Rapid Determination of Chimerism Status Using Dihydrorhodamine Assay in a Patient with X-linked Chronic Granulomatous Disease Following Hematopoietic Stem Cell Transplantation

Chronic granulomatous disease (CGD) is a rare genetic disease, which is caused by defects in the NADPH oxidase complex (gp91phox, p22phox, p40phox, p47phox, and p67phox) of phagocytes. This defect results in impaired production of superoxide anions and other reactive oxygen species (ROS), which are necessary for killing bacterial and fungal microorganisms and leads to recurrent, life-threatening bacterial and fungal infections and granulomatous inflammation. The dihydrorhodamine (DHR) flow cytometry assay is a useful diagnostic tool for CGD that can detect absent or reduced NADPH oxidase activity in stimulated phagocytes. We report a patient with X-linked CGD carrying a novel mutation of the CYBB gene whose chimerism status following hematopoietic stem cell transplantation (HSCT) has been rapidly determined using the DHR assay. The level of DHR activity correlates well with short tandem repeat PCR analysis. Considering the advantages of this simple, rapid, and cost-effective procedure, serial measurement of DHR assay would facilitate the rapid determination of a patient's engraftment status, as a supplementary monitoring tool of chimerism status following HSCT.

p47phox, the phagocyte NADPH oxidase/NOX2 organizer: structure, phosphorylation and implication in diseases

Phagocytes such as neutrophils play a vital role in host defense against microbial pathogens. The anti-microbial function of neutrophils is based on the production of superoxide anion (O2(.-)), which generates other microbicidal reactive oxygen species (ROS) and release of antimicrobial peptides and proteins. The enzyme responsible for O2(.-) production is called the NADPH oxidase or respiratory burst oxidase. This multicomponent enzyme system is composed of two transmembrane proteins (p22phox and gp91phox, also called NOX2, which together form the cytochrome b(558)) and four cytosolic proteins (p47phox, p67phox, p40phox and a GTPase Rac1 or Rac2), which assemble at membrane sites upon cell activation. NADPH oxidase activation in phagocytes can be induced by a large number of soluble and particulate agents. This process is dependent on the phosphorylation of the cytosolic protein p47phox. p47phox is a 390 amino acids protein with several functional domains: one phox homology (PX) domain, two src homology 3 (SH3) domains, an auto-inhibitory region (AIR), a proline rich domain (PRR) and has several phosphorylated sites located between Ser303 and Ser379. In this review, we will describe the structure of p47phox, its phosphorylation and discuss how these events regulate NADPH oxidase activation.

Proteinuria in a Boy with Infectious Mononucleosis, C1q Nephropathy, and Dent's Disease

C1q nephropathy is a proliferative glomerulopathy with extensive mesangial deposition of C1q. A three-year old boy presented with a nephrotic-range proteinuria during an acute phase of Epstein-Barr virus (EBV) infection, and he had a family history of Dent's disease. The renal biopsy findings were compatible with C1q nephropathy. However, EBV in situ hybridization was negative. The CLCN5 gene analysis revealed an R637X hemizygous mutation, which was the same as that detected in his maternal cousin, the proband of the family. The causal relationship between EBV infection and C1q nephropathy remains to be determined. Moreover, the effects of underlying Dent's disease in the process of C1q nephropathy has to be considered.

Specific structural features of syndecans and heparan sulfate chains are needed for cell signaling

The syndecans, heparan sulfate proteoglycans, are abundant molecules associated with the cell surface and extracellular matrix and consist of a protein core to which heparan sulfate chains are covalently attached. Each of the syndecan core proteins has a short cytoplasmic domain that binds cytosolic regulatory factors. The syndecans also contain highly conserved transmembrane domains and extracellular domains for which important activities are becoming known. These protein domains locate the syndecan on cell surface sites during development and tumor formation where they interact with other receptors to regulate signaling and cytoskeletal organization. The functions of cell surface heparan sulfate proteoglycan have been centered on the role of heparan sulfate chains, located on the outer side of the cell surface, in the binding of a wide array of ligands, including extracellular matrix proteins and soluble growth factors. More recently, the core proteins of the syndecan family transmembrane proteoglycans have also been shown to be involved in cell signaling through interaction with integrins and tyrosine kinase receptors.

Cloning and sequence analysis of the Antheraea pernyi nucleopolyhedrovirus gp64 gene.

Frequent outbreaks of the purulence disease of Chinese oak silkworm are reported in Middle and Northeast China. The disease is produced by the pathogen Antheraea pernyi nucleopolyhedrovirus (AnpeNPV). To obtain molecular information of the virus, the polyhedra of AnpeNPV were purified and characterized. The genomic DNA of AnpeNPV was extracted and digested with HindIII. The genome size of AnpeNPV is estimated at 128 kb. Based on the analysis of DNA fragments digested with HindIII, 23 fragments were bigger than 564 bp. A genomic library was generated using HindIII and the positive clones were sequenced and analysed. The gp64 gene, encoding the baculovirus envelope protein GP64, was found in an insert. The nucleotide sequence analysis indicated that the AnpeNPV gp64 gene consists of a 1,530 nucleotide open reading frame (ORF), encoding a protein of 509 amino acids. Of the eight gp64 homologues, the AnpeNPV gp64 ORF shared the most sequence similarity with the gp64 gene of Anticarsia gemmatalis NPV, but not Bombyx mori NPV. The upstream region of the AnpeNPV gp64 ORF encoded the conserved transcriptional elements for early and late stage of the viral infection cycle. These results indicated that AnpeNPV belongs to group I NPV and was far removed in molecular phylogeny from the BmNPV.

Cloning of TLR3 Isoform

Toll-like receptor (TLR) 3 is a member of the TLR family that confers innate immunity by recognizing viral pathogens. Herein, we report that the TLR3 isoform is expressed on human primary cells and cell lines. This isoform has 2, 520 bp cDNAs compared to the 2, 712 bp of full cDNA, is produced by deletion of an intron-like sequence within exon 4 and is co-expressed with wild type TLR3 in primary human astrocytes and glioblastoma cell lines. This finding suggests the TLR3 isoform in astrocytes may have a different immunological role for binding ligands during the immune response in brain.

Molecular characterization of full-length genome of Japanese encephalitis virus (KV1899) isolated from pigs in Korea

We have determined the complete nucleotide and deduced amino acid sequences of the Japanese encephalitis virus (JEV) strain KV1899, isolated from a fattening pig in Korea. In comparison with 22 fully sequenced JEV genomes currently available, we found that the 10,963-nucleotide RNA genome of KV1899 has a 13-nucelotide deletion in the 3' non-translated variable region and 53 unique nucleotide sequences including 3' non-translated region (NTR). Its single open reading frame has a total of 28 amino acid substitutions. Comparison of the KV1899 genomic sequence with those of the 21 fully sequenced JEV strains in published databases showed nucleotide homology ranging from 97.4% (Ishikawa strain) to 87.0% (CH2195 strain). Amino acid homology with KV1899 strain ranged from 96.4% (K94P05) to 91.0% (GP78). The KV1899 showed the highest nucleotide homology with Ishikawa strain and the highest amino acid homology with K94P05. We performed an extensive E gene based phylogenetic analysis on a selection of 41 JEV isolates available from the GenBank. Compared with Anyang strain, isolated from a pig in 1969, that is current live vaccine strain for swine in Korea, the homology of nucleotide sequence in envelope gene was only 87.1%. The prM gene of the isolate was closely related with those of Ishikawa and K94P05 strains, which were grouped into genotype I of JEV.

Loss of virus specific epitopes on JE virus 'E' glycoprotein by acetone treatment.

BACKGROUND & OBJECTIVES: Some Japanese encephalitis (JE) virus strains have been placed in group II based on the loss of reactivity against Hs (H = HI positive; s = JE virus specific) group of monoclonal antibodies (MAbs) in haemagglutination-inhibition (HI) test employing sucrose acetone (SA) extracted antigens. Also acetone-fixation of cells infected with some of the virus strains results in the loss of immunofluorescence (IF) against virus specific MAbs. The present study was undertaken to elucidate the effect of acetone on virus specific haemagglutination (HA) epitopes expressed on 'E' glycoprotein of group II strains of JE virus. METHODS: Porcine kidney (PS) cells were infected with JE virus strains (2 group I Indian strains, 5 group II strains and one neutralization-escape variant of 733913 group I strain). HI and complement fixation (CF) tests were carried out employing both polyethylene glycol (PEG) precipitated and SA extracted antigens of JE virus. RESULTS: Employing PEG precipitated antigens, Indian strain G9473 showed titres ranging from 1:40 to 1:160 against all the four virus specific HsMAbs and strain 641686 (1:160) with one of the four MAbs (Hs-1) by HI test whereas their SA extracted antigens did not react at all. In contrast, CF was positive employing both SA and PEG antigens in the presence of all four HsMAbs. The reactivity shown by PEG antigens in the HI test was confirmed by blocking the HA activity with the respective MAb. SA antigens, though negative in the HI test, were positive by the blocking assay. Interestingly, some of the non-HI MAbs which were negative against SA antigens, showed positive HI reaction with PEG antigens. Also, additional epitopes on Japanese (Yoken), Sri Lankan (691004) and two Indian (755468 and 641686) JE virus strains were detected either by blocking HA or surface IF. INTERPRETATION & CONCLUSIONS: It seems that the acetone treatment might result in HA property of the antigen which is no longer inhibited by an antibody in the HI test. The characterization of such labile and conformation-dependent epitopes is currently been undertaken to elucidate their role either in protection or immunopathogenesis of JE.

Expression and characterization of the flavoprotein domain of gp91phox

Truncated forms of gp91(phox) were expressed in E. coli in which the N-terminal hydrophobic transmembrane region was replaced with a portion of the highly soluble bacterial protein thioredoxin (TRX). TRX-gp91(phox) (306-569), which contains the putative FAD and NADPH binding sites, showed NADPH-dependent NBT (nitroblue tetrazolium) reductase activity, whereas TRX-gp91(phox) (304-423) and TRX-gp91(phox) (424-569) were inactive. Activity saturated at about a 1:1 molar ratio of FAD to TRX-gp91(phox) (306- 569), and showed the same Km for NADPH as that for superoxide generating activity by the intact enzyme. Activity was not inhibited by superoxide dismutase, indicating that it was not mediated by superoxide, but was blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium (DPI). In the presence of Rac1, the cytosolic regulatory protein p67(phox) stimulated the NBT reductase activity, but p47(phox) had no effect. Truncated p67(phox) containing the activation domain (residues 199- 210) stimulated activity approximately 2-fold, whereas forms mutated or lacking this region failed to stimulate the activity. Our data indicate that: 1) TRX-gp91(phox) (306-569) contains the binding sites for both pyridine and flavin nucleotides; 2) this flavoprotein domain shows NBT reductase activity; and 3) the flavin-binding domain of gp91(phox) is the target of regulation by the activation domain of p67(phox).

A novel 200 kDa plasma membrane glycoprotein with high basal tyrosine kinase activity in tumour cells.

We have detected a tyrosine phosphorylated 200 kDa glycoprotein (gp200) on the surface of two tumour cells of neural origin, namely A1235 glioma and A172 glioblastoma. gp200 (polypeptide mass of 165-170 kDa) has all the structural features of a growth factor receptor and it appears to display high basal tyrosine kinase activity, a characteristic associated with transforming proteins. Another interesting feature of gp200 is that it is immunologically highly related to the EGF receptor (polypeptide mass of 135 kDa), a member of the erb-B family of proteins; however, it lacks EGF binding activity. gp200 also differs from all other EGF-receptor-related oncogenic proteins, namely erb-B-2, erb-B-3 and erb-B-4 gene products, and hence appears to be yet another member of the erb-B family of proteins. This is further strengthened by the fact that both gp200 and the EGF receptor are also recognized by a conformation-specific anti-peptide antibody to the cytoplasmic domain of the beta-type PDGF receptor. In the EGF- and the PDGF receptors, this peptide epitope is cryptic and receptor phosphorylation unmasks this site [Panneerselvam K, Reitz H, Khan S A, and Bishayee S (1995) J Biol Chem 270, 7975-7979] indicating that this epitope might be important in biological message transmission. In this context, the expression of a novel EGF-receptor-related 200 kDa protein with high basal kinase activity in certain tumour cells of neural origin and the fact that it contains an important peptide epitope suggest its possible role in normal and abnormal cell growth.

Proteoglycans in skeletal muscle

1. Proteoglycans are macromolecules composed of a protein and one or mor chains of sulfated carbohydrates, the glycosaminoglycans. Proteoglycans are found on the cell surface and in the extracellular matrix participating in the cell-cell-extracellular matrizx interaction. In this review I present the information accumulated in the past years regarding the presence, characteristics, localization, control of expression and alteration in some pathological states of skeletal muscle proteoglycans. 2. This review presents and discusses current information in this area and some projections for the future in four sections: first, the proteoglycans present in embryonic cells and cell lines from skeletal muscle. Second, the presence of proteoglycans in adult skeletal muscles. Third, the regulation of the expression of skeletal muscle proteoglycans, and fourth, skeletal muscle proteoglycans in pathological conditions

Developmentally-regulated cell surface N-linked oligosaccharides participate in intercellular cohesion in Dictyostelium discoideum.

The ability of purified plasma membrane glycoconjugates to inhibit the EDTA-resistant agglutination between aggregation-stage cells of Dictyostelium discoideum has suggested that receptor binding of these glycoconjugates provides a basis for cell-cell cohesion during aggregation. This has been tested by analysis of a series of mutants with different defects in the assembly of N-linked oligosaccharides. Mutant HL241 lacks outer branch components of N-linked oligosaccharides and fails to aggregate or express EDTA-resistant cohesion. HL244 makes unsulphated but otherwise normal N-linked oligosaccharides, generates multiple tips on aggregated cell mounds in some clones, and shows abnormally strong EDTA-resistant cohesion. Two mutants that are temperature-sensitive for complete processing of N-linked oligosaccharides are also temperature-sensitive for expression of both aggregation ability and EDTA-resistant cohesion. A revertant that recovered essentially normal N-linked oligosaccharide processing at the restrictive temperature has also recovered its ability to aggregate and to agglutinate in EDTA.

Characterization of concanavalin A-binding neuronal and glial surface glycoproteins from human foetal brain.

Neuronal and glial surface glycoproteins have been isolated from human foetal brains by affinity chromatography on 8 M urea or 6 M guanidine-treated Con A-Sepharose 4B at 4 degrees C and three groups of glycoproteins of molecular mass 65-73 kDa, 52-63 kDa and 43-48 kDa have been identified on SDS/PAGE. These glycoproteins exhibited anomalous behaviour on SDS/PAGE, indicating the existence of a gradation of mutually interconvertible protein-SDS aggregates in dynamic equilibrium with one another. Deglycosylation and deacylation did not alter the SDS/PAGE multiple band pattern. Purified glycoproteins contained 160 +/- 90 micrograms carbohydrate/mg protein, and a sialic acid content of 25 +/- 5 nmole/mg protein. The N-terminals were blocked. The glycoproteins moved preferentially on acid/urea/PAGE. Sepharose 6B gel filtration in the absence of lipid and detergents resolved the glycoproteins into an excluded peak I and a low molecular mass peak II. Peaks I and II were non-interconvertible on Sepharose 6B gel filtration or on reversed phase HPLC in an isopropanol/water/TFA gradient system. Both peaks rendered a single fast moving band of identical mobility on acid/urea/PAGE, suggesting that peak I was possibly a micellar aggregate of the monomeric peak II. The glycoproteins were refractory to digestion by trypsin or pronase and reacted identically towards various lectins.(ABSTRACT TRUNCATED AT 250 WORDS)