The Anti-Migratory Effects of FKBPL and Its Peptide Derivative, AD-01: Regulation of CD44 and the Cytoskeletal Pathway

FK506 binding protein-like (FKBPL) and its peptide derivatives exert potent anti-angiogenic activity and and control tumour growth in xenograft models, when administered exogenously. However, the role of endogenous FKBPL in angiogenesis is not well characterised. Here we investigated the molecular effects of the endogenous protein and its peptide derivative, AD-01, leading to their anti-migratory activity. Inhibition of secreted FKBPL using a blocking antibody or siRNA-mediated knockdown of FKBPL accelerated the migration of human microvascular endothelial cells (HMEC-1). Furthermore, MDA-MB-231 tumour cells stably overexpressing FKBPL inhibited tumour vascular development suggesting that FKBPL secreted from tumour cells could inhibit angiogenesis. Whilst FKBPL and AD-01 target CD44, the nature of this interaction is not known and here we have further interrogated this aspect. We have demonstrated that FKBPL and AD-01 bind to the CD44 receptor and inhibit tumour cell migration in a CD44 dependant manner; CD44 knockdown abrogated AD-01 binding as well as its anti-migratory activity. Interestingly, FKBPL overexpression and knockdown or treatment with AD-01, regulated CD44 expression, suggesting a co-regulatory pathway for these two proteins. Downstream of CD44, alterations in the actin cytoskeleton, indicated by intense cortical actin staining and a lack of cell spreading and communication were observed following treatment with AD-01, explaining the anti-migratory phenotype. Concomitantly, AD-01 inhibited Rac-1 activity, up-regulated RhoA and the actin binding proteins, profilin and vinculin. Thus the anti-angiogenic protein, FKBPL, and AD-01, offer a promising and alternative approach for targeting both CD44 positive tumours and vasculature networks.

Regulation of Foxp3+ Inducible Regulatory T Cell Stability by SOCS2

Suppressor of cytokine signaling (SOCS) proteins are key regulators of CD4+ T cell differentiation, and in particular, we have recently shown that SOCS2 inhibits the development of Th2 cells and allergic immune responses. Interestingly, transcriptome analyses have identified SOCS2 as being preferentially expressed in both natural regulatory T cells (Tregs) and inducible Tregs (iTregs); however, the role of SOCS2 in Foxp3+ Treg function or development has not been fully elucidated. In this study, we show that despite having no effect on natural Treg development or function, SOCS2 is highly expressed in iTregs and required for the stable expression of Foxp3 in iTregs in vitro and in vivo. Indeed, SOCS2-deficient CD4+ T cells upregulated Foxp3 following in vitro TGF-ß stimulation, but failed to maintain stable expression of Foxp3. Moreover, in vivo generation of iTregs following OVA feeding was impaired in the absence of SOCS2 and could be rescued in the presence of IL-4 neutralizing Ab. Following IL-4 stimulation, SOCS2-deficient Foxp3+ iTregs secreted elevated IFN-? and IL-13 levels and displayed enhanced STAT6 phosphorylation. Therefore, we propose that SOCS2 regulates iTreg stability by downregulating IL-4 signaling. Moreover, SOCS2 is essential to maintain the anti-inflammatory phenotype of iTregs by preventing the secretion of proinflammatory cytokines. Collectively, these results suggest that SOCS2 may prevent IL-4-induced Foxp3+ iTreg instability. Foxp3+ iTregs are key regulators of immune responses at mucosal surfaces; therefore, this dual role of SOCS2 in both Th2 and Foxp3+ iTregs reinforces SOCS2 as a potential therapeutic target for Th2-biased diseases.

Regulation of cellular responses by deubiquitinating enzymes:an update

The conjugation of ubiquitin as either a monomer or as a chain has long been known to regulate the stability, localisation, trafficking and/or function of many intracellular proteins. However, the recent explosion in our knowledge of the enzymes responsible for the removal of ubiquitin suggests they also play an important role in the regulation of many processes. Here we examine what is known about the role of deubiquitinating enzymes (DUBs), with particular emphasis upon their impact on cellular responses to external stimuli. In addition, we look at the evidence that although these enzymes are heavily outnumbered by those responsible for ubiquitin conjugation, that these enzymes may still be important cellular regulators, due to their ability to play multiple roles which can be cell type and cell context specific.

Dam Methylation Participates in the Regulation of PmrA/PmrB and RcsC/RcsD/RcsB Two Component Regulatory Systems in Salmonella enterica Serovar Enteritidis

The absence of Dam in Salmonella enterica serovar Enteritidis causes a defect in lipopolysaccharide (LPS) pattern associated to a reduced expression of wzz gene. Wzz is the chain length regulator of the LPS O-antigen. Here we investigated whether Dam regulates wzz gene expression through its two known regulators, PmrA and RcsB. Thus, the expression of rcsB and pmrA was monitored by quantitative real-time RT-PCR and Western blotting using fusions with 3×FLAG tag in wild type (wt) and dam strains of S. Enteritidis. Dam regulated the expression of both rcsB and pmrA genes; nevertheless, the defect in LPS pattern was only related to a diminished expression of RcsB. Interestingly, regulation of wzz in serovar Enteritidis differed from that reported earlier for serovar Typhimurium; RcsB induces wzz expression in both serovars, whereas PmrA induces wzz in S. Typhimurium but represses it in serovar Enteritidis. Moreover, we found that in S. Enteritidis there is an interaction between both wzz regulators: RcsB stimulates the expression of pmrA and PmrA represses the expression of rcsB. Our results would be an example of differential regulation of orthologous genes expression, providing differences in phenotypic traits between closely related bacterial serovars.

Global down-regulation of HOX gene expression in PML-RARalpha + acute promyelocytic leukemia identified by small-array real-time PCR

Acute promyelocytic leukemia (APL) is associated with a reciprocal and balanced translocation involving the retinoic acid receptor-alpha (RARalpha). All-trans retinoic acid (ATRA) is used to treat APL and is a potent morphogen that regulates HOX gene expression in embryogenesis and organogenesis. HOX genes are also involved in hematopoiesis and leukemogenesis. Thirty-nine mammalian HOX genes have been identified and classified into 13 paralogous groups clustered on 4 chromosomes. They encode a complex network of transcription regulatory proteins whose precise targets remain poorly understood. The overall function of the network appears to be dictated by gene dosage. To investigate the mechanisms involved in HOX gene regulation in hematopoiesis and leukemogenesis by precise measurement of individual HOX genes, a small-array real-time HOX (SMART-HOX) quantitative polymerase chain reaction (PCR) platform was designed and validated. Application of SMART-HOX to 16 APL bone marrow samples revealed a global down-regulation of 26 HOX genes compared with normal controls. HOX gene expression was also altered during differentiation induced by ATRA in the PML-RARalpha(+) NB4 cell line. PML-RARalpha fusion proteins have been reported to act as part of a repressor complex during myeloid cell differentiation, and a model linking HOX gene expression to this PML-RARalpha repressor complex is now proposed.

Regulation of the pollen-specific actin-depolymerizing factor LIADF1

Pollen tube growth is dependent on a dynamic actin cytoskeleton, suggesting that actin-regulating proteins are involved. We have examined the regulation of the lily pollen-specific actin-depolymerizing factor (ADF) LIADF1. Its actin binding and depolymerizing activity is pH sensitive, inhibited by certain phosphoinositides, but not controlled by phosphorylation. Compared with its F-actin binding properties, its low activity in depolymerization assays has been used to explain why pollen ADF decorates F-actin in pollen grains. This low activity is incompatible with a role in increasing actin dynamics necessary to promote pollen tube growth. We have identified a plant homolog of actin-interacting protein, AIP1, which enhances the depolymerization of F-actin in the presence of LIADF1 by similar to60%. Both pollen ADF and pollen AIP1 bind F-actin in pollen grains but are mainly cytoplasmic in pollen tubes. Our results suggest that together these proteins remodel actin filaments as pollen grains enter and exit dormancy.

Apolipoprotein-defined lipoproteins and apolipoproteins:Associations with abnormal albuminuria in type 1 diabetes in the diabetes control and complications trial/epidemiology of diabetes interventions and complications cohort

Dyslipoproteinemia has been associated with nephropathy in diabetes, with stronger correlations in men than in women. We aimed to characterize and compare plasma lipoprotein profiles associated with normal and increased albuminuria in men and women using apolipoprotein-defined lipoprotein subclasses and simple apolipoprotein measures.

Trait directed de novo population transcriptome dissects genetic regulation of a balanced polymorphism in phosphorus nutrition/arsenate tolerance in a wild grass Holcus lanatus

The aim of this study was to characterize the transcriptome of a balanced polymorphism, under the regulation of a single gene, for phosphate fertilizer responsiveness/arsenate toler- ance in wild grass Holcus lanatus genotypes screened from the same habitat.

De novo transcriptome sequencing, RNAseq (RNA sequencing) and single nucleotide poly- morphism (SNP) calling were conducted on RNA extracted from H.lanatus. Roche 454 sequencing data were assembled into c. 22 000 isotigs, and paired-end Illumina reads for phosphorus-starved (P) and phosphorus-treated (P+) genovars of tolerant (T) and nontoler- ant (N) phenotypes were mapped to this reference transcriptome.

Heatmaps of the gene expression data showed strong clustering of each P+/P treated genovar, as well as clustering by N/T phenotype. Statistical analysis identified 87 isotigs to be significantly differentially expressed between N and T phenotypes and 258 between P+ and P treated plants. SNPs and transcript expression that systematically differed between N and T phenotypes had regulatory function, namely proteases, kinases and ribonuclear RNA- binding protein and transposable elements.

A single gene for arsenate tolerance led to distinct phenotype transcriptomes and SNP pro- files, with large differences in upstream post-translational and post-transcriptional regulatory genes rather than in genes directly involved in P nutrition transport and metabolism per se.

Clinical and technical factors associated with skin intrinsic fluorescence in subjects with type 1 diabetes from the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications Study

The Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and Complications(EDIC) studies have established multiyear mean hemoglobin A1c (HbA1c) as predictive of microvascular complications in persons with type 1 diabetes. However, multiyear mean HbA1c is not always available in the clinical setting. Skin advanced glycation end products (AGEs) are thought to partially reflect effects of hyperglycemia over time, and measurement of skin AGEs might be a surrogate for multiyear mean HbA1c. As certain AGEs fluoresce and skin fluorescence has been demonstrated to correlate with the concentration of skin AGEs, noninvasive measurement by skin intrinsic fluorescence(SIF) facilitates the exploration of the association of mean HbA1c and other clinical/technical factors with SIF using the detailed phenotypic database available in DCCT/EDIC.

Long-term renal outcomes of patients with type 1 diabetes mellitus and microalbuminuria:an analysis of the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications cohort

Microalbuminuria is a common diagnosis in the clinical care of patients with type 1 diabetes mellitus. Long-term outcomes after the development of microalbuminuria are variable.

Modern-day clinical course of type 1 diabetes mellitus after 30 years' duration:the diabetes control and complications trial/epidemiology of diabetes interventions and complications and Pittsburgh epidemiology of diabetes complications experience (1983-2005)

Clinical treatment goals of type 1 diabetes mellitus (T1DM) have changed since the Diabetes Control and Complications Trial (DCCT) demonstrated reduced long-term complications with intensive diabetes therapy. There have been few longitudinal studies to describe the clinical course of T1DM in the age of intensive therapy. Our objective was to describe the current-day clinical course of T1DM.

Glycation, carbonyl stress, EAGLEs, and the vascular complications of diabetes

The association between poor metabolic control and the microvascular complications of diabetes is now well established, but the relationship between long-term metabolic control and the accelerated atherosclerosis of diabetes is as yet poorly defined. Hyperglycemia is the standard benchmark by which metabolic control is assessed. One mechanism by which elevated glucose levels may mediate vascular injury is through early and advanced glycation reactions affecting a wide variety of target molecules. The "glycation hypothesis'' has developed over the past 30 years, evolving gradually into a "carbonyl stress hypothesis'' and taking into account not only the modification of proteins by glucose, but also the roles of oxidative stress, a wide range of reactive carbonyl-containing intermediates (derived not only from glucose but also from lipids), and a variety of extra- and intracellular target molecules. The final products of these reactions may now be termed "Either Advanced Glycation or Lipoxidation End-Products'' or "EAGLEs.'' The ubiquity of carbonyl stress within the body, the complexity of the reactions involved, the variety of potential carbonyl intermediates and target molecules and their differing half-lives, and the slow development of the complications of diabetes all pose major challenges in dissecting the significance of these processes. The extent of the reactions tends to correlate with overall metabolic control, creating pitfalls in the interpretation of associative data. Many animal and cell culture studies, while supporting the hypothesis, must be viewed with caution in terms of relevance to human diabetes. In this article, the development of the carbonyl stress hypothesis is reviewed, and implications for present and future treatments to prevent complications are discussed.

Serum lipoproteins in the diabetes control and complications trial/epidemiology of diabetes intervention and complications cohort:associations with gender and glycemia

To relate the nuclear magnetic resonance (NMR)-determined lipoprotein profile, conventional lipid and apolipoprotein measures, and in vitro oxidizibility of LDL with gender and glycemia in type 1 diabetes.