PED/PEA-15 controls fibroblast motility and wound closure by ERK1/2-dependent mechanisms

Cell migration is dependent on the control of signaling events that play significant roles in creating contractile force and in contributing to wound closure. We evaluated wound closure in fibroblasts from mice overexpressing (TgPED) or lacking ped/pea-15 (KO), a gene overexpressed in patients with type 2 diabetes. Cultured skin fibroblasts isolated from TgPED mice showed a significant reduction in the ability to recolonize wounded area during scratch assay, compared to control fibroblasts. This difference was observed both in the absence and in the presence of mytomicin C, an inhibitor of mitosis. In time-lapse experiments, TgPED fibroblasts displayed about twofold lower velocity and diffusion coefficient, as compared to controls. These changes were accompanied by reduced spreading and decreased formation of stress fibers and focal adhesion plaques. At the molecular level, TgPED fibroblasts displayed decreased RhoA activation and increased abundance of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2). Inhibition of ERK1/2 activity by PD98059 restored RhoA activation, cytoskeleton organization and cell motility, and almost completely rescued wound closure of TgPED fibroblasts. Interestingly, skin fibroblasts isolated from KO mice displayed an increased wound closure ability. In vivo, healing of dorsal wounds was delayed in TgPED and accelerated in KO mice. Thus, PED/PEA-15 may affect fibroblast motility by a mechanism, at least in part, mediated by ERK1/2.     

Phylogeny and classification of Leptophlebiidae (Ephemeroptera) with an emphasis on Neotropical fauna

Mayflies from the family Leptophlebiidae are cosmopolitan and highly diverse morphologically; they are also the largest family in numbers of genera and the second in number of species in the order Ephemeroptera. In spite of their broad diversity and the efforts employed to understand the evolution of this group, the internal classification of Leptophlebiidae remains controversial at all levels. More recently, important changes have been incorporated into the systematics of the family, increasing the number of subfamilies (from two to six) and recognizing several tribes. We present a phylogeny of the family based on 153 taxa (53 genera) and two molecular markers, representing 1655 bp, and verify the taxonomic status of the subfamilies, tribes and complexes. Based on these results, the number of subfamilies has been increased from six to eight and one new tribes and two new subtribes have been added. In addition, new ranks are proposed and the concept of Atalophlebiinae revised, including genera with distributions in the Australasian and Neotropical regions.     

Hematology and plasma chemistry values in free-ranging Humboldt penguins (Spheniscus humboldti) in Chile

To establish baseline hematologic and plasma biochemistry values in free-ranging Humboldt penguins (Spheniscus humboldti), heparinized blood samples were collected from 51 apparently healthy, adult Humboldt penguins residing at two colonies off the Chilean coast. Thirty samples were collected in April, 1992, from penguins inhabiting the Ex-islote de los Pájaros Niños in Algarrobo, Chile. In September, 1992, 21 samples were collected from birds inhabiting Isla de Cachagua, Chile. Hematologic values measured include packed cell volume, leucocyte count, leucocyte differential, and the presence of blood parasites. Plasma biochemistry values measured include glucose, blood urea nitrogen, creatinine, uric acid, calcium, inorganic phosphorous, sodium, potassium, chloride, total protein, albumin, globulin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total bilirubin, and creatine kinase. Only the mean values for chloride and for the number of eosinophils differed significantly between the two sample groups. No blood parasites were seen. © 1995 Wiley-Liss, Inc.     

Interferon-gamma is required for the late but not early control of Leishmania amazonensis infection in C57Bl/6 mice

The critical role of interferon-gamma (IFN-gamma) in the resistance of C57Bl/6 mice to Leishmania major is widely established but its role in the relative resistance of these animals to L. amazonensis infection is still not clear. In this work we use C57Bl/6 mice congenitally deficient in the IFN-gamma gene (IFN-gamma KO) to address this issue. We found that IFN-gamma KO mice were as resistant as their wild-type (WT) counterparts at least during the first two months of infection. Afterwards, whereas WT mice maintained lesion growth under control, IFN-gamma KO mice developed devastating lesions. At day 97 of infection, their lesions were 9-fold larger than WT controls, concomitant with an increased parasite burden. At this stage, lesion-draining cells from IFN-gamma KO mice had impaired capacity to produce interleukin-12 (IL-12) and tumour necrosis factor-a in response to parasite antigens whereas IL-4 was slightly increased in comparison to infected WT mice. Together, these results show that IFN-gamma is not critical for the initial control of L. amazonensis infection in C57Bl/6 mice, but is essential for the development of a protective Th1 type immune response in the later stages.     

Human estrogen receptor alpha gene is a target of Runx2 transcription factor in osteoblasts

Several studies into the mechanisms involved in control of osteoblast-specific gene expression have identified Runx2 and ERalpha (estrogen receptor alpha) as essential regulators of osteoblast differentiation. Recently, interactions between Runx2 and ERalpha have been described. Here, we investigate the role of Runx2 on the regulation of ERalpha expression by determining its interaction with the F promoter, one of the multiple promoters of the human ERalpha gene and the only one active in bone. We found that, in this promoter, three Runx2-like sites are present. By electrophoretic mobility shift assay in combination with supershift and ChIP experiments, we demonstrated that Runx2 preferentially binds one of the Runx2 motifs of the F promoter. To understand whether or not they are involved in influencing F promoter activity, different promoter-reporter deletion and mutation constructs were transiently transfected into human osteoblastic cells. Comparison of luciferase activities allowed the identification of a prevalent negative role of a sequence context, within the -117,877/-117,426 region, which may be under the control of Runx2 (a) site. Finally, silencing and overexpression of endogenous Runx2 provided evidence that Runx2 has a more complex role than initially expected. In fact, Runx2 (a) and Runx2 (b) sites carried out opposite roles which are conditioned by Runx2 levels in bone cells. Therefore, the resulting F promoter activity may be tightly regulated by a dynamic interplay between these two Runx2 sites, with a predominance of negative effect of the Runx2 (a) site.     

Proteolytic degradation of nitric oxide synthase isoforms by calpain is modulated by the expression levels of HSP90

Ca2+ loading of Jurkat and bovine aorta endothelium cells induces the degradation of the neuronal and endothelial nitric oxide synthases that are selectively expressed in these cell lines. For neuronal nitric oxide synthase, this process involves a conservative limited proteolysis without appreciable loss of catalytic activity. By contrast, endothelial nitic oxide synthase digestion proceeds through a parallel loss of protein and catalytic activity. The chaperone heat shock protein 90 (HSP90) is present in a large amount in Jurkat cells and at significantly lower levels in bovine aorta endothelium cells. The differing ratios of HSP90/nitric oxide synthase (NOS) occurring in the two cell types are responsible for the conservative or nonconservative digestion of NOS isozymes. Consistently, we demonstrate that, in the absence of Ca2+, HSP90 forms binary complexes with NOS isozymes or with calpain. When Ca2+ is present, a ternary complex containing the three proteins is produced. In this associated state, HSP90 and NOS forms are almost completely resistant to calpain digestion, probably due to a structural hindrance and a reduction in the catalytic efficiency of the protease. Thus, the recruitment of calpain in the HSP90-NOS complexes reduces the extent of the proteolysis of these two proteins. We have also observed that calpastatin competes with HSP90 for the binding of calpain in reconstructed systems. Digestion of the proteins present in the complexes can occur only when free active calpain is present in the system. This process can be visualized as a novel mechanism involving the association of NOS with HSP90 and the concomitant recruitment of active calpain in ternary complexes in which the proteolysis of both NOS isozymes and HSP90 is significantly reduced.     

Symmetrical α-bromoacryloylamido diaryldienone derivatives as a novel series of antiproliferative agents. Design,synthesis and biological evaluation

In a continuing study of hybrid compounds containing the α-bromoacryloyl moiety as potential anticancer drugs, we synthesized a novel series of hybrids 4a–h, in which this moiety was linked to a 1,5-diaryl-1,4-pentadien-3-one system. Many of the conjugates prepared (4b, 4c, 4e and 4g) demonstrated pronounced, submicromolar antiproliferative activity against four cancer cell lines. Moreover, compound 4b induced apoptosis through the mitochondrial pathway and activated caspase-3 in a concentration-dependent manner.     

Antiviral activity of benzimidazole derivatives. II. Antiviral activity of 2-phenylbenzimidazole derivatives

Seventy-six 2-phenylbenzimidazole derivatives were synthesized and evaluated in cell-based assays for cytotoxicity and antiviral activity against a panel of 10 RNA and DNA viruses. The most commonly affected viruses were, in decreasing order, CVB-2, BVDV, Sb-1, HSV-1, and YFV, while HIV-1 and VSV were not affected, and RSV, VV and Reo-1 were only susceptible to a few compounds. Thirty-nine compounds exhibited high activity (EC₅₀ = 0.1–10 μM) against at least one virus, and four of them were outstanding for their high and selective activity against VV (24, EC₅₀ = 0.1 μM) and BVDV (50, 51, and 53 with EC₅₀ = 1.5, 0.8, and 1.0 μM, respectively). The last compounds inhibited at low micromolar concentrations the NS5B RdRp of BVDV and also of HCV, the latter sharing structural similarity with the former. The considered compounds represent attractive leads for the development of antiviral agents against poxviruses, pestiviruses and even HCV, which are important human and veterinary pathogens.