Search

TheCEACAM5gene product [carcinoembryonic antigen (CEA)] is an attractive target for colorectal cancer because of its high expression in virtually all colorectal tumors and limited expression in most healthy adult tissues. However, highly active CEA-directed investigational therapeutics have been reported to be toxic, causing severe colitis because CEA is expressed on normal gut epithelial cells. Here, we developed a strategy to address this toxicity problem: the Tmod dual-signal integrator. CEA Tmod cells use two receptors: a chimeric antigen receptor (CAR) activated by CEA and a leukocyte Ig-like receptor 1 (LIR-1)–based inhibitory receptor triggered by human leukocyte antigen (HLA)-A*02. CEA Tmod cells exploit instances of HLA heterozygous gene loss in tumors to protect the patient from on-target, off-tumor toxicity. CEA Tmod cells potently killed CEA-expressing tumor cells in vitro and in vivo. But in contrast to a traditional CEA-specific T cell receptor transgenic T cell, Tmod cells were highly selective for tumor cells even when mixed with HLA-A*02–expressing cells. These data support further development of the CEA Tmod construct as a therapeutic candidate for colorectal cancer.

Cell therapy is an emerging therapeutic modality with the power to exploit new cancer targets and potentially achieve positive outcomes for patients with few other options. Like all synthetic treatments, cell therapy has the risk of toxicity via unpredicted off-target behavior. We describe an empirical method to model off-tumor, off-target reactivity of receptors used for investigational T cell therapies. This approach utilizes an optimal panel of diverse human cell-lines to capture the large majority of protein-coding gene expression in adult human tissues. We apply this cell-line set to test Jurkat and primary T cells engineered with a dual-signal integrator, called Tmod

BackgroundMesothelin (MSLN) and carcinoembryonic antigen (CEA) are classic tumor-associated antigens that are expressed in many solid tumors including the majority of lung, colorectal and pancreatic cancers. However, both MSLN and CEA are also expressed in vital normal organs. This normal expression creates risk of serious inflammation for CEA- or MSLN-directed therapeutics. To date all active CEA- or MSLN-targeted investigational therapeutics have been toxic when administered systemically.MethodsWe have developed a safety mechanism to protect normal tissues without abrogating sensitivity of cytotoxic T cells directed at MLSN(+) or CEA(+) tumors in a subset of patients with defined loss of heterozygosity (LOH) in their tumors (figure 1). This dual-receptor (Tmod< sup >TM) system exploits common LOH at the HLA locus in cancer cells, allowing T cells to recognize the difference between tumor and normal tissue.1 2 T cells engineered with specific Tmod constructs contain: (i) a MSLN- or CEA-activated CAR; and, (ii) an inhibitory receptor gated by HLA-A*02. HLA-A*02 binding blocks T cell cytotoxicity, even in the presence of MSLN or CEA. The Tmod system is designed to treat heterozygous HLA class I patients, selected for HLA LOH. When HLA-A*02 is absent from tumors selected for LOH, the CARs are predicted to mediate potent killing of the A*02(-) malignant cells.ResultsThe Tmod system robustly protects surrogate normal cells even in mixed-cell populations in vitro while mediating robust cytotoxicity of tumor cells in xenograft models (see example in figure 2). The MSLN CAR can also be paired with other blockers, supporting scalability of the approach to patients beyond HLA-A*02 heterozygotes.Abstract 122 Figure 1Illustration of the Tmod T cell engaging with tumor cells with somatic loss of HLA-A*02 and with normal cells.Abstract 122 Figure 2Bioluminescence measurements show the average difference between the size of the MSLN(+)A*02(+) ‘normal’ graft compared to the MSLN(+)A*02(-) tumor graft on the two flanks of mice after T cell infusion. Both tumor and normal grafts are destroyed by CAR-Ts (CAR-3 and M5 benchmark) while the MSLN Tmod cells kill the tumor but not the normal graft.ConclusionsThe Tmod mechanism may provide an alternative route to leverage solid-tumor antigens such as MSLN and CEA in safer, more effective ways than previously possible.ReferencesHamburger AE, DiAndreth B, Cui J, et al. Engineered T cells directed at tumors with defined allelic loss. Mol Immunol 2020;128:298–310.Hwang MS, Mog BJ, Douglass J, et al. Targeting loss of heterozygosity for cancer-specific immunotherapy. Proc Natl Acad Sci U S A 2021;118(12):e2022410118.

Structure-activity relationship (SAR) investigations of a novel class of triazolopyridazinone p38α mitogen activated protein kinase (MAPK) inhibitors are disclosed. From these studies, increased in vitro potency was observed for 2,6-disubstituted phenyl moieties and N-ethyl triazolopyridazinone cores due to key contacts with Leu108, Ala157 and Val38. Further investigation led to the identification of three compounds, 3g, 3j and 3m that are highly potent inhibitors of LPS-induced MAPKAP kinase 2 (MK2) phosphorylation in 50% human whole blood (hWB), and possess desirable in vivo pharmacokinetic and kinase selectivity profiles.

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.

The p38 mitogen-activated protein kinase (MAPK) 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-1beta and tumor necrosis factor alpha. Thus, inhibitors of this enzyme are postulated to have significant therapeutic potential for the treatment of rheumatoid arthritis, inflammatory bowel disease, osteoporosis, and many other diseases where aberrant cytokine signaling is the driver of disease. Herein, we describe a novel class of 3-amino-7-phthalazinylbenzoisoxazole-based inhibitors. With relatively low molecular weight, these compounds are highly potent in enzyme and cell-based assays, with minimal protein shift in 50% human whole blood. Compound 3c was efficacious (ED 50 = 0.05 mg/kg) in the rat collagen induced arthritis (CIA) model.

In this communication, we report the discovery of 1S (apremilast), a novel potent and orally active phosphodiesterase 4 (PDE4) and tumor necrosis factor-alpha inhibitor. The optimization of previously reported 3-(1,3-dioxo-1,3-dihydroisoindol-2-yl)-3-(3,4-dimethoxyphenyl)propionic acid PDE4 inhibitors led to this series of sulfone analogues. Evaluation of the structure-activity relationship of substitutions on the phthalimide group led to the discovery of an acetylamino analogue 1S, which is currently in clinical trials.

TNF-α mediates both protective and detrimental manifestations of the host immune response. Our previous work has shown thalidomide to be a relatively selective inhibitor of TNF-α production in vivo and in vitro. Additionally, we have recently reported that thalidomide exerts a costimulatory effect on T cell responses. To develop thalidomide analogues with increased anti-TNF-α activity and reduced or absent toxicities, novel TNF-α inhibitors were designed and synthesized. When a selected group of these compounds was examined for their immunomodulatory activities, different patterns of cytokine modulation were revealed. The tested compounds segregated into two distinct classes: one class of compounds, shown to be potent phosphodiesterase 4 inhibitors, inhibited TNF-α production, increased IL-10 production by LPS-induced PBMC, and had little effect on T cell activation; the other class of compounds, similar to thalidomide, were not phosphodiesterase 4 inhibitors and markedly stimulated T cell proliferation and IL-2 and IFN-γ production. These compounds inhibited TNF-α, IL-1β, and IL-6 and greatly increased IL-10 production by LPS-induced PBMC. Similar to thalidomide, the effect of these agents on IL-12 production was dichotomous; IL-12 was inhibited when PBMC were stimulated with LPS but increased when cells were stimulated by cross-linking the TCR. The latter effect was associated with increased T cell CD40 ligand expression. The distinct immunomodulatory activities of these classes of thalidomide analogues may potentially allow them to be used in the clinic for the treatment of different immunopathological disorders.

N-Phthaloyl 3-amino-3-arylpropionic acid analogs of thalidomide that are potent inhibitors of tumor necrosis factor-alpha are reported. These compounds were found to be potent inhibitors of phosphodiesterase 4.

Two forms of the monocyte chemoattractant protein-1 receptors (the type A monocyte chemoattractant protein 1 (MCP-1) receptor CCR-2A and the type B MCP-1 receptor (CCR-2B) have been recently cloned and found to differ only in their terminal carboxyl tails. Here, we report that the two isoforms are alternatively spliced variants of a single MCP-1 receptor gene. Sequencing of the gene revealed that the 47-amino acid carboxyl tail of CCR2B was located in the same exon as the seven transmembrane domains of the receptor, and the 61-amino acid tail of CCR2A was in a downstream exon. Examination of freshly isolated human monocytes by reverse transcriptase-polymerase chain reaction revealed that CCR2B was the predominant isoform and that message levels of both CCR2A and CCR2B decreased as the monocytes differentiated into macrophages. In stably transfected cell lines, CCR2B trafficked well to the cell surface, but CCR2A was found predominately in the cytoplasm. Equilibrium binding studies revealed that those CCR2A receptors that successfully trafficked to the cell surface bound MCP-1 with high affinity (Kd = 310 pM), similar to CCR2B. In signaling studies, both CCR2A and CCR2B mediated agonist-dependent calcium mobilization, as well as inhibition of adenylyl cyclase. Creation of chimeras between CCR2A and the human thrombin receptor revealed that the cytoplasmic retention of CCR2A was due to its terminal carboxyl tail. Progressive truncation of the carboxyl tail indicated that a cytoplasmic retention signal(s) was located between residues 316 and 349. These data indicate that the alternatively spliced form of the human MCP-1 receptor (CCR2A) binds MCP-1 with high affinity and is a functional receptor and that expression at the cell surface is controlled by amino acid sequences located in the terminal carboxyl tail.

The transcription of two genes, PS-IAA4/5 and PS-IAA6, in pea is induced by indoleacetic acid (IAA) and protein synthesis inhibitors such as cycloheximide (CHX) and anisomycin (ANI). Induction by IAA is rapid, taking 5 and 7.5 minutes for PS-IAA4/5 and PS-IAA6, respectively, and is independent of IAA concentration and whether IAA has a free or esterified carboxyl group (ethyl-IAA). The rate of mRNA accumulation, however, is dependent on hormone concentration, and is greater with IAA than with ethyl-IAA. The turnover rates (t1/2) of the PS-IAA4/5 and PS-IAA6 mRNAs are 60 and 75 minutes, respectively, and are not affected by IAA. CHX or ANI induce the transcription of PS-IAA4/5 and PS-IAA6 more slowly than IAA (5 to 10 minutes for PS-IAA4/5 and 20 minutes for PS-IAA6). While protein synthesis inhibitors stabilize both mRNAs, the rapidity of induction by CHX and ANI cannot be accounted for solely by mRNA stabilization. The relationship between mRNA induction and protein synthesis inhibition does not obey Michaelis-Menten kinetics, but rather is best described by a hyperbolic curve, suggesting the release of transcriptional repression by the inhibition of protein synthesis. RNA expression experiments with transgenic tobacco seedlings or with transfected pea protoplasts using PS-IAA4/5 promoter GUS or CAT fusions reveal that CHX transcriptionally activates PS-IAA4/5 gene expression. Thus, protein synthesis inhibitors have a dual effect on PS-IAA4/5 and PS-IAA6. (1) They stabilize both mRNAs (possibly by a translational arrest-linked process or by preventing the synthesis of a labile nuclease(s)). (2) They activate transcription (possibly by preventing the synthesis or function of a repressor).

The plant growth hormone indole-3-acetic acid (IAA) transcriptionally activates expression of several genes in plants. We have previously identified a 164-bp promoter region (-318 to -154) in the PS-IAA4/5 gene that confers IAA inducibility. Linker-scanning mutagenesis across the region has identified two positive domains: domain A (48 bp; -203 to -156) and domain B (44 bp; -299 to -256), responsible for transcriptional activation of PS-IAA4/5 by IAA. Domain A contains the highly conserved sequence 5'-TGTCCCAT-3' found among various IAA-inducible genes and behaves as the major auxin-responsive element. Domain B functions as an enhancer element which may also contain a less efficient auxin-responsive element. The two domains act cooperatively to stimulate transcription; however, tetramerization of domain A or B compensates for the loss of A or B function. The two domains can also mediate IAA-induced transcription from the heterologous cauliflower mosaic virus 35S promoter (-73 to +1). In vivo competition experiments with icosamers of domain A or B show that the domains interact specifically and with different affinities to low abundance, positive transcription factor(s). A model for transcriptional activation of PS-IAA4/5 by IAA is discussed.

The plant hormone auxin transcriptionally activates early genes in pea. We have developed a transient assay system using protoplasts of auxin-responsive pea seedling cells to define the auxin-responsive element, AuxRE, of the early auxin-induced PS-IAA4/5 gene. The auxin responsive protoplasts show an authentic hormonal response identical to that observed in intact pea tissue, with respect to rapidity, specificity and cycloheximide (CHX) inducibility of the PS-IAA4/5 transcript. The hormone also mediates rapid and specific induction of chloramphenicol acetyltransferase (CAT) activity in protoplasts transfected with a chimeric IAA4/5-CAT gene. The IAA-induced CAT activity is developmentally regulated and is observed only in protoplasts derived from auxin-responsive regions of the pea seedling. Extensive deletion analysis of the PS-IAA4/5 promoter defined a promoter region between -318 and -154 that confers auxin inducibility. This AuxRE mediates auxin-inducible CAT activity in pea cells driven by the non auxin-responsive CaMV 35S minimal promoter. The functionality of this promoter region as an AuxRE was further verified in tobacco plants using IAA4/5-GUS gene fusions. The AuxRE contains two domains: Domain A acts as an auxin switch; domain B has an enhancer-like activity. The A and B domains contain the highly conserved sequences found in various auxin-regulated genes (T/GGT-CCCAT (domain A) and C/AACATGGNC/AA/GTGTT/CT/CC/A (domain B)). DNase I footprinting reveals binding of nuclear proteins to the highly conserved sequence found in A and B domains. The sequence of the A domain does not correspond to any known regulatory elements found in other eukaryotic genes, and the data suggest that this conserved motif functions as an AuxRE. A model for the early transcriptional activation of the PS-IAA4/5 gene by IAA is discussed.

Fruit ripening is one of the most dramatic developmental transitions associated with extensive alteration in gene expression. The plant hormone ethylene is considered to be the causative ripening agent. Transgenic tomato plants were constructed expressing antisense or sense RNA to the key enzyme in the ethylene (C2H4) biosynthetic pathway, 1 -aminocyclopropane-1-carboxylate (ACC) synthase using the constitutive CaMV 35S and fruit specific E8 promoters. Fruits expressing antisense LE-ACS2 RNA produce less ethylene and fail to ripen only when ethylene production is suppressed by more than 99% (>0.1 nl/g fresh weight). Ethylene production is considerably inhibited (50%) in fruits expressing sense LE-ACS2 RNA. Antisense fruits accumulate normal levels of polygalacturonase (PG), ACC oxidase (pTOM13), E8, E17, J49, and phytoene desaturase (D2) mRNAs which were previously thought to be ethylene-inducible. E4 gene expression is inhibited in antisense fruits and its expression is not restored by treatment with exogenous propylene (C3H6). Antisense fruits accumulate PG mRNA, but it is not translated. Immunoblotting experiments indicate that the PG protein is not expressed in antisense fruits but its accumulation is restored by propylene (C3H6) treatment. The results suggest that at least two signal-transduction pathways are operating during tomato fruit ripening. The independent (developmental) pathway is responsible for the transcriptioncl activation of genes such as PG, ACC oxidase, E8, E17, D2, and J49. The ethylene-dependent pathway is responsible for the transcriptional and posttranscriptional regulation of genes involved in lycopene, aroma biosynthesis, and the translatability of developmentally regulated genes such as PG. © 1993Wiley-Liss, Inc.1 © 1993Wiley-Liss, Inc.

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.

The p38alpha 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-1beta and tumor necrosis factor alpha. 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 p38alpha inhibitors. In particular, compound 3f is highly potent in the enzyme and cell-based assays, selective in an Ambit kinase screen, and efficacious (ED(50)or = 0.01 mg/kg) in the rat collagen induced arthritis (CIA) model.

Extracellular cAMP serves as a chemoattractant as well as a signal which regulates gene expression during development of Dictyostelium discoideum. The cell adhesion molecule gp80 is expressed at the aggregation stage, between 6 and 10 h of development, and is known to be under cAMP regulation. Transcription of the gp80 gene is first turned on at a low, basal level at the preaggregation stage and is then greatly augmented by pulses of low levels of cAMP at the aggregation stage. Using cloned cDNA sequences, we have isolated genomic DNA fragments encompassing the gp80 gene. The gp80 gene has a single open reading frame, with multiple transcription start sites located downstream from a putative TATA box. Several short, repeated sequences in the upstream sequence have also been identified. The cloned 1.3-kilobase upstream DNA was sufficient to confer proper temporal and cAMP regulation on a gp80 minigene reporter in Dictyostelium cells. Deletional analysis of this 5'-flanking DNA led to the mapping of a cAMP-response element (CRE) in the gp80 gene to sequences between -306 and -289 base pairs upstream of the translational start site. Present within this region is a sequence we refer to as box 1 (TGGTGTG). The gp80 box 1-CRE binds specifically to a protein present in nuclear extracts, but binding is abolished when mutations are introduced in the box 1 sequence. The gp80 box 1-CRE shows little sequence homology to CREs of late developmental genes and the expression of gp80 may involve a distinct signal transduction pathway.

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.

A novel class of pyrazolopyridazine p38α 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 p38α enzyme, the secretion of TNFα in a LPS-challenged THP1 cell line and TNFα-induced production of IL-8 in the presence of 50% human whole blood (hWB). This study resulted in the discovery of several inhibitors with IC50 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 p38α inhibitors 2h, 2m, and 13h. Inhibitor 2m was found to be highly selective for p38α/β over a panel of 402 other kinases in Ambit screening, and was highly efficacious in vivo in the inhibition of TNFα production in LPS-stimulated Lewis rats with an ED50 of ca. 0.08 mg/kg.

Investigations into the structure-activity relationships (SAR) of a series of phthalazine-based inhibitors of p38 are described. These efforts originated from quinazoline 1 and through rational design led to the development of a series of orally bioavailable, potent, and selective inhibitors. Kinase selectivity was achieved by exploiting a collection of interactions with p38alpha including close contact to Ala157, occupation of the hydrophobic gatekeeper pocket, and a residue flip with Gly110. Substitutions on the phthalazine influenced the pharmacokinetic properties, of which compound 16 displayed the most desirable profile. Oral dosing (0.03 mg/kg) of 16 in rats 1 h prior to LPS challenge gave a >50% decrease in TNFalpha production.

Novel potent trisubstituted pyridazine inhibitors of p38 MAP (mitogen activated protein) kinase are described that have activity in both cell-based assays of cytokine release and animal models of rheumatoid arthritis. They demonstrated potent inhibition of LPS-induced TNF-alpha production in mice and exhibited good efficacy in the rat collagen induced arthritis model.

Thalidomide, (1), is a known inhibitor of TNF-alpha release in LPS stimulated human PBMC. Herein we describe the TNF-alpha inhibitory activity of amino substituted analogs of thalidomide (1) and its isoindolin-1-one analog, EM-12 (2). The 4-amino substituted analogs were found to be potent inhibitors of TNF-alpha release in LPS stimulated human PBMC.

Genes induced by the plant hormone auxin are probably involved in the execution of vital cellular functions and developmental processes. Experimental approaches designed to elucidate the molecular mechanisms of auxin action have focused on auxin perception, genetic dissection of the signaling apparatus and specific gene activation. Auxin-responsive promoter elements of early genes provide molecular tools for probing auxin signaling in reverse. Functional analysis of several auxin-specific promoters of unrelated early genes suggests combinatorial utilization of both conserved and variable elements. These elements are arranged into autonomous domains and the combination of such modules generates uniquely composed promoters. Modular promoters allow for auxin-mediated transcriptional responses to be revealed in a tissue- and development-specific manner.

Summary The plant growth hormone auxin typified by indoleacetic acid (IAA) transcriptionally activates early genes in pea, PS-IAA4/5 and PS-IAA6, that are members of a multigene family encoding short-lived nuclear proteins. To gain first insight into the biological role of PS-IAA4/5 and PSIAA6, promoter-β-glucuronidase (GUS) gene fusions were constructed and their expression during early development of transgenic tobacco seedlings was examined. The comparative analysis reveals spatial and temporal expression patterns of both genes that correlate with cells, tissues, and developmental processes known to be affected by auxin. GUS activity in seedlings of both transgenic lines is located in the root meristem, sites of lateral root initiation and in hypocotyls undergoing rapid elongation. In addition, mutually exclusive cell-specific expression is evident. For instance, PS-IAA4/5—GUS but not PS-IAA6—GUS is expressed in root vascular tissue and in guard cells, whereas only PS-IAA6—GUS activity is detectable in glandular trichomes and redistributes to the elongating side of the hypocotyl upon gravitropic stimulation. Expression of PS-IAA4/5 and PS-IAA6 in elongating, dividing, and differentiating cell types indicates multiple functions during development. The common and yet distinct activity patterns of both genes suggest a combinatorial code of spatio-temporal co-expression of the various PS-IAA4/ 5-like gene family members in plant development that may mediate cell-specific responses to auxin.

We have examined the ligand specificity and signal transduction pathways of a recently cloned receptor for monocyte chemoattractant protein-1 (MCP-1). In human 293 cells stably transfected with the MCP-1 receptor, MCP-1 bound specifically with high affinity (Kd = 260 pM) and induced a rapid mobilization of calcium from intracellular stores. The closely related chemokines MIP-1 alpha, MIP-1 beta, RANTES, interleukin 8 (IL-8), and Gro-alpha were inactive at concentrations as high as 300 nM. Activation of the MCP-1 receptor potently inhibited adenylyl cyclase with an IC50 = 90 pM. Activation of the MIP-1 alpha/RANTES receptor also mediated inhibition of adenylyl cyclase activity but with a different pharmacological profile: MIP-1 alpha (110 pM, IC50), RANTES (140 pM), MIP-1 beta (10 nM), and MCP-1 (820 nM). Mobilization of intracellular calcium and inhibition of adenylyl cyclase were blocked by pertussis toxin, suggesting that the MCP-1 receptor coupled to G alpha i. These results demonstrate that the MCP-1 receptor binds and signals in response to picomolar concentrations of MCP-1 in a highly specific manner. Signaling was manifested as mobilization of intracellular calcium and inhibition of adenylyl cyclase and was mediated by a pertussis toxin-sensitive G-protein(s).

Ethylene is one of the simplest organic molecules with biological activity. Its effects on plant tissue are spectacular and commercially important (1,2). This hydrocarbon gas is generally considered to be the fruit ripening hormone (2,3). Because of its effects on plant senescence, large losses of fruits and vegetables are incurred annually in the U.S. The losses are much greater in third world countries because of the lack of sufficient refrigeration and transportation. Consequently, it has always been a goal of plant biologists and of postharvest physiologists, in particular, to be able to prevent or delay fruit ripening in a reversible manner by controlling ethylene action or production. Thus, an understanding of ethylene action and biosynthesis is of fundamental as well as of applied significance. This update summarizes the recent advances in manipulating key genes in the ethylene biosynthetic pathway to prevent ethylene production and fruit ripening.

Ethylene is one of the simplest organic molecules with biological activity. Its effects on plant tissue are spectacular and commercially important (1,2). This hydrocarbon gas is generally considered to be the fruit ripening hormone (2,3). Because of its effects on plant senescence, large losses of fruits and vegetables are incurred annually in the U.S. The losses are much greater in third world countries because of the lack of sufficient refrigeration and transportation. Consequently, it has always been a goal of plant biologists and of postharvest physiologists, in particular, to be able to prevent or delay fruit ripening in a reversible manner by controlling ethylene action or production. Thus, an understanding of ethylene action and biosynthesis is of fundamental as well as of applied significance. This update summarizes the recent advances in manipulating key genes in the ethylene biosynthetic pathway to prevent ethylene production and fruit ripening.

At the aggregation stage of Dictyostelium discoideum development, a cell surface glycoprotein of Mr 80,000 (gp80) has been found to mediate the EDTA-resistant type of cell-cell adhesion via homophilic interaction (Siu, C.-H., A. Cho, and A. H. C. Choi. 1987. J. Cell Biol. 105:2523-2533). To investigate the structure-function relationships of gp80, we have isolated full length cDNA clones for gp80 and determined the DNA sequence. The deduced structure of gp80 showed three major domains. An amino-terminal globular domain composed of the bulk of the protein is supported by a short stalk region, which is followed by a membrane anchor at the carboxy terminus. Structural analysis suggested that the cell-binding domain of gp80 resides within the globular domain near the amino terminus. To investigate the relationship of the cell-binding activity to this region of the polypeptide, three protein A/gp80 (PA80) gene fusions were constructed using the expression vector pRIT2T. These PA80 fusion proteins were assayed for their ability to bind to aggregation stage cells. Binding of 125I-labeled fusion proteins PA80I (containing the Val123 to Ile514 fragment of gp80) and PA80II (Val123 to Ala258) was dosage dependent and could be inhibited by precoating cells with the cell cohesion-blocking mAb 80L5C4. On the other hand, there was no appreciable binding of PA80III (Ile174 to Ile514) to cells. Reassociation of cells was significantly inhibited in the presence of PA80I or PA80II. In addition, 125I-labeled PA80II exhibited homophilic interaction with immobilized PA80I, PA80II, or gp80. The results of these studies lead to the mapping of a cell-binding domain in the region between Val123 and Leu173 of gp80 and provide direct evidence that the cell-binding activity of gp80 resides in the protein moiety.

We have previously reported that a monoclonal antibody directed specifically against a surface glycoprotein of Mr 80 000 (gp80) inhibits the EDTA-resistant contact sites A of Dictyostelium discoideum (Siu, C.-H., and Choi, A.H.C. (1985) J. Biol. Chem. 260, 16030–16036). In this report, we describe an assay using this monoclonal antibody to quantitate the amount of gp80 expressed at different developmental stages. Under normal conditions, gp80 is detectable after 6 h of development and it rapidly accumulates between 6 and 10 h, corresponding to the time when cells acquire their EDTA-resistant binding sites. At the peak level, there are 1.5 · 105 gp80 molecules per cell. More than 90% of the cellular gp80 is located on the cell surface. When cells are given exogenous pulses of cAMP, a precocious and enhanced expression of gp80 is induced. At the peak level, the cAMP-pulsed cells accumulate five times more gp80 than the non-pulsed cells. This is preceeded by an equally rapid accumulation of gp80 transcripts, suggesting that cAMP regulates gp80 synthesis at the transcriptional level.

A cell surface glycoprotein of Dictyostelium discoideum with M r 80,000 (gp80) has been shown to mediate the formation of the developmentally acquired EDTA-resistant cell-cell binding sites termed contact sites A. We have isolated cDNA clones encoding gp80 by immunological screening of an expression library prepared in Escherichia coli . Double-stranded cDNA was prepared from poly(A) + RNA isolated from cells at 8 hr of development and cloned into the bacteriophage expression vector λgt11. Two recombinant phages containing cDNA inserts of 1.2 and 0.8 kilobases were isolated and shown to contain sequences coding for gp80 by the immunoselect assay. Partial DNA sequence analysis also confirmed that one of these cDNA clones, λDdgp80c-19, contained the coding sequence for the amino terminus of gp80. DNA-RNA hybridization showed that the insert of λDdgp80c-19 hybridized to a single mRNA transcript of approximately 2.0 kilobases. gp80 mRNA became detectable after 6 hr of development, reached its maximum level at 9 hr, and dropped to a negligible level by 15 hr. This pattern of mRNA accumulation corresponded closely to that of gp80 synthesis in D. discoideum cells, suggesting that gp80 expression is regulated at the transcriptional level.

During development of the cellular slime mold Dictyostelium discoideum, cells migrate in respose to cAMP to form aggregates, which give rise to fruiting bodies consisting of two major cell types: spores and stalk cells. Multicellularity is achieved by the expression of two types of cell–cell adhesion sites. The EDTA-sensitive binding sites are expressed at the initial stage of development. At the aggregation stage, cells acquire EDTA-resistant binding sites, which are mediated by a cell-surface glycoprotein of Mr 80 000 (gp80). gp80 is preferentially associated with cell surface filopodia, which are probably involved in the initiation of contact formation between cells. Covaspheres conjugated with gp80 bind specifically to aggregation-stage cells. The binding can be inhibited by precoating cells with an anti-gp80 monoclonal antibody, thus suggesting that gp80 mediates cell-cell binding via homophilic interaction. The structure of gp80 predicted from its cDNA sequence can be divided into three major domains: a membrane anchor, a hinge, and a globular region. An analysis of fusion proteins containing different gp80 segments shows that the cell-binding activity resides in the globular region. In the postaggregation stages, gp80 is replaced by other surface glycoproteins in maintaining cell–cell adhesion. One of them has a Mr of 150 000 (gp150). Anti-gp150 antibodies have no effect on aggregation-stage cells, but they disrupt cell–cell adhesion at subsequent stages. It becomes evident that the complex phenomena of cell adhesion and tissue organization involve the participation of a number of surface glycoproteins.