[Progress of genomic mutation spectrum in biliary tract malignant tumors].

Biliary tract cancer is characterized by high invasiveness, occult early clinical manifestations and rapid progression. Surgical resection typically fails to achieve satisfactory outcomes. Biliary tract cancer exhibits low sensitivity to radiotherapy and chemotherapy. The prognosis of patients is extremely poor. Genomics research based on next-generation sequencing technology has made some advances. The gene mutation spectrum of biliary tract cancer has been preliminarily revealed, which lays a foundation for the study of molecular typing. This review summarizes the research progress and clinical application of gene mutation spectrum of biliary tract cancer in recent years, aiming to provide reference for the clinical diagnosis, treatment and basic research.

[The role of three-dimensional chromatin structure changes in tumor progression].

The human genome is not a linear structure, but a three-dimensional structure through complex folding and assembly. Chromosome structure capture technology can detect the three-dimensional construction of chromatin. Hi-C sequencing data of various tumors indicate that the chromatin topology associated domains changed during tumor progression and is related to copy number variation. In addition, transformation of the genomic compartment is related to gene expression. However, current researches on three-dimensional structures of tumoral chromatin are still in the stage of exploration, and some conclusions are too superficial to be applied to the clinic immediately, which requires further study.

Patterns of dopamine transporter imaging in subtypes of multiple system atrophy.

OBJECTIVES: To investigate the differences in the pattern of striatal (caudate and putamen) dopamine transporter (DAT) loss in a multiple system atrophy (MSA) cohort, based on the clinical variants parkinsonian subtype (MSA-P) and cerebellar subtype (MSA-C) via (11)C-N-2-carbomethoxy-3-(4-fluorophenyl)-tropane (11 C-CFT) positron emission tomography (PET) imaging. MATERIALS AND METHODS: One hundred and six subjects (forty-one patients with probable MSA-P; forty patients with probable MSA-C; twenty-five healthy controls) underwent 11 C-CFT PET. Subregional 11 C-CFT uptake of bilateral caudate, anterior putamen, and posterior putamen was calculated respectively to measure the striatal dopaminergic function. RESULTS: Significant decrease in DAT binding in striatum was revealed in patients with MSA-C and MSA-P compared to normal controls (all regions, MSA-C vs controls, P < .0001; MSA-P vs controls, P < .0001). DAT reduction was more pronounced in MSA-P patients than that in MSA-C patients (all regions, P < .0001). Eleven of forty MSA-C patients displayed no DAT loss, whereas striatal DAT loss was evident in all MSA-P patients. MSA-P subtype showed a more obvious anteroposterior gradient of DAT loss and more asymmetric dopaminergic dysfunction compared to MSA-C patients. CONCLUSION: The subtypes of MSA studied here show significantly different spatial/anatomic patterns of striatonigral degeneration which may provide insights into their disease pathophysiology. Specifically, MSA-P patients exhibit an uneven and much greater pronounced loss of dopamine innervation, while a relatively uniform pattern is revealed in patients with the MSA-C. Furthermore, the typical reduction in DAT 11 C-CFT binding in striatum is not present in all MSA-C patients, with a minority of cases showing normal DAT binding.

Molecular cloning and tissue distribution profiles of the chicken R-spondin1 gene.

Rspo1 belongs to the Rspo family, which is composed of 4 members (Rspo1-4) that share 40 to 60% sequence homology and similar domain organizations, and regulate the WNT signaling pathway via a common mechanism. Rspo1 plays a key role in vertebrate development and is an effective mitogenic factor of gastrointestinal epithelial cells. We report the cloning of chicken Rspo1 and its gene expression distribution among tissues. It contained an open reading frame of 783 bp encoding a protein of 260 amino acids, and its molecular weight was predicted to be 28.80 kDa. Reverse transcription-polymerase chain reaction-based gene expression analysis indicated that chicken Rspo1 was highly expressed in the stomach muscle tissue, but was expressed at low levels in the lung, brain, jejunum, cecum, ileum, spleen, pancreas, kidney, and glandular stomach. These results suggest that Rspo1 plays a major role in muscular immune protection.

Lactoferrin mRNA expression in mouse mammary glands during pregnancy and lactation.

Lactoferrin (Lf) is an iron-binding glycoprotein that is produced by mucosal epithelial cells in mammals. Lf has non-immune natural defense functions and biological functions in addition to and distinct from its role in regulating inflammatory responses. Lf also improved some physiological and immunological parameters. Lf is a biomarker for monitoring medical treatment in inflammatory bowel diseases. Current LF research focuses on iron absorption, antimicrobial activity, and the modulation of iron metabolism during inflammation. No systematic research about Lf expression levels in mouse mammary glands during pregnancy and lactation exists. We investigated Lf mRNA expression levels in mouse mammary glands by collecting samples on days 1, 6, 12, and 18 of pregnancy and lactation (six mice per group). The expression levels of Lf mRNA were measured by semi-quantitative reverse transcription polymerase chain reaction using GAPDH as an internal control. Lf mRNA was not expressed in mammary glands on days 1, 6, and 12 of pregnancy, but it was expressed on day 18 (IOD: integrated optical density; Lf(IOD)/GAPDH(IOD) = 0.46). Lf expression levels were higher during lactation stages than during pregnancy stages, and it stabilized at 0.71-0.73 (Lf(IOD)/GAPDH(IOD)) from day 1 to 12 of lactation; however, the difference was not significant (P > 0.05). At day 18 of lactation, Lf expression began to decline (Lf(IOD)/GAPDH(IOD) = 0.61), but the difference was not significant (P > 0.05). Based on these results, the variation in Lf expression levels during developmental stages may be related to its regulatory role in mouse mammary gland immunity.

Endogenous galectin-3 controls experimental malaria in a species-specific manner.

Galectins are evolutionarily conserved glycan-binding proteins with pleiotropic roles in innate and adaptive immune responses. Galectin-3 has been implicated in several immunological processes as well as in pathogen recognition through specific binding to glycosylated receptors on the surface of host cells or microorganisms. In spite of considerable evidence supporting a role for galectin-3 in host-pathogen interactions, the relevance of this lectin in the regulation of the host defence mechanisms in vivo is poorly understood. In this study, we analysed the impact of galectin-3 deficiency during infection with three distinct species of rodent malaria parasites, Plasmodium yoelii 17XNL, Plasmodium berghei ANKA and Plasmodium chabaudi AS. We found that galectin-3 deficiency showed a marginal effect on the course of parasitaemia during P. chabaudi infection, but did not alter the course of parasitaemia during P. berghei infection. However, lack of galectin-3 significantly reduced P. yoelii parasitaemia. This reduced parasitaemia in Lgals3(-/-) mice was consistent with higher titres of anti-P. yoelii MSP1(19) IgG2b isotype antibodies when compared with their wild-type counterparts. Our results reflect the complexity and singularity of host-pathogen interactions, indicating a species-specific role of endogenous galectin-3 in the control of parasite infections and the modulation of antibody responses.

The bone marrow compartment is modified in the absence of galectin-3.

Galectin-3 (gal-3) is a ß-galactoside binding protein present in multivalent complexes with an extracellular matrix and with cell surface glycoconjugates. In this context, it can deliver a variety of intracellular signals to modulate cell activation, differentiation and survival. In the hematopoietic system, it was demonstrated that gal-3 is expressed in myeloid cells and surrounding stromal cells. Furthermore, exogenous and surface gal-3 drive the proliferation of myeloblasts in a granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent manner. Here, we investigated whether gal-3 regulates the formation of myeloid bone marrow compartments by studying galectin-3(-/-) mice (gal-3(-/-)) in the C57BL/6 background. The bone marrow histology of gal-3(-/-) mice was significantly modified and the myeloid compartments drastically disturbed, in comparison with wild-type (WT) animals. In the absence of gal-3, we found reduced cell density and diaphyseal disorders containing increased trabecular projections into the marrow cavity. Moreover, myeloid cells presented limited capacity to differentiate into mature myeloid cell populations in gal-3(-/-) mice and the number of hematopoietic multipotent progenitors was increased relative to WT animals. In addition, bone marrow stromal cells of these mice had reduced levels of GM-CSF gene expression. Taken together, our data suggest that gal-3 interferes with hematopoiesis, controlling both precursors and stromal cells and favors terminal differentiation of myeloid progenitors rather than proliferation.

Development of allergic inflammation in a murine model of asthma is dependent on the costimulatory receptor OX40.

Asthma is thought to result from an abnormal expansion of CD4 T cells reactive with airborne allergens, and pathology is controlled by several cytokines of the T helper type 2 (Th2) family. The exact molecules which are involved in generating allergen-reactive T cells are not clear. Studies with blocking reagents or knockout animals have shown that the CD28/B7 interaction partially controls development of allergic asthma in mouse models, but may not be the sole molecule involved. In this report, we have investigated the role of the tumor necrosis factor receptor family member OX40 in allergic inflammation using OX40-deficient mice. OX40 has been shown to participate in regulating clonal expansion and memory development of CD4 T cells and may synergize with CD28. Our studies demonstrate that OX40(-/)- mice, primed with the model allergen ovalbumin and challenged through the airways with aerosolized antigen, are severely impaired in their ability to generate a Th2 response characterized by high levels of interleukin (IL)-5, IL-4, and immunoglobulin E. Moreover, OX40(-/)- mice exhibit diminished lung inflammation, including an 80-90% reduction in eosinophilia and mucus production, less goblet cell hyperplasia, and significantly attenuated airway hyperreactivity. These studies highlight the potential importance of OX40 in development of allergic asthma and suggest that targeting OX40 may prove useful therapeutically.

IgE class-restricted tolerance induced by neonatal administration of soluble or cell-bound IgE. Cellular mechanisms.

Certain aspects of the phenomenon of IgE class-restricted tolerance induced in mice by neonatal treatment with monoclonal IgE, either in soluble form or coupled to syngeneic spleen cells, were examined. The present studies document that this tolerance results from exposure to IgE molecules, irrespective of their antigen specificity, and the resulting effects are polyclonal in nature since IgE responses directed against antigenic determinants unrelated to the tolerance-inducing IgE molecules are affected. Moreover, such findings indicate that the molecular subregion(s) responsible for inducing IgE class-restricted tolerance resides in the epsilon heavy chain constant region domain(s) of IgE. When soluble IgE is employed, tolerance induction results from neonatal treatment with doses as low as 2.5 micrograms per injection per mouse; cell-bound IgE is considerably more potent, in terms of total dose required, since tolerance results from treatment with as few as 1 X 10(6) cells per injection (per mouse), equivalent to an absolute quantity of 0.2 ng of IgE per injection. This long-term class-specific tolerance appears to be a unique feature of the IgE antibody system, since treatment of mice with monoclonal antibodies of the IgA, IgG1, or IgG2b isotypes, either in soluble or cell-bound form, does not perturb antibody responses of their corresponding isotypes or in the IgE class. By analyzing the lymphoid cells of IgE-tolerant mice after they reached adulthood, the following observations were made: (a) lymphoid cells from such tolerant mice fail to develop FcR epsilon + cells upon in vitro stimulation with IgE, as is characteristically observed with lymphoid cells from nontolerant mice; and (b) mice rendered tolerant by neonatal treatment with soluble IgE possess IgE class-restricted suppressor T cells, demonstrable in adoptive transfer experiments, whereas no such suppressor cells are evident in mice in which cell-bound IgE was used for neonatal treatment. The latter observations could mean that two different mechanisms underlie the IgE class-restricted tolerance, or both mechanisms operate coordinately to varying degrees depending upon which regimen is used for tolerance induction, as discussed herein.

IgE-mediated chemotaxis of rat basophilic leukemia cells towards specific antigen.

We evaluated chemotactic properties of four sublines of rat basophilic leukemia cells using blindwell Boyden chamber assays. After sensitization with a mouse monoclonal IgE directed against dinitrophenyl (DNP), cells from sublines 2H3-C and 926a underwent chemotaxis toward DNP-bovine serum albumin (BSA) and sublines RBL-1 and 4A did not. Chemotactic responses required specific IgE and were determined by the IgE antigen specificity used for sensitization. The threshold for chemotaxis was on the order of 10(-10) M DNP-BSA. Release of incorporated [3H]-serotonin did not always parallel chemotactic responses, which suggests that chemotaxis and secretion may be two unlinked processes that occur during basophil activation. Our results predict a possible in vivo mechanism whereby specific chemotactic responses of basophils and other FcR epsilon-bearing cells are mediated via specific IgE bound to membrane FcR epsilon.

A clonal analysis of the IgE response and its implications with regard to isotope commitment.

In a clonal analysis of the IgE response, it was found that a small proportion of primary or nonimmune B cells in spleen and mesenteric lymph nodes can be stimulated by antigen to produce IgE-secreting clones. In addition, there appears to be no substantial difference in the frequency of such cells between the classical low and high IgE responder strains. An analysis of immune, or memory, B cells revealed substantial increases in the frequency of B cells secreting IgE as compared with primary B cells, although the actual proportion of B cells secreting IgE remained relatively low. When the IgE-secreting clones derived from either primary or secondary B cells were reanalyzed for the presence of other isotypes, it was found that all clones secreting IgE were secreting at least one other isotype, with the majority secreting two or three other isotypes in addition to IgE. This demonstrates that there is no distinct subpopulation of B cells committed to IgE expression per se.

Human keratinocytes release the endogenous beta-galactoside-binding soluble lectin immunoglobulin E (IgE-binding protein) which binds to Langerhans cells where it modulates their binding capacity for IgE glycoforms.

A better understanding of the pathophysiological role of Langerhans cells (LC) in atopic diseases is dictated by the characterization of the structures involved in immunoglobulin (IgE)-binding on their cell surface. We previously reported that human LC express the high affinity receptor for IgE (Fc epsilon RI), as well as the low affinity receptor for IgE (Fc epsilon RII/CD23). In the present study, we document the presence of a third IgE-binding structure on human LC, the IgE-binding protein (epsilon BP), an endogenous soluble beta-galactoside binding lectin. Immunohistochemical studies performed on normal human skin revealed an anti-epsilon BP reactivity in the cytoplasm of keratinocytes and in that of acinous cells of eccrine sweat glands. epsilon BP was also found on the cell surface of LC, as shown by anti-epsilon BP/anti-CD1a double labeling and flow cytometric analysis. Anti-epsilon BP binding to the surface of LC was completely abolished by preincubation with lactose and restored by addition of recombinant human epsilon BP, indicating that epsilon BP binds to LC surface by virtue of its lectin property. Immunoblot analysis of anti-epsilon BP-reactive material in keratinocytes and purified LC disclosed a protein with an apparent molecular weight of 33,000 consistent with epsilon BP. Interestingly, mRNA transcripts for epsilon BP were detected only in keratinocytes but not in purified LC isolated from normal skin. epsilon BP was found to be released in culture supernatants of keratinocytes. Incubation of LC with these supernatants resulted in epsilon BP-binding to LC surface via protein-carbohydrate interaction. Most importantly, we could show that binding of human myeloma IgE to LC was inhibited by epsilon BP. In contrast, neuraminidase-treated human myeloma IgE binds to LC only in the presence of epsilon BP. In situ binding studies revealed that keratinocytes, although containing epsilon BP intracytoplasmatically, failed to exhibit any IgE-binding properties. Collectively, our results suggest that human keratinocytes produce the beta-galactoside-binding lectin epsilon BP, which subsequently binds to the surface of LC where it is functional in modulating their binding capacity for IgE glycoforms.

Secretion of leukotriene C and other arachidonic acid metabolites by macrophages challenged with immunoglobulin E immune complexes.

Resident mouse peritoneal macrophages release the slow-reacting substance leukotriene C (LTC) on exposure to particulate IgE immune complexes. Because these cells lose their responsiveness to an IgE stimulus after 4 h in culture, maximum release of 20:4 metabolites is observed before this time. However, a similar diminution in 20:4 metabolism was not observed with a zymosan stimulus. Freshly explanted cells are deficient in intracellular glutathione (GSH) (12.4 +/- 0.4 pmol/micrograms cell protein), but GSH increases to a steady state value of 30-35 pmol/micrograms of cell protein between 3 and 9 h of culture. Because GSH is required for the synthesis of LTC and prostaglandin (PG)E2, cultures challenged immediately after explanation have a diminished capacity to synthesize these 20:4 metabolites and release prostacyclin as the major product. By 4-5 h in culture, macrophages form significant amounts of LTC and PGE2. Under optimum conditions of maximum responsiveness to an IgE stimulus and GSH content (after 4 h of culture), macrophages challenged with latex beads coated with IgE immune complexes synthesize 1.0 +/- 0.3 pmol of LTC/microgram cell protein (60 +/- 18 pmol/10(6) cells) in addition to prostacyclin (8.2 +/- 0.8 pmol/micrograms cell protein) and PGE2 (4.7 +/- 1.5 pmol/micrograms cell protein). These amounts are quantitatively similar to the arachidonic acid metabolites produced by macrophages challenged with IgG immune complex-coated latex beads or zymosan. These data demonstrate that macrophages produce large quantities of LTC and other 20:4 metabolites in response to particle-bound IgE and antigen, provided that the appropriate in vitro conditions are met. The macrophage might, therefore, be a major source of slow-reacting substance and other 20:4 metabolites generated during IgE-mediated reactions in vivo.

IgE-mediated release of leukotriene C4, chondroitin sulfate E proteoglycan, beta-hexosaminidase, and histamine from cultured bone marrow-derived mouse mast cells.

Mouse bone marrow-derived mast cells differentiated in vitro and sensitized with monoclonal IgE respond to antigen-initiated activation with the release of histamine, beta-hexosaminidase, chondroitin sulfate E proteoglycan, and leukotriene C4 (LTC4). The chondroitin sulfate E nature of the glycosaminoglycan side chain was established by demonstrating that the chondroitinase ABC disaccharide digestion products were composed of equal quantities of 4-sulfated and 4,6-disulfated N-acetyl-galactosamine. The single immunoreactive sulfidopeptide leukotriene, released and quantitated with a class-specific antibody, was identified as LTC4 by its retention time on reverse-phase high-performance liquid chromatography and by its specific spasmogenic activity on the guinea pig ileum. The release of the preformed mediators, as well as of LTC4, was related in a dose-response fashion to the concentration of monoclonal IgE used during the sensitization step and to the concentration of specific antigen used to initiate the activation-secretion response. The optimal concentrations of IgE for sensitization and of antigen for challenge were the same for the release of preformed mediators and of LTC4. In addition, the time courses of their release were superimposable, with a plateau at 5 min after antigen challenge. The release of three preformed mediators and of LTC4 after fixation of IgE, washing of the sensitized cells, and antigen challenge unequivocally indicates a bone marrow-derived mast cell origin for these products. Linear regression analyses of the net percent release of beta-hexosaminidase to histamine and of 35S-chondroitin sulfate E to beta-hexosaminidase yielded straight lines that intersected at the origin, which indicates that the three preformed mediators are localized in the secretory granules of the bone marrow-derived mast cells. The concomitant generation of 23 ng of LTC4/10(6) sensitized bone marrow-derived mast cells represents the first example of IgE-dependent release of substantial amounts of LTC4, a component of slow reacting substance of anaphylaxis, from a mast cell population of greater than 95% purity. The IgE-dependent generation of LTC4, rather than prostaglandin D2, by the chondroitin sulfate E proteoglycan-containing bone marrow-derived mast cells contrasts with the predominant generation of prostaglandin D2 by heparin proteoglycan-containing mast cells. These differences together support the existence of two phenotypically different mast cell subclasses.

Transgenic mice expressing the human high-affinity immunoglobulin (Ig) E receptor alpha chain respond to human IgE in mast cell degranulation and in allergic reactions.

The high-affinity receptor for immunoglobulin (Ig) E (Fc epsilon RI) on mast cells and basophils plays a key role in IgE-mediated allergies. Fc epsilon RI is composed of one alpha, one beta, and two gamma chains, which are all required for cell surface expression of Fc epsilon RI, but only the alpha chain is involved in the binding to IgE. Fc epsilon RI-IgE interaction is highly species specific, and rodent Fc epsilon RI does not bind human IgE. To obtain a "humanized" animal model that responds to human IgE in allergic reactions, transgenic mice expressing the human Fc epsilon RI alpha chain were generated. The human Fc epsilon RI alpha chain gene with a 1.3-kb promoter region as a transgene was found to be sufficient for mast cell-specific transcription. Cell surface expression of the human Fc epsilon RI alpha chain was indicated by the specific binding of human IgE to mast cells from transgenic mice in flow cytometric analyses. Expression of the transgenic Fc epsilon RI on bone marrow-derived mast cells was 4.7 x 10(4)/cell, and the human IgE-binding affinity was Kd = 6.4 nM in receptor-binding studies using 125I-IgE. The transgenic human Fc epsilon RI alpha chain was complexed with the mouse beta and gamma chains in immunoprecipitation studies. Cross-linking of the transgenic Fc epsilon RI with human IgE and antigens led to mast cell activation as indicated by enhanced tyrosine phosphorylation of the Fc epsilon RI beta and gamma chains and other cellular proteins. Mast cell degranulation in transgenic mice could be triggered by human IgE and antigens, as demonstrated by beta-hexosaminidase release in vitro and passive cutaneous anaphylaxis in vivo. The results demonstrate that the human Fc epsilon RI alpha chain alone not only confers the specificity in human IgE binding, but also can reconstitute a functional receptor by coupling with the mouse beta and gamma chains to trigger mast cell activation and degranulation in a whole animal system. These transgenic mice "humanized" in IgE-mediated allergies may be valuable for development of therapeutic agents that target the binding of IgE to its receptor.

Targeted disruption of the galectin-3 gene results in decreased susceptibility to multiple low dose streptozotocin-induced diabetes in mice.

Galectin 3 (Gal-3) is an antiapoptotic and a proinflammatory lectin. We hypothesized that the proinflammatory properties of Gal-3 may influence disease induction in the multiple low doses of streptozotocin model of diabetes. Diabetes was induced in C57BL/6 Gal-3(+/+) and Gal-3(-/-) mice and disease monitored by blood glucose level, immuno-histology, insulin content of islets and expression of the proinflammatory cytokines, TNF-alpha, IFN-gamma, IL-17, and iNOS in pancreatic lymph nodes. Gal-3(+/+) mice developed delayed and sustained hyperglycemia, mononuclear cellular infiltration and reduced insulin content of islets accompanied with expression of proinflammatory cytokines. Gal-3(-)/(-) mice were relatively resistant to diabetogenesis as evaluated by glycemia, quantitative histology and insulin content. Further, we observed the weaker expression of IFN-gamma and complete absence of TNF-alpha, and IL-17 in draining pancreatic lymph nodes. Macrophages, the first cells that infiltrate the islet in this model of diabetes, produce less TNF-alpha and NO in Gal-3(-/-) mice. Thus, Gal-3 is involved in immune mediated beta cell damage and is required for diabetogenesis in this model of disease.

[Experimental study of toluidine blue-dextran-40 as lymphatic tracer in head and neck region].

Objective: To investigate the feasibility and application value of toluidine bule-dextran-40 (TB-Dex-40) as the tracer for lymphatic system in head and neck region. Methods: Twenty healthy adult New Zealand white rabbits were equally divided into two groups: the experimental group (TB-Dex-40 group, n=10) and the control group (TB group, n=10). Rabbits in experimental group received submucosal injection of 1.0% (0.14 mOsm/L) TB-Dex-40, and the control group received injection of 1.0% (32.60 mOsm/L) TB.The staining time and fading time of lymphatic vessels and lymphnodes in the neck region were recorded, and the diffusion ranges of the two dyes in the tongue region were measured. Lymph nodespecimen were collected for pathological examination after 10 min, 1 hour and 4 weeks of staining. The experimental animals were sacrificed before and 4 weeks after the experiment. After death, organs of heart, lung, liver and kidney were examined pathologically. Results: TB-Dex-40 reached sentinel lymph node (SLN) and stained lymphatic vessels at an average of (21.67±0.19) s after injection, while in control group was(3.22±0.34) s (P<0.01). The time difference between the two dyes reaching sentinel lymph nodes was statistically significant.The durations from lymphatic staining to marked fading were (19.70±1.34) min in experimental group and (14.30±0.95) min in control group, respectively.The difference was statistically significant (P<0.01). SLN staining by TB-Dex-40 was still evident after 4 weeks, while TB staining has completely faded after 2 d.The average ranges of diffusionin tongue were (10.50±1.08) mm in experimental group and (20.00±1.05) mm in controlgroup, respectively. The difference was statistically significant (P<0.01).No abnormalities were found in blood test and pathological examination of main organs. Conclusions: TB-Dex-40 has high specificity forstaining lymphatic vessels and is a good tracer with potential clinical value.

[Hypoxia increases chemotherapy resistance in nasopharyngeal carcinoma via inducing CDK6 deSUMOylation].

Objective: To understand the mechanism of chemotherapy resistance in nasopharyngeal carcinoma under hypoxic conditions through the perspective of protein SUMOylation modification. Methods: Cobalt chloride (CoCl(2)) was used to establish the hypoxic model of human nasopharyngeal carcinoma CNE1 cells. Then, the cell cycle was detected by flow cytometry, and the expression level of small ubiquitin-related modifier(SUMO) and cyclin-dependent kinase 6 (CDK6) proteins were detected by western blotting. MTT assay was used to determine the median lethal dose (IC(50)) of cancer cells against cisplatin, and enzyme-linked immunosorbent assay (ELISA) was used to determine lactate dehydrogenase (LDH) level. Results: The cell cycle of CNE1 induced by hypoxia was arrested in G0/G1 phase.The results of Western blot showed that the protein expression level of CDK6 in CNE1 cells was lower than that in the control group (0.83±0.25 vs. 0.43±0.21, t=14.67, P=0.003). The protein level of conjugated SUMO1 was significantly lower than that in the control group (2.69±0.48 vs. 1.38±0.31, t=17.22, P=0.001), while the level of free SUMO1 protein was significantly higher than that in the control group (2.01±0.43 vs. 2.60±0.59, t=15.45, P=0.002).The LC50 of CNE1 cells in the control group was significantly lower than that in the hypoxic group (29.44 µg/ml vs. 97.72 µg/ml, t=12.79, P=0.001). After CNE1 cells received 50 µg/ml cisplatin for 48 h, the LDH content in the supernatant of the control group was significantly higher than that in the hypoxic group ((541.49±64.59) ng/ml vs. (234.67±41.03) ng/ml, t=11.94, P=0.007)). The apoptosis rate of CNE1 cells in the control group was significantly higher than that in the hypoxic group ((76.64±5.37)% vs. (32.84±4.77) ng/ml, t=8.49, P=0.003)). Conclusion: Hypoxia can dissociate the covalent modification of CDK6 and SUMO1, inhibit cell cycle and increase the chemotherapy resistance of nasopharyngeal carcinoma.

High-resolution three-dimensional imaging of the rich membrane structures of bone marrow-derived mast cells.

Atomic force microscopy (AFM) enables high-resolution three-dimensional (3D) imaging of cultured bone marrow-derived mast cells. Cells were immobilized by a quick centrifugation and fixation to preserve their transient cellular morphologies followed by AFM characterization in buffer. This "fix-and-look" approach preserves the structural integrity of individual cells. Well-known membrane morphologies, such as ridges and microvilli, are visualized, consistent with prior electron microscopy observations. Additional information including the 3D measurements of these characteristic features are attained from AFM topographs. Filopodia and lamellopodia, associated with cell spreading, were captured and visualized in three dimensions. New morphologies are also revealed, such as high-density ridges and micro-craters. This investigation demonstrates that the "fix-and-look" approach followed by AFM imaging provides an effective means to characterize the membrane structure of hydrated cells with high resolution. The quantitative imaging and measurements pave the way for systematic correlation of membrane structural features with the biological status of individual cells.

Kinetics of mobilization and differentiation of lymphohematopoietic cells during experimental murine schistosomiasis in galectin-3 -/- mice.

Galectin-3 (gal-3), a beta-galactoside-binding animal lectin, plays a role in cell-cell and cell-extracellular matrix interactions. Extracellular gal-3 modulates cell migration and adhesion in several physiological and pathological processes. Gal-3 is highly expressed in activated macrophages. Schistosoma mansoni eggs display a large amount of gal-3 ligands on their surface and elicit a well-characterized, macrophage-dependent, granulomatous, inflammatory reaction. Here, we have investigated the acute and chronic phases of S. mansoni infection in wild-type and gal-3(-/-) mice. In the absence of gal-3, chronic-phase granulomas were smaller in diameter, displaying thinner collagen fibers with a loose orientation. Schistosoma-infected gal-3(-/-) mice had remarkable changes in the monocyte/macrophage, eosinophil, and B lymphocyte subpopulations as compared with the infected wild-type mice. We observed a reduction of macrophage number, an increase in eosinophil absolute number, and a decrease in B lymphocyte subpopulation (B220(+/high) cells) in the periphery during the evolution of the disease in gal-3(-/-) mice. B lymphopenia was followed by an increase of plasma cell number in bone marrow, spleen, and mesenteric lymph nodes of the infected gal-3(-/-) mice. The plasma IgG and IgE levels also increased in these mice. Gal-3 plays a role in the organization, collagen distribution, and mobilization of inflammatory cells to chronic-phase granulomas, niches for extramedullary myelopoiesis, besides interfering with monocyte-to-macrophage and B cell-to-plasma cell differentiation.