CD39 (ectonucleoside triphosphate diphosphohydrolase-1; ENTPD1) metabolizes extracellular ATP and ADP to AMP. AMP is subsequently metabolized by CD79 to adenosine. CD39 activity is therefore a key regulator of purinergic signalling in cancer, thrombosis, and autoimmune diseases. In this study we demonstrate that soluble, recombinant CD39 shows substrate inhibition with ADP or ATP as the substrate. Although CD39 activity initially increased with increasing substrate concentration, at high concentrations of ATP or ADP, CD39 activity was markedly reduced. Although the reaction product, AMP, inhibits CD39 activity, insufficient AMP was generated under our conditions to account for the substrate inhibition seen. In contrast, inhibition was not seen with UDP or UTP as substrates. 2-methylthio-ADP also showed no substrate inhibition, indicating the nucleotide base is an important determinant of substrate inhibition. Molecular dynamics simulations revealed that ADP can undergo conformational rearrangements within the CD39 active site that were not seen with UDP or 2-methylthio-ADP. Appreciating the existence of substrate inhibition of CD39 will help the interpretation of studies of CD39 activity, including investigations into drugs that modulate CD39 activity.
ABSTRACT: Environmental hygiene monitoring in the food processing environment has become important in current food safety programs to ensure safe food production. However, conventional monitoring of surface hygiene based on visual inspection and microbial counts is slow, tedious, and thus unable to support the current risk-based management system. Therefore, this study was conducted to assess the performance of a real-time total adenylate assay that detected ATP+ADP+AMP (A3) for food contact surface hygiene in 13 food processing plants and two commercial kitchens in Malaysia. The A3 value was compared with the microbial count (aerobic plate count [APC]) on food contact surfaces. Receiver-operating characteristic (ROC) analysis was performed to assess the reliability of the data and to determine the optimal threshold value for hygiene indication of food contact surfaces. Overall, the A3 value demonstrated a weak positive relationship with APC. However, the A3 value significantly correlated with APC for food processing environments associated with raw meat and raw food ingredients such as fruit that harbor a high microbial load. ROC analysis suggested an optimal threshold for the A3 value of 500 relative light units to balance the sensitivity and specificity at 0.728 and 0.719, respectively. The A3 assay as a hygiene indicator for food contact surfaces had an efficiency of 72.1%, indicating its reliability as a general hygiene indicator.
In general, Cordyceps sinensis is much more popular than C. militaris, though both species contain quite similar bioactive ingredients and exhibit medicinal activities. Many bioactive ingredients have been isolated from C. militaris, such as adenosine, cordycepin, D-mannitol, and exopolysaccharides. C. militaris is claimed to have extensive pharmacological properties, such as: anti-inflammatory; anti-fatigue; anti-bacterial; anti-diabetic; improve lung, liver, and kidney functions; to be beneficial for treating cancer as well as male and female sexual dysfunctions. C. militaris is fast gaining momentum for its so-called health benefits, and it is often used as a substitute for C. sinensis. In view of the growing popularity of C. militaris, nowadays C. militaris cultivation for stroma is also done. There is a great diversity of compounds from different strains of Cordyceps and different artificially cultivated products. This study is to determine the optimum culture parameters integrated with substrate of choice to bring the indoor-cultivated C. militaris to a higher and more consistent level of quality. To achieve the above objective, the resultant products after growth were analyzed for adenosine, cordycepin, and D-mannitol using the high-performance liquid chromatography method. The optimum culture condition to produce a high level of adenosine is by using millet as solid substrate. It must be cultivated in the dark for the first 7 days and harvested on day 40. The optimum culture condition to produce a high level of cordycepin is by using soybean as solid substrate. It must be cultivated in the dark for the first 14 days and harvested on day 50. While a high level of D-mannitol is achieved with millet as the solid substrate. It must be kept in the dark for the first 7 days and harvested on day 50. The adenosine level decreased and cordycepin increased from day 40 of culture to day 50 generally.
Microbial degradation of pesticide residues has the potential to reduce their hazards to human and environmental health. However, in some cases, degradation can activate pesticides, making them more toxic to microbes. Here we report on the β-cypermethrin (β-CY) toxicity to Bacillus cereus GW-01, a recently described β-CY degrader, and effects of antioxidants on β-CY degradation. GW-01 exposed to β-CY negatively affected the growth rate. The highest maximum specific growth rate (μm) appeared at 25 mg/L β-CY. β-CY induced the oxidative stress in GW-01. The activities of superoxide dismutase (SOD), catalyse (CAT), and glutathione-S-transferase (GST) were significantly higher than that in control (p
The aim of the study was to explore a propriety standardized ethanolic extract from leaves of Orthosiphon stamineus Benth in improving impairments in short-term social memory in vivo, possibly via blockade of adenosine A2A receptors (A2AR). The ethanolic extract of O. stamineus leaves showed significant in vitro binding activity of A2AR with 74% inhibition at 150 μg/ml and significant A2AR antagonist activity with 98% inhibition at 300 μg/mL. A significant adenosine A1 receptor (A1R) antagonist activity with 100% inhibition was observed at 300 μg/mL. Its effect on learning and memory was assessed via social recognition task using Sprague Dawley rats whereby the ethanolic extract of O. stamineus showed significant (p < 0.001) change in recognition index (RI) at 300 mg/kg and 600 mg/kg p.o and 120 mg/kg i.p., respectively, compared to the vehicle control. In comparison, the ethanolic extract of Polygonum minus aerial parts showed small change in inflexion; however, it remained insignificant in RI at 200 mg/kg p.o. Our findings suggest that the ethanolic extract of O. stamineus leaves improves memory by reversing age-related deficits in short-term social memory and the possible involvement of adenosine A1 and adenosine A2A as a target bioactivity site in the restoration of memory.
Adenosine A1 receptors (AA1R) have been shown to counteract N-methyl-D-aspartate (NMDA)-mediated glutamatergic excitotoxicity. In the present study, we investigated the role of AA1R in neuroprotection by trans-resveratrol (TR) against NMDA-induced retinal injury. In total, 48 rats were divided into the following four groups: normal rats pretreated with vehicle; rats that received NMDA (NMDA group); rats that received NMDA after pretreatment with TR; and rats that received NMDA after pretreatment with TR and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an AA1R antagonist. Assessment of general and visual behaviour was performed using the open field test and two-chamber mirror test, respectively, on Days 5 and 6 post NMDA injection. Seven days after NMDA injection, animals were euthanized, and eyeballs and optic nerves were harvested for histological parameters, whereas retinae were isolated to determine the redox status and expression of pro- and anti-apoptotic proteins. In the present study, the retinal and optic nerve morphology in the TR group was protected from NMDA-induced excitotoxic damage. These effects were correlated with the lower retinal expression of proapoptotic markers, lipid peroxidation, and markers of nitrosative/oxidative stress. The general and visual behavioural parameters in the TR group showed less anxiety-related behaviour and better visual function than those in the NMDA group. All the findings observed in the TR group were abolished by administration of DPCPX.
Weak organic acids are widely used as preservatives and disinfectants in the food industry. Despite their widespread use, the antimicrobial mode of action of organic acids is still not fully understood. This study investigated the effect of acetic acid on the cell membranes and cellular energy generation of four Salmonella strains. Using a nucleic acid/protein assay, it was established that acetic acid did not cause leakage of intracellular components from the strains. A scanning electron microscopy study further confirmed that membrane disruption was not the antimicrobial mode of action of acetic acid. Some elongated Salmonella cells observed in the micrographs indicated a possibility that acetic acid may inhibit DNA synthesis in the bacterial cells. Using an ATP assay, it was found that at a neutral pH, acetic acid caused cellular energy depletion with an ADP/ATP ratio in the range between 0.48 and 2.63 (p<0.05) that was apparent for the four Salmonella strains. We suggest that this effect was probably due solely to the action of undissociated acid molecules. The antimicrobial effect of acetic acid was better under acidic conditions (ADP/ATP ratio of 5.56 ± 1.27; p<0.05), where the role of both pH and undissociated acid molecules can act together. We concluded that the inhibitory effect of acetic acid is not solely attributable to acidic pH but also to undissociated acid molecules. This finding has implication for the use of acetic acid as an antimicrobial against Salmonella on food products, such as chicken meat, which can buffer its pH.
Human A3 adenosine receptor hA3AR has been implicated in gastrointestinal cancer, where its cellular expression has been found increased, thus suggesting its potential as a molecular target for novel anticancer compounds. Observation made in our previous work indicated the importance of the carbonyl group of amide in the indolylpyrimidylpiperazine (IPP) for its human A2A adenosine receptor (hA2AAR) subtype binding selectivity over the other AR subtypes. Taking this observation into account, we structurally modified an indolylpyrimidylpiperazine (IPP) scaffold, 1 (a non-selective adenosine receptors' ligand) into a modified IPP (mIPP) scaffold by switching the position of the carbonyl group, resulting in the formation of both ketone and tertiary amine groups in the new scaffold. Results showed that such modification diminished the A2A activity and instead conferred hA3AR agonistic activity. Among the new mIPP derivatives (3-6), compound 4 showed potential as a hA3AR partial agonist, with an Emax of 30% and EC50 of 2.89 ± 0.55 μM. In the cytotoxicity assays, compound 4 also exhibited higher cytotoxicity against both colorectal and liver cancer cells as compared to normal cells. Overall, this new series of compounds provide a promising starting point for further development of potent and selective hA3AR partial agonists for the treatment of gastrointestinal cancers.
Senile cataract is the most common cause of bilateral blindness and results from the loss of transparency of the lens. Maintenance of the unique tissue architecture of the lens is vital for keeping the lens transparent. Membrane transport mechanisms utilizing several magnesium (Mg)-dependent ATPases, play an important role in maintaining lens homeostasis. Therefore, in Mg-deficiency states, ATPase dysfunctions lead to intracellular depletion of K(+) and accumulation of Na(+) and Ca(2+). High intracellular Ca(2+) causes activation of the enzyme calpain II, which leads to the denaturation of crystallin, the soluble lens protein required for maintaining the transparency of the lens. Mg deficiency also interferes with ATPase functions by causing cellular ATP depletion. Furthermore, Mg deficiency enhances lenticular oxidative stress by increased production of free radicals and depletion of antioxidant defenses. Therefore, Mg supplementation may be of therapeutic value in preventing the onset and progression of cataracts in conditions associated with Mg deficiency.
The diagnostic value of adenosine deaminase (ADA) activity was studied to evaluate its use in the differential diagnosis of tuberculous meningitis in the local setting. Cerebrospinal fluid (CSF) from 119 patients with meningitis and other conditions with central nervous system symptoms were collected and ADA activity determined by the colorimetric method of Guisti read at 628 nm. The CSF was also subjected to other laboratory examinations so as to provide the aetiological diagnosis. All 14 tuberculous meningitis patients had ADA activity greater than the cut off value of 9.0 IU/L. High ADA activity was also seen in 13 of 105 non-tuberculous cases giving a specificity of 87.6%. Even though the ADA activity determination is sensitive for tuberculosis, it was not specific enough to be used as a rapid diagnostic test. However when interpreted together with clinical signs and symptoms and other laboratory tests, it is a useful adjunctive rapid marker for tuberculosis.
812 West Malaysian Orang Asli belonging to four ethnic groups were surveyed for adenosine deaminase (ADA; EC 3.5.4.4) using starch gel electrophoresis. Only the common ADA1 and ADA2 alleles were found, with the frequencies of the latter being 0.025, 0.103, 0.115 and 0.028 in the Semai, Semelai, Temuan, and Jakun groups, respectively. A new 'breeding genetic distance' was applied to these gene frequencies and the Semelai and Temuan were found to be more closely related to each other, and to have considerably more evolutionary flexibility on this scale of 'micro-evolution' than the other two groups. The Semai and Jakun were more similar to each other on the basis of these ADA gene frequencies.
944 adenosine deaminase phenotypings of Malay, Chinese, and Indian blood donors and newborns at Kuala Lumpur, Malaysia, yielded ADA1 gene frequency estimates of 0.885 for the Malays, 0.939 for the Chinese, and 0.853 for the Indians.
Epilepsy is one of the most common and disabling chronic neurological diseases affecting people of all ages. Major challenges of epilepsy management include the persistently high percentage of drug-refractoriness among patients, the absence of disease-modifying treatments, and its diagnosis and prognosis. To date, long-term video-electroencephalogram (EEG) recordings remain the gold standard for an epilepsy diagnosis. However, this is very costly, has low throughput, and in some instances has very limited availability. Therefore, much effort is put into the search for non-invasive diagnostic tests. Purinergic signalling, via extracellularly released adenosine triphosphate (ATP), is gaining increasing traction as a therapeutic strategy for epilepsy treatment which is supported by evidence from both experimental models and patients. This includes in particular the ionotropic P2X7 receptor. Besides that, other components from the ATPergic signalling cascade such as the metabotropic P2Y receptors (e.g., P2Y1 receptor) and ATP-release channels (e.g., pannexin-1), have also been shown to contribute to seizures and epilepsy. In addition to the therapeutic potential of purinergic signalling, emerging evidence has also shown its potential as a diagnostic tool. Following seizures and epilepsy, the concentration of purines in the blood and the expression of different compounds of the purinergic signalling cascade are significantly altered. Herein, this review will provide a detailed discussion of recent findings on the diagnostic potential of purinergic signalling for epilepsy management and the prospect of translating it for clinical application. This article is part of the Special Issue on 'Purinergic Signaling: 50 years'.
In recent years, shrimp aquaculture industry had grown significantly to become the major source of global shrimp production. Despite that, shrimp aquaculture production was impeded by various shrimp diseases over the past decades. Interestingly, different shrimp species demonstrated variable levels of immune strength and survival (immune-survival) ability towards different diseases, especially the much stronger immune-survival ability shown by the ancient shrimp species, Macrobrachium rosenbergii compared to other shrimp species. In this study, two important shrimp species, M. rosenbergii and Penaeus monodon (disease tolerant strain) (uninfected control and VpAHPND-infected) were compared to uncover the potential underlying genetic factors. The shrimp species were sampled, followed by RNA extraction and cDNA conversion. Five important immune-survival genes (C-type Lectin, HMGB, STAT, ALF3, and ATPase 8/6) were selected for PCR, sequencing, and subsequent genetics analysis. The overall genetic analyses conducted, including Analysis of Molecular Variance (AMOVA) and population differentiation, showed significant genetic differentiation (p<0.05) between different genes of M. rosenbergii and P. monodon. There was greater genetic divergence identified between HMGB subgroups of P. monodon (uninfected control and VpAHPND-infected) compared to other genes. Besides that, based on neutrality tests conducted, purifying selection was determined to be the main evolutionary driving force of M. rosenbergii and P. monodon with stronger purifying selection exhibited in M. rosenbergii genes. Potential balancing selection was identified for VpAHPND-infected HMGB subgroup whereas directional selection was detected for HMGB (both species) and ATPase 8/6 (only P. monodon) genes. The divergence times between M. rosenbergii and P. monodon genes were estimated through Bayesian molecular clock analysis, which were 438.6 mya (C-type Lectin), 1885.4 mya (HMGB), 432.6 mya (STAT), 448.1 mya (ALF3), and 426.4 mya (ATPase 8/6) respectively. In conclusion, important selection forces and evolutionary divergence information of immune-survival genes between M. rosenbergii and P. monodon were successfully identified.
Hsp90 is an ATP-dependent molecular chaperone that is involved in important cellular pathways such as signal transduction pathways. It is a potential cancer drug target because it plays a critical role for stabilization and activation of oncoproteins. Thus, small molecule compounds that control the Hsp90 function are useful to elucidate potential lead compounds against cancer. We studied effect of a naturally occurring styryl-lactone goniothalamin on the activity of Hsp90. Although many drugs targeting Hsp90 inhibit the ATPase activity of Hsp90, goniothalamin enhanced rather than inhibited the ATPase activity of a cyanobacterial Hsp90 (HtpG) and a yeast Hsp90. It increased both K(m) and k(cat) of the Hsp90s. Domain competition assays and tryptophan fluorescence measurements with various truncated derivatives of HtpG indicated that goniothalamin binds to the N-terminal domain of HtpG. Goniothalamin did not influence on the interaction of HtpG with a non-native protein or the anti-aggregation activity of HtpG significantly. However, it inhibited the activity of HtpG that assists refolding of a non-native protein in cooperation with the Hsp70 chaperone system. This is the first report to show that a small molecule that binds to the N-terminal domain of Hsp90 activates its ATPase activity, while inhibiting the chaperone function of Hsp90.
Like other nematodes, both L(3) and adult Teladosagia circumcincta secrete or excrete NH(3)/NH(4)(+), but the reactions involved in the production are unclear. Glutamate dehydrogenase is a significant source NH(3)/NH(4)(+) in some species, but previous reports indicate that the enzyme is absent from L(3)Haemonchus contortus. We show that glutamate dehydrogenase was active in both L(3) and adult T. circumcincta. The apparent K(m)s of the L(3) enzyme differed from those of the adult enzyme, the most significant of these being the increase in the K(m) for NH(4)(+) from 18mM in L(3) to 49mM in adults. The apparent V(max) of the oxidative deamination reaction was greater than that of the reductive reaction in L(3), but this was reversed in adults. The activity of the oxidative reaction of the L(3) enzyme was not affected by adenine nucleotides, but that of the reductive reaction was stimulated significantly by either ADP or ATP. The L(3) enzyme was more active with NAD(+) than it was with NADP(+), although the activities supported by NADH and NADPH were similar at saturating concentrations. While the activity of the oxidative reaction was sufficient to account for the NH(3)/NH(4)(+) efflux we have previously reported, the reductive amination reaction was likely to be more active.
Compounds that block the function of connexin and pannexin protein channels have been suggested to be valuable therapeutics for a range of diseases. Some of these compounds are now in clinical trials, but for many of them, the literature is inconclusive about the molecular effect on the tissue, despite evidence of functional recovery. Blocking the different channel types has distinct physiological and pathological implications and this review describes current knowledge of connexin and pannexin protein channels, their function as channels and possible mechanisms of the channel block effect for the latest therapeutic compounds. We summarize the evidence implicating pannexins and connexins in disease, considering their homeostatic versus pathological roles, their contribution to excesive ATP release linked to disease onset and progression.
Bahagian aktif bagi enzim toksin bakteria daripada Burkholderia pseudomallei, Pseudomonas aeruginosa dan difteria merupakan domain ADP-ribosilasi. Domain ini didapati terpelihara di antara ketiga-tiga mikroorganisme. Di dalam kajian ini, domain ADP-ribosilasi Burkholderia pseudomallei telah diamplifikasi daripada genom B. pseudomallei virulen dengan menggunakan pencetus-pencetus yang dibina berdasarkan kepada jujukan domain ADP ribosilasi Pseudomonas aeruginosa. Hasil DNA amplifikasi ditulenkan dan digunakan sebagai prob (HPCR2) untuk menyaring DNA selitan daripada B. pseudomallei yang diklonkan ke dalam vektor pengekspresan pSport-I. Objektif kajian ini adalah untuk menyaring lapan klon yang positif hasil daripada penyaringan awal melalui pendekatan immunoblot menggunakan antitoksin daripada arnab. Penyaringan ini juga melibatkan tiga klon yang tidak memberikan isyarat positif semasa penyaringan secara immunoblot. Keputusan menunjukkan hanya satu klon (L31) daripada lapan klon immunoblot positif mempunyai domain ADP-ribosilasi. Penjujukan DNA separa klon L31 secara manual melibatkan dua pencetus menghasilkan jujukan sepanjang 450pb. Analisis selanjutnya mendapati daripada enam kemungkinan translasi kepada polipeptida hanya satu polipeptida wujud yang tidak mempunyai sebarang kodon penamat pada jujukan kodonnya.
The transwell migration assay is commonly used for assessing cell migration. It involves the enumeration of cells that
have migrated across a pore-containing membrane. We describe a randomised approach to quantifying migrated
cells and compare it to a conventional full cell count. We used ATP as a chemoattractant and automatic cell quantification performed on all fields (Full count; FC) or 10 randomly selected fields (Randomised count; RC). The two
methods were compared by evaluating standard deviations (SD), coefficient of variation (CV) and using the Bland-Altman analysis. The dispersion of data is higher with the RC approach (3.77-6.66% CV for control; 3.89-4.48% CV
for ATP-treated wells) compared to FC (0.27-0.46% CV for control; 0.05-0.09% CV for ATP-treated wells), but are
acceptable considering that the number of migrated cells are in the thousands. Both methods verified that an ATP
migration assay for BV2 microglia was established, demonstrating that the RC approach is reliable and comparable
to a full count.