Amputation and heat induced protein synthesis in the regenerating forelimb of Notophthalmus viridescens

This thesis compares the responses of regenerating forelimb tissues of the newt Notophthalmu..f vlridescens to the stresses of hyperthermia and ID.echanical injury of amputation. In particular, both quantitative and qualitative changes in the synthesis of soluble proteins in stump tissues, including those of the heat shock protein family (HSP70-1ike) were examined. Results from SDS-PAGEfluorography indicate that the trauma of amputation mimics the heat shock response both quantitatively and temporally in its transient repression of the synthesis of most normal cellular proteins, and qualitatively. in the locaJized expression of two unique proteins (hsp30 and hsp70). Fluorography of proteins separated by twodimensional gets revealed that thelCl4:alizedt amputation induced 70kDa protein (amp70) was distinct from the more basic newt hsp/hsc70 isoforms. Although limb amputation resulted in an increase in the synthesis of HSP70 mRNA analogous to that induced by heat 3.b.OCKf amp70 did not cross-react with murine monoclonal antibodies directed against both the inducible and cognate HSP70 proteins of the human. Thus, the possible relationship of amp70 to other members of the HSP70-1ike protein family remains unclear. Western analyses indicated that the levels of the constitutive form of HSP70 (hsc70) were found to be regulated in a stage-dependent manner in the distal stump tissues of the regen,erating forelimb of the newt. The highest levels were found in the mid-late bud stage, a period during which rapidly dividing blastema cells begin to redifferentiate in a proximodistal direction. Immediately after amputation) hsc70 synthesis and accumulation was depressed below steady-state levels measured in the unamputated limb~ The results are discussed in light of a possible role for HSPs and amputatio~ induced proteins in the epimorphic regeneration of the amphibian limb.

An examination of transcriptional and translational regulation of the 70kDa heat shock proteins following amputation of the tail and forelimb in the newt Notophthalmus viridescens

Several stresses to tissues including hyperthermia, ischemia, mechanical trauma and heavy metals have been demonstrated to affect the regulation of a subset of the family of heat shock proteins of70kOa (hsp70). In several organisms following some of these traumas, the levels of hsp70 mRNA and proteins are dramatically upregulated. However, the effects of the stress on limb and tail amputation in the newt Notophthalmus viridescens, involving mechanical tissue damage, have not adequately been examined. In the present study, three techniques were utilized to quantitate the levels of hsp70 mRNA and protein in the tissues of the forelimbs and tails of newts during the early post-traumatic events following surgical resection of these:: appendages. These included quantitative Western blotting of proteins separated by both one and twodimensional SDS-polyacrylamide gel electrophoresis and quantitative Northern blot analysis of total RNA. In tissues of both the limb and tail one hour after amputation, there were no significant differences in the levels of hsp70 protein measured by one-dimensional SOSPAGE followed by Western blotting, when compared to the levels measured in the unamputated limb. A 30 minute heat shock at 35°C failed to elicit an increase in the levels of hsp70 protein in these tissues. Further analysis using the more sensitive 20 PAGE separation of stump tissue proteins revealed that at least some of the five hsp70 isoforms of the newt may be differentially regulated in limbs and tails in response to trauma. It appears also that amputation of the tail and limb tissues leads to slight 3 elevation in the levels of HSP70 mRNA when compared to those of their respective unstressed tissues.

An exploratory study for the discovery of non-invasive hepatocellular carcinoma biomarkers among high-risk hepatitis C virus infected patients

Hepatocellular Carcinoma (HCC) is a major healthcare problem, representing the third most common cause of cancer-related mortality worldwide. Chronic infections with Hepatitis B virus (HBV) and/or Hepatitis C virus (HCV) are the major risk factors for the development of HCC. The incidence of HBV -associated HCC is in decline as a result of an effective HBV vaccine; however, since an equally effective HCV vaccine has not yet been developed, there are 130 million HCV infected patients worldwide who are at a high-risk for developing HCC. Because reliable parameters and/or tools for the early detection of HCC among high-risk individuals are severely lacking, HCC patients are always diagnosed at a late stage where surgical solutions or effective treatment are not possible. Using urine as a non-invasive sample source, two different approaches (proteomic-based and genomic-based approaches) were pursued with the common goal of discovering potential biomarker candidates for the early detection of HCC among high-risk chronic HCV infected patients. Urine was collected from 106 HCV infected Egyptian patients, 32 of whom had already developed HCC and 74 patients who were diagnosed as HCC-free at the time of initial sample collection. In addition to these patients, urine samples were also collected from 12 healthy control individuals. Total urinary proteins, Trans-renal nucleic acid (Tr-NA) and microRNA (miRNA) were isolated from urine using novel methodologies and silicon carbide-loaded spin columns. In the first, "proteomic-based", approach, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to identify potential candidates from pooled urine samples. This was followed by validating relative expression levels of proteins present in urine among all the patients using quantitative real time-PCR (qRT-PCR). This approach revealed that significant over-expression of three proteins: DJ-1, Chromatin Assembly Factor-1 (CAF-1) and 11 Moemen Abdalla HCC Biomarkers Heat Shock Protein 60 (HSP60), were characteristic events among HCC-post HCV infected patients. As a single-based HCC biomarker, CAF-1 over-expression identified HCC among HCV infected patients with a specificity of 90%, sensitivity of 66% and with an overall diagnostic accuracy of 78%. Moreover, the CAF-lIHSP60 tandem identified HCC among HCV infected patients with a specificity of 92%, sensitivity of 61 % and with an overall diagnostic accuracy of 77%. In the second genomic-based approach, two different approaches were processed. The first approach was the miRNA-based approach. The expression levels of miRNAs isolated from urine were studied using the Illumina MicroRNA Expression Profiling Assay. This was followed by qRT-PCR-based validation of deregulated expression of identified miRNA candidates among all the patients. This approach shed the light on the deregulated expression of a number of miRNAs, which may have a role in either the development of HCC among HCV infected patients (i.e. miR-640, miR-765, miR-200a, miR-521 and miR-520) or may allow for a better understanding of the viral-host interaction (miR-152, miR-486, miR-219, miR452, miR-425, miR-154 and miR-31). Moreover, the deregulated expression of both miR-618 and miR-650 appeared to be a common event among HCC-post HCV infected patients. The results of the search for putative targets of these two miRNA suggested that miR-618 may be a potent oncogene, as it targets the tumor-suppressor gene Low density lipoprotein-related protein 12 (LPR12), while miR-650 may be a potent tumor-suppressor gene, as it is supposed to downregulate the TNF receptor-associated factor-4 (TRAF4) oncogene. The specificity of miR-618 and miR-650 deregulated expression patterns for the early detection of HCC among HCV infected patients was 68% and 58%, respectively, whereas the sensitivity was 64% and 72%, respectively. When the deregulated expression of both miRNAs was combined as a tandem biomarker, the specificity and the sensitivity were 75% and 58% respectively. 111 Moemen Abdalla HCC Biomarkers In the second, "Trans-renal nucleic acid-based", approach, the urinary apoptotic nucleic acid (uaNA) levels of 70ng/mL or more were found to be a good predictor of HCC among chronic HCV infected patients. The specificity and the sensitivity of this diagnostic approach were 76% and 86%, respectively, with an overall diagnostic value of 81 %. The uaNA levels positively correlated to HCC disease progression as monitored by epigenetic changes of a panel of eight tumor-suppressor genes (TSGs) using methylation-sensitive PCR. Moreover, the pairing of high uaNA levels (:::: 70 ng/mL) and CAF-1 over-expreSSIOn produced a highly specific (l 00%) multiple-based HCC biomarker with an acceptable sensitivity of 64%, and with a diagnostic accuracy of 82%. In comparison to the previous pairing, the uaNA levels (:::: 70 ng/mL) in tandem with HSP60 over-expression was less specific (89%) but highly sensitive (72%), resulting in a diagnostic accuracy of 64%. The specificities of miR-650 deregulated expression in combination with either high uaNA content or HSP 60 over-expression were 82% and 79%, respectively, whereas, the sensitivities of these combinations were 64% and 58%, respectively. The potential biomarkers identified in this study compare favorably with the diagnostic accuracy of the a-fetoprotein levels test, which has a specificity of 75%, sensitivity of 68% and an overall diagnostic accuracy of 70%. Here we present an intriguing study which shows the significance of using urine as a noninvasive sample source for the identification of promising HCC biomarkers. We have also introduced new techniques for the isolation of different urinary macromolecules, especially miRNA, from urine. Furthermore, we strongly recommend the potential biomarkers indentified in this study as focal points of any future research on HCC diagnosis. A larger testing pool will determine if their use is practical for mass population screening. This explorative study identified potential targets that merit further investigation for the development of diagnostically accurate biomarkers isolated from 1-2 mL urine samples that were acquired in a non-invasive manner.

Mode of action of FMRFamide-like peptide on drosophila body wall muscle conractions

Neuropeptides are the largest group of signalling chemicals that can convey the information from the brain to the cells of all tissues. DPKQDFMRFamide, a member of one of the largest families of neuropeptides, FMRFamide-like peptides, has modulatory effects on nerve-evoked contractions of Drosophila body wall muscles (Hewes et aI.,1998) which are at least in part mediated by the ability of the peptide to enhance neurotransmitter release from the presynaptic terminal (Hewes et aI., 1998, Dunn & Mercier., 2005). However, DPKQDFMRFamide is also able to act directly on Drosophila body wall muscles by inducing contractions which require the influx of extracellular Ca 2+ (Clark et aI., 2008). The present study was aimed at identifying which proteins, including the membrane-bound receptor and second messenger molecules, are involved in mechanisms mediating this myotropic effect of the peptide. DPKQDFMRFamide induced contractions were reduced by 70% and 90%, respectively, in larvae in which FMRFamide G-protein coupled receptor gene (CG2114) was silenced either ubiquitously or specifically in muscle tissue, when compared to the response of the control larvae in which the expression of the same gene was not manipulated. Using an enzyme immunoassay (EIA) method, it was determined that at concentrations of 1 ~M- 0.01 ~M, the peptide failed to increase cAMP and cGMP levels in Drosophila body wall muscles. In addition, the physiological effect of DPKQDFMRFamide at a threshold dose was not potentiated by 3-lsobutyl-1-methylxanthine, a phosphodiesterase inhibitor, nor was the response to 1 ~M peptide blocked or reduced by inhibitors of cAMP-dependent or cGMP-dependent protein kinases. The response to DPKQDFMRFamide was not affected in the mutants of the phosholipase C-~ (PLC~) gene (norpA larvae) or IP3 receptor mutants, which suggested that the PLC-IP3 pathway is not involved in mediat ing the peptide's effects. Alatransgenic flies lacking activity of calcium/calmodul in-dependent protein kinase (CamKII showed an increase in muscle tonus following the application of 1 JlM DPKQDFMRFamide similar to the control larvae. Heat shock treatment potentiated the response to DPKQDFMRFamide in both ala1 and control flies by approximately 150 and 100 % from a non heat-shocked larvae, respectively. Furthermore, a CaMKII inhibitor, KN-93, did not affect the ability of peptide to increase muscle tonus. Thus, al though DPKQDFMRFamide acts through a G-protein coupled FMRFamide receptor, it does not appear to act via cAMP, cGMP, IP3, PLC or CaMKl1. The mechanism through which the FMRFamide receptor acts remains to be determined.

Investigation into the relationships between molecular chaperones, mitochondrial antioxidant enzymes, and endothermic vertebrate longevity

The maximum lifespan (MLSP) of endothermic vertebrates can range from as little as a year to over two centuries, yet the underlying phenotype of aging is very similar amongst this group of organisms. One organelle that may be important in the phenotype of aging is the mitochondrion. When damaged, this organelle is thought to contribute to many of the neurodegenerative diseases of aging. For this thesis, mitochondria from brain tissues of 7 mammalian and 2 avian species were isolated to assess whether the antioxidant glutathione system and major molecular chaperone, HSP60, is correlated to species MLSP. Furthermore, HSP60, and the major endoplasmic reticulum chaperone, GRP78, were measured under basal conditions, and following the introduction of an oxidative stress (hydrogen peroxide) in cultured mammalian myoblasts from 10 different species. My results indicate that the enzymes involved in the glutathione defense system are not correlated to species MLSP in brain mitochondria; however HSP60 levels are indeed higher in the longer-lived species. HSP60 levels are also higher at the basal level in cultured mammalian myoblasts and after 1 hour of hydrogen peroxide exposure. GRP78 induction is not correlated to species MLSP at the basal level or following hydrogen peroxide exposure. Therefore, these results suggest that HSP60 is a correlate of longevity in endothermic vertebrate species, but neither the glutathione antioxidant defense system, nor GRP78, correlates to species longevity.

The study of ligand binding specificities of the lipid binding proteins : recombinant human a-tocopherol transport protein (a-ttp), supernatant protein factor (spf) and S. cerevisiae Sec 14p for vitamin e (rrr-a-tocopherol) and other hydrophobic ligands.

One of the various functions of proteins in biological systems is the transport of small molecules, for this purpose proteins have naturally evolved special mechanisms to allow both ligand binding and its subsequent release to a target site; a process fundamental to many biological processes. Transport of Vitamin E (a-tocopherol), a lipid soluble antioxidant, to membranes helps in the protection of polyunsaturated fatty acids against peroxidative damage. In this research, the ligand binding characteristics of several members of the CRALTRIO family of lipid binding proteins was examined; the recombinant human a-Tocopherol Transfer Protein (a-TIP), Supernatant Protein Factor (SPF)ffocopherol Associated Protein (TAP), Cellular Retinaldehyde Binding Protein (CRALBP) and the phosphatidylinositol transfer protein from S. cerevisiae Sec 14p. Recombinant Sec 14p was expressed and purified from E. coli for comparison of tocopherol binding to the two other recombinant proteins postulated to traffic a-tocopherol. Competitive binding assays using [3H]-a-tocopherol and Lipidex-l000 resin allowed determination of the dissociation constants ~) of the CRAL-TRIO proteins for a-tocopherol and - 20 hydrophobic ligands for evaluation of the possible biological relevance of the binding interactions observed. The KIs (nM) for RRR-a-tocopherol are: a-TIP: 25.0, Sec 14p: 373, CRALBP: 528 and SPFffAP: 615. This indicates that all proteins recognize tocopherol but not with the same affinity. Sec 14p bound its native ligand PI with a KI of381 whereas SPFffAP bound PI (216) and y-tocopherol (268) similarly in contrast to the preferential binding ofRRR-a-tocopherol by a-TIP. Efforts to adequately represent biologically active SPFff AP involved investigation of tocopherol binding for several different recombinant proteins derived from different constructs and in the presence of different potential modulators (Ca+2, Mg+2, GTP and GDP); none of these conditions enhanced or inhibited a-tocopherol binding to SPF. This work suggests that only aTTP serves as the physiological mediator of a-tocopherol, yet structural homology between proteins allows common recognition of similar ligand features. In addition, several photo-affmity analogs of a-tocopherol were evaluated for their potential utility in further elucidation of a-TTP function or identification of novel tocopherol binding proteins.

Synthesis of fluorescent analogues of a-tocopherol as ligands for the human a-tocopherol transfer protein (a-TTP) /

To further understand in vivo localization and trafficking of a-tocopherol (a-Toe), the most biologically active form of vitamin E, between lipid environments, tocopherols are required that can be followed by teclu1iques such as confocal microscopy and fluorescence resonance energy transfer (FRET) assays. To this end, sixteen fluorescent analogues of a-tocopherol (la-d [(1)anthroy loxy -a-tocopherols, A O-a-Toes], 2a-d [w-nitro benzoxadiazole-a-tocopherols, NBD-aToes], 3a-d [w-dansyl-a-tocopherols, DAN-a-Toes], and 4a-d [w-N-methylanthranilamide-atocopherols, NMA-a-TocsD were prepared by substituting fluorescent labels at the terminus of w-functionalized alkyl chains extending from C-2 of the chroman ring while retaining key binding features of the natural ligand. These compounds were prepared starting from (S)-Trolox® acid VIa esterification, protection, and reduction producing the silyl-protected (S)-Trolox aldehyde that was coupled using Wittig chemistry to different w-hydroxyalkylphosphonium bromides. Reduction of the alkene generated the w-hydroxy functionalized 2-n-alkyl intermediates 9a-d having the necessary 2R stereochemistry. A series of functional group manipulations including mesylation, substitution with azide, and hydride reduction provided w-amino functionalized intermediates 12a-d as well. Coupling intermediates 9a-d and 12a-d with the selected fluorophores (9- anthracene carboxylic acid, 4-chloro-7-nitrobenz-2-oxa-l,3-diazole, 5- dimethylaminonapthalene-l-sulfonyl chloride, and I-methyl-2H-3,1-benzoxazine-2,4(1H)dione), followed by deprotection of the phenolic silyl group, gave the desired fluorescent ligands la-d, 2a-d, 3a-d and 4a-d in good yield. Assessment of their binding affinities with recombinant human a-tocopherol transfer protein (ha-TTP) utilizing fluorescent titration binding assays identified competent ligands for further use in protein studies. Compounds Id (C9-AO-a-Toc) and 2d (C9-NBD-a-Toc) both having nonyl alkyl chain extensions between the chromanol and fluorophore were shown to bind specifically to ha-TTP with dissociation constants (KdS) of approximately 280 nM and 55 nM respectively, as compared to 25 nM for the natural ligand 2R,4'R,^'R-a-tocophQxoL.

An investigation into differences between indoleacetic acid and fusicoccin in their influence on RNA synthesis, protein synthesis and growth in Avena coleoptile tissue

Growth stimulation of Avena coleoptile tissue by indoleacetic acid (IAA) and fusicoccin (FC) was compared by measuring both their influence on RNA and protein synthesis during IAA or FC stimulated growth. FC stimulated growth more than IAA during the initial four hour exposure, after which the growth rate gradually declined to the control rate. FC, but not IAA, increased the uptake of 3H-Ieucine into tissue and the specific radioactivity of extracted protein. Cycloheximide inhibited the incorporation of 3H-Ieucine into protein by approximately 60% to 70% in all cases. In the presence of cycloheximide 3H-radioactivity accumulated in FC-treated tissue, whereas IAA did not seem to influence 3H-accumulation. These results suggest that FC stimulated leucine uptake into the tissue and that increased specific activity of coleoptile protein is due to increased leucine uptake, not an increased rate of protein synthesis. There was no measurable influence of IAA and/or FC on RNA and protein synthesis during the initial hours of a growth stimulation. Inhibitors of RNA and protein synthesis, actinomycin D and cycloheximide, respectively, severely inhibited IAA enhanced growth but only partially inhibited FC stimulated growth. The data are consistent with suggestions that a rapidly turning over protein participates in IAA stimulated growth, and that a continual synthesis of RNA and proteins is an absolute requirement for a long term growth response to IAA. On the contrary, FC-stimulated growth exhibited less dependency on the transcription and translation processes. The data are consistent with proposals suggesting different sites of action for FC and IAA stimulated growth. l?hen compared to CO2-free air, CO2 at 300 ppm had no significant influence on coleoptile growth and protein synthesis in the presence or absence of lAA or FC. Also, I mM malate, pH 6.0 did not influence growth of coleoptiles in the presence or absence of lAA. This result was obtained despite reports indicating that 300 ppm CO2 or I mM malate stimulates growth and protein synthesis. This lack of difference between CO2-treated and untreated tissue could indicate either that the interstitial space CO2 concentration is not actually different in the two treatments due to significant endogenous respiratory CO2 or else the data would suggest a very loose coupling between dark CO2 fixation and growth. IAA stimulated the in vivo fixation of 14c-bicarbonate (NaHI4c03) by about 25% and the addition of cycloheximide caused an inhibition of bicarbonate fixation within 30 min. Cycloheximide has also been reported to inhibit IAA-stimulated H+ excretion. These data are consistent with the acid growth theory and suggest that lAA stimulated growth involves dark CO2 fixation. The roles of dark CO2 fixation in lAA-stimulated growth are discussed.

GABA accumulation and the hypersensitive response in isolated mesophyll cells treated with the G protein activator mastoparan

GABA (y-amino butyric acid) is a non-protein amino acid synthesized through the a-decarboxylation of L-glutamate. This reaction is catalyzed by L-glutamate decarboxylase (EC 4.1.1.15), a cytosolic Ca2+/calmodulin-stimulated enzyme. The purpose of this study is to determine whether or not GABA accumulation is associated with the hypersensitive response of isolated Asparagus sprengeri mesophyll cells. The addition of 25 J.lM mastoparan, a G protein activator, to suspensions of isolated asparagus mesophyll cells significantly increased GABA synthesis and cell death. Cell death was assessed using Evan's blue dye and fluorescein diacetate tests for cell viability. In addition, mastoparan stimulated pH-dependent alkalinization of the external medium, and a rapid and large 02 consumption followed by a loss of photosynthetic activity. The rate of 02 consumption and the net decrease in 02 in the dark was enhanced by light. The inactive mastoparan analogue Mas17 was ineffective in stimulating GABA accumulation, medium alkalinization, 02 uptake and cell death. Accumulation of H202 in response tomastoparan was not detected, however, mastoparan caused the cell-dependent degradation of added H202. The pH dependence of mastoparan-stimulated alkalinization suggests cellular electrolyte leakage, while the consumption of 02 corresponds to the oxidative burst in which 02 at the cell surface is reduced to form various active oxygen species. The results are indicative of the "hypersensitive response" of plants to pathogen attack, namely, the death of cells in the locality of pathogen invasion. The data are compatible with a model in which mastoparan triggers G protein activity, subsequent intracellular signal transduction pathway/s, and the hypersensitive response. It is postulated that the physiological elicitation of the hypersensitive response involves G protein signal transduction. The synthesis of GABA during the hypersensitive response has not been documented previously; however the role/s of GABA synthesis in the hypersensitive response, if any, remain unclear.

Isolation and partial characterization of a complementary protein from the mycoparasite, Piptocephalis virginiana, which specifically binds to two glycoproteins b and c of the host cell surface

Presence of surface glycoprotein in Piptocephalis virginiana that recognizes the host glycoproteins band c, reported earlier from our laboratory, was detected by immunofluorescence microscopy. Germinated spores of P. virginiana treated with Mortierella pusilla cell wall protein extract, primary antibodies prepared against glycoproteins band c and FITC-goat anti-rabbit IgG conjugate showed fluorescence. This indicated that on the surfaces of the biotrophic mycoparasite P. virginiana , there might be a complementary molecule which recognizes the glycoproteins band c from M. pusilla. Immunobinding analysis identified a glycoprotein of Mr 100 kDa from the mycoparasite which binds with the host glycoproteins band c, separately as well as collectively. Purification of this glycoprotein was achieved by (i) 60% ammonium sulfate precipitation, (ii) followed by heat treatment, and (iii) Sephadex G-IOO gel filtration. The glycoprotein was isolated by preparative polyacrylamide gel electrophoresis by cutting and elution. The purity of the protein ·was ascertained by SDS-PAGE and Western blot analysis. Positive reaction to periodic acid-Schiff reagent revealed the glycoprotein nature of this 100 kDa protein. Mannose was identified as a major sugar component of this glycoprotein by using a BoehringerMannheim Glycan Differentiation Kit. Electrophoretically purified glycoprotein was used to raIse polyclonal antibody in rabbit. The specificity of the antibody was determined by dot-immunobinding test and western-blot analysis. Immunofluorescence mIcroscopy revealed surface localization of the protein on the germ tube of Piptocephalis virginiana. Fluorescence was also observed at the surfaceJ of the germinated spores and hyphae of the host, M. pusilla after treatment with complementary protein from P. virginiana, primary antibody prepared against the complementary protein and FITC-goat anti-rabbit IgG conjugate.

Role of Phosphorylation of the Oxysterol Binding Protein (OSBP) in (PI(4)P) and Sterol-Containing Membrane Recognition and Binding

Studies have demonstrated that the oxysterol binding protein (OSBP) acts as a phosphatidylinositol phosphate (PIP)-sterol exchanger at membrane contact sites (MCS) of the endoplasmic reticulum (ER) and Golgi. OSBP is known to pick up phosphatidylinositol-4-phosphate (PI(4)P) from the ER, transfer it to the trans-Golgi in exchange for a cholesterol molecule that is then transferred from the trans-Golgi to the ER. Upon further examination of this pathway by Ridgway et al. (1), it appeared that phosphorylation of OSBP played a role in the localization of OSBP. The dephosphorylation state of OSBP was linked to Golgi localization and the depletion of cholesterol at the ER. To mimic the phosphorylated state of OSBP, the mutant OSBP-S5E was designed by Ridgway et al. (1). The lipid and sterol recognition by wt-OSBP and its phosphomimic mutant OSBP-S5E were investigated using immobilized lipid bilayers and dual polarization interferometry (DPI). DPI is a technique in which the protein binding affinity to immobilized lipid bilayers is measured and the binding behavior is examined through real time. Lipid bilayers containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and varying concentrations of PI(4)Ps or sterols (cholesterol or 25-hydroxycholesterol) were immobilized on a silicon nitride chip. It was determined that wt-OSBP binds differently to PI(4)P-containing bilayers compared to OSBP-S5E. The binding behavior suggested that wt-OSBP extracts PI(4)P and the change in the binding behavior, in the case of OSBP-S5E, suggested that the phosphorylation of OSBP may prevent the recognition and/or extraction of PI(4)P. In the presence of sterols, the overall binding behavior of OSBP, regardless of phosphorylation state, was fairly similar. The maximum specific bound mass of OSBP to sterols did not differ as the concentration of sterols increased. However, comparing the maximum specific bound mass of OSBP to cholesterol with oxysterol (25-hydroxycholesterol), OSBP displayed nearly a 2-fold increase in bound mass. With the absence of the wt-OSBP-PI(4)P binding behavior, it can be speculated that the sterols were not extracted. In addition, the binding behavior of OSBP was further tested using a fluorescence based binding assay. Using 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (22-NBD cholesterol), wt-OSBP a one site binding dissociation constant Kd, of 15 ± 1.4 nM was determined. OSBP-S5E did not bind to 22-NBD cholesterol and Kd value was not obtained.