Differential effect of canagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, on slow and fast skeletal muscles from nondiabetic mice.

There has been a concern that sodium-glucose cotransporter 2 (SGLT2) inhibitors could reduce skeletal muscle mass and function. Here, we examine the effect of canagliflozin (CANA), an SGLT2 inhibitor, on slow and fast muscles from nondiabetic C57BL/6J mice. In this study, mice were fed with or without CANA under ad libitum feeding, and then evaluated for metabolic valuables as well as slow and fast muscle mass and function. We also examined the effect of CANA on gene expressions and metabolites in slow and fast muscles. During SGLT2 inhibition, fast muscle function is increased, as accompanied by increased food intake, whereas slow muscle function is unaffected, although slow and fast muscle mass is maintained. When the amount of food in CANA-treated mice is adjusted to that in vehicle-treated mice, fast muscle mass and function are reduced, but slow muscle was unaffected during SGLT2 inhibition. In metabolome analysis, glycolytic metabolites and ATP are increased in fast muscle, whereas glycolytic metabolites are reduced but ATP is maintained in slow muscle during SGLT2 inhibition. Amino acids and free fatty acids are increased in slow muscle, but unchanged in fast muscle during SGLT2 inhibition. The metabolic effects on slow and fast muscles are exaggerated when food intake is restricted. This study demonstrates the differential effects of an SGLT2 inhibitor on slow and fast muscles independent of impaired glucose metabolism, thereby providing new insights into how they should be used in patients with diabetes, who are at a high risk of sarcopenia.

Genome-Wide Identification and Expression Analysis of Tomato ADK Gene Family during Development and Stress.

Adenylate kinase (ADK) is widely distributed in organisms and plays an important role in cellular energy homeostasis. In plants, ADK has important functions in plant growth and development regulation as well as in adaptation to the environment. However, little information is available about the ADK genes in tomato (Solanum lycopersicum), an important economic crop. To investigate the characteristics and functions of ADK genes in tomato, a total of 11 ADK genes were identified and named according to their chromosomal locations. The ADK family in Arabidopsis, tomato, potato, and rice was divided into six groups, and motif analysis revealed that each SlADK protein contained five to eight conserved motifs. A total of 4 to 19 exons were identified in tomato ADK gene family members, and interestingly, most members possessed 4 exons. Several stress response elements were identified in the promoter regions of SlADKs. The 11 SlADKs were randomly distributed on 9 of the 12 tomato chromosomes. Three duplication events were observed between tomato chromosomes, and a high degree of conservation of synteny was demonstrated between tomato and potato. The online TomExpress platform prediction revealed that SlADKs were expressed in various tissues and organs, basically consistent with the data obtained from real-time quantitative PCR (qPCR). The qPCR verification was also performed to determine the expression level of SlADKs and demonstrated that the genes responded to multiple abiotic stresses, such as drought, salt, and cold. Besides, the qPCR results showed that SlADK transcription was responsive to most of the applied hormone treatment. For correlation network analysis under 44 global conditions, the results showed that the number of 17, 3, 4, and 6 coexpressed genes matched with SlADK5, 8, 9, and 11, respectively. For specific gene function analysis, expression of SlADK10 was inhibited using virus-induced gene silencing (VIGS). Compared to wild-type plants, plants with silenced SlADK10 gene had poor drought resistance, indicating SlADK10 regulated drought tolerance of tomato positively. In summary, the information provided in the present study will be helpful to understand the evolutionary relationship and their roles of tomato ADK gene family in further research.

Remote Ischemic Post-Conditioning Therapy is Protective in Mouse Model of Traumatic Optic Neuropathy.

Traumatic optic neuropathy (TON) is characterized by visual dysfunction after indirect or direct injury to the optic nerve following blunt head trauma. TON is associated with increased oxidative stress and inflammation resulting in retinal ganglion cell (RGC) death. Remote ischemic post-conditioning (RIC) has been shown to enhance endogenous protective mechanisms in diverse disease models including stroke, vascular cognitive impairment (VCI), retinal injury and optic nerve injury. However, the protective mechanisms underlying the improvement of retinal function and RGC survival after RIC treatment remain unclear. Here, we hypothesized that RIC therapy may be protective following TON by preventing RGC death, oxidative insult and inflammation in the mouse retina. To carry out the study, mice were divided in three different groups (Control, TON and TON + RIC). We harvested retinal tissue 5 days after TON induction for western blotting and histochemical analysis. We observed increased TON-induced retinal cell death compared with controls by cleaved caspase-3 immunohistochemistry. Furthermore, the TON cohort demonstrated increased TUNEL positive cells which were significantly attenuated by RIC. Immunofluorescence data showed that oxidative stress markers dihydroethidium (DHE), NOX-2 and nitrotyrosine expression were elevated in the TON group relative to controls and RIC therapy significantly reduced the expression level of these markers. Next, we found that the proinflammatory cytokine TNF-α was increased and anti-inflammatory IL-10 was decreased in plasma of TON animals, and RIC therapy reversed this expression level. Interestingly, western blotting of retinal tissue showed that RGC marker Brn3a and tight junction proteins (ZO-1 and Occludin), and AMPKα1 expression were downregulated in the TON group compared to controls. However, RIC significantly increased the expression levels of these proteins. Together these data suggest that RIC therapy activates endogenous protective mechanisms which may attenuate TON-induced oxidative stress and inflammation, and improves BRB integrity.

Pharmacological inhibition of the mitochondrial NADPH oxidase 4/PKCα/Gal-3 pathway reduces left ventricular fibrosis following myocardial infarction.

Although the initial reparative fibrosis after myocardial infarction (MI) is crucial for preventing rupture of the ventricular wall, an exaggerated fibrotic response and reactive fibrosis outside the injured area are detrimental. Although metformin prevents adverse cardiac remodeling, as well as provides glycemic control, the underlying mechanisms remain poorly documented. This study describes the effect of mitochondrial NADPH oxidase 4 (mitoNox) and protein kinase C-alpha (PKCα) on the cardiac fibrosis and galectin 3 (Gal-3) expression. Randomly rats underwent MI, received metformin or saline solution. A model of biomechanical strain and co-culturewas used to enable cross talk between cardiomyocytes and fibroblasts. Long-term metformin treatment after MIwas associated with (1) a reduction in myocardial fibrosis and Gal-3 levels; (2) an increase in adenosine monophosphate-activated protein kinase (AMPK) α1/α2 levels; and (3) an inhibition of both mRNA expression and enzymatic activities of mitoNox and PKCα. These findings were replicated in the cellular model, where the silencing of AMPK expression blocked the ability of metformin to protect cardiomyocytes from strain. The use of specific inhibitors or small interference RNA provided evidence that PKCα is downstream of mitoNox, and that the activation of this pathway results in Gal-3 upregulation.The Gal-3 secreted by cardiomyocytes has a paracrine effect on cardiac fibroblasts, inducing their activation. In conclusion, a metformin-induced increase in AMPK improves myocardial remodeling post-MI, which is related to the inhibition of the mitoNox/PKCα/Gal-3 pathway. Manipulation of this pathway might offer new therapeutic options against adverse cardiac remodeling, in terms of preventing the activation of the present fibroblast population.

Upregulation of SIRT1-AMPK by thymoquinone in hepatic stellate cells ameliorates liver injury.

Thymoquinone (TQ) is a biologically active compound isolated from the seeds of Nigella sativa L. (Ranuculaceae). This study investigated the hepato-protective effect of TQ on liver injury through AMP-activated protein kinase (AMPK) signaling in hepatic stellate cells (HSCs). In vitro, TGF-ß time-dependently attenuated liver kinase B-1 (LKB1) and AMPK phosphorylation, which were blocked by pretreatment with TQ and AICAR (an activator of AMPK). TQ significantly inhibited collagen-Ι, α-SMA, TIMP-1 and enhanced MMP-13 expression, contributing to prevent TGF-ß-induced human HSCs activation. Moreover, TQ induced peroxisome proliferator activated receptor-γ (PPAR-γ) expression, which was inhibited by genetic deletion of AMPK. In vivo, C57BL/6 mice were fed with ethanol diet for 10 days, then administering a single dose of ethanol (5g/kg body weight) via gavage. TQ (20 or 40mg/kg) were given by gavage every day. TQ attenuated the increases in serum aminotransferase and hepatic triglyceride in mice fed with ethanol, while significantly activated LKB1 and AMPK phosphorylation. In addition, TQ enhanced the sirtuin 1 (SIRT1) expression. In conclusion, we demonstrate that AMPK pathway is a key therapeutic target for controlling liver injury and TQ confers hepato-protection against TGF-ß-induced the activation of HSCs and ethanol-induced liver injury.

Induction of AMPK activity corrects early pathophysiological alterations in the subtotal nephrectomy model of chronic kidney disease.

The rat kidney ablation and infarction (A/I) model of subtotal or 5/6th nephrectomy is the most commonly studied model of nondiabetic chronic kidney disease (CKD). The A/I kidney at 1 wk exhibits reductions in kidney function, as determined by glomerular filtration rate, and diminished metabolic efficiency as determined by oxygen consumption per sodium transport (QO2/TNa). As renoprotective AMPK activity is affected by metabolic changes and cellular stress, we evaluated AMPK activity in this model system. We show that these early pathophysiological changes are accompanied by a paradoxical decrease in AMPK activity. Over time, these kidney parameters progressively worsen with extensive kidney structural, functional, metabolic, and fibrotic changes observed at 4 wk after A/I. We show that induction of AMPK activity with either metformin or 5-aminoimidazole-4-carboxamide ribonucleotide increases AMPK activity in this model and also corrects kidney metabolic inefficiency, improves kidney function, and ameliorates kidney fibrosis and structural alterations. We conclude that AMPK activity is reduced in the subtotal nephrectomy model of nondiabetic CKD, that altered regulation of AMPK is coincident with the progression of disease parameters, and that restoration of AMPK activity can suppress the progressive loss of function characteristic of this model. We propose that induction of AMPK activity may prove an effective therapeutic target for the treatment of nondiabetic CKD.

Glucocorticoid receptor is involved in the breed-dependent transcriptional regulation of mtDNA- and nuclear-encoded mitochondria genes in the liver of newborn piglets.

BACKGROUND: Mitochondria, which are essential for the functionality of eukaryotic cells, are particularly important in metabolically active tissues such as liver. Different breeds of pigs demonstrate distinct metabolic profiles in the liver, yet little is known whether the expression and transcriptional regulation of mitochondrial genes differ between breeds. RESULTS: Here we used male newborn Large White (LW) and Erhualian (EHL) piglets to delineate the difference in hepatic mitochondrial gene regulation between breeds. The hepatic content of ATP was significantly higher (p < 0.01) in EHL piglets, which was associated with lower mtDNA copy number (p < 0.05). Most of the mtDNA-encoded genes (10 of 13), however, were more abundantly expressed in EHL compared to LW piglets. We also detected 3 differentially expressed nuclear-encoded mitochondrial genes, among which isocitrate dehydrogenase 2 (IDH2) and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit d (ATP5H) were expressed significantly lower, while adenylate kinase 1 (AK1) was significantly over expressed in EHL piglets. Compared to LW, the over expression of mtDNA-encoded genes in EHL was associated with significantly higher (p < 0.01) glucocorticoid receptor (GR) binding to the control region of mtDNA with no alterations in the methylation status. For nuclear-encoded genes, however, a negative correlation was observed between GR binding and mRNA expression of AK1 and ATP5H. Moreover, higher expression of AK1 in EHL piglets was also associated with lower cytosine methylation (p < 0.05) and hydroxymethylation (p < 0.05). In the promoter region. CONCLUSIONS: These results indicate a role of the GR in the breed-dependent regulation of mitochondrial genes in the liver of newborn piglets.

A single bout of exercise increases the expression of glucose but not fatty acid transporters in skeletal muscle of IL-6 KO mice.

IL-6 is a biologically active cytokine released during exercise by contracting skeletal muscles. It appears to be highly involved in the regulation of muscles energy substrate utilization. Whether an ablation of IL-6 (IL-6 KO) in mice subjected to a single bout of exercise affects lipid and/or glucose metabolism is currently unknown. In the present study we examined fatty acid (FAT/CD36, FABPpm, FATP-1, FATP-4) as well as glucose (GLUT-1, GLUT-4) transporters expression in IL-6 KO mice. In addition, intramuscular glycogen and lipid content was also evaluated. The expression of all fatty acid transporters (FAT/CD36: +25 %; FATP-1: +31 %; FABPpm: +12.7 %; FATP-4: +7.2 %) was increased in muscles from IL-6 KO mice compared to wild type (WT) mice. Accordingly intramuscular lipid content was also increased in these muscles (FFA: +38 %; DAG: +36 % and TAG: +160 %). Interestingly, IL-6 deficiency had only minor effect on glucose transporters expression (GLUT-1: -4 %, and GLUT-4: -5.1 %), with no apparent difference in muscular glycogen content. A single bout of exercise increased the glucose transporters (GLUT-1: +8 %; GLUT-4: +15 %) as well as FA transporters (FAT/CD36: +13 %; FABPpm: +4.5 %; FATP: +2.5 %, FATP-4: +10 %) expression but only in WT skeletal muscles. In muscles from IL-6 KO mice exercise induced changes only in glucose (GLUT-1: +20 %; GLUT-4: +35 %) but not in the content of FA transporters. Concomitantly, IL-6 KO mice displayed shorter time toward exhaustion with more pronounced reductions in intramuscular lipid and glycogen content. We may speculate, that IL-6 deficiency provokes more pronounced glucose utilization over lipid oxidation during a single bout of exhausting exercise.

Dynamic expression of adenylate kinase 2 in the hippocampus of pilocarpine model rats.

Studies have shown that adenylate kinase 2 (AK2) is released from the mitochondrial inner membrane space during neuronal apoptosis, which plays an important role in temporal lobe epilepsy (TLE). We investigated the expression of AK2 in the hippocampus of a pilocarpine model rats. Using reverse transcription polymerase chain reaction and immunohistochemistry, AK2 mRNA and immune-positive cells were investigated during the entire epileptic process in pilocarpine-induced rat model of TLE. AK2 mRNA level was increased in rat hippocampus during different phases of the epileptogenesis, and reached a peak at 72 h. At 72 h time point, AK2 mRNA level was more than threefold comparing with the control. AK2-positive cells were observed in all regions of the hippocampus in model rats, but not in brain tissues of controls. The mean percentage of AK2-positive cells was increased as early as 6 h following seizure and reached a peak at 72 h. The pattern of AK2 expression over time was similar to that observed during neuronal apoptosis as detected with TUNEL staining. These results suggest that AK2 participates in the pathophysiological process of TLE and may be a marker for neuronal apoptosis induced by pathological injury in TLE.

[Expression, purification and enzymatic characterization of adenylate kinase of Thermus thermophilus HB27 in Escherichia coli].

OBJECTIVE: To clone the gene encoding adenylate kinase of Thermus thermophilus HB27, an extremely thermophilic bacterium, express the protein in Escherichia coil and study the enzymatic characterization. METHODS: The DNA fragment encoding adenylate kinase was obtained by PCR from the total DNA of Thermus thermophilus HB27 and cloned into the vector pET-28a. The recombinant plasmid was identified by PCR, restriction endonuclease digestion and sequence analysis. Enzymatic characterization of the expressed protein was carried out using spectrophotometric assays. RESULTS: The gene coding for adenylate kinase from Thermus thermophilus HB27 was cloned and the protein was overexpressed in Escherichia coli BL21(DE3). The optimum reactive pH and temperature for the enzyme were 8.5 and 90 degrees celsius;, respectively. The Km of the recombinant adenylate kinase for ADP was 68.6 micromol/L, with an V(max)ADP of 0.294 mmol/(L.min). Under the condition of environmental temperature at 70, 80, 90, or 100 degrees celsius; for 7 h, the recombinant adenylate kinase still retained the enzymatic activity with high thermostability. AP5A, a specific adenylate kinase inhibitor, inhibited the enzymatic activity of the protein by 70% at the concentration of 2.0 mmol/L, with a Ki value of 46.39 micromol/L for ADP. CONCLUSION: The gene coding for adenylate kinase of Thermus thermophilus HB27 has been successfully cloned and expressed in Escherichia coil, which provides the basis for potential use of the highly thermostable recombinant HB27 adenylate kinase.

Tumor necrosis factor alpha-induced skeletal muscle insulin resistance involves suppression of AMP-kinase signaling.

Elevated levels of tumor necrosis factor (TNFalpha) are implicated in the development of insulin resistance, but the mechanisms mediating these chronic effects are not completely understood. We demonstrate that TNFalpha signaling through TNF receptor (TNFR) 1 suppresses AMPK activity via transcriptional upregulation of protein phosphatase 2C (PP2C). This in turn reduces ACC phosphorylation, suppressing fatty-acid oxidation, increasing intramuscular diacylglycerol accumulation, and causing insulin resistance in skeletal muscle, effects observed both in vitro and in vivo. Importantly even at pathologically elevated levels of TNFalpha observed in obesity, the suppressive effects of TNFalpha on AMPK signaling are reversed in mice null for both TNFR1 and 2 or following treatment with a TNFalpha neutralizing antibody. Our data demonstrate that AMPK is an important TNFalpha signaling target and is a contributing factor to the suppression of fatty-acid oxidation and the development of lipid-induced insulin resistance in obesity.

Failure of right ventricular adaptation in children with tetralogy of Fallot.

BACKGROUND: The left ventricle (LV) adapts to chronic hypoxia by expressing protective angiogenic, metabolic, and antioxidant genes to improve O2 delivery and energy production, and to minimize reoxygenation injury. The ability of the right ventricle (RV) to adapt to hypoxia in children with tetralogy of Fallot (TOF) is unknown. METHODS AND RESULTS: Gene expression using real-time polymerase chain reaction was measured in RV myocardium obtained during surgical repair of TOF from 23 patients: 13 cyanotic and 10 acyanotic. Results were compared between the 2 groups and correlated with age at surgery, severity of cyanosis, and early postoperative course. The cyanotic patients were younger at surgery compared with acyanotic (5+/-3 versus 9+/-4 months; P=0.01), had higher hematocrit (43+/-4 versus 38+/-3 grams/dL; P=0.004), and lower O2 saturations (84+/-4% versus 98+/-2%; (P<0.001). Cyanotic patients had a significantly lower expression of vascular endothelial growth factor (VEGF), glycolytic enzymes, and glutathione peroxidase (GPX) (P<0.05), and a higher expression of collagen (P<0.01) compared with acyanotic patients. Gene expression correlated inversely with severity of cyanosis ie, preoperative hematocrit (P<0.01) and positively with preoperative saturation (P<0.05). The relationship between gene expression and cyanosis was independent of age at surgery. Ca2+ handling genes did not correlate with the severity of hypoxia. Lower angiogenic, glycolytic, and antioxidant gene expression correlated with increasing postoperative lactate (P<0.05). CONCLUSIONS: The RV fails to up regulate adaptive pathways in response to increasing hypoxia in children with TOF. The implications of an early maladaptive response of the RV on long-term RV function require further investigation.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD) alter body weight by decreasing insulin-like growth factor I (IGF-I) signaling.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) affects glycemia due to reduced gluconeogenesis; when combined with a reduction in feed intake, this culminates in decreased body weight. We investigated the effects of steady-state levels of TCDD (loading dose rates of 0.0125, 0.05, 0.2, 0.8, and 3.2 microg/kg) or approximately isoeffective dose rates of 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD) (loading dose rates of 0.3125, 1.25, 5, 20, and 80 microg/kg) on body weight, phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression and activity, and circulating concentrations of insulin, glucose, and insulin-like growth factor-I (IGF-I), and expression of hepatic phosphorylated AMP kinase-alpha (p-AMPK) protein in female Sprague-Dawley rats (approximately 250 gm) at 2, 4, 8, 16, 32, 64, and 128 days after commencement of treatment. At the 0.05 and 1.25 microg/kg loading dose rates of TCDD and HxCDD, respectively, there was a slight increase in body weight as compared to controls, whereas at the 3.2 and 80 microg/kg loading dose rates of TCDD and HxCDD, respectively, body weight of the rats was significantly decreased. TCDD and HxCDD also inhibited PEPCK activity in a dose-dependent fashion, as demonstrated by reductions in PEPCK mRNA and protein. Serum IGF-I levels of rats treated initially with 3.2 microg/kg TCDD or 80 microg/kg HxCDD started to decline at day 4 and decreased to about 40% of levels seen in controls after day 16, remaining low for the duration of the study. Eight days after initial dosing, hepatic p-AMPK protein was increased in a dose-dependent manner with higher doses of TCDD and HxCDD. There was no effect with any dose of TCDD or HxCDD on circulating insulin or glucose levels. In conclusion, doses of TCDD or HxCDD that began to inhibit body weight in female rats also started to inhibit PEPCK, inhibited IGF-I, while at the same time inducing p-AMPK.

[Expression of adenylate kinase of Schistosoma japonicum and evaluation on the immunoreactivity of the recombinant protein].

OBJECTIVE: To express and purify the Schistosoma japonicum adenylate kinase (AK) protein of which the cDNA sequence was subcloned into the pET32a(+) soluble expression plasmid, and to evaluate its immunoreactivity. METHODS: The recombinant plasmid was transformed into E. coli BL21, and induced with IPTG for expression. The bacterial lysis was conducted by ultrasonication and the supernatant was analyzed by SDS-PAGE after boiling and centrifugation. The target protein was purified with the Ni-NTA agarose. The proteins on the gel were transferred to the PVDF film and then the immunoreactivity was tested by Western blotting using anti-6-His antibody, sera from rabbits 6 weeks after infected with cercariae or UV-attenuated cercariae. The purified protein was coated for ELISA to test the cercariae infected rabbit serum and the normal rabbit serum. RESULTS: The molecular weight of the target fusion protein was Mr 40 000 after being induced with IPTG. The fused protein showed a single band when reacted with anti-6-His antibody, the cercariae infected rabbit serum and attenuated cercariae infected rabbit serum. CONCLUSION: The AK protein is expressed as a fused protein with thioredoxin and its molecular weight is about Mr 40000. This protein has a positive immunoreactivity with sera of rabbits infected with cercariae and UV-attenuated cercariae.

Activation of an ATP-dependent K(+) conductance in Xenopus oocytes by expression of adenylate kinase cloned from renal proximal tubules.

In rabbit proximal convoluted tubules, an ATP-sensitive K(+) (K(ATP)) channel has been shown to be involved in membrane cross-talk, i.e. the coupling (most likely mediated through intracellular ATP) between transepithelial Na(+) transport and basolateral K(+) conductance. This K(+) conductance is inhibited by taurine. We sought to isolate this K(+) channel by expression cloning in Xenopus oocytes. Injection of renal cortex mRNA into oocytes induced a K(+) conductance, largely inhibited by extracellular Ba(2+) and intracellular taurine. Using this functional test, we isolated from our proximal tubule cDNA library a unique clone, which induced a large K(+) current which was Ba(2+)-, taurine- and glibenclamide-sensitive. Surprisingly, this clone is not a K(+) channel but an adenylate kinase protein (AK3), known to convert NTP+AMP into NDP+ADP (N could be G, I or A). AK3 expression resulted in a large ATP decrease and activation of the whole-cell currents including a previously unknown, endogenous K(+) current. To verify whether ATP decrease was responsible for the current activation, we demonstrated that inhibition of glycolysis greatly reduces oocyte ATP levels and increases an inwardly rectifying K(+) current. The possible involvement of AK in the K(ATP) channel's regulation provides a means of explaining their observed activity in cytosolic environments characterized by high ATP concentrations.

Identification of a novel human adenylate kinase. cDNA cloning, expression analysis, chromosome localization and characterization of the recombinant protein.

Adenylate kinases have an important role in the synthesis of adenine nucleotides that are required for cellular metabolism. We report the cDNA cloning of a novel 22-kDa human enzyme that is sequence related to the human adenylate kinases and to UMP/CMP kinase of several species. The enzyme was expressed in Escherichia coli and shown to catalyse phosphorylation of AMP and dAMP with ATP as phosphate donor. When GTP was used as phosphate donor, the enzyme phosphorylated AMP, CMP, and to a small extent dCMP. Expression as a fusion protein with the green fluorescent protein showed that the enzyme is located in the cytosol. Northern blot analysis with mRNA from eight different human tissues demonstrated that the enzyme was expressed exclusively in brain, with two mRNA isoforms of 2.4 and 4.0 kb. The gene that encoded the enzyme was localized to chromosome 1p31. Based on the substrate specificity and the sequence similarity with the previously identified human adenylate kinases, we have named this novel enzyme adenylate kinase 5.

Identification of a novel adenylate kinase system in the brain: cloning of the fourth adenylate kinase.

We identify a novel subtype of adenylate kinase, which is the 4th adenylate kinase (AK4), in the vertebrate. AK4 mRNA is expressed in the mammalian central nervous system in a region-specific manner from the middle stage of embryogenesis to the adulthood in the rodent. The presence of three isozymes of adenylate kinase (AK1, AK2 and AK3) that maintains the homeostasis of adenine and guanine nucleotide composition has been reported in the vertebrate. Obtained mouse AK4 cDNA is 3667 bp in size. The predicted open reading frame consists of 223 amino acid residues. Rat AK4 cDNA is also obtained, and the predicted open reading frame is the same length as that of the mouse. The predicted rat AK4 molecule shows 97.8% homology with mouse AK4. Rat AK4 protein is distinct from rat AK3, 53.8% homologous with rat AK3, although the adenylate kinase signature and the mitochondrial energy transfer protein signature are found in both sequences. Interestingly, rat AK4 is 89.2% homologous with the human AK3 over 223 amino acid residues and rat AK3 is 53.7% homologous with the human AK3 indicating that the reported human AK3 actually belongs to the AK4 group (therefore, it should be referred to as human AK4). Although the sequence of AK4 is most similar to that of AK3 among the AK isozymes, its in vivo expression is completely different from AK3; AK4 mRNA is expressed in the pyramidal cells in the hippocampus (mainly in the subfield CA3), the granular cells in the cerebellum, nasal neuroepithelium and the liver while AK3 mRNA is expressed ubiquitously in the body. It is probable that AK4 acts on the specific mechanism of energy metabolism rather than control of the homeostasis of the ADP pool ubiquitously.

Increase of adenylate kinase isozyme 1 protein during neuronal differentiation in mouse embryonal carcinoma P19 cells and in rat brain primary cultured cells.

Adenylate kinase (AK), which catalyzes the equilibrium reaction among AMP, ADP, and ATP, is considered to participate in the homeostasis of energy metabolism in cells. Among three vertebrate isozymes, AK isozyme 1 (AK1) is present prominently in the cytosol of skeletal muscle and brain. When mouse embryonal carcinoma P19 cells were differentiated by retinoic acid into neural cells, the amount of AK1 protein and enzyme activity increased about fivefold concomitantly with neurofilament (NF). Double-immunofluorescence staining showed that both AK1 and NF were located in neuronal processes as well as the perinuclear regions in neuron-like cells, but not in glia-like cells. The amount of brain-type creatine kinase increased only twofold during P19 differentiation. The AK isozyme 2, which was not detected in adult mouse brain, was found in P19 cells and did not increase during the differentiation. Mitochondrial AK isozyme 3, which uses GTP instead of ATP as a phosphate donor, was increased significantly. Immunohistochemical analysis with the primary cultured cells from rat cerebral cortex showed similar cellular localization of AK1 to those observed with differentiated P19 cells. These results suggest an important role of this enzyme in neuronal functions and in neuronal differentiation.

Cloning and heterologous expression of Entamoeba histolytica adenylate kinase and uridylate/cytidylate kinase.

We have isolated two cDNA clones encoding Entamoeba histolytica nucleotide kinases, EhAK and EhUK, expressed them in E. coli and performed functional studies of the recombinant enzymes. Nucleotide sequence analysis showed that EhAK and EhUK genes exhibited the features characteristic of E. histolytica genes, such as transcripts with relatively short 5' and 3' untranslated flanking regions containing the conserved E. histolytica transcription promoter elements located 5' to the initiation codon and a polyadenylation signal in the 3' UTR, a distinctive codon usage bias for A or T in the third position and an AT bias greater than 75% in the flanking regions of the transcripts. At the protein level, both enzymes belong to the short variant nucleoside monophosphate (NMP) kinases, which lack a 29amino acid LID region present in the long variant isoenzymes. EhAK was 30-38% identical to the members of the adenylate kinase (AK) family while EhUK was more similar (48-49% identity) to UMP/CMP kinases. Both enzymes used ATP as preferred phosphate-group donor but each one exhibited strict specificity for the acceptor NMP, EhAK for AMP and EhUK for the pyrimidine nucleoside monophosphates UMP and CMP. Biochemical characterization of the enzymes and phylogenetic reconstruction showed that EhUK is an authentic and well conserved member of the UMP/CMP kinase group while EhAK is the most divergent member known of the AK1 isoenzymes.