Understanding the influence of trenbolone acetate and polyamines on proliferation of bovine satellite cells.

Approximately 90% of beef cattle on feed in the United States receive at least one anabolic implant, which results in increased growth, efficiency, and economic return to producers. However, the complete molecular mechanism through which anabolic implants function to improve skeletal muscle growth remains unknown. This study had 2 objectives: (1) determine the effect of polyamines and their precursors on proliferation rate in bovine satellite cells (BSC); and (2) understand whether trenbolone acetate (TBA), a testosterone analog, has an impact on the polyamine biosynthetic pathway. To address these, BSC were isolated from 3 finished steers and cultured. Once cultures reached 75% confluency, they were treated in 1% fetal bovine serum (FBS) and/or 10 nM TBA, 10 mM methionine (Met), 8 mM ornithine (Orn), 2 mM putrescine (Put), 1.5 mM spermidine (Spd), or 0.5 mM spermine (Spe). Initially, a range of physiologically relevant concentrations of Met, Orn, Put, Spd, and Spe were tested to determine experimental doses to implement the aforementioned experiments. One, 12, or 24 h after treatment, mRNA was isolated from cultures and abundance of paired box transcription factor 7 (Pax7), Sprouty 1 (Spry), mitogen-activated protein kinase-1 (Mapk), ornithine decarboxylase (Odc), and S adenosylmethionine (Amd1) were determined, and normalized to 18S. No treatment × time interactions were observed (P ≥ 0.05). Treatment with TBA, Met, Orn, Put, Spd, or Spe increased (P ≤ 0.05) BSC proliferation when compared with control cultures. Treatment of cultures with Orn or Met increased (P ≤ 0.01) expression of Odc 1 h after treatment when compared with control cultures. Abundance of Amd1 was increased (P < 0.01) 1 h after treatment in cultures treated with Spd or Spe when compared with 1% FBS controls. Cultures treated with TBA had increased (P < 0.01) abundance of Spry mRNA 12 h after treatment, as well as increased mRNA abundance of Mapk (P < 0.01) 12 h and 24 h after treatment when compared with 1% FBS control cultures. Treatment with Met increased (P < 0.01) mRNA abundance of Pax7 1 h after treatment as compared with 1% FBS controls. These results indicate that treatments of BSC cultures with polyamines and their precursors increase BSC proliferation rate, as well as abundance of mRNA involved in cell proliferation. In addition, treatment of BSC cultures with TBA, polyamines, or polyamine precursors impacts expression of genes related to the polyamine biosynthetic pathway and proliferation.

Effects of feeding birdsfoot trefoil hay on neutral detergent fiber digestion, nitrogen utilization efficiency, and lactational performance by dairy cows.

This experiment was conducted to determine effects of feeding birdsfoot trefoil hay-based diets in comparison with an alfalfa hay-based diet on N utilization efficiency, ruminal fermentation, and lactational performance by mid-lactation dairy cows. Nine multiparous lactating Holstein cows (131 ± 22.6 d in milk), 3 of which were rumen fistulated, were fed 3 experimental diets in a replicated 3 × 3 Latin square design with 3 periods of 14 d of adaptation and 7 d of data and sample collection. Within squares, cows were randomly assigned to diets as follows: alfalfa hay-based diet (AHT), alfalfa and birdsfoot trefoil hay-based diet (ABT), and birdsfoot trefoil hay-based diet (BT). Intakes of dry matter and crude protein were similar across treatments, whereas ABT and BT diets resulted in decreased fiber intake compared with AHT. Feeding BT tended to increase neutral detergent fiber digestibility compared with AHT and ABT. Milk yield tended to increase for cows consuming ABT or BT diets. Milk true protein concentration and yield were greater for cows consuming ABT relative to those fed AHT. Concentration of total volatile fatty acids tended to increase by cows fed BT compared with those fed AHT and ABT. Feeding birdsfoot trefoil hay in a total mixed ration resulted in a tendency to decrease acetate proportion, but it tended to increase propionate proportion, leading to a tendency to decrease acetate-to-propionate ratio. Whereas concentration of ammonia-N was similar across treatments, cows offered BT exhibited greater microbial protein yield relative to those fed AHT and ABT. Cows offered birdsfoot trefoil hay diets secreted more milk N than AHT, resulting in improved N utilization efficiency for milk N. The positive effects due to feeding birdsfoot trefoil hay were attributed to enhanced neutral detergent fiber digestion, and thus it could replace alfalfa hay in high-forage dairy diets while improving N utilization efficiencies and maintaining lactational performance compared with alfalfa hay.

Physicochemical evolution and molecular adaptation of the cetacean and artiodactyl cytochrome b proteins.

Cetaceans have most likely experienced metabolic shifts since evolutionarily diverging from their terrestrial ancestors, shifts that may be reflected in the proteins such as cytochrome b that are responsible for metabolic efficiency. However, accepted statistical methods for detecting molecular adaptation are largely biased against even moderately conservative proteins because the primary criterion involves a comparison of nonsynonymous and synonymous substitution rates (dN/dS); they do not allow for the possibility that adaptation may come in the form of very few amino acid changes. We apply the MM01 model to the possible molecular adaptation of cytochrome b among cetaceans because it does not rely on a dN/dS ratio, instead evaluating positive selection in terms of the amino acid properties that comprise protein phenotypes that selection at the molecular level may act upon. We also apply the codon-degeneracy model (CDM), which focuses on evaluating overall patterns of nucleotide substitution in terms of base exchange, codon position, and synonymy to estimate the overall effect of selection. Using these relatively new models, we characterize the molecular adaptation that has occurred in the cetacean cytochrome b protein by comparing revealed amino acid replacement patterns to those found among artiodactyls, the modern terrestrial mammals found to be most closely related to cetaceans. Our findings suggest that several regions of the cetacean cytochrome b protein have experienced molecular adaptation. Also, these adaptations are spatially associated with domain structure, protein function, and the structure and function of the cytochrome bc(1) complex and its constituents. We also have found a general correlation between the results of the analytical software programs TreeSAAP (which implements the MM01 model) and CDM (which implements the codon-degeneracy model).

Fabrication of plastic microfluid channels by imprinting methods.

Microfluidic devices have been fabricated on poly(methyl methacrylate) substrates by two independent imprinting techniques. First-generation devices were fabricated using a small-diameter wire to create an impression in plastics softened by low-temperature heating. The resulting devices are limited to only simple linear channel designs but are readily produced at low cost. Second-generation devices with more complex microchannel arrangements were fabricated by imprinting the plastic substrates using an inverse three-dimensional image of the device micromachined on a silicon wafer. This micromachined template may be used repeatedly to generate devices reproducibly. Fluorescent analtyes were used to demonstrate reproducible electrophoretic injections. An immunoassay was also performed in an imprinted device as a demonstration of future applications.

Certification of phencyclidine in lyophilized human urine reference materials.

The National Institute of Standards and Technology (formerly the National Bureau of Standards), in cooperation with the College of American Pathologists (CAP), has certified the concentrations of phencyclidine (PCP) in two new reference materials (RMs). One of these materials is Standard Reference Material (SRM) 1511, Multidrugs of Abuse in Freeze-dried Urine, and the other material is a CAP PCP RM. In order to minimize the possibility of undetected bias, two independent analytical methods, employing gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry, were used to certify PCP in these materials. Results from the two methods were in good agreement and were statistically combined to yield certified values of 23.8 ng/mL for PCP in SRM 1511 and 11.9, 23.4, and 49.5 ng/mL for three levels of PCP in the CAP RM. A round-robin study of SRM 1511 among five military laboratories demonstrated the suitability of the SRM for its intended purpose.

Flow immunoassay using solid-phase entrapment.

A flow injection immunoassay was performed using a column packed with reversed-phase sorbents to effect separation of the immunoreacted species by entrapping free analyte and allowing antibody-conjugated analyte to pass unretained. Fluorescein-labeled analyte was measured in a competitive assay for the anticonvulsant drug phenytoin. The simplicity of the assay was the greatest advantage of the technique, which allowed for measurement of phenytoin in a 2-min assay time. The reliable detection limit for the assay was 5 nmol L(-)(1) of phenytoin in serum. The columns were regenerated with periodic injections of ethanol solutions to remove the entrapped analyte and prepare the column for subsequent analyses.

The certification of morphine and codeine in a human urine standard reference material.

The National Institute of Standards and Technology (NIST, formerly the National Bureau of Standards) has developed and certified a Standard Reference Material, SRM 2381, for use in testing for bias in determinations of morphine and codeine in human urine. Each unit of this SRM consists of three vials with different levels of morphine and codeine in lyophilized urine. Three different analytical methods, employing GC/MS, LC/MS, and MS/MS, were used to certify the concentrations of each analyte. Results from the three methods were in good agreement and, therefore, were statistically combined to yield certified values of 138, 293, and 578 ng/mL for morphine and 134, 283, and 591 for codeine. A round-robin study on this material among nine military laboratories demonstrated the suitability of the SRM for its intended purpose.

Determination of Oltipraz in serum by high-performance liquid chromatography with optical absorbance and mass spectrometric detection.

Three methods have been developed for the analysis of Oltipraz in serum. A method suitable for routine use employs spiking with a homologous internal standard, off-line solid-phase extraction, high-performance liquid chromatographic separation, and optical absorbance detection at 450 nm. Method detection limit is about 1 ng/ml. A second method, less susceptible to bias from co-eluting interferences, uses a stable isotope-labeled internal standard, similar extraction and separation, and detection by thermospray mass spectrometry. Method detection limit is about 0.2 ng/ml. A third method was developed which can be used without specially synthesized internal standards. It uses on-line solid-phase extraction, with quantification by comparison with external standards. Method detection limit is about 3 ng/ml. Good agreement was observed between these methods and with similar and different methods run in other laboratories. Calibration curves were linear over the entire range which was investigated, i.e., up to 500 ng/ml. Coefficients of variation were similar for all three methods, being about 5%.

The certification of cocaine and benzoylecgonine in a human urine standard reference material.

The concentrations of cocaine and benzoylecgonine (BE) in Standard Reference Material (SRM) 1508, cocaine and metabolites in freeze-dried human urine, were determined at the National Institute of Standards and Technology (NIST, formerly NBS) by two independent methods. For cocaine, one method was based on gas chromatography/mass spectrometry (GC/MS); the other was based on high-performance liquid chromatography (HPLC). For BE, one method was based on GC/MS; the other was based on flow injection analysis/thermospray mass spectrometry (FIA/MS). The results for each pair of methods were statistically evaluated. Concentrations were determined in the SRM for three levels of cocaine and three levels of benzoylecgonine. Methylecgonine, although present in the material, was not determined. For cocaine, the concentrations were 90, 263, and 429 ng/mL of human urine. For BE, the concentrations were 103, 259, and 510 ng/mL of human urine.

Determination of glycyrrhetinic acid in human plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method has been developed for measuring 18 beta-glycyrrhetinic acid (GRA) in human plasma in the range of 0.1-3 micrograms/ml. The acetate ester of GRA is added to the plasma as an internal standard, plasma proteins are denatured with urea to release GRA, and the GRA and the internal standard are extracted in an ion-pairing solid-phase extraction process. An isocratic, reversed-phase HPLC separation is used, followed by ultraviolet absorbance detection at 248 nm. The results from the analysis of five GRA-fortified plasma pools show a mean relative standard deviation of 7% and are accurate to within 10%. With evaporative concentration of the extract, the limit of detection for GRA in plasma is approximately 10 ng/ml.

High-purity 4-nitrophenol: purification, characterization, and specifications for use as a spectrophotometric reference material.

We describe specifications for high-purity 4-nitrophenol, which is suitable for spectrophotometric standardization. Such a reference material is needed in clinical enzymology to establish the proper molar absorptivity of 4-nitrophenol under final reaction conditions, particularly for measuring alkaline phosphatase activity in human serum. Some lots of 4-nitrophenol available commercially met these specifications, but several did not. The latter can be purified to meet our specifications by recrystallization or sublimation. The molar absorptivity of 4-nitrophenol (35 mumol/L) IN 10 mmol/L NaOH at 25 degrees C at 401 nm is 18380 +/- 90 L.mol-1.cm-1.