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2.  Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the N-­terminal carbohydrate-recognition domain of human galectin-4 
The N-terminal carbohydrate-recognition domain of human galectin-4 has been crystallized by the vapour-diffusion technique using polyethylene glycol as the precipitant agent. A complete data set was collected from a native crystal to 2.2 Å resolution.
Galectin-4 is a tandem-repeat-type galectin that is expressed in the epithelium of the alimentary tract from the tongue to the large intestine. Additionally, strong expression of galectin-4 can also be induced in cancers in other tissues, including the breast and liver. In order to explore its potential as a target for anticancer drug design, elucidation of the structural basis of the carbohydrate-binding specificities of galectin-4 has been focused on. As an initial step, the N-­terminal carbohydrate-recognition domain of human galectin-4 (hGal4-CRD-­1) has been successfully crystallized using the vapour-diffusion technique, a complete data set has been collected to 2.2 Å resolution and the structure has been solved by the molecular-replacement technique. The crystals belonged to space group P6122, with unit-cell parameters a = b = 71.25, c = 108.66 Å. The asymmetric unit contained one molecule of hGal4-CRD-1, with a V M value of 2.34 Å3 Da−1 and a solvent content of 47.51%.
doi:10.1107/S1744309110010778
PMCID: PMC2864688  PMID: 20445255
N-terminal carbohydrate-recognition domain; human galectin-4
3.  5-Lipoxygenase Deficiency Impairs Innate and Adaptive Immune Responses during Fungal Infection 
PLoS ONE  2012;7(3):e31701.
5-lipoxygenase-derived products have been implicated in both the inhibition and promotion of chronic infection. Here, we sought to investigate the roles of endogenous 5-lipoxygenase products and exogenous leukotrienes during Histoplasma capsulatum infection in vivo and in vitro. 5-LO deficiency led to increased lung CFU, decreased nitric oxide production and a deficient primary immune response during active fungal infection. Moreover, H. capsulatum-infected 5-LO−/− mice showed an intense influx of neutrophils and an impaired ability to generate and recruit effector T cells to the lung. The fungal susceptibility of 5-LO−/− mice correlated with a lower rate of macrophage ingestion of IgG-H. capsulatum relative to WT macrophages. Conversely, exogenous LTB4 and LTC4 restored macrophage phagocytosis in 5-LO deficient mice. Our results demonstrate that leukotrienes are required to control chronic fungal infection by amplifying both the innate and adaptive immune response during histoplasmosis.
doi:10.1371/journal.pone.0031701
PMCID: PMC3308949  PMID: 22448213
4.  Expression of human protein S100A7 (psoriasin), preparation of antibody and application to human larynx squamous cell carcinoma 
BMC Research Notes  2011;4:494.
Background
Up-regulation of S100A7 (Psoriasin), a small calcium-binding protein, is associated with the development of several types of carcinomas, but its function and possibility to serve as a diagnostic or prognostic marker have not been fully defined. In order to prepare antibodies to the protein for immunohistochemical studies we produced the recombinant S100A7 protein in E. coli. mRNA extracted from human tracheal tumor tissue which was amplified by RT-PCR to provide the region coding for the S100A7 gene. The amplified fragment was cloned in the vector pCR2.1-TOPO and sub-cloned in the expression vector pAE. The protein rS100A7 (His-tag) was expressed in E. coli BL21::DE3, purified by affinity chromatography on an Ni-NTA column, recovered in the 2.0 to 3.5 mg/mL range in culture medium, and used to produce a rabbit polyclonal antibody anti-rS100A7 protein. The profile of this polyclonal antibody was evaluated in a tissue microarray.
Results
The rS100A7 (His-tag) protein was homogeneous by SDS-PAGE and mass spectrometry and was used to produce an anti-recombinant S100A7 (His-tag) rabbit serum (polyclonal antibody anti-rS100A7). The molecular weight of rS100A7 (His-tag) protein determined by linear MALDI-TOF-MS was 12,655.91 Da. The theoretical mass calculated for the nonapeptide attached to the amino terminus is 12,653.26 Da (delta 2.65 Da). Immunostaining with the polyclonal anti-rS100A7 protein generated showed reactivity with little or no background staining in head and neck squamous cell carcinoma cells, detecting S100A7 both in nucleus and cytoplasm. Lower levels of S100A7 were detected in non-neoplastic tissue.
Conclusions
The polyclonal anti-rS100A7 antibody generated here yielded a good signal-to-noise contrast and should be useful for immunohistochemical detection of S100A7 protein. Its potential use for other epithelial lesions besides human larynx squamous cell carcinoma and non-neoplastic larynx should be explored in future.
doi:10.1186/1756-0500-4-494
PMCID: PMC3278597  PMID: 22082027
S100A7 (Psoriasin); Recombinant protein; Production of a polyclonal antibody; E. coli BL21::DE3; Mass spectrometry
5.  Differential expression of immunomodulatory galectin-1 in peripheral leukocytes and adult tissues and its cytosolic organization in striated muscle 
Glycobiology  2010;20(5):507-520.
Galectin-1 (Gal-1) is important in immune function and muscle regeneration, but its expression and localization in adult tissues and primary leukocytes remain unclear. To address this, we generated a specific monoclonal antibody against Gal-1, termed αhGal-1, and defined a sequential peptide epitope that it recognizes, which is preserved in human and porcine Gal-1, but not in murine Gal-1. Using αhGal-1, we found that Gal-1 is expressed in a wide range of porcine tissues, including striated muscle, liver, lung, brain, kidney, spleen, and intestine. In most types of cells, Gal-1 exhibits diffuse cytosolic expression, but in cells within the splenic red pulp, Gal-1 showed both cytosolic and nuclear localization. Gal-1 was also expressed in arterial walls and exhibited prominent cytosolic and nuclear staining in cultured human endothelial cells. However, human peripheral leukocytes and promyelocytic HL60 cells lack detectable Gal-1 and also showed very low levels of Gal-1 mRNA. In striking contrast, Gal-1 exhibited an organized cytosolic staining pattern within striated muscle tissue of cardiac and skeletal muscle and colocalized with sarcomeric actin on I bands. These results provide insights into previously defined roles for Gal-1 in inflammation, immune regulation and muscle biology.
doi:10.1093/glycob/cwp203
PMCID: PMC2900886  PMID: 20053628
galectin-1 expression; leukocytes; monoclonal antibody; muscle; tissue localization
6.  Innate immune lectins kill bacteria expressing blood group antigen 
Nature medicine  2010;16(3):295-301.
The expression of ABO(H) blood group antigens causes deletion of cells that generate self anti-blood group antibodies, but this deletion limits adaptive immunity toward pathogens bearing cognate blood group antigens. To explore potential defense mechanisms against these pathogens, given such limitations in adaptive immunity, we screened for innate proteins that could recognize human blood group antigens. Here we report that two innate immune lectins, galectins-4 and -8, which are expressed in the intestinal tract, recognize and kill human blood group antigen-expressing E. coli, while failing to alter viability of other E. coli strains or other gram-negative or gram-positive organisms both in vitro and in vivo. Killing by both galectins-4 and -8 resides within their C-terminal domains, occurs rapidly and independently of complement, and is accompanied by disruption of membrane integrity. These results demonstrate that innate defense lectins can provide immunity against pathogens that display blood group self-antigens on their surface.
doi:10.1038/nm.2103
PMCID: PMC2853181  PMID: 20154696
7.  Galectin-1 Induces Reversible Phosphatidylserine Exposure at the Plasma Membrane 
Molecular Biology of the Cell  2009;20(5):1408-1418.
Cells normally undergo physiological turnover through the induction of apoptosis and phagocytic removal, partly through exposure of cell surface phosphatidylserine (PS). In contrast, neutrophils appear to possess apoptosis-independent mechanisms of removal. Here we show that Galectin-1 (Gal-1) induces PS exposure independent of alterations in mitochondrial potential, caspase activation, or cell death. Furthermore, Gal-1–induced PS exposure reverts after Gal-1 removal without altering cell viability. Gal-1–induced PS exposure is uniquely microdomain restricted, yet cells exposing PS do not display evident alterations in membrane morphology nor do they exhibit bleb formation, typically seen in apoptotic cells. Long-term exposure to Gal-1 prolongs PS exposure with no alteration in cell cycle progression or cell growth. These results demonstrate that Gal-1–induced PS exposure and subsequent phagocytic removal of living cells represents a new paradigm in cellular turnover.
doi:10.1091/mbc.E08-07-0786
PMCID: PMC2649277  PMID: 19116313

Results 1-7 (7)