Intracellular interference of infectious bursal disease virus

A search for dominant-negative mutant polypeptides hampering infectious bursal disease virus (IBDV) replication has been undertaken. We have found that expression of a mutant version of the VP3 structural polypeptide known as VP3/M3, partially lacking the domain responsible for the interaction with the virus-encoded RNA polymerase, efficiently interferes with the IBDV replication cycle. Transformed cells stably expressing VP3/M3 show a significant reduction (up to 96%) in their ability to support IBDV growth. Our findings provide a new tool for the characterization of the IBDV replication cycle and might facilitate the generation of genetically modified chicken lines with a reduced susceptibility to IBDV infection.     

dUTPase as a platform for antimalarial drug design: structural basis for the selectivity of a class of nucleoside inhibitors

Pyrimidine metabolism is a major route for therapeutic intervention against malaria. Here we report inhibition and structural studies on the deoxyuridine nucleotidohydrolase from the malaria parasite Plasmodium falciparum (PfdUTPase). We have identified a series of triphenylmethane derivatives of deoxyuridine with antimalarial activity in vitro which inhibit specifically the Plasmodium dUTPase versus the human enzyme. A 2.4 Angstrom crystal structure of PfdUTPase in complex with one of these inhibitors reveals an atypical trimeric enzyme in which the triphenylmethane derivative can be seen to select for PfdUTPase by way of interactions between the trityl group and the side chains of residues Phe46 and Ile117. Immunofluorescence microscopy studies of parasitized red blood cells reveal that enzyme concentrations are highest during the trophozoite/schizont stages, suggesting that PfdUTPase has a major role in DNA replication. Taken together the data show that PfdUTPase may be considered as an antimalarial drug target.     

2-Alkoxydihydrocinnamates as PPAR agonists. Activity modulation by the incorporation of phenoxy substituents

Herein we describe a series of potent and selective PPARgamma agonists with moderate PPARalpha affinity and little to no affinity for other nuclear receptors. In vivo studies in a NIDDM animal model (ZDF rat) showed that these compounds are efficacious at low doses in glucose normalization and plasma triglyceride reduction. Compound 1b (LY519818) was selected from our SAR studies to be advanced to clinical evaluation for the treatment of type II diabetes.     

Regulation of Sertoli-germ cell adherens junction dynamics in the testis via the nitric oxide synthase (NOS)/cGMP/protein kinase G (PRKG)/beta-catenin (CATNB) signaling pathway: an in vitro and in vivo study

During spermatogenesis, extensive restructuring of cell junctions takes place in the seminiferous epithelium to facilitate germ cell movement. However, the mechanism that regulates this event remains largely unknown. Recent studies have shown that nitric oxide (NO) likely regulates tight junction (TJ) dynamics in the testis via the cGMP/protein kinase G (cGMP-dependent protein kinase, PRKG) signaling pathway. Due to the proximity of TJ and adherens junctions (AJ) in the testis, in particular at the blood-testis barrier, it is of interest to investigate if NO can affect AJ dynamics. Studies using Sertoli-germ cell cocultures in vitro have shown that the levels of NOS (nitric oxide synthase), cGMP, and PRKG were induced when anchoring junctions were being established. Using an in vivo model in which adult rats were treated with adjudin [a molecule that induces adherens junction disruption, formerly called AF-2364, 1-(2,4-dichlorobenzyl)-IH-indazole-3-carbohydrazide], the event of AJ disruption was also associated with a transient iNOS (inducible nitric oxide synthase, NOS2) induction. Immunohistochemistry has illustrated that NOS2 was intensely accumulated in Sertoli and germ cells in the epithelium during adjudin-induced germ cell loss, with a concomitant accumulation of intracellular cGMP and an induction of PRKG but not cAMP or protein kinase A (cAMP-dependent protein kinase, PRKA). To identify the NOS-mediated downstream signaling partners, coimmunoprecipitation was used to demonstrate that NOS2 and eNOS (endothelial nitric oxide synthase, NOS3) were structurally associated with the N-cadherin (CDH2)/beta-catenin (CATNB)/actin complex but not the nectin-3 (poliovirus receptor-related 3, PVRL 3)/afadin (myeloid/lymphoid or mixed lineage-leukemia tranlocation to 4 homolog, MLLT4) nor the integrin beta1 (ITB1)-mediated protein complexes, illustrating the spatial vicinity of NOS with selected AJ-protein complexes. Interestingly, CDH2 and CATNB were shown to dissociate from NOS during the adjudin-mediated AJ disruption, implicating the CDH2/CATNB protein complex is the likely downstream target of the NO signaling. Furthermore, PRKG, the downstream signaling protein of NOS, was shown to interact with CATNB in the rat testis. Perhaps the most important of all, pretreatment of testes with KT5823, a specific PRKG inhibitor, can indeed delay the adjudin-induced germ cell loss, further validating NOS/NO regulates Sertoli-germ cell AJ dynamics via the cGMP/PRKG pathway. These results illustrate that the CDH2/CATNB-mediated adhesion function in the testis is regulated, at least in part, via the NOS/cGMP/PRKG/CATNB pathway.     

Evidence that osteogenic progenitor cells in the human tunica albuginea may originate from stem cells: implications for peyronie disease

Tissue ossification in Peyronie disease (commonly known as Peyronie's disease [PD]), a localized fibrotic lesion within the tunica albuginea (TA) of the penis, may result from osteogenic differentiation of fibroblasts, myofibroblasts, and/or adult stem cells in the TA, and may be triggered by chronic inflammation, oxidative stress, and profibrotic factors like transforming growth factor beta 1 (TGFB1). In this study, we have investigated whether cultures of cells from normal TA and PD plaques undergo osteogenesis, express markers for stem cells, and originate other cell lineages via processes modulated by TGFB1. We found that TA and PD cells in osteogenic medium (OM) expressed osteogenic markers, alkaline phosphatase, and osteopontin and underwent calcification. PD cells, but not TA cells, formed foci in soft agar that were positive for alkaline phosphatase and calcification and expressed the mRNAs for osteoblast-specific factors pleiotrophin and periostin and bone morphogenic protein 2. Both cultures expressed stem cell marker CD34 antigen but not protein tyrosine phosphatase, receptor type c. TA and PD cells expressed smooth-muscle cell markers smoothelin and transgelin. None of the cultures underwent adipogenesis in adipogenic medium. Incubation with TGFB1 increased osteogenesis and myofibroblast differentiation and reduced CD34 antigen expression in both cultures. TA and PD cells modulated the differentiation of the multipotent C3H 10T(1/2) cells in dual cultures, into osteoblasts and myofibroblasts. In conclusion, both TA and PD cultures contain cells, presumably stem cells, that undergo osteogenic and myofibroblast differentiation, and may induce these processes by paracrine interactions. This may explain progression of fibrosis in the PD plaque and its eventual calcification.     

A role for MCP-1/CCR2 in interstitial lung disease in children

Background

Particulate matter (PM) can exacerbate allergic airway diseases. Although health effects of PM with a diameter of less than 100 nm have been focused, few studies have elucidated the correlation between the sizes of particles and aggravation of allergic diseases. We investigated the effects of nano particles with a diameter of 14 nm or 56 nm on antigen-related airway inflammation.

Methods

ICR mice were divided into six experimental groups. Vehicle, two sizes of carbon nano particles, ovalbumin (OVA), and OVA + nano particles were administered intratracheally. Cellular profile of bronchoalveolar lavage (BAL) fluid, lung histology, expression of cytokines, chemokines, and 8-hydroxy-2'-deoxyguanosine (8-OHdG), and immunoglobulin production were studied.

Results

Nano particles with a diameter of 14 nm or 56 nm aggravated antigen-related airway inflammation characterized by infiltration of eosinophils, neutrophils, and mononuclear cells, and by an increase in the number of goblet cells in the bronchial epithelium. Nano particles with antigen increased protein levels of interleukin (IL)-5, IL-6, and IL-13, eotaxin, macrophage chemoattractant protein (MCP)-1, and regulated on activation and normal T cells expressed and secreted (RANTES) in the lung as compared with antigen alone. The formation of 8-OHdG, a proper marker of oxidative stress, was moderately induced by nano particles or antigen alone, and was markedly enhanced by antigen plus nano particles as compared with nano particles or antigen alone. The aggravation was more prominent with 14 nm of nano particles than with 56 nm of particles in overall trend. Particles with a diameter of 14 nm exhibited adjuvant activity for total IgE and antigen-specific IgG₁ and IgE.

Conclusion

Nano particles can aggravate antigen-related airway inflammation and immunoglobulin production, which is more prominent with smaller particles. The enhancement may be mediated, at least partly, by the increased local expression of IL-5 and eotaxin, and also by the modulated expression of IL-13, RANTES, MCP-1, and IL-6.     

Applicability of RAPD markers on silver-stained polyacrylamide gels to ascertain genetic diversity in Peripatus acacioi (Peripatidae; Onychophora)

RAPD (random amplification of polymorphic DNA) molecular markers can be utilized for analyzing genetic variability in populations for which only a few or no molecular markers are available. They were used in a study of an endangered species, Peripatus acacioi, found in the Tripuí Ecological Station, in Ouro Preto, MG, Brazil. The ecological station was specifically created to protect this velvet worm species, the first of this group found in Brazil. For an initial evaluation of the genetic diversity of this species, DNA samples from the lobopods of four individuals, collected at random, were analyzed using RAPD. Each reaction was run with a different primer (Operon RAPD 10-mer Kits), totaling 13 primers (OPC2, OPC3, OPC4, OPC6, OPC8, OPC10, OPC11, OPL2, OPL7, OPL11, OPL13, OPL18, and OPL19). Due to the low amplification yield, RAPD fragments were separated in polyacrylamide gels and stained with silver nitrate. Numerous bands were observed. Fifty-five of the amplified bands proved to be reproducible, both in terms of presence and intensity. Among these, 27 were variable and 28 were constant. The average number of bands per gel was 4.2. Nine of the 13 primers tested allowed the identification of constant and variable bands among these four individuals. RAPD analysis of genetic variation using silver-stained polyacrylamide gel electrophoresis provided measures of band sharing among the individuals, and therefore could be used in population genetics studies of P. acacioi.     

Analysis of the stability of the spermadhesin PSP-I/PSP-II heterodimer. Effects of Zn2+ and acidic pH

Spermadhesins are a family of 12-16 kDa proteins with a single CUB domain. PSP-I and PSP-II, the most abundant boar spermadhesins, are present in seminal plasma as a noncovalent heterodimer. Dimerization markedly affects the binding ability of the subunits. Notably, heparin and mannose 6-phosphate binding abilities of PSP-II are abolished, indicating that the corresponding binding sites may be located at (or near) the dimer interface. Pursuing the hypothesis that cryptic binding sites in PSP-I/PSP-II may be exposed in specific physiological environments, we examined the influence of Zn2+ and acidic pH on the heterodimer stability. According to near-UV CD spectra, the core native fold is preserved in the presence of physiological concentrations of Zn2+, a cation unusually abundant in boar seminal plasma. However, the thermostability of the heterodimer decreases significantly, as observed by CD and differential scanning calorimetry. The effect is Zn2+-specific and is reversed by EDTA. Destabilization is also observed at acidic pH. Gel filtration analysis using radioiodinated PSP-I/PSP-II reveals that dissociation of the heterodimer at low (nanomolar) protein concentrations is promoted by both Zn2+ and acidic pH. Although the integrity of the heterodimer in seminal plasma seems to be guaranteed by its high concentration, dissociation may be facilitated in the female genital tract because of dilution of the protein in the intraluminal fluids of the cervix and the uterus, and the acidic fluid of the uterotubal junction. Such a mechanism may be relevant in the regulation of uterine immune reactions.     

Functional homology among human and fission yeast Cdc14 phosphatases

Budding and fission yeast Cdc14 homologues, a conserved family of serine-threonine phosphatases, play a role in the inactivation of mitotic cyclin-dependent kinases (CDKs) by molecularly distinct mechanisms. Saccharomyces cerevisiae Cdc14 protein phosphatase inactivates CDKs by promoting mitotic cyclin degradation and the accumulation of a CDK inhibitor to allow budding yeast cells to exit from mitosis. Schizosaccharomyces pombe Flp1 phosphatase down-regulates CDK/cyclin activity, controlling the degradation of the Cdc25 tyrosine phosphatase for fission yeast cells to undergo cytokinesis. In the present work, we show that human Cdc14 homologues (hCdc14A and hCdc14B) rescued flp1-deficient fission yeast strains, indicating functional homology. We also show that hCdc14A and B interacted in vivo with S. pombe Cdc25 and that hCdc14A dephosphorylated this mitotic inducer both in vitro and in vivo. Our results support a Cdc14 conserved inhibitory mechanism acting on S. pombe Cdc25 protein and suggest that human cells may regulate Cdc25 in a similar manner to inactivate Cdk1-mitotic cyclin complexes.     

p21Cip1 and p27Kip1 induce distinct cell cycle effects and differentiation programs in myeloid leukemia cells

The cyclin-dependent kinase (Cdk) inhibitors p21(Cip1) and p27(Kip1) have been proposed to exert redundant functions in cell cycle progression and differentiation programs, although nonoverlapping functions have also been described. To gain further insights into the relevant mechanisms and to detect possible functional differences between both proteins, we conditionally expressed p21(Cip1) and p27(Kip1) in K562, a multipotent human leukemia cell line. Temporal ectopic expression of either p21(Cip1) or p27(Kip1) arrested proliferation, inhibited Cdk2 and Cdk4 activities, and suppressed retinoblastoma phosphorylation. However, whereas p21(Cip1) arrested cells in both G(1) and G(2) cell cycle phases, p27(Kip1) blocked the G(1)/S-phase transition. Furthermore, although both p21(Cip1) and p27(Kip1) associated with Cdk6, only p27(Kip1) significantly inhibited its activity. Most importantly, each protein promoted differentiation along a distinct pathway; p21(Cip1) triggered megakaryocytic maturation, whereas p27(Kip1) resulted in the expression of erythroid markers. Consistently, p21(Cip1) and p27(Kip1) were rapid and transiently up-regulated when K562 cells are differentiated into megakaryocytic and erythroid lineages, respectively. These findings demonstrate distinct functions of p21(Cip1) and p27(Kip1) in cell cycle regulation and differentiation and indicate that these two highly related proteins possess unique biological activities and are not functionally interchangeable.