SDS Electrophoresis on Gradient Polyacrylamide Gels as a Semiquantitative Tool for the Evaluation of Proteinuria.

Proteinuria is an important sign of kidney diseases. Different protein patterns in urine associated with glomerular, tubular and overload proteinuria may be differentiated using the immunochemical detection of indicator proteins or via urinary proteins electrophoresis. Our aim was to characterize sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using commercially available 4-20% gradient gels as a method to detect and differentiate proteinuria. Our laboratory-based study used excess urine samples collected for routine diagnostic purposes from adult patients of a tertiary-care hospital, including patients with albumin/creatinine < 30 mg/g and patients with dipstick proteinuria. The limit of albumin detection was estimated to be 3 mg/L. In 93 samples with albumin/creatinine < 30 mg/g, an albumin fraction was detected in 87% of samples with a minimum albumin concentration of 2.11 mg/L. The separation of 300 urine samples of patients with proteinuria revealed distinct protein patterns differentiated using the molecular weights of the detected proteins: glomerular (albumin and higher molecular weights) and two types of tubular proteinuria ("upper" ≥20 kDa and "lower" with lower molecular weights). These patterns were associated with different values of the glomerular filtration rate (median 66, 71 and 31 mL/min/1.72 m2, respectively, p = 0.004) and different proportions of multiple myeloma and nephrological diagnoses. As confirmed using tandem mass spectrometry and western blot, the SDS-PAGE protein fractions contained indicator proteins including immunoglobulin G, transferrin (glomerular proteinuria), α1-microglobulin, retinol-binding protein, neutrophil gelatinase-associated lipocalin, cystatin C, and ß2-microglobulin (tubular), immunoglobulin light chain, myoglobin, and lysozyme (overflow). SDS-PAGE separation of urine proteins on commercially available 4-20% gradient gels is a reliable technique to diagnose proteinuria and differentiate between its main clinically relevant types.

Effects of Ca2+ ions on the horseshoe crab coagulation cascade triggered by lipopolysaccharide.

The lipopolysaccharide (LPS)-triggered horseshoe crab coagulation cascade is composed of three protease zymogens, prochelicerase C (proC), prochelicerase B (proB) and the proclotting enzyme (proCE). In this study, we found that Ca 2+ ions increase the production of the clotting enzyme as a result of a cascade reaction reconstituted by recombinant proteins of wild-type (WT) proC, WT proB and WT proCE. We divided the cascade into three stages: autocatalytic activation of WT proC on the surface of LPS into WT α-chelicerase C (Stage 1); activation of WT proB on the surface of LPS into WT chelicerase B by WT α-chelicerase C (Stage 2) and activation of WT proce into WT CE by chelicerase B (Stage 3). Ca2+ ions enhanced the proteolytic activation in Stage 2, but not those in Stages 1 and 3. Moreover, we performed isothermal titration calorimetry to clarify the interaction of LPS or the recombinant zymogens with Ca2+ ions. LPS interacted with Ca2+ ions at an association constant of Ka = 4.7 × 104 M-1, but not with any of the recombinant zymogens. We concluded that LPS bound with Ca2+ ions facilitates the chain reaction of the cascade as a more efficient scaffold than LPS itself.

Miltirone induces cell death in hepatocellular carcinoma cell through GSDME-dependent pyroptosis.

Pyroptosis is a form of programmed cell death, and recently described as a new molecular mechanism of chemotherapy drugs in the treatment of tumors. Miltirone, a derivative of phenanthrene-quinone isolated from the root of Salvia miltiorrhiza Bunge, has been shown to possess anti-cancer activities. Here, we found that miltirone inhibited the cell viability of either HepG2 or Hepa1-6 cells, and induced the proteolytic cleavage of gasdermin E (GSDME) in each hepatocellular carcinoma (HCC) cell line, with concomitant cleavage of caspase 3. Knocking out GSDME switched miltirone-induced cell death from pyroptosis to apoptosis. Additionally, the induction effects of miltirone on GSDME-dependent pyroptosis were attenuated by siRNA-mediated caspase three silencing and the specific caspase three inhibitor Z-DEVD-FMK, respectively. Miltirone effectively elicited intracellular accumulation of reactive oxygen species (ROS), and suppressed phosphorylation of mitogen-activated and extracellular signal-regulated kinase (MEK) and extracellular regulated protein kinases 1/2 (ERK1/2) for pyroptosis induction. Moreover, miltirone significantly inhibited tumor growth and induced pyroptosis in the Hepa1-6 mouse HCC syngeneic model. These results provide a new insight that miltirone is a potential therapeutic agent for the treatment of HCC via GSDME-dependent pyroptosis.

Pharmaceutical modulation of the proteolytic profile of Transforming Growth Factor Beta induced protein (TGFBIp) offers a new avenue for treatment of TGFBI-corneal dystrophy.

Corneal dystrophies are a group of genetically inherited disorders with mutations in the TGFBI gene affecting the Bowman's membrane and the corneal stroma. The mutant TGFBIp is highly aggregation-prone and is deposited in the cornea. Depending on the type of mutation the protein deposits may vary (amyloid, amorphous powdery aggregate or a mixed form of both), making the cornea opaque and thereby decreases visual acuity. The aggregation of the mutant protein is found to be specific with a unique aggregation mechanism distinct to the cornea. The proteolytic processing of the mutant protein is reported to be different compared to the WT protein. The proteolytic processing of mutant protein gives rise to highly amyloidogenic peptide fragments. The current treatment option, available for patients, is tissue replacement surgery that is associated with high recurrence rates. The clinical need for a simple treatment option for corneal dystrophy patients has become highly essential either to prevent the protein aggregation or to dissolve the preformed aggregates. Here, we report the screening of 2500 compounds from the Maybridge RO3 fragment library using weak affinity chromatography (WAC). The primary hits from WAC were validated by 15N-HSQC NMR assays and specific regions of binding were identified. The recombinant mutant proteins (4th FAS-1 domain of R555W and H572R) were subjected to limited proteolysis by trypsin together with the lead compounds identified by NMR assays. The lead compounds (MO07617, RJF00203 and, BTB05094) were effective to delay/prevent the generation of amyloidogenic peptides in the R555W mutant and compounds (RJF00203 and BTB05094) were effective to delay/prevent the generation of amyloidogenic peptides in the H572R mutant. Thus the lead compounds reported here upon further validation and/or modification might be proposed as a potential treatment option to prevent/delay aggregation by inhibiting the formation of amyloidogenic peptides in TGFBI-corneal dystrophy.

Porcine mesothelium matrix as a biomaterial for wound healing applications.

The increasing economic burden of wound healing in healthcare systems requires the development of functional therapies. Xenografts with preserved extracellular matrix (ECM) structure and biofunctional components overcome major limitations of autografts and allografts (e.g. availability) and artificial biomaterials (e.g. foreign body response). Although porcine mesothelium is extensively used in clinical practice, it is under-investigated for wound healing applications. Herein, we compared the biochemical and biological properties of the only two commercially available porcine mesothelium grafts (Meso Biomatrix® and Puracol® Ultra ECM) to traditionally used wound healing grafts (Endoform™, ovine forestomach and MatriStem®, porcine urinary bladder) and biomaterials (Promogran™, collagen/oxidized regenerated cellulose). The Endoform™ and the Puracol® Ultra ECM showed the highest (p<0.05) soluble collagen and elastin content. The MatriStem® had the highest (p<0.05) basic fibroblast growth factor (FGFb) content, whereas the Meso Biomatrix® had the highest (p<0.05) transforming growth factor beta-1 (TGF-ß1) and vascular endothelial growth factor (VEGF) content. All materials showed tissue-specific structure and composition. The Endoform™ and the Meso Biomatrix® had some nuclei residual matter. All tissue grafts showed similar (p>0.05) response to enzymatic degradation, whereas the Promogran™ was not completely degraded by matrix metalloproteinase (MMP)-8 and was completely degraded by elastase. The Promogran™ showed the highest (p<0.05) permeability to bacterial infiltration. The Promogran™ showed by far the lowest dermal fibroblast and THP-1 attachment and growth. All tested materials showed significantly lower (p<0.05) tumor necrosis factor-alpha (TNF-α) expression than the lipopolysaccharides group. The MatriStem® and the Puracol® Ultra ECM promoted the highest (p<0.05) number of micro-vessel formation, whereas the Promogran™ the lowest (p<0.05). Collectively, these data confer that porcine mesothelium has the potential to be used as a wound healing material, considering its composition, resistance to enzymatic degradation, cytocompatibility, and angiogenic potential.

Reduced expression of carbonic anhydrase III in skeletal muscles could be linked to muscle fatigue: A rat muscle fatigue model.

BACKGROUND: Carbonic anhydrase III (CAIII) is expressed abundantly in slow skeletal muscles, adipocytes, and the liver. It plays a critical role in maintaining intracellular pH, antioxidation, and energy metabolism, which are further involved in fatigue. However, its function and mechanism in maintaining the physiological function of muscles or antifatigue are still ambiguous. We hypothesized that changes of CAIII in skeletal muscles might be related to the occurrence of muscle fatigue. METHOD: After establishing a rat soleus muscle fatigue model, we measured the protein expression of the CAIII in muscles. And the muscle intracellular biochemical indices [malondialdehyde (MDA), adenosine triphosphate (ATP), and lactic acid] were also measured using assay kits. After transfected by CAIII-overexpressing and knockdown lentiviral vectors, the rat soleus muscles were induced to fatigue to investigate the effects and possible molecular mechanisms of CAIII in antifatigue. RESULTS: The expression of CAIII in fatigued soleus muscles was significantly decreased compared with that of the control group (P â€‹< â€‹0.001). Moreover, the ATP level in the fatigued muscle also significantly decreased, whereas lactic acid and MDA levels were significantly increased (P â€‹< â€‹0.001). After posttransfection for 21 days, CAIII levels in muscles were significantly reduced in the CAIII-interfering lentivirus group, but increased in the CAIII-overexpressed lentivirus group (P â€‹< â€‹0.001). In addition, CAIII knockdown muscles showed more reduction of the maximal muscle force and ATP levels â€‹and more increase of MDA and lactic acid levels during the fatigue test than the control group, (P â€‹< â€‹0.05). On the other hand, CAIII-overexpressed muscles showed less reduction of the maximal muscle force and ATP levels and less increase of MDA and lactic acid levels during muscle fatigue than the control group (P â€‹< â€‹0.05). CONCLUSIONS: Our study showed that soleus muscle fatigue induced by electrical stimulation could result in downregulation of CAIII and ATP levels â€‹and accumulation of lactic acid and MDA. Further study showed that CAIII knockdown led to more reduction of the maximal muscle force, whereas CAIII overexpression showed less reduction of the maximal muscle force, which suggested that CAIII levels in muscles might be related to the occurrence of muscle fatigue. TRANSLATIONAL POTENTIAL: CAIII plays an important role in muscle fatigue. Up-regulating the expression of CAIII might contribute to dissipating fatigue, which would provide a new method to solve the difficulties in eliminating muscular fatigue.

Monoacylglycerol lipase inhibitors: modulators for lipid metabolism in cancer malignancy, neurological and metabolic disorders.

Monoacylglycerol lipase (MAGL) is a serine hydrolase that plays a crucial role catalysing the hydrolysis of monoglycerides into glycerol and fatty acids. It links the endocannabinoid and eicosanoid systems together by degradation of the abundant endocannabinoid 2-arachidaoylglycerol into arachidonic acid, the precursor of prostaglandins and other inflammatory mediators. MAGL inhibitors have been considered as important agents in many therapeutic fields, including anti-nociceptive, anxiolytic, anti-inflammatory, and even anti-cancer. Currently, ABX-1431, a first-in-class inhibitor of MAGL, is entering clinical phase 2 studies for neurological disorders and other diseases. This review summarizes the diverse (patho)physiological roles of MAGL and will provide an overview on the development of MAGL inhibitors. Although a large number of MAGL inhibitors have been reported, novel inhibitors are still required, particularly reversible ones.

Introducing a simple model system for binding studies of known and novel inhibitors of AMPK: a therapeutic target for prostate cancer.

Prostate cancer (PC) is one of the leading cancers in men, raising a serious health issue worldwide. Due to lack of suitable biomarker, their inhibitors and the platform for testing those inhibitors result in poor prognosis of PC. AMP-activated protein kinase (AMPK) is a highly conserved protein kinase found in eukaryotes that is involved in growth and development, and also acts as a therapeutic target for PC. The aim of the present study is to identify novel potent inhibitors of AMPK and propose a simple cellular model system for understanding its biology. Structural modelling and MD simulations were performed to construct and refine the 3D models of Dictyostelium and human AMPK. Binding mechanisms of different drug compounds were studied by performing molecular docking, molecular dynamics and MM-PBSA methods. Two novel drugs were isolated having higher binding affinity over the known drugs and hydrophobic forces that played a key role during protein-ligand interactions. The study also explored the simple cellular model system for drug screening and understanding the biology of a therapeutic target by performing in vitro experiments.

Pneumatic hydrodynamics influence transplastomic protein yields and biological responses during in vitro shoot regeneration of Nicotiana tabacum callus: Implications for bioprocess routes to plant-made biopharmaceuticals.

Transplastomic plants are capable of high-yield production of recombinant biopharmaceutical proteins. Plant tissue culture combines advantages of agricultural cultivation with the bioprocess consistency associated with suspension culture. Overexpression of recombinant proteins through regeneration of transplastomic Nicotiana tabacum shoots from callus tissue in RITA® temporary immersion bioreactors has been previously demonstrated. In this study we investigated the hydrodynamics of periodic pneumatic suspension of liquid medium during temporary immersion culture (4 min aeration every 8 h), and the impact on biological responses and transplastomic expression of fragment C of tetanus toxin (TetC). Biomass was grown under a range of aeration rates for 3, 20 and 40-day durations. Growth, mitochondrial activity (a viability indicator) and TetC protein yields were correlated against the hydrodynamic parameters, shear rate and energy dissipation rate (per kg of medium). A critical aeration rate of 440 ml min-1 was identified, corresponding to a shear rate of 96.7 s-1, pneumatic power input of 8.8 mW kg-1 and initial 20-day pneumatic energy dissipation of 127 J kg-1, at which significant reductions in biomass accumulation and mitochondrial activity were observed. There was an exponential decline in TetC yields with increasing aeration rates at 40 days, across the entire range of conditions tested. These observations have important implications for the optimisation and scale-up of transplastomic plant tissue culture bioprocesses for biopharmaceutical production.

The VTLISFG motif in the BH1 domain plays a significant role in regulating the degradation of Mcl-1.

Mcl-1 is a member of the Bcl-2 family protein; its degradation is required for the initiation of apoptosis. The mechanism, however, is not yet clearly known. Previously, it was reported that Mcl-1 is degraded through the ubiquitination-mediated pathway and the PEST domain is the motif responsible for promoting this degradation. We found evidence that this may not be true. We generated several Mcl-1 deletion mutants and examined their effects on protein stability. Deletion of the PEST domain did not prevent the degradation of Mcl-1 during apoptosis. The BH1 domain, but not the PEST, BH3 or BH2 domain, exhibited a short half-life. A peptide named "F3" (VTLISFG) in the C-terminus of the BH1 domain appears to be critical for the rapid turnover of Mcl-1. Deletion of F3 from GFP-Mcl-1-ΔPEST retarded the degradation of this mutant. F3 appeared to be the minimum functional sequence of the degradation motif, since deletion of a single residue was sufficient to abrogate its short half-life. Fusion of F3 with p32 resulted in the degradation of p32 during UV-induced apoptosis, while wild type p32 was not affected. Taken together, these findings suggest that F3 (VTLISFG), instead of PEST, is the major motif responsible for the degradation of Mcl-1 during apoptosis.

Identification and active site analysis of the 1-aminocyclopropane-1-carboxylic acid oxidase catalysing the synthesis of ethylene in Agaricus bisporus.

BACKGROUND: 1-Aminocyclopropane-1-carboxylate oxidase (ACO) is a key enzyme that catalyses the final step in the biosynthesis of the plant hormone ethylene. Recently, the first ACO homologue gene was isolated in Agaricus bisporus, whereas information concerning the nature of the ethylene-forming activity of this mushroom ACO is currently lacking. METHODS: Recombinant ACO from A. bisporus (Ab-ACO) was purified and characterised for the first time. Molecular modelling combined with site-directed mutagenesis and kinetic and spectral analysis were used to investigate the property of Ab-ACO. RESULTS: Ab-ACO has eight amino acid residues that are conserved in the Fe (II) ascorbate family of dioxygenases, including four catalytic residues in the active site, but Ab-ACO lacks a key residue, S289. In comparison to plant ACOs, Ab-ACO requires ACC and Fe (II) but does not require ascorbate. In addition, Ab-ACO had relatively low activity and was completely dependent on bicarbonate, which could be ascribed to the replacement of S289 by G289. Moreover, the ferrous ion could induce a change in the tertiary, but not the secondary, structure of Ab-ACO. CONCLUSIONS: These results provide crucial experimental support for the ability of Ab-ACO to catalyse ethylene formation in a similar manner to that of plant ACOs, but there are differences between the biochemical and catalytic characteristics of Ab-ACO and plant ACOs. GENERAL SIGNIFICANCE: This work enhances the understanding of the ethylene biosynthesis pathways in fungi and could promote profound physiological research of the role of ethylene in the regulation of mushroom growth and development.

ACE-inhibitory activity of enzymatic protein hydrolysates from lupin and other legumes.

The objective of this investigation was to compare the angiotensin converting enzyme (ACE)-inhibitory activity of the hydrolysates obtained by pepsin digestion of proteins of some legumes, such as chickpea, common bean, lentil, lupin, pea, and soybean, by using the same experimental procedure. The ACE-inhibitory activity was measured by using the tripeptide hippuryl-histidyl-leucine (HHL), as model peptide, and HPLC-DAD, as analytical method. The peptide mixtures of all legumes were active, with soybean and lupin the most efficient, with IC50 values of 224 and 226 µg/ml, respectively. Considering the promising results obtained with lupin, and aiming to identify the protein(s) that release(s) the peptides responsible for the activity, the peptides obtained from the pepsin digestion of some industrial lupin protein isolates and purified protein fractions were tested. The most active mixture, showing an IC50 value of 138 µg/ml, was obtained hydrolysing a mixture of lupin α+ß conglutin.

Modulation of liver function, antioxidant responses, insulin resistance and glucose transport by Oroxylum indicum stem bark in STZ induced diabetic rats.

A decoction of stem bark of Oroxylum indicum Vent. (OI) is taken (2-3 times/day) by the tribal people of Sikkim, India to treat diabetes but scientific validation of its overall potential is lacking. Present study was aimed to assess in vitro antihyperglycemic activity of standardized OI extract using inhibition of α-glucosidase, BSA glycation and enhancement of insulin sensitivity. Antidiabetic and antioxidant modulatory effects of OI extract along with the blood biomarkers of toxic response were studied in streptozotocin (STZ) induced diabetic rats. In vitro analysis showed strong antioxidant capacity of OI -and potential to inhibit BSA glycation and α-glucosidase activity which was comparable to standard counterparts. Extract also improved insulin sensitivity in mature 3T3-L1 adipocytes. In vivo effects of OI extract (oral 250 mg/kg b.wt.) on STZ induced type II diabetic rats normalized the antioxidant status (p≤0.01). Analysis of blood biomarkers of toxic response indicated its safety. Lowering of total cholesterol and HDL levels (p≤0.05) and restoration of glycated Hb (p≤0.01) were also found in OI treated diabetic rats. HOMA-IR, QUICKI analysis along with area under the curve analysis showed the capacity of OI extract to enhance the insulin sensitivity significantly (p≤0.01) which was confirmed by increased GLUT-4 translocation in skeletal muscles.

Treatment with the cysteine precursor l-2-oxothiazolidine-4-carboxylate (OTC) implicates taurine deficiency in severity of dystropathology in mdx mice.

Oxidative stress has been implicated in the pathology of the lethal skeletal muscle disease Duchenne muscular dystrophy (DMD), and various antioxidants have been investigated as a potential therapy. Recently, treatment of the mdx mouse model for DMD with the antioxidant and cysteine and glutathione (GSH) precursor n-acetylcysteine (NAC) was shown to decrease protein thiol oxidation and improve muscle pathology and ex vivo muscle strength. This study further investigates the mechanism for the benefits of NAC on dystrophic muscle by administering l-2-oxothiazolidine-4-carboxylate (OTC) which also upregulates intracellular cysteine and GSH, but does not directly function as an antioxidant. We observed that OTC, like NAC, decreases protein thiol oxidation, decreases pathology and increases strength, suggesting that the both NAC and OTC function via increasing cysteine and GSH content of dystrophic muscle. We demonstrate that mdx muscle is not deficient in either cysteine or GSH and that these are not increased by OTC treatment. However, we show that dystrophic muscle of 12 week old mdx mice is deficient in taurine, a by-product of disposal of excess cysteine, a deficiency that is ameliorated by OTC treatment. These data suggest that in dystrophic muscles, apart from the strong association of increased oxidative stress and protein thiol oxidation with dystropathology, another major issue is an insufficiency in taurine that can be corrected by increasing the availability of cysteine. This study provides new insight into the molecular mechanism underlying the benefits of NAC in muscular dystrophy and supports the use of OTC as an alternative drug for potential clinical applications to DMD.

The protective effects of resveratrol on Schwann cells with toxicity induced by ethanol in vitro.

Schwann cells (SCs) are the myelin forming cells in the peripheral nervous system, they play a key role in the pathology of various polyneuropathies and provide trophic support to axons via expression of various neurotrophic factors, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). Ethanol (EtOH) adversely affected both SCs proliferation and myelin formation in culture. Resveratrol (Res) has been shown to regulate many cellular processes and to display multiple protective and therapeutic effects. Whether Res has protective effects on SCs with EtOH-induced toxicity is still unclear. The protective efficacy of Res on EtOH-treated SCs in vitro was investigated in the present study. Res improved cell viability of the EtOH-treated SCs. Hoechst 33342 staining and terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick-end labeling analysis showed that the EtOH-induced apoptosis was inhibited by Res. The effects of Res were blocked by the 5'-adenosine monophosphate-activated protein kinase inhibitor Compound C and the silencing information regulator T1 inhibitor nicotinamide. Res could increase the mRNA and protein levels of BDNF and GDNF in the EtOH-treated SCs. However, the EtOH-induced increase of NGF in the SCs is inhibited by Res. The data from the present study indicate that Res protects SCs from EtOH-induced cell death and regulates the expression of neurotrophicfactors. Res and its derivative may be effective for the treatment of neuropathic diseases induced by EtOH.