Complete nucleotide sequence of mouse mammary tumor virus from jyg chinese wild mice: Absence of bacterial insertion sequences in the cloned viral gag gene

Mammary tumors of a newly isolated strain of Chinese wild mouse (JYG mouse) harbor exogenous mouse mammary tumor virus (MMTV). The complete nucleotide sequence of exogenous JYG-MMTV was determined on the proviral 5' long terminal repeat (LTR)(partial)-gag-pol-env-3' LTR (partial) fragment cloned into a plasmid vector and the cDNA sequence from JYG-MMTV producing cells. Similarly to the other MMTV species the LTR of JYG-MMTV contains an open reading frame (ORF). The amino acid sequence of the JYG-MMTV ORF resembles that of SW-MMTV (92% identity) and endogenous Mtv-7 (93% identity) especially at the C-terminal region. Thus, a functional similarity in T-cell receptor V beta recognition as a superantigen is implicated among these MMTV species. Analysis of the viral gag nucleotide sequence revealed that this gene is not disrupted by the bacterial insertion sequence IS1 or IS2, which have been reported to be present in the majority of the plasmids containing the gag region. Comparison of amino acid sequences of JYG-MMTV with those of BR6-MMTV showed that over 96% of the amino acids of gag, pol, protease and env products are identical. These results suggest the intact nature of the nucleotide sequence of the near full-length MMTV genome cloned in the plasmid.     

Delayed neurotoxicity of diisopropylfluorophosphate (DFP): autoradiographic localization of high-affinity [3H]DFP binding sites in the chicken spinal cord

The delayed neurotoxic organophosphate, diisopropylfluorophosphate (DFP) binds with high affinity to membrane-bound proteins from chicken nerve tissues. The autoradiographic distribution of [³H]DFP binding sites in spinal cord sections of chicken showed higher concentrations of binding sites in gray matter than in white matter. In the cervical region, fairly high densities of [³H]DFP binding sites were found in laminae X and to a lesser extent, in the ventral horn gray matter. To identify the membrane-associated DFP-binding proteins, detergent-solubilized membranes were labeled widi 5-10nM [³H]DFP (10pmol/mg protein) for 70 min at 37°C. Gel-exclusion chromatography of the [³H]DFP-radiolabeled membranes indicated at least two major radioactive proteins with apparent molecular weights of 150-670 kDa and 40-129 kDa. Although we could not identify the high affinity DFP binding proteins, the autoradiographic experiments clearly demonstrated that the DFP binding proteins localized on gray matter of chicken spinal cord.     

Tumor scintigraphy by the method for subtracting the initial image with technetium-99m labeled antibody

The method for subtracting the initial image from the localization image was evaluated for radioimmunoscintigraphy of tumors with technetium-99m (Tc-99m) labeled antibodies. Monoclonal antibodies were parental mouse and mouse-human chimeric antibodies to carcinoembryonic antigen (CEA), designated F11-39 and ChF11-39, respectively, both of which have been found to discriminate CEA in tumor tissues from the CEA-related antigens. After reduction of the intrinsic disulfide bonds, these antibodies were labeled with Tc-99m. In vivo studies were performed on athymic nude mice bearing the human CEA-producing gastric carcinoma xenografts. Though biodistribution results showed selective and progressive accumulation of Tc-99m labeled antibodies at the tumor site, high radioactivity in blood was inappropriate for scintigraphic visualization of the tumors within a few hours. We examined the subtraction of the initial Tc-99m image from the Tc-99m localization image after a few hours. Subtracted images of the same count reflected the in vivo behavior of the Tc-99m radioactivity. The subtracted scintigrams revealed excellent tumor images with no significant extrarenal background. Visualization of the tumor site was dependent on antigen-specific binding and nonspecific exudation. These results demonstrate that a method of subtraction of the initial image may serve as a potentially useful diagnostic method for an abnormal site for agents with a low pharmacokinetic value.     

Comparison of the axonal and glial reactions between the caudal and rostral border in the cryoinjured dorsal funiculus of the rat spinal cord

Axonal and glial reactions to traumatic injury were compared between the caudal and rostral border of the lesion after freeze-injury to the C3 dorsal funiculus by attaching a liquid nitrogen-cooled copper probe to the dorsum of the rat spinal cord. The axonal and glial changes were examined up to 60 days postoperative by light and electron microscopy and immunohistochemistry for neurofilaments. Regenerative axonal changes and the appearance of numerous undifferentiated cells were found at the caudal border 7 days after cryoinjury. In contrast, such axonal and cellular reactions were scarce at the rostral border. Undifferentiated cells clearly manifested their phenotypes by differentiating into oligodendrocytes or astrocytes 11 days postinjury. The results indicated that glial cell reactions occurred in association with regenerative axonal changes at the proximal stump of the injured nerve fibers, suggesting that regenerating and demyelinated naked axons could be responsible for the appearance of the immature glial cells.     

S-1 plus cisplatin combination chemotherapy in patients with advanced non-small cell lung cancer: a multi-institutional phase II trial.

PURPOSE: To evaluate the efficacy and toxicity of a novel combination chemotherapeutic regimen including cisplatin with an oral anticancer agent, S-1 that consisted of tegafur, 5-chloro-2, 4-dihydroxypyridine, and potassium oxonate, for non-small-cell lung cancer (NSCLC) patients. EXPERIMENTAL DESIGN: In this phase II trial, patients with locally advanced and metastatic NSCLC were treated with the oral administration of S-1 at 40 mg/m(2) twice a day for 21 consecutive days while cisplatin (60 mg/m(2)) was administered intravenously on day 8. This schedule was repeated every 5 weeks. RESULTS: Of 56 patients enrolled in the study, 55 patients were eligible and analyzed. The median number of cycles administered was 3 (range, 1-12 cycles). Among these 55 patients, one complete response and 25 partial responses were observed with an overall response rate of 47% (95% confidence interval, 34-61%). The median survival time was 11 months and the 1-year survival rate was 45%. Hematologic toxicities of grades 3 and 4 included neutropenia (29%) and anemia (22%). No grade 4 nonhematologic toxicity was observed. Grade 3 toxicity included anorexia (13%), vomiting (7%), or diarrhea (7%). CONCLUSIONS: S-1 plus cisplatin combination chemotherapy showed a promising effectiveness with acceptable toxicity rates in patients with advanced NSCLC. These results warrant further investigations of this regimen including a randomized controlled trial for its use as a first line treatment for NSCLC.     

Two proliferation-related proteins, TYMS and PGK1, could be new cytotoxic T lymphocyte-directed tumor-associated antigens of HLA-A2+ colon cancer.

PURPOSE: The purpose of this work was to provide a scientific basis for specific immunotherapy of colon cancer. EXPERIMENTAL DESIGN: This study focused on identification of colon tumor-associated antigens and HLA-A2-restricted and tumor-reactive cytotoxic T lymphocytes (CTLs) generated from tumor-infiltrating lymphocytes of a colon cancer patient. A gene expression cloning method was used to identify genes coding for tumor antigens. Fifty-six peptides with HLA-A2-binding motifs encoded by these proteins were examined for their ability to induce HLA-A2-restricted and tumor-reactive CTLs. RESULTS: We identified the following three genes coding for proliferation-related proteins: thymidylate synthase (TYMS), which is involved in chemoresistance (5-fluorouracil); 5'-aminoimidazole-4-carboxamide-1-beta-d-ribonucleotide transfolmylase/inosinicase (AICRT/I); and phosphoglycerate kinase 1 (PKG1), which was secreted by tumor cells and involved in the angiogenic process. TYMS was preferentially expressed in tumor cells, whereas AICRT/I and PKG1 were equally expressed in both cancer cells and normal tissues at the mRNA level. Among 56 peptides with HLA-A2-binding motifs encoded by these proteins, 8 peptides were recognized by the CTLs, and 5 of 8 peptides were also recognized by the CTL precursors without ex vivo activation in the peripheral blood of colon cancer patients. Furthermore, four of them (one each from TYMS and PKG1 and two from AICRT/1) possessed the ability to induce HLA-A2-restricted and peptide-specific CTLs cytotoxic to colon tumor cells in peripheral blood mononuclear cells of colon cancer patients. CONCLUSIONS: TYMS and PGK1, as well as their epitope peptides, might be appropriate target molecules for specific immunotherapy of HLA-A2(+) colon cancer patients because of the positive role of TYMS and PGK1 in chemoresistance (5-fluorouracil) and angiogenesis of tumor cells, respectively.     

Significant antitumoral activity of cationic multilamellar liposomes containing human IFN-beta gene against human renal cell carcinoma.

PURPOSE: Immunotherapy is the most effective treatment against metastatic renal cell carcinoma (RCC). However, the response rate is approximately 15%. More effective therapy is, therefore, needed for patients with metastatic RCC. We then examined the antitumor effect of cationic multilamellar liposome containing human IFN-beta (huIFN-beta) gene (IAB-1) against RCC. EXPERIMENTAL DESIGN: Concentrations of huIFN-beta protein were measured by ELISA. The cytotoxicity of IAB-1 against human RCC (NC65, ACHN, and freshly isolated RCC cells), prostate and bladder cancer cell lines, and renal proximal tubule endothelial cells (RPTEC5899) was examined by the colorimetric method using tetrazolium salt. Apoptosis was assessed by the acridine-orange staining. For in vivo study, we used NC65 cells inoculated into severe combined immunodeficiency mouse. RESULTS: The RCC cells treated with IAB-1 secreted significant amounts of huIFN-beta protein continuously. Drastic in vitro cytotoxic effect of IAB-1 against RCC was observed. In contrast, treatment with 1000 IU/ml recombinant huIFN-beta protein resulted in weak cytotoxicity. The cytotoxic effect against prostate and bladder cancer cell lines was less than that against RCC. Furthermore, no significant cytotoxicity was observed in RPTEC5899 cells. Apoptosis was observed in the cells treated with IAB-1, but recombinant huIFN-beta failed to induce apoptosis. The size of NC65 tumors transfected with IAB-1 in mice was significantly smaller than that receiving injection of empty liposome or recombinant huIFN-beta protein. CONCLUSION: These findings indicate that IAB-1 may have an antitumor activity against human RCC by inducing apoptosis, suggesting its potential clinical application for gene therapy against RCC.     

Prediction of nodal metastasis by comparative genomic hybridization in biopsy specimens from patients with superficial esophageal squamous cell carcinoma.

PURPOSE: Selection of appropriate protocols for treatment of superficial esophageal squamous cell carcinoma (SESCC) is dependent on lymph node metastasis status. Therefore, it is important to know whether lymph node metastasis is present before treatment. EXPERIMENTAL DESIGN: In this study, we examined the relation between DNA sequence copy number aberrations detected by comparative genomic hybridization and lymph node metastasis in 26 surgically resected SESCCs (training samples). We then assessed whether the genetic information is predictive for nodal status in biopsy specimens from eight newly enrolled patients with SESCC (blinded samples). RESULTS: Pathological examination revealed that 17 of 26 training samples (65.4%) did not have associated lymph node metastasis. Gains of 8q24 and/or 20q12-qter were observed in 12, including all (nine of nine) with nodal metastasis. Fourteen training samples did not have gain of either 8q24 or 20q12-qter. Of the blinded samples, two showed no gain of 8q24 or 20q12-qter, and as anticipated the postoperative pathological examination revealed no nodal metastasis. The remaining six blinded samples had gains of 8q24 and/or 20q12-qter, and lymph node metastasis was detected by postoperative examination in four of these tumors. CONCLUSIONS: Absence of gains of 8q24 and/or 20q12-qter appears to be associated with absence of lymph node metastasis in patients with SESCC; therefore, less invasive surgery can be chosen.     

Depression of long term potentiation in gerbil hippocampus following postischemic hypothermia

To investigate the mechanism of chronic cell death following postischemic hypothermia, the change of N-methyl- -aspartate receptor (NMDAR) were examined by immunohistochemistry of NMDAR1 and long-term potentiation (LTP) in the CA1 subfield of the gerbil hippocampus. At 1 week following postischemic hypothermia (32°C×4 h), all CA1 neurons survived; however, immunoreactivity of NMDAR1 increased in neuronal perikarya whereas decreased in dendrites in the CA1 neurons. The abnormality was still observed in remaining CA1 neurons at 1 month after hypothermia. LTP was also significantly depressed at 1 week after hypothermia. These results suggest that some abnormalities in the glutamate receptor may be caused by ischemia; such abnormality would persist in spite of hypothermia treatment, resulting in the depression of LTP.