OBJECTIVEIt is known that Siglec-8 is selectively expressed on human eosinophils at a high level and mediates eosinophil apoptosis when crosslinked with its antibody. The aim of our review is to elucidate the molecular and biological characteristic of Siglec-8 and then discuss the function and possible mechanisms of Siglec-8 in eosinophils. Thereby, we will expand our understanding to the regulation of eosinophil apoptosis, and provide important clues to the treatment of asthma and other hyper-eosinophilic diseases.

DATA SOURCESMost articles were identified by searching of PubMed online resources using the key term Siglecs.

STUDY SELECTIONMainly original milestone articles and critical reviews written by major pioneer investigators in the field were selected.

RESULTSSiglec-8 is selectively expressed on human eosinophil and can specifically induce eosinophil apoptosis.

CONCLUSIONThe restricted expression of Siglec-8 on human eosinophil and the rapid progress in understanding its role as cell signaling and activation of death receptors have made it an attractive target for treatment of asthma and other hyper-eosinophilic diseases.

Apoptosis ; genetics ; physiology ; Asthma ; metabolism ; therapy ; Eosinophils ; cytology ; metabolism ; Humans ; Sialic Acid Binding Immunoglobulin-like Lectins ; metabolism

Using total DNA isolated from Amaranthus caudatus as the template, a DNA fragment of about 700bp upstream of the coding sequence of Amaranthus caudatus agglutinin (ACA) gene was amplified by TAIL-PCR and cloned. To examine the regulatory function of this DNA fragment, it was inserted into a plant expression vector containing GUS gene to substitute the CaMV 35S promoter and the resulted recombinant plasmid was designated as pBpAG. The expression vector pBpAG was transferred to different tissues of plants, via Agrobacterium-mediated transformation in vacuum condition. Transient expression of GUS in the transformed tissues was detected by histochemical GUS staining and the results showed that the GUS activity was expressed specifically in seeds. These preliminary results indicate that this DNA fragment upstream of the ACA coding sequence could very possibly be a promoter with seed specificity. Some putative cis-elements within the promoter were discussed.
Amaranthus ; genetics ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Molecular Sequence Data ; Plant Lectins ; genetics ; Promoter Regions, Genetic ; genetics ; Rhizobium ; genetics ; metabolism

The complement system is a key component of innate immunity. More than 45 genes encoding the proteins of complement components or their isotypes and subunits, receptors, and regulators have been discovered. These genes are distributed throughout different chromosomes, with 19 genes comprising three significant complement gene clusters in the human genome. Genetic deficiency of any early component of the classical pathway (C1q, C1r/s, C2, C4, and C3) is associated with autoimmune diseases due to the failure of clearance of immune complexes (IC) and apoptotic materials, and the impairment of normal humoral response. Deficiencies of mannan-binding lectin (MBL) and the early components of the alternative (factor D, properdin) and terminal pathways (from C3 onward components: C5, C6, C7, C8, C9) increase susceptibility to infections and their recurrence. While the association of MBL deficiency with a number of autoimmune and infectious disorders has been well established, the effects of the deficiency of other lectin pathway components (ficolins, MASPs) have been less extensively investigated due to our incomplete knowledge of the genetic background of such deficiencies and the functional activity of those components. For complement regulators and receptors, the consequences of their genetic deficiency vary depending on their specific involvement in the regulatory or signalling steps within the complement cascade and beyond. This article reviews current knowledge and concepts about the genetic load of complement component deficiencies and their association with diseases. An integrative presentation of genetic data with the latest updates provides a background to further investigations of the disease association investigations of the complement system from the perspective of systems biology and systems genetics.
Animals ; Complement Activation ; genetics ; Complement System Proteins ; deficiency ; genetics ; HLA Antigens ; genetics ; Humans ; Immunity, Innate ; genetics ; Immunologic Deficiency Syndromes ; genetics ; Lectins ; metabolism ; Multigene Family ; Receptors, Complement ; genetics

Cell adhesive molecular P-selectin was cloned, expressed and anchored on CHO cell membrane through GPI for selection specific antibodies. Total human platelet RNA was extracted and the functional segment of P-selectin gene was cloned by RT-PCR. The P-selectin functional segment gene was cloned into a eukaryotic expression vector pMCEw2-GPI containing an attenuated neo gene together with a downstream GPI, which was synthesized by overlapping PCR. The recombinant plasmid pMCEw2-GPI-P-selectin was then transfected to CHO(dhfr-) cells and screened with G418. ELISA, western-blot and immunofluorescence were carried out to detect the stability of P-selection expression on cell membrane. These results provided a necessary basis for the following study of selection the antibodies targeting P-selectin.
Animals ; Antibodies ; metabolism ; CHO Cells ; Cell Membrane ; metabolism ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Epidermal Growth Factor ; metabolism ; Glycosylphosphatidylinositols ; metabolism ; Lectins ; metabolism ; P-Selectin ; biosynthesis ; genetics ; immunology ; RNA ; metabolism ; Transfection

OBJECTIVETo explore the mechanism of Flt3 receptor-interacting lectin (FRIL) maintains quiescence of hematopoietic stem cells (HSCs) in vitro.

METHODSCord blood CD34+ cells were cultured in suspension medium supplemented with or without FRIL and FL. Cells were collected at different time points and the expression of some cell cycle regulators, especially those involved in G0/G1 phase regulation were detected on mRNA and protein level.

RESULTSThe expressions of G0/G1 phase related cyclins or CDKs were undetectable in the newly isolated CD34+ cells, expressions of Cyclin D3, CDK6 and P27 were the lowest in FRIL cultured group after 3d's culture (FRIL group: 483 +/- 63, 553 +/- 39, 0.312 +/- 0.030; FL group: 2437 +/- 52, 3209 +/- 98, 0.787 +/- 0.024; BLANK: 914 +/- 105, 1497 +/- 55, 0.616 +/- 0.029, respectively), but the expression of P53 was the highest in FRIL group (FRIL group: 4.476 +/- 0.159; FL group: 0.581 +/- 0.099, BLANK: 2.167 +/- 0.114). The expression of positive regulators of cell cycle in FRIL group were the same as that of FL group and blank group or lower.

CONCLUSIONFRIL preserves HSCs effectively in vitro through the mechanisms of down-regulation of cyclin D3 and CDK6 and activation of P53. P27 is mostly involved in the differentiation of HSCs.

Antigens, CD34 ; Cell Cycle ; drug effects ; Cell Cycle Proteins ; genetics ; metabolism ; Cells, Cultured ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; drug effects ; metabolism ; Humans ; Mannose-Binding Lectins ; pharmacology ; Plant Lectins ; pharmacology ; RNA, Messenger ; genetics

Ex vivo maintainance of human stem cells is crucial for many clinical applications. Current culture conditions provide some level support but cytokines induce most quiescent stem cells to proliferate and differentiate. Better control of primitive cells is needed to extend the time and range of manipulation of such cells. A recently identified plant lectin Flt3 receptor-interacting lectin (FRIL) present may a special ability to preserve primitive CB progenitors for extended periods in culture without exogenous cytokines. But the mechanisms of FRIL preserving quiescent primitive cells are still unknown. Recently a novel protein HTm4 and its alternatively spliced variant HTm4S, which serve as hematopoietic cell cycle regulators, have been identified. In this report we studied the effect of FRIL on the in vitro maintenance of quiescent human cord blood stem cells and the expression of the novel hematopoietic cell cycle regulator HTm4 and HTm4S in progenitor cells cultured in FRIL. We analyzed the proliferation and the HPP-CFC proportion of CD34(+) cells treated with FRIL. The human HTm4 and HTm4S mRNA expression was detected by semi-quantitative RT-PCR, and the cell cycle status of CB CD34(+) cells was analyzed by FACS. The results showed that incubation of CD34(+) cells in FRIL resulted in a low proliferation of progenitor cells and fewer cycling cells, but FRIL selectively maintained a higher number of primitive cells with proliferative potential in suspension culture. CB CD34(+) cells cultured in FRIL showed significant diversity in the expression of HTm4 and HTm4S during 0~14 d. On d 0, HTm4 was detected at high level, downregulated on d 1, but upregulated during d 3 to d 14, and reaching the highest level on d 7. But the expression levels of HTm4S changed little in the cells cultured in FRIL except the obviously increased expression on d 7. Exogenous expression showed that HTm4 was localized around the karyon while HTm4S scatted in the cytoplasm, respectively, which may be responsible for their difference in function. Thus, FRIL can preserve quiescent primitive CD34(+), and FRIL's ability to preserve quiescent primitive cells in a reversible manner may significantly expand the time and range of ex vivo manipulations of human stem cells for clinical applications. In other words, HTm4 and HTm4S may play a crucial role in the cell cycle modulation of CD34(+) progenitor cells maintained with FRIL in vitro.
Antigens, CD20 ; biosynthesis ; genetics ; Antigens, CD34 ; metabolism ; Cell Cycle ; Cell Cycle Proteins ; biosynthesis ; genetics ; Cell Separation ; Cells, Cultured ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Mannose-Binding Lectins ; pharmacology ; Membrane Proteins ; biosynthesis ; genetics ; Plant Lectins ; pharmacology

OBJECTIVETo discover and identify differentially expressed genes associated with colorectal adenoma formation and the role of RegIV in colorectal adenoma differentiation.

METHODSA subtracted cDNA library was constructed with cDNAs that were isolated from either the normal mucosa or adenoma tissue of a single patient. Suppressive subtractive hybridization (SSH) combined with virtual northern blotting was used to characterize differentially expressed genes and contigs were assembled by electronic cloning (in silico cloning) with the EST database. Semi-quantitative RT-PCR was performed in 9 colorectal adenomas.

RESULTSThe amino acid sequence was determined with open reading frame (ORF) prediction software and was found to be 100% homologous to the protein product of RegIV (a novel gene isolated from a large inflammatory bowel disease library). RegIV was found to be highly expressed in all of the adenoma samples (9/9) compared with the normal mucosa samples, while 5/6 cases showed RegIV to be more strongly expressed in adenocarcinoma.

CONCLUSIONRegIV may play an important role in the initiation of colorectal adenoma differentiation, and its detection may be useful in the early diagnosis of colorectal adenoma formation.

Adenoma ; genetics ; metabolism ; Blotting, Northern ; Colorectal Neoplasms ; genetics ; metabolism ; Humans ; Lectins, C-Type ; biosynthesis ; genetics ; Nucleic Acid Hybridization ; Pancreatitis-Associated Proteins ; Prognosis ; Reverse Transcriptase Polymerase Chain Reaction

Pancreatic cancer is a notorious disease with a poor prognosis and low survival rates, which is due to limited advances in understanding of the molecular mechanism and inadequate development of effective treatment options over the decades. In previous studies, we demonstrated that a novel soluble protein named pancreatic adenocarcinoma up-regulated factor (PAUF) acts on tumor and immune cells and plays an important role in metastasis and progression of pancreatic cancer. Here we show that PAUF promotes adhesiveness of pancreatic cancer cells to various extracellular matrix (ECM). Our results further support a positive correlation of activation and expression of focal adhesion kinase (FAK), a key player in tumor cell metastasis and survival, with PAUF expression. PAUF-mediated adhesiveness was significantly attenuated upon blockade of the FAK pathway. Moreover, PAUF appeared to enhance resistance of pancreatic cancer cells to anoikis via modulation of FAK. Our results suggest that PAUF-mediated FAK activation plays an important role in pancreatic cancer progression.
Anoikis/genetics ; Cell Line, Tumor ; Focal Adhesion Protein-Tyrosine Kinases/*metabolism ; Focal Adhesions/genetics/*metabolism ; Humans ; Lectins/genetics/*metabolism ; Pancreatic Neoplasms/enzymology/genetics/*metabolism ; Proto-Oncogene Proteins pp60(c-src)/metabolism ; Signal Transduction/genetics

In this study, we constructed a yeast consortium surface-display expression system by using Flo1 as an anchor protein. Endoglucanase II (EGII) and cellobiohydrolase II (CBHII) from Trichoderma reesei, and β3-glucosidase 1 (BGLI) from Aspergillus aculeatus were immobilized on Saccharomyces cerevisiae Y5. We constructed the cellulose-displaying expression yeast consortium (Y5/fEGII:Y5/fCBHII:Y5/fBGLI = 1:1:1) and investigated the enzymatic ability and ethanol fermentation. The displayed cellulolytic enzymes was stabile during the 96-h fermentation. The yeast consortium produced 0.77 g/L ethanol from 10 g/L phosphoric acid swollen cellulose (PASC) within 96 h. The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.35 g/g, which correspond to 68.6% of the theoretical yield.
Aspergillus ; enzymology ; Cellulase ; genetics ; Cellulose ; metabolism ; Cellulose 1,4-beta-Cellobiosidase ; genetics ; Enzymes, Immobilized ; genetics ; Ethanol ; metabolism ; Fermentation ; Glucosidases ; genetics ; Mannose-Binding Lectins ; metabolism ; Protein Binding ; Saccharomyces cerevisiae ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; metabolism ; Trichoderma ; enzymology

OBJECTIVETo clone the recombinant human islet neogenesis-associated protein (rhINGAP) gene for its secretory expression in Pichia pastoris.

METHODSINGAP gene was amplified with PCR and inserted between Xho I and EcoR I downstream sites of the alpha factor of the recombinant plasmid alpha/pUC18. The fusion gene of alpha factor and INGAP was subsequently inserted between BamH I and EcoR I sites of the plasmid pPIC9K of P. pastoris. After confirmation with restriction enzyme digestion and sequencing, the positive recombinant plasmid that integrated INGAP gene was linearized with Sal I digestion and transformed into the yeast host strain GS115 through electroporation. The yeast transformants that harbored the INGAP gene with high copies were selected with the auxotroph medium and G418, followed then by PCR verification of the positive transformants, from which the expression of recombinant human INGAP was induced with methanol as the only carbone source. The antigenic activity of the desired protein was then detected using Western blotting and enzyme-linked immunosorbent assay (ELISA).

RESULTS AND CONCLUSIONThe recombinant expression plasmid INGAP/pPIC9K was successfully constructed, and 3 positive transformants were obtained. The expressed protein showed good antigenic activity as confirmed by Western blotting and ELISA.

Antigens, Neoplasm ; genetics ; metabolism ; Biomarkers, Tumor ; genetics ; metabolism ; Blotting, Western ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Humans ; Islets of Langerhans ; metabolism ; Lectins, C-Type ; genetics ; metabolism ; Pancreatitis-Associated Proteins ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; metabolism