Adenylate Kinase Activity Research Articles

Compartmentalization in cardiomyocytes modulates creatine kinase and adenylate kinase activities.

Intracellular molecules are transported by motor proteins or move by diffusion resulting from random molecular motion. Cardiomyocytes are packed with structures that are crucial for function, but also confine the diffusional spaces, providing cells with a means to control diffusion. They form compartments in which local concentrations are different from the overall, average concentrations. For example, calcium and cyclic AMP are highly compartmentalized, allowing these versatile second messengers to send different signals depending on their location. In energetic compartmentalization, the ratios of AMP and ADP to ATP are different from the average ratios. This is important for the performance of ATPases fuelling cardiac excitation-contraction coupling and mechanical work. A recent study suggested that compartmentalization modulates the activity of creatine kinase and adenylate kinase in situ. This could have implications for energetic signaling through, for example, AMP-activated kinase. It highlights the importance of taking compartmentalization into account in our interpretation of cellular physiology and developing methods to assess local concentrations of AMP and ADP to enhance our understanding of compartmentalization in different cell types.

Protecting Lyophilized Escherichia coli Adenylate Kinase.

Drying protein-based drugs, usually via lyophilization, can facilitate storage at ambient temperature and improve accessibility but many proteins cannot withstand drying and must be formulated with protective additives called excipients. However, mechanisms of protection are poorly understood, precluding rational formulation design. To better understand dry proteins and their protection, we examine Escherichia coli adenylate kinase (AdK) lyophilized alone and with the additives trehalose, maltose, bovine serum albumin, cytosolic abundant heat soluble protein D, histidine, and arginine. We apply liquid-observed vapor exchange NMR to interrogate the residue-level structure in the presence and absence of additives. We pair these observations with differential scanning calorimetry data of lyophilized samples and AdK activity assays with and without heating. We show that the amino acids do not preserve the native structure as well as sugars or proteins and that after heating the most stable additives protect activity best.

Better maintenance of enzymatic capacity and higher levels of substrate transporter proteins in skeletal muscle of aging female mice.

This study investigated sex-specific differences in high-energy phosphate, glycolytic, and mitochondrial enzyme activities and also metabolite transporter protein levels in the skeletal muscles of adult (5 months old), middle-aged (12 months old), and advanced-aged (24 months old) mice. While gastrocnemius glycogen content increased with age regardless of sex, gastrocnemius triglyceride levels increased only in advanced-aged female mice. Aging decreased creatine kinase and adenylate kinase activities in the plantaris muscle of both sexes and in the soleus muscle of male mice but not in female mice. Irrespective of sex, phosphofructokinase and lactate dehydrogenase (LDH) activities decreased in the plantaris and soleus muscles. Additionally, hexokinase activity in the plantaris muscle and LDH activity in the soleus muscle decreased to a greater extent in aged male mice compared with those in aged female mice. Mitochondrial enzyme activities increased in the plantaris muscle of aged female mice but did not change in male mice. The protein content of the glucose transporter 4 in the aged plantaris muscle and fatty acid translocase/cluster of differentiation 36 increased in the aged plantaris and soleus muscles of both sexes, with a significantly higher content in female mice. These findings suggest that females possess a better ability to maintain metabolic enzyme activity and higher levels of metabolite transport proteins in skeletal muscle during aging, despite alterations in lipid metabolism. Our data provide a basis for studying muscle metabolism in the context of age-dependent metabolic perturbations and diseases that affect females and males differently.

Pentaene macrolides AB023a and takanawaene C produced by Streptomyces xanthocidicus strain S3 for controlling pepper anthracnose

A natural product library consisting of the culture extracts of 814 actinomycete strains was screened for antifungal compounds that disrupt the cell integrity of plant pathogenic fungi using an adenylate kinase (AK) assay system. The culture extract of Streptomyces xanthocidicus strain S3 exhibited high AK activity against various plant pathogens. The active ingredients, AT-1 and AT-2, were isolated from the culture extract using a series of chromatographic procedures. Based on MS, UV, and NMR spectrometric analyses, the structures of AT-1 and AT-2 were determined as the pentaene macrolides, AB023a and takanawaene C. AB023a and takanawaene C displayed broad-spectrum antifungal activity against Aspergillus oryzae, Botrytis cinerea, Colletotrichum coccodes, C. gloeosporioides, C. orbiculare, Cylindrocarpon destructans, and Fusarium oxysporum f. sp. lycopersici, showing minimum inhibitory concentrations of 1–32 μg/mL. Treatment of AB023a and takanawaene C successfully inhibited anthracnose development on pepper plants in a concentration-dependent manner without phytotoxicity. The disease control efficacy of both compounds was comparable to that of the commercial fungicide chlorothalonil. Collectively, these results suggest that the polyene macrolides produced by S. xanthocidicus strain S3 can be used as natural fungicides for plant disease control.

Allosteric communication between ligand binding domains modulates substrate inhibition in adenylate kinase

Enzymes play a vital role in life processes; they control chemical reactions and allow functional cycles to be synchronized. Many enzymes harness large-scale motions of their domains to achieve tremendous catalytic prowess and high selectivity for specific substrates. One outstanding example is provided by the three-domain enzyme adenylate kinase (AK), which catalyzes phosphotransfer between ATP to AMP. Here we study the phenomenon of substrate inhibition by AMP and its correlation with domain motions. Using single-molecule FRET spectroscopy, we show that AMP does not block access to the ATP binding site, neither by competitive binding to the ATP cognate site nor by directly closing the LID domain. Instead, inhibitory concentrations of AMP lead to a faster and more cooperative domain closure by ATP, leading in turn to an increased population of the closed state. The effect of AMP binding can be modulated through mutations throughout the structure of the enzyme, as shown by the screening of an extensive AK mutant library. The mutation of multiple conserved residues reduces substrate inhibition, suggesting that substrate inhibition is an evolutionary well conserved feature in AK. Combining these insights, we developed a model that explains the complex activity of AK, particularly substrate inhibition, based on the experimentally observed opening and closing rates. Notably, the model indicates that the catalytic power is affected by the microsecond balance between the open and closed states of the enzyme. Our findings highlight the crucial role of protein motions in enzymatic activity.

Adenylate Kinase-Catalyzed Reactions of AMP in Pieces: Specificity for Catalysis at the Nucleoside Activator and Dianion Catalytic Sites.

The pressure to optimize the enzymatic rate acceleration for adenylate kinase (AK)-catalyzed phosphoryl transfer has led to the evolution of an induced-fit mechanism, where the binding energy from interactions between the protein and substrate adenosyl group is utilized to drive a protein conformational change that activates the enzyme for catalysis. The adenine group of adenosine contributes 11.8 kcal mol-1 to the total ≥14.7 kcal mol-1 adenosine stabilization of the transition state for AK-catalyzed phosphoryl transfer to AMP. The relative third-order rate constants for activation of adenylate kinase, by the C-5 truncated adenosine 1-(β-d-erythrofuranosyl)adenine (EA), for catalysis of phosphoryl transfer from ATP to phosphite dianion (HP, kcat/KHPKAct = 260 M-2 s-1), fluorophosphate (47 M-2 s-1), and phosphate (9.6 M-2 s-1), show that substitution of -F for -H and of -OH for -H at HP results, respectively, in decreases in the reactivity of AK for catalysis of phosphoryl transfer due to polar and steric effects of the -F and -OH substituents. The addition of a 5'-CH2OH to the EA activator results in a 3.0 kcal mol-1 destabilization of the transition state for AK-activated phosphoryl transfer to HP due to a steric effect. This is smaller than the 8.3 kcal mol-1 steric effect of the 5'-CH2OH substituent at OMP on HP-activated OMPDC-catalyzed decarboxylation of 1-(β-d-erythrofuranosyl)orotate. The 2'-OH ribosyl substituent shows significant interactions with the transition states for AK-catalyzed phosphoryl transfer from ATP to AMP and for adenosine-activated AK-catalyzed phosphoryl transfer from ATP to HP.

The lipopolysaccharide-transporter complex LptB2FG also displays adenylate kinase activity in vitro dependent on the binding partners LptC/LptA

Lipopolysaccharide (LPS) is an essential glycolipid that covers the surface of gram-negative bacteria. The transport of LPS involves a dedicated seven-protein transporter system called the lipopolysaccharide transport system (Lpt) machinery that physically spans the entire cell envelope. The LptB2FG complex is an ABC transporter that hydrolyzes ATP to extract LPS from the inner membrane for transport to the outer membrane. Here, we extracted LptB2FG directly from the inner membrane with its original lipid environment using styrene-maleic acid polymers. We found that styrene-maleic acid polymers–LptB2FG in nanodiscs display not only ATPase activity but also a previously uncharacterized adenylate kinase (AK) activity, as it catalyzed phosphotransfer between two ADP molecules to generate ATP and AMP. The ATPase and AK activities of LptB2FG were both stimulated by the interaction on the periplasmic side with the periplasmic LPS transport proteins LptC and LptA and inhibited by the presence of the LptC transmembrane helix. We determined that the isolated ATPase module (LptB) had weak AK activity in the absence of transmembrane proteins LptF and LptG, and one mutation in LptB that weakens its affinity for ADP led to AK activity similar to that of fully assembled complex. Thus, we conclude that LptB2FG is capable of producing ATP from ADP, depending on the assembly of the Lpt bridge, and that this AK activity might be important to ensure efficient LPS transport in the fully assembled Lpt system.

Impacts of mutations on dynamic allostery of adenylate kinase.

Escherichia coli adenylate kinase (AK) is composed of CORE domain and two branch domains: LID and AMP-binding domain (AMPbd). AK exhibits considerable allostery in a reversible phosphoryl transfer reaction, which is largely attributed to the relative motion of LID and AMPbd with respect to CORE. Such an allosteric conformational change is also evident in the absence of ligands. Recent studies showed that the mutations in branch domains can adjust dynamic allostery and alter the substrate affinity and enzyme activity. In this work, we use all-atom molecular dynamics simulation to study the impacts of mutations in branch domains on AK's dynamic allostery by comparing two double mutants, i.e., LID mutant (Val135Gly, Val142Gly) and AMPbd mutant (Ala37Gly, Ala55Gly), with wild-type. Two mutants undergo considerable conformational fluctuation and exhibit deviation from the initially extended apo state to more compact structures. The LID domain in the LID mutant adjusts its relative position and firmly adheres to CORE. More strikingly, AMPbd mutations affect the relative positions of both the AMPbd domain and remote LID domain. Both domains undergo considerable movement, especially the inherent hinge swing motion of the flexible LID domain. In both mutants, the mutations can enhance the inter-domain interaction. The results about the conformation change of AK in both mutants are in line with the experiment of AK's affinity and activity. As revealed by our findings, the flexibility of branch domains and their inherent motions, especially LID domain, is highly relevant to dynamic allostery in the AK system.

Addition of tea tree oil (Melaleuca alternifolia) in diet minimize biochemical disturbances in silver catfish Rhamdia quelen exposed to the antiparasitic amitraz

In the present study, we evaluated diets supplemented with Melaleuca alternifolia essential oil (tea tree oil - TTO) as a growth promoter and its possible hepatoprotective effects in Rhamdia quelen exposed to the insecticide amitraz. Six groups (8 per group, in triplicate) were tested: control A (basal diet), B (basal diet + TTO 1.0 mL/kg), C (basal diet +0.84 mL/kg amitraz), D (basal diet +1.19 mL/kg amitraz), E (basal diet +1.0 mL/kg TTO + 0.84 mL/kg amitraz) and F (basal diet + TTO 1.0 mL/kg + 1.19 mL/kg amitraz). Initially R. quelen received diets according to each group. After 14 days, four groups were exposed to two levels of amitraz for a period of 7 days, and were later transferred to amitraz-free water for recovery. We measured levels of reactive oxygen species (ROS) and lipoperoxidation (LOOH) in liver tissue, and activities of enzymes involved in energy metabolism, adenylate kinase (AK) and pyruvate kinase (PK), in addition to zootechnical indices such as length, weight body and fish weight gain. ROS and LOOH levels increased significantly (p < 0.05) in fish exposed to amitraz concentrations, especially in groups of fish not supplemented with TTO. By contrast, AK and PK enzyme activities were significantly lower (p < 0.05) for the groups exposed to amitraz compared to the unexposed group. Finally, fish supplemented with TTO showed better (p < 0.05) zootechnical performance. Addition of TTO minimizes biochemical disturbances in fish exposed to amitraz and serves as a natural additive to improve fish production.

ADP and ATP Promote Mitochondrial Calcium Capacity Through Distinct Roles

The ratio of ADP and ATP is a natural indicator of cellular bioenergetic state, as such these analytes have become prominent factors in metabolism research. Beyond mitochondrial oxidative phosphorylation and ATPase activities, ADP and ATP act as steric regulators of enzymes, e.g. cytochrome C oxidase, and are major factors in mitochondrial calcium storage potential. Consideration of all routes of adenylate conversion is critical to understanding the role of each individual adenylate in the realm of mitochondrial function. Mitochondrial Adenylate Kinase activity dismutates ADP into ATP and AMP via a reversible reaction. Through the use of an Adenylate Kinase inhibitor we have noted that this enzymatic activity drives stark differences in the adenylate profile of isolated mitochondria when measured by 31-P magnetic resonance spectroscopy. These differences are further noted when the Adenylate Kinase reverse reaction is blocked by calcium. Additionally, by utilizing endogenous mitochondrial Adenylate Kinase activity, we identified distinct roles for ADP and ATP in promoting mitochondrial calcium capacity. These roles are such that ADP provides a defense against permeability transition, while ATP contributes to calcium storage through the formation of dense calcium phosphate granules. Our findings showcase how adenylate kinases elicit considerable impact on the outcome of a variety of mitochondrial assays through their drastic manipulation of the adenylate profile. Cytochrome c oxidase activity, P/O ratio, and mitochondrial calcium dynamics were all affected by Adenylate Kinase activity. Of particular note, it was revealed that ATP is required for a particularly dense form of mitochondrial amorphous calcium phosphate, which in the future may further entangle mitochondria with physiological calcium events.

Effects of antibiotics on enhanced biological phosphorus removal and its mechanisms

Many kinds of antibiotics are continuously discharged into wastewater and typically cause a great decrease in sewage treatment performance, whereas mechanisms of differences in the impacts of commonly used antibiotics on phosphate removal are still elusive. Thus, an enhanced biological phosphorus removal (EBPR) system, as an effective method of phosphate removal, was developed, and its performance in the treatment of artificial wastewater containing antibiotics at short- (8 h) and long-term (15 days) exposure was investigated. The results show that phosphorus removal was consistently inhibited by the addition of antibiotics with a significant difference (P < 0.05). To interpret the phenomena, mechanistic equations were developed, and the results indicate that for short-term tests, the difference was mainly caused by the suppression of polyhydroxyalkanoate (PHA) degradation and the activity of polyphosphate kinase (PPK), resulting in the different inhibition of the soluble orthophosphorus (SOP) uptake process. For long-term tests, the difference in SOP uptake was principally caused by the inhibition of PHA degradation and the activity of PPK, whereas the difference in SOP release resulted from the inhibition of activities of exopolyphosphatase (PPX) and adenylate kinase (ADK). Moreover, micro-mechanisms of such inhibition were identified from molecular docking and electrostatic potential.

Urea as a Protein LID Opener: Overcoming Substrate Inhibition in Adenylate Kinase by Tuning Conformational Dynamics

Enzymes are designed to accelerate vital chemical reactions by multiple orders of magnitude. Many of them harness large-scale motions of domains and subunits to promote their activity. Studying structural dynamics is hence essential in order to decipher how protein machines function. Combined with H2MM, a photon-by-photon hidden Markov model analysis, single-molecule FRET studies on the enzyme adenylate kinase (AK) recently revealed very fast domain motions, two orders of magnitude faster than the turnover of the enzyme. AK may use numerous cycles of conformational rearrangement in order to find a relative orientation of its substrates that is optimal for the chemical reaction, the interconversion of adenine nucleotides. In the present work we studied the impact of urea on the domain motions of AK, particularly those of its ATP-binding LID-domain. Although urea has been commonly used as a protein denaturant, it is reported that in minor amounts it actually enhances the catalytic activity of AK. We show that this activation is caused by an effect on substrate inhibition. High concentrations of the substrate AMP decrease the rate of LID-domain opening, disturb the balance between open and closed conformations and reduce turnover. Minor amounts of urea partially revert this effect by opening the domains and allow the enzyme to regain activity. Importantly, in absence of inhibitory concentrations of AMP, the activity of AK is not enhanced by urea and can even be reduced, although urea here again favors the open conformation This suggests a delicate balance between domain closure dynamics and substrate binding, which might not be a unique feature of AK, and is likely employed by a multitude of enzymes to regulate their activity.

Ordinary Gasoline Emissions Induce a Toxic Response in Bronchial Cells Grown at Air-Liquid Interface.

Gasoline engine emissions have been classified as possibly carcinogenic to humans and represent a significant health risk. In this study, we used MucilAir™, a three-dimensional (3D) model of the human airway, and BEAS-2B, cells originating from the human bronchial epithelium, grown at the air-liquid interface to assess the toxicity of ordinary gasoline exhaust produced by a direct injection spark ignition engine. The transepithelial electrical resistance (TEER), production of mucin, and lactate dehydrogenase (LDH) and adenylate kinase (AK) activities were analyzed after one day and five days of exposure. The induction of double-stranded DNA breaks was measured by the detection of histone H2AX phosphorylation. Next-generation sequencing was used to analyze the modulation of expression of the relevant 370 genes. The exposure to gasoline emissions affected the integrity, as well as LDH and AK leakage in the 3D model, particularly after longer exposure periods. Mucin production was mostly decreased with the exception of longer BEAS-2B treatment, for which a significant increase was detected. DNA damage was detected after five days of exposure in the 3D model, but not in BEAS-2B cells. The expression of CYP1A1 and GSTA3 was modulated in MucilAir™ tissues after 5 days of treatment. In BEAS-2B cells, the expression of 39 mRNAs was affected after short exposure, most of them were upregulated. The five days of exposure modulated the expression of 11 genes in this cell line. In conclusion, the ordinary gasoline emissions induced a toxic response in MucilAir™. In BEAS-2B cells, the biological response was less pronounced, mostly limited to gene expression changes.

Inflammatory responses, energy metabolism enzymes, oxidative status in Clostridium perfringens infection in broilers

The aim of this study was to determine whether infection by Clostridium perfringens negatively interferes with the oxidant/antioxidant status and activity of energy metabolism enzymes (creatine kinase (CK), adenylate kinase (AK) and pyruvate kinase (PK)), as well as the zootechnical performance of broilers. A completely randomized design with three treatments, with five replications by treatment, and 10 birds by repetitions was used: T1: non-infected group; T2: group infected with C. perfringens; T3: group infected with C. perfringens, and basal diet with performance enhancers (antibiotic and coccidiostatic). At 21 days of age, the birds were experimentally infected orally with 4.0 x 108 CFU/mL C. perfringens. At strategic moments during the experimental period, zootechnical data and blood samples were collected. There were no significant differences among treatments in terms of weight gain, feed intake, or feed conversion. At 20 days, heterophils counts were significantly lower in the T3 group birds than in the others. A significant reduction in the number of total leukocytes, because of the lower number of lymphocytes, was observed in groups T2 and T3 on day 34. Neutrophil and monocyte counts were significantly lower in group T3 than in group T1 on day 34. On day 42, there were significantly lower levels of total protein and globulin in birds in T3 than in T1. On day 27, PK activity was significantly higher in groups T2 and T3; similar behavior observed on day 34 when CK and PK activities in T2 and T3 were significantly higher than those of T1. AK activity was significantly higher in the serum of T3 birds than in those of the other groups at 34 days. At 41 days, CK and PK activities were significantly higher only in birds in group T3 compared to other groups. The levels of reactive oxygen species and lipoperoxidation, as well as the activity of glutathione S-transferase, were significantly lower in birds from group T3 compared to other groups at age 20 days. After a challenge with C. perfringens, there were fluctuations in the behavior of oxidative stress biomarkers; in particular, at 41 days of age, birds in T2 had elevated levels of lipoperoxidation, different from what was seen in birds in T3 that consumed feed with a performance enhancer. Preliminary results suggest that clostridiosis affects serum activity of enzymes in the phosphotransfer network, requiring compensatory enzymatic changes in an attempt to maintain energy homeostasis. In addition, the infection reduces the number of inflammatory cells and causes oxidant/antioxidant imbalance that may contribute to the pathophysiology of the disease. Además, la infección reduce el número de células inflamatorias y provoca un desequilibrio en el estado oxidante / antioxidante que puede contribuir a la fisiopatología de la enfermedad.

The effects of açaí oil addition in tilapia diets on performance, hepatic energy metabolism enzymes and antioxidant responses

The aim of the present study was to determine whether açai oil (Euterpe oleraceae) addition in the diets of juvenile Nile tilapia (Oreochromis niloticus) in biofloc system would stimulate hepatic energy metabolism and antioxidant responses. Treatments are as follows: T0 (6%-soybean oil), T1 (1%-AÇAI; 5%-SOY), T3 (3% AÇAI; 3%-SOY) and T6 (6%-AÇAI). In the liver, reactive oxygen species levels were significantly lower in T1, T3 and T6 than in T0, lipid peroxidation levels were significantly lower in T3 and T6, and nitric oxide levels were significantly lower in the T6 than in T0. Glutathione peroxidase activity was significantly higher in the T3 and T6 than in T0, while superoxide dismutase activity and total antioxidant capacity levels were significantly higher in the T6 than in T0. Adenylate kinase activity was significantly lower in the T6 than in T0. These results suggest that the açai oil addition in Nile tilapia diets stimulated antioxidant responses.

Protective effects of diet containing rutin against trichlorfon-induced muscle bioenergetics disruption and impairment on fatty acid profile of silver catfish Rhamdia quelen

Trichlorfon is an organophosphate insecticide that is widely used on fish farms to control parasitic infections. It has been detected in freshwater ecosystems as well as in fishery products. There is a growing body of evidence to suggest that certain feed additives may reduce or prevent pesticide-induced toxicity in fish. The aim of the present study was to determine whether acute exposure to trichlorfon would alter bioenergetic homeostasis and alter fatty acid profiles in muscles of silver catfish (Rhamdia quelen). We also sought to determine whether rutin prevents or reduces these effects. Cytosolic and mitochondrial creatine kinase (CK) and activities of complexes II-III and IV in muscle were significantly inhibited by exposure to 11 mg/L trichlorfon for 48 h compared to effects in the unexposed group. Total content of polyunsaturated fatty acids (omega-3 and omega-6) were significantly lower in muscle of silver catfish exposed to 11 mg/L trichlorfon for 48 h than in the unexposed group. Addition of 3 mg rutin/kg feed increased CK activity and prevented inhibition of complex IV activity, as well as preventing all alterations of muscle fatty acid profiles elicited by exposure to trichlorfon. No significant differences were observed between groups with respect to muscle adenylate kinase or pyruvate kinase activities, as well as total content of saturated and monounsaturated fatty acids. Our findings suggest that exposure (48 h) to 11 mg trichlorfon/L water inhibits cytosolic and mitochondrial CK activity in muscle. Trichlorfon also affects activities of complexes II-III and IV in respiratory chain, with important consequences for adenosine triphosphate production. The pesticide alters fatty acid profiles in the fish and endangers human consumers of the product. The most important finding of the present study is that inclusion of rutin improves bioenergetic homeostasis and muscle fatty acid profiles, suggesting that it reduces trichlorfon-induced muscle damage.

Adenylate kinase 1 deficiency disrupts mouse sperm motility under conditions of energy stress†.

Mammalian spermatozoa are highly polarized cells characterized by compartmentalized cellular structures and energy metabolism. Adenylate kinase (AK), which interconverts two ADP molecules into stoichiometric amounts of ATP and AMP, plays a critical role in buffering adenine nucleotides throughout the tail to support flagellar motility. Yet the role of the major AK isoform, AK1, is still not well characterized. Here, by using a proteomic analysis of testis biopsy samples, we found that AK1 levels were significantly decreased in nonobstructive azoospermia patients. This result was further verified by immunohistochemical staining of AK1 on a tissue microarray. AK1 was found to be expressed in post-meiotic round and elongated spermatids in mouse testis and subsequent mature sperm in the epididymis. We then generated Ak1 knockout mice, which showed that AK1 deficiency did not induce any defects in testis development, spermatogenesis, or sperm morphology and motility under physiological conditions. We further investigated detergent-modeled epididymal sperm and included individual or mixed adenine nucleotides to mimic energy stress. When only ADP was available, Ak1 disruption largely compromised sperm motility, manifested as a smaller beating amplitude and higher beating frequency, which resulted in less effective forward swimming. The energy restriction/recover experiments with intact sperm further addressed this finding. Besides, decreased AK activity was observed in sperm of a male fertility disorder mouse model induced by cadmium chloride. These results cumulatively demonstrate that AK1 was dispensable for testis development, spermatogenesis, or sperm motility under physiological conditions, but was required for sperm to maintain a constant adenylate energy charge to support sperm motility under conditions of energy stress.

A potential alternative to traditional antibiotics in aquaculture: Yeast glycoprotein exhibits antimicrobial effect in vivo and in vitro on Aeromonas caviae isolated from Carassius auratus gibelio.

In aquaculture, antibiotics are commonly used to provide protection against pathogens; however, this practice has become controversial due to increased occurrences of microbial resistance, and alternatives are needed. This study aimed to investigate the antimicrobial activity of yeast glycoprotein (YG) against Aeromonas caviae. Pathogens were isolated from liver of diseased Carassius auratus gibelio. Based on morphological and biochemical analysis, together with 16S rRNA gene sequencing, the isolated strains were identified as A. caviae and concluded as clones of a single strain and named L2. Further pathogenicity analysis revealed that A. caviae possessed β‐haemolysis, and its median lethal dose for C. gibelio was 1.33 × 106 CFU/ml. Hepatic adenylate kinase and pyruvate kinase activities of C. gibelio were inhibited post–A. caviae infection. Antimicrobial drug test suggested that A. caviae was a multidrug‐resistant organism but could be inhibited by YG in vitro. Minimum inhibitory and bactericidal concentration of YG was 83.3 mg/ml and 166.7 mg/ml, respectively. Microbiota sequencing results showed that YG supplement could obviously decrease the relative abundance of Aeromonas and increase the microbial diversity. Our study revealed that A. caviae from C. gibelio was a multidrug‐resistant bacteria strain, and could be significantly inhibited by YG in vivo and in vitro, thus providing important insights into ecological control and pathogenesis of A. caviae in aquaculture.

Nanospheres as a technological alternative to suppress hepatic cellular damage and impaired bioenergetics caused by nerolidol in Nile tilapia (Oreochromis niloticus).

Nerolidol is a sesquiterpene found in essential oils of several plant species. It is found commonly in human and animal diets and is approved by the US Food and Drug Administration as a flavoring agent. Nevertheless, recent studies have suggested that nerolidol has potent hepatotoxic effects. Because use of plant-based products in human and animal food has expanded considerably, it is essential to develop approaches such as nanotechnology to avoid or reduce hepatic toxic effects. Therefore, the aim of the study was to determine whether nerolidol dietary supplementation elicited hepatic damage associated with impairment of energy homeostasis, as well as whether supplementation with nerolidol-loaded in nanospheres prevented hepatotoxic effects in Nile tilapia (Oreochromis niloticus). Nile tilapia were divided into five groups (A-E, n = 10 per group) with four replicates each, as follows: group A received basal feed (without supplementation); group B received feed containing 0.5mL free nerolidol/kg; group C received feed containing 1.0mL free nerolidol/kg; group D received feed containing 0.5mL nanospheres nerolidol/kg; and group E received feed containing 1.0mL nanospheres nerolidol/kg. All groups received experimental feed once a day (10% total biomass) at 2p.m. for 60 consecutive days. Hepatic liver weight and relative liver weight were significantly lower in fish fed 1.0mL free nerolidol/kg feed than in fish given basal diet (control group). Hepatic pyruvate kinase (1.0mL free nerolidol/kg) and adenylate kinase (0.5 and 1.0mL free nerolidol/kg) activities were significantly lower than in the control group, while hepatic reactive oxygen species and lipid damage levels were significantly higher. Finally, the comet assay revealed significant increases in the frequency of damage and the damage index in fish given 0.5 and 1.0mL free nerolidol/kg in a dose-dependent manner. Nerolidol-loaded in nanospheres prevented all alterations elicited by free nerolidol. Based on these data, we concluded that dietary supplementation with free nerolidol elicited severe impairment of hepatic bioenergetics homeostasis that appeared to be mediated by excessive ROS production and lipid damage, contributing to a genotoxic effect. Dietary supplementation with nerolidol-loaded in nanospheres did not elicit hepatic damage, and therefore, should be considered as a replacement so as to limit toxicity, permitting its continued use as a dietary supplement.

Participation of phosphoryl transfer network on branchial energetic imbalance of matrinxã (Brycon amazonicus) exposed to air: Notable involvement of creatine kinase

Concerns about common aquaculture procedures have been currently raised, including air exposure, since usually they cause a variable degree of stress and contribute to fish mortality. Energetic alterations during exposure to air have been extensively reported, but their effects linked to phosphoryl transfer network, an essential network of enzymes to precise coupling of the adenosine triphosphate (ATP)-production and ATP-consuming process for maintenance of bioenergetic homeostasis remain unknown. Thus, the aim of this study was to evaluate whether the phosphoryl transfer network could be a pathway involved in the bioenergetic imbalance of matrinxã (Brycon amazonicus) exposed to air. Branchial cytosolic and mitochondrial creatine kinase (CK) activities were significantly lower at 30 and 60 min post-stress in fish subjected to air exposure than in the control group. Branchial ATP content and sodium‑potassium pump (Na+, K+-ATPase) activity were significantly lower at 30 and 60 min post-stress in fish subjected to air exposure than in the control group, while reactive oxygen species (ROS) and lipid peroxidation levels were significantly higher in the same moments in fish subjected to air exposure than in the control group. No significant difference was observed between groups or over the time regarding adenylate kinase and pyruvate kinase activities. Based on these data, impairment of CK activity elicited by air exposure disrupted branchial energetic balance via reduction on ATP availability in the gills and provoked impairment of Na+, K +ATPase activity, which appears to be mediated by ROS overproduction and lipid peroxidation.