Yukio Nagamachi

Peptide Transporter in the Rat Small Intestine: Ultrastructural Localization and the Effect of Starvation and Administration of Amino Acids

Peptide transporter-1 is a H+/peptide cotransporter responsible for the uptake of small peptides and peptide-like drugs, and is present in the absorptive epithelial cells of the villi in the small intestine (duodenum, jejunum, and ileum). It has been localized to the apical microvillous plasma membrane of the absorptive epithelial cells of the rat small intestine using the immunogold electron microscopic technique. Digital image analysis of the jejunum revealed that the transporter protein was abundant at the tip of the villus and that the amount decreased from the tip of the villus to its base. The effect of dietary administration of amino acids and starvation on the expression of PepT1 in the jejunum was examined by immunoblotting and image analysis of immunofluorescence. Starvation markedly increased the amount of peptide transporter present, whereas dietary administration of amino acids reduced it. The gradient of the transporter protein along the crypt-villus axis was maintained under either condition. These observations show that it is specific to the microvillous plasma membrane and that its expression is regulated by the nutritional condition.

Increased phospholipase D activity in human breast cancer

Phospholipase D is believed to play an important role in cell proliferation and tumorigenesis. One of its major functions is to cause a sustained activation of protein kinase C through the primary production of phosphatidic acid from phosphatidylcholine by the enzyme, followed by dephosphorylation forming diacylglycerol. Protein kinase C is known to be activated or translocated in some tumors including breast tumors. In order to examine phospholipase D activity in breast tumors, surgical specimens of human breast tumors were obtained by mastectomy or wide excision, and their phospholipase D activities were assayed by determining the formation of phosphatidylethanol from phosphatidylcholine and ethanol. Phospholipase D activity was predominantly localized in the microsomal fraction of the tumor tissue and markedly stimulated by oleic acid. We observed a significant increase in phospholipase D activity in 17 out of 19 spontaneous human breast tumors as compared to adjacent histologically normal breast tissue. The mean specific activity in the tumors was 52.9±41.8 (SD) pmol min−1 mg protein−1 whereas the value for the normal breast tissue was 34.0±36.2 (SD) pmol min−1 mg protein−1 (P<0.01; paired Wilcoxons rank-sum test). The mean tumor/normal activity ratio was 2.37. Among prognostic factors, the nuclear grade, evaluated according to Schnitt et al., was found to be correlated with the activity ratio. Our results suggest a role for phospholipase D in human breast tumors. An elevation in phospholipase D activity is useful as a potential marker for malignant disease in the breast.

Carcinoembryonic antigen levels in peritoneal washings can predict peritoneal recurrence after curative resection of gastric cancer

Carcinoembryonic antigen (CEA) levels were determined in the peritoneal washings from 120 patients with gastric cancer and 9 patients with benign diseases. Elevated values (> 100 ng/g of protein) were observed in 20 of 25 patients with gastric cancer with visible dissemination and 16 of 25 patients with serosal invasion but no dissemination. The same elevation was found in only 9 of 70 patients with no serosal invasion and none of the patients with benign disease. The 2‐year survival rates after curative operation for the patients with and without elevation of CEA levels were 21% (19 patients) and 100% (66 patients), respectively (P < 0.001). A negative correlation was found between CEA levels and survival times after noncurative operation. These results indicate that the CEA level in peritoneal washings could be a sensitive detector of invisible peritoneal dissemination and a new predictor for the postoperative prognosis of gastric cancer.

Genomic structure and chromosomal localization of the mouse Ogg1 gene that is involved in the repair of 8-hydroxyguanine in DNA damage

Abstract8-Hydroxyguanine (7,8-dihydro-8-oxoguanine: oh8Gua) is a damaged form of guanine induced by oxygen-free radicals and causes GC to TA transversions. Previously we isolated the hOGG1 gene, a human homolog of the yeast OGG1 gene, which encodes a DNA glycosylase and lyase to excise oh8Gua in DNA. In this study, we isolated a mouse homolog (Ogg1) of the OGG1 gene, characterized oh8Gua-specific DNA glycosylase/AP lyase activities of its product, and determined chromosomal localization and exon-intron organization of this gene. A predicted protein possessed five domains homologous to human and yeast OGG1 proteins. Helix-hairpin-helix and C2H2 zinc finger-like DNA-binding motifs found in human and yeast OGG1 proteins were also retained in mouse Ogg1 protein. The properties of a GST fusion protein were identical to human and yeast OGG1 proteins in glycosylase/lyase activities, their substrate specificities, and suppressive activities against the spontaneous mutagenesis of an Escherichia coli mutM mutY double mutant. The mouse Ogg1 gene was mapped to Chromosome (Chr) 6, and consisted of 7 exons approximately 6 kb long. Two DNA-binding motifs were encoded in exons 4 through 5. These data will facilitate the investigation of the OGG1 gene to elucidate the relationship between oxidative DNA damage and carcinogenesis.

8-Hydroxydeoxyguanosine levels in DNA of human breast cancers are not significantly different from those of non-cancerous breast tissues by the HPLC-ECD method

8-Hydroxydeoxyguanosine (oh8dG) is a promutagenic DNA lesion produced by oxygen radicals, and a high level of 8-hydroxyguanine in breast cancers was previously demonstrated by the gas chromatography-mass-spectrometry method. To confirm the previous observation, the oh8dG levels of DNA of 22 breast cancers and corresponding adjacent non-cancerous breast tissues were analyzed by high performance liquid chromatography-electrochemical detector (HPLC-ECD) system, and the correlation of the oh8dG levels in breast cancer DNAs with clinical and immunohistochemical parameters was examined. However, the levels of oh8dG in DNA of breast cancers are not significantly different from those of corresponding non-cancerous breast tissues (P = 0.084) by the HPLC-ECD method. Furthermore, the oh8dG levels in breast cancers were not associated with p53 and erbB-2 immunoreactions, with expression of estrogen and progesterone receptors, and with clinical stage and histological grade. Thus, in contrast to the previous data, the present study using the HPLC-ECD method does not indicate an increase of oh8dG levels in breast cancers.

The study of tumor necrosis factor beta gene polymorphism in lung cancer patients

Background. In recent years numerous reports have discussed the relationship between the human leukocyte antigen and lung cancer. However, the genetic background of lung cancer has not yet been precisely clarified.

Quantification of serum-soluble HLA class I antigens in patients with gastric cancer

The amount of sHLA-I in serum was examined in 74 patients with gastric cancer and 15 normal healthy controls. For mAbs, W6/32 specific for HLA-A, -B, -C, and biotin IOT2 specific for HLA class I associated with beta 2 microglobulin, were used to determine the values of sHLA-I using an ELISA. The patients in stage-IV gastric cancer showed lower values of sHLA-I (445.4 +/- 247.1 ng/ml) than those in stage I (725.9 +/- 575.8 ng/ml), stage II (752.8 +/- 255.0 ng/ml), and normal controls (868.9 +/- 715.0 ng/ml) (P < 0.05). In analysis of the patients with HLA-A24, the allele that has been reported to secrete more sHLA-I than other alleles, the results were nearly the same. These results suggest that the secretion of sHLA-I is low in patients with very advanced cancer. However, there was no correlation between the sHLA-I level and the metastasis or prognosis in longitudinal studies in 11 patients.

Infrequent Overexpression of p53 Protein in Epstein‐Barr Virus‐associated Gastric Carcinomas

Epstein‐Barr virus (EBV) infection was studied in a total of 412 patients with poorly differentiated gastric adenocarcinoma by in situ hybridization for EBV‐encoded small RNA. EBV‐speciflc RNA was detected in tumor cell nuclei of 83 (20.1%) of 412 gastric carcinomas, of which 60 were histologically subclassified as gastric carcinoma with lymphoid stroma (GCLS). All EBV‐positive gastric carcinomas as well as 90 randomly selected EBV‐negative gastric carcinomas were further studied for p53 protein expression by immunohistocbemistry. The Overexpression of p53 protein was demonstrated in only 7 (8.4%) of 83 EBV‐positive gastric carcinomas. This was in marked contrast to the frequency of 34.4% in EBV‐negative gastric carcinomas. In addition, a few p53‐positive nuclei were characteristically scattered in the tumors of many EBV‐positive GCLS, but this was not regarded as p53 Overexpression arising from mutation of the gene. Our findings suggested that EBV‐associated gastric carcinomas may arise through a different mechanism from other types of gastric carcinomas without EBV infection.

Peritoneal interleukin‐6, interleukin‐8 and granulocyte elastase activity after elective abdominal surgery

In this study we investigated the interleukin‐8 concentration (IL‐8) and granulocyte elastase activity (GE) after elective abdominal surgery. Postoperative interleukin‐6 (IL‐6), IL‐8 concentrations and GE in the peritoneal fluid were examined in 27 patients who underwent various types of elective abdominal surgery. We compared these results with clinical parameters of surgical stress, operating time (OT) and blood loss during the operation (BL). P‐IL‐6 and P‐IL‐8 were significantly correlated with OT (P‐IL‐6; r=0.67, P<0.001: P‐IL‐8; r=0.59, P<0.001) and BL (P‐IL‐6; r=0.61, P<0.001: P‐IL‐8: r= 0.48, P<0.01). P‐IL‐8 was significantly correlated with P‐IL‐6 (r=0.68, P<0.001) and there was a positive correlation between GE and P‐IL‐8 (r=0.37, P<0.05). These findings indicate that IL‐8 might activate granulocytes in the peritoneal cavity after elective abdominal surgery and that assaying P‐IL‐6 and P‐IL‐8 is useful in assessing the hosts response to surgical stress.

Rupture of the Spleen in the Newborn: Treatment Without Splenectomy

In the newborn, traumatic rupture of the spleen is rare. Only 24 survivors have been reported in the world literatures, including four cases which have appeared in the Japanese literature. All were treated by splenectomy.‘-3 In view of the danger of overwhelming infection following splenectomy in early childhood,4 preserving whatever splenic tissue remains vascularized is indicated. SeraS reported two infants in whom pneumococcal meningitis occurred after splenectomy in the neonatal period for splenic rupture. The first survivor of a ruptured spleen treated by repair of the organ is reviewed.