Expression of Serum Retinol Binding Protein and Transthyretin within Mouse Gastric Ghrelin Cells

PubMed Central

Walker, Angela K.; Gong, Zhi; Park, Won-Mee

2013-01-01

Ghrelin is an orexigenic peptide hormone produced mainly by a distinct group of dispersed endocrine cells located within the gastric oxyntic mucosa. Besides secreted gene products derived from the preproghrelin gene, which include acyl-ghrelin, desacyl-ghrelin and obestatin, ghrelin cells also synthesize the secreted protein nesfatin-1. The main goal of the current study was to identify other proteins secreted from ghrelin cells. An initial gene chip screen using mRNAs derived from highly enriched pools of mouse gastric ghrelin cells demonstrated high levels of serum retinol-binding protein (RBP4) and transthyretin (TTR), both of which are known to circulate in the bloodstream bound to each other. This high expression was confirmed by quantitative RT-PCR using as template mRNA derived from the enriched gastric ghrelin cell pools and from two ghrelin-producing cell lines (SG-1 and PG-1). RBP4 protein also was shown to be secreted into the culture medium of ghrelin cell lines. Neither acute nor chronic caloric restriction had a significant effect on RBP4 mRNA levels within stomachs of C57BL/6J mice, although both manipulations significantly decreased stomach TTR mRNA levels. In vitro studies using PG-1 cells showed no effect on RBP4 release of octanoic acid, epinephrine or norepinephrine, all of which are known to act directly on ghrelin cells to stimulate ghrelin secretion. These data provide new insights into ghrelin cell physiology, and given the known functions of RBP4 and TTR, support an emerging role for the ghrelin cell in blood glucose handling and metabolism. PMID:23840311

Functions of Intracellular Retinoid Binding-Proteins.

PubMed

Napoli, Joseph L

Multiple binding and transport proteins facilitate many aspects of retinoid biology through effects on retinoid transport, cellular uptake, metabolism, and nuclear delivery. These include the serum retinol binding protein sRBP (aka Rbp4), the plasma membrane sRBP receptor Stra6, and the intracellular retinoid binding-proteins such as cellular retinol-binding proteins (CRBP) and cellular retinoic acid binding-proteins (CRABP). sRBP transports the highly lipophilic retinol through an aqueous medium. The major intracellular retinol-binding protein, CRBP1, likely enhances efficient retinoid use by providing a sink to facilitate retinol uptake from sRBP through the plasma membrane or via Stra6, delivering retinol or retinal to select enzymes that generate retinyl esters or retinoic acid, and protecting retinol/retinal from excess catabolism or opportunistic metabolism. Intracellular retinoic acid binding-proteins (CRABP1 and 2, and FABP5) seem to have more diverse functions distinctive to each, such as directing retinoic acid to catabolism, delivering retinoic acid to specific nuclear receptors, and generating non-canonical actions. Gene ablation of intracellular retinoid binding-proteins does not cause embryonic lethality or gross morphological defects. Metabolic and functional defects manifested in knockouts of CRBP1, CRBP2 and CRBP3, however, illustrate their essentiality to health, and in the case of CRBP2, to survival during limited dietary vitamin A. Future studies should continue to address the specific molecular interactions that occur between retinoid binding-proteins and their targets and their precise physiologic contributions to retinoid homeostasis and function.

Contribution of cellular retinol-binding protein type 1 to retinol metabolism during mouse development.

PubMed

Matt, Nicolas; Schmidt, Carsten K; Dupé, Valérie; Dennefeld, Christine; Nau, Heinz; Chambon, Pierre; Mark, Manuel; Ghyselinck, Norbert B

2005-05-01

Within cells, retinol (ROL) is bound to cytoplasmic proteins (cellular retinol-binding proteins [CRBPs]), whose proposed function is to protect it from unspecific enzymes through channeling to retinoid-metabolizing pathways. We show that, during development, ROL and retinyl ester levels are decreased in CRBP type 1 (CRBP1) -deficient embryos and fetuses by 50% and 80%, respectively. The steady state level of retinoic acid (RA) is also decreased but to a lesser extent. However, CRBP1-null fetuses do not exhibit the abnormalities characteristic of a vitamin A-deficiency syndrome. Neither CRBP1 deficiency alters the expression patterns of RA-responding genes during development, nor does CRBP1 availability modify the expression of an RA-dependent gene in primary embryonic fibroblasts treated with ROL. Therefore, CRBP1 is required in prenatal life to maintain normal amounts of ROL and to ensure its efficient storage but seems of secondary importance for RA synthesis, at least under conditions of maternal vitamin A sufficiency. Copyright 2005 Wiley-Liss, Inc.

Retinol Binding Protein 4 in Relation to Diet, Inflammation, Immunity, and Cardiovascular Diseases12

PubMed Central

Zabetian-Targhi, Fateme; Mahmoudi, Mohammad J; Rezaei, Nima; Mahmoudi, Maryam

2015-01-01

Retinol binding protein 4 (RBP4), previously called retinol binding protein (RBP), is considered a specific carrier of retinol in the blood. It is also an adipokine that has been implicated in the pathophysiology of insulin resistance. RBP4 seems to be correlated with cardiometabolic markers in inflammatory chronic diseases, including obesity, type 2 diabetes, metabolic syndrome, and cardiovascular diseases (CVDs). It has recently been suggested that inflammation produced by RBP4 induces insulin resistance and CVD. The clinical relevance of this hypothesis is discussed in this review. Knowledge concerning the association of RBP4 with inflammation markers, oxidative stress, and CVDs as well as concerning the role of diet and antioxidants in decreasing RBP4 concentrations are discussed. Special attention is given to methodologies used in previously published studies and covariates that should be controlled when planning new studies on this adipokine. PMID:26567199

A Polymorphism in the Retinol Binding Protein 4 Gene is Not Associated with Gestational Diabetes Mellitus in Several Different Ethnic Groups

PubMed Central

Urschitz, Johann; Sultan, Omar; Ward, Kenneth

2011-01-01

Objective Various Asian and Pacifific Islander groups have higher prevalence rates of type 2 diabetes and gestational diabetes. This increased incidence is likely to include genetic factors. Single nucleotide polymorphisms in the retinol binding protein 4 gene have been linked to the occurrence of type 2 diabetes. Hypothesizing a link between retinol binding protein 4 and gestational diabetes, we performed a candidate gene study to look for an association between an important retinol binding protein gene polymorphism (rs3758539) and gestational diabetes. Study Design Blood was collected from Caucasian, Asian, and Pacific Islander women diagnosed with gestational diabetes and from ethnically matched non-diabetic controls. DNA was extracted and real time PCR technology (TaqMan, Applied Biosystems) used to screen for the rs3758539 single nucleotide polymorphism located 5′ of exon 1 of the retinol binding protein 4 gene. Results Genotype and allele frequencies in the controls and gestational diabetes cases were tested using chi-square contingency tests. Genotype frequencies were in Hardy-Weinberg equilibrium. There was no association between the rs3758539 retinol binding protein 4 single nucleotide polymorphism and gestational diabetes in the Caucasian, Filipino, or Pacific Islander groups. Conclusion Interestingly, the rs3758539 retinol binding protein 4 single nucleotide polymorphism was not found to be associated with gestational diabetes. The absence of association suggests that gestational and type 2 diabetes may have more divergent molecular pathophysiology than previously suspected. PMID:21886308

Prognostic Importance of Vitamins A, E and Retinol-binding Protein 4 in Renal Cell Carcinoma Patients.

PubMed

Sobotka, Roman; Čapoun, Otakar; Kalousová, Marta; Hanuš, Tomáš; Zima, Tomáš; Koštířová, Milada; Soukup, Viktor

2017-07-01

To assess the prognostic importance of serum levels of retinol, retinol-binding protein 4 (RBP4) and vitamin E at the time of diagnosis in patients with renal cell carcinoma (RCC). In this prospective study, in a cohort of 102 renal cell carcinoma patients, relationships between serum levels of the aforementioned markers and recurrence-free survival (RFS), overall survival (OS), as well as cancer-specific survival (CSS), were evaluated. The vitamin A and vitamin E levels were determined by high-performance liquid chromatography (HPLC), while the RBP4 level by enzyme-linked immunosorbent assay (ELISA). The median follow-up period was 39 months. Renal cell carcinoma recurred in 9 patients; 23 patients died with 12 of them from RCC. The preoperative vitamin E level was associated to RFS (p=0.02). We found a significant relationship between OS and the level of RBP4 (p=0.002), retinol (p=0.037) and vitamin E (p=0.007). The CSS period was significantly associated with the level of RBP4 (p=0.0001) and retinol (p=0.0003). Patients with an RBP4 level less than 21.0 mg/l at the time of diagnosis had a 13.5-times higher risk of death due to RCC progression; this risk was up to 7.7-times higher with vitamin A levels under 0.52 mg/l. Low levels of vitamin A, E and RBP4 at the time of RCC diagnosis are associated with a poorer prognosis after surgery. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

An immunohistochemical study of retinol-binding protein (RBP) in livers of free-living polar bears (Ursus maritimus) from east Greenland.

PubMed

Heier, Annabelle; Sonne, Christian; Gröne, Andrea; Leifsson, Pall S; Dietz, Rune; Born, Erik W; Bacciarini, Luca N

2005-09-01

From 1999 to 2002 samples from 114 free-ranging polar bears (Ursus maritimus) were collected in the municipality of Scoresby Sound, East Greenland, to detect levels of organochlorines and potential histopathologic changes. Livers of 16 female polar bears from this group were evaluated histologically and analyzed for hepatic retinol-binding protein by immunohistochemistry. Retinol-binding protein is the main transport protein for retinol, an important vitamin A metabolite in the polar bear. Only mild pathologic changes were noted on histologic evaluation of the livers. Small lymphocytic or lymphohistiocytic infiltrates were present in all the livers. Small lipid granulomas, mild periportal fibrosis, and bile duct proliferation were found in several cases. Immunohistochemistry for retinol-binding protein of hepatic tissue from free-ranging polar bears showed no distinct difference in staining intensity by a number of criteria: age, season (fasting and nonfasting), or lactation status. The staining was diffuse to finely stippled in the cytoplasm and showed very little variation among the animals. Because of the lack of macroscopic changes and the absence of severe histologic liver lesions, these polar bears were assumed to be healthy. The diffuse cytoplasmic retinol-binding protein staining in hepatocytes of free-ranging polar bears varies markedly from the prominent granular, less intense staining of captive polar bears investigated previously.

Binding of human serum proteins to titanium dioxide particles in vitro.

PubMed

Zaqout, Mazen S K; Sumizawa, Tomoyuki; Igisu, Hideki; Higashi, Toshiaki; Myojo, Toshihiko

2011-01-01

To determine the capacity of human serum proteins to bind to titanium dioxide (TiO(2)) particles of different polymorphs and sizes. TiO(2) particles were mixed with diluted human serum, purified human serum albumin (HSA) or purified human serum gamma-globulin (HGG) solutions. After incubation at 37°C for 1 h, the particles were sedimented by centrifugation, and proteins in the supernatant, as well as those bound to the particles, were analyzed. The total protein concentration in the supernatant was lowered by TiO(2), whereas the albumin/globulin ratio was elevated by the particles. Incubation with TiO(2) also lowered the immunoglobulin, pre-albumin, beta2-microglobulin, ceruloplasmin and retinol-binding protein levels, but not ferritin levels, in the supernatant. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins in the supernatant, especially HGG, were observed to decrease, while those released from the particles (after adding 1% SDS and heating) increased, depending on the dose of TiO(2). Purified HGG and HSA were also bound to TiO(2), although the former appeared to have a higher affinity. All the proteins tested showed the highest binding potency to the amorphous particles (<50 nm) and the lowest to the rutile particles (<5,000 nm), while binding to anatase particles was intermediate. The affinity to the larger anatase was higher than that to smaller anatase particles in most cases. Human serum proteins, including the two major components, HSA and HGG, are bound by TiO(2) particles. The polymorph of the particles seems to be important for determining the binding capacity of the particles and it may affect distribution of the particles in the body.

Ligand Binding Induces Conformational Changes in Human Cellular Retinol-binding Protein 1 (CRBP1) Revealed by Atomic Resolution Crystal Structures.

PubMed

Silvaroli, Josie A; Arne, Jason M; Chelstowska, Sylwia; Kiser, Philip D; Banerjee, Surajit; Golczak, Marcin

2016-04-15

Important in regulating the uptake, storage, and metabolism of retinoids, cellular retinol-binding protein 1 (CRBP1) is essential for trafficking vitamin A through the cytoplasm. However, the molecular details of ligand uptake and targeted release by CRBP1 remain unclear. Here we report the first structure of CRBP1 in a ligand-free form as well as ultra-high resolution structures of this protein bound to either all-trans-retinol or retinylamine, the latter a therapeutic retinoid that prevents light-induced retinal degeneration. Superpositioning of human apo- and holo-CRBP1 revealed major differences within segments surrounding the entrance to the retinoid-binding site. These included α-helix II and hairpin turns between β-strands βC-βD and βE-βF as well as several side chains, such as Phe-57, Tyr-60, and Ile-77, that change their orientations to accommodate the ligand. Additionally, we mapped hydrogen bond networks inside the retinoid-binding cavity and demonstrated their significance for the ligand affinity. Analyses of the crystallographic B-factors indicated several regions with higher backbone mobility in the apoprotein that became more rigid upon retinoid binding. This conformational flexibility of human apo-CRBP1 facilitates interaction with the ligands, whereas the more rigid holoprotein structure protects the labile retinoid moiety during vitamin A transport. These findings suggest a mechanism of induced fit upon ligand binding by mammalian cellular retinol-binding proteins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Plasma Levels of Fatty Acid-Binding Protein 4, Retinol-Binding Protein 4, High-Molecular-Weight Adiponectin, and Cardiovascular Mortality Among Men With Type 2 Diabetes: A 22-Year Prospective Study.

PubMed

Liu, Gang; Ding, Ming; Chiuve, Stephanie E; Rimm, Eric B; Franks, Paul W; Meigs, James B; Hu, Frank B; Sun, Qi

2016-11-01

To examine select adipokines, including fatty acid-binding protein 4, retinol-binding protein 4, and high-molecular-weight (HMW) adiponectin in relation to cardiovascular disease (CVD) mortality among patients with type 2 diabetes mellitus. Plasma levels of fatty acid-binding protein 4, retinol-binding protein 4, and HMW adiponectin were measured in 950 men with type 2 diabetes mellitus in the Health Professionals Follow-up Study. After an average of 22 years of follow-up (1993-2015), 580 deaths occurred, of whom 220 died of CVD. After multivariate adjustment for covariates, higher levels of fatty acid-binding protein 4 were significantly associated with a higher CVD mortality: comparing extreme tertiles, the hazard ratio and 95% confidence interval of CVD mortality was 1.78 (1.22-2.59; P trend=0.001). A positive association was also observed for HMW adiponectin: the hazard ratio (95% confidence interval) was 2.07 (1.42-3.06; P trend=0.0002), comparing extreme tertiles, whereas higher retinol-binding protein 4 levels were nonsignificantly associated with a decreased CVD mortality with an hazard ratio (95% confidence interval) of 0.73 (0.50-1.07; P trend=0.09). A Mendelian randomization analysis suggested that the causal relationships of HMW adiponectin and retinol-binding protein 4 would be directionally opposite to those observed based on the biomarkers, although none of the Mendelian randomization associations achieved statistical significance. These data suggest that higher levels of fatty acid-binding protein 4 and HMW adiponectin are associated with elevated CVD mortality among men with type 2 diabetes mellitus. Biological mechanisms underlying these observations deserve elucidation, but the associations of HMW adiponectin may partially reflect altered adipose tissue functionality among patients with type 2 diabetes mellitus. © 2016 American Heart Association, Inc.

Elevated Serum Triglyceride and Retinol-Binding Protein 4 Levels Associated with Fructose-Sweetened Beverages in Adolescents

PubMed Central

Chan, Te-Fu; Lin, Wei-Ting; Chen, Yi-Ling; Huang, Hsiao-Ling; Yang, Wei-Zeng; Lee, Chun-Ying; Chen, Meng-Hsueh; Wang, Tsu-Nai; Huang, Meng-Chuan; Chiu, Yu-Wen; Huang, Chun-Chi; Tsai, Sharon; Lin, Chih-Lung; Lee, Chien-Hung

2014-01-01

Background The metabolic effect of fructose in sugar-sweetened beverages (SSB) has been linked to de novo lipogenesis and uric acid (UA) production. Objectives This study investigated the biological effects of SSB consumption on serum lipid profiles and retinol-binding protein 4 (RBP4) among Taiwanese adolescents. Methods We evaluated the anthropometric parameters and biochemical outcomes of 200 representative adolescents (98 boys and 102 girls) who were randomly selected from a large-scale cross-sectional study. Data were analyzed using multiple regression models adjusted for covariates. Results Increased SSB consumption was associated with increased waist and hip circumferences, body mass index (BMI) values and serum UA, triglyceride (TG) and RBP4 levels. Adolescents who consumed >500 ml/day of beverages half-to-heavily sweetened with high-fructose corn syrup (HFCS) exhibited TG and RBP4 levels 22.7 mg/dl and 13.92 ng/ml higher than non-drinkers, respectively. HFCS drinkers with hyperuricemia had higher TG levels than HFCS drinkers with normal UA levels (98.6 vs. 81.6 mg/dl). The intake of HFCS-rich SSBs and high value of BMI (≥24) interactively reinforced RBP4 levels among overweight/obese adolescents. Circulating RBP4 levels were significantly correlated with weight-related outcomes and TG and UA concentration among HFCS drinkers (r = 0.253 to 0.404), but not among non-drinkers. Conclusions High-quantity HFCS-rich beverage consumption is associated with higher TG and RBP4 levels. Hyperuricemia is likely to intensify the influence of HFCS-rich SSB intake on elevated TG levels, and in overweight and obese adolescents, high BMI may modify the action of fructose on higher circulating levels of RBP4. PMID:24475021

High dietary fat-induced obesity in Wistar rats and type 2 diabetes in nonobese Goto-Kakizaki rats differentially affect retinol binding protein 4 expression and vitamin A metabolism.

PubMed

Shirai, Tomomi; Shichi, Yuta; Sato, Miyuki; Tanioka, Yuri; Furusho, Tadasu; Ota, Toru; Tadokoro, Tadahiro; Suzuki, Tsukasa; Kobayashi, Ken-Ichi; Yamamoto, Yuji

2016-03-01

Obesity is a major risk factor for type 2 diabetes, which is caused mainly by insulin resistance. Retinol binding protein 4 (RBP4) is the only specific transport protein for retinol in the serum. RBP4 level is increased in the diabetic state and high-fat condition, indicating that retinol metabolism may be affected under these conditions. However, the precise effect of diabetes and high fat-induced obesity on retinol metabolism is unknown. In this study, we examined differences in retinol metabolite levels in rat models of diet-induced obesity and type 2 diabetes (Goto-Kakizaki [GK] rat). Four-week-old male Wistar and GK rats were given either a control diet (AIN-93G) or a high-fat diet (HFD, 40% fat kJ). After 15 weeks of feeding, the RBP4 levels increased by 2-fold in the serum of GK rats but not HFD-fed rats. The hepatic retinol concentration of HFD-fed rats was approximately 50% that of the controls (P < .01). In contrast, the renal retinol concentrations of GK rats increased by 70% (P < .01). However, expression of RARβ in the kidney, which was induced in a retinoic acid-dependent manner, was downregulated by 90% (P < .01) in GK rats. In conclusion, diabetes and obesity affected retinol metabolism differently, and the effects were different in different peripheral tissues. The impact of HFD may be limited to the storage of hepatic vitamin A as retinyl palmitate. In particular, our data indicate that renal retinoic acid production might represent an important target for the treatment of type 2 diabetes mellitus. Copyright © 2016 Elsevier Inc. All rights reserved.

Biochemical Properties of Purified Human Retinol Dehydrogenase 12 (RDH12): Catalytic Efficiency toward Retinoids and C9 Aldehydes and Effects of Cellular Retinol-Binding Protein Type I (CRBPI) and Cellular Retinaldehyde-Binding Protein (CRALBP) on the Oxidation and Reduction of Retinoids†

PubMed Central

Belyaeva, Olga V.; Korkina, Olga V.; Stetsenko, Anton V.; Kim, Tom; Nelson, Peter S.; Kedishvili, Natalia Y.

2008-01-01

Retinol dehydrogenase 12 (RDH12) is a novel member of the short-chain dehydrogenase/reductase superfamily of proteins that was recently linked to Leber’s congenital amaurosis 3 (LCA). We report the first biochemical characterization of purified human RDH12 and analysis of its expression in human tissues. RDH12 exhibits ~2000-fold lower Km values for NADP+ and NADPH than for NAD+ and NADH and recognizes both retinoids and lipid peroxidation products (C9 aldehydes) as substrates. The kcat values of RDH12 for retinaldehydes and C9 aldehydes are similar, but the Km values are, in general, lower for retinoids. The enzyme exhibits the highest catalytic efficiency for all-trans-retinal (kcat/Km ~900 min−1 μM−1), followed by 11-cis-retinal (450 min−1 mM−1) and 9-cis-retinal (100 min−1 mM−1). Analysis of RDH12 activity toward retinoids in the presence of cellular retinol-binding protein (CRBP) type I or cellular retinaldehyde-binding protein (CRALBP) suggests that RDH12 utilizes the unbound forms of all-trans- and 11-cis-retinoids. As a result, the widely expressed CRBPI, which binds all-trans-retinol with much higher affinity than all-trans-retinaldehyde, restricts the oxidation of all-trans-retinol by RDH12, but has little effect on the reduction of all-trans-retinaldehyde, and CRALBP inhibits the reduction of 11-cis-retinal stronger than the oxidation of 11-cis-retinol, in accord with its higher affinity for 11-cis-retinal. Together, the tissue distribution of RDH12 and its catalytic properties suggest that, in most tissues, RDH12 primarily contributes to the reduction of all-trans-retinaldehyde; however, at saturating concentrations of peroxidic aldehydes in the cells undergoing oxidative stress, for example, photoreceptors, RDH12 might also play a role in detoxification of lipid peroxidation products. PMID:15865448

A non-retinoid antagonist of Retinol-Binding Protein 4 rescues phenotype in a model of Stargardt disease without inhibiting the visual cycle.

PubMed

Racz, Boglarka; Varadi, Andras; Kong, Jian; Allikmets, Rando; Pearson, Paul G; Johnson, Graham; Cioffi, Christopher L; Petrukhin, Konstantin

2018-06-05

A primary pathological defect in the heritable eye disorder Stargardt disease is excessive accumulation of cytotoxic lipofuscin bisretinoids in the retina. Age-dependent accumulation of lipofuscin in the retinal pigment epithelium (RPE) matches the age-dependent increase in the incidence of the atrophic (dry) form of age-related macular degeneration (AMD) and therefore may be one of several pathogenic factors contributing to AMD progression. Lipofuscin bisretinoid synthesis in the retina depends on the influx of serum retinol from the circulation into the RPE. Formation of the tertiary retinol-binding protein 4 (RBP4)-transthyretin-retinol complex in the serum is required for this influx. Herein, we report the pharmacological effects of the non-retinoid RBP4 antagonist, BPN-14136. BPN-14136 dosing in the Abca4-/- mouse model of increased lipofuscinogenesis significantly reduced serum RBP4 levels and inhibited bisretinoid synthesis, and this inhibition correlated with a partial reduction in visual cycle retinoids such as retinaldehydes serving as bisretinoid precursors. BPN-14136 administration at doses inducing maximal serum RBP4 reduction did not produce changes in the rate of the visual cycle, consistent with minimal changes in dark adaptation. Abca4-/- mice exhibited dysregulation of the complement system in the retina, and BPN-14136 administration normalized the retinal levels of proinflammatory complement cascade components such as complement factors D and H, C-reactive protein, and C3. We conclude that BPN-14136 has several beneficial characteristics, combining inhibition of bisretinoid synthesis and reduction in retinaldehydes with normalization of the retinal complement system. BPN-14136, or a similar compound, may be a promising drug candidate to manage Stargardt disease and dry AMD. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

The relative dose response test based on retinol-binding protein 4 is not suitable to assess vitamin A status in very low birth weight infants.

PubMed

Schmiedchen, Bettina; Longardt, Ann Carolin; Bührer, Christoph; Raila, Jens; Loui, Andrea; Schweigert, Florian J

2014-01-01

The relative dose response (RDR) test, which quantifies the increase in serum retinol after vitamin A administration, is a qualitative measure of liver vitamin A stores. Particularly in preterm infants, the feasibility of the RDR test involving blood is critically dependent on small sample volumes. This study aimed to assess whether the RDR calculated with retinol-binding protein 4 (RBP4) might be a substitute for the classical retinol-based RDR test for assessing vitamin A status in very preterm infants. This study included preterm infants with a birth weight below 1,500 g (n = 63, median birth weight 985 g, median gestational age 27.4 weeks) who were treated with 5,000 IU retinyl palmitate intramuscularly 3 times a week for 4 weeks. On day 3 (first vitamin A injection) and day 28 of life (last vitamin A injection), the RDR was calculated and compared using serum retinol and RBP4 concentrations. The concentrations of retinol (p < 0.001) and RBP4 (p < 0.01) increased significantly from day 3 to day 28. On day 3, the median (IQR) retinol-RDR was 27% (8.4-42.5) and the median RBP4-RDR was 8.4% (-3.4 to 27.9), compared to 7.5% (-10.6 to 20.8) and -0.61% (-19.7 to 15.3) on day 28. The results for retinol-RDR and RBP4-RDR revealed no significant correlation. The agreement between retinol-RDR and RBP4-RDR was poor (day 3: Cohen's κ = 0.12; day 28: Cohen's κ = 0.18). The RDR test based on circulating RBP4 is unlikely to reflect the hepatic vitamin A status in preterm infants. © 2013 S. Karger AG, Basel.

Retinol Binding Protein 4 in children with Inflammatory Bowel Disease: a negative correlation with the disease activity.

PubMed

Roma, E; Krini, M; Hantzi, E; Sakka, S; Panayiotou, I; Margeli, A; Papassotiriou, I; Kanaka-Gantenbein, C

2012-10-01

Retinol Binding Protein-4 (RBP-4), the action of which was initially thought to be only the transport of vitamin A, is a major circulating adipocytokine involved in the inflammation. We evaluated the serum RBP-4 levels in children with inflammatory bowel disease (IBD) and correlated them with transthyretin (TTR), inflammation markers, disease activity, and body mass index (BMI). In 41 children of mean age 11.9 ± 3.6 years (range 5-17.7 y) with IBD (19 with Crohn's disease (CD) and 22 with Ulcerative colitis (UC) serum RBP-4, TTR, Amyloid A (SAA), C-Reactive Protein (CRP), Erythrocyte Sedimentation Rate (ESR), disease activity and BMI were prospectively determined and compared with those of 42 matched controls. No difference in the RBP-4 and TTR serum levels, between patients and controls as well as between active and remission state of the disease was noticed. A negative correlation of serum RBP-4 with the disease activity, SAA and ESR and a positive correlation with TTR was found, but no significant correlation with CRP or BMI was found. Inflammation markers were significantly increased in patients compared to controls and had a positive correlation with the disease activity. RBP-4 negatively correlated with disease activity of children with IBD probably indicating a protective anti-inflammatory mechanism of action in addition to transport of vitamin A.

Circulating Retinol-Binding Protein-4 Concentration Might Reflect Insulin Resistance–Associated Iron Overload

PubMed Central

Fernández-Real, José Manuel; Moreno, José María; Ricart, Wifredo

2008-01-01

OBJECTIVES—The mechanisms behind the association between retinol-binding protein-4 (RBP4) and insulin resistance are not well understood. An interaction between iron and vitamin A status, of which RBP4 is a surrogate, has long been recognized. We hypothesized that iron-associated insulin resistance could be behind the impaired insulin action caused by RBP4. RESEARCH DESIGN AND METHODS—Serum ferritin and RBP4 concentration and insulin resistance were evaluated in a sample of middle-aged men (n = 132) and in a replication independent study. Serum RBP4 was also studied before and after iron depletion in patients with type 2 diabetes. Finally, the effect of iron on RBP4 release was evaluated in vitro in adipose tissue. RESULTS—A positive correlation between circulating RBP4 and log serum ferritin (r = 0.35 and r = 0.61, respectively; P < 0.0001) was observed in both independent studies. Serum RBP4 concentration was higher in men than women in parallel to increased ferritin levels. On multiple regression analyses to predict serum RBP4, log serum ferritin contributed significantly to RBP4 variance after controlling for BMI, age, and homeostasis model assessment value. Serum RBP4 concentration decreased after iron depletion in type 2 diabetic patients (percent mean difference −13.7 [95% CI −25.4 to −2.04]; P = 0.024). The iron donor lactoferrin led to increased dose-dependent adipose tissue release of RBP4 (2.4-fold, P = 0.005) and increased RBP4 expression, while apotransferrin and deferoxamine led to decreased RBP4 release. CONCLUSIONS—The relationship between circulating RBP4 and iron stores, both cross-sectional and after iron depletion, and in vitro findings suggest that iron could play a role in the RBP4–insulin resistance relationship. PMID:18426863

Association of Serum Retinol-Binding Protein 4 Concentration With Risk for and Prognosis of Amyotrophic Lateral Sclerosis.

PubMed

Rosenbohm, Angela; Nagel, Gabriele; Peter, Raphael S; Brehme, Torben; Koenig, Wolfgang; Dupuis, Luc; Rothenbacher, Dietrich; Ludolph, Albert C

2018-05-01

Knowledge about the metabolic states of patients with amyotrophic lateral sclerosis (ALS) may provide a therapeutic approach. To investigate the association between the onset and prognosis of ALS and serum retinol-binding protein 4 (RBP4) concentration as a biomarker for insulin resistance and vitamin A metabolism. Case-control design for risk factors of ALS; cohort design for prognostic factors within ALS cases. Between October 1, 2010, and June 30, 2014, a population-based case-control study with randomly selected controls was established based on the ALS Registry Swabia in southern Germany, with a target population of 8.4 million inhabitants. Response rates were 64.8% among the cases and 18.7% among the controls. The dates of analysis were April 2016 to May 2017. Serum samples were measured for RBP4. Information on covariates was assessed by an interview-based standardized questionnaire. Main outcomes and measures were adjusted odds ratios for risk of ALS associated with serum RBP4 concentration, as well as time to death associated with RBP4 concentration at baseline in ALS cases only. Conditional logistic regression was applied to calculate multivariable odds ratios for risk of ALS. Survival models were used in cases only to appraise their prognostic value. Data from 289 patients with ALS (mean [SD] age, 65.7 [10.5] years; 172 [59.5%] male) and 504 controls (mean [SD] age, 66.3 [9.8] years; 299 [59.3%] male) were included in the case-control study. Compared with controls, ALS cases were characterized by lower body mass index, less educational attainment, smoking, light occupational work intensity, and self-reported diabetes. The median serum RBP4 concentration was lower in ALS cases than in controls (54.0 vs 59.5 mg/L). In the multivariable model, increasing RBP4 concentration was associated with reduced odds for ALS (top vs bottom quartile odds ratio, 0.36; 95% CI, 0.22-0.59; P for trend <.001), which persisted after further adjustment for renal function and

The complexity of minocycline serum protein binding.

PubMed

Zhou, Jian; Tran, Brian T; Tam, Vincent H

2017-06-01

Serum protein binding is critical for understanding the pharmacology of antimicrobial agents. Tigecycline and eravacycline were previously reported to have atypical non-linear protein binding; the percentage of free fraction decreased with increasing total concentration. In this study, we extended the investigation to other tetracyclines and examined the factors that might impact protein binding. Different minocycline concentrations (0.5-50 mg/L) and perfusion media (saline, 0.1 M HEPES buffer and 0.1 and 1 M PBS) were examined by in vitro microdialysis. After equilibration, two dialysate samples were taken from each experiment and the respective antimicrobial agent concentrations were analysed by validated LC-MS/MS methods. For comparison, the serum protein bindings of doxycycline and levofloxacin were also determined. The free fraction of minocycline decreased with increasing total concentration, and the results depended on the perfusion media used. The trends of minocycline protein binding in mouse and human sera were similar. In addition, serum protein binding of doxycycline showed the same concentration-dependent trend as minocycline, while the results of levofloxacin were concentration independent. The serum protein bindings of minocycline and doxycycline are negatively correlated with their total concentrations. It is possible that all tetracyclines share the same pharmacological property. Moreover, the specific perfusion media used could also impact the results of microdialysis. Additional studies are warranted to understand the mechanism(s) and clinical implications of serum protein binding of tetracyclines. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Vitamin A intake and serum retinol levels in children and adolescents with cystic fibrosis.

PubMed

Woestenenk, Janna W; Broos, Nancy; Stellato, Rebecca K; Arets, Hubertus G M; van der Ent, Cornelis K; Houwen, Roderick H J

2016-06-01

Pancreatic insufficient cystic fibrosis (CF) patients receive vitamin A supplementation according to CF-specific recommendations to prevent deficiencies. Whether current recommendations are optimal for preventing both deficiency and toxicity is a subject of debate. We assessed the longitudinal relation between serum retinol levels and appropriate variables. We studied vitamin A intake, and the long-term effects of vitamin A intake, coefficient of fat absorption (CFA) and immunoglobulin G (IgG) on serum retinol levels in 221 paediatrics CF patients during a seven-year follow up period. Total vitamin A intake, derived from 862 dietary assessments, exceeded the tolerable upper intake level in 30% of the assessments, mainly up to age six. Although CF patients failed to meet the CF-specific recommendations, serum retinol deficiency was found in only 17/862 (2%) of the measurements. Longitudinally, we observed no association to serum retinol levels for total vitamin A intake, CFA, gender or age but serum retinol levels were associated with serum IgG levels. Each g/L increase in serum IgG level would result in a 2.49% (95% CI -3.60 to -1.36%) reduction in serum retinol levels. In this large sample of children and adolescents with CF, serum retinol deficiency was rare despite lower than the CF-specific recommendations. However, the TUL was commonly exceeded. A reduction in CF-specific vitamin A supplementation recommendations should therefore be considered. Moreover, serum retinol levels were not associated with vitamin A intake, CFA, gender or age, although a decreased serum retinol was associated with an increased serum IgG. Copyright © 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Molecular Mechanism of Wide Photoabsorption Spectral Shifts of Color Variants of Human Cellular Retinol Binding Protein II.

PubMed

Cheng, Cheng; Kamiya, Motoshi; Uchida, Yoshihiro; Hayashi, Shigehiko

2015-10-21

Color variants of human cellular retinol binding protein II (hCRBPII) created by protein engineering were recently shown to exhibit anomalously wide photoabsorption spectral shifts over ∼200 nm across the visible region. The remarkable phenomenon provides a unique opportunity to gain insight into the molecular basis of the color tuning of retinal binding proteins for understanding of color vision as well as for engineering of novel color variants of retinal binding photoreceptor proteins employed in optogenetics. Here, we report a theoretical investigation of the molecular mechanism underlying the anomalously wide spectral shifts of the color variants of hCRBPII. Computational modeling of the color variants with hybrid molecular simulations of free energy geometry optimization succeeded in reproducing the experimentally observed wide spectral shifts, and revealed that protein flexibility, through which the active site structure of the protein and bound water molecules is altered by remote mutations, plays a significant role in inducing the large spectral shifts.

Serum Retinol Levels in Pregnant Adolescents and Their Relationship with Habitual Food Intake, Infection and Obstetric, Nutritional and Socioeconomic Variables.

PubMed

Spíndola Garcêz, Laís; de Sousa Paz Lima, Geania; de Azevedo Paiva, Adriana; Maria Rebêlo Sampaio da Paz, Suzana; Lázaro Gomes, Erica Ivana; Nunes, Valéria Sutti; Cotta de Faria, Eliana; de Barros-Mazon, Sílvia

2016-10-25

Globally, vitamin A deficiency (VAD) affects about 19.1 million pregnant women. Its occurrence is classically associated with inadequate food intake and may also be associated with socioeconomic factors and the presence of infection. The aim of this study was to determine the factors related to serum retinol levels among pregnant teenagers. The sample consisted of 89 pregnant adolescents, from whom socioeconomic, obstetric, anthropometric, and food consumption data were collected. Serum concentrations of retinol and the supposed presence of infection were determined by high-performance liquid chromatography and C-reactive protein quantification, respectively. The serum retinol concentrations were classified according to the criteria of the World Health Organization. We adopted a 5% significance level for all statistical tests. Serum retinol levels were significantly and positively associated with sanitation ( p = 0.008) and pre-gestational nutritional status ( p = 0.002), and negatively with the trimester ( p = 0.001). The appropriate sanitation conditions and pre-pregnancy body mass index (BMI) were shown to have a protective effect against VAD. Conversely, serum retinol levels were reduced with trimester progression, favoring VAD occurrence.

Sitagliptin down-regulates retinol-binding protein 4 and reduces insulin resistance in gestational diabetes mellitus: a randomized and double-blind trial.

PubMed

Sun, Xia; Zhang, Zhendong; Ning, Hui; Sun, Hong; Ji, Xianghong

2017-06-01

Gestational diabetes mellitus (GDM) is a condition that affects increasing number of pregnant women worldwide. Sitagliptin was reported to alleviate symptoms of type 2 diabetes mellitus by reducing serum levels of retinol-binding protein 4 (RBP-4). We investigated the effectiveness of sitagliptin on insulin sensitivity parameters in GDM patients. Pregnant GDM women in the 2nd trimester were recruited for this study. Participants were then assigned randomly to sitagliptin treatment group or placebo treatment group, and administered sitagliptin or placebo daily for 16 weeks. Glucose and insulin profiles, as well as serum RBP-4 level, were measured at both baseline and end of the study. After 16 weeks of treatment, participants in the STL group exhibited significantly improved levels of fasting plasma glucose and serum insulin, homeostasis model of assessment of β cell function (HOMA-β) and insulin resistance (HOMA-IR), compared with those in the placebo group. Serum levels of RBP-4 were also markedly decreased in the sitagliptin treatment group, and more importantly it was positively correlated with improved insulin resistance parameters. Our study supports a potentially promising role of sitagliptin in improving insulin resistance by decreasing RBP-4 in GDM-affected women.

Retinol Binding Protein-4 Levels and Non-alcoholic Fatty Liver Disease: A community-based cross-sectional study.

PubMed

Chen, Xuechen; Shen, Tianran; Li, Qing; Chen, Xu; Li, Yanping; Li, Dan; Chen, Gengdong; Ling, Wenhua; Chen, Yu-Ming

2017-03-23

Previous reports on the association between retinol binding protein 4 (RBP4) and nonalcoholic fatty liver disease (NAFLD) were controversial. This study aimed to investigate the association between the serum RBP4 levels and occurrence of NAFLD in Chinese population. In total, 2938 participants aged 40-75 years were involved in this community-based cross-sectional study. General information, lifestyle factors, serum levels of RBP4 and the presence of NAFLD were determined. Patients with NAFLD had significantly higher concentrations of RBP4 (37.9 ± 6.8 μg/ml) than did non-NAFLD controls (35.0 ± 6.7 μg/ml) (P < 0.001). The odds ratios (ORs) of NAFLD for the highest (vs. lowest) quartile of RBP4 were 1.884 (95% CI: 1.391, 2.551) for females (P < 0.001), and 2.107 (95% CI: 1.357, 3.273) for male participants (P < 0.01) after adjusting for related factors. The serum RBP4 levels were positively associated with the prevalence of NAFLD in middle-aged and elderly Chinese people, and Homeostatic model assessment-insulin resistance (HOMA-IR), trunk fat, the waist-to-hip ratio (WHR), systolic blood pressure (SBP), fasting insulin, high density lipoprotein cholesterol (HDL-C) and triglycerides (TG) might be implicated in the pathogenesis of RBP4 in NAFLD.

The new platinum-based anticancer agent LA-12 induces retinol binding protein 4 in vivo

PubMed Central

2011-01-01

Background The initial pharmacokinetic study of a new anticancer agent (OC-6-43)-bis(acetato)(1-adamantylamine)amminedichloroplatinum (IV) (LA-12) was complemented by proteomic screening of rat plasma. The objective of the study was to identify new LA-12 target proteins that serve as markers of LA-12 treatment, response and therapy monitoring. Methods Proteomic profiles were measured by surface-enhanced laser desorption-ionization time-of-flight mass spectrometry (SELDI-TOF MS) in 72 samples of rat plasma randomized according to LA-12 dose and time from administration. Correlation of 92 peak clusters with platinum concentration was evaluated using Spearman correlation analysis. Results We identified Retinol-binding protein 4 (RBP4) whose level correlated with LA-12 level in treated rats. Similar results were observed in randomly selected patients involved in Phase I clinical trials. Conclusions RBP4 induction is in agreement with known RBP4 regulation by amantadine and cisplatin. Since retinol metabolism is disrupted in many cancers and inversely associates with malignancy, these data identify a potential novel mechanism for the action of LA-12 and other similar anti-cancer drugs. PMID:22040120

Nitrofen interferes with trophoblastic expression of retinol-binding protein and transthyretin during lung morphogenesis in the nitrofen-induced congenital diaphragmatic hernia model.

PubMed

Kutasy, Balazs; Gosemann, Jan H; Doi, Takashi; Fujiwara, Naho; Friedmacher, Florian; Puri, Prem

2012-02-01

Retinoids play a key role in lung development. Retinoid signaling pathway has been shown to be disrupted in the nitrofen model of congenital diaphragmatic hernia (CDH) but the exact mechanism is not clearly understood. Retinol-binding protein (RBP) and transthyretin (TTR) are transport proteins for delivery of retinol to the tissues via circulation. Previous studies have shown that pulmonary retinol levels are decreased during lung morphogenesis in the nitrofen CDH model. In human newborns with CDH, both retinol and RBP levels are decreased. It has been reported that maternal RBP does not cross the placenta and the fetus produces its own RBP by trophoblast. RBP and TTR synthesized in the fetus are essential for retinol transport to the developing organs including lung morphogenesis. We hypothesized that nitrofen interferes with the trophoblastic expression of RBP and TTR during lung morphogenesis and designed this study to examine the trophoblastic expression of RBP and TTR, and the total level of RBP and TTR in the lung in the nitrofen model of CDH. Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetal lungs and placenta harvested on D21 and divided into two groups: control (n = 8) and nitrofen with CDH (n = 8). Total lung RBP and TTR levels using protein extraction were compared with enzyme linked immunoassay (ELISA). Immunohistochemistry was performed to evaluate trophoblastic RBP and TTR expression. Total protein levels of lung RBP and TTR were significantly lower in CDH (0.26 ± 0.003 and 6.4 ± 0.5 μg/mL) compared with controls (0.4 ± 0.001 and 9.9 ± 1.6 μg/mL, p < 0.05). In the control group, immunohistochemical staining showed strong immunoreactivity of RBP and TTR in the trophoblast compared to CDH group. Decreased trophoblast expression of retinol transport proteins suggest that nitrofen may interfere with the fetal retinol transport resulting in reduced pulmonary RBP and TTR levels and causing pulmonary

Effect of vitamin A supplementation on the urinary retinol excretion in very low birth weight infants.

PubMed

Schmiedchen, Bettina; Longardt, Ann Carolin; Loui, Andrea; Bührer, Christoph; Raila, Jens; Schweigert, Florian J

2016-03-01

Despite high-dose vitamin A supplementation of very low birth weight infants (VLBW, <1500 g), their vitamin A status does not improve substantially. Unknown is the impact of urinary retinol excretion on the serum retinol concentration in these infants. Therefore, the effect of high-dose vitamin A supplementation on the urinary vitamin A excretion in VLBW infants was investigated. Sixty-three VLBW infants were treated with vitamin A (5000 IU intramuscular, 3 times/week for 4 weeks); 38 untreated infants were classified as control group. On days 3 and 28 of life, retinol, retinol-binding protein 4 (RBP4), glomerular filtration rate, proteinuria, and Tamm-Horsfall protein were quantified in urine. On day 3 of life, substantial retinol and RBP4 losses were found in both groups, which significantly decreased until day 28. Notwithstanding, the retinol excretion was higher (P < 0.01) under vitamin A supplementation as compared to infants of the control group. On day 28 of life, the urinary retinol concentrations were predictive for serum retinol concentrations in the vitamin A treated (P < 0.01), but not in the control group (P = 0.570). High urinary retinol excretion may limit the vitamin A supplementation efficacy in VLBW infants. Advanced age and thus postnatal kidney maturation seems to be an important contributor in the prevention of urinary retinol losses.

All-trans retinol, vitamin D and other hydrophobic compounds bind in the axial pore of the five-stranded coiled-coil domain of cartilage oligomeric matrix protein.

PubMed Central

Guo, Y; Bozic, D; Malashkevich, V N; Kammerer, R A; Schulthess, T; Engel, J

1998-01-01

The potential storage and delivery function of cartilage oligomeric matrix protein (COMP) for cell signaling molecules was explored by binding hydrophobic compounds to the recombinant five-stranded coiled-coil domain of COMP. Complex formation with benzene, cyclohexane, vitamin D3 and elaidic acid was demonstrated through increases in denaturation temperatures of 2-10 degreesC. For all-trans retinol and all-trans retinoic acid, an equilibrium dissociation constant KD = 0.6 microM was evaluated by fluorescence titration. Binding of benzene and all-trans retinol into the hydrophobic axial pore of the COMP coiled-coil domain was proven by the X-ray crystal structures of the corresponding complexes at 0.25 and 0.27 nm resolution, respectively. Benzene binds with its plane perpendicular to the pore axis. The binding site is between the two internal rings formed by Leu37 and Thr40 pointing into the pore of the COMP coiled-coil domain. The retinol beta-ionone ring is positioned in a hydrophobic environment near Thr40, and the 1.1 nm long isoprene tail follows a completely hydrophobic region of the pore. Its terminal hydroxyl group complexes with a ring of the five side chains of Gln54. A mutant in which Gln54 is replaced by Ile binds all-trans retinol with affinity similar to the wild-type, demonstrating that hydrophobic interactions are predominant. PMID:9736606

Urinary Tamm-Horsfall protein, albumin, vitamin D-binding protein, and retinol-binding protein as early biomarkers of chronic kidney disease in dogs.

PubMed

Chacar, Fernanda; Kogika, Márcia; Sanches, Talita R; Caragelasco, Douglas; Martorelli, Cínthia; Rodrigues, Camila; Capcha, Jose Manuel C; Chew, Dennis; Andrade, Lúcia

2017-06-01

Proteinuria is a marker and mediator of chronic kidney disease (CKD). In clinical practice, the urinary protein-to-creatinine ratio (UP/C) is of limited usefulness, because it indicates only the magnitude of proteinuria and not the origin of the loss (glomerular or tubular). The complete assessment of proteinuria includes quantitative and qualitative evaluations, both of which are required in order to optimize the therapy. In addition to measuring the UP/C, we performed SDS-PAGE and western blotting to determine the expression of albumin, vitamin D-binding protein (VDBP), retinol-binding protein (RBP), and Tamm-Horsfall protein (THP) in urine samples of 49 dogs: healthy (control) dogs ( n  =   9); and dogs with CKD ( n  =   40), stratified by stage. In the dogs with stage 3 or 4 CKD, there was a predominance of tubular proteins. Neither VDBP nor RBP was observed in the urine of the control dogs. Among the dogs with stage 1 or 2 CKD, VDBP and RBP were detected in those without proteinuria or with borderline proteinuria. The expression of urinary albumin was significantly higher in the stage 4 group than in any other group ( P  ≤   0.01). In the stage 4 group, urinary THP was either undetectable or lower than in the control group ( P  ≤   0.01). In conclusion, urinary VDBP and RBP might act as early markers of kidney injury, and a decrease in urinary THP could be an indicator of CKD progression. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Alterations of Hepatic Metabolism in Chronic Kidney Disease via D-box-binding Protein Aggravate the Renal Dysfunction.

PubMed

Hamamura, Kengo; Matsunaga, Naoya; Ikeda, Eriko; Kondo, Hideaki; Ikeyama, Hisako; Tokushige, Kazutaka; Itcho, Kazufumi; Furuichi, Yoko; Yoshida, Yuya; Matsuda, Masaki; Yasuda, Kaori; Doi, Atsushi; Yokota, Yoshifumi; Amamoto, Toshiaki; Aramaki, Hironori; Irino, Yasuhiro; Koyanagi, Satoru; Ohdo, Shigehiro

2016-03-04

Chronic kidney disease (CKD) is associated with an increase in serum retinol; however, the underlying mechanisms of this disorder are poorly characterized. Here, we found that the alteration of hepatic metabolism induced the accumulation of serum retinol in 5/6 nephrectomy (5/6Nx) mice. The liver is the major organ responsible for retinol metabolism; accordingly, microarray analysis revealed that the hepatic expression of most CYP genes was changed in 5/6Nx mice. In addition, D-box-binding protein (DBP), which controls the expression of several CYP genes, was significantly decreased in these mice. Cyp3a11 and Cyp26a1, encoding key proteins in retinol metabolism, showed the greatest decrease in expression in 5/6Nx mice, a process mediated by the decreased expression of DBP. Furthermore, an increase of plasma transforming growth factor-β1 (TGF-β1) in 5/6Nx mice led to the decreased expression of the Dbp gene. Consistent with these findings, the alterations of retinol metabolism and renal dysfunction in 5/6Nx mice were ameliorated by administration of an anti-TGF-β1 antibody. We also show that the accumulation of serum retinol induced renal apoptosis in 5/6Nx mice fed a normal diet, whereas renal dysfunction was reduced in mice fed a retinol-free diet. These findings indicate that constitutive Dbp expression plays an important role in mediating hepatic dysfunction under CKD. Thus, the aggravation of renal dysfunction in patients with CKD might be prevented by a recovery of hepatic function, potentially through therapies targeting DBP and retinol. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Prediagnostic level of serum retinol in relation to reduced risk of hepatocellular carcinoma.

PubMed

Yuan, Jian-Min; Gao, Yu-Tang; Ong, Choon-Nam; Ross, Ronald K; Yu, Mimi C

2006-04-05

Retinol and its derivatives (retinoids), which have antioxidant activity and promote cell differentiation, may protect against the development of hepatocellular carcinoma (HCC) by controlling hepatocellular differentiation and reducing inflammatory responses. We examined prospectively the relationship between prediagnostic serum concentrations of retinol, alpha-carotene; beta-carotene; beta-cryptoxanthin; lutein; lycopene; zeaxanthin; alpha-, gamma-, and delta-tocopherols; and selenium and the risk of developing HCC among 213 patients with HCC and 1087 matched control subjects from a cohort of 18,244 men in Shanghai, China, who were monitored from 1986 through 2001. Odds ratios (ORs) and 95% confidence intervals (CIs) for men by quartile of serum concentrations of micronutrients were estimated by using logistic regression with adjustment for cigarette smoking status, alcohol intake, self-reported history of physician-diagnosed hepatitis or liver cirrhosis at recruitment, and seropositivity for hepatitis B surface antigen (HBsAg). All statistical tests were two-sided. Men with high prediagnostic serum retinol levels had a lower risk of HCC than men in the lowest quartile (Q2 versus Q1, OR = 0.37, 95% CI = 0.22 to 0.61; Q3 versus Q1, OR = 0.30, 95% CI = 0.17 to 0.50; and Q4 versus Q1, OR = 0.13, 95% CI = 0.06 to 0.26; Ptrend < .001). A statistically significant interaction was observed between retinol and HBsAg seropositivity on HCC risk; HBsAg-positive men in the lowest tertile of retinol had a greater than 70-fold higher risk (OR = 72.7, 95% CI = 31.6 to 167.4) of HCC than HBsAg-negative men in the highest tertile of retinol (Pinteraction = .018). No independent effect of serum levels of alpha-carotene; beta-carotene; beta-cryptoxanthin; lutein; lycopene; zeaxanthin; alpha-, gamma-, and delta-tocopherols; or selenium on HCC risk were observed. High prediagnostic serum level of retinol is associated with a decreased risk of HCC in this population.

Dose-Response Relationship between Serum Retinol Levels and Survival in Patients with Colorectal Cancer: Results from the DACHS Study.

PubMed

Maalmi, Haifa; Walter, Viola; Jansen, Lina; Owen, Robert W; Ulrich, Alexis; Schöttker, Ben; Chang-Claude, Jenny; Hoffmeister, Michael; Brenner, Hermann

2018-04-19

Current knowledge on the role of retinol in the prognosis of patients with colorectal cancer (CRC) is very limited. We investigated the association of serum retinol levels with survival outcomes in a large cohort of 2908 CRC patients from Germany. Retinol concentrations were determined in serum collected shortly after diagnosis by mass spectrometry. Associations between serum retinol levels and survival outcomes were assessed using multivariable Cox regression and dose-response analyses. The joint association of serum retinol and serum 25-hydroxyvitamin D₃ (25(OH)D₃) with survival outcomes was also examined. During a median follow-up of 4.8 years, 787 deaths occurred, 573 of which were due to CRC. Dose-response curves showed an inverse relationship between serum retinol levels and survival endpoints in the range of <2.4 µmol/L, but no associations at higher levels. Low (<1.2 µmol/L) versus high (≥2.4 µmol/L) serum retinol levels were associated with poorer overall survival (Hazard ratio (HR) = 1.46, 95% confidence interval (CI) = 1.19⁻1.78, P-trend = 0.0003) and CRC-specific survival (HR = 1.69, 95% CI = 1.33⁻2.15, P-trend < 0.0001). Joint presence of low serum retinol (<1.2 µmol/L) and low 25(OH)D₃ (<30 nmol/L) was associated with a particularly strong decrease in overall and CRC-specific survival. Low serum retinol levels were identified as a predictor of poor survival in CRC patients, in particular when co-occurring with low serum concentrations of 25(OH)D₃. The clinical implications of these findings require further investigation.

Phylogeny of lion tamarins (Leontopithecus spp) based on interphotoreceptor retinol binding protein intron sequences.

PubMed

Mundy, N I; Kelly, J

2001-05-01

The evolutionary relationships of the lion tamarins (Leontopithecus) were investigated using nuclear interphotoreceptor retinol binding protein (IRBP) intron sequences. Phylogenetic reconstructions strongly support the monophyly of the genus, and a sister relationship between the golden lion tamarin, Leontopithecus rosalia, and the black lion tamarin, L. chrysopygus, to the exclusion of the golden-headed lion tamarin, L. chrysomelas. The most parsimonious evolutionary reconstruction suggests that the ancestral lion tamarin and the common ancestor of L. rosalia and L. chrysopygus had predominantly black coats. This reconstruction is not consistent with a theory of orthogenetic evolution of coat color that was based on coat color evolution in marmosets and tamarins. An alternative reconstruction that is consistent with metachromism requires that ancestral lion tamarins had agouti hairs. Copyright 2001 Wiley-Liss, Inc.

Improved simultaneous determination method of beta-carotene and retinol with saponification in human serum and rat liver.

PubMed

Hosotani, Keisuke; Kitagawa, Masahiro

2003-07-05

Among the many simultaneous determination methods for carotenoid and retinoid, there are only a few reports including the saponification process. However, the yields of beta-carotene and retinol were higher when using this process. In this study, the analytical conditions, including saponification, were investigated. The extraction solvent was n-hexane and the sample solvent was HPLC mobile phase in the beta-carotene and retinol analysis. BHT as an antioxidant was added at concentrations of 0.125 and 0.025%, respectively, to ethanol and n-hexane phase in the extraction process for serum. The recovery rates were 99.7, 93.7 and 98.3% for beta-carotene, retinol and retinyl palmitate in serum, respectively, and 107.1, 92.8 and 98.8% for beta-carotene, retinol and retinyl palmitate in liver, respectively. The within-day coefficients of variation (C.V.) were 6.0% for serum and 4.7% for liver in the case of beta-carotene, 7.1% for serum, and 5.1% for liver in the case of retinol. The between-day coefficients of variation were 2.7% for serum and 2.7% for liver in the case of beta-carotene, and for retinol, 6.4% for serum and 2.7% for liver.

Biologically active protein fragments containing specific binding regions of serum albumin or related proteins

NASA Technical Reports Server (NTRS)

Carter, Daniel C. (Inventor)

1998-01-01

In accordance with the present invention, biologically active protein fragments can be constructed which contain only those specific portions of the serum albumin family of proteins such as regions known as subdomains IIA and IIIA which are primarily responsible for the binding properties of the serum albumins. The artificial serums that can be prepared from these biologically active protein fragments are advantageous in that they can be produced much more easily than serums containing the whole albumin, yet still retain all or most of the original binding potential of the full albumin proteins. In addition, since the protein fragment serums of the present invention can be made from non-natural sources using conventional recombinant DNA techniques, they are far safer than serums containing natural albumin because they do not carry the potentially harmful viruses and other contaminants that will be found in the natural substances.

Immunohistochemical study of retinol-binding protein in livers of polar bears (Thalarctos maritimus).

PubMed

Heier, A; Gröne, A; Völlm, J; Kübber-Heiss, A; Bacciarini, L N

2003-03-01

Liver tumors of unknown cause have frequently been described in polar bears. Concurrent decrease of vitamin A levels and chronic liver disease are associated with hepatic carcinogenesis in humans. More than 90% of the body's vitamin A is stored in the liver, where it is bound to an intracellular retinol-binding protein (RBP). Therefore, in this retrospective study, RBP was assessed by immunohistochemistry in liver sections of 11 polar bears. Two of these polar bears had hepatocellular carcinoma, four showed other chronic liver changes, and five had normal livers. In normal livers, the cytoplasm stained diffusely positive with intensely staining cytoplasmic granules. RBP staining was evaluated and the abundance of diffuse cytoplasmic staining and intracytoplasmic large granules was determined. All cases with pathologic liver changes had markedly decreased staining intensities for RBP compared with normal livers. The findings of this study suggest that in polar bears, as in humans, vitamin A metabolism may play a role in hepatic carcinogenesis.

Fusion protein of retinol-binding protein and albumin domain III reduces liver fibrosis

PubMed Central

Lee, Hongsik; Jeong, Hyeyeun; Park, Sangeun; Yoo, Wonbaek; Choi, Soyoung; Choi, Kyungmin; Lee, Min-Goo; Lee, Mihwa; Cha, DaeRyong; Kim, Young-Sik; Han, Jeeyoung; Kim, Wonkon; Park, Sun-Hwa; Oh, Junseo

2015-01-01

Activated hepatic stellate cells (HSCs) play a key role in liver fibrosis, and inactivating HSCs has been considered a promising therapeutic approach. We previously showed that albumin and its derivative designed for stellate cell-targeting, retinol-binding protein–albumin domain III fusion protein (referred to as R-III), inactivate cultured HSCs. Here, we investigated the mechanism of action of albumin/R-III in HSCs and examined the anti-fibrotic potential of R-III in vivo. R-III treatment and albumin expression downregulated retinoic acid (RA) signaling which was involved in HSC activation. RA receptor agonist and retinaldehyde dehydrogenase overexpression abolished the anti-fibrotic effect of R-III and albumin, respectively. R-III uptake into cultured HSCs was significantly decreased by siRNA-STRA6, and injected R-III was localized predominantly in HSCs in liver. Importantly, R-III administration reduced CCl4- and bile duct ligation-induced liver fibrosis. R-III also exhibited a preventive effect against CCl4-inducd liver fibrosis. These findings suggest that the anti-fibrotic effect of albumin/R-III is, at least in part, mediated by downregulation of RA signaling and that R-III is a good candidate as a novel anti-fibrotic drug. PMID:25864124

Binding of perlecan to transthyretin in vitro.

PubMed Central

Smeland, S; Kolset, S O; Lyon, M; Norum, K R; Blomhoff, R

1997-01-01

Transthyretin is one of two specific proteins involved in the transport of thyroid hormones in plasma; it possesses two binding sites for serum retinol-binding protein. In the present study we demonstrate that transthyretin also interacts in vitro with [35S]sulphate-labelled material from the medium of HepG2 cells. By using the same strategy as for purifying serum retinol-binding protein, [35S]sulphate-labelled medium was specifically eluted from a transthyretin-affinity column. Ion-exchange chromatography showed that the material was highly polyanionic, and its size and alkali susceptibility suggested that it was a proteoglycan. Structural analyses with chondroitinase ABC lyase and nitrous acid revealed that approx. 20% was chondroitin sulphate and 80% heparan sulphate. Immunoprecipitation showed that the [35S]sulphate-labelled material contained perlecan. Further analysis by binding studies revealed specific and saturable binding of 125I-transthyretin to perlecan-enriched Matrigel. Because inhibition of sulphation by treating HepG2 cells with sodium chlorate increased the affinity of the perlecan for transthyretin, and [3H]heparin was not retained by the transthyretin affinity column, the binding is probably mediated by the core protein and is not a protein-glycosaminoglycan interaction. Because perlecan is released from transthyretin in water, the binding might be due to hydrophobic interactions. PMID:9307034

Comparison of a fluorometric assay kit with high-performance liquid chromatography for the assessment of serum retinol concentration.

PubMed

Elom, Aglago Kouassivi; Imane, El Menchawy; Kaoutar, Benjeddou; Khalid, El Kari; Asmaa, El Hamdouchi; Mehdi, Azlaf; Noureddine, El Haloui; Hassan, Aguenaou

2015-06-01

Although high-performance liquid chromatography (HPLC) is the commonly used method for the analysis of retinol in biological samples, simple and rapid test kits are available. This study compared a rapid test kit (ICHECK Fluoro®) to HPLC for the assessment of serum retinol concentrations. For the analysis by HPLC, sample preparation included standard deproteinization and extraction phases. The analysis by ICHECK was performed by injecting serum into IEX reagent vials (n=89) and mixing manually for separation. After precipitation of the proteins, the vial was introduced into the chamber of the ICHECK Fluoro and analysed at 0 min (ICHECK0min) and 15 min later (ICHECK15min). Bland and Altman approach was applied to test the agreement between HPLC and ICHECK. Mean HPLC, ICHECK0min and ICHECK15min values were 421.2±106.0 µg/L, 423.1±118.3 µg/L and 413.2±107.6 µg/L, respectively. Retinol concentrations significantly decreased in the IEX solution over time (p<0.001). No significant proportional bias was observed between HPLC and ICHECK0min (r-0.038, p=0.73) and ICHECK15min (r=-0.024, p=0.82). Fixed biases (HPLC minus ICHECK) for ICHECK0min and ICHECK15min were respectively -1.9±23.1 µg/l (p=0.45) and 8.0±22.7 µg/l (p=0.002). ICHECK Fluoro may offer a reliable mean for assessing serum retinol for measurements performed with no significant time delay.

Serum carotenoid, retinol and tocopherol concentrations and risk of cervical cancer among Chinese women.

PubMed

Zhang, Yuan-Yuan; Lu, Ling; Abliz, Guzalnur; Mijit, Fatima

2015-01-01

Despite many epidemiological studies on the effects of dietary antioxidant micronutrients on risk of cervical cancer, the findings remain uncertain and little evidence is available for serum nutrient markers. The present study aimed to examine the relationship between serum carotenoid, retinol and tocopherol concentrations and risk of cervical cancer among Chinese women. We conducted a hospital-based case- control study in which 358 adults (158 incident cases and 200 controls) were recruited from Xinjiang, China. Serum levels of carotenoids (α-carotene, β-carotene, β-cryptoxanthin, lycopene and lutein/zeaxanthin), retinol, and tocopherols (α-tocopherol and γ-tocopherol) were assessed by reverse-phase high-performance liquid chromatography. We found inverse associations between serum carotenoid (α-carotene, β-carotene, and lutein/zeaxanthin) and tocopherol (α-tocopherol) concentrations and the risk of cervical cancer after adjusting for potential confounders, but a null association for retinol. The ORs for 1-SD increase were 0.71 (95% CI: 0.56- 0.92; p=0.003) for total carotenoids and 0.75 (95% CI: 0.60-0.94; p=0.008) for total tocopherols. These results show that higher serum concentrations of some carotenoids and tocopherols are associated with a lower risk of cervical cancer among Chinese women.

Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism).

PubMed

Daughaday, W H; Trivedi, B

1987-07-01

It has recently been recognized that human serum contains a protein that specifically binds human growth hormone (hGH). This protein has the same restricted specificity for hGH as the membrane-bound GH receptor. To determine whether the GH-binding protein is a derivative of, or otherwise related to, the GH receptor, we have examined the serum of three patients with Laron-type dwarfism, a condition in which GH refractoriness has been attributed to a defect in the GH receptor. The binding of 125I-labeled hGH incubated with serum has been measured after gel filtration of the serum through an Ultrogel AcA 44 minicolumn. Nonspecific binding was determined when 125I-hGH was incubated with serum in the presence of an excess of GH. Results are expressed as percent of specifically bound 125I-hGH and as specific binding relative to that of a reference serum after correction is made for endogenous GH. The mean +/- SEM of specific binding of sera from eight normal adults (26-46 years of age) was 21.6 +/- 0.45%, and the relative specific binding was 101.1 +/- 8.6%. Sera from 11 normal children had lower specific binding of 12.5 +/- 1.95% and relative specific binding of 56.6 +/- 9.1%. Sera from three children with Laron-type dwarfism lacked any demonstrable GH binding, whereas sera from 10 other children with other types of nonpituitary short stature had normal relative specific binding. We suggest that the serum GH-binding protein is a soluble derivative of the GH receptor. Measurement of the serum GH-binding protein may permit recognition of other abnormalities of the GH receptor.

High serum level of retinol and α-tocopherol affords protection against oral cancer in a multiethnic population.

PubMed

Athirajan, Vimmitra; Razak, Ishak Abdul; Thurairajah, Nalina; Ghani, Wan Maria Nabillah; Ching, Helen-Ng Lee; Yang, Yi-Hsin; Peng, Karen-Ng Lee; Abdul Rahman, Zainal Ariff; Mustafa, Wan Mahadzir Wan; Abraham, Mannil Thomas; Kiong, Tay Keng; Mun, Yuen Kar; Jalil, Norma; Zain, Rosnah Binti

2014-01-01

A comparative cross-sectional study involving oral cancer patients and healthy individuals was designed to investigate associations between retinol, α-tocopherol and β-carotene with the risk of oral cancer. This study included a total of 240 matched cases and controls where subjects were selected from the Malaysian Oral Cancer Database and Tissue Bank System (MOCDTBS). Retinol, α-tocopherol and β-carotene levels and intake were examined by high-performance liquid chromatography (HPLC) and food frequency questionnaire (FFQ) respectively. It was found that results from the two methods applied did not correlate, so that further analysis was done using the HPLC method utilising blood serum. Serum levels of retinol and α-tocopherol among cases (0.177±0.081, 1.649±1.670μg/ml) were significantly lower than in controls (0.264±0.137, 3.225±2.054μg/ml) (p<0.005). Although serum level of β-carotene among cases (0.106±0.159 μg/ml) were lower compared to controls (0.134±0.131μg/ml), statistical significance was not observed. Logistic regression analysis showed that high serum level of retinol (OR=0.501, 95% CI=0.254-0.992, p<0.05) and α-tocopherol (OR=0.184, 95% CI=0.091-0.370, p<0.05) was significantly related to lower risk of oral cancer, whereas no relationship was observed between β-carotene and oral cancer risk. High serum levels of retinol and α-tocopherol confer protection against oral cancer risk.

Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism).

PubMed Central

Daughaday, W H; Trivedi, B

1987-01-01

It has recently been recognized that human serum contains a protein that specifically binds human growth hormone (hGH). This protein has the same restricted specificity for hGH as the membrane-bound GH receptor. To determine whether the GH-binding protein is a derivative of, or otherwise related to, the GH receptor, we have examined the serum of three patients with Laron-type dwarfism, a condition in which GH refractoriness has been attributed to a defect in the GH receptor. The binding of 125I-labeled hGH incubated with serum has been measured after gel filtration of the serum through an Ultrogel AcA 44 minicolumn. Nonspecific binding was determined when 125I-hGH was incubated with serum in the presence of an excess of GH. Results are expressed as percent of specifically bound 125I-hGH and as specific binding relative to that of a reference serum after correction is made for endogenous GH. The mean +/- SEM of specific binding of sera from eight normal adults (26-46 years of age) was 21.6 +/- 0.45%, and the relative specific binding was 101.1 +/- 8.6%. Sera from 11 normal children had lower specific binding of 12.5 +/- 1.95% and relative specific binding of 56.6 +/- 9.1%. Sera from three children with Laron-type dwarfism lacked any demonstrable GH binding, whereas sera from 10 other children with other types of nonpituitary short stature had normal relative specific binding. We suggest that the serum GH-binding protein is a soluble derivative of the GH receptor. Measurement of the serum GH-binding protein may permit recognition of other abnormalities of the GH receptor. PMID:3474620

High Serum Retinol as a Relevant Contributor to Low Bone Mineral Density in Postmenopausal Osteoporotic Women.

PubMed

Navarro-Valverde, Cristina; Caballero-Villarraso, Javier; Mata-Granados, José M; Casado-Díaz, Antonio; Sosa-Henríquez, Manuel; Malouf-Sierra, Jorge; Nogués-Solán, Xavier; Rodríguez-Mañas, Leocadio; Cortés-Gil, Xavier; Delgadillo-Duarte, Joaquín; Quesada-Gómez, José Manuel

2018-06-01

There is controversial information about the impact of vitamin A on bone. Some epidemiological studies show that excessive intake of vitamin A, or an excess of serum vitamin A, has related with adverse impact on bone mass; however, other studies did not find these links, and some authors have proposed that this vitamin might promote a better bone health. The present work aims to contribute to clarify the real role of vitamin A in bone tissue. For this purpose, a cross-sectional study of 154 osteoporotic non-treated postmenopausal women (> 65 years old) was carried out. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry. We assessed concentrations of serum retinol, osteocalcin, parathyroid hormone, alkaline phosphatase, calcium, and phosphorus. We also studied demographic and anthropometric parameters. Spearman's correlations between retinol levels and other variables found negative correlations with BMD in both lumbar spine (R = - 0.162, P < 0.01) and femoral neck (R = - 0.182, P < 0.01), as well as alkaline phosphatase (R = - 0.110; P < 0.05) and phosphorus (R = - 0.110; P < 0.05). A positive correlation between retinol and fertile window was observed (R = 0.158; P < 0.01). After multivariable adjustment, we still found a negative correlation between serum retinol and BMD, both at the lumbar spine (R = - 0.210; P < 0.01) and at the femoral neck (R = - 0.324, P < 0.001). It is concluded that elevated serum-retinol levels are associated with an increased risk of low bone mass and thus with osteoporotic fractures. Therefore, osteoporosis-risk assessment should include quantification of serum metabolite of vitamin A.

Serial changes in selected serum constituents in low birth weight infants on peripheral parenteral nutrition with different zinc and copper supplements.

PubMed

Lockitch, G; Pendray, M R; Godolphin, W J; Quigley, G

1985-07-01

One hundred and five infants of birth weight 2000 g or less who received peripherally administered parenteral nutrition for periods of three or more weeks, were randomly assigned to groups receiving different amounts of zinc and copper supplement. The blood concentrations of zinc, copper, retinol-binding protein, prealbumin, alkaline phosphatase and aspartate transaminase were followed weekly. Mean serum zinc, retinol-binding protein and prealbumin declined significantly over time while alkaline phosphatase rose. Only the group receiving the highest zinc supplement maintained a mean serum zinc concentration within the normal range at seven weeks. No difference in the protein or enzyme concentrations was found between the different zinc supplement groups. No difference was seen in serum copper or ceruloplasmin between copper dose groups although one intravenous supplement was double that of the other.

Lipodystrophy, lipid profile changes, and low serum retinol and carotenoid levels in children and adolescents with acquired immunodeficiency syndrome.

PubMed

Battistini, Tânia Regina Beraldo; Sarni, Roseli Oselka Saccardo; de Souza, Fabíola Isabel Suano; Pitta, Tassiana Sacchi; Fernandes, Ana Paula; Hix, Sonia; Fonseca, Fernando Luiz Affonso; Tardini, Priscila Chemiotti; dos Santos, Valter Pinho; Lopez, Fábio Ancona

2010-06-01

To assess serum retinol and levels of carotenoids in children and adolescents with acquired immunodeficiency syndrome (AIDS) and to correlate low serum retinol and carotenoid levels with the presence of lipodystrophy, lipid profile changes, lipid peroxidation, and insulin resistance. A cross-sectional, controlled observational study was carried out with 30 children and adolescents with AIDS (mean age 9.1 y) receiving antiretroviral therapy (median length of treatment 28.4 mo), including 30 uninfected healthy controls matched for age and gender. Clinical and laboratory assessments were performed to determine nutritional status, presence of lipodystrophy, serum concentrations of retinol, beta-carotene, lycopene, lipid profile (high-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triacylglycerols), lipid peroxidation (thiobarbituric acid-reactive substances), glycemia, and serum insulin (homeostasis model assessment for insulin resistance, cutoff point >3). Statistical analysis was done with chi-square test and Student's t test. Lipodystrophy was observed in 53.3% of patients with AIDS, and dyslipidemia was detected in 60% and 23% of subjects with human immunodeficiency virus and control subjects, respectively (P = 0.004). A higher prevalence of retinol deficiency (60% versus 26.7%, P = 0.009) and beta-carotene deficiency (23.3% versus 3.3%, P = 0.026) was found in the group with human immunodeficiency virus than in the control group. No correlation was found for low retinol and beta-carotene levels, changes in lipid and glucose metabolism, or lipodystrophy in children and adolescents with AIDS. Despite the high frequency of dyslipidemia, lipodystrophy, and retinol and beta-carotene deficiencies, it was not possible to demonstrate a correlation of these findings with lipid peroxidation and insulin resistance. More studies are needed to investigate the causes of retinol and beta-carotene deficiencies in this population and the clinical

Domain-Swapped Dimers of Intracellular Lipid-Binding Proteins: Evidence for Ordered Folding Intermediates.

PubMed

Assar, Zahra; Nossoni, Zahra; Wang, Wenjing; Santos, Elizabeth M; Kramer, Kevin; McCornack, Colin; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James H

2016-09-06

Human Cellular Retinol Binding Protein II (hCRBPII), a member of the intracellular lipid-binding protein family, is a monomeric protein responsible for the intracellular transport of retinol and retinal. Herein we report that hCRBPII forms an extensive domain-swapped dimer during bacterial expression. The domain-swapped region encompasses almost half of the protein. The dimer represents a novel structural architecture with the mouths of the two binding cavities facing each other, producing a new binding cavity that spans the length of the protein complex. Although wild-type hCRBPII forms the dimer, the propensity for dimerization can be substantially increased via mutation at Tyr60. The monomeric form of the wild-type protein represents the thermodynamically more stable species, making the domain-swapped dimer a kinetically trapped entity. Hypothetically, the wild-type protein has evolved to minimize dimerization of the folding intermediate through a critical hydrogen bond (Tyr60-Glu72) that disfavors the dimeric form. Copyright © 2016 Elsevier Ltd. All rights reserved.

Serum lipopolysaccharide-binding protein prediction of severe bacterial infection in cirrhotic patients with ascites.

PubMed

Albillos, Agustín; de-la-Hera, Antonio; Alvarez-Mon, Melchor

2004-05-15

Serum lipopolysaccharide-binding protein is increased in a subset of non-infected ascitic cirrhotic patients, a finding previously related to bacterial passage from the gut to the circulation without overt infection. We prospectively analysed the risk factors associated with a first episode of severe bacterial infection in 84 ascitic cirrhotics, followed up for a median of 46 weeks. The cumulative probability of such infection in patients with raised and normal lipopolysaccharide-binding protein was 32.4% and 8.0% (p=0.004), respectively. Increased lipopolysaccharide-binding protein was the only factor independently associated with severe bacterial infection in a multivariate analysis (relative risk 4.49, 95% CI 1.42-14.1). Monitoring of serum lipopolysaccharide-binding protein could, therefore, help to target cirrhotic patients with ascites for antibiotic prophylaxis.

[Correlation of retinol binding protein 4 with 
metabolic indexes of glucose and 
lipid, bile cholesterol saturation index].

PubMed

Wang, Wen; Li, Nianfeng

2015-06-01

To measure retinol binding protein 4 (RBP4) levels in serum and bile and to analyze their relationship with insulin resistance, dyslipidemia or cholesterol saturation index (CSI).
 A total of 60 patients with gallstone were divided into a diabetes group (n=30) and a control group (n=30). The concentrations of RBP4 in serum and bile were detected by enzyme-linked immunosorbent assay (ELISA). Enzyme colorimetric method was used to measure the concentration of biliary cholesterol, bile acid and phospholipid. Biliary CSI was calculated by Carey table. Partial correlation and multiple linear regression analysis were used to evaluate the correlation between the RBP4 levels in serum or bile and the above indexes.
 The RBP4 concentrations in serum and bile in the diabetes group were significantly elevated compared with those in the control group (both P<0.01). There was no significant difference in the serum total bile acid (TBA), serum triglyceride (TG), serum high-density lipoprotein (HDL), bile TBA, bile total cholesterol (TC) , bile phospholipids and bile CSI between the 2 groups (all P>0.05); but the serum TC, low density lipoprotein (LDL), fasting blood glucose (FBG), fasting insulin (FINS), and homeostasis model assessment for insulin resistance (HOMA-IR) in the diabetes group were significantly increased compared to those in the control group (all P<0.05). The partial correlation analysis, which was adjusted by age, showed that the bile RBP4 was positively correlated with body mass index (BMI), waist circumference (WC), FINS, FBG, TC, LDL and HOMA-IR (r=0.283, 0.405, 0.685, 0.667, 0.553, 0.424 and 0.735, respectively), and the serum RBP4 was also positively correlated with the WC, FINS, FBG, TC, LDL and HOMA-IR (r=0.317, 0.734, 0.609, 0.528, 0.386 and 0.751, respectively). Stepwise multivariate linear regression analysis suggested that the HOMA-IR, BMI and WC were independently correlated with the level of bile RBP4 (multiple regression equation: Ybile RBP4

13C Natural Abundance of Serum Retinol Is a Novel Biomarker for Evaluating Provitamin A Carotenoid-Biofortified Maize Consumption in Male Mongolian Gerbils.

PubMed

Gannon, Bryan M; Pungarcher, India; Mourao, Luciana; Davis, Christopher R; Simon, Philipp; Pixley, Kevin V; Tanumihardjo, Sherry A

2016-07-01

Crops such as maize, sorghum, and millet are being biofortified with provitamin A carotenoids to ensure adequate vitamin A (VA) intakes. VA assessment can be challenging because serum retinol concentrations are homeostatically controlled and more sensitive techniques are resource-intensive. We investigated changes in serum retinol relative differences of isotope amount ratios of (13)C/(12)C (δ(13)C) caused by natural (13)C fractionation in C3 compared with C4 plants as a biomarker to detect provitamin A efficacy from biofortified (orange) maize and high-carotene carrots. The design was a 2 × 2 × 2 maize (orange compared with white) by carrot (orange compared with white) by a VA fortificant (VA+ compared with VA-) in weanling male Mongolian gerbils (n = 55), which included a 14-d VA depletion period and a 62-d treatment period (1 baseline and 8 treatment groups; n = 5-7/group). Liver VA and serum retinol were quantified, purified by HPLC, and analyzed by GC combustion isotope ratio mass spectrometry for (13)C. Treatments affected liver VA concentrations (0.048 ± 0.039 to 0.79 ± 0.24 μmol/g; P < 0.0001) but not overall serum retinol concentrations (1.38 ± 0.22 μmol/L). Serum retinol and liver VA δ(13)C were significantly correlated (R(2) = 0.92; P < 0.0001). Serum retinol δ(13)C differentiated control groups that consumed white maize and white carrots (-27.1 ± 1.2 δ(13)C‰) from treated groups that consumed orange maize and white carrots (-21.6 ± 1.4 δ(13)C‰ P < 0.0001) and white maize and orange carrots (-30.6 ± 0.7 δ(13)C‰ P < 0.0001). A prediction model demonstrated the relative contribution of orange maize to total dietary VA for groups that consumed VA from mixed sources. Provitamin A efficacy and quantitative estimation of the relative contribution to dietary VA were demonstrated with the use of serum retinol δ(13)C. This method could be used for maize efficacy or effectiveness studies and with other C4 crops biofortified with provitamin A

Comparison of a New Multiplex Immunoassay for Measurement of Ferritin, Soluble Transferrin Receptor, Retinol-Binding Protein, C-Reactive Protein and α1-Acid-glycoprotein Concentrations against a Widely-Used s-ELISA Method

PubMed Central

Henderson, Amanda M.; Samson, Kaitlyn L. I.; Aljaadi, Abeer M.; Devlin, Angela M.; Becquey, Elodie; Wirth, James P.

2018-01-01

Recently, a multiplex ELISA (Quansys Biosciences) was developed that measures ferritin, soluble transferrin receptor (sTfR), retinol-binding protein (RBP), C-reactive protein (CRP), α1-acid glycoprotein (AGP), thyroglobulin, and histidine-rich protein 2. Our primary aim was to conduct a method-comparison study to compare five biomarkers (ferritin, sTfR, RBP, CRP, and AGP) measured with the Quansys assay and a widely-used s-ELISA (VitMin Lab, Willstaett, Germany) with use of serum samples from 180 women and children from Burkina Faso, Cambodia, and Malaysia. Bias and concordance were used to describe the agreement in values measured by the two methods. We observed poor overall agreement between the methods, both with regard to biomarker concentrations and deficiency prevalence estimates. Several measurements were outside of the limit of detection with use of the Quansys ELISA (total n = 42 for ferritin, n = 2 for sTfR, n = 0 for AGP, n = 5 for CRP, n = 22 for RBP), limiting our ability to interpret assay findings. Although the Quansys ELISA has great potential to simplify laboratory analysis of key nutritional and inflammation biomarkers, there are some weaknesses in the procedures. Overall, we found poor comparability of results between methods. Besides addressing procedural issues, additional validation of the Quansys against a gold standard method is warranted for future research. PMID:29393894

Identification of V122I (Val122Ile) transthyretin cardiac amyloidosis (ATTR) using serum retinol-binding protein 4 (RBP4) and a clinical prediction model

PubMed Central

Arvanitis, Marios; Koch, Clarissa M; Chan, Gloria G.; Arancivia, Celia M.T.; LaValley, Michael; Jacobson, Daniel; Berk, John L.; Connors, Lawreen H.; Ruberg, Frederick L.

2017-01-01

Importance Transthyretin amyloid cardiomyopathy (ATTR) is an under-recognized cause of heart failure (HF) in the elderly, owing in part to difficulty in diagnosis. ATTR can result from mutant TTR protein with one of the most common mutations in the United States, V122I, present in 3.43% of African Americans. Objective To determine whether serum retinol-binding protein 4 (RBP4), an endogenous TTR ligand, could be used as a diagnostic test for ATTR V122I amyloidosis. Design Combined prospective and retrospective cohort study Setting Tertiary care referral center Participants Fifty prospectively genotyped African American patients over age 60 years with non-amyloid HF and cardiac wall thickening, and a comparator cohort of biopsy proven ATTR V122I amyloidosis patients (n=25) comprised the development cohort. Twenty-seven prospectively genotyped African American patients and 9 ATTR V122I amyloidosis patients comprised the validation cohort. Main Outcomes and Measures Circulating RBP4, TTR, B-type natriuretic peptide (BNP) and troponin I (TnI) concentrations, electrocardiography (ECG), echocardiography, and clinical characteristics were assessed in all patients. Receiver operating characteristic (ROC) analysis was performed to identify optimal thresholds for ATTR V122I amyloidosis identification. A clinical prediction rule was developed using penalized logistic regression, evaluated using ROC analysis and validated in an independent cohort of cases and controls. Results Age, gender, BNP and TnI were similar between ATTR V122I amyloidosis patients and controls. Serum RBP4 concentration was lower in patients with ATTR V122I amyloidosis compared to non-amyloid controls (31.5 vs. 49.4 ug/ml, p < 0.001) and the difference persisted after controlling for potential confounding parameters. Left ventricular ejection fraction (LVEF) was lower in ATTR V122I amyloidosis (40% vs. 57%, p<0.001), while interventricular septal diameter (IVSd) was higher (16 vs. 14 mm, p<0.001). ROC

Iron deficiency and anemia are associated with low retinol levels in children aged 1 to 5 years.

PubMed

Saraiva, Bárbara C A; Soares, Michele C C; Santos, Luana C dos; Pereira, Simone C L; Horta, Paula M

2014-01-01

To analyze the occurrence of anemia and iron deficiency in children aged 1 to 5 years and the association of these events and retinol deficiency. This was an observational analytic cross-sectional study conducted in Vitoria, ES, Brazil, between April and August of 2008, with healthy children aged 1 to 5 years (n=692) that lived in areas covered by primary healthcare services. Sociodemographic and economic conditions, dietary intake (energy, protein, iron, and vitamin A ingestion), anthropometric data (body mass index-for-age and height-for-age), and biochemical parameters (ferritin, hemoglobin, and retinol serum) were collected. The prevalence of anemia, iron deficiency, and retinol deficiency was 15.7%, 28.1%, and 24.7%, respectively. Univariate analysis showed a higher prevalence of anemia (PR: 4.62, 95% CI: 3.36, 6.34, p<0.001) and iron deficiency (PR: 4.51, 95% CI: 3.30, 6.17, p<0.001) among children with retinol deficiency. The same results were obtained after adjusting for socioeconomic and demographic conditions, dietary intake, and anthropometric variables. There was a positive association between ferritin vs. retinol serum (r=0.597; p<0.001) and hemoglobin vs. retinol serum (r=0.770; p<0.001). Anemia and iron deficiency were associated with low levels of serum retinol in children aged 1 to 5 years, and a positive correlation was verified between serum retinol and serum ferritin and hemoglobin levels. These results indicate the importance of initiatives encouraging the development of new treatments and further research regarding retinol deficiency. Copyright © 2014 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

Interphotoreceptor Retinoid-Binding Protein Protects Retinoids from Photodegradation

PubMed Central

Gonzalez-Fernandez, Federico; Betts-Obregon, Brandi; Yust, Brian; Mimun, Joshua; Sung, Dongjin; Sardar, Dhiraj; Tsin, Andrew T.

2015-01-01

Retinol degrades rapidly in light into a variety of photoproducts. It is remarkable that visual cycle retinoids can evade photodegradation as they are exchanged between the photoreceptors, retinal pigment epithelium and Müller glia. Within the interphotoreceptor matrix, all-trans retinol, 11-cis retinol and retinal are bound by interphotoreceptor retinoid-binding protein (IRBP). Apart from its role in retinoid trafficking and targeting, could IRBP have a photoprotective function? HPLC was used to evaluate the ability of IRBP to protect all-trans and 11-cis retinols from photodegradation when exposed to incandescent light (0 to 8,842 μW/cm2); time periods of 0 – 60 min, and bIRBP: retinol molar ratios of 1:1 to 1:5. bIRBP afforded a significant prevention of both all-trans and 11-cis retinol to rapid photodegradation. The effect was significant over the entire light intensity range tested, and extended to the bIRBP: retinol ratio 1:5. In view of the continual exposure of the retina to light, and the high oxidative stress in the outer retina, our results suggest IRBP may have an important protective role in the visual cycle by reducing photodegradation of all-trans and 11-cis retinols. This role of IRBP is particularly relevant in the high flux conditions of the cone visual cycle. PMID:25565073

Modulated photophysics of a cationic DNA-staining dye inside protein bovine serum albumin: study of binding interaction and structural changes of protein.

PubMed

Samanta, Anuva; Jana, Sankar; Ray, Debarati; Guchhait, Nikhil

2014-01-01

The binding affinity of cationic DNA-staining dye, propidium iodide, with transport protein, bovine serum albumin, has been explored using UV-vis absorption, fluorescence, and circular dichroism spectroscopy. Steady state and time resolved fluorescence studies authenticate that fluorescence quenching of bovine serum albumin by propidium iodide is due to bovine serum albumin-propidium iodide complex formation. Thermodynamic parameters obtained from temperature dependent spectral studies cast light on binding interaction between the probe and protein. Site marker competitive binding has been encountered using phenylbutazone and flufenamic acid for site I and site II, respectively. Energy transfer efficiency and distance between bovine serum albumin and propidium iodide have been determined using Förster mechanism. Structural stabilization or destabilization of protein by propidium iodide has been investigated by urea denaturation study. The circular dichroism study as well as FT-IR measurement demonstrates some configurational changes of the protein in presence of the dye. Docking studies support the experimental data thereby reinforcing the binding site of the probe to the subdomain IIA of bovine serum albumin. Copyright © 2013 Elsevier B.V. All rights reserved.

Modulated photophysics of a cationic DNA-staining dye inside protein bovine serum albumin: Study of binding interaction and structural changes of protein

NASA Astrophysics Data System (ADS)

Samanta, Anuva; Jana, Sankar; Ray, Debarati; Guchhait, Nikhil

2014-03-01

The binding affinity of cationic DNA-staining dye, propidium iodide, with transport protein, bovine serum albumin, has been explored using UV-vis absorption, fluorescence, and circular dichroism spectroscopy. Steady state and time resolved fluorescence studies authenticate that fluorescence quenching of bovine serum albumin by propidium iodide is due to bovine serum albumin-propidium iodide complex formation. Thermodynamic parameters obtained from temperature dependent spectral studies cast light on binding interaction between the probe and protein. Site marker competitive binding has been encountered using phenylbutazone and flufenamic acid for site I and site II, respectively. Energy transfer efficiency and distance between bovine serum albumin and propidium iodide have been determined using Förster mechanism. Structural stabilization or destabilization of protein by propidium iodide has been investigated by urea denaturation study. The circular dichroism study as well as FT-IR measurement demonstrates some configurational changes of the protein in presence of the dye. Docking studies support the experimental data thereby reinforcing the binding site of the probe to the subdomain IIA of bovine serum albumin.

Increased retinol-free RBP4 contributes to insulin resistance in gestational diabetes mellitus.

PubMed

Chen, Yanmin; Lv, Ping; Du, Mengkai; Liang, Zhaoxia; Zhou, Menglin; Chen, Danqing

2017-07-01

Retinol-binding protein 4 (RBP4) is a circulating retinol transporter that is strongly associated with insulin resistance. The aim of this study was to evaluate the RBP4 and retinol level in rat model of gestational diabetes mellitus and the relationship between retinol-free RBP4 (apo-RBP4), retinol-bound RBP4 (holo-RBP4) and insulin resistance. Pregnant rats were administered streptozotocin to induce diabetes. The RBP4 and retinol levels were evaluated in GDM and normal pregnant rats. After then, normal pregnant rats were divided into two groups to receive either apo-RBP4 or vehicle injection. The metabolic parameters and insulin signaling in adipose tissue, skeletal muscle and liver were determined in apo-RBP4 and control groups. Primary human adipocytes were cultured in vitro with different proportions of apo-RBP4 and holo-RBP4 for 24 h. The interaction between RBP4 and STRA6 was assessed by co-immunoprecipitation, and the expression of JAK-STAT pathway and insulin signaling were detected by Western blotting and immunofluorescence. We found increases in serum RBP4 levels and the RBP4:retinol ratio but not in the retinol levels in GDM rats. Exogenous apo-RBP4 injection attenuated insulin sensitivity in pregnant rats. In vitro, a prolonged interaction between RBP4 and STRA6 was observed when apo-RBP4 was present. In response to increased apo-RBP4 levels, cells showed elevated activation of the JAK2/STAT5 cascade and SOCS3 expression, decreased phosphorylation of IR and IRS1, and attenuated GLUT4 translocation and glucose uptake upon insulin stimulation. Apo-RBP4 is a ligand that activates the STRA6 signaling cascade, inducing insulin resistance in GDM.

Pharmacokinetics of warfarin in rats: role of serum protein binding and tissue distribution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheung, W.K.

The purpose of this study was to explore the role of serum protein binding and tissue distribution in the non-linear pharmacokinetics of warfarin in rats. The first phase of the research was an attempt to elucidate the causes of intersubject differences in serum protein binding of warfarin in rats. It was found that the distribution of S-warfarin between blood and liver, kidneys, muscle, or fatty tissue was non-linear. Based on the tissue distribution data obtained, a physiologically-based pharmacokinetic model was developed to describe the time course of S-warfarin concentrations in the serum and tissues of rats. The proposed model wasmore » able to display the dose-dependent pharmacokinetics of warfarin in rats. Namely a lower clearance and a smaller apparent volume of distribution with increasing dose, which appear to be due to the presence of capacity-limited, high-affinity binding sites for warfarin in various tissues. To determine if the binding of warfarin to the high-affinity binding sites in the liver of rats is reversible, concentrations of S-warfarin in the liver and serum of rats were monitored for a very long time after an intravenous injection of a 1 mg/kg dose. In another study in rats, non-radioactive warfarin was found to be able to displace tissue-bound C/sup 14/-warfarin which was administered about 200 hours before the i.v. injection of the non-radioactive warfarin, showing that the binding of warfarin to the high-affinity binding sites in the body is persistent and reversible.« less

Accounting for the influence of inflammation on retinol-binding protein in a population survey of Liberian preschool-age children.

PubMed

Larson, Leila Margaret; Addo, O Yaw; Sandalinas, Fanny; Faigao, Katherine; Kupka, Roland; Flores-Ayala, Rafael; Suchdev, Parminder S

2017-04-01

Vitamin A deficiency (VAD) is an important contributor to child morbidity and mortality. The prevalence of VAD, measured by retinol-binding protein (RBP) or retinol, is overestimated in populations with a high prevalence of inflammation. We aimed to quantify and adjust for the effect of inflammation on VAD prevalence in a nationally representative survey of Liberian children 6 to 35 months of age. We compared five approaches to adjust RBP for inflammation and estimate VAD prevalence (defined as RBP < 0.7 µmol/L): (1) ignoring inflammation; (2) excluding individuals with inflammation (C-reactive protein (CRP) >5 mg/L or alpha1-acid glycoprotein (AGP) >1 g/)L; (3) multiplying each individual's RBP by an internal correction factor; (4) by an external correction factor; and (5) using regression (corrected RBP = exp(InRBP - β 1 (lnCRP obs -lnCRP ref ) - β 2 (lnAGP obs -lnAGP ref )). Corrected RBP was based on a regression model where reference lnCRP and lnAGP were set to the maximum of the lowest decile. The unadjusted prevalence of VAD was 24.7%. Children with elevated CRP and/or AGP had significantly lower RBP concentrations than their apparently healthy peers (geometric mean RBP 0.79 µmol/L (95% CI: 0.76, 0.82) vs. 0.95 µmol/L (95% CI: 0.92, 0.97), P < 0.001). Using approaches 2-5 resulted in a prevalence of VAD of 11.6%, 14.3%, 13.5% and 7.3%, respectively. Depending on the approach, the VAD prevalence is reduced 10-17 percentage points when inflammation is taken into account. Further quantification of the influence of inflammation on biomarkers of vitamin A status from other national surveys is needed to compare and recommend the preferred adjustment approach across populations. © 2016 John Wiley & Sons Ltd.

Accounting for the influence of inflammation on retinol-binding protein in a population survey of Liberian preschool-age children

PubMed Central

Larson, Leila Margaret; Addo, O. Yaw; Sandalinas, Fanny; Faigao, Katherine; Kupka, Roland; Flores-Ayala, Rafael; Suchdev, Parminder S.

2016-01-01

Vitamin A deficiency (VAD) is an important contributor to child morbidity and mortality. The prevalence of VAD, measured by retinol-binding protein (RBP) or retinol, is overestimated in populations with a high prevalence of inflammation. We aimed to quantify and adjust for the effect of inflammation on VAD prevalence in a nationally representative survey of Liberian children 6 to 35 months of age. We compared five approaches to adjust RBP for inflammation and estimate VAD prevalence (defined as RBP <0.7 μmol/L): (1) ignoring inflammation; (2) excluding individuals with inflammation (C-reactive protein (CRP) >5 mg/L or alpha1-acid glycoprotein (AGP) >1 g/)L; (3) multiplying each individual’s RBP by an internal correction factor; (4) by an external correction factor; and (5) using regression (corrected RBP = exp(InRBP – β1(lnCRPobs-lnCRPref) – β2(lnAGPobs-lnAGPref)). Corrected RBP was based on a regression model where reference lnCRP and lnAGP were set to the maximum of the lowest decile. The unadjusted prevalence of VAD was 24.7%. Children with elevated CRP and/or AGP had significantly lower RBP concentrations than their apparently healthy peers (geometric mean RBP 0.79 μmol/L (95% CI: 0.76, 0.82) vs. 0.95 μmol/L (95% CI: 0.92, 0.97), P <0.001). Using approaches 2–5 resulted in a prevalence of VAD of 11.6%, 14.3%, 13.5% and 7.3%, respectively. Depending on the approach, the VAD prevalence is reduced 10–17 percentage points when inflammation is taken into account. Further quantification of the influence of inflammation on biomarkers of vitamin A status from other national surveys is needed to compare and recommend the preferred adjustment approach across populations. PMID:26842430

Visfatin and retinol-binding protein 4 concentrations in lean, glucose-tolerant women with PCOS.

PubMed

Yildiz, Bulent O; Bozdag, Gurkan; Otegen, Umit; Harmanci, Ayla; Boynukalin, Kubra; Vural, Zehra; Kirazli, Serafettin; Yarali, Hakan

2010-01-01

Since insulin resistance is accepted to be a common feature of polycystic ovary syndrome (PCOS), the exact molecular mechanism(s) involved in glucose and lipid metabolism have been under investigation in the syndrome. Recently, two novel adipokines, namely visfatin and retinol-binding protein 4 (RBP4), have been suggested to play a role in insulin resistance and diabetes. This study sought to determine whether plasma concentrations of visfatin and RBP4 are altered in PCOS by comparing a total of 27 lean, normal glucose-tolerant PCOS patients with 19 age- and body mass index-matched healthy controls. The mean plasma visfatin concentrations were higher in PCOS patients than those in healthy subjects (37.9+/-18.2 versus 19.8+/-17.5, P<0.01), while RBP4 concentrations were similar between the two. Both adipokines were correlated with each other in the whole (r=0.50, P<0.01) and in PCOS (r=0.52, P<0.01) groups but not in controls. The results suggest that lean, glucose-tolerant women with PCOS have increased circulating visfatin and unaltered RBP4 concentrations compared with healthy lean women. In order to clarify overlapping effects and their potential contribution to the pathophysiology of PCOS, further studies are needed. Copyright (c) 2009 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Components of metabolic syndrome in relation to plasma levels of retinol binding protein 4 (RBP4) in a cohort of people aged 65 years and older.

PubMed

Majerczyk, M; Kocełak, P; Choręza, P; Arabzada, H; Owczarek, A J; Bożentowicz-Wikarek, M; Brzozowska, A; Szybalska, A; Puzianowska-Kuźnicka, M; Grodzicki, T; Więcek, A; Olszanecka-Glinianowicz, M; Chudek, J

2018-03-09

Elevated plasma concentration of retinol binding protein 4 (RBP4) has recently emerged as a potential risk factor as a component of developing metabolic syndrome (MS). Therefore, this study aimed to analyse the relationship between components of MS and concentrations of plasma RBP4 in a population of subjects 65 years and older. The study sample consisted of 3038 (1591 male) participants of the PolSenior study, aged 65 years and older. Serum lipid profile, concentrations of RBP4, glucose, insulin, C-reactive protein, IL-6, and activity of aminotransferases were measured. Nutritional status (BMI/waist circumference) and treatment with statins and fibrates were evaluated. Glomerular filtration rate (eGFR), de Ritis ratio, and fatty liver index (FLI), as well as HOMA-IR were calculated. Our study revealed a strong relationship between components of MS and RBP4 in both sexes: plasma RBP4 levels were increased in men by at least 3×, and in women by at least 4×. Hypertriglyceridemia was most strongly associated with elevated plasma RBP4 levels. Multivariate, sex-adjusted regression analysis demonstrated that chronic kidney disease [OR 1.86 (95% CI 1.78-1.94)], hypertriglyceridemia [OR 1.52 (1.24-1.87)], hypertension [OR 1.15 (1.12-1.19)], low serum HDL cholesterol [OR 0.94 (0.92-0.97)], and age > 80 years [OR 0.86 (0.81-0.90)] were each independently associated with RBP4 concentration (all p < 0.001). In Caucasians 65 years and older, RBP4 serum levels are associated with a number of components of MS, independent of sex and kidney function. Hypertriglyceridemia as a component of MS is most significantly related to RBP4 concentration.

Erythropoietin binding protein from mammalian serum

DOEpatents

Clemons, Gisela K.

1997-01-01

Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described.

THE INFLUENCE OF SERUM BINDING PROTEINS AND CLEARANCE ON THE COMPARATIVE RECEPTOR BINDING POTENCY OF ENDOCRINE ACTIVE COMPOUNDS

EPA Science Inventory

THE INFLUENCE OF SERUM BINDING PROTEINS AND CLEARANCE ON THE COMPARATIVE RECEPTOR BINDING POTENCY OF ENDOCRINE ACTIVE COMPOUNDS. JG Teeguarden1 and HA Barton2. 1ENVIRON International, Ruston LA; 2US EPA, ORD, NHEERL, ETD, Pharmacokinetics Branch, RTP, NC.

One measure of th...

Erythropoietin binding protein from mammalian serum

DOEpatents

Clemons, G.K.

1997-04-29

Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described. 11 figs.

Promotion of Orange-Fleshed Sweet Potato Increased Vitamin A Intakes and Reduced the Odds of Low Retinol-Binding Protein among Postpartum Kenyan Women.

PubMed

Girard, Amy Webb; Grant, Frederick; Watkinson, Michelle; Okuku, Haile Selassie; Wanjala, Rose; Cole, Donald; Levin, Carol; Low, Jan

2017-05-01

Background: Orange-fleshed sweet potato (OFSP) improves vitamin A (VA) status of young children; research with pregnant and lactating women is limited. Objective: We examined the effectiveness of the Mama SASHA (Sweetpotato Action for Security and Health in Africa) program to improve nutrition knowledge, diets, and nutritional status of pregnant and lactating women (PLW) in Western Kenya. Methods: Eight health facilities were allocated to the Mama SASHA intervention or comparison arms. PLW in intervention facilities received enhanced nutrition counseling at health clinics, were linked with community-based maternal support groups, and received vouchers for OFSP vine cuttings. Control PLW received clinic-based nutrition counseling only. A total of 505 women in early and midpregnancy, attending their first antenatal care visit, and with no previous engagement in project activities were enrolled from the 8 facilities. Nutrition and health-seeking knowledge, food security, dietary patterns, and anthropometric measurements were collected at 4 time points at ≤9 mo postpartum. VA intakes were assessed with multipass 24-h recalls in a subsample of 206 mothers at 8-10 mo postpartum. VA status was assessed by using serum retinol-binding protein (RBP). Impacts were estimated with multilevel mixed models adjusted for clustering and differences at enrollment. Results: At enrollment, 22.9% of women had RBP <1.17 μmol/L. By 9 mo postpartum, intervention women had significantly higher intakes of VA [adjusted difference = 297.0 retinol activity equivalent (RAE) units; 95% CI: 82, 513 RAE units; P = 0.01; n = 206], greater consumption of VA-rich fruit and vegetables in the previous 7 d (difference-in-difference estimate: 0.40 d; 95% CI: 0.23, 0.56 d; P < 0.01), and a 45% reduction in the odds of RBP <1.17 μmol/L (OR: 0.55; 95% CI: 0.33, 0.92; P = 0.01). Conclusion: Promotion of OFSP to PLW through health services is a feasible strategy to improve women's nutrition knowledge, VA

Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression.

PubMed

Chow, Brian A; Hamilton, Jason; Alsop, Derek; Cattet, Marc R L; Stenhouse, Gordon; Vijayan, Mathilakath M

2010-06-01

Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears. Copyright 2010 Elsevier Inc. All rights reserved.

Hypophysectomy eliminates and growth hormone (GH) maintains the midpregnancy elevation in GH receptor and serum binding protein in the mouse

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sanchez-Jimenez, F.; Fielder, P.J.; Martinez, R.R.

1990-02-01

({sup 125}I)Iodomouse GH (({sup 125}I)iodo-mGH) binding to samples of serum and hepatic microsomal membranes was measured in hypophysectomized pregnant, sham-operated pregnant, intact pregnant, and intact adult virgin mice. Surgeries were carried out on day 11 of pregnancy, and the animals were killed on day 14. The binding of mGH to both serum and hepatic microsomal membranes of intact virgin mice was much lower than to those of intact pregnant mice. In hypophysectomized mice, the mGH-binding capacity of both serum and hepatic microsomes decreased to values similar to those of nonpregnant mice. No significant differences were observed between intact and sham-operatedmore » pregnant animals in the maternal serum mGH concentration, the serum GH-binding protein concentration, or the hepatic GH receptor concentration. GH receptor and binding protein-encoding mRNAs were also higher in intact and sham-operated pregnant mice than in virgin and hypophysectomized mice. Hypophysectomized mice were treated with 200 micrograms/day bovine GH, administered by osmotic minipump; after 3 days of treatment, a significant elevation of hepatic GH receptor and serum GH-binding protein levels was observed. These results demonstrate an up-regulation of hepatic GH receptors and serum GH-binding protein by GH during pregnancy in the mouse.« less

Structural and Binding Properties of Two Paralogous Fatty Acid Binding Proteins of Taenia solium Metacestode

PubMed Central

Yang, Hyun-Jong; Shin, Joo-Ho; Diaz-Camacho, Sylvia Paz; Nawa, Yukifumi; Kang, Insug; Kong, Yoon

2012-01-01

Background Fatty acid (FA) binding proteins (FABPs) of helminths are implicated in acquisition and utilization of host-derived hydrophobic substances, as well as in signaling and cellular interactions. We previously demonstrated that secretory hydrophobic ligand binding proteins (HLBPs) of Taenia solium metacestode (TsM), a causative agent of neurocysticercosis (NC), shuttle FAs in the surrounding host tissues and inwardly transport the FAs across the parasite syncytial membrane. However, the protein molecules responsible for the intracellular trafficking and assimilation of FAs have remained elusive. Methodology/Principal Findings We isolated two novel TsMFABP genes (TsMFABP1 and TsMFABP2), which encoded 133- and 136-amino acid polypeptides with predicted molecular masses of 14.3 and 14.8 kDa, respectively. They shared 45% sequence identity with each other and 15–95% with other related-members. Homology modeling demonstrated a characteristic β-barrel composed of 10 anti-parallel β-strands and two α-helices. TsMFABP2 harbored two additional loops between β-strands two and three, and β-strands six and seven, respectively. TsMFABP1 was secreted into cyst fluid and surrounding environments, whereas TsMFABP2 was intracellularly confined. Partially purified native proteins migrated to 15 kDa with different isoelectric points of 9.2 (TsMFABP1) and 8.4 (TsMFABP2). Both native and recombinant proteins bound to 11-([5-dimethylaminonaphthalene-1-sulfonyl]amino)undecannoic acid, dansyl-DL-α-amino-caprylic acid, cis-parinaric acid and retinol, which were competitively inhibited by oleic acid. TsMFABP1 exhibited high affinity toward FA analogs. TsMFABPs showed weak binding activity to retinol, but TsMFABP2 showed relatively high affinity. Isolation of two distinct genes from an individual genome strongly suggested their paralogous nature. Abundant expression of TsMFABP1 and TsMFABP2 in the canal region of worm matched well with the histological distributions of lipids

Redox proteomic analysis of serum from aortic anerurysm patients: insights on oxidation of specific protein target.

PubMed

Spadaccio, Cristiano; Coccia, Raffaella; Perluigi, Marzia; Pupo, Gilda; Schininà, Maria Eugenia; Giorgi, Alessandra; Blarzino, Carla; Nappi, Francesco; Sutherland, Fraser W; Chello, Massimo; Di Domenico, Fabio

2016-06-21

oxidative stress is undoubtedly one of the main players in abdominal aortic aneurysm (AAA) pathophysiology. Recent studies in AAA patients reported an increase in the indices of oxidative damage at the tissue level and in biological fluids coupled with the loss of counter-regulatory mechanisms of protection from oxidative stress. We recently reported, in a proteomic analysis of AAA patient sera, changes in the expression of several proteins exerting important modulatory activities on cellular proliferation, differentiation and response to damage. This study aimed to explore the involvement of protein oxidation, at peripheral levels, in AAA. a redox proteomic approach was used to investigate total and specific protein carbonylation and protein-bound 4-hydroxy-2-nonenal (HNE) in the serum of AAA patients compared with age-matched controls. our results show increased oxidative damage to protein as indexed by the total carbonyl levels and total protein-bound HNE. By redox proteomics we identified specific carbonylation of three serum proteins: serum retinol-binding protein, vitamin D-binding protein and fibrinogen α-chain HNE. We also identified increased protein-bound HNE levels for hemopexin, IgK chain C region and IgK chain V-III region SIE. In addition we found a high correlation between specific protein carbonylation and protein-bound HNE and the aortic diameter. Moreover the analysis of serum proteins with antioxidant activity demonstrates the oxidation of albumin together with the overexpression of transferrin, haptoglobin and HSPs 90, 70, 60 and 32. this study support the involvement of oxidative stress in the pathogenesis of AAA and might provide a further degree of knowledge in the cause-effect role of oxidative stress shedding new light on the molecular candidates involved in the disease.

Serum transferrin as a prognostic indicator of spontaneous closure and mortality in gastrointestinal cutaneous fistulas.

PubMed Central

Kuvshinoff, B W; Brodish, R J; McFadden, D W; Fischer, J E

1993-01-01

OBJECTIVE: This study determined whether there are any laboratory or other features that will enable prediction of spontaneous closure in patients with gastrointestinal cutaneous fistulas. SUMMARY BACKGROUND DATA: Although the anatomic criteria for spontaneous closure of gastrointestinal cutaneous fistulas have been presented by several authors, less than 50% of such fistulas tend to close, even in the most recent series. METHODS: A group of patients with gastrointestinal cutaneous fistulas with anatomical features favorable to study were investigated with respect to a series of parameters including the usual demographic parameters, plus fistula output, number of blood transfusions, presence of sepsis, as well as metabolic parameters including serum transferrin, retinol-binding protein, thyroxin-binding prealbumin, and serum albumin. RESULTS: Of 79 patients with 116 fistulas, 16 (20.3%) died. Causes of death were uncontrolled sepsis in eight patients and cancer in five patients. Postoperative fistulas constituted 80% of the group. The presence of local sepsis, systemic sepsis, remote sepsis (such as pneumonia or line sepsis), the number of fistulas, fistula output, and the number of blood transfusions were not predictive of spontaneous closure, whereas serum transferrin was predictive of spontaneous closure. Serum transferrin, retinol-binding protein, and thyroxin-binding prealbumin were predictive of mortality. CONCLUSIONS: Serum transferrin does not appear to be an entirely independent variable, but seems to identify those patients with significant remote sepsis, systemic sepsis, and neoplasia in whom these processes are clinically significant. The results, if confirmed, and provided that nutritional needs are met, suggest that short-turnover proteins, particularly serum transferrin, might be useful in predicting which patients with gastrointestinal cutaneous fistulas should undergo surgery despite anatomic criteria favorable for spontaneous closure. PMID:8507110

The structure and dynamics of rat apo-cellular retinol-binding protein II in solution: comparison with the X-ray structure.

PubMed

Lu, J; Lin, C L; Tang, C; Ponder, J W; Kao, J L; Cistola, D P; Li, E

1999-03-05

The structure and dynamics of rat apo-cellular retinol binding protein II (apo-CRBP II) in solution has been determined by multidimensional NMR analysis of uniformly enriched recombinant rat 13C, 15N-apo-CRBP II and 15N-apo-CRBP II. The final ensemble of 24 NMR structures has been calculated from 3274 conformational restraints or 24.4 restraints/residue. The average root-mean-square deviation of the backbone atoms for the final 24 structures relative to their mean structure is 1.06 A. Although the average solution structure is very similar to the crystal structure, it differs at the putative entrance to the binding cavity, which is formed by the helix-turn-helix motif, the betaC-betaD turn and the betaE-betaF turn. The mean coordinates of the main-chain atoms of amino acid residues 28-38 are displaced in the solution structure relative to the crystal structure. The side-chain of F58, located on the betaC-betaD turn, is reoriented such that it interacts with L37 and no longer blocks entry into the ligand-binding pocket. Residues 28-35, which form the second helix of the helix-turn-helix motif in the crystal structure, do not exhibit a helical conformation in the solution structure. The solution structure of apo-CRBP II exhibits discrete regions of backbone disorder which are most pronounced at residues 28-32, 37-38 and 73-76 in the betaE-betaF turn as evaluated by the consensus chemical shift index, the root-mean-square deviation, amide 1H exchange rates and 15N relaxation studies. These studies indicate that fluctuations in protein conformation occur on the microseconds to ms time-scale in these regions of the protein. Some of these exchange processes can be directly observed in the three-dimensional 15N-resolved NOESY spectrum. These results suggest that in solution, apo-CRBP II undergoes conformational changes on the microseconds to ms time-scale which result in increased access to the binding cavity. Copyright 1999 Academic Press.

Probing the binding interaction of a phenazinium dye with serum transport proteins: a combined fluorometric and circular dichroism study.

PubMed

Bose, Debosreeta; Sarkar, Deboleena; Chattopadhyay, Nitin

2010-01-01

In the present investigation, an attempt has been made to study the interaction of phenosafranin (PSF), a cationic phenazinium dye with the transport proteins, bovine serum albumin (BSA) and human serum albumin (HSA), employing steady-state and time-resolved fluorometric and circular dichroism (CD) techniques. The photophysical properties of the dye are altered on binding with the serum proteins. An explicit study with respect to the modification of the fluorescence and fluorescence anisotropy upon binding, effect of denaturant, fluorescence lifetime and CD measurements reveal that the dye binds to both BSA and HSA with almost the same affinity. Far-UV CD spectra indicate a decrease in the percentage of alpha-helicity only for BSA upon binding with the probe. Near-UV CD responses indicate an alteration in the tertiary structure of both the transport proteins because of binding.

QSAR modeling of β-lactam binding to human serum proteins

NASA Astrophysics Data System (ADS)

Hall, L. Mark; Hall, Lowell H.; Kier, Lemont B.

2003-02-01

The binding of beta-lactams to human serum proteins was modeled with topological descriptors of molecular structure. Experimental data was the concentration of protein-bound drug expressed as a percent of the total plasma concentration (percent fraction bound, PFB) for 87 penicillins and for 115 β-lactams. The electrotopological state indices (E-State) and the molecular connectivity chi indices were found to be the basis of two satisfactory models. A data set of 74 penicillins from a drug design series was successfully modeled with statistics: r2=0.80, s = 12.1, q2=0.76, spress=13.4. This model was then used to predict protein binding (PFB) for 13 commercial penicillins, resulting in a very good mean absolute error, MAE = 12.7 and correlation coefficient, q2=0.84. A group of 28 cephalosporins were combined with the penicillin data to create a dataset of 115 beta-lactams that was successfully modeled: r2=0.82, s = 12.7, q2=0.78, spress=13.7. A ten-fold 10% leave-group-out (LGO) cross-validation procedure was implemented, leading to very good statistics: MAE = 10.9, spress=14.0, q2 (or r2 press)=0.78. The models indicate a combination of general and specific structure features that are important for estimating protein binding in this class of antibiotics. For the β-lactams, significant factors that increase binding are presence and electron accessibility of aromatic rings, halogens, methylene groups, and =N- atoms. Significant negative influence on binding comes from amine groups and carbonyl oxygen atoms.

Evaluation of insulin-like growth factor (IGF-1) and retinol binding protein (RBP-4) levels in patients with newly diagnosed pancreatic adenocarcinoma (PDAC).

PubMed

Wlodarczyk, Barbara; Gasiorowska, Anita; Borkowska, Anna; Malecka-Panas, Ewa

The elevation of insulin-like growth factor 1 (IGF-1) and adipokine retinol-binding protein 4 (RBP-4) is known to be associated with the risk of many cancers. The aim of this study was to evaluate the serum concentrations of IGF-1 and RBP-4 in patients with PDAC and chronic pancreatitis (CP). The study included 43 patients with PDAC, 39 patients with CP and 10 controls. The concentrations of IGF-1 and RBP-4 were obtained using the ELISA method (Corgenix UK Ltd R&D Systems). The study protocol was approved by the Bioethics Committee at the Medical University of Lodz. In PDAC patients the serum IGF-1 level was significantly higher than in patients with CP (107.79 ± 66.40 ng/ml vs 89.91 ± 74.06 ng/ml; P < 0.05). Patients with both CP and diabetes mellitus (DM) were noted to have a significantly lower level of IGF-1 compared with those who only had CP (51.33 ± 24.30 ng/ml vs 108.42 ± 82.39 ng/ml; P = 0.01). The same result was obtained for men with and without DM (58.05 ± 32.44 ng/ml vs 98.79 ± 79.47 ng/ml, P = 0.05). As regards the serum level of RBP-4, the PDAC and CP groups were not significantly different from each other. Diabetes accompanying PDAC does not influence the level of IGF-1 as opposed to diabetes in the course of CP. The IGF-1 level can be useful for early diagnosis of PDAC. High concentration of RBP-4 is not specific to pancreatic cancer, so it does not appear to be a useful biomarker for PDAC. Copyright © 2017 IAP and EPC. Published by Elsevier B.V. All rights reserved.

THE INFLUENCE OF SERUM BINDING PROTEINS AND FEEDBACK CONTROL OF SERUM ESTRADIOL LEVELS ON THE COMPARATIVE POTENCY OF ENDOCRINE ACTIVE COMPOUNDS

EPA Science Inventory

THE INFLUENCE OF SERUM BINDING PROTEINS ON THE COMPARATIVE RECEPTOR BINDING POTENCY OF ENDOCRINE ACTIVE COMPOUNDS. JG Teeguarden1 and HA Barton2. 1ICF Consulting, Research Triangle Park NC; 2US EPA, ORD, NHEERL, ETD, Pharmacokinetics Branch, RTP, NC.

Accurate comparison of...

Unraveling the binding interaction of a bioactive pyrazole-based probe with serum proteins: Relative concentration dependent 1:1 and 2:1 probe-protein stoichiometries.

PubMed

Kundu, Pronab; Chattopadhyay, Nitin

2018-06-15

Molecular interactions and binding of probes/drugs with biomacromolecular systems are of fundamental importance in understanding the mechanism of action and hence designing of proactive drugs. In the present study, binding interactions of a biologically potent fluorophore, (E)-1,5-diphenyl-3-styryl-4,5-dihydro-1H-pyrazole (DSDP) with two serum transport proteins, human serum albumin and bovine serum albumin, have been investigated exploiting multi-spectroscopic techniques. The spectrophotometric and fluorometric studies together with fluorescence quenching, fluorescence anisotropy, urea induced denaturation studies and fluorescence lifetime measurements reveal strong binding of DSDP with both the plasma proteins. Going beyond the vast literature data mostly providing 1:1 probe-protein complexation, the present investigation portrays 2:1 probe-protein complex formation at higher relative probe concentration. A newer approach has been developed to have an estimate of the binding constants varying the concentration of the protein, instead of the usual practice of varying the probe. The binding constants for the 2:1 DSDP-protein complexes are determined to be 1.37 × 10 10  M -2 and 1.47 × 10 10  M -2 for HSA and BSA respectively, while those for the 1:1 complexation process come out to be 1.85 × 10 5  M -1 and 1.73 × 10 5  M -1 for DSDP-HSA and DSDP-BSA systems respectively. Thermodynamic analysis at different temperatures implies that the forces primarily involved in the binding process are hydrogen bonding and hydrophobic interactions. Competitive replacement studies with known site markers and molecular docking simulations direct to the possible locations and binding energies of DSDP with the two serum proteins, corroborating well with the experimental results. Copyright © 2018 Elsevier B.V. All rights reserved.

Photoactivable analogs for labeling 25-hydroxyvitamin D3 serum binding protein and for 1,25-dihydroxyvitamin D3 intestinal receptor protein

NASA Technical Reports Server (NTRS)

Kutner, A.; Link, R. P.; Schnoes, H. K.; DeLuca, H. F.

1986-01-01

3-Azidobenzoates and 3-azidonitrobenzoates of 25-hydroxyvitamin D3 as well as 3-deoxy-3-azido-25-hydroxyvitamin D3 and 3-deoxy-3-azido-1,25-dihydroxyvitamin D3 were prepared as photoaffinity labels for vitamin D serum binding protein and 1,25-dihydroxyvitamin D3 intestinal receptor protein. The compounds prepared were easily activated by short- or long-wavelength uv light, as monitored by uv and ir spectrometry. The efficacy of the compounds to compete with 25-hydroxyvitamin D3 or 1,25-dihydroxyvitamin D3 for the binding site of serum binding protein and receptor, respectively, was studied to evaluate the vitamin D label with the highest affinity for the protein. The presence of an azidobenzoate or azidonitrobenzoate substituent at the C-3 position of 25-OH-D3 significantly decreased (10(4)- to 10(6)-fold) the binding activity. However, the labels containing the azido substituent attached directly to the vitamin D skeleton at the C-3 position showed a high affinity, only 20- to 150-fold lower than that of the parent compounds with their respective proteins. Therefore, 3-deoxy-3-azidovitamins present potential ligands for photolabeling of vitamin D proteins and for studying the structures of the protein active sites.

Cyanidin 3-glucoside ameliorates hyperglycemia and insulin sensitivity due to downregulation of retinol binding protein 4 expression in diabetic mice.

PubMed

Sasaki, Rie; Nishimura, Natsumi; Hoshino, Hiromi; Isa, Yasuka; Kadowaki, Maho; Ichi, Takahito; Tanaka, Akihito; Nishiumi, Shin; Fukuda, Itsuko; Ashida, Hitoshi; Horio, Fumihiko; Tsuda, Takanori

2007-12-03

Adipocyte dysfunction is strongly associated with the development of obesity and insulin resistance. It is accepted that the regulation of adipocytokine expression is one of the most important targets for the prevention of obesity and improvement of insulin sensitivity. In this study, we have demonstrated that anthocyanin (cyanidin 3-glucoside; C3G) which is a pigment widespread in the plant kingdom, ameliorates hyperglycemia and insulin sensitivity due to the reduction of retinol binding protein 4 (RBP4) expression in type 2 diabetic mice. KK-A(y) mice were fed control or control +0.2% of a C3G diet for 5 weeks. Dietary C3G significantly reduced blood glucose concentration and enhanced insulin sensitivity. The adiponectin and its receptors expression were not responsible for this amelioration. C3G significantly upregulated the glucose transporter 4 (Glut4) and downregulated RBP4 in the white adipose tissue, which is accompanied by downregulation of the inflammatory adipocytokines (monocyte chemoattractant protein-1 and tumor necrosis factor-alpha) in the white adipose tissue of the C3G group. These findings indicate that C3G has significant potency in an anti-diabetic effect through the regulation of Glut4-RBP4 system and the related inflammatory adipocytokines.

Cellular Binding of Anionic Nanoparticles is Inhibited by Serum Proteins Independent of Nanoparticle Composition.

PubMed

Fleischer, Candace C; Kumar, Umesh; Payne, Christine K

2013-09-01

Nanoparticles used in biological applications encounter a complex mixture of extracellular proteins. Adsorption of these proteins on the nanoparticle surface results in the formation of a "protein corona," which can dominate the interaction of the nanoparticle with the cellular environment. The goal of this research was to determine how nanoparticle composition and surface modification affect the cellular binding of protein-nanoparticle complexes. We examined the cellular binding of a collection of commonly used anionic nanoparticles: quantum dots, colloidal gold nanoparticles, and low-density lipoprotein particles, in the presence and absence of extracellular proteins. These experiments have the advantage of comparing different nanoparticles under identical conditions. Using a combination of fluorescence and dark field microscopy, flow cytometry, and spectroscopy, we find that cellular binding of these anionic nanoparticles is inhibited by serum proteins independent of nanoparticle composition or surface modification. We expect these results will aid in the design of nanoparticles for in vivo applications.

Assessment of IgE binding to native and hydrolyzed soy protein in serum obtained from dogs with experimentally induced soy protein hypersensitivity.

PubMed

Serra, Montserrat; Brazís, Pilar; Fondati, Alessandra; Puigdemont, Anna

2006-11-01

To assess binding of IgE to native, whole hydrolyzed, and separated hydrolyzed fractions of soy protein in serum obtained from dogs with experimentally induced soy protein hypersensitivity. 8 naïve Beagles (6 experimentally sensitized to native soy protein and 2 control dogs). 6 dogs were sensitized against soy protein by administration of allergens during a 90-day period. After the sensitization protocol was completed, serum concentrations of soy-specific IgE were measured and intradermal skin tests were performed in all 6 dogs to confirm that the dogs were sensitized against soy protein. Serum samples from each sensitized and control dog underwent western blot analysis to assess the molecular mass band pattern of the different allergenic soy fractions and evaluate reactivities to native and hydrolyzed soy protein. In sera from sensitized dogs, a characteristic band pattern with 2 major bands (approx 75 and 50 kd) and 2 minor bands (approx 31 and 20 kd) was detected, whereas only a diffuse band pattern associated with whole hydrolyzed soy protein was detected in the most reactive dog. Reactivity was evident only for the higher molecular mass peptide fraction. In control dogs, no IgE reaction to native or hydrolyzed soy protein was detected. Data suggest that the binding of soy-specific IgE to the hydrolyzed soy protein used in the study was significantly reduced, compared with binding of soy-specific IgE to the native soy protein, in dogs with experimentally induced soy hypersensitivity.

Binding of vitamin A with milk α- and β-caseins.

PubMed

Bourassa, P; N'soukpoé-Kossi, C N; Tajmir-Riahi, H A

2013-05-01

The binding sites of retinol and retinoic acid with milk α- and β-caseins were determined, using constant protein concentration and various retinoid contents. FTIR, UV-visible and fluorescence spectroscopic methods as well as molecular modelling were used to analyse retinol and retinoic acid binding sites, the binding constant and the effect of retinoid complexation on the stability and conformation of caseins. Structural analysis showed that retinoids bind caseins via both hydrophilic and hydrophobic contacts with overall binding constants of K(retinol-)(α)(-caseins)=1.21 (±0.4)×10(5) M(-1) and K(retinol-)(β)(-caseins)=1.11 (±0.5)×10(5) M(-1) and K(retinoic acid-)(α)(-caseins)=6.2 (±0.6)×10(4) M(-1) and K(retinoic acid-)(β)(-caseins)=6.3 (±0.6)×10(4) M(-1). The number of bound retinol molecules per protein (n) was 1.5 (±0.1) for α-casein and 1.0 (±0.1) for β-casein, while 1 molecule of retinoic acid was bound in the α- and β-casein complexes. Molecular modelling showed different binding sites for retinol and retinoic acid on α- and β-caseins with more stable complexes formed with α-casein. Retinoid-casein complexation induced minor alterations of protein conformation. Caseins might act as carriers for transportation of retinoids to target molecules. Copyright © 2012 Elsevier Ltd. All rights reserved.

Retinol-Binding Protein 4 and Lipids Prospectively Measured During Early to Mid-Pregnancy in Relation to Preeclampsia and Preterm Birth Risk.

PubMed

Mendola, Pauline; Ghassabian, Akhgar; Mills, James L; Zhang, Cuilin; Tsai, Michael Y; Liu, Aiyi; Yeung, Edwina H

2017-06-01

Maternal retinol-binding protein 4 (RBP4) and lipids may relate to preeclampsia and preterm birth risk but longitudinal data are lacking. This study examines these biomarkers longitudinally during pregnancy in relation to preeclampsia and preterm birth risk. Maternal serum samples from the Calcium for Preeclampsia Prevention (CPEP) trial were analyzed at baseline: average 15 gestational weeks; mid-pregnancy: average 27 weeks; and at >34 weeks. We measured RBP4, total cholesterol, high-density lipoprotein, low-density lipoprotein, triglycerides and lipoprotein (a) (Lp(a)). Cross-sectional logistic regression analyses estimated the odds ratio (OR) and 95% confidence intervals (CI) for preterm preeclampsia (n = 63), term preeclampsia (n = 104), and preterm delivery (n = 160) associated with RBP4 and lipids at baseline and mid-pregnancy compared with controls (n = 136). Longitudinal trajectories across pregnancy were assessed using mixed linear models with fixed effects. Adjusted models included clinical and demographic factors. RBP4 concentrations at baseline and mid-pregnancy were associated with a 4- to 8-fold increase in preterm preeclampsia risk but were not associated with term preeclampsia. RBP4 measured mid-pregnancy was also associated with preterm birth (OR = 6.67, 95% CI: 1.65, 26.84). Higher triglyceride concentrations in mid-pregnancy were associated with a 2- to 4-fold increased risk for both preeclampsia and preterm birth. Longitudinal models demonstrate that both preterm preeclampsia and preterm birth cases had elevated RBP4 throughout gestation. Elevated RBP4 is detectable early in pregnancy and its strong relation with preterm preeclampsia merits further investigation and confirmation to evaluate its potential use as a predictor, particularly among high-risk women. © Published by Oxford University Press on behalf of American Journal of Hypertension Ltd 2017. This work is written by (a) US Government employees(s) and is in the public domain in the US.

Generation of therapeutic protein variants with the human serum albumin binding capacity via site-specific fatty acid conjugation.

PubMed

Cho, Jinhwan; Lim, Sung In; Yang, Byung Seop; Hahn, Young S; Kwon, Inchan

2017-12-21

Extension of the serum half-life is an important issue in developing new therapeutic proteins and expanding applications of existing therapeutic proteins. Conjugation of fatty acid, a natural human serum albumin ligand, to a therapeutic protein/peptide was developed as a technique to extend the serum half-life in vivo by taking advantages of unusually long serum half-life of human serum albumin (HSA). However, for broad applications of fatty acid-conjugation, several issues should be addressed, including a poor solubility of fatty acid and a substantial loss in the therapeutic activity. Therefore, herein we systematically investigate the conditions and components in conjugation of fatty acid to a therapeutic protein resulting in the HSA binding capacity without compromising therapeutic activities. By examining the crystal structure and performing dye conjugation assay, two sites (W160 and D112) of urate oxidase (Uox), a model therapeutic protein, were selected as sites for fatty acid-conjugation. Combination of site-specific incorporation of a clickable p-azido-L-phenylalanine to Uox and strain-promoted azide-alkyne cycloaddition allowed the conjugation of fatty acid (palmitic acid analog) to Uox with the HSA binding capacity and retained enzyme activity. Deoxycholic acid, a strong detergent, greatly enhanced the conjugation yield likely due to the enhanced solubility of palmitic acid analog.

Human CRISP-3 binds serum alpha(1)B-glycoprotein across species.

PubMed

Udby, Lene; Johnsen, Anders H; Borregaard, Niels

2010-04-01

CRISP-3 was previously shown to be bound to alpha(1)B-glycoprotein (A1BG) in human serum/plasma. All mammalian sera are supposed to contain A1BG, although its presence in rodent sera is not well-documented. Since animal sera are often used to supplement buffers in experiments, in particular such that involve cell cultures, binding proteins present in sera might interfere in the experiments. We examined sera from five different animal species for CRISP-3 binding proteins using gel filtration and ligand blotting. We developed a rapid method for isolation of proteins that bind to human CRISP-3 and identified the isolated proteins by mass spectrometry and N-terminal sequencing. We identified A1BG as a CRISP-3 binding protein in sera from cow, horse and rabbit. CRISP-3 bound kininogen 1 in mouse serum, whereas rat serum showed no CRISP-3 binding activity. In equine serum, we furthermore detected a possible CRISP, already bound to A1BG. It seems to be a common mechanism that A1BGs bind CRISPs, also across species. Apart from the possible physiological implications hereof, complex binding of CRISPs by A1BG (and other proteins) may interfere with the detection and function of CRISPs, when these are studied in the presence of animal sera. Copyright 2009 Elsevier B.V. All rights reserved.

Plasma Levels of Fatty Acid-Binding Protein 4, Retinol-Binding Protein 4, High-Molecular Weight Adiponectin, and Cardiovascular Mortality among Men with Type 2 Diabetes: A 22-Year Prospective Study

PubMed Central

Liu, Gang; Ding, Ming; Chiuve, Stephanie E.; Rimm, Eric B.; Franks, Paul W.; Meigs, James B.; Hu, Frank B.; Sun, Qi

2016-01-01

Objective To examine select adipokines, including fatty acid-binding protein 4 (FABP4), retinol-binding protein 4 (RBP4), and high-molecular weight (HMW) adiponectin in relation to cardiovascular disease (CVD) mortality among patients with type 2 diabetes (T2D). Approach and Results Plasma levels of FABP4, RBP4, and HMW adiponectin were measured in 950 men with T2D in the Health Professionals Follow-up Study. After an average of 22 years of follow up (1993–2015), 580 deaths occurred, of whom 220 died of CVD. After multivariate adjustment for covariates, higher levels of FABP4 were significantly associated with a higher CVD mortality: comparing extreme tertiles, the hazard ratio (HR) and 95% confidence interval (CI) of CVD mortality was 1.78 (1.22, 2.59; P trend=0.001). A positive association was also observed for HMW adiponectin: the HR (95% CI) was 2.07 (1.42, 3.06; P trend=0.0002), comparing extreme tertiles, whereas higher RBP4 levels were non-significantly associated with a decreased CVD mortality with an HR (95% CI) of 0.73 (0.50, 1.07; P trend=0.09). A Mendelian randomization (MR) analysis suggested that the causal relationships of HMW adiponectin and RBP4 would be directionally opposite to those observed based on the biomarkers, although none of the MR associations achieved statistical significance. Conclusions These data suggest that higher levels of FABP4 and HMW adiponectin are associated with elevated CVD mortality among men with T2D. Biological mechanisms underlying these observations deserve elucidation, but the associations of HMW adiponectin may partially reflect altered adipose tissue functionality among T2D patients. PMID:27609367

Oral isotretinoin (13-cis-retinoic acid) therapy in severe acne: drug and vitamin A concentrations in serum and skin.

PubMed

Rollman, O; Vahlquist, A

1986-04-01

The disposition of oral isotretinoin to the skin and the effects of the drug on the vitamin A levels in serum and skin were studied in 17 patients with nodulocystic acne. All patients received 0.5 mg/kg/day for 3 months and 8 patients continued treatment with 0.75 mg/kg/day for another 3 months. The parent drug, the major metabolite (4-oxo-isotretinoin), and 2 natural retinoids (retinol and dehydroretinol) were monitored in serum and biopsies of uninvolved skin, using adsorption high-pressure liquid chromatography. During the initial 3 months of treatment the mean isotretinoin level in the serum was 145 ng/ml and in the epidermis 73 ng/g. The corresponding values for 4-oxo-isotretinoin were 615 and 113 ng/g, respectively. Even at the highest dosage there was no progressive accumulation of isotretinoin in serum, epidermis, or subcutis. After discontinuation of therapy the drug disappeared from both serum and skin within 2-4 weeks. The serum transport of vitamin A, monitored by the concentrations of retinol, retinol-binding protein, and prealbumin (transthyretin), was not affected by the treatment. By contrast, the retinol level in the epidermis increased by an average of 53% (p less than 0.01) and the dehydroretinol level decreased by 79% (p less than 0.001) as a result of 3 months of treatment. Both changes were reversible. The results suggest that isotretinoin therapy interferes with the endogenous vitamin A metabolism in the skin.

Albumin-coated SPIONs: an experimental and theoretical evaluation of protein conformation, binding affinity and competition with serum proteins

NASA Astrophysics Data System (ADS)

Yu, Siming; Perálvarez-Marín, Alex; Minelli, Caterina; Faraudo, Jordi; Roig, Anna; Laromaine, Anna

2016-07-01

The variety of nanoparticles (NPs) used in biological applications is increasing and the study of their interaction with biological media is becoming more important. Proteins are commonly the first biomolecules that NPs encounter when they interact with biological systems either in vitro or in vivo. Among NPs, super-paramagnetic iron oxide nanoparticles (SPIONs) show great promise for medicine. In this work, we study in detail the formation, composition, and structure of a monolayer of bovine serum albumin (BSA) on SPIONs. We determine, both by molecular simulations and experimentally, that ten molecules of BSA form a monolayer around the outside of the SPIONs and their binding strength to the SPIONs is about 3.5 × 10-4 M, ten times higher than the adsorption of fetal bovine serum (FBS) on the same SPIONs. We elucidate a strong electrostatic interaction between BSA and the SPIONs, although the secondary structure of the protein is not affected. We present data that supports the strong binding of the BSA monolayer on SPIONs and the properties of the BSA layer as a protein-resistant coating. We believe that a complete understanding of the behavior and morphology of BSA-SPIONs and how the protein interacts with SPIONs is crucial for improving NP surface design and expanding the potential applications of SPIONs in nanomedicine.The variety of nanoparticles (NPs) used in biological applications is increasing and the study of their interaction with biological media is becoming more important. Proteins are commonly the first biomolecules that NPs encounter when they interact with biological systems either in vitro or in vivo. Among NPs, super-paramagnetic iron oxide nanoparticles (SPIONs) show great promise for medicine. In this work, we study in detail the formation, composition, and structure of a monolayer of bovine serum albumin (BSA) on SPIONs. We determine, both by molecular simulations and experimentally, that ten molecules of BSA form a monolayer around the

Vitamin D binding protein as a serum biomarker of Alzheimer's disease.

PubMed

Bishnoi, Ram J; Palmer, Raymond F; Royall, Donald R

2015-01-01

Vitamin D binding protein (VDBP), a multifunctional protein, has been found to be elevated in the cerebrospinal fluid (CSF) of neurodegenerative disorder cases, implicating it in the pathogenesis of Alzheimer's disease (AD). However, the contribution of VDBP to AD has not been fully explored. We used a Multiple Indicators Multiple Causes (MIMIC) approach to examine the relationship between serum VDBP levels and cognitive performance in a well characterized AD cohort, the Texas Alzheimer's Research and Care Consortium (TARCC). Instead of categorical diagnoses, we used a latent dementia phenotype (d), which has been validated in several prior studies using this dataset. We found that serum VDBP levels are significantly positively associated with d scores, which in turn are inversely related to cognitive performance. This suggests that d mediates the adverse effects of serum VDB on cognition and therefore that its effects are specifically dementing. d scores are also specifically related to default mode network (DMN) structure. VDBP acts as an amyloid-β (Aβ) scavenger, and Aβ deposition in the DMN is seen in the pre-clinical stages of AD. We speculate then that serum effects of VDBP are mediated through changes in DMN structure or function, most probably via Aβ. Aβ affects the DMN early in the course of AD. Therefore, raised serum VDBP levels may be a useful indicator of future dementia and/or dementia conversion. This might be confirmed through longitudinal analysis of TARCC data.

Femtosecond studies of protein-ligand hydrophobic binding and dynamics: human serum albumin.

PubMed

Zhong, D; Douhal, A; Zewail, A H

2000-12-19

In this contribution, we report studies of the nature of the dynamics and hydrophobic binding in protein-ligand complexes of human serum albumin with 2-(2'-hydroxyphenyl)-4-methyloxazole. With femtosecond time resolution, we examined the orientational motion of the ligand, its intrinsic nuclear motions, and the lifetime changes in the hydrophobic phase. For comparisons, with similar but chemical nanocavities, we also studied the same ligand in micelles and cyclodextrins. The hydrophobic interactions in the binding crevice are much stronger than those observed in cyclodextrins and micelles. The confined geometry restrains the nonradiative decay and significantly lengthens the excited-state lifetime. The observed dynamics over the femtosecond-to-nanosecond time scale indicate that the binding structure is rigid and the local motions of the ligand are nearly "frozen" in the protein. Another major finding is the elucidation of the directed dynamics by the protein. Proton transfer and intramolecular twisting of 2-(2'-hydroxyphenyl)-4-methyloxazole were observed to evolve along two routes: one involves the direct stretching motion in the molecular plane (approximately 200 fs) and is not sensitive to the environment; the second, less dominant, is related to the twisting motion (approximately 3 ps) of the two heterocyclic rings and drastically slows down in the protein hydrophobic pocket.

Serum retinol, alpha-tocopherol, and beta-carotene levels are not altered by excess ingestion of diacylglycerol-containing plant sterol esters.

PubMed

Saito, Shinichiro; Tomonobu, Kazuichi; Kudo, Naoto; Shiiba, Daisuke; Hase, Tadashi; Tokimitsu, Ichiro

2006-01-01

Diacylglycerol (DAG) suppresses the postprandial increase in serum triglycerides, and has antiobesity effects. On the other hand, plant sterol esters (PSE) lower serum cholesterol levels in hypercholesterolemia. Thus, DAG-containing PSE (PSE/DAG) would be expected to maintain an appropriate serum cholesterol level and decrease the risk of arteriosclerotic disorders. Several recent studies, however, report negative effects of PSE on serum fat-soluble (pro)vitamin levels. The objective of this study was to investigate the effect of PSE/DAG on serum retinol, beta-carotene, and alpha-tocopherol levels using a threefold excess of the effective dose obtained in our previous study. A randomized placebo-controlled double-blind parallel study was performed in healthy and mildly hypercholesterolemic subjects, in which the subjects ingested 1.2 g PSE/30 g DAG for 2 weeks in the form of mayonnaise-type products. Triacylglycerol (TAG) mayonnaise was used as a control. There were no subjective adverse effects or changes in serum retinol, alpha-tocopherol, and beta-carotene levels, abdominal symptoms, hematologic values, or blood biochemical values. Ingestion of a threefold excess of PSE/DAG for 2 weeks had no adverse effects compared to ingestion of conventional TAG mayonnaise. Copyright 2006 S. Karger AG, Basel.

Haematoporphyrin and OO'-diacetylhaematoporphyrin binding by serum and cellular proteins. Implications for the clearance of these photochemotherapeutic agents by cells.

PubMed Central

Smith, A; Neuschatz, T

1983-01-01

Haematoporphyrin derivative (HpD), a mixture of porphyrins, is currently used as a photochemotherapeutic agent in the treatment of neoplasias. The interaction of purified components of HpD with serum and cellular proteins was investigated using absorption and fluorescence spectroscopy. The interactions of haematoporphyrin and OO'-diacetylhaematoporphyrin with human albumin and with haemopexin, the two major serum porphyrin-binding proteins, show stoichiometries of 1 mol of porphyrin bound per mol of protein. The apparent dissociation constants, Kd, are in the range of 1-2 microM for albumin and 3-4 microM for haemopexin. These two major components of HpD would, after intravenous injection, bind to albumin and circulate in serum as albumin complexes. Free porphyrin rather than porphyrin bound to albumin interacts with Morris hepatoma tissue culture cells. A rapid high-affinity saturable transport system operates at free porphyrin concentrations of less than 2 microM. In addition, fluorescence spectra show that components in rat liver cytosol can bind haematoporphyrin and OO'-diacetylhaematoporphyrin and distinguish these binders from those present in rat serum. PMID:6225429

Isomer-Specific Binding Affinity of Perfluorooctanesulfonate (PFOS) and Perfluorooctanoate (PFOA) to Serum Proteins.

PubMed

Beesoon, Sanjay; Martin, Jonathan W

2015-05-05

Perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) are among the most prominent contaminants in human serum, and these were historically manufactured as technical mixtures of linear and branched isomers. The isomers display unique pharmacokinetics in humans and in animal models, but molecular mechanisms underlying isomer-specific PFOS and PFOA disposition have not previously been studied. Here, ultrafiltration devices were used to examine (i) the dissociation constants (Kd) of individual PFOS and PFOA isomers with human serum albumin (HSA) and (ii) relative binding affinity of isomers in technical mixtures spiked to whole calf serum and human serum. Measurement of HSA Kd's demonstrated that linear PFOS (Kd=8(±4)×10(-8) M) was much more tightly bound than branched PFOS isomers (Kd range from 8(±1)×10(-5) M to 4(±2)×10(-4) M). Similarly, linear PFOA (Kd=1(±0.9)×10(-4) M) was more strongly bound to HSA compared to branched PFOA isomers (Kd range from 4(±2)×10(-4) M to 3(±2)×10(-4) M). The higher binding affinities of linear PFOS and PFOA to total serum protein were confirmed when both calf serum and human serum were spiked with technical mixtures. Overall, these data provide a mechanistic explanation for the longer biological half-life of PFOS in humans, compared to PFOA, and for the higher transplacental transfer efficiencies and renal clearance of branched PFOS and PFOA isomers, compared to the respective linear isomer.

Circulating retinol binding protein 4 levels in nonalcoholic fatty liver disease: a systematic review and meta-analysis.

PubMed

Zhou, Zhongwei; Chen, Hongmei; Ju, Huixiang; Sun, Mingzhong

2017-09-20

Retinol binding protein 4 (RBP4) is implicated in obesity, insulin resistance and type 2 diabetes mellitus that are closely associated with nonalcoholic fatty liver disease (NAFLD). However, recent investigations regarding circulating RBP4 levels in NAFLD are conflicting. This meta-analysis is to determine whether NAFLD, non-alcoholic steatohepatitis (NASH) and simple steatosis (SS) patients have altered RBP4 levels. We performed a systematic search in PubMed, EMBASE and The Cochrane Library up until 18 March 2017, and 12 studies comprising a total of 4247 participants (2271 NAFLD patients and 1976 controls) were included in the meta-analysis. There were no significant differences of circulating RBP4 levels in the following comparisons: (1) NAFLD patients vs controls (standardized mean differences [SMD]: 0.08; 95% CI: -0.21, 0.38); (2) NASH patients vs controls (SMD: -0.49; 95% CI: -1.09, 0.12); (3) SS patients vs controls (SMD: -0.72; 95% CI: -1.64, 0.20) and (4) NASH vs SS patients (SMD: -0.04; 95% CI: -0.32, 0.24). The results remained essentially unchanged in the comparisons between NAFLD patients and controls after excluding single individual study or bariatric studies (n = 2). No significant publication bias was detected. However, there was significant heterogeneity among studies and the subgroup and meta-regression analyses did not find the potential sources. Circulating RBP4 levels may not be associated with NAFLD. Further prospective cohort studies are required to confirm these findings.

Newborn serum retinoic acid level is associated with variants of genes in the retinol metabolism pathway.

PubMed

Manolescu, Daniel C; El-Kares, Reyhan; Lakhal-Chaieb, Lajmi; Montpetit, Alexandre; Bhat, Pangala V; Goodyer, Paul

2010-06-01

Retinoic acid (RA) is a critical regulator of gene expression during embryonic development. In rodents, moderate maternal vitamin A deficiency leads to subtle morphogenetic defects and inactivation of RA pathway genes causes major disturbances of embryogenesis. In this study, we quantified RA in umbilical cord blood of 145 healthy full-term Caucasian infants from Montreal. Sixty seven percent of values were <10 nmol/L (84 were <0.07 nmol/L) and 33% had moderate or high levels. Variation in RA could not be explained by parallel variation in its precursor, retinol (ROL). However, we found that the (A) allele of the rs12591551 single nucleotide polymorphism (SNP) in the ALDH1A2 gene (ALDH1A2rs12591551(A)), occurring in 19% of newborns, was associated with 2.5-fold higher serum RA levels. ALDH1A2 encodes retinaldehyde dehydrogenase (RALDH) 2, which synthesizes RA in fetal tissues. We also found that homozygosity for the (A) allele of the rs12724719 SNP in the CRABP2 gene (CRABP2rs12724719(A/A)) was associated with 4.4-fold increase in umbilical cord serum RA. CRABP2 facilitates RA binding to its cognate receptor complex and transfer to the nucleus. We hypothesize that individual variation in RA pathway genes may account for subtle variations in RA-dependent human embryogenesis.

QSAR modeling of human serum protein binding with several modeling techniques utilizing structure-information representation.

PubMed

Votano, Joseph R; Parham, Marc; Hall, L Mark; Hall, Lowell H; Kier, Lemont B; Oloff, Scott; Tropsha, Alexander

2006-11-30

Four modeling techniques, using topological descriptors to represent molecular structure, were employed to produce models of human serum protein binding (% bound) on a data set of 1008 experimental values, carefully screened from publicly available sources. To our knowledge, this data is the largest set on human serum protein binding reported for QSAR modeling. The data was partitioned into a training set of 808 compounds and an external validation test set of 200 compounds. Partitioning was accomplished by clustering the compounds in a structure descriptor space so that random sampling of 20% of the whole data set produced an external test set that is a good representative of the training set with respect to both structure and protein binding values. The four modeling techniques include multiple linear regression (MLR), artificial neural networks (ANN), k-nearest neighbors (kNN), and support vector machines (SVM). With the exception of the MLR model, the ANN, kNN, and SVM QSARs were ensemble models. Training set correlation coefficients and mean absolute error ranged from r2=0.90 and MAE=7.6 for ANN to r2=0.61 and MAE=16.2 for MLR. Prediction results from the validation set yielded correlation coefficients and mean absolute errors which ranged from r2=0.70 and MAE=14.1 for ANN to a low of r2=0.59 and MAE=18.3 for the SVM model. Structure descriptors that contribute significantly to the models are discussed and compared with those found in other published models. For the ANN model, structure descriptor trends with respect to their affects on predicted protein binding can assist the chemist in structure modification during the drug design process.

Photoperiodic regulation of cellular retinol binding protein, CRBP1 [corrected] and nestin in tanycytes of the third ventricle ependymal layer of the Siberian hamster.

PubMed

Barrett, Perry; Ivanova, Elena; Graham, E Scott; Ross, Alexander W; Wilson, Dana; Plé, Helene; Mercer, Julian G; Ebling, Francis J; Schuhler, Sandrine; Dupré, Sandrine M; Loudon, Andrew; Morgan, Peter J

2006-12-01

Tanycytes in the ependymal layer of the third ventricle act both as a barrier and a communication gateway between the cerebrospinal fluid, brain and portal blood supply to the pituitary gland. However, the range, importance and mechanisms involved in the function of tanycytes remain to be explored. In this study, we have utilized a photoperiodic animal to examine the expression of three unrelated gene sequences in relation to photoperiod-induced changes in seasonal physiology and behaviour. We demonstrate that cellular retinol binding protein [corrected] (CRBP1), a retinoic acid transport protein, GPR50, an orphan G-protein-coupled receptor and nestin, an intermediate filament protein, are down-regulated in short-day photoperiods. The distribution of the three sequences is very similar, with expression located in cells with tanycyte morphology in the region of the ependymal layer where tanycytes are located. Furthermore, CRBP1 expression in the ependymal layer is shown to be independent of a circadian clock and altered testosterone levels associated with testicular regression in short photo-period. Pinealectomy of Siberian hamsters demonstrates CRBP1 expression is likely to be dependent on melatonin output from the pineal gland. This provides evidence that tanycytes are seasonally responsive cells and are likely to be an important part of the mechanism to facilitate seasonal physiology and behaviour in the Siberian hamster.

Serum heart type fatty acid binding protein levels are not changed in hyperthyroidism.

PubMed

Ozbek, Mustafa; Gungunes, Askin; Sahin, Mustafa; Ginis, Zeynep; Ucan, Bekir; Sayki, Muyesser; Tutal, Esra; Cakal, Erman; Kuşkonmaz, Serife M; Öztürk, Mehmet A; Delibasi, Tuncay

2016-09-01

Heart type fatty acid binding protein (H-FABP) is a small protein and released into the circulation when myocardial damage has occurred. Previous studies have demonstrated that H-FABP is closely associated with cardiac and some endocrinologic disorders including prediabetes, metabolic syndrome, and acromegaly. Hyperthyroism is a well-known disorder associated with cardiovascular diseases. We aimed to investigate the effect of hyperthyrodism on H-FABP levels. Forty six patients with hyperthyroidism with no known history of coronary artery disease and 40 healthy controls are involved in the study. Serum H-FABP levels are measured using sandwich enzyme-linked immunosorbent assay. There was no significant difference between serum H-FABP levels of patients with hyperthyroidism and controls (871±66 pg/mL, and 816±66 pg/mL, respectively P=0.56). There was no significant correlation between H-FABP, free triiodothyronine (fT3), free thyroxine (fT4), and thyroid stimulating hormone (TSH) levels in patients and controls. Serum H-FABP levels are not altered in patients with hyperthyroidism.

A Major Binding Protein for Leukemia Inhibitory Factor in Normal Mouse Serum: Identification as a Soluble Form of the Cellular Receptor

NASA Astrophysics Data System (ADS)

Layton, Meredith J.; Cross, Bronwyn A.; Metcalf, Donald; Ward, Larry D.; Simpson, Richard J.; Nicola, Nicos A.

1992-09-01

A protein that specifically binds leukemia inhibitory factor (LIF) has been isolated from normal mouse serum by using four successive fractionation steps: chromatography on a LIF affinity matrix, anion-exchange chromatography, size-exclusion chromatography, and preparative native gel electrophoresis. The purified LIF-binding protein (LBP) is a glycoprotein with an apparent molecular mass of 90 kDa that specifically binds 125I-labeled murine LIF with an affinity comparable to that of the low-affinity cellular LIF receptor (K_d = 600 pM). N-terminal sequencing has identified this protein as a soluble truncated form of the α chain of the cellular LIF receptor. LBP is present in normal mouse serum at high levels (1 μg/ml) and these levels are elevated in pregnant mice and reduced in neonatal mice. Since normal serum concentrations of LBP can block the biological actions of LIF in culture, LBP may serve as an inhibitor of the systemic effects of locally produced LIF.

Motion of spin label side chains in cellular retinol-binding protein: correlation with structure and nearest-neighbor interactions in an antiparallel beta-sheet.

PubMed

Lietzow, Michael A; Hubbell, Wayne L

2004-03-23

A goal in the development of site-directed spin labeling in proteins is to correlate the motion of a nitroxide side chain with local structure, interactions, and dynamics. Significant progress toward this goal has been made using alpha-helical proteins of known structure, and the present study is the first step in a similar exploration of a beta-sheet protein, cellular retinol-binding protein (CRBP). Nitroxide side chains were introduced along both interior and edge strands. At sites in interior strands, the side-chain motion is strongly influenced by interactions with side chains of neighboring strands, giving rise to a rich variety of dynamic modes (weakly ordered, strongly ordered, immobilized) and complex electron paramagnetic resonance spectra that are modulated by strand twist. The interactions giving rise to the dynamic modes are explored using mutagenesis, and the results demonstrate the particular importance of the non-hydrogen-bonded neighbor residue in giving rise to highly ordered states. Along edge strands of the beta-sheet, the motion of the side chain is simple and weakly ordered, resembling that at solvent-exposed surfaces of an alpha-helix. A simple working model is proposed that can account for the wide variety of dynamic modes encountered. Collectively, the results suggest that the nitroxide side chain is an effective probe of side-chain interactions, and that site-directed spin labeling should be a powerful means of monitoring conformational changes that involve changes in beta-sheet topology.

Sun-dried cowpeas and amaranth leaves recipe improves β-carotene and retinol levels in serum and hemoglobin concentration among preschool children.

PubMed

Nawiri, Mildred P; Nyambaka, Hudson; Murungi, Jane I

2013-03-01

Vitamin A deficiency (VAD) and anemia are major challenges among children and expecting and lactating mothers in developing countries. Intervention with locally available dark green leafy vegetables (DGLV) is more sustainable to eradicate VAD, being cost-effective and readily adaptable to local communities. DGLV contain high levels of iron and β-carotene (BC) and therefore useful in fighting VAD and anemia. Since DGLVs are season-dependent sun-drying enables their availability during low seasons. However, their contribution to the bioavailability of BC and the improvement of hemoglobin are not well understood. The study therefore investigated the effect of consuming cooked recipe consisting of sun-dried amaranth and cowpea leaves on the levels of BC, retinol, and hemoglobin in preschool children from Machakos District, a semiarid region in Kenya. Vegetables were purchased from local vegetable market, with some sun-dried in an open shade. Levels of BC and retinol in serum and BC in fresh and processed vegetables were determined by a HPLC method and hemoglobin using a portable Hemocue Analyzer. All-trans-BC levels in uncooked fresh cowpea and amaranth leaves were 806.0 μg/g and 599.0 μg/g dry matter, respectively, while the dehydration and cooking processes retained the β-carotene levels at over 60 %. Consumption of the dehydrated vegetables significantly improved both serum BC and retinol levels (p < 0.05), while the baseline hemoglobin levels improved by 4.6 %. The study has shown that intervention with locally available sun-dried vegetables improves the bioavailability of BC, retinol, and hemoglobin levels among preschool children.

Mathematical modeling of serum 13C-retinol in captive rhesus monkeys provides new insights on hypervitaminosis A.

PubMed

Escaron, Anne L; Green, Michael H; Howe, Julie A; Tanumihardjo, Sherry A

2009-10-01

Hypervitaminosis A is increasingly a public health concern, and thus noninvasive quantitative methods merit exploration. In this study, we applied the (13)C-retinol isotope dilution test to a nonhuman primate model with excessive liver stores. After baseline serum chemistries, rhesus macaques (Macaca mulatta; n = 16) were administered 3.5 mumol (13)C(2)-retinyl acetate. Blood was drawn at baseline, 5 h, and 2, 4, 7, 14, 21, and 28 d following the dose. Liver biopsies were collected 7 d before and 2 d after dosing (n = 4) and at 7, 14, and 28 d (n = 4/time) after dosing. Serum and liver were analyzed by HPLC and GC-combustion-isotope ratio MS for retinol and its enrichment, respectively. Model-based compartmental analysis was applied to serum data. Lactate dehydrogenase was elevated in 50% of the monkeys. Total body reserves (TBR) of vitamin A (VA) were calculated at 28 d. Predicted TBR (3.52 +/- 2.01 mmol VA) represented measured liver stores (4.56 +/- 1.38 mmol VA; P = 0.124). Predicted liver VA concentrations (13.3 +/- 9.7 micromol/g) were similar to measured liver VA concentrations (16.4 +/- 5.3 micromol/g). The kinetic models predict that 27-52% of extravascular VA is exchanging with serum in hypervitaminotic A monkeys. The test correctly diagnosed hypervitaminosis A in all monkeys, i.e. 100% sensitivity. Stable isotope techniques have important public health potential for the classification of VA status, including hypervitaminosis, because no other technique besides invasive liver biopsies, correctly identifies excessive liver VA stores.

Structural Basis for the Acyltransferase Activity of Lecithin:Retinol Acyltransferase-like Proteins*

PubMed Central

Golczak, Marcin; Kiser, Philip D.; Sears, Avery E.; Lodowski, David T.; Blaner, William S.; Palczewski, Krzysztof

2012-01-01

Lecithin:retinol acyltransferase-like proteins, also referred to as HRAS-like tumor suppressors, comprise a vertebrate subfamily of papain-like or NlpC/P60 thiol proteases that function as phospholipid-metabolizing enzymes. HRAS-like tumor suppressor 3, a representative member of this group, plays a key role in regulating triglyceride accumulation and energy expenditure in adipocytes and therefore constitutes a novel pharmacological target for treatment of metabolic disorders causing obesity. Here, we delineate a catalytic mechanism common to lecithin:retinol acyltransferase-like proteins and provide evidence for their alternative robust lipid-dependent acyltransferase enzymatic activity. We also determined high resolution crystal structures of HRAS-like tumor suppressor 2 and 3 to gain insight into their active site architecture. Based on this structural analysis, two conformational states of the catalytic Cys-113 were identified that differ in reactivity and thus could define the catalytic properties of these two proteins. Finally, these structures provide a model for the topology of these enzymes and allow identification of the protein-lipid bilayer interface. This study contributes to the enzymatic and structural understanding of HRAS-like tumor suppressor enzymes. PMID:22605381

A novel high-throughput method for supported liquid extraction of retinol and alpha-tocopherol from human serum and simultaneous quantitation by liquid chromatography tandem mass spectrometry.

PubMed

Hinchliffe, Edward; Rudge, James; Reed, Paul

2016-07-01

Measurement of vitamin A (retinol) and E (alpha-tocopherol) in UK clinical laboratories is currently performed exclusively by high-performance liquid chromatography with ultraviolet detection. We investigated whether retinol and alpha-tocopherol could be measured simultaneously by liquid chromatography tandem mass spectrometry. Serum samples (100 μL) were extracted using Isolute + Supported Liquid Extraction plates. Chromatography was performed on a Phenomenex Kinetex Biphenyl 2.6 μm, 50 × 2.1 mm column, and liquid chromatography tandem mass spectrometry on a Waters Acquity TQD. Injection-to-injection time was 4.3 min. The assay was validated according to published guidelines. Patient samples were used to compare liquid chromatography tandem mass spectrometry and high-performance liquid chromatography with ultraviolet detection methods. For retinol and alpha-tocopherol, respectively, the assay was linear up to 6.0 and 80.0 μmol/L, and lower limit of quantification was 0.07 and 0.26 μmol/L. Intra and interassay imprecision were within desirable analytical specifications. Analysis of quality control material aligned to NIST SRM 968e, and relative spiked recovery from human serum, both yielded results within 15% of target values. Method comparison with high-performance liquid chromatography with ultraviolet detection methodology demonstrated a negative bias for retinol and alpha-tocopherol by the liquid chromatography tandem mass spectrometry method. Analysis of United Kingdom National External Quality Assurance Scheme samples yielded mean bias from the target value of +3.0% for retinol and -11.2% for alpha-tocopherol. We have developed a novel, high-throughput method for extraction of retinol and alpha-tocopherol from human serum followed by simultaneous quantitation by liquid chromatography tandem mass spectrometry. The method offers a rapid, sensitive, specific and cost-effective alternative to high-performance liquid chromatography with

Clinical and laboratory features of neuropathies with serum IgM binding to TS-HDS.

PubMed

Pestronk, Alan; Schmidt, Robert E; Choksi, Rati M; Sommerville, R Brian; Al-Lozi, Muhammad T

2012-06-01

In this investigation we studied clinical and laboratory features of polyneuropathies in patients with serum IgM binding to the trisulfated disaccharide IdoA2S-GlcNS-6S (TS-HDS). We retrospectively compared 58 patients with selective IgM binding to TS-HDS to 41 consecutive patients with polyneuropathies without TS-HDS binding. Patients with IgM vs. TS-HDS commonly had distal, sensory, axonal neuropathies. Weakness was associated with IgM M-proteins. Hand pain and serum IgM M-proteins were more common than in control neuropathy patients. TS-HDS antibody binding was often selectively κ class. Biopsies showed capillary pathology with thickened basal lamina and C5b9 complement deposition. IgM in sera with TS-HDS antibodies often bound to capillaries. Serum IgM binding to TS-HDS is associated with painful, sensory > motor, polyneuropathies with an increased frequency of persistent hand discomfort, serum IgM M-proteins, and capillary pathology. Serum IgM binding to TS-HDS suggests a possible immune etiology underlying some otherwise idiopathic sensory polyneuropathies. Copyright © 2011 Wiley Periodicals, Inc.

CXCL4 is a novel nickel-binding protein and augments nickel allergy.

PubMed

Kuroishi, T; Bando, K; Tanaka, Y; Shishido, K; Kinbara, M; Ogawa, T; Muramoto, K; Endo, Y; Sugawara, S

2017-08-01

Nickel (Ni) is the most frequent metal allergen and induces a TH 1 -dependent type-IV allergy. Although Ni 2+ is considered to bind to endogenous proteins, it currently remains unclear whether these Ni-binding proteins are involved in Ni allergy in vivo. We previously reported the adjuvant effects of lipopolysaccharide (LPS) in a Ni allergy mouse model. As LPS induces a number of inflammatory mediators, we hypothesized that Ni-binding protein(s) are also induced by LPS. The objective of this study was to purify and identify Ni-binding protein(s) from serum taken from LPS-injected mice (referred as LPS serum) and examined the augmenting effects of these Ni-binding protein(s) on Ni allergy in an in vivo model. BALB/cA mice were sensitized with an i.p. injection of NiCl 2 and LPS. Ten days after sensitization, mice were challenged with NiCl 2 by an i.d. injection into ear pinnae. Ni-binding protein(s) were purified by Ni-affinity column chromatography and gel filtration. Lipopolysaccharide serum, but not serum taken from saline-injected mice, augmented ear swelling induced by Ni-allergic inflammation. Ni-binding, but not non-binding fraction, purified from LPS serum augmented Ni-allergic inflammation. Mass spectrometry and Western blotting detected CXCL4 in the active fraction. A batch analysis with Ni-sepharose and a surface plasmon resonance analysis revealed direct binding between CXCL4 and Ni 2+ . Recombinant CXCL4 augmented Ni-allergic inflammation and exerted adjuvant effects at the sensitization phase. These results indicate that CXCL4 is a novel Ni-binding protein that augments Ni allergy at the elicitation and sensitization phases. This is the first study to demonstrate that the Ni-binding protein augments Ni allergy in vivo. © 2017 John Wiley & Sons Ltd.

Genetic Determination of Serum Levels of Diabetes-Associated Adipokines

PubMed Central

Schleinitz, Dorit

2015-01-01

Adipose tissue secretes an abundance of proteins. Some of these proteins are known as adipokines and adipose-derived hormones which have been linked with metabolic disorders, including type 2 diabetes, and even with cancer. Variance in serum adipokine concentration is often closely associated with an increase (obesity) or decrease (lipodystrophy) in fat tissue mass, and it is affected by age, gender, and localization of the adipose tissue. However, there may be genetic variants which, in consequence, influence the serum concentration of a certain adipokine, and thereby promote metabolic disturbances or, with regard to the “protective” allele, exert beneficial effects. This review focuses on the genetic determination of serum levels of the following adipokines: adiponectin, chemerin, leptin, progranulin, resistin, retinol binding protein 4, vaspin, adipsin, apelin, and omentin. The article reports on the latest findings from genome-wide association studies (GWAS) and candidate gene studies, showing variants located in/nearby the adipokine genes and other (non-receptor) genes. An extra chapter highlights adipokine-receptor variants. Epigenetic studies on adipokines are also addressed. PMID:26859657

Monoclonal antibodies to human vitamin D-binding protein.

PubMed Central

Pierce, E A; Dame, M C; Bouillon, R; Van Baelen, H; DeLuca, H F

1985-01-01

Monoclonal antibodies to vitamin D-binding protein isolated from human serum have been produced. The antibodies obtained have been shown to be specific for human vitamin D-binding protein by three independent assays. The antibodies recognize human vitamin D-binding protein specifically in an enzyme-linked immunosorbent assay. Human vitamin D-binding protein is detected specifically in both pure and crude samples by a radiometric immunosorbent assay (RISA) and by an immunoprecipitation assay. The anti-human vitamin D-binding protein antibodies cross-react with monkey and pig vitamin D-binding protein, but not with vitamin D-binding protein from rat, mouse, or chicken, as determined by the RISA and immunoprecipitation assays. Images PMID:3936035

Nitrofen increases total retinol levels in placenta during lung morphogenesis in the nitrofen model of congenital diaphragmatic hernia.

PubMed

Kutasy, Balazs; Pes, Lara; Friedmacher, Florian; Paradisi, Francesca; Puri, Prem

2014-10-01

It has been shown that pulmonary retinol level is decreased during lung morphogenesis in the nitrofen-induced PH in congenital diaphragmatic hernia (CDH). Placenta has a major role in the retinol homeostasis in fetal life. Since there is no fetal retinol synthesis, maternal retinol has to cross the placenta. Placenta is the main fetal retinol store where retinol is stored in retinyl-ester formation. Trophoblasts have to produce its own retinol-binding protein (RBP) for retinol transport from placenta to fetus. Recently, we demonstrated that trophoblastic RBP expression is decreased in the nitrofen model of CDH. The aim of this study was to investigate the retinol transfer from mother to the placenta in nitrofen model of CDH. Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetal placenta harvested on D21 and divided into two groups: control (n = 11) and nitrofen with CDH (n = 11). Retinoid levels in placenta were measured using HPLC. Immunohistochemistry was performed to evaluate trophoblastic expression of main RSP genes. Total retinol levels in the placenta were significantly increased in CDH placenta compared to control placenta. The retinyl-ester levels were significantly increased in CDH placenta compared to control placenta. Markedly, decreased immunoreactivity of retinoid signaling pathway was observed in trophoblast cells in CDH compared to control placenta. Increased placental retinol levels show that retinol is transferred from mother to placenta and stored in the placenta in nitrofen model of CDH during lung morphogenesis. Nitrofen may disturb the mobilization of retinol from placenta to fetal circulation causing PH in CDH.

Concentration of carotenoids, retinol and alpha-tocopherol in plasma of six microchiroptera species.

PubMed

Müller, Kerstin; Voigt, Christian C; Raila, Jens; Hurtienne, Andrea; Vater, Marianne; Brunnberg, Leo; Schweigert, Florian J

2007-07-01

To adequately feed species in captivity it is necessary to know their nutritional habits and their natural availability of specific nutrients. Such essential nutrients are vitamin A, vitamin E and selected carotenoids as vitamin-A-precursors. Because their blood plasma concentration are valid biomarkers of nutritional status of dietary intake, we determined the concentrations of carotenoids, retinol and alpha-tocopherol by HPLC as well as the transport proteins for retinol, the retinol-binding protein (RBP) and transthyretin (TTR) immunologically in the plasma of six species of microchiroptera from free-ranging animals and compared it in one species (Carollia perspicillata) to a group held in captivity. Plasma concentrations of the investigated components were generally much lower compared to most other mammals. Within the bats, differences were observed for all components. As in other species retinol, RBP and TTR were present but no retinyl esters could be detected. Plasma of the insectivorous bat species Molossus molossus contained carotenoids. Within the group of carotenoids, beta-carotene was dominant and only traces of lutein were present. Phyllostomus hastatus revealed the highest alpha-tocopherol concentration. No differences in the plasma content of the investigated compounds were found between a group of Carollia perspicillata kept in captivity for 20 years and free-ranging individuals from a population in Central America. No sex related differences were obvious. In conclusion, nutritional biomarkers in bats were highly variable due to dietary and possible species-specific differences.

Bioavailability of Oral Hydrocortisone Corrected for Binding Proteins and Measured by LC-MS/MS Using Serum Cortisol and Salivary Cortisone.

PubMed

Johnson, T N; Whitaker, M J; Keevil, B; Ross, R J

2018-01-01

The assessment absolute bioavailability of oral hydrocortisone is complicated by its saturable binding to cortisol binding globulin (CBG). Previous assessment of bioavailability used a cortisol radioimmunoassay which has cross reactivity with other steroids. Salivary cortisone is a measure of free cortisol and LC-MS/MS is the gold standard method for measuring steroids. We here report the absolute bioavailability of hydrocortisone calculated using serum cortisol and salivary cortisone measured by LC-MS/MS. 14 healthy male dexamethasone suppressed volunteers were administered 20 mg hydrocortisone either intravenously or orally by tablet. Samples of serum and saliva were taken and measured for cortisol and cortisone by LC-MS/MS. Serum cortisol was corrected for saturable binding using published data and pharmacokinetic parameters derived using the program WinNonlin. The mean (95% CI) bioavailability of oral hydrocortisone calculated from serum cortisol, unbound serum cortisol and salivary cortisone was 1.00 (0.89-1.14); 0.88 (0.75-1.05); and 0.93 (0.83-1.05), respectively. The data confirm that, after oral administration, hydrocortisone is completely absorbed. The data derived from serum cortisol corrected for protein binding, and that from salivary cortisone, are similar supporting the concept that salivary cortisone reflects serum free cortisol levels and that salivary cortisone can be used as a non-invasive method for measuring the pharmacokinetics of hydrocortisone.

Biofortified yellow cassava and vitamin A status of Kenyan children: a randomized controlled trial.

PubMed

Talsma, Elise F; Brouwer, Inge D; Verhoef, Hans; Mbera, Gloria N K; Mwangi, Alice M; Demir, Ayşe Y; Maziya-Dixon, Busie; Boy, Erick; Zimmermann, Michael B; Melse-Boonstra, Alida

2016-01-01

Whereas conventional white cassava roots are devoid of provitamin A, biofortified yellow varieties are naturally rich in β-carotene, the primary provitamin A carotenoid. We assessed the effect of consuming yellow cassava on serum retinol concentration in Kenyan schoolchildren with marginal vitamin A status. We randomly allocated 342 children aged 5-13 y to receive daily, 6 d/wk, for 18.5 wk 1) white cassava and placebo supplement (control group), 2) provitamin A-rich cassava (mean content: 1460 μg β-carotene/d) and placebo supplement (yellow cassava group), and 3) white cassava and β-carotene supplement (1053 μg/d; β-carotene supplement group). The primary outcome was serum retinol concentration; prespecified secondary outcomes were hemoglobin concentration and serum concentrations of β-carotene, retinol-binding protein, and prealbumin. Groups were compared by using ANCOVA, adjusting for inflammation, baseline serum concentrations of retinol and β-carotene, and stratified design. The baseline prevalence of serum retinol concentration <0.7 μmol/L and inflammation was 27% and 24%, respectively. For children in the control, yellow cassava, and β-carotene supplement groups, the mean daily intake of cassava was 378, 371, and 378 g, respectively, and the total daily supply of provitamin A and vitamin A from diet and supplements was equivalent to 22, 220, and 175 μg retinol, respectively. Both yellow cassava and β-carotene supplementation increased serum retinol concentration by 0.04 μmol/L (95% CI: 0.00, 0.07 μmol/L); correspondingly, serum β-carotene concentration increased by 524% (448%, 608%) and 166% (134%, 202%). We found no effect on hemoglobin concentration or serum concentrations of retinol-binding protein and prealbumin. In our study population, consumption of yellow cassava led to modest gains in serum retinol concentration and a large increase in β-carotene concentration. It can be an efficacious, new approach to improve vitamin A status. This

Identification of candidate diagnostic serum biomarkers for Kawasaki disease using proteomic analysis

PubMed Central

Kimura, Yayoi; Yanagimachi, Masakatsu; Ino, Yoko; Aketagawa, Mao; Matsuo, Michie; Okayama, Akiko; Shimizu, Hiroyuki; Oba, Kunihiro; Morioka, Ichiro; Imagawa, Tomoyuki; Kaneko, Tetsuji; Yokota, Shumpei; Hirano, Hisashi; Mori, Masaaki

2017-01-01

Kawasaki disease (KD) is a systemic vasculitis and childhood febrile disease that can lead to cardiovascular complications. The diagnosis of KD depends on its clinical features, and thus it is sometimes difficult to make a definitive diagnosis. In order to identify diagnostic serum biomarkers for KD, we explored serum KD-related proteins, which differentially expressed during the acute and recovery phases of two patients by mass spectrometry (MS). We identified a total of 1,879 proteins by MS-based proteomic analysis. The levels of three of these proteins, namely lipopolysaccharide-binding protein (LBP), leucine-rich alpha-2-glycoprotein (LRG1), and angiotensinogen (AGT), were higher in acute phase patients. In contrast, the level of retinol-binding protein 4 (RBP4) was decreased. To confirm the usefulness of these proteins as biomarkers, we analyzed a total of 270 samples, including those collected from 55 patients with acute phase KD, by using western blot analysis and microarray enzyme-linked immunosorbent assays (ELISAs). Over the course of this experiment, we determined that the expression level of these proteins changes specifically in the acute phase of KD, rather than the recovery phase of KD or other febrile illness. Thus, LRG1 could be used as biomarkers to facilitate KD diagnosis based on clinical features. PMID:28262744

Longitudinal study of serum carotenoid, retinol, and tocopherol concentrations in relation to breast cancer risk among postmenopausal women12

PubMed Central

Kim, Mimi; Adams-Campbell, Lucile L; Caan, Bette J; Chlebowski, Rowan T; Neuhouser, Marian L; Shikany, James M; Rohan, Thomas E

2009-01-01

Background: Prospective studies have examined the association of serum and plasma carotenoids and micronutrients and breast cancer; however, to date, studies have only assessed exposure at one point in time. Objective: This study analyzed baseline and repeated serum measurements of carotenoids, retinol, and tocopherols to assess their associations with postmenopausal breast cancer risk. Design: Serum concentrations of α-carotene, β-carotene, β-cryptoxanthin, lycopene, lutein + zeaxanthin, retinol, α-tocopherol, and γ-tocopherol were measured in a 6% sample of women in the Women's Health Initiative clinical trials at baseline and at years 1, 3, and 6 and in a 1% sample of women in the observational study at baseline and at year 3. The association of baseline compounds and breast cancer risk was estimated by Cox proportional hazards models. In addition, repeated measurements were analyzed as time-dependent covariates. Of 5450 women with baseline measurements, 190 incident cases of breast cancer were ascertained over a median of 8.0 y of follow-up. Results: After multivariable adjustment, risk of invasive breast cancer was inversely associated with baseline serum α-carotene concentrations (hazard ratio for highest compared with the lowest tertile: 0.55; 95% CI: 0.34, 0.90; P = 0.02) and positively associated with baseline lycopene (hazard ratio: 1.47; 95% CI: 0.98, 2.22; P = 0.06). Analysis of repeated measurements indicated that α-carotene and β-carotene were inversely associated with breast cancer and that γ-tocopherol was associated with increased risk. Conclusions: The present study, which was the first to assess repeated measurements of serum carotenoids and micronutrients in relation to breast cancer, adds to the evidence of an inverse association of specific carotenoids with breast cancer. The positive associations observed for lycopene and γ-tocopherol require confirmation. This trial was registered at clinicaltrials.gov as NCT00000611. PMID:19474140

Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

PubMed

Oshiro, Satoshi; Honda, Shinya

2014-04-18

Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

Serum proteomic analysis reveals potential serum biomarkers for occupational medicamentosa-like dermatitis caused by trichloroethylene.

PubMed

Huang, Peiwu; Ren, Xiaohu; Huang, Zhijun; Yang, Xifei; Hong, Wenxu; Zhang, Yanfang; Zhang, Hang; Liu, Wei; Huang, Haiyan; Huang, Xinfeng; Wu, Desheng; Yang, Linqing; Tang, Haiyan; Zhou, Li; Li, Xuan; Liu, Jianjun

2014-08-17

Trichloroethylene (TCE) is an industrial solvent with widespread occupational exposure and also a major environmental contaminant. Occupational medicamentosa-like dermatitis induced by trichloroethylene (OMLDT) is an autoimmune disease and it has become one major hazard in China. In this study, sera from 3 healthy controls and 3 OMLDT patients at different disease stages were used for a screening study by 2D-DIGE and MALDI-TOF-MS/MS. Eight proteins including transthyretin (TTR), retinol binding protein 4 (RBP4), haptoglobin, clusterin, serum amyloid A protein (SAA), apolipoprotein A-I, apolipoprotein C-III and apolipoprotein C-II were found to be significantly altered among the healthy, acute-stage, healing-stage and healed-stage groups. Specifically, the altered expression of TTR, RBP4 and haptoglobin were further validated by Western blot analysis and ELISA. Our data not only suggested that TTR, RBP4 and haptoglobin could serve as potential serum biomarkers of OMLDT, but also indicated that measurement of TTR, RBP4 and haptoglobin or their combination could help aid in the diagnosis, monitoring the progression and therapy of the disease. Copyright © 2014. Published by Elsevier Ireland Ltd.

Alteration of human serum albumin binding properties induced by modifications: A review

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, Małgorzata; Szkudlarek, Agnieszka; Chudzik, Mariola; Pożycka, Jadwiga; Sułkowska, Anna

2018-01-01

Albumin, a major transporting protein in the blood, is the main target of modification that affects the binding of drugs to Sudlow's site I and II. These modification of serum protein moderates its physiological function, and works as a biomarker of some diseases. The main goal of the paper was to explain the possible alteration of human serum albumin binding properties induced by modifications such as glycation, oxidation and ageing, their origin, methods of evaluation and positive and negative meaning described by significant researchers.

Male mice are susceptible to high fat diet-induced hyperglycaemia and display increased circulatory retinol binding protein 4 (RBP4) levels and its expression in visceral adipose depots.

PubMed

Asha, G V; Raja Gopal Reddy, M; Mahesh, M; Vajreswari, A; Jeyakumar, S M

2016-01-01

Vitamin A and its metabolites are known to modulate adipose tissue development and its associated complications. Here, we assessed the vitamin A status and its metabolic pathway gene expression in relation to sexual dimorphism by employing 35 days old C57BL/6J male and female mice, which were fed either stock or high fat (HF) diet for 26 weeks. HF diet feeding increased body weight/weight gain and white adipose tissue (WAT) of visceral and subcutaneous regions, however, increase in vitamin A levels observed only in subcutaneous WAT. Further, the expression of most of the vitamin A metabolic pathway genes showed no sexual dimorphism. The observed HF diet-induced hyperglycaemia in male corroborates with increased retinol binding protein 4 (RBP4) levels in plasma and its expression in visceral adipose depots. In conclusion, the male mice are susceptible to high fat diet-induced hyperglycaemia and display higher plasma RBP4 levels, possibly due to its over-expression in visceral adipose depots.

Interaction of Merocyanine 540 with serum albumins: photophysical and binding studies.

PubMed

Banerjee, Mousumi; Pal, Uttam; Subudhhi, Arijita; Chakrabarti, Abhijit; Basu, Samita

2012-03-01

Photophysical studies on binding interactions of a negatively charged anti-tumor photosensitizer, Merocyanine 540 (MC 540), with serum proteins, bovine serum albumin (BSA) and human serum albumin (HSA), have been performed using absorption and steady-state as well as time-resolved fluorescence techniques. Formation of ground state complex has been confirmed from the detailed studies of absorption spectra of MC 540 in presence of SAs producing isosbestic points. Binding between the proteins and MC 540, which perturbs the existing equilibrium between the fluorescent monomer and its non-fluorescent dimer, induces a remarkable enhancement in fluorescence anisotropy and intensity of MC 540 along with a red shift of its maximum. The binding stoichiometry of MC 540 and SAs are more than 1.0 which depicts that two types of complexes, i.e., 1:1 and 2:1 are formed with addition of varied concentration of protein. Both the steady-state and time-resolved fluorescence results show that in 2:1 complex one of the MC 540 molecules is exposed towards aqueous environment with a greater extent when bound with HSA compared to BSA due to the structural flexibility of that protein. Thermodynamic analyses using van't Hoff plot indicate that the binding between MC 540 and individual SA is an entropy-driven phenomenon. The probable hydrophobic binding site has been located by denaturation of proteins, micropolarity measurement and Förster resonance energy transfer and that is further supported by molecular docking studies. Changes in circular dichroism spectra of BSA in presence of MC 540 depict secondary structural changes of the protein. The induced-CD shows that BSA due to its rigid structure generates chirality in MC 540 much more efficiently compared to HSA. Copyright © 2011 Elsevier B.V. All rights reserved.

Expression of serum insulin-like growth factors, insulin-like growth factor-binding proteins, and the growth hormone-binding protein in heterozygote relatives of Ecuadorian growth hormone receptor deficient patients.

PubMed

Fielder, P J; Guevara-Aguirre, J; Rosenbloom, A L; Carlsson, L; Hintz, R L; Rosenfeld, R G

1992-04-01

Recently, an isolated population of apparent GH-receptor deficient (GHRD) patients has been identified in the Loja province of southern Ecuador. These individuals presented many of the physical and biochemical phenotypes characteristic of Laron-Syndrome and are believed to have a defect in the GH-receptor gene. In this study, we have compared the biochemical phenotypes between the affected individuals and their parents, considered to be obligate heterozygotes for the disorder. Serum GH, insulin-like growth factor I and II (IGF-I and IGF-II) levels were measured by RIA Insulin-like growth factor binding proteins. (IGFBPs) were measured by Western ligand blotting (WLB) of serum samples, following separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and relative quantitation of serum IGFBPs was performed with a scanning laser densitometer. Serum GH-binding protein (GHBP) levels were measured with a ligand-mediated immunofunctional assay using a monoclonal antibody raised against the GHBP. These values were then compared to values obtained from normal, sex-matched adult Ecuadorian controls, to determine if the above parameters were abnormal in the heterozygotes. The serum IGF-I levels of the GHRD patients were less than 13% of control values for adults and 2% for children. However, the IGF-I levels of both the mothers and fathers were not significantly different from that of the control population. The serum IGF-II levels of the GHRD patients were approximately 20% of control values for adults and 12% for the children. The IGF-II levels of the mothers were reduced, but were not significantly different from that of the control population. However, IGF-II levels of the fathers were significantly lower than those of controls (64% of control male levels). WLB analysis of serum IGFBP levels of the affected subjects demonstrated increased IGFBP-2 and decreased IGFBP-3, suggesting an inverse relationship between these IGFBPs. The GHRD patients who had the

Goodpasture Antigen-binding Protein/Ceramide Transporter Binds to Human Serum Amyloid P-Component and Is Present in Brain Amyloid Plaques*

PubMed Central

Mencarelli, Chiara; Bode, Gerard H.; Losen, Mario; Kulharia, Mahesh; Molenaar, Peter C.; Veerhuis, Robert; Steinbusch, Harry W. M.; De Baets, Marc H.; Nicolaes, Gerry A. F.; Martinez-Martinez, Pilar

2012-01-01

Serum amyloid P component (SAP) is a non-fibrillar glycoprotein belonging to the pentraxin family of the innate immune system. SAP is present in plasma, basement membranes, and amyloid deposits. This study demonstrates, for the first time, that the Goodpasture antigen-binding protein (GPBP) binds to human SAP. GPBP is a nonconventional Ser/Thr kinase for basement membrane type IV collagen. Also GPBP is found in plasma and in the extracellular matrix. In the present study, we demonstrate that GPBP specifically binds SAP in its physiological conformations, pentamers and decamers. The START domain in GPBP is important for this interaction. SAP and GPBP form complexes in blood and partly colocalize in amyloid plaques from Alzheimer disease patients. These data suggest the existence of complexes of SAP and GPBP under physiological and pathological conditions. These complexes are important for understanding basement membrane, blood physiology, and plaque formation in Alzheimer disease. PMID:22396542

cis Retinol oxidation regulates photoreceptor access to the retina visual cycle and cone pigment regeneration

PubMed Central

Sato, Shinya

2016-01-01

Key points This study explores the nature of the cis retinol that Müller cells in the retina provide to cones for the regeneration of their visual pigment.We report that the retina visual cycle provides cones exclusively with 11‐cis chromophore in both salamander and mouse and show that this selectivity is dependent on the 11‐cis‐specific cellular retinaldehyde binding protein (CRALBP) present in Müller cells.Even though salamander blue cones and green rods share the same visual pigment, only blue cones but not green rods are able to dark‐adapt in the retina following a bleach and to use exogenous 9‐cis retinol for pigment regeneration, suggesting that access to the retina visual cycle is cone‐specific and pigment‐independent.Our results show that the retina produces 11‐cis retinol that can be oxidized and used for pigment regeneration and dark adaptation selectively in cones and not in rods. Abstract Chromophore supply by the retinal Müller cells (retina visual cycle) supports the efficient pigment regeneration required for cone photoreceptor function in bright light. Surprisingly, a large fraction of the chromophore produced by dihydroceramide desaturase‐1, the putative all‐trans retinol isomerase in Müller cells, appears to be 9‐cis retinol. In contrast, the canonical retinal pigment epithelium (RPE) visual cycle produces exclusively 11‐cis retinal. Here, we used the different absorption spectra of 9‐cis and 11‐cis pigments to identify the isoform of the chromophore produced by the visual cycle of the intact retina. We found that the spectral sensitivity of salamander and mouse cones dark‐adapted in the isolated retina (with only the retina visual cycle) was similar to that of cones dark‐adapted in the intact eye (with both the RPE and retina visual cycles) and consistent with pure 11‐cis pigment composition. However, in mice lacking the cellular retinaldehyde binding protein (CRALBP), cone spectral sensitivity contained a

Mathematical Modeling of Serum 13C-Retinol in Captive Rhesus Monkeys Provides New Insights on Hypervitaminosis A1–3

PubMed Central

Escaron, Anne L.; Green, Michael H.; Howe, Julie A.; Tanumihardjo, Sherry A.

2009-01-01

Hypervitaminosis A is increasingly a public health concern, and thus noninvasive quantitative methods merit exploration. In this study, we applied the 13C-retinol isotope dilution test to a nonhuman primate model with excessive liver stores. After baseline serum chemistries, rhesus macaques (Macaca mulatta; n = 16) were administered 3.5 μmol 13C2-retinyl acetate. Blood was drawn at baseline, 5 h, and 2, 4, 7, 14, 21, and 28 d following the dose. Liver biopsies were collected 7 d before and 2 d after dosing (n = 4) and at 7, 14, and 28 d (n = 4/time) after dosing. Serum and liver were analyzed by HPLC and GC-combustion-isotope ratio MS for retinol and its enrichment, respectively. Model-based compartmental analysis was applied to serum data. Lactate dehydrogenase was elevated in 50% of the monkeys. Total body reserves (TBR) of vitamin A (VA) were calculated at 28 d. Predicted TBR (3.52 ± 2.01 mmol VA) represented measured liver stores (4.56 ± 1.38 mmol VA; P = 0.124). Predicted liver VA concentrations (13.3 ± 9.7 μmol/g) were similar to measured liver VA concentrations (16.4 ± 5.3 μmol/g). The kinetic models predict that 27–52% of extravascular VA is exchanging with serum in hypervitaminotic A monkeys. The test correctly diagnosed hypervitaminosis A in all monkeys, i.e. 100% sensitivity. Stable isotope techniques have important public health potential for the classification of VA status, including hypervitaminosis, because no other technique besides invasive liver biopsies, correctly identifies excessive liver VA stores. PMID:19710158

Quantization of bovine serum albumin by fluorescence enhancement effects and corresponding binding of macrocyclic host-protein assembly.

PubMed

Bardhan, Munmun; Misra, Tapas; Ganguly, Tapan

2012-01-05

The present paper reports the investigations on the spectroscopic behavior of the binary complexes of the dye aurintricarboxylic acid (ATA) with protein bovine serum albumin (BSA) and 18-crown 6 (CW) (ATA·BSA, ATA·CW) and the ternary complex ATA·CW·BSA by using UV-vis steady state and time resolved fluorescence spectroscopy. The primary aim of the work is to determine the protein (BSA) quantization by fluorescence enhancement method and investigate the 'enhancer' activity of crown ether (CW) on it to increase the resolution. Steady state and time resolved fluorescence measurements demonstrated how fluorescence intensity of ATA could be used for the determination of the protein BSA in aqueous solution. The binding of dye (probe/fluorescent medicinal molecule) with protein and the denaturing effect in the polar environment of acetonitrile of the dye protein complex act as drug binding as well as drug release activity. Apart from its basic research point of view, the present study also possesses significant importance and applications in the field of medicinal chemistry. Copyright © 2011 Elsevier B.V. All rights reserved.

Structural modification of serum vitamin D3-binding protein and immunosuppression in AIDS patients.

PubMed

Yamamoto, N; Naraparaju, V R; Srinivasula, S M

1995-11-01

A serum glycoprotein, vitamin D3-binding protein (Gc protein), can be converted by beta-galactosidase of stimulated B lymphocytes and sialidase of T lymphocytes to a potent macrophage-activating factor (MAF), a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is a precursor for MAF. Treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generates an extremely high-titered MAF (GcMAF). When peripheral blood monocytes/macrophages of 46 HIV-infected patients were treated with GcMAF (100 pg/ml), the monocytes/macrophages of all patients were efficiently activated. However, the MAF precursor activity of plasma Gc protein was low in 16 (35%) of of these patients. Loss of the MAF precursor activity appeared to be due to deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase found in the patient blood stream. Levels of plasma alpha-N-acetylgalactosaminidase activity in individual patients had an inverse correlation with the MAF precursor activity of their plasma Gc protein. Thus, precursor activity of Gc protein and alpha-N-acetylgalactosaminidase activity in patient blood can serve as diagnostic and prognostic indices.

Locating the Binding Sites of Pb(II) Ion with Human and Bovine Serum Albumins

PubMed Central

Belatik, Ahmed; Hotchandani, Surat; Carpentier, Robert; Tajmir-Riahi, Heidar-Ali

2012-01-01

Lead is a potent environmental toxin that has accumulated above its natural level as a result of human activity. Pb cation shows major affinity towards protein complexation and it has been used as modulator of protein-membrane interactions. We located the binding sites of Pb(II) with human serum (HSA) and bovine serum albumins (BSA) at physiological conditions, using constant protein concentration and various Pb contents. FTIR, UV-visible, CD, fluorescence and X-ray photoelectron spectroscopic (XPS) methods were used to analyse Pb binding sites, the binding constant and the effect of metal ion complexation on HSA and BSA stability and conformations. Structural analysis showed that Pb binds strongly to HSA and BSA via hydrophilic contacts with overall binding constants of KPb-HSA = 8.2 (±0.8)×104 M−1 and KPb-BSA = 7.5 (±0.7)×104 M−1. The number of bound Pb cation per protein is 0.7 per HSA and BSA complexes. XPS located the binding sites of Pb cation with protein N and O atoms. Pb complexation alters protein conformation by a major reduction of α-helix from 57% (free HSA) to 48% (metal-complex) and 63% (free BSA) to 52% (metal-complex) inducing a partial protein destabilization. PMID:22574219

Steady-State Fluorescence Anisotropy to Investigate Flavonoids Binding to Proteins

ERIC Educational Resources Information Center

Ingersoll, Christine M.; Strollo, Christen M.

2007-01-01

The steady-state fluorescence anisotropy is employed to study the binding of protein of a model protein, human serum albumin, to a commonly used flavonoid, quercetin. The experiment describes the thermodynamics, as well as the biochemical interactions of such binding effectively.

Effect of maternal vitamin A supplementation on retinol concentration in colostrum.

PubMed

Grilo, Evellyn C; Lima, Mayara S R; Cunha, Lahyana R F; Gurgel, Cristiane S S; Clemente, Heleni A; Dimenstein, Roberto

2015-01-01

To investigate the effect of vitamin A supplementation on the retinol concentration in colostrum under fasting and postprandial conditions. This was a quasi-experimental study, with before and after assessments, conducted with 33 patients treated at a public maternity hospital. Blood and colostrum samples were collected under fasting conditions in the immediate postpartum period. A second colostrum collection occurred two hours after the first meal of the day, at which time a mega dose of 200,000 IU of retinyl palmitate was administered. On the following day, the colostrum was collected again under fasting and postprandial conditions. Serum and colostrum retinol concentrations were determined by high performance liquid chromatography. The serum retinol concentration was 37.3 (16.8-62.2) μg/dL, indicating adequate nutritional status. The colostrum retinol concentration before supplementation was 46.8 (29.7-158.9) μg/dL in fasting and 67.3 (31.1-148.7) μg/dL in postprandial condition (p < 0.05), showing an increase of 43.8%. After supplementation, the values were 89.5 (32.9-264.2) μg/dL and 102.7 (37.3-378.3) μg/dL in fasting and postprandial conditions, respectively (p < 0.05), representing an increase of 14.7%. This study demonstrated that maternal supplementation with high doses of vitamin A in postpartum resulted in a significant increase of the retinol concentration in colostrum under fasting conditions, with an even greater increase after a meal. Copyright © 2014 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

Spectral Changes of Erythrosin B Luminescence Upon Binding to Bovine Serum Albumin

NASA Astrophysics Data System (ADS)

Sablin, N. V.; Gerasimova, M. A.; Nemtseva, E. V.

2016-04-01

Changes in absorption, fluorescence, phosphorescence, and delayed fluorescence spectra of erythrosin B are studied in the presence of bovine serum albumin at room temperature. Spectral and chronoscopic characteristics of the observed photophysical processes are defined. The binding of erythrosin B with the protein followed by spectral changes is demonstrated. Absorption and fluorescence spectra of the dye in the bound state are described, the binding mechanism is analyzed. The binding parameters of the dye-protein complex are estimated.

Al cation induces aggregation of serum proteins.

PubMed

Chanphai, P; Kreplak, L; Tajmir-Riahi, H A

2017-07-15

Al cation is known to induce protein fibrillation and causes several neurodegenerative disorders. We report the spectroscopic, thermodynamic analysis and AFM imaging for the Al cation binding process with human serum albumin (HSA), bovine serum albumin (BSA) and milk beta-lactoglobulin (b-LG) in aqueous solution at physiological pH. Hydrophobicity played a major role in Al-protein interactions with more hydrophobic b-LG forming stronger Al-protein complexes. Thermodynamic parameters ΔS, ΔH and ΔG showed Al-protein bindings occur via hydrophobic and H-bonding contacts for b-LG, while van der Waals and H-bonding interactions prevail in HSA and BSA adducts. AFM clearly indicated that aluminum cations are able to force BSA and b-LG into larger or more robust aggregates than HSA, with HSA 4±0.2 (SE, n=801) proteins per aggregate, for BSA 17±2 (SE, n=148), and for b-LG 12±3 (SE, n=151). Thioflavin T test showed no major protein fibrillation in the presence of Al cation. Al complexation induced major alterations of protein conformations with the order of perturbations b-LG>BSA>HSA. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression of proteins in serum, synovial fluid, synovial membrane, and articular cartilage samples obtained from dogs with stifle joint osteoarthritis secondary to cranial cruciate ligament disease and dogs without stifle joint arthritis.

PubMed

Garner, Bridget C; Kuroki, Keiichi; Stoker, Aaron M; Cook, Cristi R; Cook, James L

2013-03-01

To identify proteins with differential expression between healthy dogs and dogs with stifle joint osteoarthritis secondary to cranial cruciate ligament (CCL) disease. Serum and synovial fluid samples obtained from dogs with stifle joint osteoarthritis before (n = 10) and after (8) surgery and control dogs without osteoarthritis (9) and archived synovial membrane and articular cartilage samples obtained from dogs with stifle joint osteoarthritis (5) and dogs without arthritis (5). Serum and synovial fluid samples were analyzed via liquid chromatography-tandem mass spectrometry; results were compared against a nonredundant protein database. Expression of complement component 3 in archived tissue samples was determined via immunohistochemical methods. No proteins had significantly different expression between serum samples of control dogs versus those of dogs with stifle joint osteoarthritis. Eleven proteins (complement component 3 precursor, complement factor I precursor, apolipoprotein B-100 precursor, serum paraoxonase and arylesterase 1, zinc-alpha-2-glycoprotein precursor, serum amyloid A, transthyretin precursor, retinol-binding protein 4 precursor, alpha-2-macroglobulin precursor, angiotensinogen precursor, and fibronectin 1 isoform 1 preproprotein) had significantly different expression (> 2.0-fold) between synovial fluid samples obtained before surgery from dogs with stifle joint osteoarthritis versus those obtained from control dogs. Complement component 3 was strongly expressed in all (5/5) synovial membrane samples of dogs with stifle joint osteoarthritis and weakly expressed in 3 of 5 synovial membrane samples of dogs without stifle joint arthritis. Findings suggested that the complement system and proteins involved in lipid and cholesterol metabolism may have a role in stifle joint osteoarthritis, CCL disease, or both.

Interaction study on bovine serum albumin physically binding to silver nanoparticles: Evolution from discrete conjugates to protein coronas

NASA Astrophysics Data System (ADS)

Guo, Jun; Zhong, Ruibo; Li, Wanrong; Liu, Yushuang; Bai, Zhijun; Yin, Jun; Liu, Jingran; Gong, Pei; Zhao, Xinmin; Zhang, Feng

2015-12-01

The nanostructures formed by inorganic nanoparticles together with organic molecules especially biomolecules have attracted increasing attention from both industries and researching fields due to their unique hybrid properties. In this paper, we systemically studied the interactions between amphiphilic polymer coated silver nanoparticles and bovine serum albumins by employing the fluorescence quenching approach in combination with the Stern-Volmer and Hill equations. The binding affinity was determined to 1.30 × 107 M-1 and the interaction was spontaneously driven by mainly the van der Waals force and hydrogen-bond mediated interactions, and negatively cooperative from the point of view of thermodynamics. With the non-uniform coating of amphiphilic polymer, the silver nanoparticles can form protein coronas which can become discrete protein-nanoparticle conjugates when controlling their molar ratios of mixing. The protein's conformational changes upon binding nanoparticles was also studied by using the three-dimensional fluorescence spectroscopy.

Comparison of methods of assessing vitamin A status in children.

PubMed

Makdani, D; Sowell, A L; Nelson, J D; Apgar, J; Gunter, E W; Hegar, A; Potts, W; Rao, D; Wilcox, A; Smith, J C

1996-10-01

A study of children (2-8 years; n = 613) in Belize, Central America, was conducted to determine what proportion of the children might be at risk of vitamin A (vit A) deficiency. The data provide an opportunity to compare results of three methods of assessing vit A status in a population which was not severely malnourished. Serum retinyl ester concentrations were also determined; their relevance to one of the tests, the relative dose response (RDR) test, is discussed. The three methods of assessing vit A status were: RDR test, fasting serum retinol concentration, and conjunctival impression cytology (CIC). Retinol-binding protein (RBP), serum retinyl esters and serum zinc concentrations were also determined. Inadequate vit A status was indicated for 17% of subjects by the RDR test (14% cutoff), for 24% by fasting serum retinol concentration (< 0.87 mumol/L), and for 49% by "abnormal" CIC score. Retinyl esters constituted 24% of serum retinoids at the time (5 hours after a retinyl palmitate dose) at which the second blood sample is taken for the RDR test. Regression tree analyses (CART) indicated ethnicity was a predictor of RDR score; ethnicity, stunting and age were predictors of fasting serum retinol concentration; ethnicity and stunting were predictors of 0-hour retinyl ester concentration. The three indices of vit A status did not identify the same individuals nor indicate the same percentage of the population to be at risk for vit A deficiency. Increased concentrations of retinyl esters at 5 hours compared to those at 0 hours suggest that insufficient retinol may have been taken up by the liver at 5 hours to release all accumulated retinol-binding protein (RBP) in deficient individuals; prevalence of vit A deficiency might therefore be underestimated by the RDR test. The selection of ethnicity as a predictor of RDR score and of 0-hour retinol and retinyl ester concentrations suggests that factors other than vit A status affect vit A metabolism and may affect

Multivalent DNA-binding properties of the HMG-1 proteins.

PubMed Central

Maher, J F; Nathans, D

1996-01-01

HMG-I proteins are DNA-binding proteins thought to affect the formation and function of transcription complexes. Each protein contains three DNA-binding motifs, known as AT-hooks, that bind in the minor groove of AT tracts in DNA. Multiple AT-hooks within a polypeptide chain should contact multiple AT tracts, but the rules governing these interactions have not been defined. In this study, we demonstrate that high-affinity binding uses two or three appropriately spaced AT tracts as a single multivalent binding site. These principles have implications for binding to regulatory elements such as the interferon beta enhancer, TATA boxes, and serum response elements. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8692884

Retinol esterification in bovine retinal pigment epithelium: reversibility of lecithin:retinol acyltransferase.

PubMed Central

Saari, J C; Bredberg, D L; Farrell, D F

1993-01-01

Esterification of all-trans-retinol is a key reaction of the vertebrate visual cycle, since it produces an insoluble, relatively non-toxic, form of the vitamin for storage and supplies substrate for the isomerization reaction. CoA-dependent and -independent pathways have been described for retinol esterification in retinal pigment epithelium (RPE). The CoA-independent reaction, catalysed by lecithin:retinol acyltransferase (LRAT) was examined in more detail in this study. Addition of retinol to RPE microsomes results in a burst of retinyl ester synthesis, followed by a rapid apparent cessation of the reaction. However, [3H]retinol, added when retinyl ester synthesis has apparently ceased, is rapidly incorporated into retinyl ester without a net increase in the amount of ester. The specific radioactivities of [3H]retinol and [3H]retinyl ester reach the same value. [14C]Palmitate from palmitoyl-CoA is incorporated into preexisting retinyl ester in the absence of net ester synthesis, too. These exchange reactions suggest that the reaction has reached equilibrium at the plateau of the progress curve and that only the accumulation of retinyl ester, and not its synthesis, has stopped during this phase of the reaction. Studies with geometrical isomers of retinol revealed that the rate of exchange of all-trans-retinol with all-trans-retinyl esters was about 6 times more rapid than exchange of 11-cis-retinol with 11-cis-retinyl ester. This is the first demonstration of the reversibility of LRAT and the first example of stereospecificity of retinyl ester synthesis in the visual system. Reversal of the LRAT reaction could contribute to the mobilization of 11-cis-retinol from 11-cis-retinyl ester pools. Images Figure 3 PMID:8489497

Clinical relevance of drug binding to plasma proteins

NASA Astrophysics Data System (ADS)

Ascenzi, Paolo; Fanali, Gabriella; Fasano, Mauro; Pallottini, Valentina; Trezza, Viviana

2014-12-01

Binding to plasma proteins highly influences drug efficacy, distribution, and disposition. Serum albumin, the most abundant protein in plasma, is a monomeric multi-domain macromolecule that displays an extraordinary ligand binding capacity, providing a depot and carrier for many endogenous and exogenous compounds, such as fatty acids and most acidic drugs. α-1-Acid glycoprotein, the second main plasma protein, is a glycoprotein physiologically involved in the acute phase reaction and is the main carrier for basic and neutral drugs. High- and low-density lipoproteins play a limited role in drug binding and are natural drug delivery system only for few lipophilic drugs or lipid-based formulations. Several factors influence drug binding to plasma proteins, such as pathological conditions, concurrent administration of drugs, sex, and age. Any of these factors, in turn, influences drug efficacy and toxicity. Here, biochemical, biomedical, and biotechnological aspects of drug binding to plasma proteins are reviewed.

Purification and characterization of a novel cytosolic NADP(H)-dependent retinol oxidoreductase from rabbit liver.

PubMed

Huang, D Y; Ichikawa, Y

1997-03-07

Rabbit liver cytosol exhibits very high retinol dehydrogenase activity. At least two retinol dehydrogenases were demonstrated to exist in rabbit liver cytosol, and the major one, a cytosolic NADP(H)-dependent retinol dehydrogenase (systematic name: retinol oxidoreductase) was purified about 1795-fold to electrophoretic and column chromatographic homogeneity by a procedure involving column chromatography on AF-Red Toyopearl twice and then hydroxyapatite. Its molecular mass was estimated to be 34 kDa by SDS-PAGE, and 144 kDa by HPLC gel filtration, suggesting that it is a homo-tetramer. The enzyme uses free retinol and retinal, and their complexes with CRBP as substrates in vitro. The optimum pH values for retinol oxidation of free retinol and CRBP-retinol were 8.8-9.2 and 8.0-9.0, respectively, and those for retinal reduction of free retinal and retinal-CRBP were the same, 7.0-7.6. Km for free retinol and Vmax for retinal formation were 2.8 microM and 2893 nmol/min per mg protein at 37 degrees C (pH 9.0) and the corresponding values with retinol-CRBP as a substrate were 2.5 microM and 2428 nmol/min per mg protein at 37 degrees C (pH 8.6); Km for free retinal and Vmax for retinol formation were 6.5 microM and 4108 nmol/min per mg protein, and the corresponding values with retinal-CRBP as a substrate were 5.1 microM and 3067 nmol/min per mg protein at 37 degrees C, pH 7.4. NAD(H) was not effective as a cofactor. 4-Methylpyrazole was a weak inhibitor (IC50 = 28 mM) of the enzyme, and ethanol was neither a substrate nor an inhibitor of the enzyme. This enzyme exhibits relatively broad aldehyde reductase activity and some ketone reductase activity, the activity for aromatic substitutive aldehydes being especially high and effective. Whereas, except in the case of retinol, oxidative activity toward the corresponding alcohols was not detected. This novel cytosolic enzyme may play an important role in vivo in maintaining the homeostasis of retinal, the substrate of retinoic

Interaction of Colloidal Gold Nanoparticles with Model Serum Proteins: The Nanoparticle-Protein 'Corona' from a PhysicoChemical Viewpoint

NASA Astrophysics Data System (ADS)

Dominguez Medina, Sergio

When nanoparticles come in contact with biological fluids they become coated with a mixture of proteins present in the media, forming what is known as the nanoparticle-protein 'corona'. This corona changes the nanoparticles' original surface properties and plays a central role in how these get screened by cellular receptors. In the context of biomedical research, this presents a bottleneck for the transition of nanoparticles from research laboratories to clinical settings. It is therefore fundamental to probe these nanoparticle-protein interactions in order to understand the different physico-chemical mechanisms involved. This thesis is aimed to investigate the exposure of colloidal gold nanoparticles to model serum proteins, particularly serum albumin, the main transporter of molecular compounds in the bloodstream of mammals. A set of experimental tools based on optical microscopy and spectroscopy were developed in order to probe these interactions in situ. First, the intrinsic photoluminescence and elastic scattering of individual gold nanoparticles were investigated in order to understand its physical origin. These optical signals were then used to measure the size of the nanoparticles while in Brownian diffusion using fluctuation correlation spectroscopy. This spectroscopic tool was then applied to detect the binding of serum albumin onto the nanoparticle surface, increasing its hydrodynamic size. By performing a binding isotherm as a function of protein concentration, it was determined that serum albumin follows an anti-cooperative binding mechanism on negatively charged gold nanoparticles. This protein monolayer substantially enhanced the stability of the colloid, preventing their aggregation in saline solutions with ionic strength higher than biological media. Cationic gold nanoparticles in contrast, aggregated when serum albumin was present at a low protein-to-nanoparticle ratio, but prevented aggregation if exposed in excess. Single-molecule fluorescence

Meloidogyne incognita Fatty Acid- and Retinol- Binding Protein (Mi-FAR-1) Affects Nematode Infection of Plant Roots and the Attachment of Pasteuria penetrans Endospores.

PubMed

Phani, Victor; Shivakumara, Tagginahalli N; Davies, Keith G; Rao, Uma

2017-01-01

Root-knot nematode (RKN) Meloidogyne incognita is an economically important pest of crops. Pasteuria penetrans , is a nematode hyperparasitic bacterium capable of suppressing the reproduction of RKN and thereby useful for its management. Secreted fatty acid and retinol-binding proteins are unique in nematodes and are engaged in nutrient acquisition, development and reproduction; they are also a component of the nematode cuticle and thought to be involved in the interface between hosts and parasites. Attachment of endospores to the cuticle of second stage juveniles of RKN is the primary step of infection and several factors have been identified to facilitate attachment. In this study, the full length of Mi-far-1 (573 bp) was cloned from M. incognita and characterized. Analysis revealed that the Mi-far-1 was rich in α-helix structure, contained a predicted consensus casein kinase II phosphorylation site and a glycosylation site. Quantitative PCR showed the highest expression in the fourth stage juveniles and in situ hybridization revealed the presence of Mi-far-1 mRNA in the hypodermis below the cuticle. Single copy insertion pattern of Mi-far-1 in M. incognita genome was detected by Southern blotting. Knockdown of Mi-far-1 showed significantly increased attachment of P. penetrans' endospores on juvenile cuticle surface and also affected host finding, root infection and nematode fecundity.

Meloidogyne incognita Fatty Acid- and Retinol- Binding Protein (Mi-FAR-1) Affects Nematode Infection of Plant Roots and the Attachment of Pasteuria penetrans Endospores

PubMed Central

Phani, Victor; Shivakumara, Tagginahalli N.; Davies, Keith G.; Rao, Uma

2017-01-01

Root-knot nematode (RKN) Meloidogyne incognita is an economically important pest of crops. Pasteuria penetrans, is a nematode hyperparasitic bacterium capable of suppressing the reproduction of RKN and thereby useful for its management. Secreted fatty acid and retinol-binding proteins are unique in nematodes and are engaged in nutrient acquisition, development and reproduction; they are also a component of the nematode cuticle and thought to be involved in the interface between hosts and parasites. Attachment of endospores to the cuticle of second stage juveniles of RKN is the primary step of infection and several factors have been identified to facilitate attachment. In this study, the full length of Mi-far-1 (573 bp) was cloned from M. incognita and characterized. Analysis revealed that the Mi-far-1 was rich in α-helix structure, contained a predicted consensus casein kinase II phosphorylation site and a glycosylation site. Quantitative PCR showed the highest expression in the fourth stage juveniles and in situ hybridization revealed the presence of Mi-far-1 mRNA in the hypodermis below the cuticle. Single copy insertion pattern of Mi-far-1 in M. incognita genome was detected by Southern blotting. Knockdown of Mi-far-1 showed significantly increased attachment of P. penetrans’ endospores on juvenile cuticle surface and also affected host finding, root infection and nematode fecundity. PMID:29209280

Influence of myristic acid on furosemide binding to bovine serum albumin. Comparison with furosemide-human serum albumin complex

NASA Astrophysics Data System (ADS)

Bojko, B.; Sułkowska, A.; Maciążek-Jurczyk, M.; Równicka, J.; Sułkowski, W. W.

2010-06-01

Fluorescence studies on furosemide (FUR) binding to bovine serum albumin (BSA) showed the existence of three or four binding sites in the tertiary structure of the protein. Two of them are located in subdomain IIA, while the others in subdomains IB and/or IIIA. Furosemide binding in subdomain IB is postulated on the basis of run of Stern-Volmer plot indicating the existence of two populations of tryptophans involved in the interaction with FUR. In turn, the significant participation of tyrosil residues in complex formation leads to the consideration of the subdomain IIIA as furosemide low-affinity binding site. The effect of increasing concentration of fatty acid on FUR binding in all studied binding sites was also investigated and compared with the previous results obtained for human serum albumin (HSA). For BSA the lesser impact of fatty acid on affinity between drug and albumin was observed. This is probably a result of more significant role of tyrosines in the complex formation and different polarity of microenvironment of the fluorophores when compared HSA and BSA. The most distinct differences between FUR-BSA and FUR-HSA binding parameters are observed when third fatty acid molecule is bound with the protein and rotation of domains I and II occurs. However these structural changes mostly affect FUR low affinity binding sites.

Posaconazole in Human Serum: a Greater Pharmacodynamic Effect than Predicted by the Non-Protein-Bound Serum Concentration ▿

PubMed Central

Lignell, Anders; Löwdin, Elisabeth; Cars, Otto; Chryssanthou, Erja; Sjölin, Jan

2011-01-01

It is generally accepted that only the unbound fraction of a drug is pharmacologically active. Posaconazole is an antifungal agent with a protein binding of 98 to 99%. Taking into account the degree of protein binding, plasma levels in patients, and MIC levels of susceptible strains, it can be assumed that the free concentration of posaconazole sometimes will be too low to exert the expected antifungal effect. The aim was therefore to test the activity of posaconazole in serum in comparison with that of the calculated unbound concentrations in protein-free media. Significant differences (P < 0.05) from the serum control were found at serum concentrations of posaconazole of 1.0 and 0.10 mg/liter, with calculated free concentrations corresponding to 1× MIC and 0.1× MIC, respectively, against one Candida lusitaniae strain selected for proof of principle. In RPMI 1640, the corresponding calculated unbound concentration of 0.015 mg/liter resulted in a significant effect, whereas that of 0.0015 mg/liter did not. Also, against seven additional Candida strains tested, there was an effect of the low posaconazole concentration in serum, in contrast to the results in RPMI 1640. Fluconazole, a low-grade-protein-bound antifungal, was used for comparison at corresponding concentrations in serum and RPMI 1640. No effect was observed at the serum concentration, resulting in a calculated unbound concentration of 0.1× MIC. In summary, there was a substantially greater pharmacodynamic effect of posaconazole in human serum than could be predicted by the non-protein-bound serum concentration. A flux from serum protein-bound to fungal lanosterol 14α-demethylase-bound posaconazole is suggested. PMID:21502622

Deglycosylation of serum vitamin D3-binding protein leads to immunosuppression in cancer patients.

PubMed

Yamamoto, N; Naraparaju, V R; Asbell, S O

1996-06-15

Serum vitamin D3-binding protein (Gc protein) can be converted by beta-galactosidase of B cells and sialidase of T cells to a potent macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is the precursor of the macrophage activating factor (MAF). Treatment of Gc protein with immobilized beta-galactosidase and sialidase generates an extremely high titered MAF, Gc-MAF. When peripheral blood monocytes/macrophages of 52 patients bearing various types of cancer were incubated with 100 pg/ml of GcMAF, the monocytes/macrophages of all patients were efficiently activated. However, the MAF precursor activity of patient plasma Gc protein was found to be severely reduced in about 25% of this patient population. About 45% of the patients had moderately reduced MAF precursor activities. Loss of the precursor activity was found to be due to deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase detected in the patient's bloodstream. The source of the enzyme appeared to be cancerous cells. Radiation therapy decreased plasma alpha-N-acetylgalactosaminidase activity with concomitant increase of precursor activity. This implies that radiation therapy decreases the number of cancerous cells capable of secreting alpha-N-acetylgalactosaminidase. Both alpha-N-acetylgalactosaminidase activity and MAF precursor activity of Gc protein in patient bloodstream can serve as diagnostic and prognostic indices.

Kinetic studies of retinol addition radicals.

PubMed

El-Agamey, Ali; Fukuzumi, Shunichi; Naqvi, K Razi; McGarvey, David J

2011-03-07

Retinol neutral radicals (RS-retinol˙), generated from the reaction of retinol with 4-pyridylthiyl and 2-pyridylthiyl radicals in argon-saturated methanol, undergo β-elimination, which can be monitored via the slow secondary absorption rise at 380 nm attributed to the rearrangement of the unstable retinol neutral addition radicals to the more stable addition radicals. Rate constants for the β-elimination reactions (k(β)) of 4-PyrS-retinol˙ were measured at different temperatures and the Arrhenius equation for the reaction is described by log (k(β)/s(-1)) = (12.7 ± 0.2) - (54.3 ± 1.3)/θ, where θ = 2.3RT kJ mol(-1). The reactivities of retinol addition radicals (RS-retinol˙), generated from the reaction of retinol with various thiyl radicals, towards oxygen have also been investigated in methanol. In the presence of oxygen, the decay of RS-retinol˙ fits to biexponential kinetics and both observed rate constants for the RS-retinol˙ decay are oxygen-concentration dependent. This suggests that at least two thiyl addition radicals, formed from the reaction of RS˙ with retinol, undergo oxygen addition reactions. In light of the estimated rate constants for oxygen addition to RS-retinol˙ and RS-CAR˙ (CAR: carotenoid), the antioxidant-prooxidant properties of retinol are discussed.

Computing the binding affinity of a ligand buried deep inside a protein with the hybrid steered molecular dynamics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Villarreal, Oscar D.; Yu, Lili; Department of Laboratory Medicine, Yancheng Vocational Institute of Health Sciences, Yancheng, Jiangsu 224006

Computing the ligand-protein binding affinity (or the Gibbs free energy) with chemical accuracy has long been a challenge for which many methods/approaches have been developed and refined with various successful applications. False positives and, even more harmful, false negatives have been and still are a common occurrence in practical applications. Inevitable in all approaches are the errors in the force field parameters we obtain from quantum mechanical computation and/or empirical fittings for the intra- and inter-molecular interactions. These errors propagate to the final results of the computed binding affinities even if we were able to perfectly implement the statistical mechanicsmore » of all the processes relevant to a given problem. And they are actually amplified to various degrees even in the mature, sophisticated computational approaches. In particular, the free energy perturbation (alchemical) approaches amplify the errors in the force field parameters because they rely on extracting the small differences between similarly large numbers. In this paper, we develop a hybrid steered molecular dynamics (hSMD) approach to the difficult binding problems of a ligand buried deep inside a protein. Sampling the transition along a physical (not alchemical) dissociation path of opening up the binding cavity- -pulling out the ligand- -closing back the cavity, we can avoid the problem of error amplifications by not relying on small differences between similar numbers. We tested this new form of hSMD on retinol inside cellular retinol-binding protein 1 and three cases of a ligand (a benzylacetate, a 2-nitrothiophene, and a benzene) inside a T4 lysozyme L99A/M102Q(H) double mutant. In all cases, we obtained binding free energies in close agreement with the experimentally measured values. This indicates that the force field parameters we employed are accurate and that hSMD (a brute force, unsophisticated approach) is free from the problem of error amplification

A Comparison of the Effects of Aerobic and Intense Exercise on the Type 2 Diabetes Mellitus Risk Marker Adipokines, Adiponectin and Retinol Binding Protein-4

PubMed Central

Phillips, Amy

2014-01-01

With a more sedentary population comes growing rates of obesity and increased type 2 diabetes mellitus (T2DM) risk. Exercise generally induces positive changes in traditional T2DM risk markers such as lipids, glucose tolerance, and insulin sensitivity; however alterations in concentrations of many circulating cytokines and their respective receptors are also becoming apparent. These cytokines may be early-response health risk factors otherwise overlooked in traditional T2DM risk marker analysis. Plasma levels of two adipocyte-originating cytokines, adiponectin and retinol binding protein 4 (RBP-4), alter following exercise. Adiponectin has anti-inflammatory, anti-atherosclerotic, and anti-insulin resistance roles and its secretion increases with physical activity, whilst elevated RBP-4 leads to increased insulin resistance, and secretion decreases with increasing physical activity; thus these plasma adipokine levels alter favourably following exercise. Although current data are limited, they do suggest that the more intense the exercise, the greater the positive effect on plasma RBP-4 levels, whilst lower intensity aerobic exercise may positively improve adiponectin concentrations. Therefore short-duration, high intensity training may provide a time-efficient alternative to the recommended 150 min moderate aerobic exercise per week in providing positive changes in RBP-4 and other traditional T2DM risk markers and due to increased compliance give greater health benefits over the longer term. PMID:26464853

Comparison of plasma pigment epithelium-derived factor (PEDF), retinol binding protein 4 (RBP-4), chitinase-3-like protein 1 (YKL-40) and brain-derived neurotrophic factor (BDNF) for the identification of insulin resistance.

PubMed

Toloza, F J K; Pérez-Matos, M C; Ricardo-Silgado, M L; Morales-Álvarez, M C; Mantilla-Rivas, J O; Pinzón-Cortés, J A; Pérez-Mayorga, M; Arévalo-García, M L; Tolosa-González, G; Mendivil, C O

2017-09-01

To evaluate and compare the association of four potential insulin resistance (IR) biomarkers (pigment-epithelium-derived factor [PEDF], retinol-binding-protein-4 [RBP-4], chitinase-3-like protein 1 [YKL-40] and brain-derived neurotrophic factor [BDNF]) with objective measures of IR. We studied 81 subjects with different metabolic profiles. All participants underwent a 5-point OGTT with calculation of multiple IR indexes. A subgroup of 21 participants additionally underwent a hyperinsulinemic-euglycemic clamp. IR was defined as belonging to the highest quartile of incremental area under the insulin curve (iAUCins), or to the lowest quartile of the insulin sensitivity index (ISI). PEDF was associated with adiposity variables. PEDF and RBP4 increased linearly across quartiles of iAUCins (for PEDF p-trend=0.029; for RBP-4 p-trend=0.053). YKL-40 and BDNF were not associated with any adiposity or IR variable. PEDF and RBP-4 levels identified individuals with IR by the iAUCins definition: A PEDF cutoff of 11.9ng/mL had 60% sensitivity and 68% specificity, while a RBP-4 cutoff of 71.6ng/mL had 70% sensitivity and 57% specificity. In multiple regression analyses simultaneously including clinical variables and the studied biomarkers, only BMI, PEDF and RBP-4 remained significant predictors of IR. Plasma PEDF and RBP4 identified IR in subjects with no prior diagnosis of diabetes. Copyright © 2017 Elsevier Inc. All rights reserved.

The primary structure of fatty-acid-binding protein from nurse shark liver. Structural and evolutionary relationship to the mammalian fatty-acid-binding protein family.

PubMed

Medzihradszky, K F; Gibson, B W; Kaur, S; Yu, Z H; Medzihradszky, D; Burlingame, A L; Bass, N M

1992-02-01

The primary structure of a fatty-acid-binding protein (FABP) isolated from the liver of the nurse shark (Ginglymostoma cirratum) was determined by high-performance tandem mass spectrometry (employing multichannel array detection) and Edman degradation. Shark liver FABP consists of 132 amino acids with an acetylated N-terminal valine. The chemical molecular mass of the intact protein determined by electrospray ionization mass spectrometry (Mr = 15124 +/- 2.5) was in good agreement with that calculated from the amino acid sequence (Mr = 15121.3). The amino acid sequence of shark liver FABP displays significantly greater similarity to the FABP expressed in mammalian heart, peripheral nerve myelin and adipose tissue (61-53% sequence similarity) than to the FABP expressed in mammalian liver (22% similarity). Phylogenetic trees derived from the comparison of the shark liver FABP amino acid sequence with the members of the mammalian fatty-acid/retinoid-binding protein gene family indicate the initial divergence of an ancestral gene into two major subfamilies: one comprising the genes for mammalian liver FABP and gastrotropin, the other comprising the genes for mammalian cellular retinol-binding proteins I and II, cellular retinoic-acid-binding protein myelin P2 protein, adipocyte FABP, heart FABP and shark liver FABP, the latter having diverged from the ancestral gene that ultimately gave rise to the present day mammalian heart-FABP, adipocyte FABP and myelin P2 protein sequences. The sequence for intestinal FABP from the rat could be assigned to either subfamily, depending on the approach used for phylogenetic tree construction, but clearly diverged at a relatively early evolutionary time point. Indeed, sequences proximately ancestral or closely related to mammalian intestinal FABP, liver FABP, gastrotropin and the retinoid-binding group of proteins appear to have arisen prior to the divergence of shark liver FABP and should therefore also be present in elasmobranchs

Characterization of the binding of metoprolol tartrate and guaifenesin drugs to human serum albumin and human hemoglobin proteins by fluorescence and circular dichroism spectroscopy.

PubMed

Duman, Osman; Tunç, Sibel; Kancı Bozoğlan, Bahar

2013-07-01

The interactions of metoprolol tartrate (MPT) and guaifenesin (GF) drugs with human serum albumin (HSA) and human hemoglobin (HMG) proteins at pH 7.4 were studied by fluorescence and circular dichroism (CD) spectroscopy. Drugs quenched the fluorescence spectra of HSA and HMG proteins through a static quenching mechanism. For each protein-drug system, the values of Stern-Volmer quenching constant, bimolecular quenching constant, binding constant and number of binding site on the protein molecules were determined at 288.15, 298.15, 310.15 and 318.15 K. It was found that the binding constants of HSA-MPT and HSA-GF systems were smaller than those of HMG-MPT and HMG-GF systems. For both drugs, the affinity of HMG was much higher than that of HSA. An increase in temperature caused a negative effect on the binding reactions. The number of binding site on blood proteins for MPT and GF drugs was approximately one. Thermodynamic parameters showed that MPT interacted with HSA through electrostatic attraction forces. However, hydrogen bonds and van der Waals forces were the main interaction forces in the formation of HSA-GF, HMG-MPT and HMG-GF complexes. The binding processes between protein and drug molecules were exothermic and spontaneous owing to negative ∆H and ∆G values, respectively. The values of binding distance between protein and drug molecules were calculated from Förster resonance energy transfer theory. It was found from CD analysis that the bindings of MPT and GF drugs to HSA and HMG proteins altered the secondary structure of HSA and HMG proteins.

Reversible covalent binding of neratinib to human serum albumin in vitro.

PubMed

Chandrasekaran, Appavu; Shen, Li; Lockhead, Susan; Oganesian, Aram; Wang, Jianyao; Scatina, JoAnn

2010-12-01

Neratinib (HKI-272), an irreversible inhibitor of Her 2 tyrosine kinase, is currently in development as an alternative for first and second line therapy in metastatic breast cancer patients who overexpress Her 2. Following incubation of [(14)C]neratinib in control human plasma at 37°C for 6 hours, about 60% to 70% of the radioactivity was not extractable, due to covalent binding to albumin. In this study, factors that could potentially affect the covalent binding of neratinib to plasma proteins, specifically to albumin were investigated. When [(14)C]neratinib was incubated at 10 μg/mL in human serum albumin (HSA) or control human plasma, the percent binding increased with time; the highest percentages of binding (46 and 67%, respectively) were observed at 6 hours, the longest duration of incubation examined. Binding increased with increasing temperature; the highest percentages of binding to HSA or human plasma (59 and 78%) were observed at 45°C, the highest temperature tested. The binding also increased with increasing pH of incubation; the highest percentages of binding (56 and 65%) were observed at pH 8.5, the highest pH value tested. The percentages of binding were similar (53% to 57%) when a wide range of concentrations of [(14)C]neratinib (50 ng/mL to 10 μg/mL) were incubated with human plasma at 37°C for 6 hours, indicating that the binding was independent of the substrate concentration, especially in the therapeutic range (50 to 200 ng/mL). When human plasma proteins containing covalently bound [(14)C]neratinb were suspended in a 10 fold volume of phosphate buffer at pH 4.0, 6.0, 7.4, and 8.5, and further incubated at 37°C for ~ 16 hours, about 45%, 44%, 32%, and 12% of the total radioactivity, respectively, was released as unchanged [(14)C]neratinib, indicating that the binding is reversible in nature, with more released at pH 7.4 and below. In conclusion, the covalent binding of neratinib to serum albumin is pH, time and temperature dependent, but

Vitamin A in human nutrition.

PubMed

Sklan, D

1987-01-01

Vitamin A, an unsaturated 20 carbon cyclic alcohol, has a variety of physiological functions including a role in vision, reproduction, growth and maintenance of epithelial and bone structures. The main sources of vitamin A are from preformed vitamin A in animal foods or from -carotene in green plants. Carotene is cleaved in the intestinal mucosa to retinol, which is transported in chylomicrons mainly to the liver which is the major storage site of vitamin A, where stores are mainly of retinyl palmitate. Utilization of vitamin A appears to be a highly regulated process; retinol is transported in the serum bound to a specific binding protein. There also may be some control of the level of retinol in cells possibly through membrane receptors or of excretion from the cell. Intracellular cytosolic retinol binding proteins transport retinol to the nucleus where specific receptors for retinol have been found. Intracellular binding proteins for retinoic acid and retinal, metabolites of retinol have also been found, and an interstitial protein transporting retinol is present in the eye. Vitamin A deficiency causes cessation of growth, night blindness, and renders the organism more susceptible to infection, and vitamin A supplementation has been shown to enhance immune response. Epidemiological studies have shown low vitamin A and carotene to be correlated with incidence of cancer. Excess vitamin A intake results in hypervitaminosis with severe detrimental effects. Vitamin A requirements appear to be met in most developed countries although in the populations at greatest risk, newborns and pregnant and nursing women, cases of deficiency are observed. However, in large areas of the world vitamin A deficiency is endemic, causing widespread blindness and mortality.

Serum corticosteroid binding globulin expression is modulated by fasting in polar bears (Ursus maritimus).

PubMed

Chow, Brian A; Hamilton, Jason; Cattet, Marc R L; Stenhouse, Gordon; Obbard, Martyn E; Vijayan, Mathilakath M

2011-01-01

Polar bears (Ursus maritimus) from several subpopulations undergo extended fasting during the ice-free season. However, the animals appear to conserve protein despite the prolonged fasting, though the mechanisms involved are poorly understood. We hypothesized that elevated concentrations of corticosteroid binding globulin (CBG), the primary cortisol binding protein in circulation, lead to cortisol resistance and provide a mechanism for protein conservation during extended fasting. The metabolic state (feeding vs. fasting) of 16 field sampled male polar bears was determined based on their serum urea to creatinine ratio (>25 for feeding vs. <5 for fasting). There were no significant differences in serum cortisol levels between all male and female polar bears sampled. Serum CBG expression was greater in lactating females relative to non-lactating females and males. CBG expression was significantly higher in fasting males when compared to non-fasting males. This leads us to suggest that CBG expression may serve as a mechanism to conserve protein during extended fasting in polar bears by reducing systemic free cortisol concentrations. This was further supported by a lower serum glucose concentration in the fasting bears. As well, a lack of an enhanced adrenocortical response to acute capture stress supports our hypothesis that chronic hunger is not a stressor in this species. Overall, our results suggest that elevated serum CBG expression may be an important adaptation to spare proteins by limiting cortisol bioavailability during extended fasting in polar bears. Copyright © 2010 Elsevier Inc. All rights reserved.

Greatly enhanced binding of a cationic porphyrin towards bovine serum albumin by cucurbit[8]uril.

PubMed

Lei, Wanhua; Jiang, Guoyu; Zhou, Qianxiong; Zhang, Baowen; Wang, Xuesong

2010-10-28

Binding affinity towards serum albumin and intracellular proteins is of importance for a photodynamic therapy (PDT) sensitizer to selectively localize in tumours and efficiently induce cell death. In this paper, it was found that cucurbit[8]uril (CB8) can greatly improve the binding affinity of 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate) (TMPyP), a promising PDT photosensitizer, towards bovine serum albumin (BSA). Absorption, fluorescence emission, (1)H NMR, dynamic light scattering, atomic force microscope, as well as protein photocleavage measurements suggest that the binding enhancement originates from the formation of a ternary complex of CB8·TMPyP·tryptophan residues. This finding opens up a new approach for the development of more efficient PDT agents.

Maternal serum fatty acid binding protein 4 (FABP4) and the development of preeclampsia.

PubMed

Scifres, Christina M; Catov, Janet M; Simhan, Hyagriv

2012-03-01

Serum fatty acid binding protein 4 (FABP4) is associated with components of the metabolic syndrome in nonpregnant individuals, including dyslipidemia and insulin resistance. Preeclampsia shares many features with the metabolic syndrome, but the relationship between early pregnancy serum FABP4 levels and the development of preeclampsia is unknown. The aim of the study was to test the hypothesis that FABP4 is elevated in women who develop preeclampsia before the onset of disease. This was a nested case-control study within a larger prospective cohort of healthy women with singleton gestations. Cases included 22 women who developed preeclampsia, and a random sample of 72 unmatched controls delivered without preeclampsia was identified. Maternal serum FABP4 was measured at less than 13 wk gestation and 24-28 wk gestation, which was before the onset of preeclampsia in all patients. The main outcome measure was preeclampsia (new-onset gestational hypertension and proteinuria for the first time after 20 wk gestation). Maternal serum FABP4 concentrations were higher in women who ultimately developed preeclampsia both at 8-13 wk (20.4±12.3 vs. 10.1±4.7 ng/ml; P<0.01) and at 24-28 wk (20.7±11.7 vs. 9.9±4.5 ng/ml; P<0.01). After controlling for first trimester body mass index, systolic blood pressure, and nulliparity, FABP4 was associated with the development of preeclampsia (adjusted odds ratio, 1.2; 95% confidence interval, 1.1-1.3; P<0.01). Maternal serum FABP4 levels are elevated before the clinical onset of preeclampsia, and this increase occurs independently of maternal body mass index.

Identification of C1q as a Binding Protein for Advanced Glycation End Products.

PubMed

Chikazawa, Miho; Shibata, Takahiro; Hatasa, Yukinori; Hirose, Sayumi; Otaki, Natsuki; Nakashima, Fumie; Ito, Mika; Machida, Sachiko; Maruyama, Shoichi; Uchida, Koji

2016-01-26

Advanced glycation end products (AGEs) make up a heterogeneous group of molecules formed from the nonenzymatic reaction of reducing sugars with the free amino groups of proteins. The abundance of AGEs in a variety of age-related diseases, including diabetic complications and atherosclerosis, and their pathophysiological effects suggest the existence of innate defense mechanisms. Here we examined the presence of serum proteins that are capable of binding glycated bovine serum albumin (AGEs-BSA), prepared upon incubation of BSA with dehydroascorbate, and identified complement component C1q subcomponent subunit A as a novel AGE-binding protein in human serum. A molecular interaction analysis showed the specific binding of C1q to the AGEs-BSA. In addition, we identified DNA-binding regions of C1q, including a collagen-like domain, as the AGE-binding site and established that the amount of positive charge on the binding site was the determining factor. C1q indeed recognized several other modified proteins, including acylated proteins, suggesting that the binding specificity of C1q might be ascribed, at least in part, to the electronegative potential of the ligand proteins. We also observed that C1q was involved in the AGEs-BSA-activated deposition of complement proteins, C3b and C4b. In addition, the AGEs-BSA mediated the proteolytic cleavage of complement protein 5 to release C5a. These findings provide the first evidence of AGEs as a new ligand recognized by C1q, stimulating the C1q-dependent classical complement pathway.

Serum protein mediators of dementia and aging proper.

PubMed

Royall, Donald R; Al-Rubaye, Safa; Bishnoi, Ram; Palmer, Raymond F

2016-12-03

The latent variable "δ" (for "dementia") appears to be uniquely responsible for the dementing aspects of cognitive impairment. Age, depressive symptoms, gender and the apolipoprotein E (APOE) ε4 allele are independently associated with δ. In this analysis, we explore serum proteins as potential mediators of age's specific association with δ in a large, ethnically diverse longitudinal cohort, the Texas Alzheimer's Research and Care Consortium (TARCC). 22 serum proteins were recognized as partial mediators of age's association with δ. These include Insulin-like Growth Factor-Binding Protein 2 (IGF-BP2), which we had previously associated with age-specific cognitive change, and both Pancreatic Polypeptide (PP) and von Willebrand Factor (vWF), previously associated with δ. Nine other δ-related proteins were not confirmed by this ethnicity adjusted analysis. Our findings suggest that age's association with the disabling fraction of cognitive performance is partially mediated by serum proteins, somatomedins and hormones. Those proteins may offer targets for the specific treatment of age-related effects on dementia severity and conversion risk.

Protein-protein binding before and after photo-modification of albumin

NASA Astrophysics Data System (ADS)

Rozinek, Sarah C.; Glickman, Randolph D.; Thomas, Robert J.; Brancaleon, Lorenzo

2016-03-01

Bioeffects of directed-optical-energy encompass a wide range of applications. One aspect of photochemical interactions involves irradiating a photosensitizer with visible light in order to induce protein unfolding and consequent changes in function. In the past, irradiation of several dye-protein combinations has revealed effects on protein structure. Beta lactoglobulin, human serum albumin (HSA) and tubulin have all been photo-modified with meso-tetrakis(4- sulfonatophenyl)porphyrin (TSPP) bound, but only in the case of tubulin has binding caused a verified loss of biological function (loss of ability to form microtubules) as a result of this light-induced structural change. The current work questions if the photo-induced structural changes that occur to HSA, are sufficient to disable its biological function of binding to osteonectin. The albumin-binding protein, osteonectin, is about half the molecular weight of HSA, so the two proteins and their bound product can be separated and quantified by size exclusion high performance liquid chromatography. TSPP was first bound to HSA and irradiated, photo-modifying the structure of HSA. Then native HSA or photo-modified HSA (both with TSPP bound) were compared, to assess loss in HSA's innate binding ability as a result of light-induced structure modification.

ANALYSIS OF DRUG-PROTEIN BINDING BY ULTRAFAST AFFINITY CHROMATOGRAPHY USING IMMOBILIZED HUMAN SERUM ALBUMIN

PubMed Central

Mallik, Rangan; Yoo, Michelle J.; Briscoe, Chad J.; Hage, David S.

2010-01-01

Human serum albumin (HSA) was explored for use as a stationary phase and ligand in affinity microcolumns for the ultrafast extraction of free drug fractions and the use of this information for the analysis of drug-protein binding. Warfarin, imipramine, and ibuprofen were used as model analytes in this study. It was found that greater than 95% extraction of all these drugs could be achieved in as little as 250 ms on HSA microcolumns. The retained drug fraction was then eluted from the same column under isocratic conditions, giving elution in less than 40 s when working at 4.5 mL/min. The chromatographic behavior of this system gave a good fit with that predicted by computer simulations based on a reversible, saturable model for the binding of an injected drug with immobilized HSA. The free fractions measured by this method were found to be comparable to those determined by ultrafiltration, and equilibrium constants estimated by this approach gave good agreement with literature values. Advantages of this method include its speed and the relatively low cost of microcolumns that contain HSA. The ability of HSA to bind many types of drugs also creates the possibility of using the same affinity microcolumn to study and measure the free fractions for a variety of pharmaceutical agents. These properties make this technique appealing for use in drug binding studies and in the high-throughput screening of new drug candidates. PMID:20227701

Contribution of Streptococcus mutans Strains with Collagen-Binding Proteins in the Presence of Serum to the Pathogenesis of Infective Endocarditis

PubMed Central

Otsugu, Masatoshi; Matayoshi, Saaya; Teramoto, Noboru; Nakano, Kazuhiko

2017-01-01

ABSTRACT Streptococcus mutans, a major pathogen of dental caries, is considered one of the causative agents of infective endocarditis (IE). Recently, bacterial DNA encoding 120-kDa cell surface collagen-binding proteins (CBPs) has frequently been detected from S. mutans-positive IE patients. In addition, some of the CBP-positive S. mutans strains lacked a 190-kDa protein antigen (PA), whose absence strengthened the adhesion to and invasion of endothelial cells. The interaction between pathogenic bacteria and serum or plasma is considered an important virulence factor in developing systemic diseases; thus, we decided to analyze the pathogenesis of IE induced by S. mutans strains with different patterns of CBP and PA expression by focusing on the interaction with serum or plasma. CBP-positive (CBP+)/PA-negative (PA−) strains showed prominent aggregation in the presence of human serum or plasma, which was significantly greater than that with CBP+/PA-positive (PA+) and CBP-negative (CBP−)/PA+ strains. Aggregation of CBP+/PA− strains was also observed in the presence of a high concentration of type IV collagen, a major extracellular matrix protein in serum. In addition, aggregation of CBP+/PA− strains was drastically reduced when serum complement was inactivated. Furthermore, an ex vivo adherence model and an in vivo rat model of IE showed that extirpated heart valves infected with CBP+/PA− strains displayed prominent bacterial mass formation, which was not observed following infection with CBP+/PA+ and CBP−/PA+ strains. These results suggest that CBP+/PA− S. mutans strains utilize serum to contribute to their pathogenicity in IE. PMID:28947650

The hydroxyl-functionalized magnetic particles for purification of glycan-binding proteins.

PubMed

Sun, Xiuxuan; Yang, Ganglong; Sun, Shisheng; Quan, Rui; Dai, Weiwei; Li, Bin; Chen, Chao; Li, Zheng

2009-12-01

Glycan-protein interactions play important biological roles in biological processes. Although there are some methods such as glycan arrays that may elucidate recognition events between carbohydrates and protein as well as screen the important glycan-binding proteins, there is a lack of simple effectively separate method to purify them from complex samples. In proteomics studies, fractionation of samples can help to reduce their complexity and to enrich specific classes of proteins for subsequent downstream analyses. Herein, a rapid simple method for purification of glycan-binding proteins from proteomic samples was developed using hydroxyl-coated magnetic particles coupled with underivatized carbohydrate. Firstly, the epoxy-coated magnetic particles were further hydroxyl functionalized with 4-hydroxybenzhydrazide, then the carbohydrates were efficiently immobilized on hydroxyl functionalized surface of magnetic particles by formation of glycosidic bond with the hemiacetal group at the reducing end of the suitable carbohydrates via condensation. All conditions of this method were optimized. The magnetic particle-carbohydrate conjugates were used to purify the glycan-binding proteins from human serum. The fractionated glycan-binding protein population was displayed by SDS-PAGE. The result showed that the amount of 1 mg magnetic particles coupled with mannose in acetate buffer (pH 5.4) was 10 micromol. The fractionated glycan-binding protein population in human serum could be eluted from the magnetic particle-mannose conjugates by 0.1% SDS. The methodology could work together with the glycan microarrays for screening and purification of the important GBPs from complex protein samples.

Innovative approaches to vitamin A assessment.

PubMed

Craft, N E

2001-05-01

The health and sight of millions of children are compromised each year as a consequence of vitamin A (VA) deficiency. Serum retinol is the most commonly used indicator of VA status. Unfortunately, its use is impractical for national surveys because it involves collection of venous blood, centrifugation and frozen storage before analysis. To make VA assessment more practical, we have developed approaches incorporating dried blood spots (DBS) or portable instrumentation. DBS have been used as a sample matrix to screen neonates for many biochemical compounds. Until recently, it was not thought that VA was stable in DBS. However, we demonstrated that the measure of DBS retinol correlates well with serum retinol in both healthy adults (r(2) = 0.88-0.90) and compromised populations (r(2) = 0.73-0.84). Compared with serum retinol, the sensitivity and specificity of detecting VA deficiency by DBS retinol range from 73 to 93% and from 90 to 100%, respectively. Although few data are available, retinol binding protein (RBP) can also be measured in DBS. RBP has been used as a surrogate marker for serum retinol. Correlations coefficients (r(2)) between serum RBP and serum retinol range from 0.4 to 0.8. In addition, work has been done to develop portable instrumentation to measure VA status in the field. A fluorometer has been optimized for VA fluorescence and is linear into the deficient range for the direct fluorimetric measurement of serum holo-RBP. Progress is being made to use the instrument to directly measure holo-RBP in a drop of whole blood.

Alterations in chemically induced tissue injury related to all-trans-retinol pretreatment in rodents.

PubMed

Sauer, J M; Hooser, S B; Badger, D A; Baines, A; Sipes, I G

1995-01-01

Retinol (vitamin A) is an essential nutrient which has many physiological effects throughout the body. Our studies have demonstrated that retinol modulation of immune response, through alteration of macrophage and neutrophil function, can have dramatic effects on the toxicity of some compounds. Based on these studies, our current hypothesis for retinol potentiation of chemical-induced liver injury is that retinol administered to rats prior to the hepatotoxicant (CCl4 and AA in rats; and AA, APAP, and GalN in mice) primes the Kupffer cells to a more active state. This may occur in part as a result of increases in chemical mediators such as TNF from these Kupffer cells. Following hepatocyte damage by a toxicant, Kupffer cells are activated to release reactive oxygen species, immune mediators, and chemotactic factors which all serve to enhance the inflammatory response. This increased inflammatory response then results in increased injury to the already toxicant-damaged hepatocytes. In addition, retinol modulation of toxicant activation and detoxification may also make important contributions to the potentiation of some toxicants such as AA. Retinol protection of CCl4 hepatotoxicity in mice is more difficult to explain at this time but is possibly related to alterations in CCl4 metabolism in this species. Differences in response between pulmonary and liver macrophages (Kupffer cells) may explain the retinol protection from 1-NN pulmonary toxicity. Retinol may decrease the inflammatory response through downregulation of pulmonary macrophage function, thus resulting in decreased pulmonary injury. Finally, since retinol protection of cadmium toxicity in the liver and testis requires 7 days of retinol pretreatment, we suspect that retinol is inducing protective protein(s) in these organs. Aside from its normal biological role in rhe body, clinical medicine has found new uses for retinol in the treatment and prevention of some cancers, and in the treatment of certain

The Binding Constant of Estradiol to Bovine Serum Albumin: An Upper-Level Experiment Utilizing Tritium-Labeled Estradiol and Liquid Scintillation Counting

ERIC Educational Resources Information Center

Peihong Liang; Adhyaru, Bhavin; Pearson, Wright L.; Williams, Kathryn R.

2006-01-01

The experiment used [to the third power]H-labeled estradiol to determine the binding constant of estradiol to bovine serum albumin. Estradiol must complex with serum proteins for the transport in the blood stream because of its low solubility in aqueous systems and estradiol-protein binding constant, where K[subscript B] is important to understand…

Behind the scenes of vitamin D binding protein: more than vitamin D binding.

PubMed

Delanghe, Joris R; Speeckaert, Reinhart; Speeckaert, Marijn M

2015-10-01

Although being discovered in 1959, the number of published papers in recent years reveals that vitamin D binding protein (DBP), a member of the albuminoid superfamily, is a hot research topic. Besides the three major phenotypes (DBP1F, DBP1S and DBP2), more than 120 unique variants have been described of this polymorphic protein. The presence of DBP has been demonstrated in different body fluids (serum, urine, breast milk, ascitic fluid, cerebrospinal fluid, saliva and seminal fluid) and organs (brain, heart, lungs, kidneys, placenta, spleen, testes and uterus). Although the major function is binding, solubilization and transport of vitamin D and its metabolites, the name of this glycoprotein hides numerous other important biological functions. In this review, we will focus on the analytical aspects of the determination of DBP and discuss in detail the multifunctional capacity [actin scavenging, binding of fatty acids, chemotaxis, binding of endotoxins, influence on T cell response and influence of vitamin D binding protein-macrophage activating factor (DBP-MAF) on bone metabolism and cancer] of this abundant plasma protein. Copyright © 2015 Elsevier Ltd. All rights reserved.

Spectroscopic and molecular modelling studies of binding mechanism of metformin with bovine serum albumin

NASA Astrophysics Data System (ADS)

Sharma, Deepti; Ojha, Himanshu; Pathak, Mallika; Singh, Bhawna; Sharma, Navneet; Singh, Anju; Kakkar, Rita; Sharma, Rakesh K.

2016-08-01

Metformin is a biguanide class of drug used for the treatment of diabetes mellitus. It is well known that serum protein-ligand binding interaction significantly influence the biodistribution of a drug. Current study was performed to characterize the binding mechanism of metformin with serum albumin. The binding interaction of the metformin with bovine serum albumin (BSA) was examined using UV-Vis absorption spectroscopy, fluorescence, circular dichroism, density functional theory and molecular docking studies. Absorption spectra and fluorescence emission spectra pointed out the weak binding of metformin with BSA as was apparent from the slight change in absorbance and fluorescence intensity of BSA in presence of metformin. Circular dichroism study implied the significant change in the conformation of BSA upon binding with metformin. Density functional theory calculations showed that metformin has non-planar geometry and has two energy states. The docking studies evidently signified that metformin could bind significantly to the three binding sites in BSA via hydrophobic, hydrogen bonding and electrostatic interactions. The data suggested the existence of non-covalent specific binding interaction in the complexation of metformin with BSA. The present study will certainly contribute to the development of metformin as a therapeutic molecule.

A comparative study of the effects of retinol and retinoic acid on histological, molecular, and clinical properties of human skin.

PubMed

Kong, Rong; Cui, Yilei; Fisher, Gary J; Wang, Xiaojuan; Chen, Yinbei; Schneider, Louise M; Majmudar, Gopa

2016-03-01

All-trans retinol, a precursor of retinoic acid, is an effective anti-aging treatment widely used in skin care products. In comparison, topical retinoic acid is believed to provide even greater anti-aging effects; however, there is limited research directly comparing the effects of retinol and retinoic acid on skin. In this study, we compare the effects of retinol and retinoic acid on skin structure and expression of skin function-related genes and proteins. We also examine the effect of retinol treatment on skin appearance. Skin histology was examined by H&E staining and in vivo confocal microscopy. Expression levels of skin genes and proteins were analyzed using RT-PCR and immunohistochemistry. The efficacy of a retinol formulation in improving skin appearance was assessed using digital image-based wrinkle analysis. Four weeks of retinoic acid and retinol treatments both increased epidermal thickness, and upregulated genes for collagen type 1 (COL1A1), and collagen type 3 (COL3A1) with corresponding increases in procollagen I and procollagen III protein expression. Facial image analysis showed a significant reduction in facial wrinkles following 12 weeks of retinol application. The results of this study demonstrate that topical application of retinol significantly affects both cellular and molecular properties of the epidermis and dermis, as shown by skin biopsy and noninvasive imaging analyses. Although the magnitude tends to be smaller, retinol induces similar changes in skin histology, and gene and protein expression as compared to retinoic acid application. These results were confirmed by the significant facial anti-aging effect observed in the retinol efficacy clinical study. © 2015 Wiley Periodicals, Inc.

Increased uptake of dietary retinoids at the maternal-fetal barrier in the nitrofen model of congenital diaphragmatic hernia.

PubMed

Kutasy, Balazs; Friedmacher, Florian; Pes, Lara; Paradisi, Francesca; Puri, Prem

2014-06-01

The retinol signaling pathway is disrupted in congenital diaphragmatic hernia (CDH). Since there is no fetal retinol synthesis, maternal retinol has to cross the placenta. Nitrofen interferes with the retinol-binding protein (RBP) transfer pathway in CDH. However, in RBP knockout mice, retinol has been shown to be present. In this model, increased uptake of maternal dietary retinyl ester (RE) bounded in low-dense-lipoprotein (LDL) through low-density-lipoprotein-receptor 1 (LRP1) and increased activity of RE hydrolysis by lipoprotein-lipase (LPL) have been found. The aim of this study was to investigate the RE transfer pathway in the nitrofen CDH model. Pregnant rats were treated with nitrofen or vehicle on gestational day (D9) and sacrificed on D21. Immunohistochemistry was performed to evaluate LRP1 and LPL protein expression. Serum LDL levels were measured by ELISA. Pulmonary and serum retinoid levels were measured using HPLC. Markedly increased trophoblastic and pulmonary LRP1 and LPL immunoreactivity were observed in CDH compared to controls. Significantly increased serum LDL and RE levels were observed in CDH compared to controls. The increased uptake of dietary retinoids at the maternal-fetal barrier in the nitrofen CDH model suggests that the RE transfer pathway may be the main source of retinol in this model. Copyright © 2014 Elsevier Inc. All rights reserved.

Contribution of Streptococcus mutans Strains with Collagen-Binding Proteins in the Presence of Serum to the Pathogenesis of Infective Endocarditis.

PubMed

Otsugu, Masatoshi; Nomura, Ryota; Matayoshi, Saaya; Teramoto, Noboru; Nakano, Kazuhiko

2017-12-01

Streptococcus mutans , a major pathogen of dental caries, is considered one of the causative agents of infective endocarditis (IE). Recently, bacterial DNA encoding 120-kDa cell surface collagen-binding proteins (CBPs) has frequently been detected from S. mutans -positive IE patients. In addition, some of the CBP-positive S. mutans strains lacked a 190-kDa protein antigen (PA), whose absence strengthened the adhesion to and invasion of endothelial cells. The interaction between pathogenic bacteria and serum or plasma is considered an important virulence factor in developing systemic diseases; thus, we decided to analyze the pathogenesis of IE induced by S. mutans strains with different patterns of CBP and PA expression by focusing on the interaction with serum or plasma. CBP-positive (CBP + )/PA-negative (PA - ) strains showed prominent aggregation in the presence of human serum or plasma, which was significantly greater than that with CBP + /PA-positive (PA + ) and CBP-negative (CBP - )/PA+ strains. Aggregation of CBP + /PA - strains was also observed in the presence of a high concentration of type IV collagen, a major extracellular matrix protein in serum. In addition, aggregation of CBP + /PA - strains was drastically reduced when serum complement was inactivated. Furthermore, an ex vivo adherence model and an in vivo rat model of IE showed that extirpated heart valves infected with CBP + /PA - strains displayed prominent bacterial mass formation, which was not observed following infection with CBP + /PA + and CBP - /PA + strains. These results suggest that CBP + /PA - S. mutans strains utilize serum to contribute to their pathogenicity in IE. Copyright © 2017 American Society for Microbiology.

Protein microarray analysis reveals BAFF-binding autoantibodies in systemic lupus erythematosus

PubMed Central

Price, Jordan V.; Haddon, David J.; Kemmer, Dodge; Delepine, Guillaume; Mandelbaum, Gil; Jarrell, Justin A.; Gupta, Rohit; Balboni, Imelda; Chakravarty, Eliza F.; Sokolove, Jeremy; Shum, Anthony K.; Anderson, Mark S.; Cheng, Mickie H.; Robinson, William H.; Browne, Sarah K.; Holland, Steven M.; Baechler, Emily C.; Utz, Paul J.

2013-01-01

Autoantibodies against cytokines, chemokines, and growth factors inhibit normal immunity and are implicated in inflammatory autoimmune disease and diseases of immune deficiency. In an effort to evaluate serum from autoimmune and immunodeficient patients for Abs against cytokines, chemokines, and growth factors in a high-throughput and unbiased manner, we constructed a multiplex protein microarray for detection of serum factor–binding Abs and used the microarray to detect autoantibody targets in SLE. We designed a nitrocellulose-surface microarray containing human cytokines, chemokines, and other circulating proteins and demonstrated that the array permitted specific detection of serum factor–binding probes. We used the arrays to detect previously described autoantibodies against cytokines in samples from individuals with autoimmune polyendocrine syndrome type 1 and chronic mycobacterial infection. Serum profiling from individuals with SLE revealed that among several targets, elevated IgG autoantibody reactivity to B cell–activating factor (BAFF) was associated with SLE compared with control samples. BAFF reactivity correlated with the severity of disease-associated features, including IFN-α–driven SLE pathology. Our results showed that serum factor protein microarrays facilitate detection of autoantibody reactivity to serum factors in human samples and that BAFF-reactive autoantibodies may be associated with an elevated inflammatory disease state within the spectrum of SLE. PMID:24270423

Selective binding of pyrene in subdomain IB of human serum albumin: Combining energy transfer spectroscopy and molecular modelling to understand protein binding flexibility

NASA Astrophysics Data System (ADS)

Ling, Irene; Taha, Mohamed; Al-Sharji, Nada A.; Abou-Zied, Osama K.

2018-04-01

The ability of human serum albumin (HSA) to bind medium-sized hydrophobic molecules is important for the distribution, metabolism, and efficacy of many drugs. Herein, the interaction between pyrene, a hydrophobic fluorescent probe, and HSA was thoroughly investigated using steady-state and time-resolved fluorescence techniques, ligand docking, and molecular dynamics (MD) simulations. A slight quenching of the fluorescence signal from Trp214 (the sole tryptophan residue in the protein) in the presence of pyrene was used to determine the ligand binding site in the protein, using Förster's resonance energy transfer (FRET) theory. The estimated FRET apparent distance between pyrene and Trp214 was 27 Å, which was closely reproduced by the docking analysis (29 Å) and MD simulation (32 Å). The highest affinity site for pyrene was found to be in subdomain IB from the docking results. The calculated equilibrium structure of the complex using MD simulation shows that the ligand is largely stabilized by hydrophobic interaction with Phe165, Phe127, and the nonpolar moieties of Tyr138 and Tyr161. The fluorescence vibronic peak ratio I1/I3 of bound pyrene inside HSA indicates the presence of polar effect in the local environment of pyrene which is less than that of free pyrene in buffer. This was clarified by the MD simulation results in which an average of 5.7 water molecules were found within 0.5 nm of pyrene in the binding site. Comparing the fluorescence signals and lifetimes of pyrene inside HSA to that free in buffer, the high tendency of pyrene to form dimer was almost completely suppressed inside HSA, indicating a high selectivity of the binding pocket toward pyrene monomer. The current results emphasize the ability of HSA, as a major carrier of several drugs and ligands in blood, to bind hydrophobic molecules in cavities other than subdomain IIA which is known to bind most hydrophobic drugs. This ability stems from the nature of the amino acids forming the binding

Calcium-dependent interaction of monomeric S100P protein with serum albumin.

PubMed

Kazakov, Alexei S; Shevelyova, Marina P; Ismailov, Ramis G; Permyakova, Maria E; Litus, Ekaterina A; Permyakov, Eugene A; Permyakov, Sergei E

2018-03-01

S100 proteins are multifunctional (intra/extra)cellular mostly dimeric calcium-binding proteins engaged into numerous diseases. We have found that monomeric recombinant human S100P protein interacts with intact human serum albumin (HSA) in excess of calcium ions with equilibrium dissociation constant of 25-50nM, as evidenced by surface plasmon resonance spectroscopy and fluorescent titration by HSA of S100P labelled by fluorescein isothiocyanate. Calcium removal or S100P dimerization abolish the S100P-HSA interaction. The interaction is selective, since S100P does not bind bovine serum albumin and monomeric human S100B lacks interaction with HSA. In vitro glycation of HSA disables its binding to S100P. The revealed selective and highly specific conformation-dependent interaction between S100P and HSA shows that functional properties of monomeric and dimeric forms of S100 proteins are different, and raises concerns on validity of cell-based assays and animal models used for studies of (patho)physiological roles of extracellular S100 proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Protein Binding Capacity of Different Forages Tannin

NASA Astrophysics Data System (ADS)

Yusiati, L. M.; Kurniawati, A.; Hanim, C.; Anas, M. A.

2018-02-01

Eight forages of tannin sources(Leucaena leucocephala, Arachis hypogaea, Mimosa pudica, Morus alba L, Swietenia mahagoni, Manihot esculenta, Gliricidia sepium, and Bauhinia purpurea)were evaluated their tannin content and protein binding capacity. The protein binding capacity of tannin were determined using precipitation of bovine serum albumin (BSA). Swietenia mahagonihas higest total tannin level and condensed tannin (CT) compared with other forages (P<0.01). The Leucaena leucocephala has highest hydrolysable tannin (HT) level (P<0.01). The total and condensed tannin content of Swietenia mahagoni were 11.928±0.04 mg/100 mg and 9.241±0.02mg/100mg dry matter (DM) of leaves. The hydrolysable tannin content of Leucaena leucocephala was 5.338±0.03 mg/100 mg DM of leaves. Binding capacity was highest in Swietenia mahagoni and Leucaena leucocephala compared to the other forages (P<0.01). The optimum binding of BSA to tannin in Leucaena leucocephala and Swietenia mahagoniwere1.181±0.44 and 1.217±0.60mg/mg dry matter of leaves. The present study reports that Swietenia mahagoni has highest of tannin content and Leucaena leucocephala and Swietenia mahagoni capacity of protein binding.

Association Between Serum Levels of Adipocyte Fatty Acid-binding Protein and Free Thyroxine

PubMed Central

Tseng, Fen-Yu; Chen, Pei-Lung; Chen, Yen-Ting; Chi, Yu-Chao; Shih, Shyang-Ron; Wang, Chih-Yuan; Chen, Chi-Ling; Yang, Wei-Shiung

2015-01-01

Abstract Adipocyte fatty acid-binding protein (AFABP) has been shown to be a biomarker of body weight change and atherosclerosis. Changes in thyroid function are associated with changes in body weight and risks of cardiovascular diseases. The association between AFABP and thyroid function status has been seldom evaluated. The aim of this study was to compare the serum AFABP concentrations in hyperthyroid patients and those in euthyroid individuals, and to evaluate the associations between serum AFABP and free thyroxine (fT4) levels. For this study, 30 hyperthyroid patients and 30 euthyroid individuals at a referral medical center were recruited. The patients with hyperthyroidism were treated with antithyroid regimens as clinically indicated. No medication was given to the euthyroid individuals. The body weight, body mass index, thyroid function, serum levels of AFABP, and biochemical data of both groups at baseline and at the 6th month were compared. Associations between AFABP and fT4 levels were also analyzed. At the baseline, the hyperthyroid patients had significantly higher serum AFABP levels than the euthyroid individuals (median [Q1, Q3]: 22.8 [19.4, 30.6] ng/mL vs 18.6 [15.3, 23.2] ng/mL; P = 0.038). With the antithyroid regimens, the AFABP serum levels of the hyperthyroid patients decreased to 16.6 (15.0, 23.9) ng/mL at the 6th month. No difference in the AFABP level was found between the hyperthyroid and the euthyroid groups at the 6th month. At baseline, sex (female vs male, ß = 7.65, P = 0.022) and fT4 level (ß = 2.51, P = 0.018) were significantly associated with AFABP levels in the univariate regression analysis. At the 6th month, sex and fT4 level (ß = 8.09, P < 0.001 and ß = 3.61, P = 0.005, respectively) were also significantly associated with AFABP levels. The associations between sex and fT4 level with AFABP levels remained significant in the stepwise multivariate regression analysis, both at baseline and at

Enhanced protective antibody to a mutant meningococcal factor H-binding protein with low-factor H binding

PubMed Central

Granoff, Dan M.; Giuntini, Serena; Gowans, Flor A.; Lujan, Eduardo; Sharkey, Kelsey; Beernink, Peter T.

2016-01-01

Meningococcal factor H-binding protein (FHbp) is an antigen in 2 serogroup B meningococcal vaccines. FHbp specifically binds human and some nonhuman primate complement FH. To investigate the effect of binding of FH to FHbp on protective antibody responses, we immunized infant rhesus macaques with either a control recombinant FHbp antigen that bound macaque FH or a mutant antigen with 2 amino acid substitutions and >250-fold lower affinity for FH. The mutant antigen elicited 3-fold higher serum IgG anti-FHbp titers and up to 15-fold higher serum bactericidal titers than the control FHbp vaccine. When comparing sera with similar IgG anti-FHbp titers, the antibodies elicited by the mutant antigen gave greater deposition of complement component C4b on live meningococci (classical complement pathway) and inhibited binding of FH, while the anti-FHbp antibodies elicited by the control vaccine enhanced FH binding. Thus, the mutant FHbp vaccine elicited an anti-FHbp antibody repertoire directed at FHbp epitopes within the FH binding site, which resulted in greater protective activity than the antibodies elicited by the control vaccine, which targeted FHbp epitopes outside of the FH combining site. Binding of a host protein to a vaccine antigen impairs protective antibody responses, which can be overcome with low-binding mutant antigens. PMID:27668287

Preclinical characterization of anticancer gallium(III) complexes: solubility, stability, lipophilicity and binding to serum proteins.

PubMed

Rudnev, Alexander V; Foteeva, Lidia S; Kowol, Christian; Berger, Roland; Jakupec, Michael A; Arion, Vladimir B; Timerbaev, Andrei R; Keppler, Bernhard K

2006-11-01

The discovery and development of gallium(III) complexes capable of inhibiting tumor growth is an emerging area of anticancer drug research. A range of novel gallium coordination compounds with established cytotoxic efficacy have been characterized in terms of desirable chemical and biochemical properties and compared with tris(8-quinolinolato)gallium(III) (KP46), a lead anticancer gallium-based candidate that successfully finished phase I clinical trials (under the name FFC11), showing activity against renal cell cancer. In view of probable oral administration, drug-like parameters, such as solubility in water, saline and 0.5% dimethyl sulfoxide, stability against hydrolysis, measured as the rate constant of hydrolytic degradation in water or physiological buffer using a capillary zone electrophoresis (CZE) assay, and the octanol-water partition coefficient (logP) providing a rational estimate of a drug's lipophilicity, have been evaluated and compared. The differences in bioavailability characteristics between different complexes were discussed within the formalism of structure-activity relationships. The reactivity toward major serum transport proteins, albumin and transferrin, was also assayed in order to elucidate the drug's distribution pathway after intestinal absorption. According to the values of apparent binding rate constants determined by CZE, both KP46 and bis(2-acetylpyridine-4,4-dimethyl-3-thiosemicarbazonato-N,N,S)gallium(III) tetrachlorogallate(III) (KP1089) bind to transferrin faster than to albumin. This implies that transferrin would rather mediate the accumulation of gallium antineoplastic agents in solid tumors. A tendency of being faster converted into the protein-bound form found for KP1089 (due possibly to non-covalent binding) seems complementary to its greater in vitro antiproliferative activity.

Characterization of the complex between native and reduced bovine serum albumin with aquacobalamin and evidence of dual tetrapyrrole binding.

PubMed

Dereven'kov, Ilia A; Hannibal, Luciana; Makarov, Sergei V; Makarova, Anna S; Molodtsov, Pavel A; Koifman, Oskar I

2018-05-02

Serum albumin binds to a variety of endogenous ligands and drugs. Human serum albumin (HSA) binds to heme via hydrophobic interactions and axial coordination of the iron center by protein residue Tyr161. Human serum albumin binds to another tetrapyrrole, cobalamin (Cbl), but the structural and functional properties of this complex are poorly understood. Herein, we investigate the reaction between aquacobalamin (H 2 OCbl) and bovine serum albumin (BSA, the bovine counterpart of HSA) using Ultraviolet-Visible and fluorescent spectroscopy, and electron paramagnetic resonance. The reaction between H 2 OCbl and BSA led to the formation of a BSA-Cbl(III) complex consistent with N-axial ligation (amino). Prior to the formation of this complex, the reactants participate in an additional binding event that has been examined by fluorescence spectroscopy. Binding of BSA to Cbl(III) reduced complex formation between the bound cobalamin and free cyanide to form cyanocobalamin (CNCbl), suggesting that the β-axial position of the cobalamin may be occupied by an amino acid residue from the protein. Reaction of BSA containing reduced disulfide bonds with H 2 OCbl produces cob(II)alamin and disulfide with intermediate formation of thiolate Cbl(III)-BSA complex and its decomposition. Finally, in vitro studies showed that cobalamin binds to BSA only in the presence of an excess of protein, which is in contrast to heme binding to BSA that involves a 1:1 stoichiometry. In vitro formation of BSA-Cbl(III) complex does not preclude subsequent heme binding, which occurs without displacement of H 2 OCbl bound to BSA. These data suggest that the two tetrapyrroles interact with BSA in different binding pockets.

Serum concentrations of lipids, vitamin d metabolites, retinol, retinyl esters, tocopherols and selected carotenoids in twelve captive wild felid species at four zoos.

PubMed

Crissey, Susan D; Ange, Kimberly D; Jacobsen, Krista L; Slifka, Kerri A; Bowen, Phyllis E; Stacewicz-Sapuntzakis, Maria; Langman, Craig B; Sadler, William; Kahn, Stephen; Ward, Ann

2003-01-01

Serum concentrations of several nutrients were measured in 12 captive wild felid species including caracal (Felis caracal), cheetah (Acinonyx jubatus), cougar (Felis concolor), fishing cat (Felis viverrinus), leopard (Panthera pardus), lion (Panthera leo), ocelot (Felis pardalis), pallas cat (Felis manul), sand cat (Felis margarita), serval (Felis serval), snow leopard (Panthera uncia) and tiger (Panthera tigris). Diet information was collected for these animals from each participating zoo (Brookfield Zoo, Fort Worth Zoo, Lincoln Park Zoological Gardens and North Carolina Zoological Park). The nutritional composition of the diets at each institution met the probable dietary requirements for each species except for the pallas cat. Blood samples were collected from each animal (n = 69) and analyzed for lipids (total cholesterol, triacylglycerides, HDL cholesterol and LDL cholesterol), vitamin D metabolites [25-hydroxycholecalciferol (25(OH)D) and 1,25-dihydroxycholecalciferol (1,25(OH)(2)D)], vitamin A (retinol, retinyl stearate and retinyl palmitate), vitamin E (alpha- and gamma-tocopherol) and selected carotenoids. Species differences were found for all except triacylglycerides and 1,25(OH)(2)D. Genus differences were found for retinol, retinyl palmitate, retinyl stearate, gamma-tocopherol and beta-carotene. Circulating nutrient concentrations for many of the species in this study have not been reported previously and most have not been compared with the animals' dietary intakes. The large number of animals analyzed provides a substantial base for comparing the serum nutrient concentrations of healthy animals, for both wild and captive exotic species.

Characterization of binding of N'-nitrosonornicotine to protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hughes, M.F.

1986-01-01

The NADPH-dependent activation of the carcinogenic nitrosamine, N'-nitrosonornicotine (NNN) to a reactive intermediate which binds covalently to protein was assessed using male Sprague-Dawley rat liver and lung microsomes. The NADPH-dependent covalent binding of (/sup 14/C)NNN to liver and lung microsomes was linear with time up to 90 and 45 min, respectively and was also linear with protein concentrations up to 3.0 and 2.0 mg/ml, respectively. The apparent K/sub m/ and V/sub max/ of the NADPH-dependent binding to liver microsomes were determined from the initial velocities. Addition of the thiols glutathione, cystein, N-acetylcysteine or 2-mercapthoethanol significantly decreased the non-NADPH-dependent binding tomore » liver microsomal protein, but did not affect the NADPH-dependent binding. Glutathione was required in order to observe any NADPH-dependent binding to lung microsomal protein. In lung microsomes, SKF-525A significantly decreased the NADPH-dependent binding by 79%. Replacement of an air atmosphere with N/sub 2/ or CO:O/sub 2/ (8:2) significantly decreased the NADPH-dependent binding of (/sup 14/C)NNN to liver microsomal protein by 40% or 27% respectively. Extensive covalent binding of (/sup 14/C)NNN to liver and muscle microsomal protein occurred in the absence of an NADPH-generating system, in the presence of 50% methanol and also to bovine serum albumin, indicating a nonenzymatic reaction. These data indicate that cytochrome P-450 is at least in part responsible for the metabolic activation of the carcinogen NNN, but also suggest additional mechanisms of activation.« less

A spectroscopic and molecular docking approach on the binding of tinzaparin sodium with human serum albumin

NASA Astrophysics Data System (ADS)

Abdullah, Saleh M. S.; Fatma, Sana; Rabbani, Gulam; Ashraf, Jalaluddin M.

2017-01-01

Protein bound toxins are poorly removed by conventional extracorporeal therapies. Venous thromboembolism (VTE) is a major cause of morbidity and mortality in patients with cancer. The interaction between tinzaparin, an inhibitor of angiotensin converting enzyme and human serum albumin, a principal plasma protein in the liver has been investigated in vitro under a simulated physiological condition by UV-vis spectrophotometry and fluorescence spectrometry. The intrinsic fluorescence intensity of human serum albumin was strongly quenched by tinzaparin (TP). The binding constants and binding stoichiometry can be calculated from the data obtained from fluorescence quenching experiments. The negative value of ΔG° reveals that the binding process is a spontaneous process. Thermodynamic analysis shows that the HSA-TP complex formation occurs via hydrogen bonds, hydrophobic interactions and undergoes slight structural changes as evident by far-UV CD. It indicated that the hydrophobic interactions play a main role in the binding of TP to human serum albumin. In addition, the distance between TP (acceptor) and tryptophan residues of human serum albumin (donor) was estimated to be 2.21 nm according to the Förster's resonance energy transfer theory. For the deeper understanding of the interaction, thermodynamic, and molecular docking studies were performed as well. Our docking results suggest that TP forms stable complex with HSA (Kb ∼ 104) and its primary binding site is located in subdomain IIA (Sudlow Site I). The results obtained herein will be of biological significance in pharmacology and clinical medicine.

Changes in serum proteins after endotoxin administration in healthy and choline-treated calves.

PubMed

Yilmaz, Z; Eralp Inan, O; Kocaturk, M; Baykal, A T; Hacariz, O; Hatipoglu, I; Tvarijonaviciute, A; Cansev, M; Ceron, J; Ulus, I H

2016-09-20

This study aimed to investigate the possible serum protein changes after endotoxin administration in healthy and choline-treated calves using proteomics. These results are expected to contribute to the understanding of the pathophysiological mechanisms of endotoxemia and the beneficial effect of choline administration in this clinical situation. Healthy-calves (n = 20) were divided into 4 groups: Control, Choline treated (C), Lipopolysaccharide administered (LPS), and LPS + C. Control calves received 0.9 % NaCl injection. Calves in C and LPS + C groups received choline chloride (1 mg/kg/iv). Endotoxin (LPS) was injected (2 μg/kg/iv) to the calves in LPS and LPS + C groups. Serum samples were collected before and after the treatments. Differentially expressed proteins (> 1.5 fold-change relative to controls) were identified by LC-MS/MS. After LPS administration, 14 proteins increased, and 13 proteins decreased within 48 h as compared to controls. In the LPS group, there were significant increases in serum levels of ragulator complex protein (189-fold) and galectin-3-binding protein (10-fold), but transcription factor MafF and corticosteroid binding globulin were down regulated (≥ 5 fold). As compared with the LPS group, in LPS + C group, fibrinogen gamma-B-chain and antithrombin were up-regulated, while hemopexin and histone H4 were down-regulated. Choline treatment attenuated actin alpha cardiac muscle-1 overexpression after LPS. LPS administration produces changes in serum proteins associated with lipid metabolism, immune and inflammatory response, protein binding/transport, cell adhesion, venous thrombosis, cardiac contractility and blood coagulation. The administration of choline is associated with changes in proteins which can be related with its beneficial effect in this clinical situation.

Serum growth hormone (GH)-binding protein/receptor: an important determinant of GH responsiveness.

PubMed

Martha, P M; Reiter, E O; Dávila, N; Shaw, M A; Holcombe, J H; Baumann, G

1992-12-01

Individual growth rates (or responses to GH therapy) and adult heights vary over a wide range. The reasons for this variation are poorly understood. Based on the reciprocal relationship between GH production and serum GH-binding protein/receptor (GH-BP), we hypothesized that genetic growth potential was achieved by a specific combination of GH-BP/receptor and GH production in each individual. To address the question whether GH production regulates GH-BP, or vice versa, we studied GH-deficient children, where one of the parameters, GH exposure, could be controlled through exogenous administration. Forty-three untreated prepubertal GH-deficient children were studied before and after 6 and 12 months of GH replacement therapy (0.18 mg/kg.week). Growth velocity, height, bone age, weight and their respective Z scores, serum GH-BP, and serum insulin-like growth factor I (IGF-I) were measured at each time point. The patients responded with significant increases in serum IGF-I, age-adjusted growth velocity, and height (P < 10(-6) for all). Before therapy, GH-BP correlated directly with chronologic and bone age (P < 10(-4), but not with either growth velocity or IGF-I. In contrast, GH-BP correlated strongly with the response to therapy whether assessed as the incremental change in IGF-I (P < 10(-6)) or as the increase in growth velocity (P approximately 0.003). GH treatment had no consistent effect on GH-BP/receptor levels. These findings support the concept that the GH-BP/receptor endowment is characteristic for an individual and plays a pivotal role in somatic growth. The GH-BP/receptor system and its ontogeny appears relatively independent of regulation by GH. Differences in individual GH-BP/GH receptor complement account for some of the variability in the response to GH, and GH-BP levels may serve as a predictor for the degree of response. The reciprocal relationship between GH production and GH-BP in normal subjects probably results from adjustment of GH secretion to

Characterization of human short chain dehydrogenase/reductase SDR16C family members related to retinol dehydrogenase 10.

PubMed

Adams, Mark K; Lee, Seung-Ah; Belyaeva, Olga V; Wu, Lizhi; Kedishvili, Natalia Y

2017-10-01

All-trans-retinoic acid (RA) is a bioactive derivative of vitamin A that serves as an activating ligand for nuclear transcription factors, retinoic acid receptors. RA biosynthesis is initiated by the enzymes that oxidize retinol to retinaldehyde. It is well established that retinol dehydrogenase 10 (RDH10, SDR16C4), which belongs to the 16C family of the short chain dehydrogenase/reductase (SDR) superfamily of proteins, is the major enzyme responsible for the oxidation of retinol to retinaldehyde for RA biosynthesis during embryogenesis. However, several lines of evidence point towards the existence of additional retinol dehydrogenases that contribute to RA biosynthesis in vivo. In close proximity to RDH10 gene on human chromosome 8 are located two genes that are phylogenetically related to RDH10. The predicted protein products of these genes, retinol dehydrogenase epidermal 2 (RDHE2, SDR16C5) and retinol dehydrogenase epidermal 2-similar (RDHE2S, SDR16C6), share 59% and 56% sequence similarity with RDH10, respectively. Previously, we showed that the single ortholog of the human RDHE2 and RDHE2S in frogs, Xenopus laevis rdhe2, oxidizes retinol to retinaldehyde and is essential for frog embryonic development. In this study, we explored the potential of each of the two human proteins to contribute to RA biosynthesis. The results of this study demonstrate that human RDHE2 exhibits a relatively low but reproducible activity when expressed in either HepG2 or HEK293 cells. Expression of the native RDHE2 is downregulated in the presence of elevated levels of RA. On the other hand, the protein encoded by the human RDHE2S gene is unstable when expressed in HEK293 cells. RDHE2S protein produced in Sf9 cells is stable but has no detectable catalytic activity towards retinol. We conclude that the human RDHE2S does not contribute to RA biosynthesis, whereas the low-activity RA-sensitive human RDHE2 may have a role in adjusting the cellular levels of RA in accord with

Evidence that Chemical Chaperone 4-Phenylbutyric Acid Binds to Human Serum Albumin at Fatty Acid Binding Sites

PubMed Central

James, Joel; Shihabudeen, Mohamed Sham; Kulshrestha, Shweta; Goel, Varun; Thirumurugan, Kavitha

2015-01-01

Endoplasmic reticulum stress elicits unfolded protein response to counteract the accumulating unfolded protein load inside a cell. The chemical chaperone, 4-Phenylbutyric acid (4-PBA) is a FDA approved drug that alleviates endoplasmic reticulum stress by assisting protein folding. It is found efficacious to augment pathological conditions like type 2 diabetes, obesity and neurodegeneration. This study explores the binding nature of 4-PBA with human serum albumin (HSA) through spectroscopic and molecular dynamics approaches, and the results show that 4-PBA has high binding specificity to Sudlow Site II (Fatty acid binding site 3, subdomain IIIA). Ligand displacement studies, RMSD stabilization profiles and MM-PBSA binding free energy calculation confirm the same. The binding constant as calculated from fluorescence spectroscopic studies was found to be kPBA = 2.69 x 105 M-1. Like long chain fatty acids, 4-PBA induces conformational changes on HSA as shown by circular dichroism, and it elicits stable binding at Sudlow Site II (fatty acid binding site 3) by forming strong hydrogen bonding and a salt bridge between domain II and III of HSA. This minimizes the fluctuation of HSA backbone as shown by limited conformational space occupancy in the principal component analysis. The overall hydrophobicity of W214 pocket (located at subdomain IIA), increases upon occupancy of 4-PBA at any FA site. Descriptors of this pocket formed by residues from other subdomains largely play a role in compensating the dynamic movement of W214. PMID:26181488

An odorant-binding protein as a new allergen from Siberian hamster (Phodopus sungorus).

PubMed

Torres, J A; Pastor-Vargas, C; de las Heras, M; Vivanco, F; Cuesta, Javier; Sastre, J

2012-01-01

A case of anaphylaxis following a bite from a Siberian hamster (SH; Phodopus sungorus) is described. Skin prick tests with hair, urine and salivary gland extracts from SH were positive, while the tests were negative for hair extracts from other rodents. IgE immunoblotting with the patient serum revealed 3 IgE-binding bands of about 18, 21 and 23 kDa. When the patient's serum was preincubated with rabbit, mouse and gerbil hair extracts, no inhibition of the 3 SH IgE-binding bands was demonstrated. Proteins extracted from the 3 bands were analyzed by N-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry, and peptides were sequenced. IgE-binding bands were identified as being an odorant-binding protein belonging to the lipocalin family. Analysis of the 3 IgE-binding bands found in the hair, urine and salivary glands of SH showed a new allergenic protein lacking cross-reactivity with allergens from other rodents. The 3 bands likely correspond to isoforms of a single allergen. Copyright © 2011 S. Karger AG, Basel.

Characterizing Active Pharmaceutical Ingredient Binding to Human Serum Albumin by Spin-Labeling and EPR Spectroscopy.

PubMed

Hauenschild, Till; Reichenwallner, Jörg; Enkelmann, Volker; Hinderberger, Dariush

2016-08-26

Drug binding to human serum albumin (HSA) has been characterized by a spin-labeling and continuous-wave (CW) EPR spectroscopic approach. Specifically, the contribution of functional groups (FGs) in a compound on its albumin-binding capabilities is quantitatively described. Molecules from different drug classes are labeled with EPR-active nitroxide radicals (spin-labeled pharmaceuticals (SLPs)) and in a screening approach CW-EPR spectroscopy is used to investigate HSA binding under physiological conditions and at varying ratios of SLP to protein. Spectral simulations of the CW-EPR spectra allow extraction of association constants (KA ) and the maximum number (n) of binding sites per protein. By comparison of data from 23 SLPs, the mechanisms of drug-protein association and the impact of chemical modifications at individual positions on drug uptake can be rationalized. Furthermore, new drug modifications with predictable protein binding tendency may be envisaged. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

HPLC/UV quantitation of retinal, retinol, and retinyl esters in serum and tissues

PubMed Central

Kane, Maureen A.; Folias, Alexandra E.; Napoli, Joseph L.

2008-01-01

We report robust HPLC/UV methods for quantifying retinyl esters (RE), retinol (ROL) and retinal (RAL) applicable to diverse biological samples, with lower limits of detection of 0.7 pmol, 0.2 pmol, and 0.2 pmol, respectively, and linear ranges >3 orders of magnitude. These assays function well with small, complex biological samples (10–20 mg tissue). Coefficients of variation range from: intra-day, 5.9–10.0%; inter-day, 5.9–11.0%. Quantification of endogenous RE, ROL, and RAL in mouse serum and tissues (liver, kidney, adipose, muscle, spleen, testis, skin, brain, and brain regions) reveals utility. Ability to discriminate spatial concentrations of ROL and RE is illustrated with C57BL/6 mouse brain loci (hippocampus, cortex, olfactory bulb, thalamus, cerebellum, and striatum.) We also developed a method to distinguish isomeric forms of ROL to investigate precursors of retinoic acid. The ROL isomer assay has limits of detection between 3.5–4.5 pmol and a similar linear range and % CV as the ROL/RE and RAL assays. The assays described here provide for sensitive and rigorous quantification of endogenous RE, ROL, and RAL to elucidate retinoid homeostasis in disease states, such as Alzheimer’s disease, type 2 diabetes, obesity, and cancer. PMID:18410739

Purification of a protein from serum of cattle with hepatic lipidosis, and identification of the protein as haptoglobin.

PubMed

Yoshino, K; Katoh, N; Takahashi, K; Yuasa, A

1992-06-01

A protein that has 2 subunits with molecular weight of 35,000 and 23,000 was detected in serum of cattle with hepatic lipidosis (fatty liver). The protein was purified from serum obtained from a cow with fatty liver, and was identified as haptoglobin, which is known to have hemoglobin-binding capacity and to be an acute-phase protein. To assess the relevance of haptoglobin in fatty liver, cattle were classified in 3 groups (healthy control, haptoglobin-positive, and haptoglobin-negative); liver triglyceride content and several serum biochemical variables were evaluated for the 3 groups. Compared with the control and haptoglobin-negative cattle, haptoglobin-positive cattle had significantly (P less than 0.01) higher liver triglyceride content, serum bilirubin concentration, and aspartate transaminase activity. Serum haptoglobin concentration was high in slaughter cattle (27 of 40 cattle tested), particularly in cows (20/28).

The interaction of human serum albumin with selected lanthanide and actinide ions: Binding affinities, protein unfolding and conformational changes.

PubMed

Ali, Manjoor; Kumar, Amit; Kumar, Mukesh; Pandey, Badri N

2016-04-01

Human serum albumin (HSA), the most abundant soluble protein in blood plays critical roles in transportation of biomolecules and maintenance of osmotic pressure. In view of increasing applications of lanthanides- and actinides-based materials in nuclear energy, space, industries and medical applications, the risk of exposure with these metal ions is a growing concern for human health. In present study, binding interaction of actinides/lanthanides [thorium: Th(IV), uranium: U(VI), lanthanum: La(III), cerium: Ce(III) and (IV)] with HSA and its structural consequences have been investigated. Ultraviolet-visible, Fourier transform-infrared, Raman, Fluorescence and Circular dichroism spectroscopic techniques were applied to study the site of metal ions interaction, binding affinity determination and the effect of metal ions on protein unfolding and HSA conformation. Results showed that these metal ions interacted with carbonyl (CO..:)/amide(N..-H) groups and induced exposure of aromatic residues of HSA. The fluorescence analysis indicated that the actinide binding altered the microenvironment around Trp214 in the subdomain IIA. Binding affinity of U(VI) to HSA was slightly higher than that of Th(IV). Actinides and Ce(IV) altered the secondary conformation of HSA with a significant decrease of α-helix and an increase of β-sheet, turn and random coil structures, indicating a partial unfolding of HSA. A correlation was observed between metal ion's ability to alter HSA conformation and protein unfolding. Both cationic effects and coordination ability of metal ions seemed to determine the consequences of their interaction with HSA. Present study improves our understanding about the protein interaction of these heavy ions and their impact on its secondary structure. In addition, binding characteristics may have important implications for the development of rational antidote for the medical management of health effects of actinides and lanthanides. Copyright © 2016 Elsevier

Super-sensitive time-resolved fluoroimmunoassay for thyroid-stimulating hormone utilizing europium(III) nanoparticle labels achieved by protein corona stabilization, short binding time, and serum preprocessing.

PubMed

Näreoja, Tuomas; Rosenholm, Jessica M; Lamminmäki, Urpo; Hänninen, Pekka E

2017-05-01

Thyrotropin or thyroid-stimulating hormone (TSH) is used as a marker for thyroid function. More precise and more sensitive immunoassays are needed to facilitate continuous monitoring of thyroid dysfunctions and to assess the efficacy of the selected therapy and dosage of medication. Moreover, most thyroid diseases are autoimmune diseases making TSH assays very prone to immunoassay interferences due to autoantibodies in the sample matrix. We have developed a super-sensitive TSH immunoassay utilizing nanoparticle labels with a detection limit of 60 nU L -1 in preprocessed serum samples by reducing nonspecific binding. The developed preprocessing step by affinity purification removed interfering compounds and improved the recovery of spiked TSH from serum. The sensitivity enhancement was achieved by stabilization of the protein corona of the nanoparticle bioconjugates and a spot-coated configuration of the active solid-phase that reduced sedimentation of the nanoparticle bioconjugates and their contact time with antibody-coated solid phase, thus making use of the higher association rate of specific binding due to high avidity nanoparticle bioconjugates. Graphical Abstract We were able to decrease the lowest limit of detection and increase sensitivity of TSH immunoassay using Eu(III)-nanoparticles. The improvement was achieved by decreasing binding time of nanoparticle bioconjugates by small capture area and fast circular rotation. Also, we applied a step to stabilize protein corona of the nanoparticles and a serum-preprocessing step with a structurally related antibody.

Dye-binding protein assay using a long-wave-absorbing cyanine probe.

PubMed

Zheng, Hong; Mao, Yu Xia; Li, Dong Hui; Zhu, Chang Qing

2003-07-01

A simple and fast protein assay that involves the binding of water-soluble sulfonate heptamethylene cyanine to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 778 to 904 nm, and the increase in absorption at 904 nm is monitored. This assay is very reproducible, of good color stability for at least 80 min, and sensitive at the 100 ng/mL level of human serum albumin (HSA) when a spectrophotometer with near-infrared wavelength is used to measure absorbance. Few chemicals except ionic surfactants such as cetyltrimethylammonium bromide and sodium dodecyl sulfonate interfere with the assay. Purified proteins have different capacities to interact with the dye; under the experimental conditions, the linear ranges of bovine serum albumin (BSA), HSA and gamma-IgG were 200-2000, 100-2400, and 200-3000 ng/mL, respectively. The relative standard deviation for the five replicate determinations of 1200 ng/mL BSA is 2.1%.

Interaction entropy for protein-protein binding

NASA Astrophysics Data System (ADS)

Sun, Zhaoxi; Yan, Yu N.; Yang, Maoyou; Zhang, John Z. H.

2017-03-01

Protein-protein interactions are at the heart of signal transduction and are central to the function of protein machine in biology. The highly specific protein-protein binding is quantitatively characterized by the binding free energy whose accurate calculation from the first principle is a grand challenge in computational biology. In this paper, we show how the interaction entropy approach, which was recently proposed for protein-ligand binding free energy calculation, can be applied to computing the entropic contribution to the protein-protein binding free energy. Explicit theoretical derivation of the interaction entropy approach for protein-protein interaction system is given in detail from the basic definition. Extensive computational studies for a dozen realistic protein-protein interaction systems are carried out using the present approach and comparisons of the results for these protein-protein systems with those from the standard normal mode method are presented. Analysis of the present method for application in protein-protein binding as well as the limitation of the method in numerical computation is discussed. Our study and analysis of the results provided useful information for extracting correct entropic contribution in protein-protein binding from molecular dynamics simulations.

Interaction entropy for protein-protein binding.

PubMed

Sun, Zhaoxi; Yan, Yu N; Yang, Maoyou; Zhang, John Z H

2017-03-28

Protein-protein interactions are at the heart of signal transduction and are central to the function of protein machine in biology. The highly specific protein-protein binding is quantitatively characterized by the binding free energy whose accurate calculation from the first principle is a grand challenge in computational biology. In this paper, we show how the interactionentropy approach, which was recently proposed for protein-ligand binding free energy calculation, can be applied to computing the entropic contribution to the protein-protein binding free energy. Explicit theoretical derivation of the interactionentropy approach for protein-protein interaction system is given in detail from the basic definition. Extensive computational studies for a dozen realistic protein-protein interaction systems are carried out using the present approach and comparisons of the results for these protein-protein systems with those from the standard normal mode method are presented. Analysis of the present method for application in protein-protein binding as well as the limitation of the method in numerical computation is discussed. Our study and analysis of the results provided useful information for extracting correct entropic contribution in protein-protein binding from molecular dynamics simulations.

Effect of Proteolysis with Alkaline Protease Following High Hydrostatic Pressure Treatment on IgE Binding of Buckwheat Protein.

PubMed

Lee, Chaeyoon; Lee, Wonhui; Han, Youngshin; Oh, Sangsuk

2017-03-01

Buckwheat is a popular food material in many Asian countries and it contains major allergenic proteins. This study was performed to analyze the effects of hydrolysis with alkaline protease following high hydrostatic pressure (HHP) treatment on the IgE binding of buckwheat protein. Extracted buckwheat protein was treated with HHP at 600 MPa for 30 min and hydrolyzed with alkaline protease for 240 min. IgE binding was examined using an enzyme-linked immunosorbent assay (ELISA) with serum samples from 14 patients who were allergic to buckwheat. Depending on the serum samples, HHP treatment of buckwheat protein without enzymatic hydrolysis decreased the IgE binding by 8.9% to 73.2% or increased by 31% to 78%. The IgE binding of buckwheat protein hydrolyzed with alkaline protease decreased by 73.8% to 100%. The IgE binding of buckwheat protein hydrolyzed with alkaline protease following HHP treatment decreased by 83.8% to 100%. This suggested that hydrolysis with alkaline protease following HHP treatment could be applied to reduce the IgE binding of buckwheat protein. © 2017 Institute of Food Technologists®.

The interaction of albumin and fatty-acid-binding protein with membranes: oleic acid dissociation.

PubMed

Catalá, A

1984-10-01

Bovine serum albumin or fatty-acid-binding protein rapidly lose oleic acid when incubated in the presence of dimyristoyl lecithin liposomes. The phenomenon is dependent on vesicle concentration and no measurable quantities of protein are found associated with liposomes. Upon gel filtration on Sepharose CL-2B of incubated mixtures of microsomes containing [1-14C] oleic acid and albumin or fatty-acid-binding protein, association of fatty acid with the soluble proteins could be demonstrated. Both albumin and fatty-acid-binding protein stimulated the transfer of oleic acid from rat liver microsomes to egg lecithin liposomes. These results indicate that albumin is more effective in the binding of oleic acid than fatty-acid-binding protein, which allows a selective oleic acid dissociation during its interaction with membranes.

Mathematical modeling as accounting: predicting the fate of serum proteins and therapeutic monoclonal antibodies.

PubMed

Gurbaxani, Brian

2007-02-01

This article reviews current efforts to mathematically model the half lives of serum proteins, especially antibodies. While it is recognized that the neonatal Fc receptor, FcRn, is necessary for longer serum persistence of certain proteins, particularly the high abundance IgGs and albumin, it is not clear that it is sufficient to completely determine the half lives of these proteins. More specifically, it is unclear why the high avidity (bivalent), high affinity FcRn-IgG interaction, with half saturation in the 10 to 100 nM range (at endosomal pH according to the currently proposed mechanism), would result in a salvage mechanism that saturates at serum concentrations in the 10 to 100 mg/ml range--a discrepancy of 4 to 5 orders of magnitude. Alternative explanations include the proposal that the very low affinity binding between FcRn and IgG at blood pH is also relevant to the salvage mechanism, and that factors in addition to FcRn binding modulate the maintenance and clearance of IgG from serum.

Complement proteins bind to nanoparticle protein corona and undergo dynamic exchange in vivo

NASA Astrophysics Data System (ADS)

Chen, Fangfang; Wang, Guankui; Griffin, James I.; Brenneman, Barbara; Banda, Nirmal K.; Holers, V. Michael; Backos, Donald S.; Wu, Linping; Moghimi, Seyed Moein; Simberg, Dmitri

2017-05-01

When nanoparticles are intravenously injected into the body, complement proteins deposit on the surface of nanoparticles in a process called opsonization. These proteins prime the particle for removal by immune cells and may contribute toward infusion-related adverse effects such as allergic responses. The ways complement proteins assemble on nanoparticles have remained unclear. Here, we show that dextran-coated superparamagnetic iron oxide core-shell nanoworms incubated in human serum and plasma are rapidly opsonized with the third complement component (C3) via the alternative pathway. Serum and plasma proteins bound to the nanoworms are mostly intercalated into the nanoworm shell. We show that C3 covalently binds to these absorbed proteins rather than the dextran shell and the protein-bound C3 undergoes dynamic exchange in vitro. Surface-bound proteins accelerate the assembly of the complement components of the alternative pathway on the nanoworm surface. When nanoworms pre-coated with human plasma were injected into mice, C3 and other adsorbed proteins undergo rapid loss. Our results provide important insight into dynamics of protein adsorption and complement opsonization of nanomedicines.

Polymorphisms affecting vitamin D-binding protein modify the relationship between serum vitamin D (25[OH]D3) and food allergy.

PubMed

Koplin, Jennifer J; Suaini, Noor H A; Vuillermin, Peter; Ellis, Justine A; Panjari, Mary; Ponsonby, Anne-Louise; Peters, Rachel L; Matheson, Melanie C; Martino, David; Dang, Thanh; Osborne, Nicholas J; Martin, Pamela; Lowe, Adrian; Gurrin, Lyle C; Tang, Mimi L K; Wake, Melissa; Dwyer, Terry; Hopper, John; Dharmage, Shyamali C; Allen, Katrina J

2016-02-01

There is evolving evidence that vitamin D insufficiency may contribute to food allergy, but findings vary between populations. Lower vitamin D-binding protein (DBP) levels increase the biological availability of serum vitamin D. Genetic polymorphisms explain almost 80% of the variation in binding protein levels. We sought to investigate whether polymorphisms that lower the DBP could compensate for adverse effects of low serum vitamin D on food allergy risk. From a population-based cohort study (n = 5276) we investigated the association between serum 25-hydroxyvitamin D3 (25[OH]D3) levels and food allergy at age 1 year (338 challenge-proven food-allergic and 269 control participants) and age 2 years (55 participants with persistent and 50 participants with resolved food allergy). 25(OH)D3 levels were measured using liquid chromatography-tandem mass spectrometry and adjusted for season of blood draw. Analyses were stratified by genotype at rs7041 as a proxy marker of DBP levels (low, the GT/TT genotype; high, the GG genotype). Low serum 25(OH)D3 level (≤50 nM/L) at age 1 years was associated with food allergy, particularly among infants with the GG genotype (odds ratio [OR], 6.0; 95% CI, 0.9-38.9) but not in those with GT/TT genotypes (OR, 0.7; 95% CI, 0.2-2.0; P interaction = .014). Maternal antenatal vitamin D supplementation was associated with less food allergy, particularly in infants with the GT/TT genotype (OR, 0.10; 95% CI, 0.03-0.41). Persistent vitamin D insufficiency increased the likelihood of persistent food allergy (OR, 12.6; 95% CI, 1.5-106.6), particularly in those with the GG genotype. Polymorphisms associated with lower DBP level attenuated the association between low serum 25(OH)D3 level and food allergy, consistent with greater vitamin D bioavailability in those with a lower DBP level. This increases the biological plausibility of a role for vitamin D in the development of food allergy. Copyright © 2015 American Academy of Allergy, Asthma

Paracetamol and cytarabine binding competition in high affinity binding sites of transporting protein

NASA Astrophysics Data System (ADS)

Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

2006-07-01

Paracetamol (acetaminophen, AA) the most popular analgesic drug is commonly used in the treatment of pain in patients suffering from cancer. In our studies, we evaluated the competition in binding with serum albumin between paracetamol (AA) and cytarabine, antyleukemic drug (araC). The presence of one drug can alter the binding affinity of albumin towards the second one. Such interaction can result in changing of the free fraction of the one of these drugs in blood. Two spectroscopic methods were used to determine high affinity binding sites and the competition of the drugs. Basing on the change of the serum albumin fluorescence in the presence of either of the drugs the quenching ( KQ) constants for the araC-BSA and AA-BSA systems were calculated. Analysis of UV difference spectra allowed us to describe the changes in drug-protein complexes (araC-albumin and AA-albumin) induced by the presence of the second drug (AA and araC, respectively). The mechanism of competition between araC and AA has been proposed.

Plasma binding of an alpha-blocking agent, nicergoline--affinity for serum albumin and native and modified alpha 1-acid glycoprotein.

PubMed

Robert, L; Migne, J; Santonja, R; Zini, R; Schmid, K; Tillement, J P

1983-06-01

The binding of nicergoline, an alpha-blocking drug, by human plasma proteins was studied using gel filtration, polyacrylamide gel electrophoresis, and equilibrium dialysis techniques. 3H-labeled nicergoline added to plasma was eluted together with two major protein fractions, one containing mainly serum albumin, the other glycoproteins such as alpha 1-acid glycoprotein (alpha 1-AG). Equilibrium dialysis experiments with pure human serum albumin and alpha 1-AG as well as with its chemically modified forms, desialylated, carboxymethylated, and both desialylated and carboxymethylated alpha 1-AG gave the following results: nicergoline has about a 4-fold higher affinity for alpha 1-AG than for serum albumin. There are two binding sites per molecule on serum albumin and one on alpha 1-AG. The binding parameters of alpha 1-AG were not significantly modified by desialylation or carboxymethylation. Only desialylated and carboxymethylated alpha 1-AG showed a decreased binding for nicergoline, suggesting conformational modifications induced by these combined treatments. The fact that desialylated alpha 1-AG keeps its affinity for nicergoline suggests the possibility of a selective introduction of this drug in cells possessing the Ashwell-type specific receptor for desialylated alpha 1-AG, for instance hepatocytes. Increased serum alpha 1-AG concentration induced by inflammatory reactions will also modify the distribution of bound nicergoline between serum albumin and alpha 1-AG and as a consequence its half-life and cell distribution.

Lipid A binding proteins in macrophages detected by ligand blotting

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hampton, R.Y.; Golenbock, D.T.; Raetz, C.R.H.

1987-05-01

Endotoxin (LPS) stimulates a variety of eukaryotic cells. These actions are involved in the pathogenesis of Gram-negative septicemia. The site of action of the LPS toxic moiety, lipid A (LA), is unclear. Their laboratory has previously identified a bioactive LA precursor lipid IV/sub A/, which can be enzymatically labeled with /sup 32/P/sub i/ (10/sup 9/ dpm/nmole) and purified (99%). They now show that this ligand binds to specific proteins immobilized on nitrocellulose (NC) from LPS-sensitive RAW 264.7 cultured macrophages. NC blots were incubated with (/sup 32/P)-IV/sub A/ in a buffer containing BSA, NaCl, polyethylene glycol, and azide. Binding was assessedmore » using autoradiography or scintillation counting. Dot blot binding of the radioligand was inhibited by excess cold IV/sub A/, LA, or ReLPS but not by phosphatidylcholine, cardiolipin, phosphatidylinositol, or phosphatidic acid. Binding was trypsin-sensitive and dependent on protein concentration. Particulate macrophage proteins were subjected to SDS-PAGE and then electroblotted onto NC. Several discrete binding proteins were observed. Identical treatment of fetal bovine serum or molecular weight standards revealed no detectable binding. By avoiding high nonspecific binding of intact membranes, this ligand blotting assay may be useful in elucidating the molecular actions of LPS.« less

Volatile anesthetics compete for common binding sites on bovine serum albumin: a 19F-NMR study.

PubMed Central

Dubois, B W; Cherian, S F; Evers, A S

1993-01-01

There is controversy as to the molecular nature of volatile anesthetic target sites. One proposal is that volatile anesthetics bind directly to hydrophobic binding sites on certain sensitive target proteins. Consistent with this hypothesis, we have previously shown that a fluorinated volatile anesthetic, isoflurane, binds saturably [Kd (dissociation constant) = 1.4 +/- 0.2 mM, Bmax = 4.2 +/- 0.3 sites] to fatty acid-displaceable domains on serum albumin. In the current study, we used 19F-NMR T2 relaxation to examine whether other volatile anesthetics bind to the same sites on albumin and, if so, whether they vary in their affinity for these sites. We show that three other fluorinated volatile anesthetics bind with varying affinity to fatty acid-displaceable domains on serum albumin: halothane, Kd = 1.3 +/- 0.2 mM; methoxyflurane, Kd = 2.6 +/- 0.3 mM; and sevoflurane, Kd = 4.5 +/- 0.6 mM. These three anesthetics inhibit isoflurane binding in a competitive manner: halothane, K(i) (inhibition constant) = 1.3 +/- 0.2 mM; methoxyflurane, K(i) = 2.5 +/- 0.4 mM; and sevoflurane, K(i) = 5.4 +/- 0.7 mM--similar to each anesthetic's respective Kd of binding to fatty acid displaceable sites. These results illustrate that a variety of volatile anesthetics can compete for binding to specific sites on a protein. PMID:8341659

Protein metabolism in obese patients during very low-calorie mixed diets containing different amounts of proteins and carbohydrates.

PubMed

Pasquali, R; Casimirri, F; Melchionda, N

1987-12-01

To assess long-term nitrogen sparing capacity of very low-calorie mixed diets, we administered two isoenergetic (2092KJ) liquid formula regimens of different composition for 8 weeks to two matched groups of massively obese patients (group 1: proteins 60 g, carbohydrate 54 g; group 2: proteins 41 g, carbohydrates 81 g). Weight loss was similar in both groups. Daily nitrogen balance (g) during the second month resulted more a negative in group 2 with respect to group 1. However, within the groups individual nitrogen sparing capacity varied markedly; only a few in group 1 and one in group 2 were able to attain nitrogen equilibrium throughout the study. Daily urine excretion of 3-methylhistidine fell significantly in group 1 but did not change in group 2. Unlike total proteins, albumins, and transferrin, serum levels of retinol-binding protein, thyroxin-binding globulin, and complement-C3 fell significantly in both groups but per cent variations of complement-C3 were more pronounced in the first group. Prealbumin levels fell persistently in group 1 and transiently in group 2. The results indicate that even with this type of diet an adequate amount of dietary protein represents the most important factor in minimizing whole body protein catabolism during long-term semistarvation in massively obese patients. Moreover, they confirm the possible role of dietary carbohydrates in the regulation of some visceral protein metabolism.

Binding of ring-substituted indole-3-acetic acids to human serum albumin.

PubMed

Soskić, Milan; Magnus, Volker

2007-07-01

The plant hormone, indole-3-acetic acid (IAA), and its ring-substituted derivatives have recently attracted attention as promising pro-drugs in cancer therapy. Here we present relative binding constants to human serum albumin for IAA and 34 of its derivatives, as obtained using the immobilized protein bound to a support suitable for high-performance liquid chromatography. We also report their octanol-water partition coefficients (logK(ow)) computed from retention data on a C(18) coated silica gel column. A four-parameter QSPR (quantitative structure-property relationships) model, based on physico-chemical properties, is put forward, which accounts for more than 96% of the variations in the binding affinities of these compounds. The model confirms the importance of lipophilicity as a global parameter governing interaction with serum albumin, but also assigns significant roles to parameters specifically related to the molecular topology of ring-substituted IAAs. Bulky substituents at ring-position 6 increase affinity, those at position 2 obstruct binding, while no steric effects were noted at other ring-positions. Electron-withdrawing substituents at position 5 enhance binding, but have no obvious effect at other ring positions.

Comparison and analysis on the serum-binding characteristics of aspirin-zinc complex and aspirin.

PubMed

Zhang, Hua-Xin; Zhang, Qun; Wang, Hong-Lin; Li, Li-Wei

2017-09-01

This study was designed to compare the protein-binding characteristics of aspirin-zinc complex (AZN) with those of aspirin itself. AZN was synthesized and interacted with a model transport protein, human serum albumin (HSA). Three-dimensional fluorescence, ultraviolet-visible and circular dichroism (CD) spectra were used to characterize the interaction of AZN with HSA under physiological conditions. The interaction mechanism was explored using a fluorescence quenching method and thermodynamic calculation. The binding site and binding locality of AZN on HSA were demonstrated using a fluorescence probe technique and Förster non-radiation energy transfer theory. Synchronous fluorescence and CD spectra were employed to reveal the effect of AZN on the native conformation of the protein. The HSA-binding results for AZN were compared with those for aspirin under consistent experimental conditions, and indicated that aspirin acts as a guide in AZN when binding to Sudlow's site I, in subdomain IIA of the HSA molecule. Moreover, compared with aspirin, AZN showed greater observed binding constants with, but smaller changes in the α-helicity of, HSA, which proved that AZN might be easier to transport and have less toxicity in vivo. Copyright © 2017 John Wiley & Sons, Ltd.

SDS-binding assay based on tyrosine fluorescence as a tool to determine binding properties of human serum albumin in blood plasma

NASA Astrophysics Data System (ADS)

Zhdanova, Nadezda; Shirshin, Evgeny; Fadeev, Victor; Priezzhev, Alexander

2016-04-01

Among all plasma proteins human serum albumin (HSA) is the most studied one as it is the main transport protein and can bind a wide variety of ligands especially fatty acids (FAs). The concentration of FAs bound to HSA in human blood plasma differs by three times under abnormal conditions (fasting, physical exercises or in case of social important diseases). In the present study a surfactant sodium dodecyl sulfate (SDS) was used to simulate FAs binding to HSA. It was shown that the increase of Tyr fluorescence of human blood plasma due to SDS addition can be completely explained by HSA-SDS complex formation. Binding parameters of SDS-HSA complex (average number of sites and apparent constant of complex formation) were determined from titration curves based on tyrosine (Tyr) fluorescence.

Thermometric sensing of peroxide in organic media. Application to monitor the stability of RBP-retinol-HRP complex.

PubMed

Ramanathan, K; Jönsson, B R; Danielsson, B

2000-08-01

The stability of horseradish peroxidase (HRP) in aqueous and organic solvents is applied to develop a simple thermometric procedure to detect the binding of retinoic acid-HRP conjugate to retinol binding protein (RBP). Butanone peroxide (BP) in organic phase and hydrogen peroxide in aqueous phase is detected thermometrically on a HRP column, immobilized by cross-linking with glutaraldehyde on controlled pore glass (CPG). Acetone, acetonitrile, methanol, and 2-butanol are used for detection of BP, in the flow injection analysis (FIA) mode. A linear range between 1 and 50 mM BP is obtained in all the organic solvents with a precision of 5-7% (CV%). The magnitude and nature of the thermometric response is significantly different in each organic solvent. The stability of HRP in the organic phase is used to study the stability of a retinoic acid-HRP conjugate bound to immobilized RBP. The response of HRP (to 20 mM BP) in the retinoic acid-HRP conjugate is used as an indicator of the stability of the RBP-retinoic acid-HRP complex, after challenges with various organic/aqueous solvents. Both immobilized HRP and RBP are stable at least for 6 months. The effect of o-phenylene diamine on the thermometric response of HRP is also investigated. A scheme for the design of a thermometric retinol (vitamin A) biosensor is proposed.

Temperature-responsive release of cortisol from its binding globulin: a protein thermocouple.

PubMed

Cameron, Angus; Henley, David; Carrell, Robin; Zhou, Aiwu; Clarke, Anthony; Lightman, Stafford

2010-10-01

Only 5% of circulating cortisol is active and unbound to carrier proteins. Because cortisol levels vary rapidly due to the pulsatile nature of cortisol secretion, the dynamics of cortisol binding are critical determinants of tissue levels of free cortisol and consequent hormonal signaling. The major glucocorticoid carrier protein is corticosteroid binding globulin (CBG), a member of the serpin family that undergoes conformational changes to bind and release hormones. This mechanism has been noted to be temperature responsive, and we have now investigated the effects of temperature on the binding of human CBG to both cortisol and progesterone. Recombinant human CBG was synthesized and used for binding studies with cortisol and progesterone between 34 and 43 C. Binding was monitored by recording the change in intrinsic protein fluorescence. Binding of the steroids to the other major carrier, serum albumin, was measured in a similar manner. There was no effect of temperature on the interaction between human serum albumin and either cortisol or progesterone. The association of both cortisol and progesterone with CBG is more than three orders of magnitude greater than that with HSA, and this interaction was extremely responsive to changes in temperature. The affinity of both cortisol and progesterone for CBG drops approximately 16-fold as temperature increases from 35 to 42 C. This study clearly shows that even within the clinically relevant range of temperatures found in humans, CBG acts as a protein thermocouple that is exquisitely sensitive to temperature change and will release cortisol in response to fever or external sources of heat. This has major implications for our understanding of cortisol regulation in febrile patients.

Association of subcutaneous and visceral fat mass with serum concentrations of adipokines in subjects with type 2 diabetes mellitus.

PubMed

Saito, Tomoyuki; Murata, Miho; Otani, Taeko; Tamemoto, Hiroyuki; Kawakami, Masanobu; Ishikawa, San-E

2012-01-01

The goal of the study was to examine the association of subcutaneous and visceral fat mass with serum concentrations of adipokines in 130 subjects with type 2 diabetes mellitus. The levels of serum high sensitivity C-reactive protein (HS-CRP), adiponectin, high-molecular-weight (HMW) adiponectin, interleukin-18, and retinol-binding protein 4 were measured. Percentage body fat was determined by dual energy X-ray absorptiometry, and subcutaneous and visceral fat areas were measured by abdominal CT. HS-CRP had significant positive correlations with percentage body fat and subcutaneous fat area, and a particularly significant positive correlation with visceral fat area. Serum adiponectin had a negative correlation with the subcutaneous and visceral fat areas, with the strongest correlation with the visceral fat area. Similar results were obtained for HMW adiponectin. Serum adiponectin had a negative correlation with visceral fat area in subjects with a visceral fat area < 100 cm², but not in those with a visceral fat area ≥ 100 cm². In contrast, serum HS-CRP showed a positive correlation with visceral fat area in subjects with visceral fat area ≥ 100 cm², but not in those with a visceral fat area < 100 cm². These findings indicate that an increased visceral fat area is associated with inflammatory changes, and that inflammatory reactions may alter the functional properties of visceral fat in type 2 diabetes mellitus.

Insights into the binding behavior of bovine serum albumin to black carbon nanoparticles and induced cytotoxicity

NASA Astrophysics Data System (ADS)

Wu, Hai; Chen, Miaomiao; Shang, Mengting; Li, Xiang; Mu, Kui; Fan, Suhua; Jiang, Shuanglin; Li, Wenyong

2018-07-01

Black carbon (BC) is a main component of particulate matter (PM2.5). Due to their small size (<100 nm), inhaled ultrafine BC nanoparticles may penetrate the lung alveoli, where they interact with surfactant proteins and lipids, causing more serious damage to human health. Here, BC was analyzed to investigate the binding mechanism of its interaction with protein and induction of cytotoxicity changes. The binding process and protein conformation between BC and a serum protein (bovine serum albumin, BSA) were monitored by using a fluorescence quenching technique and UV-vis absorption, Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies. The experimental results revealed that the fluorescence quenching of BSA induced by BC was a static quenching process and the hydrophobic force played the critical role in the interaction. The native conformation of BSA on the BC surface was slightly disturbed but obvious structural unfolding of the secondary structure did not occur. In the cytotoxicity study, BC nanoparticles with low concentrations exhibited strong toxicity towards BEAS-2B cells. However, the toxicity of BC nanoparticles could be mitigated by the presence of BSA. Therefore, proteins in biological fluids likely reduce the toxic effect of BC on human health. These findings delineated the binding mechanism and the toxicity between BC and the BSA-BC system, contributing to the understanding of the biological effects of BC exposure on human health in polluted atmospheres.

The Application of a Three-Step Proteome Analysis for Identification of New Biomarkers of Pancreatic Cancer

PubMed Central

Abulaizi, Mayinuer; Tomonaga, Takeshi; Satoh, Mamoru; Sogawa, Kazuyuki; Matsushita, Kazuyuki; Kodera, Yoshio; Obul, Jurat; Takano, Shigetsugu; Yoshitomi, Hideyuki; Miyazaki, Masaru; Nomura, Fumio

2011-01-01

We searched for novel tumor markers of pancreatic cancer by three-step serum proteome analysis. Twelve serum abundant proteins were depleted using immunoaffinity columns followed by fractionation by reverse-phase high-performance liquid chromatography. Proteins in each fraction were separated by two-dimensional gel electrophoresis. Then the gel was stained by Coomassie Brilliant Blue. Protein spots in which the expression levels were significantly different between cancer and normal control were identified by LC-MS/MS. One hundred and two spots were upregulated, and 84 spots were downregulated in serum samples obtained from patients with pancreatic cancers, and 58 proteins were identified by mass spectrometry. These candidate proteins were validated using western blot analysis and enzyme-linked immunosorbent assay (ELISA). As a result of these validation process, we could confirm that the serum levels of apolipoprotein A-IV, vitamin D-binding protein, plasma retinol-binding protein 4, and tetranectin were significantly decreased in patients with pancreatic cancer. PMID:22091389

Analysis and stability of retinol in plasma.

PubMed

Peng, Y M; Xu, M J; Alberts, D S

1987-01-01

A simple, precise, and specific high-performance liquid chromatography (HPLC) method was developed for the simultaneous measurement of retinol (ROH), 13-cis-retinoic acid (13-cRA), and 4-oxo-13-cRA. The average recovery of ROH from serum or plasma was 95%, and the precision of the assay was less than 5%. With this HPLC method, a series of studies was carried out to evaluate the stability of ROH in various matrices. ROH was stable under our HPLC assay conditions as well as in plasma- and in serum-enriched culture media; however, ROH was not stable in aqueous matrices. Serum or heparinized plasma may be routinely used for measurement of ROH concentrations, providing EDTA, oxalate, and citrate are not used as anticoagulants. Because of ROH stability, blood samples can be kept on ice in the dark for at least 24 hours prior to separation of plasma. In addition, plasma samples containing ROH can be stored for up to 1 year at -20 degrees C without loss of stability.

Unravelling the binding mechanism and protein stability of human serum albumin while interacting with nefopam analogues: a biophysical and insilico approach.

PubMed

Gokara, Mahesh; Narayana, Vidadala V; Sadarangani, Vineet; Chowdhury, Shatabdi Roy; Varkala, Sreelaxmi; Ramachary, Dhevalapally B; Subramanyam, Rajagopal

2017-08-01

In this study, molecular binding affinity was investigated for Nefopam analogues (NFs), a functionalized benzoxazocine, with human serum albumin (HSA), a major transport protein in the blood. Its binding affinity and concomitant changes in its conformation, binding site and simulations were also studied. Fluorescence data revealed that the fluorescence quenching of HSA upon binding of NFs analogues is based on a static mechanism. The three analogues of NFs binding constants (K A ) are in the order of NF3 > NF2 > NF1 with values of 1.53 ± .057 × 10 4 , 2.16 ± .071 × 10 4 and 3.6 ± .102 × 10 5  M -1 , respectively. Concurrently, thermodynamic parameters indicate that the binding process was spontaneous, and the complexes were stabilized mostly by hydrophobic interactions, except for NF2 has one hydrogen bond stabilizes it along with hydrophobic interactions. Circular dichroism (CD) studies revealed that there is a decrease in α-helix with an increase in β-sheets and random coils signifying partial unfolding of the protein upon binding of NFs, which might be due to the formation of NFs-HSA complexes. Further, molecular docking studies showed that NF1, NF2 and NF3 bound to subdomains IIIA, IB and IIA through hydrophobic interactions. However, NF1 have additionally formed a single hydrogen bond with LYS 413. Furthermore, molecular simulations unveiled that NFs binding was in support with the structural perturbation observed in CD, which is evident from the root mean square deviation and R g fluctuations. We hope our insights will provide ample scope for engineering new drugs based on the resemblances with NFs for enhanced efficacy with HSA.

Identification and immunological characterization of the ligand domain of Plasmodium vivax reticulocyte binding protein 1a.

PubMed

Ntumngia, Francis B; Thomson-Luque, Richard; Galusic, Sandra; Frato, Gabriel; Frischmann, Sarah; Peabody, David S; Chackerian, Bryce; Ferreira, Marcelo U; King, Christopher L; Adams, John H

2018-05-07

Erythrocyte invasion by malaria parasites is essential for blood-stage development. Consequently, parasite proteins critically involved in erythrocyte invasion such as the Plasmodium vivax reticulocyte-binding proteins (RBPs) that mediate preferential invasion of reticulocytes are considered potential vaccine targets. Thus, targeting the RBPs could prevent blood-stage infection and disease. The RBPs are large and little is known about their functional domains and whether individuals naturally exposed to P. vivax acquire binding-inhibitory antibodies to these critical binding regions. This study aims at functionally and immunologically characterize Plasmodium vivax RBP1a. Recombinant proteins of overlapping fragments of RBP1a were used to determine binding specificity to erythrocytes and immunogenicity in laboratory animals. Naturally-acquired antibody response to these proteins was evaluated using serum samples from individuals in endemic regions. The N-terminal extracellular region, RBP1157-650 (RBP1:F8) was determined to bind both reticulocytes and normocytes, with a preference for immature reticulocytes. Antibodies elicited against rRBP1:F8 blocked RBP1:F8-erythrocyte binding. Naturally-acquired anti-RBP1 binding-inhibitory antibodies were detected in serum of P. vivax exposed-individuals from Papua New Guinea and Brazil. Recombinant RBP1:F8 binds human erythrocytes, elicits artificially-induced functional blocking antibodies and is a target of naturally acquired binding-inhibitory antibodies.

Distinct loops in arrestin differentially regulate ligand binding within the GPCR opsin

PubMed Central

Sommer, Martha E.; Hofmann, Klaus Peter; Heck, Martin

2012-01-01

G-protein-coupled receptors are universally regulated by arrestin binding. Here we show that rod arrestin induces uptake of the agonist all-trans-retinol in only half the population of phosphorylated opsin in the native membrane. Agonist uptake blocks subsequent entry of the inverse agonist 11-cis-retinal (that is, regeneration of rhodopsin), but regeneration is not blocked in the other half of aporeceptors. Environmentally sensitive fluorophores attached to arrestin reported that conformational changes in loopV−VI (N-domain) are coupled to the entry of agonist, while loopXVIII−XIX (C-domain) engages the aporeceptor even before agonist is added. The data are most consistent with a model in which each domain of arrestin engages its own aporeceptor, and the different binding preferences of the domains lead to asymmetric ligand binding by the aporeceptors. Such a mechanism would protect the rod cell in bright light by concurrently sequestering toxic all-trans-retinol and allowing regeneration with 11-cis-retinal. PMID:22871814

[Insulin-like growth factor-binding protein-1: a new biochemical marker of nonalcoholic fatty liver disease?].

PubMed

Graffigna, Mabel Nora; Belli, Susana H; de Larrañaga, Gabriela; Fainboim, Hugo; Estepo, Claudio; Peres, Silvia; García, Natalia; Levalle, Oscar

2009-03-01

to assess the presence of nonalcoholic fatty liver disease in patients with risk factors for this pathology (obesity, dyslipidemia, metabolic syndrome and diabetes type 2) and to determine the role of insulin, HOMA index, insulin-like growth factor-binding protein-1, sex hormone-binding globulin and plasminogen activator inhibitor type 1, as biochemical markers. Ninety-one patients with risk factors for nonalcoholic fatty liver disease were evaluated. Serum transaminases, insulin, sex hormone-binding globulin, insulin-like growth factor-binding protein-1 and plasminogen activator inhibitor type 1 were measured. The diagnosis of fatty liver was performed by ultrasonography and liver biopsies were performed to 31 subjects who had steatosis by ultrasonography and high alanine aminotransferase. Nonalcoholic fatty liver disease was present in 65 out of 91 patients (71,4%). Liver biopsy performed to 31 subjects confirmed nonalcoholic steatohepatitis. Twenty-five patients had different degrees of fibrosis. Those individuals with fatty liver had higher waist circumference, serum levels of triglycerides, insulin and HOMA index, and lower serum insulin-like growth factor-binding protein-1 concentration. The degree ofhepatic steatosis by ultrasonography was positively correlated to waist circumference, triglycerides, insulin and HOMA index (p<0,003; p<0,003; p<0,002 and p<0,001, respectively), and was negatively correlated to HDL-cholesterol and insulin-like growth factor-binding protein-1 (p<0,025 and p<0,018, respectively). We found a high prevalence of NAFLD in patients with risk factors, most of them overweight or obese. Although SHBG and PAI-1 have a closely relationship to insulin resistance, they did not show to be markers of NAFLD. Regardless of low IGFBP-1 levels associated with NAFLD, serum IGFBP-1 measure is less accessible than insulin and triglycerides levels, HOMA index and waist circumference. Moreover, it is not a better marker for NAFLD than the above

On the interaction of luminol with human serum albumin: Nature and thermodynamics of ligand binding

NASA Astrophysics Data System (ADS)

Moyon, N. Shaemningwar; Mitra, Sivaprasad

2010-09-01

The mechanism and thermodynamic parameters for the binding of luminol (LH 2) with human serum albumin was explored by steady state and picosecond time-resolved fluorescence spectroscopy. It was shown that out of two possible LH 2 conformers present is solution, only one is accessible for binding with HSA. The thermodynamic parameters like enthalpy (Δ H) and entropy (Δ S) change corresponding to the ligand binding process were also estimated by performing the experiment at different temperatures. The ligand replacement experiment with bilirubin confirms that LH 2 binds into the sub-domain IIA of the protein.

Both HIV-infected and HIV-exposed uninfected children living in Brazil, Argentina and Mexico have similar rates of low concentrations of retinol, β-carotene and vitamin E

PubMed Central

Monteiro, Jacqueline P.; Freimanis-Hance, Laura; Faria, Lidiane B.; Mussi-Pinhata, Marisa M; Korelitz, James; Vannucchi, Hélio; Queiroz, Wladimir; Succi, Regina CM; Hazra, Rohan

2009-01-01

Our objective was to describe the prevalence of low concentrations of retinol, β-carotene, and vitamin E in a group of HIV-infected Latin American children and a comparison group of HIV-exposed, uninfected children. Our hypothesis was that the rates of low concentrations of these micronutrients would be higher in the HIV-infected group than those in the HIV-exposed, uninfected group. This was a cross-sectional substudy of a larger cohort study at clinical pediatric HIV centers in Latin America. Serum levels of micronutrients were measured in the first stored sample obtained after each child’s first birthday by high-performance liquid chromatography. Low concentrations of retinol, β-carotene and vitamin E were defined as serum levels below 0.70 μmol/L, 0.35 μmol/L and 18.0 μmol/L, respectively. The population for this analysis was 336 children (124 HIV-infected, 212 HIV-exposed, uninfected) aged ≥ 1 to < 4 years of age. Rates of low concentrations were 74% for retinol, 27% for β-carotene, and 89% for vitamin E. These rates were not affected by HIV status. Among the HIV-infected children those treated with antiretrovirals were less likely to have retinol deficiency, but no other HIV-related factors correlated with micronutrient low serum levels. Low concentrations of retinol, β-carotene and vitamin E are very common in children exposed to HIV living in Brazil, Argentina and Mexico, regardless of HIV-infection status. PMID:19917451

The promiscuous protein binding ability of erythrosine B studied by metachromasy (metachromasia).

PubMed

Ganesan, Lakshmi; Buchwald, Peter

2013-04-01

The present study aims to elucidate aspects of the protein binding ability of erythrosine B (ErB), a poly-iodinated xanthene dye and an FDA-approved food colorant (FD&C Red No. 3), which we have identified recently as a promiscuous inhibitor of protein-protein interactions (PPIs) with a remarkably consistent median inhibitory concentration (IC50 ) in the 5- to 30-μM range. Because ErB exhibits metachromasy, that is, color change upon binding to several proteins, we exploited this property to quantify its binding to proteins such as bovine serum albumin (BSA) and CD40L (CD154) and to determine the corresponding binding constants (Kd ) and stoichiometry (nb ) using spectrophotometric methods. Binding was reversible, and the estimated affinities for both protein targets obtained here (Kd values of 14 and 20 μM for BSA and CD40L, respectively) were in good agreement with that expected from the PPI inhibitory activity of ErB. A stoichiometry greater than one was observed both for CD40L and BSA binding (nb of 5-6 and 8-9 for BSA and CD40L, respectively), indicating the possibility of nonspecific binding of the flat and rigid ErB molecule at multiple sites, which could explain the promiscuous PPI inhibitory activity if some of these overlap with the binding site of the protein partner and interfere with the binding. Copyright © 2013 John Wiley & Sons, Ltd.

The Promiscuous Protein Binding Ability of Erythrosine B Studied by Metachromasy (Metachromasia)

PubMed Central

Ganesan, Lakshmi; Buchwald, Peter

2013-01-01

The present study aims to elucidate aspects of the protein binding ability of erythrosine B (ErB), a poly-iodinated xanthene dye and an FDA-approved food colorant (FD&C Red No. 3), which we have identified recently as a promiscuous inhibitor of protein–protein interactions (PPI) with a remarkably consistent median inhibitory concentration (IC50) in the 5–30 µM range. Because ErB exhibits metachromasy, i.e., color change upon binding to several proteins, we exploited this property to quantify its binding to proteins such as bovine serum albumin (BSA) and CD40L (CD154) and to determine the corresponding binding constants (Kd) and stoichiometry (nb) using spectrophotometric methods. Binding was reversible and the estimated affinities for both protein targets obtained here (Kd values of 14 and 20 µM for BSA and CD40L, respectively) were in good agreement with that expected from the protein–protein interaction (PPI) inhibitory activity of ErB. A stoichiometry greater than one was observed both for CD40L and BSA binding (nb of 5–6 and 8–9 for BSA and CD40L, respectively) indicating the possibility of nonspecific binding of the flat an rigid ErB molecule at multiple sites, which could explain the promiscuous PPI inhibitory activity if some of these overlap with the binding site of the protein partner and interfere with the binding. PMID:23456742

Exploring the affinity binding of alkylmaltoside surfactants to bovine serum albumin and their effect on the protein stability: A spectroscopic approach.

PubMed

Hierrezuelo, J M; Carnero Ruiz, C

2015-08-01

Steady-state and time-resolved fluorescence together with circular dichroism (CD) spectroscopic studies was performed to examine the interactions between bovine serum albumin (BSA) and two alkylmaltoside surfactants, i.e. n-decyl-β-D-maltoside (β-C10G2) and n-dodecyl-β-D-maltoside (β-C12G2), having identical structures but different tail lengths. Changes in the intrinsic fluorescence of BSA from static as well as dynamic measurements revealed a weak protein-surfactant interaction and gave the corresponding binding curves, suggesting that the binding mechanism of surfactants to protein is essentially cooperative in nature. The behavior of both surfactants is similar, so that the differences detected were attributed to the more hydrophobic nature of β-C12G2, which favors the adsorption of micelle-like aggregates onto the protein surface. These observations were substantially demonstrated by data derived from synchronous, three-dimensional and anisotropy fluorescence experiments. Changes in the secondary structure of the protein induced by the interaction with surfactants were analyzed by CD to determine the contents of α-helix and β-strand. It was noted that whereas the addition of β-C10G2 appears to stabilize the secondary structure of the protein, β-C12G2 causes a marginal denaturation of BSA for a protein:surfactant molar ratio as high as 1 to 100. Copyright © 2015 Elsevier B.V. All rights reserved.

Definition of IgG- and albumin-binding regions of streptococcal protein G.

PubMed

Akerström, B; Nielsen, E; Björck, L

1987-10-05

Protein G, the immunoglobin G-binding surface protein of group C and G streptococci, also binds serum albumin. The albumin-binding site on protein G is distinct from the immunoglobulin G-binding site. By mild acid hydrolysis of the papain-liberated protein G fragment (35 kDa), a 28-kDa fragment was produced which retained full immunoglobulin G-binding activity (determined by Scatchard plotting) but had lost all albumin-binding capacity. A protein G (65 kDa), isolated after cloning and expression of the protein G gene in Escherichia coli, had comparable affinity to immunoglobulin G (5-10 X 10(10)M-1), but much higher affinity to albumin than the 35- and 28-kDa protein G fragments (31, 2.6, and 0 X 10(9)M-1, respectively). The amino-terminal amino acid sequences of the 65-, 35-, and 28-kDa fragments allowed us to exactly locate the three fragments in an overall sequence map of protein G, based on the partial gene sequences published by Guss et al. (Guss, B., Eliasson, M., Olsson, A., Uhlen, M., Frej, A.-K., Jörnvall, H., Flock, J.-I., and Lindberg, M. (1986) EMBO J. 5, 1567-1575) and Fahnestock et al. (Fahnestock, S. R., Alexander, P., Nagle, J., and Filpula, D. (1986) J. Bacteriol. 167, 870-880). In this map could then be deduced the location of three homologous albumin-binding regions and three homologous immunoglobulin G-binding regions.

Conserved chemosensory proteins in the proboscis and eyes of Lepidoptera.

PubMed

Zhu, Jiao; Iovinella, Immacolata; Dani, Francesca Romana; Liu, Yu-Ling; Huang, Ling-Qiao; Liu, Yang; Wang, Chen-Zhu; Pelosi, Paolo; Wang, Guirong

2016-01-01

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are endowed with several different functions besides being carriers for pheromones and odorants. Based on a previous report of a CSP acting as surfactant in the proboscis of the moth Helicoverpa armigera , we revealed the presence of orthologue proteins in two other moths Plutella xylostella and Chilo suppressalis , as well as two butterflies Papilio machaon and Pieris rapae , using immunodetection and proteomic analysis. The unusual conservation of these proteins across large phylogenetic distances indicated a common specific function for these CSPs. This fact prompted us to search for other functions of these proteins and discovered that CSPs are abundantly expressed in the eyes of H. armigera and possibly involved as carriers for carotenoids and visual pigments. This hypothesis is supported by ligand-binding experiments and docking simulations with retinol and β-carotene. This last orange pigment, occurring in many fruits and vegetables, is an antioxidant and the precursor of visual pigments. We propose that structurally related CSPs solubilise nutritionally important carotenoids in the proboscis, while they act as carriers of both β-carotene and its derived products 3-hydroxyretinol and 3-hydroxyretinal in the eye. The use of soluble olfactory proteins, such as CSPs, as carriers for visual pigments in insects, here reported for the first time, parallels the function of retinol-binding protein in vertebrates, a lipocalin structurally related to vertebrate odorant-binding proteins.

Effects of an endurance cycling competition on resting serum insulin-like growth factor I (IGF-I) and its binding proteins IGFBP-1 and IGFBP-3

PubMed Central

Chicharro, J; Lopez-Calderon, A; Hoyos, J; Martin-Velasco, A; Villa, G; Villanua, M; Lucia, A

2001-01-01

Objectives—To determine whether consecutive bouts of intense endurance exercise over a three week period alters serum concentrations of insulin-like growth factor I (IGF-I) and/or its binding proteins. Methods—Seventeen professional cyclists (mean (SEM) VO2MAX, 74.7 (2.1) ml/kg/min; age, 27 (1) years) competing in a three week tour race were selected as subjects. Blood samples were collected at each of the following time points: t0 (control, before the start of competition), t1 (end of first week), and t3 (end of third week). Serum levels of both total and free IGF-I and IGF binding proteins 1 and 3 (IGFBP-1 and IGFBP-3) were measured in each of the samples. Cortisol levels were measured in nine subjects. Results—A significant (p<0.01) increase was found in total IGF-I and IGFBP-1 at both t1 and t3 compared with to (IGF-I: 110.9 (17.7), 186.8 (12.0), 196.9 (14.7) ng/ml at t0, t1, and t3 respectively; IGFBP-1: 54.6 (6.6), 80.6 (8.0), and 89.2 (7.9) ng/ml at t0, t1, and t3 respectively). A significant (p<0.01) decrease was noted in free IGF-I at t3 compared with both to and t1 (t0: 0.9 (0.1) ng/ml; t1: 0.9 (0.1) ng/ml; t3: 0.7 (0.1) ng/ml); in contrast, IGFBP-3 levels remained stable throughout the race. Conclusions—It would appear that the increase in circulating levels of both IGF-I and its binding protein IGFBP-1 is a short term (one week) endocrine adaptation to endurance exercise. After three weeks of training, total IGF-I and IGFBP-1 remained stable, whereas free IGF-I fell below starting levels. Key Words: cycling; insulin-like growth factor; exercise; endurance; binding proteins PMID:11579061

Characterization of diverse subvariants of the meningococcal factor H (fH) binding protein for their ability to bind fH, to mediate serum resistance, and to induce bactericidal antibodies.

PubMed

Seib, Kate L; Brunelli, Brunella; Brogioni, Barbara; Palumbo, Emmanuelle; Bambini, Stefania; Muzzi, Alessandro; DiMarcello, Federica; Marchi, Sara; van der Ende, Arie; Aricó, Beatrice; Savino, Silvana; Scarselli, Maria; Comanducci, Maurizio; Rappuoli, Rino; Giuliani, Marzia M; Pizza, Mariagrazia

2011-02-01

Neisseria meningitidis is a commensal of the human nasopharynx but is also a major cause of septicemia and meningitis. The meningococcal factor H binding protein (fHbp) binds human factor H (fH), enabling downregulation of complement activation on the bacterial surface. fHbp is a component of two serogroup B meningococcal vaccines currently in clinical development. Here we characterize 12 fHbp subvariants for their level of surface exposure and ability to bind fH, to mediate serum resistance, and to induce bactericidal antibodies. Flow cytometry and Western analysis revealed that all strains examined expressed fHbp on their surface to different extents and bound fH in an fHbp-dependent manner. However, differences in fH binding did not always correlate with the level of fHbp expression, indicating that this is not the only factor affecting the amount of fH bound. To overcome the issue of strain variability in fHbp expression, the MC58ΔfHbp strain was genetically engineered to express different subvariants from a constitutive heterologous promoter. These recombinant strains were characterized for fH binding, and the data confirmed that each subvariant binds different levels of fH. Surface plasmon resonance revealed differences in the stability of the fHbp-fH complexes that ranged over 2 orders of magnitude, indicating that differences in residues between and within variant groups can influence fH binding. Interestingly, the level of survival in human sera of recombinant MC58 strains expressing diverse subvariants did not correlate with the level of fH binding, suggesting that the interaction of fHbp with fH is not the only function of fHbp that influences serum resistance. Furthermore, cross-reactive bactericidal activity was seen within each variant group, although the degree of activity varied, suggesting that amino acid differences within each variant group influence the bactericidal antibody response.

IRF5 haplotypes demonstrate diverse serological associations which predict serum interferon alpha activity and explain the majority of the genetic association with systemic lupus erythematosus

PubMed Central

Niewold, Timothy B; Kelly, Jennifer A; Kariuki, Silvia N; Franek, Beverly S; Kumar, Akaash A; Kaufman, Kenneth M; Thomas, Kenaz; Walker, Daniel; Kamp, Stan; Frost, Jacqueline M; Wong, Andrew K; Merrill, Joan T; Alarcón-Riquelme, Marta E; Tikly, Mohammed; Ramsey-Goldman, Rosalind; Reveille, John D; Petri, Michelle A; Edberg, Jeffrey C; Kimberly, Robert P; Alarcón, Graciela S; Kamen, Diane L; Gilkeson, Gary S; Vyse, Timothy J; James, Judith A; Gaffney, Patrick M; Moser, Kathy L; Crow, Mary K; Harley, John B

2012-01-01

haematopoietic systems. Humoral autoimmunity is a hallmark of SLE, and patients frequently have circulating auto-antibodies directed against dsDNA, as well as RNA binding proteins (RBP). Anti-RBP autoantibodies include antibodies which recognize Ro, La, Smith (anti-Sm), and ribonucleoprotein (anti-nRNP), collectively referred to as anti-retinol-binding protein). Anti-retinol-binding protein and anti-dsDNA auto-antibodies are rare in the healthy population.1 These auto-antibodies can be present in sera for years preceding the onset of clinical SLE illness2 and are likely pathogenic in SLE.34 PMID:22088620

Plasma concentrations of retinol in obese children and adolescents: relationship to metabolic syndrome components

PubMed Central

Teske, Márcia; Melges, Ana Paula B.; de Souza, Fabíola Isabel S.; Fonseca, Fernando Luiz A.; Sarni, Roseli Oselka S.

2014-01-01

Objective: To evaluate obese children and adolescents' retinol plasma levels and to correlate them with metabolic syndrome components. Methods: Cross-sectional study with 61 obese children and adolescents (body mass index Z score - ZBMI>+2). Pubertal development, arterial blood pressure, body weight and height for nutritional classification and waist circumference were obtained. A 15mL blood sample was collected (after a 12-hour fasting in a low luminosity room) for retinol determination (cut-off inadequate if <30µg/dL), lipid profile (HDL-c, LDL-c, and triglycerides), oral glucose tolerance test (fasting and 120 minutes) and for high sensitivity C-reactive protein. Spearman correlation and multiple linear regression were used in the statistical analysis. Results: Mean age was 10.7±2.7 years. There was a predominance of male gender 38/61 (62%) and pre-pubertal 35/61 (57%) subjects. The average plasmatic retinol was 48.5±18.6ug/dL. Retinol deficiency and severe obesity were observed in 6/61 (10%) and 36/61 (59%), respectively. Glucose level at 120 minutes was the independent and predictive variable of plasma retinol levels [β=-0.286 (95%CI -0.013 - -0.001)]. Conclusions: An independent and inverse association between plasma retinol levels and glucose tolerance was observed, suggesting an important contribution of this vitamin in the morbidities associated to obesity in children and adolescents. PMID:24676190

Antiaging action of retinol: from molecular to clinical.

PubMed

Bellemère, G; Stamatas, G N; Bruère, V; Bertin, C; Issachar, N; Oddos, T

2009-01-01

The antiaging efficacy of retinol (ROL) has been explored mainly clinically in photoprotected skin sites and for high doses of ROL (0.4-1.6%). The objective of the study was to demonstrate the antiaging action of a low and tolerable dose of ROL (0.1%) ex vivo by measuring the expression of cellular retinoic-acid-binding protein II (CRABP2) and heparin-binding epidermal growth factor (HBEGF) by a histological evaluation of the epidermis and in vivo by assessing major aging signs and performing three-dimensional profilometry and digital imaging during a 9-month double-blind placebo-controlled study involving 48 volunteers. Finally, epidermal cell proliferation was evaluated using tryptophan fluorescence spectroscopy. Our results demonstrate that 0.1% ROL induced CRABP2 and HBEGF gene expression and increased keratinocyte proliferation and epidermal thickness. In human volunteers, topical application of a ROL-containing product improved all major aging signs assessed in our study (wrinkles under the eyes, fine lines and tone evenness). Moreover, tryptophan fluorescence increased in the active-agent-treated group and not in the placebo-treated group, indicating that cell proliferation was accelerated in vivo. These data demonstrate that a product containing a low dose (0.1%) of ROL promotes keratinocyte proliferation ex vivo and in vivo, induces epidermal thickening ex vivo and alleviates skin aging signs, without any significant adverse reaction. Copyright 2009 S. Karger AG, Basel.

Human granulocyte/pollen-binding protein. Recognition and identification as transferrin.

PubMed Central

Sass-Kuhn, S P; Moqbel, R; Mackay, J A; Cromwell, O; Kay, A B

1984-01-01

Normal human serum was found to contain a heat-stable protein which promoted the binding of granulocytes to timothy grass pollen (granulocyte/pollen-binding protein [GPBP]). GPBP was purified by gel filtration, anion exchange, and affinity chromatography. Virtually all of the granulocyte/pollen-binding activity was associated with a beta-1-protein having a molecular mass of approximately 77,000 D and an isoelectric point of between 5.5 and 6.1. By immunoelectrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein was identified as transferrin. Monospecific antisera raised against either GPBP or transferrin removed biological activity from GPBP preparations, and GPBP and transferrin gave lines of identity with these two antisera. The apparent heterogeneity in the molecular size and charge of GPBP observed during progressive purification was minimal when GPBP was saturated with ferric ions before the separation procedures. These experiments indicate that granulocyte/pollen binding is a hitherto unrecognized property of transferrin which appears to be unrelated to iron transport and raises the possibility that transferrin might have a physiological role in the removal of certain organic matter. Images PMID:6690479

BindML/BindML+: Detecting Protein-Protein Interaction Interface Propensity from Amino Acid Substitution Patterns.

PubMed

Wei, Qing; La, David; Kihara, Daisuke

2017-01-01

Prediction of protein-protein interaction sites in a protein structure provides important information for elucidating the mechanism of protein function and can also be useful in guiding a modeling or design procedures of protein complex structures. Since prediction methods essentially assess the propensity of amino acids that are likely to be part of a protein docking interface, they can help in designing protein-protein interactions. Here, we introduce BindML and BindML+ protein-protein interaction sites prediction methods. BindML predicts protein-protein interaction sites by identifying mutation patterns found in known protein-protein complexes using phylogenetic substitution models. BindML+ is an extension of BindML for distinguishing permanent and transient types of protein-protein interaction sites. We developed an interactive web-server that provides a convenient interface to assist in structural visualization of protein-protein interactions site predictions. The input data for the web-server are a tertiary structure of interest. BindML and BindML+ are available at http://kiharalab.org/bindml/ and http://kiharalab.org/bindml/plus/ .

Binding of trivalent chromium to serum transferrin is sufficiently rapid to be physiologically relevant.

PubMed

Deng, Ge; Wu, Kristi; Cruce, Alex A; Bowman, Michael K; Vincent, John B

2015-02-01

Transferrin, the major iron transport protein in the blood, also transports trivalent chromium in vivo. Recent in vitro studies have, however, suggested that the binding of chromic ions to apotransferrin is too slow to be biologically relevant. Nevertheless, the in vitro studies have generally failed to adequately take physiological bicarbonate concentrations into account. In aqueous buffer (with ambient (bi)carbonate concentrations), the binding of chromium to transferrin is too slow to be physiologically relevant, taking days to reach equilibrium with the protein's associated conformational changes. However, in the presence of 25mM (bi)carbonate, the concentration in human blood, chromic ions bind rapidly and tightly to transferrin. Details of the kinetics of chromium binding to human serum transferrin and conalbumin (egg white transferrin) in the presence of bicarbonate and other major potential chromium ligands are described and are consistent with transferrin being the major chromic ion transporter from the blood to tissues. Copyright © 2014 Elsevier Inc. All rights reserved.

Insights into the binding behavior of bovine serum albumin to black carbon nanoparticles and induced cytotoxicity.

PubMed

Wu, Hai; Chen, Miaomiao; Shang, Mengting; Li, Xiang; Mu, Kui; Fan, Suhua; Jiang, Shuanglin; Li, Wenyong

2018-07-05

Black carbon (BC) is a main component of particulate matter (PM 2.5 ). Due to their small size (<100nm), inhaled ultrafine BC nanoparticles may penetrate the lung alveoli, where they interact with surfactant proteins and lipids, causing more serious damage to human health. Here, BC was analyzed to investigate the binding mechanism of its interaction with protein and induction of cytotoxicity changes. The binding process and protein conformation between BC and a serum protein (bovine serum albumin, BSA) were monitored by using a fluorescence quenching technique and UV-vis absorption, Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies. The experimental results revealed that the fluorescence quenching of BSA induced by BC was a static quenching process and the hydrophobic force played the critical role in the interaction. The native conformation of BSA on the BC surface was slightly disturbed but obvious structural unfolding of the secondary structure did not occur. In the cytotoxicity study, BC nanoparticles with low concentrations exhibited strong toxicity towards BEAS-2B cells. However, the toxicity of BC nanoparticles could be mitigated by the presence of BSA. Therefore, proteins in biological fluids likely reduce the toxic effect of BC on human health. These findings delineated the binding mechanism and the toxicity between BC and the BSA-BC system, contributing to the understanding of the biological effects of BC exposure on human health in polluted atmospheres. Copyright © 2018 Elsevier B.V. All rights reserved.

Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

NASA Technical Reports Server (NTRS)

Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

2000-01-01

Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography.

PubMed

Hu, S; Brady, S R; Kovar, D R; Staiger, C J; Clark, G B; Roux, S J; Muday, G K

2000-10-01

Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

Interactions of poly(amidoamine) dendrimers with human serum albumin: binding constants and mechanisms.

PubMed

Giri, Jyotsnendu; Diallo, Mamadou S; Simpson, André J; Liu, Yi; Goddard, William A; Kumar, Rajeev; Woods, Gwen C

2011-05-24

The interactions of nanomaterials with plasma proteins have a significant impact on their in vivo transport and fate in biological fluids. This article discusses the binding of human serum albumin (HSA) to poly(amidoamine) [PAMAM] dendrimers. We use protein-coated silica particles to measure the HSA binding constants (K(b)) of a homologous series of 19 PAMAM dendrimers in aqueous solutions at physiological pH (7.4) as a function of dendrimer generation, terminal group, and core chemistry. To gain insight into the mechanisms of HSA binding to PAMAM dendrimers, we combined (1)H NMR, saturation transfer difference (STD) NMR, and NMR diffusion ordered spectroscopy (DOSY) of dendrimer-HSA complexes with atomistic molecular dynamics (MD) simulations of dendrimer conformation in aqueous solutions. The binding measurements show that the HSA binding constants (K(b)) of PAMAM dendrimers depend on dendrimer size and terminal group chemistry. The NMR (1)H and DOSY experiments indicate that the interactions between HSA and PAMAM dendrimers are relatively weak. The (1)H NMR STD experiments and MD simulations suggest that the inner shell protons of the dendrimers groups interact more strongly with HSA proteins. These interactions, which are consistently observed for different dendrimer generations (G0-NH(2)vs G4-NH(2)) and terminal groups (G4-NH(2)vs G4-OH with amidoethanol groups), suggest that PAMAM dendrimers adopt backfolded configurations as they form weak complexes with HSA proteins in aqueous solutions at physiological pH (7.4).

Evaluation of the binding interaction between bovine serum albumin and dimethyl fumarate, an anti-inflammatory drug by multispectroscopic methods

NASA Astrophysics Data System (ADS)

Jattinagoudar, Laxmi; Meti, Manjunath; Nandibewoor, Sharanappa; Chimatadar, Shivamurti

2016-03-01

The information of the quenching reaction of bovine serum albumin with dimethyl fumarate is obtained by multi-spectroscopic methods. The number of binding sites, n and binding constants, KA were determined at different temperatures. The effect of increasing temperature on Stern-Volmer quenching constants (KD) indicates that a dynamic quenching mechanism is involved in the interaction. The analysis of thermodynamic quantities namely, ∆H° and ∆S° suggested hydrophobic forces playing a major role in the interaction between dimethyl fumarate and bovine serum albumin. The binding site of dimethyl fumarate on bovine serum albumin was determined by displacement studies, using the site probes viz., warfarin, ibuprofen and digitoxin. The determination of magnitude of the distance of approach for molecular interactions between dimethyl fumarate and bovine serum albumin is calculated according to the theory of Förster energy transfer. The CD, 3D fluorescence spectra, synchronous fluorescence measurements and FT-IR spectral results were indicative of the change in secondary structure of the protein. The influence of some of the metal ions on the binding interaction was also studied.

Thermodynamic and kinetic analyses of curcumin and bovine serum albumin binding.

PubMed

Hudson, Eliara Acipreste; de Paula, Hauster Maximiler Campos; Ferreira, Guilherme Max Dias; Ferreira, Gabriel Max Dias; Hespanhol, Maria do Carmo; da Silva, Luis Henrique Mendes; Pires, Ana Clarissa Dos S

2018-03-01

Bovine serum albumin (BSA)/curcumin binding and dye photodegradation stability were evaluated. BSA/curcumin complex showed 1:1 stoichiometry, but the thermodynamic binding parameters depended on the technique used and BSA conformation. The binding constant was of the order of 10 5 L·mol -1 by fluorescence and microcalorimetric, and 10 3 and 10 4 L·mol -1 by surface plasmon resonance (steady-state equilibrium and kinetic experiments, respectively). For native BSA/curcumin, fluorescence indicated an enthalpic and entropic driven process based on the standard enthalpy change (ΔH ○ F =-8.67kJ·mol -1 ), while microcalorimetry showed an entropic driven binding process (ΔH ○ cal =29.11kJ·mol -1 ). For the unfolded BSA/curcumin complex, it was found thatp ΔH ○ F =-16.12kJ·mol -1 and ΔH ○ cal =-42.63kJ·mol -1 . BSA (mainly native) increased the curcumin photodegradation stability. This work proved the importance of using different techniques to characterize the protein-ligand binding. Copyright © 2017 Elsevier Ltd. All rights reserved.

Binding of mitomycin C to blood proteins: A spectroscopic analysis and molecular docking

NASA Astrophysics Data System (ADS)

Jang, Jongchol; Liu, Hui; Chen, Wei; Zou, Guolin

2009-06-01

Mitomycin C (MMC) was the first recognized bioreductive alkylating agent, and has been widely used clinically for antitumor therapy. The binding of MMC to two human blood proteins, human serum albumin (HSA) and human hemoglobin (HHb), have been investigated by fluorescence quenching, synchronous fluorescence, circular dichroism (CD) spectroscopy and molecular docking methods. The fluorescence data showed that binding of MMC to proteins caused strong fluorescence quenching of proteins through a static quenching way, and each protein had only one binding site for the drug. The binding constants of MMC to HSA and HHb at 298 K were 2.71 × 10 4 and 2.56 × 10 4 L mol -1, respectively. Thermodynamic analysis suggested that both hydrophobic interaction and hydrogen bonding played major roles in the binding of MMC to HSA or HHb. The CD spectroscopy indicated that the secondary structures of the two proteins were not changed in the presence of MMC. The study of molecular docking showed that MMC was located in the entrance of site I of HSA, and in the central cavity of HHb.

Determination of human serum alpha1-acid glycoprotein and albumin binding of various marketed and preclinical kinase inhibitors.

PubMed

Zsila, Ferenc; Fitos, Ilona; Bencze, Gyula; Kéri, György; Orfi, László

2009-01-01

There are about 380 protein kinase inhibitors in drug development as of today and 15 drugs have been marketed already for the treatment of cancer. This time 139 validated kinase targets are in the focus of drug research of pharmaceutical companies and big efforts are made for the development of new, druglike kinase inhibitors. Plasma protein binding is an important factor of the ADME profiling of a drug compound. Human serum albumin (HSA) and alpha(1)-acid glycoprotein (AAG) are the most relevant drug carriers in blood plasma. Since previous literature data indicated that AAG is the principal plasma binding component of some kinase inhibitors the present work focuses on the comprehensive evaluation of AAG binding of a series of marketed and experimental kinase inhibitors by using circular dichroism (CD) spectroscopy approach. HSA binding was also evaluated by affinity chromatography. Protein binding interactions of twenty-six kinase inhibitors are characterized. The contribution of AAG and HSA binding data to the pharmacokinetic profiles of the investigated therapeutic agents is discussed. Structural, biological and drug binding properties of AAG as well as the applicability of the CD method in studying drug-protein binding interactions are also briefly reviewed.

Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

PubMed Central

Choe, Weonu; Durgannavar, Trishaladevi A.; Chung, Sang J.

2016-01-01

The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed. PMID:28774114

Effective binding of perhalogenated closo-borates to serum albumins revealed by spectroscopic and ITC studies

NASA Astrophysics Data System (ADS)

Kuperman, Marina V.; Losytskyy, Mykhaylo Yu.; Bykov, Alexander Yu.; Yarmoluk, Sergiy M.; Zhizhin, Konstantin Yu.; Kuznetsov, Nikolay T.; Varzatskii, Oleg A.; Gumienna-Kontecka, Elzbieta; Kovalska, Vladyslava B.

2017-08-01

The interactions of boron cluster compounds closo-borates with biomolecules are widely studied due to their efficiency as agents for boron neutron capture therapy of cancer. In present work the binding abilities of anionic halogen closo-borates [B10Hal10]2- (Hal = Cl, Br, I) and [B12Hal12]2- (Hal = Cl, I) towards bovine and human serum albumins were investigated by spectroscopic and isothermal titration calorimetry (ITC) methods. The protein fluorescence quenching method and ITC studies confirmed the complex formation. The degree of protein fluorescence quenching increased from chlorine to iodine boron derivatives that is attributed to external heavy atom effect. The ITC data point on the existence in the protein structure of two types of binding sites: with higher and lower affinity to closo-borates. Albumin-closo-borate complex binding ratio, n (4-5 anions per protein molecule) is higher than for the parent hydrogen closo-borates (2 anions per protein molecule). Binding constants estimated by fluorescent and ITC methods indicate higher affinity of halogen closo-borates to albumins (K in the range of 104-106 M-1) comparing to that of the hydrogen closo-borate (K about 103 M-1). Due to their high affinity and high binding ratio to albumins halogen closo-borates are proposed for further studies as agents for boron neutron capture therapy.

A possible structural model of members of the CPF family of cuticular proteins implicating binding to components other than chitin

PubMed Central

Papandreou, Nikos C.; Iconomidou, Vassiliki A.; Willis, Judith H.; Hamodrakas, Stavros J.

2010-01-01

The physical properties of cuticle are determined by the structure of its two major components, cuticular proteins (CPs) and chitin, and, also, by their interactions. A common consensus region (extended R&R Consensus) found in the majority of cuticular proteins, the CPRs, binds to chitin. Previous work established that β-pleated sheet predominates in the Consensus region and we proposed that it is responsible for the formation of helicoidal cuticle. Remote sequence similarity between CPRs and a lipocalin, bovine plasma retinol binding protein (RBP), led us to suggest an antiparallel β-sheet half-barrel structure as the basic folding motif of the R&R Consensus. There are several other families of cuticular proteins. One of the best defined is CPF. Its four members in Anopheles gambiae are expressed during the early stages of either pharate pupal or pharate adult development, suggesting that the proteins contribute to the outer regions of the cuticle, the epi- and/or exocuticle. These proteins did not bind to chitin in the same assay used successfully for CPRs. Although CPFs are distinct in sequence from CPRs, the same lipocalin could also be used to derive homology models for one Anopheles gambiae and one Drosophila melanogaster CPF. For the CPFs, the basic folding motif predicted is an eight-stranded, antiparallel β-sheet, full-barrel structure. Possible implications of this structure are discussed and docking experiments were carried out with one possible Drosophila ligand, 7(Z), 11(Z)-heptacosadiene. PMID:20417215

Serum levels of Mac-2 binding protein increase with cardiovascular risk and reflect silent atherosclerosis.

PubMed

Sugiura, Tomonori; Dohi, Yasuaki; Takase, Hiroyuki; Yamashita, Sumiyo; Murai, Shunsuke; Tsuzuki, Yuji; Ogawa, Shintaro; Tanaka, Yasuhito; Ohte, Nobuyuki

2016-08-01

Mac-2 binding protein (M2BP) was reported to be a useful biomarker for liver fibrosis and malignant tumors. We hypothesized that expression of M2BP might also change in the process of atherosclerosis. This study included subjects who visited our hospital for a physical checkup. The M2BP levels in subjects with hypertension, dyslipidemia, or abnormal glucose metabolism were higher than those in subjects without such risk factors. Moreover, the M2BP levels were associated with severity of cardiovascular risk. Subdivision of M2BP levels into quartiles revealed that M2BP was significantly associated with reactive oxygen metabolites, central systolic blood pressure, and radial augmentation index (AI). Logistic regression analysis with the endpoint of high radial AI (above mean value) showed that high radial AI was independently associated with high M2BP. Although the spectrum was narrow as compared to that in cases of hepatic fibrosis, serum M2BP may reflect silent atherosclerosis in apparently healthy subjects. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Serum adipocyte-fatty acid binding protein (FABP4) levels in women from Mexico exposed to polycyclic aromatic hydrocarbons (PAHs).

PubMed

Ochoa-Martínez, Ángeles C; Ruíz-Vera, Tania; Pruneda-Álvarez, Lucia G; González-Palomo, Ana K; Almendarez-Reyna, Claudia I; Pérez-Vázquez, Francisco J; Pérez-Maldonado, Iván N

2017-01-01

Recent studies indicate that exposure to polycyclic aromatic hydrocarbons (PAHs) is a very important risk factor for the development of cardiovascular diseases (CVDs). Correspondingly, adipocyte-fatty acid binding protein (FABP4, also known as aP2 and AFABP) has been proposed as a new, meaningful and useful biomarker to predict metabolic and cardiovascular diseases. Therefore, the aim of this study was to evaluate serum FABP4 levels in Mexican women exposed to PAHs. Urinary 1-hydroxypyrene ((1-OHP), exposure biomarker for PAHs) levels were quantified using a high-performance liquid chromatography (HPLC) technique, and serum FABP4 concentrations were analyzed using a commercially available ELISA kit. The mean urinary 1-OHP level found in women participating in this study was 1.30 ± 1.10 μmol/mol creatinine (2.45 ± 2.10 μg/g creatinine). Regarding serum FABP4 concentrations, the levels ranged from 3.80 to 62.5 ng/mL in the assessed population. Moreover, a significant association (p < 0.001) was found between urinary 1-OHP levels and serum FABP4 concentrations in women after adjusting for potential confounding variables. The presented data in this study can be considered only as a starting point for further studies. Then, in order to elucidate whether FABP4 represents a risk factor for CVD disease in humans exposed to air contaminants (such as PAHs), large epidemiological studies are necessary.

Newly designed modifier prolongs the action of short-lived peptides and proteins by allowing their binding to serum albumin.

PubMed

Shechter, Yoram; Sasson, Keren; Lev-Goldman, Vered; Rubinraut, Sara; Rubinstein, Menachem; Fridkin, Mati

2012-08-15

We found that human serum albumin (HSA) contains a single binding domain for derivatives of long-chain fatty acid (LCFA)-like molecules in which the carboxylate is replaced by sulfonate. Accordingly, we have synthesized 16-sulfo-hexadecanoic acid-N-hydroxysuccinimide ester [HO(3)S-(CH(2))(15)-CONHS], an agent that reacts selectively with the amino side chains of peptides and proteins. A macromolecule containing a single 16-sulfohexadecanoate moiety associating with albumin with a K(a) value of 0.83 ± 0.08 × 10(6) M(-1), a sufficient affinity to extend the actions in vivo of such short-lived peptides and proteins. Subcutaneous administration of insulin-NHCO-(CH(2))(15)-SO(3)(-) into mice facilitated a glucose-lowering effect 4.3 times in duration and 6.6 times in area under the curve (AUC) as compared to an in vitro equipotent amount of Zn(2+)-free insulin. Similarly, subcutaneous and intravenous administration of exendin-4-NHCO-(CH(2))(15)-SO(3)(-) to mice yielded prolonged and stable reduction in glucose level, 5-9-fold longer than that of exendin-4. Also, a single subcutaneous administration of human interferon-α2-[NH-CO-(CH(2))(15)-SO(3)(-)](3) to mice yielded circulating antiviral activity over a period of 40 h. In conclusion, a simple, hydrophilic reagent has been engineered, synthesized, and studied. Its linkage to peptides and proteins in a monomodified fashion yielded hydrophilic, prolonged acting derivatives, due to their acquired ability to associate with serum albumin after administration.

Plasma proteins of rainbow trout (Oncorhynchus mykiss) isolated by binding to lipopolysaccharide from Aeromonas salmonicida.

PubMed

Hoover, G J; el-Mowafi, A; Simko, E; Kocal, T E; Ferguson, H W; Hayes, M A

1998-07-01

In an attempt to find plasma proteins that might be involved in the constitutive resistance of rainbow trout to furunculosis, a disease caused by Aeromonas salmonicida (AS), we purified serum and plasma proteins based on their calcium- and carbohydrate-dependent affinity for A. salmonicida lipopolysaccharide (LPS) coupled to an epoxy-activated synthetic matrix (Toyopearl AF Epoxy 650M). A multimeric family of high molecular weight (96 to 200-kDa) LPS-binding proteins exhibiting both calcium and mannose dependent binding was isolated. Upon reduction the multimers collapsed to subunits of approximately 16-kDa as estimated by 1D-PAGE and exhibited pI values of 5.30 and 5.75 as estimated from 2D-PAGE. Their N-terminal sequences were related to rainbow trout ladderlectin (RT-LL), a Sepharose-binding protein. Polyclonal antibodies to the LPS-purified 16-kDa subunits recognized both the reduced 16-kDa subunits and the non-reduced multimeric forms. A calcium- and N-acetylglucosamine (GlcNAc)-dependent LPS-binding multimeric protein (approximately 207-kDa) composed of 34.5-kDa subunits was purified and found to be identical to trout serum amyloid P (SAP) by N-terminal sequence (DLQDLSGKVFV). A protein of 24-kDa, in reduced and non-reduced conditions, was isolated and had N-terminal sequence identity with a known C-reactive protein (CRP) homologue, C-polysaccharide-binding protein 2 (TCBP2) of rainbow trout. A novel calcium-dependent LPS-binding protein was purified and termed rainbow trout lectin 37 (RT-L37). This protein, composed of dimers, tetramers and pentamers of 37 kDa subunits (pI 5.50-6.10) with N-terminal sequence (IQE(D/N)GHAEAPGATTVLNEILR) showed no close homology to proteins known or predicted from cDNA sequences. These findings demonstrate that rainbow trout have several blood proteins with lectin properties for the LPS of A. salmonicida; the biological functions of these proteins in resistance to furunculosis are still unknown.

Interaction between valproic acid and aspirin in epileptic children: serum protein binding and metabolic effects.

PubMed

Orr, J M; Abbott, F S; Farrell, K; Ferguson, S; Sheppard, I; Godolphin, W

1982-05-01

In five of six epileptic children who were taking 18 to 49 mg/kg/day valproic acid (VPA), the steady-state serum free fractions of VPA rose from 12% to 43% when antipyretic doses of aspirin were also taken. Mean total VPA half-life (t1/2) rose from 10.4 +/- 2.7 to 12.9 +/- 1.8 hr and mean free VPA t1/2 rose from 6.7 +/- to 2.1 to 8.9 +2- 3.0 hr when salicylate was present in the serum. The in vitro albumin binding association constant (ka) for VPA was decreased by salicylate, but the in vivo ka value was not affected. The 12-hr (trough) concentrations of both free and total VPA were higher in the presence of serum salicylate in five of six patients. Renal excretion of unchanged VPA decreased in five of six patients, but the VPA carboxyl conjugate metabolite-excretion patterns were not consistently affected. Salicylate appeared to displace VPA from serum albumin in vivo, but the increased VPA t1/2 and changes in VPA elimination patterns suggest that serum salicylate also altered VPA metabolism.

Serum Mannose-binding Lectin in Patients on Peritoneal Dialysis Compared With Healthy Individuals.

PubMed

Akbari, Roghayeh; Najafi, Iraj; Maleki, Suzan; Alizadeh-Navaei, Reza

2016-01-01

The increased susceptibility to infection in patients with end-stage renal disease is probably secondary to the impaired immune defense in uremia. Mannose-binding lectin (MBL) has an important role in host defense through activation of the lectin complement pathway. The aim of this study was to measure serum MBL level in peritoneal dialysis patients and compare it with a healthy group. Seventy peritoneal dialysis patients and 70 healthy individuals were enrolled in this study. Serum MBL levels were measured by an enzyme-linked immunosorbent assay kit using the mannan molecule. In addition, serum C-reactive protein and albumin levels were measured to determine whether there is a correlation between serum MBL level and these two parameters. The mean serum MBL level was 2.32 ± 2.54 µg/mL (range, zero to 6.93 µg/mL) in the patients group and 1.80 ± 2.14 µg/mL (range, zero to 6.97µg/mL) in the control group (P = .19). No significant correlation was detected between age and serum MBL level in either the groups. In the patients group, no significant correlation was found between serum MBL and C-reactive protein levels or MBL and albumin levels. There were no correlation between duration of peritoneal dialysis and MBL or dialysis adequacy and MBL, either. This study did not find MBL deficiency in peritoneal dialysis patients as compared to the healthy individuals.

Associations of serum adipocyte fatty acid binding protein with body composition and fat distribution in nondiabetic Chinese women.

PubMed

Hao, Yaping; Ma, Xiaojing; Luo, Yuqi; Hu, Xiang; Pan, Xiaoping; Xiao, Yunfeng; Bao, Yuqian; Jia, Weiping

2015-05-01

Previous studies have demonstrated evidence of a positive relationship between serum adipocyte fatty acid binding protein (A-FABP) and obesity. However, associations of A-FABP with body composition and ectopic fat accumulation remain unclear. This study aimed to elucidate the effect of body composition, visceral fat area (VFA), and subcutaneous fat area (SFA) on serum A-FABP levels in a cohort of Chinese women without diabetes mellitus. A total of 2108 women without diabetes (760 premenopausal and 1348 postmenopausal women; age, 20-78 y) selected from the Shanghai Obesity Study were enrolled. VFA and SFA were measured by magnetic resonance imaging, and body composition was determined by bioelectrical impedance analysis. A high VFA was defined as ≥ 80 cm(2). A high SFA was defined as that above the 75th percentile cutoff point of the menopause-specific population, respectively. Serum A-FABP levels were higher in postmenopausal than premenopausal women (P < .001). Both premenopausal and postmenopausal women with an isolated high VFA had higher A-FABP levels than did those with an isolated high SFA (P = .017 and .002, respectively). In both body mass index (BMI) groups (< 25 and ≥ 25 kg/m(2)), women with a high VFA had higher serum A-FABP levels regardless of their menopausal status. Multiple stepwise regression analysis showed that A-FABP was independently associated with fat mass (Standardized β = 0.417 and 0.252 for premenopausal and postmenopausal status, respectively, both P < .001). Moreover, VFA was identified as an independent risk factor for A-FABP in postmenopausal women (Standardized β = 0.114, P = .001). Application of the same regression analyses model to the two BMI groups produced similar results in both BMI categories. Serum A-FABP levels were associated with fat mass, and were also influenced by VFA after menopause in Chinese women without diabetes mellitus.

Binding of volatile anesthetics to serum albumin: measurements of enthalpy and solvent contributions.

PubMed

Sawas, Abdul H; Pentyala, Srinivas N; Rebecchi, Mario J

2004-10-05

This study directly examines the enthalpic contributions to binding in aqueous solution of closely related anesthetic haloethers (desflurane, isoflurane, enflurane, and sevoflurane), a haloalkane (halothane), and an intravenous anesthetic (propofol) to bovine and human serum albumin (BSA and HSA) using isothermal titration calorimetry. Binding to serum albumin is exothermic, yielding enthalpies (DeltaH(obs)) of -3 to -6 kcal/mol for BSA with a rank order of apparent equilibrium association constants (K(a) values): desflurane > isoflurane approximately enflurane > halothane >or= sevoflurane, with the differences being largely ascribed to entropic contributions. Competition experiments indicate that volatile anesthetics, at low concentrations, share the same sites in albumin previously identified in crystallographic and photo-cross-linking studies. The magnitude of the observed DeltaH increased linearly with increased reaction temperature, reflecting negative changes in heat capacities (DeltaC(p)). These -DeltaC(p) values significantly exceed those calculated for burial of each anesthetic in a hydrophobic pocket. The enhanced stabilities of the albumin/anesthetic complexes and -DeltaC(p) are consistent with favorable solvent rearrangements that promote binding. This idea is supported by substitution of D(2)O for H(2)O that significantly reduces the favorable binding enthalpy observed for desflurane and isoflurane, with an opposing increase of DeltaS(obs). From these results, we infer that solvent restructuring, resulting from release of water weakly bound to anesthetic and anesthetic-binding sites, is a dominant and favorable contributor to the enthalpy and entropy of binding to proteins.

No major role for binding by salivary proteins as a defense against dietary tannins in Mediterranean goats.

PubMed

Hanovice-Ziony, Michal; Gollop, Nathan; Landau, Serge Yan; Ungar, Eugene David; Muklada, Hussein; Glasser, Tzach Aharon; Perevolotsky, Avi; Walker, John Withers

2010-07-01

We investigated whether Mediterranean goats use salivary tannin-binding proteins to cope with tannin-rich forages by determining the affinity of salivary or parotid gland proteins for tannic acid or quebracho tannin. Mixed saliva, sampled from the oral cavity, or parotid gland contents were compared to the intermediate affinity protein bovine serum albumin with a competitive binding assay. Goats that consume tannin-rich browse (Damascus) and goats that tend to avoid tannins (Mamber) were sequentially fed high (Pistacia lentiscus L.), low (vetch hay), or zero (wheat hay) tannin forages. Affinity of salivary proteins for tannins did not differ between goat breeds and did not respond to presence or absence of tannins in the diet. Proteins in mixed saliva had slightly higher affinity for tannins than those in parotid saliva, but neither source contained proteins with higher affinity for tannins than bovine serum albumin. Similarly, 3 months of browsing in a tannin-rich environment had little effect on the affinity of salivary proteins for tannin in adult goats of either breed. We sampled mixed saliva from young kids before they consumed forage and after 3 months of foraging in a tannin-rich environment. Before foraging, the saliva of Mamber kids had higher affinity for tannic acid (but not quebracho tannin) than the saliva of Damascus kids, but there was no difference after 3 months of exposure to tannin-rich browse, and the affinity of the proteins was always similar to the affinity of bovine serum albumin. Our results suggest there is not a major role for salivary tannin-binding proteins in goats. Different tendencies of goat breeds to consume tannin-rich browse does not appear be related to differences in salivary tannin-binding proteins.

Fluorescence analysis of competition of phenylbutazone and methotrexate in binding to serum albumin in combination treatment in rheumatology

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, M.; Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

2009-04-01

Combination of several drugs is often necessary especially during long-them therapy. The competition between drugs can cause a decrease of the amount of a drug bound to albumin. This results in an increase of the free, biological active fraction of the drug. The aim of the presented study was to describe the competition between phenylbutazone (Phe) and methotrexate (MTX), two drugs recommended for the treatment of rheumatology in binding to bovine (BSA) and human (HSA) serum albumin in the high affinity binding site. Fluorescence analysis was used to estimate the effect of drugs on the protein fluorescence and to define the binding and quenching properties of drugs-serum albumin complexes. The effect of the displacement of one drug from the complex of the other with serum albumin has been described on the basis of the comparison of the quenching curves and binding constants for the binary and ternary systems. The conclusion that both Phe and MTX form a binding site in the same subdomain (IIA) points to the necessity of using a monitoring therapy owning to the possible increase of the uncontrolled toxic effects.

The Association of Serum Iron-Binding Proteins and the Antioxidant Parameter Levels in Age-Related Macular Degeneration.

PubMed

Čolak, Emina; Žorić, Lepša; Radosavljević, Aleksandra; Ignjatović, Svetlana

2018-05-01

Age-related macular degeneration (AMD) is the leading cause of the irreversible central visual loss among the elderly in the developed countries. Iron is considered a potent generator of the oxidative damage whose levels increase with age, potentially exacerbating the age-related diseases. The aim of this study was to assess the serum values of iron, and iron-binding proteins (transferrin, ferritin, and haptoglobin) in patients with AMD along with the parameters of the antioxidant defense: superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase, and total antioxidant status (TAS), in order to analyze the possible impact of iron and iron-binding proteins to the development of oxidative stress in AMD patients, and the association of the selected parameters with the AMD. In addition, the aim was to examine the gender differences and calculate the cutoff points of tested parameters that could be associated with AMD. A cross-sectional study included 55 AMD patients aged 71.7 ± 7.36 years and 65 aged-matched control subjects aged 70.25 ± 6.46 years. Significantly lower ferritin (P = 0.025), SOD (P = 0.026), GPx (P = 0.019), and TAS (P < 0.004) values were found in patients with AMD compared to the controls (P < 0.05). Significant association of GPx < 27 U/gHb (odds ratio [OR]: 1.13; 95% confidence interval [CI] 0.78-2.10; P = 0.049), TAS < 1.25 mmol/L (OR: 5.77; 95% CI 0.98-367.0; P < 0.000), ferritin < 84.8 pg/mL (OR: 2.52; 95% CI 1.37-4.62; P = 0.002), and haptoglobin<1.51 g/L (OR: 1.94; 95% CI 1.05-3.56; P = 0.031) was found with the AMD. According to receiver operating characteristic curve analysis, ferritin concentration <84.8 pg/L, GPx < 27 U/gHb, and TAS < 1.25 mmol/L have sufficient predictive ability for AMD. Significantly reduced capacity of the antioxidant defense system and iron-binding storage proteins (ferritin) found in AMD could have an important role in the development of increase oxidative stress

Volatile anesthetic binding to proteins is influenced by solvent and aliphatic residues.

PubMed

Streiff, John H; Jones, Keith A

2008-10-01

The main objective of this work was to characterize VA binding sites in multiple anesthetic target proteins. A computational algorithm was used to quantify the solvent exclusion and aliphatic character of amphiphilic pockets in the structures of VA binding proteins. VA binding sites in the protein structures were defined as the pockets with solvent exclusion and aliphatic character that exceeded minimum values observed in the VA binding sites of serum albumin, firefly luciferase, and apoferritin. We found that the structures of VA binding proteins are enriched in these pockets and that the predicted binding sites were consistent with experimental determined binding locations in several proteins. Autodock3 was used to dock the simulated molecules of 1,1,1,2,2-pentafluoroethane, difluoromethyl 1,1,1,2-tetrafluoroethyl ether, and sevoflurane and the isomers of halothane and isoflurane into these potential binding sites. We found that the binding of the various VA molecules to the amphiphilic pockets is driven primarily by VDW interactions and to a lesser extent by weak hydrogen bonding and electrostatic interactions. In addition, the trend in Delta G binding values follows the Meyer-Overton rule. These results suggest that VA potencies are related to the VDW interactions between the VA ligand and protein target. It is likely that VA bind to sites with a high degree of solvent exclusion and aliphatic character because aliphatic residues provide favorable VDW contacts and weak hydrogen bond donors. Water molecules occupying these sites maintain pocket integrity, associate with the VA ligand, and diminish the unfavorable solvation enthalpy of the VA. Water molecules displaced into the bulk by the VA ligand may provide an additional favorable enthalpic contribution to VA binding. Anesthesia is a component of many health related procedures, the outcomes of which could be improved with a better understanding of the molecular targets and mechanisms of anesthetic action.

Potential Protein Toxicity of Synthetic Pigments: Binding of Poncean S to Human Serum Albumin☆

PubMed Central

Gao, Hong-Wen; Xu, Qing; Chen, Ling; Wang, Shi-Long; Wang, Yuan; Wu, Ling-Ling; Yuan, Yuan

2008-01-01

Using various methods, e.g., spectrophotometry, circular dichroism, and isothermal titration calorimetry, the interaction of poncean S (PS) with human serum albumin (HSA) was characterized at pH 1.81, 3.56, and 7.40 using the spectral correction technique, and Langmuir and Temkin isothermal models. The consistency among results concerning, e.g., binding number, binding energy, and type of binding, showed that ion pair electrostatic attraction fixed the position of PS in HSA and subsequently induced a combination of multiple noncovalent bonds such as H-bonds, hydrophobic interactions, and van der Waals forces. Ion pair attraction and H-bonds produced a stable PS-HSA complex and led to a marked change in the secondary structure of HSA in acidic media. The PS-HSA binding pattern and the process of change in HSA conformation were also investigated. The potentially toxic effect of PS on the transport function of HSA in a normal physiological environment was analyzed. This work provides a useful experimental strategy for studying the interaction of organic substances with biomacromolecules, helping us to understand the activity or mechanism of toxicity of an organic compound. PMID:17905844

Corona-directed nucleic acid delivery into hepatic stellate cells for liver fibrosis therapy.

PubMed

Zhang, Zhengping; Wang, Chunming; Zha, Yinhe; Hu, Wei; Gao, Zhongfei; Zang, Yuhui; Chen, Jiangning; Zhang, Junfeng; Dong, Lei

2015-03-24

Strategies to modify nanoparticles with biological ligands for targeted drug delivery in vivo have been widely studied but met with limited clinical success. A possible reason is that, in the blood circulation, serum proteins could rapidly form a layer of protein "corona" on the vehicle surface, which might block the modified ligands and hamper their targeting functions. We speculate that strategies for drug delivery can be designed based upon elegant control of the corona formation on the vehicle surfaces. In this study, we demonstrate a retinol-conjugated polyetherimine (RcP) nanoparticle system that selectively recruited the retinol binding protein 4 (RBP) in its corona components. RBP was found to bind retinol, and direct the antisense oligonucleotide (ASO)-laden RcP carrier to hepatic stellate cells (HSC), which play essential roles in the progression of hepatic fibrosis. In both mouse fibrosis models, induced by carbon tetrachloride (CCl4) and bile duct ligation (BDL), respectively, the ASO-laden RcP particles effectively suppressed the expression of type I collagen (collagen I), and consequently ameliorated hepatic fibrosis. Such findings suggest that this delivery system, designed to exploit the power of corona proteins, can serve as a promising tool for targeted delivery of therapeutic agents for the treatment of hepatic fibrosis.

Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography.

PubMed

Arakawa, Tsutomu; Tsumoto, Kouhei; Ejima, Daisuke; Kita, Yoshiko; Yonezawa, Yasushi; Tokunaga, Masao

2007-04-10

In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%-60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.

Effects of glycation on meloxicam binding to human serum albumin

NASA Astrophysics Data System (ADS)

Trynda-Lemiesz, Lilianna; Wiglusz, Katarzyna

2011-05-01

The current study reports a binding of meloxicam a pharmacologically important new generation, non-steroidal anti-inflammatory drug to glycated form of the human serum albumin (HSA). The interaction of the meloxicam with nonglycated and glycated albumin has been studied at pH 7.4 in 0.05 M sodium phosphate buffer with 0.1 M NaCl, using fluorescence quenching technique and circular dichroism spectroscopy. Results of the present study have shown that the meloxicam could bind both forms of albumin glycated and nonglycated at a site, which was close to the tryptophan residues. Similarly, how for native albumin glycated form has had one high affinity site for the drug with association constants of the order of 10 5 M -1. The glycation process of the HSA significantly has affected the impact of the meloxicam on the binding of other ligands such as warfarin and bilirubin. The affinity of the glycated albumin for bilirubin as for native albumin has been reduced by meloxicam but observed effect was weaker by half (about 20%) compared with nonglycated albumin. In contrast to the native albumin meloxicam binding to glycated form of the protein only slightly affected the binding of warfarin. It seemed possible that the effects on warfarin binding might be entirely attributable to the Lys 199 modification which was in site I.

Spectroscopic and molecular modeling studies on the binding of the flavonoid luteolin and human serum albumin.

PubMed

Jurasekova, Zuzana; Marconi, Giancarlo; Sanchez-Cortes, Santiago; Torreggiani, Armida

2009-11-01

Luteolin (LUT) is a polyphenolic compound, found in a variety of fruits, vegetables, and seeds, which has a variety of pharmacological properties. In the present contribution, binding of LUT to human serum albumin (HSA), the most abundant carrier protein in the blood, was investigated with the aim of describing the binding mode and parameters of the interaction. The application of circular dichroism, UV-Vis absorption, fluorescence, Raman and surface-enhanced Raman scattering spectroscopy combined with molecular modeling afforded a clear picture of the association mode of LUT to HSA. Specific interactions with protein amino acids were evidenced. LUT was found to be associated in subdomain IIA where an interaction with Trp-214 is established. Hydrophobic and electrostatic interactions are the major acting forces in the binding of LUT to HSA. The HSA conformations were slightly altered by the drug complexation with reduction of alpha-helix and increase of beta-turns structures, suggesting a partial protein unfolding. Also the configuration of at least two disulfide bridges were altered. Furthermore, the study of molecular modeling afforded the binding geometry. 2009 Wiley Periodicals, Inc.

Probing the binding of fluoxetine hydrochloride to human serum albumin by multispectroscopic techniques

NASA Astrophysics Data System (ADS)

Katrahalli, Umesha; Jaldappagari, Seetharamappa; Kalanur, Shankara S.

2010-01-01

The interaction between human serum albumin (HSA) and fluoxetine hydrochloride (FLX) have been studied by using different spectroscopic techniques viz., fluorescence, UV-vis absorption, circular dichroism and FTIR under simulated physiological conditions. Fluorescence results revealed the presence of static type of quenching mechanism in the binding of FLX to HSA. The values of binding constant, K of FLX-HSA were evaluated at 289, 300 and 310 K and were found to be 1.90 × 10 3, 1.68 × 10 3 and 1.45 × 10 3 M -1, respectively. The number of binding sites, n was noticed to be almost equal to unity thereby indicating the presence of a single class of binding site for FLX on HSA. Based on the thermodynamic parameters, Δ H0 and Δ S0 nature of binding forces operating between HSA and FLX were proposed. Spectral results revealed the conformational changes in protein upon interaction. Displacement studies indicated the site I as the main binding site for FLX on HSA. The effect of common ions on the binding of FLX to HSA was also investigated.

/sup 3/H-retinol derived photopigment in chick pineal membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wallingford, J.; Zatz, M.

Pineal glands display a day-night rhythm in the synthesis and secretion of melatonin. Dispersed chick pinealocytes retain their ability to respond to light in vitro for at least a week. Pinealocytes incubated overnight with /sup 3/H-retinol in the dark incorporate radioactivity predominantly into retinyl esters. To identify the chick pineal photopigment, SDS-PAGE was performed on radiolabelled preparations of pinealocytes and (intraocularly injected) rat retina. When intact cells or membrane preparations of cultured cells were incubated with NaCNBH/sub 3/, in the dark, a single radioactive peak with an apparent molecular weight of 32,000 daltons was observed. Rat retina preparations revealed amore » major peak at approximately 40,000 daltons. Protease inhibitors were present in the workup, and radioactivity corresponding to the smaller peak from pineal was not observed in retina. There was no radioactive peak when NaCNBH/sub 3/ was omitted. When samples were boiled in SDS the radioactivity shifted to the origin. These data suggest a protein in pinealocyte membranes which binds retinoid via a Schiff's base. Exposure to light of deoxycholate solubilized pineal membranes reduced the radioactivity associated with the protein. These findings raise the possibility that this protein is the pinealocyte's photopigment. Photopigments smaller than those observed in mammals have been reported in invertebrates.« less

How Proteins Bind Macrocycles

PubMed Central

Villar, Elizabeth A.; Beglov, Dmitri; Chennamadhavuni, Spandan; Porco, John A.; Kozakov, Dima; Vajda, Sandor; Whitty, Adrian

2014-01-01

The potential utility of synthetic macrocycles as drugs, particularly against low druggability targets such as protein-protein interactions, has been widely discussed. There is little information, however, to guide the design of macrocycles for good target protein-binding activity or bioavailability. To address this knowledge gap we analyze the binding modes of a representative set of macrocycle-protein complexes. The results, combined with consideration of the physicochemical properties of approved macrocyclic drugs, allow us to propose specific guidelines for the design of synthetic macrocycles libraries possessing structural and physicochemical features likely to favor strong binding to protein targets and also good bioavailability. We additionally provide evidence that large, natural product derived macrocycles can bind to targets that are not druggable by conventional, drug-like compounds, supporting the notion that natural product inspired synthetic macrocycles can expand the number of proteins that are druggable by synthetic small molecules. PMID:25038790

Decreased serum fatty acid binding protein 4 concentrations are associated with sarcopenia in chronic hemodialysis patients.

PubMed

Lin, Yu-Li; Liou, Hung-Hsiang; Lai, Yu-Hsien; Wang, Chih-Hsien; Kuo, Chiu-Huang; Chen, Shu-Yuan; Hsu, Bang-Gee

2018-06-21

Fatty acid binding protein 4 (FABP4) is found to play a role in skeletal muscle homeostasis. Since the dysregulation of FABP4 and sarcopenia are both highly prevalent in patients on chronic hemodialysis (HD), the correlation between them remains unknown. We aimed to examine this relationship in a cross-sectional study. A total of 120 chronic HD patients were recruited, and whose skeletal muscle mass, handgrip strength, and gait speed were assessed and blood samples were obtained. We grouped these participants into sarcopenia (n = 20) and non-sarcopenia groups according to European Working Group on Sarcopenia in Older People criteria. The sarcopenia group exhibited lower weight (P < 0.001), height (P = 0.019), waist circumference (P < 0.001), body mass index (P < 0.001), body fat mass (P = 0.004), and lower serum triglycerides (P = 0.009), creatinine (P < 0.001), phosphorus (P = 0.013), intact parathyroid hormone (P = 0.012), and FABP4 concentrations (P = 0.005), and higher malnutrition-inflammation scores (MIS) (P = 0.031), urea reduction rates (P < 0.001), and fractional clearance index for urea (Kt/V) values (P < 0.001). Serum FABP4 concentrations (odds ratio (OR): 0.98, 95% confidence interval (CI): 0.96-0.99, P = 0.043), body fat mass (OR: 0.86, 95% CI: 0.77-0.97, P = 0.013), MIS (OR: 6.90, 95% CI: 1.31-36.36, P = 0.023), and Kt/V (each increase of 0.1, OR: 2.15, 95% CI: 1.29-3.57, P = 0.003) were independent predictors of sarcopenia in chronic HD patients. We delineated the association between serum FABP4 concentrations and sarcopenia in chronic HD patients. Copyright © 2018. Published by Elsevier B.V.

The alternative complement pathway control protein H binds to immune complexes and serves their detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nydegger, U.E.; Corvetta, A.; Spaeth, P.J.

1983-01-01

During solubilization of immune complexes C3b becomes fixed to the immunoglobulin part and serves as a receptor for the alternative complement pathway control protein H. The H-C3b immune complex interaction can be made detectable using 4% polyethyleneglycol to separate free from bound /sup 125/I-H. Tetanus toxoid (Te)/anti-Te complexes kept soluble with fresh serum and containing 125 IU of specific antibody bound 18% of /sup 125/I-H; when fresh serum was chelated with 10 mM EDTA, /sup 125/I-H binding was only 5%. On sucrose density gradients, the H-binding material sedimented in the range of 12 to 30 S. In 36 serum samplesmore » from rheumatoid arthritis (RA) patients and in 12 serum samples from patients with systemic lupus erythematosus (SLE), /sup 125/I-H binding was significantly elevated to 9.5 +/- 4.7% (mean +/- 1 SD) and 13.3 +/- 5.6%, respectively, while /sup 125/I-H binding by 36 normal human sera was 4 +/- 2%. RA samples (17/36, 47%) and SLE samples (9/12, 75%) had H-binding values increased by more than 2 SD above the normal mean. The serum samples were also assessed for conglutinin- and C1q-binding activities; a significant correlation between H and C1q binding was observed (P less than 0.001); there was no correlation between H and conglutinin binding. Although binding to immune complexes through its interaction with C3b, H clearly detects a population of complexes other than conglutinin, thus expanding the possibilities of further characterizing pathological complexes.« less

Serum Vitamin D, Vitamin D Binding Protein, and Risk of Colorectal Cancer

PubMed Central

Anic, Gabriella M.; Weinstein, Stephanie J.; Mondul, Alison M.; Männistö, Satu; Albanes, Demetrius

2014-01-01

Background We previously reported a positive association between serum 25-hydroxyvitamin D (25(OH)D) and colorectal cancer risk. To further elucidate this association, we examined the molar ratio of 25(OH)D to vitamin D binding protein (DBP), the primary 25(OH)D transport protein, and whether DBP modified the association between 25(OH)D and colorectal cancer risk. Methods In a nested case-control study within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study, controls were 1∶1 matched to 416 colorectal cancer cases based on age and date of blood collection. Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CI) for quartiles of 25(OH)D, DBP, and the molar ratio of 25(OH)D:DBP, a proxy for free, unbound circulating 25(OH)D. Results Comparing highest to lowest quartiles, DBP was not associated with colorectal cancer risk (OR = 0.91; 95% CI: 0.58, 1.42, p for trend  = 0.58); however, a positive risk association was observed for the molar ratio of 25(OH)D:DBP (OR = 1.44; 95% CI: 0.92, 2.26, p for trend  = 0.04). In stratified analyses, the positive association between 25(OH)D and colorectal cancer was stronger among men with DBP levels above the median (OR = 1.89; 95% CI: 1.07, 3.36, p for trend  = 0.01) than below the median (OR = 1.20; 95% CI: 0.68, 2.12, p for trend  = 0.87), although the interaction was not statistically significant (p for interaction  = 0.24). Conclusion Circulating DBP may influence the association between 25(OH)D and colorectal cancer in male smokers, with the suggestion of a stronger positive association in men with higher DBP concentrations. This finding should be examined in other populations, especially those that include women and non-smokers. PMID:25036524

Guidance for selecting the measurement conditions in the dye-binding method for determining serum protein: theoretical analysis based on the chemical equilibrium of protein error.

PubMed

Suzuki, Y

2001-11-01

A methodology for selecting the measurement conditions in the dye-binding method for determining serum protein has been studied by a theoretical calculation. This calculation was based on the fact that a protein error occurs because of a reaction between the side chains of a positively charged amino acid residue in a protein molecule and a dissociated dye anion. The calculated characteristics of this method are summarized as follows: (1) Although the reaction between the dye and the protein occurs up to about pH 12, a change in the color shade, called protein error, is observed only in a pH region restricted within narrow limits. (2) Although the apparent absorbance (the absorbance of the test solution measured against a reagent blank) is lower than the true absorbance indicated by the formed dye-protein complex, the apparent absorbance correlates with the true absorbance with a correlation coefficient of 1.0. (3) At a higher dye concentration, the calibration curve is more linear at a higher pH than at a lower pH. Most of these characteristics were similarly observed experimentally in the reactions of BPB, BCG and BCP with human and bovine albumins. It is concluded that in order to ensure the linearity of the calibration curve, the measurement should be performed at a higher dye concentration and sufficiently high pH where the detection sensitivity is satisfied.

Detecting cis-regulatory binding sites for cooperatively binding proteins

PubMed Central

van Oeffelen, Liesbeth; Cornelis, Pierre; Van Delm, Wouter; De Ridder, Fedor; De Moor, Bart; Moreau, Yves

2008-01-01

Several methods are available to predict cis-regulatory modules in DNA based on position weight matrices. However, the performance of these methods generally depends on a number of additional parameters that cannot be derived from sequences and are difficult to estimate because they have no physical meaning. As the best way to detect cis-regulatory modules is the way in which the proteins recognize them, we developed a new scoring method that utilizes the underlying physical binding model. This method requires no additional parameter to account for multiple binding sites; and the only necessary parameters to model homotypic cooperative interactions are the distances between adjacent protein binding sites in basepairs, and the corresponding cooperative binding constants. The heterotypic cooperative binding model requires one more parameter per cooperatively binding protein, which is the concentration multiplied by the partition function of this protein. In a case study on the bacterial ferric uptake regulator, we show that our scoring method for homotypic cooperatively binding proteins significantly outperforms other PWM-based methods where biophysical cooperativity is not taken into account. PMID:18400778

Serum levels of insulin-like growth factor binding protein-1 and ovulatory responses to clomiphene citrate in women with polycystic ovarian disease.

PubMed

Tiitinen, A E; Laatikainen, T J; Seppälä, M T

1993-07-01

To study the serum levels of insulin, insulin-like growth factor I (IGF-I), and insulin-like growth factor binding protein-1 (IGFBP-1) in relation to clomiphene citrate (CC) responsiveness in women with polycystic ovarian disease (PCOD). Prospective. PATIENTS, SETTING: Twenty-three women with PCOD admitted consecutively to the University Infertility Clinic, a tertiary referral center. Blood samples were taken at fasting state and during oral glucose tolerance test (OGTT) for the determination of insulin, IGF-I, and IGFBP-1. A dose of 50 to 200 mg/d CC was given for ovulation induction. With CC treatment, ovulation was achieved in 13 of 23 PCOD patients. The IGFBP-1 concentration was lower in CC nonresponders than in CC responders (20.5 +/- 4.0 ng/mL versus 41.0 +/- 8.5 ng/mL) (P < 0.05). This difference was accentuated in 13 lean PCOD patients. Lean CC nonresponders (n = 7) had almost threefold lower serum IGFBP-1 levels than lean CC responders (n = 6) (24.0 +/- 3.1 ng/mL versus 61.8 +/- 8.6 ng/mL) (P < 0.01). By contrast, among 10 obese PCOD patients, the IGFBP-1 levels were low irrespective of CC responsiveness (14.8 +/- 8.0 ng/mL versus 16.7 +/- 7.2 ng/mL). The differences remained during OGTT. The concentrations of IGF-I, insulin, sex hormone-binding globulin, LH, FSH, and androgens showed no significant differences between CC responders and nonresponders. There was an inverse correlation between serum insulin and IGFBP-1 levels in obese PCOD patients, whereas this was not seen in lean patients. In lean PCOD patients, low serum IGFBP-1 concentration is related to CC unresponsiveness by a mechanism unrelated to insulin.

Binding of plasma proteins to titanium dioxide nanotubes with different diameters

PubMed Central

Kulkarni, Mukta; Flašker, Ajda; Lokar, Maruša; Mrak-Poljšak, Katjuša; Mazare, Anca; Artenjak, Andrej; Čučnik, Saša; Kralj, Slavko; Velikonja, Aljaž; Schmuki, Patrik; Kralj-Iglič, Veronika; Sodin-Semrl, Snezna; Iglič, Aleš

2015-01-01

Titanium and titanium alloys are considered to be one of the most applicable materials in medical devices because of their suitable properties, most importantly high corrosion resistance and the specific combination of strength with biocompatibility. In order to improve the biocompatibility of titanium surfaces, the current report initially focuses on specifying the topography of titanium dioxide (TiO2) nanotubes (NTs) by electrochemical anodization. The zeta potential (ζ-potential) of NTs showed a negative value and confirmed the agreement between the measured and theoretically predicted dependence of ζ-potential on salt concentration, whereby the absolute value of ζ-potential diminished with increasing salt concentrations. We investigated binding of various plasma proteins with different sizes and charges using the bicinchoninic acid assay and immunofluorescence microscopy. Results showed effective and comparatively higher protein binding to NTs with 100 nm diameters (compared to 50 or 15 nm). We also showed a dose-dependent effect of serum amyloid A protein binding to NTs. These results and theoretical calculations of total available surface area for binding of proteins indicate that the largest surface area (also considering the NT lengths) is available for 100 nm NTs, with decreasing surface area for 50 and 15 nm NTs. These current investigations will have an impact on increasing the binding ability of biomedical devices in the body leading to increased durability of biomedical devices. PMID:25733829

Photolysis of retinol in liposomes and its protection with tocopherol and oxybenzone.

PubMed

Young, A M; Gregoriadis, G

1996-03-01

In this work the stabilities of retinol in methanolic solutions and liposomal suspensions exposed to UV light were compared using absorbance spectroscopy and the ability of alpha-tocopherol and the sunscreen additive, oxybenzone, to reduce the rate of retinol decomposition assessed. Retinol in methanol decolorized almost completely within a few minutes of exposure to a 6 W 350 nm wavelength lamp. From the concentration dependence of the reaction rates it appears that retinol activated by light can decomposed either directly or after collision with a second retinol molecule. Several reaction products are formed. alpha-Tocopherol solutions were unaffected by 350 nm light but they did darken when irradiated with 250 nm wavelength light. Addition of alpha-tocopherol or removal of oxygen from the retinol in methanol solutions reduced only slightly the rates of retinol photolysis. When dispersed in water within liposomes made of equimolar egg phosphatidylcholine (PC) and cholesterol, up to six-fold increases in the decomposition rate of the retinol were observed. The reaction rate could be reduced but only slightly by increasing the ratio of PC to retinol. A mechanism that explains the concentration dependence of the retinol photolysis is that the reduction in reaction rate on diluting the retinol concentration within a given liposome was due to the prevention of the reaction between one light-activated retinol molecule with another within the same liposome. Incorporation of oxybenzone into the liposomes reduced the reaction rates. The results suggested that most of the protection in this case arises through the oxybenzone closet to the light source absorbing the light, thereby preventing it reaching retinol much further into the sample. Incorporation of alpha-tocopherol into the liposomes could also reduce substantially the photolysis rate of co-entrapped retinol. The mechanism of protection in this case appears to be via the tocopherol quenching activated retinol

Alteration of methotrexate binding to human serum albumin induced by oxidative stress. Spectroscopic comparative study

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, M.; Sułkowska, A.; Równicka-Zubik, J.

2016-01-01

Changes of oxidative modified albumin conformation by comparison of non-modified (HSA) and modified (oHSA) human serum albumin absorption spectra, Red Edge Excitation Shift (REES) effect and fluorescence synchronous spectra were investigated. Studies of absorption spectra indicated that changes in the value of absorbance associated with spectral changes in the region from 200 to 250 nm involve structural alterations related to variations in peptide backbone conformation. Analysis of the REES effect allowed for the observation of changes caused by oxidation in the region of the hydrophobic pocket containing the tryptophanyl residue. Synchronous fluorescence spectroscopy confirmed changes of the position of the tryptophanyl and tyrosil residues fluorescent band. Effect of oxidative stress on binding of methotrexate (MTX) was investigated by spectrofluorescence, UV-VIS and 1HNMR spectroscopy. MTX caused the fluorescence quenching of non-modified (HSA) and modified (oHSA) human serum albumin molecule. The values of binding constants, Hill's coefficients and a number of binding sites in the protein molecule in the high affinity binding site were calculated for the binary MTX-HSA and MTX-oHSA systems. For these systems, qualitative analysis in the low affinity binding sites was performed with the use of the 1HNMR technique.

Retinoid Pathway and Cancer Therapeutics

PubMed Central

Bushue, Nathan; Wan, Yu-Jui Yvonne

2010-01-01

The retinoids are a class of compounds that are structurally related to vitamin A. Retinoic acid, which is the active metabolite of retinol, regulates a wide range of biological processes including development, differentiation, proliferation, and apoptosis. Retinoids exert their effects through a variety of binding proteins including cellular retinol binding protein (CRBP), retinol-binding proteins (RBP), cellular retinoic acid-binding protein (CRABP), and nuclear receptors i.e. retinoic acid receptor (RAR) and retinoid × receptor (RXR). Because of the pleiotropic effects of retinoids, understanding the function of these binding proteins and nuclear receptors assists us in developing compounds that have specific effects. This review summarizes our current understanding of how retinoids are processed and act with the emphasis on the application of retinoids in cancer treatment and prevention. PMID:20654663

Elucidation of the binding mechanism of coumarin derivatives with human serum albumin.

PubMed

Garg, Archit; Manidhar, Darla Mark; Gokara, Mahesh; Malleda, Chandramouli; Suresh Reddy, Cirandur; Subramanyam, Rajagopal

2013-01-01

Coumarin is a benzopyrone which is widely used as an anti-coagulant, anti-oxidant, anti-cancer and also to cure arthritis, herpes, asthma and inflammation. Here, we studied the binding of synthesized coumarin derivatives with human serum albumin (HSA) at physiological pH 7.2 by using fluorescence spectroscopy, circular dichroism spectroscopy, molecular docking and molecular dynamics simulation studies. By addition of coumarin derivatives to HSA the maximum fluorescence intensity was reduced due to quenching of intrinsic fluorescence upon binding of coumarin derivatives to HSA. The binding constant and free energy were found to be 1.957±0.01×10(5) M(-1), -7.175 Kcal M(-1) for coumarin derivative (CD) enamide; 0.837±0.01×10(5) M(-1), -6.685 Kcal M(-1) for coumarin derivative (CD) enoate, and 0.606±0.01×10(5) M(-1), -6.49 Kcal M(-1) for coumarin derivative methylprop (CDM) enamide. The CD spectroscopy showed that the protein secondary structure was partially unfolded upon binding of coumarin derivatives. Further, the molecular docking studies showed that coumarin derivatives were binding to HSA at sub-domain IB with the hydrophobic interactions and also with hydrogen bond interactions. Additionally, the molecular dynamics simulations studies contributed in understanding the stability of protein-drug complex system in the aqueous solution and the conformational changes in HSA upon binding of coumarin derivatives. This study will provide insights into designing of the new inspired coumarin derivatives as therapeutic agents against many life threatening diseases.

Binding site and affinity prediction of general anesthetics to protein targets using docking.

PubMed

Liu, Renyu; Perez-Aguilar, Jose Manuel; Liang, David; Saven, Jeffery G

2012-05-01

The protein targets for general anesthetics remain unclear. A tool to predict anesthetic binding for potential binding targets is needed. In this study, we explored whether a computational method, AutoDock, could serve as such a tool. High-resolution crystal data of water-soluble proteins (cytochrome C, apoferritin, and human serum albumin), and a membrane protein (a pentameric ligand-gated ion channel from Gloeobacter violaceus [GLIC]) were used. Isothermal titration calorimetry (ITC) experiments were performed to determine anesthetic affinity in solution conditions for apoferritin. Docking calculations were performed using DockingServer with the Lamarckian genetic algorithm and the Solis and Wets local search method (http://www.dockingserver.com/web). Twenty general anesthetics were docked into apoferritin. The predicted binding constants were compared with those obtained from ITC experiments for potential correlations. In the case of apoferritin, details of the binding site and their interactions were compared with recent cocrystallization data. Docking calculations for 6 general anesthetics currently used in clinical settings (isoflurane, sevoflurane, desflurane, halothane, propofol, and etomidate) with known 50% effective concentration (EC(50)) values were also performed in all tested proteins. The binding constants derived from docking experiments were compared with known EC(50) values and octanol/water partition coefficients for the 6 general anesthetics. All 20 general anesthetics docked unambiguously into the anesthetic binding site identified in the crystal structure of apoferritin. The binding constants for 20 anesthetics obtained from the docking calculations correlate significantly with those obtained from ITC experiments (P = 0.04). In the case of GLIC, the identified anesthetic binding sites in the crystal structure are among the docking predicted binding sites, but not the top ranked site. Docking calculations suggest a most probable binding site

Binding Site and Affinity Prediction of General Anesthetics to Protein Targets Using Docking

PubMed Central

Liu, Renyu; Perez-Aguilar, Jose Manuel; Liang, David; Saven, Jeffery G.

2012-01-01

Background The protein targets for general anesthetics remain unclear. A tool to predict anesthetic binding for potential binding targets is needed. In this study, we explore whether a computational method, AutoDock, could serve as such a tool. Methods High-resolution crystal data of water soluble proteins (cytochrome C, apoferritin and human serum albumin), and a membrane protein (a pentameric ligand-gated ion channel from Gloeobacter violaceus, GLIC) were used. Isothermal titration calorimetry (ITC) experiments were performed to determine anesthetic affinity in solution conditions for apoferritin. Docking calculations were performed using DockingServer with the Lamarckian genetic algorithm and the Solis and Wets local search method (https://www.dockingserver.com/web). Twenty general anesthetics were docked into apoferritin. The predicted binding constants are compared with those obtained from ITC experiments for potential correlations. In the case of apoferritin, details of the binding site and their interactions were compared with recent co-crystallization data. Docking calculations for six general anesthetics currently used in clinical settings (isoflurane, sevoflurane, desflurane, halothane, propofol, and etomidate) with known EC50 were also performed in all tested proteins. The binding constants derived from docking experiments were compared with known EC50s and octanol/water partition coefficients for the six general anesthetics. Results All 20 general anesthetics docked unambiguously into the anesthetic binding site identified in the crystal structure of apoferritin. The binding constants for 20 anesthetics obtained from the docking calculations correlate significantly with those obtained from ITC experiments (p=0.04). In the case of GLIC, the identified anesthetic binding sites in the crystal structure are among the docking predicted binding sites, but not the top ranked site. Docking calculations suggest a most probable binding site located in the

Counterion-Release Entropy Governs the Inhibition of Serum Proteins by Polyelectrolyte Drugs.

PubMed

Xu, Xiao; Ran, Qidi; Dey, Pradip; Nikam, Rohit; Haag, Rainer; Ballauff, Matthias; Dzubiella, Joachim

2018-02-12

Dendritic polyelectrolytes constitute high potential drugs and carrier systems for biomedical purposes. Still, their biomolecular interaction modes, in particular those determining the binding affinity to proteins, have not been rationalized. We study the interaction of the drug candidate dendritic polyglycerol sulfate (dPGS) with serum proteins using isothermal titration calorimetry (ITC) interpreted and complemented with molecular computer simulations. Lysozyme is first studied as a well-defined model protein to verify theoretical concepts, which are then applied to the important cell adhesion protein family of selectins. We demonstrate that the driving force of the strong complexation, leading to a distinct protein corona, originates mainly from the release of only a few condensed counterions from the dPGS upon binding. The binding constant shows a surprisingly weak dependence on dPGS size (and bare charge) which can be understood by colloidal charge-renormalization effects and by the fact that the magnitude of the dominating counterion-release mechanism almost exclusively depends on the interfacial charge structure of the protein-specific binding patch. Our findings explain the high selectivity of P- and L-selectins over E-selectin for dPGS to act as a highly anti-inflammatory drug. The entire analysis demonstrates that the interaction of proteins with charged polymeric drugs can be predicted by simulations with unprecedented accuracy. Thus, our results open new perspectives for the rational design of charged polymeric drugs and carrier systems.

[Expression and significance of adipocyte fatty acid-binding protein in placenta, serum and umbilical cord blood in preeclampsia].

PubMed

Yan, Jian-Ying; Wang, Xiao-Juan

2010-12-01

To investigate the change of adipocyte fatty acid-binding protein (FABP4) in maternal serum and umbilical cord blood and FABP4 mRNA placental expression in patients with preeclampsia (PE). A total of 60 women with PE and 60 normal pregnant women as control participated in this study.All are admitted to Fujian Maternity and Children Health Hospital for delivery from December 2008 to October 2009. Patients with PE were divided into early-onset group (n = 30, presented at ≤ 34 weeks of gestation) and late-onset group (n = 30, presented at > 34 weeks of gestation), with 30 normal pregnant women as early control group (≤ 34 weeks of gestation) and 30 as late control group (> 34 weeks of gestation). Enzyme-linked immunosorbent assay (ELISA) was used to detect FABP4, fasting serum glucose, fasting insulin (FINS) in maternal serum and FABP4 in umbilical cord blood. Real-time fluorescent quantitative reverse transcription PCR was used to detect placental FABP4 mRNA expression. Furthermore, clinical and biochemical parameters were recorded, such as body mass index (BMI), systolic pressure (SP), diastolic pressure (DP), mean arterial pressure (MAP), total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), high density lipoprotein (HDL), creatinine (Cr), uric acid (UA), glomerular filtration rate (GFR), 24 hours urine protein in pregnant women and neonatal weight. (1) Maternal serum FABP4 was (176 ± 9) ng/L in early-onset PE group and (170 ± 9) ng/L in late-onset PE group, significantly elevated as compared to (81 ± 13) ng/L in early control group and (94 ± 15) ng/L in late control group. (2) Mean maternal FINS, homeostasis model of assessment for insulin resistence index (HOMA-IR) were significantly elevated in the early-onset PE group and late-onset PE group as compared to control groups, respectively. (3) Mean placental FABP4 mRNA expression were significantly elevated in the early-onset PE group and late-onset PE group as compared to late control

Synthesis, characterization and serum albumin binding studies of vitamin K3 derivatives.

PubMed

Suganthi, Murugesan; Elango, Kuppanagounder P

2017-01-01

Synthesis, characterization and bovine serum albumin (BSA) binding properties of three derivatives of vitamin K3 have been described. Results of UV-Vis and fluorescence spectra indicate complexation between BSA and the ligands with conformational changes in protein, which is strongly supported by synchronous and three dimensional fluorescence studies. Addition of the ligands quenches the fluorescence of BSA which is accompanied by reduction in quantum yield (Ф) from 0.1010 to 0.0775-0.0986 range. Thermodynamic investigations reveal that hydrophobic interaction is the major binding force in the spontaneous binding of these ligands with BSA. The binding constants obtained depend on the substituent present in the quinone ring, which correlates linearly with the Taft's field substituent constant (σ F ). The results show that compound with strong electron withdrawing nitro-group forms relatively stronger complex with BSA than amino and thioglycolate substituted ones. Circular dichroism studies show that the α-helical content of the protein, upon complexation with the ligands, decreases in the case of amino and nitro substituted vitamin K3 while increases in thioglycolate substituted compound. Molecular docking studies indicated that the vitamin K3 derivatives are surrounded by hydrophobic residues of the BSA molecule, which is in good agreement with the results of fluorescence spectral and thermodynamic studies. Copyright © 2016 Elsevier B.V. All rights reserved.

Titration calorimetry of anesthetic-protein interaction: negative enthalpy of binding and anesthetic potency.

PubMed

Ueda, I; Yamanaka, M

1997-04-01

Anesthetic potency increases at lower temperatures. In contrast, the transfer enthalpy of volatile anesthetics from water to macromolecules is usually positive. The transfer decreases at lower temperature. It was proposed that a few selective proteins bind volatile anesthetics with negative delta H, and these proteins are involved in signal transduction. There has been no report on direct estimation of binding delta H of anesthetics to proteins. This study used isothermal titration calorimetry to analyze chloroform binding to bovine serum albumin. The calorimetrically measured delta H cal was -10.37 kJ.mol-1. Thus the negative delta H of anesthetic binding is not limited to signal transduction proteins. The binding was saturable following Fermi-Dirac statistics and is characterized by the Langmuir adsorption isotherms, which is interfacial. The high-affinity association constant, K, was 2150 +/- 132 M-1 (KD = 0.47 mM) with the maximum binding number, Bmax = 3.7 +/- 0.2. The low-affinity K was 189 +/- 3.8 M-1 (KD = 5.29 mM), with a Bmax of 13.2 +/- 0.3. Anesthetic potency is a function of the activity of anesthetic molecules, not the concentration. Because the sign of delta H determines the temperature dependence of distribution of anesthetic molecules, it is irrelevant to the temperature dependence of anesthetic potency.

Phenanthrene binding by humic acid-protein complexes as studied by passive dosing technique.

PubMed

Zhao, Jian; Wang, Zhenyu; Ghosh, Saikat; Xing, Baoshan

2014-01-01

This work investigated the binding behavior of phenanthrene by humic acids (HA-2 and HA-5), proteins (bovine serum albumin (BSA)), lysozyme and pepsin), and their complexes using a passive dosing technique. All sorption isotherms were fitted well with Freundlich model and the binding capability followed an order of HA-5 > HA-2 > BSA > pepsin > lysozyme. In NaCl solution, phenanthrene binding to HA-BSA complexes was much higher than the sum of binding to individual HA and BSA, while there was no enhancement for HA-pepsin. Positively charged lysozyme slightly lowered phenanthrene binding on both HAs due to strong aggregation of HA-lysozyme complexes, leading to reduction in the number of binding sites. The binding enhancement by HA-BSA was observed under all tested ion species and ionic strengths. This enhancement can be explained by unfolding of protein, reduction of aggregate size and formation of HA-BSA complexes with favorable conformations for binding phenanthrene. Copyright © 2013 Elsevier Ltd. All rights reserved.

Determination of the affinity of drugs toward serum albumin by measurement of the quenching of the intrinsic tryptophan fluorescence of the protein.

PubMed

Epps, D E; Raub, T J; Caiolfa, V; Chiari, A; Zamai, M

1999-01-01

Binding of new chemical entities to serum proteins is an issue confronting pharmaceutical companies during development of potential therapeutic agents. Most drugs bind to the most abundant plasma protein, human serum albumin (HSA), at two major binding sites. Excepting fluorescence spectroscopy, existing methods for assaying drug binding to serum albumin are insensitive to higher-affinity compounds and can be labour-intensive, time-consuming, and usually require compound-specific assays. This led us to examine alternative ways to measure drug-albumin interaction. One method described here uses fluorescence quenching of the single tryptophan (Trp) residue in HSA excited at 295 nm to measure drug-binding affinity. Unfortunately, many compounds absorb, fluoresce, or both, in this UV wavelength region of the spectrum. Several types of binding phenomenon and spectral interference were identified by use of six structurally unrelated compounds and the equations necessary to make corrections mathematically were derived and applied to calculate binding constants accurately. The general cases were: direct quenching of Trp fluorescence by optically transparent ligands with low or high affinities; binding of optically transparent, non-fluorescent ligands to two specific sites where both sites or only one site result in Trp fluorescence quenching; and chromophores whose absorption either overlaps the Trp emission and quenches by energy transfer or absorbs light at the Trp fluorescence excitation wavelength producing absorptive screening as well as fluorescence quenching. Unless identification of the site specificity of drug binding to serum albumin is desired, quenching of the Trp fluorescence of albumin by titration with ligand is a rapid and facile method for determining the binding affinities of drugs for serum albumin.

Molecular characterization of an aldo-keto reductase from Marivirga tractuosa that converts retinal to retinol.

PubMed

Hong, Seung-Hye; Nam, Hyun-Koo; Kim, Kyoung-Rok; Kim, Seon-Won; Oh, Deok-Kun

2014-01-01

A recombinant aldo-keto reductase (AKR) from Marivirga tractuosa was purified with a specific activity of 0.32unitml(-1) for all-trans-retinal with a 72kDa dimer. The enzyme had substrate specificity for aldehydes but not for alcohols, carbonyls, or monosaccharides. The enzyme turnover was the highest for benzaldehyde (kcat=446min(-1)), whereas the affinity and catalytic efficiency were the highest for all-trans-retinal (Km=48μM, kcat/Km=427mM(-1)min(-1)) among the tested substrates. The optimal reaction conditions for the production of all-trans-retinol from all-trans-retinal by M. tractuosa AKR were pH 7.5, 30°C, 5% (v/v) methanol, 1% (w/v) hydroquinone, 10mM NADPH, 1710mgl(-1) all-trans-retinal, and 3unitml(-1) enzyme. Under these optimized conditions, the enzyme produced 1090mgml(-1) all-trans-retinol, with a conversion yield of 64% (w/w) and a volumetric productivity of 818mgl(-1)h(-1). AKR from M. tractuosa showed no activity for all-trans-retinol using NADP(+) as a cofactor, whereas human AKR exhibited activity. When the cofactor-binding residues (Ala158, Lys212, and Gln270) of M. tractuosa AKR were changed to the corresponding residues of human AKR (Ser160, Pro212, and Glu272), the A158S and Q270E variants exhibited activity for all-trans-retinol. Thus, amino acids at positions 158 and 270 of M. tractuosa AKR are determinant residues of the activity for all-trans-retinol. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Complement-mediated bactericidal activity of anti-factor H binding protein monoclonal antibodies against the meningococcus relies upon blocking factor H binding.

PubMed

Giuntini, Serena; Reason, Donald C; Granoff, Dan M

2011-09-01

Binding of the complement-downregulating protein factor H (fH) to the surface of the meningococcus is important for survival of the organism in human serum. The meningococcal vaccine candidate factor H binding protein (fHbp) is an important ligand for human fH. While some fHbp-specific monoclonal antibodies (MAbs) block binding of fH to fHbp, the stoichiometry of blocking in the presence of high serum concentrations of fH and its effect on complement-mediated bactericidal activity are unknown. To investigate this question, we constructed chimeric antibodies in which the human IgG1 constant region was paired with three murine fHbp-specific binding domains designated JAR 3, JAR 5, and MAb502. By surface plasmon resonance, the association rates for binding of all three MAbs to immobilized fHbp were >50-fold higher than that for binding of fH to fHbp, and the MAb dissociation rates were >500-fold lower than that for fH. While all three MAbs elicited similar C1q-dependent C4b deposition on live bacteria (classical complement pathway), only those antibodies that inhibited binding of fH to fHbp (JAR 3 and JAR 5) had bactericidal activity with human complement. MAb502, which did not inhibit fH binding, had complement-mediated bactericidal activity only when tested with fH-depleted human complement. When an IgG1 anti-fHbp MAb binds to sparsely exposed fHbp on the bacterial surface, there appears to be insufficient complement activation for bacteriolysis unless fH binding also is inhibited. The ability of fHbp vaccines to elicit protective antibodies, therefore, is likely to be enhanced if the antibody repertoire is of high avidity and includes fH-blocking activity.

Short-term increase of serum troponin I and serum heart-type fatty acid-binding protein (H-FABP) in dogs following administration of formoterol.

PubMed

Strauss, Volker; Wöhrmann, Thomas; Frank, Ilona; Hübel, Ulrich; Luft, Jörg; Bode, Gerd; Germann, Paul-Georg

2010-07-01

In this paper, changes in serum levels of the cardiac biomarkers troponin I and the heart-type fatty acid-binding protein (H-FABP) following administration of a long-acting beta(2)-sympathicomimeticum (long-acting beta-agonist, LABA) to dogs were measured. We measured troponin I in dogs in a 4-week repeated-dose study with inhalative administration of formoterol (13microg/kgd) and a glucocorticoid/formoterol combination (143/16microg/kgd). The medians of troponin I increased within 3 days in both groups, far beyond the cut-off level (0.1microg/L), but returned to baseline levels on study day 9. The increase was more pronounced in the formoterol-only group (3.29microg/L) compared to the glucocorticoid/formoterol combination group (1.32microg/L). In a second study, we measured serum troponin I as well as serum H-FABP levels in several samples over 7 days in dogs, receiving a single inhalative dose of a glucocorticoid/formoterol combination (120/12mug/kgd). The median of the troponin I concentration increased above the cut-off level within 2h and that of H-FABP within 4h. The medians of both parameters were temporarily above the cut-off levels even on study day 7. Both studies were conducted according to national animal welfare guidelines. To our knowledge, this is the first report that shows a corresponding increase of troponin I and H-FABP in dogs treated with formoterol. Both parameters are more sensitive in detecting a drug-induced cardiac injury compared to total LDH, total CK as well as CK MB activity. However, it is recommended to take at least three blood samples per day to assess a temporary increase of troponin I.

Anisotropy of fluctuation dynamics of proteins with an elastic network model.

PubMed Central

Atilgan, A R; Durell, S R; Jernigan, R L; Demirel, M C; Keskin, O; Bahar, I

2001-01-01

Fluctuations about the native conformation of proteins have proven to be suitably reproduced with a simple elastic network model, which has shown excellent agreement with a number of different properties for a wide variety of proteins. This scalar model simply investigates the magnitudes of motion of individual residues in the structure. To use the elastic model approach further for developing the details of protein mechanisms, it becomes essential to expand this model to include the added details of the directions of individual residue fluctuations. In this paper a new tool is presented for this purpose and applied to the retinol-binding protein, which indicates enhanced flexibility in the region of entry to the ligand binding site and for the portion of the protein binding to its carrier protein. PMID:11159421

Identification of Tetranectin as a Potential Biomarker for Metastatic Oral Cancer

PubMed Central

Arellano-Garcia, Martha E.; Li, Roger; Liu, Xiaojun; Xie, Yongming; Yan, Xiaofei; Loo, Joseph A.; Hu, Shen

2010-01-01

Lymph node involvement is the most important predictor of survival rates in patients with oral squamous cell carcinoma (OSCC). A biomarker that can indicate lymph node metastasis would be valuable to classify patients with OSCC for optimal treatment. In this study, we have performed a serum proteomic analysis of OSCC using 2-D gel electrophoresis and liquid chromatography/tandem mass spectrometry. One of the down-regulated proteins in OSCC was identified as tetranectin, which is a protein encoded by the CLEC3B gene (C-type lectin domain family 3, member B). We further tested the protein level in serum and saliva from patients with lymph-node metastatic and primary OSCC. Tetranectin was found significantly under-expressed in both serum and saliva of metastatic OSCC compared to primary OSCC. Our results suggest that serum or saliva tetranectin may serve as a potential biomarker for metastatic OSCC. Other candidate serum biomarkers for OSCC included superoxide dismutase, ficolin 2, CD-5 antigen-like protein, RalA binding protein 1, plasma retinol-binding protein and transthyretin. Their clinical utility for OSCC detection remains to be further tested in cancer patients. PMID:20957082

Comparative study of flavins binding with human serum albumin: a fluorometric, thermodynamic, and molecular dynamics approach.

PubMed

Sengupta, Abhigyan; Sasikala, Wilbee D; Mukherjee, Arnab; Hazra, Partha

2012-06-04

Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are derivatives of riboflavin (RF), a water-soluble vitamin, more commonly known as vitamin B(2). Flavins have attracted special attention in the last few years because of the recent discovery of a large number of flavoproteins. In this work, these flavins are used as extrinsic fluorescence markers for probing the microheterogeneous environment of a well-known transport protein, human serum albumin (HSA). Steady-state and time-resolved fluorescence experiments confirm that both FMN and FAD bind to the Sudlow's site-1 (SS1) binding pocket of HSA, where Trp214 resides. In the case of RF, a fraction of RF molecules binds at the SS1, whereas the major fraction of RF molecules remains unbound or surface bound to the protein. Moreover, flavin(s)-HSA interactions are monitored with the help of isothermal titration calorimetry, which provides free energy, enthalpy, and entropy changes of binding along with the binding constants. The molecular picture of binding interaction between flavins and HSA is well explored by docking and molecular dynamics studies. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

PubMed Central

Nilvebrant, Johan; Åstrand, Mikael; Georgieva-Kotseva, Maria; Björnmalm, Mattias; Löfblom, John; Hober, Sophia

2014-01-01

The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein. PMID:25089830

Absolute quantitation of NAPQI-modified rat serum albumin by LC-MS/MS: monitoring acetaminophen covalent binding in vivo.

PubMed

LeBlanc, André; Shiao, Tze Chieh; Roy, René; Sleno, Lekha

2014-09-15

Acetaminophen is known to cause hepatoxicity via the formation of a reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI), as a result of covalent binding to liver proteins. Serum albumin (SA) is known to be covalently modified by NAPQI and is present at high concentrations in the bloodstream and is therefore a potential biomarker to assess the levels of protein modification by NAPQI. A newly developed method for the absolute quantitation of serum albumin containing NAPQI covalently bound to its active site cysteine (Cys34) is described. This optimized assay represents the first absolute quantitation of a modified protein, with very low stoichiometric abundance, using a protein-level standard combined with isotope dilution. The LC-MS/MS assay is based on a protein standard modified with a custom-designed reagent, yielding a surrogate peptide (following digestion) that is a positional isomer to the target peptide modified by NAPQI. To illustrate the potential of this approach, the method was applied to quantify NAPQI-modified SA in plasma from rats dosed with acetaminophen. The resulting method is highly sensitive (capable of quantifying down to 0.0006% of total RSA in its NAPQI-modified form) and yields excellent precision and accuracy statistics. A time-course pharmacokinetic study was performed to test the usefulness of this method for following acetaminophen-induced covalent binding at four dosing levels (75-600 mg/kg IP), showing the viability of this approach to directly monitor in vivo samples. This approach can reliably quantify NAPQI-modified albumin, allowing direct monitoring of acetaminophen-related covalent binding.

Serum Antioxidant Concentrations and Metabolic Syndrome Are Associated among U.S. Adolescents in Recent National Surveys123

PubMed Central

Beydoun, May A.; Canas, J. Atilio; Beydoun, Hind A.; Chen, Xiaoli; Shroff, Monal R.; Zonderman, Alan B.

2012-01-01

Specific micronutrients, including retinol, retinyl esters, carotenoids [α-carotene, β-carotene (cis+trans), β-cryptoxanthin, lutein+zeaxanthin, and total lycopene], vitamin E, and vitamin C have antiinflammatory and antioxidant effects, properties shown to reduce oxidative stress, a process that accompanies the pathogenesis of many chronic diseases. It is still largely unknown whether they are associated with the occurrence of metabolic syndrome (MetS) in the adolescent U.S. population. MetS was defined by the International Diabetes Federation (IDF) criteria. Other non-MetS outcomes relying on blood measurements were elevated HOMA-IR, C-reactive protein (CRP), and hyperuricemia. We tested associations between serum antioxidants and MetS outcomes among adolescents aged 12–19 y using cross-sectional data from NHANES 2001–2006 (n = 782–4285). IDF MetS prevalence was estimated at 7% among boys and 3% among girls. In adjusted models, adolescents with MetS had consistently lower carotenoid concentrations compared with their counterparts without MetS. Total carotenoids were also inversely related to HOMA-IR and CRP. Vitamin C was inversely related to uric acid level and MetS binary outcome. Retinol+retinyl esters exhibited an inverse relationship with CRP and a positive relationship with uric acid and HOMA-IR as well as MetS binary outcome. Vitamin E had no association with MetS, particularly after controlling for serum cholesterol and TG. In conclusion, among U.S. adolescents, serum carotenoid concentrations were inversely associated with MetS status, HOMA-IR, and CRP, whereas serum vitamin C was inversely related to MetS status and serum uric acid. Vitamin E had no consistent association with MetS, whereas retinol+retinyl esters had a positive relationship with HOMA-IR, uric acid, and MetS, while being inversely related to CRP. These associations need further study. PMID:22810988

Discovery of binding proteins for a protein target using protein-protein docking-based virtual screening.

PubMed

Zhang, Changsheng; Tang, Bo; Wang, Qian; Lai, Luhua

2014-10-01

Target structure-based virtual screening, which employs protein-small molecule docking to identify potential ligands, has been widely used in small-molecule drug discovery. In the present study, we used a protein-protein docking program to identify proteins that bind to a specific target protein. In the testing phase, an all-to-all protein-protein docking run on a large dataset was performed. The three-dimensional rigid docking program SDOCK was used to examine protein-protein docking on all protein pairs in the dataset. Both the binding affinity and features of the binding energy landscape were considered in the scoring function in order to distinguish positive binding pairs from negative binding pairs. Thus, the lowest docking score, the average Z-score, and convergency of the low-score solutions were incorporated in the analysis. The hybrid scoring function was optimized in the all-to-all docking test. The docking method and the hybrid scoring function were then used to screen for proteins that bind to tumor necrosis factor-α (TNFα), which is a well-known therapeutic target for rheumatoid arthritis and other autoimmune diseases. A protein library containing 677 proteins was used for the screen. Proteins with scores among the top 20% were further examined. Sixteen proteins from the top-ranking 67 proteins were selected for experimental study. Two of these proteins showed significant binding to TNFα in an in vitro binding study. The results of the present study demonstrate the power and potential application of protein-protein docking for the discovery of novel binding proteins for specific protein targets. © 2014 Wiley Periodicals, Inc.

PNPLA3 I148M Variant Influences Circulating Retinol in Adults with Nonalcoholic Fatty Liver Disease or Obesity.

PubMed

Mondul, Alison; Mancina, Rosellina M; Merlo, Andrea; Dongiovanni, Paola; Rametta, Raffaela; Montalcini, Tiziana; Valenti, Luca; Albanes, Demetrius; Romeo, Stefano

2015-08-01

Retinol is a lipid-soluble essential nutrient that is stored as retinyl esters in lipid droplets of hepatic stellate cells. Patatin-like phospholipase domain-containing 3 (PNPLA3), through its retinyl-palmitate lipase activity, releases retinol from lipid droplets in hepatic stellate cells in vitro and ex vivo. We have shown that the genetic variant I148M (rs738409) reduces the PNPLA3 retinyl-palmitate lipase activity. The aim of the present genetic association study was to test whether overweight/obese carriers of the PNPLA3 148M mutant allele had lower circulating concentrations of retinol than individuals who are homozygous for the 148I allele. PNPLA3 I148M (rs738409) was genotyped by Taqman assay in 76 overweight/obese individuals [BMI (kg/m(2)) ≥25; mean ± SD age: 59.7 ± 11.4 y; male gender: 70%] with a histologic diagnosis of nonalcoholic fatty liver disease (NAFLD; namely the Milan NAFLD cohort) and in 413 obese men (BMI ≥30; mean ± SD age: 57.1 ± 4.9 y) from the α-Tocopherol, β-Carotene Cancer Prevention (ATBC) Study. Serum concentrations of retinol and α-tocopherol were measured by HPLC in both cohorts. β-Carotene concentrations in the ATBC study were measured by using HPLC. The PNPLA3 148M mutant allele was associated with lower fasting circulating concentrations of retinol (β = -0.289, P = 0.03) in adults with NAFLD (Milan NAFLD cohort). The PNPLA3 148M mutant allele was also associated with lower fasting circulating concentrations of retinol in adults with a BMI ≥30 (ATBC study; β = -0.043, P = 0.04). We showed for the first time, to our knowledge, that carriers of the PNPLA3 148M allele with either fatty liver plus obesity or obesity alone have lower fasting circulating retinol concentrations. © 2015 American Society for Nutrition.

Human sex hormone-binding globulin binding affinities of 125 structurally diverse chemicals and comparison with their binding to androgen receptor, estrogen receptor, and α-fetoprotein.

PubMed

Hong, Huixiao; Branham, William S; Ng, Hui Wen; Moland, Carrie L; Dial, Stacey L; Fang, Hong; Perkins, Roger; Sheehan, Daniel; Tong, Weida

2015-02-01

One endocrine disruption mechanism is through binding to nuclear receptors such as the androgen receptor (AR) and estrogen receptor (ER) in target cells. The concentration of a chemical in serum is important for its entry into the target cells to bind the receptors, which is regulated by the serum proteins. Human sex hormone-binding globulin (SHBG) is the major transport protein in serum that can bind androgens and estrogens and thus change a chemical's availability to enter the target cells. Sequestration of an androgen or estrogen in the serum can alter the chemical elicited AR- and ER-mediated responses. To better understand the chemical-induced endocrine activity, we developed a competitive binding assay using human pregnancy plasma and measured the binding to the human SHBG for 125 structurally diverse chemicals, most of which were known to bind AR and ER. Eighty seven chemicals were able to bind the human SHBG in the assay, whereas 38 chemicals were nonbinders. Binding data for human SHBG are compared with that for rat α-fetoprotein, ER and AR. Knowing the binding profiles between serum and nuclear receptors will improve assessment of a chemical's potential for endocrine disruption. The SHBG binding data reported here represent the largest data set of structurally diverse chemicals tested for human SHBG binding. Utilization of the SHBG binding data with AR and ER binding data could enable better evaluation of endocrine disrupting potential of chemicals through AR- and ER-mediated responses since sequestration in serum could be considered. Published by Oxford University Press on behalf of the Society of Toxicology 2014. This work is written by US Government employees and is in the public domain in the US.

Streptococcus pyogenes collagen type I-binding Cpa surface protein. Expression profile, binding characteristics, biological functions, and potential clinical impact.

PubMed

Kreikemeyer, Bernd; Nakata, Masanobu; Oehmcke, Sonja; Gschwendtner, Caroline; Normann, Jana; Podbielski, Andreas

2005-09-30

The Streptococcus pyogenes collagen type I-binding protein Cpa (collagen-binding protein of group A streptococci) expressed by 28 serotypes of group A streptococci has been extensively characterized at the gene and protein levels. Evidence for three distinct families of cpa genes was found, all of which shared a common sequence encoding a 60-amino acid domain that accounted for selective binding to type I collagen. Surface plasmon resonance-based affinity measurements and functional studies indicated that the expression of Cpa was consistent with an attachment role for bacteria to tissue containing collagen type I. A cpa mutant displayed a significantly decreased internalization rate when incubated with HEp-2 cells but had no effect on the host cell viability. By utilizing serum from patients with a positive titer for streptolysin/DNase antibody, an increased anti-Cpa antibody titer was noted for patients with a clinical history of arthritis or osteomyelitis. Taken together, these results suggest Cpa may be a relevant matrix adhesin contributing to the pathogenesis of S. pyogenes infection of bones and joints.

In Situ Protein Binding Assay Using Fc-Fusion Proteins.

PubMed

Padmanabhan, Nirmala; Siddiqui, Tabrez J

2017-01-01

This protocol describes an in situ protein-protein interaction assay between tagged recombinant proteins and cell-surface expressed synaptic proteins. The assay is arguably more sensitive than other traditional protein binding assays such as co-immunoprecipitation and pull-downs and provides a visual readout for binding. This assay has been widely used to determine the dissociation constant of binding of trans-synaptic adhesion proteins. The step-wise description in the protocol should facilitate the adoption of this method in other laboratories.

Binding of Complement Factor H (FH) Decreases Protective Anti-FH Binding Protein Antibody Responses of Infant Rhesus Macaques Immunized With a Meningococcal Serogroup B Vaccine

PubMed Central

Granoff, Dan M.; Costa, Isabella; Konar, Monica; Giuntini, Serena; Van Rompay, Koen K. A.; Beernink, Peter T.

2015-01-01

Background. The meningococcal vaccine antigen, factor H (FH)–binding protein (FHbp), binds human complement FH. In human FH transgenic mice, binding decreased protective antibody responses. Methods. To investigate the effect of primate FH binding, we immunized rhesus macaques with a 4-component serogroup B vaccine (4CMenB). Serum FH in 6 animals bound strongly to FHbp (FHbp-FHhigh) and, in 6 animals, bound weakly to FHbp (FHbp-FHlow). Results. There were no significant differences between the respective serum bactericidal responses of the 2 groups against meningococcal strains susceptible to antibody to the NadA or PorA vaccine antigens. In contrast, anti-FHbp bactericidal titers were 2-fold lower in FHbp-FHhigh macaques against a strain with an exact FHbp match to the vaccine (P = .08) and were ≥4-fold lower against 4 mutants with other FHbp sequence variants (P ≤ .005, compared with FHbp-FHlow macaques). Unexpectedly, postimmunization sera from all 12 macaques enhanced FH binding to meningococci. In contrast, serum anti-FHbp antibodies elicited by 4CMenB in mice whose mouse FH did not bind to the vaccine antigen inhibited FH binding. Conclusions. Binding of FH to FHbp decreases protective anti-FHbp antibody responses of macaques to 4CMenB. Even low levels of FH binding skew the antibody repertoire to FHbp epitopes outside of the FH-binding site, which enhance FH binding. PMID:25676468

Esterification of all-trans-retinol in normal human epithelial cell strains and carcinoma lines from oral cavity, skin and breast: reduced expression of lecithin:retinol acyltransferase in carcinoma lines.

PubMed

Guo, X; Ruiz, A; Rando, R R; Bok, D; Gudas, L J

2000-11-01

When exogenous [(3)H]retinol (vitamin A) was added to culture medium, normal human epithelial cells from the oral cavity, skin, lung and breast took up and esterified essentially all of the [(3)H]retinol within a few hours. As shown by [(3)H]retinol pulse-chase experiments, normal epithelial cells then slowly hydrolyzed the [(3)H]retinyl esters to [(3)H]retinol, some of which was then oxidized to [(3)H]retinoic acid (RA) over a period of several days. In contrast, cultured normal human fibroblasts and human umbilical vein endothelial cells (HUVEC) did not esterify significant amounts of [(3)H]retinol; this lack of [(3)H]retinol esterification was correlated with a lack of expression of lecithin:retinol acyltransferase (LRAT) transcripts in normal fibroblast and HUVEC strains. These results indicate that normal, differentiated cell types differ in their ability to esterify retinol. Human carcinoma cells (neoplastically transformed epithelial cells) of the oral cavity, skin and breast did not esterify much [(3)H]retinol and showed greatly reduced LRAT expression. Transcripts of the neutral, bile salt-independent retinyl ester hydrolase and the bile salt-dependent retinyl ester hydrolase were undetectable in all of the normal cell types, including the epithelial cells. These experiments suggest that retinoid-deficiency in the tumor cells could develop because of the lack of retinyl esters, a storage form of retinol.

Stability of selected serum proteins after long-term storage in the Janus Serum Bank.

PubMed

Gislefoss, Randi E; Grimsrud, Tom K; Mørkrid, Lars

2009-01-01

Human serum from biobanks is frequently used in prospective epidemiological studies. Long-term storage may modify its composition. A better understanding of the stability of the serum components may improve the interpretation of future studies. The concentrations of selected proteins; immunoglobulins, carrier proteins and enzymes in samples stored at -25 degrees C for 25 years and 2 years were compared with 1-month-old samples. For each length of storage time, 130 specimens were randomly selected from apparently healthy male blood donors aged 40-49 years. We examined the distribution of values, compared dispersion and localization of central tendency, and established reference intervals for each component. The study demonstrated non-significant or numerically small group differences in the concentrations of albumin, aspartate amino transferase, cystatin C, immunoglobulin E, immunoglobulin G, and sex hormone binding globulin. Mean values between fresh and 25-year-old samples suggested larger differences during storage for alanine amino transferase (-73.4%), creatinine kinase (-96.1%), insulin C-peptide (-98.7%), ferritin (-18.5%) and transferrin (+8.2%). The findings showed that long-term storage can introduce a considerable bias for vulnerable components.

Ultra-Performance Liquid Chromatographic Determination of Tocopherols and Retinol in Human Plasma

PubMed Central

Bell, Edward C.; John, Mathew; Hughes, Rodney J.; Pham, Thu

2014-01-01

A rapid, selective and sensitive ultra-performance liquid chromatography method has been developed for the detection and quantification of tocopherols and retinol in human plasma. Alpha-tocopherol, gamma-tocopherol and retinol are assayed using fluorescence detection. Excitation/emission wavelengths are 295/330 nm and 325/470 nm for the analysis of both tocopherols and retinol, respectively. Retinol acetate is employed as the internal standard. The reversed-phase method incorporates gradient elution with a mobile phase consisting of methanol and acetonitrile. Separation of vitamin compounds is achieved using a bridged ethyl hybrid C18 column. The retention times for retinol, retinol acetate, gamma-tocopherol and alpha-tocopherol are 1.6, 1.8, 3.9 and 4.3 min, respectively. The limits of quantification for retinol, gamma-tocopherol and alpha-tocopherol were 0.02, 0.02 and 0.1 µg/mL, respectively. The assay method is suitable for the analysis of tocopherols and retinol in human plasma. The method may be applied following the ingestion of foods fortified with these fat-soluble vitamins. PMID:24170122

Development of optical immunosensors for detection of proteins in serum.

PubMed

Kyprianou, Dimitris; Chianella, Iva; Guerreiro, Antonio; Piletska, Elena V; Piletsky, Sergey A

2013-01-15

The detection of proteins in biological samples such as blood, serum or plasma by biosensors is very challenging due to the complex nature of the matrix, which contains a high level of many interfering compounds. Here we show the application of a novel polymeric immobilisation matrix that helps in the detection of specific protein analytes in biological samples by surface plasmon resonance (SPR) immunosensors. This polymer matrix contains thioacetal functional groups included in the network, and these groups do not require any further activation in order to react with proteins, making it attractive for sensor fabrication. The protein prostate specific antigen (PSA) was selected as a model target analyte. A sandwich format with two primary antibodies recognising different parts (epitopes) of the analyte was used for the detection of PSA in serum. The efficiency of the reduction of non-specific binding achieved with novel polymer was compared with those of other techniques such as coating of sensor surface with polyethylene glycol (PEG), use of charged hydrophilic aspartic acid and surfactants such as Tween20. The detection limit of the polymer based immunosensor was 0.1 ng ml(-1) for free form PSA (f-PSA) in buffer and 5 ng ml(-1) in 20% serum. This is an improvement compared with similar devices reported on literature, indicating the potential of the immunosensor developed here for the analysis of real samples. Copyright © 2012 Elsevier B.V. All rights reserved.

Penicillin binding protein 3 of Staphylococcus aureus NCTC 8325-4 binds and activates human plasminogen.

PubMed

Kylväjä, Riikka; Ojalehto, Tuomas; Kainulainen, Veera; Virkola, Ritva; Westerlund-Wikström, Benita

2016-08-04

Staphylococcus aureus is a versatile pathogen expressing a number of virulence-associated adhesive molecules. In a previous study, we generated in a secretion-competent Escherichia coli strain a library of random FLAG-tag positive (FTP) polypeptides of S. aureus. To identify adhesive proteins and gain additional knowledge on putative virulence factors of S. aureus, we here screened the FTP library against human serum proteins. Staphylococcus aureus NCTC 8325-4, origin of the FTP library, adhered to immobilized plasminogen in vitro. In an enzyme-linked immunoassay a C-terminal part of penicillin binding protein 3 (PBP3), included in the FTP library, bound to immobilized plasminogen. We expressed and purified full-length PBP3 and its C-terminal fragments as recombinant proteins. In a time-resolved fluorometry-based assay the PBP3 polypeptides bound to immobilized plasminogen. The polypeptides enhanced formation of plasmin from plasminogen as analyzed by cleavage of a chromogenic plasmin substrate. The present findings, although preliminary, demonstrate reliably that S. aureus NCTC 8325-4 adheres to immobilized plasminogen in vitro and that the adhesion may be mediated by a C-terminal fragment of the PBP3 protein. The full length PBP3 and the penicillin binding C-terminal domain of PBP3 expressed as recombinant proteins bound plasminogen and activated plasminogen to plasmin. These phenomena were inhibited by the lysine analogue ε-aminocaproic acid suggesting that the binding is mediated by lysine residues. A detailed molecular description of surface molecules enhancing the virulence of S. aureus will aid in understanding of its pathogenicity and help in design of antibacterial drugs in the future.

Analysis of the expression pattern of the carrier protein transthyretin and its receptor megalin in the human scalp skin and hair follicles: hair cycle-associated changes.

PubMed

Adly, Mohamed A

2010-12-01

Transthyretin is a serum and cerebrospinal fluid protein synthesized early in development by the liver, choroid plexus and several other tissues. It is a carrier protein for the antioxidant vitamins, retinol, and thyroid hormones. Transthyretin helps internalize thyroxine and retinol-binding protein into cells by binding to megalin, which is a multi-ligand receptor expressed on the luminal surface of various epithelia. We investigated the expression of transthyretin and its receptor megalin in the human skin; however, their expression pattern in the hair follicle is still to be elucidated. This study addresses this issue and tests the hypothesis that "the expression of transthyretin and megalin undergoes hair follicle cycle-dependent changes." A total of 50 normal human scalp skin biopsies were examined (healthy females, 53-62 years) using immunofluorescence staining methods and real-time PCR. In each case, 50 hair follicles were analyzed (35, 10, and 5 follicles in anagen, catagen, and telogen, respectively). Transthyretin and megalin were prominently expressed in the human scalp skin and hair follicles, on both gene and protein levels. The concentrations of transthyretin and megalin were 0.12 and 0.03 Ul/ml, respectively, as indicated by PCR. The expression showed hair follicle cycle-associated changes i.e., strong expression during early and mature anagen, very weak expression during catagen and moderate expression during telogen. The expression values of these proteins in the anagen were statistically significantly higher than those of either catagen or telogen hair follicles (P ≤ 0.001). This study provides the first morphologic indication that transthyretin and megalin are variably expressed in the human scalp skin and hair follicles. It also reports variations in the expression of these proteins during hair follicle cycling. The clinical ramifications of these findings are open for further investigations.

Selective binding of proteins on functional nanoparticles via reverse charge parity model: an in vitro study

NASA Astrophysics Data System (ADS)

Ghosh, Goutam; Panicker, Lata; Barick, K. C.

2014-03-01

The conformation of proteins absorbed on nanoparticles surface plays a crucial role in applications of nanoparticles in biomedicine. The surface protein conformation depends on several factors, namely, nature of protein-nanoparticles interaction, chemical composition of the surface of nanoparticles etc. A model of the electrostatic binding of proteins on charged surface nanoparticles has been proposed earlier (Ghosh et al 2013 Colloids Surf. B 103 267). Also, the irreversible denaturation of the protein conformation due to binding of counterions was reported. In this paper, we have used this model, involving reverse charge parity, to show selective binding of proteins on charged surface iron oxide nanoparticles (IONPs). IONPs were surface functionalized with cetylpyridinium chloride (CPC), cetyl(trimethyl)ammonium bromide (CTAB) and cetylpyridinium iodide (CPI). The effect of counterions (Cl-, Br- and I-) on protein conformation has also been investigated. Several proteins such as α-lactalbumin (ALA), β-lactoglobulin (BLG), ovalbumin (OVA), bovin serum albumin (BSA) and HEWL were chosen for this investigation.

A peptide affinity column for the identification of integrin alpha IIb-binding proteins.

PubMed

Daxecker, Heide; Raab, Markus; Bernard, Elise; Devocelle, Marc; Treumann, Achim; Moran, Niamh

2008-03-01

To understand the regulation of integrin alpha(IIb)beta(3), a critical platelet adhesion molecule, we have developed a peptide affinity chromatography method using the known integrin regulatory motif, LAMWKVGFFKR. Using standard Fmoc chemistry, this peptide was synthesized onto a Toyopearl AF-Amino-650 M resin on a 6-aminohexanoic acid (Ahx) linker. Peptide density was controlled by acetylation of 83% of the Ahx amino groups. Four recombinant human proteins (CIB1, PP1, ICln and RN181), previously identified as binding to this integrin regulatory motif, were specifically retained by the column containing the integrin peptide but not by a column presenting an irrelevant peptide. Hemoglobin, creatine kinase, bovine serum albumin, fibrinogen and alpha-tubulin failed to bind under the chosen conditions. Immunodetection methods confirmed the binding of endogenous platelet proteins, including CIB1, PP1, ICln RN181, AUP-1 and beta3-integrin, from a detergent-free platelet lysate. Thus, we describe a reproducible method that facilitates the reliable extraction of specific integrin-binding proteins from complex biological matrices. This methodology may enable the sensitive and specific identification of proteins that interact with linear, membrane-proximal peptide motifs such as the integrin regulatory motif LAMWKVGFFKR.

Serum levels of fatty acid binding protein 4 and fat metabolic markers in relation to catecholamines following exercise.

PubMed

Iso, Tatsuya; Sunaga, Hiroaki; Matsui, Hiroki; Kasama, Shu; Oshima, Naomi; Haruyama, Hikari; Furukawa, Nozomi; Nakajima, Kiyomi; Machida, Tetsuo; Murakami, Masami; Yokoyama, Tomoyuki; Kurabayashi, Masahiko

2017-11-01

Lipolysis is stimulated by activation of adrenergic inputs to adipose tissues. Our recent study showed that serum concentrations of fatty acid binding protein 4 (FABP4) are robustly elevated in patients with acute myocardial infarction and ventricular tachyarrhythmia, that display a marked activation of the sympathetic nervous system (SNS). However, it remains unknown whether circulating FABP4 concentrations are associated with exercise-induced SNS activation. Thirty one healthy volunteers underwent cardiopulmonary exercise testing on a cycle ergometer up to the workload levels below and above anaerobic threshold, low- and high-intensity exercise, respectively. Serial blood samplings were performed before and after exercise. High-intensity exercise significantly increased serum concentrations of FABP4 and catecholamines, and their concentrations declined fast thereafter in a similar fashion. These changes were accompanied by little, if any, changes in other metabolic markers. Regardless of adiposity, percent change from baseline to peak FABP4 levels (%FABP4) was comparable in all subjects. Stepwise regression analysis revealed that %FABP4 was highly correlated with that in norepinephrine. Our study reveals the significant correlation between circulating FABP4 and norepinephrine levels during exercise testing. Together with the fact that FABP4 is secreted from adipocytes via β-adrenergic-mediated lipolytic mechanisms, this study suggests FABP4 as a potential biomarker for adrenergic overdrive. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Deglycosylation of serum vitamin D3-binding protein by alpha-N-acetylgalactosaminidase detected in the plasma of patients with systemic lupus erythematosus.

PubMed

Yamamoto, N; Naraparaju, V R; Moore, M; Brent, L H

1997-03-01

A serum glycoprotein, Gc protein (vitamin D3-binding protein), can be converted by beta-galactosidase of B cells and sialidase of T cells to a potent macrophage-activating factor (MAF), a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is the precursor for MAF. Treatment of Gc protein with immobilized beta-galactosidase and sialidase generates a remarkably high titered macrophage-activating factor (GcMAF). When peripheral blood monocytes/ macrophages (designated macrophages) of 33 systemic lupus erythematosus patients were incubated with GcMAF (100 pg/ml), the macrophages of all patients were activated as determined by superoxide generation. However, the precursor activity of patient plasma Gc protein was lost or reduced in these patients. Loss of the precursor activity was the result of deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase activity found in the patient plasma. Levels of plasma alpha-N-acetylgalactosaminidase activity in individual patients had an inverse correlation with the MAF precursor activity of their plasma Gc protein. Deglycosylated Gc protein cannot be converted to macro-phage-activating factor. The resulting defect in macro-phage activation may lead to an inability to clear pathogenic immune complexes. Thus, elevated plasma alpha-N-acetylgalactosaminidase activity resulting in the loss of MAF precursor activity and reduced macro-phage activity may play a role in the pathogenesis of systemic lupus erythematosus.

Titration calorimetry of anesthetic-protein interaction: negative enthalpy of binding and anesthetic potency.

PubMed Central

Ueda, I; Yamanaka, M

1997-01-01

Anesthetic potency increases at lower temperatures. In contrast, the transfer enthalpy of volatile anesthetics from water to macromolecules is usually positive. The transfer decreases at lower temperature. It was proposed that a few selective proteins bind volatile anesthetics with negative delta H, and these proteins are involved in signal transduction. There has been no report on direct estimation of binding delta H of anesthetics to proteins. This study used isothermal titration calorimetry to analyze chloroform binding to bovine serum albumin. The calorimetrically measured delta H cal was -10.37 kJ.mol-1. Thus the negative delta H of anesthetic binding is not limited to signal transduction proteins. The binding was saturable following Fermi-Dirac statistics and is characterized by the Langmuir adsorption isotherms, which is interfacial. The high-affinity association constant, K, was 2150 +/- 132 M-1 (KD = 0.47 mM) with the maximum binding number, Bmax = 3.7 +/- 0.2. The low-affinity K was 189 +/- 3.8 M-1 (KD = 5.29 mM), with a Bmax of 13.2 +/- 0.3. Anesthetic potency is a function of the activity of anesthetic molecules, not the concentration. Because the sign of delta H determines the temperature dependence of distribution of anesthetic molecules, it is irrelevant to the temperature dependence of anesthetic potency. PMID:9083685

Effect of the Protein Corona on Antibody-Antigen Binding in Nanoparticle Sandwich Immunoassays.

PubMed

de Puig, Helena; Bosch, Irene; Carré-Camps, Marc; Hamad-Schifferli, Kimberly

2017-01-18

We investigated the effect of the protein corona on the function of nanoparticle (NP) antibody (Ab) conjugates in dipstick sandwich immunoassays. Ab specific for Zika virus nonstructural protein 1 (NS1) were conjugated to gold NPs, and another anti-NS1 Ab was immobilized onto the nitrocellulose membrane. Sandwich immunoassay formation was influenced by whether the strip was run in corona forming conditions, i.e., in human serum. Strips run in buffer or pure solutions of bovine serum albumin exhibited false positives, but those run in human serum did not. Serum pretreatment of the nitrocellulose also eliminated false positives. Corona formation around the NP-Ab in serum was faster than the immunoassay time scale. Langmuir binding analysis determined how the immobilized Ab affinity for the NP-Ab/NS1 was impacted by corona formation conditions, quantified as an effective dissociation constant, K D eff . Results show that corona formation mediates the specificity and sensitivity of the antibody-antigen interaction of Zika biomarkers in immunoassays, and plays a critical but beneficial role.

Alpha-amylase inhibitor, CS-1036 binds to serum amylase in a concentration-dependent and saturable manner.

PubMed

Honda, Tomohiro; Kaneno-Urasaki, Yoko; Ito, Takashi; Kimura, Takako; Matsushima, Nobuko; Okabe, Hiromi; Yamasaki, Atsushi; Izumi, Takashi

2014-03-01

(2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl 4-O-(6-deoxy-β-D-glucopyranosyl)-α-D-glucopyranoside (CS-1036), which is an α-amylase inhibitor, exhibited biphasic and sustained elimination with a long t1/2 (18.4-30.0 hours) in rats and monkeys, but exhibited a short t1/2 (3.7-7.9 hours) in humans. To clarify the species differences in the t1/2, the plasma protein binding of CS-1036 was evaluated by ultrafiltration. A concentration-dependent and saturable plasma protein binding of CS-1036 was observed in rats and monkeys with the dissociation rate constant (KD) of 8.95 and 27.2 nM, and maximal binding capacity (Bmax) of 52.8 and 22.1 nM, respectively. By the assessments of the recombinant amylase and immunoprecipitation, the major binding protein of CS-1036 in rats was identified as salivary amylase (KD 5.64 nM). CS-1036 also showed concentration-dependent and saturable binding to human salivary and pancreatic amylase, with similar binding affinity in rats. However, the protein binding of CS-1036 was constant in human plasma (≤10.2%) due to the lower serum amylase level compared with rats and monkeys. From the calculation of the unbound fraction (fu) in plasma based on in vitro KD and Bmax, the dose-dependent increase in fu after oral administration is speculated to lead to a dose-dependent increase in total body clearance and a high area under the curve/dose at lower doses, such as 0.3 mg/kg in rats.

A novel antilithiatic protein from Tribulus terrestris having cytoprotective potency.

PubMed

Aggarwal, Anshu; Tandon, Simran; Singla, Surinder Kumar; Tandon, Chanderdeep

2012-08-01

Adhesion of calcium oxalate (CaOx) crystals to kidney cells is a key event in kidney stones associated with marked hyperoxaluria. As the propensity of stone recurrence and persistent side effects are not altered by surgical techniques available, phytotherapeutic agents could be useful as an adjuvant therapy. The present study is aimed at examining the antilithiatic potency of the protein biomolecules of Tribulus terrestris, a plant which is a common constituent of herbal marketed preparations to treat urolithiasis. Various biochemical methods with mass spectrometry were used to purify and characterize the purified protein. The protective potency of the protein was tested on the oxalate induced injury on renal epithelial cell lines (NRK 52E). An antilithiatic protein having molecular weight of ~ 60kDa was purified. This purified protein showed similarities with Carotenoid cleavage dioxygenase 7 (CCD7) of Arabidopsis thaliana after matching peptide mass fingerprints in MASCOT search engine. An EF hand domain was identified in CCD7 by SCAN PROSITE. Presence of an EF hand domain, a characteristic feature of calcium binding proteins and a role in the synthesis of retinol which is transported by retinol binding protein, a protein found in kidney stone matrix; of CCD7 support the role of TTP as an antilithiatic protein. The protective potency of TTP on NRK 52E was quite comparable to the aqueous extract of cystone. Our findings suggest that this purified protein biomolecule from Tribulus terrestris could open new vista in medical management of urolithiasis.

Predicting protein-binding RNA nucleotides with consideration of binding partners.

PubMed

Tuvshinjargal, Narankhuu; Lee, Wook; Park, Byungkyu; Han, Kyungsook

2015-06-01

In recent years several computational methods have been developed to predict RNA-binding sites in protein. Most of these methods do not consider interacting partners of a protein, so they predict the same RNA-binding sites for a given protein sequence even if the protein binds to different RNAs. Unlike the problem of predicting RNA-binding sites in protein, the problem of predicting protein-binding sites in RNA has received little attention mainly because it is much more difficult and shows a lower accuracy on average. In our previous study, we developed a method that predicts protein-binding nucleotides from an RNA sequence. In an effort to improve the prediction accuracy and usefulness of the previous method, we developed a new method that uses both RNA and protein sequence data. In this study, we identified effective features of RNA and protein molecules and developed a new support vector machine (SVM) model to predict protein-binding nucleotides from RNA and protein sequence data. The new model that used both protein and RNA sequence data achieved a sensitivity of 86.5%, a specificity of 86.2%, a positive predictive value (PPV) of 72.6%, a negative predictive value (NPV) of 93.8% and Matthews correlation coefficient (MCC) of 0.69 in a 10-fold cross validation; it achieved a sensitivity of 58.8%, a specificity of 87.4%, a PPV of 65.1%, a NPV of 84.2% and MCC of 0.48 in independent testing. For comparative purpose, we built another prediction model that used RNA sequence data alone and ran it on the same dataset. In a 10 fold-cross validation it achieved a sensitivity of 85.7%, a specificity of 80.5%, a PPV of 67.7%, a NPV of 92.2% and MCC of 0.63; in independent testing it achieved a sensitivity of 67.7%, a specificity of 78.8%, a PPV of 57.6%, a NPV of 85.2% and MCC of 0.45. In both cross-validations and independent testing, the new model that used both RNA and protein sequences showed a better performance than the model that used RNA sequence data alone in

Evaluation of the binding effect of human serum albumin on the properties of granules.

PubMed

Kristó, Katalin; Bajdik, János; Eros, István; Pintye-Hódi, Klára

2008-11-01

The main objective of this study was the application of a solution of human serum albumin as a granulating fluid. The properties of the granules formed were evaluated and compared with those when a conventional binder was applied in the same concentration. The powder mixture contained a soluble (mannitol) and an insoluble component (different types of cellulose). The protein solution applied exerted an appropriate aggregating effect if the system contained microcrystalline celluloses. Powdered cellulose was not suitable for the granulation with human serum albumin solution. As compared with the same concentration of the conventionally applied cellulose ethers as binder, the prepared granules exhibited a larger particle size, a significantly better compressibility, a higher breaking hardness and a favourable deformation process. These findings mainly reflect the good adhesive properties of the protein. The best compressibility and mechanical behaviour were attained on the application of the microcrystalline cellulose Vivapur type 105. This favourable behaviour may be connected with the wettability of cellulose. These results suggest that the formulation of tablets may be easier from an active agent in the serum that binds to albumin (e.g. interferon) since the amount of additives (binder) can be reduced.

Off surface matrix based on-chip electrochemical biosensor platform for protein biomarker detection in undiluted serum.

PubMed

Arya, Sunil K; Kongsuphol, Patthara; Park, Mi Kyoung

2017-06-15

The manuscript describes a concept of using off surface matrix modified with capturing biomolecule for on-chip electrochemical biosensing. 3D matrix made by laser engraving of polymethyl methacrylate (PMMA) sheet as off surface matrix was integrated in very close vicinity of the electrode surface. Laser engraving and holes in PMMA along with spacing from surface provide fluidic channel and incubation chamber. Covalent binding of capturing biomolecule (anti-TNF-α antibody) on off-surface matrix was achieved via azide group activity of 4-fluoro-3-nitro-azidobenzene (FNAB), which act as cross-linker and further covalently binds to anti-TNF-α antibody via thermal reaction. Anti-TNF-α/FNAB/PMMA matrix was then integrated over comb structured gold electrode array based sensor chip. Separate surface modification followed by integration of sensor helped to prevent the sensor chip surface from fouling during functionalization. Nonspecific binding was prevented using starting block T20 (PBS). Results for estimating protein biomarker (TNF-α) in undiluted serum using Anti-TNF-α/FNAB/PMMA/Au reveal that system can detect TNF-α in 100pg/ml to 100ng/ml range with high sensitivity of 119nA/(ng/ml), with negligible interference from serum proteins and other cytokines. Thus, use of off surface matrix may provide the opportunity to electrochemically sense biomarkers sensitively to ng/ml range with negligible nonspecific binding and false signal in undiluted serum. Copyright © 2016 Elsevier B.V. All rights reserved.

Ultra-performance liquid chromatographic determination of tocopherols and retinol in human plasma.

PubMed

Bell, Edward C; John, Mathew; Hughes, Rodney J; Pham, Thu

2014-10-01

A rapid, selective and sensitive ultra-performance liquid chromatography method has been developed for the detection and quantification of tocopherols and retinol in human plasma. Alpha-tocopherol, gamma-tocopherol and retinol are assayed using fluorescence detection. Excitation/emission wavelengths are 295/330 nm and 325/470 nm for the analysis of both tocopherols and retinol, respectively. Retinol acetate is employed as the internal standard. The reversed-phase method incorporates gradient elution with a mobile phase consisting of methanol and acetonitrile. Separation of vitamin compounds is achieved using a bridged ethyl hybrid C18 column. The retention times for retinol, retinol acetate, gamma-tocopherol and alpha-tocopherol are 1.6, 1.8, 3.9 and 4.3 min, respectively. The limits of quantification for retinol, gamma-tocopherol and alpha-tocopherol were 0.02, 0.02 and 0.1 µg/mL, respectively. The assay method is suitable for the analysis of tocopherols and retinol in human plasma. The method may be applied following the ingestion of foods fortified with these fat-soluble vitamins. © The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Importance of neonatal FcR in regulating the serum half-life of therapeutic proteins containing the Fc domain of human IgG1: a comparative study of the affinity of monoclonal antibodies and Fc-fusion proteins to human neonatal FcR.

PubMed

Suzuki, Takuo; Ishii-Watabe, Akiko; Tada, Minoru; Kobayashi, Tetsu; Kanayasu-Toyoda, Toshie; Kawanishi, Toru; Yamaguchi, Teruhide

2010-02-15

The neonatal FcR (FcRn) binds to the Fc domain of IgG at acidic pH in the endosome and protects IgG from degradation, thereby contributing to the long serum half-life of IgG. To date, more than 20 mAb products and 5 Fc-fusion protein products have received marketing authorization approval in the United States, the European Union, or Japan. Many of these therapeutic proteins have the Fc domain of human IgG1; however, the serum half-lives differ in each protein. To elucidate the role of FcRn in the pharmacokinetics of Fc domain-containing therapeutic proteins, we evaluated the affinity of the clinically used human, humanized, chimeric, or mouse mAbs and Fc-fusion proteins to recombinant human FcRn by surface plasmon resonance analysis. The affinities of these therapeutic proteins to FcRn were found to be closely correlated with the serum half-lives reported from clinical studies, suggesting the important role of FcRn in regulating their serum half-lives. The relatively short serum half-life of Fc-fusion proteins was thought to arise from the low affinity to FcRn. The existence of some mAbs having high affinity to FcRn and a short serum half-life, however, suggested the involvement of other critical factor(s) in determining the serum half-life of such Abs. We further investigated the reason for the relatively low affinity of Fc-fusion proteins to FcRn and suggested the possibility that the receptor domain of Fc-fusion protein influences the structural environment of the FcRn binding region but not of the FcgammaRI binding region of the Fc domain.

Spectroscopic characterization of effective components anthraquinones in Chinese medicinal herbs binding with serum albumins

NASA Astrophysics Data System (ADS)

Bi, Shuyun; Song, Daqian; Kan, Yuhe; Xu, Dong; Tian, Yuan; Zhou, Xin; Zhang, Hanqi

2005-11-01

The interactions of serum albumins such as human serum albumin (HSA) and bovine serum albumin (BSA) with emodin, rhein, aloe-emodin and aloin were assessed employing fluorescence quenching and absorption spectroscopic techniques. The results obtained revealed that there are relatively strong binding affinity for the four anthraquinones with HSA and BSA and the binding constants for the interactions of anthraquinones with HSA or BSA at 20 °C were obtained. Anthraquinone-albumin interactions were studied at different temperatures and in the presence of some metal ions. And the competition binding of anthraquinones with serum albumins was also discussed. The Stern-Volmer curves suggested that the quenching occurring in the reactions was the static quenching process. The binding distances and transfer efficiencies for each binding reactions were calculated according to the Föster theory of non-radiation energy transfer. Using thermodynamic equations, the main action forces of these reactions were also obtained. The reasons of the different binding affinities for different anthraquinone-albumin reactions were probed from the point of view of molecular structures.

Calcium-binding proteins and development

NASA Technical Reports Server (NTRS)

Beckingham, K.; Lu, A. Q.; Andruss, B. F.; McIntire, L. V. (Principal Investigator)

1998-01-01

The known roles for calcium-binding proteins in developmental signaling pathways are reviewed. Current information on the calcium-binding characteristics of three classes of cell-surface developmental signaling proteins (EGF-domain proteins, cadherins and integrins) is presented together with an overview of the intracellular pathways downstream of these surface receptors. The developmental roles delineated to date for the universal intracellular calcium sensor, calmodulin, and its targets, and for calcium-binding regulators of the cytoskeleton are also reviewed.

The effect of glycation on bovine serum albumin conformation and ligand binding properties with regard to gliclazide

NASA Astrophysics Data System (ADS)

Żurawska-Płaksej, Ewa; Rorbach-Dolata, Anna; Wiglusz, Katarzyna; Piwowar, Agnieszka

2018-01-01