Protein absorbance (A₂₈₀) and HCTLase activity of column fractions from the chromatographic purification of human liver HCTLase (A–E) and recombinant HCTLase (F, G).

<p>A₂₈₀ nm, solid lines; HCTL activity, solid lines with open circles. (A) Carboxymethyl (CM) column 1, (B) ceramic hydroxyapatite (HA) column, (C) Superdex 200 column 1, (D) CM column 2, (E) Superdex 200 column 2; (F) HA column, (G) Superdex 200 column. Ticks represent fraction changes.</p

Inhibition of human liver HCTL activity.

<p>* HCTLase activity was measured at 10 mM concentration of substrate.</p><p>† Iodoacetamide inhibited rBPHL to the same extent.</p><p>Inhibition of human liver HCTL activity.</p

IEF activity and Coomassie stain of liver BPHL and rBPHL.

<p>(A) HCTLase activity stain of rBHPL (lane 2) and human liver BPHL (lane 3). Hemoglobin shows a faint band as well (lane 1). (B) Coomassie Blue stain of hemoglobin, rBPHL and human liver BPHL (lanes 1–3, respectively).</p

Purification of recombinant human BPHL (HCTLase).

<p>DEAE indicates diethylaminoethyl; and HA, hydroxyapatite.</p><p>* Units of HCTLase activity are µmol NTB produced per minute measured at ≈K_m concentration of substrate.</p><p>Purification of recombinant human BPHL (HCTLase).</p

LC-MS chromatograms showing rBPHL-mediated HCTL cleavage at (A) t = 5 min and (B) t = 0 of reaction time, and valacyclovir ester cleavage at (C) t = 5 min and (D) t = 0.

<p>Chromatograms A and B represent the sum of two MRM channels monitoring the following transitions: <i>m/z</i> 117 > 89 (for HCTL, Rt  =  10.7 min) and <i>m/z</i> 136 > 89 (for Hcy, Rt  =  5.4 min). The inset shows the daughter ion spectrum (for <i>m/z</i> 136) of the product peak at Rt  =  5.4 min, and is consistent with the spectrum obtained from commercial Hcy. Chromatograms C and D also represent the sum of two MRM channels monitoring the following transitions: <i>m/z</i> 326 > 152 (for VC, Rt  =  18.3 min) and <i>m/z</i> 226 > 152 (for acyclovir, Rt  =  6.1 min). The inset shows the daughter ion spectrum (for <i>m/z</i> 226) of the product peak at Rt  =  6.1 min, and is consistent with the spectrum obtained from commercial acyclovir. The small product peak at t  =  0 in chromatogram D reflects the extremely rapid metabolism of VC by rBPHL, which generated a detectable amount of acyclovir in even the very short period of time (∼10 sec) between initiation and quenching of the enzymatic reaction.</p

BPHL substrates.

<p>* <i>V</i>_max from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110054#pone-0110054-g004" target="_blank">Figure 4B</a>.</p><p>† from this study.</p><p>‡ from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110054#pone.0110054-Puente2" target="_blank">[27]</a>.</p><p>BPHL substrates.</p

Substrate dependence and pH optimum of HCTL hydrolysis by BPHL.

<p>Substrate dependence of HCTL hydrolysis by (A) purified human liver HCTLase (BPHL) and (B) rBPHL. Closed symbols, velocity (v) vs substrate concentration [S]; open symbols, [S]/v vs [S]. K_m for HCTL hydrolysis by purified liver BPHL =  3.92 mM and for rBPHL K_m  =  3.18 mM. (C) HCTLase activity of rBPHL (% maximal activity) as a function of pH. Open squares, □―□ 50 mM citrate/Na₂HPO₄; open circles ○―○, 50 mM Tris-HCl; open triangle, ▵―▵ 50 mM Na₂PO₄.</p

SDS-PAGE analysis of pooled column fractions from (A) human liver and (B) recombinant HCTLase purification.

<p>(A) Lane 1, molecular weight markers (kDa); lane 2, liver extract; lane 3, diethylaminoethyl (DEAE) pool; lane 4, CM-1 pool; lane 5, ceramic hydroxyapatite (HA) pool; lane 6, Superdex 200 (#1) pool; lane 7, CM-2 pool; lane 8, Superdex 200 (#2) pool. Gel was stained with silver stain kit (Pierce Chemical). (B) Lane 1, molecular weight markers (kDa); lane 2, <i>E. coli</i> extract; lane 3, DEAE pool; lane 4, HA pool; lane 5, Superdex 200 pool. Gel was stained with Imperial Protein Stain (Pierce Chemical).</p

Paraoxonase‑3 Is Depleted from the High-Density Lipoproteins of Autoimmune Disease Patients with Subclinical Atherosclerosis

Patients with autoimmune diseases have a significantly increased risk of developing cardiovascular disease. In disease, high-density lipoprotein (HDL) particles lose their anti-inflammatory and antioxidant properties and become dysfunctional. The purpose of this study was to test the hypothesis that alterations in the HDL proteomic profile are associated with subclinical atherosclerosis and HDL dysfunction in patients with autoimmune diseases such as systemic lupus erythematosus (SLE) and type 1 diabetes. Targeted proteomics was used to quantify the relative abundance of 18 proteins in HDL from SLE patients with and without atherosclerotic plaque detectable by carotid ultrasound. Changes in the proteomic profile were compared against the in vitro ability of HDL to protect against lipid oxidation. The same proteins were quantified in HDL from patients with type 1 diabetes with or without coronary artery calcification as determined by computed tomography. In each population, paraoxonase-3 (PON3), a potent antioxidant protein, was depleted from the HDL of patients with subclinical atherosclerosis. PON3 expression in HDL was positively correlated with HDL antioxidant function. These results suggest that PON3 may be an important protein in preventing atherosclerosis and highlight the importance of antioxidant proteins in the prevention of atherosclerosis in vivo

Semi-Automated, Occupationally Safe Immunofluorescence Microtip Sensor for Rapid Detection of <i>Mycobacterium</i> Cells in Sputum

<div><p>An occupationally safe (biosafe) sputum liquefaction protocol was developed for use with a semi-automated antibody-based microtip immunofluorescence sensor. The protocol effectively liquefied sputum and inactivated microorganisms including <i>Mycobacterium tuberculosis</i>, while preserving the antibody-binding activity of <i>Mycobacterium</i> cell surface antigens. Sputum was treated with a synergistic chemical-thermal protocol that included moderate concentrations of NaOH and detergent at 60°C for 5 to 10 min. Samples spiked with <i>M. tuberculosis</i> complex cells showed approximately 10⁶-fold inactivation of the pathogen after treatment. Antibody binding was retained post-treatment, as determined by analysis with a microtip immunosensor. The sensor correctly distinguished between <i>Mycobacterium</i> species and other cell types naturally present in biosafe-treated sputum, with a detection limit of 100 CFU/mL for <i>M. tuberculosis</i>, in a 30-minute sample-to-result process. The microtip device was also semi-automated and shown to be compatible with low-cost, LED-powered fluorescence microscopy. The device and biosafe sputum liquefaction method opens the door to rapid detection of tuberculosis in settings with limited laboratory infrastructure.</p></div