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Protein Carboxyl Methylation and Sperm Motility
Published in Claude Gagnon, Controls of Sperm Motility, 2020
Further evidence for a role for protein-carboxyl methylation in sperm function was provided by experiments with inhibitors of protein methylation in intact spermatozoa. The combination of adenosine, homocysteine, and 3-deazaadenosine has been shown to block intracellular methylation reaction.58 The addition of adenosine, homocysteine, and 3-deazaadenosine caused a time-dependent progressive inhibition of sperm protein-carboxyl methylation. This inhibition was associated with a concomitant decrease in sperm motility (Table 4). Similar time-dependent inhibition in sperm motility was also observed when erythro-9-(2-hydroxyl-3-nonyl) adenine (EHNA), in combination with homocysteine and adenosine, were incubated with spermatozoa.59 In the latter studies, EHNA was used at a sufficiently low concentration (0.1 mM) to eliminate the possibility of a direct action on dynein ATPase as the cause for the decrease in sperm motility.60,61 On the other hand, Goh and Hoskins62 concluded from their studies with EHNA (0.5 mM), adenosine, and homocysteine that the relationship between sperm motility, levels of S-adenosylhomocysteine (the methylation reaction natural intracellular inhibitor), and protein-carboxyl methylation was not always clear. Nevertheless, they were the first to show directly that agents that elevate S-adenosylhomocysteine inhibit sperm motility. Another report did not support the conclusion that PCM activity levels in spermatozoa were linked to sperm motility. Hetch et al.63 reported that in the population of infertile men studied, a higher level of PCM activity above that of controls was detected. It is worth noting here that these patients did not suffer from oli-gozoospermia or asthenospermia, but were unable to conceive after 1 year of unprotected intercourse. This population of infertile patients is definitely completely different from the population studied by Gagnon et al.56,57 in which a direct relationship between sperm motility and PCM activity was established. The same group also reported that severely asthenospermic patients had levels of PCM activity that were not consistently low. However, they added that their activity was influenced by the quantity of immature germ cells (also possibly leukocytes) present in the sperm pellets analyzed.63 Considering the high levels of PCM activity in spermatids52 and leukocytes,64 and the fact that the elimination of immature germ cells, cell debris, and leukocytes is necessary to establish a good correlation between PCM activity and sperm motility, their results could be explained by the presence of immature germ cells and other round cells in some of the washed pellet assayed.
Elucidating the pathogenesis of adenosine deaminase 2 deficiency: current status and unmet needs
Published in Expert Opinion on Orphan Drugs, 2021
Teresa K Tarrant, Susan J. Kelly, Michael S Hershfield
From the perspective of this review, the Ado/NETs hypothesis rests on the premise that ADA2, as the major ADA isozyme in plasma, regulates levels of extracellular Ado, whereas ADA1 is primarily involved with intracellular Ado. This premise also underlies some published notions of how ADA2 might function as an extracellular growth factor. It should be appreciated that ADA2 is found to be the major ADA isozyme in plasma when reaction rates are measured at a substrate (Ado) concentration saturating for both isozymes, i.e. far higher than Ado levels in vivo. Use of this condition yields the maximal velocity (Vmax) for each enzyme, and if other critical assay variables are also comparable, it allows results from different laboratories to be compared. But even under this non-physiologic assay condition, substantial ADA1 activity is found in plasma. For example, in studies of healthy adults in Japan, Iran, and the US conducted over a 30-year span using assays with 6–12 mM Ado, mean ADA1 (EHNA-inhibitable) and ADA2 (EHNA-resistant) activities in plasma ranged from 3 to 5 U/L and 9 to 13 U/mL, respectively [48–50] (and unpublished).
Approaches for designing and discovering purinergic drugs for gastrointestinal diseases
Published in Expert Opinion on Drug Discovery, 2020
Diego Dal Ben, Luca Antonioli, Catia Lambertucci, Andrea Spinaci, Matteo Fornai, Vanessa D’Antongiovanni, Carolina Pellegrini, Corrado Blandizzi, Rosaria Volpini
Enzymes able to modify Ado (like ADA and ADK) directly or indirectly modulate the extracellular concentration of this nucleoside. Ado is modified to inosine by ADA, and this degradation occurs also intracellularly. Inhibitors of ADA have been developed based on nucleoside or non-nucleoside scaffolds [38]. Among the nucleoside-based ADA inhibitors are 2ʹ-deoxycoformycin (or pentostatin, Figure 4) and cladribine (or leustatin, Figure 4), approved by FDA for the treatment of hairy cell leukemia. Coformycin analogues were developed also as inhibitors of ADA and adenosine 5ʹ-monophosphate deaminase (AMPDA) [78]. Further Ado analogues are 1-deazaadenosine and 8-azaadenosine. Classical non-nucleoside inhibitors of ADA are EHNA (a purine derivative with a long 9-substituent; Figure 4) and the imidazole-based compound FR221647 (Figure 4) [38].
Repurposing of Streptomyces antibiotics as adenosine deaminase inhibitors by pharmacophore modeling, docking, molecular dynamics, and in vitro studies
Published in Journal of Receptors and Signal Transduction, 2020
K. G. Arun, C. S. Sharanya, J. Abhithaj, C. Sadasivan
ADA (bovine), antibiotics, and adenosine were purchased from Sigma-Aldrich. The ADA inhibitory activity of selected antibiotics was determined through Berthelot reaction [14]. In this reaction, ADA converts adenosine to inosine and produce ammonia, and this ammonia reacts with phenol and sodium hypochlorite to form indophenol. The reaction was initiated by the addition of 10 µL enzyme solution (0.40 units/mL) to a reaction mixture containing 50 mM potassium phosphate buffer of pH 7.4 and 20 µM adenosine. After five minutes, the reaction was terminated by the addition of 400 µL phenol-nitroprusside solution. Then, 500 µL of sodium hypochlorite in 0.6 M NaOH was added and incubated for 30 min. The quantity of indophenol produced was determined by measuring the optical density (OD) at 635 nm. A solution without the enzyme was taken as the blank for the measurement of OD. Erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA), a potent inhibitor of ADA, was used as positive control. The ADA activity was expressed in micromoles of ammonia liberated in one minute. The assay was repeated in triplicate under same conditions. The enzyme kinetics studies were carried out with different substrate concentrations (10–60 µM) and fixed concentration (50 µM) of test compounds. The Lineweaver–Burk plot was created, and Michaelis-Menten constant (Km) and maximal velocity (Vmax) were determined from the graph.