New Strategy to Preserve Phosphate by Ionic Liquid Matrices in Matrix-Assisted Laser Desorption/Ionization: A Case of Adenosine Nucleotides

Adenosine -5′-triphosphate (ATP) plays a valuable role in metabolic activity to produce adequate energy in a biosystem. A high ATP/AMP ratio has a correlation with diabetes that induces suppression of AMP-activated protein kinase (AMPK). Matrix-assisted laser desorption/ionization (MALDI)–mass spectrometry (MS) has outstanding potential in determining the ratio of several types of adenosine phosphates in a sample to rapidly understand the primary energy transfer in metabolism. Although MALDI is viewed as a soft ionization technique for MS analysis, excess photon energy might crack the phosphate bonds leading to misinterpretation of the ATP level. In this work, ionic liquid matrices (ILMs) were employed to reduce fragmentation and increase the detection efficiency during the MALDI process. This study demonstrated for the first time that 2,5-dihydroxybenzoic acid pyridine (DHBP) is one of the most effective matrices for further quantitative analysis of adenosine nucleotides. This systematic screening of ILMs also enhances the fundamental understanding of MALDI.

ENERGY HOMEOSTASIS OF THE ARTIFICIAL BLADDER IN EXPERIMENTAL CONDITIONS

Patients with malignant neoplasms of the bladder or severe anatomical and functional bladder abnormalities are the candidates for cystectomy and orthotopic urine diversion. The distal portion of the ileum is the most frequent anatomical organ, which is used for the formation of the urinary conduit. The formation of a urinary reservoir from the ileum that is unable to perform new functions, and mainly programmed to isolate enzymes, digest food and absorb nutrients, salts and minerals changes the energy exchange in the ileum wall under the influence of urine. The aim of the research is the examination of energy metabolism changes in the ileum wall during the formation of artificial bladder in experimental conditions conducted on mini-pigs. Experimental studies were performed on 21 female mini-pigs with an average age of 6 to 14 months. The duration of the experiment was 12 months. An imbalance of the ATP-ADP-AMP system was observed in the transplant tissue of the graft in different postoperative periods. Changes in the level of ATP are characteristics of the development of tissue ischemia. Results. The values of the adenosine nucleotides sum (the energy potential indicative of the respiration rate of mitochondria) and the phosphorylation index were lower at different times of observation in the transplant tissue. The energy charge value was lower to a lesser extent relative to the control. Conclusion. The revealed changes in the level of adenosine nucleotides confirm the severity of the disturbances of the energy homeostasis in the transplant tissue at all stages of the observation.

Molecular structure of human KATP in complex with ATP and ADP

In many excitable cells, KATP channels respond to intracellular adenosine nucleotides: ATP inhibits while ADP activates. We present two structures of the human pancreatic KATP channel, containing the ABC transporter SUR1 and the inward-rectifier K+ channel Kir6.2, in the presence of Mg2+ and nucleotides. These structures, referred to as quatrefoil and propeller forms, were determined by single-particle cryo-EM at 3.9 Å and 5.6 Å, respectively. In both forms, ATP occupies the inhibitory site in Kir6.2. The nucleotide-binding domains of SUR1 are dimerized with Mg2+-ATP in the degenerate site and Mg2+-ADP in the consensus site. A lasso extension forms an interface between SUR1 and Kir6.2 adjacent to the ATP site in the propeller form and is disrupted in the quatrefoil form. These structures support the role of SUR1 as an ADP sensor and highlight the lasso extension as a key regulatory element in ADP’s ability to override ATP inhibition.

Molecular structure of human KATP in complex with ATP and ADP

ABSTRACTIn many excitable cells KATP channels respond to intracellular adenosine nucleotides: ATP inhibits while ADP activates. We present two structures of the human pancreatic KATP channel, containing the ABC transporter SUR1 and the inward-rectifier K+ channel Kir6.2, in the presence of Mg2+ and nucleotides. These structures, referred to as quatrefoil and propeller forms, were determined by single-particle cryo-EM at 3.9 Å and 5.6 Å, respectively. In both forms ATP occupies the inhibitory site in Kir6.2. The nucleotide-binding domains of SUR1 are dimerized with Mg2+-ATP in the degenerate site and Mg2+-ADP in the consensus site. A lasso extension forms an interface between SUR1 and Kir6.2 adjacent to the ATP site in the propeller form, and is disrupted in the quatrefoil form. These structures support the role of SUR1 as an ADP sensor and highlight the lasso extension as a key regulatory element in ADP’s ability to override ATP inhibition.

Rapid generation of hypomorphic mutations

Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels.