Application of thermoresponsive HPLC to forensic toxicology: determination of barbiturates in human urine

A high-performance liquid chromatography (HPLC) method has been developed for the assays of five barbiturates in human urine using a new thermoresponsive polymer separation column, which is composed of N-isopropylacrylamide polymer. According to elevating the column temperature from 10 ℃ to 50 ℃, five barbiturates, such as metharbital, primidone, phenobarbital, mephobarbital and pentobarbital, became well separated by this method. Five barbiturates showed good linearity in the range of 0.2-10 mg/ml. Good accuracy, precision and recoveries for these drugs were obtained at 1 and 5 μg/ml urine. The method with the new-type column seems to have high potential to be extensively used in forensic toxicology for analysis of many drugs and poisons by HPLC and HPLC-mass spectrometry (MS) (-MS)

Plasma angiotensin-converting enzyme 2 (ACE2) is a marker for renal outcome of diabetic kidney disease (DKD) (U-CARE study 3)

Introduction ACE cleaves angiotensin I (Ang I) to angiotensin II (Ang II) inducing vasoconstriction via Ang II type 1 (AT1) receptor, while ACE2 cleaves Ang II to Ang (1-7) causing vasodilatation by acting on the Mas receptor. In diabetic kidney disease (DKD), it is still unclear whether plasma or urine ACE2 levels predict renal outcomes or not. Research design and methods Among 777 participants with diabetes enrolled in the Urinary biomarker for Continuous And Rapid progression of diabetic nEphropathy study, the 296 patients followed up for 9 years were investigated. Plasma and urinary ACE2 levels were measured by the ELISA. The primary end point was a composite of a decrease of estimated glomerular filtration rate (eGFR) by at least 30% from baseline or initiation of hemodialysis or peritoneal dialysis. The secondary end points were a 30% increase or a 30% decrease in albumin-to-creatinine ratio from baseline to 1 year. Results The cumulative incidence of the renal composite outcome was significantly higher in group 1 with lowest tertile of plasma ACE2 (p=0.040). Group 2 with middle and highest tertile was associated with better renal outcomes in the crude Cox regression model adjusted by age and sex (HR 0.56, 95% CI 0.31 to 0.99, p=0.047). Plasma ACE2 levels demonstrated a significant association with 30% decrease in ACR (OR 1.46, 95% CI 1.044 to 2.035, p=0.027) after adjusting for age, sex, systolic blood pressure, hemoglobin A1c, and eGFR. Conclusions Higher baseline plasma ACE2 levels in DKD were protective for development and progression of albuminuria and associated with fewer renal end points, suggesting plasma ACE2 may be used as a prognosis marker of DKD.Trial registration number UMIN000011525

Macrophage Receptor with Collagenous Structure (MARCO) Is Processed by either Macropinocytosis or Endocytosis-Autophagy Pathway

<div><p>The Macrophage Receptor with COllagenous structure (MARCO) protein is a plasma membrane receptor for un-opsonized or environmental particles on phagocytic cells. Here, we show that MARCO was internalized either by ruffling of plasma membrane followed by macropinocytosis or by endocytosis followed by fusion with autophagosome in CHO-K1 cells stably transfected with GFP-MARCO. The macropinocytic process generated large vesicles when the plasma membrane subsided. The endocytosis/autophagosome (amphisome) generated small fluorescent puncta which were visible in the presence of glutamine, chloroquine, bafilomycin, ammonia, and other amines. The small puncta, but not the large vesicles, co-localized with LC3B and lysosomes. The LC3-II/LC3-I ratio increased in the presence of glutamine, ammonia, and chloroquine in various cells. The small puncta trafficked between the peri-nuclear region and the distal ends of cells back and forth at rates of up to 2–3 μm/sec; tubulin, but not actin, regulated the trafficking of the small puncta. Besides phagocytosis MARCO, an adhesive plasma membrane receptor, may play a role in incorporation of various extracellular materials into the cell via both macropinocytic and endocytic pathways.</p></div

Detection of Necroptosis in Ligand-Mediated and Hypoxia-Induced Injury of Hepatocytes Using a Novel Optic Probe-Detecting Receptor-Interacting Protein (RIP)1/RIP3 Binding

Liver injury is often observed in various pathological conditions including posthepatectomy state and cancer chemotherapy. It occurs mainly as a consequence of the combined necrotic and apoptotic types of cell death. In order to study liver/hepatocyte injury by the necrotic type of cell death, we studied signal-regulated necrosis (necroptosis) by developing a new optic probe for detecting receptor-interacting protein kinase 1(RIP)/RIP3 binding, an essential process for necroptosis induction. In the mouse hepatocyte cell line, TIB-73 cells, TNF-alpha/cycloheximide (T/C) induced RIP1/3 binding only when caspase activity was suppressed by the caspase-specific inhibitor z-VAD-fmk (zVAD). T/C/zVAD-induced RIP1/3 binding was inhibited by necrostatin-1 (Nec-1), an allosteric inhibitor of RIP1. The reduced cell survival by T/C/zVAD was improved by Nec-1. These facts indicate that T/C induces necroptosis of hepatocytes when the apoptotic pathway is inhibited/unavailable. FasL also induced cell death, which was only partially inhibited by zVAD, indicating the possible involvement of necroptosis rather than apoptosis. FasL activated caspase 3 and, similarly, induced RIP1/3 binding when the caspases were inactivated. Interestingly. FasL-induced RIP1/3 binding was significantly suppressed by the antioxidants Trolox and N-acetyl cysteine (NAC), suggesting the involvement of reactive oxygen species (ROS) in FasL-induced necroptotic cellular processes. H2O2 . by itself. induced RIP1/3 binding that was suppressed by Nec-1. but not by zVAD. Hypoxia induced RIP1/3 binding after reoxygenation, which was suppressed by Nec-1 or by the antioxidants. Cell death induced by hypoxia/ reoxygenation (H/R) was also improved by Nec-1. Similar to H2O2 , H/R did not require caspase inhibition for RIP1/3 binding, suggesting the involvement of a caspase-independent mechanism for non-ligand-induced and/or redox-mediated necroptosis. These data indicate that ROS can induce necroptosis and mediate the FasL- and hypoxia-induced necroptosis via a molecular mechanism that differs from a conventional caspase-dependent pathway. In conclusion. necroptosis is potentially involved in liver/hepatocyte injury induced by oxidative stress and FasL in the absence of apoptosis

Effects of cytochalasin D and nocodazole on fluorescent autophagic puncta in GFP-MARCO-CHO cells.

<p>(A) Fluorescence staining of actin in GFP-MARCO-CHO cells. The cells were cultured for 15 hr in the absence (a) or presence of 4 mM (NH₄)₃CO₃ (b-d), as follows: (b) without further stimulation; (c) with 1 μM nocodazole; or (d) with 1 μg/mL cytochalasin D. They were fixed with formalin solution and stained with rhodamine phalloidin and DAPI using standard immunofluorescence techniques. (B) Fluorescence staining of tubulin in GFP-MARCO-CHO cells. The cells were cultured and treated with chemicals as shown in Fig 5(A). They were fixed with formalin solution and treated with anti-α-tubulin followed by Alexa Fluor^® 594-conjugated secondary antibody and DAPI using standard immunofluorescence techniques. (C) The cells were cultured in the presence of 4 mM (NH₄)₃CO₃ for 15 hr as follows; (a) without further stimulation, (b) with 1 μg/mL cytochalasin D, or (c) with 1 μM nocodazole. Fig 5C(d) provides a higher magnification image of the boxed area in (c). The arrows and arrowheads indicate autophagic puncta and interrupted macropinocytosis, respectively.</p

Processing of GFP-MARCO in lysosomes.

<p>(A) GFP-MARCO-CHO cells were incubated with or without (NH₄)₂CO₃ and the cells were loaded with pHrode Red AM. The cells were washed and cultured in HEPES-buffered HBSS for 2h. The acidity of lysosomes was neutralized by (NH₄)₂CO₃. (B) Macropinocytic inclusions (arrows) did not co-localize with lysosomes. The control GFP-MARCO-CHO cells were fixed and stained with anti-LAMP2 antibody (2^nd Ab: Alexa Fluor® 594-labeled goat anti-mouse IgG). (C) Co-localization of GFP-MARCO and LAMP2 in autophagic puncta. The cells were grown for 15 hr in the presence of 50 μM chloroquine. See also the legend to Fig 4 (B) for immunofluorescent staining. (D) GFP-MARCO-CHO cells were cultured for 15 hr in fresh F12 medium in the presence or absence (Control) of 4 mM (NH₄)₂CO₃. The supernatant of the cellular lysate was analyzed by SDS-PAGE followed by western blotting for the detection of GFP. The membrane was reprobed with POD-tagged anti-tubulin to serve loading control.</p

L-Glutamine and ammonia induce fluorescent puncta in GFP-MARCO-CHO cells.

<p>(A) The cells were cultured in either F12 complete medium or DMEM complete medium and the fluorescence images were captured by confocal microscopy. Many puncta (200–600 nm in diameter) appeared when the cells were culture in DMEM. (B) The cells were cultured for 12 hr in (a) F12 complete medium (containing 1 mM L-glutamine), (b) F12 medium supplemented with 3 mM L-glutamine (to a final concentration of 4 mM L-glutamine), (c) F12 medium supplemented with 3 mM L-alanyl-L-glutamine (Ala-Gln), or (d) F12 medium supplemented with 4 mM (NH₄)₂CO₃. (C) Proliferation of GFP-MARCO-CHO cells in different culture media. The cells were suspended in F12 medium at 2.0 x 10⁴ cells /mL, aliquotted at 100 μL/well in a 96-well culture dish, and pre-cultured overnight. The medium then was replaced with fresh medium as follows: F12 medium; F12 medium supplemented with 4 or 8 mM (NH₄)₂CO₃; F12 containing 4 mM L-glutamine, F12 without 10% FBS; DMEM; or L-glutamine-free DMEM. Cells then were cultured for 24 h or 48 h. The viable cells were assayed using WST-8. Data are presented as mean ± SEM of 6 wells per medium per time point. *, Significantly different from control (F12) value. (D) Loss of ammonia-induced fluorescent small puncta upon inhibition of the endocytic pathway. The cells were cultured in F12 culture medium and exposed to 4 mM ammonium carbonate for 6 hr in the absence or presence of 100 μM dynasore, a dynamin inhibitor.</p

Autophagy in GFP-MARCO-HEK cells is induced by L-glutamine and amines.

<p>(A) The cells were cultured (a) in DMEM complete medium for 6 hr; (b) in L-glutamine-free DMEM for 6 hr; or (c) in L-glutamine-free DMEM for 6 hr and then in DMEM (4 mM L-glutamine) for 4 hr. Fluorescent puncta appeared at 4 hr in the presence of 4 mM L-glutamine. The images were captured by fluorescence microscopy. (B) Western blot analysis of LC3 and ATG5 in GFP-MARCO-HEK cells. The cells were cultured in complete DMEM (4 mM L-glutamine) (left three lanes), L-glutamine-free DMEM for 6 hr (middle three lanes), or L-glutamine-free DMEM for 6 hr followed by culturing in complete DMEM (4 mM L-glutamine) (right three lanes). M, Western marker lane. α-Tubulin was adopted as loading control and the amount of LC3-I, LC3-II and tubulin were measured by densitometry. LC3-I/tubulin, LC3-II/tubulin, and LC3-II/LC3-I ratios were presented as mean ± SEM (N = 3). *, Significantly different from glutamine (+). #, Significantly different from glutamine (-).</p

Effects of ammonia and amine compounds on conversion of LC3-I to LC3-II (lipidated LC3-I) and autophagic puncta formation.

<p>(A) Western blot analysis for the detection of LC3-I and LC3-II. GFP-MARCO-CHO cells were cultured in F12 culture medium and exposed to (NH₄)₃CO₃. (a) The cells were cultured in the presence of 4 mM (NH₄)₂CO₃ for 0, 2, 6, and 20 hr. (b) The cells were cultured for 6 hr in the presence of 0, 0.1, 0.4, 1, and 4 mM (NH₄)₂CO₃. α-Tubulin was adopted as loading control. The band for LC3-I was not clear in GFP-MARCO-CHO cells. (B) Jurkat (human T cell leukemia) and J774.1 (murine macrophages) cells were exposed for 6 hr to 4 mM (NH₄)₂CO₃ or 50 μM chloroquine. (C) GFP-MARCO-CHO cells were grown for 8 hr in the absence (Control) or presence of 4 mM (NH₄)₂CO₃, 8 mM trimethylamine hydrochloride (TMA-HCl), or 8 mM trimethylamine (TMA). M, Western marker lane. α-Tubulin was adopted as loading control and the amount of LC3-I, LC3-II and tubulin were measured by densitometry. LC3-I/tubulin, LC3-II/tubulin, and LC3-II/LC3-I ratios were presented as mean ± SEM (N = 3). *, Significantly diferent from the control value. (D) Formation of fluorescent autophagic puncta by amines. GFP-MARCO-CHO cells were cultured for 8 hr in complete F12 culture medium in the absence (Control) or presence of 8 mM trimethylamine hydrochloride (TMA-HCl) or of 8 mM trimethylamine (TMA).</p

Poly(ADP-ribose) Polymerase (PARP) is Critically Involved in Liver Ischemia/Reperfusion-injury

Background: Poly(ADP-ribose) polymerase (PARP) is a DNA-repairing enzyme activated by extreme genomic stress, and therefore is potently activated in the remnant liver suffering from ischemia after surgical resection. However, the impact of PARP on post-ischemic liver injury has not been elucidated yet. Materials and methods: We investigated the impact of PARP on murine hepatocyte/liver injury induced by hypoxia/ischemia, respectively. Results: PJ34, a specific inhibitor of PARP, markedly protected against hypoxia/reoxygenation (H/R)-induced cell death, though z-VAD-fmk, a pan-caspase inhibitor similarly showed the protective effect. PJ34 did not affect H/R-induced caspase activity or caspase-mediated cell death. z-VAD-fmk also did not affect the production of PAR (i.e., PARP activity). Therefore, PARPand caspase-mediated cell death occurred in a mechanism independent of each other in H/R. H/R immediately induced activation of PARP and cell death afterwards, both of which were suppressed by PJ34 or Trolox, an antioxidant. This suggests that H/R-induced cell death occurred redox-dependently through PARP activation. H/R and OS induced nuclear translocation of apoptosis inducing factor (AIF, a marker of parthanatos) and RIP1-RIP3 interaction (a marker of necroptosis), both of which were suppressed by PJ34. H/R induced PARPmediated parthanatos and necroptosis redox-dependently. In mouse experiments, PJ34 significantly reduced serum levels of AST, ALT & LDH and areas of hepatic necrosis after liver ischemia/reperfusion, similar to z-VAD-fmk or Trolox. Conclusion: PARP, activated by ischemic damage and/or oxidative stress, may play a critical role in post-ischemic liver injury by inducing programmed necrosis (parthanatos and necroptosis). PARP inhibition may be one of the promising strategies against post-ischemic liver injury