An industry perspective approach and control strategy for implementation of ready-to-use cells in bioassays: survey outcome and recommendations.

The BioPhorum Development Group is an industry collaboration enabling the sharing of common practices for the development of biopharmaceuticals. Bioassays are an important part of an analytical control system. Utilization of ready-to-use cells can increase operational flexibility and improve efficiency by providing frozen cell banks uniform stock while removing challenges associated with maintaining cultured cells. The BioPhorum Development Group-Bioassay workstream conducted an intercompany benchmarking survey and group discussions around the use of ready-to-use cells for bioassays. The results of the collaboration provide alignment on nomenclature, production, qualification and implementation of ready-to-use cells to support the assay life cycle.

A Novel Cell-based Luciferase Reporter Platform for the Development and Characterization of T-Cell Redirecting Therapies and Vaccine Development.

T-cell immunotherapies are promising strategies to generate T-cell responses towards tumor-derived or pathogen-derived antigens. Adoptive transfer of T cells genetically modified to express antigen receptor transgenes has shown promise for the treatment of cancer. However, the development of T-cell redirecting therapies relies on the use of primary immune cells and is hampered by the lack of easy-to-use model systems and sensitive readouts to facilitate candidate screening and development. Particularly, testing T-cell receptor (TCR)-specific responses in primary T cells and immortalized T cells is confounded by the presence of endogenous TCR expression which results in mixed alpha/beta TCR pairings and compresses assay readouts. Herein, we describe the development of a novel cell-based TCR knockout (TCR-KO) reporter assay platform for the development and characterization of T-cell redirecting therapies. CRISPR/Cas9 was used to knockout the endogenous TCR chains in Jurkat cells stably expressing a human interleukin-2 promoter-driven luciferase reporter gene to measure TCR signaling. Reintroduction of a transgenic TCR into the TCR-KO reporter cells results in robust antigen-specific reporter activation compared with parental reporter cells. The further development of CD4/CD8 double-positive and double-negative versions enabled low-avidity and high-avidity TCR screening with or without major histocompatibility complex bias. Furthermore, stable TCR-expressing reporter cells generated from TCR-KO reporter cells exhibit sufficient sensitivity to probe in vitro T-cell immunogenicity of protein and nucleic acid-based vaccines. Therefore, our data demonstrated that TCR-KO reporter cells can be a useful tool for the discovery, characterization, and deployment of T-cell immunotherapy.

The clinical role of serum cell division control 42 in coronary heart disease.

Cell division control 42 (CDC42) regulates blood lipids, atherosclerosis, T cell differentiation and inflammation, which is involved in the process of coronary heart disease (CHD). This study aimed to evaluate the CDC42 level and its correlation with clinical features, the T-helper 17 (Th17)/regulatory-T (Treg) cell ratio and prognosis in CHD patients. In total, 210 CHD patients, 20 healthy controls and 20 disease controls were enrolled. Serum CDC42 levels of all participants were measured by enzyme-linked immunosorbent assay. In CHD patients, Th17 and Treg cells were discovered by flow cytometry; CHD patients were followed-up for a median of 16.9 months (range of 2.5-38.2 months). CDC42 level was lowest in CHD patients (median (interquartile range (IQR)): 402.5 (287.3-599.0) pg/mL), moderate in disease controls (median (IQR): 543.5 (413.0-676.3) pg/mL) and highest in healthy controls (median (IQR): 668.0 (506.5-841.3) pg/mL) (p < .001). Moreover, in CHD patients, lower CDC42 level was related to more prevalent diabetes mellitus (p = .021), and higher levels of C-reactive protein (p = .001), Gensini score (p = .006), Th17 cells (p = .001) and Th17/Treg ratio (p < .001) but was associated with lower Treg cells (p = .018). Furthermore, CDC42 low level [below the median level (402.5 pg/mL) of CDC42 in CHD patients] was correlated with higher accumulating major adverse cardiovascular event (MACE) risk (p = .029), while no correlation was found between the quartile of CDC42 level and accumulating MACE risk in CHD patients (p = .102). The serum CDC42 level is decreased and its low level is related to higher Th17/Treg ratio and increased accumulating MACE risk in CHD patients.

Pseudo-mono-axial ligand fields that support high energy barriers in triangular dodecahedral Dy(iii) single-ion magnets.

The synthesis of air-stable, high-performance single-molecule magnets (SMMs) is of great significance for their practical applications. Indeed, Ln complexes with high coordination numbers are satisfactorily air stable. However, such geometries easily produce spherical ligand fields that minimize magnetic anisotropy. Herein, we report the preparation of three air-stable eight-coordinate mononuclear Dy(iii) complexes with triangular dodecahedral geometries, namely, [Dy(BPA-TPA)Cl](BPh4)2 (1) and [Dy(BPA-TPA)(X)](BPh4)2·nCH2Cl2 (X = CH3O- and n = 1 for 2; L = PhO- and n = 2 for 3), using a novel design concept in which the bulky heptadentate [2,6-bis[bis(2-pyridylmethyl)amino]methyl]-pyridine (BPA-TPA) ligand enwraps the Dy(iii) ion through weak coordinate bonds leaving only a small vacancy for a negatively charged (Cl-), methoxy (CH3O-) or phenoxy (PhO-) moiety to occupy. Magnetic measurements reveal that the single-molecule magnet (SMM) property of complex 1 is actually poor, as there is almost no energy barrier. However, complexes 2 and 3 exhibit fascinating SMM behavior with high energy barriers (U eff = 686 K for 2; 469 K for 3) and magnetic hysteresis temperatures up to 8 K, which is attributed to the pseudolinear ligand field generated by one strong, highly electrostatic Dy-O bond. Ab initio calculations were used to show the apparent difference in the magnetic dynamics of the three complexes, confirming that the pseudo-mono-axial ligand field has an important effect on high-performance SMMs compared with the local symmetry. This study not only presents the highest energy barrier for a triangular dodecahedral SMM but also highlights the enormous potential of the pseudolinear Dy-L ligand field for constructing promising SMMs.

Tuning the Equatorial Negative Charge in Hexagonal Bipyramidal Dysprosium(III) Single-Ion Magnets to Improve the Magnetic Behavior.

Taking advantage of the pentaethylene glycol (EO5) and deprotonation of EO5, a family of new structurally hexagonal bipyramidal Dy(III) complexes, [Dy(EO5)(2,6-dichloro-4-nitro-PhO)2](2,6-dichloro-4-nitro-PhO) (1), [Dy(EO5-BPh2)(2,6-dichloro-4-nitro-PhO)2] (2), and [Dy(EO5-BPh2)(2,6-dichloro-4-nitro-PhO)Cl] (3), were controbllably synthesized and structurally characterized. Magnetic measurements show that complex 1 is a zero-field SIM and has an observable hysteresis opening up to 4 K. Conversely, only under extra magnetic field is slow magnetic relaxation observed in 2 and 3. This considerable difference in the magnetic behavior is mainly caused by the change of the equatorial negative charge. Detailed ab initio calculations further elucidate that the quantum tunneling is induced by the presence of equatorial negative charge, and the magnetic anisotropy depends on the axial ligands. This work demonstrates that the absence of the equatorial negative charge should also be considered in the rational design of promising single molecular magnets based on the oblate ions.

Determining ADCC Activity of Antibody-Based Therapeutic Molecules using Two Bioluminescent Reporter-Based Bioassays.

Antibody Fc effector function is one of the main mechanisms of action (MoA) for therapeutic monoclonal antibodies. Measurement of antibody-dependent cellular cytotoxicity (ADCC) is critical for understanding the Fc effector function during monoclonal antibody development. This article covers two cell-based ADCC bioassays which can quantitatively measure the antibody potency in ADCC. Basic Protocol 1 describes the ADCC reporter bioassay using engineered ADCC effector cells which measures the FcγRIIIa-mediated luciferase reporter activation upon the binding of antibody-coated target cells. Basic Protocol 2 describes the PBMC ADCC bioassay using primary peripheral blood mononuclear cells (PBMC) as effector cells and engineered HiBiT target cells in an assay that measures the release of HiBiT from target cells upon antibody-mediated target lysis. Optimization of several key assay parameters including cell handling, effector:target (E:T) ratios, assay plate, and plate reader requirement, and how these parameters impact assay performance are discussed. © 2021 Promega Corporation. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: ADCC reporter bioassay using engineered ADCC bioassay effector cells Basic Protocol 2: PBMC ADCC bioassay using primary PBMC and engineered HiBiT target cells.

Own-race faces capture attention faster than other-race faces: evidence from response time and the N2pc.

Studies have shown that people are better at recognizing human faces from their own-race than from other-races, an effect often termed the Own-Race Advantage. The current study investigates whether there is an Own-Race Advantage in attention and its neural correlates. Participants were asked to search for a human face among animal faces. Experiment 1 showed a classic Own-Race Advantage in response time both for Chinese and Black South African participants. Using event-related potentials (ERPs), Experiment 2 showed a similar Own-Race Advantage in response time for both upright faces and inverted faces. Moreover, the latency of N2pc for own-race faces was earlier than that for other-race faces. These results suggested that own-race faces capture attention more efficiently than other-race faces.

Development of a robust reporter-based ADCC assay with frozen, thaw-and-use cells to measure Fc effector function of therapeutic antibodies.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is one of the main mechanisms of action for many therapeutic antibodies. Classic ADCC assays measure antibody-dependent target cell cytotoxicity induced by primary effector cells that are isolated from human blood. They suffer from high assay variability due to the genetic and immune-status-mediated variation from blood donors. Here we report the development of a robust reporter-based ADCC assay that uses an engineered Jurkat stable cell line as the source of effector cells. These engineered effector cells were further developed as frozen, thaw-and-use format that can be plated for assay immediately after thaw. We demonstrate that frozen, thaw-and-use Jurkat effector cells showed appropriate Fc effector function similar to fresh cells from continuous culture, with added benefits of convenience and consistency. This robust assay is able to measure antibody potency for several therapeutic antibodies targeted to hematopoietic or solid tumors. The assay can distinguish effector functions for different antibody IgG isotypes in two antibody model systems: anti-CD20 and anti-EGFR. It is able to detect changes in ADCC biological activity for heat-stressed rituximab and trastuzumab, demonstrating that it possesses proper stability-indicting property. When compared with a classic PBMC-based ADCC assay, the ADCC reporter assay showed better assay precision and similar correlation of antibody glycosylation with ADCC biological activity for a panel of glyco-modified trastuzumab mixtures. Together these data demonstrate that this robust ADCC reporter assay meets the requirement of a potency bioassay that can quantify antibody Fc effector function in ADCC mechanism of action during drug discovery and development.

Characterization of in vitro antibody-dependent cell-mediated cytotoxicity activity of therapeutic antibodies - impact of effector cells.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action implicated in the clinical efficacy of several therapeutic antibodies. In vitro ADCC assays employing effector cells capable of inducing lysis of target cells bound by antibodies are routinely performed to support the research and development of therapeutic antibodies. ADCC assays are commonly performed using peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells or engineered cell lines as effector cells. In this study we evaluated the impact of different effector cell types including primary PBMCs, primary NK cells, engineered NK cell lines, and an engineered reporter cell line, on the in vitro ADCC activity of two glycoforms of a humanized IgG1 antibody. The results of this study show the differential effects on both the efficacy and potency of the antibodies by different effector cells and the finding that both the allotype and the expression level of CD16a affect the potency of effector cells in ADCC assays. Our results also show that engineered NK or reporter cell lines provide reduced variability compared to primary effector cells for in vitro ADCC assays.

Smaller holistic processing of faces associated with face drawing experience.

The type of experience involved with an object category has been regarded as one important factor in shaping of the human object recognition system. Laboratory training studies have shown that different kinds of learning experience with the same set of novel objects resulted in different perceptual and neural changes. Whether this applies to natural real-world objects remains to be seen. We compared two groups of observers who had different learning experiences with faces, using holistic processing as a dependent measure. We found that, while ordinary observers had extensive individuation experience with faces and displayed typical holistic face processing, art students who had acquired additional experience in drawing faces, and thus in attending to parts of a face, showed less holistic processing than did ordinary observers. These results converge with laboratory training studies on the role of type of experience in the development of different perceptual markers for different object categories. It is thus insufficient to categorize expertise simply in terms of object domains (e.g., expertise with faces). Instead, perceptual expertise should be classified in terms of the underlying process or task demand.

Co-regulation of caveolar and Cdc42-dependent fluid phase endocytosis by phosphocaveolin-1.

Several clathrin-independent endocytosis mechanisms have been identified that can be distinguished by specific requirements for certain proteins, such as caveolin-1 (Cav1) and the Rho GTPases, RhoA and Cdc42, as well as by specific cargo. Some endocytic pathways may be co-regulated such that disruption of one pathway leads to the up-regulation of another; however, the underlying mechanisms for this are unclear. Cav1 has been reported to function as a guanine nucleotide dissociation inhibitor (GDI), which inhibits Cdc42 activation. We tested the hypothesis that Cav1 can regulate Cdc42-dependent, fluid phase endocytosis. We demonstrate that Cav1 overexpression decreases fluid phase endocytosis, whereas silencing of Cav1 enhances this pathway. Enhancement of Cav1 phosphorylation using a phosphatase inhibitor reduces Cdc42-regulated pinocytosis while stimulating caveolar endocytosis. Fluid phase endocytosis was inhibited by expression of a putative phosphomimetic mutant, Cav1-Y14E, but not by the phospho-deficient mutant, Cav1-Y14F. Overexpression of Cav2, or a Cav1 mutant in which the GDI region was altered to the corresponding sequence in Cav2, did not suppress fluid phase endocytosis. These results suggest that the Cav1 expression level and phosphorylation state regulates fluid phase endocytosis via the interaction between the Cav1 GDI region and Cdc42. These data define a novel molecular mechanism for co-regulation of two distinct clathrin-independent endocytic pathways.

Stimulation of GLUT4 (glucose transporter isoform 4) storage vesicle formation by sphingolipid depletion.

Insulin stimulates glucose transport in fat and skeletal muscle cells primarily by inducing the translocation of GLUT4 (glucose transporter isoform 4) to the PM (plasma membrane) from specialized GSVs (GLUT4 storage vesicles). Glycosphingolipids are components of membrane microdomains and are involved in insulin-regulated glucose transport. Cellular glycosphingolipids decrease during adipocyte differentiation and have been suggested to be involved in adipocyte function. In the present study, we investigated the role of glycosphingolipids in regulating GLUT4 translocation. We decreased glycosphingolipids in 3T3-L1 adipocytes using glycosphingolipid synthesis inhibitors and investigated the effects on GLUT4 translocation using immunocytochemistry, preparation of PM sheets, isolation of GSVs and FRAP (fluorescence recovery after photobleaching) of GLUT4-GFP (green fluorescent protein) in intracellular structures. Glycosphingolipids were located in endosomal vesicles in pre-adipocytes and redistributed to the PM with decreased expression at day 2 after initiation of differentiation. In fully differentiated adipocytes, depletion of glycosphingolipids dramatically accelerated insulin-stimulated GLUT4 translocation. Although insulin-induced phosphorylation of IRS (insulin receptor substrate) and Akt remained intact in glycosphingolipid-depleted cells, both in vitro budding of GLUT4 vesicles and FRAP of GLUT4-GFP on GSVs were stimulated. Glycosphingolipid depletion also enhanced the insulin-induced translocation of VAMP2 (vesicle-associated membrane protein 2), but not the transferrin receptor or cellubrevin, indicating that the effect of glycosphingolipids was specific to VAMP2-positive GSVs. Our results strongly suggest that decreasing glycosphingolipid levels promotes the formation of GSVs and, thus, GLUT4 translocation. These studies provide a mechanistic basis for recent studies showing that inhibition of glycosphingolipid synthesis improves glycaemic control and enhances insulin sensitivity in animal models of Type 2 diabetes.

Luciferase Reporter Assay System for Deciphering GPCR Pathways.

The G protein coupled receptors (GPCR) represent the target class for nearly half of the current therapeutic drugs and remain to be the focus of drug discovery efforts. The complexity of receptor signaling continues to evolve. It is now known that many GPCRs are coupled to multiple G-proteins, which lead to regulation of respective signaling pathways downstream. Deciphering this receptor coupling will aid our understanding of the GPCR function and ultimately developing drug candidates. Here, we report the development of four homogenous bioluminescent reporter assays using improved destabilized luciferases and various response elements: CRE, NFAT-RE, SRE, and SRF-RE. These assays allowed measurement of major GPCR pathways including cAMP production, intracellular Ca(2+) mobilizations, ERK/MAPK activ-ity, and small G protein RhoA activity, respectively using the same reporter assay format. We showed that we can decipher G protein activation profiles for exogenous m(3) muscarinic receptor and endogenous ß(2)-adrenergic receptors in HEK293 cells by using these four reporter assays. Furthermore, we demonstrated that these assays can be readily used for potency rankings of agonists and antagonists, and for high throughput screening.

Inhibition of caveolar uptake, SV40 infection, and beta1-integrin signaling by a nonnatural glycosphingolipid stereoisomer.

Caveolar endocytosis is an important mechanism for the uptake of certain pathogens and toxins and also plays a role in the internalization of some plasma membrane (PM) lipids and proteins. However, the regulation of caveolar endocytosis is not well understood. We previously demonstrated that caveolar endocytosis and beta1-integrin signaling are stimulated by exogenous glycosphingolipids (GSLs). In this study, we show that a synthetic GSL with nonnatural stereochemistry, beta-D-lactosyl-N-octanoyl-L-threo-sphingosine, (1) selectively inhibits caveolar endocytosis and SV40 virus infection, (2) blocks the clustering of lipids and proteins into GSLs and cholesterol-enriched microdomains (rafts) at the PM, and (3) inhibits beta1-integrin activation and downstream signaling. Finally, we show that small interfering RNA knockdown of beta1 integrin in human skin fibroblasts blocks caveolar endocytosis and the stimulation of signaling by a GSL with natural stereochemistry. These experiments identify a new compound that can interfere with biological processes by inhibiting microdomain formation and also identify beta1 integrin as a potential mediator of signaling by GSLs.

Transmembrane segment peptides can disrupt cholecystokinin receptor oligomerization without affecting receptor function.

Oligomerization of the G protein-coupled cholecystokinin (CCK) receptor has been demonstrated, but its molecular basis and functional importance are not clear. We now examine contributions of transmembrane (TM) segments to oligomerization of this receptor using a peptide competitive inhibition strategy. Oligomerization of CCK receptors tagged at the carboxyl terminus with Renilla luciferase or yellow fluorescent protein was quantified using bioluminescence resonance energy transfer (BRET). Synthetic peptides representing TM I, II, V, VI, and VII of the CCK receptor were utilized as competitors. Of these, only TM VI and VII peptides disrupted receptor BRET. Control studies established that the beta2-adrenergic receptor TM VI peptide that disrupts oligomerization of that receptor had no effect on CCK receptor BRET. Notably, disruption of CCK receptor oligomerization had no effect on agonist binding, biological activity, or receptor internalization. To gain insight into the face of TM VI contributing to oligomerization, we utilized analogous peptides with alanines in positions 315, 319, and 323 (interhelical face) or 317, 321, and 325 (external lipid-exposed face). The Ala317,321,325 peptide eliminated the disruptive effect on CCK receptor BRET, whereas the other mutant peptide behaved like wild-type TM VI. This suggests that the lipid-exposed face of the CCK receptor TM VI most contributes to oligomerization and supports external contact dimerization of helical bundles, rather than domain-swapped dimerization. Fluorescent CCK receptor mutants with residues 317, 321, and 325 replaced with alanines were also prepared and failed to yield significant resonance transfer signals using either BRET or a morphological FRET assay, further supporting this interpretation.

Distinct mechanisms of clathrin-independent endocytosis have unique sphingolipid requirements.

Sphingolipids (SLs) play important roles in membrane structure and cell function. Here, we examine the SL requirements of various endocytic mechanisms using a mutant cell line and pharmacological inhibitors to disrupt SL biosynthesis. First, we demonstrated that in Chinese hamster ovary cells we could distinguish three distinct mechanisms of clathrin-independent endocytosis (caveolar, RhoA, and Cdc42 dependent) which differed in cargo, sensitivity to pharmacological agents, and dominant negative proteins. General depletion of SLs inhibited endocytosis by each clathrin-independent mechanism, whereas clathrin-dependent uptake was unaffected. Depletion of glycosphingolipids (GSLs; a subgroup of SLs) selectively blocked caveolar endocytosis and decreased caveolin-1 and caveolae at the plasma membrane. Caveolar endocytosis and PM caveolae could be restored in GSL-depleted cells by acute addition of exogenous GSLs. Disruption of RhoA- and Cdc42-regulated endocytosis by SL depletion was shown to be related to decreased targeting of these Rho proteins to the plasma membrane and could be partially restored by exogenous sphingomyelin but not GSLs. Both the in vivo membrane targeting and in vitro binding to artificial lipid vesicles of RhoA and Cdc42 were shown to be dependent upon sphingomyelin. These results provide the first evidence that SLs are differentially required for distinct mechanisms of clathrin-independent endocytosis.

Membrane microdomains, caveolae, and caveolar endocytosis of sphingolipids.

Caveolae are flask-shape membrane invaginations of the plasma membrane that have been implicated in endocytosis, transcytosis, and cell signaling. Recent years have witnessed the resurgence of studies on caveolae because they have been found to be involved in the uptake of some membrane components such as glycosphingolipids and integrins, as well as viruses, bacteria, and bacterial toxins. Accumulating evidence shows that endocytosis mediated by caveolae requires unique structural and signaling machinery (caveolin-1, src kinase), which indicates that caveolar endocytosis occurs through a mechanism which is distinct from other forms of lipid microdomain-associated, clathrin-independent endocytosis. Furthermore, a balance of glycosphingolipids, cholesterol, and caveolin-1 has been shown to be important in regulating caveolae endocytosis.

The glycosphingolipid, lactosylceramide, regulates beta1-integrin clustering and endocytosis.

Glycosphingolipids are known to play roles in integrin-mediated cell adhesion and migration; however, the mechanisms by which glycosphingolipids affect integrins are unknown. Here, we show that addition of the glycosphingolipid, C8-lactosylceramide (C8-LacCer), or free cholesterol to human fibroblasts at 10 degrees C causes the formation of glycosphingolipid-enriched plasma membrane domains as shown by visualizing a fluorescent glycosphingolipid probe, BODIPY-LacCer, incorporated into the plasma membrane of living cells. Addition of C8-LacCer or cholesterol to cells initiated the clustering of beta1-integrins within these glycosphingolipid-enriched domains and the activation of the beta1-integrins as assessed using a HUTS antibody that only binds activated integrin. On warming to 37 degrees C, beta1-integrins were rapidly internalized via caveolar endocytosis in cells treated with C8-LacCer or cholesterol, whereas little beta1-integrin was endocytosed in untreated fibroblasts. Incubation of cells with C8-LacCer or cholesterol followed by warm-up caused src activation, a reorganization of the actin cytoskeleton, translocation of RhoA GTPase away from the plasma membrane as visualized using total internal reflection fluorescence microscopy, and transient cell detachment. These studies show that LacCer can regulate integrin function both by modulating integrin clustering in microdomains and by regulating integrin endocytosis via caveolae. Our findings suggest the possibility that aberrant levels of glycosphingolipids found in cancer cells may influence cell attachment events by direct effects on integrin clustering and internalization.

Analysis of the cellular and molecular mechanisms of trophic action of a misspliced form of the type B cholecystokinin receptor present in colon and pancreatic cancer.

Gastrin and cholecystokinin (CCK) have trophic action on cells expressing wild type A or B CCK receptors. Potential relevance to pancreatic and colonic cancers was raised by the demonstration of a misspliced type B CCK receptor that, when expressed in Balb3T3 cells, had constitutive activity to stimulate intracellular calcium. We attempted to confirm and extend this observation in CHO cells by establishing lines expressing similar densities of variant or wild type B CCK receptor. While both were capable of normal binding and agonist-induced signaling, neither expressed constitutive signaling and both had similar basal growth. Agonist stimulation of cells expressing misspliced receptor had greater increases in calcium and greater growth rates than control cells despite similar MAP kinase phosphorylation responses. Thus, this variant receptor can potentiate peptide-stimulated signaling and trophic action and may contribute to the proliferation of neoplasms expressing it.

HIV Rev self-assembly is linked to a molten-globule to compact structural transition.

By regulating the differential expression of proviral pre mRNA in the host cell, Rev plays a crucial role in the HIV-1 life cycle. The capacity of Rev to function is intimately linked to its ability to self-associate. Nevertheless, little is known about the exact role of self-association in the molecular mechanism defining its biological activity. A prerequisite knowledge is a definition of the molecular events undertaken by Rev during the process of self-assembly. Thus, this study was initiated to monitor the structure of Rev as a function of protein concentration. Rev undergoes a structural transition as a consequence of self-assembly. This structural transition was monitored by three spectroscopic methods. The accessibility of the single tryptophan in Rev monomer to acrylamide quenching increases with decreasing protein concentration. At very low concentration of Rev, the tryptophan accessibility is close to that of an unfolded Rev. As evaluated by circular dichroism, the secondary structure of Rev is protein concentration dependent as evidenced by an increase in the magnitude of ellipticity with increasing protein concentration. Further, results from ANS binding studies indicate that the ANS binding sites in Rev experience an apparent increase in hydrophobicity as the Rev concentration was increased. These concentration dependent changes seem to reach a maximum above 5 microM Rev monomer concentration. In order to define the mode of Rev self-association sedimentation velocity and equilibrium experiments were conducted. There are evidently two consecutive progressive association processes. At protein concentrations below 0.5 mg/ml, the data from sedimentation studies can be fitted to a single isodesmic model. Simulation of velocity sedimentation profile indicates that free Rev monomer that has not entered into the association processes can best be described to exhibit a value of S(20,w) that is substantially smaller than 1.4 S, a value needed to fit the rest of the data. The latter value is consistent for a Rev monomer with the expected molecules weight and if it were to assume a compact globular shape. These spectroscopic and hydrodynamic results imply that monomeric Rev is in a molten globule state, which becomes more compact upon self-association.