DNA binding-independent transcriptional activation of the vascular endothelial growth factor gene (VEGF) by the Myb oncoprotein

Myb is a key transcription factor that can regulate proliferation, differentiation, and apoptosis, predominantly in the haemopoietic system. Abnormal expression of Myb is associated with a number of cancers, both haemopoietic and non-haemopoietic. In order to better understand the role of Myb in normal and tumorigenic processes, we undertook a cDNA array screen to identify genes that are regulated by this factor. In this way, we identified the gene encoding vascular endothelial growth factor (VEGF) as being potentially regulated by the Myb oncoprotein in myeloid cells. To determine whether this was a direct effect on VEGF gene transcription, we examined the activity of the murine VEGF promoter in the presence of either wild-type (WT) or mutant forms of Myb. It was found that WT Myb was able to activate the VEGF promoter and that a minimal promoter region of 120 bp was sufficient to confer Myb responsiveness. Surprisingly, activation of the VEGF promoter was independent of DNA binding by Myb. This was shown by the use of DNA binding-defective Myb mutants and by mutagenesis of a potential Myb-binding site in the minimal promoter. Mutation of Sp1 sites within this region abolished Myb-mediated regulation of a reporter construct, suggesting that Myb DNA binding-independent activation of VEGF expression occurs via these Sp1 binding elements. Regulation of VEGF production by Myb has implications for the potential role of Myb in myeloid leukaemias and in solid tumours where VEGF may be functioning as an autocrine growth factor. (c) 2006 Elsevier Inc. All rights reserved.

Giant protein kinases: Domain interactions and structural basis of autoregulation

The myosin-associated giant protein kinases twitchin and titin are composed predominantly of fibronectin- and immunoglobulin-like modules, We report the crystal structures of two autoinhibited twitchin kinase fragments, one from Aplysia and a larger fragment from Caenorhabditis elegans containing an additional C-terminal immunoglobulin-like domain, The structure of the longer fragment shoes that the immunoglobulin domain contacts the protein kinase domain on the opposite side from the catalytic cleft, laterally exposing potential myosin binding residues, Together, the structures reveal the cooperative interactions between the autoregulatory region and the residues from the catalytic domain involved in protein substrate binding, ATP binding, catalysis and the activation loop, and explain the differences between the observed autoinhibitory mechanism and the one found in the structure of calmodulin-dependent kinase I.

Processing of mutated human proinsulin to mature insulin in the non-endocrine cell line, CHO

Heterologous genes encoding proproteins, including proinsulin, generally produce mature protein when expressed in endocrine cells while unprocessed or partially processed protein is produced in non-endocrine cells. Proproteins, which are normally processed in the regulated pathway restricted to endocrine cells, do not always contain the recognition sequence for cleavage by furin, the endoprotease specific to the constitutive pathway, the principal protein processing pathway in non-endocrine cells. Human proinsulin consists of B-Chain-C-peptide-A-Chain and cleavage at the B/C and C/A junctions is required for processing. The B/C, but not the C/A junction, is recognised and cleaved in the constitutive pathway. We expressed a human proinsulin and a mutated proinsulin gene with an engineered furin recognition sequence at the C/A junction and compared the processing efficiency of the mutant and native proinsulin in Chinese Hamster Ovary cells. The processing efficiency of the mutant proinsulin was 56% relative to 0.7% for native proinsulin. However, despite similar levels of mRNA being expressed in both cell lines, the absolute levels of immunoreactive insulin, normalized against mRNA levels, were 18-fold lower in the mutant proinsulin-expressing cells. As a result, there was only a marginal increase in absolute levels of insulin produced by these cells. This unexpected finding may result from preferential degradation of insulin in non-endocrine cells which lack the protection offered by the secretory granules found in endocrine cells.

MHCPEP, a database of MHC-binding peptides: Update 1996

MHCPEP is a curated database comprising over 9000 peptide sequences known to bind MHC molecules. Entries are compiled from published reports as well as from direct submissions of experimental data. Each entry contains the peptide sequence, its MHC specificity and, when available, experimental method, observed activity, binding affinity, source protein, anchor positions and publication references. The present format of the database allows text string matching searches but can easily be converted for use in conjunction with sequence analysis packages. The database can be accessed via Internet using WWW, FTP or Gopher.

Nuclear localization of RelB is associated with effective antigen-presenting cell function

Dendritic cells (DC) are potent APCs that enter resting tissues as precursors and, after Ag exposure, differentiate and migrate to draining lymph nodes. The phenotype of RelB knockout mice implicates this member of the NF kappa B/Rel family in DC differentiation. To further elucidate the role of RelB in DC differentiation, mRNA, intracellular protein expression, and DNA binding activity of RelB were examined in immature and differentiated human DC, as well as other PB mononuclear cell populations. RelB protein and mRNA were detected constitutively in lymphocytes and in activated monocytes, differentiated DC, and monocyte-derived DC. Immunohistochemical staining demonstrated RelB within the differentiated lymph node interdigitating DC and follicular DC, but not undifferentiated DC in normal skin. Active nuclear RelB was detected by supershift assay only in differentiated DC derived from either PB precursors or monocytes and in activated B cells. These RelB+ APC were potent stimulators of the MLR. The data indicate that RelB expression is regulated both transcriptionally and post-translationally in myeloid cells. Within the nucleus, RelB may specifically transactivate genes that are critical for APC function.

Biologically active conformer of the effector region of human C5a and modulatory effects of N-terminal receptor binding determinants on activity

A conformationally biased decapeptide agonist of human C5a (C5a(65-74)Y65,F67,P69,P71,D-Ala73 or YSFKPMPLaR) was used as a functional probe of the C5a receptor (C5aR) in order to understand the conformational features in the C-terminal effector region of C5a that are important for C5aR binding and signal transduction. YSFKPMPLaR was a potent, full agonist of C5a, but at higher concentrations had a superefficacious effect compared to the natural factor. The maximal efficacy of this analogue was 216 +/- 56% that of C5a in stimulating the release of beta-glucuronidase from human neutrophils. C5aR activation and binding curves both occurred in the same concentration range with YSFKPMPLaR, characteristics not observed with natural C5a or more conformationally flexible C-terminal agonists. YSFKPMPLaR was then used as a C-terminal effector template onto which was synthesized various C5aR binding determinants from the N-terminal core domain of the natural factor. In general, the presence of N-terminal binding determinants had little effect on either potency or binding affinity when the C-terminal effector region was presented to the C5aR in this biologically active conformation. However, one peptide, C5a(12-20)-Ahx-YSFKPMPLaR, expressed a 100-fold increase in affinity for the neutrophil C5aR and a 6-fold increase in potency relative to YSFKPMPLaR. These analyses showed that the peptides used in this study have up to 25% of the potency of C5a in human fetal artery and up to 5% of the activity of C5a in the PMN enzyme release assay.

Immediate early gene expression and delayed cell death in limbic areas of the rat brain after kainic acid treatment and recovery in the cold

Systemic injection of kainic acid (KA) results in characteristic behaviors and programmed cell death in some regions of the rat brain. We used KA followed by recovery at 4 degrees C to restrict damage to limbic structures and compared patterns of immediate early gene (IEG) expression and associated DNA binding activity in these damaged areas with that in spared brain regions. Male Wistar rats were injected with BA (12 mg/kg, ip) and kept at 4 degrees C for 5 h. This treatment reduced the severity of behaviors and restricted damage (observed by Nissl staining) to the CA1 and CA3 regions of the hippocampus and an area including the entorhinal cortex. DNA laddering, characteristic of apoptosis, was first evident in the hippocampus and the entorhinal cortex 18 and 22 h after RA, respectively. The pattern of IEG mRNA induction fell into three classes: IEGs that were induced in both damaged and spared areas (c-fos, fos B, jun B, and egr-1), IEGs that were induced specifically in the damaged areas (fra-2 and c-jun), and an IEG that was significantly induced by saline injection and/or the cold treatment (jun D). The pattern of immunoreactivity closely followed that of mRNA expression. Binding to the AP-1 and EGR DNA consensus sequences increased in all three regions studied. This study describes a unique modification of the animal model of ICA-induced neurotoxicity which may prove a useful tool for dissecting the molecular cascade that ultimately results in programmed cell death. (C) 1997 Academic Press.

Nuclear localization of RelB is associated with effective antigen-presenting cell function

Dendritic cells (DC) are potent APCs that enter resting tissues as precursors and, after Ag exposure, differentiate and migrate to draining lymph nodes. The phenotype of RelB knockout mice implicates this member of the NF kappa B/Rel family in DC differentiation. To further elucidate the role of RelB in DC differentiation, mRNA, intracellular protein expression, and DNA binding activity of RelB were examined in immature and differentiated human DC, as well as other PB mononuclear cell populations. RelB protein and mRNA were detected constitutively in lymphocytes and in activated monocytes, differentiated DC, and monocyte-derived DC. Immunohistochemical staining demonstrated RelB within the differentiated lymph node interdigitating DC and follicular DC, but not undifferentiated DC in normal skin. Active nuclear RelB was detected by supershift assay only in differentiated DC derived from either PB precursors or monocytes and in activated B cells. These RelB(+) APC were potent stimulators of the MLR. The data indicate that RelB expression is regulated both transcriptionally and post-translationally in myeloid cells. Within the nucleus, RelB may specifically transactivate genes that are critical for APC function.

Substrate-bound carbohydrates stimulate signal transduction and neurite outgrowth in an olfactory neuron cell line

SUBPOPULATIONS of olfactory receptor neurons, which are dispersed throughout the olfactory neuroepithelium, express specific cell surface carbohydrates and project to discrete regions of the olfactory bulb. Cell surface carbohydrates such as N-acetyl-lactosamine have been postulated to mediate sorting and selective fasciculation of discrete axon subpopulations during development of the olfactory pathway. Substrate-bound N-acetyl-lactosamine promotes neurite outgrowth by both clonal olfactory receptor neuron cell lines and olfactory receptor neurons in vitro, indicating that cell surface carbohydrates may be ligands for receptor-mediated stimulation of axon growth in vivo. In the present study, the role of transmembrane signaling in N-acetyl-lactosamine-stimulated neurite outgrowth was examined in the clonal olfactory neuron cell line 4.4.2. Substrate-bound N-acetyl-lactosamine stimulated neurite outgrowth which was specifically inhibited by antagonists to N- and L-type calcium channels and to tyrosine kinase phosphorylation. These results indicate that N-acetyl-lactosamine can evoke transmembrane receptor-mediated responses capable of influencing neurite outgrowth.

Non-expression of insulin-like growth factor-I receptor is associated with apoptosis: an ultrastructural study on rat ameloblasts

Insulin-like growth factor-I (IGF-I) is a preiotrophic polypeptide which appears to have roles both as a circulating endocrine hormone and as a locally synthesized paracrine or autocrine tissue factor. IGF-I plays a major role in regulating the growth of cells in vivo and in vitro and initiates metabolic and mitogenic processes in a wide variety of cell types by binding to specific type I receptors in the plasma membrane, In this study, we report the distribution of IGF-I receptors in odontogenic cells at the ultrastructural level using the high resolution protein A-gold technique, In the pre-secretory stage, very little gold label was visible over the ameloblasts and odontoblasts, During the secretory stage the label was mostly seen in association with the cell membranes and endoplasmic reticulum of the ameloblasts. Lysosome-like elements in the post-secretory stage were labelled as well as multivesicular dense bodies, Very little labelling was encountered in the ameloblasts in the transitional stage, where apoptotic bodies were clearly visible, The maturation stage also exhibited labelling of the secretory-like granules in the distal surface. The presence of gold particles over the plasma membrane is an indication that IGF-I receptor is a membrane-bound receptor. Furthermore, the intracellular distribution of the label over the endoplasmic reticulum supports the local synthesis of the IGF-I receptor. The absence of labelling over the transitional ameloblasts suggests that the transitional stage may require the non-expression of IGF-I as a prerequiste or even a trigger for apoptosis.

The structure of versutoxin (delta-atracotoxin-Hv1) provides insights into the binding of site 3 neurotoxins to the voltage-gated sodium channel

Background: Versutoxin (delta-ACTX-Hv1) is the major component of the venom of the Australian Blue Mountains funnel web spider, Hadronyche versuta. delta-ACTX-Hv1 produces potentially fatal neurotoxic symptoms in primates by slowing the inactivation of voltage-gated sodium channels; delta-ACTX-Hv1 is therefore a useful tool for studying sodium channel function. We have determined the three-dimensional structure of delta ACTX-Hv1 as the first step towards understanding the molecular basis of its interaction with these channels. Results: The solution structure of delta-ACTX-Hv1, determined using NMR spectroscopy, comprises a core beta region containing a triple-stranded antiparallel beta sheet, a thumb-like extension protruding from the beta region and a C-terminal 3(10) helix that is appended to the beta domain by virtue of a disulphide bond. The beta region contains a cystine knot motif similar to that seen in other neurotoxic polypeptides. The structure shows homology with mu-agatoxin-l, a spider toxin that also modifies the inactivation kinetics of vertebrate voltage-gated sodium channels. More surprisingly, delta-ACTX-Hv1 shows both sequence and structural homology with gurmarin, a plant polypeptide. This similarity leads us to suggest that the sweet-taste suppression elicited by gurmarin may result from an interaction with one of the downstream ion channels involved in sweet-taste transduction. Conclusions: delta-ACTX-Hv1 shows no structural homology with either sea anemone or alpha-scorpion toxins, both of which also modify the inactivation kinetics of voltage-gated sodium channels by interacting with channel recognition site 3. However, we have shown that delta-ACTX-Hv1 contains charged residues that are topologically related to those implicated in the binding of sea anemone and alpha-scorpion toxins to mammalian voltage-gated sodium channels, suggesting similarities in their mode of interaction with these channels.

Growth factors and cytokines modulate gene expression of cell-surface proteoglycans in human periodontal ligament cells

Cell-surface proteoglycans have been known to be involved in many functions including interactions with components of the extracellular microenvironment, and act as co-receptors which bind and modify the action of various growth factors and cytokines. The purpose of this study was to determine the regulation by growth factors and cytokines on cell-surface proteoglycan gene expression in cultured human periodontal ligament (PDL) cells. Subconfluent, quiescent PDL cells were treated with various concentrations of serum, bFGF, PDGF-BB, TGF-beta1, IL-1 beta, and IFN-gamma. RT-PCR technique was used, complemented with Northern blot for syndecan-1, to examine the effects of these agents on the mRNA expression of five cell-surface proteoglycans (syndecan-1, syndecan-2, syndecan-4, glypican and betaglycan). Syndecan-1 mRNA levels increased in response to serum, bFGF and PDGF-BB, but decreased in response to TGF-beta1, IL-1 beta and IFN-gamma. In contrast, syndecan-2 mRNA levels were upregulated by TCF-beta1 and IL-1 beta stimulation, but remained unchanged with the other agents. Betaglycan gene expression decreased in response to serum, but was upregulated by TCF-beta1 and unchanged by the other stimulants. Additionally, syndecan-4 and glypican were not significantly altered in response to the regulator molecules studied, with the exception that glypican is decreased in response to IFN-gamma. These data demonstrate that the gene expression of the five cell-surface proteoglycans studied is differentially regulated in PDL cells lending support to the nation of distinct functions for these cell-surface proteoglycans. (C) 2001 Wiley-Liss, inc.

Localization of neurotransmitters and calcium binding proteins to neurons of salamander and mudpuppy retinas

We wished to identify the different types of retinal neurons on the basis of their content of neuroactive substances in both larval tiger salamander and mudpuppy retinas, favored species for electrophysiological investigation. Sections and wholemounts of retinas were labeled by immunocytochemical methods to demonstrate three calcium binding protein species and the common neurotransmitters, glycine, GABA and acetylcholine. Double immunostained sections and single labeled wholemount retinas were examined by confocal microscopy. Immunostaining patterns appeared to be the same in salamander and mudpuppy. Double and single cones, horizontal cells, some amacrine cells and ganglion cells were strongly calbindin-immunoreactive (IR). Calbindin-IR horizontal cells colocalized GABA. Many bipolar cells, horizontal cells, some amacrine cells and ganglion cells were strongly calretinin-IR. One type of horizontal cell and an infrequently occurring amacrine cell were parvalbumin-IR. Acetylcholine as visualized by ChAT-immunoreactivity was seen in a mirror-symmetric pair of amacrine cells that colocalized GABA and glycine. Glycine and GABA colocalized with calretinin, calbindin and occasionally with parvalbumin in amacrine cells. (C) 2001 Elsevier Science Ltd. All rights reserved.