Deleterious acute and chronic effects of bradycardic right ventricular apex pacing: consequences for arrhythmic outcome.

In the chronic complete atrioventricular (AV) block dog (CAVB) model, both bradycardia and altered ventricular activation due to the uncontrolled idioventricular rhythm contribute to ventricular remodeling and the enhanced susceptibility to Torsade de Pointes (TdP) arrhythmias. We investigated the effect of permanent bradycardic right ventricular apex (RVA) pacing on mechanical and electrical remodeling and TdP. In 23 anesthetized dogs, serial experiments were performed at sinus rhythm (SR), acutely after AV block (AAVB) and 3 weeks of remodeling CAVB at a fixed pacing rate of 60/min. ECG, and left (LV) and right ventricular (RV) monophasic action potentials durations (MAPD) were recorded; activation time (AT) and activation recovery interval (ARI) were determined from ten distinct LV electrograms; interventricular mechanical delay (IVMD) and time-to-peak strain (TTP) of the LV septal and lateral wall (ΔTTP: lateral wall minus septal wall) were obtained echocardiographically. Dofetilide (25 µg/kg/5 min) was infused to study TdP inducibility. In baseline AAVB, in comparison to SR, RVA bradypacing acutely increased QT interval, LV, and RVMAPD. Echocardiographic IVMD and ΔTTP were initially increased, which was partially corrected after 3 weeks of RVA pacing (IVMD: 22 ± 13 vs. 42 ± 11 vs. 31 ± 6 ms; ΔTTP: -2 ± 47 vs. -114 ± 38 vs. -36 ± 22 ms). QT interval (362 ± 23 vs. 373 ± 29 ms), LVMAPD (245 ± 18 vs. 253 ± 22 ms), RVMAPD (226 ± 26 vs. 238 ± 31 ms), and mean LV-ARI (268 ± 5 vs. 267 ± 6 ms) were not significantly changed after 3 weeks of RVA pacing. During AAVB, dofetilide increased mean LV-ARI (381 ± 11 ms) with largest increases in the later activated basal areas (slope AT-ARI: +0.96). In contrast with acute RVA pacing, 3 week pacing increased TdP inducibility (0/13 vs. 11/21) and mean LV-ARI (484 ± 18 ms), while the slope of AT-ARI responded differently on dofetilide (-2.37), with larger APD increases in the early region. The latter was supported at the molecular level: reduced RNA expressions of three repolarization-related ion channel genes in early (KCNQ1, KCNH2, and KCNJ2) versus two in late regions (KNCQ1 and KCNJ2). In conclusion, bradycardic RVA pacing acutely induced LV intra- and interventricular mechanical dyssynchrony, which was partially reversed after 3 weeks of pacing (remodeling). The latter occurred without apparent baseline electrical effects. However, dofetilide clearly unmasked (region-specific) arrhythmic consequences of remodeling.

Stimulation of mouse bone marrow cells with kit ligand, FLT3 ligand, and thrombopoietin leads to efficient retrovirus-mediated gene transfer to stem cells, whereas interleukin 3 and interleukin 11 reduce transduction of short- and long-term repopulating cells.

The effects of cytokine stimulation during retroviral transduction on in vivo reconstitution of mouse hematopoietic stem cells was tested in a murine competitive repopulation assay with alpha-thalassemia as a marker to distinguish donor and recipient red blood cells (RBCs) and the enhanced green fluorescent protein (EGFP) as a marker for gene transfer. After transplantation, EGFP was detected in up to 90% of circulating RBCs, platelets, and leukocytes, and in primitive progenitors in bone marrow (BM), spleen, and thymus of individual transplanted mice for observation periods of more than 6 months. Large quantitative differences in reconstitution were observed after transplantation with graded numbers (1000-30, 000) of EGFP(+) cells preconditioned with various combinations of Kit ligand (KL), FLT-3 ligand (FL), thrombopoietin (TPO), interleukin 3 (IL-3), and IL-11. Relative to nonmanipulated BM cells, repopulation of EGFP(+) cells was maintained by KL/FL/TPO stimulation, but approximately 30-fold reduced after KL/FL/TPO/IL-3, or KL/FL/IL-3/IL-11. These differences were not caused by changes in the ability of immature hematopoietic cells to home to the BM, which was only moderately reduced. In conclusion, these quantitative transplantation studies of mice demonstrate the importance of optimal ex vivo cytokine stimulation for gene transfer to stem cells with retention of their in vivo hematopoietic potential, and also emphasize that overall in vitro transduction frequency does not predict gene transfer to repopulating stem cells.

Characterization of the rat connexin40 promoter: two Sp1/Sp3 binding sites contribute to transcriptional activation.

OBJECTIVES: The gap junction protein connexin40 (Cx40) is differentially expressed during embryonic development and in adult tissues, for which the molecular basis is unknown. In order to elucidate the molecular mechanisms controlling Cx40 expression, we set out to map and characterize its promoter. METHODS: The transcriptional activity of individual rat Cx40 (rCx40)-derived promoter fragments fused to the luciferase reporter gene was determined by transfection/reporter assays in Cx40-expressing (A7r5, rat smooth muscle embryonic thoracic aorta cells, and BWEM, v-myc transformed rat fetal cardiomyocytes) and Cx40-nonexpressing cells (N2A, mouse neuroblastoma cells). The nature of DNA-protein interactions was investigated by a combination of standard electrophoretic-mobility-shift assays (EMSA) and EMSA/antibody supershift assays. RESULTS: Quantification of luciferase activity in cell lysates revealed that a 235-base-pair fragment, in between map positions -150 and +85 relative to the transcription initiation site, is able to provide for a significant level of transcription in both Cx40-expressing (A7r5, BWEM) and -nonexpressing (N2A) cells. These results indicate that this region contains the basal promoter but is not sufficient to completely determine the endogenous Cx40-expression pattern within these cell types. In search for the responsible transcriptional regulatory element(s), additional segments of the (-150, +85) region were deleted and the remaining fragments were tested for transcriptional activity. These studies established that the regions in between map positions (-96, -71) and (+58, +85) contribute to promoter activity. EMSA with these regions revealed that predominantly two DNA-protein complexes are formed upon incubation with either A7r5, BWEM or N2A nuclear extracts, which could be both inhibited by including excess oligonucleotide containing the Sp1 consensus binding site in the binding reaction. Purified recombinant human Sp1 provided also for a shift in the EMSA using these promoter regions as target fragments. When the DNA-protein complexes formed with nuclear extract were subsequently incubated with either an anti-Sp1 or an anti-Sp3 antibody clear supershifts in the EMSA were obtained, indicating Sp1 and Sp3 binding to both the (-98, -64) and (+53, +87) regions. The introduction of mutations within the core sequence of the putative Sp1/Sp3 binding sites present in these regulatory elements reduced the level of transcriptional activity and abrogated Sp1/Sp3 binding to these sites. CONCLUSION: The results indicate that at least two Sp1/Sp3 binding sites in the rCx40 promoter contribute to the transcriptional activation of its gene in cultured cells.

Efficient detection and selection of immature rhesus monkey and human CD34+ hematopoietic cells expressing the enhanced green fluorescent protein (EGFP).

The feasibility of using the enhanced green fluorescent protein (EGFP) as a selectable reporter molecule of retroviral-mediated gene transfer in immature rhesus monkey and human CD34+ hematopoietic cells was examined. Retroviral transduction with the MFG-EGFP retroviral vector resulted in readily detectable EGFP expression in 27% of human and 11-35% of rhesus monkey bone marrow cells, and in 17-38% of rhesus monkey peripheral blood cells mobilized with FLT3 ligand (FL) and granulocyte colony-stimulating factor (G-CSF). In addition, we used the human CD34+ KG1A cell line as a model to study viability and growth of successfully transduced cells. Cultures of mock- and EGFP-transduced KG1A cells generated equal viable cell numbers for at least 1 month, indicating the absence of a cytotoxic effect of EGFP expression in these cells. FACS selection on the basis of EGFP and CD34 expression resulted in enriched subsets (> or = 87%) of CD34+ EGFP-negative and CD34+ EGFP-positive KG1A, rhesus monkey and human bone marrow cells, demonstrating the potential of obtaining almost pure populations of transduced immature hematopoietic cells. EGFP expression was also readily demonstrated in erythroid and granulocyte/macrophage colonies derived from the CD34+ EGFP-positive rhesus monkey and human bone marrow cells by either inverted fluorescence microscopy or flow cytometry. Using four-color flow cytometry, EGFP expression could also be demonstrated in viable and phenotypically defined immature subpopulations of the CD34+ cells, ie those expressing little or no HLA-DR (rhesus monkey) or CD38 (human) antigens at the cell surface. These results demonstrate that EGFP is a very useful marker to monitor gene transfer efficiency in phenotypically defined immature rhesus monkey and human hematopoietic cell types and to select for these cells by multicolor flow cytometry prior to transplantation.

Simultaneous infection with retroviruses pseudotyped with different envelope proteins bypasses viral receptor interference associated with colocalization of gp70 and target cells on fibronectin CH-296.

Several factors are thought to limit the efficiency of retroviral transduction in clinical gene therapy protocols that target hematopoietic stem cells. For example, the level of expression of the amphotropic receptor Pit-2, a phosphate symporter, appears to be low in human and murine hematopoietic stem cells. We have previously demonstrated that transduction of hematopoietic cells in the presence of the fibronectin (FN) fragment CH-296 is extremely efficient (H. Hanenberg, X. L. Xiao, D. Dilloo, K. Hashino, I. Kato, and D. A. Williams, Nat. Med. 2:876-882, 1996). To examine functionally whether the retrovirus receptor is a limiting factor in transduction of hematopoietic cells, we performed competition experiments in the presence of FN CH-296 with retrovirus vectors pseudotyped with the same or a different envelope protein. We demonstrate in both human erythroleukemia (HEL) cells and primary human CD34(+) hematopoietic cells inhibition of efficient infection due to receptor interference when two vectors targeting the amphotropic receptor are used simultaneously. Receptor interference lasted up to 24 h. No interference was demonstrated when vectors targeting the amphotropic receptor and the gibbon ape leukemia virus (GALV) receptor Pit-1 were used concurrently. In contrast, simultaneous infection with vectors targeting both Pit-1 and Pit-2 yielded transduction efficiencies consistently higher than with either vector alone in both HEL cells and human CD34(+) hematopoietic cells. These data demonstrate that the use of FN CH-296 leads to amphotropic receptor saturation in these cells. Simultaneous infection with vectors targeting both amphotropic and GALV receptors may prove to be of additional benefit in the design of gene therapy protocols.

Highly efficient transduction of the green fluorescent protein gene in human umbilical cord blood stem cells capable of cobblestone formation in long-term cultures and multilineage engraftment of immunodeficient mice.

Purified CD34(+) and CD34(+)CD38(-) human umbilical cord blood (UCB) cells were transduced with the recombinant variant of Moloney murine leukemia virus (MoMLV) MFG-EGFP or with SF-EGFP, in which EGFP expression is driven by a hybrid promoter of the spleen focus-forming virus (SFFV) and the murine embryonic stem cell virus (MESV). Infectious MFG-EGFP virus was produced by an amphotropic virus producer cell line (GP+envAm12). SF-EGFP was produced in the PG13 cell line pseudotyped for the gibbon ape leukemia virus (GaLV) envelope proteins. Using a 2-day growth factor prestimulation, followed by a 2-day, fibronectin fragment CH-296-supported transduction, CD34(+) and CD34(+)CD38(-) UCB subsets were efficiently transduced using either vector. The use of the SF-EGFP/PG13 retroviral packaging cell combination consistently resulted in twofold higher levels of EGFP-expressing cells than the MFG-EGFP/Am12 combination. Transplantation of 10(5) input equivalent transduced CD34(+) or 5 x 10(3) input equivalent CD34(+)CD38(-) UCB cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in median engraftment percentages of 8% and 5%, respectively, which showed that the in vivo repopulating ability of the cells had been retained. In addition, mice engrafted after transplantation of transduced CD34(+) cells using the MFG-EGFP/Am12 or the SF-EGFP/PG13 combination expressed EGFP with median values of 2% and 23% of human CD45(+) cells, respectively, which showed that the NOD/SCID repopulating cells were successfully transduced. EGFP+ cells were found in all human hematopoietic lineages produced in NOD/SCID mice including human progenitors with in vitro clonogenic ability. EGFP-expressing cells were also detected in the human cobblestone area-forming cell (CAFC) assay at 2 to 6 weeks of culture on the murine stromal cell line FBMD-1. During the transduction procedure the absolute numbers of CAFC week 6 increased 5- to 10-fold. The transduction efficiency of this progenitor cell subset was similar to the fraction of EGFP+ human cells in the bone marrow of the NOD/SCID mice transplanted with MFG-EGFP/Am12 or SF-EGFP/PG13 transduced CD34(+) cells, ie, 6% and 27%, respectively. The study thus shows that purified CD34(+) and highly purified CD34(+)CD38(-) UCB cells can be transduced efficiently with preservation of repopulating ability. The SF-EGFP/PG13 vector/packaging cell combination was much more effective in transducing repopulating cells than the MFG-EGFP/Am12 combination.

Enhanced green fluorescent protein as selectable marker of retroviral-mediated gene transfer in immature hematopoietic bone marrow cells.

The further improvement of gene transfer into hematopoietic stem cells and their direct progeny will be greatly facilitated by markers that allow rapid detection and efficient selection of successfully transduced cells. For this purpose, a retroviral vector was designed and tested encoding a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP). Murine cell lines (NIH 3T3, Rat2) and bone marrow cells transduced with this retroviral vector demonstrated a stable green fluorescence signal readily detectable by flow cytometry. Functional analysis of the retrovirally transduced bone marrow cells showed EGFP expression in in vitro clonogenic progenitors (GM-CFU), day 13 colony-forming unit-spleen (CFU-S), and in peripheral blood cells and marrow repopulating cells of transplanted mice. In conjunction with fluorescence-activated cell sorting (FACS) techniques EGFP expression could be used as a marker to select for greater than 95% pure populations of transduced cells and to phenotypically define the transduced cells using antibodies directed against specific cell-surface antigens. Detrimental effects of EGFP expression were not observed: fluorescence intensity appeared to be stable and hematopoietic cell growth was not impaired. The data show the feasibility of using EGFP as a convenient and rapid reporter to monitor retroviral-mediated gene transfer and expression in hematopoietic cells, to select for the genetically modified cells, and to track these cells and their progeny both in vitro and in vivo.

Green fluorescent protein variants as markers of retroviral-mediated gene transfer in primary hematopoietic cells and cell lines.

Retroviral vectors are widely used for the introduction of exogenous genetic material into hematopoietic cells. Here we report the generation of retroviral vectors containing the Aequorea victoria green fluorescent protein (GFP) gene and improved versions thereof. Murine fibroblasts transduced with the mutant GFP genes demonstrated a distinct green fluorescent signal in fluorescence-activated cell sorter (FACS) analysis. The relative intensities of peak green fluorescence observed with different GFP mutants were in the order EGFP>hGFP(S65T)> GFP-PTS1 or RSGFP>wildtype GFP (wtGFP). Furthermore, GFP-PTS1 expression was observed in murine (3T3, Rat2, and freshly-cultured bone marrow) and human (K562) cells transduced with the corresponding retroviral vector. The GFP-PTS1 positive phenotype could be selected for by FACS and appeared to be stable for at least 1 month in murine fibroblasts and human K562 cells. Therefore, these GFP variants are convenient selectable markers to monitor retroviral-mediated gene transfer and expression in mammalian hematopoietic cells.

Expression cloning of glycosyltransferases.

Cloning of cDNAs encoding glycosyltransferases enters into a new era because of the advent of expression cloning for glycosyltransferases. This mini-review summarizes a short historical view on the development of this method and surveys various improvements over the original method. This review also emphasizes the advantages of those recently developed improvements with a brief description on their background. Finally, the review provides some prospects based on these developments in the methodology. It is expected that increasing numbers of newly cloned glycosyltransferases will become critical tools for understanding the roles of carbohydrates.

Isolation and characterization of a pseudogene related to human core 2 beta-1,6-N-acetylglucosaminyl-transferase.

In a previous study, we isolated genomic clones encoding core 2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT) and blood group IGnT and proposed that these two genes were produced from a common ancestral gene by duplication, diversion and intron insertion. In the present study, we have isolated a pseudogene which is highly related to the gene of C2GnT. The sequence analysis of this pseudogene indicated that the pseudogene was produced by duplication of a common precursor gene for C2GnT. These results taken together strongly suggest that the ancestral gene was first duplicated and one of the duplicated genes directly evolved into the IGnT gene. The other duplicated gene was further duplicated to produce the C2GnT gene and the pseudogene.

Genomic organization of core 2 and I branching beta-1,6-N-acetylglucosaminyltransferases. Implication for evolution of the beta-1,6-N-acetylglucosaminyltransferase gene family.

Two human beta-1,6-N-acetylglucosaminyltransferases forming the core 2 O-glycan branch, C2GnT and the I antigen, IGnT, are homologous to each other in three regions of the catalytic domain (A, B, C) and their genes reside at the same locus, chromosome 9, band q21 (Bierhuizen,M.F.A., Mattei, M.-G. and Fukuda,M., Genes Dev., 7, 468-478, 1993). In order to investigate how these two enzymes are related at the genomic level, and how this gene family evolved, we have elucidated their genomic structures. It was found that C2GnT is coded by two exons, of which the second exon encodes the whole translation product. In contrast, the complete coding sequence for IGnT is divided over three exons. Importantly, the highly homologous region B is encoded entirely by exon 2 in the C2GnT gene, while the same region is split between exons 1 and 2 in the IGnT gene. The other highly homologous regions, A and C, are also encoded by exon 2 in the C2GnT gene, while they are encoded by exon 1 and exon 3, respectively, in the IGnT gene. These results strongly suggest that the common ancestral gene was first duplicated and then each duplicated gene evolved into the C2GnT or IGnT gene by intron insertion and divergence following the duplication. The sequences upstream from the transcription initiation sites of the C2GnT and IGnT genes have promoter activity and contain TATA-like sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of a differentiation antigen and poly-N-acetyllactosaminyl O-glycans directed by a cloned core 2 beta-1,6-N-acetylglucosaminyltransferase.

Chinese hamster ovary (CHO) cells do not contain detectable amounts of core 2 beta-1,6-N-acetylglucosaminyltransferase, C2GnT, and thus lack various modifications in their branched O-linked oligosaccharides. In the present study, the O-linked oligosaccharides and the occurrence of a differentiation antigen were analyzed in CHO cells stably transfected with cDNA encoding human leukosialin alone (CHO-leu) or with cDNAs encoding both leukosialin and C2GnT (CHO-leu.C2GnT). The analysis of O-glycans, released from [3H]glucosamine-labeled cells, revealed that CHO-leu cells synthesize O-glycans with a Gal beta 1-->3GalNAc backbone, whereas CHO-leu.C2GnT cells synthesize in addition O-glycans with a Gal beta 1-->3(Gal beta 1-->4GlcNAc beta 1-->6)GalNAc backbone. Moreover, CHO-leu.C2GnT cells express poly-N-acetyllactosaminyl extensions from the GlcNAc beta 1-->6 branch in O-glycans, while CHO-leu cells express no detectable amount of poly-N-acetyllactosaminyl O-glycans. It was also demonstrated that leukosialin in CHO-leu.C2GnT cells is recognized by the T305 monoclonal antibody, while the same antibody did not react at all with CHO-leu cells. In addition, the transient expression cloning scheme using the T305 monoclonal antibody as a selectin marker and COS-1 cells, which endogenously express C2GnT as recipient cells, resulted in the isolation of cDNA encoding leukosialin. These results indicate that C2GnT determines the expression of poly-N-acetyllactosamines in O-glycans and together with leukosialin, an onco-differentiation antigen recognized by the T305 antibody.

Expression of the developmental I antigen by a cloned human cDNA encoding a member of a beta-1,6-N-acetylglucosaminyltransferase gene family.

The blood group i/I antigens were the first identified alloantigens that display a dramatic change during human development. The i and I antigens are determined by linear and branched poly-N-acetyllactosaminoglycans, respectively. In human erythrocytes during embryonic development, the fetal (i) antigen is replaced by the adult (I) antigen as a result of the appearance of a beta-1,6-N-acetylglucosaminyltransferase, the I-branching enzyme. Here, we report the cDNA cloning and expression of this branching enzyme that converts linear into branched poly-N-acetyllactosaminoglycans, thus introducing the I antigen in transfected cells. The cDNA sequence predicts a protein with type II membrane topology as has been found for all other mammalian glycosyltransferases cloned to date. The Chinese hamster ovary cells that stably express the isolated cDNA acquire I-branched structures as evidenced by the structural analysis of glycopeptides from these cells. Comparison of the amino acid sequence with those of other glycosyltransferases revealed that this I-branching enzyme and another beta-1,6-N-acetylglucosaminyltransferase that forms a branch in O-glycans are strongly homologous in the center of their putative catalytic domains. Moreover, the genes encoding these two beta-1,6-N-acetylglucosaminyltransferases were found to be located at the same locus on chromosome 9, band q21. These results indicate that the I-branching enzyme represents a member of a beta-1,6-N-acetylglucosaminyltransferase gene family of which expression is controlled by developmental programs.

Expression cloning of a cDNA encoding UDP-GlcNAc:Gal beta 1-3-GalNAc-R (GlcNAc to GalNAc) beta 1-6GlcNAc transferase by gene transfer into CHO cells expressing polyoma large tumor antigen.

A cDNA encoding UDP-GlcNAc:Gal beta 1-3GalNAc-R (GlcNAc to GalNAc) beta 1-6GlcNAc transferase (EC 2.4.1.102), which forms critical branches in O-glycans, has been isolated by an expression cloning approach using Chinese hamster ovary (CHO) cells. Increased activity of this enzyme and the concomitant occurrence of the O-glycan core 2 structure [Gal beta 1-3(GlcNAc beta 1-6)GalNAc] has been observed in a variety of biological processes, such as T-cell activation and immunodeficiency due to the Wiskott-Aldrich syndrome and AIDS. Since CHO cells do not express this enzyme, CHO cell lines were established to stably express polyoma large tumor (T) antigen, which enables transient expression cloning. Because the antibody used was found to detect most efficiently the oligosaccharide products attached to leukosialin, the CHO cells were also stably transfected with leukosialin cDNA. By using this particular CHO cell line, a cDNA that encodes a protein determining the formation of the core 2 structure was isolated from an HL-60 cDNA library. The cDNA sequence predicts a protein with type II membrane topology, as has been found for all other mammalian glycosyltransferases cloned to date. The expression of the presumed catalytic domain as a fusion protein with the IgG binding domain of protein A enabled us to demonstrate unequivocally that the cDNA encodes the core 2 beta-1,6-N-acetylglucosaminyltransferase, the enzyme responsible for the formation of Gal beta 1-3(GlcNAc beta 1-6)GalNAc structures. No activity with this enzyme was detected toward the acceptors for other beta 1-6GlcNAc transferases.

Con A affinity of rat alpha 1-acid glycoprotein (rAGP): changes during inflammation, dexamethasone or phenobarbital treatment as detected by crossed affino immunoelectrophoresis (CAIE) are not only a reflection of biantennary glycan content.

Using crossed affino immunoelectrophoresis (CAIE), the secretion of the Con A most reactive form (CAIE-3) of rat alpha 1-acid glycoprotein (rAGP) has been shown to be increased in sera of Wistar and Sprague Dawley rats during inflammation and treatment with dexamethasone or phenobarbital. Primary hepatocyte cultures prepared from experimentally treated Wistar rats reflect these in vivo findings, since rAGP as present in corresponding secretion media shows similar changes in Con A reactivity. In this study, the relation of this increase towards the amount of biantennary glycans was investigated for both differently treated rat strains. For this purpose, metabolically labelled rAGP, secreted by isolated hepatocytes under the various conditions, was separated on Con A-Sepharose into four fractions. For each fraction of rAGP its behaviour in CAIE was established, revealing a positive correlation for Con A reactivity between the two methods. However, the enormous increase in Con A reactivity of rAGP in CAIE during inflammation and other conditions (increase in CAIE-3), could not be shown using Con A-Sepharose chromatography. Glycopeptides of each fraction were prepared and the amount of biantennary glycans was assessed. Contrary to expectations, an increase of the total amount of biantennary glycans of rAGP, secreted during conditions associated with an increase in CAIE-3 was not found. The independency of the results with regard to rat strain and procedures used underlined the generality of these findings. Consequently, not only the biantennary glycan content is responsible for the separation of rAGP in CAIE. The importance of other differences in glycosylation, e.g. sialylation, for the increase of rAGP CAIE-3 during various experimental conditions is discussed.

Structural assessment of the N-linked oligosaccharides of cell-CAM 105 by lectin-agarose affinity chromatography.

The N-linked oligosaccharides of cell-CAM 105, a glycoprotein involved in the intercellular adhesion between rat hepatocytes, were studied by sequential lectin-agarose affinity chromatography of desialylated, [14C]-labelled glycopeptides. These glycopeptides were obtained by extensive pronase digestion followed by N-[14C]acetylation of the peptide moieties and desialylation by mild acid hydrolysis. Assuming that all glycopeptides were radiolabelled to the same specific radioactivity, Concanavalin A-Sepharose chromatography indicated that the majority of the glycans (84%) were of the complex-type of which approximately half were bi-antennary structures. The remainder of the glycans comprised oligomannose-type structures and/or incomplete bi-antennary structures. Pisum sativum lectin-agarose chromatography revealed that part of the bi-antennary glycans contained a fucose residue alpha(1-6)-linked to the N-acetylglucosamine which is attached to asparagine. Furthermore, the presence of tri-, and tetra- and/or tri'-antennary complex-type glycans was demonstrated by chromatography on immobilized Phaseolus vulgaris leukoagglutinating phytohemagglutinin and Aleuria aurantia lectin (AAL). AAL-agarose chromatography furthermore indicated the presence of alpha(1-3)-linked fucose in part of these glycopeptides, whereas no alpha(1-6)-linked fucose could be detected in these structures. The degree of beta-galactosylation of the complex-type glycans was investigated by chromatography on Ricinus communis agglutinin-agarose. The results indicated that only part of the bi-antennary glycans were completely beta-galactosylated. Similarly, at least three beta-galactose residues were present in only a part of the tri-, and tetra- and/or tri'-antennary glycans.

Glycosylation of three molecular forms of human alpha 1-acid glycoprotein having different interactions with concanavalin A. Variations in the occurrence of di-, tri-, and tetraantennary glycans and the degree of sialylation.

Human alpha 1-acid glycoprotein (AGP) was separated into a non-bound (AGP-A; 46%), a retarded (AGP-B; 39%) and a bound fraction (AGP-C; 15%) using concanavalin A (ConA)-Sepharose chromatography. The apparent molecular masses, as determined by SDS-PAGE, of the three fractions were 43.5, 42.3 and 41.2 kDa, respectively. The occurrence of N-linked di-, tri- and tetraantennary glycans on these three molecular forms (AGP-A, -B, and -C) was studied by sequential lectin-affinity chromatography of the 14C-labelled glycopeptides. These were obtained by extensive pronase treatment followed by N-[14C]acetylation of the peptide moieties. The glycopeptides of AGP-A did not bind to ConA-Sepharose whereas for AGP-B and AGP-C 18% and 44%, respectively, of the glycopeptides were bound as diantennary structures. Glycopeptide fractions of all three forms of AGP which were not bound to ConA-Sepharose were shown to contain equal amounts of both tri- and tetraantennary glycans by chromatography with Phaseolus vulgaris leukoagglutinating lectin (L-PHA). With the assumption that each molecule contains five glycosylation sites, it could be shown that AGP-A contains no diantennary structures whereas AGP-B and AGP-C contain one and two diantennary structures, respectively. In addition each of the molecular forms contains equal amounts of tri- and tetraantennary structures on the remaining glycosylation sites. The results of this study, therefore, exclude a uniformity of glycan chains in the three molecular forms of AGP. The degree of sialylation of each of the molecular forms was investigated by chromatography on L-PHA-agarose and Ricinus communis agglutinin-I--agarose both before and after desialylation of the glycopeptides. It was shown that about 90% of the biantennary glycans of both AGP-B and AGP-C were disialylated while the remainder were monosialylated. The degree of sialylation of the tri- and tetraantennary glycans was identical for the three molecular forms. In each case, one or more terminal galactose residues occurred on at least 20% of the tri- and 65% of the tetraantennary chains. It is suggested that the decrease in the exposure of galactose residues from AGP-A to AGP-C is related to the concomittant decrease in branching of the glycans of the three molecular forms. The relevance of these findings to studies on the function of AGP during inflammatory and liver diseases is discussed.

Removal of the N-terminal part of alfalfa mosaic virus coat protein interferes with the specific binding to RNA 1 and genome activation.

Trypsinized coat protein of alfalfa mosaic virus lacking 25 amino acids at its N terminus still has the capability to form complexes with RNA which are detectable by sedimentation in sucrose gradients. However, it does not protect specific sites on the RNA against degradation by ribonuclease, as the native coat protein does (D. Zuidema, M. F. A. Bierhuizen, B. J. C. Cornelissen, J. F. Bol, and E. M. J. Jaspars (1983) Virology 125, 361-369.). The trypsinized coat protein has lost the capacity of the native coat protein to make the genome RNAs of alfalfa mosaic virus infectious or to interfere with the infectivity brought about by the native coat protein. These findings suggest that genome activation occurs via binding of the N-terminal part of the coat protein to specific sites on the RNAs.

Coat protein binding sites on RNA 1 of alfalfa mosaic virus.

The largest genome segment, RNA 1, of alfalfa mosaic virus forms complexes with viral coat protein. These complexes were subjected to digestion with ribonucleases T1 or A and filtered onto Millipore filters. Specific fragments were collected from the filters by phenol extraction. After electrophoretic separation in denaturing polyacrylamide gels, these fragments were sequenced. Besides extracistronic fragments originating from the 3'-terminal region of RNA 1, fragments were found originating from an intracistronic region of the RNA. A striking phenomenon is that the intracistronic fragments were not found when ribonuclease A was used to degrade RNA/protein complexes. The findings are in agreement with the postulation of Houwing and Jaspars (1978), that a conformational change at the 3' ends of the genome RNAs induced by the coat protein is a prerequisite to start an infection cycle.