Signal Transduction Elements Research Articles

The role of cytosolic phospholipase A(2) in insulin secretion.

Cytosolic phospholipase A(2) (cPLA(2)) comprises a widely expressed family of enzymes, some members of which have the properties required of signal transduction elements in electrically excitable cells. Thus, alpha- and beta-isoforms of cPLA(2) are activated by the increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) achieved in depolarized cells. Activation is associated with a redistribution of the enzyme within the cell; activation of cPLA(2) generates arachidonic acid (AA), a biologically active unsaturated fatty acid that can be further metabolized to generate a plethora of biologically active molecules. Studies using relatively nonselective pharmacological inhibitors have implicated cPLA(2) in insulin secretory responses to stimuli that elevate beta-cell [Ca(2+)](i); therefore, we have investigated the role of cPLA(2) in beta-cell function by generating beta-cell lines that under- or overexpress the alpha-isoform of cPLA(2). The functional phenotype of the modified cells was assessed by observation of cellular ultrastructure, by measuring insulin gene expression and insulin protein content, and by measuring the effects of insulin secretagogues on cPLA(2) distribution, on changes in [Ca(2+)](i), and on the rate and pattern of insulin secretion. Our results suggest that cPLA(2) is not required for the initiation of insulin secretion from beta-cells, but that it plays an important role in the maintenance of beta-cell insulin stores. Our data also demonstrate that excessive production of, or exposure to, AA is deleterious to normal beta-cell secretory function through metabolic dysfunction.

Bone morphogenetic protein 2 is expressed by, and acts upon, mature epithelial cells in the colon

Background & Aims: The recent findings of bone morphogenetic protein (BMP) receptor Ia mutations in juvenile polyposis and frequent Smad4 mutations in colon cancer suggest a role for BMPs in the colonic epithelium and colon cancer. We investigated the role of BMP2 in the colon. Methods: We assessed BMP receptor expression in cell lines using the reverse-transcribed polymerase chain reaction and immunoblotting. We investigated the effect of BMP2 on cell lines using the MTT assay and by immunoblotting for markers of differentiation, proliferation, and apoptosis. We assessed the expression of BMP2, its receptors, and signal transduction elements in mouse and human colon tissue using immunohistochemistry. We also investigated the effect of the BMP antagonist noggin in vivo in mice by assessing colon tissue with immunohistochemistry and immunoblotting. Finally, we investigated the expression of BMP2 in microadenomas from familial adenomatous polyposis patients. Results: BMP receptors (BMPR) Ia, BMPR Ib, and BMPR II are all expressed in colonic epithelial cell lines. BMP2 inhibits colonic epithelial cell growth in vitro, promoting apoptosis and differentiation and inhibiting proliferation. BMP2, BMPRIa, BMPRIb, BMPRII, phosphorylated Smad1, and Smad4 are expressed predominantly in mature colonocytes at the epithelial surface in normal adult human and mouse colon. Noggin inhibits apoptosis and proliferation in mouse colonic epithelium in vivo. BMP2 expression is lost in the microadenomas of familial adenomatous polyposis patients. Conclusions: These data suggest that BMP2 acts as a tumor suppressor promoting apoptosis in mature colonic epithelial cells.

Ovarian carcinoma cells inhibit T cell proliferation: suppression of IL-2 receptor β and γ expression and their JAK-STAT signaling pathway

Deficient T cell immune function and intracellular signaling in cancer patients may result from effects of tumors or their products on lymphocytes. Recently, it was demonstrated that several ovarian carcinoma cell lines could produce soluble factors that inhibited T cell proliferation. The aim of this study is to assess the effect of supernatants from 3 ovarian carcinoma cell lines (OVCAR3, CAOV3, SKOV3) on signal transduction elements that are linked to the IL-2R and its JAK-STAT pathway. A profound inhibition of proliferation, lower level of IFN-γ and higher level of IL-10 gene expression were observed when CD8 + T cells were co-cultured with supernatants from 3 ovarian carcinoma cell lines. Cell cycle studies on inhibited CD8 + T cells showed most of them were growth arrested in G 0/G 1 phase. Western blot analysis showed that tumor supernatants suppressed expression of JAK3 and tyrosine phosphorylation of STAT5. JAK1 was not altered and the inhibition of STAT3 only appeared in OVCAR3 cells. Tumor supernatants also partially blocked induction of IL-2R β and γ chains expression. These findings suggest that ovarian carcinoma cells may suppress T cell proliferation through inhibition IL-2 dependent signaling pathways, which may be a mechanism of ovarian carcinoma induced immunosuppression.

Signal Events: Cell Signal Transduction and Its Inhibition in Cancer

After completing this course, the reader will be able to: Recognize the importance of identifying new molecular targets for cancer therapy and how they relate to the development of novel therapeutic agents with high therapeutic indices and low toxicity. Explain the process of signal transduction (specifically the ErbB family of receptors and the mTOR pathways) and how it relates to cell growth, differentiation, and survival, and describe the effects of aberrations in signal transduction on the development of neoplasms. Describe some of the novel targeted therapies that have been developed and explain the mechanisms by which signal transduction inhibitors inhibit tumor growth and induce tumor regression in patients with cancer. Access and take the CME test online and receive one hour of AMA PRA category 1 credit at CME.TheOncologist.com Signal transduction refers to communication processes used by regulatory molecules to mediate the essential cell processes of growth, differentiation, and survival. Signal transduction elements interact through complex biochemically related networks. Aberrations in signal transduction elements can lead to increased proliferative potential, sustained angiogenesis, tissue invasion and metastasis, and apoptosis inhibition. Most human neoplasms have aberrant signal transduction elements. Several compounds that target aberrant signal transduction elements, such as those in the ErbB family of tyrosine kinase receptors and mammalian target of rapamycin, are in development. To date, commercially available signal-transduction-targeting compounds include trastuzumab, a monoclonal antibody against the ErbB-2 receptor for the treatment of metastatic breast cancer overexpressing the ErbB-2 (HER-2) receptor, and gefitinib, an inhibitor of the ErbB-1 receptor tyrosine kinase that recently received regulatory approval for the treatment of patients with non-small cell lung cancer. In contrast to traditional cytotoxic treatments, although signal transduction inhibitors are capable of inducing tumor regression, particularly in malignancies that are principally driven by specific target aberrations, preclinical and early clinical investigations suggest that their predominant beneficial effects are growth inhibitory in nature; therefore, new clinical trial designs and evaluation end points may be required to ultimately assess their value. Prospective profiling of patients and tumors to determine treatment response is also essential to the success of these clinical trials. However, responsiveness to these novel therapies is dependent on a multitude of factors that ultimately determine the robustness and quality of the downstream response.

Signal events: Cell signal transduction and its inhibition in cancer.

Signal transduction refers to communication processes used by regulatory molecules to mediate the essential cell processes of growth, differentiation, and survival. Signal transduction elements interact through complex biochemically related networks. Aberrations in signal transduction elements can lead to increased proliferative potential, sustained angiogenesis, tissue invasion and metastasis, and apoptosis inhibition. Most human neoplasms have aberrant signal transduction elements. Several compounds that target aberrant signal transduction elements, such as those in the ErbB family of tyrosine kinase receptors and mammalian target of rapamycin, are in development. To date, commercially available signal-transduction-targeting compounds include trastuzumab, a monoclonal antibody against the ErbB-2 receptor for the treatment of metastatic breast cancer overexpressing the ErbB-2 (HER-2) receptor, and gefitinib, an inhibitor of the ErbB-1 receptor tyrosine kinase that recently received regulatory approval for the treatment of patients with non-small cell lung cancer. In contrast to traditional cytotoxic treatments, although signal transduction inhibitors are capable of inducing tumor regression, particularly in malignancies that are principally driven by specific target aberrations, preclinical and early clinical investigations suggest that their predominant beneficial effects are growth inhibitory in nature; therefore, new clinical trial designs and evaluation end points may be required to ultimately assess their value. Prospective profiling of patients and tumors to determine treatment response is also essential to the success of these clinical trials. However, responsiveness to these novel therapies is dependent on a multitude of factors that ultimately determine the robustness and quality of the downstream response.

Gravitactic signal transduction elements in Astasia longa investigated during parabolic flights.

Euglena gracilis and its close relative Astasia longa show a pronounced negative gravitactic behavior. Many experiments revealed that gravitaxis is most likely mediated by an active physiological mechanism. The goal of the present study was to examine elements in the sensory transduction by means of inhibitors of gravitaxis and the intracellular calcium concentration during short microgravity periods. During the course of six parabolic flights (ESA 31th parabolic flight campaign and DLR 6th parabolic flight campaign) the effects of trifluoperazine (calmodulin inhibitor), caffeine (phosphodiesterase inhibitor) and gadolinium (blocks mechano-sensitive ion channels) was investigated. Due to the extreme parabolic flight maneuvers of the aircraft alternating phases of 1.8 x g(n) (about 20 s) and microgravity (about 22 s) were achieved (g(n): acceleration of Earth's gravity field). The duration of the microgravity periods was sufficient to detect a loss of cell orientation in the samples. In the presence of gadolinium impaired gravitaxis was found during acceleration, while caffeine-treated cells showed, compared to the controls, a very precise gravitaxis and faster reorientation in the 1.8 x g(n) period following microgravity. A transient increase of the intracellular calcium upon increased acceleration was detected also in inhibitor-treated samples. Additionally, it was found that the cells showed a higher calcium signal when they deviated from the vertical swimming direction. In the presence of trifluoperazine a slightly higher general calcium signal was detected compared to untreated controls, while gadolinium was found to decrease the intracellular calcium concentration. In the presence of caffeine no clear changes of intracellular calcium were detected compared to the control.

Biosensors for environmental monitoring of endocrine disruptors: a review article.

This article provides an overview of the applications of biosensors in analysis and monitoring of endocrine-disrupting compounds (EDCs) in the environment. Special attention is devoted to the various types of physical-chemical signal transduction elements, biological mechanisms employed as sensing elements and techniques used for immobilisation of the bioreceptor molecules on the transducer surface. Two different classes of biosensors for EDCs are considered: biosensors that measure endocrine-disrupting effects, and biosensors that respond to the presence of a specific substance (or group of substances) based on the specific recognition of a biomolecule. Several examples of them are presented to illustrate the power of the biosensor technology for environmental applications. Future trends in the development of new, more advanced devices are also outlined.

CaMKII-dependent Phosphorylation Regulates SAP97/NR2A Interaction

Synapse-associated protein 97 (SAP97), a member of membrane-associated guanylate kinase protein family, has been implicated in the processes of targeting ionotropic glutamate receptors at postsynaptic sites. Here we show that SAP97 is enriched at the postsynaptic density where it co-localizes with both ionotropic glutamate receptors and downstream signaling proteins such as Ca2+/calmodulin-dependent protein kinase II (CaMKII). SAP97 and alphaCaMKII display a high co-localization pattern in hippocampal neurons as well as in transfected COS-7 cells. Metabolic labeling of hippocampal cultures reveals that N-methyl-D-aspartic acid (NMDA) receptor activation induces CaMKII-dependent phosphorylation of SAP97; co-incubation with the CaMKII-specific inhibitor KN-93 reduces SAP97 phosphorylation to basal levels. Our results show that SAP97 directly interacts with the NR2A subunit of NMDA receptor both in an in vitro "pull-out" assay and in co-immunoprecipitation experiments from homogenates and synaptosomes purified from hippocampal rat tissue. Interestingly, in the postsynaptic density fraction, SAP97 fails to co-precipitate with NR2A. We show here that SAP97 is directly associated with NR2A through its PDZ1 domain, and CaMKII-dependent phosphorylation of SAP97-Ser-232 disrupts NR2A interaction both in an in vitro pull-out assay and in transfected COS-7 cells. Moreover, expression of SAP97(S232D) mutant has effects similar to those observed upon constitutively activating CaMKII. Our findings suggest that SAP97/NR2A interaction is regulated by CaMKII-dependent phosphorylation and provide a novel mechanism for the regulation of synaptic targeting of NMDA receptor subunits.

Targeted therapy of solid malignancies via HLA class II antigens: a new biotherapeutic approach?

Intracellular signals, delivered in professional antigen-presenting cells following the engagement of major histocompatibility complex (MHC) class II molecules, activate a variety of cellular functions that also contribute to efficient antigen presentation. As far as human malignancies, the signaling ability of human leukocyte antigens (HLA) class II molecules is a rather well-characterized event in hematologic tumors; in contrast, very limited evidences are available in solid neoplasias of different histotypes that may constitutively express HLA class II antigens. Among solid malignancies, a significant proportion of human cutaneous melanomas have been shown to express HLA class II molecules, and cutaneous melanoma undoubtedly represents a 'model disease' to investigate tumor immunobiology, to unveil the molecular basis underlying the interactions between neoplastic cells and host's immune system, and ultimately to set up new bio-immunotherapeutic approaches. Upcoming preclinical evidences unveil a signaling potential of HLA-DR antigens expressed on melanoma cells, and suggest for the clinical implication of HLA class II molecules as novel therapeutic targets. Therefore, in this review, we will focus on the emerging role of HLA class II antigens as intracellular signal transducing elements in neoplastic cells of the melanocytic lineage, emphasizing their foreseeable role in targeted therapy of human melanoma and potentially of HLA class II antigens-positive tumors of different histology.

Tumour necrosis factor-alpha- vs. growth factor deprivation-promoted cell death: distinct converging pathways.

Perturbations of neuronal physiological homeostasis are likely to underscore neuronal demise/impairments that are reportedly associated with aging of the central nervous system and age-related neurodegenerative diseases such as Alzheimer's disease (AD). A number of age- and/or disease-associated neurotoxic events has been described. These include abnormally modified proteins such as beta amyloid and hyper-phosphorylated Tau, cytokines such as tumour necrosis factor-alpha (TNFalpha), high levels of free radicals conducive to oxidative stress, and impaired/decreased neuronal trophic support by neurotrophic factors. Overall, it could be argued that toxic events in the aged brain are either active, such as those due to a direct action of cytokines, or passive, such as those due to lack of growth factor support. It is therefore conceivable that cellular responses to such diverse toxic stimuli are different, suggesting that interventions should be targeted accordingly. In order to begin answering this question, we determined in PC12 cells the time course of activity, in response to TNFalpha (active) or growth factor withdrawal (passive), of protein kinase c-zeta (PKCzeta), nuclear factor kappa B (NFkappaB), caspases 3 and 8, and poly (ADP-ribose) polymerase (PARP), key signal transduction elements associated with modulation of cell death/survival in PC12 cells. We found that the overall activity of PKCzeta, NFkappaB and caspase 8 was significantly different depending on the apoptotic initiator. The pattern of caspase 3 and PARP activity, however, was not statistically different between serum-free- and TNFalpha-induced cell death conditions. This suggests that two distinct cell responses are elicited that converge at caspase 3, which then induces downstream events involved in the execution of a common apoptotic programme. These results contribute to the aim of differentially targeting neuronal death in the aged brain (characterized by neurotrophic factor impairments) or in the diseased brain (e.g. AD, characterized by elevated levels of pro-inflammatory cytokines).

CDNA-AFLP analysis of inducible gene expression in rice seminal root tips under a water deficit

The seminal roots of an upland rice variety, Azucena, showed accelerated elongation in response to a water deficit. The elongation of cortical cells in the elongation zone is rapidly stimulated within 16 h by the water deficit. cDNA-AFLP analysis was used to examine gene expression in seminal root tips at four time points (4, 16, 48 and 72 h) during the water deficit. One hundred and six unique genes induced by the water deficit were obtained. The expression patterns of these genes were confirmed by Northern blot analysis based on 21 selected genes representing different patterns. The 106 upregulated genes were composed of 60 genes of known function, 28 genes of unknown function and 18 novel genes. Sixty genes of known functions were involved in transport facilitation, metabolism and energy, stress- and defense-related proteins, cellular organization and cell-wall biogenesis, signal transduction, expression regulator and transposable element, suggesting that seminal root tips undergo a complex adaptive process in response to the water deficit. Expression of 22 genes reached a maximum within 16 h of water deficit treatment. These included aquaporin ( PIP2a), 9- cis-epoxycarotenoid dioxygenase ( NCED1) and a negative regulator of gibberellin signal transduction ( SPY); eight other genes participated in cell wall loosening or vesicle traffic.

Threonine phosphorylation sites in the beta 2 and beta 7 leukocyte integrin polypeptides.

The cytoplasmic domains of integrins play a key role in a variety of integrin-mediated events including adhesion, migration, and signaling. The molecular mechanisms that enhance integrin function are still incompletely understood. Because protein kinases are known to be involved in the signaling and the activation of integrins, the role of phosphorylation has been studied by several groups. The beta(2) leukocyte integrin subunit has previously been shown to become phosphorylated in leukocytes on cytoplasmic serine and functionally important threonine residues. We have now mapped the phosphorylated threonine residues in activated T cells. After phorbol ester stimulation, all three threonine residues (758-760) of the threonine triplet became phosphorylated but only two at a time. CD3 stimulation leads to a strong threonine phosphorylation of the beta(2) integrin, but differed from phorbol ester activation in that phosphorylation occurred only on threonine 758. The other leukocyte-specific integrin, beta(7), has also been shown to need the cytoplasmic domain and leukocyte-specific signal transduction elements for integrin activation. Cell activation with phorbol ester, and interestingly, through the TCR-CD3 complex, caused beta(7) integrin binding to VCAM-1. Additionally, cell activation led to increased phosphorylation of the beta(7) subunit, and phosphoamino acid analysis revealed that threonine residues became phosphorylated after cell activation. Sequence analysis by manual radiosequencing by Edman degradation established that threonine phosphorylation occurred in the same threonine triplet as in beta(2) phosphorylation.

HIV-1 Nef control of cell signalling molecules: multiple strategies to promote virus replication.

HIV-1 has at its disposal numerous proteins encoded by its genome which provide the required arsenal to establish and maintain infection in its host for a considerable number of years. One of the most important and enigmatic of these proteins is Nef. The Nef protein of HIV-1 plays a fundamental role in the virus life cycle. This small protein of approximately 27 kDa is required for maximal virus replication and disease progression. The mechanisms by which it is able to act as a positive factor during virus replication is an area of intense research and although some controversy surrounds Nef much has been gauged as to how it functions. Its ability to modulate the expression of key cellular receptors important for cell activation and control signal transduction elements and events by interacting with numerous cellular kinases and signalling molecules, including members of the Src family kinases, leading to an effect on host cell function is likely to explain at least in part its role during infection and represents a finely tuned mechanism where this protein assists HIV-1 to control its host.

Hydroxamate Based Inhibitors of Adenylyl Cyclase. Part 1. The Effect of Acyclic Linkers on P‐Site Binding.

AbstractFor Abstract see ChemInform Abstract in Full Text.

EXPRESSION AND LOCATION OF VASP IN HUMAN ENDOTHELIAL CELLS: EFFECT OF A LAMINAR SHEAR STRESS

Recent elucidation of the primary VASP (vasodilator-simulated phosphoprotein) and identity of VASP binding proteins suggests that VASP is an important component of focal contacts which links signal transduction pathways and elements controlling cell motility. The aim of our study was to evaluate shear-stress-induced changes of VASP expression and localization in ECs. We showed, by western blotting measurements, that the shear stress involve some modifications on the expression of phosphorilated (50 kD) and non phosphorilated (46kD) VASP. Moreover, a fluorescence double-labeling shows the location of VASP on the actin fibers. At rest, ECs showed an array of microfilament bundles of the actin fibers and VASP were along their entire length. After exposure to shear stress, the stress fibers appeared and were oriented along with the flow. There were a thicker expression of VASP than in control, targeted to the ends of stress fibres. This seems to be an adjustment of the cells towards the mechanical stresses. These results suggest that VASP is a potential important component which participates in the regulation of cell actin remodelling induced by shear flow. VASP were involved in the mechano-transduction pathways.

P38 Mitogen-activated Protein Kinase and Extracellular Signal-regulated Kinases Play Distinct Roles in the Activation of Dendritic Cells by Two Representative Haptens, NiCl2 and 2,4-dinitrochlorobenzene

Previous studies have demonstrated that haptens induce several phenotypic and functional changes of dendritic cells in vivo as well as in vitro. Although recently, the crucial role of p38 mitogen-activated protein kinase has been reported in the activation of dendritic cells by haptens, the signal transduction elements involved in each phenotypic and functional changes that occur in the activation of dendritic cells by haptens remain unknown. Therefore, we examined the role of mitogen-activated protein kinases and nuclear factor-kappaB in the signal transduction of dendritic cells stimulated with two representative haptens, i.e., NiCl2 and 2,4-dinitrochlorobenzene. Human monocyte-derived dendritic cells stimulated with 2,4-dinitrochlorobenzene induced the phosphorylation of p38 and stress-activated protein kinase/c-jun N-terminal kinases, whereas NiCl2 induced that of p44/42 extracellular signal-regulated kinases, p38, and stress-activated protein kinase/c-jun N-terminal kinases. In addition, NiCl2 phosphorylated inhibitor kappaB and activated nuclear factor-kappaB. In contrast, primary irritants, e.g., benzalkonium chloride, or sodium lauryl sulfate, did not activate these signal transduction pathways. By using specific inhibitors for extracellular signal-regulated kinases and p38 pathways, PD98059 and SB203580, respectively, we demonstrated that the augmentation of CD86, HLA-DR, and CD83, and the production of interleukin-8 along with its increased mRNA expression by monocyte-derived dendritic cells stimulated with 2,4-dinitrochlorobenzene, and the augmentation of CD83 and the interleukin-12 p40 production by monocyte-derived dendritic cells stimulated with NiCl2, were suppressed by SB203580, whereas PD98059 suppressed the production of interleukin-1beta and tumor necrosis factor-alpha, together with their increased mRNA expression by monocyte-derived dendritic cells treated with NiCl2. On the other hand, in spite of the activation of nuclear factor-kappaB by monocyte-derived dendritic cells stimulated with NiCl2, nuclear factor-kappaB inhibitor did not significantly affect the phenotypic and functional changes in the activation of monocyte-derived dendritic cells. These data indicate that NiCl2 and 2,4-dinitrochlorobenzene stimulate different signal transduction pathways in monocyte-derived dendritic cells, and subsequently induce different phenotypic and functional changes in them.

The role of octadecanoids and functional mimics in soybean defense responses.

Oxylipins of the jasmonate pathway and synthetic functional analogs have been analyzed for their elicitor-like activities in an assay based on the induced accumulation of glyceollins, the phytoalexins of soybean (Glycine max L.), in cell suspension cultures of this plant. Jasmonic acid (JA) and its methyl ester showed weak phytoalexin-inducing activity when compared to an early jasmonate biosynthetic precursor, 12-oxo-phytodienoic acid (OPDA), as well as to the bacterial phytotoxin coronatine and certain 6-substituted indanoyl-L-isoleucine methyl esters, which all were highly active. Interestingly, different octadecanoids and indanoyl conjugates induced the accumulation of transcripts of various defense-related genes to different degrees, indicating distinct induction competencies. Therefore, these signaling compounds and mimics were further analyzed for their effects on signal transduction elements, such as the transient enhancement of the cytosolic Ca2+ concentration and MAP kinase activation, which are known to be initiated by a soybean pathogen-derived beta-glucan elicitor. In contrast to the beta-glucan elicitor, none of the other compounds tested triggered these early signaling elements. Moreover, endogenous levels of OPDA and JA in soybean cells were shown to be unaffected after treatment with beta-glucans. Thus, OPDA and JA, which are functionally mimicked by coronatine and a variety of 6-substituted derivatives of indanoyl-L-isoleucine methyl ester, represent highly efficient signaling compounds of a lipid-based pathway not deployed in the beta-glucan elicitor-initiated signal transduction.

Signal transduction mechanism leading to enhanced proliferation of primary cultured adult rat hepatocytes treated with royal jelly 57-kDa protein.

A 57-kDa protein in royal jelly (RJ) was previously shown to stimulate hepatocyte DNA synthesis and prolongs the proliferation of hepatocytes as well as increasing albumin production [Kamakura, M., Suenobu, N., and Fukushima, M. (2001) Biochem. Biophys. Res. Commun. 282, 865-874]. In this study, I investigated the signal transduction mechanisms involved in the induction of hepatocyte DNA synthesis and the promotion of cell survival by this 57-kDa protein in primary cultures of adult rat hepatocytes. Hepatocyte DNA synthesis induced by the 57-kDa protein was not influenced by several alpha- and beta-adrenoceptor antagonists, but was dose-dependently abolished by an inhibitor of a tyrosine-specific protein kinase, genistein. A phospholipase C inhibitor (U-73122) and a protein kinase C (PKC) inhibitor (sphingosine) inhibited 57-kDa protein-stimulated he-patocyte DNA synthesis, whereas a protein kinase A inhibitor (H-89) did not. The 57-kDa protein also activated PKC in rat hepatocytes. Various inhibitors of intracellular signal transduction elements (PD98059, p21 ras farnesyltransferase inhibitor, wortmannin and rapamycin) also blocked hepatocyte DNA synthesis induced by the 57-kDa protein. Furthermore, the 57-kDa protein activated mitogen-activated protein (MAP) kinase in rat hepatocytes. The activation of MAP kinase by the 57-kDa protein was inhibited by PD98059 and sphingosine. The 57-kDa protein also activated protein kinase B, which is a key regulator of cell survival. These results suggest that, like growth factors, the 57-kDa protein activates several important intracellular signaling factors involved in the stimulation of hepatocyte DNA synthesis and the protection of cells from apoptosis.

Hydroxamate based inhibitors of adenylyl cyclase. Part 2: The effect of cyclic linkers on P-site binding

The adenylyl cyclases (ACs) are a family of enzymes that are key elements of signal transduction by virtue of their ability to convert ATP to cAMP. The catalytic mechanism of this transformation proceeds through initial binding of ATP to the purine binding site (P-site) followed by metal mediated cyclization with loss of pyrophosphate. Previous work in our group identified novel inhibitors which possess an adenine ring joined to a metal-coordinating hydroxamic acid through flexible linkers. Considering the spatial positioning of the metals with respect to the adenine binding site coupled with potentially favorable entropic factors, conformational restriction of the tether through a stereochemistry based SAR employing a rigid cyclic scaffold was explored.

Hydroxamate based inhibitors of adenylyl cyclase. Part 1: The effect of acyclic linkers on P-site binding

The adenylyl cyclases (ACs) are a family of enzymes that are key elements of signal transduction by virtue of their ability to convert ATP to cAMP. The catalytic mechanism of this transformation proceeds through initial binding of ATP to the purine binding site (P-site) followed by metal mediated cyclization with loss of pyrophosphate. Crystallographic analysis of ACs with known inhibitors reveals the presence of two metals in the active site. Presently, nine isoforms of adenylyl cyclase are known and unique isoform combinations are expressed in a tissue specific manner. The development of isoform specific inhibitors of adenylyl cyclase may prove to be a useful strategy toward the design of novel therapeutic agents. In order to develop novel AC inhibitors, we have chosen a design approach utilizing molecules with the adenine ring system joined to a metal-coordinating hydroxamic acid via flexible acyclic linkers. The designed inhibitors were assayed against type V AC with the size and heteroatom content of the linkers varied to probe the interaction of the nucleotide and metal binding sites within the enzyme.