Role In Intracellular Metabolism Research Articles

Citrate exporter enhances both extracellular and intracellular citric acid accumulation in the koji fungi Aspergillus luchuensis mut. kawachii and Aspergillus oryzae

Citrate exporter CexA plays a key role in the production of citric acid in fungi; however, its role in intracellular metabolism has remained unclear. In this study, we comparably characterized homologous cexA genes in the white koji fungus Aspergillus luchuensis mut. kawachii and the yellow koji fungus Aspergillus oryzae, which exhibit high and low abilities, respectively, to produce citric acid. Disruption of cexA caused a significant decline of both extracellular and intracellular citric acid accumulation in Aspergillus kawachii, while overexpression of the A.kawachii cexA gene (AkcexA) into A.oryzae significantly enhanced both extracellular and intracellular citric acid accumulation in A.oryzae to a level comparable to that of A.kawachii. In addition, overexpression of two intrinsic cexA homologs (AocexA and AocexB) in A.oryzae also enhanced its extracellular and intracellular citric acid accumulation. Comprehensive analysis of intracellular metabolites from an AkcexA-overexpressing strain of A.oryzae compared with its control strain identified metabolic changes associated with intracellular citric acid accumulation via the glycolytic pathway, pentose phosphate pathway, and tricarboxylic acid cycle. Our results indicate that citric acid export enhances not only extracellular citric acid accumulation but also intracellular metabolic fluxes to generate citric acid.

CYPminer: an automated cytochrome P450 identification, classification, and data analysis tool for genome data sets across kingdoms

BackgroundCytochrome P450 monooxygenases (termed CYPs or P450s) are hemoproteins ubiquitously found across all kingdoms, playing a central role in intracellular metabolism, especially in metabolism of drugs and xenobiotics. The explosive growth of genome sequencing brings a new set of challenges and issues for researchers, such as a systematic investigation of CYPs across all kingdoms in terms of identification, classification, and pan-CYPome analyses. Such investigation requires an automated tool that can handle an enormous amount of sequencing data in a timely manner.ResultsCYPminer was developed in the Python language to facilitate rapid, comprehensive analysis of CYPs from genomes of all kingdoms. CYPminer consists of two procedures i) to generate the Genome-CYP Matrix (GCM) that lists all occurrences of CYPs across the genomes, and ii) to perform analyses and visualization of the GCM, including pan-CYPomes (pan- and core-CYPome), CYP co-occurrence networks, CYP clouds, and genome clustering data. The performance of CYPminer was evaluated with three datasets from fungal and bacterial genome sequences.ConclusionsCYPminer completes CYP analyses for large-scale genomes from all kingdoms, which allows systematic genome annotation and comparative insights for CYPs. CYPminer also can be extended and adapted easily for broader usage.

Autophagy regulation as a promising approach for improving cancer immunotherapy.

Autophagy plays a critical role in intracellular metabolism and maintaining cellular homeostasis. Certain tumor cells present a higher basal autophagy rate and autophagy inhibition can lead to impaired metabolic dysfunction in autophagy-dependent tumor cells. Autophagy status in immune cells dictates their fate and response to antigen; however, autophagy in immune cells may be beneficial or detrimental depending on the developmental stage of the cell and more specifically its degree of differentiation. Autophagy-deficient hosts present variations in many metabolites, proteins and enzymes that may have tumor-promoting or -inhibiting effects. The centrality of autophagy in the metabolism of some cancers and immune cells poses as a critical target whose mechanisms must be further unraveled to optimize patient response and prevent tumor recurrence.

Ivermectin-induced programmed cell death and disruption of mitochondrial membrane potential in bovine mammary gland epithelial cells

Ivermectin (IVM) is a commercially well-known antiparasitic agent derived from the natural fermentation product avermectin. Originally used as a veterinary drug, IVM has been studied for its pharmacokinetic advantages, such as anticancer, antimigration, and antiproliferative effects, using several cell types. In the present study, we verified that IVM suppressed bovine mammary gland epithelial cell proliferation and induced the arrest of the cell cycle from the sub-G1 to the G2/M phase in these cells. Due to IVM treatment, the homeostasis of calcium ions, which play a crucial role in intracellular metabolism, deteriorated, leading to the loss of the mitochondrial membrane potential (MMP). To underpin these results, further studies using inhibitors of Ca2+ signaling were performed; combination treatment with IVM and these factors, including 2-APB, BAPTA-AM, or ruthenium red, inhibited the IVM-induced MMP disruption. Furthermore, following IVM treatment, the relationships among various cell signaling mediators were altered, and the balance between diverse cellular processes associated with cell survival or death was disturbed. In conclusion, we assessed the anti-survival effects of IVM on mammary gland epithelial cells; IVM may impede normal lactation in dairy cows.

Mitochondrial translation requires folate-dependent tRNA methylation

Folates enable the activation and transfer of one-carbon units for biosynthesis of purines, thymidine and methionine1–3. Antifolates are important immunosuppressive4 and anticancer agents5. In proliferating lymphocytes6 and human cancers7,8, folate enzymes localizing to the mitochondria are particularly strongly upregulated. This in part reflects the need for mitochondria to generate one-carbon units and export them to the cytosol for anabolic metabolism2,9. The full range of uses of folate-bound one-carbon units in the mitochondrial compartment itself, however, has not been thoroughly explored. Here we show that loss of catalytic activity of the mitochondrial folate enzyme serine hydroxymethyltransferase 2 (SHMT2), but not other folate enzymes, leads to defective oxidative phosphorylation due to impaired mitochondrial translation. We find that SHMT2, presumably by generating mitochondrial 5,10-methylenetetrahydrofolate, provides methyl donors for producing the taurinomethyluridine base at the wobble position of select mitochondrial tRNAs. Mitochondrial ribosome profiling reveals that SHMT2 knockout cells, due to lack of this modified base, suffer from defective translation with preferential mitochondrial ribosome stalling at certain lysine (AAG) and leucine (UUG) codons. This results in impaired respiratory chain enzyme expression. Stalling at these specific codons also occurs in certain mitochondrial inborn errors of metabolism. Disrupting whole-cell folate metabolism, by folate deficiency or antifolate therapy, also impairs the respiratory chain. In summary, mammalian mitochondria use folate-bound one-carbon units to methylate tRNA, and this modification is required for respiratory chain translation and thus oxidative phosphorylation.

Intracellular absorption of transdermal magnesium demonstrated by ESEM-EDS

Magnesium, the second most abundant intracellular cation in human body, plays a fundamental role in intracellular metabolism. As a cofactor in a huge number of key enzimatic reactions, magnesium is especially important for those enzymes that use nucleotides as cofactors or substrates (phosphotransferases, phosphohydrolases, etc.). The majority of magnesium studies were performed by detecting serum magnesium concentration, measure that doesn’t consider the intracellular uptake. In the present study, a different approach has been carried out. By means of an Environmental Scanning Electron Microscope (ESEM) equipped with an Energy Dispersive Spectroscopy (EDS) detector, the intracellular magnesium presence and uptake has been measured in a semiquantitative approach on samples of exfoliating epithelial cells from the oral mucosa [1]. The intracellular magnesium uptake has been experimentally induced by applying, twice a day and for 4 months, on the skin of healthy volunteers, a spray of a magnesium chlorate supersaturated solution. Epithelial cells and blood samples were collected at time zero and every 2 months of treatment. Despite the constant serum concentration of magnesium along the treatment and in absence of any side effect, the EDS analysis reveals a progressive and regular intracellular magnesium increasing of about 100% of the values at each experimental step. Results can confirm the high specificity and, more in general, the high reliability of the EDS analysis about a parameter that can result neglect utilizing only the serum concentration. Moreover, transdermal absorption of magnesium can represent an efficient way of magnesium administration, low dose and side effect free, to be utilize in chronic clinical magnesium deficiency [2].

The Mechanism of the Negative Chronotropic and Dromotropic Actions of Adenosine 5'-triphosphate in the Heart: An Update

Adenosine 5'-triphosphate (ATP) plays a critical role in intracellular metabolism and energetics. Extracellular ATP is rapidly degraded to adenosine by ectoenzymes. Both ATP and adenosine suppress cardiac pacemakers' automaticity and atrioventricular nodal conduction, albeit via the different mechanism of actions. This historical update summarizes the current knowledge regarding the negative chronotropic and dromotropic actions of ATP and discusses the clinical implications regarding the utility of ATP as a diagnostic and therapeutic agent in the management of neutrally mediated syncope and paroxysmal supra ventricular tachycardia.

Thyroid Hormone Transport by the Human Monocarboxylate Transporter 8 and Its Rate-Limiting Role in Intracellular Metabolism

Cellular entry of thyroid hormone is mediated by plasma membrane transporters. We have identified rat monocarboxylate transporter 8 (MCT8) as an active and specific thyroid hormone transporter. The MCT8 gene is located on the X-chromosome. The physiological relevance of MCT8 has been demonstrated by the identification of hemizygous mutations in this gene in males with severe psychomotor retardation and elevated serum T(3) levels. We have characterized human (h) MCT8 by analysis of iodothyronine uptake and metabolism in cell lines transiently transfected with hMCT8 cDNA alone or together with cDNA coding for iodothyronine deiodinase D1, D2, or D3. MCT8 mRNA was detected by RT-PCR in a number of human cell lines as well as in COS1 cells but was low to undetectable in other cell lines, including JEG3 cells. MCT8 protein was not detected in nontransfected cell lines tested by immunoblotting using a polyclonal C-terminal hMCT8 antibody but was detectable in transfected cells at the expected size (61 kDa). Transfection of COS1 and JEG3 cells with hMCT8 cDNA resulted in 2- to 3-fold increases in uptake of T(3) and T(4) but little or no increase in rT(3) or 3,3'-diiodothyronine (3,3'-T(2)) uptake. MCT8 expression produced large increases in T(4) metabolism by cotransfected D2 or D3, T(3) metabolism by D3, rT(3) metabolism by D1 or D2, and 3,3'-T(2) metabolism by D3. Affinity labeling of hMCT8 protein was observed after incubation of intact transfected cells with N-bromoacetyl-[(125)I]T(3). hMCT8 also facilitated affinity labeling of cotransfected D1 by bromoacetyl-T(3). Our findings indicate that hMCT8 mediates plasma membrane transport of iodothyronines, thus increasing their intracellular availability.

Preventing the Development of Complex Regional Pain Syndrome after Surgery

COMPLEX regional pain syndrome (CRPS), previously known as reflex sympathetic dystrophy (RSD), is used to describe a syndrome of pain and sudomotor or vasomotor instability. This pain syndrome usually has an initiating noxious event in the periphery, is not limited to the distribution of a single nerve, and is disproportionate to the inciting event. The Consensus Conference of the International Association for the Study of Pain has subclassified CRPS into two forms: CRPS I (formerly RSD) and CRPS II (formerly causalgia). According to the International Association for the Study of Pain, the diagnosis of CRPS I requires (1) continuing pain, allodynia, or hyperalgesia disproportionate to the injury; (2) evidence at some time of edema, changes in skin blood flow, or abnormal sudomotor activity in the region of pain; and (3) no other conditions that would otherwise account for the degree of pain and dysfunction. Motor disturbances and trophic changes, such as altered nail and hair growth, may be observed in some cases. CRPS II is a pain syndrome that starts after a nerve injury and is not necessarily limited to the distribution of the injured nerve. The diagnostic criteria are the same as those of CRPS I. Patients with CRPS I or CRPS II can have sympathetically maintained pain or sympathetically independent pain. Sympathetically maintained pain, a term introduced in 1986 by Roberts, is pathologic pain that is supported by sympathetic efferent activity, circulating catecholamines, and/or increased sensitivity of -adrenergic receptors. Sympathetically maintained pain is identified by the ability to lessen the pain by sympatholytic blocks or interventions. Sympathetically independent pain has components of pain from sources other than sympathetic innervation and is believed to be most commonly observed in advanced cases of CRPS that do not respond to sympathetic blocks. Patients with CRPS may present with components of only sympathetically maintained pain or sympathetically independent pain or, more commonly, a combination of pain from each. Despite increasing research interest, little is known regarding which patients are at increased risk for development of postoperative CRPS and what the optimal perioperative treatment strategy is for those patients undergoing surgery who have a previous history of CRPS. This review outlines the surgical procedures that are believed to increase risk for development of CRPS and describes pharmacologic and regional analgesic techniques that may be of benefit for preventing the development of CRPS after surgery.

Autotaxin, tumor motility-stimulating exophosphodiesterase

While nucleotides have a well-established role in intracellular metabolism, ATP and other nucleotides also have important extracellular roles in receptormediated signal transduction (34, 35). Extracellular or cell surface proteins capable of binding ATP and hydrolyzing phosphoester bonds of nucleotides are known to exist but their function has remained obscure. Our recent data point to a structure-function correlation between PDE activity and motility stimulation by ATX, indicating a biologically important functional role for the ecto/exophosdiesterases in the stimulation of cellular motility. Data from studies with PC-1 and gp130 RB13-6 have suggested that cell surface PDE's may also play roles in cellular differentiation. Extracellular PDE activities, in combination with other nucleotidases, may result in ecto-nucleotidase cascades (36–38). These data suggest that ecto-/exo-enzymes may catalyze extracellular biochemical reactions that are important in the regulation of cell behavior.

Effect of mucosal potassium on the intestinal glucose transport.

The extracellular space (inulin or raffinose space) and the intracellular concentration of sodium and potassium were determined in the intestinal mucosal scrapings of rats following a perfusion of the intestine with isosmotic solutions of electrolytes or non-electrolytes. Following a perfusion with K2SO4 or mannitol, the intracellular concentration of potassium increased significantly. Perfusion with sorbitol or raffinose did not produce such change. Mannitol (but not sorbitol or raffinose) increased the rate of mediated diffusion (but not that of active transport) of glucose from the gut lumen. It is concluded that the increased glucose absorption is due to the high intracellular concentration of potassium which causes an enhanced metabolic disappearance of the free intracellular glucose. In light of modern concepts regarding the intestinal absorption of glucose and of the findings of present and related studies, Verzar's theory concerning the role of intracellular metabolism (phosphorylation) in the absorption is reassessed.