The characterisation of novel secreted Ly-6 proteins from rat urine by the combined use of two-dimensional gel electrophoresis, microbore high performance liquid chromatography and expressed sequence tag data

A proteomic study of rat urine was undertaken using two-dimensional gel electrophoresis, microbore high performance liquid chromatography, mass spectrometry and N-terminal sequencing. Five known urinary proteins were identified but two novel peptide fragments matched a large number of rat expressed sequence tags (ESTs) from a liver library. By combining protein chemical and nucleotide data, two 101-residue open reading frames with 90% amino acid identity were determined, rat urinary protein 1 (RUP-1) and RUP-2. The data established signal peptide removal and provided evidence for N-glycosylation. A third related sequence, rat spleen protein (RSP-1) was confirmed from EST searches. These three proteins have been submitted to SWISS-PROT as P81827, P81828 and Q9QXN2, respectively. A fourth novel homologue was found in porcine and bovine ESTs from embryo libraries. Alignment with known homologues showed conserved cysteine positions characteristic of a secreted subfamily of Ly-6 proteins. In two cases, antineoplastic urinary protein and caltrin, these homologues have unverified functional annotations. The RUP sequences showed high scoring matches to three unrelated rat mRNAs subsequently established to be chimeric. Two of these share extended sectional identity to RUP-1 but the third may represent another novel Ly-6 homologue. These chimeras have caused serious annotation errors in secondary databases.     

Costimulatory molecule-targeted antibody therapy of a spontaneous autoimmune disease

Humans and mice deficient in Fas, a tumor necrosis factor (TNF)-receptor family member, cannot induce apoptosis of autoreactive cells, and consequently develop progressive lymphoproliferative disorders and lupus-like autoimmune diseases. Previous studies have shown that short-term administrations of agonistic monoclonal antibodies against CD137, another TNF-receptor family member, activate T cells and induce rejection of allografts and established tumors. Here we report that treatment with an agonistic monoclonal antibody to CD137 (2A) blocks lymphadenopathy and spontaneous autoimmune diseases in Fas-deficient MRL/lpr mice, ultimately leading to their prolonged survival. Notably, 2A treatment rapidly augments IFN-gamma production, and induces the depletion of autoreactive B cells and abnormal double-negative T cells, possibly by increasing their apoptosis through Fas- and TNF receptor-independent mechanisms. This study demonstrates that agonistic monoclonal antibodies specific for costimulatory molecules can be used as novel therapeutic agents to delete autoreactive lymphocytes and block autoimmune disease progression.     

Development of a 1-microl scale assay for mitogen-activated kinase kinase 7 using 2-D fluorescence intensity distribution analysis anisotropy

This paper describes the development of a robust, miniaturizable, and quantitative fluorescence-based assay for mitogen-activated protein kinase kinase 7 (MKK7). As a first step, the basic steady-state kinetics of the MKK7-catalyzed phosphorylation of c-Jun N-terminal kinases (JNKs) 1, 2, and 3 were defined using standard radiometric methods. Subsequently, the authors found that in addition to the holo JNKs, a series of novel small peptides (based on the region around the JNK phosphorylation site) are also substrates, provided that these were prephosphorylated on the Y residue of the TPY motif. One of these peptide substrates was used in the development of a fluorescence polarization-based assay using an antibody as a sensor. The assay was successfully miniaturized for use with conventional fluorescence polarization (FP) reader technology in 8.5 microl and on the single microl scale using Evotec proprietary 2-dimensional fluorescence intensity distribution analysis (2D-FIDA) anisotropy and liquid handling technology. The steady-state kinetic parameters derived using the FP or 2D-FIDA anisotropy format assays correlated well with those generated using a radiometric assay. Moreover, the quantitative sensitivity to known inhibitors was maintained independent of the format and assay volume. In addition, the authors found that the 2D-FIDA anisotropy assay exhibited superior performance statistics (typical Z' = approximately 0.5) relative to conventional FP (typical Z' = 0.3) and yielded the additional benefit of order-of-magnitude savings in terms of reagent costs. The 2D-FIDA anisotropy assay was used to carry out a successful high-throughput screening in 1-microl final volume against company file compounds.     

Live-cell imaging of endocytosis during conidial germination in the rice blast fungus,Magnaporthe grisea

Although there is growing evidence that endocytosis is important in hyphal tip growth, it has not previously been shown to occur during fungal spore germination. We have analysed and characterized endocytosis during the germination of living conidia of the rice blast fungus, Magnaporthe grisea. Conidia treated with the endocytic markers Lucifer Yellow carbohydrazide, FITC-dextran, and FM4-64 were imaged by confocal microscopy. Internalization of these fluorescent marker dyes by conidia was blocked by chemical and temperature treatments that inhibit endocytosis, and the sequential staining of organelles by the membrane-selective dye FM4-64 was consistent with dye internalization by endocytosis. FM4-64 uptake occurred within 2-3 min of conidial hydration, more than 40 min before the emergence of the germ tube. The times at which each of the three conidial cells initiated dye internalization were different as were the rates of dye uptake by each cell. Using these techniques we have demonstrated for the first time that ungerminated and germinated spores of filamentous fungi undergo endocytosis. Furthermore, internalization of FITC-dextran and Lucifer Yellow carbohydrazide by germinating conidia provides the first direct evidence for fluid-phase endocytosis in a filamentous fungus. FM4-64 was internalized by both ungerminated conidia and conidial germlings on the rice leaf suggesting that endocytosis might play a significant role in spore germination and germ tube growth during the pre-penetration phase of infection.     

The hyaluronan receptor, CD44

CD44 is a widely expressed cell surface hyaluronan receptor which plays a key role in mediating cell migration. A number of recent papers demonstrating an interplay between CD44 and matrix metalloproteinases (MMPs) have shed important insights into the molecular mechanisms underlying these events. This has important implication for understanding how mis-regulation of CD44 can contribute to disease pathologies.     

Project Grow-2-Gether: a study of the genetic and environmental influences on child eating and obesity.

"Project Grow-2-Gether" is a child nutrition study of same-sex, 3- to 7-year-old monozygotic and dizygotic twin pairs. The study attempts to bridge two bodies of literature that have rarely interfaced with respect to obesity and ingestive behavior: the first being behavioral genetic approaches to obesity-related traits, and the second being developmental approaches focusing on parent-child relationships. The overarching aim of Project Grow-2-Gether is to disentangle genetic from potential home-environmental influences on child eating behavior and body fat. This paper reviews the rationale for Project Grow-2-Gether, its procedures, and core phenotypic measurement battery. A focus of the study is acquisition of controlled food intake measurements obtained in the laboratory, measurement of specific home environmental variables, and multi-method evaluation of parent-child feeding relations. Future directions may involve longitudinal assessment of child growth and molecular analyses for specific genes that influence child eating behavior.     

Structured RNA upstream of insect cap distal iron responsive elements enhances iron regulatory protein-mediated control of translation

Iron regulatory protein (IRP) blocks ribosomal assembly by binding to an iron responsive element (IRE) located proximal (<60 nts) to the mRNA cap, thereby repressing translation. Constructs with IREs located 60–100 nts from the cap permit ribosomal assembly but the ribosomes pause at IRE/IRP complexes resulting in partial repression of translation. However, insect ferritin mRNAs have cap-distal IREs located 90–156 nts from the cap. Because iron can be toxic, it seems unlikely that insects would be unable to fully regulate ferritin synthesis at the level of translation. Calpodes ferritin consists of two subunits, S and G. In vitro translation of Calpodes ferritin and IRP1 from fat body mRNA yields only G subunits suggesting that IRP1 more efficiently represses translation of the S subunit than the G. When repression is removed by the addition of IRE competitor RNA, the synthesis of both subunits is greatly increased. S and G ferritin mRNAs have identical IREs in similar far cap-distal positions. While both ferritin mRNAs are predicted to have stem-loops between the IRE and the RNA cap, in general insect S mRNAs have more cap-proximal RNA structure than G mRNAs. Therefore, we examined the effect of upstream secondary structure on ribosomal assembly onto S ferritin mRNA constructs using sucrose gradient analysis of translation initiation complexes. We found no evidence for ribosomal assembly on wild type Calpodes S ferritin mRNA in the presence of IRP1 while constructs lacking the wild type secondary structure showed ribosomal pausing. Constructs with wild type secondary structure preceded by an unstructured upstream leader assemble ribosomes in the presence or absence of IRP1. Sequence and RNA folding analyses of other insect ferritins with cap-distal IREs failed to identify any common sequences or IRE-like structures that might bind to IRP1 with lower affinity or to another RNA binding protein. We propose that stem-loops upstream from the IRE act like pleats that shorten the effective distance between the IRE and cap and allow full translational repression by IRP1. In this way some cap-distal IREs may function like cap-proximal ones.     

Gamma-adaptin appendage domain: structure and binding site for Eps15 and gamma-synergin

The AP1 complex is one of a family of heterotetrameric clathrin-adaptor complexes involved in vesicular trafficking between the Golgi and endosomes. The complex has two large subunits, gamma and beta1, which can be divided into trunk, hinge, and appendage domains. The 1.8 A resolution structure of the gamma appendage is presented. The binding site for the known gamma appendage ligand gamma-synergin is mapped through creation of point mutations designed on the basis of the structure. We also show that Eps15, a protein believed to be involved in vesicle formation at the plasma membrane, is also a ligand of gamma appendage and binds to the same site as gamma-synergin. This observation explains the demonstrated brefeldinA (BFA)-sensitive colocalization of Eps15 and AP1 at the Golgi complex.     

Impairment of macrophage survival by NaCl: implications for early pulmonary inflammation in cystic fibrosis

Inflammation, characterized by the presence of proinflammatory chemokines and neutrophils, is a hallmark of early airway disease in infants with cystic fibrosis (CF), although the underlying mechanisms remain poorly defined. In this study, we evaluated the role of NaCl and the ensuing hyperosmolar effect on tumor necrosis factor (TNF)-alpha signaling and apoptosis in macrophages. Incubation of mouse macrophages with NaCl activated p38(mapk) and the p46(jnk) and p54(jnk) c-jun NH(2)-terminal kinase isoforms, but not p42(mapk/erk2) or Akt. Similar results were obtained with sorbitol, suggesting a general response to hyperosmolarity. Strikingly, the activation of p42(mapk/erk2) and Akt by TNF-alpha was also inhibited in the presence of NaCl. Because the activation of p42(mapk/erk2) and Akt has been associated with survival responses, we investigated the effect of NaCl on macrophage apoptosis. The results indicated a synergistic increase in apoptosis when macrophages were exposed to TNF-alpha in the presence of NaCl compared with stimulation with TNF-alpha alone or NaCl alone. Furthermore, pharmacological inhibition of p42(mapk/erk2) and Akt mimicked the effect of NaCl. Collectively, these findings indicate that modest elevations in NaCl differentially regulate the activation of mitogen-activated protein kinases and Akt and potentiate macrophage apoptosis. We speculate that augmentation of macrophage apoptosis in CF airways may result in decreased clearance of neutrophils and in deficiencies in the elimination of common CF pathogens.     

Characterisation of a monoclonal antibody to carp IL-1beta and the development of a sensitive capture ELISA

A carp IL-1beta gene was identified from a subtraction hybridisation technology based cDNA library from activated carp leucocytes. This gene was cloned into pQE vector carrying 6xHis tag and the protein was expressed. Recombinant IL-1beta was used to produce hybridomas specific for carp IL-1beta. Monoclonal antibodies were purified by affinity column and a sandwich ELISA for IL-1beta was developed with a detection limit of 10 ng of the recombinant protein. Using the capture ELISA, the presence of native IL-1beta in culture supernatant of PHA-stimulated leucocytes from carp was identified, which was confirmed by SDS-PAGE and Western blot. Since IL-1beta is known to stimulate proliferation of T & B cells and macrophages, its ability to stimulate proliferation of carp leucocytes was studied using tritiated thymidine. The recombinant protein was found to significantly stimulate proliferation of head kidney and spleen cells from carp.