A silicon-based device, dubbed a microphysiometer, can be used to detect and monitor the response of cells to a variety of chemical substances, especially ligands for specific plasma membrane receptors. The microphysiometer measures the rate of proton excretion from 10(4) to 10(6) cells. This article gives an overview of experiments currently being carried out with this instrument with emphasis on receptors with seven transmembrane helices and tyrosine kinase receptors. As a scientific instrument, the microphysiometer can be thought of as serving two distinct functions. In terms of detecting specific molecules, selected biological cells in this instrument serve as detectors and amplifiers. The microphysiometer can also investigate cell function and biochemistry. A major application of this instrument may prove to be screening for new receptor ligands. In this respect, the microphysiometer appears to offer significant advantages over other techniques.

The trkB family of transmembrane proteins serves as receptors for BDNF and NT-4/5. The family is composed of a tyrosine kinase-containing isoform as well as several alternatively spliced "truncated receptors" with identical extracellular ligandbinding domains but very small intracellular domains. The two best-characterized truncated trkB receptors, designated as trkB.T1 and trkB.T2, contain intracellular domains of only 23 and 21 amino acids, respectively. Although it is known that the tyrosine kinase isoform (trkB.FL) is capable of initiating BDNF and NT-4/5-induced signal transduction, the functional role or roles of the truncated receptors remain enigmatic. At the same time, the potential importance of the truncated receptors in the development, maintenance, and regeneration of the nervous system has been highlighted by recent developmental and injury paradigm investigations. Here we have used trkB cDNA transfected cell lines to demonstrate that both trkB.T1 and trkB.T2 are capable of mediating BDNF-induced signal transduction. More specifically, BDNF activation of either trkB.T1 or trkB.T2 increases the rate of acidic metabolite release from the cell, a common physiological consequence of many signaling pathways. Further, these trkB.T1-and trkB.T2-mediated changes occur with kinetics distinct from changes mediated by trkB.FL, suggesting the participation of at least some unique rate-limiting component or components. Mutational analysis demonstrates that the isoform-specific sequences within the intracellular domains of each receptor are essential for signaling capability. Finally, inhibitor studies suggest that kinases are likely to be involved in the trkB.T1 and trkB.T2 signaling pathways.

The early steps that lead to the rise in calcium and egg activation at fertilization are unknown but of great interest--particularly with the advent of in vitro fertilization techniques for treating male infertility and whole-animal cloning by nuclear transfer. This calcium rise is required for egg activation and the subsequent events of development in eggs of all species. Injection of intact sperm or sperm extracts can activate eggs, suggesting that sperm-derived factors may be involved. Here we show that nitric oxide synthase is present at high concentration and active in sperm after activation by the acrosome reaction. An increase in nitrosation within eggs is evident seconds after insemination and precedes the calcium pulse of fertilization. Microinjection of nitric oxide donors or recombinant nitric oxide synthase recapitulates events of egg activation, whereas prior injection of oxyhaemoglobin, a physiological nitric oxide scavenger, prevents egg activation after fertilization. We conclude that nitric oxide synthase and nitric-oxide-related bioactivity satisfy the primary criteria of an egg activator: they are present in an appropriate place, active at an appropriate time, and are necessary and sufficient for successful fertilization.

This is the second publication of Clinical Development Plans from the National Cancer Institute, Division of Cancer Prevention and Control, Chemoprevention Branch and Agent Development Committee. The Clinical Development Plans summarize the status of promising chemopreventive agents regarding evidence for safety and chemopreventive efficacy in preclinical and clinical studies. They also contain the strategy for further development of these drugs, addressing pharmacodynamics, drug effect measurements, intermediate biomarkers for monitoring efficacy, toxicity, supply and formulation, regulatory approval, and proposed clinical trials. Sixteen new Clinical Development Plans are presented here: curcumin, dehydroepiandrosterone, folic acid, genistein, indole‐3‐carbinol, perillyl alcohol, phenethyl isothiocyanate, 9‐cis‐retinoic acid, 13‐cis‐retinoic acid, l‐selenomethionine and 1,4‐phenylenebis(methylene)selenocyanate, sulindac sulfone, tea, ursodiol, vitamin A, and (+)‐vorozole. The objective of publishing these plans is to stimulate interest and thinking among the scientific community on the prospects for developing these and future generations of chemopreventive drugs. © 1997 Wiley‐Liss, Inc.

The TF-1 human erythroleukemic cell line exhibits opposing physiological responses toward tumor necrosis factor-␣ (TNF) treatment, dependent upon the mitotic state of the cells. Mitotically active cells in log growth respond to TNF by rapidly undergoing apoptosis whereas TNF exposure stimulates cellular proliferation in mitotically quiescent cells. The concentrationdependent TNF-induced apoptosis was monitored by cellular metabolic activity and confirmed by both DNA epifluorescence and DNA fragmentation. Moreover, these responses could be detected by measuring extracellular acidification activity, enabling rapid prediction (within ϳ 1.5 h of TNF treatment) of the fate of the cell in response to TNF. Growth factor resupplementation of quiescent cells, resulting in reactivation of cell cycling, altered TNF action from a proliferative stimulus to an apoptotic signal. Expression levels of the type II TNF receptor subtype (p75TNFR) were found to correlate with sensitivity to TNF-induced apoptosis. Pretreatment of log growth TF-1 cells with a neutralizing antip75TNFR monoclonal antibody inhibited TNF-induced apoptosis by greater than 80%. Studies utilizing TNF receptor subtype-specific TNF mutants and neutralizing antisera implicated p75TNFR in TNF-dependent apoptotic signaling. These data show a bifunctional physiological role for TNF in TF-1 cells that is dependent on mitotic activity and controlled by the p75TNFR.Cells have the capability of responding to a multitude of signals that it encounters in its extracellular environment. One such signal with widespread pleiotropic actions is the cytokine tumor necrosis factor-␣ (TNF) 1 (1). TNF has been shown to modulate proliferation, differentiation, and apoptotic or necrotic cell death in a number of different cell types (2-4). These disparate responses to TNF are mediated by TNF binding to specific cell surface receptors. Two distinct TNF receptors, type I (p55TNFR) and type II (p75TNFR) (M r 55,000 -60,000 and 70,000 -80,000 in human cells, respectively), have been identified (5, 6), although it remains unclear which of the many responses reported for TNF can be attributed to a specific receptor subtype (4). Moreover, the precise signal transduction pathways for each of these receptor subtypes have yet to be fully delineated. One action of TNF, the induction of apoptosis, is characterized by a discrete set of cellular events regulated by gene expression (7,8). The physiological events accompanying apoptosis include condensation of the chromatin, degradation of DNA through the activation of endogenous nucleases, and dissolution of the cell into small membrane-bound apoptotic vesicles (9, 10). In vivo, these vesicles are phagocytosed by macrophages or other phagocytic cells. Cell death by apoptosis is essential in many physiological processes, including embryonic development of the nervous system (11), oncogenic pathology (12), and clonal selection of hematopoietic cells (13).Conversely, TNF has also been shown to stimulate cellular proliferation in a variety of systems...

The regulatory guanine nucleotide binding protein (G protein) activators cholera toxin and the GTP analog 5-guanylyl imidophosphate, the second messenger diacylglycerol, and certain diamino acids all facilitate (amplify) the settlement and metamorphic responses of planktonic larvae of Haliotis rufescens (marine mollusc) to morphogenetic chemical stimuli. In contrast, the G protein-inhibiting analog guanosine 5'-0-[,B-thioldiphosphate inhibits facilitation by L-a,4-diaminopropionic acid but does not block facilitation by diacylglycerol. Diacylglycerol, cholera toxin, and the guanine nucleotide analogs alone neither induce the settlement and metamorphosis of the larvae nor do they inhibit induction of metamorphosis by y-ammnobutyric acid. These results thus establish the existence of separate regulatory and inductive pathways controlling larval metamorphosis in response to two classes of exogenous chemical signals from the environment. The regulatory pathway, operating independently through a G protein-diacylglycerol cascade apparently controlled by facilitating diamino acids in the water column, can amplify the larval responsiveness to inducers of metamorphosis. This mechanism may have adaptive significance in the recognition and selection of favorable habitats for metamorphosis of the larvae. Similar regulatory pathways, based on exogenous control of a G protein-diacylglycerol cascade, may govern responsiveness to stimuli in other sensory and developmental systems.Planktonic larvae of the marine gastropod mollusc Haliotis rufescens are induced to settle from the water column, attach to substrate, and metamorphose in response to exogenous y-aminobutyric acid (GABA)-mimetic peptides that are found at the surfaces of specific algae; GABA and GABA analogs also induce this metamorphosis (1-5). The morphogenetic peptides, GABA, and GABA analogs all are recognized by a discrete population of larval chemosensory receptors that have been characterized directly by radioligand binding (5-7). The settlement and metamorphosis of Haliotis larvae in response to low concentrations of any of these inducers can be facilitated, or amplified, by several diamino acids structurally related to lysine, including L-a,3-diaminopropionic acid (DAPA) (see Fig. 1A), without altering the number or affinity of the GABA chemoreceptors (6)(7)(8).Recent evidence has shown that this facilitation of the morphogenetic response occurs at a postreceptor level independent of inducer binding at the GABA chemoreceptors (6). We present evidence here that transduction of the facilitating DAPA signal is mediated by a regulatory pathway that is separate from the morphogenetic pathway. Our evidence suggests that this regulatory pathway involves a diacylglycerol second messenger and that the level of this second messenger is regulated by a guanine nucleotide binding protein. MATERIALS AND METHODS, and rifampicin were purchased from Sigma. DAPA was from CalbiochemBehring. The cyanobacterial GABA-mimetic peptide fraction was obtained as described (9). P...

The early events that occur after treatment of the highly interferon a (IFN-a)-sensitive human lymphoblastoid Daudi cell line with human leukocyte IFN-a have been examined. IFN-a treatment ofDaudi cells results in a rapid and transient increase in the cellular content of diacylglycerol, which occurs in the absence of inositol phospholipid turnover, or an increase in intracellular calcium concentration. Furthermore, IFN-a treatment results in a selective, time-dependent activation of the Ca2+-independent E isoform of protein kinase C (PKC), while the a isoform is unaffected by IFN-a treatment. In contrast, IFN-a treatment of an IFN-resistant subclone of Daudi cells had no effect on the diacylglycerol content of cells and on the activation of PKC-e. The selective PKC inhibitor staurosporine blocked the transcriptional activation of IFN-a-stimulated genes, the cytoplasmic accumulation of mRNAs for these genes, and the induction of antiviral activity by IFN-a against vesicular stomatitis virus in IFN-sensitive cells. These observations suggest that transmembrane signaling of IFN-a involves diacylglycerol production and activation of PKC-E in Daudi cells.-,411, and -y) and Ca2+-independent (PKC-8, -E, -s, and -ti) enzymes. Furthermore, PKC-E has been shown to have Ca2+-independent phorbol ester binding activities and exhibits substrate specificity distinct from other characterized PKC isoforms (10, 11).We have investigated the early events that occur upon treatment of human Daudi lymphoblastoid cells with IFN-a in order to identify the biochemical pathways of transmembrane signaling. IFN-a treatment of these cell lines results in cessation of cell growth, protection against viral infection, and the rapid transcriptional activation of ISGs (12, 13). We report that the outstanding features of the signal transduction pathway of IFN-a in Daudi cells are the generation of DAG in the absence of inositol phospholipid turnover or Ca2+ elevation and the activation of the calcium-independent E isoform of PKC. Furthermore, staurosporine, a potent inhibitor of PKC activity blocks the transcriptional activation of ISGs, the cytoplasmic accumulation of ISG mRNA, and the induction of antiviral activity against vesicular stomatitis virus (VSV) in Daudi cells by IFN-a.

Abstract. A 40-kD protein kinase C (PKC)E related activity was found to associate with human epithelial specific cytokeratin (CK) polypeptides 8 and 18. The kinase activity coimmunoprecipitated with CK8 and 18 and phosphorylated immunoprecipitates of the CK. Immunoblot analysis of CK8/18 immunoprecipitates using an anti-PKCE specific antibody showed that the 40-kD species, and not native PKCE (90 kD) associated with the cytokeratins. Reconstitution experiments demonstrated that purified CK8 or CK18 associated with a 40-kD tryptic fragment of purified PKCE, or with a similar species obtained from cells that express the fragment constitutively but do not express CK8/18. C YTOKERATINS (CK)' are a group of intermediate filament (IF) proteins which are expressed primarily in epithelial tissues (Lazarides, 1982 ;Steinert and Roop, 1988 ;Franke et al ., 1981;Osborn and Weber, 1986) . The 30 or so polypeptides which make up the family ofcytokeratin proteins are divided into acidic (type I) and basic/ neutral (type II) keratins. In epithelial cells, CK are found as mosaic noncovalent polymers with an assembly consisting of at least one type I and one type II CK (Steinert and Roop, 1988). For example, "simple" single layer epithelial cells such as intestinal epithelia express CK8 (type II) and CK18 (type I), whereas esophageal epithelial cells express CK4 (type II) and CK13 (type I) predominantly. Cytokeratins are not only important as tissue-specific markers, they also form important markers of cell differentiation .IF proteins, including cytokeratins, undergo several posttranslational modifications such as N112-terminal acetylation (Steinert and Idler, 1975), glycosylation (King and Hounsell, 1989;Roberts and Brunt, 1986), and serine/threonine phosphorylation (Steinert, 1988;Gilmartin et al ., 1984 ;Yeagle et al., 1990;Baribault et al., 1989) . The functional role of IF protein phosphorylation is not well under- A peptide pseudosubstrate specific for PKCE inhibited phosphorylation of CK8/18 in intact cells or in a kinase assay with CK8/18 immunoprecipitates . Tryptic peptide map analysis -of the cytokeratins that were phosphorylated by purified rat brain PKCE or as immunoprecipitates by the associated kinase showed similar phosphopeptides . Furthermore, PKCE immunoreactive species and CK8/18 colocalized using immunofluorescent double staining . We propose that a kinase related to the catalytic fragment of PKCE physically associates with and phosphorylates cytokeratins 8 and 18.