Mutagenesis of RpoE-like sigma factor genes in Bdellovibrio reveals differential control of groEL and two groES genes

BACKGROUND: Bdellovibrio bacteriovorus HD100 must regulate genes in response to a variety of environmental conditions as it enters, preys upon and leaves other bacteria, or grows axenically without prey. In addition to "housekeeping" sigma factors, its genome encodes several alternate sigma factors, including 2 Group IV-RpoE-like proteins, which may be involved in the complex regulation of its predatory lifestyle.

RESULTS: We find that one sigma factor gene, bd3314, cannot be deleted from Bdellovibrio in either predatory or prey-independent growth states, and is therefore possibly essential, likely being an alternate sigma 70. Deletion of one of two Group IV-like sigma factor genes, bd0881, affects flagellar gene regulation and results in less efficient predation, although not due to motility changes; deletion of the second, bd0743, showed that it normally represses chaperone gene expression and intriguingly we find an alternative groES gene is expressed at timepoints in the predatory cycle where intensive protein synthesis at Bdellovibrio septation, prior to prey lysis, will be occurring.

CONCLUSIONS: We have taken the first step in understanding how alternate sigma factors regulate different processes in the predatory lifecycle of Bdellovibrio and discovered that alternate chaperones regulated by one of them are expressed at different stages of the lifecycle.

Transforming Growth Factor-beta superfamily:evaluation as breast cancer biomarkers and preventive agents

The Transforming Growth Factor-beta (TGFbeta) superfamily of cytokines is comprised of a number of structurally-related, secreted polypeptides that regulate a multitude of cellular processes including proliferation, differentiation and neoplastic transformation. These growth regulatory molecules induce ligand-mediated hetero-oligomerization of distinct type II and type I serine/threonine kinase receptors that transmit signals predominantly through receptor-activated Smad proteins but also induce Smad-independent pathways. Ligands, receptors and intracellular mediators of signaling initiated by members of the TGFbeta family are expressed in the mammary gland and disruption of these pathways may contribute to the development and progression of human breast cancer. Since many facets of TGFbeta and breast cancer have been recently reviewed in several articles, except for discussion of recent developments on some aspects of TGFbeta, the major focus of this review will be on the role of activins, inhibins, BMPs, nodal and MIS-signaling in breast cancer with emphasis on their utility as potential diagnostic, prognostic and therapeutic targets.

Nuclear translocation and functions of growth factor receptors

Cellular signal transduction in response to environmental signals involves a relay of precisely regulated signal amplifying and damping events. A prototypical signaling relay involves ligands binding to cell surface receptors and triggering the activation of downstream enzymes to ultimately affect the subcellular distribution and activity of DNA-binding proteins that regulate gene expression. These so-called signal transduction cascades have dominated our view of signaling for decades. More recently evidence has accumulated that components of these cascades can be multifunctional, in effect playing a conventional role for example as a cell surface receptor for a ligand whilst also having alternative functions for example as transcriptional regulators in the nucleus. This raises new challenges for researchers. What are the cues/triggers that determine which role such proteins play? What are the trafficking pathways which regulate the spatial distribution of such proteins so that they can perform nuclear functions and under what circumstances are these alternative functions most relevant?

Novel functional interaction between the plasma membrane Ca2+ pump 4b and the proapoptotic tumor suppressor Ras-associated factor 1 (RASSF1)

Plasma membrane calmodulin-dependent calcium ATPases (PMCAs) are enzymatic systems implicated in the extrusion of calcium from the cell. We and others have previously identified molecular interactions between the cytoplasmic COOH-terminal end of PMCA and PDZ domain-containing proteins. These interactions suggested a new role for PMCA as a modulator of signal transduction pathways. The existence of other intracellular regions in the PMCA molecule prompted us to investigate the possible participation of other domains in interactions with different partner proteins. A two-hybrid screen of a human fetal heart cDNA library, using the region 652-840 of human PMCA4b (located in the catalytic, second intracellular loop) as bait, revealed a novel interaction between PMCA4b and the tumor suppressor RASSF1, a Ras effector protein involved in H-Ras-mediated apoptosis. Immunofluorescence co-localization, immunoprecipitation, and glutathione S-transferase pull-down experiments performed in mammalian cells provided further confirmation of the physical interaction between the two proteins. The interaction domain has been narrowed down to region 74-123 of RASSF1C (144-193 in RASSF1A) and 652-748 of human PMCA4b. The functionality of this interaction was demonstrated by the inhibition of the epidermal growth factor-dependent activation of the Erk pathway when PMCA4b and RASSF1 were co-expressed. This inhibition was abolished by blocking PMCA/RASSSF1 association with an excess of a green fluorescent protein fusion protein containing the region 50-123 of RASSF1C. This work describes a novel protein-protein interaction involving a domain of PMCA other than the COOH terminus. It suggests a function for PMCA4b as an organizer of macromolecular protein complexes, where PMCA4b could recruit diverse proteins through interaction with different domains. Furthermore, the functional association with RASSF1 indicates a role for PMCA4b in the modulation of Ras-mediated signaling.

Nerve Growth Factor Sensitisation of TRPA1 on Sensory Neurones

Background: Sensory neurones from the trigeminal nerve innervate the oro-facial region and teeth. Transient receptor potential channels (TRPs) expressed by these neurones are responsible for relaying sensory information such as changes in ambient temperature, mechanical sensations and pain. Study of TRP channel expression and regulation in human sensory neurones therefore merits investigation to improve our understanding of allodynia and hyperalgesia. Objective: The objective of this study was to differentiate human dental pulp stem cells (hDPSCs) towards a neuronal phenotype (peripheral neuronal equivalents; PNEs) and employ this model to study TRP channel sensitisation. Method: hDPSCs were enriched by preferential adhesion to fibronectin, plated on coverslips (thickness 0) coated with poly-l-ornithine and laminin and then differentiated for 7 days in neurobasal A medium with additional supplementation. A whole cell patch clamp technique was used to investigate whether TRP channels on PNE membranes were modulated in the presence of nerve growth factor (NGF). PNEs were treated with NGF for 20 minutes immediately before experimentation and then stimulated for TRPA1 activity using cinnamaldehyde. Peak currents were read at 80 mV and -80 mV and compared to peak currents recorded in untreated PNEs. Data were analysed and plotted using Clampfit9 software (Molecular Devices, Sunnyvale, California, USA). Result: Results showed for the first time that pre-treatment of PNEs by NGF produced significantly larger inward and outward currents demonstrating that TRPA1 channels on PNE membranes were capable of becoming sensitised following treatment with NGF. Conclusion: Sensitisation of TRPA1 by NGF provides evidence of a mechanism for rapid neuronal sensitisation that is independent of TRPA1 gene expression