Seeing Keratinocyte Proteins through the Looking Glass of Intrinsic Disorder.

Epidermal keratinocyte proteins include many with an eccentric amino acid content (compositional bias), atypical ultrastructural fate (built-in protease sensitivity), or assembly visible at the light microscope level (cytoplasmic granules). However, when considered through the looking glass of intrinsic disorder (ID), these apparent oddities seem quite expected. Keratinocyte proteins with highly repetitive motifs are of low complexity but high adaptation, providing polymers (e.g., profilaggrin) for proteolysis into bioactive derivatives, or monomers (e.g., loricrin) repeatedly cross-linked to self and other proteins to shield underlying tissue. Keratohyalin granules developing from liquid-liquid phase separation (LLPS) show that unique biomolecular condensates (BMC) and proteinaceous membraneless organelles (PMLO) occur in these highly customized cells. We conducted bioinformatic and in silico assessments of representative keratinocyte differentiation-dependent proteins. This was conducted in the context of them having demonstrated potential ID with the prospect of that characteristic driving formation of distinctive keratinocyte structures. Intriguingly, while ID is characteristic of many of these proteins, it does not appear to guarantee LLPS, nor is it required for incorporation into certain keratinocyte protein condensates. Further examination of keratinocyte-specific proteins will provide variations in the theme of PMLO, possibly recognizing new BMC for advancements in understanding intrinsically disordered proteins as reflected by keratinocyte biology.

The Developing Balance of Thrombosis and Hemorrhage in Pediatric Surgery: Clinical Implications of Age-Related Changes in Hemostasis.

Bleeding and thrombosis in critically ill infants and children is a vexing clinical problem. Despite the relatively low incidence of bleeding and thrombosis in the overall pediatric population relative to adults, these critically ill children face unique challenges to hemostasis due to extreme physiologic derangements, exposure of blood to foreign surfaces and membranes, and major vascular endothelial injury or disruption. Caring for pediatric patients on extracorporeal support, recovering from solid organ transplant or invasive surgery, and after major trauma is often complicated by major bleeding or clotting events. As our ability to care for the youngest and sickest of these children increases, the gaps in our understanding of the clinical implications of developmental hemostasis have become increasingly important. We review the current understanding of the development and function of the hemostatic system, including the complex and overlapping interactions of coagulation proteins, platelets, fibrinolysis, and immune mediators from the neonatal period through early childhood and to young adulthood. We then examine scenarios in which our ability to effectively measure and treat coagulation derangements in pediatric patients is limited. In these clinical situations, adult therapies are often extrapolated for use in children without taking age-related differences in pediatric hemostasis into account, leaving clinicians confused and impacting patient outcomes. We discuss the limitations of current coagulation testing in pediatric patients before turning to emerging ideas in the measurement and management of pediatric bleeding and thrombosis. Finally, we highlight opportunities for future research which take into account this developing balance of bleeding and thrombosis in our youngest patients.

Functional and structural basis of E. coli enolase inhibition by SF2312: a mimic of the carbanion intermediate.

Many years ago, the natural secondary metabolite SF2312, produced by the actinomycete Micromonospora, was reported to display broad spectrum antibacterial properties against both Gram-positive and Gram-negative bacteria. Recent studies have revealed that SF2312, a natural phosphonic acid, functions as a potent inhibitor of human enolase. The mechanism of SF2312 inhibition of bacterial enolase and its role in bacterial growth and reproduction, however, have remained elusive. In this work, we detail a structural analysis of E. coli enolase bound to both SF2312 and its oxidized imide-form. Our studies support a model in which SF2312 acts as an analog of a high energy intermediate formed during the catalytic process. Biochemical, biophysical, computational and kinetic characterization of these compounds confirm that altering features characteristic of a putative carbanion (enolate) intermediate significantly reduces the potency of enzyme inhibition. When SF2312 is combined with fosfomycin in the presence of glucose-6 phosphate, significant synergy is observed. This suggests the two agents could be used as a potent combination, targeting distinct cellular mechanism for the treatment of bacterial infections. Together, our studies rationalize the structure-activity relationships for these phosphonates and validate enolase as a promising target for antibiotic discovery.

Structural Basis of Protein Kinase R Autophosphorylation.

The RNA-activated protein kinase, PKR, is a key mediator of the innate immunity response to viral infection. Viral double-stranded RNAs induce PKR dimerization and autophosphorylation. The PKR kinase domain forms a back-to-back dimer. However, intermolecular ( trans) autophosphorylation is not feasible in this arrangement. We have obtained PKR kinase structures that resolves this dilemma. The kinase protomers interact via the known back-to-back interface as well as a front-to-front interface that is formed by exchange of activation segments. Mutational analysis of the front-to-front interface support a functional role in PKR activation. Molecular dynamics simulations reveal that the activation segment is highly dynamic in the front-to-front dimer and can adopt conformations conducive to phosphoryl transfer. We propose a mechanism where back-to-back dimerization induces a conformational change that activates PKR to phosphorylate a "substrate" kinase docked in a front-to-front geometry. This mechanism may be relevant to related kinases that phosphorylate the eukaryotic initiation factor eIF2α.

Structural and Functional Studies of Bacterial Enolase, a Potential Target against Gram-Negative Pathogens.

Enolase is a glycolytic metalloenzyme involved in carbon metabolism. The advantage of targeting enolase lies in its essentiality in many biological processes such as cell wall formation and RNA turnover and as a plasminogen receptor. We initially used a DARTS assay to identify enolase as a target in Escherichia coli. The antibacterial activities of α-, ß-, and γ-substituted seven-member ring tropolones were first evaluated against four strains representing a range of Gram-negative bacteria. We observed that the chemical properties and position of the substituents on the tropolone ring play an important role in the biological activity of the investigated compounds. Both α- and ß-substituted phenyl derivatives of tropolone were the most active with minimum inhibitory concentrations in the range of 11-14 µg/mL. The potential inhibitory activity of the synthetic tropolones was further evaluated using an enolase inhibition assay, X-ray crystallography, and molecular docking simulations. The catalytic activity of enolase was effectively inhibited by both the naturally occurring ß-thujaplicin and the α- and ß-substituted phenyl derivatives of tropolones with IC50 values in range of 8-11 µM. Ligand binding parameters were assessed by isothermal titration calorimetry and differential scanning calorimetry techniques and agreed with the in vitro data. Our studies validate the antibacterial potential of tropolones with careful consideration of the position and character of chelating moieties for stronger interaction with metal ions and residues in the enolase active site.

Post-Glycosylation Diversification (PGD): An Approach for Assembling Collections of Glycosylated Small Molecules.

Many small molecule natural products with antibiotic and antiproliferative activity are adorned with a carbohydrate residue as part of their molecular structure. The carbohydrate moiety can act to mediate key interactions with the target, attenuate physicochemical properties, or both. Facile incorporation of a carbohydrate group on de novo small molecules would enable these valuable properties to be leveraged in the evaluation of focused compound libraries. While there is no universal way to incorporate a sugar on small molecule libraries, techniques such as glycorandomization and neoglycorandomization have made signification headway toward this goal. Here, we report a new approach for the synthesis of glycosylated small molecule libraries. It puts the glycosylation early in the synthesis of library compounds. Functionalized aglycones subsequently participate in chemoselective diversification reactions distal to the carbohydrate. As a proof-of-concept, we prepared several desosaminyl glycosides from only a few starting glycosides, using click cycloadditions, acylations, and Suzuki couplings as diversification reactions. New compounds were then characterized for their inhibition of bacterial protein translation, bacterial growth, and in a T-cell activation assay.

Cardiolipin mediates membrane and channel interactions of the mitochondrial TIM23 protein import complex receptor Tim50.

The phospholipid cardiolipin mediates the functional interactions of proteins that reside within energy-conserving biological membranes. However, the molecular basis by which this lipid performs this essential cellular role is not well understood. We address this role of cardiolipin using the multisubunit mitochondrial TIM23 protein transport complex as a model system. The early stages of protein import by this complex require specific interactions between the polypeptide substrate receptor, Tim50, and the membrane-bound channel-forming subunit, Tim23. Using analyses performed in vivo, in isolated mitochondria, and in reductionist nanoscale model membrane systems, we show that the soluble receptor domain of Tim50 interacts with membranes and with specific sites on the Tim23 channel in a manner that is directly modulated by cardiolipin. To obtain structural insights into the nature of these interactions, we obtained the first small-angle x-ray scattering-based structure of the soluble Tim50 receptor in its entirety. Using these structural insights, molecular dynamics simulations combined with a range of biophysical measurements confirmed the role of cardiolipin in driving the association of the Tim50 receptor with lipid bilayers with concomitant structural changes, highlighting the role of key structural elements in mediating this interaction. Together, these results show that cardiolipin is required to mediate specific receptor-channel associations in the TIM23 complex. Our results support a new working model for the dynamic structural changes that occur within the complex during transport. More broadly, this work strongly advances our understanding of how cardiolipin mediates interactions among membrane-associated proteins.

Current preclinical models for the advancement of translational bladder cancer research.

Bladder cancer is a common disease representing the fifth most diagnosed solid tumor in the United States. Despite this, advances in our understanding of the molecular etiology and treatment of bladder cancer have been relatively lacking. This is especially apparent when recent advances in other cancers, such as breast and prostate, are taken into consideration. The field of bladder cancer research is ready and poised for a series of paradigm-shifting discoveries that will greatly impact the way this disease is clinically managed. Future preclinical discoveries with translational potential will require investigators to take full advantage of recent advances in molecular and animal modeling methodologies. We present an overview of current preclinical models and their potential roles in advancing our understanding of this deadly disease and for advancing care.

Nuclear magnetic resonance structure and dynamics of the response regulator Sma0114 from Sinorhizobium meliloti.

Receiver domains control intracellular responses triggered by signal transduction in bacterial two-component systems. Here, we report the solution nuclear magnetic resonance structure and dynamics of Sma0114 from the bacterium Sinorhizobium meliloti, the first such characterization of a receiver domain from the HWE-kinase family of two-component systems. The structure of Sma0114 adopts a prototypical α(5)/ß(5) Rossman fold but has features that set it apart from other receiver domains. The fourth ß-strand of Sma0114 houses a PFxFATGY sequence motif, common to many HWE-kinase-associated receiver domains. This sequence motif in Sma0114 may substitute for the conserved Y-T coupling mechanism, which propagates conformational transitions in the 455 (α4-ß5-α5) faces of receiver domains, to prime them for binding downstream effectors once they become activated by phosphorylation. In addition, the fourth α-helix of the consensus 455 face in Sma0114 is replaced with a segment that shows high flexibility on the pico- to nanosecond time scale by (15)N relaxation data. Secondary structure prediction analysis suggests that the absence of helix α4 may be a conserved property of the HWE-kinase-associated family of receiver domains to which Sma0114 belongs. In spite of these differences, Sma0114 has a conserved active site, binds divalent metal ions such as Mg(2+) and Ca(2+) that are required for phosphorylation, and exhibits micro- to millisecond active-site dynamics similar to those of other receiver domains. Taken together, our results suggest that Sma0114 has a conserved active site but differs from typical receiver domains in the structure of the 455 face that is used to effect signal transduction following activation.

Loss of the urothelial differentiation marker FOXA1 is associated with high grade, late stage bladder cancer and increased tumor proliferation.

Approximately 50% of patients with muscle-invasive bladder cancer (MIBC) develop metastatic disease, which is almost invariably lethal. However, our understanding of pathways that drive aggressive behavior of MIBC is incomplete. Members of the FOXA subfamily of transcription factors are implicated in normal urogenital development and urologic malignancies. FOXA proteins are implicated in normal urothelial differentiation, but their role in bladder cancer is unknown. We examined FOXA expression in commonly used in vitro models of bladder cancer and in human bladder cancer specimens, and used a novel in vivo tissue recombination system to determine the functional significance of FOXA1 expression in bladder cancer. Logistic regression analysis showed decreased FOXA1 expression is associated with increasing tumor stage (p<0.001), and loss of FOXA1 is associated with high histologic grade (p<0.001). Also, we found that bladder urothelium that has undergone keratinizing squamous metaplasia, a precursor to the development of squamous cell carcinoma (SCC) exhibited loss of FOXA1 expression. Furthermore, 81% of cases of SCC of the bladder were negative for FOXA1 staining compared to only 40% of urothelial cell carcinomas. In addition, we showed that a subpopulation of FOXA1 negative urothelial tumor cells are highly proliferative. Knockdown of FOXA1 in RT4 bladder cancer cells resulted in increased expression of UPK1B, UPK2, UPK3A, and UPK3B, decreased E-cadherin expression and significantly increased cell proliferation, while overexpression of FOXA1 in T24 cells increased E-cadherin expression and significantly decreased cell growth and invasion. In vivo recombination of bladder cancer cells engineered to exhibit reduced FOXA1 expression with embryonic rat bladder mesenchyme and subsequent renal capsule engraftment resulted in enhanced tumor proliferation. These findings provide the first evidence linking loss of FOXA1 expression with histological subtypes of MIBC and urothelial cell proliferation, and suggest an important role for FOXA1 in the malignant phenotype of MIBC.

Stress-activated kinase pathway alteration is a frequent event in bladder cancer.

OBJECTIVES: The stress-activated MAP kinases (SAPK) signaling pathways play a critical role in the cellular response to toxins and physical stress, mediate inflammation, and modulate carcinogenesis and tumor metastasis. The stress-activated MAP kinases (MAPK) c-Jun N-terminal kinase (JNK) and p38 are activated upon phosphorylation by a widely expressed and conserved family of upstream MAP kinase kinases (MAP2K). Signaling mediated by p38 and JNK has well-established importance in cancer, yet the contribution of this pathway in urothelial bladder cancer is not understood. This study evaluated stress-activated MAP kinase pathway expression in cell lines derived from human urothelial carcinomas. MATERIALS AND METHODS: Total protein lysates from a panel of human urothelial bladder cancer cell lines (RT4, T24, UMUC-3, J82, 5637, 253J, and 253J-BV) were analyzed by immunoblotting for the JNK and p38 MAPKs, as well as MKK3, MKK4, MKK6, and MKK7. Quantitative real time PCR was utilized to determine mRNA expression levels of the MAP2Ks. Stress stimuli (sorbitol, hydrogen peroxide, and UV irradiation) were used to active p38, which was measured by phospho-antibody. RESULTS: Although protein levels were variable, all cell lines expressed p38 and JNK. On the other hand, with the exception of the well-differentiated cell line RT4, each cell line had a reduction or absence of expression of one or more MAP2K. 253J and 253J-BV exhibited no expression of MKK6, even when an excess of protein was queried. mRNA levels indicated that both transcriptional and post-transcriptional mechanisms are involved in the regulation of MAP2Ks. Decreased MAP2K expression correlated with decreased ability to activate p38 in response to stress stimuli. CONCLUSIONS: Aberrant MAP2K protein expression indicates that altered cellular signal transduction mediated via JNK and p38 may be common in bladder cancer. Down-regulation of MAP2Ks likely occurs at both the transcriptional and post-transcriptional levels. Consistent with the known function of p38 and JNK in apoptosis, defects in normal pathway function caused by decreased expression of upstream MAP2Ks may provide a survival advantage to bladder cancer cells. Further investigations should focus on identifying a functional role for these pathways in bladder cancer development.

NMR assignments for the Sinorhizobium meliloti response regulator Sma0114.

Response regulators are terminal ends of bacterial two-component systems that undergo extensive structural reorganization in response to phosphoryl transfer from their cognate histidine kinases. The response regulator encoded by the gene sma0114 of Sinorhizobium meliloti is a part of a unique class of two-component systems that employ HWE histidine kinases. The distinct features of Sma0114 include a PFxFATGY motif that houses the conserved threonine in the "Y-T coupling" conformational switch which mediates output response through downstream protein-protein interactions, and the replacement of the conserved phenylalanine/tyrosine in Y-T coupling by a leucine. Here we present (1)H, (15)N, and (13)C NMR assignments for Sma0114. We identify the secondary structure of the protein based on TALOS chemical shift analysis, (3)J(HNHα) coupling constants and hydrogen-deuterium exchange. The secondary structure determined by NMR is in good agreement with that predicted from the sequence. Both methods suggest that Sma0114 differs from standard CheY-like folds by missing the fourth α-helix. Our initial NMR characterization of Sma0114 paves the way to a full investigation of the structure and dynamics of this response regulator.

Regulation of response regulator autophosphorylation through interdomain contacts.

DNA-binding response regulators (RRs) of the OmpR/PhoB subfamily alternate between inactive and active conformational states, with the latter having enhanced DNA-binding affinity. Phosphorylation of an aspartate residue in the receiver domain, usually via phosphotransfer from a cognate histidine kinase, stabilizes the active conformation. Many of the available structures of inactive OmpR/PhoB family proteins exhibit extensive interfaces between the N-terminal receiver and C-terminal DNA-binding domains. These interfaces invariably involve the α4-ß5-α5 face of the receiver domain, the locus of the largest differences between inactive and active conformations and the surface that mediates dimerization of receiver domains in the active state. Structures of receiver domain dimers of DrrB, DrrD, and MtrA have been determined, and phosphorylation kinetics were analyzed. Analysis of phosphotransfer from small molecule phosphodonors has revealed large differences in autophosphorylation rates among OmpR/PhoB RRs. RRs with substantial domain interfaces exhibit slow rates of phosphorylation. Rates are greatly increased in isolated receiver domain constructs. Such differences are not observed between autophosphorylation rates of full-length and isolated receiver domains of a RR that lacks interdomain interfaces, and they are not observed in histidine kinase-mediated phosphotransfer. These findings suggest that domain interfaces restrict receiver domain conformational dynamics, stabilizing an inactive conformation that is catalytically incompetent for phosphotransfer from small molecule phosphodonors. Inhibition of phosphotransfer by domain interfaces provides an explanation for the observation that some RRs cannot be phosphorylated by small molecule phosphodonors in vitro and provides a potential mechanism for insulating some RRs from small molecule-mediated phosphorylation in vivo.

BCAN Think Tank session 2: Molecular detection of bladder cancer: the path to progress.

Initial detection of bladder cancer and surveillance for cancer recurrence and progression are central topics in the field of urologic oncology. A session at the 3rd Annual Bladder Cancer Think Tank Meeting focused on urine-based markers for bladder cancer screening and surveillance. Here, we review the key points from the presentations, as well as the recommendations from a working group tasked with critically evaluating the information discussed in the session. Although the practicality of markers has been challenged, the resounding consensus was that continued research efforts directed at developing and evaluating markers for screening and early detection are warranted.

A novel domain in translational GTPase BipA mediates interaction with the 70S ribosome and influences GTP hydrolysis.

BipA is a universally conserved prokaryotic GTPase that exhibits differential ribosome association in response to stress-related events. It is a member of the translation factor family of GTPases along with EF-G and LepA. BipA has five domains. The N-terminal region of the protein, consisting of GTPase and beta-barrel domains, is common to all translational GTPases. BipA domains III and V have structural counterparts in EF-G and LepA. However, the C-terminal domain (CTD) of the protein is unique to the BipA family. To investigate how the individual domains of BipA contribute to the biological properties of the protein, deletion constructs were designed and their GTP hydrolysis and ribosome binding properties assessed. Data presented show that removal of the CTD abolishes the ability of BipA to bind to the ribosome and that ribosome complex formation requires the surface provided by domains III and V and the CTD. Additional mutational analysis was used to outline the BipA-70S interaction surface extending across these domains. Steady state kinetic analyses revealed that successive truncation of domains from the C-terminus resulted in a significant increase in the intrinsic GTP hydrolysis rate and a loss of ribosome-stimulated GTPase activity. These results indicate that, similar to other translational GTPases, the ribosome binding and GTPase activities of BipA are tightly coupled. Such intermolecular regulation likely plays a role in the differential ribosome binding by the protein.

Knockdown of the Drosophila GTPase nucleostemin 1 impairs large ribosomal subunit biogenesis, cell growth, and midgut precursor cell maintenance.

Mammalian nucleostemin (NS) is a nucleolar guanosine triphosphate-binding protein implicated in cell cycle progression, stem cell proliferation, and ribosome assembly. Drosophila melanogaster contains a four-member nucleostemin family (NS1-4). NS1 is the closest orthologue to human NS; it shares 33% identity and 67% similarity with human NS. We show that NS1 has intrinsic GTPase and ATPase activity and that it is present within nucleoli of most larval and adult cells. Endogenous NS1 and lightly expressed green fluorescent protein (GFP)-NS1 enrich within the nucleolar granular regions as expected, whereas overexpressed GFP-NS1 localized throughout the nucleolus and nucleoplasm, and to several transcriptionally active interbands of polytene chromosomes. Severe overexpression correlated with the appearance of melanotic tumors and larval/pupal lethality. Depletion of 60% of NS1 transcripts also lead to larval and pupal lethality. NS1 protein depletion>95 correlated with the loss of imaginal island (precursor) cells in the larval midgut and to an apparent block in the nucleolar release of large ribosomal subunits in terminally differentiated larval midgut polyploid cells. Ultrastructural examination of larval Malpighian tubule cells depleted for NS1 showed a loss of cytoplasmic ribosomes and a concomitant appearance of cytoplasmic preautophagosomes and lysosomes. We interpret the appearance of these structures as indicators of cell stress response.

Key concerns about the current state of bladder cancer: a position paper from the Bladder Cancer Think Tank, the Bladder Cancer Advocacy Network, and the Society of Urologic Oncology.

Bladder cancer is the fifth most common cancer in the United States and, on a per capita basis, is the most expensive cancer from diagnosis to death. Unfortunately, National Cancer Institute funding for bladder cancer is quite low when compared with other common malignancies. Limited funding has stifled research opportunities for new and established investigators, ultimately encouraging them to redirect research efforts to other organ sites. Waning interest of scientists has further fueled the cycle of modest funding for bladder cancer. One important consequence of this has been a lack of scientific advancement in the field. Patient advocates have decidedly advanced research efforts in many cancer sites. Breast, prostate, pancreatic, and ovarian cancer advocates have organized highly successful campaigns to lobby the federal government and the medical community to devote increased attention and funding to understudied malignancies and to conduct relevant studies to better understand the therapy, diagnosis, and prevention of these diseases. Bladder cancer survivors have lacked a coordinated advocacy voice until recently. A concerted effort to align bladder cancer advocates, clinicians, and urologic organizations is essential to define the greatest needs in bladder cancer and to develop related solutions. This position paper represents a collaborative discussion to define the most concerning trends and greatest needs in the field of bladder cancer as outlined by the Bladder Cancer Think Tank, the Bladder Cancer Advocacy Network, and the Society of Urologic Oncology.

Rethinking the central dogma: noncoding RNAs are biologically relevant.

Non-coding RNAs (ncRNAs) are a large class of functional molecules with over 100 unique classes described to date. ncRNAs are diverse in terms of their function and size. A relatively new class of small ncRNA, called microRNAs (miRNA), have received a great deal of attention in the literature in recent years. miRNAs are endogenously encoded gene families that demonstrate striking evolutionary conservation. miRNAs serve essential and diverse physiological functions such as differentiation and development, proliferation, maintaining cell type phenotypes, and many others. The discovery and ongoing investigation of miRNAs is part of a revolution in biology that is changing the basic concepts of gene expression and RNA functionality. A single miRNA can participate in controlling the expression of up to several hundred protein-coding genes by interacting with mRNAs, generally in 3' untranslated regions. Our new and developing understanding of miRNAs, and other ncRNAs, promises to lead to significant contributions to medicine. Specifically, miRNAs are likely to serve as the basis for novel therapies and diagnostic tools.

FYN is overexpressed in human prostate cancer.

OBJECTIVE: To test the hypothesis that FYN, a member of the SRC family of kinases (SFKs), is up-regulated in prostate cancer, as FYN is functionally distinct from other SFKs, and interacts with FAK and paxillin (PXN), regulators of cell morphology and motility. MATERIALS AND METHODS: Through data-mining in Oncomine (http://www.oncomine.org), cell-line profiling with immunoblotting, quantitative reverse transcription and polymerase chain reaction (RT-PCR) and immunohistochemical analysis, we described FYN expression in prostate cancer. The analysis included 32 cases of prostate cancer, nine of prostatic intraepithelial neoplasia (PIN) and 19 normal prostates. Samples were scored for the percentage of stained glands and intensity of staining (from 0 to 3). Each sample was assigned a composite score generated by multiplying percentage and intensity. RESULTS: Data-mining showed an eight times greater FYN expression in prostate cancer than in normal tissue; this was specific to FYN and not present for other SFKs. Expression of FYN in prostate cancer cell lines (LNCaP, 22Rv1, PC3, DuPro) was detected using quantitative RT-PCR and immunoblotting. Expression of FYN and its signalling partners FAK and PXN was detected in human tissue. Comparing normal with cancer samples, there was a 2.1-fold increase in median composite score for FYN (P < 0.001) 1.7-fold increase in FAK (P < 0.001), and a doubling in PXN (P < 0.05). There was a 1.7-fold increase in FYN (P < 0.05) and a 1.6-fold increase in FAK (P < 0.01) in cancer compared with PIN. CONCLUSIONS: These studies support the hypothesis that FYN and its related signalling partners are up-regulated in prostate cancer, and support further investigation into the role of the FYN as a therapeutic target.

Salmonella enterica serovar Typhimurium BipA exhibits two distinct ribosome binding modes.

BipA is a highly conserved prokaryotic GTPase that functions to influence numerous cellular processes in bacteria. In Escherichia coli and Salmonella enterica serovar Typhimurium, BipA has been implicated in controlling bacterial motility, modulating attachment and effacement processes, and upregulating the expression of virulence genes and is also responsible for avoidance of host defense mechanisms. In addition, BipA is thought to be involved in bacterial stress responses, such as those associated with virulence, temperature, and symbiosis. Thus, BipA is necessary for securing bacterial survival and successful invasion of the host. Steady-state kinetic analysis and pelleting assays were used to assess the GTPase and ribosome-binding properties of S. enterica BipA. Under normal bacterial growth, BipA associates with the ribosome in the GTP-bound state. However, using sucrose density gradients, we demonstrate that the association of BipA and the ribosome is altered under stress conditions in bacteria similar to those experienced during virulence. The data show that this differential binding is brought about by the presence of ppGpp, an alarmone that signals the onset of stress-related events in bacteria.