Requirement of mitogen-activated protein kinase kinase 3 (MKK3) for activation of p38alpha and p38delta MAPK isoforms by TGF-beta 1 in murine mesangial cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix (ECM) synthesis that leads to renal fibrosis. Intracellular signaling mechanisms involved in this process remain incompletely understood. Mitogen-activated protein kinase (MAPK) is a major stress signal-transducing pathway, and we have previously reported activation of p38 MAPK by TGF-beta1 in rat mesangial cells and its role in the stimulation of pro-alpha1(I) collagen. In this study, we further investigated the mechanism of p38 MAPK activation by TGF-beta1 and the role of MKK3, an upstream MAPK kinase of p38 MAPK, by examining the effect of targeted disruption of the Mkk3 gene. We first isolated glomerular mesangial cells from MKK3-null (Mkk3-/-) and wild-type (Mkk3+/+) control mice. Treatment with TGF-beta1 induced rapid phosphorylation of MKK3 as well as p38 MAPK within 15 min in cultured wild-type (Mkk3+/+) mouse mesangial cells. In contrast, TGF-beta1 failed to induce phosphorylation of either MKK3 or p38 MAPK in MKK3-deficient (Mkk3-/-) mouse mesangial cells, indicating that MKK3 is required for TGF-beta1-induced p38 MAPK activation. TGF-beta1 selectively activated the p38 MAPK isoforms p38alpha and p38delta in wild-type (Mkk3+/+) mesangial cells, but not in MKK3-deficient (Mkk3-/-) mesangial cells. Thus, activation of p38alpha and p38delta is dependent on the activation of upstream MKK3 by TGF-beta1. Furthermore, MKK3 deficiency resulted in a selective disruption of TGF-beta1-stimulated up-regulation of pro-alpha1(I) collagen expression but not TGF-beta1 induction of fibronectin and PAI-1. These data demonstrate that the MKK3 is a critical component of the TGF-beta1 signaling pathway, and its activation is required for subsequent p38alpha and p38delta MAPK activation and collagen stimulation by TGF-beta1.     

Cyclosporine A enhances leukocyte binding by human intestinal microvascular endothelial cells through inhibition of p38 MAPK and iNOS. Paradoxical proinflammatory effect on the microvascular endothelium

The calcineurin inhibitor cyclosporine A (CsA) modulates leukocyte cytokine production but may also effect nonimmune cells, including microvascular endothelial cells, which regulate the inflammatory process through leukocyte recruitment. We hypothesized that CsA would promote a proinflammatory phenotype in human intestinal microvascular endothelial cells (HIMEC), by inhibiting inducible nitric-oxide synthase (iNOS, NOS2)-derived NO, normally an important mechanism in limiting endothelial activation and leukocyte adhesion. Primary cultures of HIMEC were used to assess CsA effects on endothelial activation, leukocyte interaction, and the expression of iNOS as well as cell adhesion molecules. CsA significantly increased leukocyte binding to activated HIMEC, but paradoxically decreased endothelial expression of cell adhesion molecules (E-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule-1). In contrast, CsA completely inhibited the expression of iNOS in tumor necrosis factor-alpha/lipopolysaccharide-activated HIMEC. CsA blocked p38 MAPK phosphorylation in activated HIMEC, a key pathway in iNOS expression, but failed to inhibit NFkappaB activation. These studies demonstrate that CsA exerts a proinflammatory effect on HIMEC by blocking iNOS expression. CsA exerts a proinflammatory effect on the microvascular endothelium, and this drug-induced endothelial dysfunction may help explain its lack of efficacy in the long-term treatment of chronically active inflammatory bowel disease.     

SUMO-1 targets RanGAP1 to kinetochores and mitotic spindles

RanGAP1 was the first documented substrate for conjugation with the ubiquitin-like protein SUMO-1. However, the functional significance of this conjugation has not been fully clarified. We sought to examine RanGAP1 behavior during mitosis. We found that RanGAP1 associates with mitotic spindles and that it is particularly concentrated at foci near kinetochores. Association with kinetochores appeared soon after nuclear envelope breakdown and persisted until late anaphase, but it was lost coincident with nuclear envelope assembly in telophase. A mutant RanGAP1 protein lacking the capacity to be conjugated to SUMO-1 no longer associated with spindles, indicating that conjugation was essential for RanGAP1's mitotic localization. RanBP2, a nuclear pore protein that binds SUMO-1-conjugated RanGAP1 during interphase, colocalized with RanGAP1 on spindles, suggesting that a complex between these two proteins may be involved in mitotic targeting of RanGAP1. This report shows for the first time that SUMO-1 conjugation is required for mitotic localization of RanGAP1, and suggests that a major role of SUMO-1 conjugation to RanGAP1 may be the spatial regulation of the Ran pathway during mitosis.     

The mechanism of inhibition of Ran-dependent nuclear transport by cellular ATP depletion

Rran-dependent nuclear transport requires a nuclear pool of RanGTP both for the assembly of export complexes and the disassembly of import complexes. Accordingly, in order for these processes to proceed, Ran-dependent nuclear import and export assays in vitro require the addition of GTP to produce RanGTP. Notably, no ATP requirement can be detected for these transport processes in vitro. But in vivo, when cells are depleted of ATP by the addition of sodium azide and 2-deoxyglucose to block ATP production by oxidative phosphorylation and glycolysis, respectively, Ran-dependent nuclear import and export are rapidly inhibited. This raised the question of whether there is an ATP requirement for these nuclear transport pathways in an intact cell that has remained undetected in vitro. Here we report that the free (but not total) GTP concentration rapidly drops to an undetectable level upon ATP depletion as does the availability of RanGTP. Our conclusion is that the inhibition of Ran-dependent nuclear transport observed upon ATP depletion in vivo results from a shortage of RanGTP rather than the inhibition of some ATP-dependent process.     

Identification of cytoskeletal regulatory proteins required for efficient phagocytosis in Drosophila

Phagocytosis is a complex and apparently evolutionarily conserved process that plays a central role in the immune response to infection. By ultrastructural and functional criteria, Drosophila hemocyte (macrophage) phagocytosis resembles mammalian phagocytosis. Using a non-saturated forward genetic screen for larval hemocyte phagocytosis mutants, D-SCAR and profilin were identified as important regulators of phagocytosis in Drosophila. In both hemocytes ex vivo and the macrophage-like S2 cell line, lack of D-SCAR significantly decreased phagocytosis of Escherichia coli and Staphylococcus aureus. In contrast, profilin mutant hemocytes exhibited increased phagocytic activity. Analysis of double mutants suggests that D-SCAR and profilin interact during phagocytosis. Finally, RNA interference studies in S2 cells indicated that the D-SCAR homolog D-WASp also participates in phagocytosis. This study demonstrates that Drosophila provides a viable model system in which to dissect the complex interactions that regulate phagocytosis.     

Zea mays ontology--a database of international terms

Data retrieval, comprehension and sharing within and between plant-based databases are essential to exploit comparative genomic information to elucidate functional aspects of plant biology and to conduct studies of synteny and homology. However, the functionality is often hampered by the variability of terms used to describe comparable objects. The Zea mays Plant Structure Ontology database is designed to overcome this problem via the provision of a controlled vocabulary that facilitates knowledge sharing. It comprises international botanical terms, references, synonyms, and phylogenetic information and is open-source.     

A two-component modular approach for enhancing T-cell activation utilizing a unique anti-FcgammaRI-streptavidin construct and microspheres coated with biotinylated-antigen

The professional antigen presenting cell (APC) plays an essential role in the initiation and propagation of the acquired immune response. Thus, much work has been done in designing strategies that target vaccine antigen (Ag) to APC. Utilizing recombinant DNA technology, we have created a unique two-component system that delivers biotinylated Ag to the Fc gamma receptor type I (FcgammaRI) on APC. Our studies demonstrate that we can successfully engineer FcgammaRI-specific targeting element proteins that simultaneously bind both biotin and recognize FcgammaRI. Additionally, we are able to engineer biotinylated Ag, which form functional elements when adsorbed onto latex microspheres. Furthermore, the targeting and functional element components bind to each other and successfully form two-component immunogens. T-cell activation in response to targeted Ag-laden microspheres is 10- to 100-fold greater than the response to the non-targeted Ag-laden microspheres. This enhancement is 100- to 1000-fold greater than the responses generated to soluble Ag. Thus, our results suggest that specific targeting of Ag-laden microspheres to FcgammaRI may significantly enhance the adjuvant properties of microparticulate delivery systems. Further development of this system may help to elucidate the mechanisms involved in generating enhanced responses to APC-targeted vaccines and significantly advance vaccine technology.     

Temporal dynamics of three culturable γ-Proteobacteria taxa in salt marsh sediments

Temporal dynamics of three marine -Proteobacteria taxa withculturable members were followed in sediment communities in southeastern USsaltmarshes using a whole-genome hybridization approach. Labeled DNA from threebacterial isolates was used to probe sediment community DNA from two saltmarshes on Sapelo Island, GA at multiple time points over a three-year period.The relative abundance of the three isolates (and their close relatives)accounted for up to 28% of the total sediment community DNA. Hybridizationsignals for the taxon represented by isolate SIGA28M (with a 16S rRNA sequencesimilarity of 97% to Vibrio proteolyticus) showedsignificant temporal variation in both marshes, varying from 1% to 28% over thethree-year period. The taxa represented by isolates SIGA172a and SIGA198 (16SrRNA sequence similarities of 90% and 92% to Shewanellafrigidimarina and Vibrio nigripulchritudo,respectively) accounted for less than 6% of the sediment community at any timepoint. The variation in relative abundance of these three groups did not appearto follow clear seasonal trends, and could not be readily correlated withenvironmental variables.     

Retinoblastoma: from bench to bedside

Retinoblastoma (Rb) is the most common primary ocular malignancy of children and is caused by a mutation in the gene RB1. Approximately 40% of cases are associated with one or more constitutional mutations, and are therefore heritable, whereas the other 60% are sporadic. Rb is exclusively found in young children. In some cases, Rb tumours metastasise to extraocular organs including bone, lung and brain. Although there is no effective treatment for metastatic disease, non-metastatic cases can be cured by removal of the eye(enucleation). Newer treatment strategies emphasise salvaging the affected eye whenever possible. Animal models of Rb have been developed with xenograft and transgenic techniques. Each model has both strengths and weaknesses for exploring the mechanisms of disease development and progression and the efficacy of new treatment strategies.