Global profiling of the cell surface proteome of cancer cells uncovers an abundance of proteins with chaperone function

There is currently limited data available pertaining to the global characterization of the cell surface proteome. We have implemented a strategy for the comprehensive profiling and identification of surface membrane proteins. This strategy has been applied to cancer cells, including the SH-SY5Y neuroblastoma, the A549 lung adenocarcinoma, the LoVo colon adenocarcinoma, and the Sup-B15 acute lymphoblastic leukemia (B cell) cell lines and ovarian tumor cells. Surface membrane proteins of viable, intact cells were subjected to biotinylation then affinity-captured and purified on monomeric avidin columns. The biotinylated proteins were eluted from the monomeric avidin columns as intact proteins and were subsequently separated by two-dimensional PAGE, transferred to polyvinylidene difluoride membranes, and visualized by hybridization with streptavidin-horseradish peroxidase. Highly reproducible, but distinct, two-dimensional patterns consisting of several hundred biotinylated proteins were obtained for the different cell populations analyzed. Identification of a subset of biotinylated proteins among the different cell populations analyzed using matrix-assisted laser desorption ionization and tandem mass spectrometry uncovered proteins with a restricted expression pattern in some cell line(s), such as CD87 and the activin receptor type IIB. We also identified more widely expressed proteins, such as CD98, and a sushi repeat-containing protein, a member of the selectin family. Remarkably, a set of proteins identified as chaperone proteins were found to be highly abundant on the cell surface, including GRP78, GRP75, HSP70, HSP60, HSP54, HSP27, and protein disulfide isomerase. Comprehensive profiling of the cell surface proteome provides an effective approach for the identification of commonly occurring proteins as well as proteins with restricted expression patterns in this compartment.     

Destruction of Cucumber green mottle mosaic virus by heat treatment and rapid detection of virus inactivation by RT-PCR

Heat treatment is commonly used to control viral contamination of seeds. To study virus inactivation, virus was purified from seeds contaminated with Cucumber green mottle mosaic virus (CGMMV) after various heat treatments. CGMMV particles were observed to be physically disrupted by high temperature. Analysis of viral RNA revealed that the 5' and 3' termini of the genome were protected whereas regions between 2-2.5, 3.2-3.7 and 4-4.8 kb from the 5' terminus were not. Heat inactivation of virus on seeds was confirmed by RT-PCR using primers for a heat-sensitive region. The RT-PCR approach developed here may prove preferable to time- and labor-intensive bioassays for assessing virus heat inactivation.     

Nucleotide sequences of two Korean isolates of Cucumber green mottle mosaic virus

The nucleotide sequences of the genomic RNAs of Cucumber green mottle mosaic virus Korean watermelon isolate (CGMMV-KW) and Korean oriental melon isolate (CGMMV-KOM) were determined and compared to the sequences of other tobamoviruses including CGMMV strains W and SH. Each CGMMV isolate had a genome of 6,424 nucleotides. Each also had 60 and 176 nucleotides of 5' and 3' untranslated regions (UTRs), respectively, and four open reading frames (ORF1-4). ORFs 1 to 4 encode proteins of 129, 186, 29, and 17.4 kDa, respectively. The nucleotide and deduced amino acid sequences of CGMMV-KOM and CGMMV-KW were more than 98.3% identical. When compared to other CGMMV strains in a phylogenetic analysis they were found to form a distinct virus clade, and were more distantly related to other tobamoviruses (23.5-56.7% identity).     

Molecular cloning,expression pattern and chromosomal mapping of pig CD69

CD69 is a type II membrane protein belonging to C-type lectin family receptor, and expressed on activated leukocytes. Pig CD69 was cloned by RT-PCR using degenerate primers. Pig CD69 cDNA contains a 600 bp open reading frame with its predicted polypeptide sequence of 200 amino acids. Pig CD69 has 75%, 67%, and 57% sequence identity with cow, human, and mouse CD69, respectively. A splicing isoform, which lacks exon 2 encoding the transmembrane domain, was detected. Pig CD69 gene is located on Chromosome (Chr) 5q25 where the NKG2D gene was mapped. In RT-PCR analysis, pig CD69 mRNA was detected in activated PBL, NK cells, macrophages, monocytes, and granulocytes, but not in resting cells. The inducers for CD69 gene expression were PMA, PHA, LPS, G7 mAb, PNK-E mAb, PM16-6 mAb and the K562 cell line. Moreover, CD69 mRNA is expressed in bone marrow, spleen, thymus and lymph nodes but not in muscle, mammary gland, or the pig kidney cell line (LLC-PK(1)). These results indicate that pig Chr 5q25 contains the NK gene complex and CD69 can be used as an activation marker in pig cells of innate as well as acquired immune systems.     

Histone peptide-induced nasal tolerance: suppression of murine lupus

Induced mucosal tolerance has been shown to be beneficial in preventing or treating a number of murine and human autoimmune disorders. However, this particular form of therapy has not been thoroughly tested in systemic lupus erythematosus. In this study, we investigated the conditions for induction of nasal tolerance using a histone peptide named H471 expressing a dominant T cell epitope in the histone protein H4 of mononucleosome in lupus-prone SNF(1) female mice. We also tested the effect of chronic peptide nasal treatment on the development of autoimmune reactivities in these mice. Results demonstrated that a dose-dependent nasal tolerance to peptide H471 can be achieved before or after peptide sensitization in SNF(1) mice. In addition, tolerance to mononucleosomes was induced by nasal instillation of SNF(1) mice with H471. This was accompanied by an increase in IL-10 and suppression of IFN-gamma production by lymph node cells. Suppression of Th1-type cytokines was also observed in SNF(1) mice that were nasally administered with H471 before intradermal injection with the peptide. Finally, chronic nasal instillation of mice with the H471 peptide not only suppressed the development of autoantibodies, but also altered the severity of glomerulonephritis in lupus-prone SNF(1) mice.     

Ultra scaledown to predict filtering centrifugation of secreted antibody fragments from fungal broth

Extracellularly expressed anti-hen egg lysozyme single-chain antibody fragments (scFv) produced by Aspergillus awamori were recovered using filtering centrifugation. Two filtering centrifuges with 0.5- and 30-L capacities were used to represent laboratory- and pilot-scale equipment, respectively. Critical regime analysis using the computational fluid dynamics (CFD) technique provided information about the local energy dissipation rates in both units. Experimental data indicated loss of scFv activity for energy dissipation rates above about 2.0 x 10(4) W kg(-1). This loss of activity increased in the presence of gas-liquid interfaces during filtering centrifugation. An ultra scaledown filtering centrifuge with a maximum working volume of 35 mL was designed to mimic the operating conditions identified by the critical regime analysis for the laboratory- and pilot-plant-scale units. The recovered scFv activity levels and the separation performance of the three units were comparable when operated at equal maximum energy dissipation rates.     

Biochemical characterization of oligomerization of Escherichia coli GTP cyclohydrolase I

GTP cyclohydrolase I (E.C. 3.5.4.16) is a homodecameric protein that catalyzes the conversion of GTP to 7,8- dihydroneopterin triphosphate (H(2)NTP), the initial step in the biosynthesis of pteridines. It was proposed that the enzyme complex could be composed of a dimer of two pentamers, or a pentamer of tightly associated dimers; then the active site of the enzyme was located at the interface of three monomers (Nar et al. 1995a, b). Using mutant enzymes that were made by site-directed mutagenesis, we showed that a decamer of GTP cyclohydrolase I should be composed of a pentamer of five dimers, and that the active site is located between dimers, as analyzed by a series of size exclusion chromatography and the reconstitution experiment. We also show that the residues Lys 136, Arg139, and Glu152 are of particular importance for the oligomerization of the enzyme complex from five dimers to a decamer.     

Suprageneric classification of peptidoglycan group B actinomycetes by nucleotide sequencing of 5S ribosomal RNA

5S ribosomal RNA sequences were determined for thirteen actinomycetes mainly representatives with the rare group B type peptidoglycan. The primary and secondary structure of the resultant sequences were of the type characteristic of Gram-positive bacteria with DNA rich in guanine plus cytosine. The sequencing and associated chemotaxonomic data provide compelling grounds for classifying actinomycetes with a group B type peptidoglycan in a single family. The familyMicrobacteriaceae fam. nov. is proposed to accommodate actinomycetes classified in the generaAgromyces, Aureobacterium, Clavibacter, Curtobacterium andMicrobacterium.     

A comparison between primary and secondary toe-to-hand transplantation

Although primary toe-to-hand transplantation is performed with increasing frequency, its use is still controversial because of the lack of any comparative studies documenting its safety and efficacy. Between August of 1990 and December of 1993, 175 consecutive toe-to-hand transplantations for crush and avulsion injuries were performed in 122 patients. The average interval between injury and primary reconstruction was 7 days, and the average interval between injury and secondary reconstruction was 10.7 months. Follow-up ranged from 18 to 91 months, with an average follow-up of 58 months. There were 31 primary transplantations and 144 secondary transplantations. The survival rate was 96.8 percent (30 of 31) for primary reconstruction and 96.5 percent (139 of 144) for secondary reconstruction. Intraoperative anastomotic revision was necessary in 3.2 percent (one of 31) of primary transplantations and 7.6 percent (11 of 144) of secondary transplantations. Three primary toe-to-hand transplantations (9.7 percent) and 17 secondary toe-to-hand transplantations (11.8 percent) were re-explored in the postoperative period. Each group had one superficial infection. The infection rate was 6.5 percent and 0.7 percent in the primary and secondary groups, respectively. Other complications included partial skin loss, which occurred in one patient (3.2 percent) in the primary group and six patients (4.2 percent of 144 transplantations) in the secondary group. Secondary procedures to improve function were necessary in six secondary transplantations (4.2 percent) and in none of the primary transplantations. There was no statistical difference between the two groups in terms of survival, intraoperative anastomotic revision, re-exploration, future secondary procedure, infection, and complications. This series demonstrates that primary toe-to-hand transplantation can be performed in the suitable candidate safely with as much success as secondary reconstruction. Primary toe transplantation can potentially reduce the overall period of recovery and rehabilitation, allowing the patient to return to work sooner. Further study to evaluate and compare the final functional outcome and return to work time between primary and secondary toe-to-hand transplantation is needed.