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1.
Summary The glycosylation and subsequent processing of native and recombinant glycoproteins expressed in established insect cell lines
and insect larvae were compared. TheSpodoptera frugiperda (Sf21) andTrichoplusia ni (TN-368 and BTI-Tn-5B1-4) cell lines possessed several intrinsic glycoproteins that are modified with both N- and O-linked
oligosaccharides. The N-linked oligosaccharides were identified as both the simple (high mannose) and complex (containing
sialic acid) types. Similarly, theT. ni larvae also possessed intrinsic glycoproteins that were modified with O-linked and simple and complex N-linked oligosaccharides.
Additionally, human placental, secreted alkaline phosphatase (SEAP) produced during replication of a recombinant baculovirus
inT. ni larvae was modified with complex oligosaccharide having sialic acid linked α(2–6) to galactose. 相似文献
2.
T. R. Davis M. L. Shuler R. R. Granados H. A. Wood 《In vitro cellular & developmental biology. Animal》1993,29(11):842-846
Summary The processing of the N-linked oligosaccharide modifying a secreted alkaline phosphatase glycoprotein (SEAP) expressed with
a recombinantAutographa californica nuclear polyhedrosis virus was evaluated in insect cell lines established fromSpodoptera frugiperda, Trichoplusia ni, andMamestra brassicae. Studies with Endoglycosidase H (Endo H), which removes high-mannose oligosaccharides, revealed that 79% of the intracellular
SEAP produced in theM. brassicae-derived MB0503 cell line was Endo H resistant. The commonly usedS. frugiperda Sf21 and Sf9 cell lines produced 44 and 21% Endo H-resistant intracellular SEAP, respectively. Detection of oligosaccharide
moieties with lectins, which selectively recognize terminal sugars, identified only mannose residues on SEAP expressed in
the six insect cell lines. However, the oligosaccharide moiety of SEAP expressed in a Chinese hamster ovary cell line contained
sialic acid. Therefore, when expressed in mammalian cells, the oligosaccharide present on SEAP is processed into complex oligosaccharide,
but in insect cells it is of the high-mannose type. Studies with inhibitors of the initial oligosaccharide processing steps
demonstrated that all six cell lines possessed glycosidase I/II and mannosidase I activity and that glycosylation was required
for secretion. 相似文献
3.
N-Linked glycosylation of a baculovirus-expressed recombinant glycoprotein in insect larvae and tissue culture cells 总被引:5,自引:3,他引:2
The potential of insect cell cultures and larvae infected with recombinant
baculoviruses to produce authentic recombinant glycoproteins cloned from
mammalian sources was investigated. A comparison was made of the N-linked
glycans attached to secreted alkaline phosphatase (SEAP) produced in four
species of insect larvae and their derived cell lines plus one additional
insect cell line and larvae of one additional species. These data survey
N-linked oligosaccharides produced in four families and six genera of the
order Lepidoptera. Recombinant SEAP expressed by recombinant isolates of
Autographa californica and Bombyx mori nucleopolyhedroviruses was purified
from cell culture medium, larval hemolymph or larval homogenates by
phosphate affinity chromatography. The N-linked oligosaccharides were
released with PNGase-F, labeled with 8- aminonaphthalene-1-3-6-trisulfonic
acid, fractionated by polyacrylamide gel electrophoresis, and analyzed by
fluorescence imaging. The oligosaccharide structures were confirmed with
exoglycosidase digestions. Recombinant SEAP produced in cell lines of
Lymantria dispar (IPLB-LdEIta), Heliothis virescens (IPLB-HvT1), and Bombyx
mori (BmN) and larvae of Spodoptera frugiperda, Trichoplusia ni ,
H.virescens , B.mori , and Danaus plexippus contained oligosaccharides that
were structurally identical to the 10 oligosaccharides attached to SEAP
produced in T.ni cell lines. The oligosaccharide structures were all
mannose-terminated. Structures containing two or three mannose residues,
with and without core fucosylation, constituted more than 75% of the
oligosaccharides from the cell culture and larval samples.
相似文献
4.
T. R. Davis T. J. Wickham K. A. McKenna R. R. Granados M. L. Shuler H. A. Wood 《In vitro cellular & developmental biology. Animal》1993,29(5):388-390
Summary A recombinantAutographa californica baculovirus expressing secreted alkaline phosphatase (SEAP) gene was used to evaluate the expression of a secreted glycoprotein
in eight insect cell lines derived fromSpodoptera frugiperda, Trichoplusia ni, Mamestra brassicae andEstigmene acrea. Because cell density was found to influence protein production, SEAP production was evaluated at optimal cell densities
for each cell line on both a per cell and per milliliter basis. On a per cell basis, theT. ni-derived BTI-TN-5B1-4 cells produced a minimum of 20-fold more SEAP than theS. frugiperda-derived Sf9 or Sf21 cell lines and a minimum of 9-fold more than any of the other cell lines growing in serum-containing
medium. On a per milliliter basis, BTI-TN-5B1-4 cells produced a minimum of fivefold more SEAP than any of the other cell
lines tested. Using cell lines that were adapted to serum-free medium, SEAP yields were the same or better than their counterparts
in serum-containing medium. At 3 days postinoculation, extracellular SEAP activity ranged from 59 to 85% of total SEAP activity
with cell lines grown in serum-free and serum-containing media. 相似文献
5.
Summary The production of recombinant secreted alkaline phosphatase protein in virally infected insect cells was studied in shaker
flask and high aspect rotating-wall vessel (HARV) culture. Two commonly used cell lines, Spodoptera frugiperda Sf-9 (Sf-9) and a nonaggregating isolate of the Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) cell line, Trichoplusia ni Tn-5B1-4-NA (TN-5B1-4-NA), were used and monitored for 120-h postinfection. Different responses to culture in the HARV were
seen in the two cell lines. While the Sf-9 cell line was able to produce slightly greater amounts of recombinant protein in
the HARV than in shaker flask controls, the Tn-5B1-4-NA cell line produced significantly lesser amounts in the HARV than in
the shaker flasks. Both cell lines exhibited longer life spans and longer periods of protein production in HARV culture than
in shaker flask culture, presumably due to lower levels of shear encountered in the HARV. The important difference was in
the protein production rate responses of the two cell lines. While the protein production rates of Sf-9 cells were comparable
in both HARV and shaker flask cultures, the protein production rates of Tn-5B1-4-NA cells were much lower in HARV culture
than in shaker flask cultures. The conclusion is drawn that cell line-specific adaptation to the HARV strongly influences
recombinant protein production. 相似文献
6.
James E. Maruniak Alejandra Garcia-Canedo Jaqueline J. S. Rodrigues 《In vitro cellular & developmental biology. Animal》1994,30(4):283-286
Summary Four insect cell lines were used to isolate two recombinant baculoviruses which had theβ-galactosidase (β-gal) gene for colorimetric assay purposes. Plaque assays were performed using twoTrichoplusia ni cell lines: BTI-TN-5B1-4 and TN-368, and twoSpodoptera frugiperda cell lines: IPLB-SF-21AE and SF9. The number of plaques (occlusion positive and blueβ-gal+ recombinants) formed in theTrichoplusia cells was higher than in theSpodoptera cells. The appearance ofAutographa californica NPV polyhedra was also faster in theT. ni cell lines. The effect of cell passage on the plaque formation proved to be critical when two different passages of the SF9
cells were tested. The higher passage produced a lower viral titration. The size and time of appearance of the plaques was
also different. 相似文献
7.
Bo Liu Xin Gong Shaohong Chang Yili Yang Miao Song Demin Duan Lina Wang Qingjun Ma Jun Wu 《Journal of biotechnology》2009,143(2):95-102
Glycoproteins secreted by the yeast Kluyveromyces lactis are usually modified by the addition at asparagines-linked glycosylation sites of heterogeneous mannan residues. The secreted glycoproteins in K. lactis that become hypermannosylated will bear a non-human glycosylation pattern and can adversely affect the half-life, tissue distribution and immunogenicity of a therapeutic protein. Here, we describe engineering a K. lactis strain to produce non-hypermannosylated glycoprotein, decreasing the outer-chain mannose residues of N-linked oligosaccharides. We investigated and developed the method of two-step homologous recombination to knockout the OCH1 gene, encoding α1,6-mannosyltransferase and MNN1 gene, which is homologue of Saccharomyces cerevisiae MNN1, encoding a putative α1,3-mannosyltransferase. We found the Kloch1 mutant strain has a defect in hyperglycosylation, inability in adding mannose to the core oligosaccharide. The N-linked oligosaccharides assembled on a secretory glycoprotein, HSA/GM–CSF in Kloch1 mutant, contained oligosaccharide Man13–14GlcNAc2, and in Kloch1 mnn1 mutant, contained oligosaccharide Man9–11GlcNAc2, whereas those in the wild-type strain, consisted of oligosaccharides with heterogeneous sizes, Man>30GlcNAc2. Taken together, these results indicated that KlOch1p plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in K. lactis. The KlMnn1p, was proved to be certain contribution to the outer hypermannosylation, most possibly encodes α1,3-mannosyltransferase. Therefore, the Kloch1 and Kloch1 mnn1 mutants can be used as a foundational host to produce glycoproteins lacking the outer-chain hypermannoses and further maybe applicable to be a promising system for yeast therapeutic protein production. 相似文献
8.
Comparative studies were performed on the replication of the Autographa californica nuclear polyhedrosis virus in cell lines from Estigmene acrea, BTI-EAA; Lymantria dispar, IPLB-LD64BA; Mamestra brassicae, IZD-MB0503; Spodoptera frugiperda, IPLB-SF1254; and Trichoplusia ni, TN-368. Significant differences were observed in the amount of virus obtained from the cell lines, with M. brassicae and T. ni producing more polyhedra than the other lines. These two cell lines also produced nonoccluded virus most rapidly, followed by S. frugiperda, E. acrea, and L. dispar. Sensitivities of the cell lines to infection by the virus, as determined by plaque formation, followed the same pattern, with M. brassicae being most sensitive and L. dispar least so. The T. ni cell line produced polyhedra which were more pathogenic to T. ni larvae than those from the other cells. These differences have important implications in the application of cell cultures in the development of microbial insecticides. 相似文献
9.
G. M. S. Lima R. W. S. Aguiar R. F. T. Corrêa E. S. Martins A. C. M. Gomes T. Nagata M. T. De-Souza R. G. Monnerat B. M. Ribeiro 《World journal of microbiology & biotechnology》2008,24(12):2941-2948
The cry2Aa and cry2Ab genes from a Brazilian Bacillus thuringiensis strain were introduced into the genome of the baculovirus Autographa californica
multiple nucleopolyhedrovirus (AcMNPV) in order to evaluate the heterologous proteins expression in insect cells and their toxicity to different insects.
The recombinant viruses (vAcCry2Aa and vSynCry2Ab) were amplified in Trichoplusia ni (BTI-Tn5B1-4) cells and used to infect Spodoptera frugiperda larvae. Total extracts from S. frugiperda infected with the recombinant viruses were analysed by SDS-PAGE, which detected the presence of polypeptides around 65 kDa.
Cuboid-shaped protein crystals were observed in insect extracts by light and scanning electron microscopy. Bioassays, using
the heterologous proteins showed toxicity against second instar A. gemmatalis larvae (Cry2Aa) with a LC50 of 1.03 μg/ml and second instar S. frugiperda larvae (Cry2Ab) with a LC50 of 3.45 μg/ml. No toxic activity was detected for Aedes aegypti and Culex quinquenfaciatus. 相似文献
10.
We have expanded on the suitability ofp-aminobenzoic acid ethyl ester as an ultraviolet-absorbing reagent [Wanget al., (1984) Anal Biochem 141:366–81] for the analysis of asparagine-linked oligosaccharides derived from glycoproteins. The oligosaccharides released from glycoproteins by hydrazinolysis/N-reacetylation were derivatized withp-aminobenzoic acid ethyl ester and the derivatives were purified and separated into neutral and acidic oligosaccharides on a PRE-SEP C18 cartridge. The acidic oligosaccharides could be further separated into a few species by high-voltage paper electrophoresis.
p-Aminobenzoic acid ethyl ester derivatives of neutral oligosaccharides were analyzed by gel permeation chromatography on Bio-Gel P-4 and HPLC on a silica-based amide column. The elution profile and the proportion of the oligosaccharides were in agreement with literature values. The overall yield of oligosaccharides from glycoproteins was approximately 70%. Fifty pmol of oligosaccharide were detectable on Bio-Gel P-4 and 4–5 pmol on HPLC.Abbreviations HPLC
high performance liquid chromatography
- NABEE
p-aminobenzoic acid ethyl ester
- FAB-MS
fast-atom bombardment mass spectrometry
- (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, (GlcNAc)5 and (GlcNAc)6
chito-oligosaccharides containing 2,3,4,5 and 6 residues ofN-acetylglucosamine 相似文献
11.
William J. McCarthy 《In vitro cellular & developmental biology. Animal》1994,30(10):690-695
Summary Comparison of lytic-dose response behavior of seven lepidopteran cell lines to the activated delta-endotoxin polypeptides
ofBacillus thuringiensis subspecieskurstaki (HD-263) andaizawai (HD-112) indicated distinct differences among the lines. The lines derived fromSpodoptera speciesS. exigua (URC-SE-1A) andS. littoralis (UIV-SL-575) were more susceptible to lysis byaizawai towin (Bta) thankurstaki toxin (Btk) as were cells from theLymantria dispar line (IPLB-LD652Y). However, the concentrations of Bta required for lysis of 50% of URC-SE-1A and IPLB-LD652Y cells (LC50) were 0.2 to 0.8 μg/ml compared to 5 to 9 μg/ml for UIV-SL-575 cells. In comparison, Btk LC50 concentrations for the three lines were similar (14 to 19 μg/ml). Cells fromS. frugiperda (IPLB-SF-21AE) andTrichoplusia ni (TN368) were similar in their response to Bta (LC50=2.5 to 3.7 μg/ml) and Btk (LC50=1.0 to 2.8 μg/ml) whereas the lines derived fromHeliothis spp. were the least susceptible to both toxins. The LC50 concentrations for Bta with theH. zea line (IPLB-HA-1075) andH. virescens line (BCIRL-HV-AM1) were >50 μg/ml and for Btk were >50 μg/ml and 42 to 50 μg/ml, respectively, yet for both lines Btk was
the more cytolytic.
Cytolysis of TN368 cells could be inhibited to varying extents by preincubation of the toxins with the aminosugars of galactose,
mannose, and glucose and theirN-acetyl derivatives. The unsubstituted hexoses were not inhibitory. The order of decreasing inhibitory effectiveness was the
same for both toxins regardless of the derivative species and followed the order galactose, mannose, and glucose. Also, inhibition
of cytolysis could be achieved to varying extents by assaying cells grown in medium with tunicamycin. Lysis with Btk was inhibited
68 and 37% using treated cells of TN368 and IPLB-LD652Y, respectively; however, no inhibition was observed with URC-SE-1A
cells. Further, no inhibition of Bta-mediated lysis was obtained with tunicamycin-grown cells of the three lines. 相似文献
12.
Haruko Ogawa Ako Hijikata Maho Amano Kyoko Kojima Hisako Fukushima Ineo Ishizuka Yoshie Kurihara Isamu Matsumoto 《Glycoconjugate journal》1996,13(4):555-566
The oligosaccharide structures ofCry j I, a major allergenic glycoprotein ofCryptomeria japonica (Japanese cedar, sugi), were analysed by 400 MHz1H-NMR and two-dimensional sugar mapping analyses. The four major fractions comprised a series of biantennary complex type N-linked oligosaccharides that share a fucose/xylose-containing core and glucosamine branches including a novel structure with a nongalactosylated fucosylglucosamine branch.Rabbit polyclonal anti-Cry j I IgG antibodies cross-reacted with three different plant glycoproteins having the same or shorter N-linked oligosaccharides asCry j I. ELISA and ELISA inhibition studies with intact glycoproteins, glycopeptides and peptides indicated that both anti-Cry j I IgGs and anti-Sophora japonica bark lectin II (B-SJA-II) IgGs included oligosaccharide-specific antibodies with different specificities, and that the epitopic structures against anti-Cry j I IgGs include a branch containing 1–6 linked fucose and a core containing fucose/xylose, while those against anti-B-SJA-II IgGs include nonreducing terminal mannose residues. The cross-reactivities of human allergic sera to miraculin andClerodendron Trichotomum lectin (CTA) were low, and inhibition studies suggested that the oligosaccharides onCry j I contribute little or only conformationally to the reactivity of specific IgE antibodies.Abbreviations
Cry j I
a major allergenic glycoprotein ofCryptomeria japonica
- B-SJA-II
Sophora japonica bark lectin II
- CTA
Clerodendron trichotomum lectin
- TFMS
trifluoromethanesulfonic acid
- HRP
horseradish peroxidase 相似文献
13.
Transfection of insect cell lines using polyethylenimine 总被引:1,自引:0,他引:1
I. D. Ogay O. A. Lihoradova Sh. S. Azimova A. A. Abdukarimov J. M. Slack D. E. Lynn 《Cytotechnology》2006,51(2):89-98
Insect cell lines have been widely used in recombinant baculovirus expression systems and transient gene expression studies. Critical to these applications have been the transfection of foreign DNA. This has been frequently done using labor intensive and cytotoxic liposome-based transfection reagents. In the current study we have optimized a new kind of polyethylenimine-based DNA transfection reagent on the Spodoptera frugiperda Sf9 insect cell line. A plasmid vector that transiently expresses green fluorescent protein (GFP) was effectively delivered into Sf9 cells. A transfection efficiency of 54% and cell viability of 85–90% were obtained for Sf9 cells. The developed transfection protocol has now been successfully used to transfect eight insect cell lines derived from Bombyx mori, Trichoplusia ni, Helicoverpa zea, Heliothis virescens and S. frugiperda with GFP and GUS with transfection efficiencies of at least 45%. This method provides high heterologous protein expression levels, transfection efficacy and cell viability, and could be used for transient gene expression in other lepidopteran cell lines.
相似文献
D. E. LynnEmail: Phone: 301-504-6328Fax: 301-504-5104 |
14.
Encapsulation and development of the endoparasitoid,Microplitis croceipes (Cresson), were studied in six atypical lepidopteran host species whose usual host isHelicoverpa zea (Boddie). The candidate hosts examined were: the fall armywormSpodoptera frugiperda (J. E. Smith); the beet armyworm,Spodoptera exigua (Hübner); the cabbage looper,Trichoplusia ni (Hübner); the greater wax moth,Galleria mellonella (L.); the Indian meal moth,Plodia interpunctella (Hübner); and the diamondback moth,Plutella xylostella (L.). BothS. exigua andT. ni were completely unsuitable forM. croceipes development due to the high rate of eggs that were encapsulated within three days after parasitism. Encapsulation inS. frugiperda included mainly parasitoid eggs and was first detected six days after parasitization at 25°C and two days at 30°C. Encapsulation inG. mellonella occurred only in the larval stage of the parasitoid. InP. interpunctella, parasitoid larvae reached the 3rd stadium, but none of them pupated. OnlyS. frugiperda andG. mellonella supported successful development ofM. croceipes from egg to adult. The percentage of parasitoids reaching the adult stage in these hosts was higher at 30°C than at 25°C (13% vs. 4% inS. frugiperda, and 21% vs. 3% inG. mellonella, respectively). However, these percentages were too low to substitute them as a more economical host for rearingM. croceipes. This biological information will be useful in additional laboratory studies directed toward reducing the rate of encapsulation (e.g., manipulation of host rearing temperature) to increase production ofM. croceipes on these hosts. 相似文献
15.
Unlike their counterparts in budding yeast Saccharomyces cerevisiae, the glycoproteins of Schizosaccharomyces pombe contain, in addition to α-d-mannose (Man), a large number of α-d-galactose (Gal) residues. In both yeasts, large outer chains are attached to the oligosaccharide cores of glycoproteins during
export via Golgi. Formation of the yeast-specific large outer chain is initiated by α-1,6-mannosylatransferase encoded by
the och1
+ gene, the disruption of which blocked outer chain elongation. We previously reported that N-linked oligosaccharide structures of S. pombe och1Δ mutant consisted of Gal2–6Man9GlcNAc2 with α-linked Gal residues attached to the core oligosaccharide moiety. The disruption of gms1
+, a gene encoding the UDP-galactose transporter required for the synthesis of galactomannan, abolished cell surface galactosylation
in S. pombe. In this study, we constructed a gms1Δoch1Δ double mutant and determined the N- and O-linked oligosaccharide structures present on the cell surface. Oligosaccharides were liberated from glycoproteins by hydrazinolysis
and labeled with the fluorophore, 2-aminopyridine. The pyridylaminated N-linked oligosaccharides were analyzed by high-performance liquid chromatography in combination with α1,2-mannosidase digestion
and partial acetolysis. These analyses revealed that the N-linked oligosaccharides of gms1Δoch1Δ cells consisted of α1,2-linked Man-extended core oligosaccharides (Man8–12GlcNAc2) from which the fission yeast-specific α-linked Gal residues were completely absent. 相似文献
16.
The yeast extract (of unknown origin) present in the commercially available serum-free medium ‘Express Five’ contains factors
(‘yeast extract factors’) up to 35 kDa which are essential for growth of Trichoplusia ni insect cells. A yeast extract brand lacking these components could not support growth of T. ni cells. However, cell proliferation was restored by adding chromatographic fractions containing the yeast extract factors.
The yeast extract factors were not solely responsible for the growth enhancing effect of yeast extract but some other components,
which seem to be generally present in yeast extracts, are also required for T. ni proliferation. 相似文献
17.
Insect cell cultures have been extensively utilised for means of production for heterologous proteins and biopesticides. Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five™) cell lines have been widely used for the production of recombinant proteins, thus metabolism of these cell lines
have been investigated thoroughly over recent years. The Helicoverpa zea cell line has potential use for the production of a biopesticide, specifically the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV). The growth, virus production, nutrient consumption and waste production
of this cell line was investigated under serum-free culture conditions, using SF900II and a low cost medium prototype (LCM).
The cell growth (growth rates and population doubling time) was comparable in SF900II and LCM, however, lower biomass and
cell specific virus yields were obtained in LCM. H. zea cells showed a preference for asparagine over glutamine, similar to the High Five™ cells. Ammonia was accumulated to significantly
high levels (16 mM) in SF900II, which is an asparagine and glutamine rich medium. However, given the absence of asparagine
and glutamine in the medium (LCM), H. zea cells adapted and grew well in the absence of these substrates and no accumulation of ammonia was observed. The adverse effect
of ammonia on H. zea cells is unknown since good production of biologically active HaSNPV was achieved in the presence of high ammonia levels.
H. zea cells showed a preference for maltose even given an abundance supply of free glucose. Accumulation of lactate was observed
in H. zea cell cultures.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
18.
Evidence that specific high mannose structures directly regulate multiple cellular activities 总被引:4,自引:0,他引:4
Neeraja Sathyamoorthy Jean M. Decker Anne P. Sherblom Andrew Muchmore 《Molecular and cellular biochemistry》1991,102(2):139-147
Previous studies have demonstrated that much of the immunomodulatory activity of the glycoprotein uromodulin can be attributed to attached oligosaccharides. Structural studies of isolated and purified saccharides derived from uromodulin suggest that the structure Man6GlcNAc2-asn can inhibit in vitro assays of antigen driven T cell proliferation. Based on these observations, we isolated a series of high mannose glycopeptides from a variety of natural sources and tested them for biologic activity in a number of assays. We found that purified mannose rich glycopeptides are able to activate the hexose monophosphate (HMP)shunt, induce prostaglandin synthesis, and directly stimulate IL-1 synthesis. These in vitro effects appear to have in vivo counterparts. Thus in a species-restricted fashion, high mannose compounds are able to directly activate a delayed mononuclear cell infiltrate after intradermal injection. Our data suggest that specific mannose oligosaccharides may activate as well as inhibit cellular immune responses at several different levels. These findings support the hypothesis that specific saccharide structures could participate in the physiologic regulation of the immune response. 相似文献
19.
Shainn-Wei Wang William J. McCarthy 《In vitro cellular & developmental biology. Animal》1997,33(4):315-323
Summary A sensitive lactate dehydrogenase (LDH) assay was modified to determine the cytolytic activity of Bacillus thuringiensis CryIC and CryIAc delta endotoxins to viable collagenase-dissociated midgut epithelial cells (MEC) from larvae of Spodoptera frugiperda and Spodoptera exigua. The MEC preparations from these Spodoptera sp. consisted predominantly of columnar cells (65–75%) and goblet cells (25–35%). Time course microscopy experiments indicated
that only the columnar cells became swollen during CryIC toxin incubation. Also, comparative cytotoxicity studies were run
with cell lines of nonmidgut origin established from S. frugiperda (SF21AE) and S. exigua (SEUCR1A). Optimum conditions for the cytotoxicity assay were similar for MEC and cell lines of both species, and were met
in an assay in which 0.1-ml cell concentrations (8.5±0.5×104 cells) were incubated with toxin dilutions (0.01–20 μg) for 1 h at 24° C at a final pH of 7.8.
The Spodoptera sp. MEC were twofold more sensitive to CryIC (68% lysis) than CryIAc (32% lysis) at optimum toxin levels (2.5–5 μg). Also,
the SEUCR1A cells were more sensitive (2.3-fold) to CryIC (70% lysis) than CryIAc (30% lysis) at optimum toxin levels of 5–10
μg. The SF21AE cells, however, were twofold less sensitive to CryIC (30% lysis) than SEUCR1A cells and response to CryIAc
and CryIC was similar.
Immunoblot analysis of either Spodoptera sp. MEC or brush border membrane vesicles (BBMV) identified seven CryIC binding proteins with molecular mass of 137, 120,
115, 68, 65, 63, and 45 kDa. Occasionally, a 148-kDa protein band was observed. The CryIAc toxin bound to two proteins on
MEC and BBMV with molecular mass of 137 and 120 kDa. 相似文献
20.
Summary In this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal
surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal β1,3 GalNAc and (GlcNAc β1,4)
n
oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal β1-3 GalNAc, Gal-αGal,
and (GlcNAc β1,4)
n
oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal β1,3
GalNAc, (GlcNAc β1,4)
n
oligomers and fucose linked α1,6 or terminal α1,3 or α1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system
contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-αGal, Gal β1-3 GalNAc and (GlcNAc β1,4)
n
oligomers; Fuc linked α1,2 to Gal, α1,3 to GlcNAc in (poly)lactosamine chains and α1,6 to GlcNAc in N-linked glycans. Most
of these glycoproteins probably corresponds to the H+K+-ATPase β-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose,
(GlcNAc β1,4)
n
oligomers and fucose linked α1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive
tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly
in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier. 相似文献