Visualization and quantification of dynamic STAT3 homodimerization in living cells using homoFluoppi.

Dimerization in signal transduction is a dynamically regulated process and a key regulatory mechanism. Signal transducer and activator of transcription 3 (STAT3) dimerizes after tyrosine phosphorylation upon cytokine stimulation. Because only the STAT3 dimer possesses the trans-activation activity, dimerization is an indispensable process for cytokine signaling. Here we report the detection of dynamic STAT3 dimerization in living cells using the homoFluoppi system. This method allowed us to validate the presence of an intact Src homology 2 domain and STAT3 Tyr705 phosphorylation, which facilitate puncta formation and homodimerization. Puncta formation was reversible, as determined by a decreased punctate signal after washout of oncostatin M. We analyzed STAT3 mutants, which have been reported in patients with hyper IgE syndrome and inflammatory hepatocellular adenoma (IHCA). Analysis of the IHCA mutants using homoFluoppi revealed constitutive activity independent of cytokine stimulation and novel insight into kinetics of dimer dissociation process. Next, we used homoFluoppi to screen for inhibitors of STAT3 dimerization, and identified 3,4-methylenedioxy-ß-nitrostyrene as a novel inhibitor. The results of this study show that homoFluoppi is a useful research tool for the analysis of proteins like STAT3 that dynamically dimerize, and is applicable for the screening of dimerization modulators.

A self-consistent GW approach to the van der Waals potential for a helium dimer.

van der Waals interaction between two helium (He) atoms is studied by calculating the total energy as a function of the He-He distance within the self-consistent GW approximation, which is expected to behave correctly in the long wavelength limit. In the Born-Oppenheimer (BO) approximation, the pair potential curve has its minimum value at 2.87 Å, which is somewhat larger than the local density approximation result, 2.40 Å, and is closer to previous quantum chemistry results. The expectation value for the interatomic distance, calculated by solving the Schrödinger equation for the two nuclei problem using the BO potential energy curve, is 30 Å, which is smaller but of the same order as previous experimental and theoretical results.

Constitutively expressed Siglec-9 inhibits LPS-induced CCR7, but enhances IL-4-induced CD200R expression in human macrophages.

Siglecs recognize the sialic acid moiety and regulate various immune responses. In the present study, we compared the expression levels of Siglecs in human monocytes and macrophages using a quantitative real-time reverse transcription-polymerase chain reaction analysis. The differentiation of monocytes into macrophages by macrophage colony-stimulating factor or granulocyte macrophage colony-stimulating factor enhanced the expression of Siglec-7 and Siglec-9. The differentiated macrophages were stimulated by lipopolysaccharide (LPS) plus interferon (IFN)-γ or interleukin (IL)-4. The expression of Siglec-10 was enhanced by IL-4, whereas that of Siglec-7 was reduced by LPS plus IFN-γ. The expression of Siglec-9 was not affected by these stimuli. The knockdown of Siglec-9 enhanced the expression of CCR7 induced by the LPS or the LPS plus IFN-γ stimulation, and decreased the IL-4-induced expression of CD200R. These results suggest that Siglec-9 is one of the main Siglecs in human blood monocytes/macrophages and modulates innate immunity.

Siglec-9 modulated IL-4 responses in the macrophage cell line RAW264.

Siglecs, an immunoglobulin-like lectin family that recognizes the sialic acid moiety, regulate various aspects of immune responses. In the present study, we investigated the effects of Siglecs on the macrophage cell line RAW264, which was stimulated with interleukin-4 (IL-4). The induction of arginase-1 (Arg1) by IL-4 was stronger in Siglec-9-expressing cells than in mock cells. Mutations in the cytoplasmic tyrosine-based inhibitory motifs in Siglec-9 markedly reduced the expression of Arg1. The phosphorylation of Akt by IL-4 and extracellular signal-regulated kinase (ERK) without IL-4 was stronger in Siglec-9-expressing cells, indicating the enhanced activation of the phosphatidylinositol 3 kinase (PI-3K) and mitogen-activated protein kinase kinase (MEK)/ERK pathways, respectively. The enhanced expression of Arg1 was inhibited by MEK inhibitors, but not by PI-3K inhibitor. These results indicate that Siglec-9 affects several different signaling pathways in IL-4-stimulated macrophages, which resulted in enhanced induction of Arg1 in Siglec-9-expressing RAW264 cells.

Effects of Siglec on the expression of IL-10 in the macrophage cell line RAW264.

Interleukin-10 (IL-10) expression was significantly elevated upon stimulation with lipopolysaccharide (LPS) when the sialic acid-recognizing Ig-superfamily lectin Siglec-5 or -9 was overexpressed in RAW264 cells. During the course to clarify the mechanism for this activation, we found that IL-10 promoter proximal region up to -500 bp led to transactivation similar to that up to -1,500 bp. Among the transcription factors that activate the mouse IL-10 promoter so far reported, the level of C/EBPß was increased in Siglec-9-expressing cells. Transient expression of the C/EBPß major isoform LAP led to an increase in the expression of IL-10 in Siglec-9-expressing cells, but not in mock-transfected control RAW264 cells upon stimulation with LPS, as assessed by either a luciferase assay or the production of IL-10. Without LPS, the IL-10 promoter was activated by transiently expressed LAP in Siglec-9-expressing cells, however, the magnitude of transactivation was less than that with the LPS stimulation. The knockdown of C/EBPß down-regulated the production of IL-10. Taken together, these results suggest that one of the reasons for the stimulation of IL-10 expression in Siglec-9-expressing cells may be an increase in intracellular C/EBPß level.

Lectin-dependent localization of cell surface sialic acid-binding lectin Siglec-9.

Siglecs are immunoglobulin lectin group proteins that recognize the sialic acid moiety. We previously reported that the expression of Siglec-9 on the macrophage cell line RAW264 markedly enhanced Toll-like receptor (TLR)-induced interleukin (IL)-10 production and inhibited the production of proinflammatory cytokines. In this study, we examined the lectin-dependent anti-inflammatory activities of Siglec-9. IL-10 production was modestly reduced by a mutation that disrupted the lectin activity of Siglec-9, while the reduction in tumor necrosis factor-α was not affected. Membrane fractionation experiments revealed that a part of Siglec-9 resided in the detergent-insoluble microdomain, the so-called lipid raft fraction. The amount of Siglec-9 in the lipid raft fraction rapidly increased following TLR2 stimulation by peptidoglycan and peaked after 3-10 min. This time course was similar to that of TLR2. The double tyrosine mutant in immunoreceptor tyrosine-based inhibitory motifs moved to lipid rafts in a similar manner, while lectin-defective Siglec-9 was not detected in the lipid raft fraction. The production of IL-10 was partially reduced by cholesterol oxidase that disturbed lipid raft organization. Taken together, these results suggest that Siglecs exhibit lectin-dependent changes in cellular localization, which may be partly linked to its control mechanism that increases the production of IL-10.

Enhanced lentiviral vector production in 293FT cells expressing Siglec-9.

Siglecs, sialic acid-recognizing Ig-superfamily lectins, regulate various aspects of immune responses, and have also been shown to induce the endocytosis of binding materials such as anti-Siglec antibodies or sialic acid-harboring bacteria. In this study, we demonstrated that the expression of Siglec-9 enhanced the transfection efficiency of several cell lines such as macrophage RAW264 and non-hematopoietic 293FT cells. We applied this finding to the production of a lentiviral vector in which cells were transfected simultaneously with multiple vectors, and achieved a twice increase in viral production levels. Furthermore, 293FT cells expressing lectin-defective Siglec-9 produced three- to seven-fold higher titer of viral vector compared with parental 293FT cells. These results suggest that Siglec-9 enhanced lentiviral vector production in a lectin-independent manner.

Usefulness of the final filter of the IV infusion set in intravenous administration of drugs--contamination of injection preparations by insoluble microparticles and its causes.

The purpose of this study is to clarify the presence of so much insoluble microparticles in the injections and to confirm the usefulness of the final filter for its removal. The test drugs used were Doyle 1 g vial, Cefotax 1 g vial, Minomycin 100 mg vial, Omegacin 0.3 g vial, Maxipime 1 g vial, Rocephin 1 g vial, Isovorin 25 mg vial, Diamox 500 mg vial and Fungizone 50 mg vial. 1) An appropriate volume of physiological saline was poured into the test drug vial from the 500 ml physiological saline bag. 2) The dissolution of the preparation was checked. 3) That test drug solution was returned into the same 500 ml physiological saline bag. 4) 5 ml of test drug solution was extracted from the inside of the 500 ml physiological saline bag. 5) The number of insoluble microparticles in the each test drug solution at pre- and post-filtrations were counted by using a particle counter for the solution. Two results were shown as follows (microparticle size: 5 microm or greater), (a) Doyle 1 g (ASPC): The number of insoluble microparticles in pre- and post-filtrations of a Doyle 1 g vial were 250 +/- 45 and 0/5 ml, respectively (microparticle size: < or =5 microm 1662/5 ml, 5-10 microm: 238/5 ml, >10 microm: 11/5 ml). (b) Diamox 500 mg (acetazolamide): The number of insoluble microparticles in pre- and post-filtrations of Diamox were 158 +/- 53 and 0.3 +/- 0.47/5 ml, respectively (microparticle size: < or =5 microm 4030/5 ml, 5-10 microm: 144/5 ml, >10 microm: 14/5 ml). The presence of great numbers of insoluble microparticles in several injection preparations was clarified. Although the all test drugs used cleared the criteria of the number of insoluble microparticles of the Japanese Pharmacopoeia, it was suggested to be not suitable that great numbers of insoluble microparticles were administrated in the body fluid of patients, because a possibility to occlude capillaries and/or to injure the tissues by them was been thought. But we could remove them nearly completely by passing the solutions of drugs through an infusion filter. Otherwise, in this examination, we found that so much insoluble microparticles derived from the disposable syringe (10 ml) were used for dissolving freeze-dried preparations routinely (microparticle size: < or =5 microm 329/5 ml, 5-10 microm: 125/5 ml, >10 microm: 39/5 ml). These results suggest that incorporation of a final filter in the IV line is extremely necessary not only for the prevention of bacterial infections, but also for elimination of insoluble microparticles.

Carcinoma cuniculatum of the esophagus.

AIMS: Extremely well-differentiated squamous cell carcinoma with the features of so-called carcinoma cuniculatum (CC) is a rare neoplasm. We describe the clinicopathologic findings of the first 2 cases of CC of the esophagus. METHODS AND RESULTS: Two elderly men presented with symptoms and clinical signs of esophageal malignancy. Repeated endoscopic biopsies of their esophageal tumors were inconclusive. Resection revealed CC of the esophagogastric junction in both cases. The tumors extended into the adventitia but no lymph node metastases were present. In situ hybridization for human papillomavirus HPV subtypes was negative. CONCLUSION: Carcinoma cuniculatum is reported for the first time in the esophagus. The diagnosis of this tumor variant is difficult by means of cytological examination or by endoscopic biopsies alone. Carcinoma cuniculatum in this location shows biologic features similar to verrucous carcinoma (deep penetration, no lymph nodes metastases, and location at one end of the esophagus). No evidence of human papillomavirus could be demonstrated.