Expression of cannabinoid receptors CB1 and CB2 in canine cutaneous mast cell tumours.

Cannabinoid receptors (CB1 and CB2) belong to endocannabinoid system (ECS), which is also composed from endocannabinoids and the enzymatic systems involved in their biosynthesis and degradation. The expression of CB1 and CB2 have been previously identified in normal canine mast cell and in atopic dermatitis. Canine cutaneous mast cell tumours (cMCTs) are among the most common cutaneous neoplasms in dogs and have a highly variable clinical behaviour. Expression of CB1-CB2 was assessed by means of immunohistochemistry in thirty-seven dogs (from 2019 to 2021) with proven histological diagnosis of cMCT. Dogs were divided in two groups according to the Kiupel's grading system: high-grade (HG) cMCT and low-grade (LG) cMCT. A semiquantitative (score 0-3) and quantitative assessment of immunoreactivity (IR) was performed for each case. Our results show that there CB1 and CB2 are highly expressed in LG- cMCT, in contrast to HG- cMCT.

Plin5/p-Plin5 Guards Diabetic CMECs by Regulating FFAs Metabolism Bidirectionally.

BACKGROUND: Hyper-free fatty acidemia (HFFA) impairs cardiac capillaries, as well as type 2 diabetes mellitus (T2DM). Perilipin 5 (Plin5) maintains metabolic balance of free fatty acids (FFAs) in high oxidative tissues via the states of nonphosphorylation and phosphorylation. However, when facing to T2DM-HFFA, Plin5's role in cardiac microvascular endothelial cells (CMECs) is not defined. METHODS: In mice of WT or Plin5-/-, T2DM models were rendered by high-fat diet combined with intraperitoneal injection of streptozocin. CMECs isolated from left ventricles were incubated with high glucose (HG) and high FFAs (HFFAs). Plin5 phosphorylation was stimulated by isoproterenol. Plin5 expression was knocked down by small interfering RNA (siRNA). We determined cardiac function by small animal ultrasound, apoptotic rate by flow cytometry, microvessel quantity by immunohistochemistry, microvascular integrity by scanning electron microscopy, intracellular FFAs by spectrophotometry, lipid droplets (LDs) by Nile red staining, mRNAs by quantitative real-time polymerase chain reaction, proteins by western blots, nitric oxide (NO) and reactive oxygen species (ROS) by fluorescent dye staining and enzyme-linked immunosorbent assay kits. RESULTS: In CMECs, HFFAs aggravated cell injury induced by HG and activated Plin5 expression. In mice with T2DM-HFFA, Plin5 deficiency reduced number of cardiac capillaries, worsened structural incompleteness, and enhanced diastolic dysfunction. Moreover, in CMECs treated with HG-HFFAs, both ablation and phosphorylation of Plin5 reduced LDs content, increased intracellular FFAs, stimulated mitochondrial ß-oxidation, added ROS generation, and reduced the expression and activity of endothelial nitric oxide synthase (eNOS), eventually leading to increased apoptotic rate and decreased NO content, all of which were reversed by N-acetyl-L-cysteine. CONCLUSION: Plin5 preserves lipid balance and cell survival in diabetic CMECs by regulating FFAs metabolism bidirectionally via the states of nonphosphorylation and phosphorylation.

Identification of the mass-silent post-transcriptionally modified nucleoside pseudouridine in RNA by matrix-assisted laser desorption/ionization mass spectrometry.

A new method using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for the direct analysis of the mass-silent post-transcriptionally modified nucleoside pseudouridine in nucleic acids has been developed. This method utilizes 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide to derivatize pseudouridine residues. After chemical derivatization all pseudouridine residues will contain a 252 Da 'mass tag' that allows the presence of pseudouridine to be identified using mass spectrometry. Pseudouridine residues can be identified in intact nucleic acids by obtaining a mass spectrum of the nucleic acid before and after derivatization. The mass difference (in units of 252 Da) will denote the number of pseudouridine residues present. To determine the sequence location of pseudouridine, a combination of enzymatic hydrolysis and mass spectrometric steps are used. Here, MALDI analysis of RNase T1 digestion products before and after modification are used to narrow the sequence location of pseudouridine to specific T1 fragments in the gene sequence. Further mass spectrometric monitoring of exonuclease digestion products from isolated T1 fragments is then used for exact sequence placement. This approach to pseudouridine identification is demonstrated using Escherichia coli tRNAS: This new method allows for the direct determination of pseudouridine in nucleic acids, can be used to identify modified pseudouridine residues and can be used with general modification mapping approaches to completely characterize the post-transcriptional modifications present in RNAs.

Mutations at position A960 of E. coli 23 S ribosomal RNA influence the structure of 5 S ribosomal RNA and the peptidyltransferase region of 23 S ribosomal RNA.

The proximity of loop D of 5 S rRNA to two regions of 23 S rRNA, domain II involved in translocation and domain V involved in peptide bond formation, is known from previous cross-linking experiments. Here, we have used site-directed mutagenesis and chemical probing to further define these contacts and possible sites of communication between 5 S and 23 S rRNA. Three different mutants were constructed at position A960, a highly conserved nucleotide in domain II previously crosslinked to 5 S rRNA, and the mutant rRNAs were expressed from plasmids as homogeneous populations of ribosomes in Escherichia coli deficient in all seven chromosomal copies of the rRNA operon. Mutations A960U, A960G and, particularly, A960C caused structural rearrangements in the loop D of 5 S rRNA and in the peptidyltransferase region of domain V, as well as in the 960 loop itself. These observations support the proposal that loop D of 5 S rRNA participates in signal transmission between the ribosome centers responsible for peptide bond formation and translocation.

Chemical modification of S-adenosylhomocysteinase by a water-soluble carbodiimide.

S-Adenosylhomocysteinase (EC 3.3.1.1) from rat liver is inactivated by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC) in a pseudo-first-order fashion. The rate of inactivation is linearly related to the concentration of the reagent, and a second-order rate constant of 4.94 +/- 0.27 M-1 min-1 is obtained at pH 5.5 and 25 degrees C. The inactivation does not involve change in the quaternary structure of the enzyme nor modification or release of the enzyme-bound NAD. Lack of modification at tyrosine, serine, cysteine, histidine, and lysine residues and the fact that the inactivation is favored at low pH suggest that the inactivation is caused by the modification of a carboxyl group. Statistical analysis of the relationship between the residual enzyme activity and the extent of modification, and comparison of the number of residues modified in the presence and absence of the substrate adenosine show that, among four reactive residues per enzyme subunit, only one residue which reacts more rapidly with the reagent than the rest is critical for activity. The CMC-modified enzyme binds adenosine and S-adenosylhomocysteine and is able to oxidize the 3' hydroxyl of these substrates, but apparently fails to catalyze the abstraction of the 4' proton of adenosine.