Intra-Abdominal Cooling System Limits Ischemia-Reperfusion Injury During Robot-Assisted Renal Transplantation.

Robot-assisted kidney transplantation is feasible; however, concerns have been raised about possible increases in warm ischemia times. We describe a novel intra-abdominal cooling system to continuously cool the kidney during the procedure. Porcine kidneys were procured by standard open technique. Groups were as follows: Robotic renal transplantation with (n = 11) and without (n = 6) continuous intra-abdominal cooling and conventional open technique with intermittent 4°C saline cooling (n = 6). Renal cortex temperature, magnetic resonance imaging, and histology were analyzed. Robotic renal transplantation required a longer anastomosis time, either with or without the cooling system, compared to the open approach (70.4 ± 17.7 min and 74.0 ± 21.5 min vs. 48.7 ± 11.2 min, p-values < 0.05). The temperature was lower in the robotic group with cooling system compared to the open approach group (6.5 ± 3.1°C vs. 22.5 ± 6.5°C; p = 0.001) or compared to the robotic group without the cooling system (28.7 ± 3.3°C; p < 0.001). Magnetic resonance imaging parenchymal heterogeneities and histologic ischemia-reperfusion lesions were more severe in the robotic group without cooling than in the cooled (open and robotic) groups. Robot-assisted kidney transplantation prolongs the warm ischemia time of the donor kidney. We developed a novel intra-abdominal cooling system that suppresses the noncontrolled rewarming of donor kidneys during the transplant procedure and prevents ischemia-reperfusion injuries.

High levels of ceruloplasmin in the serum of transgenic mice developing hepatocellular carcinoma.

Transgenic mice expressing the Simian virus 40 large T antigen under the control of the liver-specific human antithrombin-III promoter all develop well-differentiated hepatocellular carcinoma. During tumour development serum ceruloplasmin (Cp) increases gradually until it reaches 30 times control levels in all transgenic mice at 6 months of age. The accumulation of Cp in the serum is due to the increased transcription of the Cp gene as well as to the increase in Cp mRNA stability in the livers of the transgenic mice. One-half of the overproduced Cp is charged with copper and Cp-associated serum oxidase activity increases in parallel with the holo-Cp concentration. Through its ferroxidase activity Cp is involved prominently in iron metabolism. Analysis of copper and iron in serum and liver revealed increased copper levels in the serum of tumour-bearing animals and which increased in parallel with Cp concentration; the amounts of copper in the liver were unchanged. In contrast, serum iron remained constant during tumour development whereas the iron concentration in the livers of the transgenic mice decreased.

The sugar binding activity of MR60, a mannose-specific shuttling lectin, requires a dimeric state.

MR60 is an intracellular membrane protein which has been shown to act as a mannoside specific lectin and to be identical to ERGIC-53, a protein characteristic of the endoplasmic reticulum-Golgi apparatus-intermediate compartment, acting as a shuttle. According to its primary sequence, this MR60/ERGIC-53 protein contains a luminal domain including the carbohydrate recognition domain, a stem, a transmembrane segment and a cytosolic domain. The endogenous MR60/ERGIC-53 protein is spontaneously oligomeric, (dimers and hexamers). In this paper, we study the relationship between the oligomerization state and the sugar binding capacity by using recombinant proteins. The expression of the recombinant proteins was evidenced by immunocytochemistry and by immunoprecipitation followed by SDS-PAGE analysis. The full size recombinant protein binds mannosides and is oligomeric, up to the hexameric form. Two truncated proteins lacking the transmembrane and the cytosolic domains were prepared and characterized. A long one, containing the cysteine 466 close to the C-terminal end of the recombinant protein but lacking the cysteine 475, close to the C-terminal end of the native protein, does bind mannosides and forms dimers but no higher oligomeric forms. A shorter one, lacking both the cysteines 466 and 475, does not bind mannosides and does not form dimers or higher polymers. The two cysteines in the carbohydrate recognition domain (C190 and C230) are not involved in the stabilization of oligomers. In conclusion, this study shows that the luminal moiety of MR60/ERGIC-53 contains a device allowing both its oligomeric pattern and its sugar binding capability.

Increased alpha2,6 sialylation of N-glycans in a transgenic mouse model of hepatocellular carcinoma.

Liver cancer is one of the most frequent and lethal malignancies worldwide. Early detection is hampered by the absence of reliable markers. Mice transgenic for the SV40 large T antigen under the control of a liver-specific promoter spontaneously develop well-differentiated hepatocellular carcinomas between 8 to 10 weeks of age. They are excellent models to investigate the alterations of protein expression in the early stages of tumor development and to follow these changes during tumor progression. In the present study, we analyzed the glycosylation changes occurring during tumor development in transgenic mice expressing the SV40 T antigen under the control of the antithrombin III promoter. The analysis of serum and liver glycoproteins by an ELISA type assay, using the lectin from Sambucus nigra (SNA) as a probe, revealed the presence of increased levels of Neu5Ac alpha2,6Gal beta1,4GlcNAc on N-glycans in the tumor-bearing transgenic mice as compared to controls. On serum glycoproteins the increase in alpha2,6 sialylation followed tumor progression, reaching up to 10 times control levels. However, significantly higher SNA binding (2-fold) could already be observed on serum glycoproteins from mice exhibiting only microscopically small neoplastic foci. On liver membrane glycoproteins, the increase in alpha2,6 sialylation was less pronounced, reaching two to three times control values in 6-month-old mice. Western blotting of serum and liver proteins with radiolabeled SNA showed that all glycoproteins that bind the lectin in controls exhibit larger amounts of Neu5Ac alpha2,6Gal beta1,4GlcNAc on N-glycans in the tumor-bearing mice. This general increase in alpha2,6 sialylation on all glycoproteins is due to the increased activity of the galactoside:alpha2,6 sialyltransferase (ST6Gal I), which specifically transfers Neu5Ac residues in alpha2,6 linkage to Gal beta1,4GlcNAc units on N-glycans. As for the structures synthesized by the enzyme, the increase of ST6Gal I activity in the serum as well as in liver microsomes of the transgenic mice followed tumor progression. Interestingly, the activity of the galactoside:alpha2,3 sialyltransferase (ST3Gal III), which uses the same acceptor substrate (Gal beta1,4GlcNAc), was unchanged in the earlier stages of tumor development but decreased in the serum and in liver microsomes from later stages. Using a rat ST6Gal I cDNA as a probe, Northern blots of total RNA extracted from the livers of control and transgenic mice revealed an increased (4-fold) expression of the ST6Gal I gene. The single transcripts detected in both normal and cancerous liver showed identical size.

Fold recognition and molecular modeling of a lectin-like domain in UDP-GalNac:polypeptide N-acetylgalactosaminyltransferases.

By use of threading methods, the C-terminal region of uridine diphospho-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-transferases) was predicted to have the same fold as the lectin-domain of the plant cytotoxins ricin and abrin-a, for which crystal structure are available. The sequence identities are very low. Nevertheless, the amino acids involved in the hydrophobic core essential for the structure stability and the cysteine residues are conserved. In addition, the amino-acids involved in carbohydrate binding are conserved in ppGalNAc-transferases. The extra C-terminal domain of these enzymes is therefore a putative glycan-binding domain. A model of the lectin-like domain of human ppGalNAc-transferase T1 was built using knowledge based methods. Geometry optimization of the complex with galactose allowed prediction that this domain could bind this monosaccharide. However, the interaction seems to be rather weak, and at the moment there is no evidence that ppGalNAc-transferases displays a lectin activity in vivo.

Comparison of the carbohydrate-binding specificities of seven N-acetyl-D-galactosamine-recognizing lectins.

Seven plant lectins, Dolichos biflorus agglutinin (DBA), Griffonia simplicifolia agglutinin (GSA, isolectin A4), Helix pomatia agglutinin (HPA), soybean (Glycine max) agglutinin (SBA), Salvia sclarea agglutinin (SSA), Vicia villosa agglutinin (VVA, isolectin B4) and Wistaria floribunda agglutinin (WFA), known to be specific for N-acetyl-D-galactosamine-(GalNAc) bearing glycoconjugates, have been compared by the binding of their radiolabelled derivatives, to eight well-characterized synthetic oligosaccharides immobilized via a spacer on an inert silica matrix (Synsorb). The eight oligosaccharides included the Forssman, the blood group A and the T antigens, as well as alpha GalNAc coupled directly to the support (Tn antigen) and also structures with GalNAc linked alpha or beta to positions 3 or 4 of an unsubstituted Gal. The binding studies clearly distinguished the lectins into alpha GalNAc-specific agglutinins like DBA, GSA and SSA, and lectins which recognize alpha- as well as beta-linked GalNAc residues like HPA, VVA, WFA and SBA. HPA was the only lectin which bound to the beta Gal1----3 alpha GalNAc-Synsorb adsorbent (T antigen) indicating that it also recognizes internal GalNAc residues. Among the alpha GalNAc-specific lectins, DBA strongly recognized blood group A structures while GSA displayed weaker recognition, and SSA bound only slightly to this affinity matrix. In addition, DBA and SSA were able to distinguish between GalNAc linked alpha 1----3 and GalNAc linked alpha 1----4, to the support, the latter being a much weaker ligand. These results were corroborated by the binding of the lectins to biological substrates as determined by their hemagglutination titers with native and enzyme-treated red blood cells carrying known GalNAc determinants, e.g. blood group A, and the Cad and Tn antigens. For SSA, the binding to the alpha GalNAc matrix was inhibited by a number of glycopeptides and glycoproteins confirming the strong preference of this lectin for alpha GalNAc-Ser/Thr-bearing glycoproteins.

Biosynthesis of truncated O-glycans in the T cell line Jurkat. Localization of O-glycan initiation.

Glycoproteins from the human T leukemia cells Jurkat were found to bind to the GalNAc alpha 1----Ser/Thr-specific lectin from Salvia sclarea seeds. The analysis of the O-linked saccharides of immunopurified leukosialin, the major [3H]glucosamine-labeled glycoprotein in Jurkat cell lysate, revealed the presence of mainly GalNAc alpha 1----Ser/Thr with only minor amounts (approximately 17%) of more complex O-glycans. A comparison between Jurkat and K562 cell glycosyltransferase involved in the biosynthesis of O-linked carbohydrates showed that a markedly lower activity of UDP-Gal:GalNAc alpha 1----Ser/Thr beta 1----3galactosyltransferase is apparently responsible for the presence of truncated O-glycans in the Jurkat cell line. The O-glycosylation defect makes Jurkat cells an ideal model to study the initiation of O-linked saccharides. Pulse-chase experiments with [35S] methionine showed that the addition of GalNAc to leukosialin is responsible for the decreased mobility of the mature glycoprotein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, no biosynthetic intermediates between the O-glycan-free precursor and the fully O-glycosylated form could be detected either with an anti-leukosialin antiserum or with the GalNAc-specific lectin. Lowering the chase temperature to 15 degrees C completely inhibited the transfer of GalNAc to the peptide core indicating that O-glycan initiation takes place in the first Golgi elements and not in transitional vesicles between endoplasmic reticulum and Golgi. In addition, treatment of the cells with monensin did not inhibit GalNAc transfer to leukosialin apoprotein. These results indicate that the initiation of O-glycosylation in Jurkat cells starts in the cis-Golgi stacks.

Phosphorylation of the major leukocyte surface sialoglycoprotein, leukosialin, is increased by phorbol 12-myristate 13-acetate.

Leukosialin (CD43) is a heavily O-glycosylated membrane glycoprotein present on all leukocytes and on platelets. We found that leukosialin is phosphorylated in erythroid, myeloid, and T-lymphoid cell lines, as well as in platelets and peripheral blood lymphocytes. Leukosialin phosphorylation was increased 2.5-15-fold following phorbol ester treatment. The phosphorylation could be inhibited with the protein kinase C inhibitor staurosporine but not with HA 1004 that inhibits cAMP- or cGMP-dependent protein kinases. The phosphoamino acid analysis showed that serine residues were exclusively phosphorylated, either with or without phorbol ester treatment. Two-dimensional peptide maps of phosphorylated leukosialin from K562 and Jurkat cells gave almost identical patterns. The number of labeled peptides increased after treatment with phorbol ester, indicating that new sites were phosphorylated. The major phosphorylation site on leukosialin was identified as Ser-332 in a region of the cytoplasmic domain located 73 amino acids from the transmembrane portion.

O-glycosylation of leukosialin in K562 cells. Evidence for initiation and elongation in early Golgi compartments.

The O-glycosylation of leukosialin, a major sialoglycoprotein found on leukocytes, has been studied in the human erythroleukemic cell line K562. The appearance of its O-linked chains has been followed in pulse-chase experiments with [35S]methionine by immunoprecipitation with an anti-peptide antiserum as well as with a lectin from Salvia sclarea seeds (SSA) specific for GalNAc-Ser/Thr and the peanut (Arachis hypogaea) agglutinin (PNA) which recognizes Gal beta 1----3GalNAc-Ser/Thr structures. An O-glycan-free precursor was converted into the fully O-glycosylated mature form within the 10-min labeling period and no intermediates carrying only GalNAc-Ser/Thr structures could be detected. The ionophore monensin was used in order to slow down intracellular traffic and thus O-glycan synthesis. The drug partly inhibited the transport from rough endoplasmic reticulum (RER) to the Golgi and also the cell-surface expression of leukosialin. It was found to have a marked effect on the synthesis of O-linked carbohydrate structures of leukosialin since the amount of O-glycans containing only GalNAc or NeuNAc alpha 2----6GalNAc was significantly increased after monensin treatment. Under these conditions the biosynthesis of the N-glycan on leukosialin was completely arrested in an endoglycosidase-H-sensitive step of processing, whereas the O-glycans already contained galactose and sialic acid although at a reduced level. On the other hand, the small amounts of leukosialin expressed on the cell surface of monensin-treated cells carried the same glycans as those remaining blocked inside the cell. In addition, immunocytochemical studies using SSA and PNA on untreated K562 cells suggested the absence of detectable amounts of GalNAc-Ser/Thr-bearing glycoproteins in the RER as well as in the Golgi. In contrast Gal beta 1----3GalNAc structures could be detected on intracellular membranes which were tentatively identified as the cis-Golgi. Together these results lead us to the following conclusions: N-glycan transfer occurs in the RER before the initiation of O-glycans which takes place at the entrance of the protein into the Golgi; further elongation of O-glycans with galactose and sialic acid follows very rapidly, probably before the final processing of N-glycans to complex-type structures.

Human T-lymphocyte activation is associated with changes in O-glycan biosynthesis.

The activation of human T-lymphocytes by anti-CD3 antibodies and interleukin-2 results in a marked increase in apparent molecular weight of the major cell-surface sialoglycoprotein. Both forms of the sialoglycoprotein were identified as leukosialin by a monospecific antiserum, and the differences in molecular weight were found to be due to changes in the carbohydrate structures. Our results suggest that resting T-lymphocytes express on leukosialin the disialotetrasaccharides NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)Gal-NAc-Ser/Thr, whereas activated human T-cells carry on leukosialin exclusively the more complex structures NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6)GalNAc-Ser/Thr. The radical shift in the biosynthetic pathway of O-glycans in activated T-lymphocytes compared to resting cells is apparently caused by a decrease of alpha 2----6 sialyltransferase activity and by the parallel dramatic stimulation of the beta 1----6GlcNAc-transferase. Since both enzymes compete for the same precursor substrate, the coordinate changes in their activities are most likely responsible for the complete change of the carbohydrate structures on leukosialin during the activation of human T-lymphocytes.

Isolation and characterization of an N-acetylgalactosamine specific lectin from Salvia sclarea seeds.

Crude extracts from Salvia sclarea seeds were known to contain a lectin which specifically agglutinates Tn erythrocytes (Bird, G. W. G., and Wingham, G. (1974) Vox Sang. 26, 163-166). We have purified the lectin to homogeneity by ion-exchange chromatography and affinity chromatography. The agglutinin was found to be a glycoprotein of Mr = 50,000, composed of two identical subunits of Mr = 35,000 linked together by disulfide bonds. The purified lectin agglutinates specifically Tn erythrocytes and, at higher concentrations, also Cad erythrocytes. Native A, B, or O red blood cells are not agglutinated by the lectin and, even after treatment with sialidase or papain, these cells are not recognized. Tn red cells present 1.45 X 10(6) accessible sites to the lectin which binds to these erythrocytes with an association constant of 1.8 X 10(6) M-1. On Cad red cells, 1.73 X 10(6) sites are accessible to the lectin which binds with an association constant of 1.0 X 10(6) M-1. The carbohydrate specificity of the S. sclarea lectin has been determined in detail, using well defined monosaccharide, oligosaccharide, and glycopeptide structures. The lectin was found to be specific for terminal N-acetylgalactosamine (GalNAc) residues. It binds preferentially alpha GalNAc determinants either linked to Ser or Thr (as in Tn structures) or linked in 1-3 to a beta GalNAc or to an unsubstituted beta Gal. Although more weakly, the lectin binds beta GalNAc residues linked in 1-4 to a beta Gal (as in Cad structures). It does not recognize beta GalNAc determinants linked in 1-3 to a Gal (as in globoside) or the alpha GalNAc residues of blood group A structures.