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albumin human [keywords]
- A Systematic Evaluation of Solubility Enhancing Excipients to Enable the Generation of Permeability Data for Poorly Soluble Compounds in Caco-2 Model. [JOURNAL ARTICLE]
- Drug Metab Lett 2014 Nov 26.
The study presented here identified and utilized a panel of solubility enhancing excipients to enable the generation of flux data in the Human colon carcinoma (Caco-2) system for compounds with poor solubility. Solubility enhancing excipients Dimethyl acetamide (DMA) 1 % v/v, polyethylene glycol (PEG) 400 1% v/v, povidone 1% w/v, poloxamer 188 2.5% w/v and bovine serum albumin (BSA) 4% w/v did not compromise Caco-2 monolayer integrity as assessed by trans-epithelial resistance measurement (TEER) and Lucifer yellow (LY) permeation. Further, these excipients did not affect P-glycoprotein (P-gp) mediated bidirectional transport of digoxin, permeabilities of high (propranolol) or low permeability (atenolol) compounds, and were found to be inert to Breast cancer resistant protein (BCRP) mediated transport of cladribine. This approach was validated further using poorly soluble tool compounds, atazanavir (poloxamer 188 2.5% w/v) and cyclosporine A (BSA 4% w/v) and also applied to new chemical entity (NCE) BMS-A in BSA 4% w/v, for which Caco-2 data could not be generated using the traditional methodology due to poor solubility (<1 µM) in conventional Hanks balanced salt solution (HBSS). Poloxamer 188 2.5% w/v increased solubility of atazanavir by >8 fold whereas BSA 4% w/v increased the solubility of cyclosporine A and BMS-A by >2-4 fold thereby enabling permeability as well as efflux liability estimation in the Caco-2 model with reasonable recovery values. To conclude, addition of excipients such as poloxamer 188 2.5% w/v and BSA 4% w/v to HBSS leads to a significant improvement in the solubility of the poorly soluble compounds resulting in enhanced recoveries without modulating transporter-mediated efflux, expanding the applicability of Caco-2 assays to poorly soluble compounds.
- Evaluation of HTLV-1 activity in HAM/TSP patients using proviral load and Tax mRNA expression after In Vitro lymphocyte activation. [JOURNAL ARTICLE]
- Iran J Basic Med Sci 2014 Jul; 17(7):531-536.
HTLV-1 is the first human retrovirus that has been recognized and is associated with HAM/TSP and ATLL. Studies have shown that less than five percent of HTLV-1 infected carriers develop HAM/TSP or ATLL and about ninety-five percent remain asymptomatic. Therefore, the proviral load with Tax may affect cellular genes such as cytokines and oncogenes, as well as involve in pathogenicity.Thirty HAM/TSP patients, thirty HTLV-1 healthy carriers, and MT-2 cell line were evaluated for HTLV-1 activity. PBMCs were isolated and activated using PMA and ionomycine. Real-time PCR and TaqMan methods were performed using specific primers and fluorescence probes for Tax expression and proviral load assessment. B2microglobulin (β2m) and albumin were used as controls in Tax expression and in proviral load, respectively.An insignificant increase in Tax expression was observed in rest PBMCs of HAM/TSP patients compared to healthy carriers. However, after lymphocyte activation there was a significant increase in Tax expression in HAM/TSP patients (P=0.042). The Proviral load in patients was significantly higher than in carriers. Moreover, there was a significant correlation between Tax mRNA expression in activated PBMCs and proviral load (R=0.37, P=0.012).Although proviral load had been addressed as a valuable index for monitoring HTLV-1 infected subjects, the results of this study demonstrated that Tax expression in activated PBMCs along with proviral load assessment in HAM/TSP patients are a more reliable factor for determining the prognosis and monitoring healthy carriers and HAM/TSP patients.
- Genetic modulation of diabetic nephropathy among mouse strains with Ins2 Akita mutation. [JOURNAL ARTICLE]
- Physiol Rep 2014 Nov 1; 2(11)
Diabetic nephropathy (DN) is a major complication of diabetes and the leading cause of end-stage renal disease. DN is characterized by changes in kidney structure and function but the underlying genetic and molecular factors are poorly understood. We used a mouse diversity panel to explore the genetic basis of DN traits in mice carrying the Ins2 Akita mutation. Twenty-eight Akita strains were generated by breeding this panel to DBA/2.Akita mice. Male F1 diabetic and nondiabetic littermates were evaluated for DN-related traits. Urine albumin-to-creatinine ratios (ACRs), volume and cystatin C as well as blood urea nitrogen and lipoprotein levels varied significantly among the diabetic strains. For most Akita strains, ACR values increased 2- to 6-fold over euglycemic control values. However, six strains exhibited changes in ACR exceeding 10-fold with two strains (NOD/ShiLt and CBA) showing 50- to 83- fold increases. These increases are larger than previously reported among available DN mouse models establishing these strains as useful for additional studies of renal function. ACRs correlated with cystatin C (P = 0.0286), a measure of hyperfiltration and an interstitial tubular marker associated with DN onset in humans suggesting that tubule damage as well as podocyte-stress contributed to reduced kidney function assessed by ACR. Although large changes were seen for ACRs, severe nephropathology was absent. However, glomerular hypertrophy and collagen IV content were found to vary significantly among strains suggesting a genetic basis for early onset features of DN. Our results define the range of DN phenotypes that occur among common inbred strains of mice.
- Effect of different glycation agents on Cu(II) binding to human serum albumin, studied by liquid chromatography, nitrogen microwave-plasma atomic-emission spectrometry, inductively-coupled-plasma mass spectrometry, and high-resolution molecular-mass spectrometry. [JOURNAL ARTICLE]
- Anal Bioanal Chem 2014 Nov 27.
The ability of human serum albumin to capture unbound copper under different clinical conditions is an important variable potentially affecting homeostasis of this element. Here, we propose a simple procedure based on size-exclusion chromatography with on-line UV and nitrogen microwave-plasma atomic-emission spectrometry (MP-AES) for quantitative evaluation of Cu(II) binding to HSA upon its glycation in vitro. The Cu-to-protein molar ratio for non-glycated albumin was 0.98 ± 0.09; for HSA modified with glyoxal (GO), methylglyoxal (MGO), oxoacetic acid (GA), and glucose (Glc), the ratios were 1.30 ± 0.22, 0.72 ± 0.14, 0.50 ± 0.06, and 0.95 ± 0.12, respectively. The results were confirmed by using ICP-MS as an alternative detection system. A reduced ability of glycated protein to coordinate Cu(II) was associated with alteration of the N-terminal metal-binding site during incubation with MGO and GA. In contrast, glycation with GO seemed to generate new binding sites as a result of tertiary structural changes in HSA. Capillary reversed-phase liquid chromatography with electrospray-ionization quadrupole-time-of-flight tandem mass spectrometry enabled detection and identification of Cu(II) coordinated to the N-terminal metal-binding site (Cu(II)-DAHK) in all tryptic digests analyzed. This is the first report confirming Cu(II)-DAHK species in HSA by means of high-resolution tandem mass spectrometry, and the first report on the use of MP-AES in combination with chromatographic separation.
- Sirtuin1 Maintains Actin Cytoskeleton by Deacetylation of Cortactin in Injured Podocytes. [JOURNAL ARTICLE]
- J Am Soc Nephrol 2014 Nov 25.
Recent studies have highlighted the renoprotective effect of sirtuin1 (SIRT1), a deacetylase that contributes to cellular regulation. However, the pathophysiologic role of SIRT1 in podocytes remains unclear. Here, we investigated the function of SIRT1 in podocytes. We first established podocyte-specific Sirt1 knockout (SIRT1(pod-/-)) mice. We then induced glomerular disease by nephrotoxic serum injection. The increase in urinary albumin excretion and BUN and the severity of glomerular injury were all significantly greater in SIRT1(pod-/-) mice than in wild-type mice. Western blot analysis and immunofluorescence showed a significant decrease in podocyte-specific proteins in SIRT1(pod-/-) mice, and electron microscopy showed marked exacerbation of podocyte injury, including actin cytoskeleton derangement in SIRT1(pod-/-) mice compared with wild-type mice. Protamine sulfate-induced podocyte injury was also exacerbated by podocyte-specific SIRT1 deficiency. In vitro, actin cytoskeleton derangement in H2O2-treated podocytes became prominent when the cells were pretreated with SIRT1 inhibitors. Conversely, this H2O2-induced derangement was ameliorated by SIRT1 activation. Furthermore, SIRT1 activation deacetylated the actin-binding and -polymerizing protein cortactin in the nucleus and facilitated deacetylated cortactin localization in the cytoplasm. Cortactin knockdown or inhibition of the nuclear export of cortactin induced actin cytoskeleton derangement and dissociation of cortactin from F-actin, suggesting the necessity of cytoplasmic cortactin for maintenance of the actin cytoskeleton. Taken together, these findings indicate that SIRT1 protects podocytes and prevents glomerular injury by deacetylating cortactin and thereby, maintaining actin cytoskeleton integrity.
- Sensitive and site-specific identification of carboxymethylated and carboxyethylated peptides in tryptic digests of proteins and human plasma. [JOURNAL ARTICLE]
- J Proteome Res 2014 Nov 25.
Glycation refers to a non-enzymatic post-translational modification formed by the reaction of amino groups and reducing sugars. Consecutive oxidation and degradation can produce advanced glycation end-products (AGEs), such as Nε-(carboxyethyl)- (CEL)- and Nε-(carboxymethyl)lysine (CML). Although CEL and CML are considered as markers of arteriosclerosis, diabetes mellitus, and ageing, the modified proteins and the exact modification sites are mostly unknown due to their low frequency and the lack of their enrichment strategies. Here we report characteristic fragmentation patterns of CML- and CEL-containing peptides and two modification-specific reporter ions for each modification (CML: m/z 142.1 and 187.1; CEL: m/z 156.1 and 201.1). The protocol allowed sensitive and selective precursor ion scans to detect the modified peptides in complex sample mixtures. The corresponding m/z-values identified eight CEL/CML-modification sites in glycated human serum albumin (HSA) by targeted nano-RPC-MS/MS. The same strategy revealed 21 CML-sites in 17 different proteins, including modified lysine residues 88 and 396 of human serum albumin, in a pooled plasma sample that was obtained from patients with type 2 diabetes mellitus.
- C3a Receptor Antagonist Ameliorates Inflammatory and Fibrotic Signals in Type 2 Diabetic Nephropathy by Suppressing the Activation of TGF-β/smad3 and IKBα Pathway. [JOURNAL ARTICLE]
- PLoS One 2014; 9(11):e113639.
Diabetic nephropathy (DN) is a serious complication for patients with diabetes mellitus (DM). Emerging evidence suggests that complement C3a is involved in the progression of DN. The aim of this study was to investigate the effect of C3a Receptor Agonist (C3aRA) on DN and its potential mechanism of action in rats with type 2 diabetes mellitus (T2DM).T2DM was induced in SD rats by a high fat diet (HFD) plus repeated low dose streptozocin (STZ) injections. T2DM rats were treated with vehicle or C3aRA for 8 weeks. Biochemical analysis, HE and PAS stains were performed to evaluate the renal function and pathological changes. Human renal glomerular endothelial cells (HRGECs) were cultured and treated with normal glucose (NG), high glucose (HG), HG+C3a, HG+C3a+C3aRA and HG+C3a+BAY-11-7082 (p-IKBα Inhibitor) or SIS3 (Smad3 Inhibitor), respectively. Real-time PCR, immunofluorescent staining and western blot were performed to detect the mRNA and protein levels, respectively.T2DM rats showed worse renal morphology and impaired renal function compared with control rats, including elevated levels of serum creatinine (CREA), blood urea nitrogen (BUN) and urine albumin excretion (UACR), as well as increased levels of C3a, C3aR, IL-6, p-IKBα, collagen I, TGF-β and p-Smad3 in the kidney of T2DM rats and C3a-treated HRGECs. In contrast, C3aRA treatment improved renal function and morphology, reduced CREA, UACR and the intensity of PAS and collagen I staining in the kidney of T2DM rats, and decreased C3a, p-IKBα, IL-6, TGF-β, p-Smad3 and collagen I expressions in HRGECs and T2DM rats.C3a mediated pro-inflammatory and pro-fibrotic responses and aggravated renal injury in T2DM rats. C3aRA ameliorated T2DN by inhibiting IKBα phosphorylation and cytokine release, and also TGF-β/Smad3 signaling and ECM deposition. Therefore, complement C3a receptor is a potential therapeutic target for DN.
- Association of aldosterone and arginine vasopressin concentrations and clinical markers of hypoperfusion in neonatal foals. [JOURNAL ARTICLE]
- Equine Vet J 2014 Nov 24.
Critically ill foals often present to veterinary hospitals with impaired organ perfusion, which can be demonstrated by increased blood L-lactate concentrations. As a compensatory mechanism to low blood pressure and electrolyte abnormalities, aldosterone and arginine vasopressin (AVP) are released to restore organ perfusion and function. Several studies have investigated the ability of blood L-lactate concentrations to predict severity of disease and outcome in critically ill humans, adult horses and foals. However, information on the aldosterone and AVP response to hypoperfusion and its association with L-lactate concentrations in neonatal foals is limited.To determine the association between clinical hypoperfusion and endocrine markers of reduced tissue perfusion in normo- and hypoperfused foals.Prospective, multicentre, cross-sectional observational study.Blood samples were collected on admission from 72 clinically hypoperfused, 110 normoperfused (73 hospitalised and 37 healthy) foals of ≤4 days of age. Foals were considered clinically hypoperfused if they had L-lactate concentrations ≥2.5 mmol/L and one of the 3 following findings: heart rate >120 beats/min; packed cell volume (PCV) > 0.44 L/L, or azotaemia (increased creatinine and BUN). Blood concentrations of aldosterone and AVP were determined by radioimmunoassays.Aldosterone, AVP, creatinine, and BUN concentrations and heart rate, PCV, and blood osmolality were higher in clinically hypoperfused compared to normoperfused foals (P<0.05). Risk of hypoperfusion increased with the presence of hypothermic extremities (OR = 5.26) and with each one unit increase in albumin concentrations (OR = 3.5) (P<0.05). The proposed admission L-lactate cutoff value above which non-survival could be reliably predicted in hospitalised foals was 10.6 mmol/L with 82% of sensitivity and 74% of specificity.Hyperaldosteronaemia and hypervasopressinaemia as well as hypothermic extremities and increased albumin concentrations are potent predictors of hypoperfusion in hospitalised foals.
- Enhancing the functional maturity of iPSC-derived human hepatocytes via controlled presentation of cell-cell interactions in vitro. [JOURNAL ARTICLE]
- Hepatology 2014 Nov 25.
Induced pluripotent stem cell-derived human hepatocyte-like cells (iHeps) could provide a powerful tool for studying the mechanisms underlying human liver development and disease, testing the efficacy and safety of pharmaceuticals across different patients (i.e. personalized medicine), and enabling cell-based therapies in the clinic. However, current in vitro protocols that rely upon growth factors and extracellular matrices (ECM) alone yield iHeps with low levels of liver functions relative to adult primary human hepatocytes (PHHs). Moreover, these low hepatic functions in iHeps are difficult to maintain for prolonged times (weeks to months) in culture. Here, we engineered a micropatterned co-culture (iMPCC) platform in a multi-well format that, in contrast to conventional confluent cultures, significantly enhanced the functional maturation and longevity of iHeps in culture for at least 4 weeks in vitro when benchmarked against multiple donors of PHHs. In particular, iHeps were micropatterned onto collagen-coated domains of empirically optimized dimensions, surrounded by 3T3-J2 murine embryonic fibroblasts, and then sandwiched with a thin layer of ECM gel (Matrigel™). We assessed iHep maturity via global gene expression profiles, hepatic polarity, secretion of albumin and urea, basal CYP450 activities, phase-II conjugation, drug-mediated CYP450 induction, and drug-induced hepatotoxicity. Conclusion: Controlling both homotypic interactions between iHeps and heterotypic interactions with stromal fibroblasts significantly matures iHep functions and maintains them for several weeks in culture. In the future, iMPCCs could prove useful for drug screening, studying molecular mechanisms underlying iHep differentiation, modeling liver diseases, and integration into human-on-a-chip systems being designed to assess multi-organ responses to compounds. This article is protected by copyright. All rights reserved.
- Release of uremic retention solutes from protein binding by hypertonic predilution hemodiafiltration. [JOURNAL ARTICLE]
- ASAIO J 2014 Nov 21.
Protein-bound uremic retention solutes accumulate in patients suffering from chronic kidney disease and its removal by hemodialysis is hampered. Therefore, we developed a dialysis technique where the protein-bound uremic retention solutes are removed more efficiently under high ionic strength.Protein-bound uremic solutes like phenylacetic acid, indoxyl sulfate and p-cresyl sulfate was combined with plasma in the presence of increased ionic strength. The protein integrity of proteins and enzymatic activities were analysed. In-vitro dialysis of albumin solution was performed to investigate the clearance of the bound uremic retention solutes. In-vitro hemodiafiltrations of human blood were performed to investigate the influence of increased ionic strength on blood cell survival. The protein-bound fraction of phenylacetic acid, indoxyl sulfate and p-cresyl sulfate was significantly decreased from 59.4 ± 3.4%, 95.7 ± 0.6%, 96.9 ± 1.5% to 36.4 ± 3.7%, 87.8 ± 0.6% and 90.8 ± 1.3% respectively. The percentage of phenylacetic acid, indoxyl sulfate and p-cresyl sulfate released from protein was 23.0 ± 5.7%, 7.9 ± 1.1% and 6.1 ± 0.2%. The clearance during in-vitro dialysis was increased by 13.1 ± 3.6 %, 68.8 ± 15.1 % and 53.6 ± 10.2% respectively. There was no difference in NaCl concentrations at the outlet of the dialyser using isotonic and hypertonic solutions. In conclusion, this study forms the basis for establishing a novel therapeutic approach to remove protein-bound retention solutes.