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Biomedical subjects

A T Remaley

Publications and source records attributed to A T Remaley.

At least 37 records · Page 2Linked to original sources
PubMed2000

Pressure cycling technology: a novel approach to virus inactivation in plasma.

BACKGROUND: Hydrostatic-pressure virus inactivation is a novel approach to the inactivation of pathogens in plasma and blood-derived components, that retains the therapeutic properties of these products. STUDY DESIGN AND METHODS: A custom-built apparatus was used to pressurize human plasma samples spiked with lambda phage. Phage titer and plasma protein activities were monitored after pressure treatment. RESULTS: Pressure-mediated inactivation of lambda phage was found to be an effective means for virus inactivation, particularly when performed at near-zero (0 degrees C) temperatures, rather than at temperatures above 20 degrees C and below -40 degrees C. The efficiency of inactivation was improved by an increase in applied pressure and repeated cycling from atmospheric to high pressure. In contrast, activities of plasma proteins alkaline phosphatase and total amylase did not vary with temperature and remained within 29 percent and 6 percent, respectively, of starting values after the same pressure treatments. By combining cycling, near-zero temperatures, and high pressure, phage titers in serum were reduced approximately 6 log after 10 to 20 minutes of treatment. Activities of plasma proteins IgG, IgM, and factor X were at 104 percent, 89 percent, and 80 percent, respectively, of starting values after 20 minutes of the same temperature and pressure treatment. CONCLUSION: High-pressure procedures may be useful for the inactivation of viruses in blood and other protein-containing components.

Antibodies, Viral↗
PubMed2000

Dual HDL/total cholesterol test: a single-tube, homogeneous assay for sequential measurement of HDL cholesterol and total cholesterol.

The quantitation of cholesterol in lipoprotein subfractions is valuable in estimating the risk for coronary artery disease, but requires multiple tests. We describe a relatively simple procedure, referred to as the dual HDL/total cholesterol (DHT) assay, which allows the sequential measurement of HDL cholesterol (HDL-C) and total cholesterol (total-C) in a single tube. HDL-C is first measured using a homogeneous assay that utilizes an anti-apolipoprotein B [apo(B)] antibody, which sterically blocks the enzymatic measurement of cholesterol on the non-HDL subfractions. Next, deoxycholate is added, which disrupts the antibody-apo(B) complex and allows the subsequent enzymatic measurement of the remaining cholesterol in the non-HDL subfractions. The DHT assay has an acceptable analytical performance and yields results similar to standard methods: for HDL-C, y(DHT) = 0.98x + 0.19, r=0.90; for total-C, y(DHT) = 1.11x - 0.09, r=0.99. In summary, the DHT assay is a homogeneous assay for both HDL-C and total-C, and provides a simple and cost-effective method for screening for hyperlipidaemia.

Antibodies↗
PubMed1999

Human ATP-binding cassette transporter 1 (ABC1): genomic organization and identification of the genetic defect in the original Tangier disease kindred.

Tangier disease is characterized by low serum high density lipoproteins and a biochemical defect in the cellular efflux of lipids to high density lipoproteins. ABC1, a member of the ATP-binding cassette family, recently has been identified as the defective gene in Tangier disease. We report here the organization of the human ABC1 gene and the identification of a mutation in the ABC1 gene from the original Tangier disease kindred. The organization of the human ABC1 gene is similar to that of the mouse ABC1 gene and other related ABC genes. The ABC1 gene contains 49 exons that range in size from 33 to 249 bp and is over 70 kb in length. Sequence analysis of the ABC1 gene revealed that the proband for Tangier disease was homozygous for a deletion of nucleotides 3283 and 3284 (TC) in exon 22. The deletion results in a frameshift mutation and a premature stop codon starting at nucleotide 3375. The product is predicted to encode a nonfunctional protein of 1,084 aa, which is approximately half the size of the full-length ABC1 protein. The loss of a Mnl1 restriction site, which results from the deletion, was used to establish the genotype of the rest of the kindred. In summary, we report on the genomic organization of the human ABC1 gene and identify a frameshift mutation in the ABC1 gene of the index case of Tangier disease. These results will be useful in the future characterization of the structure and function of the ABC1 gene and the analysis of additional ABC1 mutations in patients with Tangier disease.

ATP Binding Cassette Transporter 1↗
PubMed1999

Kinetic analysis of the rapid intraoperative parathyroid hormone assay in patients during operation for hyperparathyroidism.

BACKGROUND: Rapid intraoperative parathyroid hormone (RI-PTH) assay is used to guide adequacy of resection during operation for hyperparathyroidism. We compared the RI-PTH assay (15 minutes) with a standard PTH assay, determined whether the PTH half-life varied between patients, and constructed a kinetic analysis of the RI-PTH data. METHODS: Forty-five patients with hyperparathyroidism had blood sampled at baseline and at times after parathyroid resection. Intact PTH was determined using RI-PTH and a standard assay. Values were fitted to an exponential decay curve using the baseline and the follow-up time points. PTH half-life and the new postexcision baseline value were calculated from the decay curve. RESULTS: The RI-PTH assay and the standard PTH assay correlated well. Average PTH half-life was 1.68 +/- 0.94 minutes (0.42 to 3.81 minutes). A kinetic analysis yielded a formula for the generation of a PTH decay curve. Using a 50% reduction in RI-PTH at 5 minutes as the criterion for adequate resection, 2 patients were incorrectly classified as not being cured. These patients were correctly classified using the kinetic analysis. CONCLUSIONS: PTH half-life can vary substantially. A kinetic analysis may be more accurate in assessing adequacy of resection. This method allows the surgeon to interpret RI-PTH data independent of the timing of samples.

Adult↗
PubMed1999

The differential effect of food intake and beta-adrenergic stimulation on adipose-derived hormones and cytokines in man.

We determined whether the physiologic changes that accompany food intake or sympathetic activation by beta-adrenergic stimulation result in alterations in the secretion of leptin, tumor necrosis factor-alpha (TNF alpha), or interleukin-6 (IL-6) by serially sampling sc abdominal adipose interstitial fluid by open-flow microperfusion before and after a standardized meal and in response to isoproterenol (1 micromol/L) delivered locally. Post cibum IL-6 rose up to 5-fold, whereas leptin and TNF alpha secretion did not change; TNF alpha, but not IL-6, correlated positively with indices of lipolysis. Isoproterenol-induced lipolysis was accompanied by a transient 40% reduction in leptin and a parallel 85% elevation of TNF alpha concentration, whereas IL-6 levels did not change; again, TNF alpha correlated positively with lipolysis. These data show that secretion of some, but not all, metabolically relevant polypeptides by adipose tissue is modulated within a short time frame by food or stress stimuli, suggesting a role of these peptides in local autocrine/paracrine or distant endocrine effects on fat metabolism. TNF alpha's close correlation with lipolysis suggests that this cytokine participates in a local positive autocrine feedback loop, potentiating lipolysis and inhibiting insulin's antilipolytic actions. The regulations of adipose leptin, TNF alpha, and IL-6 secretion seem distinct from each other and different in the fed vs. fasting state.

Adipose Tissue↗
PubMed1999

Prevalence-value-accuracy plots: a new method for comparing diagnostic tests based on misclassification costs.

The clinical accuracy of diagnostic tests commonly is assessed by ROC analysis. ROC plots, however, do not directly incorporate the effect of prevalence or the value of the possible test outcomes on test performance, which are two important factors in the practical utility of a diagnostic test. We describe a new graphical method, referred to as a prevalence-value-accuracy (PVA) plot analysis, which includes, in addition to accuracy, the effect of prevalence and the cost of misclassifications (false positives and false negatives) in the comparison of diagnostic test performance. PVA plots are contour plots that display the minimum cost attributable to misclassifications (z-axis) at various optimum decision thresholds over a range of possible values for prevalence (x-axis) and the unit cost ratio (UCR; y-axis), which is an index of the cost of a false-positive vs a false-negative test result. Another index based on the cost of misclassifications can be derived from PVA plots for the quantitative comparison of test performance. Depending on the region of the PVA plot that is used to calculate the misclassification cost index, it can potentially lead to a different interpretation than the ROC area index on the relative value of different tests. A PVA-threshold plot, which is a variation of a PVA plot, is also described for readily identifying the optimum decision threshold at any given prevalence and UCR. In summary, the advantages of PVA plot analysis are the following: (a) it directly incorporates the effect of prevalence and misclassification costs in the analysis of test performance; (b) it yields a quantitative index based on the costs of misclassifications for comparing diagnostic tests; (c) it provides a way to restrict the comparison of diagnostic test performance to a clinically relevant range of prevalence and UCR; and (d) it can be used to directly identify an optimum decision threshold based on prevalence and misclassification costs.

Apolipoprotein A-I↗
PubMed1999

Telomerase activity in microdissected human gliomas.

Future improvements in the diagnosis and treatment of human gliomas might rely on obtaining more specific information concerning the biologic characteristics of individual tumor cells. Telomerase, a ribonucleoprotein that synthesizes telomeres, has been reported to be expressed in a majority of human tumors, including several subtypes of brain tumor. We hypothesized that a quantitative assay for telomerase activity, combined with selective microdissection of tumor or normal brain cells, might reveal telomerase gain-of-function to be important in the pathogenesis of gliomas and that telomerase levels might have prognostic significance. We used tissue microdissection for selective analysis of tumor cells obtained from eight patients with glioma, one with a meningioma, and one with a primary B-cell lymphoma of the central nervous system. Normal brain tissue microdissected from another patient was used as a control. Telomerase activity was screened by an electrophoretic method and then assayed by a quantitative ELISA method. All of the eight gliomas had positive telomerase activity, as did the lymphoma. The meningioma and normal brain were negative. Quantitative analysis of telomerase activity did not correlate with tumor grade nor predict outcome. Selective tissue microdissection, combined with qualitative and quantitative telomerase assays, permits rapid and reliable detection of telomerase activity in diverse brain tumor tissues. These preliminary findings suggest that telomerase reactivation is a frequent event in glioma tumorigenesis that can be sensitively and specifically detected in gliomas of all histologic grades. Furthermore, specific detection of telomerase reactivation represents another mechanism by which tumor formation and progression might become the target of novel therapeutics.

Adult↗
PubMed1998

Differential rate of cholesterol efflux from the apical and basolateral membranes of MDCK cells.

Epithelial cells contain two distinct membrane surfaces, the apical and basolateral plasma membranes, which have different lipid and protein compositions. In order to assess the effect of the compositional differences of the apical and basolateral membranes on their ability to undergo cholesterol efflux, MDCK cells were radiolabeled with [3H]cholesterol and grown as a polarized monolayer on filter inserts, that separate the upper apical compartment from the lower basolateral compartment. The rate of cholesterol efflux from the basolateral membrane into media containing HDL in the basolateral compartment was 6.3%/h +/-0.7, whereas HDL-mediated efflux from the apical membrane was approximately 3-fold slower (1.9%/h +/-0.3). In contrast, Fu5AH cells, which do not form distinct polarized membrane domains, had a similar rate of HDL-mediated cholesterol efflux into the apical and basolateral compartments. Similar to HDL, other cholesterol acceptors, namely LDL, bovine serum albumin, and a lipid emulsion, also showed a decreased rate of cholesterol efflux from the apical membrane surface versus the basolateral membrane. Compared to the basolateral membrane, the apical membrane was also found to be more resistant to cholesterol oxidase treatment, to bind less HDL, and to take up less cholesterol from the medium. In conclusion, cholesterol efflux occurred less readily from the apical membrane than from the basolateral membrane for all types of acceptors tested. These results suggest that differences in the composition of the apical and basolateral membrane lead to a relative decrease in cholesterol desorption from the apical membrane and hence a reduced rate of cholesterol efflux.

Animals↗
PubMed1997

Decreased reverse cholesterol transport from Tangier disease fibroblasts. Acceptor specificity and effect of brefeldin on lipid efflux.

Tangier disease is characterized by HDL hypercatabolism and increased deposition of cholesterol in tissues. Tangier disease skin fibroblasts have decreased apoA-I-mediated cholesterol and phospholipid efflux, which may lead to the excess accumulation of cellular cholesterol. The mechanism of apolipoprotein-mediated cholesterol efflux and the apolipoprotein acceptor specificity for cholesterol efflux from normal and Tangier disease fibroblasts was investigated. Normal cells readily effluxed cholesterol and phospholipid to apoA-I and to all of the other apolipoproteins tested (apoA-II, AIV, C-I, C-II, C-III). In contrast, Tangier cells were almost completely defective in cholesterol efflux to apoA-I and to all of the other apolipoproteins tested. HDL was also less effective, by approximately 50%, in stimulating cholesterol efflux from Tangier cells compared with normal cells. In addition, Tangier cells also showed significantly reduced phospholipid efflux to both apolipoproteins and HDL. A similar rate of cholesterol efflux, however, was observed from normal and Tangier cells when phospholipid vesicles or cyclodextrin were used as acceptors. In contrast to normal cells, only phospholipid vesicles and cyclodextrin and not apoA-I or HDL depleted intracellular cholesteryl esters from Tangier cells. Brefeldin, an inhibitor of intracellular vesicular trafficking, decreased HDL-mediated cholesterol efflux by approximately 40% but almost completely blocked both cholesterol and phospholipid efflux to apoA-I from normal cells. Brefeldin also inhibited cholesteryl ester depletion by apoA-I and HDL from normal cells. Brefeldin, however, had no significant effect on cholesterol efflux from Tangier cells to HDL. In summary, Tangier cells were found to be defective in both cholesterol and phospholipid efflux to HDL and apoA-I. The defect in apolipoprotein-mediated lipid efflux was not specific for apoA-I but also occurred for other apolipoproteins, and brefeldin blocked HDL-mediated lipid efflux from normal but not Tangier disease cells. On the basis of these results, a model is proposed whereby decreased cholesterol efflux by apolipoproteins in Tangier cells is the result of a defect in a brefeldin-sensitive pathway of lipid efflux.

Adult↗
PubMed1995

Carboxyl-terminal domain truncation alters apolipoprotein A-I in vivo catabolism.

Apolipoprotein A-I (apoA-I), the major protein of high density lipoproteins, facilitates reverse cholesterol transport from peripheral tissue to liver. To determine the structural motifs important for modulating the in vivo catabolism of human apoA-I (h-apoA-I), we generated carboxyl-terminal truncation mutants at residues 201 (apoA-I201), 217 (apoA-I217), and 226 (apoA-I226) by site-directed mutagenesis. ApoA-I was expressed in Escherichia coli as a fusion protein with the maltose binding protein, which was removed by factor Xa cleavage. The in vivo kinetic analysis of the radioiodinated apoA-I in normolipemic rabbits revealed a markedly increased rate of catabolism for the truncated forms of apoA-I. The fractional catabolic rates (FCR) of 9.10 +/- 1.28/day (+/- S.D.) for apoA-I201, 6.34 +/- 0.81/day for apoA-I217, and 4.42 +/- 0.51/day for apoA-I226 were much faster than the FCR of recombinant intact apoA-I (r-apoA-I, 0.93 +/- 0.07/day) and h-apoA-I (0.91 +/- 0.34/day). All the truncated forms of apoA-I were associated with very high density lipoproteins, whereas the intact recombinant apoA-I (r-apoA-I) and h-apoA-I associated with HDL2 and HDL3. Gel filtration chromatography revealed that in contrast to r-apoA-I, the mutant apoA-I201 associated with a phospholipid-rich rabbit apoA-I containing particle. Analysis by agarose gel electrophoresis demonstrated that the same mutant migrated in the pre-beta position, but not within the alpha position as did r-apoA-I. These results indicate that the carboxyl-terminal region (residue 227-243) of apoA-I is critical in modulating the association of apoA-I with lipoproteins and in vivo metabolism of apoA-I.

Animals↗
PubMed1995

Identification of novel differentially expressed hepatic genes in cholesterol-fed rabbits by a non-targeted gene approach.

Several key genes involved in cholesterol metabolism are known to be directly regulated by cholesterol. The possible indirect effect, however, of increased levels of cellular cholesterol on gene expression and its possible role in cholesterol metabolism and atherosclerosis has not been thoroughly explored. In order to determine the overall effect of cholesterol on gene expression, we isolated differentially expressed genes from a PCR-based subtraction library prepared from the liver of chow-fed and cholesterol-fed rabbits. A total of nine upregulated and four down-regulated cDNA fragments were isolated. As determined by Northern blot analysis, the expression of the isolated cDNAs began to change as early as the first week on the cholesterol-rich diet or as late as 4 weeks, which corresponded with hepatic cholesterol accumulation. Three of the cDNAs were identified by DNA sequence homology, whereas the remaining cDNAs had no significant homology match. CYP1A1, a cytochrome P450 isoenzyme, was found to be down-regulated in hepatocytes by cholesterol feeding. Osteopontin and Mac-2, which are produced by macrophages, were found to be up-regulated in Kupffer cells by cholesterol feeding. Overall these results demonstrate the usefulness of the subtraction library approach for identifying new candidate genes for exploring the pathogenesis of atherosclerosis.

Animals↗
PubMed1995

Polarized secretion of apoA-I and apoA-II by transfected MDCK cells.

Apolipoproteins (apo) are secreted preferentially from the basolateral surface of hepatocytes and enterocytes. The polarized secretion of proteins is either mediated by a protein-dependent sorting signal or by a cell-dependent default pathway. In order to determine the mechanism for the polarized secretion of apolipoproteins, we examined the secretion of apoA-I and apoA-II in transfected Madin-Darby canine kidney (MDCK) cells. Transfected MDCK cells and Caco-2 cells were grown as a polarized monolayer on tissue culture inserts, which separate an upper apical compartment from the lower basolateral compartment, and the secretion of apoA-I and apoA-II into the apical and basolateral compartments was quantitated by immunoprecipitation. Caco-2 cells almost exclusively secreted apoA-I and apoA-II basolaterally, with an apical to basolateral ratio of 18:82 for apoA-I, and 11:89 for apoA-II. In contrast, transfected MDCK cells secreted significant amounts of apoA-I and apoA-II into both compartments, but with a bias toward apical secretion and an apical to basolateral ratio of 66:34 and 68:32, respectively. The polarized secretion of MDCK cells was not due to transcytosis, diffusion, or differential recovery. As assessed by density gradient ultracentrifugation, apoA-I and apoA-II secreted from either the apical or basolateral surface were relatively lipid-poor. Overall, these results suggest that the polarized secretion of apoA-I and apoA-II does not occur by a protein-dependent sorting signal, but by a cell-dependent default pathway that leads to preferential basolateral secretion by Caco-2 cells and both apical and basolateral secretion in MDCK cells, but with a bias toward apical secretion.

Animals↗
PubMed1993

O-linked glycosylation modifies the association of apolipoprotein A-II to high density lipoproteins.

O-linked glycosylation is a common post-translational modification of apolipoproteins, but no structural or functional role for it has been identified. We examined the biosynthesis of apolipoprotein (apo) A-II in Hep G2 cells and in glycosylation-defective Chinese hamster ovary (CHO) cell mutants transfected with apoA-II cDNA. Three monomeric isoforms of apoA-II with an apparent molecular mass of 8.5, 9.8, and 11.4 kDa were synthesized by Hep G2 cells and transfected wild-type CHO cells. The 9.8- and 11.4-kDa isoforms were sialylated but not the 8.5-kDa isoform. Transfected 1dlD cells, which are defective in the biosynthesis of galactose and N-acetylgalactosamine, only produced the 8.5-kDa isoform; however, when grown in media supplemented with these sugars, ldlD cells produced all three isoforms of apoA-II. Pulse-chase analysis of ldlD cells showed that glycosylation was not necessary for secretion of apoA-II. Glycosylation did modify the association of apoA-II with nascent high density lipoprotein (HDL) secreted by Hep G2 cells. The sialylated isoforms were lipid-poor and were present in the lipoprotein-deficient density range, whereas the nonsialylated 8.5-kDa isoform was associated with LpA-I, A-II lipoprotein particles in the HDL density range. ApoA-II from transfected ldlD cells, regardless of glycosylation, were lipid-poor. When preincubated with HDL from serum, however, sialylated apoA-II from both ldlD cells and Hep G2 cells associated with lipoprotein particles within the HDL3 density, whereas nonsialylated apoA-II was found throughout the HDL density range. In summary, O-linked glycosylation is not necessary for the secretion of apoA-II but does modify the association of apoA-II to HDL and may, therefore, play an important role in the metabolism of HDL.

Animals↗
PubMed1992

N-linked glycosylation of rabies virus glycoprotein. Individual sequons differ in their glycosylation efficiencies and influence on cell surface expression.

Many eukaryotic proteins are modified by N-linked glycosylation, a process in which oligosaccharides are added to asparagine residues in the sequon Asn-X-Ser/Thr. However, not all such sequons are glycosylated. For example, rabies virus glycoprotein (RGP) contains three sequons, only two of which appear to be glycosylated in virions. To examine further the signals in proteins which regulate N-linked core glycosylation, the glycosylation efficiencies of each of the three sequons in the antigenic domain of RGP were compared. For these studies, mutants were generated in which one or more sequons were deleted by site-directed mutagenesis. Core glycosylation of these mutants was studied using two independent systems: 1) in vitro translation in rabbit reticulocyte lysate supplemented with dog pancreatic microsomes, and 2) transfection into glycosylation-deficient Chinese hamster ovary cells. Parallel results were obtained with both systems, demonstrating that the sequon at Asn37 is inefficiently glycosylated, the sequons at Asn247 and Asn319 are efficiently glycosylated, and the glycosylation efficiency of each sequon is not influenced by glycosylation at other sequons in this protein. High levels of cell surface expression of RGP in Chinese hamster ovary cells are seen with any mutant containing an intact sequon at Asn247 or Asn319, whereas low levels of cell surface expression are seen when the sequon at Asn37 is present alone; deletion of all three sequons completely blocks RGP cell surface expression. Thus, although core glycosylation at Asn37 is inefficient, it is still sufficient to support a biological function, cell surface expression. Future studies using mutagenesis of this model protein and its expression in these two well defined systems will aim to begin to unravel the rules governing core glycosylation of glycoproteins.

Antigens, Viral↗
PubMed1991

Expression of human glycophorin A in wild type and glycosylation-deficient Chinese hamster ovary cells. Role of N- and O-linked glycosylation in cell surface expression.

Glycophorin A, the most abundant sialoglycoprotein on human red blood cells, carries several medically important blood group antigens. To study the role of glycosylation in surface expression and antigenicity of this highly glycosylated protein (1 N-linked and 15 O-linked oligosaccharides), glycophorin A cDNA (M-allele) was expressed in Chinese hamster ovary (CHO) cells. Both wild type CHO cells and mutant CHO cells with well defined glycosylation defects were used. Glycophorin A was well expressed on the surface of transfected wild type CHO cells. On immunoblots, the CHO cells expressed monomer (approximately 38 kDa) and dimer forms of glycophorin A which co-migrated with human red blood cell glycophorin A. The transfected cells specifically expressed the M blood group antigen when tested with mouse monoclonal antibodies. Tunicamycin treatment of these CHO cells did not block surface expression of glycophorin A, indicating that, in the presence of normal O-linked glycosylation, the N-linked oligosaccharide is not required for surface expression. To study O-linked glycosylation, glycophorin A cDNA was transfected into the Lec 2, Lec 8, and ldlD glycosylation-deficient CHO cell lines. Glycophorin A with truncated O-linked oligosaccharides was well expressed on the surface of ldlD cells (cultured in the presence of N-acetylgalactosamine alone), Lec 2 cells, and Lec 8 cells with monomers of approximately 25 kDa, approximately 33 kDa, and approximately 25 kDa, respectively. In contrast, non-O-glycosylated glycophorin A (approximately 19-kDa monomers) was poorly expressed on the surface of ldlD cells cultured in the absence of both galactose and N-acetylgalactosamine. Thus, under these conditions, in the absence of O-linked glycosylation, the N-linked oligosaccharide itself is not able to support appropriate surface expression of glycophorin A in transfected CHO cells.

Animals↗
PubMed1991

Stable expression of rabies virus glycoprotein in Chinese hamster ovary cells.

The rabies virus glycoprotein (G protein) has several important functions and is a major antigenic stimulus of the host immune system following rabies virus infection or vaccination. We developed a model system for studying the role of N-linked glycosylation in the intracellular transport and antigenicity of this molecule. The full-length cDNA of the G protein of the ERA strain of rabies virus was inserted into the eukaryotic shuttle vector pSG5 and then stably transfected into wild-type Chinese hamster ovary (CHO) cells and mutant CHO cell lines defective in glycosylation. Transfected wild-type CHO cells expressed the G protein (detected by immunofluorescence) on the cell surface in a manner similar to rabies virus-infected cells. The transfected wild-type CHO cells were shown by immunoprecipitation to produce a protein of 67K that comigrated with the fully glycosylated G protein isolated from virus-infected cells or purified virions. Treatment of the transfected cell lines with tunicamycin completely blocked surface expression and resulted in the intracellular accumulation of the G protein, suggesting that the presence of N-linked oligosaccharides is important for transport of this glycoprotein to the plasma membrane. The G protein cDNA was also expressed in the lectin-resistant CHO cell lines Lec 1, Lec 2 and Lec 8. In these cells initial N-linked glycosylation does occur, but later steps in processing of the oligosaccharides are blocked. In each case, the G protein was expressed on the surface of lectin-resistant CHO cells in a similar manner to expression on wild-type CHO cells. This suggests that various different N-linked oligosaccharide structures support intracellular transport of this glycoprotein. Thus, stably transfected CHO cell lines will provide a useful model system for further studies of the role of N-linked glycosylation in trafficking and antigenicity of the rabies virus G protein.

Animals↗