Identification of genes encoding N‐glycan processing β‐N‐acetylglucosaminidases in Trichoplusia ni and Bombyx mori: Implications for glycoengineering of baculovirus expression systems

Glycoproteins produced by non‐engineered insects or insect cell lines characteristically bear truncated, paucimannose N‐glycans in place of the complex N‐glycans produced by mammalian cells. A key reason for this difference is the presence of a highly specific N‐glycan processing β‐N‐acetylglucosaminidase in insect, but not in mammalian systems. Thus, reducing or abolishing this enzyme could enhance the ability of glycoengineered insects or insect cell lines to produce complex N‐glycans. Of the three insect species routinely used for recombinant glycoprotein production, the processing β‐N‐acetylglucosaminidase gene has been isolated only from Spodoptera frugiperda. Thus, the purpose of this study was to isolate and characterize the genes encoding this important processing enzyme from the other two species, Bombyx mori and Trichoplusia ni. Bioinformatic analyses of putative processing β‐N‐acetylglucosaminidase genes isolated from these two species indicated that each encoded a product that was, indeed, more similar to processing β‐N‐acetylglucosaminidases than degradative or chitinolytic β‐N‐acetylglucosaminidases. In addition, over‐expression of each of these genes induced an enzyme activity with the substrate specificity characteristic of processing, but not degradative or chitinolytic enzymes. Together, these results demonstrated that the processing β‐N‐acetylglucosaminidase genes had been successfully isolated from Trichoplusia ni and Bombyx mori. The identification of these genes has the potential to facilitate further glycoengineering of baculovirus‐insect cell expression systems for the production of glycosylated proteins. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Gene Control in Somatic Embryos of Digitalis lanata: Expression of the β-Glucuronidase Gene Fused to a Plastocyanin Promoter

Digitalis lanata was transformed by agrobacteria-mediated gene transfer with a chimeric reporter gene encoding for β-glucuronidase (CUS) from Escherichia coll under the control of the plastocyanin 3 (Pc3) promoter from Spinada oleracea (Pc3::uidA fusion gene). Transformed cell lines were regenerated to plants via somatic embryos. CUS activity was determined fluorometrically and histochemically. The Pc3::uidA fusion gene was expressed in the late globular and bipolar stages of somatic embryos. Expression started in globular embryos (stage-1-globules) in that part of the parenchymatic tissue which later on formed the cotyledons. No GUS activity was detectable in the parenchymatic tissue forming the root pole, in cells of the developing procambium or in epidermal cells. These tissues were free of GUS activity also in bipolar embryos. The parenchymatic cells of the cotyledons and the primary cortex of the hypocotyl of germinating embryos showed GUS activity, in contrast to the epidermal cells and the cells of the central cylinder.     

In vivo deglycosylation of recombinant glycoproteins in tobacco BY-2 cells

Production of recombinant pharmaceutical glycoproteins has been carried out in multiple expression systems. However, N-glycosylation, which increases heterogeneity and raises safety concerns due to the presence of non-human residues, is usually not controlled. The presence and composition of N-glycans are also susceptible to affect protein stability, function and immunogenicity. To tackle these issues, we are developing glycoengineered Nicotiana tabacum Bright Yellow-2 (BY-2) cell lines through knock out and ectopic expression of genes involved in the N-glycosylation pathway. Here, we report on the generation of BY-2 cell lines producing deglycosylated proteins. To this end, endoglycosidase T was co-expressed with an immunoglobulin G or glycoprotein B of human cytomegalovirus in BY-2 cell lines producing only high mannose N-glycans. Endoglycosidase T cleaves high mannose N-glycans to generate single, asparagine-linked, N-acetylglucosamine residues. The N-glycosylation profile of the secreted antibody was determined by mass spectrometry analysis. More than 90% of the N-glycans at the conserved Asn297 site were deglycosylated. Likewise, extensive deglycosylation of glycoprotein B, which possesses 18 N-glycosylation sites, was observed. N-glycan composition of gB glycovariants was assessed by in vitro enzymatic mobility shift assay and proven to be consistent with the expected glycoforms. Comparison of IgG glycovariants by differential scanning fluorimetry revealed a significant impact of the N-glycosylation pattern on the thermal stability. Production of deglycosylated pharmaceutical proteins in BY-2 cells expands the set of glycoengineered BY-2 cell lines.     

Differences between normal and scaleless (sc/sc) chicken epidermal cell surfaces detected with wheat germ agglutinin

Dissociated epidermal cells derived from the backskin of scaleless chick embryos (stage 34 or 35) form larger agglutinates with wheat germ agglutinin (WGA) than epidermal cells from normal embryonic skin. [³H]Acetyl WGA binding to the scaleless cells is twice as great as to normal epidermal cells. Treatment of these cells with concanavalin A (conA) results in equivalent agglutination of both mutant and normal epidermal cells, whereas neither scaleless nor normal epidermal cells are agglutinated by Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA) or Ulex europeus agglutinin (UEA). This alteration in cell surface carbohydrates may be related to the failure of the scaleless mutant embryonic epidermis to undergo normal morphogenesis.     

Stereoselective hydrolysis of xenobiotic esters by different cell lines from rat liver and hepatoma and its application to chiral prodrugs for designated growth suppression of cancer cells

Stereoselectivity in the hydrolysis of racemic ethyl 2-phenylacetate derivatives by cultured cells of noncancerous cell lines from rat liver (BRL, BRL 3A, Clone 9, and ARLJ301–3), spontaneously or oncogene transformed rat liver cell lines (ARLJ301–3TR1, Anr4, Anr9–1, and Anr13–1), and cancer cell lines from rat hepatoma (H4-II-E, McA-RH7777, and MH₁C₁) and sarcoma (XC) was studied. A strong (R)-enantiomer preference was found in the hydrolysis of ethyl 2-hydroxy- ( 2c ) and 2-methoxy-2-phenylacetate ( 3c ) by the noncancerous and oncogene-transformed cells and an (S)-enantiomer preference for ethyl N-acylphenylalaninates with all the present cell lines. These inclinations were, however, not recognized with ethyl 2-methoxy-2-phenylpropanoate and ethyl N-difluoroacetyl-or N-trifluoroacetylphenylalaninate. Moreover, the R preference for 3c was reversed in the reaction by hepatoma cells. Thus, the stereoselectivity is influenced by both structure of acyl group and species of cell lines. The hepatoma cells were considerably different from the noncancerous or oncogene-transformed cells in stereoselectivity. This fact was consistent with the order of colony formation in soft agar cultures (index of malignancy) and the resemblance in actively stained esterase patterns in gel electrophoresis. The stereoselective hydrolysis leads to cell-specific activation of anticancer prodrugs. This has been confirmed for the first time by the stereoselectivity of Anr4 and H4-II-E cells in the hydrolysis of a chiral mustard ester, bis(2-chloroethyl)aminophenyl 2-methoxy-2-phenylacetate ( 14 ) and by the difference of IC₅₀ values of (R)- and (S)- 14 against the two cell lines. © 1995 Wiley-Liss, Inc.     

The nephrotoxin ochratoxin A induces apoptosis in cultured human proximal tubule cells

To test the apoptotic potential of the nephrotoxic mycotoxin ochratoxin A (OTA), we exposed human proximal tubule-derived cells (IHKE cells) for various times to OTA concentrations close to those occurring during dietary exposure (from 2 to 100 nmol/L) and investigated caspase 3 activation, chromatin condensation, and DNA fragmentation. OTA induced a time- and concentration-dependent activation of caspase 3: concentrations as low as 5 nmol/L OTA caused a slight but significant increase in caspase 3 activity after 7 days of OTA exposure. Exposure to 10 nmol/L OTA for 72 or 24 h led to a significantly increased activity of caspase 3 in human proximal tubule-derived cells. Radical scavengers such as N-acetylcysteine had no effect on OTA-induced caspase 3 activation. Chelation of intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethylester) (BAPTA-AM) also showed no effect. Exposure to 30 nmol/L or more OTA led to DNA fragmentation and chromatin condensation in IHKE cells. Cultured renal epithelial MDCK-C7 and MDCK-C11 or OK cells also showed increased caspase 3 activity after OTA exposure. We conclude that exposure to low OTA concentrations can lead to direct or indirect caspase 3 activation and subsequently to apoptosis in cultured human proximal tubule cells and in other renal epithelial cell lines of different origins. This revised version was published online in July 2006 with corrections to the Cover Date.     

Developmental changes in the amount of pigments,inorganic material and uric acid inLocusta migratoria cinerascens Fabr. (Orthoptera,Acrididae) epidermis,during the last larval instar and the imaginal life

Summary Locusta epidermal cells accumulate in their cytoplasm various inclusions which give rise to tegumentary patterns of brown spots and lines.Five major types of inclusions are identified. Ultrastructural examination and electron microprobe studies show that types 1, 2 and 3 correspond to successive steps of concentric pigment and mineral deposition on a matrix. Type 3 is the richest in ommochromes (acridiommatins 1 and 2), and mineralized with calcium phosphate. Type 4 is characterized by pigment and mineral disappearance, which in type. 5 are substituted by uric microcrystals. From the end of the 4^th instar to the beginning of the imaginal life, three generations of inclusions are observed to succeed each other in the different epidermal cells. Tergal, pleural and sternal areas are characterized by the morphology and biochemical composition of the inclusions they accumulate. Despite cellular asynchronism, ecdysis is accompanied by a phase of pigment and cation elimination followed by the storage of uric microcrystals.Abbreviations L larval instar (numbered I to V) Abbreviations used in Figs 2, 3 and 4 B basal membrane - Cu cuticle - E epidermis - Gly glycogen - Ly lysosome - M muscle cell - N nucleus - P pigment (ommochrome) granule - SC secretory cell - tu microtubule - Ur uric acid microcrystal     

The Components of the CO2-Indluced Stomatal Movements

Leaf discs of Vicia Faba were allowed to float on water in glass dishes placed in vessels containing KOH. The vessels were kept in darkness at constant temperature. The stomatal width and osmotic values of the guard cells and epidermal cells were measured, generally at one-hour intervals. When the CO₂ content of the air surrounding the leaf specimen falls, it causes a disturbance in the osmotic equilibrium between guard cells and epidermal cells. Sometimes the changes start in the form of falling osmotic values in both kinds of cell. In other cases the values rise, and in still others the changes may be confined chiefly to one of these kinds of cell. Since the changes are not the same in guard cells and epidermal cells, the osmotic difference between them rises or falls. The difference rises during the time immediately after removal of CO₂ from the surrounding air. This causes an osmotic surplus to arise or increase in the guard cells. Later, this change may take place in the opposite direction. The stomatal movements occurring simultaneously follow, on broad lines, the osmotic surplus of the guard cells. Consequently, the CO₂-induced stomatal movement is the result of an interaction between an active component—i.e., the intrinsic osmotic changes in the guard cells—and an osmopassive component, by which is meant here the osmotic changes in the epidermal cells.