Elements Of Signal Transduction Pathways Research Articles

G\u03b13 subunit Thga3 positively regulates conidiation, mycoparasitism, chitinase activity, and hydrophobicity of Trichoderma harzianum

Heterotrimeric G-proteins are key elements of signal transduction pathways, which participate in regulating multiple biological processes in fungi including growth, conidiation, antagonism, and mycoparasitism. Among G protein subunits, Gα3 showed diverse regulatory functions in different fungi. In this study, we cloned a Gα3 subunit coding gene thga3 from T. harzianum Th33 that can antagonize Rhizoctonia solani and some other plant pathogenic fungi. A thga3 deletion strain Δthga3 was generated using the double-crossover homologous recombination strategy, and Rthga3 was generated by transforming thga3-expressing vector into the protoplasts of Δthga3 by the PEG/CaCl2-mediated method. The biological characteristics of wild-type Th33, Δthga3 and Rthga3 were evaluated. Compared with wild-type Th33, Δthga3 showed 15%, 94%, and 23% decrease in hyphal growth, conidia yield, and chitinase activity, respectively, and Δthga3 showed lower antagonistic and mycoparasitism abilities, while there were no significant differences between wild-type Th33 and Rthga3. The hyphal surface hydrophobicity of Δthga3 significantly decreased compared with those of the wild-type Th33 and Rthga3. qRT-PCR analysis revealed that transcript abundance of the hydrophobin gene (tha_09745) of Δthga3 decreased by 80% compared with that of wild-type Th33 and Rthga3. The results showed that thga3 positively regulates the growth, conidiation, hydrophobicity, chitinase activities, and mycoparasitism of Th33 towards R. solani. We hence deduced that the expression level of Tha_09745 is correlated to the hyphal hydrophobicity of Th33 and therefore affects the other biological characteristics of Th33. The findings of this report provide a foundation for elucidating the G-protein signal regulatory mechanisms of fungi.

Tightening the Screws on PsbA in Cyanobacteria

Cyanobacterial genomes encode several isoforms of the D1 (PsbA) subunit of Photosystem II (PSII). The distinct regulation of each isoform ensures adaptation under changing environmental conditions. Uncovering the missing elements of signal transduction pathways and psbA gene expression could open new avenues in engineering programs of cyanobacterial strains.

Harnessing Environmental Ca2+ for Extracellular Protein Thermostabilization.

Ca2+ is the third-most prevalent metal ion in the environment. EF hands are common Ca2+-binding motifs found in both extracellular and intracellular proteins of eukaryotes and prokaryotes. Cytoplasmic EF hand proteins often mediate allosteric control of signal transduction pathway components in response to intracellular Ca2+ concentration fluctuations by coupling Ca2+ binding to changes in protein structure. We show that an extracellular structural Ca2+-binding site mediates protein thermostabilization by such conformational coupling as well. Binding Ca2+ to the EF hand of the extracellular (periplasmic) Escherichia coli glucose-galactose binding protein thermostabilizes this protein by ∼17 K relative to its Ca2+-free form. Using statistical thermodynamic analysis of a fluorescent conjugate of ecGGBP that reports simultaneously on ligand binding and multiple conformational states, we found that its Ca2+-mediated stabilization is determined by conformational coupling mechanisms in two independent conformational exchange reactions. Binding to folded and unfolded states determines the maximum Ca2+-mediated stability. A disorder → order transition accompanies the formation of the Ca2+ complex in the folded state and dictates the minimum Ca2+ concentration at which the Ca2+-bound state becomes dominant. Similar transitions also encode the structural changes necessary for Ca2+-mediated control elements in signal transduction pathways. Ca2+-mediated thermostabilization and allosteric control, therefore, share a fundamental conformational coupling mechanism, which may have implications for the evolution of EF hands.

Overexpression of Two Upstream Phospholipid Signaling Genes Improves Cold Stress Response and Hypoxia Tolerance, but Leads to Developmental Abnormalities in Barley

Phosphatidylinositol transfer protein (PITP) and phosphatidylinositol 4-kinase (PI4K) are very upstream regulatory elements of the phospholipid signaling pathway in the signal transduction network. Unlike in animal systems, their role in stress signaling is poorly understood in plants. To study this area, PITP- and PI4K-overexpressing transgenic barley lines were developed. Morphological and developmental abnormalities were surveyed and characterized. It was revealed that the overexpression of the upstream signaling genes led to more phenotypic abnormalities than in other transgenic studies working with effector genes or even transcription factors. We hypothesize that this high level of abnormalities is the consequence of the modulation of the very upstream signal transduction pathway elements. On the other hand, we also revealed that overexpression of the PITP and PI4K genes increased stress tolerance during hypoxic cold stress, but not during salinity stress. Differences were also found in the level of frost tolerance between the transgenic overexpression plants and the recipient Golden Promise line. Molecular analysis showed that this improvement was not related to the most important cold responsive transcription factors, the CBF genes. We conclude that the transgenic method may be useful to prove the role of an upstream signaling element; however, due to the many developmental consequences that occur as side effects, it is a less advisable approach to achieve improved stress tolerance.

Gα proteins Gvm2 and Gvm3 regulate vegetative growth, asexual development, and pathogenicityon apple in Valsa mali.

In fungi, heterotrimeric guanine-nucleotide binding proteins (G-proteins) are key elements of signal transduction pathways, which control growth, asexual and sexual development, as well as virulence. In this study, we have identified two genes encoding heterotrimeric G protein alpha subunits, named Gvm2 and Gvm3, from Valsa mali, the causal agent of apple Valsa canker. Characterization of Gvm2 and Gvm3 mutants indicates that Gvm3 may be a crucial regulator of vegetative growth. Deletion of the corresponding gene results in a 20% reduction in growth rate. Besides, Gvm2 and Gvm3 seem to be involved in asexual reproduction, and mutants are hypersensitive to oxidative and cell membrane stresses. Interestingly, both G protein alpha subunits were most probably involved in V. mali virulence. In infection assays using Malus domestica cv. ‘Fuji’ leaves and twigs, the size of lesions caused by deletion mutants △Gvm2, or △Gvm3 are significantly reduced. Furthermore, many genes encoding hydrolytic enzymes—important virulence factors in V. mali—are expressed at a lower level in these deletion mutants. Our results suggest that Gvm2 and Gvm3 play an important role in virulence probably by regulation of expression of cell wall degrading enzymes. △Gvm2, and △Gvm3 mutants were further analyzed with respect to their impact on the transcript levels of genes in the cAMP/PKA pathway. The expression of the genes encoding adenylate cyclase VmAC, protein kinase A (PKA) regulatory subunit VmPKR, and PKA catalytic subunit VmPKA1 are down-regulated in both mutants. Further analyses indicated that intracellular cAMP level and PKA activity are down-regulated in the △Gvm3 mutant, but are basically unchanged in the △Gvm2 mutant. Overall, our findings indicate that both Gvm2 and Gvm3 play diverse roles in the modulation of vegetative growth, asexual development, and virulence in V. mali.

P3.03-006 Optical Control of Growth Factor Receptors to Advance Signal Transduction Research and Drug Screening: Topic: Mesothelioma Transitional

Growth factor receptors are central elements of signal transduction pathways and increasingly important targets for anticancer drugs. In recent years naturally occurring light sensitive protein domains (LSPDs) from different kingdoms of life have been used to generate genetically encoded chimeric signaling molecules that can be activated reversibly and with spatiotemporal precision by light. The development of such optogenetic tools has led to a plethora of new discoveries in the neurosciences but has received comparably little attention in cancer research — partly due to a lack of appropriate tools. Our aim was therefore to generate synthetic growth factor receptors that can be activated with light and allow fine-tuned control of growth factor-associated signal transduction pathways. To generate receptor tyrosine kinases (RTKs) that can be optically activated (Opto-RTKs), intracellular domains of RTKs were fused to LSPDs of the light-oxygen voltage (LOV) family from various species. The resulting chimeric receptors were tested for light-dependent activation of signal transduction by reporter gene assays, immunoblotting and various cell biological tests assessing DNA synthesis, epithelial mesenchymal transition (EMT) and angiogenesis. Three of the tested LOV domains enabled light-dependent receptor dimerization and activation of the corresponding signal transduction pathways when fused to the intracellular domains of FGFR1, EGFR, RET, c-Met or ROS1. Opto-RTKs enabled stringent control of the MAPK, PI3K and PLCγ pathways. Signal activation could be spatially confined to illuminated regions of culture plates and signals rapidly subsided after cessation of illumination. Light was able to replace FGF2 for the induction of cell proliferation and EMT in mesothelioma cells and VEGF for the stimulation of angiogenic sprouting in endothelial cells. Moreover, Opto-RTKs enabled light-assisted screening for small molecule inhibitors of EGFR, FGFR1 and the orphan RTK ROS1. Our optogenetic approach allows light-mediated control of growth factor receptors representing clinically relevant drug targets. Opto-RTKs enable dissection of dynamic signals with increased spatiotemporal resolution and open new possibilities for drug screening. Transfer of the design principle to additional membrane receptors is ongoing.

Role of amplification in phospholipase Cγ2 activation in modulation of gastric mucosal inflammatory responses to Helicobacter pylori: effect of ghrelin.

Phosphoinositide-specific phospholipase C (PLC) enzymes are crucial elements of signal transduction pathways that provide a common link of communication integrating specific receptor responses to a variety of hormones, growth factors, and bacterial endotoxins with the intended intracellular targets. Here, we examined the involvement of PLC in modulation of gastric mucosal inflammatory responses to Helicobacter pylori LPS by peptide hormone, ghrelin. We show that stimulation of gastric mucosal cells with the LPS leads to the activation and membrane translocation of the γ2 isoform of PLC, phosphorylated on Tyr as well as Ser, while the effect of ghrelin is reflected in the translocation and phosphorylation of membrane-associated PLCγ2 on Tyr mainly. Moreover, we demonstrate that PLCγ2 phosphorylation on Tyr remains under the control of the Src family protein tyrosine kinases (SFK-PTKs), and is intimately linked to PLCγ2 membrane localization, while the LPS-induced phosphorylation of membrane-recruited PLCγ2 on Ser displays dependence on protein kinase Cδ (PKCδ) and leads to the amplification in PLCγ2 activation. Thus, our findings link the extent of H. pylori-elicited gastric mucosal inflammatory involvement to the PKCδ-mediated amplification in PLCγ2 activation through phosphorylation on Ser.

Asymmetric Distribution of Anionic Phospholipids in Supported Lipid Bilayers

Lipid bilayers with a controlled content of anionic lipids are a prerequisite for the quantitative study of hydrophobic-electrostatic interactions of proteins with lipid bilayers. Here, the asymmetric distribution of zwitterionic and anionic lipids in supported lipid bilayers is studied by neutron reflectometry. We prepare POPC/POPS (3:1) unilamellar vesicles in a high-salt-concentration buffer. Initially, no fusion of the vesicles to a SiO(2) surface is observed over hours and days. Once the isotonic buffer is exchanged with hypotonic buffer, vesicle fusion and bilayer formation occur by osmotic shock. Neutron reflectivity on the bilayers formed this way reveals the presence of anionic lipids (d(31)-POPS) in the outer bilayer leaflet only, and no POPS is observed in the leaflet facing the SiO(2) substrate. We argue that this asymmetric distribution of POPS is induced by the electrostatic repulsion of the phosphatidylserines from the negatively charged hydroxy surface groups of the silicon block. Such bilayers with controlled and high contents of anionic lipids in the outer leaflet are versatile platforms for studying anionic lipid protein interactions that are key elements in signal transduction pathways in the cytoplasmic leaflet of eukaryotic cells.

Abstract 1070: Differential response dynamic of leukemia cells to GM-CSF and IL-3 stimulation in signal transduction pathways

Abstract GM-CSF and IL-3 regulate the survival, proliferation, differentiation, and functional activation of hematopoietic cells. GM-CSF also contributes to controlling the function of dendritic cells and T-cells. Aberrant function of GM-CSF has been linked to multiple diseases including juvenile myelomonocytic leukemia (JMML), chronic myelomonocytic leukemia, rheumatoid arthritis, and alveolar proteinosis. GM-CSF and IL-3 share a common βc subunit on their receptor. Many studies have revealed that dys-regulation of Ras/PI3K/Akt and Ras/Raf/Mek/Erk pathways are responsible for hypersensitivities of GM-CSF and IL-3 in various diseases. However, few data have distinguished the difference of signal transduction pathways between the two cytokines. We previously reported that JMML cells are selectively hypersensitive to GM-CSF in vitro, while they show normal sensitivity to IL-3. Others documented that there was a time gap between CREB responded to GM-CSF and IL-3 stimulation. In the present study, we hypothesized that the signal transduction pathway of GM-CSF has differential response dynamic from that of IL-3. We first tested the CFU-GM growth pattern of a leukemia cell line, TF-1, in response to GM-CSF and IL-3 stimulation. We found that the GM-CSF dose-response curve was markedly shifted leftward, indicating that TF-1 cells were more sensitive to GM-CSF than to IL-3. This pattern is very similar to that observed in primary JMML cells. We next investigated the activities of the elements downstream in Ras pathway. After serum starving in medium with 0.5% BSA for 16 hours, TF-1 cells were stimulated with GM-CSF or IL-3 at a concentration range of 0.01-500pM. We found that the total protein levels of CREB were consistently unchanged. On the other hand, significant phosphorylation of CREB on serine 133 began to occur at concentrations of GM-CSF at 10pM. Significant phosphorylation of Erk was observed even at a concentration as low as 1 pM of GM-CSF. However, this phosphorylation pattern was not seen until 100pM of IL-3, and the level of phosphorylation was lower. The same dynamic, GM-CSF vs. IL-3, was also found in pAkt and pSTAT5. In conclusion, our data demonstrate that leukemia cells respond to GM-CSF and IL-3 stimulation in different dynamic patterns in signal transduction pathways. This indicates that the selective hypersensitivity to GM-CSF in some leukemia cells may be caused by aberrant elements in signal transduction pathways that are responsive to the low concentrations of GM-CSF stimulation. We raise a concern that any study related to responsiveness of GM-CSF or IL-3 stimulation should be interpreted cautiously; with specific attention paid to the concentration levels and the length of stimulation, in order to precisely characterize the function of GM-CSF and IL-3. This observed differential response dynamic might apply to a broader phenomenon in other cytokines or hormones in other tissues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1070. doi:1538-7445.AM2012-1070

Developmental transcriptome of Aplysia californica.

Genome-wide transcriptional changes in development provide important insight into mechanisms underlying growth, differentiation, and patterning. However, such large-scale developmental studies have been limited to a few representatives of Ecdysozoans and Chordates. Here, we characterize transcriptomes of embryonic, larval, and metamorphic development in the marine mollusc Aplysia californica and reveal novel molecular components associated with life history transitions. Specifically, we identify more than 20 signal peptides, putative hormones, and transcription factors in association with early development and metamorphic stages-many of which seem to be evolutionarily conserved elements of signal transduction pathways. We also characterize genes related to biomineralization-a critical process of molluscan development. In summary, our experiment provides the first large-scale survey of gene expression in mollusc development, and complements previous studies on the regulatory mechanisms underlying body plan patterning and the formation of larval and juvenile structures. This study serves as a resource for further functional annotation of transcripts and genes in Aplysia, specifically and molluscs in general. A comparison of the Aplysia developmental transcriptome with similar studies in the zebra fish Danio rerio, the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, and other studies on molluscs suggests an overall highly divergent pattern of gene regulatory mechanisms that are likely a consequence of the different developmental modes of these organisms.

Reprint of “Cell-free production of G protein-coupled receptors for functional and structural studies” [J. Struct. Biol. 158 (2007) 482–493

G-protein coupled receptors (GPCRs) are key elements in signal transduction pathways of eukaryotic cells and they play central roles in many human diseases. So far, most structural and functional approaches have been limited by the immense difficulties in the production of sufficient amounts of protein samples in conventional expression systems based on living cells. We report the high level production of six different GPCRs in an individual cell-free expression system based on Escherichia coli extracts. The open nature of cell-free systems allows the addition of detergents in order to provide an artificial hydrophobic environment for the reaction. This strategy defines a completely new technique for the production of membrane proteins that can directly associate with detergent micelles upon translation. We demonstrate the efficient overproduction of the human melatonin 1B receptor, the human endothelin B receptor, the human and porcine vasopressin type 2 receptors, the human neuropeptide Y4 receptor and the rat corticotropin releasing factor receptor by cell-free expression. In all cases, the long chain polyoxyethylene detergent Brij78 was found to be highly effective for solubilization and milligram amounts of soluble protein could be generated in less than 24 h. Single particle analysis indicated a homogenous distribution of predominantly protein dimers of the cell-free expressed GPCR samples, with dimensions similar to the related rhodopsin. Ligand interaction studies with the endothelin B receptor and a derivative of its peptide ligand ET-1 gave further evidence of a functional folding of the cell-free produced protein.

Virus-induced hypovirulence in Cryphonectria parasitica: still an unresolved conundrum.

In this paper we review some of the most recent literature about the interactions between Cryphonectria parasitica; the ascomycete fungus that causes chestnut blight; and viruses in the species Cryphonectria hypovirus 1 (family Hypoviridae); which; when present in the fungus; render the fungus incapable of causing the severe disease that is normally the result of infection of European and American chestnut trees (Castanea sativa and C. dentata; respectively). We provide an overview of the findings accumulated over the last few decades; in an attempt to link them to the technological advances made during this time. In particular; we focus on what is known about the molecular aspects of the virus-fungus interaction; detailing; and critically reviewing some recent findings and their significance in understanding the phenomenon of virus-induced hypovirulence. After describing the diversity of virus species that infect C. parasitica; we detail various aspects of the effects the presence of virus has on secretory pathways; and on various elements of signal transduction pathways. We also discuss the recent finding of a silencing suppressor encoded by the genome of Cryphonectria hypovirus 1; and recent results from studies of differential expression profiling using cDNA microarray analysis.

Cell-free production of G protein-coupled receptors for functional and structural studies

G-protein coupled receptors (GPCRs) are key elements in signal transduction pathways of eukaryotic cells and they play central roles in many human diseases. So far, most structural and functional approaches have been limited by the immense difficulties in the production of sufficient amounts of protein samples in conventional expression systems based on living cells. We report the high level production of six different GPCRs in an individual cell-free expression system based on Escherichia coli extracts. The open nature of cell-free systems allows the addition of detergents in order to provide an artificial hydrophobic environment for the reaction. This strategy defines a completely new technique for the production of membrane proteins that can directly associate with detergent micelles upon translation. We demonstrate the efficient overproduction of the human melatonin 1B receptor, the human endothelin B receptor, the human and porcine vasopressin type 2 receptors, the human neuropeptide Y4 receptor and the rat corticotropin releasing factor receptor by cell-free expression. In all cases, the long chain polyoxyethylene detergent Brij78 was found to be highly effective for solubilization and milligram amounts of soluble protein could be generated in less than 24 h. Single particle analysis indicated a homogenous distribution of predominantly protein dimers of the cell-free expressed GPCR samples, with dimensions similar to the related rhodopsin. Ligand interaction studies with the endothelin B receptor and a derivative of its peptide ligand ET-1 gave further evidence of a functional folding of the cell-free produced protein.

Temporal response of liver signal transduction elements during in vivo endotoxin challenge in cattle: Effects of growth hormone treatment

We quantified the changes in abundance of inducible nitric oxide synthase (iNOS) and associated tissue signal transduction pathway elements (STPEs) in the bovine liver in response to lipopolysaccharide (LPS) challenge and further assessed the impact on the LPS-driven variable responses as affected by daily treatment with recombinant growth hormone (GH) prior to LPS challenge. Twenty-four cross-bred beef steers were divided into GH-treated (recombinant bovine GH, Monsanto Inc., St. Louis, MO; 0.1 mg/kg BW, i.m., daily for 12 days) and non-GH-treatment (control) groups ( n = 12/group). Liver biopsy samples were obtained from all animals at 0, 3, 6, and 24 h after LPS challenge ( E. coli 055:B5, 2.5 μg/kg BW, i.v. bolus) for Western blot analyses of iNOS and STPEs. In response to LPS, tissue levels of iNOS increased significantly ( P < 0.001) in the first 3 h and persisted at levels greater than those at time 0 until 24 h. GH further augmented levels of iNOS at 0, 3, and 6 h resulting in an overall significant increase in the iNOS protein level ( P < 0.01). AKT/protein kinase B (AKT/PKB) phosphorylation levels at time 0 were not different between GH-treated and control animals; LPS increased the phosphorylation of AKT/PKB with GH treatment stimulating a four-fold further increase of AKT/PKB phosphorylation. Effects similar to those on AKT/PKB were also observed on signal transducer and activator of transcription 5b (STAT5b). The family of mitogen-activated protein kinase (MAPK) showed different pattern of response. ERK1/2 phosphorylation increased 3 h after LPS challenge but only in GH-treated group ( P < 0.01). Compared to 0 h, SAPK/JUN phosphorylation increased in both experimental groups 3, 6 h ( P < 0.01), and 24 h ( P < 0.05) after LPS. However, at 3 h the increase was greater ( P < 0.01) in GH-treated than in control animals. No effect of LPS challenge or GH treatment on p38 MAPK was observed. These results suggest that GH treatment has a significant impact on the differential activation of STPEs in the clinical response to LPS.

PKC-independent signal transduction pathways increase SERCA2 expression in adult rat cardiomyocytes

Catecholamines seem to play a major role in the initial response of the heart to pressure overload. The mechanisms by which α 1A-adrenoceptor stimulation increases protein synthesis and subsequently cell size have been worked out in the past. However, little is known about the functional consequence of this type of hypertrophy. Recent transgenic work seems to indicate an adaptive character of this response, but mechanistic insights have yet to be established. The present study investigates whether chronic (overnight) exposure of cardiomyocytes to phenylephrine, an α-adrenoceptor agonist, modifies the expression of calcium-handling proteins and identifies key elements of signal transduction pathways leading to such alterations. Cardiomyocytes exposed to phenylephrine had elevated expression of SR-calcium ATPase (SERCA), but not of the sodium–calcium exchanger (NCX). SERCA induction persisted in the presence of protein kinase C (PKC) inhibitors, but required an increase in diastolic cell calcium levels via activation of the sodium–proton exchanger (NHE) and the reverse mode of the NCX. Downstream of an increase in resting cell calcium concentrations an activation of the calcineurin/NFAT pathway was found to be responsible for SERCA2 induction. Transfection of cardiomyocytes with decoys directed against NFAT activity inhibited the increase in SERCA2 expression. Decoys did not inhibit the concomitant PKC-dependent increase in hypertrophic growth. In the absence of SERCA up-regulation, hypertrophied cardiomyocytes were unable to maintain normal, load-free cell shortening. In conclusion, our data give mechanistic insights into the adaptional process during α-adrenoceptor-dependent myocardial hypertrophy.

Interleukin-6 and oncostatin M stimulation of proliferation of prostate cancer 22Rv1 cells through the signaling pathways of p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase

Interleukin-6 (IL-6) is a pleiotropic regulator of prostate cancer cell growth. Oncostatin M (OSM), an IL-6-type cytokine, affects the growth of prostate cancers in a paracrine and autocrine manner. In order to understand better the mechanisms controlling proliferation and intracellular signaling by these cytokines in advanced prostate carcinoma, we performed studies in 22Rv1 cells derived from the relapsed xenograft CWR22R. Expression of IL-6 and OSM receptors (OSMR-beta) and elements of signal transduction pathways in 22Rv1 cells were investigated by RT-PCR. Proliferation was assessed by cell counting after treatment with either IL-6 or OSM. IL-6 secretion was measured in conditioned medium from 22Rv1 cells by ELISA. Expression and phosphorylation status of signal transducers and activators of transcription factor (STAT) 3, mitogen-activated protein kinases (MAPK) p44/p42 and p38, and protein kinase B (Akt) was investigated by Western blot. 22Rv1 cells express both subunits of the IL-6 receptor (gp80 and gp130) and leukemia inhibitory factor receptor-beta (LIFR-beta) but not OSMR-beta. Their proliferation was stimulated by IL-6 or OSM and the maximal effect was observed at a concentration of 10 ng/ml of either cytokine. Interestingly, neither IL-6 nor OSM induced phosphorylation of STAT3. OSM modestly increased the phosphorylation of p38 and both cytokines exerted an effect on Akt phosphorylation. IL-6 and OSM stimulate proliferation of 22Rv1 cells, at least in part through activation of the phosphatidylinositol 3-kinase (PI 3-K) signaling pathway. Our data provide additional evidence for the growth-stimulatory role of IL-6 and related cytokines in advanced prostate cancer and may serve as a basis for the development of novel experimental therapies.

Cellular scaffolds in mammalian eggs.

Cellular scaffolds serve as structural components to which various elements of signal transduction pathways can be associated. The association of components on a scaffold can have several important functions, for example they can: 1) associate upstream regulatory components in a cascade that can increase the speed of response to a stimulus; 2) restrict access of substrates to enzymes associated with the scaffold; 3) permit cross talk between distinct signaling pathways, and; 4) aid in the establishment of cellular polarity. The conversion of the mammalian egg into the zygote requires many rapid alterations during a distinct time frame to mediate the biochemical and structural changes that occur. Cellular scaffolds provide a mechanism that can perform these rapid, highly orchestrated changes. They can permit interaction between distinct calcium-dependent pathways and also can provide a means for the calcium signal, that is initiated by fertilization, to act on calcium-independent pathways. This review considers various lines of evidence suggesting that in the mammalian egg, the meiotic spindle serves as a cellular scaffold that permits coordination among several signaling pathways essential for fertilization and the initiation of early development.

Apoptosis induced by Rac GTPase correlates with induction of FasL and ceramides production.

Rho proteins, members of the Ras superfamily of GTPases, are critical elements in signal transduction pathways governing cell proliferation and cell death. Different members of the family of human Rho GTPases, including RhoA, RhoC, and Rac1, participate in the regulation of apoptosis in response to cytokines and serum deprivation in different cell systems. Here, we have characterized the mechanism of apoptosis induced by Rac1 in NIH 3T3 cells. It requires protein synthesis and caspase-3 activity, but it is independent of the release of cytochrome c from mitochondria. Moreover, an increase in mitochondria membrane potential and the production of reactive oxygen species was observed. Rac1-induced apoptosis was related to the simultaneous increase in ceramide production and synthesis of FasL. Generation of FasL may be mediated by transcriptional regulation involving both c-Jun amino terminal kinase as well as nuclear factor-kappa B-dependent signals. None of these signals, ceramides or FasL, was sufficient to induce apoptosis in the parental cell line, NIH 3T3 cells. However, any of them was sufficient to induce apoptosis in the Rac1-expressing cells. Finally, inhibition of FasL signaling drastically reduced apoptosis by Rac1. Thus, Rac1 seems to induce apoptosis by a complex mechanism involving the generation of ceramides and the de novo synthesis of FasL. These results suggest that apoptosis mediated by Rac1 results from a signaling mechanism that involves biochemical and transcriptional events under control of Rac1.

Immunohistochemical localization of Galphaq, PLCbeta, Galphai1-2, PKA, and the endothelin B and extracellular Ca2+-sensing receptors during early amelogenesis.

Antibodies specific to Galphaq, PLCbeta, Galphai 1-2, and PKA were immunohistochemically (IHC) localized in the pre-ameloblasts up to initial dentin matrix deposition and continued in the distal ends of the pre-secretory ameloblasts to the beginning of enamel matrix secretion. It was hypothesized that the endothelin B receptor (ETBR) and/or the extracellular Ca2+-sensing receptor (CaR) would localize in the same locations as their known downstream signal transduction pathway (STP) effectors during events related to early amelogenesis. Localization was similar for the 4 signal transduction pathway elements and the CaR. The ETBR was not localized in any of the cells of the enamel organ. These findings indicate that the CaR and its related STPs are expressed in the pre-ameloblasts and pre-secretory ameloblasts in positions where they may be able to detect increases in extracellular Ca2+ concentrations observed in the pre-dentin matrix in a previous study. These observations are consistent with the hypothesis that increased levels of free Ca2+ in the pre-dentin matrix serve as a primary signal for modification of gene expression important to amelogenesis.

Modulation of odor-induced increases in [Ca 2+] i by inhibitors of protein kinases A and C in rat and human olfactory receptor neurons

Protein kinases A and C have been postulated to exert multiple effects on different elements of signal transduction pathways in olfactory receptor neurons. However, little is known about the modulation of olfactory responses by protein kinases in intact olfactory receptor neurons. To further elucidate the details of the modulation of odorant responsiveness by these protein kinases, we investigated the action of two protein kinase inhibitors: H89, an inhibitor of protein kinase A, and N-myristoylated EGF receptor, an inhibitor of protein kinase C, on odorant responsiveness in intact olfactory neurons. We isolated individual olfactory neurons from the adult human and rat olfactory epithelium and measured responses of the isolated cells to odorants or biochemical activators that have been shown to initiate cyclic AMP or inositol 1,4,5-trisphospate production in biochemical preparations. We employed calcium imaging techniques to measure odor-elicited changes in intracellular calcium that occur over several seconds. In human olfactory receptor neurons, the protein kinase A and C inhibitors affected the responses to different sets of odorants. In rats, however, the protein kinase C inhibitor affected responses to all odorants, while the protein kinase A inhibitor had no effect. In both species, the effect of inhibition of protein kinases was to enhance the elevation and block termination of intracellular calcium levels elicited by odorants. Our results show that protein kinases A and C may modulate odorant responses of olfactory neurons by regulating calcium fluxes that occur several seconds after odorant stimulation. The effects of protein kinase C inhibition are different in rat and human olfactory neurons, indicating that species differences are an important consideration when applying data from animal studies to apply to humans.