Correlation of cyclooxygenase 2 expression and inflammatory cells infiltration in colorectal cancer.

This study want to determine correlation of cyclooxygenans 2 (Cox-2) expression and inflammatory reaction in colorectal carcinoma. Archival H and E slides of 150 patients with primary colorectal carcinoma were reviewed to confirm pathological feature and to select suitable tissue blocks for immunohistochemical staining with mouse monoclonal antibody against human Cox-2 and various inflammatory cells. After scoring, statistical analysis were carried out with SPSS software, chi2 methods and bivariate pearson correlation analysis. The expression of Cox-2 (grade 3 to 7) in 71.3% of patients associated with mast cells, neutrophils, eosinophils, macrophages, CD3+ lymphocytes infiltration was significant (p = 0.001). Correlation of Cox-2 expression associated with CD8+ lymphocytes infiltration was not significant (p = 0.569). Also CD3+ lymphocytes show severe infiltration when the expression of Cox-2 is negative (p<0.05). The main purpose of this study was to evaluate the interaction between the Cox-2 expression and inflammatory cells infiltration. This study showed that there is a close relationship of Cox-2 expression and mast cells, neutrophils, eosinophils, macrophages and CD3+ lymphocytes. The only exception was CD8+ lymphocytes. It is may be due to independent role of anti tumoral effect of this inflammatory cells.

Novel type Arabidopsis thaliana H(+)-PPase is localized to the Golgi apparatus.

Vacuolar H(+)-PPase, a membrane bound proton-translocating pyrophosphatase found in various species including plants, some protozoan and prokaryotes, has been demonstrated to be localized to the vacuolar membrane in plants. Using a GUS reporter system and a green fluorescent protein (GFP) fusion protein, we investigated the tissue distribution and the subcellular localization, respectively, of a novel type H(+)-PPase encoded by AVP2/AVPL1 identified in the Arabidopsis thaliana genome. We showed that AVP2/AVPL1 is highly expressed at the trichome and the filament of stamen. Furthermore, the fluorescence of GFP-tagged AVP2/AVPL1 showed small dot-like structures that were observed throughout the cytoplasm of various Arabidopsis cells under a fluorescent microscope. The distribution of this dot-like fluorescent pattern was apparently affected by a treatment with brefeldin A. Moreover, we demonstrated that most dot-like fluorescent structures colocalized with a Golgi resident protein. These findings suggest that this novel type H(+)-PPase resides on the Golgi apparatus rather than the vacuolar membrane.

Characterization of influenza virus NS1 protein by using a novel helper-virus-free reverse genetic system.

We have developed a novel helper-virus-free reverse genetic system to genetically manipulate influenza A viruses. The RNPs, which were purified from the influenza A/WSN/33 (WSN) virus, were treated with RNase H in the presence of NS (nonstructural) cDNA fragments. This specifically digested the NS RNP. The NS-digested RNPs thus obtained were transfected into cells together with the in vitro-reconstituted NS RNP. The NS-digested RNPs alone did not rescue viruses; however, cotransfection with the NS RNP did. This protocol was also used to rescue the NP transfectant. We obtained two NS1 mutants, dl12 and N110, using this protocol. The dl12 NS gene contains a deletion of 12 amino acids at positions 66 to 77 near the N terminus. This virus was temperature sensitive in Madin-Darby bovine kidney (MDBK) cells as well as in Vero cells. The translation of all viral proteins as well as cellular proteins was significantly disrupted during a later time of infection at the nonpermissive temperature of 39 degrees C. The N110 mutant consists of 110 amino acids which are the N-terminal 48% of the WSN virus NS1 protein. Growth of this virus was significantly reduced at any temperature. In the virus-infected cells, translation of the M1 protein was reduced to 10 to 20% of that of the wild-type virus; however, the translation of neither the nucleoprotein nor NS1 was significantly interfered with, indicating the important role of NS1 in translational stimulation of the M1 protein.

Influenza virus hemagglutinin and neuraminidase glycoproteins stimulate the membrane association of the matrix protein.

We have analyzed the mechanism by which the matrix (M1) protein associates with cellular membranes during influenza A virus assembly. Interaction of the M1 protein with the viral hemagglutinin (HA) or neuraminidase (NA) glycoprotein was extensively analyzed by using wild-type and transfectant influenza viruses as well as recombinant vaccinia viruses expressing the M1 protein, HA, or NA. Membrane binding of the M1 protein was significantly stimulated at the late stage of virus infection. Using recombinant vaccinia viruses, we found that a relatively small fraction (20 to 40%) of the cytoplasmic M1 protein associated with cellular membranes in the absence of other viral proteins, while coexpression of the HA and the NA stimulated membrane binding of the M1 protein. The stimulatory effect of the NA (>90%) was significant and higher than that of the HA (>60%). Introduction of mutations into the cytoplasmic tail of the NA interfered with its stimulatory effect. Meanwhile, the HA may complement the defective NA and facilitate virus assembly in cells infected with the NA/TAIL(-) transfectant. In conclusion, the highly conserved cytoplasmic tails of the HA and NA play an important role in virus assembly.

Influenza virus NS1 protein stimulates translation of the M1 protein.

The influenza virus NS1 protein was shown to stimulate translation of the M1 protein. M-CAT RNA, which contains the chloramphenicol acetyltransferase (CAT) reporter gene and the terminal noncoding sequence of segment 7 (coding for the M1 and M2 proteins), was ribonucleoprotein transfected into clone 76 cells expressing the influenza virus RNA polymerase and NP proteins required for the transcription and replication of influenza virus ribonucleoproteins. When the cells were superinfected with a recombinant vaccinia virus which expresses the NS1 protein, CAT expression from the M-CAT RNA was significantly stimulated but transcription was not altered. The expression of NS-CAT RNA, which contains noncoding sequences of segment 8 (coding for the NS1 and NS2 proteins), was not altered by the NS1 protein. Site-directed mutagenesis showed that the sequence GGUAGAUA upstream of the initiation codon on segment 7 was required for stimulation.

An influenza virus temperature-sensitive mutant defective in the nuclear-cytoplasmic transport of the negative-sense viral RNAs.

An influenza A virus mutant ts-51, which contains a temperature-sensitive (ts) defect in the genome encoding the M1 and M2 proteins, was characterized. Nucleotide sequencing of the M segment revealed a predicted single amino acid change of phenylalanine to serine at amino acid position 79 in the M1 protein. The nuclear-cytoplasmic transport of the negative-sense viral RNAs (vRNAs) was then investigated using an in situ hybridization technique. At 6 hr after the ts-51 virus infection, approximately 95% of the vRNAs were accumulated in the nucleus at a non-permissive temperature, when approximately 50% of the vRNAs were transported into the cytoplasm in the wild-type virus-infected cells. The M1 protein of the ts-51 virus was also accumulated in the nucleus under the same conditions. Therefore, the M1 protein of the ts-51 virus may be associated with the vRNPs, but the possible M1-vRNP complex thus formed was defective in the nuclear-cytoplasmic transport and the single amino acid change in the M1 protein was responsible for this defect.

Biological function of recombinant IL-6 expressed in a baculovirus system.

The cDNA of human interleukin-6 (IL-6) was cloned into baculovirus DNA. Insect cells (Spodoptera frugiperda cells) infected with the recombinant baculovirus secreted a large amount of 22K protein into the culture medium. This culture fluid contained high biological activity of growth stimulation of a mouse myeloid cell line (MH-60). The IL-6 was purified by a one-step procedure employing immunoaffinity chromatography of monoclonal antibody to IL-6. The specific activities (BSF-2 reference units/mg protein) of the original culture medium and the purified material from the one-step purification were 0.3-1 x 10(6) and greater than 2 x 10(7), respectively. The highly purified IL-6 could not induce an antiviral state in human diploid fibroblast (FS-4) cells.

Proposal of standardized methods and reference for assaying recombinant human tumor necrosis factor.

Two assay methods for recombinant human tumor necrosis factor (rH-TNF) were developed, one a biological L-cell assay and the other an enzyme-linked immunosorbent assay. The accuracy and reproducibility of each and the correlation between the two were studied. As a result of this investigation, the two assay methods were found appropriate for standardization of rH-TNF. A freeze-dried reference was prepared, and examination of its potency and stability showed it to be suitable for use as a reference standard for rH-TNF assays.