A comparison of the isoelectric points of mouse liver UDP-glucuronosyltransferase enzymes conjugating the twelve benzo[a]pyrene phenols

Solubilized mouse liver microsomes were subjected to chromatofocusing using a pH 9.5 to 6.0 gradient. UDP-glucuronosyltransferase activity was assayed using 12 benzo[a]pyrene phenols as substrates. The rank of microsomal activity for the phenols was as follows: 12 > 10 > 4 > 1 > 7 > 5 > 8 > 9 > 3 > 11 > 6 > 2. Fractions separated on chromatofocusing according to isoelectric point indicated that 3-, 10-, 11-, and 12-hydroxybenzo[a]pyrene were conjugated primarily by a high pI (～8.5) activity(s), 2-, 6-, 8-, and 9-hydroxybenzo[a]pyrene were conjugated primarily by a low pI (～6.7) activity(s), and 1-, 4-, 5-, and 7-hydroxybenzo[a]pyrene were conjugated equally well by high and low pI forms.     

High-performance liquid chromatographic analysis of biogenic amines in biological materials as o-phthalaldehyde derivatives

A remarkably sensitive, simple and selective reversed-phase high-performance liquid chromatographic (HPLC) method has been developed, allowing, for the first time, the direct measurement of histamine, norepinephrine, octopamine, normetanephrine, dopamine, serotonin and tyramine in a single sample of plasma (2 ml), tissue (0.2 g), or urine. The biogenic amines were modified by pre-column derivatization with o-phthalaldehyde which stabilizes the molecules, aids in extraction, and improves HPLC detection at the nanogram level. To minimize losses during the sampling procedure a careful collection procedure was designed.     

Reductive carboxylation and 2-hydroxyglutarate formation by wild-type IDH2 in breast carcinoma cells

Mitochondrial NADPH-dependent isocitrate dehydrogenase, IDH2, and cytosolic IDH1, catalyze reductive carboxylation of 2-oxoglutarate. Both idh2 and idh1 monoallelic mutations are harbored in grade 2/3 gliomas, secondary glioblastomas and acute myeloid leukemia. Mutant IDH1/IDH2 enzymes were reported to form an oncometabolite r-2-hydroxyglutarate (2HG), further strengthening malignancy. We quantified CO₂-dependent reductive carboxylation glutaminolysis (RCG) and CO₂-independent 2HG production in HTB-126 and MDA-MB-231 breast carcinoma cells by measuring ¹³C incorporation from 1-¹³C-glutamine into citrate, malate, and 2HG. For HTB-126 cells, ¹³C-citrate, ¹³C-malate, and ¹³C-2-hydroxyglutarate were enriched by 2-, 5-, and 15-fold at 5 mM glucose (2-, 2.5-, and 13-fold at 25 mM glucose), respectively, after 6 h. Such enrichment decreased by 6% with IDH1 silencing, but by 30–50% upon IDH2 silencing while cell respiration and ATP levels rose up to 150%. Unlike 2HG production RCG declined at decreasing CO₂. At hypoxia (5% O₂), IDH2-related and unrelated ¹³C-accumulation into citrate and malate increased 1.5–2.5-fold with unchanged IDH2 expression; whereas hypoxic 2HG formation did not. ¹³C–2HG originated by ∼50% from other than IDH2 or IDH1 reactions, substantiating remaining activity in IDH1&2-silenced cells. Relatively high basal ¹²C–2HG levels existed (5-fold higher vs. non-tumor HTB-125 cells) and ¹³C–2HG was formed despite the absence of any idh2 and idh1 mutations in HTB-126 cells. Since RCG is enhanced at hypoxia (frequent in solid tumors) and 2HG can be formed without idh1/2 mutations, we suggest 2HG as an analytic marker (in serum, urine, or biopsies) predicting malignancy of breast cancer in all patients.     

Stimulation of splenic T cells on syngeneic monolayers of epidermal basal cells (EBC) in mice. Regulation by Lyt 1+ and Lyt 2+ cell subsets

Syngeneic proliferative response of splenic T cells against monolayers of epidermal basal cells (EBC) was obtained with C57BL/6 and DBA/2 mice. Optimal response, as assessed by [³H]thymidine uptake, occurred on the 6th day of coculture. The level of [^3H]thymidine uptake by unseparated spleen cells was lower than by fractionated T cells from C57BL/6 mice, and null for DBA/2 mice. It was not significantly different when lymphocytes were cocultured with syngeneic or allogeneic epidermal cells. Ia antigens did not appear to be involved in the syngeneic response, since it was not prevented by pretreating stimulator monolayers with monoclonal anti-Ia^k antibody or by adding this antibody directly to the cultures. When the proliferative responses of separated Lyt 1⁺ and Lyt 2⁺ cell subsets were compared, the prominent role of Lyt 1⁺ cells was demonstrated. Enhancement of the T-cell reactivity by eliminating Lyt 2⁺ cells and suppression of the response of a constant number of Lyt 1⁺ cells by adding Lyt 2⁺ cells suggested that Lyt 2⁺ cells could suppress and modulate the Lyt 1⁺ cell proliferation.     

MiR‐126a‐5p limits the formation of abdominal aortic aneurysm in mice and decreases ADAMTS‐4 expression

Abdominal aortic aneurysm (AAA) is a serious vascular disease featured by inflammatory infiltration in aortic wall, aortic dilatation and extracellular matrix (ECM) degradation. Dysregulation of microRNAs (miRNAs) is implicated in AAA progress. By profiling miRNA expression in mouse AAA tissues and control aortas, we noted that miR‐126a‐5p was down‐regulated by 18‐fold in AAA samples, which was further validated with real‐time qPCR. This study was performed to investigate miR‐126a‐5p's role in AAA formation. In vivo, a 28‐d infusion of 1 μg/kg/min Angiotensin (Ang) II was used to induce AAA formation in Apoe^‐/‐ mice. MiR‐126a‐5p (20 mg/kg; MIMAT0000137) or negative control (NC) agomirs were intravenously injected to mice on days 0, 7, 14 and 21 post‐Ang II infusion. Our data showed that miR‐126a‐5p overexpression significantly improved the survival and reduced aortic dilatation in Ang II‐infused mice. Elastic fragment and ECM degradation induced by Ang II were also ameliorated by miR‐126a‐5p. A strong up‐regulation of ADAM metallopeptidase with thrombospondin type 1 motif 4 (ADAMTS‐4), a secreted proteinase that regulates matrix degradation, was observed in smooth muscle cells (SMCs) of aortic tunica media, which was inhibited by miR‐126a‐5p. Dual‐luciferase results demonstrated ADAMTS‐4 as a new and valid target for miR‐126a‐5p. In vitro, human aortic SMCs (hASMCs) were stimulated by Ang II. Gain‐ and loss‐of‐function experiments further confirmed that miR‐126‐5p prevented Ang II‐induced ECM degradation, and reduced ADAMTS‐4 expression in hASMCs. In summary, our work demonstrates that miR‐126a‐5p limits experimental AAA formation and reduces ADAMTS‐4 expression in abdominal aortas.