Emulating a crowded intracellular environment in vitro dramatically improves RT-PCR performance

Lareu, Ricky R.; Harve, Karthik S.; Raghunath, Michael

doi:10.1016/j.bbrc.2007.08.156

Cited by 45 publications

(37 citation statements)

References 22 publications

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“…3, slots l, m). This is consistent with the evidence that efficiency of PCR can be enhanced by allowing the template to interact with a non-soluble structure (Lareu et al, 2007;Richter et al, 2008).…”

Section: Comparative Kinetics Of Dnase I Digestion Of Loop Dna In Nucsupporting

confidence: 89%

The organization of a large transcriptional unit (Fyn) into structural DNA loops is cell-type specific and independent of transcription

Trevilla-García

Aranda‐Anzaldo

2012

Gene

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Section: Comparative Kinetics Of Dnase I Digestion Of Loop Dna In Nucsupporting

confidence: 89%

The organization of a large transcriptional unit (Fyn) into structural DNA loops is cell-type specific and independent of transcription

Trevilla-García

Aranda‐Anzaldo

2012

Gene

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“…24,25,32 Excluded volume effects caused by macromolecules and thermodynamically unfavorable interactions between proteins and small-molecule osmolytes induce protein folding. 26,27,33 In this study, we examined the in-solution behavior of BoNT LCs by examining their structures and functions in buffers that are known to promote protein folding. Overall, we found the structures and activities of BoNT LCs were extremely sensitive to solvent conditions; this sensitivity provides insight into the solution behavior of these enzymes and has implications for analytical assay development and inhibitor design.…”

Section: Discussionmentioning

confidence: 99%

The Osmolyte Trimethylamine N-Oxide (TMAO) Increases the Proteolytic Activity of Botulinum Neurotoxin Light Chains A, B, and E: Implications for Enhancing Analytical Assay Sensitivity

et al. 2010

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Botulism, the disease caused by botulinum neurotoxins (BoNTs), secreted by the spore-forming, anaerobic bacteria Clostridium botulinum, has been associated with food poisoning for centuries. In addition, the potency of BoNTs coupled with the current political climate has produced a threat of intentional, malicious poisoning by these toxins. The ability to detect and measure BoNTs in complex matrixes is among the highest research priorities. However, the extreme potency of these toxins necessitates that assays be capable of detecting miniscule quantities of these proteins. Thus, signal-boosting strategies must be employed. A popular approach uses the proteolytic activity of the BoNT light chain (LC) to catalyze the cleavage of synthetic substrates; reaction products are then analyzed by the analytical platform of choice. However, BoNT LCs are poor catalysts. In this study, the authors used the osmolyte trimethylamine N-oxide (TMAO) to increase the proteolytic activities of BoNT LCs. Their data suggest that concentrated solutions of TMAO induce complete folding of the LCs, resulting in increased substrate affinity and enhanced enzyme turnover. The authors observed increases in catalysis for BoNT serotypes A, B, and E, and this increased proteolytic activity translated into substantial increases in analytical assay sensitivity for these medically relevant toxins. (Journal of Biomolecular Screening 2010:928-936)

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“…Primers were used at a concentration of 0.2 lM, SYBR Green (Molecular Probes, Eugene, OR) at 1:40 000 of stock, MgCl 2 at 3 mM, and 0.5 U of Immolase enzyme per reaction. Ficoll supplementation (2.5 mg/ml of Ficoll 400 and 7.5 mg/ml of Ficoll 70) was used to improve PCR amplification efficiency [35]. Cycling conditions included an initial denaturation at 958C for 10 min to activate the Immolase enzyme, followed by amplification for 45 cycles of the specific profiles indicated ( Table 1).…”

Section: Quantitative Rt-pcrmentioning

confidence: 99%

“…RNA integrity was assessed by agarose gel electrophoresis (data not shown). Total RNA (5 lg) was used to synthesize cDNA using M-MLV Reverse Transcriptase RNase H Point Mutant and random hexamer primers (Promega, Madison, WI) according to the manufacturer's instructions and containing 2.5 mg/ml of Ficoll 400 and 7.5 mg/ml of Ficoll 70 [35]. The resultant cDNAs were purified using the Ultraclean PCR Cleanup kit (MoBio Industries, Solana Beach, CA).…”

Section: Rna Sample Preparationmentioning

confidence: 99%

Changes in the Placental Glucocorticoid Barrier During Rat Pregnancy: Impact on Placental Corticosterone Levels and Regulation by Progesterone1

Mark

Augustus

Lewis

et al. 2009

Biology of Reproduction

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Glucocorticoid excess in utero inhibits fetal growth and programs adverse outcomes in adult offspring. Access of maternal glucocorticoid to the glucocorticoid receptor (NR3C1) in the placenta and fetus is regulated by metabolism via the 11beta-hydroxysteroid dehydrogenase (HSD11B) enzymes, as well as multidrug resistance P-glycoprotein (ABCB1)-mediated efflux of glucocorticoids from the syncytiotrophoblast. This study determined expression of genes encoding the two HSD11B isoforms (Hsd11b1 and Hsd11b2), the two ABCB1 isoforms (Abcb1a and Abcb1b), and Nr3c1 in the junctional and labyrinth zones of rat placentas at Days 16 and 22 of normal gestation (Day 23 is term). To assess possible regulation of the Hsd11b and Abcb1 isoforms by glucocorticoids and progesterone, their placental expression was also measured at Day 22 after partial progesterone withdrawal from Day 16 (maternal ovariectomy plus full estrogen and partial progesterone replacement) or after treatment with dexamethasone acetate (1 microg/ml of drinking water from Day 13). Expression of Hsd11b1 mRNA increased in the labyrinth zone (the site of maternal-fetal exchange) from Day 16 to Day 22, whereas that of Hsd11b2 fell dramatically. Consistent with these changes, corticosterone levels increased 10-fold in the labyrinth zone over this period. Expression of both Abcb1a and Abcb1b was markedly higher in the labyrinth zone compared with the junctional zone on both days, consistent with the proposed barrier role of ABCB1 in the placenta. Nr3c1 mRNA expression was similar in the two placental zones at Day 16 but increased 3-fold in the labyrinth zone by Day 22. Partial progesterone withdrawal increased Hsd11b1 mRNA and protein expression in the labyrinth zone but decreased Nr3c1 mRNA expression. These data show that the dynamic expression patterns of the placental HSD11Bs in late gestation are associated with dramatic shifts in placental corticosterone. Moreover, the late gestational rise in labyrinthine Hsd11b1 seems to be driven by the normal prepartum fall in progesterone level.

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Emulating a crowded intracellular environment in vitro dramatically improves RT-PCR performance

Cited by 45 publications

References 22 publications

The organization of a large transcriptional unit (Fyn) into structural DNA loops is cell-type specific and independent of transcription

The organization of a large transcriptional unit (Fyn) into structural DNA loops is cell-type specific and independent of transcription

The Osmolyte Trimethylamine N-Oxide (TMAO) Increases the Proteolytic Activity of Botulinum Neurotoxin Light Chains A, B, and E: Implications for Enhancing Analytical Assay Sensitivity

Changes in the Placental Glucocorticoid Barrier During Rat Pregnancy: Impact on Placental Corticosterone Levels and Regulation by Progesterone1

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