthermore, phosphorylation of cPLA(2), which is the key enzyme for pro-inflammatory lipid generation, was detected in CD11b(+) splenocytes of WT and TLR2(-/-) mice but not in TLR4(-/-) mice. Subsequently, cPLA(2) activation was suppressed by inhibiting the TLR4-directed p38 and p44/42 MAPK pathways. However, T.g.HSP70-induced anaphylactic reaction was observed in TRIF(-/-) mice, but not in MyD88(-/-) mice. These findings indicate the cPLA(2) activated-PAF production via TLR4/MyD88-dependent, but not TRIF-dependent, signaling pathway in T.g.HSP70-induced anaphylactic reaction in T. gondii-infected mice.     

Immunoregulatory effects of transforming growth factor-beta in a prolonged period of culture.

It is well known that transforming growth factor-beta (TGF-beta) strikingly inhibits numerous immune functions in short-term cultures. In this study we investigated the effects of TGF-beta on the immune responses of murine spleen cells in a prolonged period of culture. The addition of exogenous TGF-beta (0.1 ng/ml) inhibited the proliferation of Con A- or LPS-stimulated spleen cells, polyclonal IgM and IgG antibody production, and NK cell activity during 4 days of the initial culture and subsequently enhanced their responses on Day 10. The augmented polyclonal IgM and IgG responses in murine spleen cells induced by LPS and TGF-beta on Day 10 were suppressed by the secondary addition of TGF-beta on Day 6. These results suggest that TGF-beta acts as an immunoregulator in prolonged period responses by immunoactivators.     

Elevation of KL-6, a lung epithelial cell marker,in plasma and epithelial lining fluid in acute respiratory distress syndrome

KL-6 is a pulmonary epithelial mucin more prominently expressed on the surface membrane of alveolar type II cells when these cells are proliferating, stimulated, and/or injured. We hypothesized that high levels of KL-6 in epithelial lining fluid and plasma would reflect the severity of lung injury in patients with acute lung injury (ALI). Epithelial lining fluid was obtained at onset (day 0) and day 1 of acute respiratory distress syndrome (ARDS)/ALI by bronchoscopic microsampling procedure in 35 patients. On day 0, KL-6 and albumin concentrations in epithelial lining fluid were significantly higher than in normal controls (P < 0.001), and the concentrations of KL-6 in epithelial lining fluid (P < 0.002) and in plasma (P < 0.0001) were higher in nonsurvivors than in survivors of ALI/ARDS. These observations were corroborated by the immunohistochemical localization of KL-6 protein expression in the lungs of nonsurvivors with ALI and KL-6 secretion from cultured human alveolar type II cells stimulated by proinflammatory cytokines. Because injury to distal lung epithelial cells, including alveolar type II cells, is important in the pathogenesis of ALI, the elevation of KL-6 concentrations in plasma and epithelial lining fluid could be valuable indicators for poor prognosis in clinical ALI.     

Administration of ANG II induces iron deposition and upregulation of TGF-beta1 mRNA in the rat liver

We previously found that ANG II infusion into rats causes iron deposition in the kidney and heart, which may have a role in the regulation of profibrotic gene expression and tissue fibrosis. In the present study, we have investigated whether ANG II can also induce iron accumulation in the liver. Prussian blue staining detected frequent iron deposition in the interstitium of the liver of rats treated with pressor dose ANG II for 7 days, whereas iron deposition was absent in the livers of control rats. Immunohistochemical and histological analyses showed that some iron-positive nonparenchymal cells were positive for ferritin and heme oxygenase-1 (HO-1) protein and TGF-beta1 mRNA and were judged to be monocytes/macrophages. It was shown that ANG II infusion caused about a fourfold increase in ferritin and HO-1 protein expression by Western blot analysis and about a twofold increase in TGF-beta1 mRNA expression by Northern blot analysis, which were both suppressed by treating ANG II-infused rats with losartan and deferoxamine. In addition, mild interstitial fibrosis was observed in the liver of rats that had been treated with pressor dose ANG II for 7 days or with nonpressor dose ANG II for 30 days, the latter of which also caused loss of hepatocytes and intrahepatic hemorrhage in the liver. Taken together, our data suggest that ANG II infusion induces aberrant iron homeostasis in the liver, which may have a role in the ANG II-induced upregulation of profibrotic gene expression in the liver.