Immunological Properties of TtT/M-87 Cell Line Established from Murine Pituitary Tumor-Associated Macrophages

TtT/M-87 cell is a macrophage cell line established from thyrotropic pituitary tumor tissues in mouse. In this paper, we report the immunological properties of M-87 cells as a model of tumor-associated macrophage. Contrasting with resident peritoneal macrophages, M-87 cells constitutively secreted small but significant amounts of TNF-α and IL-1α, which were detectable in both biological assays (cytotoxic activity for L929 and co-mitogenic activity for Con A-induced T cell proliferation, respectively) and ELISA, and produced larger amounts of these cytokines upon stimulation with LPS. They expressed MHC class II molecules on their cell surface without stimulation by IFN-γ. The accessory or antigen-presenting cell activity in antibody-producing response of spleen lymphocytes to sheep red blood cells was shown to be much higher in M-87 cells than normal peritoneal macrophages. In addition, when normal spleen lymphocytes were cultured with allogeneic tumor cells, such as EL-4 and S-180, in the presence of M-87 cells, lymphocytes reactive to stimulator cells were activated to manifest inhibitory effect on the tumor cell growth and also to manifest specific cytotoxic effect on the allogeneic tumor cells. These results show that M-87 cells derived from tumor-associated tissue are activated macrophages and that they are inhibitory to tumor cell growth and augmentative in the induction of T-cell-mediated immune responses.     

RseP (YaeL), an Escherichia coli RIP protease, cleaves transmembrane sequences

Escherichia coli RseP (formerly YaeL) is believed to function as a 'regulated intramembrane proteolysis' (RIP) protease that introduces the second cleavage into anti-sigma(E) protein RseA at a position within or close to the transmembrane segment. However, neither its enzymatic activity nor the substrate cleavage position has been established. Here, we show that RseP-dependent cleavage indeed occurs within predicted transmembrane sequences of membrane proteins in vivo. Moreover, RseP catalyzed the same specificity proteolysis in an in vitro reaction system using purified components. Our in vivo and in vitro results show that RseP can cleave transmembrane sequences of some model membrane proteins that are unrelated to RseA, provided that the transmembrane region contains residues of low helical propensity. These results show that RseP has potential ability to cut a broad range of membrane protein sequences. Intriguingly, it is nevertheless recruited to the sigma(E) stress-response cascade as a specific player of RIP.     

Effects of humid heat exposure in later sleep segments on sleep stages and body temperature in humans

This study sought to investigate the effects of humid heat exposure in later sleep segments on sleep stages and body temperature in humans. The subjects were eight healthy males, from whom informed consent had been obtained. The experiments were carried out under three different sets of conditions: a control climate [air temperature (Ta)=26°C, relative humidity (RH)=50%] (C); a humid heat climate (Ta=32°C, RH=80%) (H); and a humid heat exposure in later sleep segments (C for the first 3 h 45 min, followed by a 30-min transition to H, which was then maintained for the last 3 h 45 min) (C–H). Electroencephalogram, EOG, and mental electromyogram, rectal temperature (Tre), and skin temperature (Tsk) were continuously measured. The total amount of wakefulness was significantly increased in H compared to C–H or C. Compared to C, wakefulness in C–H and H was significantly increased during later sleep segments. Tre and mean Tsk were significantly higher in H than in C–H or C. In C–H, Tsk and Tre increased to levels equal to those observed in H after Ta and RH increase. Whole body sweat loss was significantly lower in C–H and C than in H. These results suggest that humid heat exposure in the later sleep segment reduces thermal load as compared to full-night humid heat exposure. In daily life, the use of air conditioning in the initial sleep hours can protect sleep and thermoregulation.     

Mannose-binding lectin is a regulator of inflammation that accompanies myocardial ischemia and reperfusion injury

The mannose-binding lectin (MBL), a circulating pattern recognition molecule, recognizes a wide range of infectious agents with resultant initiation of the complement cascade in an Ab-independent manner. MBL recognizes infectious non-self and altered self in the guise of apoptotic and necrotic cells. In this study, we demonstrate that mice lacking MBL, and hence are devoid of MBL-dependent lectin pathway activation but have fully active alternative and classical complement pathways, are protected from cardiac reperfusion injury with resultant preservation of cardiac function. Significantly, mice that lack a major component of the classical complement pathway initiation complex (C1q) but have an intact MBL complement pathway, are not protected from injury. These results suggest that the MBL-dependent pathway of complement activation is a key regulator of myocardial reperfusion ischemic injury. MBL is an example of a pattern recognition molecule that plays a dual role in modifying inflammatory responses to sterile and infectious injury.     

An efficient system to establish multiple embryonic stem cell lines carrying an inducible expression unit

The growing use of mouse embryonic stem (ES) cells in research emphasizes their importance in studies of molecular mechanisms that maintain pluripotency and direct cellular differentiation. Although systems for regulatable transgene expression are essential for fine analysis of cellular processes at the molecular level, a strategy for the establishment of multiple ES cell lines carrying any of these systems has not yet been described. Here, we report our development of the ROSA-TET system, an effective system for the establishment of multiple ES cell lines carrying a tetracycline (Tc)-regulatable transgene at the Gt (ROSA)26asSor (ROSA26) locus. This system contains a knock-in step of a construct carrying both loxP and its mutant sequences into the ROSA26 locus, followed by a subsequent exchange step that introduces a cDNA to be Tc-regulated to the locus using the recombinase-mediated cassette exchange reaction. Both steps are demonstrated to give desired clones with high efficiency, suggesting that this system can be introduced readily into any ES cell lines, leading to the simultaneous establishment of multiple cell lines carrying different Tc-regulated cDNAs. We believe that use of this system will strongly accelerate molecular biological research using ES cells.     

Chimeric structure of omp2 of Brucella from Pacific common minke whales (Balaenoptera acutorostrata)

In the Pacific common minke whale (Balaenoptera acutorostrata ), a new variant of Brucella has been detected using the polymerase chain reaction. Detailed analysis of the porin protein genes, omp2a and omp2b from the whale Brucella showed that these two genes have some motifs in common with Atlantic marine strains in the 5'-terminal one-third region. On the other hand, the nucleotide sequences in the 3'-terminal two-thirds region of the two genes were almost identical to the respective genes of terrestrial strains. Thus, Pacific whale Brucella omp2 genes are chimeras between marine and terrestrial strains.     

Mutation study of antithrombin: the roles of disulfide bonds in intracellular accumulation and formation of russell body-like structures

Antithrombin (AT) is a major plasma protease inhibitor with three intramolecular disulfide bonds and a deficiency of it is associated with venous thrombosis. Recently, we prepared CHO cells overexpressing a novel mutant, AT(C95R), with a disulfide bond removed, and revealed that this mutant remained for a long time in the endoplasmic reticulum (ER) without being degraded and also accumulated in newly formed membrane structures that resembled Russell bodies (RB) [Tanaka, Y. et al. (2002) J. Biol. Chem. 277, 51058-51067]. In this study, we replaced each of the individual cysteine residues of AT with an arginine and also two paired cysteine residues with arginines. We stably expressed these mutant ATs in CHO cells, and examined the roles of each cysteine residue or disulfide bond in the accumulation of mutant ATs and the formation of RB-like structures. In pulse-chase experiments, the secretion of mutant ATs with single mutations decreased approximately 1/5-1/50 times compared to that of the wild type AT. All of the mutant ATs were retained in the ER and were also found to accumulate in the RB-like structures. On the other hand, the fates of mutant ATs with double mutations fell into two categories. Secretion of mutant AT(C8R,C128R) decreased only approximately 1/2 times and no RB-like structures appeared. Mutants AT(C21R,C95R) and AT(C247R,C430R) exhibited similar secretion kinetics to the mutant ATs with the single mutations and were found in RB-like structures. On a sucrose gradient, all of the mutant ATs that induced RB-like structures migrated as oligomeric structures, whereas wild type AT and AT(C8R,C128R) migrated as monomers. Further, to clarify the morphological pathway through which RB-like structures are formed, we prepared CHO cells in which the expression of AT(C95R) was controlled by the Tet-On system. During expression of AT(C95R), RB-like structures formed through expansion of the ER. These results suggest that the correct folding with each disulfide bond is essential for the secretion of AT and oligomerization of mutant ATs in the ER is involved in the formation of RB-like structures.     

Mannose-binding lectin-deficient mice display defective apoptotic cell clearance but no autoimmune phenotype

Mannose-binding lectin (MBL) is a circulating serum protein that is sequestered to sites of inflammation and infection. MBL is a member of the collectin family with structural similarities to the lung collectins and functional similarities to C1q. Both MBL and C1q activate complement; C1q activates the classical pathway and MBL the lectin pathway. Here we demonstrate that MBL binds apoptotic cells in vitro and confirm a role for MBL in clearance of apoptotic cells in vivo. Despite MBL null mice demonstrating defective apoptotic cell clearance they did not develop spontaneous autoimmunity, lymphoproliferation, or germinal center expansion although increased numbers of peritoneal B1 cells were detected. These data demonstrate an important in vivo role for MBL in clearance of dying cells and adds the MBL null animals to the few animals with demonstrable in vivo apoptotic cell clearance defects. Moreover, it demonstrates that failure of apoptotic cell clearance can be dissociated from autoimmunity.     

Quantitative GSL-glycome analysis of human whole serum based on an EGCase digestion and glycoblotting method

Glycosphingolipids (GSLs) are lipid molecules linked to carbohydrate units that form the plasma membrane lipid raft, which is clustered with sphingolipids, sterols, and specific proteins, and thereby contributes to membrane physical properties and specific recognition sites for various biological events. These bioactive GSL molecules consequently affect the pathophysiology and pathogenesis of various diseases. Thus, altered expression of GSLs in various diseases may be of importance for disease-related biomarker discovery. However, analysis of GSLs in blood is particularly challenging because GSLs are present at extremely low concentrations in serum/plasma. In this study, we established absolute GSL-glycan analysis of human serum based on endoglycoceramidase digestion and glycoblotting purification. We established two sample preparation protocols, one with and the other without GSL extraction using chloroform/methanol. Similar amounts of GSL-glycans were recovered with the two protocols. Both protocols permitted absolute quantitation of GSL-glycans using as little as 20 μl of serum. Using 10 healthy human serum samples, up to 42 signals corresponding to GSL-glycan compositions could be quantitatively detected, and the total serum GSL-glycan concentration was calculated to be 12.1–21.4 μM. We further applied this method to TLC-prefractionated serum samples. These findings will assist the discovery of disease-related biomarkers by serum GSL-glycomics.     

Photochemical Synthesis of 6-Aryluridines1)

Abstract

Synthesis of 6-aryluridines was effected by photochemical arylation of 6-iodo-2′,3′-O-isopropylidene-5′-O-methoxymethyluridine.