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41.
Stable expression of mammalian beta 1,4-galactosyltransferase extends the N-glycosylation pathway in insect cells 总被引:5,自引:2,他引:3
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.
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42.
The impact of gender and/or hormone variations on a wide variety of neural functions makes the choice between studying males or females (or both) of a given species difficult. Although female rats are widely used experimentally, few studies control for the stage of estrus. More detailed information about how to distinguish the various stages of the estrous cycle is needed. For the present study, vaginal smears were obtained once a day and stained using an adaptation of the Papanicolaou (PAP) procedure. Images are provided of unstained “wet” samples and the corresponding PAP stained smears illustrating the cellular profile for each stage of the cycle as well as post-ovariectomy. The different cell populations across the cycle were quantified and ratios determined to show trends between the predominant and other cell types in each stage of the estrous cycle. Both stained and unstained images and cell quantification data provide valuable guidelines for distinguishing the stages of the estrous cycle. 相似文献
43.
Dadley-Moore Davina L. Lightowlers Marshall W. Gauci Charles G. Rothel James S. Jackson David C. 《International journal of peptide research and therapeutics》1999,6(5-6):303-312
Summary Two peptides corresponding to putative protective regions located at the N- and C-termini of the host-protectiveT. ovis recombinant antigen, 45W, were synthesized. Antibodies raised against 45W and 45WB/X, a truncated from of 45W, were found
to react strongly with the N-terminal peptide. When sheep were immunised with each peptide alone, the N-terminal peptide was
found to be highly immunogenic, whereas the C-terminal peptide required conjugation to a carrier protein to be immunogenic.
Both these immunogens elicited antibodies that cross-reacted with the parent protein; however, only antibodies directed toward
the N-terminal peptide were able to bind antigens from theT. ovis oncosphere. Significant protection against challenge infection was not provided by any of the peptide immunogens used. 相似文献
44.
Molecular and genetic characterisation of the host-protective oncosphere antigens of taeniid cestode parasites 总被引:8,自引:0,他引:8
Lightowlers MW Gauci CG Chow C Drew DR Gauci SM Heath DD Jackson DC Dadley-Moore DL Read AJ 《International journal for parasitology》2003,33(11):1207-1217
Highly effective recombinant vaccines have been developed against Taenia ovis infection in sheep, Taenia saginata infection in cattle, Taenia solium infection in pigs, Echinococcus granulosus and Echinococcus multilocularis infections in a variety of intermediate host species. These vaccines have been based on the identification and expression in Escherichia coli of antigens derived from the oncosphere life cycle stage, contained within the parasites' eggs. Investigation of the molecular aspects of these proteins and the genes encoding them have revealed a number of common features, including the presence of a predicted secretory signal sequence, and one or two copies of a fibronectin type III domain, each encoded by separate exons within the associated gene. Evidence has been obtained to confirm glycosylation of some of these antigens. Ongoing investigations will shed light on the biological roles played by the proteins within the parasites and the mechanism by which they make the parasites vulnerable to vaccine-induced immune responses. 相似文献
45.
Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4° C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample. 相似文献
46.
The impact of gender and/or hormone variations on a wide variety of neural functions makes the choice between studying males or females (or both) of a given species difficult. Although female rats are widely used experimentally, few studies control for the stage of estrus. More detailed information about how to distinguish the various stages of the estrous cycle is needed. For the present study, vaginal smears were obtained once a day and stained using an adaptation of the Papanicolaou (PAP) procedure. Images are provided of unstained “wet” samples and the corresponding PAP stained smears illustrating the cellular profile for each stage of the cycle as well as post-ovariectomy. The different cell populations across the cycle were quantified and ratios determined to show trends between the predominant and other cell types in each stage of the estrous cycle. Both stained and unstained images and cell quantification data provide valuable guidelines for distinguishing the stages of the estrous cycle. 相似文献
47.
Lisa?M?VallelyEmail author Angela?Kelly Martha?Kupul Ruthy?Neo Voletta?Fiya John?M?Kaldor Glen?DL?Mola Heather?Worth 《International breastfeeding journal》2013,8(1):6
Background
Interventions to prevent mother to child transmission of human immunodeficiency virus (HIV) during childbirth and breastfeeding can reduce HIV infections in infants to less than 5% in low and middle income countries. The World Health Organization (WHO) recommends all mothers, regardless of their HIV status, practice exclusive breastfeeding for the first six months of an infant’s life. In line with these recommendations and to protect, promote and support breastfeeding, in 2009 the PNG National Department of Health revised their National HIV infant feeding guidelines, reinforcing the WHO recommendation of exclusive breastfeeding for the first six months followed by the introduction of other food and fluids, while continuing breastfeeding.The overall aim of this paper is to explore health care workers’ knowledge regarding infant feeding options in PNG, specifically as they relate to HIV exposed infants.Methods
As part of a study investigating women’s and men’s experiences of prevention of mother to child transmission (PMTCT) services in two sites in PNG, 28 key informant interviews were undertaken. This paper addresses one theme that emerged from thematic data analysis: Health care workers’ knowledge regarding infant feeding options, specifically how this knowledge reflects the Papua New Guinea National HIV Care and Treatment Guidelines on HIV and infant feeding (2009).Results
Most informants mentioned exclusive breastfeeding, the majority of whom reflected the most up-to-date National Guidelines of exclusive breastfeeding for six months. The importance of breastfeeding continuing beyond this time, along with the introduction of food and fluids was less well understood. The most senior people involved in PMTCT were the informants who most accurately reflected the national guidelines of continuing breastfeeding after six months.Conclusion
Providing advice on optimal infant feeding in resource poor settings is problematic, especially in relation to HIV transmission. Findings from our study reflect those found elsewhere in identifying that key health care workers are not aware of up-to-date information relating to infant feeding, especially within the context of HIV. Greater emphasis needs to be placed on ensuring the most recent feeding guidelines are disseminated and implemented in clinical practice in PNG.48.
Clifford DL Folmes Grzegorz Sawicki Virgilio JJ Cadete Grant Masson Amy J Barr Gary D Lopaschuk 《Proteome science》2010,8(1):38
Background
During and following myocardial ischemia, glucose oxidation rates are low and fatty acids dominate as a source of oxidative metabolism. This metabolic phenotype is associated with contractile dysfunction during reperfusion. To determine the mechanism of this reliance on fatty acid oxidation as a source of ATP generation, a functional proteomics approach was utilized. 相似文献49.
Andrew J Holloway Alicia Oshlack Dileepa S Diyagama David DL Bowtell Gordon K Smyth 《BMC bioinformatics》2006,7(1):1-20
Background
It is one of the ultimate goals for modern biological research to fully elucidate the intricate interplays and the regulations of the molecular determinants that propel and characterize the progression of versatile life phenomena, to name a few, cell cycling, developmental biology, aging, and the progressive and recurrent pathogenesis of complex diseases. The vast amount of large-scale and genome-wide time-resolved data is becoming increasing available, which provides the golden opportunity to unravel the challenging reverse-engineering problem of time-delayed gene regulatory networks.Results
In particular, this methodological paper aims to reconstruct regulatory networks from temporal gene expression data by using delayed correlations between genes, i.e., pairwise overlaps of expression levels shifted in time relative each other. We have thus developed a novel model-free computational toolbox termed TdGRN (Time-delayed Gene Regulatory Network) to address the underlying regulations of genes that can span any unit(s) of time intervals. This bioinformatics toolbox has provided a unified approach to uncovering time trends of gene regulations through decision analysis of the newly designed time-delayed gene expression matrix. We have applied the proposed method to yeast cell cycling and human HeLa cell cycling and have discovered most of the underlying time-delayed regulations that are supported by multiple lines of experimental evidence and that are remarkably consistent with the current knowledge on phase characteristics for the cell cyclings.Conclusion
We established a usable and powerful model-free approach to dissecting high-order dynamic trends of gene-gene interactions. We have carefully validated the proposed algorithm by applying it to two publicly available cell cycling datasets. In addition to uncovering the time trends of gene regulations for cell cycling, this unified approach can also be used to study the complex gene regulations related to the development, aging and progressive pathogenesis of a complex disease where potential dependences between different experiment units might occurs. 相似文献50.
A nuclear gene for higher level phylogenetics: phosphoenolpyruvate carboxykinase tracks mesozoic-age divergences within Lepidoptera (Insecta) 总被引:3,自引:0,他引:3
The sequence of phosphoenolpyruvate carboxykinase (PEPCK) has been
previously identified as a promising candidate for reconstructing
Mesozoic-age divergences (Friedlander, Regier, and Mitter 1992, 1994). To
test this hypothesis more rigorously, 597 nucleotides of aligned PEPCK
coding sequence (approximately 30% of the coding region) were generated
from 18 species representing Mesozoic-age lineages of moths (Insecta:
Lepidoptera) and outgroup taxa. Relationships among basal Lepidoptera are
well established by morphological analysis, providing a strong test for the
utility of a gene which has not previously been used in systematics.
Parsimony and other phylogenetic analyses were conducted on nucleotides by
codon positions (nt1, nt2, nt3) separately and in combination, and on amino
acids, for comparison to the test phylogeny. The highest concordance was
achieved with nt1 + nt2, for which one of two most-parsimonious trees was
identical to the test phylogeny, and with all nucleotides when nt3 was
down-weighted sevenfold or higher, for which a single most-parsimonious
tree identical to the test phylogeny resulted. Substitutions in nt3
approached saturation in many, but not all, pairwise comparisons and their
exclusion or severe downweighting greatly increased the degree of
concordance with the test phylogeny. Neighbor-joining analysis confirms
this finding. The utility of PEPCK for phylogenetics is demonstrated over a
time span for which few other suitable genes are currently available.
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