Dissection of recognition determinants of Escherichia coli σ32 suggests a composite −10 region with an 'extended −10' motif and a core −10 element

σ³² controls expression of heat shock genes in Escherichia coli and is widely distributed in proteobacteria. The distinguishing feature of σ³² promoters is a long −10 region (CCCCATNT) whose tetra-C motif is important for promoter activity. Using alanine-scanning mutagenesis of σ³² and in vivo and in vitro assays, we identified promoter recognition determinants of this motif. The most downstream C (−13) is part of the −10 motif; our work confirms and extends recognition determinants of −13C. Most importantly, our work suggests that the two upstream Cs (−16, −15) constitute an 'extended −10' recognition motif that is recognized by K130, a residue universally conserved in β- and γ-proteobacteria. This residue is located in the α-helix of σDomain 3 that mediates recognition of the extended −10 promoter motif in other σs. K130 is not conserved in α- and δ-/ε-proteobacteria and we found that σ³² from the α-proteobacterium Caulobacter crescentus does not need the extended −10 motif for high promoter activity. This result supports the idea that K130 mediates extended −10 recognition. σ³² is the first Group 3 σ shown to use the 'extended −10' recognition motif.     

QSAR analysis of pyrazolidine-3,5-diones derivatives as Dyrk1A inhibitors

Individuals with Down syndrome (DS) suffer from mental retardation. Overexpression and the resulting increased specific activity of Dyrk1A kinase located on chromosome 21 cause a learning and memory deficit in Dyrk1A transgenic mice. To search for therapeutic agents with Dyrk1A inhibition activity, previously we obtained HCD160 as a new hit compound for Dyrk1A inhibition. In the present study, we synthesized 34 HCD160 derivatives to investigate the quantitative structure–activity relationship (QSAR). This analysis could provide important information for novel drug discovery for treatment of DS related learning and memory deficits.     

Disseminated prostate cancer cells can instruct hematopoietic stem and progenitor cells to regulate bone phenotype

Prostate cancer metastases and hematopoietic stem cells (HSC) frequently home to the bone marrow, where they compete to occupy the same HSC niche. We have also shown that under conditions of hematopoietic stress, HSCs secrete the bone morphogenetic proteins (BMP)-2 and BMP-6 that drives osteoblastic differentiation from mesenchymal precursors. As it is not known, we examined whether metastatic prostate cancer cells can alter regulation of normal bone formation by HSCs and hematopoietic progenitor cells (HPC). HSC/HPCs isolated from mice bearing nonmetastatic and metastatic tumor cells were isolated and their ability to influence osteoblastic and osteoclastic differentiation was evaluated. When the animals were inoculated with the LNCaP C4-2B cell line, which produces mixed osteoblastic and osteolytic lesions in bone, HPCs, but not HSCs, were able to induced stromal cells to differentiate down an osteoblastic phenotype. Part of the mechanism responsible for this activity was the production of BMP-2. On the other hand, when the animals were implanted with PC3 cells that exhibits predominantly osteolytic lesions in bone, HSCs derived from these animals were capable of directly differentiating into tartrate-resistant acid phosphatase-positive osteoclasts through an interleukin-6-mediated pathway. These studies for the first time identify HSC/HPCs as novel targets for future therapy involved in the bone abnormalities of prostate cancer.     

GIGANTEA and EARLY FLOWERING 4 in Arabidopsis exhibit differential phase-specific genetic influences over a diurnal cycle

The endogenous circadian clock regulates many physiological processes related to plant survival and adaptability. GIGANTEA (GI), a clock-associated protein, contributes to the maintenance of circadian period length and amplitude, and also regulates flowering time and hypocotyl growth in response to day length. Similarly, EARLY FLOWERING 4 (ELF4), another clock regulator, also contributes to these processes. However, little is known about either the genetic or molecular interactions between GI and ELF4 in Arabidopsis. In this study, we investigated the genetic interactions between GI and ELF4 in the regulation of circadian clock-controlled outputs. Our mutant analysis shows that GI is epistatic to ELF4 in flowering time determination, while ELF4 is epistatic to GI in hypocotyl growth regulation. Moreover, GI and ELF4 have a synergistic or additive effect on endogenous clock regulation. Gene expression profiling of gi, elf4, and gi elf4 mutants further established that GI and ELF4 have differentially dominant influences on circadian physiological outputs at dusk and dawn, respectively. This phasing of GI and ELF4 influences provides a potential means to achieve diversity in the regulation of circadian physiological outputs, including flowering time and hypocotyl growth.     

Turning CALM into excitement: AP180 and CALM in endocytosis and disease

Dynamic flux of membrane between intracellular compartments is a key feature of all eukaryotic cells. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a crucial role in membrane dynamics by facilitating membrane fusion, for example at synapses where small synaptic vesicles (SVs) undergo activity-regulated neuroexocytosis, followed by the endocytic re-cycling of SV proteins and lipids. Recent work shows that the assembly protein 180 (AP180) N-terminal homology (ANTH) domain containing proteins AP180 and clathrin assembly lymphoid myeloid leukaemia (CALM) not only regulate the assembly of the endocytic machinery but also act as sorters for a subset of SNAREs, the vesicle-associated membrane proteins (VAMPs), most notably VAMP/synaptobrevin 2 at synapses. In this review, we summarise the current state of knowledge about the roles of AP180 and CALM family members in clathrin-dependent membrane traffic, the molecular mechanistic basis for their activities and their potential involvement in human disease.     

Improved control of tuberculosis and activation of macrophages in mice lacking protein kinase R

Host factors that microbial pathogens exploit for their propagation are potential targets for therapeuic countermeasures. No host enzyme has been identified whose genetic absence benefits the intact mammalian host in vivo during infection with Mycobacterium tuberculosis (Mtb), the leading cause of death from bacterial infection. Here, we report that the dsRNA-dependent protein kinase (PKR) is such an enzyme. PKR-deficient mice contained fewer viable Mtb and showed less pulmonary pathology than wild type mice. We identified two potential mechanisms for the protective effect of PKR deficiency: increased apoptosis of macrophages in response to Mtb and enhanced activation of macrophages in response to IFN-gamma. The restraining effect of PKR on macrophage activation was explained by its mediation of a previously unrecognized ability of IFN-gamma to induce low levels of the macrophage deactivating factor interleukin 10 (IL10). These observations suggest that PKR inhibitors may prove useful as an adjunctive treatment for tuberculosis.     

Control of anti-thrombogenic properties: surface-induced self-assembly of fibrinogen fibers

Wound healing is a complex process initiated by the formation of fibrin fibers and endothelialization. Normally, this process is triggered in a wound by thrombin cleavage of fibrinopeptides on fibrinogen molecules, which allows them to self spontaneously-assemble into large fibers that provide the support structure of the clot and promote healing. We have found that the fibrous structures can also form without thrombin on most polymer or metal surfaces, including those commonly used for stents. We show that the relatively hydrophobic E and D regions of the fibrinogen molecule are adsorbed on these surfaces, exposing the αC domains, which in turn results in the formation of large fiber structures that promote endothelial cell adhesion. We show that the entire process can be suppressed when stents or other substrates are coated with polymers that are functionalized to bind the αC domains, leading to the development of potentially nonthrombogenic implant materials.     

Decreased circulating C3 levels and mesangial C3 deposition predict renal outcome in patients with IgA nephropathy

Background and Aims

Mesangial C3 deposition is frequently observed in patients with IgA nephropathy (IgAN). However, the role of complement in the pathogenesis or progression of IgAN is uncertain. In this observational cohort study, we aimed to identify the clinical implications of circulating C3 levels and mesangial C3 deposition and to investigate their utility as predictors of renal outcomes in patients with IgAN.

Methods

A total of 343 patients with biopsy-proven IgAN were enrolled between January 2000 and December 2008. Decreased serum C3 level (hypoC3) was defined as C3 <90 mg/dl. The study endpoint was end-stage renal disease (ESRD) and a doubling of the baseline serum creatinine (D-SCr).

Results

Of the patients, there were 66 patients (19.2%) with hypoC3. During a mean follow-up of 53.7 months, ESRD occurred in 5 patients (7.6%) with hypoC3 compared with 9 patients (3.2%) with normal C3 levels (P = 0.11). However, 12 patients (18.2%) with hypoC3 reached D-SCr compared with 17 patients (6.1%) with normal C3 levels [Hazard ratio (HR), 3.59; 95% confidence interval (CI), 1.33–10.36; P = 0.018]. In a multivariable model in which serum C3 levels were treated as a continuous variable, hypoC3 significantly predicted renal outcome of D-SCr (per 1 mg/dl increase of C3; HR, 0.95; 95% CI, 0.92–0.99; P = 0.011). The risk of reaching renal outcome was significantly higher in patients with mesangial C3 deposition 2+ to 3+ than in patients without deposition (HR 9.37; 95% CI, 1.10–80.26; P = 0.04).

Conclusions

This study showed that hypoC3 and mesangial C3 deposition were independent risk factors for progression, suggesting that complement activation may play a pathogenic role in patients with IgAN.     

A critical role for the Wnt effector Tcf4 in adult intestinal homeostatic self-renewal

Throughout life, intestinal Lgr5+ stem cells give rise to proliferating transient amplifying cells in crypts, which subsequently differentiate into one of the five main cell types and migrate along the crypt-villus axis. These dynamic processes are coordinated by a relatively small number of evolutionarily conserved signaling pathways, which includes the Wnt signaling pathway. The DNA-binding proteins of the T-cell factor family, Tcf1/Tcf7, Lef, Tcf3/Tcf7l1, and Tcf4/Tcf7l2, constitute the downstream effectors of the Wnt signaling pathway. While Tcf4 is the major member active during embryogenesis, the role of these Wnt effectors in the homeostasis of the adult mouse intestinal epithelium is unresolved. Using Tcf1-/-, Tcf3(flox), and novel Tcf4(flox) mice, we demonstrate an essential role for Tcf4 during homeostasis of the adult mouse intestine.     

Coexpressing shRNA with fluorescence tags for quantification of cell migration studies

Understanding migration of cells has many implications in human physiology; some examples include developmental biology, healing, immune responses and tissue remodeling. On the other hand, invasive migration by tumor cells is pathological and is a major cause of mortality amongst cancer sufferers. Cell migration assays have been widely used to quantify potentially metastatic genes. In recent years, the use of RNAi has significantly increased the tools available in cell migration research due to its specific gene targeting for knockdown. The inability to ensure 100% transfection/transduction efficiency reduces the sensitivity of cell migration assays because cells not successfully transfected/transduced with the RNAi are also included in the calculations. This study introduces a different experimental setup mathematically expressed in our named normalized relative infected cell count (N-RICC) that analyses cell migration assays by co-expressing retrovirally transduced shRNA with fluorescence tags from a single vector. Vectors transduced into cells are visible under fluorescence, thus alleviating the problems involved with transduction efficiency by individually identifying cells with targeted genes. Designed shRNAs were targeted against a list of potentially metastatic genes in a highly migratory breast cancer cell line model, MDA-MB-231. We have successfully applied N-RICC analysis to show greater sensitivity of integrin alpha5 (ITGA5) and Ras homologue A (RhoA) in cell metastasis over conventional methods in scratch-wound assays and migration chambers assays.