Preface

Ausubel, Frederick M.; Brent, Roger; Kingston, Robert E.; Moore, David D.; Seidman, J. G.; Smith, John A.; Struhl, Kevin

doi:10.1002/0471142727.mbprefs90

Cited by 724 publications

(878 citation statements)

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“…Total RNA concentrations were determined spectrophotometerically (Helio γ, Thermo Electron, Milford, MA) by optical density (OD) at 260 nm using an OD260 equivalent to 40 μg/μl (Ausubel et al 2002), and the final concentration expressed relative to muscle wet weight. Aliquots of total RNA were separated with 1% agarose gel electrophoresis, ethidium bromide stained, and monitored under an ultraviolet light (Chemi-Doc XRS, Bio-Rad, Hercules, CA) to verify RNA integrity and absence of RNA degradation, indicated by prominent 28s and 18s ribosomal RNA bands, as well as an OD260/OD280 ratio of approximately 2.0.…”

Section: Skeletal Muscle Total Rna Isolation and Quantitation And Cdnmentioning

confidence: 99%

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Co-ingestion of carbohydrate with branched-chain amino acids or l-leucine does not preferentially increase serum IGF-1 and expression of myogenic-related genes in response to a single bout of resistance exercise

Ferreira

Cooke

et al. 2015

Amino Acids

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. (2015). Coingestion of carbohydrate with branched-chain amino acids or L-leucine does not preferentially increase serum IGF-1 and expression of myogenic-related genes in response to a single bout of resistance exercise. Amino Acids 47(6), pp. 1203-1213. http://dx.doi.org/ 10.1007/s00726-015-1947-8 AbstractThis study determined if the co-ingestion of carbohydrate (CHO) with branched-chain amino acids (BCAA) or L-leucine (LEU) preferentially affected serum IGF-1 and the expression of myogenic-related genes in response to resistance exercise (RE). Forty one, college-age males were randomly assigned to 1 of 4 groups: CHO, CHO-BCAA, CHO-LEU, or placebo (PLC). Resistance exercise consisted of 4 sets of leg press and leg extension at 80% 1RM. Supplements were ingested peri-exercise, and venous blood and muscle biopsies were obtained preexercise (PRE), and at 30, 120, and 360 min post-exercise. Serum IGF-1 was determined with ELISA, and skeletal muscle mRNA expression of myostatin, ActRIIB, p21kip, p27kip, CDK2, cyclin B1, cyclin D1, Myo-D, myogenin, MRF-4, and myf5 was determined using real-time PCR. Results were determined with two-way ANOVA for serum IGF-1 and two-way MANOVA for mRNA expression. Serum IGF-1 in CHO and CHO+BCAA was greater than PLC (p < 0.05) but was not affected by RE (p > 0.05). Significant differences were detected between groups for myostatin, ActIIB, MyoD, and myf5 mRNA expression showing CHO to be significantly different than CHO+BCAA, CHO+LEU, and PLC (p < 0.05). At 30, 120 and 360 min post-exercise, p21cip was significantly less than PRE, whereas cyclin D1 was greater than PRE at 120 and 360 min post-exercise (p < 0.05). The co-ingestion of CHO with either BCAA or L-leucine in conjunction with RE had no preferential effect on serum IGF-1 or pre-translational markers indicative of myogenesis.

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Section: Skeletal Muscle Total Rna Isolation and Quantitation And Cdnmentioning

confidence: 99%

“…The cDNA concentration was determined by using an OD260 equivalent to 50 μg/μl (Ausubel et al 2002), and the starting cDNA template concentration was standardized by adjusting all samples to 200 ng prior to PCR amplification (Wilborn et al 2009;Willoughby et al 2007). …”

Section: Skeletal Muscle Total Rna Isolation and Quantitation And Cdnmentioning

confidence: 99%

Co-ingestion of carbohydrate with branched-chain amino acids or l-leucine does not preferentially increase serum IGF-1 and expression of myogenic-related genes in response to a single bout of resistance exercise

Ferreira

Cooke

et al. 2015

Amino Acids

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“…Transfectants, called mCherry-Li or dTomato-Li respectively, were grown to stationary phase in HOMEM. Genomic DNA was extracted from transfectants using a described procedure, subjected to restriction enzyme digestion with SmaI, and analyzed on a Southern blot [31]. Briefly, DNA digests were separated on a 0.8% agarose gel, transferred to Nylon (Roche, Mannheim, Germany) and UV cross–linked.…”

Section: Methodsmentioning

confidence: 99%

Infection and Activation of Human Neutrophils with Fluorescent Leishmania infantum

Cj²,

2016

J Immunol Tech Infect Dis

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Neutrophils (PMNs) are recruited in high numbers to sites of host infection by the protozoan parasites of the genus Leishmania. Although PMNs are capable of phagocytizing Leishmania parasites and are potent producers of anti-microbial compounds including reactive oxygen species (ROS), they are unable to control the establishment of infection. Prior studies document production of ROS in isolated PMNs incubated with Leishmania under conditions allowing phagocytosis, but without a measure of single cells’ responses it cannot be discerned whether PMN activation and ROS production is suppressed or ineffective in the cells that internalize the parasite. To address these interactions, we engineered a strain of fluorescent, mCherry-expressing Leishmania infantum (mCherry-Li). By infecting isolated human PMNs in vitro with mCherry-Li, we observed ready association of the parasites with PMNs in a time- and dose-dependent fashion. We also examined production of PMN ROS (using the fluorescent compound DHR123) and PMN activation (as evidence by loss of surface CD62L expression). Whereas many Li-associated (mCherry+) PMNs responded to parasite interactions and uptake with ROS production and/or activation, a proportion exhibited neither response. Furthermore, a large proportion of mCherry - “bystander” PMNs displayed both ROS production and activation. The heterogeneous response of PMNs to Leishmania exposure leads us to hypothesize, first, that some PMNs exhibit decreased activation upon phagocytosis of Leishmania, and could support their maintenance. Second, responses of bystander PMNs may contribute to a local inflammatory environment that is ineffective at parasite clearance.

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“…(MATa ura3Δ0) and BY4704 (MATa ade2::hisG his3Δ200 leu2Δ0 lys2Δ0 met15Δ0 trp1Δ63) was used as template for DNA amplification (Brachmann et al, 1998 (Ausubel et al, 1999). MM supplemented with 20 mg/l uracil (MMU) was used for auxotrophic mutant isolation.…”

Section: Strains and Mediamentioning

confidence: 99%

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

Yarimizu

Nonklang

Nakamura

et al. 2013

Yeast

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The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus.

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Preface

Cited by 724 publications

References 0 publications

Co-ingestion of carbohydrate with branched-chain amino acids or l-leucine does not preferentially increase serum IGF-1 and expression of myogenic-related genes in response to a single bout of resistance exercise

Co-ingestion of carbohydrate with branched-chain amino acids or l-leucine does not preferentially increase serum IGF-1 and expression of myogenic-related genes in response to a single bout of resistance exercise

Infection and Activation of Human Neutrophils with Fluorescent Leishmania infantum

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

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