Helen McBride

ABP 798 is a biosimilar to Rituxan® (rituximab reference product [RP]). Non-clinical assessments relevant to the primary and secondary mechanisms of action (MOA) contribute to the totality of the evidence (TOE) in supporting biosimilarity and are critical in providing scientific evidence for extrapolation of indications. Similarity of ABP 798 with rituximab RP was investigated across a range of biological activities which have potential impact on pharmacokinetics and clinical efficacy with… Show more

ABP 798 is a biosimilar to Rituxan® (rituximab reference product [RP]). Non-clinical assessments relevant to the primary and secondary mechanisms of action (MOA) contribute to the totality of the evidence (TOE) in supporting biosimilarity and are critical in providing scientific evidence for extrapolation of indications. Similarity of ABP 798 with rituximab RP was investigated across a range of biological activities which have potential impact on pharmacokinetics and clinical efficacy with non-clinical assessments relevant to MOA such as CD20 internalization, trogocytosis, binding to primary human natural killer (NK) cells as well as the ability to induce antibody-dependent cellular phagocytosis (ADCP) in peripheral blood mononuclear cells. Additionally, in vitro synergy of ABP 798 or RP with chemotherapeutic agents, in vivo xenograft studies in mice, and toxicological assessments in cynomolgus monkeys (including B cell depletion and toxicokinetics) were also conducted. Results from these non-clinical assessments contribute to the TOE supporting the biosimilarity between ABP 798 and rituximab RP across a range of primary and secondary MOAs and support justification for extrapolation to all indications of use for ABP 798 for which the RP is approved. Show less

The assessments in our nonclinical animal studies showed no unexpected safety or toxicity findings for either the biosimilar candidate or the respective RP; in each case, the in vivo PK/PD, toxicology, and toxicokinetics findings were similar. In conclusion, to date in vivo animal studies of proposed bio-similars developed/under development at Amgen have provided little information to meaningfully inform clinical efficacy, safety, or immunogenicity. For some biosimilars, the value of… Show more

The assessments in our nonclinical animal studies showed no unexpected safety or toxicity findings for either the biosimilar candidate or the respective RP; in each case, the in vivo PK/PD, toxicology, and toxicokinetics findings were similar. In conclusion, to date in vivo animal studies of proposed bio-similars developed/under development at Amgen have provided little information to meaningfully inform clinical efficacy, safety, or immunogenicity. For some biosimilars, the value of conducting such studies in the future may be questionable. Comparative clinical studies between a proposed biosimilar and RP are the best way to confirm similarity in PK/ PD, efficacy, safety, and immunogenicity. Show less

Antibody-based therapeutics targeting membrane proteins have evolved as a major modality for the treatment of cancer, inflammation and autoimmune diseases. There are numerous challenges, ranging from desired epitope expression to reliable binding/functional assays which are associated with developing antibodies for this target class. Specifically, having a robust methodology for characterizing antibody interaction with a membrane protein target is essential for providing guidance on dosing… Show more

Antibody-based therapeutics targeting membrane proteins have evolved as a major modality for the treatment of cancer, inflammation and autoimmune diseases. There are numerous challenges, ranging from desired epitope expression to reliable binding/functional assays which are associated with developing antibodies for this target class. Specifically, having a robust methodology for characterizing antibody interaction with a membrane protein target is essential for providing guidance on dosing, potency and thus expected efficacy. Fluorescence-activated cell sorting (FACS) has been commonly used to characterize antibodies binding to membrane protein targets. FACS provides information about the antibody-receptor complex (antibody bound to cells) and the apparent equilibrium dissociation constant () is elucidated by fitting the antibody-receptor binding isotherm as a function of total antibody concentration to a nonlinear regression model. Conversely, Kinetic Exclusion Assay (KinExA) has been used to measure solution-based equilibrium dissociation constant (KD) of antibodies. Here, KD is determined by measuring the free antibody concentration at equilibrium in a series of solutions in which the antibody is at constant concentration and the receptor (either in the membrane or the cell) is titrated. We measured the binding affinity of the anti-CD20 antibody, Rituximab, using both FACS and KinExA. There was ~25-fold difference in the binding affinity measured by these two techniques. We have explored this discrepancy through additional experiments around the mathematical framework involved in the analysis of these two different binding assays. Finally, our study concluded that KinExA enables accurate measurement of the KD for strong protein-protein interactions (sub-nanomolar values) compared to FACS. Show less

Neutrophils are essential for host defense against pathogens. Although calcium signals direct numerous cell functions, the molecular machinery that orchestrates calcium influx into neutrophils is poorly defined. We demonstrate that ORAI1 and ORAI2 are key components of the mouse neutrophil calcium release-activated calcium channel and are essential for neutrophil bactericidal function. We find that neutrophils separate into two populations with distinct regulation of the membrane potential… Show more

Neutrophils are essential for host defense against pathogens. Although calcium signals direct numerous cell functions, the molecular machinery that orchestrates calcium influx into neutrophils is poorly defined. We demonstrate that ORAI1 and ORAI2 are key components of the mouse neutrophil calcium release-activated calcium channel and are essential for neutrophil bactericidal function. We find that neutrophils separate into two populations with distinct regulation of the membrane potential during calcium influx, which influences the magnitude of SOCE and the effect of ORAI isoform deficiency. These findings advance understanding of the mechanics of neutrophil calcium signaling, identify neutrophil subpopulations where the cell-membrane potential modulates the calcium response, and suggest mechanisms that regulate neutrophil function during infection and inflammation. Show less

Purpose: ABP 710 has been developed as a biosimilar to infliximab reference product (RP). The objective of this study was to assess analytical similarity (structural and functional) between ABP 710 and infliximab RP licensed by the United States Food and Drug Administration (infliximab [US]) and the European Union (infliximab [EU]), using sensitive, state-of-the-art analytical methods capable of detecting minor differences in product quality attributes.
Methods: Comprehensive analytical… Show more

Purpose: ABP 710 has been developed as a biosimilar to infliximab reference product (RP). The objective of this study was to assess analytical similarity (structural and functional) between ABP 710 and infliximab RP licensed by the United States Food and Drug Administration (infliximab [US]) and the European Union (infliximab [EU]), using sensitive, state-of-the-art analytical methods capable of detecting minor differences in product quality attributes.
Methods: Comprehensive analytical characterization utilizing orthogonal techniques was performed with 14 to 28 unique lots of ABP 710 or infliximab RP, depending on the assay. Comparisons were used to investigate the primary structure related to amino acid sequence; post-translational modifications (PTMs) including glycans; higher order structure; particles and aggregates; primary biological properties mediated by target and receptor binding; product-related substances and impurities; and general properties.
Results: ABP 710 had the same amino acid sequence, primary structure, higher order structure, PTM profiles and biological activities as infliximab RP. The finished drug product had the same strength (protein content and concentration) as infliximab RP.
Conclusions: Based on the comprehensive analytical similarity assessment, ABP 710 was found to be highly analytically similar to infliximab RP for all biological activities relevant for clinical efficacy and safety.
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Purpose

The in vitro and in vivo pharmacologic assessment of ABP 980 similarity to its reference product is intended to compare the activity of ABP 980 and trastuzumab and support the overall conclusion of similarity based on a comprehensive analytical and functional evaluation.
Methods

This work complements the primary assessment of functional similarity with additional in vitro assays, binding studies, and non-clinical studies including human epidermal growth factor… Show more

Purpose

The in vitro and in vivo pharmacologic assessment of ABP 980 similarity to its reference product is intended to compare the activity of ABP 980 and trastuzumab and support the overall conclusion of similarity based on a comprehensive analytical and functional evaluation.
Methods

This work complements the primary assessment of functional similarity with additional in vitro assays, binding studies, and non-clinical studies including human epidermal growth factor receptor-2 (HER2) kinetic binding, HER2 signaling, HER2 internalization, synergy with docetaxel chemotherapy, FcγR kinetic binding, primary natural killer and monocyte cell binding, antibody-dependent cellular phagocytosis activity, in vivo xenograft studies, and toxicokinetic parameters.
Results

The results contribute to the totality of evidence with respect to functional similarity and support that ABP 980 is similar to trastuzumab in all primary and secondary mechanisms of action.
Conclusions

These results also support the scientific justification of extrapolation to all approved indications of trastuzumab given the established functional similarity of the two products and the same mechanisms of action across all conditions of use. Show less

ABP 215 (MVASI™) is the first approved biosimilar to Avastin® (bevacizumab). It is approved in the USA and the European Union (EU) for all bevacizumab indications in these jurisdictions except for ovarian cancer in the USA due to orphan drug exclusivity. ABP 215 was shown to be structurally, functionally and clinically (pharmacokinetic, efficacy and safety) similar to the bevacizumab reference product; the pharmacokinetic study was conducted in healthy adult men (n = 202); safety and efficacy… Show more

ABP 215 (MVASI™) is the first approved biosimilar to Avastin® (bevacizumab). It is approved in the USA and the European Union (EU) for all bevacizumab indications in these jurisdictions except for ovarian cancer in the USA due to orphan drug exclusivity. ABP 215 was shown to be structurally, functionally and clinically (pharmacokinetic, efficacy and safety) similar to the bevacizumab reference product; the pharmacokinetic study was conducted in healthy adult men (n = 202); safety and efficacy were evaluated in patients with advanced nonsquamous non-small-cell lung cancer (n = 642). Together, these results comprise the totality of evidence that provides scientific justification for extrapolation to all approved indications of the reference product and supports the clinical value of ABP 215 as an additional treatment option. Show less

ABP 501 [United States: AMJEVITA™ (adalimumab-atto); European Union: AMGEVITA® (adalimumab)] is the first approved biosimilar to adalimumab [reference product (RP)], a monoclonal antibody (mAb) targeting tumor necrosis factor-alfa (TNF-α). ABP 501 has received approval for use in indications that adalimumab RP is approved for, except those protected by regulatory exclusivity. A systematic step-wise totality of evidence (TOE) approach formed the basis of approval of ABP 501; this involved… Show more

ABP 501 [United States: AMJEVITA™ (adalimumab-atto); European Union: AMGEVITA® (adalimumab)] is the first approved biosimilar to adalimumab [reference product (RP)], a monoclonal antibody (mAb) targeting tumor necrosis factor-alfa (TNF-α). ABP 501 has received approval for use in indications that adalimumab RP is approved for, except those protected by regulatory exclusivity. A systematic step-wise totality of evidence (TOE) approach formed the basis of approval of ABP 501; this involved methodical accumulation of scientifically robust comparative data supporting similarity in analytical, preclinical, and clinical [pharmacokinetics (PK)], efficacy, safety and immunogenicity) evaluations. As a foundational first step, comprehensive analytical assessments demonstrated that ABP 501 is structurally and functionally similar to adalimumab RP in critical quality attributes. Preclinical assessments confirmed similar activity in assessing mechanisms of action and toxicology. Clinical evaluation included a phase 1 PK equivalence study in healthy subjects and two comparative phase 3 studies that evaluated ABP 501 and adalimumab RP in two sensitive patient populations, plaque psoriasis (PsO) and rheumatoid arthritis (RA). The PK profiles of ABP 501 and adalimumab RP were similar in healthy subjects as well as patients with PsO and RA. The pivotal phase 3 study in patients with PsO demonstrated that ABP 501 was clinically similar to adalimumab RP in terms of efficacy, safety and immunogenicity in both the primary and transition phases. The pivotal phase 3 study in patients with RA also established clinical similarity between ABP 501 and adalimumab RP; an open-label extension of this study demonstrated sustained efficacy over an additional 72 weeks, with no new safety or immunogenicity concerns with ABP 501 treatment. Show less

Trastuzumab and pertuzumab are monoclonal antibodies that bind to distinct subdomains of the extracellular domain of human epidermal growth factor receptor 2 (HER2). Adding these monoclonal antibodies to the treatment regimen of HER2-positive breast cancer has changed the paradigm for treatment in that form of cancer. Synergistic activity has been observed with the combination of these two antibodies leading to hypotheses regarding the mechanism(s) and to the development of bispecific… Show more

Trastuzumab and pertuzumab are monoclonal antibodies that bind to distinct subdomains of the extracellular domain of human epidermal growth factor receptor 2 (HER2). Adding these monoclonal antibodies to the treatment regimen of HER2-positive breast cancer has changed the paradigm for treatment in that form of cancer. Synergistic activity has been observed with the combination of these two antibodies leading to hypotheses regarding the mechanism(s) and to the development of bispecific antibodies to maximize the clinical effect further. Although the individual crystal structures of HER2-trastuzumab and HER2-pertuzumab revealed the distinct binding sites and provided the structural basis for their anti-tumor activities, detailed structural information on the HER2-trastuzumab-pertuzumab complex has been elusive. Here we present the cryo-EM structure of HER2-trastuzumab-pertuzumab at 4.36 Å resolution. Comparison with the binary complexes reveals no cooperative interaction between trastuzumab and pertuzumab, and provides key insights into the design of novel, high-avidity bispecific molecules with potentially greater clinical efficacy. Show less

ntibody therapeutics with poor solubility in the subcutaneous matrix may carry unintended risks when administered to patients. The objective of this work was to estimate the risk of antibodies that precipitate in vitro at neutral pH by determining the impact of poor solubility on distribution of the drug from the injection site as well as immunogenicity in vivo. Using fluorescence imaging in a mouse model, we show that one such precipitation-prone antibody is retained at the injection site in… Show more

ntibody therapeutics with poor solubility in the subcutaneous matrix may carry unintended risks when administered to patients. The objective of this work was to estimate the risk of antibodies that precipitate in vitro at neutral pH by determining the impact of poor solubility on distribution of the drug from the injection site as well as immunogenicity in vivo. Using fluorescence imaging in a mouse model, we show that one such precipitation-prone antibody is retained at the injection site in the subcutaneous space longer than a control antibody. In addition, we demonstrate that retention at the injection site through aggregation is concentration-dependent and leads to macrophage association and germinal center localization. Although there was delayed disposition of the aggregated antibody to draining lymph nodes, no overall impact on the immune response in lymph nodes, systemic exposure of the antibody, or enhancement of the anti-drug antibody response was evident. Unexpectedly, retention of the precipitated antibody in the subcutaneous space delayed the onset of the immune response and led to an immune suppressive response. Thus, we conclude that precipitation due to poor solubility of high doses of antibody formulations delivered subcutaneously may not be of special concern in terms of exposure or immunogenicity. Show less

ABP 215 is a biosimilar product to bevacizumab. Bevacizumab acts by binding to vascular endothelial growth factor A, inhibiting endothelial cell proliferation and new blood vessel formation, thereby leading to tumor vasculature normalization. The ABP 215 analytical similarity assessment was designed to assess the structural and functional similarity of ABP 215 and bevacizumab sourced from both the United States (US) and the European Union (EU). Similarity assessment was also made between the… Show more

ABP 215 is a biosimilar product to bevacizumab. Bevacizumab acts by binding to vascular endothelial growth factor A, inhibiting endothelial cell proliferation and new blood vessel formation, thereby leading to tumor vasculature normalization. The ABP 215 analytical similarity assessment was designed to assess the structural and functional similarity of ABP 215 and bevacizumab sourced from both the United States (US) and the European Union (EU). Similarity assessment was also made between the US- and EU-sourced bevacizumab to assess the similarity between the two products. The physicochemical properties and structural similarity of ABP 215 and bevacizumab were characterized using sensitive state-of-the-art analytical techniques capable of detecting small differences in product attributes. ABP 215 has the same amino acid sequence and exhibits similar post-translational modification profiles compared to bevacizumab. The functional similarity assessment employed orthogonal assays designed to interrogate all expected biological activities, including those known to affect the mechanisms of action for ABP 215 and bevacizumab. More than 20 batches of bevacizumab (US) and bevacizumab (EU), and 13 batches of ABP 215 representing unique drug substance lots were assessed for similarity. The large dataset allows meaningful comparisons and garners confidence in the overall conclusion for the analytical similarity assessment of ABP 215 to both US- and EU-sourced bevacizumab. The structural and purity attributes, and biological properties of ABP 215 are demonstrated to be highly similar to those of bevacizumab. Show less

The eyes may be the windows to the soul, but a window into the bone—specifically bone marrow—would be useful for studying bone development and disease. Greenbaum et al. developed a method of whole-bone optical clearing, using a series of reagents under continuous flow to delipidate and decalcify bone tissue. This process renders the entire bone transparent but does not affect endogenous fluorescence, making this compatible with reporter mice. Using light sheet fluorescence microscopy, the… Show more

The eyes may be the windows to the soul, but a window into the bone—specifically bone marrow—would be useful for studying bone development and disease. Greenbaum et al. developed a method of whole-bone optical clearing, using a series of reagents under continuous flow to delipidate and decalcify bone tissue. This process renders the entire bone transparent but does not affect endogenous fluorescence, making this compatible with reporter mice. Using light sheet fluorescence microscopy, the authors counted and mapped the number of fluorescently labeled osteoprogenitors within cleared mouse tibia, vertebral column, and femur bones treated with sclerostin antibody. With reduced variability compared to standard section analysis, this Bone CLARITY and computational analysis will be a useful tool for bone research. Show less

Designing drugs to treat diseases associated with articular joints, particularly those targeting chondrocytes, is challenging due to unique local environmental constraints including the avascular nature of cartilage, the absence of a closed joint compartment, and a highly cross‐linked extracellular matrix. In an effort to address these challenges, we developed a novel strategy to prolong residence time of intra‐articularly administered protein therapeutics. Avimer domains are naturally found in… Show more

Designing drugs to treat diseases associated with articular joints, particularly those targeting chondrocytes, is challenging due to unique local environmental constraints including the avascular nature of cartilage, the absence of a closed joint compartment, and a highly cross‐linked extracellular matrix. In an effort to address these challenges, we developed a novel strategy to prolong residence time of intra‐articularly administered protein therapeutics. Avimer domains are naturally found in membrane polypeptides and mediate diverse protein–protein interactions. Screening of a phage Avimer domain library led to identification of several low affinity type II collagen‐binding Avimers. Following several rounds of mutagenesis and reselection, these initial hits were transformed to high affinity, selective type II collagen‐binding Avimers. One such Avimer (M26) persisted in rat knees for at least 1 month following intra‐articular administration. Fusion of this Avimer to a candidate therapeutic payload, IL‐1Ra, yielded a protein construct which simultaneously bound to type II collagen and to IL‐1 receptor. In vitro, IL‐1Ra_M26 bound selectively to cartilage explants and remained associated even after extensive washing. Binding appeared to occur preferentially to pericellular regions surrounding chondrocytes. An acute intra‐articular IL‐1‐induced IL‐6 challenge rat model was employed to assess in vivo pharmacodynamics. Whereas both IL‐1Ra_M26 and native IL‐1Ra inhibited IL‐6 output when co‐administered with the IL‐1 challenge, only IL‐1Ra_M26 inhibited when administered 1 week prior to IL‐1 challenge. Collagen‐binding Avimers thus represent a promising strategy for enhancing cartilage residence time of protein therapeutics. Show less

Traditional in vitro human liver cell culture models lose key hepatic functions such as metabolic activity during short-term culture. Advanced 3D liver co-culture platforms offer the potential for extended hepatocyte functionality, allowing for the study of more complex biological interactions that can improve and refine human drug safety evaluation. Here, we utilize a perfusion flow 3D microreactor platform for the co-culture of cryopreserved primary human hepatocytes and Kupffer cells to… Show more

Traditional in vitro human liver cell culture models lose key hepatic functions such as metabolic activity during short-term culture. Advanced 3D liver co-culture platforms offer the potential for extended hepatocyte functionality, allowing for the study of more complex biological interactions that can improve and refine human drug safety evaluation. Here, we utilize a perfusion flow 3D microreactor platform for the co-culture of cryopreserved primary human hepatocytes and Kupffer cells to study the regulation of CYP3A4 activity by chronic IL-6-mediated inflammation over two weeks. Hepatocyte cultures remained stable over two weeks, with consistent albumin production and basal IL-6 levels. Direct IL-6 stimulation that mimics an inflammatory state induced a dose-dependent suppression of CYP3A4 activity, an increase in C-reactive protein (CRP) secretion, and a decrease in shed soluble IL-6R levels, indicating expected hepatic IL-6 bioactivity. Tocilizumab, an anti-IL-6R monoclonal antibody used to treat rheumatoid arthritis, has been demonstrated clinically to impact small molecule drug pharmacokinetics by modulating cytochrome P450 enzyme activities, an effect not observed in traditional hepatic cultures. We have now recapitulated the clinical observation in a 3D bioreactor system. Tocilizumab was shown to de-suppress CYP3A4 activity while reducing CRP concentration after 72 hours in the continued presence of IL-6. This change in CYP3A4 activity decreased the half-life and AUClast of the small molecule CYP3A4 substrate simvastatin hydroxy acid, measured before and after tocilizumab treatment. We conclude that next-generation in vitro liver culture platforms are well-suited for these types of long-term treatment studies and show promise for improved drug safety assessment. Show less

Orai1 is a transmembrane protein that forms homomeric, calcium-selective channels activated by stromal interaction molecule 1 (STIM1) after depletion of intracellular calcium stores. In adult skeletal muscle, depletion of sarcoplasmic reticulum calcium activates STIM1/Orai1-dependent store-operated calcium entry. Here, we used constitutive and inducible muscle-specific Orai1-knockout (KO) mice to determine the acute and long-term developmental effects of Orai1 ablation on muscle structure and… Show more

Orai1 is a transmembrane protein that forms homomeric, calcium-selective channels activated by stromal interaction molecule 1 (STIM1) after depletion of intracellular calcium stores. In adult skeletal muscle, depletion of sarcoplasmic reticulum calcium activates STIM1/Orai1-dependent store-operated calcium entry. Here, we used constitutive and inducible muscle-specific Orai1-knockout (KO) mice to determine the acute and long-term developmental effects of Orai1 ablation on muscle structure and function. Skeletal muscles from constitutive, muscle-specific Orai-KO mice exhibited normal postnatal growth and fiber type differentiation. However, a significant reduction in fiber cross-sectional area occurred by 3 mo of age, with the most profound reduction observed in oxidative, fatigue-resistant fiber types. Soleus muscles of constitutive Orai-KO mice exhibited a reduction in unique type I fibers, concomitant with an increase in hybrid fibers expressing both type I and type IIA myosins. Additionally, ex vivo force measurements showed reduced maximal specific force and in vivo exercise assays revealed reduced endurance in constitutive muscle-specific Orai-KO mice. Using tamoxifen-inducible, muscle-specific Orai-KO mice, these functional deficits were found to be the result of the delayed fiber changes resulting from an early developmental loss of Orai1 and not the result of an acute loss of Orai1-dependent store-operated calcium entry. Show less

The function of CD4+ T cells is dependent on Ca2+ influx through Ca2+ release–activated Ca2+ (CRAC) channels formed by ORAI proteins. To investigate the role of ORAI1 in proinflammatory Th1 and Th17 cells and autoimmune diseases, we genetically and pharmacologically modulated ORAI1 function. Immunization of mice lacking Orai1 in T cells with MOG peptide resulted in attenuated severity of experimental autoimmune encephalomyelitis (EAE). The numbers of T cells and innate immune cells in the CNS… Show more

The function of CD4+ T cells is dependent on Ca2+ influx through Ca2+ release–activated Ca2+ (CRAC) channels formed by ORAI proteins. To investigate the role of ORAI1 in proinflammatory Th1 and Th17 cells and autoimmune diseases, we genetically and pharmacologically modulated ORAI1 function. Immunization of mice lacking Orai1 in T cells with MOG peptide resulted in attenuated severity of experimental autoimmune encephalomyelitis (EAE). The numbers of T cells and innate immune cells in the CNS of ORAI1-deficient animals were strongly reduced along with almost completely abolished production of IL-17A, IFN-γ, and GM-CSF despite only partially reduced Ca2+ influx. In Th1 and Th17 cells differentiated in vitro, ORAI1 was required for cytokine production but not the expression of Th1- and Th17-specific transcription factors T-bet and RORγt. The differentiation and function of induced regulatory T cells, by contrast, was independent of ORAI1. Importantly, induced genetic deletion of Orai1 in adoptively transferred, MOG-specific T cells was able to halt EAE progression after disease onset. Likewise, treatment of wild-type mice with a selective CRAC channel inhibitor after EAE onset ameliorated disease. Genetic deletion of Orai1 and pharmacological ORAI1 inhibition reduced the leukocyte numbers in the CNS and attenuated Th1/Th17 cell-mediated cytokine production. In human CD4+ T cells, CRAC channel inhibition reduced the expression of IL-17A, IFN-γ, and other cytokines in a dose-dependent manner. Taken together, these findings support the conclusion that Th1 and Th17 cell function is particularly dependent on CRAC channels, which could be exploited as a therapeutic approach to T cell–mediated autoimmune diseases. Show less

ORAI1 is the pore-forming component of calcium release-activated calcium (CRAC) channels. CRAC channels are the primary route for calcium ion (Ca2+) entry into T-cells following antigen stimulation. This Ca2+ entry induces proliferation and cytokine production through activation of calcineurin and the nuclear factor of activated T-cells (NFAT) transcription factor along with subsequent cytokine-related genes. It was hypothesized that the in vivo inhibition of T-cell function by blocking ORAI1… Show more

ORAI1 is the pore-forming component of calcium release-activated calcium (CRAC) channels. CRAC channels are the primary route for calcium ion (Ca2+) entry into T-cells following antigen stimulation. This Ca2+ entry induces proliferation and cytokine production through activation of calcineurin and the nuclear factor of activated T-cells (NFAT) transcription factor along with subsequent cytokine-related genes. It was hypothesized that the in vivo inhibition of T-cell function by blocking ORAI1 or calcineurin would lead to similar functional consequences. To test this hypothesis the activity of 2C1.1, a fully human anti-ORAI1 monoclonal antibody, and cyclosporin A (CsA) were tested in vivo for their suppressive effect on T-cell-derived cytokine production and a T-cell-dependent antibody response (TDAR) using sheep red blood cells (SRBC) in cynomolgus monkeys. Despite showing similar inhibition of ex vivo interleukin (IL)-2 production by stimulated T-cells, both molecules exhibited different pharmacologic effects on the SRBC antibody response. CsA blocked the development of SRBC-specific antibodies, while 2C1.1 failed to inhibit the antigen-specific antibody response. These surprising observations suggest that full inhibition of the CRAC channel is required to inhibit a functional immune response, consistent with findings from human patients with loss of function mutations in ORAI1. Show less

We determined the expression of ORAI1 protein in rodent and non-rodent tissues using a monoclonal antibody directed against an extracellular loop of the protein. Previous reports using antibodies directed at the C-terminus of ORAI1 have not detected central nervous system (CNS) expression. Our results demonstrate broad tissue expression that includes the CNS using a unique monoclonal antibody specific to an extracellular loop of ORAI1. In addition, we present in situ hybridization (ISH) results… Show more

We determined the expression of ORAI1 protein in rodent and non-rodent tissues using a monoclonal antibody directed against an extracellular loop of the protein. Previous reports using antibodies directed at the C-terminus of ORAI1 have not detected central nervous system (CNS) expression. Our results demonstrate broad tissue expression that includes the CNS using a unique monoclonal antibody specific to an extracellular loop of ORAI1. In addition, we present in situ hybridization (ISH) results using a probe within the middle of the mouse coding region showing CNS expression of Orai1 RNA. We contrast the patterns of rodent and human tissue expression and conclude that rodents have similar expression of ORAI1 in most tissue types when compared to primates, with an important exception being the male reproductive system, where human-specific expression is observed. Show less

alcium signals regulate many critical processes during vertebrate brain development including neurogenesis, neurotransmitter specification, and axonal outgrowth. However, the identity of the ion channels mediating Ca2+ signaling in the developing nervous system is not well defined. Here, we report that embryonic and adult mouse neural stem/progenitor cells (NSCs/NPCs) exhibit store-operated Ca2+ entry (SOCE) mediated by Ca2+ release-activated Ca2+ (CRAC) channels. SOCE in NPCs was blocked by… Show more

alcium signals regulate many critical processes during vertebrate brain development including neurogenesis, neurotransmitter specification, and axonal outgrowth. However, the identity of the ion channels mediating Ca2+ signaling in the developing nervous system is not well defined. Here, we report that embryonic and adult mouse neural stem/progenitor cells (NSCs/NPCs) exhibit store-operated Ca2+ entry (SOCE) mediated by Ca2+ release-activated Ca2+ (CRAC) channels. SOCE in NPCs was blocked by the CRAC channel inhibitors La3+, BTP2, and 2-APB and Western blots revealed the presence of the canonical CRAC channel proteins STIM1 and Orai1. Knock down of STIM1 or Orai1 significantly diminished SOCE in NPCs, and SOCE was lost in NPCs from transgenic mice lacking Orai1 or STIM1 and in knock-in mice expressing the loss-of-function Orai1 mutant, R93W. Therefore, STIM1 and Orai1 make essential contributions to SOCE in NPCs. SOCE in NPCs was activated by epidermal growth factor and acetylcholine, the latter occurring through muscarinic receptors. Activation of SOCE stimulated gene transcription through calcineurin/NFAT (nuclear factor of activated T cells) signaling through a mechanism consistent with local Ca2+ signaling by Ca2+ microdomains near CRAC channels. Importantly, suppression or deletion of STIM1 and Orai1 expression significantly attenuated proliferation of embryonic and adult NPCs cultured as neurospheres and, in vivo, in the subventricular zone of adult mice. These findings show that CRAC channels serve as a major route of Ca2+ entry in NPCs and regulate key effector functions including gene expression and proliferation, indicating that CRAC channels are important regulators of mammalian neurogenesis. Show less

Calcium entry into T cells following antigen stimulation is crucial for nuclear factor of
activated T-cells (NFAT) mediated T cell activation. The movement of calcium is
mediated by calcium release-activated calcium (CRAC) channels. There are two key
components of this channel; Orai1 is the pore-forming subunit located in the plasma
membrane and stromal interaction molecule 1 (STIM1) functions as a Ca2+ sensor in the
endoplasmic reticulum. A subset of human patients carry… Show more

Calcium entry into T cells following antigen stimulation is crucial for nuclear factor of
activated T-cells (NFAT) mediated T cell activation. The movement of calcium is
mediated by calcium release-activated calcium (CRAC) channels. There are two key
components of this channel; Orai1 is the pore-forming subunit located in the plasma
membrane and stromal interaction molecule 1 (STIM1) functions as a Ca2+ sensor in the
endoplasmic reticulum. A subset of human patients carry mutations in either STIM1 or
Orai1 that affect protein function or expression resulting in defective store-operated
Ca2+ influx and CRAC channel function, and impaired T cell activation. These patients
suffer from a hereditary form of severe combined immune deficiency (SCID) syndrome,
highlighting the importance of the CRAC channel for T lymphocyte function in humans.
Since autoreactive T cells play an important role in the development of autoimmune
diseases like rheumatoid arthritis, multiple sclerosis and organ transplantation, Orai1
becomes an attractive therapeutic target for ameliorating autoimmune disease. We
developed a novel approach to inhibiting CRAC function by generating high-affinity fully
human monoclonal antibodies (mAbs) to human Orai1. These antibodies inhibited ICRAC
current, store-operated Ca2+ influx, NFAT transcription and cytokine release. These fully
human antibodies to human Orai1 may represent a novel therapeutic approach for the
treatment of autoimmunity. Show less

Structure-based rational design led to the synthesis of a novel series of potent PI3K inhibitors. The optimized pyrrolopyridine analogue 63 was a potent and selective PI3Kβ/δ dual inhibitor that displayed suitable physicochemical properties and pharmacokinetic profile for animal studies. Analogue 63 was found to be efficacious in animal models of inflammation including a keyhole limpet hemocyanin (KLH) study and a collagen-induced arthritis (CIA) disease model of rheumatoid arthritis. These… Show more

Structure-based rational design led to the synthesis of a novel series of potent PI3K inhibitors. The optimized pyrrolopyridine analogue 63 was a potent and selective PI3Kβ/δ dual inhibitor that displayed suitable physicochemical properties and pharmacokinetic profile for animal studies. Analogue 63 was found to be efficacious in animal models of inflammation including a keyhole limpet hemocyanin (KLH) study and a collagen-induced arthritis (CIA) disease model of rheumatoid arthritis. These studies highlight the potential therapeutic value of inhibiting both the PI3Kβ and δ isoforms in the treatment of a number of inflammatory diseases. Show less

Changes in cytochrome P450 expression incurred by inflammatory disease were studied in a murine collagen antibody induced arthritis (CAIA) model and compared to bacterial lipopolysaccharide-treated mice and to cytochrome P450 changes in primary mouse hepatocytes following combination treatments with cytokines IL-1β, IL-6, or TNFα. CAIA in female mice increased serum IL-1β, IL-6 and hepatic serum amyloid A (SAA) mRNA and significantly altered cytochrome P450 mRNA and activity levels. Most… Show more

Changes in cytochrome P450 expression incurred by inflammatory disease were studied in a murine collagen antibody induced arthritis (CAIA) model and compared to bacterial lipopolysaccharide-treated mice and to cytochrome P450 changes in primary mouse hepatocytes following combination treatments with cytokines IL-1β, IL-6, or TNFα. CAIA in female mice increased serum IL-1β, IL-6 and hepatic serum amyloid A (SAA) mRNA and significantly altered cytochrome P450 mRNA and activity levels. Most cytochrome P450 isoforms were down-regulated, although some, such as Cyp3a13, were up-regulated. Cytokine effects on cytochrome P450 levels in mouse hepatocytes were compared at in vitro cytokine concentrations similar to those measured in CAIA mouse serum in vivo. In vivo and in vitro cytochrome P450 suppression by cytokines was congruent for some cytochrome P450 isoforms (Cyp1a2, Cyp2c29, and Cyp3a11) but not for others (cytochrome P450 oxidoreductase (POR) and Cyp2e1). In mouse hepatocytes, IL-6 and IL-1β in combination in vitro caused a synergistic increase in SAA mRNA expression, but not in cytochrome P450 suppression. IL-1β and IL-6 were equipotent in the suppression of cytochrome P450 gene expression, while TNFα caused mild suppression only at the highest concentrations used. TNFα in combination with IL-1β, IL-6, or both had a protective effect against IL-1β and IL-6-mediated cytochrome P450 suppression. When IL-1β or IL-6 was combined with low concentrations of TNFα, several P450 isoforms were induced rather than suppressed. These data highlight the complexities of performing in vitro–in vivo comparisons using disease models for cytochrome P450 regulation by cytokines. Show less

The p38α mitogen-activated protein (MAP) kinase is a central signaling molecule in many proinflammatory pathways, regulating the cellular response to a multitude of external stimuli including heat, ultraviolet radiation, osmotic shock, and a variety of cytokines especially interleukin-1β and tumor necrosis factor α. Thus, inhibitors of this enzyme are postulated to have significant therapeutic potential for the treatment of rheumatoid arthritis, inflammatory bowel disease, and Crohn’s disease… Show more

The p38α mitogen-activated protein (MAP) kinase is a central signaling molecule in many proinflammatory pathways, regulating the cellular response to a multitude of external stimuli including heat, ultraviolet radiation, osmotic shock, and a variety of cytokines especially interleukin-1β and tumor necrosis factor α. Thus, inhibitors of this enzyme are postulated to have significant therapeutic potential for the treatment of rheumatoid arthritis, inflammatory bowel disease, and Crohn’s disease, as well as other diseases where aberrant cytokine signaling is the driver of disease. In this communication, we describe a novel class of 7-alkyl-1,5-bis-aryl-pyrazolopyridinone-based p38α inhibitors. In particular, compound 3f is highly potent in the enzyme and cell-based assays, selective in an Ambit kinase screen, and efficacious (ED50 ≤ 0.01 mg/kg) in the rat collagen induced arthritis (CIA) model. Show less

The p38 mitogen-activated protein kinase (MAPK) plays an important role in the production of proinflammatory cytokines, making it an attractive target for the treatment of various inflammatory diseases. A series of pyridazinopyridinone compounds were designed as novel p38 kinase inhibitors. A structure−activity investigation identified several compounds possessing excellent potency in both enzyme and human whole blood assays. Among them, compound 31 exhibited good pharmacokinetic properties and… Show more

The p38 mitogen-activated protein kinase (MAPK) plays an important role in the production of proinflammatory cytokines, making it an attractive target for the treatment of various inflammatory diseases. A series of pyridazinopyridinone compounds were designed as novel p38 kinase inhibitors. A structure−activity investigation identified several compounds possessing excellent potency in both enzyme and human whole blood assays. Among them, compound 31 exhibited good pharmacokinetic properties and showed excellent selectivity against other related kinases. In addition, 31 demonstrated efficacy in a collagen-induced arthritis disease model in rats. Show less

There is a need for methods to improve the diagnosis, patient staging and evaluation of therapeutic response in patients with autoimmune conditions to improve patient care. Inflammatory bowel disease (IBD) and rheumatoid arthritis (RA) are two inflammatory diseases characterized by involvement of innate and adaptive immune components that change the metabolic state of their respective target tissues, thus providing an opportunity for molecular imaging probes to detect such changes. Optimally… Show more

There is a need for methods to improve the diagnosis, patient staging and evaluation of therapeutic response in patients with autoimmune conditions to improve patient care. Inflammatory bowel disease (IBD) and rheumatoid arthritis (RA) are two inflammatory diseases characterized by involvement of innate and adaptive immune components that change the metabolic state of their respective target tissues, thus providing an opportunity for molecular imaging probes to detect such changes. Optimally, such probes and the imaging methods employed would be non-invasive, robust and reproducible, give a quantitative result, report on the status of the affected tissue(s) and respond to the effects of a therapeutic molecule. Positron emission tomography (PET) and single photon emission computed tomography (SPECT) are nuclear imaging approaches that have the potential to satisfy such requirements. In this review, the work to date and the potential of PET and SPECT imaging probes in these two inflammatory conditions, IBD and RA, are discussed. Show less

A novel class of fused pyrazole-derived inhibitors of p38alpha mitogen-activated protein kinase (MAPK) is disclosed. These inhibitors were evaluated for their ability to inhibit the p38alpha enzyme, the secretion of TNFalpha in a LPS-challenged THP1 cell line and TNFalpha-induced production of IL-8 in 50% human whole blood. This series was optimized through a SAR investigation to provide inhibitors with IC(50) values in the low single-digit nanomolar range in whole blood. Further investigation… Show more

A novel class of fused pyrazole-derived inhibitors of p38alpha mitogen-activated protein kinase (MAPK) is disclosed. These inhibitors were evaluated for their ability to inhibit the p38alpha enzyme, the secretion of TNFalpha in a LPS-challenged THP1 cell line and TNFalpha-induced production of IL-8 in 50% human whole blood. This series was optimized through a SAR investigation to provide inhibitors with IC(50) values in the low single-digit nanomolar range in whole blood. Further investigation of their pharmacokinetic profiles led to the identification of two potent and orally bioavailable p38 inhibitors 10 m and 10 q. Inhibitor 10 m was found to be efficacious in vivo in the inhibition of TNFalpha production in LPS-stimulated Lewis rats with an ED(50) of 0.1mg/kg while 10 q was found to have an ED(50) of 0.05-0.07 mg/kg. Show less

A novel class of pyrazolopyridazine p38alpha mitogen-activated protein kinase (MAPK) inhibitors is disclosed. A structure activity relationship (SAR) investigation was conducted driven by the ability of these compounds to inhibit the p38alpha enzyme, the secretion of TNFalpha in a LPS-challenged THP1 cell line and TNFalpha-induced production of IL-8 in the presence of 50% human whole blood (hWB). This study resulted in the discovery of several inhibitors with IC(50) values in the single-digit… Show more

A novel class of pyrazolopyridazine p38alpha mitogen-activated protein kinase (MAPK) inhibitors is disclosed. A structure activity relationship (SAR) investigation was conducted driven by the ability of these compounds to inhibit the p38alpha enzyme, the secretion of TNFalpha in a LPS-challenged THP1 cell line and TNFalpha-induced production of IL-8 in the presence of 50% human whole blood (hWB). This study resulted in the discovery of several inhibitors with IC(50) values in the single-digit nanomolar range in hWB. Further investigation of the pharmacokinetic profiles of these lead compounds led to the identification of three potent and orally bioavailable p38alpha inhibitors 2h, 2m, and 13h. Inhibitor 2m was found to be highly selective for p38alpha/beta over a panel of 402 other kinases in Ambit screening, and was highly efficacious in vivo in the inhibition of TNFalpha production in LPS-stimulated Lewis rats with an ED(50) of ca. 0.08mg/kg. Show less

Wnt signal transduction has emerged as an increasingly complex pathway due to the numerous ligands, receptors, and modulators identified in multiple developmental systems. Wnt signaling has been implicated in the renewal of the intestinal epithelium within adult animals and the progression of cancer in the colon. The Wnt family, however, has not been explored for function during embryonic gut development. Thus, to dissect the role of Wnt signaling in the developing gastrointestinal tract, it is… Show more

Wnt signal transduction has emerged as an increasingly complex pathway due to the numerous ligands, receptors, and modulators identified in multiple developmental systems. Wnt signaling has been implicated in the renewal of the intestinal epithelium within adult animals and the progression of cancer in the colon. The Wnt family, however, has not been explored for function during embryonic gut development. Thus, to dissect the role of Wnt signaling in the developing gastrointestinal tract, it is necessary to first obtain a complete picture of the spatiotemporal expression of the Wnt signaling factors with respect to the different tissue layers of the gut. Here, we offer an in depth in situ gene expression study of Wnt ligands, frizzled receptors, and frizzled related modulators over several days of chicken gut development. These data show some expected locations of Wnt signaling as well as a surprising lack of expression of factors in the hindgut. This paper describes the first comprehensive characterization of the dynamic expression of Wnt signaling molecules during gut development. These data form the basis for future studies to determine the role of Wnt signaling in the developing gastrointestinal tract. Show less

Modern biology is faced with the challenge of understanding the specification, generation, and maintenance of structures ranging from cells and tissues to organs and organisms. By acquiring images directly from the block face of an embedded sample, surface imaging microscopy (SIM) generates high-resolution volumetric images of biological specimens across all of these scales. Surface imaging microscopy expands our range of imaging tools by generating three-dimensional reconstructions of embryo… Show more

Modern biology is faced with the challenge of understanding the specification, generation, and maintenance of structures ranging from cells and tissues to organs and organisms. By acquiring images directly from the block face of an embedded sample, surface imaging microscopy (SIM) generates high-resolution volumetric images of biological specimens across all of these scales. Surface imaging microscopy expands our range of imaging tools by generating three-dimensional reconstructions of embryo samples at high resolution and high contrast. SIM image quality is not limited by depth or the optical properties of overlying tissue, and intrinsic or extrinsic alignment markers are not required for volume reconstruction. These volumes are highly isotropic, enabling them to be virtually sectioned in any direction without loss of image quality. Surface imaging microscopy provided a more accurate three-dimensional representation of a chick embryo than confocal microscopy of the same sample. SIM offers excellent imaging of embryos from three major vertebrate systems in developmental biology: mouse, chicken, and frog. Immediate applications of this technology are in visualizing and understanding complex morphogenetic events and in making detailed comparisons between normal and genetically modified embryos. Show less

The Ash1 protein is a daughter cell-specific repressor of HO gene transcription in Saccharomyces cerevisiae. Both ASH1 mRNA and protein are localized to the incipient daughter cell at the end of mitosis; Ash1 then inhibits HO transcription in the daughter cell after cytokinesis. Mother cells, in contrast, contain little or no Ash1 and thus are able to transcribe HO. We show that deletion of PHO85, which encodes a cyclin-dependent protein kinase, causes reduced transcription of HO and that this… Show more

The Ash1 protein is a daughter cell-specific repressor of HO gene transcription in Saccharomyces cerevisiae. Both ASH1 mRNA and protein are localized to the incipient daughter cell at the end of mitosis; Ash1 then inhibits HO transcription in the daughter cell after cytokinesis. Mother cells, in contrast, contain little or no Ash1 and thus are able to transcribe HO. We show that deletion of PHO85, which encodes a cyclin-dependent protein kinase, causes reduced transcription of HO and that this reduction is dependent on ASH1. In pho85 mutants, Ash1 protein is no longer asymmetrically localized and is present, instead, in both mother and daughter cells. Initially, it appears to be localized properly but then persists as daughter cells mature into mother cells. In contrast, ASH1 mRNA is localized appropriately to daughter cells in pho85 mutants. We observe that Ash1 protein is phosphorylated by Pho85 in vitro and that Ash1 stability increases in a pho85 mutant. These data suggest that phosphorylation of Ash1 by Pho85 governs stability of Ash1 protein. Show less

Gcn4, a yeast transcriptional activator that promotes the expression of amino acid and purine biosynthesis genes, is rapidly degraded in rich medium. Here we report that SCF(CDC4), a recently characterized protein complex that acts in conjunction with the ubiquitin-conjugating enzyme Cdc34 to degrade cell cycle regulators, is also necessary for the degradation of the transcription factor Gcn4. Degradation of Gcn4 occurs throughout the cell cycle, whereas degradation of the known cell cycle… Show more

Gcn4, a yeast transcriptional activator that promotes the expression of amino acid and purine biosynthesis genes, is rapidly degraded in rich medium. Here we report that SCF(CDC4), a recently characterized protein complex that acts in conjunction with the ubiquitin-conjugating enzyme Cdc34 to degrade cell cycle regulators, is also necessary for the degradation of the transcription factor Gcn4. Degradation of Gcn4 occurs throughout the cell cycle, whereas degradation of the known cell cycle substrates of Cdc34/SCF(CDC4) is cell cycle regulated. Gcn4 ubiquitination and degradation are regulated by starvation for amino acids, whereas the degradation of the cell cycle substrates of Cdc34/SCF(CDC4) is unaffected by starvation. We further show that unlike the cell cycle substrates of Cdc34/SCF(CDC4), which require phosphorylation by the kinase Cdc28, Gcn4 degradation requires the kinase Pho85. We identify the critical target site of Pho85 on Gcn4; a mutation of this site stabilizes the protein. A specific Pho85-Pcl complex that is able to phosphorylate Gcn4 on that site is inactive under conditions under which Gcn4 is stable. Thus, Cdc34/SCF(CDC4) activity is constitutive, and regulation of the stability of its various substrates occurs at the level of their phosphorylation. Show less

Pho85 is a cyclin-dependent protein kinase (Cdk) in budding yeast with roles in cell metabolism and cell cycle progression. Activation of Pho85 occurs through association with Pho85 cyclins (Pcls), of which 10 are known. When complexed with the G1 cyclins, Pcl1 and Pcl2, Pho85 is required for cell cycle progression in the absence of the Cdc28-dependent cyclins, Cln1 and Cln2. To identify potential targets of Pcl2-Pho85, we performed a two-hybrid screen using the Pcl2 cyclin as bait and… Show more

Pho85 is a cyclin-dependent protein kinase (Cdk) in budding yeast with roles in cell metabolism and cell cycle progression. Activation of Pho85 occurs through association with Pho85 cyclins (Pcls), of which 10 are known. When complexed with the G1 cyclins, Pcl1 and Pcl2, Pho85 is required for cell cycle progression in the absence of the Cdc28-dependent cyclins, Cln1 and Cln2. To identify potential targets of Pcl2-Pho85, we performed a two-hybrid screen using the Pcl2 cyclin as bait and recovered the transcription factor Swi5 as a Pcl2-interacting protein. We performed both biochemical and genetic tests to discover the biological significance of the interaction between Pcl2 and Swi5 seen in the two-hybrid assay. We found that Swi5 interacts in vitro with Pho85 cyclins and is phosphorylated in vitro by the Pho80-Pho85 kinase. We discovered that a subset of genes that are controlled by Swi5 and a homologous transcription factor, Ace2, was misregulated in a pho85 deletion strain; expression of the ASH1 and CTS1 genes was reduced in an ace2 deletion strain, whereas expression of both genes was increased in an ace2Delta pho85Delta double mutant. We also found that overexpression of SWI5 caused cell lethality in a pho85 deletion strain. Our results are consistent with misregulation of Swi5 activity in vivo in the absence of Pho85 and implicate Swi5 as a potential substrate of Pho85 cyclin-dependent kinase complexes. Show less

Swi5 and Ace2 are cell cycle-regulated transcription factors that activate expression of early G(1)-specific genes in Saccharomyces cerevisiae. Swi5 and Ace2 have zinc finger DNA-binding domains that are highly conserved, and the two proteins bind to the same DNA sequences in vitro. Despite this similarity in DNA binding, Swi5 and Ace2 activate different genes in vivo, with Swi5 activating the HO gene and Ace2 activating CTS1 expression. In this report we have used chimeric fusions between Swi5… Show more

Swi5 and Ace2 are cell cycle-regulated transcription factors that activate expression of early G(1)-specific genes in Saccharomyces cerevisiae. Swi5 and Ace2 have zinc finger DNA-binding domains that are highly conserved, and the two proteins bind to the same DNA sequences in vitro. Despite this similarity in DNA binding, Swi5 and Ace2 activate different genes in vivo, with Swi5 activating the HO gene and Ace2 activating CTS1 expression. In this report we have used chimeric fusions between Swi5 and Ace2 to determine what regions of these proteins are necessary for promoter-specific activation of HO and CTS1. We have identified specific regions of Swi5 and Ace2 that are required for activation of HO and CTS1, respectively. The Swi5 protein binds HO promoter DNA cooperatively with the Pho2 homeodomain protein, and the HO specificity region of Swi5 identified in the chimeric analysis coincides with the region of Swi5 previously identified that interacts with Pho2 in vitro. Swi5 and Ace2 also activate expression of a number of other genes expressed in G(1) phase of the cell cycle, including ASH1, CDC6, EGT2, PCL2, PCL9, RME1, and SIC1. Analysis of the Swi5/Ace2 chimeras shows that distinct regions of Swi5 and Ace2 contribute to the transcriptional activation of some of these other G(1)-regulated genes. Show less

The SWI5 gene encodes a zinc finger DNA-binding protein required for the transcriptional activation of the yeast HO gene. There are two Swi5p binding sites in the HO promoter, site A at -1800 and site B at -1300. Swi5p binding at site B has been investigated in some detail, and we have shown that Swi5p binds site B in a mutually cooperative fashion with Pho2p, a homeodomain protein. In this report, we demonstrate that Swi5p and Pho2p bind cooperatively to both sites A and B but that there are… Show more

The SWI5 gene encodes a zinc finger DNA-binding protein required for the transcriptional activation of the yeast HO gene. There are two Swi5p binding sites in the HO promoter, site A at -1800 and site B at -1300. Swi5p binding at site B has been investigated in some detail, and we have shown that Swi5p binds site B in a mutually cooperative fashion with Pho2p, a homeodomain protein. In this report, we demonstrate that Swi5p and Pho2p bind cooperatively to both sites A and B but that there are differences in binding to these two promoter sites. It has been shown previously that point mutations in either Swi5p binding site only modestly reduce HO expression in a PHO2 strain. We show that these mutant promoters are completely inactive in a pho2 mutant. We have created stronger point mutations at the two Swi5p binding sites within the HO promoter, and we show that the two binding sites, separated by 500 bp, are both absolutely required for HO expression, independent of PHO2. These results create an apparent dilemma, as the strong mutations at the Swi5p binding sites show that both binding sites are required for HO expression, but the earlier binding site mutations allow Swi5p to activate HO, but only in the presence of Pho2p. To explain these results, a model is proposed in which physical interaction between Swi5p proteins bound to these two sites separated by 500 bp is required for activation of the HO promoter. Experimental evidence is presented that supports the model. In addition, through deletion analysis we have identified a region near the amino terminus of Swi5p that is required for PHO2-independent activation of HO, suggesting that this region mediates the long-range interactions between Swi5p molecules bound at the distant sites. Show less

nfectious RNA transcripts were generated from full-length cDNA clones of the tobacco etch potyvirus genome containing an insertion of the bacterial beta-glucuronidase (GUS) gene between the polyprotein-coding sequences for the N-terminal 35-kDa proteinase and the helper component-proteinase. The recombinant virus was able to spread systemically in plants and accumulated to a level comparable with wild-type tobacco etch potyvirus. Proteolytic processing mediated by the 35-kDa proteinase and… Show more

nfectious RNA transcripts were generated from full-length cDNA clones of the tobacco etch potyvirus genome containing an insertion of the bacterial beta-glucuronidase (GUS) gene between the polyprotein-coding sequences for the N-terminal 35-kDa proteinase and the helper component-proteinase. The recombinant virus was able to spread systemically in plants and accumulated to a level comparable with wild-type tobacco etch potyvirus. Proteolytic processing mediated by the 35-kDa proteinase and helper component-proteinase resulted in production of an enzymatically active GUS-helper component-proteinase fusion protein. A virus passage line that retained the GUS insert after numerous plant-to-plant transfers, as well as a line that sustained a deletion of the GUS sequence, was recovered. Use of an in situ histochemical GUS assay in time-course experiments allowed the visualization of virus activity in single, mechanically inoculated leaf epidermal cells, in neighboring epidermal and mesophyll cells, in phloem-associated cells after long-distance transport, and in cells surrounding vascular tissues of organs above and below the site of inoculation. This system represents a powerful tool to study plant virus replication, short- and long-distance virus movement, and virus-host interactions. Additionally, we show that potyviruses may serve as highly efficient, autonomously replicating vectors for the expression of foreign genes in plants. Show less