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61.
The herpes simplex virus (HSV) ribonucleotide reductase comprises two nonidentical subunits, R1 and R2, which associate to form the active holoenzyme. A sensitive binding assay was developed to measure the affinity of inhibitory peptides for the HSV R1 subunit. The assay involved the use of a photoreactive radioligand [4′-azido-Phe328,3′,5′-125I-Tyr329] HSV R2-(328-337), an analogue of the decapeptide Ser-Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu which corresponds to the C-terminal sequence (328-337) of the HSV R2 protein. As the radioligand binds covalently to the HSV R1 subunit upon uv irradiation, the affinity of peptide inhibitors can be easily determined by measuring their ability to compete with this highly specific binding. The method, which did not require any pure preparation of R1, was tested at 25 and 4°C and showed a significant increase in the affinity of the peptide inhibitors at 4°C. The relative affinity of these peptides was in agreement with their relative potency to inhibit reductase activity. The affinity of R2 subunit for R1 was also determined, and an IC50 of 0.05 μM was measured. Altogether, this assay represents a precise and reliable tool with which to study more potent HSV ribonucleotide reductase peptide inhibitors, and the method could be applied to the study of other protein-protein and peptide-protein interactions. 相似文献
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In the present study, cell death was investigated in cultures of NS/0 myelomas and SP2/0-derived D5 hybridomas through morphological examination of cells stained with acridine orange and ethidium bromide. The relative contribution of elevated levels of lactic acid and ammonia, as well as deprivation of glutamine, cystine, and glucose on the induction of necrosis or apoptosis, was investigated. In batch culture of D5 hybridoma cells, induction of apoptotic cell death correlated with the exhaustion of glutamine, while in the case of NS/0 myelomas, it coincided with exhaustion of cystine. To determine whether limiting nutrients were the actual triggering factors for apoptosis in batch culture, exponentially growing cells were resuspended in glutamine or cystine-free media. Within 30 to 40 h, viability decreased to 50% and the nonviable cell population displayed typical apoptotic morphology, with crescents of condensed chromatin around the periphery of the nucleus, or with the entire nucleus present as one or a group of featureless, brightly staining spherical beads. Similarly, D5 hybridomas and NS/0 myelomas cultivated in glucose-free medium died mainly from apoptosis. Cells were also cultivated in fresh medium supplemented with elevated concentrations of ammonia (3.0 mM) and/or lactate (35 mM, 50 mM). This resulted in decreased viabilities and necrotic death in both cell lines. From these results, we conclude that D5 hybridomas and NS/0 myelomas deprived of essential nutrients die by apoptosis, whereas incubation in the presence of elevated levels of metabolic byproducts such as ammonia and lactate will induce necrotic cell death in these cells. (c) 1994 John Wiley & Sons, Inc. 相似文献
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The flavonols from each of 38 poinsettia cultivars were quantitatively resolved by HPLC. Careful selection of samples revealed significant quantitative differences in flavonol content. While all cultivars of seedling origin could be differentiated on the basis of their flavonols, several families of sports could not. Most new commercial poinsettia cultivars have been selected from among somatic mutants exhibiting different bract color. Such changes in color were usually the results of change in anthocyanin content rather than flavonol content. The flavonol content increased in color sports with less anthocyanin. Three of the flavonols were absent from a few of the cultivars but most differences between cultivars were only quantitative. Genetic data suggested independent assortment of control of quercetin 3-rhamnoside and quercetin 3-galactoside synthesis but linkage of quercetin 3-rhamnoside and kaempferol 3-rhamnoside synthesis. 相似文献
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Béatrice Assouline-Thomas Amélie Pilotte Maria Petropavlovskaia Julia Makhlin Jieping Ding David McLeod Stephen Hanley Bernard Massie Lawrence Rosenberg 《Protein expression and purification》2010,69(1):1-8
Islet Neogenesis Associated Protein (INGAP) is implicated in pancreatic islet neogenesis. INGAP peptide, a pentadecapeptide comprising amino acids 104–118, reverses diabetes in rodents and improves glucose homeostasis in patients with diabetes. The mechanism of INGAP action is unknown, but such studies would benefit from the availability of the full-length recombinant protein (rINGAP).Here we report the production of rINGAP from 293-SF cells following lentiviral transduction, and its characterization by MALDI-TOF and Q-TOF Mass Spectrometry, and HPLC.Importantly, we show that rINGAP exhibits 100× the bioactivity of INGAP peptide on a molar basis in an in vitro assay of human islet regeneration. 相似文献
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Aurélie Girard Élodie Roques Marie-Claude St-Louis Bernard Massie Denis Archambault 《Molecular biotechnology》2013,54(3):1010-1020
The aim of this study was to evaluate the usefulness of replicating but non disseminating adenovirus vectors (AdVs) as vaccine vector using human rotavirus (HRV) as a model pathogen. HRV VP7, VP4, or VP4Δ (N-terminal 336 amino acids of VP4) structural proteins as well as the VP4Δ::VP7 chimeric fusion protein were expressed in mammalian cells when delivered with the AdVs. A preliminary experiment demonstrated that VP4Δ was able to induce a HRV-specific IgG response in BALB/c mice inoculated intramuscularly with AdVs expressing the rotaviral protein. Moreover, an AdV-prime/plasmid DNA-boost regimen of vectors resulted in VP4Δ-specific antibody (Ab) titers ~4 times higher than those obtained from mice immunized with AdVs alone. Subsequently, the various HRV protein-encoding AdVs were compared using the AdV-prime/plasmid DNA-boost regimen. Higher IgG and IgA responses to HRV were obtained when VP4Δ::VP7 fusion protein was used as an immunogen as compared to VP7 or VP4 alone or to a mix of both proteins delivered independently by AdVs. A synergetic effect in terms of Ab was obtained with VP4Δ::VP7. In conclusion, this study demonstrated for the first time the suitability of using replicating but non disseminating AdVs as vaccine vector and the VP4Δ::VP7 fusion protein as an immunogen for vaccination against HRV. 相似文献
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Carla GS Saad Ana CM Ribeiro Julio CB Moraes Liliam Takayama Celio R Goncalves Marcelo B Rodrigues Ricardo M de Oliveira Clovis A Silva Eloisa Bonfa Rosa MR Pereira 《Arthritis research & therapy》2012,14(5):R216