Biomedical subjects

D L Jarvis

Publications and source records attributed to D L Jarvis.

At least 19 recordsLinked to original sources

PubMed2005

Indoor nitrous acid and respiratory symptoms and lung function in adults.

BACKGROUND: Nitrogen dioxide (NO2) is an important pollutant of indoor and outdoor air, but epidemiological studies show inconsistent health effects. These inconsistencies may be due to failure to account for the health effects of nitrous acid (HONO) which is generated directly from gas combustion and indirectly from NO2. METHODS: Two hundred and seventy six adults provided information on respiratory symptoms and lung function and had home levels of NO2 and HONO measured as well as outdoor levels of NO2. The association of indoor HONO levels with symptoms and lung function was examined. RESULTS: The median indoor HONO level was 3.10 ppb (IQR 2.05-5.09), with higher levels in homes with gas hobs, gas ovens, and in those measured during the winter months. Non-significant increases in respiratory symptoms were observed in those living in homes with higher HONO levels. An increase of 1 ppb in indoor HONO was associated with a decrease in forced expiratory volume in 1 second (FEV1) percentage predicted (-0.96%; 95% CI -0.09 to -1.82) and a decrease in percentage FEV1/forced vital capacity (FVC) (-0.45%; 95% CI -0.06 to -0.83) after adjustment for relevant confounders. Measures of indoor NO2 were correlated with HONO (r = 0.77), but no significant association of indoor NO2 with symptoms or lung function was observed. After adjustment for NO2 measures, the association of HONO with low lung function persisted. CONCLUSION: Indoor HONO levels are associated with decrements in lung function and possibly with more respiratory symptoms. Inconsistencies between studies examining health effects of NO2 and use of gas appliances may be related to failure to account for this association.

Adult↗

PubMed2001

Improved glycosylation of a foreign protein by Tn-5B1-4 cells engineered to express mammalian glycosyltransferases.

The major advantages of using the baculovirus-insect cell system for recombinant protein production are its ability to produce large amounts of recombinant proteins and its ability to provide eucaryotic modifications, such as glycosylation. However, the glycans linked to recombinant glycoproteins produced by this system typically differ from those found on native mammalian products. This is an important problem because glycans on mammalian glycoproteins can influence their functions in many different ways. The inability of baculovirus-infected insect cells to produce glycans identical to those found on native mammalian glycoproteins is due, in part, to the absence of functional levels of certain glycosyltransferases in insect cells. Thus, the purpose of this study was to engineer these activities into Tn-5B1-4, an established insect cell line that is widely used as a host for baculovirus-mediated protein production. Expression plasmids were constructed in which cDNAs encoding mammalian beta1,4-galactosyltransferase and alpha2,6-sialyltransferase were placed under the transcriptional control of a baculovirus immediate early promoter. These plasmids were then used to isolate two different transgenic Tn-5B1-4 derivatives and the biological and biochemical properties of these cell lines were examined. The results show that both of the engineered insect cell lines have improved glycoprotein-processing capabilities, relative to the parental cell line.

Animals↗

PubMed2001

Biosynthesis and subcellular localization of a lepidopteran insect alpha 1,2-mannosidase.

Like lower and higher eucaryotes, insects have alpha 1,2-mannosidases which function in the processing of N-glycans. We previously cloned and characterized an insect alpha 1,2-mannosidase cDNA and demonstrated that it encodes a member of a family of N-glycan processing alpha 1,2-mannosidases (Kawar, Z., Herscovics, A., Jarvis, D.L., 1997. Isolation and characterisation of an alpha 1,2-mannosidase cDNA from the lepidopteran insect cell line Sf9. Glycobiology 7, 433-443). These enzymes have similar protein sequences, require calcium for their activities, and are sensitive to 1-deoxymannojirimycin, but can have different substrate specificities and intracellular distributions. We recently determined the substrate specificity of the insect alpha 1,2-mannosidase, SfManI (Kawar, Z., Romero, P., Herscovics, A., Jarvis, D.L., 2000. N-glycan processing by a lepidopteran insect and 1,2-mannosidase. Glycobiology 10, 347-355). Now, we have examined the biosynthesis and subcellular localization of SfManI. We found that SfManI is partially N-glycosylated and that N-glycosylation is dramatically enhanced if the wild type sequon is changed to one that is highly utilized in a mammalian system. We also found that an SfManI-GFP fusion protein had a punctate cytoplasmic distribution in insect cells. Colocalization studies indicated that this fusion protein is localized in the Golgi apparatus, not in the endoplasmic reticulum or lysosomes. Finally, N-glycosylation had no influence over the substrate specificity or subcellular localization of SfManI.

Animals↗

PubMed2001

Insect cells encode a class II alpha-mannosidase with unique properties.

Previously, we cloned and characterized an insect (Sf9) cell cDNA encoding a class II alpha-mannosidase with amino acid sequence and biochemical similarities to mammalian Golgi alpha-mannosidase II. Since then, it has been demonstrated that other mammalian class II alpha-mannosidases can participate in N-glycan processing. Thus, the present study was performed to evaluate the catalytic properties of the Sf9 class II alpha-mannosidase and to more clearly determine its relationship to mammalian Golgi alpha-mannosidase II. The results showed that the Sf9 enzyme is cobalt-dependent and can hydrolyze Man(5)GlcNAc(2) to Man(3)GlcNAc(2), but it cannot hydrolyze GlcNAcMan(5)GlcNAc(2). These data establish that the Sf9 enzyme is distinct from Golgi alpha-mannosidase II. This enzyme is not a lysosomal alpha-mannosidase because it is not active at acidic pH and it is localized in the Golgi apparatus. In fact, its sensitivity to swainsonine distinguishes the Sf9 enzyme from all other known mammalian class II alpha-mannosidases that can hydrolyze Man(5)GlcNAc(2). Based on these properties, we designated this enzyme Sf9 alpha-mannosidase III and concluded that it probably provides an alternate N-glycan processing pathway in Sf9 cells.

Animals↗

PubMed2001

Mammalian glycosyltransferase expression allows sialoglycoprotein production by baculovirus-infected insect cells.

The baculovirus-insect cell expression system is widely used to produce recombinant mammalian glycoproteins, but the glycosylated end products are rarely authentic. This is because insect cells are typically unable to produce glycoprotein glycans containing terminal sialic acid residues. In this study, we examined the influence of two mammalian glycosyltransferases on N-glycoprotein sialylation by the baculovirus-insect cell system. This was accomplished by using a novel baculovirus vector designed to express a mammalian alpha2,6-sialyltransferase early in infection and a new insect cell line stably transformed to constitutively express a mammalian beta1,4-galactosyltransferase. Various biochemical assays showed that a foreign glycoprotein was sialylated by this virus-host combination, but not by a control virus-host combination, which lacked the mammalian glycosyltransferase genes. Thus, this study demonstrates that the baculovirus-insect cell expression system can be metabolically engineered for N-glycoprotein sialylation by the addition of two mammalian glycosyltransferase genes.

Animals↗

PubMed2001

Engineering lepidopteran insect cells for sialoglycoprotein production by genetic transformation with mammalian beta 1,4-galactosyltransferase and alpha 2,6-sialyltransferase genes.

Recombinant mammalian glycoproteins produced by the baculovirus-insect cell expression system usually do not have structurally authentic glycans. One reason for this limitation is the virtual absence in insect cells of certain glycosyltransferases, which are required for the biosynthesis of complex, terminally sialylated glycoproteins by mammalian cells. In this study, we genetically transformed insect cells with mammalian beta 1,4-galactosyltransferase and alpha 2,6-sialyltransferase genes. This produced a new insect cell line that can express both genes, serve as hosts for baculovirus infection, and produce foreign glycoproteins with terminally sialylated N-glycans.

Animals↗

PubMed2001

Novel baculovirus expression vectors that provide sialylation of recombinant glycoproteins in lepidopteran insect cells.

This report describes novel baculovirus vectors designed to express mammalian beta1,4-galactosyltransferase and alpha2,6-sialyltransferase genes at early times after infection. Sf9 cells infected with these viral vectors, unlike cells infected with a wild-type baculovirus, produced a sialylated viral glycoprotein during the late phase of infection. Thus, the two mammalian glycosyltransferases encoded by these viral vectors are necessary and sufficient for sialylation of a foreign glycoprotein in insect cells under the conditions used in this study. While some of the new baculovirus vectors described in this study produced less, one produced wild-type levels of infectious budded virus progeny.

Animals↗

PubMed2001

Glycoproteins from insect cells: sialylated or not?

Our growing comprehension of the biological roles of glycan moieties has created a clear need for expression systems that can produce mammalian-type glycoproteins. In turn, this has intensified interest in understanding the protein glycosylation pathways of the heterologous hosts that are commonly used for recombinant glycoprotein expression. Among these, insect cells are the most widely used and, particularly in their role as hosts for baculovirus expression vectors, provide a powerful tool for biotechnology. Various studies of the glycosylation patterns of endogenous and recombinant glycoproteins produced by insect cells have revealed a large variety of O- and N-linked glycan structures and have established that the major processed O- and N-glycan species found on these glycoproteins are (Gal beta1,3)GalNAc-O-Ser/Thr and Man3(Fuc)GlcNAc2-N-Asn, respectively. However, the ability or inability of insect cells to synthesize and compartmentalize sialic acids and to produce sialylated glycans remains controversial. This is an important issue because terminal sialic acid residues play diverse biological roles in many glycoconjugates. While most work indicates that insect cell-derived glycoproteins are not sialylated, some well-controlled studies suggest that sialylation can occur. In evaluating this work, it is important to recognize that oligosaccharide structural determination is tedious work, due to the infinite diversity of this class of compounds. Furthermore, there is no universal method of glycan analysis; rather, various strategies and techniques can be used, which provide glycobiologists with relatively more or less precise and reliable results. Therefore, it is important to consider the methodology used to assess glycan structures when evaluating these studies. The purpose of this review is to survey the studies that have contributed to our current view of glycoprotein sialylation in insect cell systems, according to the methods used. Possible reasons for the disagreement on this topic in the literature, which include the diverse origins of biological material and experimental artifacts, will be discussed. In the final analysis, it appears that if insect cells have the genetic potential to perform sialylation of glycoproteins, this is a highly specialized function that probably occurs rarely. Thus, the production of sialylated recombinant glycoproteins in the baculovirus-insect cell system will require metabolic engineering efforts to extend the native protein glycosylation pathways of insect cells.

Animal Diseases↗

PubMed2000

N-Glycan processing by a lepidopteran insect alpha1,2-mannosidase.

Protein glycosylation pathways are relatively poorly characterized in insect cells. As part of an overall effort to address this problem, we previously isolated a cDNA from Sf9 cells that encodes an insect alpha1,2-mannosidase (SfManI) which requires calcium and is inhibited by 1-deoxymannojirimycin. In the present study, we have characterized the substrate specificity of SfManI. A recombinant baculovirus was used to express a GST-tagged secreted form of SfManI which was purified from the medium using an immobilized glutathione column. The purified SfManI was then incubated with oligosaccharide substrates and the resulting products were analyzed by HPLC. These analyses showed that SfManI rapidly converts Man(9)GlcNAc(2)to Man(6)Glc-NAc(2)isomer C, then more slowly converts Man(6)GlcNAc(2)isomer C to Man(5)GlcNAc(2). The slow step in the processing of Man(9)GlcNAc(2)to Man(5)GlcNAc(2)by SfManI is removal of the alpha1,2-linked mannose on the middle arm of Man(9)GlcNAc(2). In this respect, SfManI is similar to mammalian alpha1,2-mannosidases IA and IB. However, additional HPLC and(1)H-NMR analyses demonstrated that SfManI converts Man(9)GlcNAc(2)to Man(5)GlcNAc(2)primarily through Man(7)GlcNAc(2)isomer C, the archetypal Man(9)GlcNAc(2)missing the lower arm alpha1,2-linked mannose residues. In this respect, SfManI differs from mammalian alpha1,2-mannosidases IA and IB, and is the first alpha1,2-mannosidase directly shown to produce Man(7)GlcNAc(2)isomer C as a major processing intermediate.

Acetylglucosamine↗

PubMed2000

N-glycan patterns of human transferrin produced in Trichoplusia ni insect cells: effects of mammalian galactosyltransferase.

The N-glycans of human serum transferrin produced in Trichopulsia ni cells were analyzed to examine N-linked oligosaccharide processing in insect cells. Metabolic radiolabeling of the intra- and extracellular protein fractions revealed the presence of multiple transferrin glycoforms with molecular weights lower than that observed for native human transferrin. Consequently, the N-glycan structures of transferrin in the culture medium were determined using three-dimensional high performance liquid chromatography. The attached oligosaccharides included high mannose, paucimannosidic, and hybrid structures with over 50% of these structures containing one fucose, alpha(1,6)-, or two fucoses, alpha(1,6)- and alpha(1,3)-, linked to the Asn-linked N-acetylglucosamine. Neither sialic acid nor galactose was detected on any of the N-glycans. However, when transferrin was coexpressed with beta(1,4)-galactosyltransferase three additional galactose-containing hybrid oligosaccharides were obtained. The galactose attachments were exclusive to the alpha(1, 3)-mannose branch and the structures varied by the presence of zero, one, or two attached fucose residues. Furthermore, the presence of the galactosyltransferase appeared to reduce the number of paucimannosidic structures, which suggests that galactose attachment inhibits the ability of hexosaminidase activity to remove the terminal N-acetylglucosamine. The ability to promote galactosylation and reduce paucimannosidic N-glycans suggests that the oligosaccharide processing pathway in insect cells may be manipulated to mimic more closely that of mammalian cells.

Animals↗

PubMed1999

Electrophoretic analysis of glycoprotein glycans produced by lepidopteran insect cells infected with an immediate early recombinant baculovirus encoding mammalian beta1,4-galactosyltransferase.

Glycosylation, the most extensive co- and post-translational modification of eukaryotic cells, can significantly affect biological activity and is particularly important for recombinant glycoproteins in human therapeutic applications. The baculovirus-insect cell expression system is a popular tool for the expression of heterologous proteins and has an excellent record of producing high levels of biologically active eukaryotic proteins. Insect cells are capable of glycosylation, but their N-glycosylation pathway is truncated in comparison with the pathway of mammalian cells. A previous study demonstrated that an immediate early recombinant baculovirus could be used to extend the insect cell N-glycosylation pathway by contributing bovine beta-1,4 galactosyltransferase (GalT) immediately after infection. Lectin blotting assays indicated that this ectopically expressed enzyme could transfer galactose to an N-linked glycan on a foreign glycoprotein expressed later in infection. In the current study, glycans were isolated from total Sf-9 cell glycoproteins after infection with the immediate early recombinant baculovirus encoding GalT, fluorescently conjugated and analyzed by electrophoresis in combination with exoglycosidase digestion. These direct analyses clearly demonstrated that Sf-9 cells infected with this recombinant baculovirus can synthesize galactosylated N-linked glycans.

Animals↗

PubMed1998

Engineering N-glycosylation pathways in the baculovirus-insect cell system.

The inability to produce eukaryotic glycoproteins with complex N-linked glycans is a major limitation of the baculovirus-insect cell expression system. Recent studies have demonstrated that metabolic engineering can be used to extend the glycoprotein processing capabilities of lepidopteran insect cells. This approach is being used to develop new baculovirus-insect cell expression systems that can produce more authentic recombinant glycoproteins and obtain new information on insect N-glycosylation pathways.

Animals↗

PubMed1998

Stable expression of mammalian beta 1,4-galactosyltransferase extends the N-glycosylation pathway in insect cells.

An established lepidopteran insect cell line (Sf9) was cotransfected with expression plasmids encoding neomycin phosphotransferase and bovine beta 1,4-galactosyltransferase. Neomycin-resistant transformants were selected, assayed for beta 1,4-galactosyltransferase activity, and the transformant with the highest level of enzymatic activity was characterized. Southern blots indicated that this transformed Sf9 cell derivative contained multiple copies of the galactosyltransferase-encoding expression plasmid integrated at a single site in its genome. One-step growth curves showed that these cells supported normal levels of baculovirus replication. Baculovirus infection of the transformed cells stimulated beta 1,4-galactosyltransferase activity almost 5-fold by 12 h postinfection. This was followed by a gradual decline in activity, but the infected cells still had about as much activity as uninfected controls as late as 48 h after infection and they were able to produce a beta 1,4-galactosylated virion glycoprotein during infection. Infection of the transformed cells with a conventional recombinant baculovirus expression vector encoding human tissue plasminogen activator also resulted in the production of a galactosylated end-product. These results demonstrate that stable transformation can be used to add a functional mammalian glycosyltransferase to lepidopteran insect cells and extend their N-glycosylation pathway. Furthermore, stably-transformed insect cells can be used as modified hosts for conventional baculovirus expression vectors to produce foreign glycoproteins with "mammalianized" glycans which more closely resemble those produced by higher eucaryotes.

Animals↗

PubMed1998

Mutational analysis of the N-linked glycans on Autographa californica nucleopolyhedrovirus gp64.

gp64 is the major envelope glycoprotein in the budded form of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). gp64 is essential for AcMNPV infection, as it mediates penetration of budded virus into host cells via the endocytic pathway. In this study, we used site-directed mutagenesis to map the positions of the N-linked glycans on AcMNPV gp64, characterize their structures, and evaluate their influence on gp64 function. We found that four of the five consensus N-glycosylation sites in gp64 are used, and we mapped the positions of those sites to amino acids 198, 355, 385, and 426 in the polypeptide chain. Endoglycosidase H sensitivity assays showed that N-linked glycans located at different positions are processed to various degrees. Lectin blotting analyses showed that each N-linked glycan on gp64 contains alpha-linked mannose, all but one contains alpha-linked fucose, and none contains detectable beta-linked galactose or alpha2,6-linked sialic acid. The amounts of infectious progeny produced by AcMNPV mutants lacking one, two, or three N-linked glycans on gp64 were about 10- to 100-fold lower than wild-type levels. This reduction did not correlate with reductions in the expression, transport, or inherent fusogenic activity of the mutant gp64s or in the gp64 content of mutant budded virus particles. However, all of the mutant viruses bound more slowly than the wild type. Therefore, elimination of one or more N-glycosylation sites in AcMNPV gp64 impairs binding of budded virus to the cell, which explains why viruses containing these mutant forms of gp64 produce less infectious progeny.

Animals↗

PubMed1997

Isolation and characterization of a class II alpha-mannosidase cDNA from lepidopteran insect cells.

Lepidopteran insect cells are used routinely as hosts for foreign glycoprotein expression by recombinant baculoviruses, but the precise nature of their N-glycosylation pathway remains poorly defined. These cells clearly have processing glucosidases and mannosidases that can convert precursors to Man3GlcNAc2 structures and fucosyltransferases that can add fucose to the oligosaccharide core. However, their ability to extend these structures to produce complex side chains like those found in mammalian cells remains to be determined. To begin to examine this pathway at the molecular genetic level, we isolated and characterized a class II alpha-mannosidase (alpha-mannosidase II) cDNA from Sf9, a lepidopteran insect cell line. In mammalian cells, this enzyme catalyzes the committed step in the pathway converting N-linked carbohydrates to complex forms. Degenerate primers against conserved regions in known class II alpha-mannosidase protein sequences were used to generate an alpha-mannosidase II-specific PCR product from Sf9 cell DNA. Sequence information from this product was used to isolate a partial cDNA clone, the 5' end was isolated by ligation-anchored PCR, and the full length alpha-mannosidase II cDNA was assembled. This cDNA contained a long open reading frame predicted to encode an 1130 amino acid protein with 37% identity to human Golgi alpha-mannosidase II and with a type II membrane topology, a feature of all known Golgi processing enzymes. Southern blotting indicated that alpha-mannosidase II is a single copy gene in Sf9 cells. Other Lepidoptera had related alpha-mannosidase II genes, but there was variation among different genera, and the Sf9 alpha-mannosidase II cDNA did not cross-hybridize with DNA from animals outside Lepidoptera. Steady-state levels of alpha-mannosidase II RNA were low in uninfected Sf9 cells and even lower after baculovirus infection. The in vitro-translated Sf9 alpha-mannosidase II protein had the expected size and was translocated and N-glycosylated by microsomal membranes. Expression of the Sf9 alpha-mannosidase II cDNA in the baculovirus system produced large amounts of a protein with the expected size and swainsonine-sensitive alpha-mannosidase II activity towards an aryl-alpha-mannoside substrate. These results demonstrate that Sf9 cells encode and express an alpha-mannosidase II with properties similar to those of the mammalian enzyme.

Amino Acid Sequence↗

PubMed1997

Isolation and characterization of an alpha 1,2-mannosidase cDNA from the lepidopteran insect cell line Sf9.

As part of our ongoing efforts to characterize the N-glycosylation pathway of lepidopteran insect cells, we have isolated an alpha 1,2-mannosidase homolog from an Sf9 cDNA library. This cDNA contains an open reading frame which encodes a 670 amino acid protein with a calculated molecular weight of 75,225 Da. This protein has two potential N-glycosylation sites, two consensus calcium binding sequences, and is predicted to be a type II integral membrane protein with a 22 amino acid transmembrane domain (residues 31-52). The amino acid sequence of this protein is 35-57% identical to Drosophila, human, murine, and yeast alpha 1,2-mannosidases. A transcript of approximately 6 kilobases was detected by Northern blot analysis of Sf9 mRNA. Genomic Southern blots probed with an intron-free fragment of the alpha 1,2-mannosidase gene indicated that there are at least two copies or cross-hybridizing variants of this gene in the Sf9 genome. In vivo expression of the cDNA using a recombinant baculovirus produced a protein that released [3H]mannose from [3H]Man9GlcNAc. This activity required calcium, but not magnesium, and was inhibited by 1-deoxymannojirimycin. These results indicate that Sf9 cells encode and express an alpha 1,2-mannosidase with properties similar to those of other eukaryotic processing alpha 1,2-mannosidases.

Amino Acid Sequence↗

PubMed1997

Variation in nebulizer aerosol output and weight output from the Mefar dosimeter: implications for multicentre studies.

The active aerosol component of nebulizers is less than 100% of output by weight, and may vary between nebulizers in different batches from the same manufacturer. A measure of bronchial responsiveness to methacholine, which can overcome this problem, is required. One hundred and sixty nebulizers from 21 centres in the European Community Respiratory Health Survey (ECRHS) were calibrated for aerosol and weight output. Methacholine challenge data were obtained for 1,021 subjects in three English centres of the ECRHS. The dose producing a 20% fall in forced expiratory volume in one second (PD20), and log-slope, the regression slope of percentage decline in FEV1 with log (dose), were calculated, with and without calibration of nebulizers by weight. Within-centre variation in nebulizer percentage aerosol output had a coefficient of variation of less than 10%. Unlike PD20, log-slope is unaffected by constant percentage overestimation of nebulizer output. Variation in output by weight of nebulizers of 10% had little affect on log-slope. It is, however, affected by the scheduled range of doses. Log-slope shows advantages in analysis, and is less affected by variation in nebulizer output. It can be used for multicentre comparisons, with restriction to a common dose protocol.

Aerosols↗

PubMed1996

The role of the AcMNPV 25K gene, "FP25," in baculovirus polh and p10 expression.

A previous study showed that an Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) 25K mutant produced less polyhedrin protein than wild-type (Jarvis et al., J. Virol. 66, 6903-6911, 1992). In this study, the role of the 25K gene product (AcMNPV ORF 61) in baculovirus gene expression was further investigated. Five different viral 25K mutants expressed lower levels of polyhedrin protein and less CAT activity under the control of the polh promoter compared to wild-type. Polh RNA was equally stable in wild-type and mutant virus-infected cells while the rate of polh transcription was significantly reduced in mutant-infected cells. In comparison, steady-state levels of p10 RNA were not reduced in 25K mutant-infected cells, indicating that the reduction in polh RNA did not reflect a general effect on very late gene transcription. Expression of ie-1, which also appears to influence polh expression (Choi and Guarino, Virology 209, 90-98, 1995), was not influenced by 25K mutation. These results show that the 25K protein is important for maintaining optimal levels of polh transcription by a mechanism that does not involve maintaining ie-1 expression.

Animals↗