Strongyloides ratti: the role of interleukin-5 in protection against tissue migrating larvae and intestinal adult worms

To determine the role of interleukin-5 (IL-5) and eosinophils in protection against Strongyloides ratti, mice treated with anti-IL-5 monoclonal antibody (mAb) were infected with S. ratti larvae. Strongyloides ratti egg numbers in faeces (EPG) in mAb treated mice were higher than those in control mice on days 6 and 7 after inoculation. The numbers of migrating worms in mAb treated mice 36 h after inoculation were higher than those observed in control mice. Intestinal worm numbers in mAb treated mice 5 days after inoculation were higher than those in control mice. These results show that eosinophils effectively protected the host against S. ratti infection by mainly the larval stage in primary infections. The involvement of eosinophils in protection against secondary infection was also examined. Before secondary infection, mice were treated with anti-IL-5 mAb and infected with S. ratti. Patent infections were not observed in either mAb treated or control Ab treated mice. The numbers of migrating worms in the head and lungs of mAb treated mice increased to 60% of that in primary infected mice. Intestinal worms were not found in mAb treated mice or in control mice after oral implantation of adult worms. Eosinophils were therefore mainly involved in protection against tissue migrating worms in secondary infections.     

Regulation of the Ca2+ pump ATPase by cAMP-dependent phosphorylation of phospholamban

Ca2+ transients in myocardial cells are modulated by cyclic AMP-dependent phosphorylation of a protein in the sarcoplasmic reticulum. This protein, termed phospholamban, serves to regulate the Ca2+ pump ATPase of this membrane, thus altering the mode of Ca2+ transients and the myocardial contractile response. Elucidating the structure of phospholamban and its intimate interaction with the Ca2+ pump ATPase should provide the basis for understanding, at the molecular level, how the cAMP system contributes to excitation-contraction coupling in muscle cells.     

Thymic atrophy characteristic in transgenic mice that harbor pX genes of human T-cell leukemia virus type I

The human T-cell leukemia viruses (HTLV) are associated with T-cell malignancies in humans. The malignant transformation occurs after a long latency in some carriers, and its mechanism appears to be distinct from that of other classes of retroviruses which induce transformation through viral or cellular oncogenes. A widely postulated explanation is that the products of novel pX genes transactivate endogenous cellular genes which lead to tumor development in T cells. To directly examine the pathological effects of pX genes in vivo, we produced transgenic mice harboring the HTLV type I pX genes under several regulatory units: HTLV type I long terminal repeat, immunoglobulin enhancer-simian virus 40 promoter, and mouse mammary tumor virus long terminal repeat. Atrophy of the thymus was characteristic in these mice no matter which regulatory unit directed the expression of the genes.     

Transient increase in proteinuria,poly-ubiquitylated proteins and ER stress markers in podocyte-specific autophagy-deficient mice following unilateral nephrectomy

Previous studies have revealed that podocytes normally can be associated with a very high degree of autophagic activity, and that a lack of autophagic activity in podocytes is associated with susceptibility to disease and to late-onset glomerulosclerosis. In the present study, we conducted unilateral nephrectomy as a surgical model for acute nephron reduction. First, using GFP-LC3 transgenic mice to monitor autophagy, we found that glomerular autophagy could be transiently suppressed by surgery, but that it was restored quickly. To further explore the significance of podocyte autophagy after unilateral nephrectomy, we investigated podocyte-specific Atg7-deficient mice. The knockout mice exhibited no pathological phenotype compared with wild-type mice before nephrectomy. However, 1 day after nephrectomy, significantly higher levels of proteinuria and ultrastructural changes that included foot process effacement and a significant reduction in podocyte number were detected in mice harboring Atg7-deficient podocytes. Moreover, biochemical and immunohistochemical analyses showed a robust increase in polyubiquitin levels and ER stress markers in the glomeruli of the mice with autophagy-deficient podocytes. These results show the importance of the autophagic process in podocytes for maintaining a normal degree of filtration function during the adaptation to compensatory kidney hypertrophy following unilateral nephrectomy.     

Physical interactions between phospholamban and sarco(endo)plasmic reticulum Ca2+-ATPases are dissociated by elevated Ca2+, but not by phospholamban phosphorylation, vanadate, or thapsigargin, and are enhanced by ATP

Previous co-immunoprecipitation studies (Asahi, M., Kimura, Y., Kurzydlowski, K., Tada, M., and MacLennan, D. H. (1999) J. Biol. Chem. 274, 32855-32862) revealed that physical interactions between phospholamban (PLN) and the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA1a) were retained, even with PLN monoclonal antibody 1D11 bound to an epitope lying between PLN residues 7 and 17. Because the 1D11 antibody relieves inhibitory interaction between the two proteins, it was of interest to determine whether PLN phosphorylation or elevation of Ca(2+), which also relieves inhibitory interactions between PLN and SERCA, would disrupt physical interactions. Co-immunoprecipitation was measured in the presence of increasing concentrations of Ca(2+) or after phosphorylation of PLN by protein kinase A. Physical interactions were dissociated by elevated Ca(2+) but not by PLN phosphorylation. The addition of ATP enhanced interactions between PLN and SERCA. The further addition of vanadate and thapsigargin, both of which stabilize the E(2) conformation, did not diminish binding of PLN to SERCA. These data suggest that physical interactions between PLN and SERCA are stable when SERCA is in the Ca(2+)-free E(2) conformation but not when it is in the E(1) conformation and that phosphorylation of PLN does not dissociate physical interactions between PLN and SERCA.     

Structure of porcine pancreatic elastase complexed with FR901277, a novel macrocyclic inhibitor of elastases, at 1.6 A resolution

Human leukocyte elastase (HLE) is a serine protease that contributes to tissue destruction in various disease states-for example, in emphysema. FR901277 is a natural product isolated from the culture filtrate of Streptomyces resistomicificus and is a potent inhibitor of both HLE and porcine pancreatic elastase (PPE). FR901277 consists of four normal amino acids and three unusual amino acids, and is a unique bicyclic peptide compound. The crystal structure of PPE complexed with FR901277 has been determined at 1.6 A resolution. The Ogamma atom of Ser-195 in PPE did not form a covalent bond with FR901277, but formed a hydrogen bond with the Nvarepsilon atom of His-57. On the other hand, the portion from L-Orn(1) through dehydroxyThr(3) in FR901277 formed an antiparallel beta-sheet structure with the backbone of the active site in PPE. The S4 through S2' binding subsites in PPE were all occupied by the hydrophobic side chains of the inhibitor molecule. Especially, the ethylidene moiety of FR901277 occupied the S1 specific pocket, indicating a CH/pi interaction. In addition, the isopropyl side chain of L-Val(7) was located at the enzyme surface between the S2 and S1' pockets with several van der Waals contacts. However, the amino acid (4) residue was not involved in a significant interaction with PPE. Comparison of inhibitor structures in different environments showed that FR901277 has a highly rigid bicyclic framework; however, it can slightly change its conformation according to the circumstances. The binding mode of FR901277 at the active site of PPE was directly applicable to that in HLE, after consideration of induced fit. The structure of the PPE-FR901277 complex provided much information regarding potential sites for modification of the physicochemical properties of FR901277.     

WRNIP1 functions upstream of DNA polymerase η in the UV-induced DNA damage response

WRNIP1 (WRN-interacting protein 1) was first identified as a factor that interacts with WRN, the protein that is defective in Werner syndrome (WS). WRNIP1 associates with DNA polymerase η (Polη), but the biological significance of this interaction remains unknown. In this study, we analyzed the functional interaction between WRNIP1 and Polη by generating knockouts of both genes in DT40 chicken cells. Disruption of WRNIP1 in Polη-disrupted (POLH^−/−) cells suppressed the phenotypes associated with the loss of Polη: sensitivity to ultraviolet light (UV), delayed repair of cyclobutane pyrimidine dimers (CPD), elevated frequency of mutation, elevated levels of UV-induced sister chromatid exchange (SCE), and reduced rate of fork progression after UV irradiation. These results suggest that WRNIP1 functions upstream of Polη in the response to UV irradiation.