Evidence that Smith-McCort dysplasia and Dyggve-Melchior-Clausen dysplasia are allelic disorders that result from mutations in a gene on chromosome 18q12

Smith-McCort dysplasia is a rare autosomal recessive osteochondrodysplasia characterized by short limbs and a short trunk with a barrel-shaped chest. The radiographic phenotype includes platyspondyly, generalized abnormalities of the epiphyses and metaphyses, and a distinctive lacy appearance of the iliac crest. We performed a genomewide scan in a consanguineous family from Guam and found evidence of linkage to loci on chromosome 18q12. Analysis of a second, smaller family was also consistent with linkage to this region, producing a maximum combined two-point LOD score of 3.04 at a recombination fraction of 0 for the marker at locus D18S450. A 10.7-cM region containing the disease gene was defined by recombination events in two affected individuals in the larger family. Furthermore, all affected children in the larger family were homozygous for a subset of marker loci within this region, defining a 1.5-cM interval likely to contain the defective gene. Analysis of three small, unrelated families with Dyggve-Melchior-Clausen syndrome, a radiographically identical disorder with the additional clinical finding of mental retardation, provided evidence of linkage to the same region, a result consistent with the hypothesis that the two disorders are allelic.     

Calnexin from Pisum sativum: cloning of the cDNA and characterization of the encoded protein

A full-length cDNA of 1951 bp encoding a calnexin (CNX) protein was cloned from a Pisum sativum expression library. The open reading frame (ORF) within this cDNA encodes a 551-amino acid protein with a calculated molecular mass of 62.47 kDa that exhibits extensive homology with the CNX proteins from soybean (80%), Arabidopsis thaliana (70%), maize (70%), and dog (39%). The characteristic CNX signature motifs, KPEDWDE and GXW, generally found in molecular chaperones, are present in pea CNX (PsCNX), along with putative sites for Ca2+ binding and phosphorylation. In PsCNX, a signal sequence and a single transmembrane domain are also present at the N- and C-terminal ends, respectively. The PsCNX protein is expressed constitutively at the RNA level in vegetative and flowering tissues, as was evident from Northern analysis. Expression of PsCNX was light independent. In vitro translation of PsCNX cDNA yielded a 75-kDa precursor, which, in the presence of canine microsomal membranes, was cotranslationally processed into a 72.5-kDa product and was imported and localized to the endoplasmic reticulum. Trypsin treatment of the in vitro translated PsCNX in the presence of canine microsomes generated a further processed 67-kDa intraluminal form. The results with PsCNX also showed that the plant protein is a phosphoprotein containing phosphoserine residues, as evidenced by immunoprecipitation of PsCNX with anti-phosphoserine antibody. The PsCNX protein was also phosphorylated by endogenous kinases of pea microsomes.     

Integrating spatially resolved three-dimensional MALDI IMS with in vivo magnetic resonance imaging

We have developed a method for integrating three dimensional-volume reconstructions of spatially resolved matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) ion images of whole mouse heads with high-resolution images from other modalities in an animal-specific manner. This approach enabled us to analyze proteomic profiles from MALDI IMS data with corresponding in vivo data provided by magnetic resonance imaging.     

Phosphorylation of slit diaphragm proteins NEPHRIN and NEPH1 upon binding of HGF promotes podocyte repair

Phosphorylation (activation) and dephosphorylation (deactivation) of the slit diaphragm proteins NEPHRIN and NEPH1 are critical for maintaining the kidney epithelial podocyte actin cytoskeleton and, therefore, proper glomerular filtration. However, the mechanisms underlying these events remain largely unknown. Here we show that NEPHRIN and NEPH1 are novel receptor proteins for hepatocyte growth factor (HGF) and can be phosphorylated independently of the mesenchymal epithelial transition receptor in a ligand-dependent fashion through engagement of their extracellular domains by HGF. Furthermore, we demonstrate SH2 domain–containing protein tyrosine phosphatase-2–dependent dephosphorylation of these proteins. To establish HGF as a ligand, purified baculovirus-expressed NEPHRIN and NEPH1 recombinant proteins were used in surface plasma resonance binding experiments. We report high-affinity interactions of NEPHRIN and NEPH1 with HGF, although NEPHRIN binding was 20-fold higher than that of NEPH1. In addition, using molecular modeling we constructed peptides that were used to map specific HGF-binding regions in the extracellular domains of NEPHRIN and NEPH1. Finally, using an in vitro model of cultured podocytes and an ex vivo model of Drosophila nephrocytes, as well as chemically induced injury models, we demonstrated that HGF-induced phosphorylation of NEPHRIN and NEPH1 is centrally involved in podocyte repair. Taken together, this is the first study demonstrating a receptor-based function for NEPHRIN and NEPH1. This has important biological and clinical implications for the repair of injured podocytes and the maintenance of podocyte integrity.     

Expression and functional activity of pro-oxidants and antioxidants in murine heart exposed to acute hypobaric hypoxia

Under hypobaric hypoxia, antioxidant defenses of the heart are stressed by the enhanced production of ROS. Mammalian heart acclimatizes to hypoxia through altered gene expression, which we studied in murine heart exposed to 10h of acute hypobaric hypoxia (AHH), equivalent to 15000ft, using cDNA arrays. Functional classification of genes with a > or =2-fold change revealed a number of pro-oxidants like Cyba, Xdh, Txnip, Ppp1r15b and antioxidants like Cat, Gpx1, Mt1, Mgst1. Interestingly, the protein level of Cyba, a subunit of NADPH oxidase, was markedly decreased in AHH exposed heart, suggesting the involvement of some stress response pathways. The AHH exposure also caused a significant reduction (50%) in the level of GSH (P<0.05). The present study provides a retrospective insight on the cellular antioxidant defense mechanisms under AHH.     

Induction of a CD4+ T regulatory type 1 response by cyclooxygenase-2-overexpressing glioma

PGE(2), synthesized by cyclooxygenase-2 (COX-2)-overexpressing tumor, is known to contribute to cellular immune suppression in cancer patients, but the mechanism remains unclear. We report the mechanism of a CD4(+) T regulatory type 1 (Tr1) induction by CD11c(+) mature dendritic cells (DCs) that phagocytose allogeneic and autologous COX-2-overexpressing glioma. A human glioma cell line, U-87MG, and primary cultured glioblastoma cells (MG-377) overexpressed COX-2. We did not detect IL-10Ralpha expression in these gliomas, and rIL-10 did not suppress their COX-2 expression. Exposure to COX-2-overexpressing glioma induced mature DCs to overexpress IL-10 and decreased IL-12p70 production. These DCs induced a Tr1 response, which is characterized by robust secretion of IL-10 and TGF-beta with negligible IL-4 secretion by CD4(+) T cells, and an inhibitory effect on admixed lymphocytes. Peripheral CD4(+) T cell populations isolated from an MG-377 patient also predominantly demonstrated a Tr1 response against MG-377 cells. Selective COX-2 inhibition in COX-2-overexpressing gliomas at the time of phagocytic uptake by DCs abrogated this regulatory response and instead elicited Th1 activity. COX-2 stable transfectants in LN-18 (LN-18-COX2) also induced a Tr1 response. The effect of a COX-2 inhibition in LN-18-COX2 is reversible after administration of PGE(2). Taken together, robust levels of PGE(2) from COX-2-overexpressing glioma, which is unresponsive to IL-10 within the local microenvironment, may cause DCs to secrete high levels of IL-10. These results indicate that COX-2-overexpressing tumors induce a Tr1 response, which is mediated by tumor-exposed, IL-10-enhanced DCs.     

Expression and characterization of Rv2430c, a novel immunodominant antigen of Mycobacterium tuberculosis

About 10% of the coding sequence of Mycobacterium tuberculosis corresponds to hitherto unknown members of the PE and PPE protein families which display significant sequence and length variation at their C-terminal region. It has been suggested that this could possibly represent a rich source of antigenic variation within the pathogen. We describe the purification and biophysical characterization of the recombinant PPE protein coded by hypothetical ORF Rv2430c, a member of the PPE gene family that was earlier shown to induce a strong B cell response. Expression of the recombinant PPE protein in Escherichia coli led to its localization in inclusion bodies and subsequent refolding using dialysis after its extraction from the same resulted in extensive precipitation. Therefore, an on-column refolding strategy was used, after which the protein was found to be in the soluble form. CD spectrum of the recombinant protein displayed predominantly alpha helical content (81%) which matched significantly with in silico and web-based secondary structure predictions. Furthermore, fluorescence emission spectra revealed that aromatic amino acids are buried inside the protein, which are exposed to aqueous environment under 8M urea. These results, for the first time, provide evidence on the structural features of PPE family protein which, viewed with its reported immunodominant characteristics, have implications for other proteins of the PE/PPE family.     

Shwachman-Diamond syndrome with exocrine pancreatic dysfunction and bone marrow failure maps to the centromeric region of chromosome 7

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by exocrine pancreatic insufficiency and hematologic and skeletal abnormalities. A genomewide scan of families with SDS was terminated at approximately 50% completion, with the identification of chromosome 7 markers that showed linkage with the disease. Finer mapping revealed significant linkage across a broad interval that included the centromere. The maximum two-point LOD score was 8.7, with D7S473, at a recombination fraction of 0. The maximum multipoint LOD score was 10, in the interval between D7S499 and D7S482 (5.4 cM on the female map and 0 cM on the male map), a region delimited by recombinant events detected in affected children. Evidence from all 15 of the multiplex families analyzed provided support for the linkage, consistent with a single locus for SDS. However, the presence of several different mutations is suggested by the heterogeneity of disease-associated haplotypes in the candidate region.