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Analysis of Modified Amino Acids
Published in Ajit S. Bhown, Protein/Peptide Sequence Analysis: Current Methodologies, 1988
The retained amino acids, lysine, arginine, and their methylated derivatives, were eluted with 20 mℓ of 3 N NH4OH. The eluate was evaporated to dryness. This dried sample could be used for analysis by paper, thin-layer, or ion-exchange chromatography. The sample could also be treated to remove lysine and arginine as follows. The sample was dissolved in 3 mℓ of 0.2 M phosphate buffer, pH 6.0, and 5 mg of lysine decarboxylase (specific activity, 15 μmol of lysine decarboxylated per 20 min per milligram enzyme protein), and 50 μg of pyridoxal phosphate in 0.25 mℓ of water were added. The mixture was incubated at 37°C for 2 hr. The reaction was terminated by immersing the tube in boiling water for 3 min. After cooling, 10 mg of the arginine deiminase (EC 3.5.3.6) (specific activity, 5 μmol of arginine deiminated per 20 min per milligram enzyme protein) was added and incubated at 37°C for 2 hr. Then 10 mℓ of 10% trichloroacetic acid was added, and the supernatant was passed through a 1 × 3-cm column of AmbeR1ite IR-120 (100 to 200 mesh, H+ form).
Regulation of flagellar motility and biosynthesis in enterohemorrhagic Escherichia coli O157:H7
Published in Gut Microbes, 2022
Hongmin Sun, Min Wang, Yutao Liu, Pan Wu, Ting Yao, Wen Yang, Qian Yang, Jun Yan, Bin Yang
The cadA gene encodes a lysine decarboxylase that plays a role in EHEC O157:H7 motility and flagellar biosynthesis.39 A gene array analysis revealed that 1464 genes (132 upregulated and 1332 downregulated) were differentially regulated by more than twofolds (with 99% confidence) in the ΔcadA mutant versus the wild-type strain.39 Interestingly, among these most upregulated genes, 16 genes, including fliA, flgABCDEFGH, and fliEFGJLMN, are involved in flagellar biosynthesis and motility. The qRT-PCR results confirmed the elevated expression of flagellar basal body and hook-encoding genes flgB and flgE, in concordance with the microarray results. In accordance, an analysis of multiple electron microscopic fields revealed that the ΔcadA mutant strain possesses more flagellar filaments than the wild-type strain grown under the same conditions. Interestingly, the motilities of both wild-type EHEC O157:H7 and ΔcadA mutant strains were similar on motility agar plates.39 The regulatory mechanisms of CadA on EHEC O157:H7 flagellar biosynthesis remain unclear. Considering that most upregulated flagellar genes in ΔcadA mutant are under the control of FliA, and the expression of fliA was also significantly increased in the ΔcadA mutant, the regulation of flagellar genes by CadA may be dependent on FliA.
Polyamine biomarkers as indicators of human disease
Published in Biomarkers, 2021
Mohsin Amin, Shiying Tang, Liliana Shalamanova, Rebecca L. Taylor, Stephen Wylie, Badr M. Abdullah, Kathryn A. Whitehead
Cadaverine and putrescine have been identified as upregulated biomolecules in periodontal disease and their role in the pathogenesis of periodontal disease have been suggested in a number of previous studies (Lamster et al.1987, Mariggiò et al.2004, Lohinai et al.2012). Despite their abundance in cells, polyamine levels are tightly regulated. However, there are some differences in the concentrations of certain polyamines such as cadaverine when measured in the saliva of individuals. Tábi et al. (2008) measured average cadaverine levels that increased to 11.8 ± 8.30 µM, from 7.9 ± 6.48 µM, after oral hygiene was restricted and they concluded that such an increase in cadaverine concentration could potentially contribute to an increase in the development of periodontal diseases. Fine and Mandel (1986) described increases of up to 10 fold of cadaverine in patients whose plaque index score was measured from 1 to 2, leading to the suggestion that cadaverine was the best indicator to measure the metabolic activity of plaque, which is associated with the onset of periodontal disease. The upregulation of cadaverine has been observed by Lohinai et al. (2012), through the assessment of human dental biofilms. The concentrations of cadaverine, lysine and lysine decarboxylase in dental biofilms after one week of oral hygiene restriction were measured as a determination of damage to the gingival sulci. Cadaverine in this instance was produced as a result of lysine decarboxylation and the cells attached to the dental tissues become lysine deprived. This results in the release of pro-inflammatory cytokines, which act on the sub-epithelial blood vessels to become permeabilised, or experience autophagy. This enables the dental biofilm constituents to access the gingival stroma to release cytokines and initiate GCF exudation.
Balance of saccharolysis and proteolysis underpins improvements in stool quality induced by adding a fiber bundle containing bound polyphenols to either hydrolyzed meat or grain-rich foods
Published in Gut Microbes, 2019
Matthew I. Jackson, Dennis E. Jewell
Similar to fecal free amino acids and branched chain SCFAs, polyamines are indicators of putrefaction; putrescine and cadaverine are representative of the putrefaction of arginine and lysine, respectively. The degree to which polyamine levels, as well as their precursor amino acids and catabolic intermediates, were changed when fiber was added to either the HM or GR background foods were evaluated. When assessed in a multivariate fashion by multivariate analysis of variance (MANOVA), the pathway consisting of polyamines, as well as their detected anabolic and catabolic intermediates (Table 2) was significantly altered by addition of fiber to the HM (p < 0.001) and GR (p < 0.001) foods. Substrates for polyamine biosynthesis were surveyed in a univariate manner. Lysine, the precursor to cadaverine via a one-step reaction catalyzed by lysine decarboxylase, was decreased by fiber addition to either food. Arginine, a precursor to putrescine and subsequently spermidine via the intermediacy of agmatine or ornithine, was not significantly changed in either food when fiber was added (Table 2, Table S3). Agmatine, an intermediate of the arginine decarboxylase pathway, was significantly decreased in both healthy dogs and those with chronic enteritis/gastroenteritis only when fiber was added to the HM food. When fiber was added to the GR food, agmatine decreased in the feces of only healthy dogs (p = 0.013), a response that differed by health status (p = 0.002 for difference by health status). Ornithine, an intermediate of the ornithine decarboxylase pathway, was significantly increased in all dogs when fiber was added to the HM food, but addition of fiber to GR had no significant effect on fecal ornithine levels for dogs when pooled or assessed by health status. Putrescine and spermidine, their mono- and di-acetylated catabolic intermediates, and the near-terminal oxidation product carboxyethyl-GABA were decreased by fiber addition to either food. In support of perturbation of polyamine homeostasis by fiber, although lysine-derived cadaverine and its acetylated derivative were statistically unchanged by fiber addition, they trended to decreased levels with the same magnitude of fiber-induced difference as for other members of the pathway with significant differences, but with greater individual variation of response that may have belied significance (SE of matched pairs assessing fiber influence for individual dogs; Table 2). Fiber-induced changes to polyamines were not dependent on health status.