Matching Items (3)

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Incorporation, Expression, and Retained Enzymatic Activity of Six Tryptophan Analogues in Dihydrofolate Reductase at Positions 30 and 47

Description

Due to a continued interest in the fundamental properties of dihydrofolate reductase (DHFR) and its enzymatic activities, this study employed the use of six fluorescent tryptophan derivatives, for single site

Due to a continued interest in the fundamental properties of dihydrofolate reductase (DHFR) and its enzymatic activities, this study employed the use of six fluorescent tryptophan derivatives, for single site amino acid replacements. The two positions 30 and 47 within DHFR were studied to discover the rate at which these larger tryptophan analogues may be incorporated. Additionally, it was to be determined how much activity the mutated DHFR’s could retain when compared to their wild type counterpart. Through a review of literature, it was shown that previous studies have illustrated successful incorporation and toleration of unnatural amino acids.
Each of the six analogues A through F were relatively efficiently incorporated into the enzyme and well tolerated. Each maintained at least a third of their catalytic activity, measured through the consumption of β-nicotinamide adenine dinucleotide phosphate. Primarily, derivatives B, C, and D were able to retain the highest amount of activity in each position; B and D were the most tolerated in positions 30 and 47 with respective values of 68 ± 6.1 and 80 ± 12. The findings in this study illustrate that single tryptophan derivatives are able to be incorporated into Escherichia coli DHFR while still allowing the maintenance of a significant portion of its enzymatic activity.

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  • 2015-05

Supplementation of Vitamin B6 and Tryptophan: The Effects on Mood States Among College Club Sport Athletes

Description

There are many studies on vitamin B6 (pyrodoxine) or tryptophan (Trp) as a way to increase mood but there are little to no studies with these two nutrients supplemented together.

There are many studies on vitamin B6 (pyrodoxine) or tryptophan (Trp) as a way to increase mood but there are little to no studies with these two nutrients supplemented together. Trp is the precursor to serotonin that requires the cofactor pyridoxal phosphate (PLP). Serotonin plays a role in mood, sleep, appetite, and other wellbeing aspects and it is believed that low levels of serotonin is associated with the risk of developing depression, anxiety, and bipolar disorders. The amount of free Trp that can pass the blood-brain barrier (BBB) is influenced by factors such as cortisol, insulin, and competition from branch chain amino acids (BCAA). College students who exercise on a regular basis and participate in club sports may experience higher cortisol levels from stress of college and higher physical activity. Cortisol decreases the Trp levels in the plasma while BCAAs compete with Trp to pass through the BBB. Insulin promotes the passage of free Trp through the BBB. In the present study, 28 healthy active college students (21.0 ± 2.1 years, 24.5± 3.1 kg/m2) were divided into three groups: vitamin B6 (n=11), Trp (n=10), or both (n=10) (2 did not complete study). Blood serum pyridoxine levels and mood states were measured at baseline and at 4 weeks with Profile of Mood States (POMS), Depression Anxiety Stress Survey (DASS), Life Orientation Test-Revised (LOT-R), and Epworth sleep scale. In the combined sample, the total POMS score improved during the study (p=0.039) and the total DASS score tended to improve during the study (p=0.068). Thus, mean depression scores for all participants decreased during the 4-week supplementation study. However, there were no time x treatment effects noted at study completion. At baseline 18% of the participants were marginally deficient in vitamin B6 (serum pyridoxine <30nmol/L), and their total POMS score was raised 78% in comparison to participants with adequate vitamin B6 status (p=0.08). DASS scores were raised 48% in vitamin B6 deficient participants versus those with adequate vitamin B6 status (p=0.243). There were no significant changes (time or time x treatment) during the course of the study for the LOT-R or sleep scores. In summary, vitamin B6 deficiency in college student athletes was remarkably high (18%) compared to the national average reported by the CDC in 2012 (10.5%), and participants with vitamin B6 deficiency displayed heightened unfavorable mood states. Moreover, supplementation with vitamin B6, tryptophan, or vitamin B6 and tryptophan improved mood state in college student athletes, but there were no differences between treatments.

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  • 2016-05

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Factors that modulate production of tryptophan by Gut Bacteria

Description

Microorganisms can produce metabolites in the gut including short chain fatty acids, vitamins, and amino acids. Certain metabolites produced in the gut can affect the brain through changes in

Microorganisms can produce metabolites in the gut including short chain fatty acids, vitamins, and amino acids. Certain metabolites produced in the gut can affect the brain through changes in neurotransmitter concentrations. Serotonin, a neurotransmitter, is associated with mood, appetite, and sleep. Up to 90% of serotonin synthesis is located in the gut, by human enterochromaffin cells. Bacteria known to biosynthesize tryptophan, precursor to serotonin, include Escherichia coli, Enterococcus and Streptococcus. Tryptophan is synthesized by bacteria with the enzyme tryptophan synthase and requires Vitamin B6 (Pyridoxal). We hypothesize that gut isolates from surgical weight loss patients can enhance tryptophan production, which relies on vitamin B6 availability. Our goal was to isolate bacteria in order to test for tryptophan production and to determine how Vitamin B6 concentrations could affect tryptophan production. We isolated gut bacteria was from successful surgical weight loss patient with selective pressures for Enterobacter isolates and Enterococcus isolates. We tested the isolates were tested to determine if they could biosynthesize tryptophan in-vitro. Bacterial cultures were enriched with yeast and enriched with serine and indole, substrates necessary for tryptophan biosynthesis. We analyzed the supernatant samples for tryptophan production using GC-FID. Bacterial isolates most closely related to E. coli and Klebsiella based on 16S rRNA gene sequences, produced tryptophan in vitro. While under serine & indole media conditions, R1, the isolate most similar to Klebsiella produced more tryptophan than R14, the isolate most similar to E. coli. We tested the R1 isolate with a gradient of vitamin B6 concentrations from 0.02 µg/mL to 0.2 µg/mL to determine its effect on tryptophan production. When less than 0.05 µg/mL of Vitamin B6 was added, tryptophan production at 6 hours was higher than tryptophan production with Vitamin B6 concentrations at 0.05 µg/mL and above. The production and consumption of tryptophan by Klebsiella under 0 µg/mL and 0.02 µg/mL concentrations of Vitamin B6 occurred at a faster rate when compared to concentrations 0.05 µg/mL or higher of Vitamin B6.

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  • 2016-05