B→O blood conversion

To maintain a constant supply of “universal blood type”,or group O red blood cells,has benefit in specialized transfusion condition.In this study, α-galactosidase cDNA was cloned from Catimor coffee bean grown in Hainan island,China,by the RT-PCR method.We have constructed a vector for α-galactosidase cDNA expression and transferred α-galactosidase cDNA into Pichia pastoris GS115 cells by electroporation.The recombinant α-galactosidase(r-α-GalE)was purified by cation ion exchange chromatography.After studying the biochemical characters of r-α-GalE we have succeeded in converting human erythrocytes from group B to group O.The animal experiment showed that transfusion of enzymetically converted group O red blood cells(ECORBC)was perfectly safe.     

Down-regulation of αGal epitopes by co-transfection of α1,3-galactosidase gene and α1,2–fucosyltransferase gene

The polycarbohydrate structure of Galα1- 3Ga1β1-4GluNAc-R （known as αGal epitopes of xenoantigen）, produced by α1-3-galactosyltransferase （α1,3-GT） in the course of animal development, is the major xenoantigen on the cell surface of porcine which causes hyperacute rejection in pig-to-human xenotransplantation. Alpha-1,3-galactosidase （AGL）, a hydrolytic enzyme, can remove the terminal α1,3-galactosyi from the Galα1-3Galβ1-4GluNAc-R structure resulting in cleaning αGai epitopes from the porcine cells. Aipha-1,2-fucosyitransferase （HT） can modify the surface carbohydrate phenotype of porcine cells, bringing about reduction of αGai epitopes expression. In this study, human AGL and HT gene were co-transfected to porcine fetal fibroblast （PFFb） in equimolar concentration to reduce the xenoantigen. Gene and protein of hAGL and HT were both detected to express at high level by RT-PCR and Western blot, respectively. There was an 84% reduction in αGai xenoantigen and an 82% increase in H antigen as assayed by flow cytometry in the AGL and HT gene co-transfected PFFb. The number and morphology of transgenic PFFb chromosome were normal. Findings indicate that Galα1-3Gal epitopes of PFFb could be down regulated by AGL and HT co-transfection without deleterious effects on the chromosomal profile of the transgenic ceil.     

Human RBCs blood group conversion from A to O using a novel α-N-acetylgalactosaminidase of high specific activity

α-N-acetylgalactosaminidase （αNAGA） can convert group A human red blood cells （RBCs） to group O. One novel αNAGA gene was cloned by PCR from Elizabethkingia meningosepticum Isolated from a domestic clinical sample. Pure recombinant αNAGA was obtained by genetic engineering and protein purification with a calculated molecule of 49.6 kD. αNAGA was selective for terminal α-N-acetylgalacto- samine residue with a high specific activity, αNAGA could completely remove A antigens of 1 U （about 100 mL） group A1 or A2 RBCs in 1 h at pH 6.8 and 25℃ with s consumption of 1.5 or 0.4 mg recombinant enzyme. Enzyme-converted group A RBCs did not agglutinate after being mixed with monoclonal snti-A or sere of groups A, B, AB and O. Other blood group antigens except ABO had no change. FCM analysis showed that A antigens and A1 antigens disappeared while H antigens increased. It indicated that αNAGA successfully converted human blood group A RBCs to universally transfusable group O RBCs without the risk of ABO-incompaUble transfusion reactions. This αNAGA was suitable for producing universal RBCs to increase clinical transfusion safety, improve the RBCs supply, and to decrease transfusion cost and support transfusion service in case of emergency.     

Down-regulation of αGal epitopes by co-transfection of α1,3-galactosidase gene and α1,2–fucosyltransferase gene

Pig is considered the primary alternative species for xenotransplantation because of ethical considerations, breeding characteristics, infectious disease concerns, and its compatible size and physiology[1,2].A major barrier of pig-to-primate transplantation is the presence of terminal αGal epitopes on the surface of pig cells. αGal epitopes were formed by α1,3-galactosyltransferase (α1,3-GT) catalysis[3,4]. Humans and Old World monkeys have lost the corresponding α1,3-GT activity in th…     

Prediction of shear stress-related hemolysis in centrifugal blood pumps by computational fluid dynamics

A quantitative evaluation of shear stress-related hemolysis in centrifugal blood pumps with different impeller designs has been investigated. Computational fluid dynamics (CFD) is applied to track the shear stress history of the streamlines of red cells. The power law model of the relations among the hemolysis, shear stress and exposure time is used to evaluate the hemolysis in the pumps. Hemolysis tests are also conducted to verify the estimations. Both the estimations and experimentally measured hemolysis levels show that the hemolysis in the streamlined impeller pump developed by the authors is lower than the pump with straight-vane under the same boundary conditions. The approach is proved to be acceptable and practical to predict hemolysis levels of blood pumps.     

Distribution of a-MSH-like immunoreactive cells in the nervous system,Hatschek's pit and other tissues of amphioxus,Branchiostoma belcheri

Immunohistochemical localization of α-melanocyte-stimulating hormone （α-MSH） in the nervous system, Hatschek＇s pit and other tissues of amphioxus （Branchiostorna belcheri） was performed using the antibody against synthetic α-MSH. The results revealed that α-MSH-like immunoreactive cells were distributed at the dorsal side and ventral side of brain vesicle, the dorsal side and the surrounding of nerve tube, and in the epithelial cells of Hatschek＇s pit, the zone 1, 3, and 6 of endostyle and gut. The immunoreactive substance was also found in the primary oocytes of the small and large growth stage of ovary and early stage spermatogenic cells in testis. These findings indicate that α-MSH is an ancient and highly conserved hormone and it is extensively distributed in amphioxus. Although Hatschek＇s pit in amphioxus does not have a structure of the intermediate lobe of vertebrate adenohypophysis, it has already hosted α-MSH-like endocrine cells, implying that the functional differentiation of α-MSH-like cells occurred earlier than the differentiation of the tissue structure. The results of the present study provided a new evidence for the endocrinology of Hatschek＇s pit and for the origin and evolution of vertebrate adenohypophysis.     

Genistein inhibits human TNF-α-induced porcine endothelial cell adhesiveness for human monocytes and natural killer cells

Cellular immune response is a major barrier to xenotransplantation. Human tumor necrosis factor-α (hTNF-α) possesses cross-species activity and directly amplifies the immune rejection via the upregulation of adhesion molecules on porcine endothelium. We investigated the role of protein tyrosine phosphorylation in the induction of expression of E-sclectin and vascular cell adhesion molecule-1 (VCAM-1), and the augmentation of adhesion of human peripheral blood monocytes (PBMo) and natural killer cells (PBNK), after rhTNF-α-stimulation of porcine aortic endothelial cells (PAEC) in vitro, rhTNF-α-increased adhesiveness of PAEC for both PBMo and PBNK was dose-dependently reduced by pretreatment of PAEC with the selective protein tyrosine kinase (PTK) inhibitor genistein. The inhibitory effect occurred at the early time of PAEC activation triggered by rhTNF-α, and was completely reversible. PTK activity assay indicated that genistein also suppressed rhTNF-α stimulated activation of protein tyrosine kinases (PTKs) in PAEC in a dose-dependent manner. Flow cytometric analysis showed that genistein inhibited the upregulation of E-selectin and VCAM-1 by rhTNF-α. These results suggest that PTKs may regulate the expression of E-selectin and VCAM-1 on PAEC and the adherence of PBMo and PBNK induced by rhTNF-α. Moreover, dietary genistein, used as an adhesion antagonist, may contribute to managing the cell-mediated rejection in the clinical application.     

A new type of triploid crucian crap?red crucian carp (♀)×allotetraploid (♂)

A new type of triploid hybrids (TC) was produced by crossing red crucian carp (♀) with allotetraploid hybrids (♂). This type of triploids with no barbel was spindle-shaped and gray in color. Compared with their parents, some data of the morphological traits in the triploid hybrids were intermediate to those of their parents, and some were beyond their parents with heterozygous traits. Under the same culture conditions, the growth rate of the new type triploids was faster than their maternal red crucian carp (RC). The gonadal development of triploids was obviously slower than that of diploids, and the germ cells in them were degenerated. Compared with another type of triploids produced by Japanese crucian carp (♀) with allotetraploid hybrids (♂), the new type triploids not only kept faster growth rate and sterility, but also had no barbel that was similar to the common carp. The absence of the barbel in the new type of triploids has the important significance in both the inheritance and fish breeding.     

Distribution of α-MSH -like immunoreactive cells in the nervous system, Hatschek’s pit and other tissues of amphioxus, Branchiostoma belcheri

Immunohistochemical localization of α-melanocyte-stimulating hormone (α-MSH) in the nervous system, Hatschek’s pit and other tissues of amphioxus (Branchiostoma belcheri) was performed using the antibody against synthetic α-MSH. The results revealed that α-MSH-like immunoreactive cells were distributed at the dorsal side and ventral side of the brain vesicle, the dorsal side and the surrounding of central tube in the nerve tube, the epithelial cells of Hatschek’s pit, and zones 1, 3 and 6 in endostyle and hindgut. The immunoreactive substance was also found in the primary oocytes of the small and large growth stage of ovary and early stage spermatogenic cells in testis. These findings indicate that α-MSH is an ancient and highly conserved hormone and it is extensively distributed in amphioxus. Although Hatschek’s pit in amphioxus do not have a structure of the intermediate lobe of vertebrate adenohypophysis, it has already hosted α-MSH-like endocrine cells, implying that the functional differentiation of α-MSH-like cells occurred earlier than the differentiation of the tissue structure. The results of present study provided new evidence for the endocrinology of Hatschek’s pit and the origin and evolution of vertebrate adenphypophysis.     

Single-cell discrimination based on optical tweezers Raman spectroscopy

The ability to discriminate between single cells in a label-free and noninvasive fashion is important for the classification of cells, and for the identification of similar cells from different origins. In this paper, we present the Raman spectroscopy-based identifi- cation of different types of single cells in aqueous media, and discrimination between the same types of cells from different donors using a novel Laser Tweezers Raman Spectroscopy (LTRS) technique, which combines laser trapping and micro-Raman spectroscopy. First, we measured the spectra of individual living human erythrocytes, i.e. red blood cells, and leucocytes (U937 cancer cells). High-quality Raman spectra with low fluorescence were obtained using a home-LTRS apparatus and 20 cells were measured for each cell type. The smoothing, baseline subtraction, and normalization of the data were followed by a principal components analysis (PCA). The PCA loading plots showed that the two different types of cells could be completely separated based only on the first component (PC1) (i.e. the peaks at 1300 cm1 ); the discrimination accuracy could therefore reach 100%. More than 50 spectra were taken for each erythrocyte obtained from the four healthy volunteers. The average discrimination accuracy was 84.5% for two random individuals taken from the four volunteers, according to the first and second PCs. This work demonstrates that LTRS is a powerful tool for the accurate identification and discrimination of single cells, and it has the potential to be applied for the highly sensitive identification of cells in clinical diagnosis and medical jurisprudence.     

IBCIS: intelligent blood cell identification system

The analysis of blood cells in microscope images can provide useful information concerning the health of patients. There are three major blood cell types, namely, erythrocytes （red）, leukocytes （white）, and platelets. Manual classification is time consuming and suscep- tible to error due to the different morphological features of the cells. This paper presents an intelligent system that simulates a human visual inspection and classification of the three blood cell types, The proposed system comprises two phases： The image preprocessing phase where blood cell features are extracted via global pattern averaging, and the neural network arbitration phase where training is the first and then classification is carried out. Experimental results suggest that the proposed method performs well in identifying blood cell types regardless of their irregular shapes, sizes and orientation, thus providing a fast, simple and efficient rotational and scale invariant blood cell identification system which can be used in automating laboratory reporting.     

The expression of N-cadherin /catenins /actin complex in human lung normal and carcinoma cells

The difference between human normal and carcinoma lung cells was studied with regard to the protein expression level and localization of the cadherin/catenin/actin complex. Results demonstrated that normal lung cell RF expressed high levels of N-cadherin, β-catenin, α-catenin. These 3 proteins were colocalized at AJs and their submembrane adhesion plaques where they link the Rho-phalloidin-positive actin stress fibers, indicating the existence of N-cadherin/catenin/actin complexes at the AJs. Aberrant expression of AJ proteins and the actin cytoskelecton in carcinoma PG cells was observed: (1) inhibition of N-cadherin and to a degree of inhibition of α-catenin protein expression; (2) varied protein modification of β-catenin in cytoplasm soluble fraction and altered distribution of immunofluorescence: majorly in the cytoplasm and minorly on the membrane; (3) disassembly of actin stress fibers and formation of actin bodies in the cytoplasm. The data suggest that inhibited expression of AJ proteins is correlated with the disruption of the AJ complexes and the actin cytoskeleton in carcinoma PG cells, and responsible for its metastasis behaviors.     

Comparative study on the effect of integrin αvβ3 on secretion of matrix metalloproteinases in human normal and carcinoma trophoblasts

The invasion of cytotrophoblast cells into the maternal endometrium during embryonic implantation is very similar to the metastasis of carcinoma cells. However, the significant difference is that the former is a highly controlled process. In this report, the effects of integrin αvβ3 on the secretion of matrix metalloproteinase (MMP) were compared between normal cytotrophoblast cells and choriocarcinoma cells by RT-PCR, gelatin zymography and immunocytochemistry. The results reveal that both normal cytotrophoblast cells and choriocarcinoma cells can express integrin αvβ3. The secretion of MMPs in normal cytotrophoblast cells is up-regulated by anti-αvβ3 antibody, whereas, decreased in choriocarcinoma cells. It was suggested that αvβ3 can modulate the expression of MMPs in trophoblasts, and this action is carried out through distinct mechanisms in normal and carcinoma cytotrophoblast cells.     

Effects of blocking LeY oligosaccharide on cell surface to MMPs secreted by blastocysts and epithelial cells in mousein vitro

In order to understand the role of Le+Y oligosaccharide antigen (Le+Y) during implantation, the relationship of Le+Y on the cell surface with matrix metalloproteinase (MMPs) secreted by blastocysts and monolayer epithelial cells during implantation in the mouse %in vitro% was studied by monoclonal antibody (mAb) AH-6, directed to Le+Y[Fuc α1-2 Gal β1-4 (Fuc α1-3) GlcNAc-], and gelatin zymography. The results showed that MMPs secretion was reduced after Le+Y on the cell surface of either epithelial cells or trophoblasts was blocked. It indicated that MMPs expression which played an important function during the process of implantation were regulated by Le+Y. Therefore, it was considered that Le+Y could regulate embryos invasion by some mechanism.     

Distribution of α-MSH-like immunoreactive cells in the nervous system, Hatschek''''s pit and other tissues of amphioxus, Branchiostoma belcheri

Immunohistochemical localization of a-melanocyte-stimulating hormone (α-MSH) in the nervous system, Hatschek's pit and other tissues of amphioxus (Branchiostoma belcheri) was performed using the antibody against synthetic α-MSH. The results revealed that α-MSH-like immunoreactive cells were distributed at the dorsal side and ventral side of brain vesicle, the dorsal side and the surrounding of nerve tube, and in the epithelial cells of Hatschek's pit, the zone 1, 3, and 6 of endostyle and gut. The immunoreactive substance was also found in the primary oocytes of the small and large growth stage of ovary and early stage spermatogenic cells in testis. These findings indicate that α-MSH is an ancient and highly conserved hormone and it is extensively distributed in amphioxus. Although Hatschek's pit in amphioxus does not have a structure of the intermediate lobe of vertebrate adenohypophysis, it has already hosted α-MSH-like endocrine cells, implying that the functional differentiation of α-MSH-like cells occurred earlier than the differentiation of the tissue structure. The results of the present study provided a new evidence for the endocrinology of Hatschek's pit and for the origin and evolution of vertebrate adenohypophysis.     

Determination of the genus-specific antigens in outer membrane proteins from the strains of Leptospira interrogans and Leptospira biflexa with different virulence

Objective:To determine the existence of genus-specific antigens in outer membrane proteins (OMPs) of leptospira with different virulence. Methods: Microscope agglutination test (MAT) was applied to detect the agglutination between commercial rabbit antiserum against leptospiral genus-specific TR/Patoc I antigen and 17 strains of Leptospira interrongans belonging to 15 serogroups and 2 strains of Leptospira biflexa belonging to 2 serogroups.The outer envelopes (OEs) of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain lai (56601) with strong virulence and serogroup Pomona serovar pomona strain Luo (56608) with low virulence,and L.biflexa serogroup Semaranga serovar patoc strain Patoc I without virulence were prepared by using the method reported in Auran et al.(1972).OMPs in the OEs were obtained by treatment with sodium deoxycholate. SDS-PAGE and western blot were used for analyzing the features of the OMPs on electrophoretic pattern and the immunoreactivity to the antiserum against TR/Patoc I antigen, respectively. Results:All the tested strains belonging to different leptospiral serogroups agglutinated to the antiserum against leptospiral genus-specific TR/Patoc I antigen with agglutination titers ranging from 1:256-1:512. A similar SDS-PAGE pattern of the OMPs from the three strains of leptospira with different virulence was shown and the molecular weight of a major protein fragment in the OMPs was found to be approximately 60 KDa.A positive protein fragment with approximately 32 KDa confirmed by Western blot,was able to react with the antiserum against leptospiral genus-specific TR/Patoc I antigen, and was found in each the OMPs of the three stains of leptospira.Conclusion: There are genus-specific antigens on the surface of L.interrogans and L.biflexa. The OMP with molecular weight of 32 KDa may be one of the genus-specific protein antigens of leptospira.     

Effect of polychlorinated biphenyls on spermatogenesis and testosterone secretion in adult cocks

The effects ofpolychlorinated biphenyls (PCBs) on reproduction of adult cocks were studied by gavaging peanut oil or PCBs (Aroclor 1254, 50 mg/kg) once a week for six consecutive weeks. Physiological parameters were recorded and gonads were removed at the end of experiment for histological examination. The results showed that there was no significant difference between the control and treatment group in body weight, respiration rate, heart rate, body temperature, and the numbers of red and white blood cells. However, there was a marked decrease in the testicular weight and serum testosterone level after PCB treatment. Morphological studies manifested severe damage of the seminiferous tubules by PCB. The number of the germ cells at the different developmental stages was decreased and condensed nuclei were observed in most of these cells. This study revealed that the reproductive function of the adult cocks is sensitive to PCBs, which inhibited mainly spermatogenesis and testosterone secretion.     

Analysis of porcine MHC expression profile

The porcine major histocompatibility complex (MHC, also named swine leukocyte antigen, SLA) is associated not only with immune responsibility and disease susceptibility, but also with some reproductive and productive traits such as growth rate and carcass composition. As yet systematical research on SLA expression profile is not reported. In order to illustrate SLA expression comprehensively and deepen our understanding of its function, we outlined the expression profile of SLA in 51 tissues of Landrace by analyzing a large amount of ESTs produced by ““Sino-Danish Porcine Genome Project““. In addition, we also compared the expression profile of SLA in several tissues from different development stages and from another breed (Erhualian). The result shows: (i) classical SLA genes are highly expressed in immune tissues and middle part of intestine; (ii) although SLA-3 is an SLA la gene, its expression abundance and pattern are quite different from those of the other two SLA la genes. The same phenomenon is seen in HLA-C expression, suggesting that the two genes may function similarly and undergo convergent evolution; (iii) except in jejunum, the antigen presenting genes are more highly expressed in breed Erhualian than in Landrace. The difference might associate with the higher resistance to bad conditions (including pathogens) of Erhualian and higher growth rates of Landrace.