Ion exchange chromatography – basic principles and application

Ion-Exchange Chromatography (IEC) allows for the separation of ionizable molecules on the basis of differences in charge properties. Its large sample-handling capacity, broad applicability (particularly to proteins and enzymes), moderate cost, powerful resolving ability, and ease of scale-up and automation have led to it becoming one of the most versatile and widely used of all liquid chromatography (LC) techniques. In this chapter, we review the basic principles of IEC, as well as the broader criteria for selecting IEC conditions. By way of further illustration, we outline protocols necessary to partially purify a serine peptidase from bovine whole brain cytosolic fraction, covering crude tissue extract preparation through to partial purification of the target enzyme using anion-exchange chromatography. Protocols for assaying total protein and enzyme activity in both pre- and post-IEC fractions are also described. The target serine peptidase, prolyl oligopeptidase (POP, EC3.4.21.26), is an 80 kDa enzyme with endopeptidase activity towards peptide substrates of ≤30 amino acids. POP is a ubiquitous post-proline cleaving enzyme with particularly high expression levels in the mammalian brain, where it participates in the metabolism of neuroactive peptides and peptide-like hormones (e.g. thyroliberin, gonadotropin-releasing hormone)

The purification and characterisation of a prolyl oligopeptidase from the cytosolic fraction of bovine whole brain

Cytosolic bovine brain prolyl oligopeptidase was purified from whole brain using ammonium suphate precipitation and chnitnjtography with DEAE sepharose, S200 gel filtration, chromatofocusmg and phenyl sepharose An overall recovery of 23% and purification factor of 253 was achieved. The relative molecular mass of the brain PO, determined by gel filtration chromatography, was found to be 69 5 kDa This was confirmed by SDS PAGE The purified enzyme was relatively unstable under assay conditions However the presence of 0 5% w/v BSA improved its stability and the assay linearity Activity was also inhibited strongly by solvents with DMF being the most inhibitory DMSO was found to be the optimal solvent in terms of enzyme activity and substrate solubility Optimal enzyme activity was observed at 37°C with complete inactivation occurring at temperatures of 50°C or more A pH optimum of 7 4 and a preference for phosphate buffer was found for the enzyme with complete inactivation of activity below pH 4 5 and above pH 10 The brain PO was confirmed to be a serine protease based on its sensitivity to AEBSF The enzyme was also inhibited strongly by some cysteine protease inhibitors such as NEM and DTNB and was activated by DTT Sensitivity to these agents would suggest the presence of a cysteme residue in close proximity to the active site Some divalent metal salts also exerted some inhibitory effects on activity with Hg2+, Cu2+, Cd2+, Co2+ and Ni2+ being the most potent Substrate spcUilcity studies performed on the purified bovine brain, partially purified bovine serum and recombinant Flavobacterium meningosepticum PO activity revealed that this oligopeptidase could hydrolyse a range of proline containing peptides including TRH, LHRH, ADNF-14, substance P, neurotensin, CLIP, a 45 amino acid residue Gly-Pro-Ala polymer and the APP fragment 708-715 N-blocked prolme containing dipeptides, including Z-Pro-Pro-OH were not hydrolysed by any of the enzyme The smallest synthetic sequences hydrolysed were an N-blocked tripeptide and a tetrapeptide The brain, serum and bacterial activities hydrolysed Z-Gly-Pro-MCA, with Km values of 62 5, 14 6 and 38 5(iM respectively The TRH analog pGlu-His-Pro-MCA was also hydrolysed by all three activities with higher Km values of 99 8, 52 1 and 73 5(xM for the brain serum and recombinant Flavobacterium meningosepticum enzyme respectively A number of prolme-containing peptides were also found to competitively inhibit all three activities Of these angiotensins I, II and II were the most potent The TRH analogs, Glu2TRH and Phe1TRH exhibiting the lowest inhibitory potency Inhibition studies performed using a range of PO-specific inhibitors revealed a-ketobenzothiazole to be the most potent inhibitor of bovine brain PO with an IC50 value of 63pM The classical PO inhibitor Z-Pro-prolinal inhibited all three activities with IC50 values of 7-10nM With regard to the majority of inhibitors tested, the brain, serum and bacterial enzymes were similar m their sensitivity However the brain and serum activities were approximately 4000 times less sensitive than the bacterial enzyme to inhibition by the N-blocked dipeptide analog, Z-Phe-Pro-methylketone An investigation into the time course inhibition of bram and bacterial activities by Z-Phe-Alachloromethylketone found that while the bacterial enzyme was completely inhibited by lxlO'5 M of this inhibitor alter 60 minutes, the bram enzyme was completely insensitive to inhibitio

Nicotinic Acetylcholine Receptor Agonists Attenuate Septic Acute Kidney Injury in Mice by Suppressing Inflammation and Proteasome Activity

Sepsis is one of the leading causes of acute kidney injury (AKI). Septic patients who develop acute kidney injury (AKI) are at increased risk of death. To date there is no effective treatment for AKI or septic AKI. Based on their anti-inflammatory properties, we examined the effects of nicotinic acetylcholine receptor agonists on renal damage using a mouse model of lipopolysaccharide (LPS)-induced AKI where localized LPS promotes inflammation-mediated kidney damage. Administration of nicotine (1 mg/kg) or GTS-21 (4 mg/kg) significantly abrogated renal leukocyte infiltration (by 40%) and attenuated kidney injury. These renoprotective effects were accompanied by reduced systemic and localized kidney inflammation during LPS-induced AKI. Consistent with these observations, nicotinic agonist treatment significantly decreased renal IκBα degradation and NFκB activation during LPS-induced AKI. Treatment of human kidney cells with nicotinic agonists, an NFκB inhibitor (Bay11), or a proteasome inhibitor (MG132) effectively inhibited their inflammatory responses following stimulation with LPS or TNFα. Renal proteasome activity, a major regulator of NFκB-mediated inflammation, was enhanced by approximately 50% during LPS-induced AKI and elevated proteasome activity was significantly blunted by nicotinic agonist administration in vivo. Taken together, our results identify enhanced renal proteasome activity during LPS-induced AKI and the suppression of both proteasome activity and inflammation by nicotinic agonists to attenuate LPS-induced kidney injury

Position change during colonoscopy improves caecal intubation rate, mucosal visibility, and adenoma detection in patients with suboptimal caecal preparation

Introduction : Most colonoscopies are completed in the left lateral (LL) position but in cases of suboptimal caecal preparation, changing the patient’s position to supine (S) and, if needed, to right lateral (RL) improves caecal intubation rate, mucosal visibility, and adenoma detection. Aim : To determine if position change during colonoscopy facilitates optimal visualisation of the caecum. Material and methods : A total of 359 patients were grouped into three categories based on the initial caecal intubation position. After caecal intubation, caecal visibility was scored on a four-point scale depending on the number of imaginary quadrants of the caecum completely visualized – Arya Caecal Prep Score. A score of 1 or 2 was unsatisfactory, while 3 or 4 was considered satisfactory. In patients with unsatisfactory score, position was changed from LL to S and then RL and visibility was scored again. Results : The initial caecal intubation in the LL position was achieved in 66.8% of patients, S in 28.5%, and RL in 4.8% of patients. 84.5% (300/355) of patients had an acceptable visualisation score at the initial caecal intubation position. Of the 55 patients with unsatisfactory caecum visualisation scores in the initial intubation position, 30 (8.5%) had satisfactory scores after the first position change (95% CI: 5.77–11.84). Twenty-five (7.04%) subjects required two position changes (95% CI: 4.61–10.22%). An additional 9.3% (11/118) of adenomas were detected in caecum and ascending colon following position change. Conclusions : Changing patient position improves caecal intubation rate, mucosal visibility, and adenoma detection

Remote video auditing with real-time feedback in an academic surgical suite improves safety and efficiency metrics: a cluster randomised study

Importance Compliance with the surgical safety checklist during operative procedures has been shown to reduce inhospital mortality and complications but proper execution by the surgical team remains elusive. Objective: We evaluated the impact of remote video auditing with real-time provider feedback on checklist compliance during sign-in, time-out and sign-out and case turnover times. Design, setting Prospective, cluster randomised study in a 23-operating room (OR) suite. Participants: Surgeons, anaesthesia providers, nurses and support staff. Exposure ORs were randomised to receive, or not receive, real-time feedback on safety checklist compliance and efficiency metrics via display boards and text messages, followed by a period during which all ORs received feedback. Main outcome(s) and measure(s) Checklist compliance (Pass/Fail) during sign-in, time-out and sign-out demonstrated by (1) use of checklist, (2) team attentiveness, (3) required duration, (4) proper sequence and duration of case turnover times. Results: Sign-in, time-out and sign-out PASS rates increased from 25%, 16% and 32% during baseline phase (n=1886) to 64%, 84% and 68% for feedback ORs versus 40%, 77% and 51% for no-feedback ORs (p<0.004) during the intervention phase (n=2693). Pass rates were 91%, 95% and 84% during the all-feedback phase (n=2001). For scheduled cases (n=1406, 71%), feedback reduced mean turnover times by 14% (41.4 min vs 48.1 min, p<0.004), and the improvement was sustained during the all-feedback period. Feedback had no effect on turnover time for unscheduled cases (n=587, 29%). Conclusions and relevance Our data indicate that remote video auditing with feedback improves surgical safety checklist compliance for all cases, and turnover time for scheduled cases, but not for unscheduled cases

Nicotine and GTS-21 treatment inhibits IκBα degradation and NFκB activation within kidneys during septic AKI.

<p>Mice were treated with saline alone (i.p.) or LPS (5 mg/kg, i.p.) plus either saline, nicotine (1 mg/kg, i.p.), or GTS-21 (4 mg/kg, i.p.). Kidney tissues were analyzed for (A) IκBα degradation and (B) NFκB activation. (A) Cytoplasmic IκBα and GAPDH levels were assessed by Western blotting (representative blot is shown) and the density of the bands were analyzed. The data are shown as the ratio of IκBα:GAPDH densities. (B) NFκB activation was determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035361#s4" target="_blank">Methods</a> section. Data are shown as NFκB activation (expressed as relative light units, RLU)(mean ± SD). *p<0.05, **p<0.001 vs. Sal+LPS. n = 5 for saline alone group and n = 8 for all other groups.</p

Nicotinic agonist administration reduces systemic and local renal inflammation during septic AKI.

<p>Mice were treated with saline alone (Sal, i.p.) or LPS (5 mg/kg, i.p.) plus either saline (i.p.), nicotine (Nic, 1 mg/kg, i.p.), or GTS-21 (GTS, 4 mg/kg, i.p.). Serum TNFαlevels (A) and renal TNFα, CCL2 and CXCL10 levels (B–D) were determined by ELISA. Data are shown as mean±SD, as pg/ml (A) or corrected for protein concentration, pg/mg kidney tissue (B-D). *p<0.05, **p<0.001 vs. Sal+LPS-treated mice. n = 5 for saline alone group and n = 8 for all other groups.</p

Proteasome inhibition blunts inflammatory responses by human kidney cell populations.

<p>(A) Human HK-2 renal tubular epithelial cells, (B) human renal glomerular endothelial cells (HRGECs), and (C) human mesangial cells (HMCs) were treated with either vehicle (filled bars) or proteasome inhibitor MG132 (10 µM, open bars) and then either LPS (1 µg/ml), or TNFα (20 ng/ml). Cell-free supernatants were analyzed for CXCL8 (upper panel) or CCL2 (lower panel) by ELISA 18 hrs later. Data are shown as cytokine levels (pg/ml) (mean±SEM) of 3 independent experiments. *p<0.05, **p<0.001 vs. LPS- or TNFα-treated (vehicle) cells.</p

Human Kidney Cells Express nAChR subunits.

<p>To assess nAChR protein levels, (A) HK-2, (B) HRGECs, and (C) HMCs were analyzed by flow cytometry using specific antibodies against the α7nAChR (green, thick solid line), α4nAChR (pink, dotted line), and β2nAChR (blue, thin solid line) vs. isotype control (shaded plot). Histogram plots (FL-nAChR subunits vs. counts) are shown for each cell type. (D) Graph shows the average geometric mean MFI (±SD) for each cell type and each nAChR subunit (isotype control = Is; α7nAChR = α7; α4nAChR = α4; and β2nAChR = β2). n = 3 samples per each condition and cell type.</p